Development and characterization of a probiotic functional beverage obtained by

fermentation from kefir with fruit and vegetable juice rich in bioactive compounds.

Development and characterization of a probiotic functional beverage obtained by

fermentation of a vegetable juice rich in bioactive compounds with kefir

A new Kefir fermented beverage from fruit and vegetable juice: development and
characterization of bioactive compounds

A new fuctional drink obtained by fermentation of a fruit and vegetable juice with kefir:
development and characterization of bioactive compounds.

Jorge Paredes, María L Escudero, Isabel M Vicario.

Abstract

A juice containing Aa mixture of extracts fruits (apple (70%) and strawberry (9%)), and vegetables
(carrot (12%) and beet (9%)) was) was evaluated as a potential substrate for production of a novel
probiotic beverage by using kefirkefir. Effects of the kefir grain amount (1- 4%, w/v) and
fermentation time (12, 24 and 48 hours) on the beverage properties were investigated. evaluating t
The organoleptic, physical-chemical and colorimetric properties were measured. The results
indicated that the amount of kefir grains have a significant effect in the variation of organic acids
content (lactic, acetic, citric, succinic and malic acid), the Cchromatic parameters (Claritylightness
(L *), Tonehue (h ab) and Chroma (C∗¿ ab ¿,)), CO2 production, acidity and viscosity. On the other
hand, fermentation time significantly affects all the parameters analyzed in the samples. The
evaluation of the organoleptic properties allowed determination that tThe most suitable conditions to
achieve the highest overall acceptability for thea fermented beverage based on mix of fruits and
vegetables juice iswas: 2% (w/v) kefir inoculumkefir inoculum at a time of 24 hours.

Keywords: kefir, kefir granules, antioxidant, probiotic, lactic acid bacteria, yeast.

Introduction

Currently the consumers´ interest in incorporating functional foods in their diet haveinterest in
incorporating functional foods in their diet has motivatedboosted a greaterthe development of the
“healthy food products” industry. Among these products are probiotics which exert a beneficial
effect on the intestinal functions of the host after consumption, and may be able to prevent various
several diseases due to ; this effect is attributed to the living microorganisms (Reig y Anesto, 2008).
Kefir is an ancient food to which beneficial properties for health have been attributed such as
reduction of symptoms of lactose intolerance, stimulation of the immune system, lowering of
cholesterol, anti-mutagenic and anti-carcinogenic properties (Guzel-Seydim et al. .,2006) also in
the Caucasus it is associated with longevity (Cevikbas et al., 1994). Kefir is characterized by
presenting a mass composed of proteins, lipids and a soluble polysaccharide called kefiran
where its microbiota is found. This symbiotic medium of microorganisms belongs to
different spectra of lactic acid bacteria, yeasts and acetic bacteria. , tThey are sugar-
fermenting ferment sugar substrates and microorganism gives it the possibility of so they can
growing both in a dairy medium or in a non-dairy medium with sugar substrates. Microbial
metabolism produces lactic acid, alcohol, CO2, B-complex vitamins and other organic acids.
Kefir has become a very important functional product, and consequently its research has
increased in the last decade (Stadie et al., 2013).
Among the foods rich in sugars that can be a fermentative substrate offor kefir are the juices of
fruits and vegetables. Many studies have linked their use to protection against several chronic
diseases associated with aging, cancer, cardiovascular diseases, cataracts, and immune
dysfunction. (Baines y Seal, 2012). @@@ These natural protective effects have been attributed to
the antioxidant potential of several components, such as carotenoids, lycopenes, betalains,
vitamins, polyphenols and other phytochemicals (Baines and Seal, 2012). Some of these
compounds, including betaines or carotenoids, have health claims authorized under Article 13 (1) of
Regulation (EC) No 1924/2006 and the scientific opinion on the justification of health claims . of the
European Food Safety Authority (EFSA). For example, Bbetaine which can be found in beetroot
"contributes to the normal metabolism of homocysteine" (Regulation (EU) 432/2012). Beta-
carotene is related to the protection of the skin against UV-induced damage (including
photo-oxidative) (ID 178, 197, 1263, 1461, 1968, 2320) and maintenance of normal function of
the immune system (ID 200, 1462).

Vegetable fFermentsed vegetables and fruit juices containing probiotics are a new and
promising approach for therapeutic products foods (Mattila-Sandholm et al., 2002). Fruits and
vegetables are a rich sources of nutrients such as that contain minerals, vitamins, dietary
fibers and antioxidants that could become which iare ideal substrates for the growth of
probiotics (Yoon, Woodams and Hang, 2004). Currently, the development of functional
beverages based on fruit juices with probiotics has increased because of they provide a
health benefits beyond traditional nutrients and showing them and have a good acceptability ofby
consumers of all ages (Sheehan, Ross and Fitzgerald, 2007).

Although the The best substrates for the administration of probiotics are dairy products,..
However, milk can generate several food hypersensitivities, these that can be classified as
allergies or intolerance frecuently occur. It is estimated that in Europe there is an average
prevalence of milk protein allergy to milk protein in children between 6 and 8%, in children while
in adults is 2% (Berciano and Caro, 2008). In addition, one of the main components of milk,
lactose, is one of the main causes of intolerances to milk, and affects one third of the world
population. On the other hand, it is estimated that there are 600 million vegetarian people in
the world. Unlike vegetarianism, veganism is a subject that does not have much information,
it is estimated at 2% of the population of the US. UU, 4% of the Swedish population and 1%
in Germany are vegans. The vegetarian and vegan product industries are growing around
10% per year (Choudhary y Jadoun, 2014).

These are some of the reasons why fermented non-dairy beverages have begun to play an
important role in the diet of consumers who are hypersensitive to milk proteins (Berciano
and Caro, 2008), lactose intolerant (Gregorio et al., 2013), vegetarians and vegans
(Choudhary and Jadoun, 2014).

The aim of this work was to elaborate, characterize and analyze the acceptance of a kefir
drink made from a commercial fruit and vegetable juice (Veggie) selected among products
which contain bioactive compounds with an authorized health declaration.

Materials and Methods

Kefir grains and inoculum preparation

Kefir grains were obtainedKefir grains were obtained locally from a donor from Sevilla families that
traditionally consumed kefir. TThe samples of Kefir were preserved in sterilized milk. To
preserve the kefir grains the substrate was renewed daily for a period of two months. The
grains were then washed with sterile distilled water and subsequently used to inoculate on
the commercial fruit and vegetable juice selected from the Veggie brand (Lot 120738) which
is composed of apple extract (70%), carrot (12%), beet (9%), and strawberry (9%).

Sampling
Each sample (commercial Veggi juices inoculated with Kefir under different conditions Table 1)
was analyzed in triplicate using the raw commercial juice without fermentation as reference
sample. The different tests were organized chronologically with fermentation times of 12, 24
and 48 hours each one with the 4 types of inoculations of kefir (1%, 2%, 3% and 4% w / v).
After the fermentation the samples were takenwere taken to evaluate theevaluate the physico-
chemical parameters. A part of the samples were kept froze for the measurement of
antioxidant activity, total phenols, organic acid (acetic, lactic, succinic, malic and citric acid).
To reduce possible changes in the organoleptic properties, the sensory evaluation samples
were kept under refrigeration at 4 ° C and in modified atmosphere (addition of nitrogen).

Physical chemical determinations

Physico-chemical analyses of pH, total titratable acidity (TTA) and soluble solid content
(SSC) were performed according to the methodology proposed by the AOAC (2000).
Viscosity was determined according to ASTM D 445 and 2515 standards. The density was
determined in a 25 ml volumetric flask. The 25 ml sample is weighed and the weight is
divided by volume of the sample, expressed as g / ml.
The content of acids lactic, acetic, citric, malic and succinic acids were detected ermined by
apply the HPLC method (reference).
Carbon dioxide was indirectly estimated by measuring the weight loss before and after the
fermentations and expressed as g/ 100 mL (Marenghi et al., 2004).
Total phenolic compounds (TPs) were analysed according to the method of Folin Ciocalteu
(Singleton, Rossi Jr. and Rossi J A Jr., 1965) with some modifications.
The antioxidant activity was analysed according to ABTS method and was determined as
Trolox equivalent antioxidant activity (TEAC: Trolox-equivalent antioxidant capacity, mols of
Trolox with the same antioxidant activity as 100 g of dry sample).
Colours of juices wereas measured, before and after fermentation, with an
spectrophotometer (CM-5 KONICA MINOLTA OPTICS INC) recording CIElab chromaticity
coordinates (L*,C ab*, h ab).

Sensory Evaluation

The fermented beverages were subjected to sensory evaluation following the UNE 87 023 standard.
The samples were kept at 10 ° C and 10 ml aliquots were served, in random order, in clear cups
(volume of 30 ml) covered with Petri dishes and marked with three-digit random numbers.

The fermented beverages were subjected to sensory evaluation following the UNE 87 023 standard.
The samples were kept at 10 ° C and 10 ml aliquots were served, in random order, in clear cups
(volume of 30 ml) covered with Petri dishes and marked with three-digit random numbers.

Two different sensory evaluation sessions were carried out. In the first stage The sensory test was
performed with fifteen untrained tasters (9 women and 6 men, between 23 and 55 years old)
ranked the 12 samples according to sweetness, acidity and alcohol. Samples were presented in
random order in 7 different sessions.

, the samples were kept at 10 ° C and 10 ml aliquots were served, in random order, in clear cups
(volume of 30 ml) covered with Petri dishes and marked with three-digit random numbers . To
determine the best perception from consumers about attributes of beverage, two sensory
evaluationstests were carried out:

The first sensory test evaluation was is carried out with the objective toof selecting 3 products
with the best acceptance of by thefrom the 12 possible onesconsumers. In the development of
the sensory test, 7 analyzes are performed, in each one a preference test is carried out and
an ordination test in attributes such as acidity, texture and alcohol perception. sweetness, acidity
and alcohol. For this, the effect of the fermentation time and percentage of inoculation of kefir
on the juice was taken into account.

The second sensory evaluation is carried out with the objetive to select the product with the
best acceptance by the consumers. For this, The sensory test was performed with thirty
untrained tasters (18 women and 12 men, between 21 and 54 years old), the samples were
kept with the same conditions before descrited.

In this sensory evaluation, it were done two tests, one hedonic test and other ordination test. The
hedonic test was performed using 7 point scale to determine if there are differences between
products in judges evaluation (Drake, 2007). Additionally, an ordination and preference test was
carried out on attributes such as arome, texture and color.2 tests were done, one hedonic and other
ordination tests. The hedonic test was performed using a 7 point scale to determine if there are
differences between products in judges acceptance (Drake, 2007). Additionally, an ordination and
preference test was carried out on attributes such as arome, texture and color.

Fig 1. Procedure Sensory evaluation of kefir drink based on fruits and vegetables
Abbreviations: K1HV: kefir at 1%, 24 hours fermentation, K2HV: kefir at 2%, 24 hours fermentation, K3HV: kefir at 3%, 24
hours fermentation.

Experimentation and analysis

All experiments were replicated in three samples done in triplicate and the data are presented as
the mean and standard error deviation of three independentthree independent experiments.
ANOVA method (P<0.05) was used to determine significative differencessignificative differences
among the differents variables in each experiment done. Statistical data wasdata were
analized with statistic program Excel 2010, It was taken into account fermentation time (12h,
24h and 48h) and kefirand kefir inoculum concentration (1%, 2%, 3% and 4% w/v).

The significative differences among judges perception on attributes as: sweetness, acidity,
alcohol, arome, texture and color were analyzed by the Friedman (P<0,05) method.

Results and Discussion

The results of the Pphysico -chemical determinations are shown in Table 1.

Solubles Total Solids Content (° Brix)

The rate of metabolism of fermentable sugars increases with increasing fermentation time,
decreasing the content of soluble solids (° Brix). The highest decrease in °Brix value was
observed in the beverage fermented with 1% w/v kefir grain in 48 hours of fermentation with
a decreased (from 10,91± 0.21 to 3,62 ± 0.04 (7,29 °Brix) from its initial value of 10,91± 0.21) and
the. The lowest reduction of total soluble solids was obtained in the samples of 12 hours of
fermentationfermented during 12 h, , reducing between 0.64 and 1.12 ° Brix (10.27 ± 0.13 and 9.79
± 0.11)..

Corona et al. (2016) reported reductions of that the highest reduction of sugars from happened in
melon (6.22 ° Brix), in melon, 4.77 ° Brix in carrot (4.77 ° Brix) and 3.48 °Brix in strawberry (3.48
°Brix) juices submitted to kefir fermentation???. Similar results were reported by While in the
results of Randazzo et al. (2016): highlights reductions from 6.46 ° Brix in the grape juices (6.46 °
Brix), 6.36 ° Brix in pomegranate to 5.8 ° Brix.(6.36 ° Brix) and in quince. (5.8 ° Brix). The above
results show an evident reduction in the content of total soluble solids in the non-dairy
beverages. This can be attributed to the fact that kefir microorganisms use these sugars as a
substrate for their survival.

Production of Carbon Dioxide

The production of carbon dioxide (CO2) is directly related to the fermentation time, besides it
shows significativent differences between the samples of among 12, 24 and 48 hours of
fermentation samples. The beverage fermented with 4% w/v of kefir grains in a fermentation
time of 48 hours, it hadshowed the highest CO2 production with a value of 4.31 g of CO 2 / 100
ml. This behavior may being be?? Or isis related with the decrease of soluble total solids
content in the fermentation time.

In a comparison with the results obtained by Corona et al. (2016) and Randazzo et al. (2016)
on similar condition of fermentatiton conditions, the melon and pomegranate juices produced
thea greaterst amount of CO2 (3.39 g CO2/100 ml and 3.21 g CO2/100 ml respectively). This
result is an important property for the final product because CO2 is athe major derivative of the
fermentation product which contributes to of the yeast and provides the product with desirable
exotic notes and yeast flavor (Guzel-Seydim, Seydim and Greene, 2000).
pH and Titulable Total Acidity (TTA)

The results indicate that there are significant differences (p <0.05) in the variation of pH and ATT
between among samples with different at 12, 24 and 48 hours of fermentation time. The highest
increase in ATT and decrease in pH value were observed after 48 hours of fermentation with 3%
w/v of kefir grains with 6,95 g ac. malic/l and 3,4 of pH. Similar results were obtained byf Corona et
al. (2016) for fig juice
In the results were observed that the fermentation time is directly proportional to decrease in pH
and the increase in ATT. Therefore, when fermentation time increases, the pH decreases. Tthe
production of organic acids during fermentation which reduces the pH value of fermented foods and
increases total titratable acidity (Puerari, Magalhaytild, Guedes and Schwan, 2015). According to
Anton et al. (2016), Tthe low pH values prevent the growth of theof the most waste and pathogenic
organisms, also create a suitable environment for the growth of yeasts and probiotic lactic acid
bacteria.
The highest increase in ATT and decrease pH value were observed during 48 hours of fermentation
with 3% w/v of kefir grains with 6,95 g ac. malic/l and 3,4 of pH. The results of Corona et al. (2016)
from whom his research was previously described, only the fig juice showed the same behavior
obtained in this research.
Organics Acid

Lactic Acid

It is observed that as the fermentation time and the content of kefir granules increase, the
concentration of lactic acid increases in the comercial juice substrate.. Therefore, in a fermentation
time of 48 hours and 4% w/v of kefir granules, the lactic acid hasreached the highest concentration
with a value of 3250.5 mg / L.

The production of lactic acid during fermentation is attributed to the metabolism of lactic acid
bacteria. that a According to Puerari, Magalha, Guedes and Schwan (2015), a high final value of
lactic acid is the a result of homofermentative metabolism. Consequently Magalhães, de Pereira,
Dias, and Schwan (2010) reported that the production of lactic acid during fermentation of kefir is of
great importance due to its inhibitory effect on pathogenic microorganisms. Consequently
Magalhães, de Pereira, Dias, and Schwan (2010) reported that the production of lactic acid during
fermentation of kéfir i and it is of great importance due to the inhibitory effect on pathogenic
microorganisms. (Magalhães, de Pereira, Dias, and Schwan (2010

Acetic Acid

The first traces of acetic acid were evidentdetected after at 12 hours of fermentation, in samples
atwith 3% kefir w/v (62.1 mg acetic acid / L). In samples larger than Above concentrations of 2% w/v
of kefir, the production of acetic acid was directly proportional to the fermentation time. Therefore,
the maximum concentration of acetic acid was presenteddetected in kefir at 4% w / v, afterin 48
hours of fermentation (, with a value of 266.8 mg / L.) Bellow In samples of 1% w/v of kefir
concentration, acetic acid production iwass not observed during the 48 hours of fermentation,
detected in accordance with this behavior is observed in the results of Bensmira and Jiang (2011)
and Magalhães et al. (2010) who verifiedreported that the average concentration of acetic acid was
practically zero during the first 18 and to 24 hours of fermentation of peanut milk and whey.in kefir
on the basi.s of peanut milk and whey.

According to Magalhães et al. (2010) the heterofermentative route used by the lactic acid and acetic
acid bacteria present in the kefir microflora leads to an increase in the amount of acetic acid in
fermented beverages and in smaller proportion succinic, formic acid and carbon dioxide, among
others. In this case, it is likely that the preferred metabolic route of fermentation of the commercial
product is homofermentative.
Citric Acid

The citric acid decreases its concentration as the concentration of kefir granules increases and the
effect of the fermentation time followed this trend only in 1 and 4% w/v kefir.The sample at 4% w/v
of kefir during the first 48 hours of fermentation decreased the concentration of citric acid to a value
of 532.8 mg / L, representing the lowest registered value. Similarly, Sabokbar, Moosavi-Nasab and
Khodaiyan (2015) reported that level of acid citric decrease during fermentation apple juice and
whey with kefir .Similarly, Sabokbar, Moosavi-Nasab and Khodaiyan (2015) in aan study of apple
juice based on fermented whey with kefir grains reported that the levels of citric acid decreased
from 5.8 ± 0.22 to 1 ± 0.15 g / L in a fermentation time of 48 hours and a variation of kefir between
2% and 8% w / v. Bensmira and Jiang (2011) showed that the level of citric acid decreases due to
some lactic acid bacteria that prefer citric acid as a substrate for the production of acetoin and
diacetyl.

Succinic Acid

The variation of succinic acid during fermentation shows an oscillating tendency where, with the
exception of 2% w/v kefir, succinic acid increases its concentration between 12 and 48 hours of
fermentation. The values of succinic acid range between 264 and 380 mg / L, in which the kefir at
1% w/v in 48 hours of fermentation has the highest concentration. In accordance with the
provisions of Magalhães et al. (2010), the lactic acid and acetic acid bacteria present in
the kefir microflora can produce succinic acid in less than acetic amounts via the heterofermentative
route.

Malic Acid

According to the results obtained, the malic acid concentration tends to increase as the
fermentation time increases. However, when increasing the concentration of kefir, there is no clear
tendency in the variation of malic acid. Therefore, after 48 hours of fermentation the highest malic
acid concentrations was obtained, (2244 mg / l) for the sample fermented with kefir at 2% w / v.

Sabokbar, Moosavi-Nasab and Khodaiyan (2015), reported a reduction in malic acid concentration
in a a mixture of apple juice and whey fermented with kefir. However, Laureys et al. (2018) in his
research on the influence of oxygen and various nutrients in the fermentation process in water kefir,
found that the source of nutrients has an immediate impact on the substrate and the production of
metabolites during the fermentation process with water- kefir. this impact is made even more
pronounced probably due to the change in microbial communities.
Therefore, it could be assumed that the increase in malic acid obsecved could be due to the
presence of Saccharomyces cerevisiae in the microbiota of the selected kefir since SC is one of the
microorganism capable of producing malic acid (Chi, Wang, Wang, Khan and Chi, 2016).

The reduction of pH and increase of ATT is related to the production of organic acids during
fermentation which reduces the pH value of fermented foods and increases total titratable acidity
(Puerari, Magalhaytild, Guedes and Schwan, 2015). According to Anton et al. (2016), The low pH
values prevent the growth of the most waste and pathogenic organisms, also create a suitable
environment for the growth of yeasts and probiotic lactic acid bacteria.

Absolute viscosity

The results shown in the table 1 indicate that the viscosity increased only in the first 12
hours of fermentation., aAfter 12 hours of fermentation, the viscosity tends to decrease as
the fermentation time increases. Consequently, the highest viscosity value was observed in
the sample at 1% w/v of kefir grains during the first 12 hours of fermentation with a value of
3.47 CP. On the other hand, the variation of content of kefir grain also havead a significativent
effect in the viscosity.

The increase in viscosity in the first 12 hours of fermentation is related to the effect of
lactobacilli which improve the consistency of kefir during the fermentation process and reform the
internal structure of beverages after fermentation, improving the consistency of kefir and this
causesing greater resistance of the inner layer of the drink and consequently produce a
higher viscosity (Irigoyen, Arana, Castiella, Torre and Ibáñez, 2005). However, Degeest,
Mozzi, and De Vuyst (2002) stated the decrease in viscosity can be explained that
glycohydrolasesby the may hydrolysis of exopolysaccharide (EPS) material in their monomers
by glycohydrolasesexplaining the decrease in viscosity detected afterwards. These g
Glycohydrolases are capable of decreasing the viscosity of the polymers produced by
Lactobacillus rhamnosus, as well as of releasing some reducing sugars (Pham, Dupont,
Roy, Lapointe and Cerning, 2000).

Density

The results obtained show that the density of the juices decreases as the fermentation time
increases, this decrease being significant (p <0.05) in some cases, except between the
samples between 12 and 24 hours of fermentation. These results may be related to the
metabolization of fermented sugars which are metabolized into lactic acid, acetic acid,
alcohol and carbon dioxide, and other compounds which may cause changing in the density
of the beverage.

Total phenols (TP)

All samples maintained over or close at initial TP value (1051.97± 14.47) during the 48 hours of
fermentation except the sample at 4% w/v of kéfir grains tando 48 hours of fermentation which
reduced significatly the TP concentration (853.39 mg / l) . The sample at 2% w/v of kefir grains
in 24 hours of fermentation reached the highest concentration of TP (1381 mg / L). Randazzo
et al. (2016) reported a decrease in total phenols in kiwi, pomegranate, strawberry, apple,
grape and quince juices in similar fermentation conditionas as in of fermentation with this
research (4% w/v kéfir, 48 hours of fermentation). The pomegranate juice showned the
greatest decrease in FTP. This result is in accordance with On the other hand, C Corona et al.
(2016) have similar results with this investigation since who indicated that carrot and tomato
juices showed a slight increase in TP FT.Mccue and Shetty (2005) affirm that the decrease of
the phenolicin TP content could be the result of the degradation of phenolic structures as
possible mechanisms of antimicrobial detoxification of yeasts and bacteria.

Antioxidant activity (AA)

The effect of tThe fermentation time had a significant effect on the AA values of the samples was
significant (p <0.05), except in the case of samples between corresponding to 12 and 24 hours of
fermentation. The results reported in the Table 1 show a decrease between 28% (490,69 ± 5,06)
and 37% (427,87 ± 7,53) duringafter 48 hours of fermentation on the AA compared to the reference
sample (684,33 ± 7,51). The information reported by Randazzo et al. (2016) reported similar
results, agree with the results obtained in this research, they observed a with a greater decrease of
AA in pomegranate, grape, apple, kiwi and fig juice, only the quince juice showed a slight increase
in AA. According to Mccue and Shetty (2005) there is an invesrse relation bewteen the data trend
for the total phenolic content and the antioxidant activity of the kefir fermented beverage with kefir
seem to be reverse, as observed this type of trend is observed in the in the present investigation in
the first 12 hours of fermentation. Mccue and Shetty (2005) affirm that the decrease of the phenolic
content could be the result of the degradation of phenolic structures as possible mechanisms of
antimicrobial detoxification of yeasts and bacteria.

Color Analysis

The results obtained show that the fermentation time has a significant effect on the Color
parameters of C∗¿ ab ¿, L * and h ab, were significantly affected by the ferementation time, while
the variation of the variation of the concentration of kefir grains during the fermentation only
hasd a significant effect in the first 12 hours of fermentation for the parameters of L * and
C∗¿ ab ¿ and for h ab., only a significant effect is observed after 48 hours of fermentation.

Randazzo et al. (2016) state that the reduction in clarity and the increase in red shades in
drinks with kefir could be explained by the browning processes that occur during
fermentation. This phenomenon is due to the activation of certain oxidases, such as
polyphenol oxidase, when the environments are not completely anaerobic (Corona et al.,
2016). The above confirms the variation of color observed by the effect of the fermentation in
the samples, in Clarity (L *) as well as in Chroma (C∗¿ ab ¿) and Tone (h ab).

Organics Acid

Lactic Acid

It is observed that as the fermentation time and the content of kefir granules increase, the
concentration of lactic acid increases in the comercial juice substrate. Therefore, in a fermentation
time of 48 hours and 4% w/v of kefir granules, the lactic acid has the highest concentration with a
value of 3250.5 mg / L.

The production of lactic acid during fermentation is attributed to the metabolism of lactic acid
bacteria that according to Puerari, Magalha, Guedes and Schwan (2015), a high final value of lactic
acid is a result of homofermentative metabolism. Consequently Magalhães, de Pereira, Dias, and
Schwan (2010) reported that the production of lactic acid during fermentation of kefir is of great
importance due to its inhibitory effect on pathogenic microorganisms. Consequently Magalhães, de
Pereira, Dias, and Schwan (2010) reported that the production of lactic acid during fermentation of
kéfir is of great importance due inhibitory effect on pathogenic microorganisms.

Acetic Acid

The first traces of acetic acid were evident at 12 hours of fermentation, in samples at 3% kefir w/v
(62.1 mg acetic acid / L). In samples larger than 2% w/v of kefir, the production of acetic acid was
directly proportional to the fermentation time. Therefore, the maximum concentration of acetic acid
was presented in kefir at 4% w / v, in 48 hours of fermentation, with a value of 266.8 mg / L. In
samples of 1% w/v kefir, acetic acid production is not observed during the 48 hours of fermentation,
this behavior is observed in the results of Bensmira and Jiang (2011) and Magalhães et al. (2010)
who verified that the average concentration of acetic acid was practically zero during the first 18 and
24 hours of fermentation in kefir on the basis of peanut milk and whey.

According to Magalhães et al. (2010) the heterofermentative route used by the lactic acid and acetic
acid bacteria present in the kefir microflora leads to an increase in the amount of acetic acid in
fermented beverages and in smaller proportion succinic, formic acid and carbon dioxide, among
others. In this case, it is likely that the preferred metabolic route of fermentation of the commercial
product is homofermentative.
Table 1
Physico-chemical analysis of fermented commercial juice with different concentrations of inoculums of kefir and fermentation times
Mean values of three measurements for each replicate
Abbreviations: SSC, soluble solid content; CO2, carbon dioxide; TTA, total titratable acidity; TP, total phenol (gallic acid equivalent mg/L); ABTS,
Trolox equivalent antioxidant activity (TEAC: Trolox-equivalent antioxidant capacity, umoles de Trolox/l); L*, lightness; h ab, hue;C ab*,chroma; n.d., not
detectable. SD, Significant differences the numbers represent significant differences between samples with different concentration fermentation inoculum of
kefir times and letters represent significant differentces between samples with different fermentation times concentration inoculum of kefir for each sample and
each physico-chemical determination: p< 0.05. Data are means ± standard deviation.
Citric Acid

The citric acid decreases its concentration as the concentration of kefir granules increases, the
effect of the fermentation time continues this trend only in 1 and 4% w/v kefir.The sample at 4% w/v
of kefir during the first 48 hours of fermentation decreased the concentration of citric acid to a value
of 532.8 mg / L, representing the lowest registered value. Sabokbar, Moosavi-Nasab and
Khodaiyan (2015) in their work on the preparation and characterization of apple juice based on
fermented whey with kefir grains report that the levels of citric acid decreased from 5.8 ± 0.22 to 1 ±
0.15 g / L in a fermentation time of 48 hours and a variation of kefir between 2% and 8% w / v.
Bensmira and Jiang (2011) showed that the level of citric acid decreases due to some lactic acid
bacteria that prefer citric acid as a substrate for the production of acetoin and diacetyl.

Succinic Acid

The variation of succinic acid during fermentation shows an oscillating tendency where, with the
exception of 2% w/v kefir, succinic acid increases its concentration between 12 and 48 hours of
fermentation. The values of succinic acid range between 264 and 380 mg / L, in which the kefir at
1% w/v in 48 hours of fermentation has the highest concentration. In accordance with the
provisions of Magalhães et al. (2010), the lactic acid and acetic acid bacteria present in
the kefir microflora can produce succinic acid in less than acetic amounts via the heterofermentative
route.

Malic Acid

According to the results obtained, the malic acid concentration tends to increase as the
fermentation time increases. However, when increasing the concentration of kefir, there is no clear
tendency in the variation of malic acid. Therefore, after 48 hours of fermentation the highest malic
acid concentrations are obtained, being 2244 mg / l the most value high registered in kefir at 2% w /
v.

The variation of the malic acid content presents a different behavior to the results obtained in the
investigation of Sabokbar, Moosavi-Nasab and Khodaiyan (2015), where the malic acid of a mixture
of apple juice and whey is reduced after a fermentation process. However, Laureys et al. (2018) in
his research on the influence of oxygen and various nutrients in the fermentation process in water
kefir, found that the source of nutrients has an immediate impact on the substrate and the
production of metabolites during the fermentation process of kefir. water, and this impact is made
even more pronounced probably due to the change in microbial communities. Therefore, it is
possible that the results of this work is related to the presence of Saccharomyces cerevisiae in the
microbiota of the selected kefir, which is one of the eukaryotic microorganisms capable of producing
malic acid (Chi, Wang, Wang, Khan and Chi, 2016).

Sensory analysis

The sensory analysis is carried out in a tasting room with a space of 10 stalls, sufficient
lighting, very well ventilated, isolated from noise, odors and free of residues.

First Sensory Test

An Ordination Test and a Preference Test were carried out on the total of the samples in
order to select 3 products with the best acceptance. For this, it was done a preference and
an ordination test, where were tested attributes such as: sweety, acidity and alcohol
perception, this allowed to analize the attributes affected by the fermentation time and the
variation of concentrations kefir.An Ordination Test and a Preference Test were carried out
on the total of the samples in order to select 3 products with the best acceptance in their
attributes of 12 possible ones. In the development of the sensory test 7 analyzes are
carried out, each one a preference and an ordination test are carried outFor the last
evaluation, it is tested attributes such as: sweet, acidity and alcohol this allowed to analize
the perception of attributes affected by the fermentation time and the different
concentrations of kefir.

The results shown in Table 2 indicate that regardless of the concentration of kefir inoculuma,
judges do not perceive sweetness in the samples with fermentation times of 48 hours. While,
the judges have greater perception of sweetness in samples of 12 and 24 hours of
fermentation. The sample with a fermentation time of 24 hours and a concentration of kefir at
2% presented the highest score in terms of sweetness perception.
Table 2.

Ordenation Test Results in attributes as sweet, acidity and alcohol.

Abbreviations: K1HD: kefir at 1%, 12 hours fermentation, K2HD: kefir at 2%, 12 hours fermentation, K3HD: kefir at
3%, 12 hours fermentation, K4HD: kefir at 4%, 12 hours fermentation, K1HV: kefir at 1%, 24 hours fermentation,
K2HV: kefir at 2%, 24 hours fermentation, K3HV: kefir at 3%, 24 hours fermentation, K4HV: kefir at 4%, 24 hours
fermentation, K1HC: kefir at 1%, 48 hours fermentation, K2HC: kefir at 2%, 48 hours fermentation, K3HC: kefir at 3%,
48 hours fermentation, K4HC: kefir at 4%, 48 hours fermentation.

The results obtained about ordination test indicate that regardless of the concentration of
kefir inoculaums, judges do not perceive sweetness in the samples with fermentation times
of 48 hours. However, they have greater perception of sweetness in samples of 12 and 24
hours of fermentation. The sample with a fermentation time of 24 hours and a concentration
of kefir at 2% presented the highest score in terms of sweetness perception.

Only significant differences were observed (p <0.05) in the perception of acidity (Table 2), in
samples between 12, 24 and 48 hours of fermentation at 2% w/v kéfir. The results of the
ordination test related to the perception of the acidity of the product allow to determine that
the judges perceive greater acidity in the samples with 48 hours of fermentation. This result
is related to the production of organic acids, since in this time of fermentation the highest
concentration occurred.

Significant differences were found (p <0.05) in the samples with kefir inoculated at 2% w/v
between fermentation times 12, 24 and 48 hours (Table 2). The samples with a fermentation
time of 48 hours independent of the concentration of kefir have the highest score in alcohol
perception. On the other hand, the sample with the lowest perception of alcohol by the
tasters was the sample at 2% kefir for 24 hours of fermentation.

Figure 1
Preference Test Result

Product Preference
20

16
Preference Notes

12

0
K4HD K2HD K1HD K3HD K1HV K4HV K3HV K2HV K2HC K4HC K1HC K3HC
Samples

Fig 2. Preference Test Result

Abbreviations: K1HD: kefir at 1%, 12 hours fermentation, K2HD: kefir at 2%, 12 hours fermentation, K3HD: kefir at 3%, 12
hours fermentation, K4HD: kefir at 4%, 12 hours fermentation, K1HV: kefir at 1%, 24 hours fermentation, K2HV: kefir at 2%,
24 hours fermentation, K3HV: kefir at 3%, 24 hours fermentation, K4HV: kefir at 4%, 24 hours fermentation, K1HC: kefir at
1%, 48 hours fermentation, K2HC: kefir at 2%, 48 hours fermentation, K3HC: kefir at 3%, 48 hours fermentation, K4HC: kefir
at 4%, 48 hours fermentation.

The result of preference test (Figure 1) indicate that the tasters have a greater preference for
the samples that have been fermented for 24 hours at 1%, 2% and 3% w/v kefir. Therefore,
these results show that the judges has preferences for a product with an acidity between
4.78 and 5.64 g / L of Malic Acid, pH between 3.65 and 3.7, refractive index between 8.69 and
9.93 ° Brix. In addition, the judges prefered a product with low perception of alcohol.

Second Sensory Test

The second sensory test is begining with samples at 1%, 2% and 3% w/v and 24 hours of
fermentation.

The arome perception (Table 3) of the samples by judges shows a clear preference for the
sample at 1 and 2 % kefir. Regarding the color attribute, there are significant differences (p
<0.05) between 1% and 3% w/v kefir samples, and between the reference sample and the 3%
w/v kefir sample.
Tablea 3.
Ordenation Test Results in attributes as arome, texture and color

Experiment ba F FCRITICAL FCRITICAL

Attributes Samples* N° Judges
%Inoculum Time baa (Friedman) (α=0,05) (α=0,01)
1% AROME [K1HV] aba
a [K2HV] be [K3HV] de [REF] bd 30 9,66 7,81 11,34
2% 24 TEXTURE [K1HV]abdab [K2HV] a [K3HV] bc [REF] cd 30 8,69 7,81 11,34
3% COLOR [K1HV]ac b
aa
[K2HV] abd [K3HV] b [REF] cd 30 5,23 7,81 11,34
REF ab
Abbreviations: K1HV: kefir at 1%, 24 hours fermentation, K2HV: kefir at 2%, 24 hours fermentation, K3HV: kefir at
3%, 24 hours fermentation, RE: reference sample

Figura 2.
Hedonic Test Results

Preference
16
14
12
10
N° Judges

8
6
4
2
0
1 2 3 4 5 6 7
Preference Notes
K1HV K3HV K2HV REF

Fig 3. Hedonic Test Results on 7 points (1 point is the worst cualification, 7 points is the best
cualification)

The results of the preferred hedonic test show that the best juice scored by judges is
fermented with 1% and 2% kéfir.

The products at 1% and 2% w/v of kefir had a score of 4.87 and 5.37. (Figure 2). The
preference analysis of these products show that only in the 1% kefir sample, 6.7% of judges
give a low score (score = 3). While, for a score of 4, the judges perception is distributed
between 36.7% and 13.3% for kefir at 1% and 2% respectively. Similarly, 30% and 43.3% of
judges attribute to the samples products a grade of 5. Likewise, 16.7% and 36.7% perceive
the fermented product with a score of 6. Finally, the order 10% and 6.7% of judges perceive
the samples with the highest possible score (score = 7). Therefore, taking into account that
kefir at 2% did not have a minimum rating (score=3) and it was rated with the highest rating
(score= 7), besides it is resalted over other samples analized on attributes such as low
acidity, high perception of sweetness, low alcohol perception and better preference about
texture. Therefore this product has a higher probability of having a better acceptance in
consumers.

Conclusions

Taking into account the increasing complexity of the needs of different kind of consumers,
including vegan, vegetarian and people with intolerance/allergy to dairy products it is
developed new functional non dairy beverage with physico-chemical and organoleptic
 properties based well met the expectations and tastes of panellists composed with fruit and
vegetable rich in bioactive compounds with water kefir.

Fermentation promoted considerable changes in the comercial juice such as: sugar content
consumption, acidity production; increase in: total phenols, carbon dioxide, organic acid
(lactic acid, acetic acid and succinic acid), lightness (L*), chroma ¿*) value; decrease in: pH,
density, antioxidant activity, citric acid, hue ( h ab) value, these changes are limited on the
level of kefir grains inoculum and fermentation time. The changing in the physico chemical
properties revealed good results in sensory evaluation with 2% kéfir and 24 hours of
fermentation as the best alternative, this can result in the production of a healthy fermented
non dairy fruit juice drink.

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