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Agro FOOD Industry Hi Tech is a peer-reviewed journal

devoted to functional food and nutraceuticals

addressed to a readership belonging to the industry.
The 2013 impact factor is 0.294, according to the
Thomson Reuters Journal Citation Reports.

Agro FOOD Industry Hi Tech

vol 25(5), September/October 2014

The forgotten nutrient? Vitamin E and the latest science
Functional foods and their clinical implications

U. N. Das
Healthy gut microbiota and long term health

Y. Vandenplas

Probiotics and Prebiotics to save human microbiota enhancing health and well-being. Note I
P. Morganti, U. Cornelli, G. Gazzaniga

15 Enrichment of infant formula with docosahexaenoic acid (DHA) and arachidonic acid (ARA)

S. T. Gautier
19 Sensory and instrumental properties of cookies enriched with "vitalplant" - extract

M. V. Pestori, A. . Mian, O. D. imurina, D. J. Jambrec, M. M. Belovi, J. M. Gubi, N. M. Nedeljkovi
24 Effects of solvent extraction system on concentration and antioxidant activity of strawberry phenolics

M. N. Miti, D. A. Kosti, A. N. Pavlovi, D. S. Dimitrijevic, J. N. Veljkovic
30 Antibacterial activity of donkey milk against Salmonella
L. ari, B. ari, A. Mandi, J. Tomi, A. Torbica, N. Nedeljkovi, B. Ikoni

The neurobiology and marketing of mood drinks

R. Cheatham

39 Hyperspectral imaging in plant based food safety - Possibility of hyperspectral imaging application
in safety control of plant based food that is converted to by-products and used as feed

D. olovi, B. Koki, I. abarkapa, J. Levi, O. Djuragi, K. Teichmann, D. Jdrejek

Rapid authentication of white wines. - Part 1: Classification by designation of origin

M. J. Martelo-Vidal, M. Vzquez

49 Feed undesirable substances as food contaminants - Part 2: Dioxins

P. I. Natskoulis, P. E. Zoiopoulos
53 New product development: carbonated beverage with different protein and creatine for
sportsmen and physically active people

M. Tomczyska-Mleko
57 Traditional and social media in food risk communication

M. Friel, J. M. Wills
61 Effect of modified atmosphere packaging on persimmon fruit (cv. Karaj) - Physical, chemical and
mechanical properties

A. Dadashpour, M. Rahemi, M. Jouki



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Editorial Director
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Marketing & Sales
Giulio Fezzardini -
Silvia Baldina -
Simona Rivarollo -
Marketing & Events
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Production Manager
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Production Assistant
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Administrative & Account
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AGC - Arti Grafiche Colombo
(Gessate MI, Italia). Authorization from
Tribunale di Milano n. 343 of May 1990.
Autorizzazione del Tribunale di Milano
n. 343 del Maggio 1990.
Anton Alldrick (CCFRA)
Sybille Buchwald-Werner (Vital Solutions GmbH)
Undurti N Das (UND Life Sciences)
Peter Engel (DSM)
Inga Koehler (Analyze & Realize GmbH)
Ray A. Matulka (Burdock Group)
Vicente Micol (University Miguel Hernndez)
Arthur Ouwehand (University of Turku)
Demosthenes B. Panagiotakos (Harokopio University)
Denis Poncelet (ONIRIS and Capsulae sarl)
Igor Pravst (Nutrition Institute Slovenia)
Joe Romano (Waters)
Yang Zhu (Wageningen University)
Ralf Zink (DMK - Deutsches Milchkontor)


Agro FOOD Industry Hi Tech customer's publication

The forgotten nutrient?

Vitamin E and the latest science

Global Marketing Manager

Vitamin E plays a vital role in supporting brain, eye,

cardiovascular, maternal and infant health, as well as
protecting the skin. The importance of vitamin E in humans
was not recognized until relatively late in the history of
vitamins and it is estimated that more than 90 per cent of
the population in the United States does not meet the
dietary intake recommendations for vitamin E.
The outcomes of studies to investigate the possible effect of
vitamin E on cardiovascular disease have been inconsistent
and subsequent media coverage has taken attention
away from the fundamental role that vitamin E plays in
supporting human health. The impact of low vitamin E
intake globally should be a serious public health concern
and, while the micronutrient is not currently high on the
agenda of scientists and funding organis ations, there is an
urgent requirement for additional research.
E is for Essential
Vitamin E is a generic term for eight fat-soluble compounds
found in nature, of which alpha-tocopherol has the
highest biological activity and is the most abundant in the
human body. The most important sources of vitamin E are
vegetable oils, nuts, wholegrains and wheatgerm and there
is also a limited supply in seeds and green leafy vegetables.
Due to changes in modern eating habits, it can be difficult
to obtain the required amount of vitamin E through diet

alone. Indeed, a recent study by the University of Vienna

found that vitamin E has low stability in vegetable oils and
levels decline throughout the shelf life of a product as a
result (1). This could be a major factor behind the
widespread vitamin E insufficiency that is currently observed
in population groups across the globe.
The vitamin E content in food and beverages is often
reported as a-tocopherol equivalents (a-TE). This term was
established to account for the differences in biological
activity of the various forms of vitamin E. The
Recommended Daily Allowance (RDA) for adults is 15mg
RRR-a-tocopherol/day (FNB, 2000) in the US. In Europe,
adult recommendations range from 4 to 25mg
a-tocopherol equivalents (a-TE)/day for men and from 3 to
12mg a-TE/day for women. In fact, there is research to
suggest that the RDA should be raised to 30mg a-TE/day.
The role of vitamin E
The main biological function of vitamin E is as an
antioxidant, preventing the propagation of free-radical
reactions and playing a critical role in cell signalling, gene
expression and the regulation of other cell functions. The
European Commission has authorized an Article 13.1 health
claim stating that vitamin E contributes to the protection of
cell constituents from oxidative damage. The damaging
effects caused by free radical reactions can lead to
various health conditions such as heart disease, cancer
and inflammatory conditions.
Until recently, little new scientific effort has been devoted
to studying the role of vitamin E in the human body. There is
however emerging evidence to demonstrate that vitamin E
supplementation can reduce the negative health

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

implications of fatty liver disease (2). This is a potentially

significant development as obesity-related conditions such
as fatty liver disease and diabetes become key public
health concerns on a global scale. There is also research to
suggest that vitamin E intake reduces and normalizes the
risk of cardiovascular events in a group of diabetic patients,
in particular those which have a specific genotype (3). In
addition, the role of vitamin E supplementation in the
treatment of neurodegenerative diseases such as
Alzheimers disease is under investigation (4) and there is
ongoing research into the role that vitamin E plays in
protecting omega-3 polyunsaturated fatty acids from
oxidative damage (5).
Need for action
Vitamin E is essential as it is a key element in cell
membranes to protect against the damaging effects
caused by oxidation and it offers numerous health benefits.
DSM has an ongoing commitment to advance research
into vitamin E and is calling on consumers, healthcare
professionals, scientists and governments to take note of
emerging science. It is also leading an initiative - Vitamins in
Motion - to highlight the critical role vitamins play in overall
nutrition and health. For more information and to access
the latest news on vitamin E, visit

1. Pignitter et al, Cold fluorescent light as major inducer
of lipid oxidation in soybean oil stored at household
conditions for eight weeks, J Agric Food Chem. Mar
12, 62(10), 2297-305 (2014)
2. N. Chalasani et al, The Diagnosis and Management of
Non-Alcoholic Fatty Liver Disease, Heptatology, June
3. S. Yusuf et al, Effects of an Angiotensin-Converting
Enzyme Inhibitor, Ramipril, on Cardiovascular Events in
High-Risk Patients, New England Journal of Medicine,
Mar 9, 342(10), 748 (2000).
4. M. Dyksen et al, Effect of Vitamin E and Memantine on
Functional Decline in Alzheimer Disease, Journal of the
American Medical Association, 311(1), (2014).
5. Food and Nutrition Board, Institute of Medicine Dietary
reference intakes for vitamin C, vitamin E, selenium,
and carotenoids, 186 283 (2000), National Academy
Press, Washington.

For more information

DSM Nutritional Products
External Communications
Alex Filz
tel. +41 61 815 83 67

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

UND Life Sciences, 2020 S 360th St, # K-202, Federal Way, WA 98003, USA
Member of AgroFood industry Hi Tech Scientific Advisory Board

Undurti N. Das

Functional foods
and their clinical implications
It is generally agreed that diet may modify the risk of developing or exacerbating certain chronic diseases. This observation
is not new since for centuries it is known that diet and foods have a functional role in health. What is new is the scientific
evidence as well as the terminology.
Our understanding of the relationships between food, physiological function and disease has progressed in recent years
especially, with regard to the role of nutrition in our state of well being. This led many manufacturers to develop and market
products derived from foods that could be used to promote good health. This resulted to the development and marketing
of a growing spectrum of products called nutraceuticals and functional foods.
Although the terms nutraceutical and functional food are used commonly around the world, there is no consensus on
their meaning. Bureau of Nutritional Sciences, of the Food Directorate of Health Canada, has proposed the following
A nutraceutical is a product isolated or purified from foods that is generally sold in medicinal forms not usually associated
with food. A nutraceutical is demonstrated to have a physiological benefit or provide protection against chronic disease.
A functional food is similar in appearance to, or may be, a conventional food, is consumed as part of a usual diet, and is
demonstrated to have physiological benefits and/or reduce the risk of chronic disease beyond basic nutritional functions.
As commercial interest in the marketing of these foods and components grows, regulatory agencies face new challenges.
Current understanding of the benefits and risks related to health claims on such products is more qualitative than
quantitative. Hence, regulators need to exhibit caution and explore this issue from a variety of perspectives. For instance,
How should nutraceuticals/functional foods be defined with precision for regulatory purposes?
Should such products remain as either foods or drugs under the Food and Drugs Act?
What kinds of health claims, if any, should be allowed on food labels?
What standards of evidence would be necessary and sufficient to prove a health benefit?
How can nutraceuticals/functional foods be regulated without unduly compromising the right of citizens to take
greater responsibility for their own health?
Current regulatory environment is said to discourage innovation and marketing of nutraceuticals/functional foods. Under
the Food and Drugs Act, only a specified range of claims may be made for foods; otherwise, they are classed as drugs. The
food regulations currently permit:
positioning the food as part of healthy eating,
claiming that a nutrient or nutritive substance is generally recognized as an aid or factor in maintaining the functions of
the body, or necessary for the maintenance of good health and normal growth and development(also known as
biological role claims and nutrient function claims).
Under the current regulatory framework nutraceuticals/functional foods appear to have an awkward fit. Although some
may appear to consumers as ordinary foods, they are known to produce physiological effects.

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

One needs to note that foods are consumed for

wellness whereas drugs are necessary to fight
illness. Moreover, drugs need to meet stringent
regulations governing Good Manufacturing
Practices, testing procedures and post-market
surveillance. In view of this dichotomy between
functional foods and drugs caution needs to be
Since, the terminology and definitions for
nutraceuticals/functional foods are not consistent
worldwide, and there are no international standards
for regulation of nutraceuticals/functional foods to
serve as baselines, it is important that (i) health
claims on products are truthful and not misleading,
(ii) different claims will require various levels of
evidence and the quality of this evidence must be
consistent, (iii) health claims on foods would require
a common scientific basis to provide consistent and
credible messages to the public, (iv) health claims
on products should be established in the context of
the total diet and other relevant lifestyle factors, and
finally (v) the possibility that addition of high doses of
nutrients or other food components to a diet - either
through fortification or non-food sources such as
nutrient supplements and over-the-counter
pharmaceutical preparations - may result in adverse
health effects that needs to be kept in mind.
In view of these controversies, more research and
discussion among all stake holders and a common
theme under which labeling claims are agreed upon
needs to be reached. This needs cooperation and
coordination from one and all.
Furthermore, one important question that needs to
be addressed is whether foods that are produced
based on recombinant technology such that they
contain higher amounts of phytochemicals, vitamins,
infection resistant chemicals, etc., can be labelled
as foods or food medicine or frankly as drugs. This is
especially so in the context of foods that may
contain super amounts of certain nutrients/vitamins/
phytochemicals, etc., that may exceed the
permissible amounts of daily recommended amount.
Though this is a theoretical possibility, while defining
functional foods and permitting them to be freely
available for human consumption it is important to
consider this possibility. It is also essential that public
is reassured about the safety and usefulness of foods
produced by recombinant biotechnology. This needs
more scientific studies that are transparent.

Table 1. Example of functional food components that have health benefits.

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

An example of functional food components that

have health benefits are given in Table 1. One big
question is how long and how much of these
functional food components can be consumed by
humans without significant side-effects. Thus, good
pharmacokinetic, phamacodynamic and
pharmacovigilance studies and relevant
toxicological studies are needed both for the sake
of science and human health and to ensure the
public that functional foods and their components
are safe.

Vrije Universiteit Brussel, Department of Pediatrics, UZ Brussel, 1090 Brussels, Belgium

Yvan Vandenplas

Healthy gut microbiota

and long term health
KEYWORDS: microbiota, microbiome, gut flora, cesarean section


This review summarizes how the composition of the gastro-intestinal microbiota depends on pre- and
postnatal factors, and birth itself. The impact of method of delivery, feeding during infancy and
medications, such as antibiotics and anti-acid medication, on the composition of the gastro-intestinal microbiota has clearly been
shown. However, the duration of the impact of these factors is not well established. The gastro-intestinal microbiome composition is
associated with many auto-immune mediated diseases. Although causality has not been obviously demonstrated, there is a strong
tendency in this direction. Nevertheless, results of the manipulation of the gastro-intestinal microbiome composition in these conditions
are often disappointing. A better understanding on factors determining the long-term composition of the gastro-intestinal microbiome
and its health consequences are a priority research topic. A better understanding of the association between the microbiome and
the immune system may have a tremendous impact on general health.

The microbiome is the ecological community of commensal,
symbiotic, and pathogenic microorganisms that literally
share our body space. This term was originally coined
by Joshua Lederberg, who argued the importance of
microorganisms inhabiting the human body in health and
disease. A dysbiosis is condition with microbial imbalances
on or inside the body. Dysbiosis is most prominent in
the digestive tract or on the skin, but can also occur on
any exposed surface or mucous membrane such as lungs,
mouth, nose, sinuses, ears, nails, or eyes. Dysbiosis has been
associated with different illnesses, such as gastroenteritis,
allergy, inflammatory bowel disease, etc.


Current evidence does not support a major role for maternal
dietary restrictions during pregnancy or lactation (1).
However, there is some evidence that manipulation of the
maternal gastrointestinal microbiota during pregnancy
may have a health benefit for the offspring. A group of
256 women was randomized during the first trimester of
pregnancy (2). Intensive dietary counseling provided
by a nutritionist was done in the intervention group, with
additional randomization to receive probiotics (L. rhamnosus
GG and B. animalis ssp. lactis Bb12) or not. The third group
was a control group. The probiotic intervention resulted in

a decreased incidence of gestational diabetes mellitus (13

percent versus 36 percent (diet/placebo) and 34 percent
(control); P = 0.03) (2). Duration of pregnancy was normal
and there were no adverse events in mothers in mothers
or infants. The dietary counseling resulted in an increased
birth weight (2). Probiotics given during the last trimester
of pregnancy and to mothers during lactation or to infants
if formula fed seem to reduced atopic dermatitis (3, 4).
However, when the probiotics are started after birth, the
outcome is most of the time negative (5, 6).
Prenatal exposure to proton pump inhibitors (PPI) and/orH2
receptor antagonists (H2RAs)As taken during pregnancy
predispose the offspring for allergic disease: the incidence of
atopic dermatitis (RR 1.32 (95 percent CI 1.06-1.64)), asthma
(RR 1.57 (95 percent CI 1.20-2.05)) and allergic rhinitis (RR 2.40
(95 percent CI 1.42-4.04))(7).

The way of delivery, vaginally versus caesarean section (C.
section) does influence colonization of the gastro-intestinal
tract. The gastro-intestinal (GI) microbiota of a baby born
through C. sections is very similar to the skin microbiota of the
mother, whereas after vaginal delivery, the GI microbiota
is very similar to the vaginal colonisation (8). EscherichiaShigella and Bacteroides species are underrepresented in
infants born by C. delivery. Infants born by elective C. delivery
have a particularly low bacterial richness and diversity (9).

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Already 15 years ago, Grnlund et al showed that on the

third day of life, vaginally born infants have a much higher
count of bifidobacteria, lactobacilli and Bacteroides thans
those born through C. section, while the number of clostridia is
comparable (10). But according to Salminen and coworkers,
the number of clostridia is still different at the age of seven
years, with vaginally born infants having higher counts for
clostridia (11).
According to the same authors, the difference in colonization
disappears around the age of one month (10). Children
born after C. section have a significant higher risk to develop
allergic disease such as allergic rhinoconjunctivitis and
asthma (12). A meta-analysis clearly identified C. section
as a risk factor to develop asthma (13). C. section is also
associated with other auto-immune diseases, such as
diabetes type 1 (14). Infants born by caesarean section
(147/865, 17 percent) had a greater risk of diarrhoea (OR(adj)
1.46, 95 percent CI 1.022 to 2.10) and sensitisation to food
allergens, both in adjusted (OR(adj) 2.06, 95 percent CI 1.123
to 3.80) and stratified analyses (by cord blood IgE) (15).
Caesarean delivery was not associated with colicky pain and
atopic dermatitis (15). C. section was also identified as a risk
factor for obesity, even in adults (13).

The fecal microbiota among infants is characterized by
a high variability. The profiles are generally dominated by
Actinobacteria (mainly the genus Bifidobacterium) and
Firmicutes (with diverse representation from numerous
genera) (9). Differences in the neonatal gut microbiota
precede the development of atopy, suggesting a crucial
role of the balance of indigenous intestinal bacteria for the
maturation of human immunity to a nonatopic mode (16,
17). At the age of 1 month, the presence of Echerichia coli
and Clostridium difficile is associate with an increased risk
to develop later in life eczema, recurrent wheeze, atopic
dermatitis and allergic sensitization (16). However, a recent
meta-analysis reported to have found no evidence to
support a protective association between perinatal use
of probiotics and doctor diagnosed asthma or childhood
wheeze (18). Randomized controlled trials to date have not
yielded sufficient evidence to recommend probiotics for the
primary prevention of these disorders (18). Of course, every
infection, also a viral gastroenteritis disrupts the equilibrium of
the GI microbiota (19). Bifidobacterium colonization is lower
in patients with type 1 diabetes compared to the control
group, whereas Candida albicans and Enterobacteriaceae
other than E. coli colonization were increased (20). High
intestinal Bacteroides fragilis and low Staphylococcus
concentrations in infants between the age of 3 weeks and
1 year are associated with a higher risk of obesity later in life
(21). Experimental data in animals, but also observational
studies in obese patients, suggest that the composition of
the gut microbiota differs in obese v. lean individuals, in
diabetic v. non-diabetic patients or in patients presenting
other diseases associated with obesity or nutritional
disbalance, such as non-alcoholic steatohepatitis (22).
Microbial studies have elaborated the normal composition
of the gut microbiome and its perturbations in the setting of
inflammatory bowel disease (23). This altered microbiome
or dysbiosis is a key player in the protracted course of
inflammation in inflammatory bowel disease (23). Fecal

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

transplantation is becoming more and more accepted as an

effective treatment of recurrent C. difficile disease (24). The
first reports are published on successful fecal transplantation
in inflammatory bowel disease, both in colitis ulcerosa and
Crohns disease (25, 26). It is becoming obvious that the GI
microbiota may play a determinant role in many, if not all,
auto-immune driven diseases since a relation has also been
reported with coeliac diseases and spondylarthritis (27, 28).
Finally, the role of microbiome in central nervous system
disorders is more and more investigated in depth (29). The
role that enteric short-chain fatty acids, particularly propionic
(also called propanoic) acid, produced from ASD-associated
GI bacteria, may play in the etiology of some forms of autism
spectrum disorders (30). Human populations that are partial
metabolizers of propionic acid are more common than
previously thought. The results from pre-clinical laboratory
studies show that propionic acid-treated rats display ASDlike repetitive, perseverative, and antisocial behaviours
and seizure (30). Neurochemical changes, consistent and
predictive with findings in autism spectrum disorder patients,
including neuroinflammation, increased oxidative stress,
mitochondrial dysfunction, glutathione depletion, and altered
phospholipid/acylcarnitine profiles, have been observed (30).
The role of the environment on the development of atopic
disease is still heavily debated. It is not impossible that the
presence or absence of pets in a household exercise a
certain effect on atopic disease through changes in the
GI microbiota. Microbiota richness and diversity tends
to be increased in infants living with pets, whereas these
measures were decreased in infants with older siblings
(31). Infants living with pets exhibited under-representation
of Bifidobacteriaceae and over-representation of
Peptostreptococcaceae; infants with older siblings exhibited
under-representation of Peptostreptococcaceae (31).

Compared with breastfed infants, formula-fed infants have
an increased richness of species, with overrepresentation
of C. difficile (9). The differences in composition between
breast milk and formula are the major reason to explain
the difference in GI microbiota composition. Prebiotic
oligosaccharides, a well identified bifidogenic factor, are the
third most important component in mothers milk, and they
are virtually absent in cow milk (32). It took some time before
enough evidence was accumulated, but it has been shown
that mothers milk contains also small amounts of potentially
probiotic bacteria, which colonize the breast canaliculi (33).
During the last decades, the use of more sophisticated culturedependent and -independent techniques, and the steady
development of the -omic approaches are opening up the
new concept of the milk microbiome, a complex ecosystem
with a greater diversity than previously anticipated (33). Human
breast milk is a source of diverse, active bacteria, including
bifidobacteria, lactobacilli. Bacteria in human breast milk
colonize the gut of breast-fed infants (microbiota transfer
from the mother to the infant). Human breast milk contains
nutritive factors supporting intestinal growth of bifidobacteria.
But many other differences in the composition of mothers
milk or formula contribute to the bifidogenic effect of
mothers milk: a low protein content, which is rich is whey
and alpha-lactalbumin, a high lactose content, and low
phosphor and low iron

contents. Phosphorus increases the buffering capacity of

the stools and as a consequence it lowers the number of
bifidobacteria. Breast milk has a high lactose content. The
metabolisation of lactose results in the production of acid,
what induces a low buffering capacity of the stools, a low
fecal pH and thus an increase of bifidobacateria. Therefore,
formula fed infants have a different GI microbiota than
breastfed infants. In infants fed classic standard infant
formula, the GI microbiota is similar to that in adults (34). The
addition of prebiotics oligosaccharides to infant formula
results in a change in GI microbiota composition, resulting
in a similar microbiota in formula and breast fed infants (35).
However, prebiotic oligosaccharides are not generic. Not all
oligosaccharides have a similar effect. A new prebiotic blend
of polydextrose and galacto-oligosaccharides may have a
smaller bifidogenic effect in young infants than a mixture of
galactose- and fructo-olgosaccharides (GOS/FOS) (36). The
counts of bifidobacteria in the infants fed the polydextrose/
galactose mixture were closer to the breast-fed group, but
only tended to be higher than control for total bifidobacteria
(P=0.069) and Bifidobacterium longum (P=0.057) at 30 days
(36). The same GOS/FOS mixture did result in higher levels
of sIgA in the stools compares to standard infant formula
(37), while this could not be confirmed with the poydextrose
mixture (36). A comparison between the physiological and
bifidogenic effects of different prebiotic supplements in infant
formulae clearly demonstrates that prebiotic mixtures are
specific (38). Supplementation of an infant formula enriched
with FOS at either 2.0 or 3.0g/L did not appear to have any
significant prebiotic effect with respect to bifidobacteria,
lactobacilli, C. difficile, E. coli or Bacteroides (39). Another
study with the specific GOS/FOS mixture showed that the
differences in GI microbiota between the groups were
maintained during the second half of the first year, even if
the prebiotic supplementation was stopped at the age of 6
months (40). It is well know from probiotics that they disappear
from the GI microbiota between 1 and 2 weeks after stopping
the administration. A prebiotic mixture and LCUPFAs given to
toddlers reduces the risk to develop infections (41). Although
of interest, the number of studies with symbiotic mixtures
(pre- and probiotics) is relatively limited. Sazawal showed
interesting effects of a mixture with Bifidobacterium lactis
HN019 and GOS 2.5 g/100 g in 634 healthy children aged
1- 3 years, for 12 months, showing a decreased incidence of
dysentery, days with severe illness days, days with fever, ear
infections, iron deficient anaemia, and as a result a better
growth and weight gain (42, 43).
Micronutrients such as Vitamin D are reported to also regulate
the gut microbiome and to protect mice from dextran sodium
sulphate-induced colitis (44).

It is well known, and logic, that the administration of
antibiotics changes the GI microbiota composition. Antibiotic
associated diarrhea (AAD) is a frequent adverse event of
administration of antibiotics, occurring in 10 to 20 percent (45).
Among many other factors, the type of antibiotic given, the
dosage, duration and the age of the patient are influencing
the incidence of AAD. According to a review, the incidence
of AAD varies between 6 and 80 percent (46). However, most
studies report an incidence of 10 to 20 (47). Also acid-blocking
medications such as H2 receptor antagonists and PPI, change

the GI microbiota to a pathogenic flora with yeast (48). Small

bowel bacterial overgrowth occurs in up to 25 percent of
the children on proton pump inhibitors (49). The American
Academy of Pediatrics reported that PPI are known to favour
the development of C. difficile (50). The lack of gastric acidity
induced by PPI is also a risk factor for the development of
pathogens in the respiratory tract, since broncho-alveolar
lavage culture is more frequently positive in patients on PPI
(51). As mentioned before, prenatal exposure to PPI increases
the risk of the offspring to develop allergic disease (7).

The composition of the GI microbiota is influence by many
external factors such as feeding and medication. A healthy,
balanced microbiota is important for general health. Many,
is not all, immune mediated diseases can be related to a
dysbiosis. Whether this regards an association, a cause or
a consequence needs further evaluation. Future research
should focus on the question if manipulation of the GI
microbiota can induce a better healing or control of autoimmune diseases.



Greer FR, Sicherer SH, Burks AW. American Academy of

Pediatrics. Committee on Nutrition; AAP Section on Allergy
and Immunology. Effects of early nutritional interventions on
the development of atopic disease in infants and children:
the role of maternal dietary restriction, breastfeeding, timing
of introduction of complementary foods, and hydrolyzed
formulas. Pediatrics 2008;121:183-91.
2. Luoto R, Laitinen K, Nermes M, et al. Impact of maternal
probiotic-supplemented dietary counseling on pregnancy
outcome and prenatal and postnatal growth: a double
blind, placebo controlled. Br J Nutr. 2010;103:1792-9.
3. Kalliomki M, Salminen S, Poussa T, et al. Probiotics during the
first 7 years of life: a cumulative risk reduction of eczema in a
randomized, placebo-controlled trial. J Allergy Clin Immunol.
4. Niers L, Martn R, Rijkers G, et al. The effects of selected
probiotic strains on the development of eczema (the PandA
study). Allergy 2009;64:1349-58.
5. Soh SE 1 , Aw M, Gerez I, et al. Probiotic supplementation in
the first 6 months of life in at risk Asian infants--effects on
eczema and atopic sensitization at the age of 1 year. Clin
Exp Allergy. 2009;39:571-8.
6. Taylor AL, Dunstan JA, Prescott SL. Probiotic supplementation
for the first 6 months of life fails to reduce the risk of atopic
dermatitis and increases the risk of allergen sensitization in
high-risk children: a randomized controlled trial. J Allergy Clin
Immunol. 2007;119:184-91.
7. Mulder B, Schuiling-Veninga CC, Bos HJ, et al. Prenatal
exposure to acid-suppressive drugs and the risk of allergic
diseases in the offspring: a cohort study. Clin Exp Allergy.
8. Johnson CL, Versalovic J. The human microbiome and its
potential importance to pediatrics. Pediatrics 2012;129:95060.
9. Azad MB, Konya T, Maughan H, et al. CHILD Study
Investigators. Gut microbiota of healthy Canadian infants:
profiles by mode of delivery and infant diet at 4 months.
CMAJ 2013;185:385-94.
10. Grnlund MM, Lehtonen OP, Eerola E, et al. Fecal microflora
in healthy infants born by different methods of delivery:
permanent changes in intestinal flora after cesarean

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delivery. J Ped Gastroenterol Nutr 1999;28:19-25.

11. Salminen S, Gibson GR, McCartney AL, Isolauri E. Influence of
mode of delivery on gut microbiota composition in seven
year old children. Gut. 2004;53:1388-9.
12. Renz-Polster H, David MR, Buist AS, et al. Caesarean section
delivery and the risk of allergic disorders in childhood Clin
Exp Allergy 2005;35:1466-72.
13. Thavagnanam S, Fleming J, Bromley A, et al. A meta-analysis
of the association between Caesarean section and
childhood asthma. Clin Exp Allergy 2008;38:629-33.
14. Cardwell CR, Stene LC, Joner G, et al. Caesarean section is
associated with an increased risk of childhood-onset type 1
diabetes mellitus: a meta-analysis of observational studies.
Diabetologia 2008;51:726-35.
15. Laubereau B, Filipiak-Pittroff B, von Berg A, et al. GINI Study
Group. Caesarean section and gastrointestinal symptoms,
atopic dermatitis, and sensitisation during the first year of life.
Arch Dis Child 2004;89:993-7.
16. Penders J, Thijs C, van den Brandt PA, et al. Gut microbiota
composition and development of atopic manifestations in
infancy: the KOALA Birth Cohort Study. Gut 2007;56:661-7.
17. Penders J, Thijs C, van den Brandt PA, et al. Gut microbiota
composition and development of atopic manifestations in
infancy: the KOALA Birth Cohort Study. Gut 2007;56:661-7.
18. Azad MB, Coneys JG, Kozyrskyj AL, et al. Probiotic
supplementation during pregnancy or infancy for the
prevention of asthma and wheeze: systematic review and
meta-analysis. BMJ. 2013;347:f6471.
19. Nelson AM, Walk ST, Taube S, et al. Disruption of the human
gut microbiota following Norovirus infection. PLoS One.
20. Soyucen E, Gulcan A, Aktuglu-Zeybek AC, et al., Differences
in the gut microbiota of healthy children and those with type
1 diabetes. Pediatr Int. 2013.
21. Vael C, Verhulst SL, Nelen V, et al Intestinal microflora and









body mass index during the first three years of life: an

observational study. Gut Pathog. 2011;3:8.
Delzenne NM, Neyrinck AM, Cani PD. Gut microbiota and
metabolic disorders: How prebiotic can work? Br J Nutr.
2013;109 Suppl 2:S81-5.
Hold GL, Smith M, Grange C, et al. Role of the gut microbiota
in inflammatory bowel disease pathogenesis: What have we
learnt in the past 10 years? World J Gastroenterol.
Jorup-Rnstrm C, Hkanson A, Sandell S, et al. Fecal
transplant against relapsing Clostridium difficile-associated
diarrhea in 32 patients. Scand J Gastroenterol. 2012;47:54852.
Vandenplas Y, Veereman G, van der Werff Ten Bosch J, et
al. Fecal microbial transplantation in a one-year-old girl with
early onset colitis - caution advised. J Pediatr Gastroenterol
Nutr. 2014.
Kao D, Hotte N, Gillevet P, et al. Fecal microbiota
transplantation inducing remission in Crohns colitis and the
associated changes in fecal microbial profile. J Clin
Gastroenterol. 2014.
Stoll ML, Kumar R, Morrow C, et al. Dysbiosis of the enteric
microbiota in pediatric spondyloarthritis. Arthritis Rheumatol.
De Palma G, Nadal I, Medina M, et al. Intestinal dysbiosis
and reduced immunoglobulin-coated bacteria associated
with coeliac disease in children. BMC Microbiol. 2010;10:63.
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Wang Y, Kasper LH. The role of microbiome in central nervous
system disorders. Brain Behav Immun. 2013.
Readers interested in a full list of references are invited to
visit our website at

*Corresponding author
1. 2nd University of Napoli, Dermatology Institute, Italy
2. Loyola University Medical School, Chicago, USA
3. ISCD Consultant, Via Innocenzo XI, 41, 00195 Roma, Italy

Pierfrancesco Morganti

Probiotics and Prebiotics

to save human microbiota enhancing health and well-being.

Note I
KEYWORDS: microbiome, microbiota, probiotics, prebiotics, human health, regulatory status, strategy of treatment


It is well established that human microbiota and a balanced diet influence health of the individual.
The microbiota has a physiological role, and its modification, e.g. by unbalanced nutrition or use of
antibiotics, can cause colonization by non-resident microorganisms, leading to different diseases. According to recent
studies, the correlation between microorganisms and humans has to be considered as a mutualistic-symbiotic relationship
and not a merely commensal activity.
High-resolution spatial, temporal and functional microarrays of the human microbiota are still needed, and the effects of
environmental perturbations, the change in diet habits, and the today use of probiotics/prebiotics has to be deeper elucidated.
Hence the necessity to further international rules to coordinate the production and distribution on the market of prebiotics and
probiotics, determining their composition, labels and advertisements for obtaining a sure safeness and effectiveness of this new
category of special foods. This is the challenge for our future.

Human body is the social network where trillions of
microorganisms living on the skin, saliva, oral mucosa,
gastrointestinal tract and genital area, participate in
maintaining the health (1, 2). Most of these microbial
inhabitants, classified into 3 major phyla and referred to as the
microbiome/microbiota, perform tasks that appear notharmful at all, but rather assist in maintaining the key
physiological processes necessary for our wellbeing (Figure 1).
It has been calculated that a human adult houses about 1012
colony forming units (CFU) species of bacteria on the skin, 1010
in the mouth, and 1014 in the gastrointestinal tract. The latter
number is far in excess of the number of eukaryotic cells in all
the tissues and organs which comprise the human body (3, 4).
These microorganisms are more than 10 times the human cells
accounting for 1-3 precent total body mass. The 3.3 million of
genes of the gastrointestinal tract (GI) result about 150 times
more than the global human genome (5, 6).
The purpose of the article is to share some informations
connecting the human microbiota with health and regular
use of probiotics.


The normal human microbiota consists of more than 1000
species that are influenced by various factors, including


Figure 1. Microbiota/
microbiome on human skin
body classified in 4 phyla.

genetics, age, sex, stress, nutrition and diet. Despite their

role is part of normal healthy physiology, once grow
beyond their typical ranges, or populate atypical areas of
the body, they cause a diseases and/or even simply
modify the GI biochemical environment.
The Bacteroides thetaiotaomicron can be a peculiar
example, since it can convert enzymatically the
carbohydrates vegetal-derived into glucose (6). Human
genome, in fact, does not possess these enzymes, so that

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

B. thetaiotaomicron interference allows obtaining nutrients

from fruits and vegetables.
It is becoming increasingly clear that the effect of the
microbial ecology in the body goes beyond the immune
system, being implicated in immune-related disorders,
such as diabetes, inflamm-aging, or obesity. Blaser et al.
(7) have shown that Helicobacter pylori, the pathogenic
bacterium causing gastric ulcers, is one of the normal
commensal of human stomach regulating both the level
of acidity and the production of ghrelin. This hormone
stimulates the brain centre of appetite in competition
with the appetite inhibitor leptin. The excess of acidity for
the disequilibrium of H. pylori provokes inflammation, and
gastric ulcer, but its elimination with antibiotics stimulates
the sensation of appetite and may cause obesity bound
to the increase of ghrelin production
According to Lee and other authors, Bacteroides fragilis
contributes to maintain in equilibrium the immune system
(8, 9), since it stimulates the production of both T-cells
regulators, and other pro-inflammatory T-cells through
the polysaccharide -A positioned on its surface. The
equilibrium between these two kinds of T-cells reduces
the inflammatory process, avoiding damages to the
immune system.
Unfortunately, due to the different way of living, in the
last century both B. fragilis and H. pylori are disappearing
(together with other anti-inflammatory commensal
microorganisms) causing a decrease of the T-cell
regulators. This could be the reason of the notably
increase of the auto-immune diseases, such as type I
diabetes, atopic dermatitis, and multiple sclerosis, all
connected with genetic and environmental components
under regulation by the human microbiota.
Research suggests that the relationship between
microorganisms and humans is not merely commensal,
but rather a mutualistic symbiotic one (9-12). On one
hand, the microbiota of the human GI has been shown to
produce vitamins, thus contributing to nutrition, digestion,
protection from establishment by alien microbes, and
stimulation of the immunological tissues (12-14).
Disorder and impairment of microbiota communities are
associated with conditions such as obesity, inflammatory
bowel disease, and other critical illness (15-17). The
alteration of microbiota due to poor nutrition or
antibiotics misuse can cause shifts in populations and
colonization by non residents microorganisms, leading to
gastrointestinal diseases.

The skin is host of numerous bacterial and fungal species,
either commensals or mutualistic, including transient
pathogenic (18). The beneficial bacteria prevent
colonization of pathogenic microorganisms, competing
for nutrients, secreting chemicals or stimulating the skins
immune system (19).
The skin is the first line of defence against infection, and a
physical and immunologic barrier (Figure 2) that
recognizes beneficial and pathogenic microbes. It
produces antimicrobial peptides, such as cathelicidins,
that control the proliferation of skin microbiota and
regulates cytokines inducing inflammation, angiogenesis,
and skin re-epitheliziation (20). In psoriasis and rosacea,

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

the self-created DNA and cathelicidin peptides cause

auto-inflammation of the skin while, at the opposite
cathelicidin suppression was shown in atopic dermatitis.
In case of eczema and psoriasis an imbalance of skin
commensals and potentially harmful microbes can take
place influencing the immune system (20).
Emerging evidences indicate that commensal microbes
inhabiting skin, airway, and gut, protect these structures
against inflammatory disorders, despite little is known
about determinants of their microbiota.
A group of study from Helsinki University has shown how
the decline of biodiversity may contribute to the rise of
asthma, allergies and other chronic inflammatory diseases
among people living in cities worldwide (21). Subjects
living on farms or near forests had more diverse bacteria
on their skin and lower allergen sensitivity than individuals
living in areas with less environmental biodiversity, such as
urban areas or near bodies of water. Furthermore, in this
study allergen- sensitive individuals have shown diversity
on their skin of gamma-proteobacteria compared to
healthy subjects. The presence of gamma-proteobacteria,
was associated with the expression of IL-10 in the blood of
the healthy subjects indicating its role in immune
tolerance (21).
These findings suggest how the increasing prevalence of
inflammatory diseases may be associated with the

Figure 2. Microbiome, antimicrobial peptides and skin immunology.

changing biodiversity of both environment and

commensal skin bacteria. Moreover, ecological statistical
approaches have revealed previously unrecognized
organizations in architecture of the body complex
microbial communities.

The gi microbiota starts in the mouth with a viable count
of 108-1010 cfu of bacteria /g saliva, which reduces in
stomach 103, duodenum and jejunum102-109, increases
again in ileum and colon to about 1010-1012
respectively cfu/g (16). The large intestine is, therefore,
the most diversely colonized and metabolically active
organ in the human body,where colonic environment is


favourable for bacterial growth due to its slow transit

time, readily available nutrients ,and favourable ph.
together with the gut immune system ,the mucosal
microflora contributes significantly to the barrier that
prevents pathogenic bacteria invasion. The gi
microbiota ecosystem may be disturbed ,in fact, by
pathogenic agents leading to a decreased bacterial
diversity and/or an unusual overgrowth of opportunistic
pathogenic residential bacteria (23, 24). Recognition of
commensal or pathogenic bacteria is of great
importance to the mucosal immune system in eliciting
positive immune activation or negative response. In
any way, a number of factors influence the
composition of microbiota, such as the physiological
condition of the host ( age, stress, healthy status, etc.),
composition of the diet and environmental
circumstances (antibiotic or antiseptic therapies,
personal hygiene, etc.) (23, 24). At this purpose the use
of prebiotic fibers and diet rich in plant-based food
seem to have health-promoting property ,beneficially
altering the microbial community (25). On the other
hand, probiotics show the ability to adhere in the
surface of digestive system and persisting longer in the
intestinal tract ,stabilize the intestinal mucosal barrier
and provide competitive exclusion of pathogen
bacteria (26).


A high-resolution spatial-temporal and functional
microarrays of the global human microbiota, is
necessary to elucidate in a deeper way the effects of
environmental perturbations, such as the use of
antimicrobials and antibiotics, the changes in diet
habitudes, and the today use of probiotics/prebiotics.
Probably by the Second Human Microbe Project can be
a way to improve the understanding the organization of
the human microbiota system (27-29).
This project, launched to survey microbial content
across 242 healthy adults by a metagenomic approach,
has developed a reference catalogue of microbial
genome sequences aimed to understand how the
specific habitats in the gut, genitourinary system, and
skin may contribute to healthy and diseased state (28,
29). Thus metagenomic studies using 16S rRNA
sequencing in adults show that the vast majority of skin
bacteria and gut flora fall into 4 major phyla (Figure 1)
(22,30). Advances in DNA amplification and sequencing
technology has given the possibility to bypass the
normal culture steps, allowing more complete views of
human microbiota and their genetic content,
collectively called microbiome. Defining in this way the
normal microbiological population on skin healthy sites
(by metagenomics of skin microbiota), it will be possible
to provide a framework for investigating bacteria and
fungi in different skin conditions, underlining new
perspective to pathogenic factors and new therapeutic
targets (Figure 3) (22,31).
For all these reasons, many therapeutic strategies
aiming to restore a normal microbiota status by the
selective use of probiotics and prebiotics should be
useful to rebalance the disordered microbial community
influenced by diseases or stress factors (17,32-34).


Figure 3. Metagenomics of skin microbiome.


Probiotics are living non-pathogenic microorganisms that may
confer health benefits on the host (35, 36), while prebiotics are
generally defined as nondigestible food ingredients that
beneficially affect the host by selectively stimulating the
growth and/or activity of one or a limited number of bacterial
species already established in the colon, and thus in effect
improve host health (37, 38). Mechanisms of probiotic activity
include (a) adhesion to the intestinal-lumen interface; (b)
competition with pathogens for receptor binding, nutrients
and colonization;(c) enhancement of mucosal barrier
function; (d) promotion of innate immune response; (e)
production of bacteriocins; (f) and modulation of cell kinetics,
with further mechanisms of action likely to be identified (36).
On the other hand prebiotics are non-digestible and
selectively fermented ingredients that allow specific changes,
both in the composition and/or activity in the gastrointestinal
microflora, conferring benefits(38, 39).
Although all prebiotics are fibres, not all fibres are prebiotics.
Thus, some known prebiotics, as inulin, are low digestible
carbohydrates, some times associated with impaired
gastrointestinal tolerance when consumed in large quantity,
while other prebiotic fibres, such as wheat dextrin and
polydextrose, exhibit high gastrointestinal tolerability (40-43).
Generally, probiotics, playing an important role in barrier
defence, may modulate the activity of many cells of the
immune system, including innate system cytokines,
macrophages, epithelial cells and granulocytes, as well as
adaptive system (33, 34). In any way probiotic bacterial
strains, having an almost exclusive saccharolytic metabolism,
typical for lactobacilli and bifidobacteria, can be considered
potentially beneficial exerting immune-activation, -deviation
or -regulation/suppression responses. Thus, probiotic and
prebiotic can significantly influence human health through a
range of effects which include: (a)detoxification of
xenobiotics (42); (b) biosynthesis of vitamin K (43); (c)
metabolic effects of fermentation of indigestible fibre (44); (d)
positive influence on transit of luminal contents by peristalsis
(45); (e) competition with pathogenic microbes for nutrients
and binding sites on mucosal epithelial cells (46); and (f)
modulation of the hosts immune response (47). In any way,
according to the joint FAO/WHO expert consultation,
probiotics have been redefined as Live microorganisms
which when administered in adequate amounts confer a

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

health benefits on the host while prebiotics confer benefits

allowing specific changes in the composition and activity of
the GI microflora (23). Thus, selection and combination of
both probiotics and prebiotics seem to represent an
efficacious approach to the control of some immune
hypersensitivity/allergy reactions, acting also as regimen for
long health and well-being. The experts agreed, in fact, that
adequate scientific evidence exists to indicate [...] health
benefits from consuming food containing probiotics, [...]
specific strains of probiotics are safe and able to confer some
health benefits on the host; and these benefits include
condition such as gastrointestinal infections, certain bowel
disorders, allergy, urogenital infection, also if their
application to prevent and treat these disorders should be
more widely considered by the medical community. In
addition, there is emerging evidence to indicate that
probiotics can be taken by healthy people to prevent certain
diseases and modulate host immunity and supporting also a
gut-microbiome brain connection may be of help in human
neurodevelopment disorders (49).







In conclusion prebiotics have essentially the same aim as
probiotics, which is to improve host health via modulation of
the intestinal flora, although by a different mechanism (48).
However, on one hand determining the precise composition
of secreted products from probiotic bacteria is challenging
because of its dependence from species, strain, micro
environment, culture conditions and their adhesivity on
human epithelium. On the other hand, prebiotics exhibit a
diverse range of physiological effects depending on their
physico-chemical characteristics, such as solubility,
fermentability, and viscosity.
Moreover, there is the necessity to further rules because the
regulatory status of probiotics and prebiotics as a component
in food is currently not established on an international basis.
In addition, even if these products are considered safe, there
is a need for refinement of in vitro and in vivo tests to better
predict their function in humans. This goal will be addressed
by the application of good manufacturing practices and the
definition of the shelf-life conditions reported on the label,
together with verifiable health claims. Finally, surveillance
system will be necessary as well as trace-back and post
marketing surveillance to control the eventual side-effects
associated with the consume of prebiotics and probiotics in
the long period of time (48). These are the future challenges
to save the human microbiota enhancing well-being and the
quality of our life.














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Readers interested in a full list of references are invited to visit our

website at

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Industry perspective


DSM, 6480 Dobbin Road,
Columbia, MD
21045, USA
indirizzo 1
indirizzo 2

Enrichment of infant formula with

docosahexaenoic acid (DHA)
and arachidonic acid (ARA)
KEYWORDS: breastfeeding, formula feeding, infant nutrition, arachidonic acid, docosahexaenoic acid


Breastfeeding is the most natural way to feed an infant. Moreover global experts recommend exclusive
breastfeeding for the first six months of life. DHA and ARA are, respectively, omega-3 and omega-6, longchain polyunsaturated fatty acids. These important fatty acids are always present in breast milk. In many instances, however, there are
compelling reasons why breastfeeding or exclusive breastfeeding is not possible. The article reviews the numerous benefits reported for
exclusive breastfeeding as well as other studies documenting the benefits of DHA- and ARA-supplemented infant formulas.

Paediatricians and nutritionists recommend exclusive
breastfeeding of infants until 6 months of age. It is helpful for
new mothers to have current information regarding
breastfeeding as well as for formula feeding. This brief article
reviews recent key published literature addressing the benefits
of DHA and ARA when provided in breast milk or in the case
of those who cannot breast feed, in infant formula.

Breastfeeding is the gold standard and the natural way to
feed an infant. Paediatricians and nutritionists are unanimous
in recommending that mothers exclusively breastfeed their
infants for about six months. In its 2012 policy statement, the
American Academy of Paediatrics reaffirmed this advice,
adding that complementary foods can be introduced at 6
months with the continuation of breastfeeding for one year or
longer as mutually desired by the mother and infant.(1) The
World Health Organization also recommends exclusive
breastfeeding for 6 months.(2)
The advantages of exclusive breastfeeding for 3 months or
more include significantly lower risks of respiratory tract and
ear infections, asthma, bronchiolitis, atopic dermatitis,
gastroenteritis, sudden infant death syndrome and several
other diseases.(1) Breastfeeding also confers greater
protection against infections because it contains many
immunological and protective factors.(3, 4) Human milk also
promotes the maturation of the infants immune system. Other

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

advantages of breastfeeding for 4 months or longer may

include higher scores for fine motor skills and communication
at age 3 compared with infants breastfed less than 4
months(5) and higher academic achievement at 10 years of
age, especially in boys.(6) The American Academy of
Paediatrics noted that the health benefits of breastfeeding
are greater when the infant is fed longer than 3 months.(1)
Evidence suggests that breastfeeding may have beneficial
effects on the long-term health of the offspring, though not
all studies are conclusive. Breastfeeding has been
associated with a lower risk of developing insulindependent (Type 1) diabetes,(7) metabolic syndrome,(8)
non-insulin-dependent (Type 2) diabetes,(9) inflammatory
bowel disease(10) and a slightly lower risk of heart
disease(11) and stroke,(12) although the risk of heart
disease and stroke has not been confirmed by others.(13)
Children who were exposed to diabetes during fetal life
had less fat tissue if they were breastfed for at least 6
months.(14) There is also evidence that children and
adolescents who were breastfed for at least 6 months were
less likely to develop mental health problems.(15) The links
between breastfeeding and risks of allergic disease are
inconsistent and vary with the type of allergy, family history
of allergic disease and other factors.(16) Environmental
exposures, changes in gene expression, maternal and
infant gene types, and various dietary factors have all been
linked to the risk of allergy.(17-19)
Whether breastfeeding partly protects against the
development of overweight and obesity later in life is
controversial. Several studies have confirmed that rapid
growth in early infancy and weight are(20) associated with


being overweight and obese in childhood and

adolescence.(21, 22) However, the evidence is inconclusive
whether breastfeeding protects against the later
development of being overweight and obese in the
offspring.(23-27) Breastfeeding is associated with a slower
rate of weight gain in infancy and lower plasma insulin levels,
which are associated with decreased fat storage.(28) Faster
infant growth and weight gain have been attributed to the
higher protein content of infant formula compared with
breast milk.(29) Breastfeeding or formula with lower protein
content restored the growth rate to that observed in
breastfed infants.(30, 31) Others suggested that the higher
energy density and volume of formula consumed contribute
to the faster growth rate of formula-fed infants.(32)

(41) Many studies have demonstrated that infants

consuming formula grow as well(48) as breastfed infants and
have comparable neurodevelopmental scores, without
adverse effects.(42)
Mothers who do not breastfeed have many choices among
formulas. Brands differ by the source of protein(s) and the
types of fat and carbohydrates they have. The main proteins
are soy, various cows milk proteins or hydrolyzed proteins,
which have been broken down to increase their digestibility.
Some formulas are free of the sugar lactose. Most
paediatricians recommend a formula with added iron to
prevent anaemia. Special formulas are available for preterm
infants and those with medical needs. All infant formulas
marketing in the U.S. must meet federal nutrient requirements
and are regulated by the Food and Drug Administration.

Long-chain PUFAs
Until the development of fatty-acid-supplemented infant
formula, breastfeeding was the only way to ensure that an
Long-chain PUFAs
infant received long-chain polyunsaturated fatty acids
Since 2001, infant formula manufacturers have been permitted
(PUFAs), including both DHA and ARA. Breast milk naturally
to add two long-chain polyunsaturated fatty acids to their
contains both of these
products. These are DHA
PUFAs, which are
and ARA. The amounts
necessary for brain
added are similar to the
development and
worldwide averages of
function,(33) visual
these fatty acids that are
function, immune
found naturally in human
system development(34)
milk. The addition of DHA
and other activities.
and ARA ensures that the
DHA must be obtained
infant receives sufficient
from the diet in a
amounts of these fatty
preformed state
acids for healthy brain
because the usual
structure and function
Western diet provides
and visual development.
too little from conversion
The only DHA used in
of its fatty acid
infant formula in the U.S.
precursor alphacomes from a vegetarian
linolenic acid or from
and sustainable source,
body stores.(35, 36)
Mothers who eat
seafood regularly or
Visual and
take fish or algal oil
supplements have
higher levels of DHA in
There is evidence
Credit for the photographs goes to (c) DSM Nutritional Products Ltd.
their breast milk than
showing that term or
women who do not
preterm infants
have dietary sources of these fatty acids.(37) Women who
supplemented with DHA in infancy may have improved
do not eat fish, vegetarians and vegans,(38) have lower
visual acuity, attention and cognitive performance in
levels of DHA in their breast milk compared with omnivore
childhood compared with infants fed unsupplemented
women or those who eat fish. Most, but not all, infant
formula, but results vary with the dose, time of assessment
formulas now contain added DHA and arachidonic acid
after birth and for cognition in particular, the measurement
(ARA), an omega-6 fatty acid that also helps support infant
tool(s). Term infants fed different amounts of DHA with ARA
had higher visual acuity scores at 12 months of age
compared with those consuming the unsupplemented
formula.(43) The same study also reported that
supplemented infants fed the lower doses of DHA with ARA
spent longer processing an active stimulus than
Mothers may choose not to breastfeed their infant because
unsupplemented infants.(44) In contrast to these reports, a
of medical conditions, work-related issues, personal
meta-analysis of four large randomized trials concluded that
preferences, social and cultural perceptions, lack of
DHA and ARA supplementation of infant formula had no
appropriate support and other circumstances,(39) but there
clinically meaningful effect on infant neurodevelopment at
are few medical reasons not to breastfeed.(40) Mothers who
18 months as assessed with the Bayley Scales of Infant
decide not to breastfeed, regardless of the reason, can be
Development.(45) Similarly, 1 percent DHA with ARA
reassured that their infant will be healthy, well-nourished and
supplementation of preterm infants born at less than 33
have normal scores for growth and cognitive development.
weeks gestation was not associated with any effect on


Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

mental development scores on the Bayley Scales at 18

months, but scores were significantly higher among the DHAsupplemented girls.(46) These authors also reported other
benefits of high-dose DHA.
Cognitive Outcomes
A concern with global cognitive tests measured at a single
time point is their lack of sensitivity to the different
developmental time courses for discrete cognitive functions.
(47) Thus, different aspects of cognition mature at different
rates. A recent study showed that in infants whose scores did
not differ on the Bayley Scales of cognitive performance at 18
months, exhibited positive effects of DHA and ARA
supplementation at 3 to 6 years of age when assessed with a
battery of cognitive tests at 36, 42, 48, 60 and 72 months of
age.(48) The results suggested that the benefits of DHA and
ARA supplementation were observed in early measures of
attention, preschool measures of rule learning and
implementation and later measures of verbal ability. These
findings suggest that more sophisticated measures of cognitive
development over longer time may yield more information
about the effects of DHA and ARA on cognitive development.

There are considerable benefits related to exclusive
breastfeeding during the first 6 months of an infants life.
However in situations in which a mother is unable to
breastfeed her infant exclusively for 6 months, there are
currently available commercial infant formulas containing
the long-chain polyunsaturated fatty acids, DHA and ARA
from algal single cell oils which can provide similar benefits
to human milk. The information in this brief review may help
parents make an informed choice about their infants
nutrition and also help them to realize that breastfeeding
versus formula feeding need not be mutually exclusive.








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of Human Milk, J Nutr, 138 (9), 1801S-1806S (2008).
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1308-1312 (2005).
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and Early Child Development: A Prospective Cohort Study, Acta
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and Academic Achievement at 10 Years, Pediatrics, 127 (1), e137145 (2011).
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the Associations of Breastfeeding, Infant Formula, and Cows Milk
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Syndrome Risk Is Increased with Higher Infancy Weight Gain and
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Cardiovascular Risk Factors and Outcomes in Later Life: Evidence

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from Epidemiological Studies, Proc Nutr Soc, 70 (4), 478-484 (2011).

10. Klement, E., Cohen, R. V., Boxman, J., et al. Breastfeeding and Risk
of Inflammatory Bowel Disease: A Systematic Review with MetaAnalysis, Am J Clin Nutr, 80 (5), 1342-1352 (2004).
11. Singhal, A. The Early Origins of Atherosclerosis, Adv Exp Med Biol,
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12. Rich-Edwards, J. W., Stampfer, M. J., Manson, J. E., et al.
Breastfeeding During Infancy and the Risk of Cardiovascular
Disease in Adulthood, Epidemiology, 15 (5), 550-556 (2004).
13. Martin, R. M., Davey Smith, G., Mangtani, P., et al. Breastfeeding
and Cardiovascular Mortality: The Boyd Orr Cohort and a
Systematic Review with Meta-Analysis, Eur Heart J, 25 (9), 778-786
14. Crume, T. L., Ogden, L., Maligie, M., et al. Long-Term Impact of
Neonatal Breastfeeding on Childhood Adiposity and Fat
Distribution among Children Exposed to Diabetes in Utero,
Diabetes Care, 34 (3), 641-645 (2011).
15. Oddy, W. H., Kendall, G. E., Li, J., et al. The Long-Term Effects of
Breastfeeding on Child and Adolescent Mental Health: A
Pregnancy Cohort Study Followed for 14 Years, J Pediatr, 156 (4),
568-574 (2010).
16. Jennings, S., Prescott, S. L. Early Dietary Exposures and Feeding
Practices: Role in Pathogenesis and Prevention of Allergic
Disease?, Postgrad Med J, 86 (1012), 94-99 (2010).
17. Karmaus, W., Dobai, A. L., Ogbuanu, I., et al. Long-Term Effects of
Breastfeeding, Maternal Smoking During Pregnancy, and Recurrent
Lower Respiratory Tract Infections on Asthma in Children, J
Asthma, 45 (8), 688-695 (2008).
18. Jones, A. P., Tulic, M. K., Rueter, K., et al. Vitamin D and Allergic
Disease: Sunlight at the End of the Tunnel?, Nutrients, 4 (1), 13-28
19. West, C. E., Videky, D. J., Prescott, S. L. Role of Diet in the
Development of Immune Tolerance in the Context of Allergic
Disease, Curr Opin Pediatr, 22 (5), 635-641 (2010).
20. Andersen, L. G., Holst, C., Michaelsen, K. F., et al. Weight and
Weight Gain During Early Infancy Predict Childhood Obesity: A
Case-Cohort Study, Int J Obes (Lond), (2012).
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Childhood and Obesity in Later Life--a Systematic Review, Obes
Rev, 6 (2), 143-154 (2005).
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and Childhood Overweight Status in a Multicenter, Cohort Study,
Pediatrics, 109 (2), 194-199 (2002).
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Children: Will There Ever Be Conclusive Empirical Evidence for a
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and Obesity at 11 Years: Prospective Birth Cohort Study, Int J
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Readers interested in full list of references are invited to visit our
website at



Industry perspective

indirizzo 1
Sad, Institute of Food Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
indirizzo 2

V. Pestoric

Sensory and instrumental properties

of cookies enriched with
"vitalplant" - extract
KEYWORDS: cookies quality, medicinal plants, functional food, sensory evaluation, colour, texture


Vitalplant extract, composed of non-toxic medicinal plants to enhance metabolism, can be considered
functional food ingredient because of its biologically active constitutes, antioxidant and antimicrobial
properties. Sensory and instrumental properties of new cookie formulations, developed by supplementing the basic cookie formula
with 2 percent, 4 percent, and 6 percent Vitalplant extract were tested in this study. Referring to the results, the extract addition
caused statistically significant (P < 0.05) differences in sensory properties and instrumentally measured colour and texture parameters
of the cookies. Based on their overall good sensory acceptability and previously confirmed antioxidant and antimicrobial properties of
Vitalplant extract, new cookie formulations can broaden the utilization of these extracts and may be regarded as health-promoting
functional foods.

Being a rich source of biologically active substances including
antioxidants and antimicrobials, medicinal plants can be
considered functional food ingredients (1). In our previous
works, a selection of non-toxic medicinal plants with proven
pharmacological action, including parsley fruit (Petroselini
fructus), buckthorn bark (Frangulae cortex), peppermint
leaves (Mentha piperitae folium) and caraway fruit (Carvi
fructus) was made in order to compose a mixture aimed for
metabolism stimulation and body weight regulation (2).
Ethanolic extracts of peppermint leaves, buckthorn bark,
parsley fruit and caraway fruit were shown to be a rich source
of plant phenolics (2) and were demonstrated to possess
antioxidant activity, which was tested by six different direct
and indirect tests (3) and to possess antimicrobial activity (4).
With respect to the obtained results, Vitalplant mixture was
composed and shown to possess a relatively high antioxidant
activity in most of the tests, which was explained by synergistic
effects of its components (3). What is even more relevant to
this study is the fact that Vitalplant extract addition
improved antioxidant activity and oxidative stability of the
cookies dose-dependently (3).
Short dough cookies are widely consumed food products,
appreciated for their versatility, convenience, conservation,
and especially for their attractive sensory attributes (5), and
for these reasons they can be considered suitable carrier
products for functional bakery formulations. Development of
new products by adding functional ingredients to carrier food
provides not only new market opportunities for food

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

producers, but also potential benefits for consumers (6).

However, the addition of some functional ingredients may
change sensory attributes of the product, which may affect
the consumers acceptability (7). Concerns about the
reductions in taste quality can interfere with the adoption of
healthy diets, since consumers emphasize sensory experiences
during consumption (appearance, texture, flavour), with the
pleasure derived from consumption as an important motivator
in eating (8). In respect of the above facts, descriptive sensory
analysis remains an important method in the evaluation of
different foods, including cookies which are usually applied
before the usual consumer tests (9). When integrated within
the product development process, sensory testing allows costeffective delivery of acceptable products to consumers and
thus reduces the risk of failure (10).
Referring to all mentioned above, sensory evaluation, colour
measurements and instrumental texture analysis of Vitalplant
supplemented cookies was performed as an integral part of
functional food product development.


The commercial plant mixture Vitalplant (Frangulae cortex
35 percent, Petroselini fructus 25 percent, Menthae pip. folium
20 percent, and Carvi fructus 20 percent) was obtained from
the Institute for Medicinal Plants Research Dr Josif Pani
(Belgrade, Serbia). The mixture was in the form of powder with
granulation of up to 3 mm. Commercially available refined


wheat flour (13.3 percent water content, 0.39 percent db ash

content, and 10.5 percent db protein content), obtained
from Zitko (Mill Baka Topola, Serbia) was used for the
formulation of the cookies. Vegetable fat was obtained from
Puratos, Serbia. Salt, powdered sugar and baking powder
were purchased in a local food store.
Preparation of plant extracts for the formulation of cookies
As explained elsewhere (2), crude plant extracts were
obtained by maceration with ethanol/water mixture (80:20,
v/v), with the ratio of raw materials to ethanol solution of 1:10,
for 24 h at room temperature and subsequently extracted in
an ultrasonic bath at room temperature for 10 min. After
filtration through a filter paper (Whatman, Grade 4 Chr, UK)
and vacuum-evaporation of the solvent at 40C, the extracts
were stored at -4C until further use.

Cookie surface colour measurements

Colour values of cookies were determined by chromameter
MINOLTA, CR-400 (Minolta Co., Ltd., Osaka, Japan). At five
measured points of top and bottom (centre and at the
corners of cookies), surface colour of cookies was recorded
for 10 randomly chosen cookies per batch and averaged.
The results were expressed as lightness (L*), rednessgreenness (a*), and yellowness-blueness (b*), according to
CIE L*a*b* system.
Textural analysis
Textural analysis of cookies was conducted using a TA.XTPlus
Texture Analyzer (Stable Micro Systems, England, UK),
equipped with 3-point bending rig (HDP/3PB), and a 5 kg load
cell. Ten measurements per each cookies type were made.

Statistical analysis
Preparation of cookies
Results were expressed as the mean of replications SD for
Baking trials were conducted under laboratory conditions.
all measurements. Analysis of variance (ANOVA) and
Dough mixing, processing and baking were performed on
Duncans multiple range tests was used to compare
laboratory-scale equipment. The basic cookie dough
means at 5 percent significance level by using the
formulation (control sample) contained 100 g of refined
statistical data analysis software system XLSTAT, version
wheat flour, 40 g of vegetable fat, 30 g of powdered sugar, 1
(2012.2.02) (
g of salt, 1 g of baking powder. By adding the ethanolic
extract (suspended in approximately 5 g of water) of herbal
mixture Vitalplant to the basic formulation, cookies were
prepared to provide three variations: 2 percent, 4 percent,
and 6 percent addition (supplementation with extract
Sensory quality of enriched cookies was defined in the terms
obtained from 2, 4, and 6 g of Vitalplant mixture,
of appearance, texture, odour and taste. The obtained results
respectively). The amount of water added varied to obtain
are shown in Table 1.
cohesive dough suitable for the production of mouldcut cookies. Ingredients were mixed in Diosna mixer as
per all-in method. All ingredients were mixed
together in one phase for 15 min. The formed dough
was packed in polyethylene bags and left to rest 0.5
hours at ambient temperature (22-24C). Following the
rest time, the dough was sheeted to a final thickness,
approximately 5 mm on a pastry break, and cut out
Table 1. Sensory evaluation of cookies.
using a rectangular cutter. Dough pieces were baked
Values are means SD of six panellists. Values with the different superscript
within a raw are statistically different (P < 0.05).Quality category was
for 12 min in a deck oven at 170C. After cooling, the
determined in dependence on scores: unacceptable (<2.5), good (2.5 - 3.5),
cookies were placed in polyethylene bags and stored
very good (3.5 - 4.5) and excellent (>4.5).
at ambient temperature until further examination (1).
Physical characteristics and sensory evaluation
Sensory evaluation was conducted by six experienced
panellists (35-50 years old), 24 h after baking. Samples were
evaluated in two replications and presented separately in a
balanced order. Sensory profiling was performed using a
generic descriptive analysis technique, which included
selected representative properties of cookies (11, 12), using
a 5-points method. Each mark was described with words,
using previously prepared standard cards (12, 13, 14). For
each sensory property, fixed importance coefficient and (IC)
was standardized by the panel. The importance coefficients
were balanced in the way that their sum equals 4.0. The
obtained marks were multiplied by the following IC: for
appearance (shape and surface) (IC = 0.8); for structure
and break (IC = 0.8); for chewiness (IC = 0.8); for odour (IC =
0.6); and for taste (IC = 1.0). The quantitative expression of
the total product quality is obtained as the weighted mean
value of the scores for each evaluated property. Quality
category was determined in dependence on scores:
unacceptable (<2.5), good (2.5 - 3.5), very good (3.5 - 4.5)
and excellent (>4.5).


Because of the experts were highly familiar with this type of

product they evaluated the samples in their general concept
of the cookies quality defined based on evaluation of three
primary sensory properties that determined the overall quality
of the cookies depending on the actual scores:
unacceptable (<2.5), good (2.5-3.5), very good (3.5-4.5) and
excellent (>4.5). Sensory evaluation showed that the cookie
enriched with 4 percent extract had almost excellent overall
quality (4.47)(Table 1). Scores for individual sensory properties,
except for odour and taste of control sample, indicated very
good or excellent quality of all cookies. In addition to, data
analysis of the odour and taste scores revealed the
statistically significant difference between the control sample
and all enriched cookies. The visual aspect of appearance
was evaluated in a similar way, comprehensive with different
levels of quality. Although it was possible to easily differentiate
between samples by colour due to different levels of the
ethanolic extract, overall perceived appearance by panel
showed that there was no statistical difference between the
samples in terms of appearance, taking into account the
complexity of this property. Similar results were obtained for

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

structure and break, noting that the sample with the addition of 4
percent extract, achieved the highest score (4.50). The addition of 2
percent extract resulted in the significantly softer product compared
to the addition of 6 percent, while there was no significant difference
in the remaining two samples. In addition, data analysis revealed a
statistically significant difference between the scores for odour and
taste of all enriched cookies and control sample. The addition of
ethanolic extract resulted in no significant differences between
relevant sensory properties of all enriched cookies, but cookies with
higher levels of enrichment were evaluated higher scores, in relation
to odour in favour of 6 percent (4.55), and to taste in favour of 4
percent (4.67) (Table 1). This is confirmed by the fact that
Vitalplant mixture extract, applied at three levels of
supplementation significantly improved the odour and the taste of
the cookies. In general, the obtained results showed that
Vitalplant mixture in the form of extract may be used in cookie
formulations without producing a negative impact on the sensory
The surface colour of a baked product is, together with texture and
flavour, a very important property for the acceptability of baked
goods by consumers (15). The obtained colour values of cookie
samples are presented in Table 2.

Table 2. Colour values of cookies

Values are means SD of twenty-five measurements.
Values with the different superscript within a column are statistically
different (P < 0.05).

According to Chevallier et al., (16) the colour of the cookies top

surface is under the influence of non-enzymatic browning during the
baking process, caused by the reaction between reducing sugars and
amino acids, as well as by starch dextrinisation and sugar
caramelisation. Regarding colour, the specific additional components
with which colour was associated in the cookies, contributed to
significant differentiation of the enriched cookies to each other and
relative to the control sample.
The obtained L* values of the top and bottom surface showed that
lightness decreased significantly (P < 0.05) , from 78.86 to 58.73 for top
surface, and from 72.23 to 55.12 for bottom surface, with the increase in
the level of supplementation. These results were in accordance with
previously published statements of the other authors (17, 18, 19), who
found that the incorporation of different compound enrichments may
contribute to darker top surface. On the other hand, the incorporation
of different amount of ethanolic extract contributed to increase in the
positive a* and b* values. There was a statistically significant increase
(from 0.06 to 6.39) in the positive a* value of the top surface in all
enriched cookies compared to the control sample. In addition, the
amount of 4 and 6 percent enrichment led to the significant increase in
the red tonality compared to the control (+a = 0.50) and the sample
with 2 percent enrichment (+a = 6.84) in the case of determining the
colour of the bottom surface (Table 2). Furthermore, yellow tonality

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

(positive b* values) significantly (P < 0.05) increased (from 27.09

to 37.75) with the addition of the extract in the case of the top
surface. The positive b* values of the bottom surface indicated
the significant increase in yellow tonality in all enriched cookies
compared to the control sample.
In general, the incorporation of different amount of supplements
led to darker bottom surface and higher values of red and
yellow tonality in relation to top surface which can certainly be
attributed to the impact of the baking process (16). Since the
addition of Vitalplant extract significantly changed colour of
cookies, further research should be focused on its acceptability
by consumers.
In addition, textural properties of cookies were analyzed

bakery producers. The addition of the medicinal plant mixture

extract may be used in the cookie formulations without
adversely affecting their sensory properties. Cookies
supplemented with 4 percent Vitalplant mixture extract were
estimated as the best sample. Further research needs to be
done in order to examine consumers acceptance of new
products with appropriate substitutions relative to liking,
especially in the terms of colour, but without neglecting the
necessary quantities that would contribute to health benefits.


Table 3. Textural properties of cookies

Values are means SD of ten measurements.
Values with the different superscript within a raw are statistically different (P < 0.05).

applying 3-point bending rig based on the hardness and

resistance of cookies to bend (Table 3). The hardness is
explained as the maximum force needed for cookies fracture,
while the fracturability is related to the resistance of cookies to
The obtained measurements showed that the cookies with the
addition 6 percent Vitalplant extract were significantly (P <
0.05) softer (measured value expressed in units of 972.0 g) in
comparison with the other investigated samples , except the
cookies with 4 percent enrichment. They were softer, but the
obtained values did not lead to statistically significant (P < 0.05)
difference this sample in terms of instrumentally determined
hardness. Referring to earlier studies (20, 21, 22, 23), the most
important factors influencing the instrumental measurements of
mechanical properties were shape, size and type and amount
of fat in the formulations). Regarding that, these properties were
considered responsible for the difference in results between the
sensory evaluation and instrumental measurements in our
experiment. However, because of the fact that the controlwheat cookies were prepared with the same amount of fat,
distinction in mechanical properties could have been related to
the difference in size (width and thickness) as well as spread ratio
Cookies fracturability is expressed as a distance (mm) at the
point of break and, as the resistance of cookies to bend. Smaller
distance value denotes higher fracturability. As far as the
fracturability of the samples is concerned, statistically significant
differences (P < 0.05) between the investigated cookies existed
(Table 3). The sample with the addition of 2 percent extract had
significantly higher fracturability (measured value expressed in
units of 0.60 mm) in comparison to the control (measured value
expressed in units of 0.60 mm) and the sample with 6 percent
enrichment (measured value expressed in units of 0.27 mm). The
obtained results were in agreement with above mentioned

Development of new products by adding Vitalplant mixture
extract to cookies may provide new market opportunities for


This paper is a result of the researches within

the Project No. TR 31029 ( The Ministry of
Education, Science and and Technological
Development, Republic of Serbia) and the
Project No. 114-451-4382 (the Provincial
Secretariat for Science and Technological
Development, Autonomous Province of
Vojvodina, Republic of Serbia)





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Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

*Corresponding author
University of Ni, Faculty of Sciences and Mathematics, Department of Chemistry, Viegradska 33,
P.O.Box 224, 18000 Ni, Serbia

Danica S. Dimitrijevic

Effects of solvent extraction system

on concentration and antioxidant activity of strawberry phenolics

KEYWORDS: strawberry, solvent extraction, phenolic compounds, antioxidant activity


Strawberry fruits contain phenolic compounds that exhibit antioxidant, anticancer, antitherosclerotic,
antiinflammatory and anti-neurodegenerative properties. This study was aimed to evaluate and
characterize the phenolic composition and antioxidant activities of wild and cultivated strawberries native to Serbia. Three solvent
systems were used for phenolic extraction (methanol, ethanol, and acetone) at the same concentrations (70 percent) and with 100
percent deionized water in presence 0.1 percent HCl. The efficiency of the solvents used to extract phenols from the 4 dried
strawberry samples varied considerably. The polyphenol content of the dried strawberry samples was 9.54 to 20.38 mg gallic acid
equivalent/g dried fruit. Photodiode-array detection (DAD) has been used for screening of the different classes of phenolic
compounds. The total phenolics and antioxidant activities in extracts of a wild strawberry were higher than those cultivated samples.



Berries such as strawberres are widely appreciated for its

characteristic aroma, bright red colour, juicy texture and
sweetness. They are used as processed food materials for
juice, jam, dried fruits, ice cream, etc., and therefore are
quite prevalent in our lives. Berries contain many phenolic
substances, and attention is being paid to health
promoting foods that have phenolic bioactivities (1).
Strawberries are a very rich source of polyphenols in the
human diet containing polyphenols from all classes. The
variety and high content makes strawberries a very
interesting sample for studies of polyphenols (2-4).
The critical point in studying polyphenols in fruit materials is
the extraction procedure used since it dictates the nature
and quantity of polyphenols that will be transferred to the
extract and futher characterized. Their polarity makes them
soluble in several types of polar solvents, such as methanol,
ethanol, acetone, and water. Solvent extraction of
polyphenolics is the initial step prior to quantification,
purification, separation, and characterisation and generally
involved the use of an acidified extragent.
The main objective of this study were to determine the
polyphenolic content and antioxidant capacity of wild
and cultivated strawberries from two different
geographical regions and to examine the efficiency of
different solvent systems for the extraction of polyphenols.
The phenolic compounds were extracted from the fruits by
using three conventional solvents, namely methanol,
ethanol and acetone.

Standards of catechin, quercetin, kaemferol, and phenolic
acid standards, such as gallic, p-coumaric, and ellagic acids,
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Cyaniding 3-glucoside and pelargonidin 3-glucoside were
purchased from Extrasynthese (Ganay, France). DPPH were
purchased from Sigma-Aldrich (Steinheim, Germany). Trolox
and Folin-Ciocalteu,s phenol reagent were obtained from
Merck (Darmstadt, Germany). Other chemicals and solvent
were of analytical grade.


Fruit samples
Strawberry cultivar Roxana from two different geographical
regions: Velika Plana (sample 1) and Vladicin Han (sample 2)
and wield strawberry from two different geographical regions:
Jastrebac (sample 3) and Vlasina (sample 4) were analyzed.
The strawberries were harvested at commercial ripeness. The
strawberries were harvested on May 2009, 500g was randomly
sampled and samples were taken from 20 bushes and dried
at room temperature until content weight.
Firstly, strawberry samples (10g) in blender were homogenous,
and samples (each weighted 3.00 g) extracted with the
methanol-water system, ethanol-water system, and acetonewater system (80 percent methanol, ethanol, or acetone)
containing 0.1 percent HCl volumes (20, 10 and 10 ml,
respectively) 3 times in the further course. The samples were
mixed in an ultrasound bath during the extraction procedure.
Such obtained extracts were filtered using the Bucher funnel

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

and Watman No.1 filter paper. The solid residues were rinsed
for several times in order to gain transparent extracts. Finally,
the obtained strawberry extracts were collected in a
graduated flask of the same volume of 50 ml.
Determination of the total phenolic compounds
Folin-Ciocalteu reagent was used to determined the total
phenolic compounds (TP) (5). A volume of 1 mL of dried
strawberry extract, diluted 5-6 times with some solvent
(to obtain absorbance within the range of the prepared
calibration curve), was mixed with 0.5 mL of Folin-Ciocalteu
reagent previous diluted with distilled water (1:2). A volume
of 2 mL of 20 percent sodium carbonate solution was
added to the mixture, shaken thoroughly and diluted to 10
mL by adding the distilled water. The mixture was to stand
for 120 min and the blue colour formed was measured at
760 nm with a spectrophotometer (UV/Vis spectrometer
agilent 8454; agilent, Santa Clara, CA, USA). Gallic acid
(GA) was used as a standard for the calibration curve. The
concentrations of gallic acid in the solution from which
the curve was prepared were 0,50, 100, 150, 250, and 500
mg/L (R2=0,996). The content of TP was expressed as mg
of gallic acid equivalent (GAE)/g of dried fruit (d.f.). All
measurements were carried out in 3 repetitions.
Determination of the total flavonoid content
The total flavonoids (TF) assay was performed as
previously described by Yang et al. (2004) (6) with minor
modifications. A volume of 1 mL of diluted extracts or
standard solution of gallic acid (50-500 mg/L) was placed
in a 10-mL volumetric flask, then 4 mL of deionized water,
after 5 min 0.3 ml of NaNO2 (5 percent) and 1.5 mL of AlCl3
(2 percent) were added. The mixture was shaken and 5 min
later 2 ml of 1M solution of NaOH were added, again well
shaken. The absorbance was measured at 510 nm against
the blank. The results were calculated according to the
calibration curve for gallic acid (R2=0,998). The content of
TF was expressed as mg of catechin equivalent (GAE)/g d.f.
All samples were analyzed in triplicate.
Measurements of DPPH scavenging activity
The free radical scavenging capacity of dried strawberry
extracts was determined according to the previously
reported procedure using the stable (DPPH) radicals (7).
The method was based on the reduction of stable DPPH
nitrogen radicals in the presence of antioxidants. An
aliquot of vineyard peach extracts or methanol solution of
Trolox (10-30 mM) was mixed with 2.5 mL of 0.1 mM DPPH
methanolic solution. The mixture was thoroughly vortexed,
kept in the dark for 30 min, and after that the absorbance
was measured at 515 nm against a blank of methanol
without DPPH. The results were calculated according to the
calibration curve for Trolox (R2=0.994). DPPH values, derived
from triplicate analysis, were expressed as mmol of TE/g d.f.
HPLC-DAD determination of phenolics composition
The individual phenolics were analyzed by the direct
injection of the extracts (previously filtered through a
0.45 m pore size membrane filter) into a Agilent 1200
chromatographic system equipped with a quaternary
pump, and Agilent 1200 DAD with radiofrequency
identification tracking technology for flow cells, a UV
lamp, an 8 L flow cell, and automatic injector and
ChemStation softwere. The columm temperature was 30C.

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

After injecting 5 L of sample extract, the separation was

performed in the Agilent/eclipse XDBC-18 4.6x150 mm
column. Two solvents were used for the gradient elution:
A-(H2O+5 percent HCOOH) and B-(80 percent ACN+5
percentHCOOH+H2O). The elution program used was as
follows: from 0 to 10 min 0 percent B, from 10 to 28 min
gradually increases 0-25 percent B, from 28 to 30 min 25
percent B, from 30 to 35 min gradually increases 25-50
percent B, from 35 to 40 min gradually increases 50-80
percent B, and finally for the last 5 min gradually decreases
80-0 percent B. The detection wavelengths were 320, 360
and 520 nm. The identification and quantitation of the
various phenolic compounds were performed by means
of calibration curves obtained with standard solutions of
cyanidin 3-glucoside, pelargonidin 3-glucoside, quercetin,
kaemferol, and p-coumaric acid. The results are expressed
as mg/100 g of d.f.
Statistical analysis
The data were reported as meanstandard deviation
(SD) with triplicate determinations. The significance of
inter-group differences was determined by the analysis of
variance (ANOVA). The p value of p<0.05 was considered
statistically significant.

Strawberry fruits contain compounds that exhibit
antioxidant, anticancer, antiatherosclerotic,
antiinflammatory and anti-neurodegenerative properties.
The total phenolics, flavonoids, and the antioxidant
capacity determined by spectrophotometry in each dried
strawberry sample grown in the Serbia presented in Table 1.
The content of total phenolics in strawberry extracts was
determined spectrophotometrically using Folin-Ciocalteau
reagent and calculated as Galic Acig Equivalent (GAE).
The highest quantity of total phenolics was achieved in
acetone extracts (14.93 20.38 mg GAE/g d.f.) and the
lowest in methanol extracts (9.59 15.02 mg GAE/g d.f.). In
our study we observed that the solvent used in the
extraction of strawberry samples had a significant effect
(p<0.05) on the total phenols content of the extracts. Some
differences were found between wild strawberries grown in
different location in Serbia (Vlasina and Jastrebac), also,
and between cv. Roxana grown in different location
(Velika Plana, Vladicin Han). All of these differences were
statistically significant.
The total flavonoid content of dried strawberries was also
determined. The amounts of total flavonoid content in
different solvent extracts of strawberries is shown in Table 1.
The results revealed that extractability of flavonoids was also
affected by the solvent used. The acetone was the best
solvent for extracting flavonoid compounds (mean 10.52 mg
CE/g d.f.). The methanol and ethanol were not good solvent
for extraction of flavonoids from strawberries. The flavonoids
were found in high concentrations ranging from 52.8 to 65.2
percent of total phenolic content in samples.
The radical scavenging activity of dried strawberries was
measured using the DPPH radical assay. The method was
based on the reaction of stable DPPH nitrogen radicals in
the presence of antioxidants. The total antioxidant
activity of the dried strawberry extracts tested varied.


d.f.; pelargonidin 3-rutinoside, 30.18-53.42 mg/100g d.f.).

These anthocyanins represented together >65 percent of
total anthocyanin content. Jakobek et al. (2007) (15)
showed that the pelargonidin derivatives represented
together >96 percent of total anthocyanin amount and 4
percent cyaniding 3-glucoside, which contrary with our
results (35 percent).
The HPLC chromatogram of dried strawberry extract,
recorded at 360 nm with a diode array detector, is shown
in Figure 2. These compound were identified by
comparison of their retention times and UV-Visible spectra
with those of standards.

Table 1. Total phenolics (TP), total flavonoids (TF) and total

antioxidant capacity (TAC) of strawberry extracts obrained from
different solvent extraction systems. The data are reported as
meanstandard deviation (n=3). 1Mean in a column followed by
different letters are significantly different using analysis of variance
at the level of<0.05. 2Different letters a significant difference
between samples in some solvent extraction system.

The strawberry extracts showed higher antioxidant activity,

102.15 m TE/g d.f. for acetonic, 79.83 m TE/g d.f. for
ethanolic and 69.96 m TE/g d.f. for methanolic extracts.
Changing the extraction solvent aqueous methanol,
aqueous ethanol and aqueous acetone altered the
phenolic composition of the samples. The data in Table 2
and 3 show that acetone was the best solvent for
extracting flavonol compound, followed by methanol and
ethanol. The highest anthocyanin content were observed in
extracts with methanol, while the lowest levels were found
in extracts with acetone.
Various solvent systems have been used for the extraction
of polyphenols from plant materials (8). Extraction yield is
dependent on the solvent and the method of extraction.
Furthermore, solvent polarity will play a key role in
increasing phenolic solubility. Water and aqueous mixtures
of ethanol, methanol and acetone are commonly used in
plant extraction (9). Alothman et al. (2009) (10) reported
that aqueous acetone was superior in compare to
methanol and ethanol for extracting flavonoids in tropical
fruits. Kajdzanoska et al. 2011 (2) reported that pure
acetone is an efficient extraction solvent for anthocyanins
and flavonols in strawberries, but for hydroxycinnamic acid
derivatives, the acetone/water/acetic acid mixture was
found as the most efficient. On the other hand, methanolwater mixtures are good solvent system for the extraction of
anthocyanins from red grapes (11, 12).
The HPLC chromatogram of methanolic extract of
strawberry recorded at 520 nm are present in Figure 1. The
amounts of anthocyanins in strawberry extracts are shown
in Table 2. Anthocyanins were found in the highest
concentrations in strawberries grown in Serbia (150.86
207.75 mg/100g d.f.). Wild strawberry-Vlasina (sample 1)
had the highest total anthocyanin content, followed wild
strawberry Jastrebac (sample 3), cv. Roxana strawberries
(sample 1 and 2).
Studies have shown that numerous factors such as harvest
season, variety, stage of harvesting, climatic conditions and
growing season can affect the composition and
concentration of individual as well as total anthocyanins
(13, 14). Pelargonidin derivatives predominanted in
strawberry (pelargonidin 3-glucoside, 69.16-98.35 mg/100g


Figure 1. HPLC chromatogram recorded at 520 nm a diode array

detector, of anthocyanins. Peaks 1, 2, 3 are identified in Table 2.

Table 2. Anthocyanin contents (mg/100 g d.f.) of strawberry samples:

cyanidin-3-sophoroside and cyanidin-3-glucosyl-rutinoside at 520 nm.
The level of pelargonidin-3-rutinoside is expressed as pelargonidin-3glucoside equivalent. Values are meanstandard deviation (n=3).

Figure 2. HPLC chromatogram recorded at 360 nm a diode

array detector, of flavanols. Peaks 1, 2 are identified in Table 3.
Ellagic acid is identified and quantified at 370 nm.

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Table 3. Flavonol content (mg/100 g d.f.) of strawberry samples:

quercetin and kaempferol at 360 nm. Values are meanstandard
deviation (n=3).

Table 3 shows the levels of the flavonols determined in dried

strawberries samples. The concentration of the total flavonols
was the lowest in sample 1 with a mean content 10.46
mg/100g d.f. and highest in sample 2, with a mean content
29.41 mg/100g d.f. in this study, quercetin was the main
flavonol (>79.9 percent of total flavonol amount), followed by
kaemferol. Quercetin content ranged from 4.85 to 32.92
mg/100g d.f. Myrecetin was no detected in all samples. The
different results may be explained by differences in growing
conditions. Also, the different extraction conditions influenced
the results.
The HPLC chromatogram of dried strawberry extract,
recorded at 320 nm with a diode array detector, is shown in
Figure 3. Content of phenolic acids is given in Table 4.
In strawberry, the concentration of phenolic acids was higher
than concentration of flavonoids. The main phenolic acid
found in strawberry was ellagic acid (mean 31.58 mg/100g d.f
in methanol extracts), followed by p-coumaric acid (mean
20.26 mg/100g d.f.).

Figure 3. HPLC chromatogram recorded at 320 nm a diode array

detector, of hydroxycinnamic acids. Peak 1 is identified in Table 4.

Previous studies indicated a similar trend whereby the most typical
polyphenolics were significantly extracted into the polar solvents
(16). Also, the present study revealed that the wild strawberries to
Serbia contained higher total phenolics (mean 19.60 mg GAE/g
d.f.) compared to cv. Roxana (mean 17.97 mg GAE/g d.f).
Generrally, strawberry fruits grown in the Serbia are a rich source of

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014


present study revealed that the wild strawberries contained higher

total phenolics and antioxidant activities compare to cv.

This work was supported by the Serbian Ministry of Education
and Science Protection (grant number 174007).


Table 4. Phenolic acid contents (mg/100 g d.f.) of strawberry
samples: ellagic acid at 370 nm and p-coumaric acid at 320 nm.
Values are meanstandard deviation (n=3).

poliphenols. Khknen et al. (2001) (17) found similar content of

total phenolics in strawberries (16.0 24.1 mg GAE/g d.f. using 70
percent acetone as extraction solvent).
Flavonoids are a group of polyphenolic compounds naturally
present in most edible fruit and vegetable plants. They constitute
most of the yellow, red and blue colours in the fruit (18).
Flavonoids from the fruit and vegetables are currently widely
studied as components that have the potential to provide
multiple health benefits.
Methanol-water with 0.1 percent HCl mixture has been used for
extraction systematical study at anthocyanins in fruits and berries
(2, 3). Also, the highest phenolic acid content were observed in
extracts with methanol. The results are in accordance with those
reported by Beevi et al. (2010) (16).
The results of this study of flavonols agree will with the literature (3,
19). Among with flavonols quercetin and kaemferol, some authors
reported low level of myricetin (20, 21).
According to previous study, the main representative of
phenolic acids in strawberry was ellagic acid or its derivatives,
followed by p-coumaric acid with is consistent with the results
in this study (15, 19). Some authors reported that the
p-coumaric acid was the dominant phenolic acid in
strawberries, with ellagic acid being second (20).


Y. Amakura, Y. Umino et al., J. Agric. Food Chem., 48, pp. 6292-6297

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M.P. Khknen, A.I. Hopia, M. Heinonen. J. Agric Food Chem., 49, pp.
4076-4082 (2001).
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99-104 (2010).
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S. Hkkinen, M. Heinonen et al., Food Res Int., 32, pp. 345-353. (1999).

As a continuation of our study on strawberry extracts
were prepared using different organic solvents. As
observed, extracts with higher antioxidant activity
also had higher polyphenolic content.
Acetone showed slightly better characteristics
than ethanol and methanol as a solvent for
total phenolic compounds, total flavonoids
and flavonols. The highest anthocyanin and
phenolic acid content were observed in
extract with methanol. Our results
can contributed to the selection of
the most efficient extraction
solvents to be use when analysis
of total extractable polyphenols
or specific groups of polyphenols
are to be made. Also, the


Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

*Corresponding author
1. University of Novi Sad, Institute of Food Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
2. University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia

Ljubisa Saric

Antibacterial activity of donkey milk

against Salmonella
KEYWORDS: donkey milk, antibacterial activity, Salmonella, lysozyme, lactoferrin


The aim of this study was to investigate the effect of initial contamination and lactation stage on the
antibacterial properties of raw donkey milk against three Salmonella species in artificially contaminated
milk samples during five days of storage at 4C. Tested samples showed antibacterial activity against all three strains at three
examined contamination levels (102, 103 and 104 CFU /ml). At the lowest level of contamination milk samples exhibited bactericidal
activity. Milk from the early and middle lactation period showed a stronger antibacterial activity in comparison to the milk from the
late lactation period. Lysozyme and lactoferrin content decreased during lactation. Antibacterial effect of donkey milk was confirmed
by scanning electron microscopy.

Salmonella is one of the principal causes of foodborne illness
worldwide and is equally present in developed and
developing countries (2). Infections of Salmonella spp. occur
in humans in two forms, as a typhoid fever, a systemic disease,
and as salmonellosis, a self-limiting gastrointestinal illness (24).
Milk and dairy products are a good medium for the growth of
Salmonella spp., whose presence in milk is associated with the
management of dairy animals, animal feed and storage
conditions (15). Recent investigations showed that Salmonella
was present in 3-6 percent of raw bovine milk samples (20),
mostly at level of 200 CFU/ml, although there are reports
about the raw milk samples with even 105 CFU /ml of
Salmonella spp. Unlike bovine milk, there are much less
literature data on Salmonella spp. in non-bovine milk,
especially on donkey milk, which has been long time
attributed significant medicinal and therapeutic properties
(28, 29). Low casein content, high percentage of essential
amino acids, protein and lipid profiles similar to those of
human milk (28) are in favour of donkey milk as a potential
new dietetic food and a good alternative for infant nutrition in
the case of cows milk protein allergy (CMPA) (16) and of
multiple food allergy (30 A). Donkey milk is mainly consumed
in Africa, Asia, Eastern Europe (29) and Italy (30 B), where
donkeys are traditionally bred. Donkey milk has been
traditionally used in Serbia as a natural remedy for treatment
of asthma and bronchitis. According to World Health
Organization estimated deaths by respiratory diseases per
100000 population in Serbia is higher than Europe and world
average (4). Considering this fact, there has been a growing
demand for donkey milk on the Serbian market, recently.


Despite the limited literature sources on donkey milk a number

of newer studies have shown the absence of this pathogen in
donkey milk (29, 23, 30 B). Antibacterial activity of donkey milk
(29, 25, 23) is attributed to the significant content of lysozyme
and lactoferrin (28, 29).
Usually milk and dairy products are submitted to
pasteurization process, which effectively eliminates
Salmonella spp. Milk-borne salmonellosis is therefore often
related to the consumption of raw or improper pasteurized
milk and dairy products (11). Local consumers in Serbia usually
consumeraw donkey milk cooled at 4C immediately after
milking. Previous investigations of raw Domestic Balkan
donkey milk (23) showed that microbiological quality of the
tested milk was in accordance with the requirements of
European Regulation n. 853/2004 for raw milk from species
other than bovine (5) after 5 days of storage at 4C.
Furthermore, during this period, the presence of Salmonella
spp. as well as other pathogens was not registered. However,
Salmonella species could be present in raw milk in different
initial number (20), depending on dairy farming practice.
Although most Salmonella serotypes are unable to
propagation at 4C, these pathogens can survive for
extended periods at this temperature (17).
The objective of this study was to investigate the impact of
level of contamination and lactation period on the
antibacterial properties of raw donkey milk against three
Salmonella (S) species: S. typhimurium, S. enteritidis, and S.
livingstone, during five days of storage at 4C. The content of
lactoferrin and lysozyme were determined in donkey milk
samples during three periods of lactation. Scanning electron
microscopy analysis was performed to determine the effect of
donkey milk on cell morphology of the tested microorganisms.

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014


Sample collection
Milk samples of 6 clinically healthy donkeys (Domestic Balkan
donkey breed) were collected immediately after the morning
hand-milking from a Special Nature Reserve Zasavica located
in the northwest of Serbia. The samples were collected in three
different lactation periods: early stage (70-90 days post-partum),
middle stage (140-160 days) and the late stage (190-210 days).
Milk samples were immediately cooled to 4C and transported
to the laboratory where the samples were frozen at 20C.
Donkeys lactation process lasts about seven months (25). At the
Special Nature Reserve Zasavica, the milking of donkeys
begins after 2.5-3 months post-partum.
Antibacterial assay
The antibacterial activity of six individual raw milk samples was
investigated against Salmonella enteritidis ATCC 13076,
Salmonella typhimurium ATCC 14028 and Salmonella livingstone
strains taken from a collection of bacteria at the Department of
Microbiology, Institute of Food Technology, University of Novi
Sad, Serbia. Bacterial strains were frozen and kept at 80C.
Samples of donkey milk were artificially contaminated with these
three Salmonella species at the three following contamination
levels: 2 log10 CFU /ml, 3 log10 CFU /ml and 4 log10 CFU /ml. After
incubation at 37C of each test microorganism on a nutrient
agar, well isolated colonies of each test microorganism were
inoculated into sterile saline and vortexed thoroughly. The
density of the bacterial suspension was adjusted to the 0.5
McFarland standard using DEN-1 densitometer (Biosan, Riga,
Latvia). From these initial suspensions further decimal dilutions
were prepared in a sterile saline solution. From each artificially
contaminated sample, 25 ml was placed into a sterile
stomacher bag and stored at 4C during five days. Changes in
the number of tested bacteria were monitored on a daily basis
according to the international standard (13). The number of
viable bacteria was expressed as log10 CFU /ml. Nutrient broth
(Himedia, India) was artificially contaminated with three
bacterial levels and it was used as positive control, while non
inoculated donkeys milk was used as negative control. All
samples were analyzed in triplicate.

the milk proteins, bovine serum albumin was used as a standard.

All samples were analyzed in triplicate.
Scanning electron microscopy (SEM)
Bacterial cells were obtained by centrifugation of milk samples
and positive controls at 4500 x g for 15 min at 4C, followed by
washing three times and resuspension in an equal volume of PBS
(pH 7.4) (22). The obtained suspension (50 l) was applied to 12
mm diameter glass coverslips (27). After 30 seconds, the
coverslips were carefully dipped into a 2.5 percent
glutaraldehyde/PBS solution (0.265 M). Fixed bacterial cells were
then dehydrated in three ethanol solutions [50 percent and 80
percent (v/v) in water, and absolute ethanol], dried and
mounted onto stubs using double sided carbon tape, coated
with a thin layer of gold. All samples were analysed by SEM (JEOL
JSM-646OLV, Japan).
Statistical analysis
Results were expressed as mean along with standard deviations,
of triplicate analyses for all measurements. Analysis of variance
was followed by Duncans multiple comparison tests using
STATISTICA version 10 (StatSoft Inc., Tulsa, OK, USA).


Antibacterial activity of donkey milk
Changes in bacterial counts are given as the mean value of
tested individual samples in each lactation phase (Tables 1, 2
and 3).
The number of viable cells of the tested Salmonella strains
decreased constantly throughout the storage period in all milk
samples from the early and middle lactation period (Tables 1, 2
and 3). The count of the tested bacteria in milk from these two
lactation periods was not statistically different (P>0.05) after five
days of storage. The results of antibacterial activity at three
different contamination levels showed that the number of the
tested microorganisms decreased below the detection limit (<1
log10 CFU /ml) only in the samples with the lowest level of
contamination (2 log10 CFU/ml). Milk from the early and middle
lactation period had a stronger antibacterial potential
compared to the milk from late lactation period, which could be
attributed to its higher content of lysozyme and lactoferrin. The
slightly weaker antimicrobial activity towards S. typhimurium in
comparison to other two tested bacteria was observed in the
samples at the contamination levels of 3 log10 CFU /ml and 4

Determination of protein profile during lactation period

A modified method of Tidona et al. (2011) was used for the
preparation of samples. After diluting the milk samples in buffer
(0.125 M Tris-HCl, 4 percent SDS, 2 percent glycerol, 2 percent
-mercaptoethanol, pH 6.8) in the ratio 1:1.5 (v/v) sample:
buffer, these milk dilutions were heated at 100C for five minutes.
The Agilent 2100 Bioanalyzer
(Agilent Technologies, Santa Clara,
CA, USA) in combination with the
Protein 80 Plus LabChip kit and the
dedicated Protein 80 software
assay on 2100 expert software was
used for the chip-based
separations. The Protein 80
LabChip kit served as a protocol
for preparing the chips. According
to the convention for SDS-PAGE
(26) fractioning is size-based, and
the profiles show the smallest
proteins emerging first in the
profiles but at the bottom of the
Table 1. Changes in S. enteritidis count (log10 CFU/ml) in raw donkeys milk during the storage at 4C.
gel patterns. For quantification of

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014


(data not shown). Salmonella

species were not detected in
negative controls during the
storage period at 4C.

Table 2. Changes in S. typhimurium count (log10 CFU/ml) in raw donkeys milk during the storage at 4C.

Donkey milk protein characterization

Lysozyme and lactoferrin were
identified as proteins with
molecular weights of 15 and
78.1 kDa, respectively (25). A
similar pattern was noted
between the results of chipbased separation and literature
data for donkey milk (25). The
concentrations of lysozyme and
lactoferrin in donkey milk
decreased during lactation in all

tested samples (Table 4).

Observed values of lysozyme and lactoferrin were
significantly different (P<0.05) between individual
samples from the same lactation stage. The differences in
the contents of these two proteins between the early and
middle lactation stage were
much lower than the differences
between middle and late
lactation period. In donkey milk,
a higher values of lysozyme
content is obtained in the first 60
days post parturition, followed
by a slight decrease until 180.
days, while in the following two
months a significant decrease
was observed, according to Pilla
and Dapr (2010) (19). Previous
investigations (23) showed that
the lysozyme and lactoferrin
were present in Domestic Balkan
Table 3. Changes in S. livingstone count (log10 CFU/ml) in raw donkeys milk during the storage at 4C.
donkey milk in amount of 1.31 g
L -1 and 4.80 mg L -1 , respectively.
That study was performed on
mixed milk samples in short time period and stage of
typical for lysozyme (9). However donkey milk lysozyme
lactation was not taken into account. Regarding to
probably has the ability to bind with calcium ions like lysozyme
results obtained in this work, milk samples from previous
from equine milk (1) since this two species belong to the same
study probably belonged to late period of lactation.
family (Equidae) (12). This ability makes lysozyme stable and
Observed different values of lysozyme and lactoferrin in
enhances its antimicrobial activity towards Gram negative
milk samples belonging to the same lactation stage can
bacteria (1). A synergistic activity between lysozyme and
be related to differences between individual animals
lactoferrin which contributes to overall antimicrobial activity of
such as age, physiological condition etc. Decrease of
donkey milk should not be excluded. It is well known that
enzymes content in donkey milk during the lactation (21),
interaction of lactoferrin with lipopolysaccharide (LPS) layer
was consistent with the results obtained for bovine milk
can cause structural changes in bacteria outer membrane,
(10). This decrease may be related to the differential
increased membrane permeability and enhanced
expression of milk protein genes during lactation in the
susceptibility of Gram negative bacteria to lysozyme (7). Also,
brushtail possum (3).
the smaller size of bacterial cells at lower temperatures reduces
log10 CFU /ml. Although it is not clear whether the lysozyme of
donkey milk acts alone or together with some other milk
component, or is its mechanism of action is known, many
authors assert that it is the main antimicrobial agent in donkey
milk (28, 29). Activity against Gram negative bacteria is not

the amount of biomass for the same number of cells, making

them more sensitive to lysozyme (14). Donkey milk possess
bactericidal effect against Salmonella species at the lowest
level of contamination, according to the definition of
bactericidal activity as the effect which leads to destruction of
99.9 percent of present bacteria (18). In the remaining milk
samples the antimicrobial activity could be defined as
bacteriostatic since the decrease of bacterial number at the
end of storage was 66 - 96 percent. The count of S. enteritidis,
S. typhimurium and S. livingstone was not significantly changed
(P>0.05) in positive control tests, during the storage at 4C


Scanning electron microscopy (SEM)

SEM results clearly indicate that morphological changes
occurred on the cells of all examined Salmonella species
in donkey milk after five days of storage at 4C.
The presence of bacterial cells with leaking cellular
content displayed by SEM (Figure 2) indicating a damage
of cell walls and confirming the antimicrobial activity of
donkey milk. This phenomenon was noted for all three
examined Salmonella species. Control cells were
morphologically regular and typical (Figure 1).

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Table 4. Changes in lysozyme (LYZ) and lactoferrin (LF) content in raw donkeys milk
during lactation period.

Figure 1. Scanning electron microscopy (SEM) of Salmonella in donkey

milk after 5 days at 4C

Domestic Balkan donkey milk showed antimicrobial activity against three tested
Salmonella species. Stronger antimicrobial activity observed in milk samples from
early and middle lactation period indicate that antibacterial potential of donkey
milk is dependent on lactation stage. This phenomenon could possibly be
attributed to the higher concentrations of lysozyme and lactoferrin in milk from
the first two periods of lactation. Antibacterial potential of donkey milk was also
influenced by the initial number of bacteria. At the lowest level of contamination
(102 CFU /ml) raw donkey milk exhibited bactericidal activity which resulted in
destruction of 99.9 percent of bacteria after five days of storage at 4C.
Antimicrobial activity of donkey milk could be described as bacteriostatic in milk
samples with higher contamination levels (103 CFU /ml and 104 CFU /ml). The
serious damage of bacterial cell walls and the leak of the cellular content
observed by SEM confirmed antimicrobial effects of Domestic Balkan donkey
milk against the tested Salmonella species.

This work is a part of the National Project (TR31029) financially supported by the
Ministry of Education and Science, Republic of Serbia. Authors are grateful to
Slobodan Simi and Nikola Nili (Special Nature Reserve Zasavica, Serbia) for
providing the milk samples.





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Demmer J., Ross I.K., et al., Differential expression of milk protein genes during lactation
in the common brushtail possum (Trichosurus vulpecula), J. Mol. Endocrinol., 20, 37-44
Easthorn Clinical Services in Central and Eastern Europe, Serbia, URL. http://www. 15.04.2013.

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council of 29 April 2004 laying down specific hygiene rules for food of
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powdered dairy products in Victoria, Aust. J. Dairy Technol., 65, 58-62
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proteins and derivatives thereof, Curr. Pharm. Design, 9, 1257-1275
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quality, Edited by Smit G., CRC Press, Washington DC, USA (2003).
Freitas N.O.C., Penha F.R.A.C., et al., Sources of human non-typhoid
salmonellosis: a review, Brazil. J. Poult.Sci., 12, 1-11 (2010).
Grubb P., Order perissodactyla. In: Mammal species of the world: A
taxonomic and geographic references, Edited by Wilson D.E. &
Reeder D.M., Johns Hopkins University Press, Baltimore, MD, USA
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growth of Listeria monocytogenes, J. Food
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bacterial genomics on determining quality
and safety in the dairy production chain, Int.
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donkeys milk in treating highly problematic
cows milk allergic children: an in vivo and in
vitro study, Pediatr. Allergy Immu., 18, 258-264
Oscar T.P., Development and validation of a
predictive microbiology model for survival
and growth of Salmonella on chicken stored
at 4 to 12C, J. Food Protect., 74, 279-284
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relevance of bacteriostatic versus
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treatment of Gram-positive bacterial
infections, Clin. Infect. Dis., 38, 864-870 (2004).
Pilla R., Dapr V., et al., Hygienic and health
characteristics of donkey milk during a followup study, J. Dairy Res., 77, 392-397 (2010).
Poppe C., Diseases of Dairy Animals |
Infectious Diseases: Salmonellosis. In:
Encyclopedia of Dairy Sciences, Edited by
Fuquay J.W., Fox P.F. & Sweeney P.H.L.,
Academic Press (2011).
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consumption, Int. Dairy J., 24, 130-142 (2012).
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vitro and in situ antimicrobial action and
mechanism of glycinin and its basic subunit,
Int. J. Food Microbiol., 154, 19-29 (2012).
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properties of Domestic Balkan donkeys milk,
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fraction from milk fermented with Lactobacillus helveticus protects

mice against Salmonella infection, Int. Dairy J., 21, 607-614 (2011).
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digested in vitro with human gastrointestinal enzymes, Int. Dairy J., 21,
158-165 (2011).
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method in determination of Kunitz trypsin inhibitor in soybean
varieties, J. Agr. Food Chem., 58, 7980-7985 (2010).
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Escherichia coli K-12 in apple juice treated with radio frequency
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Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014


Industry perspective

LLC. 360 West Illinois, #101 Chicago, 60654 Illinois, USA
indirizzo 1Assistant Professor, Tufts University, Friedman School of Nutrition, Boston, MA, USA
indirizzo 2


The neurobiology and marketing

of mood drinks
KEYWORDS: mood, beverages, marketing, neurobiology, nutrition, consumers


Consumers are increasingly over-worked, under-rested and often experiencing high levels of daily stress.
Combine this with the need for mobility and convenience, and the concept of mood-modulating
beverages becomes an appealing proposition for formulators and marketers alike. Historically, the focus has been more on the
energy-producing stimulant side with tea or coffee drinks providing caffeine. However, there is increasing interest in the relaxant
side of the equation in terms of beverage ingredients than can offer a calming effect. Unfortunately, there is often limited published
data on the safety and/or efficacy of these ingredients in beverage formulations specifically, especially when used in multi-ingredient
formulations. This article will focus first briefly on caffeine and then on a select handful of relaxant ingredients commonly chosen for
mood beverages marketed in the United States as dietary supplements.

To date, much of the mood beverage market has focused
on caffeine in the form of tea, coffee and increasingly in the
category of energy drinks. This category is projected to grow
to a value of $21.5 billion by 2017 with the target consumer
being males aged 18 to 34 years (1). The category growth
continues, often in double digits year over year, despite it
becoming increasingly challenged with lawsuits in the United
States, especially within the context of caffeine intake and
children. Many energy drinks though actually have less caffeine
than some standard coffee drinks like those purchased at
common coffee house chains. Either way, the amount of
acceptable daily caffeine intake for different age groups
remains a hotly debated topic.
The relaxing mood beverages market is thought to be closer
to $32 million in the United States, which is a mere fraction of the
energizing side. This market imbalance is likely the result of a few
factors. First, more consumers are familiar and comfortable with
caffeine and the idea of a daily stimulant beverage, especially
in the morning to wake up with and start the day. On the other
hand, the concept of a daily beverage, perhaps in the evenings,
that could have a calming or relaxing effect is less well engrained
in the minds of consumers. Certainly, anecdotally, there may be
suggestions of warm milk or chamomile tea before bed type as a
means to induce calm leading to restful sleep. This lesser defined
category of relaxing beverages presents an opportunity for
both definition and for future product formulations.
The definition of a mood beverage is a bit tenuous because
mood has different meanings to different people. Mood is often
defined as a diffuse and global state or quality of feeling over
time, while emotion is a complex set of interrelated sub-events

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

concerned with a specific object. Mood changes over time

and throughout the day and for many consumers is closely
linked to energy levels. Importantly, mood and correspondent
energy levels can be a perceived state versus an actual state,
which is further complicated by the fact there is no biochemical
marker of relaxation. A noteworthy aspect of mood is that it is
often defined in the negative, meaning a state of sullenness,
gloom, low energy or even a bad temper. For these reasons, it is
important to point out that the effects of any beverage marketed
towards a mood impact - whether to drive increased energy or
to promote calmness - is as dependent on the potential efficacy
of any ingredients contained in a given formulation in as much as
the perceived efficacy. Despite such challenges, there is great
interest in this area because consumers tend to regularly consume
beverages throughout the day, and virtually all consumers deal
with fluctuating mood and energy levels at the same time.


In terms of ingredients, there are really two halves of
neurobiological impacts stimulants and relaxants. On
the stimulant side, some of the more common beverage
formulations include caffeine, yerba mate (caffeine +
theobromine), guarana, taurine and b-vitamins. On the
relaxant side, GABA, melatonin, 5-HTP, valerian, chamomile and
L-theanine are often used in formulations.
With approximately 20,000 peer review studies in OVID Medline
(1946-present), caffeine is one of the most researched ingredients
when it comes to mood, cognition and energy enhancement
effects. In fact, so much attention has been paid to caffeine
that current research is often more focused on examination of


possible interactive effects with other stimulating ingredients.

Based on the review written by McLellan and Lieberman, it is well
documented that caffeine can act as a thermogenic, ergogenic
and cognitive aid and that its effects are consistent with the
claims of energy drink manufacturers (2). The review goes on to
state the following when it comes to possible interactive effects
with other ingredients (Table 1).

While some ingredients have rather limited data and other

ingredients a larger literature base, the challenge is that there
is often limited to no peer reviewed evidence which directly
tests the individual efficacy of a relaxing ingredient in a
beverage or beverage shot in humans. Plus, when a given
formulation includes three or more of these ingredients in
combination it is impossible to know if there are interactive
Here is a brief
overview of
select ingredients
commonly appearing
in relaxant drinks in
the U.S. market:

referred to as
natures valium,
GABA or gammaaminobutyric acid is
the chief inhibitory
neurotransmitter in
Table 1. Possible interactive effects with other ingredients
the central nervous
system. GABA is
Taken together, it suggests that there may be limited evidence
synthesized in the brain from glutamine, and does not
that interactive or synergistic effects of caffeine are happening
cross the blood brain barrier. GABA is widely distributed
in multi-ingredient beverage formulations. However, this doesnt
in the brain and is present at about a third of all brain
necessarily mean caffeine levels in a given energy drink will not
synapses. A commonly recommended dose of GABA is
cause unwanted side effects. The age, weight and caffeine
100-250 milligrams, 2-3 times daily, but no published peer
consumption patterns of the individual must be considered.
reviewed data on the dosage for GABA in beverages or
Further, given the legal and regulatory attention being paid
drink shots is publicly available. This is further complicated
to energy drinks it may be time for the concept of caffeine
by the fact that if oral GABA dietary supplements are not
equivalents to be created. This would be not unlike how vitamin
able to cross the blood brain barrier, then how are they
A equivalents are being employed nutritionally to figure out
acting on the central nervous system to have a relaxing
contributions from vitamin A precursors like beta-carotene along
effect? To overcome this challenge, GABA-like supplements
with the active-form vitamin A.
and analogues have been devised that can cross the
While the energy drink debates ensue, there is a growing interest
blood brain barrier. One GABA analogue is gammain the relaxant market. GABA, melatonin, 5-HTP, valerian,
hydroxybutyrate or GHB, also known as liquid ecstasy or the
chamomile and L-theanine all appear in various beverages
date rape drug which is obviously entirely inappropriate
in the marketplace geared towards relaxation or chilling
for product inclusion.
out to use the marketing parlance. In terms of peer review,
Apart from the inclusion of GABA or even a GABA-like
the research volumes based on an OVID Medline search
compound, there is reason to believe the GABA pathway
(1946-present) are shown in the following graph (Figure 1).
in the form of the GABA receptor itself is a reasonable
target. For instance,
compounds have an
affinity for adenosine
and benzodiazepine
(GABAa) receptors,
meaning their
ingestion can have a
calming effect. One
study that tested a
dark chocolate drink
mix standardized to
500, 250 or 0 milligrams
of cocoa polyphenols
found the highest
dose group reported
improved calmness
and contentedness
after a 30 day
Figure 1. Research volumes based on an OVID Medline search (1946-present)
intervention (3).



The key point here though is that a supplemental

beverage containing GABA is an entirely different
proposition than a product with cocoa polyphenols aimed
at the GABA receptor itself.
Melatonin is a hormone produced in the pineal gland with
circulating levels that regulate circadian rhythms. In the
United States, melatonin is categorized by the Food and Drug
Administration (FDA) as a dietary supplement. It can easily cross
cell membranes as well as the blood brain barrier. Interestingly,
melatonin is found in a wide array of edible plants, with plant
tissue concentrations varying from pictograms to micrograms
per gram (4). For example, melatonin has been detected in
tomatoes (up to 114 ng/g), olive oil (up to 119 pg/ml) and in
other foods like rice, corn, strawberries and cherries.
Melatonin is quite potent and has an extremely short plasma
half-life, which is in part why dosing proves challenging. To
treat jet leg symptoms, one study found a 5 mg immediaterelease melatonin to be most effective compared to a 2 mg
slow-release formulation, but that was only marginally more
effective than a 0.5 mg immediate-release formulation (5).
Given such uncertainties on dosing and timing, the National
Centre for Complementary and Alternative Medicine
(NCCAM) has concluded that important questions remain
about [melatonins] usefulness, how much to take and when
to take it, and long-term safety.
5-HTP or 5 hydroxytryptophan is an aromatic amino acid
that functions as a neurotransmitter. It is produced naturally
in the body from the essential amino acid L-tryptophan
and produced commercially by extraction from the seeds
of the African plant, Griffonia simplicifolia. It has been used
clinically for over 30 years and crosses the blood brain barrier.
Commonly recommended doses are to take 100 mg of 5-HTP
3 times daily, but there are no available published peer review
data for beverage and/or shot dosages.
Valerian root is sold as a dietary supplement in the United
States. According to NCCAM, the constituents of valerian
have been shown to have a sedative effect in animals, but
there is no scientific agreement on mechanism of action.
They further state that research suggests valerian may be
helpful for insomnia, but there is not enough evidence from
well-designed studies to confirm this.
Chamomile is the common name for several daisy-like plants
of the family Asteracea. The most commonly used species
include German or wild chamomile (Matricaria chamomilla)
and Roman, English or garden chamomile (Chamaemelum
nobile). A randomized controlled 8-week trial with 57 participants
was performed to test the effects of chamomile extract in
patients diagnosed with ng odour that may have been difficult
to mask in the placebo. Nonetheless, dosages were adjusted
incrementally week over week, leading up to 5 capsules daily.
The intervention group experienced a reduction in anxiety
symptoms as measured by the Hamilton Anxiety Rating (HAM-A).
Suggesting potential efficacious effects for chamomile as a

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

L-theanine or gamma-ethylamino-L-glutamic acid is a
neurologically-active amino acid found almost exclusively in tea
plants. It constitutes 1-2 percent dry weight of tea leaves and
gives green tea is characteristic umami taste. Animal studies
have revealed that L-theanine affects dopamine and serotonin
concentrations in the brain, suggesting anxiolytic or calming
effects. Research has also found increased alpha brain wave
activity in humans after L-theanine administration, meaning it
could lead to a state of effortless alertness (7-9).
This brief summary review of select relaxants and there
emerging evidence base points to the need for further
evidence-building efforts which parallel the scientific attention
given towards the stimulant side of the equation. Such efforts
will ideally consider questions like:
Is the ingredient a neurotransmitter, hormone, or
amino acid?

What is the mechanism of action? Does it act centrally or
peripherally or both?

Is there human research or only animal models?

Does it cross the blood brain barrier?

Can the body synthesize the compound on its own?

Are there downstream metabolites or interconverting
compounds to consider?
What format of delivery encapsulated extract.
beverage or beverage shot?

Sub-clinical versus clinical effects?

Interaction effects? Synergistic or antagonistic?

Timing and dosage?
Marketing angles
Despite the emergent status of research about the relaxant
ingredients, the marketplace in the United States presents
multiple options available, most all of which are marketed
as dietary supplements. Some of the brands with various
combinations of ingredients like GABA, melatonin, 5-HTP,
valerian, chamomile, L-theanine or others include Just ChillTM,
UnwindTM, Koma UnwindTM, Marleys Mellow MoodTM, NeuroTM,
SolixirTM, Body WorksTM, iChillTM and more. The options include
carbonated and uncarbonated beverages. As well as calming
beverage shots. All designed to create a relaxed but alert
Unlike energy drinks which tout vitality and energy especially as
a start to the day or as an afternoon pick-me-up, beverages
designed to calm have a less clear usage occasion or
specific day part. Plus, unpublished consumer data suggests
consumers find the term relaxation to be associated more
with drowsiness or sleepiness instead of a desirable calm.
This could mean a chamomile-based tea drink targeting a
chilled out afternoon could be lacking brand distinction from
a melatonin shot to be taken before bed for immediate sleep.
One way marketers are looking to crack this code is through
associative messaging. For example, the idea of chillaxation
coffee or all natural relaxation are touted as product
value propositions coupled with activities like surfing, yoga or
other chilled out activities. Some manufacturers are utilizing
celebrities which evoke a chilled out status like Bob Marley.
The hope is that consumers, through associating learning,
will come to realize that beverages can provide a desirable
calmness just like energy drinks provide a burst of energy when
needed. Certainly though brand messaging must be chosen
very carefully, especially in light of the number of lawsuits around
the word natural which lacks formal regulatory definition at


Effective and credible messaging can ultimately be achieved

through careful consideration of the continuum of messaging
around mood and energy; that is, an effortless calm is different
than drowsy sleepiness and the chosen lexicon to explain
those differences will matter. Other marketing tactics including
packaging, beverage colour and product name, all of which
contribute to the consumers understanding of the value
proposition. One marketing tenant of course is for manufacturers
not to overpromise specific results or functional benefits that are
not documented by credible, peer reviewed science relevant
to the specific product.

Caffeine continues to lead the way on the stimulant side of
beverage formulations. There is evolving research on moodmodulating ingredients intended to provide a calming, relaxing
effect. Moreover, there is research on beverages more broadly
that suggests mood-enhancing benefits are achievable. For
instance, a recent study found that the mere act of drinking
tea is associated with relaxation, refreshment and feelings of
satisfaction (10). Hot temperature, sensory properties like smell,
color and mouth feel were cited as contributing variables.
All said, the opportunity for mood-enhancing beverages may
be lesser defined on the calming side than the energizing
side, but that doesnt mean it is lesser important to a segment
of consumers. The key will be identifying and documenting
individual ingredients with mood-enhancing properties,
that when delivered in liquid form have recognizable and
efficacious effects. In turn, beverages and/or beverage
shots which contain these ingredients at appropriate levels
verified and listed on the container will find their audience.
Such conclusions are based on the simple assumption that any
consumer looking for a pick-me-up at one point will likely need
a calm-me-down counter at a later point. Future formulations
also need to avoid unnatural ingredients and artificial colours
or preservatives whenever possible to meet consumer
demands for naturality. Ultimately, as the applications science
catches up with present formulation and marketing efforts,

Find out more...

in our past monographic
supplement series!
Visit our website:

beverages will likely play an increasingly useful, safe and

efficacious role in both the energizing and relaxing sides of the
mood equation.



Energy Drinks and Shots: U.S. Market Trends, Packaged Facts,

Jan 29, 2013.
2. McLellan T, Lieberman HR. Do energy drinks contain active
components other than caffeine? Nutrition Reviews. 2012
3. Pase MP, Scholey AB, Pipingas A, Kras M, Nolidin K, Gibbs A,
Wesnes K, Stough C. Cocoa polyphenols enhance positive
mood states but not cognitive performance: a randomized,
placebo-controlled trial. J of Psychopharmacol. 2013
4. Peuhkuri K, Sihvola N, Korpela R. Dietary factors and
fluctuating levels of melatonin. Food & Nutrition Research
2012 July. 56:17252.
5. Morgenthaler T et al. Practice parameters for the clinical
evaluation and treatment of circadian rhythm sleep disorders.
Sleep. 2007:30(11):1445-1459.
6. Amsterdam JD, Yimei L, Soeller I. A randomized, double-blind,
placebo-controlled trial of oral Matricaria recutita
(chamomile) extract therapy for generalized anxiety disorder.
J of Clin Psychopharmacology. 2009; 29(4):378382
7. Scheid L, Ellinger S, Alteheld B, Herholz H, Ellinger J, Henn T,
Helfrich HP, Stehle P. Kinetics of L-theanine uptake and
metabolism in healthy participants are comparable after
ingestion of L-theanine via capsules and green tea. J Nutr.
2012 Dec;142(12):2091-2096.
8. Yamada T, Terashima T, Okubo T, Juneja LR, Yokogoshi H:
Effects of theanine, r-glutamylethylamide, on neurotransmitter
release and its relationship with glutamic acid
neurotransmission. Nutr Neurosci2005;8:219-226.
9. Juneja LR, Chu DC, Okubo T, Nagato Y, Yokogoshi H:
L-theaninea unique amino acid of green tea and its
relaxation effect in humans.Trends Food Sci Technol
10. Einother SJ, Martens VE. Acute effects of tea consumption on
attention and mood. Am J Clin Nutr. 2013 Dec;98(6


Industry perspective

1. University
2 of Novi Sad, Institute of Food Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
2. Biomin Holding GmbH, Technopark 1, 3430 Tulln, Austria
3. State Research Institute, Institute of Soil Science and Plant Cultivation, ul. Czartoryskich 8,
24-100 Pulawy, Poland

Hyperspectral imaging in plant

based food safety

Possibility of hyperspectral imaging application

in safety control of plant based food that is converted
to by-products and used as feed
KEYWORDS: food, by-products, plant, feed, hyperspectral imaging, safety control


Latest requirements in terms of food safety control have become extremely strict. This is reasonable, given
that each year up to 30 percent of the worlds population suffers from some food-borne disease. However,
feed safety is no less important, since only safe feed can ensure secure human food. Therefore, it is important to develop methods for
rapid and accurate assessments of hazards on food and feed. Hyperspectral imaging spectroscopy is a novel computer technology
with great potential in rapid and precise food and feed hazard detection. This paper will give a review of possible usage of this
technique in safety control of plant based food that is converted to by-product and used as animal feed.

Quality and safety control are probably the most important
requirements of contemporary food and feed production. World
health organization (WHO) reported that each year up to 30
percent of the worlds population suffers from some food-borne
disease. In response to this, producers and food control services
recognize a need to create an effective feed control system,
which will lead to safe and secure food. On the other hand,
ensuring a healthy and secure feed quality, as well as cost
competitiveness, is often crucial to survive the competition.
Nowadays, food and feed safety management systems are
designed on a detailed understanding of all inputs to the
production process. Therefore, it is of great importance to develop
effective methods for on-line detection of hazards in order to
prevent further contamination.
Traditional methods of food monitoring involve analytical
techniques, which are time consuming, expensive and often
require sample destruction. A good example of a nondestructive
analytical method for quality analysis of samples is near infrared
spectroscopy (NIRS) (1). Nevertheless, NIR spectrometers do not
have possibility to capture internal constituent gradients in specific
food or food material, which often cause considerable differences
between predicted and measured chemical composition.
Recent advances in computer technology have led to the
development of imaging systems capable of identifying quality
problems rapidly (2). Hyperspectral Imaging Spectroscopy (HIS)
was primarily developed for remote sensing applications utilizing
satellite imaging data of the earth, but today its implementation in
agriculture and food industry and science, pharmaceuticals,

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

medical diagnostics and polymer science is frequently increasing.

In the last few years the system has been developed for laboratory
use and there are several examples of its usage in other areas such
as industrial production. It can be applied on the processing line,
with the minimum of human intervention. This technology
integrates conventional imaging and moderate resolution
spectroscopy. The key preference in this technique is that entire
spectra is collected for each pixel within the image, reducing
measurement time due to the simultaneous acquisition without the
need for scanning mechanics (3). This optical method is fast and
can be readily automated, which are its major advantages in
comparison with laboratory analyses.
Usage of HIS in quality control of food is widespread, and this
paper will give a short review of possible application in detection
of most frequent contaminants of plant based food that is
converted to by-products and used as feed. Agro-industrial
by-products and crop residues represent a huge animal feed
resource, which is as yet largely unexplored potential.
Considerable research has been carried out on the potential of
these by-products and crop residues but to date, very little
effective practical application has been achieved. The
environmental and hygienic impact of agro-processing industries
in the cities has been strong due to the production of large
amounts of waste, which is a problem that urgently requires
solutions (4).
Food industry by-products and agricultural wastes used in feeding
of animals can be divided into three main groups: by-products of
animal origin, by-products of plant origin and by-products of the
fermentation industry (5). Table 1 gives short review of food
by-products of plant origin, which are commonly used as feed.


spectrographs (8). A
volume of hyperspectral
images consists of three
dimensional data that
contain spatial information
along with a high spectral
resolution (10 nm) spectrum
at each pixel location (9).
Spatial resolutions can be
adapted to the
application, which range
from microscopic to
landscapes (8).
Hyperspectral images are
made up of numerous of
contiguous wavebands for
each spatial position of an
analyzed object. Usual term
for hyperspectral image in
literature is hypercube. Each
pixel in a hypercube
contains the spectrum of
that specific position.
Obtained spectrum is a sort
of print, which
characterises the
composition of that
particular pixel. Those are
three-dimensional blocks of
data, involving two spatial
Table 1. Main groups of by-products of plant origin (5) and potential hazards
and one wavelength
dimension (Figure 1).
Biochemical constituents of
a sample are separated into particular areas of the image,
Using agro-industrial by-products as a part of feed for
since regions of a sample with similar spectral properties
livestock reduces the cost of production, improve the quality
have similar chemical composition. In that way, chemical
of feed, ensure regular feed supply even during slump period
composition of sample is visualised (10).
(December-January, and May-June) and ultimately increase
the profit margin of livestock farmers. The following
characteristics of the by-products mentioned above are most
important: protein concentration and its biological value,
quantitative and qualitative composition of amino acids,
digestibility, level of energy, fats and carbohydrates, vitamin
and mineral content (6). Application of by-products as a part
of feed for livestock must be done carefully, because they
may contain many potential hazardous
compounds, which are deleterious to
animal health and performance (7).

Application of HIS in detection of fungi and mycotoxins

Development and growth of fungi present significant
problem in feed and food industry, especially in cereal
grain products. Major risk lies in production of mycotoxins
by fungi. However, not all fungal growth results in

Hyperspectral images are analogous to
a stack of images, each acquired at a
narrow spectral band. Generally, these
systems measure reflectance with
spectral regions ranging from the visible
(VIS) to shortwave infrared (SWIR)
ranges of the solar spectrum. In these
regions reflectance, transmission,
photoluminescence, luminescence or
Raman scattering can be recorded by
hyperspectral cameras with a spectral
resolution similar to miniature


Figure 1. Hypercube with two spatial (x, y) and one wavelength () dimension

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

mycotoxin formation, and the detection of fungi does not

necessarily imply the presence of mycotoxins (11). Feed
may be contaminated by fungi during processing, storage
or transport (12). Moulds that produce toxins may also
contaminate human foods and animal feeds through
fungal growth prior to and during harvest, or during
(improper) storage.
The mycotoxins that are commonly found in cereal grains
and other products are not completely destroyed during
food processing operations (sorting, trimming, cleaning,
milling, brewing, cooking, baking, frying, roasting,
canning, flaking, extrusion etc.). Cleaning removes broken
and mouldy grain kernels. The milling processes dilute and
distribute mycotoxins into certain fractions that most
commonly become animal feed. Therefore, cereal
by-products that may be diverted to animal feed can
contain mycotoxins at concentrations greater than raw
cereals due to processing (13). Rapid field detection
methods can be of great help in control of mycotoxin
presence in food and food by-products. In that manner,
HIS has remarkable potential in protection and prevention
of fungi and mycotoxins contamination.
Relatively simple, non-invasive method to rapidly identify
fungi by means of hyperspectral images was developed
by Yao et al. (14). The study used visible-NIR hyperspectral
images taken by a pushbroom hyperspectral sensor to
identify different toxin and non-toxin producing fungal
isolates. It was assumed that the specific spectral
signatures associated with each fungus would provide
enough information for discrimination between different
fungi, including different species within a single genus.
Five different fungi, Penicillium chrysogenum, Fusarium
moniliforme (verticillioides), Aspergillus parasiticus,
Trichoderma viride, and Aspergillus flavus, were used in
the present study. The study consisted of two parts, A and
B. In experiment part A, each fungus was cultured in
individual Petri dishes. In experiment part B, all five
species were cultured in a single dish. The overall fungal
classification accuracy in experiment part A was 97.7
percent while in experiment part B the accuracy dropped
to 71.5 percent. A possible reason could be that the rapid
growth of Trichoderma in experiment part B
contaminated spectral reflectance features of the other
four isolates (14).
Del Fiore et al. (15) used HIS for the early detection of
toxigenic fungi on maize. They reported early detection of
fungal contaminants, starting at 48 h from inoculation and
incubation at 30C for A. flavus and A. niger. Further, they
achieved the early discrimination between contaminated
and uncontaminated maize samples on the basis of the
changes produced by fungal contamination in the
spectral profile of the maize. Williams et al. (16) also
confirmed the possibility to use this technique for early
detection of Fusarium verticillioides in maize kernels.
Another important usage of hyperspectral imaging is in
detection of toxic products of fungi mycotoxins. Yao et
al. (17) worked on determination of the relationship
between fluorescence emission of corn kernels inoculated
with A. flavus and aflatoxin contamination levels within the
corn (17). The kernel fluorescence emission data were
measured with a fluorescence hyperspectral imaging
system when corn kernel was excited with 365nm UV light.
The aflatoxin contamination level of the imaged single
corn kernels was subsequently chemically measured using

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

affinity column chromatography. A fluorescence peak

shift phenomenon was noted among different groups of
kernels with different aflatoxin contamination levels. The
fluorescence peak shift was found to move more toward
the longer wavelength in the blue region for the highly
contaminated kernels and toward the shorter wavelengths
for the clean kernels. Highly contaminated kernels were
also found to have a lower fluorescence peak magnitude
compared with the less contaminated kernels. It was also
noted that a general negative correlation exists between
measured aflatoxin and the fluorescence image bands in
the blue and green regions. The multivariate analysis of
variance found that the fluorescence means of four
aflatoxin groups were significantly different from each
other at the 0.01 level of alpha. Overall, the results
indicate that fluorescence hyperspectral imaging may be
applicable in estimating aflatoxin content in individual
corn kernels.
HIS for determination of pesticide residue
Fruit and vegetables are mainly consumed raw or semi
processed and it is expected that they contain higher
pesticide residue levels compared to other food groups of
plant origin. During processing of fruits and vegetables,
skin is usually peeled and rejected, which leads to the
reduction of pesticide in human food. In this way, they
remain in the byproducts used for animal feed in higher
For instance, tomato pomace is a by-product of tomato
juice, paste, or ketchup processing. It is mainly consisted
of water, tomato peels, crushed seeds and small amount
of pulp that remain after processing. It has been fed wet
for decades to sheep and cattle (18). Several authors
presented their work concerning tomato pomace as an
animal feed. Di Francia et al. (19) investigated its
application in dairy ewes nutrition and Weiss et al. (20)
used it for feeding of dairy cows. Also, tomato skin is
considered as a useful source of lycopene and some other
micronutrients. It is reasonable to expect that these food
by-products, used as a feed, could contain considerable
amounts of pesticides.
Laboratory methods for analysis of pesticides in plants are
well established. However, none of the methods is fast
enough and adequate for emergency demands or on-line
application. Time consumption problem can be
potentially solved by application of HIS for determination
of pesticide residue.
Many pesticides possess a certain level of auto fluorescence. This characteristic can be used for their
detection in plants. Peng et al. (21) developed
methodology for determination of chlorpyrifos, a
compound which can be found in commercial pesticides.
For the purpose of the experiment, hyperspectral imaging
was combined with fluorescence stimulate technology.
The methodology was based on the fact that chlorpyrifos
has strong fluorescence characteristics with peak emission
at the wavelength of 437 nm. Different concentrations of
this compound had different fluorescence emission
spectral intensity at the peak. Authors presented
satisfactory results in analyses of rape leafs and also
reported that peak emission at the wavelength of 524
might be the effect of other organic elements present in
the commercial composite pesticide. They suggested that
the same principle can be used for development of rapid


detection instrument for other vegetable pesticide residue

(21). In other study, Cheng et al. (22) also determined
traces of chlorpyrifos in vegetable samples, using the
same technology. The results from both investigations can
provide theoretical basis for developing rapid detection
instrument for pesticide residue in plant materials.
Detection of fecal contamination using HIS
Contamination of agro-industrial products and
by-products with bacterial food borne pathogens can
potentially occur at any time during production,
harvesting, processing, distribution and preparation for
consumption. Pre-harvest contamination is considered
likely to account for many outbreaks, as it is extremely
difficult to prevent. Pathogens can arrive via animal
manure used for fertilization and soil conditioning, fecal
tainted irrigation water, wild and domestic animals, and
floodwaters from a contaminated site such as a cattle
stock yard.
Fecal contamination of food products and by-products
with bacterial food borne pathogens contributing to
outbreaks of food borne illness is associated with fresh and
minimally processed products. Therefore, development of
automatic inspection system that is fast and accurate has
been required to detect the fecal contamination on fresh
products. Hyperspectral imaging technique over the last
decade has been applied to food quality and safety to
detect the defects or fecal contamination on apples,
cantaloupes, strawberries, leafy greens (lettuce, spinach),
etc. Only some of them will be mentioned.
In a two-part study, the reflectance and fluorescence
applications of multispectral detection of fecal
contamination on apples using the HRFIS were described
(23, 24). Apple images were acquired in the visible to NIR
spectral regions. This spectral range has been previously
identified as a powerful tool identifying and detecting
defects and contamination on agricultural produce (25,
26). Results indicate that fecal contamination can be
detected using reflectance imaging; however, in the
second article, results indicate that fecal contamination
detection on apples is more sensitive using fluorescence
imaging techniques.
Vargas et al. (27) investigated whether it was possible to
classify fecal matter at different concentrations on the
surface of cantaloupes and strawberries using
hyperspectral imaging, given that the other chemical and
physical aspects of the fruit surface may present similar
spectral characteristics (27). Results of these researches
are reported in two different articles for both types of
produce indicate that it is feasible to classify fecal matter
on the surface of cantaloupes and strawberries.
Liu et al. (28) presented their experiment which involved
hyperspectral imaging as a real-time method for
inspection of fecal contaminants for apple. At least one
fecal contaminant (in black circles) was clearly identified
on each apple, with black circled spots corresponding to
the larger amount of feces (50 percent w/w) on apple
surfaces and white circled spots to the lower amount of
feces (5 percent w/w) on apples. Noticeably, few spots (in
broken black circles) resulting from the depositions of 0.5
percent w/w fecal solution were indicated, probably due
to the sensitivity and detection limit of camera in this
spectral range. In other words, appearance of fecal spots
in images was consistent with that from visual observation.


Yang et al. (29) developed hyperspectral fluorescence

imaging system, and used it to obtain several twowaveband spectral ratios on leafy green vegetables,
represented by romaine lettuce and baby spinach in this
study (29). The ratios were analyzed to determine the
proper one for detecting bovine fecal contamination on
romaine lettuce and baby spinach. Two wavebands
corresponding to fluorescence emission peaks for fecal
matter and chlorophyll a were considered useful to detect
fecal contamination on samples of relatively high
chlorophyll content such as leafy green vegetables. The
results showed that the fluorescence-based hyperspectral/
multispectral imaging system can successfully detect fecal
contamination on leafy green vegetables. Similar
experiment was presented by Kang et al. (30). This
experiment involved hyperspectral fluorescence line-scan
imaging system for the detection of fecal contamination
on the leaf surface of fresh produce. In this study, romaine
lettuce (Lactua sativa L.) and baby spinach leaves
(Spinacia olerace L.) were contaminated with six bovine
fecal spots and two soil spots by applying them on surface
of vegetable leafs. Results of this research indicated that
the principal component analysis method can be used for
bovine fecal contamination detection.

The presented review paper summarises some of the potential
application of HIS in safety control and management of plant
based food that is converted to by-products. As it was
pointed out, these by-products have a great potential to be
used as animal feed. Not only that they reduce the cost of
breeding animals, but also solve the problem of waste from
the food industries, and ensure regular feed supply even
during slump period. However, these materials are often
highly contaminated with various hazardous substances, since
they are rejected during food production. Animal feed have
to be secure for use, as well as human food. Only that will
guarantee that meat and other animal products are safe for
human consumption. Therefore, methods for detection of
potential hazards need to be highly precise and fast. Food
and feed process control requires real-time monitoring at
critical processing points. HIS can easily found its place in this
industry, but since the technique is relatively new and
unexplored, a lot of investigations in this direction are needed.
HIS are ideal for finding the specific wavelengths for assessing
different types of quality determining parameters, and, what
is more important the system has showed remarkable high
accuracy. All facts indicate one: it would be desirable that
food and feed industry start to adopt hyperspectral
technology for safety and quality control. Further research
should be focused on facilitating industrial adoption of
hyperspectral technology.

This paper is a part of FOODSEG international project,
funded by the 7th Framework Programme for Research,
Technology and Demonstration of the European Union. The
authors thank for support to Integrated and Interdisciplinary
Research Project No III46012, funded by Serbian Ministry of
Education, Science and Technological Development.

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014






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Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014


*Corresponding author
University of Santiago de Compostela, Faculty of Veterinary Science, Department of Analytical Chemistry,
27002 Lugo, Spain

Manuel Vazquez

Rapid authentication of white wines.

Part 1: Classification by designation of origin
KEYWORDS: designation of origin, UV-VIS, NIR, spectral data, classification


Artificial neural networks (ANN) combined with ultraviolet (UV), visible (VIS) and near-infrared (NIR) spectral
analysis were evaluated as a rapid method to classify wines. Wines belong to Designation of Origin (DO)
Ras Baixas, Ribeira Sacra, Monterrei, Ribeiro and Valdeorras (Northwest of Spain) were classified. Classification inside a DO were also
studied for subzones of DO Ras Baixas. Results showed that DO Ribeiro, Valdeorras, Monterrei and Ribeira Sacra were 100 percent
classified using the ANN architectures developed. Ras Baixas were the DO with a worst classification. Inside DO Ras Baixas, Rosal was
100 percent classified. Results showed the feasibility of applying ANN and UV-VIS-NIR analysis to the authentication of DO.

Wine composition depends of soil, culture conditions,
microclimate, macroclimate and winemaking techniques (1,
2). The wine analysis is not an easy task due to its heterogeneity
and complexity. The economic value of wine made the wine
authentication or classification an important task worldwide.
This importance is related to quality, prevention of adulterations,
food safety and control of winemaking process. Wine quality
in import-export markets should be also guaranteed (3).
Winemakers and consumers demand analytical low-cost and
effective tools to determine the quality of wine (4).
The controlled designation of origin (DO) is the name of a
region or place recognized to produce foods with special
characteristic regarding other places and with different
manufacture and/or materials (5, 6). Wines produced in regions
with DO have Regulatory Councils that control the growing
and production conditions (2, 7). The authentication of wines
from DO is an important task for DO Regulatory Councils. They
have also the difficult task to determine the origin between the
production subzones inside a DO (8).
Different classification methods have been applied in
analytical chemistry, such as factor analysis, cluster analysis,
linear discrimination analysis (LDA) (7, 9), Partial least squares
regression (PLS) and soft independent modelling of class
analogy (SIMCA) (10, 11).
The Artificial Neural Networks (ANN) is a model of a biological
neural network and brain functioning for data processing, which
exhibits the main capabilities of the real neuron system (12).
Each neuron is connected to certain neighbours with varying
coefficients of connectivity that represent the strengths (the
weights) of these connections (13-15).
The use of ANN is increasing for many chemical applications. The


ANN can provide capacity of solve supervised and unsupervised

problems, such as clustering and modelling of data. There is
different ANN architectures and strategy of solving data. ANN is
a flexible modelling method. They can use linear and non-linear
functions in processing units (15). The construction of ANN is not
significantly affected by the sample number in each category
and data structure (16). The ANN consists of interconnected
neurons with input layers, one or more hidden layers and output
layers (14) and these neurons are developed through one or
more forms of training (17).
Wine varieties and wines from different regions have been
successfully classified using near-infrared (NIR) spectroscopy
and chemometric tools (7). However, there are scarce studies
using UV-VIS-NIR spectroscopic techniques and ANN tools
for the classification between wines of close DO or wines of
subzones inside a DO.
The aim of this work was to evaluate the feasibility of using UVVIS-NIR spectroscopic techniques combined with ANN to classify
wines. As a case of study, it was studied wines from Spanish DO
(Ras Baixas, Ribera Sacra, Ribeiro, Valdeorras and Monterrei).
The feasibility of classifying wines from subzones of DO Ras
Baixas (Condado, Salns, Rosal and Ribeira de Ulla) were also


Wine samples
White wines from Galician (northwest of Spanish) were studied.
Commercial monovarietal white wines from DO Ras Baixas,
Ribeira Sacra, Ribeiro, Valdeorras and Monterrei of 2009 and 2012
vintage obtained from different local markets were analyzed
(Table 1). All samples were stored at 5C until analysis (18).

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Table 1. Wines analysed in this study.wavelength () dimension

For the study of subzones, commercial monovarietal Albario

white wines of 2009 vintage from DO Ras Baixas were
analyzed (Table 1). Samples were collected from winemakers
in the Condado, Rosal, Salns, Soutomaior and Ribeira do Ulla
Spectral measurements
Samples were maintained at 30-35C before spectral analyses
(19). Samples were measured in Transmittance mode (T) with
2 nm band resolution (20) using a quartz cuvette with 1 mm
path length.. All samples were analyzed in UV-VIS-NIR spectral
range (190 - 2500 nm).
Wines from the studied DO (Ras Baixas, Ribeira Sacra, Ribeiro,
Monterrei and Valdeorras) were analyzed by duplicate or
triplicate (144 spectra). Wines for the study of subzones of
DO Ras Baixas were analyzed by triplicate (99 spectra). All
replicates were used as independent spectra. The analysis
was performed with a V-670 spectrophotometer (Jasco Inc.,
Japan) and the spectral data were collected with Spectra
ManagerTM II software (Jasco Inc., Japan).

regions and peak group was based on

that a small number of variables selected
from the original could provide an easier
interpretation (15). The following spectral
regions and group of peaks were selected
for calibration purposes: UV region (190400 nm), VIS region (400-780 nm), UV-VIS
region (190-780 nm). A set of UV-VISNIR wavelengths for the classification
for DO classification and other for the
classification of subzones into DO Ras
Baixas were also selected.
For DO classification: region A (spectral
ranges covering different regions of higher
absorption: 2348-2344, 2329-2325, 2305-2301,
2284-2280, 2267-2263, 2244-2240, 1883-1879,
1860-1856, 1788-1784, 1763-1759, 1745-1741,
1730-1726, 1710-1706, 1693-1989, 1665-1661,
1464-1460, 1407-1403, 1382-1378, 1343-1339,
1152-1148, 1134-1130, 964-960, 859-855, 845-841, 369-365, 338334, 305-301, 276-272, 246-242, 228-224 and 206-202 nm); peak
group B (spectral peaks with higher absorption: 2346, 2327,
2303, 2282, 2265, 2242, 1881, 1858, 1786, 1761, 1743, 1728,
1708, 1691, 1663, 1462, 1405, 1380, 1341, 1150, 1132, 962, 857,
843, 367, 336, 303, 274, 244, 226 and 204 nm).
For subzones of DO Ras Baixas classification: region C
(spectral ranges covering different regions of higher
absorption: 2261-2257, 1879-1870, 1392-1376, 856-850, 348332, 237-224, 202-190 nm); peak group D (spectral peaks with
higher absorption: 2257, 1870, 1390, 335, 223, 202 nm).
Artificial Neural Networks (ANN)
Neuro XL Clusterizer Ver. 3.1.2 (OLSOFT LLC. Tashkent, Uzbekistan)
was used to find the clusters. The clustering method used in
this study followed hierarchical techniques. NeuroXL Clusterizer
uses the agglomerative method wherein the clusterizer starts
with each object being a separate cluster by itself and then
successively clusters groups according to their similarity. The
clustering finishes when similar objects are in the same group
(24). For clustering, five groups or clusters were used for the
classification of DO wines, four clusters were used for the
classification of subzones of DO Ras Baixas wines. Network
architectures were tested to select the best classification. The
ANN trainings were unsupervised and done with all wines. Input
data were pre-treated spectral data and raw measures of
different spectral zones or peaks. The studied parameters of
neural networks were shown in Table 2.

Data pretreatment and multivariate data analysis

Spectral data were exported from Spectral ManagerTM II
software to Unscrambler software (version X 10.2, CAMO
ASA, Oslo, Norway). For the study of subzones into DO Ras
Baixas, the Soutomaior subzone data were excluded from
the analy sis by insufficient number of samples (2). For study of
classification of Galician DO, Soutomaior subzone wine data
were included in the analysis.
The spectra were pre-processed
using the standard normal
variate (SNV) transformation,
Normalize transformation,
smoothing Savitzky-Golay
and derived Savitzky-Golay
derivation transformation with
25 points, second-order filtering
operation (4, 21, 22).
Principal component analysis
(PCA) was developed using raw
and pre-processed data. These
models of PCA are utilized as
a first step of analysis to detect
patterns and outliers in data (23).
Table 2. Evaluated values for the neural network parameters.
The manual selection of spectral

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014



Raw UV-VIS-NIR spectra
Figure 1 shows the map of the DO areas
studied in the northwest of Spain. This figure
shows that the wines of Ras Baixas, Ribeira
Sacra, Ribeiro, Monterrei and Valdeorras
and Monterrei were produces in no more
than 200 km of distance. DO Ras Baixas
and Ribeiro are adjacent areas. The
same applied for DO Ribeira Sacra and
Valdeorras. The DO Ras Baixas included 5
subzones. The wines of subzones Salns and
Condado are much separate that subzones
Ribeira do Ulla and Rosal.
Figure 2a shows raw UV-VIS-NIR spectral
data of all wines studied. There are clear
differences in UV and NIR zones of spectra.
The raw UV-VIS-NIR spectral data of subzones
Condado, Rosal, Salns, Soutomaior and
Ribeira do Ulla from DO Rias Baixas are shown
in Figure 2b. A visual inspection of spectra
shows some differences mainly in UV zone but
also in NIR zone. Figure 3a shows score plot
of raw data from PCA for two first principal
components for the DO wines studied. The
91 percent of total variance are explained
by two first principal components. There is a
clear trend of separation of samples by DO.
Samples of Ras Baixas and Valdeorras are
clearly separated. The principal cause of this
separation could be the climate or the grape
Figure 2. UV-VIS-NIR raw spectral data from a) DO Ras Baixas, Ribeira Sacra, Ribeiro, Monterrei
variety. In DO Ras Baixas, there is a coastal
and Valdeorras white wines and b) subzones of DO Ras Baixas white wines.
climate and the wines are performed with
albario variety. On the other hand, in DO
the spectra in wines analyzed. Separation of subzones of DO
Valdeorras, there is a continental climate
Ras Baixas is not clear because the samples are overlapped.
and grape variety is godello. Monterrei, Ribeiro and Ribeira
However, there is a trend to separation in wines samples from
Sacra white wines are slightly mixed.
Condado and Salns. The different microclimate of these
subzones could be cause of this trend (2, 25).

Figure 1. Map of Galician DO including subzones of DO Ras Baixas: (1)

Ribeira do Ulla, (2) Salns, (3) Soutomaior, (4) Condado, and (5) Rosal.

Figure 3b shows score plot of PCA of raw data for two first
principal components for subzones of DO Ras Baixas. Two first
principal components explain 87 percent of total variance of


Classification with Artificial Neural Networks

An ANN was trained for classifying the different DO wines
and for classifying wines from subzones of DO Ras Baixas
using the spectral data. A combination of different neural
network parameters were assayed to classify samples of
Galician DO and samples of subzones of DO Ras Baixas
(Table 2).
Start learning rate is a value between 0 and 1 that affects
the rate at which the network starts learning. It was
assayed values of 0.3 and 0.5. Epoch was evaluated in the
range 100-1000. Epoch is the number of complete passes
through the neural network of the entire set of sample
patterns. Values of 0.3-0.5 of initial weights of synapses
were assayed. Learning radius is the number of neurons
that will be affected and learned during training stage.
The start learning radius was evaluated in the range of
20-900. The activation functions Log-sigmoid, threshold,
hyperbolic and sigmoid functions were assayed.
The parameters selected after the optimization are shown
in Table 3. Epoch was fixed in 10000. A start learning rate
and initial weight were fixed in 0.3. The start learning radius
selected was 900. The log-sigmoid function with convex
combination and values scaled were selected to the

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Figure 3. Score plot of PCA raw data of a) Galician DO and b) Subzones of DO Ras Baixas.

The classification of DO Ras Baixas could

be affected by the existence of subzones
with different soil and microclimate
conditions due to the geographical
separation. Therefore it was tried to
classified the subzones of DO Ras Baixas.
Table 5 shows the best ANN classification
of wines according their subzones in
DO Ras Baixas for each spectral range
studied. The best classification was 100
percent for Rosal using VIS spectral
data and 2 nd derived and zone D with
raw data. For Ribeira do Ulla, the best
classification was 100 percent in UV-VIS
zone and raw data. For Condado wines,
it was obtained a 93.33 percent using
the spectral zone D. For Salns subzone,
76.67 percent of right classification was
obtained using the spectral zone C with
raw and smoothing data.
This is the first time that ANN was used
successfully to classify wines using
spectral data. The results showed that it
was not possible to classify all the wines
into their DO using ANN and spectral
data. However, the methodology can be
applied to many DO. In these cases, ANN
coupled with spectral analysis is rapid
method and it can be applied by wine
industry or DO councils for authentication

Table 3. Selected neural network parameters.

classification of all DO white wines studied.

The classifications of DO are showing in Table 4. The
best classification of wines with spectral data and ANN
according DO were determined and the most of the
wines were correctly classified in their DO using some pretreatments and some spectral zones. Valdeorras wines
were the best classified, using the UV zone, raw data and
all pre-treatments, obtaining 100 percent of classification.
The worst classification of DO Ras Baixas wines were
in UV-VIS zone and raw data (30 percent). Using other
pre-treatments, the classification was mostly good with
values of 80-90 percent. Ribeira Sacra wines are correctly
classified in their DO with 100 percent for UV with raw
data, smoothing and SNV pre-treatment. DO Monterrei
wines were 100 percent classified in UV, VIS, UV-VIS and
region B of spectra and 2nd derived as pre-treated.

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Table 4. Proportion ( percent) of ANN classification of white wines

according to Galician DO.


preliminary. More studies increasing the number of samples

for each region and from different years are needed.



Table 5. Proportion ( percent) of ANN classification of wines according to

subzone of DO Ras Baixas

UV-VIS-NIR spectra coupled with ANN can be useful to
classify wines from different origins. DO Ribeiro, Valdeorras,
Monterrei and Ribeira Sacra can be 100 percent classified
using the ANN architectures developed. Ras Baixas were the
DO with a worst classification. Different combinations of UVVIS-NIR spectra can be used to classify different subzones of
DO Ras Baixas using ANN. However, only the subzone Rosal
and Ribeira do Ulla can be 100 percent classified with the
ANN developed. These conclusions should be considered as


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The technical experts at BENEO have conducted technical trials to improve the shelf-life and quality of glazed and iced,
freshly and frozen packed donuts. The results show that with the partial replacement of sucrose with BENEOs functional
carbohydrate Palatinose in glazings or icings, shelf-life and overall product quality can be significantly improved. Findings
showed that a partial replacement of sucrose with Palatinose in freshly packed donuts leads to an extended shelf life
stability through the unique carbohydrates ability to control water activity and moisture migration. Furthermore, the glaze
with Palatinose maintains transparency, even throughout the extended shelf life. Thanks to the low hygroscopicity of
Palatinose, the glaze does not become sticky. In addition, the Palatinose glazed, freshly packed donuts exhibit a more
pleasant sweetness in comparison to using sucrose alone. The trials also demonstrated that partial replacement of sucrose
with Palatinose in frozen packed donuts ensures a maintained glaze transparency throughout the freezing process
and after defrosting. It prevents the glaze from sticking to the foil during frosting and, of even greater importance, after
defrosting. Furthermore, Palatinose provides a slightly reduced and more pleasant sweetness in frozen packed donuts, as
with pure sucrose glazing.


Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014


Industry perspective

*Corresponding author
University of Athens, Department of Food Science and Human Nutrition, 75 Iera Odos,
indirizzo 2 Athens, Greece
2. University of Western Greece, Department of Agro-Food Enterprises Management, 2 Seferi, GR-30100
Agrinio, Greece

I. Natskoulis

Feed undesirable substances as

food contaminants
Part 2: Dioxins

KEYWORDS: Toxic substances, foods, feeds, animal nutrition, Community legislation, dioxins


This work focuses on another outstanding toxic substance of the Community legislation, namely dioxins.
Concerning dioxins, the historical background for certain pollution incidents with animal feeds as potential
food contaminants is briefly given, arriving at the dioxin episode in Belgium. Reference is also made to legislative tools available at
Community level to tackle the dioxin problem and the difficulties involved in practice, particularly with feeds of marine origin.
Furthermore, the environmental implications associated with dioxin pollution of animal feeds are explained, whereas the political will,
required in preventing and solving contamination problem in practice, is also stressed.

This article constitutes the second part of an account
dedicated to feed toxic substances as precursors of food
contaminants. Part 1 dealt with mycotoxins (30). The transfer
of chemicals from feed to animal products, as well as the risk
of food contamination by toxic substances present in animal
feed, had been documented (1, 2). Toxic substances such as
dioxins, mycotoxins, heavy metals, pesticides, veterinary drugs
are almost ubiquitous in the environment. In recent years,
increasing attention has been paid to the risk of consumers
posed by toxic substances or residues in animal feeds. This
was caused by various animal products contaminated by
environmental pollutants. The best-known examples include
contamination of animal products with dioxin as a result of
industrial activities. In addition, animal feed has been found
adulterated with hormones, antibiotics, dioxins and other
chemicals either deliberately or from malpractice, or from
sloppy manufacturing practices. The use of pesticides is an
example of controlled contamination of crops which might
reach the consumer.
Various researchers have reported the grouping of undesirable
substances in the feeding sector (3, 4). The importance of
animal feeds in relation to feed safety has been demonstrated
(5). The present work focuses on dioxins in feeds as potential
food contaminants. With reference to dioxins following the
dioxin episode in Belgium, further works are still published on
various aspects of this issue (6, 7). It should be stressed however
that, reference to provisions of the Community legislation in
the present article, in no case replaces legislation itself. For the
exact wording, the reader is directed to the Official Journal of
the European Union or the EFSA Journal.

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

The purpose of this work is to emphasize on the importance

of dioxins in animal feeds as potential contaminants of the
food industry. Furthermore, as regards the objective, the
paper focuses on the experience gathered from the history
of incidents involving dioxin contamination in the food sector,
paying particular attention to the Dioxins episode in Belgium.

The subject under consideration will be viewed through the
prism of serious incidences in the past and the tools to tackle
this question. A major food contaminant is dioxin. A large
number of compounds are referred to under the name dioxin,
some of them being highly toxic and confirmed carcinogens
(8). They are formed as by-products in a number of industrial
and thermal processes and enter the environment in various
ways, one of the most important being through the release of
polychlorinated biphenyls (PCBs) in which the two classes of
compounds referred to as dioxins, polychlorinated dibenzo-pdioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs),
are formed as by-products during synthesis. The greatest
part of environmental contamination by dioxin comes from
energy production and industrial activity. Some PCBs have
similar toxicological properties to dioxins and are often
referred to as dioxin-like PCBs and are considered together
with dioxins. Fifteen years ago, the EU experienced a series
of incidents involving dioxin pollution of the food chain. It is
clear that one source of human exposure to dioxins is food,
with food from animal origin being the predominant source
of contamination. It also appears that for dioxins, food
contamination is directly related to feed contamination.


Therefore, if one wants to reduce dioxin contamination in

the food chain, it is important to adopt measures controlling
animal feedstuffs in agricultural practice.
History of dioxin incidents
A number of accidents have taken place resulting in the
exposure of humans to dioxins. The most notable being the
Yusho and Yucheng rice oil poisonings from Japan in 1968
and Taiwan in 1979, respectively (9). Although, it was neither
the biggest nor the most severe, the Belgian PCB dioxin
crisis was one of the most mediatized food poisoning
events (10). However Covaci et al. (9) state that the literature
is generous with dioxin food environmental contamination
episodes, including the pollution with dioxin contaminated
Agent Orange of large areas during the Vietnam war (11), as
well as the large scale environmental pollution following the
Seveso incident in Italy 1976 (12), where a chemical factory
blew up and released
2,3,7,8-tetrachlorodibenzop-dioxin (TCDD), one
of most toxic synthetic
compounds known, to the
environment. There were
also various incidents that
occurred between 1996
and 2002 in Germany,
Brazil, USA and other
countries and where
feed ingredients were
contaminated with dioxins
(13). In fact, in 1998, in
Germany, increased levels
of dioxin were found in
cows milk. This was due to
an ingredient of the dairy
complementary feed, namely citrus pulp pellets from Brazil
(14), which had been exported to a number of Community
Member States. The exact cause of contamination took some
time to discover. Initially, it was attributed to an agent added
to the fuel to increase heating efficiency during dehydration
processing of the pulp. However, the actual cause was later
found to be the lime used to increase the pH and facilitate
the release of water from the hydrophilic pectins of the fresh
citrus pulp during the drying process. The EU, soon after this
incident, started to face the dioxin issue in animal feeds. Firstly,
it took measures for the disposal of contaminated citrus pulp
exported to EU territory and, secondly, it introduced, for the
first time, a maximum permitted level for dioxins in dried citrus
pulp (500 pg I-TEQ/kg upper bound limit of detection) in the
Annex of the undesirable substances Directive 1999/29/
EC. This was the first time to note that only very few
laboratories, not only in Europe but also in the world, were
competent to perform reliable dioxin analyses involving
the whole range of congeners.
The Belgian dioxin episode
While the Mad Cow Disease case in Britain, due to
feeding animal proteins to ruminants, became known
as the BSE scandal, since it was rumoured to be the
result of a deliberate action by the relevant industry to
save money by lowering the temperature and time for
sterilization of meat waste in the production of meat
and bone meal, the case of the dioxin incidence in
Belgium in first half of 1999, became known as the Dioxin


episode. Dioxin contaminated fat was inadvertently

incorporated into the diets of various species of farm
animals, predominantly poultry (15). Schepens et al.
(16) give a concise but precise account on the Belgian
episode of dioxins. These authors mentioned that in
1999, about 50 kg polychlorobiphenyls (PCBs) and 1
g dioxins were introduced into the animal food chain
through approximately 1,500 tons of animal feed
containing 60 tons of contaminated fat from a Belgian
fat-melting company. This incident caused widespread
concern both in and outside Belgium and obliged the
Belgian government to take drastic measures to protect
public health, including a large-scale food-monitoring
program with measurements of PCBs and dioxins in
over 20,000 and 450 samples, respectively, from animal
feed, animal fat, and various fat-containing food items
(17). All samples were analyzed in officially accredited
laboratories. Analysis of
contaminated foodstuff
showed a pattern of PCBs
closely matched with a
mixture of Aroclor 1254
and 1260 and a consistent
pattern of dioxin-like
compounds, dominated by
These patterns were
virtually identical to that
in the 1969 Yusho rice
poisoning, caused by
heat-degraded PCBs.
In addition, a rather
extended account on
the Belgian PCBs and
dioxin incident of JanuaryJune 1999 was released by Van Larebeke et al. (18).
These authors write that in Belgium, approximately 20
companies, collect animal fat from slaughterhouses and
melt it into a homogenous substance, which is sold to
animal-food producers. It is a common practice to include
household waste fat collected at community waste
recycling centres in this product. In January 1999, at the
Flemish fat-melting company Verkest, 4050 kg of mineral
oil containing polychlorinated biphenyls (PCBs; most likely
oil from discarded transformers originating from a waste
recycling centre) was admixed to the fat delivered to
10 animal-feed producers. Between 15 and 31 January,
the resulting 500 tons of contaminated animal feed,
containing approximately 6080 tons of fat contaminated
with 4050 kg of PCBs and almost 1 g of dioxins, were
distributed to poultry farms and to a lesser extent also to
rabbit, calf, cow, and pig breeding and raising farms,
mostly in Belgium. All started there.
In addition, Covaci et al. (9) mentioned that the
consequences of the food crisis were: (i) the introduction
in 1999 of norms for PCBs in feedstuffs and food in
Belgium followed by the introduction in 2002 of European
harmonized norms for PCDD/Fs in animal feed and food
of animal origin; (ii) the systematic national monitoring
of food of animal origin; and (iii) the creation of the
Federal Agency for Food Safety in Belgium. The human
health risk following this major incident was assessed with
contradictory results. It was suggested that, since only a
limited proportion of the food chain was contaminated, it

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

is unlikely that adverse effects were inflicted in the Belgian

population. However, another assessment suggests that
neurotoxic and behavioural effects in neonates, together
with an increase in the number of cancers, may be
observed. The same authors concluded that due to poor
crisis management of the government, the PCB incident
had dramatic political and economic consequences.
However, it has made both the policy makers and the
general public aware of the fact that food safety is a
priority issue. Moreover, the PCB crisis has enforced a
better system for effective and rapid surveillance of the
food chain, an effective communication about the nature
of risk, credible, open and responsive regulations and a
reduction of risk levels. Lastly, it has urged the introduction
of Community maximum residual limits for PCBs and dioxins
in animal feeds. It should be underlined at this point that
twelve years following the dioxin episode in Belgium,
another major incidence involving dioxin contamination of
meat took place in 2011 in Germany.
Means for tackling dioxin feed problem
Two main tools, within EU legislation for animal nutrition
were available at the time (1999) to tackle the problem
arising from the major
dioxin episode with
polluted recycled
oil in Belgium. Firstly,
the Decision on
prohibited ingredients
for animal feeding
and, secondly, the
Directive 1999/29/
EC 91/516/EEC
for undesirable
substances in animal
feeds. However, the
legislative action
to include frying oil
in the catalogue
of prohibited feed
ingredients was
not put into effect,
since it appeared
that the majority
of Member States believed that frying oils themselves
were not to blame, but rather the mistaken way in
which they were collected, allowing PCB-containing
transformer oils to enter the food chain. The application
of strict manufacturing conditions, including Good
Manufacturing Practice (GMP) and Hazard Analysis
Critical Control Points (HACCP) was suggested instead.
The second option for the Community was to put
maximum permitted levels for dioxins in the Undesirable
Substances Directive in order to control the circulation
of feed materials with dioxin exceeding this level. One
might think that the level of 500 pg/kg set for citrus pulp
in 1999 could be extended to cover all raw materials
used in animal feeding, knowing that this is a difficult
task taking into account that these levels are variable
between different feedingstuffs. However, this has proved
to be extremely difficult in practice, because a number
of unexpected obstacles emerged when investigating
the consequences of applying such levels. It has taken
two and a half years, and this after issuing the General

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Food Law (19), to adopt maximum permitted levels for

dioxin in all types of animal feeds with Directive 2002/32/
EC (20), with the observation that the proposed levels
should be reviewed in the future.
Subsequent problems
Before adopting maximum permitted levels, EU at the
time carried out investigations to acquire an indication of
background levels for the various potential feed materials.
However, the outcome of this study was surprising. The
background level for dioxin in feed materials of marine
origin, particularly that from closed seas was noticeably
high. The background level of dioxins in fish oil was much
higher compared to that of fish meal. The reason for this is
the lipophilic character of these contaminants. European
fish meal and fish oil, which showed an average content
of dioxins of the order of 1.2 and 4.8 ng WHO-TEQ /kg DM
respectively (21) are more heavily contaminated than those
from the South Pacific (Chile or Peru), which contained 0.14
and 0.61 ng WHO-TEQ/kg DM, respectively.
The problem is mainly centred on farmed fish feeds (22).
About 20 percent of the total world production of fish meal
and fish oil was used in aqua-feeds (23). In general, the diets
of farm animals (cattle,
sheep, pigs and poultry)
are formulated at a level
of protein of around
15-20 percent, whereas
the protein required by
carnivorous fish is almost
threefold. The protein
content of fish meal
can go up to about 70
percent. Fish oil has high
energy content and is
also a good source of
omega-3 type essential
fatty acids. In order to
provide the required
nutrients to farmed
fish, it is necessary that
approximately two thirds
of their diets consist
of fish meal and fish
oil. The circulation of a great number of feed materials was
authorized by the EU at the time. Maximum permitted levels
for dioxins were eventually adopted for various types of feed
materials (9 in total), but they differentiated between different
feed materials i.e. 0.75 for feed materials of plant origin, 2.25
for fish meal and 6.0 ng WHO-PCDD /F-TEQ/kg for fish oil,
which means that the latter values are 3 and 8 fold those of
plant feed materials. The EU also proposed that both target
and lower action levels be set for dioxins in feed materials
(24). As regards the issue of carry-over of dioxins through the
animal organism an excellent review has been produced
recently by Kan and Meijer (2).
Environmental issues
A school of thought exists within Europe advocating
that the problem of dioxin pollution of animal feeds
cannot be solved only by fixing maximum permitted
levels and controlling the circulation of feed materials,
and that these efforts are a time wasting and money
consuming operation, due to the cost of a complete


dioxin analysis which is quite high, because of the huge

quantities of feed marketed worldwide. One wonders
who is going to pay the price? of routine dioxin
analyses. The contamination of the environment by
dioxins is primarily caused by the aerial distribution and
deposition of emissions from various sources such as
municipal wastes incineration, production of chemicals,
transportation, etc. (25). In addition, the application
or disposal of chemicals contributes to more severe
and localized contamination, of feed materials in
particular. Accidental pollution of feed materials by
dioxins may also occur through localized dispersal into
the agricultural environment of PCBs normally used
as heat exchangers but recycled as lubricants. The
uncontrolled combustion of waste plastic or rubber
materials, which contain chlorinated compounds, may
also cause localized pollution. Considering the impact
of environmental pollution on the contamination of feed
materials, measures implemented aiming at reducing
the general dioxin burden should be actively pursued.
Therefore, there is a view that one should tackle the
problem of contamination at its real source i.e. at
environmental level. However, this is primarily an issue of
political decision to be effective in practice.
The dioxin analytical side
It should be mentioned at this point that the requirements
for the determination of levels of dioxins and dioxin-like PCBs
in feeds established with Commission Directive 2002/70/EC
were transferred to the consolidated Commission Regulation
152/2009/EC (2009) laying down the method of sampling
and analysis for the official control of feeds. However, more
recently, results of the monitoring of dioxins on Community level
were published by EFSA (27), followed by an updated version
of the latter (28), whereas, very recently, Liu et al. (29) provided
a detailed overview of last decade progress in capacity
building, source identification and quantification of dioxin
emission, environmental monitoring of dioxin contamination
and risk assessment of human exposure to dioxins in China.

As regards dioxins, fish meal and especially fish oil are
the more heavily contaminated feed materials and
fish farming is the most critical sector in this context.
Raw materials, particularly recycled ones, should be
controlled for quality and safety. The application of Good
Agricultural Practice (GAP), Good Manufacturing Practice
(GMP) and Hazard Analysis and Critical Control Points
(HACCP) should be generalized in the production and
manufacture of food and feed raw materials. Monitoring
programmes should be maintained on feed material
contamination. Research is also needed to study carryover and transfer factors for dioxins from the environment
and in particular from soil, to animal tissues and products.
Finally, emphasis should be placed on the development
of reliable methods of dioxin analysis of foods for control
purposes. Pollution of the environment with dioxins, which
subsequently enter the human food chain, is an important
problem on which the scientific community should focus
its attention. However, the problem of environmental
contamination with dioxins is more complex, since it has a
strong political component as well.










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B17/1999, Kuopio, Finland (1999).
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408-413 (2006).
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the Belgian contaminated food chain: sources, profiles and
correlations, Final Report, Edited by Broeckaert F., Bernard A.,
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109, 101-103 (2001).
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Perspect., 109, 265-273 (2007).
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25(3), 15-18 (2014).

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Institute of Plant Genetics, Breeding and Biotechnology, University of Life Sciences in Lublin, Akademicka
Street 15, 20-950 Lublin, Poland

Marta Tomczynska-Mleko

New product development:

carbonated beverage with different protein and creatine

for sportsmen and physically active people
KEYWORDS: creatine, beverage, supplement, protein, physical activity


Protein dispersions with or without sucrose were prepared with addition of different sources of protein:
defatted milk, full milk, whey protein concentrate, whey protein isolate and egg white isolate. Carbonated
beverages based on sparkling mineral water were obtained (4% protein content). Viscosity and surface tension of the dispersions and
stability of creatine in the beverages were measured. Sensory analysis of the beverages was performed. Negative linear correlation
between viscosity and flavour perception (R2 = 0.96) and positive correlation (R2 = 0.87) between viscosity and perceived consistency
were noted. Storage of protein dispersions and beverages for 30 days at 4C caused degradation of creatine in the range 21.3-28.9%
in comparison to 46.1% in water. A 500 ml serving of designed new beverage would supply 20 g of high quality protein and 5 g of
creatine for sportsmen and physically active people.

Well-balanced nutrition is crucial for sportsmen and physically
active people. According to rules created by nutritionists,
humans should consume 5-6 meals per day with appropriate
amount of carbohydrate, protein, fat, minerals and vitamins
(1, 2). It is considerably easy to find foods being a good
source of carbohydrates with low glycemic index, but foods
with high protein content are less numerous. Very often
consuming protein is connected with increased
carbohydrates intake. To prevent this, and to create very
convenient source of protein, many different protein
supplements are produced. Usually the following proteins are
used: whey proteins, casein and caseinates, egg white
proteins, soy proteins and beef proteins. To fulfil better
digestibility and less allergenicity, different protein
hydrolysates are used (3). Protein supplements are usually
produced as powders to dissolve in water or juices. Lately
ready to drink supplements are for sale containing 25-60
grams of protein in one container. In some countries (USA,
Japan) it is possible to buy these kinds of beverages in drink
automats. In South Korea and Japan carbonated beverages
with addition of skimmed milk powder are produced.
According to nutritional facts on the label, one can
(250 cm3) of this drink contains 130 kcal, 31 grams of
carbohydrates and does not contain fat and protein (4).
Certainly it is not true, as some low protein concentration is
present, which originates from skimmed milk. This beverage is
very popular in South Korea and Japan. There is no produced
carbonated beverage with considerable concentration of

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

protein, which could serve as a source of protein and

substitute a full meal.
One of the most important supplements is creatine. Its positive
influence on strength and muscle mass was proved in many
scientific research projects (5). Supplementation with creatine
has shown to increase strength, muscle mass, and muscle
morphology with concurrent heavy resistance training more
than resistance training alone. It promotes a faster
regeneration of adenosine triphosphate between high
intensity exercises (6). Similarly to other supplements, it is sold
in a form of powder dissolved in water, sugared water or
juices. A typical creatine supplementation involves a loading
phase of 20 g creatine/day or 0.3 g creatine/kg/day in 4 daily
intakes of 5 g, followed by a maintenance phase of 3-5 g
creatine/day or 0.03 g creatine/kg/day (7). Water dispersions
of creatine are produced and sold, but research showed that
at the storage most of the creatine is transformed into
creatinine (8). Some of the creatine liquid products contained
lower contents of creatine than in their label claims. Greater
degradation occurred in the samples kept at room
temperature as compared with the refrigerated ones (9).
Stability of creatine is highly affected by pH, being more
unstable at acidic condition (10). The effects of creatinine on
human health remain unclear, but lower concentration of
creatine diminishes the effect of supplementation. Uzzan et al.
(11) found, that a creatine milk-based dietary supplement
can be heat treated by any of the typical thermal processes
for beverages with little loss in processing. Processed and
refrigerated product was characterized by high shelf-life
stability. Such a product can have a shelf life of


approximately 3 months under refrigeration, with an initial 30%

to 40% over-run during formulation. There is no research on the
influence of different protein water dispersions on stability of
creatine. On the market there is no carbonated beverage
with substantial concentration of protein and creatine.
The aim of the research was to develop new products:
carbonated protein beverages with creatine and to
investigate their properties. Additionally properties of the
basic protein dispersions with or without sucrose were
evaluated. Viscosity, surface tension, creatine stability of the
beverages and protein dispersions, and sensory analysis of the
beverages were performed.


The following materials were used as the source of protein:
defatted milk powder, whole milk powder, whey protein
concentrate - WPC (all SM Spomlek, Radzy Podlaski, Poland),
whey protein isolate - WPI (Arla Foods Ingredients, Viby,
Denmark) and egg white protein isolate EWPI (Kewpie
Corporation, Tokyo, Japan). The composition of the protein
components was provided by the manufacturers and is
shown in table 1. Creatine monohydrate was purchased from
Olimp Laboratories (Dbica, Poland).

Table 1. Composition of protein components used for the beverages

production (%)

Protein dispersions preparation

Different protein source materials were dissolved in distilled
water to obtain 4 (%w/w) protein dispersions. For some
samples sucrose was added in concentration 5%. For creatine
stability measurements, 1% of creatine monohydrate was
added. The dispersions were used for viscosity, surface tension
and creatine stability measurements.

Rheological measurements
The measurements were carried out in a concentric
cylinder viscometer HAAKE model RS300, using a double
gap sensor DG41 controlled by the RheoWin Haake
Software (Thermo Fisher Scientific, Waltham, USA). Shear
stress was determined at shear rates changed from 1 to
300 s -1 in 180 s. Measurements were done in duplicate at
a controlled temperature of 24 0.1C.
Surface tension
The surface tension of investigated dispersions was
measured at 24C by the hanging drop method using
tensiometer Theta Lite (Attension/Bioline Scientific,
Espoo, Finland). Theta Optical Tensiometer captures
drop images and analyzes the drop shape as a function
of time using Young-Laplace equation implemented in
OneAttension Software (Attension/Bioline Scientific,
Espoo, Finland). Each sample was subjected to six
measurements and the average was presented.
Creatine degradation
The samples were stored at the refrigerator at 4C for 30
days and creatine degradation was measured every 5
days. Creatine degradation in investigated samples was
determined by measurements of creatinine
concentration using the BioVision creatinine assay kit
(#K625-100) (BioVision, Milpitas, U.S.A) and the
manufacturers instruction (12). The absorbance was
measured at 570 nm using spectrophotometer Specord
M42 (Carl Zeiss Jena, Germany). The standard curve
reproducibility was shown in 4 independent assays.
Sensory evaluation of the beverages
The sensory evaluation of the beverages was carried out
by 16 female and male subjects between 20 and 24
years of age. Forty subjects were trained and 16 final
panellists were selected based on the performance in
the screening tests. The samples were served immediately
after removing from the refrigerator in random order to
avoid position effect. Sensory evaluation was conducted
based on 5-levels scale for 4 different attributes. The
following attributes were evaluated: Colour (from +5
white, uniform colour without sediment to +1 yellow
colour, sediment); Taste (from +5 very pleasant, milky and
sweet, without off-tastes to +1 unpleasant, sour or bitter
with different off-tastes; Flavour (from +5 very pleasant
intensive milky aroma to +1 low intensity milky aroma or
strange odour; Consistency (from +5 full viscous
homogenous texture to +1 watery thin texture). The
panellists had very accurate tables for every sensory
attribute score which are not presented. Each test sample
was presented in duplicate.

Preparation of carbonated beverages

Carbonated beverages were prepared as follows. Basic solution
was prepared.containing protein, sucrose, vanilla sucrose,
maple syrup and creatine monohydrate. Four different
beverages were prepared with different protein source
(defatted milk powder/WPC 50%/50%, WPC, WPI and EWPI).
Basic solution was added to sparkling mineral water
(Naczowianka, Naczw,
Poland) in 500 ml plastic bottles to
obtain in the final product: 4%
protein, 2% sucrose, 2% vanilla
sucrose, 2% maple syrup and 1%
creatine monohydrate. Both were
cooled to 4oC before mixing. The
bottle was closed and the
beverages were stored at 4C for 30
days. For rheological and surface
Figure 1. Influence of shear rate on apparent viscosity
tension analysis, the beverages were
for WPI beverage prepared in distilled water.
prepared in distilled water.


Figure 2. Influence of shear rate on shear stress

for beverages prepared in distilled water.

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014


Apparent viscosity
Apparent viscosity of investigated dispersions was measured
at shear rate up to 100 s-1 for 180 s. Dispersions acted as
weakly pseudo-plastic liquids as a small decrease in apparent
viscosity with increased shear rate was noted using accurate
double-gap geometry (Figure 1). Relationship between shear
rate and shear stress fitted to linear function with R2 in the
range 0.9797-0.9998 (Figure 2). The dispersions were treated as
Newtonian liquids and the viscosity was taken from the slopes
of the lines of linear correlation (Table 2). Addition of sucrose
caused an increase in viscosity. The highest viscosity was
noted for beverage with defatted milk and WPC.
Surface tension
The surface tension of investigated dispersions was lower than
that of water due to the presence of surface active proteins
(Table 2) (13). Surface tension of the beverage with WPC was
the lowest because of the highest fat content. Increasing the
fat content was found to have an inverse effect on the
surface tension (14). Surface and interfacial tensions affect
the rheology and stability of dispersed systems (15). Surface
tension is also related to sensory food properties such as taste,
flavour perception and mouthfeel (16).

compounds at the interface. Increased fat content induces

significant retention of hydrophobic flavour compounds
resulting in effects on flavour perception (17). Decreased
viscosity and surface tension can be linked to the increased
water mobility in the medium, which leads to an
enhancement of sweet taste (18). It is generally considered
that an increase in viscosity leads to a decrease in flavour
although the magnitude of the effect depends on the
presence of other ingredients (19; 20). Also, despite the
general fact that an increase in dispersion viscosity leads to a
decrease in sweetness and in other taste attribute intensities,
the taste suppression power depends on different diluted
constituents (20, 21). Lower surface tension indicates the
formation of non-polar regions able to trap low-polar
compounds responsible for aroma (22). Present research
showed high positive correlation (R2 = 0.87) between viscosity
and perceived consistency. Pankiewicz and Jamroz (23)
found the correlation between the perceived and physical
viscosities of vodkas.

Figure 3. Sensory analysis parameters scores for

carbonated beverages.

Table 2. Parameters for the rheological linear model: shear stress =

a(shear rate) +b, surface tension and creatine degradation for different
protein source dispersions without or with sucrose and beverages
(protein concentration 4.0% w/w; sucrose concentration 5% w/w).

Sensory analysis
Figure 3 presents sensory scores for different beverages. The
lowest score value of colour was noted for beverage with
WPC, because of the yellowish colour of the concentrate. The
highest taste score was noted for beverage with defatted milk
powder and WPC, probably because of the highest
sweetness originating from the highest concentration of
lactose in this drink. Linear correlations were calculated for
viscosity and sensory analysis parameters and for surface
tension and sensory analysis parameters (charts are not
shown). High negative linear correlation between viscosity
and flavour perception (R2 = 0.96) was noted. No correlation
was found for the relationship between surface tension and
flavour, because increased fat content decreases surface
tension, but also increases concentration of flavour

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Stability of creatine
Stability of creatine was carried out over a period of 30 days.
For water dispersions 46.1% of creatine transformed into
creatinine. For different protein dispersions and the
beverages, the degradation of creatine was in the range
21.3-28.9% (Table 2). These are very good results as some
marketed formulations were found to contain less than 2% of
creatine comparing to 100% content label claim (24). Sucrose
had a little protective effect, as lower creatine degradation
was observed in sugared dispersions and beverages. A
ln(creatine concentration) versus time plot was linear,
indicating that degradation of creatine in solution to
creatinine followed rst-order kinetics (Figure 4). Similar
relationship was observed for other samples (data not shown).
The same kinetics order was observed by Dash and Sawhney
(24). The rst-order degradation rate constant was 0.0205 for
water and 0.0082 for WPC beverage. In the first order
reaction, degradation rate is the highest at the beginning of
the process. Average degradation rates per day were found
from the slopes of the lines of linear correlation. For our
research it was 0.151 mg/day/ml for water and 0.073 mg/day/
ml for WPC beverage. In practice storage of 5 g creatine in
500 ml container for 30 days resulted in 2.3 g loss of creatine in


water in comparison to 1.1 g loss in WPC beverage. To fulfil

creatine maintenance phase needs, 500 ml container of the
beverage should contain about 6.4 g of creatine. Our
research shows that creatine liquid supplements should have
a shelf-life calculated based on the degradation rate
constant. Steenge et al. (25) proved, that if creatine is
ingested together with protein, a reduced dosage and
shorter loading period are sufficient. Pittas et al. (26) reported
that a combination of protein, carbohydrate and creatine
was optimum for insulin response and creatine retention. In
another research a beneficil role of combining whey protein
isolate and creatine was shown (27, 28). Present results show
that the positive role of whey protein in mixture with creatine is
not only connected with optimization of insulin mediated
creatine retension but also with a protective role of whey
protein against creatine conversion to creatinine.

creatine degradation, protein quality, price and high sensory

analysis scores, beverage with WPC addition could be the best
candidate for the industrial production process.



Figure 4. Influence of time of storage of at 4C on degradation of

creatine in carbonated water and in carbonated WPC beverage.

Carbonated beverages with different protein and creatine
addition can be an interesting offer for food industry. There is not
such a product on the market. A 500 ml serving would supply 20
g of high quality protein and 5 g of creatine for sportsmen and
physically active people. Refrigerated storage revealed low
creatine degradation. It seems that because of the lowest


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Ahuja S., et al., Agro Food Ind. Hi Tec., 23(2), 4-5 (2012).
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Milk beverage composition: (last checked on Jan. 2nd 2014).
Persky A.M., and Rawson E.S., Agro Food Ind. Hi Tec., 21(4), 17-19
Cooper R., et al., J. Int. Soc. Sports Nutr., 33(9), 1-11 (2012).
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Nigel J.F. and Marinos E., Clin. Chim. Acta, 175(3), 199-210 (1988).
Uzzan M., et al., J. Food Sci., 72(3), 109-114 (2007).
Creatinine assay kit manual:
K625-100.pdf (last checked on Jan. 6th 2014).
Tomczyska-Mleko M., et al., Czech J. Food Sci., 32(1), 82-89
Mukherjee N., et al., Int. J. Food Eng., 1(2), art.2 (2005).
Kiani H., et al., Aust. J. Dairy Technol., 63(3), 87-92 (2008).
Tarzia A., et al., Int. J. Food Sci. Tech., 45(10), 21672175 (2010).
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Secouard S., et al., Flavour Frag. J., 21(1), 8-12 (2006).
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945 (2002).
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Pittas G., et al., J. Sports Sci., 28(1) 67-74, (2010).
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28. Hayes L.D., et al., J. Sports Med. Doping Stud., 3(3), 1-7 (2013).

Technical problems associated with using real fruit in long shelf life dry products can easily be addressed with fruit
ingredients from Taura Natural Ingredients. Manufacturers of products such as cookies, cakes, breakfast cereals and
snack bars have traditionally had to contend with the difficulties caused by moisture transfer when using fruit ingredients.
Introducing additional moisture to the product matrix in such applications poses a threat to the texture and shelf life of the
finished product. Fruit pieces, pastes and flakes from Taura Natural Ingredients eradicate this problem because they are
made using the proprietary Ultra Rapid Concentration (URC ) process. URC technology concentrates fruit pures and
blends to below 10 percent moisture in less than 60 seconds. This process enables Taura to control the water activity of the
final ingredient. Water activity is a measure of the ability of water to migrate from a given ingredient into the surrounding
product matrix and is a relative measure of the capacity for moisture transfer. Taura Natural Ingredients has the ability
to tailor the water activity of its fruit pieces and flakes to each application, opening up a world of product development
opportunities. Taura Natural Ingredients has created a free White Paper for food manufacturers addressing the challenges
associated with incorporating real fruit into long shelf life dry products. Entitled Extending the shelf life of products containing
fruit, the White Paper is available.


Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014


Industry perspective

*Corresponding author
indirizzo 1 Food Information Council, Tassel House, rue Paul-Emile Janson 6, B-1000 Brussels, Belgium
indirizzo 2


Traditional and social media in food

risk communication
KEYWORDS: social media, traditional media, communication, risk, monitoring, FoodRisC


Social media offers an approach to communicating which enforces many of the key principles of effective
risk communication, such as timeliness and openness. However, the use of social media is not without its
challenges. To ensure effective messages are communicated to the public during times of food risk/crises, it is imperative that sources
of such information are cognisant of the role played by both traditional and social media. This paper discusses these issues and
highlights some results from the recent European Commission-funded FoodRisC project - Perceptions and communication of food risk/
benefits across Europe.


Food plays a central role in all our lives and because it is
inherently personal it makes compelling news. Public interest
in nutrition, food safety and health has increased significantly
over the past years, and within the general sphere of media
communications, food risks and benefits are common topics.
The practice of journalism has
undergone significant change. The
ability to report on news is no longer
solely the remit of professionally
employed journalists. Nowadays
anyone can write and disseminate
news as a citizen journalist (e.g. hobby
and self-publishing journalists). This has
been facilitated by the emergence
of Web 2.0 technologies and the rapid developments in
digital connectivity via the internet which allows people to
easily generate, broadcast and share information via blogs,
microblogs (e.g. Twitter), content communities (e.g. YouTube
and web forums), social networking sites (e.g. Facebook) and
so on. These developments have also altered the manner by
which food safety agencies and other food risk information
sources (e.g. institutes, food industry and consumer groups)
can communicate. Traditionally information generated by
these sources was communicated to the public via journalists
operating within the traditional media arena resulting in a
communicative process which was hierarchical and one
way (1). Nowadays these sources can engage in a two-way
conversation with their stakeholders. Thus social media reflects
the views from various sectors of the population, including

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

those traditionally less likely to be heard (2). Considering the

nature of social media it can be considered an instantaneous
snapshot of public opinion (3).
By its nature, social media offers an approach to
communicating which enforces many of the key principles
of effective risk communication, such as timeliness and
openness (4). However, the use of social media is not without
its challenges, for example, it may amplify or attenuate
public perceptions of risk and shape
peoples behaviour (4-6). Although the
amplification of risk is also a possibility
in traditional media, it is likely that
social media plays an increasingly
important role given its pervasive nature
in the public domain. Members of the
public are also more likely to identify
with content on many social media
sites which has been generated by citizen reviewers and
commentators who share their experience and opinions as a
peer or collectively via various networks. The power of such
connections is evident from a recent study by researchers
at the University of California, Yale, and Facebook which
analysed postings from millions of Facebook users and found
that mood spreads virally through this social networking site (7).


In the new media scene, the broadening of topics and the
hybridisation of channels gives rise to new roles that shape
content and create new discourses and meanings regarding


the risks and benefits of food. It is important for food risk

communicators to be aware of and understand the content
presented in all media channels (2) and to understand the
inter-relationships between them. This was one of the aims of the
European Commission-funded FoodRisC project - Perceptions
and communication of food risk and benefits across Europe,
a three-and-a-half-year project which commenced in March
2010 (8). In this research project, Work Package 2 Media
involvement in communication in the food chain: classical and
new media, set out to map the media discourse occurring on
different media platforms during a number of pan-European
food crises. These included the 2008 Irish Dioxin crisis in pork, the
2011 German Dioxin crisis in pork, chicken and eggs and the
2011 German EHEC (enterohaemorrhagic Escherichia coli) crisis
in sprouted seeds (specifically fenugreek seeds). Analysis of the
first crisis was retrospective, while analyses of the other two crises
were conducted in real-time. For each crisis, media discourse
was monitored across a range of countries and social media
One of the initial challenges was to identify appropriate media
monitoring tools. Although traditional media monitoring tools
are well established, significant
developments have been made
over the last decade in the area of
social media monitoring and this has
now become one of the primary
forms of intelligence gathering. In
terms of food risk communication,
social media monitoring can be
used to determine engagement,
outreach, tone and share-of-voice
and to understand consumer
perceptions and behaviours. The
data generated can be leveraged
for different uses. For example, food
agencies may be interested in public
reaction and responses to official
announcements, while food businesses may be interested in
negative perceptions which could cause collateral damage.
As at September 2010, the project had identified over 100 social
media monitoring tools. Since then the market has expanded,
and new tools have been developed while others have
increased their functionality. Even the platforms themselves, such
as Twitter, Facebook and blogging platforms, are now providing
more analytic and management functions. The online wiki of
social media monitoring currently lists 230 tools (9). Selection of
the most appropriate tool requires a clear definition of what
is required and the findings from the FoodRisC work highlight
the importance of thoroughly researching these tools prior to
selection. Radian 6 was considered the most appropriate at
the time; however, if this research was conducted again today,
an alternative tool may be selected. Factiva and LexisNexis
were selected for traditional media monitoring. Monitoring was
conducted to evaluate reporting metrics and content. The latter
was conducted using text mining techniques based on a lexicon
of keywords that were selected for each crisis.
To gain a better understanding of how journalists go about
communicating on food risks, semi-structured interviews were
also conducted with both professional and citizen journalists
(bloggers) in four European countries that had communicated
on at least one of the two major food crises evaluated in the
project (i.e. 2011 German dioxin crisis and/or the 2011 German
EHEC crisis). In many respects, similarities were observed


between professional journalists and citizen journalists who have

expertise in their subject area (e.g. expert bloggers), compared
to citizen journalists who have no specific expertise (10). The key
findings are discussed, as appropriate, throughout the paper.


Media analysis showed that social media responded quicker
to the crises than traditional media, although there was less
social media channels and use in the 2008 Irish Dioxin Crisis than
in 2011 (80 posts UK/Ireland per day at the peak of the 2008
crisis compared with 9,000 in Germany in 2011). This finding is
expected considering the potential for immediate publication
in social media compared to the time lag which is inherent in
the traditional media editorial process. In the case of the 2008
Irish dioxin crisis, social media surged and reached a high point
earlier than traditional media but did not climb to its highest
point until traditional media had peaked (2). Given that social
media very often builds on news feeds from traditional media
(11-13), it is reasonable to hypothesise that traditional media
stimulated the second peak of social
media coverage, by providing
abundant resources. For both crises,
coverage in social media diminished
quicker than traditional media.
A strong correlation was observed
between media coverage and
communications from official sources
(e.g. national food safety authorities).
Media coverage of the 2008 Irish
dioxin crisis rose dramatically and
peaked between 8th and 10th
December before dropping to its
lowest level one day later (2). Most
official press releases and ministerial
statements were announced during the peak (14). Similarly, the
2011 German EHEC crisis was dominated by countless official
announcements (predominantly from the German Federal
Institute for Risk Assessment, BfR) as authorities tried to identify
the source of the outbreak which caused more than 3,100
cases of bloody diarrhoea, 850 cases of haemolytic syndrome
and 53 deaths. Analysis of Spanish tweets (Spanish cucumbers
were initially implicated) revealed peaks and troughs in the
volume of tweets, with the peaks corresponding to the release
of notifications from the German Federal Institute for Risk
Assessment (BfR) (15).
These findings highlight the strong emphasis placed on official
agencies as sources for food risk information and such findings
have previously been noted (16) and also emerged from the
interviews conducted with journalists. However, official sources
must not become complacent and must continually strive
to communicate with journalists in an effective and timely
manner. Failure to do so may force journalists to communicate
information from alternative sources. The impact of this may
be significant considering the potential for proliferation and
retention of information within the social media arena and
particularly if incorrect or misrepresented information is reported
in the media. Regarding the retention of information within the
social media arena, research conducted in FoodRisC has shown
that information pertaining to the German Dioxin crisis was
prevalent and easily found on the Web 20 months after the crisis
(a search was conducted using the term German Pork

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

in the Google search engine of 7 countries, which showed that

the dioxin crisis featured on the first page of a Google search in
6 countries).

Within the social media arena, reporting of food crises most
commonly occurred via Twitter, online news and blogs;
however, the order of ranking varied not only between crises
but also between countries reporting on that crisis. In addition,
Twitter and online news tended to be used at the early stages
of a crisis, while blogs tended to become more prevalent as the
crisis progressed. Twitter was primarily used to inform readers of
breaking news and to refer them to more detailed information,
usually online news. This use of Twitter is in line with insights from
other crisis events (17, 18) and reinforces the importance of
Twitter for sending fast, topic-related alerts, referring readers
to additional online information and enabling dissemination
of the original message (19). This may explain its dominance
at the early stage of a crisis. Blogs,
on the other hand, are good for
sharing reflective, opinion pieces
that provide situational overviews. In
fact, research suggests that during
crises more people read blogs and in
some cases, assign a higher degree
of credibility to blog crisis coverage
than to conventional mainstream
media coverage (20, 21). Other
communication channels such as
forums, videos or Facebook were
used to a much lesser extent during
the crises; however, it should be noted
that Facebook has privacy settings
which can stop conversations being
public so this may be a reflection of
these restrictions, rather than a lack of
As outlined earlier, professional
journalists and to some extent
professional bloggers rely on food
agencies as sources for food risk information. However, social
media is overcrowded with several sources of information
and one of the biggest challenges for organisations is to stand
out from the crowd and to be a trusted and effective source
in times of crisis. This can only be achieved by developing a
strong online profile and connectivity with the audience during
peace times. It has been reported that while private businesses
are investing in social media, other food risk and benefit
communicators such as food safety authorities have not (22).
Research conducted as part of the FoodRisC project (between
October 2012 and January 2013) also revealed a low level of
engagement by government food agencies across Europe
with social media, although there are notable exceptions. The
study assessed 37 food agencies in 30 countries, this included
two European organisations. Of the organisations studied, 30
percent had their own Twitter account, 39 percent had their
own YouTube channel, and 50 percent had their own Facebook
page. Furthermore, many organisations did not promote their
social media activities via the relevant social media icons on
their website. This highlights the potential for these agencies to
increase their profile and level of engagement with social media

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

and their intended target audience. However, it is also important

that official agencies do not lose sight of traditional channels
such as official websites, email alerts and telephone contact,
as these are particularly important channels for journalists when
validating source information.


Not only do the media transmit food risk and crisis messages with
the use of a variety of sources, they can also interpret the story
for the reader by selecting what elements (topics) to emphasise
and what sentiments to use in delivering the message (23, 24).
This interpretation may not only reflect concerns of the media
operators, but also influence readers perceptions of different
issues and the severity of the incident. The essential component
of influence is that it inspires action, i.e. the public may alter
their behaviour as a response to this information (25). Therefore
a thorough understanding of the tone and type of information
communicated during food crises is essential.
Social media have penetrated into
the practice of crisis communication
(26). It is interesting to know how
social media differ or resemble
traditional media in shaping the
discourse around food crisis. A
systematic analysis of newspaper
articles and social media postings
during the 2008 Irish dioxin crisis and
the 2011 German dioxin crisis shows
that social media users selected a
different angle in reporting crisis. For
instance, in the 2008 dioxin crisis,
in comparison with newspaper
reporters, social media users put
more focus on global reactions and
public perceptions of this incident,
but manifested less interest in topics
like the cause of the contamination
and the impact on national
economy and food industry (2).
In times of the 2011 dioxin crisis, in
Germany, there were more discussions in social media around
health facts relating to dioxin, but less conversations about
governments handling and the influence on the food supply
chain when compared to newspaper articles. These results
echo the findings from Liu (2010) that showed social media
can challenge traditional media by introducing different story
angles (27). It is worth noting that individuals vary in exposure
to media channels and their sense-making of the dioxin crisis
might be influenced by story angles held by the media (28).
In relation to tone of voice, one important measure of food
crisis management is the amount of negative media attention
it receives (27, 29). With the rise of social media, now food
managers need to monitor negative discourse on social
media as well. Analysis of the coverage of two dioxin crises
in the FoodRisC project showed there was no significant
difference between social and traditional media in terms of
tone of delivery, with more than half of newspaper articles
and social media posts employing neutral or mixed tones,
rather than purely negative tones. This is in contrast with the
perception of social media as a platform where people tend
to grumble or complain.




Monitoring behavioural intentions to a food crisis is essential

considering the potential public health and financial impacts.
For example, the recent pan-European horsemeat incident
(adulterated beef products containing undeclared horsemeat)
resulted in changes to consumer purchasing and consumption
patterns (30). Traditionally behavioural intention has been
collated using methodologies such as interviews and focus
groups. However, FoodRisC research highlights the potential of
microblogging channels such as Twitter for assessing individuals
behavioural intentions/coping strategies (15). The study involved
a quantitative analysis of 11,411 tweets collected in Spain
during the 2011 German EHEC crisis (Spain was initially thought
to be the source of the outbreak). A qualitative analysis was
conducted on 2,099 of these tweets. Behavioural intentions/
coping strategies were found to be dynamic and flexible, with
a predominance of accommodation (e.g. acceptance),
information seeking (e.g. further study) and opposition strategies.

The authors thank Aleksandar Sokolovic, Susan Rowntree and

Christine Shan for their assistance with the manuscript and
acknowledge the work of partners involved in WP2 of FoodRisC,
Aine McConnon, Monique Raats, Charo Hodgkins, Adrian
Moss, Markus Lehmkuhl, Jordi Farre, Jordi Prades Tena and Aine
Regan. The FoodRisC project has received funding from the
European Unions Seventh Framework Programme for research,
technological development and demonstration. Grant
agreement number 245124.
For further information on the project please see:





It is vital that sources of food risk information maximise the

potential of both traditional and social media to ensure their
messages are communicated in a timely and efficient manner
thereby enabling consumers to make informed decisions and
use food safely. Social media can be used as a feedback
loop, to see how the message is being received and relayed,
and so make adjustments and correct misinformation and
misunderstandings. That direct interactivity is not available via
traditional media except through readers writing to editors and
being re-published, either in print or on-line.


Considering the myriad of information which is now available

online, one of the biggest challenges is to stand out from the
crowd and to be a trusted and effective source in times of crisis.
To do this, organisations must build a strong online presence and
connectivity with their key stakeholders during peace times
and not just when a crisis is in flow.


Whilst engagement with professional journalists is essential as

they are involved not only in the dissemination of messages
to a wider audience but they are also involved in framing the
message and agenda building, the optimal approach is to
engage with key influencers in both traditional and social
media. However, this is an area requiring more research
because to date there is no industry or academic gold standard
for their identification.


Social media has democratised the process of information

generation and sharing and thus reflects the views from various
sectors of the population, including those traditionally less likely
to be heard. It therefore provides a massive database of
information which can be mined to provide intelligence on
the buzz surrounding a specific topic, consumer perceptions,
consumer intentions etc. Sources of food risk information should
maximise this resource and utilise the information generated
to optimise their message. In addition, consideration should
be given to rectifying any mis-information at the end of a
crisis period to ensure the discourse is appropriately resolved.
This is particularly important considering the length of time
information is retained and accessible on-line and within the
social media arena.





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Cultural Studies, 5(2), 285-293 (2013).
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March 24th 2014).

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014


Industry perspective

*Corresponding author
Shiraz 1University, Faculty of Agriculture, Department of Horticulture, Iran
Young2Researchers Club, Islamic Azad University, Shahr-e-Qods Branch, Tehran, Iran

????? Jouki

Effect of modified atmosphere packaging

on persimmon fruit (cv. Karaj)
Physical, chemical and mechanical properties

KEYWORDS: persimmon, MAP, shelf-life


The effects of modified atmosphere packaging (MAP) on some quality parameters of persimmon
(Diospyros kaki L.) cv. Karaj were examined over a period of 28 days. Persimmons were segmented and
stored under vacuum packaging (VP), Air packaging (21 percent O2 +79 percent N2 as AP) and two modified atmospheres
packaging systems (20 percent CO2 + 10 percent O2+70 percent N2 as active MAP1 and 30 percent CO2 + 70 percent N2 as active
MAP2) in polyethylene (PE) trays. The results indicated that the samples stored in MAP2 had the lowest changes about TSS, SF, and pH
but AP stored samples had the most changes in investigated traits. Also the samples stored in MAP2 showed the highest L* (lightness)
with lowest modifications in a*, b* and E.

Modified atmosphere packaging (MAP) combined with
refrigeration is a widely used food preservation method. The
gases normally used for preservation include combinations
of O2, CO2 and N2. Modified atmosphere packing has been
used for increased distribution range and longer shelf-life.
The effects and roles of the gases normally used in the
modified atmospheres (O2, CO2, and N2) have been
extensively reported (1, 2). Adverse physiological changes
included weight loss due to transpiration and respiration, flesh
softening and diminish of resistance to microbial pollution are
contributed to decay of fruits (3). Treatments of high CO2 may
affect on visual quality of persimmon slices (4). Fruits are
nutritious and interesting foods, because of having
attractive colour, shape, unique taste and smell and also
they are rich about minerals, vitamin and other beneficial
ingredients (5). As a useful conservation method, MAP can
be introduced for extending the shelf-life of different
perishable food products such as fruits (6- 8). Increased CO2
and/or decreased O2 levels diminish the rate of respiration
and ethylene production resulting to inhibition or delay
enzymatic reactions and alleviate physiological disorders,
and also that conserve the product from losses in quality (9).
Among of packaging materials, polyethylene has been
widely used in for modified atmosphere packaging of fruits
and vegetables (10-14). A study showed that MAP
minimized loss of water in artichoke (15). Cocci et al. (16)
showed that MAP had preservative effect on the color.
Under to exceed O2 and CO2 levels out of tolerance limits
can lead to anaerobic respiration and it may cause to the

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

production of unpleasant compounds such as ethanol and

also create other physiological disorders (17). Changes/
stability in physical attributes such as colour, firmness,
juiciness, absence of decay, and chemical attributes such
as total soluble sugars (TSS), pH and titratable acidity (TA)
are often used to assessing the postharvest shelf-life of sliced
or minimally processed packaged fruit and vegetables.
These criteria exhibit visual acceptance and
physicochemical characteristics correlated to produce
quality (18, 19). The benefits of modified atmosphere
packaging have been extensively studied in extending
shelf-life of many fruits and vegetables. However, there are
only limited studies regarding ready-to-eat permission fruits.
The objective of this work is to evaluate the potential of
modified atmosphere packaging in preserving some quality
parameters of persimmon samples and to determine the
quality losses for 28 days at 4C.


Plant materials
Persimmons grown in Karaj area in Alborz province, Iran,
were harvested from persimmons plants in the morning in
September 2012. They were transported in air-conditioned
vans to the laboratory of packaging and postharvest
physiology of fruits at the University of Tehran after
harvesting and sorting (medium size and ripe persimmons).
Packages of 30 cm 20 cm of polyethylene (PE) rolls were
used for packaging. Polypropylene of selective
permeability; permeability at 25C to O2: 69 cm3/m2/24 h/


atm; permeability at 25C to CO2: 251 cm 3/

m2/24 h/atm; permeability at 25C to H2O: 3.2 g/
m2/24 h/atm; thickness: 62 m. The data
collection and analyses were done at 1th, 7th,
14th, 21th, and 28th days. Segmented
persimmons were packed under vacuum
packaging (VP), Air packaging (21 percentO2
+79 percent N2 as AP) and two modified
atmospheres (20 percent CO2 + 10 percent
O 2+70 percent N2 as MAP1 and 30 percent CO2
+ 70 percent N2 as MAP2) in polyethylene (PE)
trays in three replicates.
Shear force
Shear force (SF) of fruits was determined using a
Table 1. Effect of different atmosphere packaging systems on some physicochemical parameters of Karajs persimmon fruits stored at 4C.
Testometric Machine M350-10CT (Testometric Co.
a-d values in the same column with different superscripts are significantly different (p <0.05).
Ltd., Rochdale, Lancashire, England) according to
w-z values in the same row with different superscripts are significantly different (p <0.05).
the method mentioned by Jouki et al. (12).
MAP: modified atmosphere packaging [MAP1 (20 percent CO2 + 10 percent O2+70
percent N2); MAP2 (30 percent CO2 + 70 percent N2); AP: Air packaging; VP:
Cutting of segments at the centre was done by
Vacuum packaging.
speed of 20 mm/s and penetration distance of 6
mm, and the shear force was exhibited as
maximum cutting force (N). Segments by Similar
sickness were selected for shear force calculation. Two
parallel packages (10 segments/ package) included six
Shear Force (SF)
segments were measured for each treatment on each
The samples stored in MAP2 had the lowest changes about
sampling day. The data were recorded in N force. The
shear force (SF) during storage. SF values were 0.549, 0.552,
duration of storage was defined for each treatment.
0.556 and 0.550 for persimmon samples stored in AP, MAP1,
MAP2 and VP, respectively (day1). At 21th day of storage the
Weight loss
most and lowest SF were related to MAP2 (0.394 N) and
Weight loss was measured by the following equation:
MAP1 (0.297 N), respectively (Table 1). Lower decrease of SF
Weight loss (g/100 g) = (W0-Wd)/W0100 (W0 = 1th day
in MAP2 can be related to higher CO2. As we know CO2 is an
weight; Wd = desired day after storage). The net weight of
anti-ethylene component that retards the softening of fruit.
each sample was registered on day 1, 7, 14, 21 and 28.
At the initial stage of conservation, protopectin (the main
Three replicate packages were analyzed.
pectic component in persimmon), is closely accompanied
with the cell wall cellulose. During storage, protopectin is
transformed into pectin by pectinase and isolated from the
The pH of the strawberry persimmon was analyzed by a pH
cell wall cause to softening and the texture would become
meter (Metrohm, Herisau, Switzerland) in duplicate
softer and the fruit would rot and loose its edible and
measurements on day 1, 7, 14, 21 and 28.
commercial values. It can be said that MAP2 samples had
the lowest pectinase activity during storage. The obtained
Colour, TA, TSS and vitamin C
results in this study are in agreement with other studies, which
The colour of poured homogenate into petri dishes was
have generally reported that fruits stored in MAP are firmer
measured using a colorimeter (Minolta Spectramatch,
than those stored in air (21, 22).
Tokyo, Japan). Three measurements were made on each
sample. Analyses were done day 1, 7, 14, 21 and 28. Three
random locations were used to measuring and the
As shown in Table 1, the pH of all the samples was in range
parameters of CIELAB L*, a*, b* were recorded. Total colour
5.53-5.68 at first day of storage time. During the storage
change (E) was measured using equation:
period, reduction on fruits pH was observed between the first
E = ((L0 - L)2 + (a0 - a)2 + (b0 b)2)1/2.
and the 21th day. The reduction of pH in persimmons
5ml of dilute fruit juice was pipetted into a boiling tube
beginning at the seventh day of its storage must have been a
and 1ml of glacial acetic acid was added and titrated
result of acid conversion to sugar or acid oxidation during the
with the dye solution to a faint permanent pink colour. The
Krebs cycle which constitutes an excellent energy reserve for
titre (TA) was recorded. The titration was repeated with
the fruit. These results are in agreement with obtained by
5ml of water for the blank (B1) and 5ml of ascorbic acid
Siriphanich (23), who reported that high-CO2 treatments
stand solution (st) and vitamin C content of the test
resulted in higher pH values than those of air control
sample was calculated using the relationship: Vitamin C
strawberries. In fact, 30 percent CO2 maintained the total
Test (mg/100ml) = [(T - B 1)/(st - B 1)] x Dilution factor. Total
acids (actors of pH and acidity) more than other treatments,
soluble solids (TSS) were measured with a Bausch and
confirming previous study (24). In general, the total acidities
Lomb Abbe 3L refractometer (20). The analysis of variance
decrease during ripening and after harvest.
(ANOVA) was carried out to test the possibility of
significance of treatment effect. LSD was used to perform
all possible pair comparisons between means of different
The percent total soluble solids (TSS) for all treatments
treatments. Significance of differences was represented at
decreased, while MAP2 resulted to slight decrease. Treatment
5 percent (P= 0.05).


Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

AP showed the highest decrease in TSS. There was significant

effect of storage time on total soluble solids (P< 0.05) (Table 1).
The variations can be explained by the hydrolysis of
carbohydrates leading to an increase in the reductive sugar
on one hand, and on the other hand, to a continuous
respiratory activity consuming low molecular weight of
reductive sugar followed by the drop observed at the end.
The smaller this variation, the slower the after-ripening process
and the longer the shelf-life (25) is in agreement with the
results of this study. Many researchers have reported that TSS
has not been influenced significantly by CA with high CO2
and low O2 or with high O2 atmospheres in apples and pears
(26), cherries (27) or longan (28).
Vitamin C content
Treatment VP had the highest maintenance vitamin
C during storage times (21 days). Also AP resulted to
maximum decrease in amount of vitamin C
throughout storage time. In general, MAP
decomposes the vitamin C content, diminishing the
initial values by between 5 and 6 percent under the
different atmospheres (Table 1). Minimum loss of
vitamin C was observed for those persimmons kept
with lower CO2 levels whereas in the other treatment
with higher CO2 levels, large losses occurred during
storage. Lower decrease of vitamin C in VP can be
attributed to lower oxygen reducing respiration rate.
Recent studies by other products showed that higher
CO2 has been resulted to higher decrease of vitamin
C (15) is in accordance with these results. In addition
temperature management after harvest is one of the
most important factors to maintain vitamin C of fruits
and vegetables whereas losses are accelerated at
higher temperatures and with longer storage
durations (29).

studies showed that loss of water after harvest decreased by

MAP, reducing weight loss and avoids wilting and shrivelling (15).
Wilting was related to the loss of smoothness and firmness and
was particularly observed in VP samples at during the storage
period, whereas slight weight loss was found in MAP2 samples.
Weight loss is due to the consumption of substances through
respiration however dehydration through evaporation remains
the main reason (25, 30). In the present study, the water loss rate is
very low (Fig. 1). MAP2 showed the lowest weight loss (10 percent)
whereas VP had the highest weight loss (35 percent). The losses of
water during storage can be contributed to the decrease of cell
expansion pressure, confirming previous study by Zhang et al. (25).

Table 2. Effect of different atmosphere packaging systems on color of Karajs

persimmon stored for 21 days at 4C.
a-d values in the same column with different superscripts are significantly different (p <0.05).
w-z values in the same row with different superscripts are significantly different (p <0.05).
MAP: modified atmosphere packaging [MAP1 (20 percent CO2 + 10 percent O2+70
percent N2); MAP2 (30 percent CO2 + 70 percent N2); AP: Air packaging; VP:
Vacuum packaging.

Figure 1. Effect of different atmosphere packaging systems on the

weight loss of Karajs persimmon stored at 4C.

Weight loss of persimmon

At the assayed temperature and during the storage period, slight
weight loss was found by MAP2 samples (Figure 1). Studies
showed that loss of weight after harvest is the main factor of
product ruin such as persimmon and artichoke. The rate of
transpiration can be reduced by during and after harvest. Last

Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014

Colour changes
Colour of persimmon is one of the most important quality
factors of fresh persimmon f or consumer preference. The L
colour value (L*) of all treatments tended to decrease over 21
days; storage of fruit in vacuum resulted in a great decrease,
while storage in MAP2 resulted in a slight decrease from day 1,
with significant difference among four treatments. The a-value
(a*) tended to increase over 21 days for all treatments; the fruit
stored in MAP2 showed the lowest changes (most
maintenance) but that stored under the VP showed a greater
decrease. The b-value (b*) increased significantly by day 7 for
all treatments, and continued to increase through day 14 and
21; with significant differences among treatments. MAP2
showed the lowest increase (lowest alterations) during 21 days
(Table 2). Colour deterioration in the persimmon pericarp can
be due to browning reactions resulting from many enzymes
activity such as polyphenol oxidase and peroxidase in the fruit
(31, 32). Also surface blackening was related to the process of
browning resulted from bruising (15). MAP1, AP and VP showed
a significant increase in external blackening when compared
with values at harvest (Table 2). The a* value increase
significantly during 7 days of storage, which is probably
supported by the accumulation of anthocyanin pigments and
high degree of red fruit. Obtained results, which demonstrated
that MAP2 was able to retard discoloration and the samples
under MAP showed lower decrease in L value, confirming
previous studies (12, 15).


Persimmon kept in modified atmosphere maintained their
weight and appearance better than those that packaged
under air. Fruits colour was affected by storage. Persimmon
colour parameters moved toward a more intense red colour
(a*) and a less intense yellow colour (b*) as not changes
during storage; also the L* value decreased in both MAP and
air packaging during storage fruit becoming darker in colour.
Firmness decreased during storage of persimmon samples in
MAP and air packed. The conclusion was drawn by
considering the parameters such as firmness, colour, weight
loss and sensory evaluation. The results showed that the
above mentioned parameters can be used as indicator for
the quality of persimmon. MAP protected persimmon from
spoilage for up to 3 weeks at 4C. MAP may offer a new
technique for spoilage prevention of horticultural
commodities. Its effectiveness, low price, and ready
availability without obvious off flavours is encouraging; it
could significantly extend the shelf life of fresh produce.


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There are signs that the global food additives market is returning to growth as the worst effects of the worldwide economic downturn
have passed and consumer spending levels are starting to increase again. As the economic situation improves, the processed foods
industry continues to expand within the developing world in particular, with high rates of growth observed in countries such as China,
India and Brazil. In turn, this is driving demand for many types of food additives, as a result of which some of the leading suppliers are
now establishing production bases in the Asian and Latin American regions. Another trend influencing the market at present is the
move away from artificial food additives and ingredients. With consumers and food manufacturers demonstrating an ever-greater
preference for products positioned on a natural or clean label platform, artificial and synthetic food additives have been falling out
of favour. Meanwhile, health demands are forcing food and beverage manufacturers to reformulate their products, mostly through
reductions in sugar, salt and saturated fat levels. This, in turn, is opening up opportunities for manufacturers of food additives. The Global
Food Additives Market 6th Edition is a new publication from Leatherhead Food Research, which updates the previous edition last
published in 2011. The report discusses and reviews the global market for food additives, notable examples of which include flavours,
sweeteners, hydrocolloids, enzymes, colours, preservatives and antioxidants. The report includes market size data for the last five years,
as well as an extensive review of the competitive landscape and discussion of likely future directions for the industry.


Agro FOOD Industry Hi Tech - vol 25(5) - September/October 2014


A cup of tea a day could keep the doctor

away: earl grey tea 'reduces heart disease'
Earl Grey tea could be highly influential in reducing the
risk of heart disease, according to scientists. New research
has found that bergamot extract, which is an important
component of the tea blend, helps to lower cholesterol.
This could help to protect against cardiovascular
illness, which are responsible for over 25 per cent of UK
deaths. Scientists from the University of Calabria, Italy,
found that bergamot contains hydroxy methyl glutaryl
flavonoids (HMGF) enzymes, which attack a number of
proteins that can contribute to heart disease. Bergamot
is the ingredient within Earl Grey tea that gives it its
distinctive flavour, meaning that having a daily dose of
the beverage could provide protection. Published in the
'Journal of functional Foods', the study suggested that
taking a daily dose of HMGF could help to lower bad
cholesterol - low-density proteins (LDL) - and be just as
effective at statins in doing so. HMGF was also found to
increase the amount of good cholesterol - high-density
lipoproteins (HDL). Bergamot has long been part of the

Mediterranean diet, which is known to be one of the

healthiest diets and to help cut down the chance of
cardiovascular disease. It is often used in traditional forms
of medicine for the treatment of inflammation, wounds
and as an antiseptic, as well as for heart protection.
While the extract is available in supplement form, it could
also be beneficial for those at risk of heart disease to
drink several cups of Earl Grey tea each day in order to
benefit from its protective qualities. The study follows on
from previous research, performed by the University of
Catanzaro, Italy, in 2012, which found that bergamot
could help to protect against diabetes and aid with
weight loss. This means that the fruit could have a number
of health benefits. Despite the fruit being likened to statins
in terms of heart protection by lowering LDL, it is thought
that statins also have other health benefits, including
being an effective treatment for men suffering from
erectile dysfunction.


Symrise AG has secured the financing for acquiring the Diana Group by successfully issuing a bond. The 500 million bond was
oversubscribed several times. Part of a comprehensive financing concept totalling 1.3 billion, it boasts highly attractive conditions
and will be used by Symrise to finance the largest acquisition in the companys history over the long term. Due to a capital increase
based on authorized capital, which was equally well received by the market, Symrise achieved proceeds from the issue of around
400 million in May 2014. In June 2014, the company secured short and medium- term borrowings from its primary banks amounting
to 400 million. The bond financing has a term of 5 years and a coupon of 1.75 percent. The issuance was supported by Mitsubishi
UFJ Securities International plc and UniCredit Bank AG as active book runners together with Banco Santander S.A., BNP Paribas,
Landesbank Hessen-Thringen and J.P. Morgan Securities plc. The bond shall be listed and admitted for trading on the official list of
the Luxembourg Stock Exchange (ISIN DE000SYM7704). As part of its refinancing activities, Symrise successfully issued its first corporate
bond in October 2010 with a maturity of seven years.
Exocyan, is a unique brand of cranberry (Vaccinium Macrocarpon) extract, a small berry grown exclusively in North and South
America. The Exocyan product line is standardized on proanthocyanidines (PACs) content, a type of flavonoid, with antioxidant
and other health activities. This berry is naturally packed with polyphenols and especially ProAnthoCyanidins (PACs). Most other
plants and berries, like grapes and green tea, contain B-Type PACs. However, cranberry contains only A-Type PACs, which have
healthy ageing properties. The A-Type PACs in cranberry are unique because they are the only PACs that have antibacterial
activity. The anti-adhesion property of A-Type PACs is the basis of cranberrys extraordinary ability to improve urinary tract health.

Agro FOOD Industry Hi Tech - vol 25(5) - September October 2014


Scientists uncover why major cow milk

allergen is actually allergenic
Cow milk allergy occurs in children and in adults. Scientists
at Messerli Research Institute at the Vetmeduni Vienna, the
Medical University of Vienna, and the University of Vienna
investigated what actually makes the milk allergenic. A
specific protein in milk known as beta-lactoglobulin is able
to initiate an allergy only when being devoid of iron. Loaded
with iron, the protein is harmless. The scientists discovered
the same mechanism recently with regard to birch pollen
allergy. Their findings help to decipher allergic reactions and
were published in the journal PLOS ONE.
Milk allergy is frequently confused with lactose intolerance.
However, these are two entirely different mechanisms that
occur in the body. People with lactose intolerance do
not digest lactose properly because they lack an enzyme
known as lactase. In the case of the potentially much more
dangerous cow milk allergy, however, the body's immune
system attacks milk proteins with its own IgE antibodies.
According to statistics, about two to three percent of
children in Europe suffer from a genuine milk allergy. Less
adults are diagnosed with the disease. The formation of
so-called Th2 lymphocytes is initiated in these patients. Th2
lymphocytes contribute in great measure to the production
of IgE antibodies to milk proteins. Hence, people develop an
allergic reaction to milk.
Such an allergy may cause swelling of the mouth
and mucous membranes, diarrhea, exacerbation of
neurodermitis, and in rare cases even an allergic shock.
Precise diagnostic investigation helps to differentiate
between allergy and intolerance and thus avoid incorrect
diets which, under certain circumstances, may cause

Lack of iron load transforms milk protein into allergen

One of the most important milk allergens, the so-called betalactoglobulin, belongs to the protein family of lipocalins. Lipocalins
possess molecular pockets which are able to accommodate
iron complexes. Iron is bound to the protein by so-called
siderophores. The first author Franziska Roth-Walter and her
colleagues now show that an "empty" milk protein, one without
iron and siderophores, helps to activate Th2 lymphocytes. As
a consequence, the production of IgE antibodies against the
milk protein is stimulated. The patient gets sensitized and may
develop an allergic reaction to milk. Roth-Walter, working at the
department of Comparative Medicine at the Messerli Research
Institute says: "Knowledge of the molecular structure of allergens
has contributed very significantly to our conclusion about milk
allergy. This is of enormous practical relevance."

Investigating the difference between organic and

conventional milk
As the next step the scientists want to find out, what
contributes to the iron load of milk proteins. The lead
investigator Erika Jensen-Jarolim explains: "One of the most
burning questions we want to answer is: Why are these milk
proteins loaded to a greater or lesser extent with iron?
The manner of keeping and feeding cows may be a factor
involved in this phenomenon. Iron loading may depend on
whether the milk is produced organically or conventionally.
This will be one of our major interests in the future. Lipocalins
exist in all mammals. We assume that our conclusions will be
applicable to the milk of other mammals as well."

PLT Health Solutions, Inc. and Oakshire Naturals, LP have announced the launch of Earthlight Whole Food Vitamin D. The patented
mushroom powder ingredient delivers 40,000 International Units (IU) of Vitamin D per gram from a non-GMO, clean-label,
natural source. Earthlights high concentration of Vitamin D will allow food, beverage and supplement producers to offer Good,
Excellent and High Potency source label claims with only a few milligrams of the ingredient. This low level of addition means that
Earthlight will not affect the organoleptic properties of the products in which it is included and contributes to the cost-effectiveness
of this Vitamin D solution. A minimally processed ingredient, Earthlight is considered a whole food form of nutrition responding to
consumer desire for cleaner labels on their food, beverages and supplements. This ingredient solution is being introduced to the
market at a time when concern about adequate levels of Vitamin D is at an all-time high in the nutrition and medical communities
a concern that is gaining awareness among consumers as well. According to Devin Stagg, Director of Corporate Strategy for PLT
Health Solutions, Earthlight Whole Food Vitamin D allows food, beverage and supplement producers to address a convergence of
consumer wants. First it was natural. Then it was clean label, and often consumers want vegetarian or vegan on their labels.
Todays answer to all of these consumer preferences is whole food nutrition.
Effective June 15, 2014, or as contracts allow, Stepan will increase prices for STEPANPOL Aromatic Polyester Polyols by $0.07 per
pound. The increase is necessary due to rising raw material costs.


Agro FOOD Industry Hi Tech - vol 25(5) - September October 2014

Are you as old as what you eat?

Researchers learn how to rejuvenate aging
immune cells
Researchers from UCL (University College London) have
demonstrated how an interplay between nutrition,
metabolism and immunity is involved in the process of ageing.
The two new studies, supported by the Biotechnology and
Biological Sciences Research Council (BBSRC), could help to
enhance our immunity to disease through dietary intervention
and help make existing immune system therapies more
As we age our immune systems decline. Older people suffer
from increased incidence and severity of both infections and
cancer. In addition, vaccination becomes less efficient with
In previous BBSRC funded work, Professor Arne Akbar's group
at UCL showed that ageing in immune system cells known
as 'T lymphocytes' was controlled by a molecule called
'p38 MAPK' that acts as a brake to prevent certain cellular
They found that this braking action could be reversed by using
a p38 MAPK inhibitor, suggesting the possibility of rejuvenating
old T cells using drug treatment.
In a new study published today in Nature Immunology the
group shows that p38 MAPK is activated by low nutrient levels,
coupled with signals associated with age, or senescence,
within the cell.
It has been suspected for a long time that nutrition,
metabolism and immunity are linked and this paper provides
a prototype mechanism of how nutrient and senescence
signals converge to regulate the function of T lymphocytes.
The study also suggests that the function of old T lymphocytes
could be reconstituted by blocking one of several molecules
involved in the process. The research was conducted at UCL
alongside colleagues from Complejo Hospitalario de Navarra,
Pamplona, Spain.

The second paper, published in The Journal of Clinical

Investigation, showed that blocking p38 MAPK boosted the
fitness of cells that had shown signs of ageing; improving
the function of mitochondria (the cellular batteries) and
enhancing their ability to divide.
Extra energy for the cell to divide was generated by the
recycling of intracellular molecules, a process known as
autophagy. This highlights the existence of a common
signaling pathway in old/senescent T lymphocytes that
controls their immune function as well as metabolism, further
underscoring the intimate association between ageing and
metabolism of T lymphocytes.
This study was conducted by researchers from UCL, Cancer
Research UK, University of Oxford and University of Tor
Vergata, Rome, Italy.
Professor Arne Akbar said: "Our life expectancy at birth is now
twice as long as it was 150 years ago and our lifespans are
on the increase. Healthcare costs associated with ageing
are immense and there will be an increasing number of older
people in our population who will have a lower quality of
life due in part to immune decline. It is therefore essential to
understand reasons why immunity decreases and whether it is
possible to counteract some of these changes.
"An important question is whether this knowledge can
be used to enhance immunity during ageing. Many drug
companies have already developed p38 inhibitors in
attempts to treat inflammatory diseases. One new possibility
for their use is that these compounds could be used to
enhance immunity in older subjects. Another possibility is
that dietary instead of drug intervention could be used to
enhance immunity since metabolism and senescence are
two sides of the same coin."
Biotechnology and Biological Sciences Research Council

Tate & Lyle has released key findings from consumer taste-test research conducted by Ipsos in May 2014. The research
revealed that Tate & Lyles proprietary TASTEVA Stevia Sweetener is preferred by nearly two out of three consumers (63
percent) over rebaudioside A, a prevalent stevia ingredient often associated with bitter aftertastes. Participants in the study
tasted two fruit-drink beverages. One was sweetened with TASTEVA Stevia Sweetener, and the other was sweetened with
rebaudioside A. In addition to overall taste preference, nearly two out of three consumers (63 percent) found the fruit drink
sweetened with TASTEVA Stevia Sweetener to have a pleasant aftertaste or no aftertaste, while more than half (54
percent) found the fruit drink sweetened with rebaudioside A to have an unpleasant aftertaste. This research is important
because it shows manufacturers they can bring those on-trend, better-for-you products to market without sacrificing clean,
sweet taste said Amy Lauer, Marketing Manager, North America, Tate & Lyle and continued: Consumers are demanding
low-sugar and low-calorie products but taste is always going to be their top priority. With TASTEVA, manufacturers can
meet that demand with a lower cost in use because masking ingredients arent necessary. TASTEVA Stevia Sweetener is a
zero-calorie sweetener that meets consumer demand for low-calorie and low-sugar products and achieves 50 percent or
more sugar-reduction levels. Easy to formulate with, TASTEVA is ideal for a wide range of applications including beverage,
dairy, baked goods, dressings, sauces, frozen foods, processed fruits, processed vegetables, snacks and cereals.

Agro FOOD Industry Hi Tech - vol 25(5) - September October 2014


MU researchers find boron facilitates

stem cell growth and development in corn
Boron deficiency is one of the most widespread causes of
reduced crop yield. Missouri and the eastern half of the
United States are plagued by boron deficient soil and, often,
corn and soybean farmers are required to supplement
their soil with boron; however, little is known about the
ways in which corn plants utilize the essential nutrient. Now,
researchers at the University of Missouri have found that boron
plays an integral role in development and reproduction in
corn plants. Scientists anticipate that understanding how corn
uses the nutrient can help farmers make informed decisions in
boron deficient areas and improve crop yields.
Boron deficiency was already known to cause plants to
stop growing, but our study showed that a lack of boron
actually causes a problem in the meristems, or the stem cells
of the plant, said Paula McSteen, associate professor in the
Division of Biological Sciences and a researcher in the Bond
Life Sciences Center at MU. That was completely unknown
before. Through a series of experiments involving scientists
from several disciplines at MU, we were able to piece
together the puzzle and reach a new conclusion.
Meristems comprise the growing points for each plant, and
every organ in the plant is developed from these specialized
stem cells. Insufficient boron causes these growing points to
disintegrate, affecting corn tassels and kernels adversely. When
tassels are stunted, crop yields are reduced, McSteen said.
The research evaluated a group of plants stunted by its
ability to grow tassels. Kim Phillips, a graduate student in
McSteens lab, mapped the corn plants genome and found

that a genetic mutation stunted tassel growth because it

was unable to transport boron across the plant membranes,
inhibiting further growth in the plants.
Amanda Durbak, a post-doctoral fellow in the College of Arts
and Science at MU, also helped prove borons usefulness to
meristems. She treated two groups of tassel-less corn, one
with a boron fertilizer and the other with only water. The group
that was treated with boron grew normally, while the group
treated with water withered.
Further testing revealed that, at the cellular level, the affected
plants meristems had altered pectin which is strengthened
with boron and stabilizes the plant cell. Without the pectin,
plant meristems disintegrate.
By using various techniques and expertise at MU, including
genomics, translational experiments with frog eggs,
research in the field, cellular testing, and evaluations at
the MU Research Reactor Analytical Chemistry facility and
at MU Plant and Soil Analysis Facility, the study team drew
conclusions that will help corn producers make informed
decisions about raising crops in boron deficient zones,
McSteen said.
Researchers at the University of Georgia and at California
State University, Long Beach also contributed to this study.
The paper, Transport of boron by the tassel-less 1 aquaporin
is critical for vegetative and reproductive development in
maize, was published in The Plant Cell and was funded in
part by the National Science Foundation.

Since 1969 STAUBER has consistently offered the finest quality ingredients to the nutritional, food, pharmaceutical, cosmetic and pet care
industries. The company was founded on the principle of partnering with manufacturers that provide the highest level of product and
service. From their corporate office in Fullerton, California, manufacturing capabilities under their control in New York, warehouses in four
strategic geographic locations and alliances with major freight companies, STAUBER is able to react quickly and deliver customer needs
by being a complete "solution provider". STAUBERs success is due to a passion for innovation. STAUBER is a one-stop, forward-thinking
supplier of a broad spectrum of solutions for the ingredient industry. The ingredients they use lead to breakthrough products for their
customers. Innovation is also reflected in the areas of Quality Assurance, lower minimums, abundant inventory, agility, responsiveness,
in-house proprietary capabilities, and full transparency in how STAUBER does business. STAUBER Performance Ingredients will represent
Jungbunzlauers complete product line and concepts to the nutritional industry throughout the United States. Jungbunzlauer and
STAUBER have been working together for decades and have jointly expanded their presence in the nutritional and dietary market
segments in the US. STAUBERs excellent market knowledge and intimate customer relationships across the US, along with Jungbunzlauers
high quality standards, provide the basis for the continued success. STAUBERs manufacturing capabilities in Florida, NY provides additional
customized and tailor made solutions for our customer base in North America says Peter Luck, Director Sales for Jungbunzlauer. STAUBER
is pleased to strengthen our twenty-plus year relationship with Jungbunzlauer. Both companies are very uniquely aligned in the philosophy
of providing high quality naturally originated ingredients to the ever-growing and dynamic nutritional industry. As innovation and new
product & delivery vehicles arise, and consolidation of much of the industry continues, it is very humbling to be associated with such a fine
company as Jungbunzlauer. Our teams look forward to continuing to work together to serve our end customers more effectively into the
future! adds Dan Stauber, CEO. As consumer demand for healthier and more natural goods continues to increase and producers seek
to find and exploit opportunities to become more efficient in all aspects of their , business, Jungbunzlauer together with its channel partner
STAUBER Performance Ingredients will provide customer oriented solutions Luck said.


Agro FOOD Industry Hi Tech - vol 25(5) - September October 2014