E3S Web of Conferences 247, 01003 (2021) https://doi.org/10.

1051/e3sconf/202124701003
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Optimization of production of the fermented milk beverage based

on Tibetan kefir grains
Rustem Khabibullin1,*, Mahmud Adylov1, and Galina Ezhkova1
1Kazan National Research Technological University, K. Marks St. 68, Kazan, 420015, Russian Federation

Abstract. In recent years we have seen interest in Tibetan kefir grains (TKG) for production of fermented
milk beverages. The article presents the results of the work on expanding the range of fermented milk
beverages based on TKG, studying the influence of fermentation conditions on quality characteristics of
beverages and their shelf life. The possibility of their optimization is shown. Using the method of Central
Composite Design (CCD), response functions were obtained in the form of 2nd-order polynomials, and the
numerical values of the coefficient of these dependencies were calculated. The conditions of milk
fermentation by Tibetan kefir grains were optimized. The numerical values of the influencing factors are:
the dose of inoculate is (60 ±2) g per 100 ml of the milk, fermentation duration -(30±3) h; fermentation
temperature - (35±4)°C. We also studied the effect of sea buckthorn, chokeberry and lingonberry juice
additives on the quality of fermented milk drinks and their shelf life. Using the same method CCD we
determined the functional dependencies of sensory indicators and shelf life of the beverage on the added
amount of berry juice and sucrose. According to the results of the conducted studies, the recipe of a
fermented milk beverage was modified. The optimal content of the components (per 100 ml of the drink) is:
juice – (12±2) ml, sucrose - (10±1) g.

1 Introduction A well-known kefir is a traditional Middle Eastern

fermented milk drink. It is a product of fermentation of
Fermentation processes used to be used only to increase milk with kefir grains or mother cultures made from
shelf life of products and improve their taste, but in grains. It consists of various species of Lactobacilli,
recent years fermented foods have become associated Lactococci, Leuconostoc, Acetobacter and yeasts (both
with a healthy diet and have attracted scientific interest lactose- and galactose-fermenting). Kefir has a fizzy,
[1]. This role of fermented foods brought to life a new sour taste, tart and refreshing aroma, it contains essential
class of foods called functional nutrition [2]. amino acids, and has healing and supporting functions
The microorganisms that provide fermentation [11]. Some products, such as yogurt, yogurt powder, and
process are also becoming the object of study, because cottage cheese powder (dahi), are very popular and
they have a wide range of biological activity: they produced [3].
control the growth of probiotic microorganisms, In recent years we have seen interest in Tibetan kefir
synthesize vitamins, specific proteins, highly digestible grains (TKG) for production of fermented milk
calcium, bacteriocins, sphingolipids and many others. As beverages. The microbial composition of TKG varies
a result, fermented foods provide many health benefits depending on its origin and substrate.
such as anti-oxidant, anti-microbial, anti-fungal, anti- Its microbial composition differs from Russian kefir,
inflammatory, anti-diabetic, anti-carcinogenic activity Irish kefir, Taiwanese kefir, Turkish fermented drink
[1], prevention of gastrointestinal infections, lowering with kefir. Tibetan kefir consists also of Lactobacilli
serum cholesterol and antimutagenic activity. Fermented (Lactobacillus casei, Lactobacillus hilgardii,
foods are recommended for people with lactose Lactobacillus delbrueckii spp., Lactobacillus plantarum,
intolerance and atherosclerosis [3]. L. Acidophilus and Lactobacillus kefir, Leuconostoc mesenteroides spp.
Bifidobacterium spp. are also known for their probiotic dextranicum), Lactococci (Streptococcus lactis), yeast
properties, but fermented foods should contain them in (Zygosaccharomyces cerevisiae, Torulospora
the amount of at least 106 cfu/g [4]. pretoriensis, Candida lambica, Candida valida), and a
At the end of the millennium hypotensive effect of small amount of acetic bacteria.
fermented products due to biologically active peptides The special grain structure is due to the ability of the
was discovered. The peptides are released during bacteria Lactobacillus kefiranofaciens and the yeast
fermentation or gastrointestinal digestion. Such Saccharomyces turicensis to self-aggregate into small
fermented dairy products exist, they are few in number, granules. The biofilms-producers Lactobacillus kefiri,
and are based mainly on Lactobacillus helveticus [5-10]. Kluyveromyces marxianus and Pichia fermentans adhere

*
Corresponding author: [email protected]
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution
License 4.0 (http://creativecommons.org/licenses/by/4.0/).
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ICEPP-2021

to the granules surface, which turns them into kefir gastrointestinal tract, should be taken into account.
grains. This process is facilitated by the symbiotic These factors include physiological state of the
relationship between bacteria and yeasts of kefir grains. introduced probiotics, storage conditions of a product, its
Later, Lactobacillus kefiri, L. kefiranofaciens, L. chemical composition, including acidity, carbohydrate
kefirgranum, Lactobacillus parakefir, and Candida kefir content, nitrogen sources, mineral content, water activity
were classified using the product name [12]. and oxygen content, and possible interactions between
The high antioxidant potential (AP) of dairy products probiotics and starter cultures [23]. It was shown that
allows us to overcome the negative effects of oxidative Lactobacillus acidophilus and Bifidobacterium animalis
stress. This high AP is explained by the breakdown of subsp. lactis ratio strongly affects the properties of a
milk protein during fermentation or digestion and the fermented milk product, including acidification profile,
release of antioxidant peptides, which increase AP by 2- organic acid production, lactose consumption, shelf life
5 times. Fat milk has higher AP than low-fat and skim at 4°C, bacterial viability, sensory properties, organic
milk due to lipophilic antioxidants, and Lactobacillus acid content and viability under gastrointestinal
casei/acidophilus provides the highest AP among tested conditions [4]. Another paper describes the optimizing
in the LAB [13]. method of the TKG culture growing using the response
Lactobacillus plantarum MA2 strains isolated from surface analyzing. As a result, the optimal values of
Chinese TKG have high antioxidant activity in vitro and skimmed milk quantity, temperature, time of
in vivo, acid and bile salt tolerance, cell surface fermentation and inoculate amount were determined
hydrophobicity and auto-aggregation, which confirmed [24].
the probiotic characteristics of these bacteria of TKG The enrichment of dairy products with natural
[14]. The high cellular antioxidant activity (CAA) has ingredients, containing a considerable amount of
been studied both at different stages of cell growth and essential nutrients, is used to develop the technology of
for different components of fermentation media - cell- fermented milk products. Spirulina, chickpea, St. John's
free extract and fermentation supernatant, and CAA wort, tarragon, mint, sage, stevia, chili pepper, black
method was recommended to use for lactic acid bacteria pepper, cinnamon, allspice, thyme, cumin and many
antioxidant potential evaluation [15]. It was shown that others are used as additives [25-29]. Use of these spicy
the level of total cholesterol, triglycerides, and low- and aromatic additives improves and diversifies the taste
density lipoproteins in the blood serum of rats, which and aromatic properties of fermented products, enrich
received a certain amount of LAB strains isolated from them with biologically active substances, increase their
TKG, reduced significantly. It was recommended to use stability when stored.
these strains as probiotic starter cultures for production Given the above, broadening the range of fermented
of fermented products [9]. Similar results were obtained milk drinks based on the TKG, studying of the influence
for other microbial strains from TKG [16]. of fermentation conditions, formation of the
Some components synthesized by microbial strains characteristics of fermented milk beverages, their
from TKG also have antioxidant and gut microbiota- changes during the storage and optimization of these
regulating activity. For example, exopolysaccharide factors should be recognized as important areas of
(EPS) produced by Lactobacillus plantarum YW11 research.
reduced oxidative stress in aging mice with increased Therefore, this paper aims:
levels of glutathione peroxidase, superoxide dismutase, To obtain the second-order polynomial response
catalase and total serum antioxidant capacity. Moreover, surfaces both of quality and shelf life indicators on the
EPS can restore the diversity of the microbiota of main fermentation parameters using Central Composite
experimental animals. The fermented milk drink Design (CCD);
containing EPS also showed a beneficial antioxidant To optimize the resulting polynomial responses, i.e.
effect [17]. to determine conditions providing the extremal
It was found that different substances with antifungal (maximal) level of these functions;
activity are formed during different stages of TKG To study the effect of plant additives on the quality
microbial community cultivation. Starting from 36 h of of fermented beverages and their shelf life by obtaining
fermentation antifungal activity is connected mainly with corresponding response surfaces;
sensitive to heat peptides and proteolytic enzymes. To optimize beverage formulation to provide
Starting from 3-4 days of cultivation and later organic maximum of these function.
acids and/or substances active in acidic environment are
accumulated. At a later stage of cultivation, a certain
contribution to the antifungal effect is also made by 2 Materials and methods
thermally-stable, protease-resistant substances [18]. Experimental studies were conducted in laboratory of the
It was shown that TKG can be used for other dairy Meat and Dairy Products Technologies Department at
products manufacturing, for example, Camembert type Kazan National Research Technological University
cheese [19, 20], and traditional Chinese Bod ljong using generally accepted methods [30] and equipment.
cheese [21]. The fermenting activity of TKG has been The following materials and objects were used and
shown not only for milk, but also for a mixture of studied. Pasteurized milk with 3.2% fat content TM
mulberry juice and whey [22]. “Very important cow” (Zelenodolsk Dairy Plant,
Several key factors, which determine the probiotics Republic of Tatarstan, Russia); Certified Organic Live
ability to survive in the product and human

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Milk Kefir Grains Tibetan Starter by Kombucha

Organic®; fresh sea buckthorn, chokeberry and
lingonberry. Juices were prepared from berries by (3)
grinding in a blender, followed by sedimentation in a The experimental data were expressed as mean ±
laboratory ELMI CM-50 centrifuge, Latvia. standard deviation (SD) from three replicates for each
The Tibetan kefir grains were preserved in sample. The analysis of variance (ANOVA) of the data
pasteurized milk at 4°C, and activated at 28°C for 24 h. was performed using the Statistica 6.0 software package.
A day later the fermented product was poured into a
container through a plastic sieve, while being gently
stirred, separated kefir grains were washed under a 3 Results and discussion
stream of cold water so that they were completely clean
for the next fermentation. Dishes used were thoroughly
3.1 Constructing Central Composite Design
washed with water, and it was forbidden to use synthetic
detergents. Fermentation conditions whose influence was evaluated
The key analytical techniques used were titrimetric and selected for consideration in the experiment were the
method according to GOST 3624-67 (acidity following: the inoculate (TKG) amount (25-75 g/100 g
determination); butyrometric method according to GOST of milk), fermentation temperature (20-45°C),
3622-67 (weight fraction of fat); Kjeldahl method fermentation time (8-40 h). We have constructed a
according to GOST R ISO 2446-2011 (weight fraction of central composite plan for three variables on 5 levels, so
protein). In some experimental series physic-chemical
in accordance with formulas (2) and (3), α =√3=1.73,
parameters such as fat content, protein, lactose, mass
N=17. The levels of these factors according to CCD are
fraction of dry milk residue, skim milk residue, shown in Table 1.
homogenization degree and freezing point were
determined on the “Clover-2M” milk analyzer. Table 1. Range of CCD values for fermentation conditions
The titratable acidity and pH values were measured optimization.
during the propagation. The pH value was determined by
using a digital desktop pH meter Hanna Instruments HI Levels Factor 1 Factor 2 Factor 3
2216. TGK amount, Fermentation Fermentation
g/100 g of temperature, time, h°C
Descriptive sensory analysis was conducted with the milk °C
participation of 5 qualified trained evaluators using the -1.73 20 15 8
consensus method in accordance with the relevant -1 25 20 12
standards (ISO 13299:2003; ISO 11035:1994; ISO 0 50 32.5 24
4121:2003, ISO 8587:2006) and was used as a tool for 1 75 45 36
evaluating and comparing the quality of fermented 1.73 80 50 40
beverages with berry juices. The score was given on a 9-
point scale. The effect of the components on the sensory
In order to optimize the fermentation process, we indicators of fermented beverages was evaluated and the
applied the response surface analysis methodology amount of berry juice (5-15 g/100 g of the beverage) and
(RSM) [24, 31] that provides for the Central Composite sucrose (5-15 g/100 g of the beverage) was selected
Design (CCD). As a result the second-order dependence (Table 2). According to formulas (2) and (3), in this case
of process efficiency indicators on the influencing k=2, α = √2 = 1.44, N =9.
factors was obtained in the following form:
Table 2. Range of CCD values for optimal formulation of
beverages with berry juice (2 factors, 5 levels).

Levels Factor 1 Factor 2

(1) Berry juice amount, Sucrose amount,
g/100 g of beverage g/100 g of beverage
where Y is a dependent variable (process efficiency or
-1.44 2.5 2.5
product quality indicators); β0, βi, βii, βij are the -1 5 5
regression equation coefficients; xi, xj are the levels of 0 10 10
the influencing factors. The total number of experiments 1 15 15
N was calculated using the formula: 1.44 17.5 17.5
𝑁𝑁 = 𝑁𝑁0 + 2 ∙ 𝑘𝑘 + 𝑛𝑛0 (2) As a response function, a comprehensive quality
criterion (CQC) was used, including taste, color, smell,
where n0 is the number of experiments in the center of
and consistency.
the plan, k is the number of factors, and N0 is the number
of experiments in the full factor experiment. The value CQC = (T+Cl+S+Cn)/4, (4)
of the distance to the axial point, also called the star
point, α is calculated by the formula: where T- evaluation of taste, Cl - rating of color, S -
score of smell, Cn - assessment of consistency, 4 –
number of indicators used.

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As the second response function, the product shelf Table 4. Sensory indicators, CQC and shelf life of fermented
life index was used, which was determined by the milk.
dynamics of the pH value changes during product
№ T S Cl Cn Y1 Y2
storage at a temperature of +18°C. The time when the
1 8.5 7.5 8.0 8.0 8.0 7.0
pH changes to an upward trend, is rounded to 12 hours 2 8.0 7.0 7.0 6.0 7.0 6.0
and expressed in days, and is considered the storage 3 8.0 7.5 7.5 8.0 7.7 6.5
time. 4 7.0 7.0 7.5 7.5 7.2 5.5
5 8.5 8.0 8.0 8.0 8.1 7.0
3.2 Optimization of fermentation conditions 6 7.5 7.0 7.0 7.5 7.25 6.0
7 8.0 7.0 8.0 8.0 7.75 6.5
In order to optimize the process of milk fermentation by 8 7.0 6.5 7.0 6.5 6.75 5.0
TKG, a graph of the dependence of the process 9 8.0 7.0 7.0 7.0 7.25 8.0
efficiency indicators on the influencing independent 10 7.0 6.0 6.0 7.0 6.5 5.0
parameters (factors) was obtained based on the results of 11 9.0 9.0 8.0 9.0 8.75 7.5
12 9.0 8.0 8.0 9.0 8.5 8.0
the laboratory studies. The main parameters which
13 9.0 8.0 8.0 8.0 8.25 7.0
influence the quality indicators of the fermented milk
14 9.0 8.0 7.0 8.0 8.0 7.0
were varied: the amount of inoculate (introduced starter 15 9.0 8.0 9.0 9.0 8.75 8.0
culture) from 25 to 75 g/l; fermentation time from 12 to 16 9.0 8.0 9.0 8.0 8.5 7.5
36 hours; fermentation temperature from 20 to 45°C. 17 9.0 8.0 9.0 9.0 8.75 8.0
The Central Composite matrix is presented in Table 3.
Based on the obtained data, the numerical values of
Table 3. Matrix of the central composite design for optimizing
the regression coefficients for Y1 and Y2 were calculated
fermentation conditions.
and these regression equations can be presented as
№ The levels of factors follows.
Factor X1 Factor X2 Factor X3
Inoculate Fermentation Fermentation Y1=8.90+0.37∙a1+0.11∙a2+ 0.03∙a3-1.05∙a11-0.09∙a22-
amount, g/l time, h temperature, °C 0.38∙a33+0.04∙a12-0.04∙a13-0.09∙a23
1 75 36 45
2 25 36 45 Y2=8.20+0.73∙b1+0.16∙b2+ 0.04∙b3-0.98∙b11-0.30∙b22-
3 75 12 45 0.71∙b33-0.06∙b12-0.06∙b13-0.06∙b23
4 25 12 45
5 75 36 20
During the optimization process, we determined the
6 25 36 20 values of the influencing factors X1 and X2, which
7 75 12 20 ensure the maximum of the response surface function.
8 25 12 20 The obtained values of the variables are (in
9 80 24 32.5 dimensionless quantities): for CQC X1=0.19, X2=0.63,
10 20 24 32.5 X3=0.01; for the function «Shelf life» X1=0.36, X2=0.22,
11 50 40 32.5 X3=-0.002. When moving from dimensionless to real
12 50 8 32.5 values, we get the optimal values of fermentation
13 50 24 15 conditions: inoculate amount (60±2) g/l; fermentation
14 50 24 50 time (30±3) h; fermentation temperature (35±4) °C.
15 50 24 32.5 Thus, carrying out the process under optimal conditions
16 50 24 32.5 provides the highest value of CQC, which is 8.9 points,
17 50 24 32.5 and the longest shelf life of the resulting fermented milk,
equal to 8.2 days.
The values of sensory indicators T, Cl, S, Cn,
summarized as CQC, as well as the shelf life are
presented in Table 4. 3.3 Optimization of the recipe of fermented
beverages with berry-juice

On the base of fermented cow milk, in order to expand

the range and improve the sensory characteristics, a
certain amount of berry juice and sucrose was added.
The main parameters influencing the quality indicators
of fermented beverages with berry-juice, berry juice and
sucrose amount, were varied at five levels. The number
of experiments conducted, determined by the formula
(2), was 9 points. The orthogonal planning matrix for 2
independent variables is presented in Table 5.

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Table 5. Matrix of the central composite design for optimizing Table 8. Sensory indicators, CQC and shelf life of a fermented
the recipe of fermented beverages. beverage with lingonberry juice.

№ The levels of factors № T S Cl Cn Y1 Y2

Factor X1 Factor X2 1 8.0 7.5 7.5 7.0 7.5 6.2
Berry juice amount, Sucrose amount, g/100 2 8.5 8.0 7.5 7.5 8.0 8.0
g/100 g of beverage g of beverage 3 8.5 7.0 7.5 7.0 7.5 6.5
1 5 5 4 8.5 8.0 8.5 7.0 8.0 7.7
2 15 5 5 8.5 8.0 8.0 8.0 8.12 7.75
3 5 15 6 9.0 8.0 9.0 7.5 8.75 8.5
4 15 15 7 8.0 7.0 7.5 7.0 7.0 6.25
5 10 10 8 7.0 8.0 8.0 7.0 7.5 8.0
6 15 10 9 9.0 8.0 8.0 7.0 7.0 7.5
7 5 10
8 10 15 Based on the obtained data, the values of the
9 10 5 regression coefficients for Y1 and Y2 were calculated,
and these regression equations are as follows.
The sensory properties of the described variants of The beverage with sea buckthorn juice:
the beverage were evaluated during laboratory tests, as
described above in the section "Materials and methods". Y1=9.56+0.38∙a1+0.15∙a2+0.45∙a11+0.06∙a22-0.18∙a12
Comprehensive quality criterion (CQC) is a function of
the response Y1. Y2=7.19+0.96∙b1+0,16∙b2+0.02∙b11+0.10∙b22-0.18∙b12
After the sensory evaluation, the shelf-life time was
With chokeberry juice:
determined and taken as the Y2 response function. The
increase in pH during storage at a warm temperature is Y1=10.53+0.46∙a1+0.13∙a2+0.45∙a11+0.06∙a22-0.13∙a12
explained by the accumulation of microscopic fungi
(molds, micromycetes) and putrefactive microorganisms, Y2=7.28+0.96∙b1+0.18∙b2+0.02∙b11+0.10∙b22-0.19∙b12
accompanied by enzymatic hydrolysis of milk proteins
and followed by deamination and decarboxylation of With lingonberry juice:
amino acids produced. Since the solubility of the Y1=9.42+0.46∙a1+0.45∙a11+0.06∙a22-0.02∙a12
released ammonia is much higher than that of carbon
dioxide, there is a pronounced leaching of the medium. Y2=7.21+0.87∙b1+0.20∙b2+0.02∙b11+0.10∙b22-0.15∙b12
The choice of temperature is determined by the operation
convenience. At lower temperatures, the shelf life is up During the optimization process, we determined the
to 10-14 days, at higher temperatures- accelerates to 3-5 numerical values of the influencing factors X1 and X2,
days. which ensure the maximum of the response surface
Sensory indicators, CQC and shelf life of the function.
fermented beverage with juices are shown in Tables 6-8. The obtained numerical values of the variables in
dimensionless quantities are as follows.
Table 6. Sensory indicators, CQC and shelf life of a fermented For the CQC function with sea buckthorn X1=0.72,
beverage with sea buckthorn juice. X2=0.48, chokeberry X1=0.84, X2=0.65, lingonberry
№ T S Cl Cn Y1 Y2 X1=0.67, X2=0.56.
1 8.0 7.0 7.0 6.0 7.0 6.0 For the function "Shelf life" with sea buckthorn
2 8.5 8.0 8.0 7.5 8.0 8.0 X1=0.85, X2=0.02, chokeberry X1=0.78, X2=0.06,
3 8.0 7.5 7.5 8.0 7.7 6.25 lingonberry X1=0.8, X2=0.1.
4 8.0 8.0 8.5 7.5 8.0 7.5 In natural values, the optimal recipes of fermented
5 8.5 8.0 8.0 8.0 8.2 8.0 beverages are: 12.2 g of sea buckthorn juice and 9.3 g of
6 8.5 9.0 8.5 8.5 8.75 8.5 sucrose per 100 ml of beverage; 13.131 g of chokeberry
7 8.0 7.0 8.0 8.0 7.75 6.0 juice and 11.3 g of sucrose per 100 ml of beverage;
8 8.0 8.5 7.0 7.5 8.0 8.2 11.632 g of lingonberry juice and 10.1 g of sucrose per
9 8.0 7.0 8.0 8.0 7.75 7.0 100 ml of beverage.
Table 7. Sensory indicators, CQC and shelf life of a fermented
beverage with chokeberry juice. 4 Conclusions
№ T S Cl Cn Y1 Y2 Using the method of Central Composite Design (CCD),
1 8.0 7.0 7.5 6.0 7.2 6.2 we optimized the conditions for milk fermentation by
2 8.5 8.0 8.5 7.5 8.2 8.2
Tibetan kefir grains. The numerical values of the
3 8.5 7.0 7.5 8.0 7.7 6.5
influencing factors are: the dose of the introduced starter
4 8.5 8.0 8.5 7.5 8.2 7.75
5 8.5 8.0 8.0 7.5 8.0 8.0
culture is (60 ±2) g per 100 ml of milk; fermentation
6 8.5 8.0 9.0 7.5 8.5 8.7 time is (30±3) h; fermentation temperature is (35±4) °C.
7 8.0 7.0 7.5 8.0 7.25 6.2 The effect of berry juices on the quality indicators of
8 7.0 8.5 8.0 7.5 8.0 8.0 fermented milk beverages and on their shelf life was
9 8.0 8.0 8.0 7.0 7.75 6.75 studied.

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