Biomedicine & Preventive Nutrition 4 (2014) 603–605

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Original article

Insignificant viability of the granules of probiotic and prebiotic with

skimmed milk powder
Chayanika Sahni a , Rajinder K. Gupta a , Pratibha Nand b,∗
a
University School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi 110078, India
b
Maharaja Surajmal Institute of Pharmacy, Janakpuri, New Delhi 110058, India

a r t i c l e i n f o a b s t r a c t

Article history: Currently, there is an escalating demand of people for probiotic health-based products. Further, the
Received 20 July 2014 survival of these bacteria in the human gastrointestinal system is questionable. Viability of probiotic in
Accepted 10 August 2014 food and food products is a challenge for the food processing industry. Providing probiotic living cells
with a physical barrier against adverse environmental conditions is therefore an approach currently
Keywords: receiving considerable interest to achieve longer shelf-life of the product. In the present study, skimmed
Probiotics milk powder along with probiotic (Lactobacillus acidophilus) and prebiotic (Fructooligosaccharide) were
Prebiotics
used to make granules by wet granulation method and the viability of the granulated probiotic organism
Skimmed milk powder
Lactobacillus acidophilus
was investigated. Results indicated poor survival of the L. acidophilus after granulation. Physico-chemical
Fructooligosaccharide characteristics of two optimised F1 and F2 formulations were assessed for pH, water holding capacity
Granules and moisture content determination. Major future challenges are also spotlighted.
© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction figure for adequate probiotic consumption, equating to 100 g of a

Probiotics are live microorganisms which when administered in
adequate amounts confer a health benefit on the host, whereas pre-
biotic is a non-viable food component that confers a health benefit
on the host associated with modulation of the microbiota [1,2]. Mix-
ture of pro- and prebiotics is synbiotic which beneficially affects the
host by improving the survival and implementation of live micro-
bial dietary supplements in the gastrointestinal tract by selectively
stimulating the growth and/or by activating the metabolism of
health promoting bacteria and thus improving host welfare. The
therapeutic value of any probiotic food normally depends on the
viability of these bacteria. Analysis of probiotic products in many
different countries has confirmed that probiotic strains exhibit poor
survival [3,4].
International Dairy Federation (IDF) has suggested that a min-
imum of 107 probiotic bacterial cells should be alive at the time
of consumption per gram of the product. The consumption of pro-
biotics at a level of 108 –109 CFU/g per day is a commonly quoted
∗ Corresponding author. Maharaja Surajmal Institute of Pharmacy, Janakpuri, New
Delhi 110058, India. Tel.: +91 8 800 514 668.
E-mail address: pratibha.msip@gmail.com (P. Nand).
food product with 106 –107 CFU/g [5,6].
Lactobacillus is a genus of Gram-positive facultative anaerobic
or micro-aerophilic found in traditional fermented dairy products
[3,4]. Probiotic L. acidophilus is aero-tolerant, rod-shaped bacte-
ria fermenting sugars into lactic acid, and grows readily at rather
low pH values (< pH 5.0) with optimum growth temperature of
around 37 ◦ C [7]. It occurs naturally in the human and animal gas-
trointestinal tract and mouth. Strains of L. acidophilus have been
studied extensively for health effects and it was observed that some
of them may be considered to have probiotic characteristics [8]
whereas others were capable of surviving in gastro-intestinal tran-
sit even at low pH, being resistant to bile and digestive enzymes.
They can also adhere to human epithelial cell lines and human
intestinal mucus [9]. There is adequate scientific evidence to indi-
cate that probiotic preparations, such as tablets, powders, etc. may
contain lower viable counts [10]. Hence, to stabilize and main-
tain viability of probiotics during storage, to protect against harsh
gastrointestinal environment, to protect sensitive ingredients from
its environment (e.g. O2 , H2 O, light) and for delivery of bioactives
into functional food, granulation technique is generally used. In the
present study, skimmed milk powder was used because it is nat-
ural food grade material with excellent natural properties, such as
emulsifier, film former viscosity builder and provides protective
matrix.

http://dx.doi.org/10.1016/j.bionut.2014.08.005
2210-5239/© 2014 Elsevier Masson SAS. All rights reserved.
604 C. Sahni et al. / Biomedicine & Preventive Nutrition 4 (2014) 603–605

2. Materials and methods

2.1. Materials

Skimmed milk powder of Mother Dairy was obtained from Khari

Baoli, Chandni Chowk, Delhi. Probiotics: Lactobacillus acidophilus
and Prebiotic: fructooligosaccharide (Hi Tech Bio Sciences India
Ltd.) were obtained from Space Labs Pvt. Ltd., Kirti Nagar, Delhi.

2.2. Granulation of probiotics and prebiotics

The probiotic, prebiotic and skimmed milk powder were mixed

in a sterilised mortar after optimisation using geometric dilution
method. Starch mucilage (2.5% w/v) was added slowly to the pow-
der mix to make wet coherent mass for F1 and F2 compositions
(Table 1) and was then passed through sieve #20 to get granules
of uniform size. These granules were dried in an oven at 60 ◦ Cfor
5 minutes and stored in an airtight container for further analysis.
Fig. 1. Cell count of F1 granules composition.
2.3. Enumeration of probiotic population in granules (CFU/g)

Water holding capacity (g of water per g of sample) was calcu-

Probiotic count in the granules was enumerated (CFU/g) by
spread plate method on MRS (De Man, Rogosa and Sharpe) medium.
An amount of 1.0 g of granules was suspended in the 10 mL auto-
claved 0.15% peptone water inside the laminar air flow (LAF) bench
(HAIER). The sample was vortexed at high speed followed by prepa-
ration of several dilutions of sample. Then, 0.1 mL of the sample
was plated in triplicate on MRS agar using spreader and incubated
at 37◦ C for 48 h [11]. Plates containing 30–300 colonies were enu-
merated and recorded as colony forming units (CFU) per millilitre or
cell density using formula: (number of colonies counted, rounded
appropriately) × (dilution factor).
2.4. Physico-chemical analysis of the granules
2.4.1. pH
The granules were dissolved in distilled water by vortexing at
high speed to make solution or suspension and pH was measured
using pH meter (Deluxe, MODEL 151-R).
2.4.2. Moisture determination
For moisture determination, pre-weighed glass Petri dish was
taken (W1) and to this added 5 g sample (W2). The Petri dish was
then kept in a hot air oven for 5 h at 105 ◦ C and was then cooled and
(W3) reweighed [12]. Moisture (%) was calculated using formula:
lated using formula:
WHC = (W2 – W1)/W0
where W0: weight of the dry sample; W1: weight of the tube
and dry sample; W2: weight of tube and sediment.
3. Results and discussion
3.1. Cell count
For probiotic effect, the probiotic strains should be present
in sufficiently large number in the final F1 and F2 granules
compositions. Results indicated that initial cell count of the pro-
biotic bacteria L. acidophilus was 20 billion CFU/mL but number
of colonies decreased in F1 and F2 granules composition. Num-
ber of colonies in 10−1 dilution (30 ± 1.1) of F1 composition gave
the cell count as 3 × 104 CFU/mL whereas cell count of F2 com-
position was 8 × 104 CFU/mL as number of colonies showed slight
increase (80 ± 1.2) (Figs. 1 and 2). Sample was plated in triplicate.
The skimmed milk powder (blank) showed no growth as probiotic
bacteria was absent. Wet granulation is one of the most com-
mon methods for granulation in which liquid solution is added to
powders to make a wet mass. Results revealed that only 0.0004%
L. acidophilus could survive after the formation of granules. It clearly

Moisture (%) = (W2 − W3)/(W2 − W1) × 100

2.4.3. Water holding capacity (WHC)

An amount of 1.0 g of sample was added to pre-weighed 15 mL
centrifuge tubes. For each tube, 10 mL distilled water was added
and vortexed for 2 minutes. After the mixture was thoroughly
wetted, samples were allowed to stand at room temperature for
30 minutes and then centrifuged at 3000 g for 20 minutes. The
supernatant was decanted and the centrifuge tube containing sed-
iment was weighed [13].

Table 1
Composition of granules formulations.

S. No. Ingredient F1 F2

1 Lactobacillus acidophilus 0.05% 7%

2 Fructooligosaccharide 2% 3%
3 Skimmed milk powder 90% 90%
4 Starch mucilage (2.5%) qs qs

qs: Quantity sufficient Fig. 2. Cell count of F2 granules composition.

 C. Sahni et al. / Biomedicine & Preventive Nutrition 4 (2014) 603–605
indicated that introduction of probiotics into granular form is
not an easy approach because microorganisms are most often
destroyed during the granulation process due to high thermal,
mechanical and moisture stresses. Hence, viability of probiotic
in food and food products is a challenge for the food processing
industry.
3.2. pH measurement
Viability of encapsulated probiotic cells depends on the physico-
chemical properties of the capsules. pH of F1 and F2 granules
compositions was 6.3 and 6.4, respectively, near to neutral pH. The
pH, however, did not affect much the viability of the probiotic cells.
3.3. Moisture content
Moisture (%) of the F1 and F2 granules compositions was 5.8%
and 5.65%, respectively. Although after the formation of granules,
they were dried in an oven at 60 ◦ C for 5 minutes. Minimum mois-
ture should be there in such food products as they might affect the
shelf-life of the product.
3.4. Water holding capacity (WHC)
Water holding capacity is the ability of the food to hold its own
or added water during the application of forces, pressing, centrifu-
gation, or heating. It is the ability to retain water against gravity and
plays a major role in formation of food texture. During ambient stor-
age condition of the granules, any change in probiotic viability and
in biochemical characteristics significantly affect quality as well as
shelf-life. Therefore, minimum moisture and water activity should
be present to enhance the shelf-life of the product. Results indi-
cated that the WHC of F1 and F2 granules composition was 0.565 g
of water/g of sample and 0.583 g of water/g of sample, respectively.
Water activity values (less than 0.3) is good for powder stability
since it represents less free water available for biochemical reac-
tions to occur and provides longer shelf-life of the composition
[14].
4. Conclusions
Probiotic therapy or microbial intervention is based on the con-
cept of healthy gut microflora and nowadays used increasingly
to treat patients with medical problems. Probiotics are generally
605
considered to be safe for people with weakened immune systems
having gastric problem, and diarrhoea. Though probiotics do not
offer a panacea for all the problems, they do offer properties, which
cannot be achieved by other routes and should be in the reper-
toire of those charged with the development of new and better
products. There is limited scientific evidence for compositions com-
prising of probiotics and prebiotics in stable granule forms. Results
of the present study clearly indicated poor viability of probiotics in
both the granular compositions, which could be due to thermal and
mechanical stress. Hence, an important goal for the future should
be to formulate and encapsulate, carefully, the probiotics to prevent
mortality and maintain good granulation operating conditions for
development of stable product.
Disclosure of interest
The authors declare that they have no conflicts of interest con-
cerning this article.
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