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Development of probiotic drink production technology with the addition of
citrus juice
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AGRITECH-IV-2020
IOP Conf. Series: Earth and Environmental Science 677 (2021) 032044
IOP Publishing
doi:10.1088/1755-1315/677/3/032044
1
Development of probiotic drink production technology with
the addition of citrus juice
I V Smotraeva1,3, A S Gargalyk1, P E Balanov1, O B Ivanchenko2 and
T A Kuznetsova2
1 Faculty of Biotechnologies (BioTech), ITMO University, 49 Kronverksky Ave.,
Saint Petersburg, 197101, Russia
2 Peter the Great St. Petersburg Polytechnic University (SPbPU), 29
Politekhnicheskaya St., St. Petersburg, 195251, Russia
3 E-mail: irinasmotraeva@yandex.ru
Abstract. The study examines the possibility of expanding the international market for low-
alcohol products by modernizing the traditional probiotic drink made from grain raw materials,
which is popular in Eastern Europe - kvass. In particular, it is proposed to solve this problem by
developing a fundamentally new version of kvass based on orange juice, honey and spices.
During the work, 5 samples were made using various strains of yeast and lactic acid bacteria.
During the physical and chemical analyses and tasting evaluation of the samples obtained, it was
revealed that all samples comply with the norms established in interstate standards. It is
noteworthy that the samples made using the bacteria Lactobacillus acidophillus, which are
usually used in the production of lactic acid products, and not in plant-based drinks, received the
highest consumer rating.
1. Introduction
Currently, there is a steady trend towards a healthy lifestyle in the world. The Russian Federation, as
one of the largest world powers, also pursues a policy aimed at strengthening and developing the values
of a rational approach to health. That is why the modern beverage market is reorienting and creating
more favourable conditions for the creation and production of new types of non-alcoholic and low-
alcohol products. The policy of using domestic raw materials, actively developed by the government of
our country, forces many entrepreneurs to create new products that have no analogues in the world.
However, one should remember about the development of traditional Slavic drinks, which are very
popular among consumers.
One of these drinks is kvass, known to the Eastern Slavs for over a thousand years. It has a pleasant
sweet and sour refreshing taste, improves metabolism [1], has a beneficial effect on the cardiovascular
system, contains a large amount of vitamins, micro- and macroelements [2], has a beneficial effect on
the human body [3].
Despite all the positive characteristics of kvass, the European market does not offer consumers a
wide range of types of this product, while other options, such as flavoured soft drinks, are represented
by hundreds of different tastes and aromas. In addition, many customers buy kvass only in the hot
season as a thirst quencher, associating it with a “summer” drink. At the same time, there are all the
prerequisites for the active consumption of probiotic drinks based on grain raw materials to be year-
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IOP Conf. Series: Earth and Environmental Science 677 (2021) 032044
IOP Publishing
doi:10.1088/1755-1315/677/3/032044
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round. These tasks can be solved by creating kvass based on orange juice with the addition of honey
and spices, which will not only be interesting from the point of view of novelty, but also useful for the
prevention of colds in cold periods, due to the content of biologically active substances of citrus fruits
and honey [4, 5].
Thus, the relevance of creating a recipe for this kvass is as follows:
• it is a suitable drink for people leading a healthy lifestyle, due to its low sugar content (about
2.5 times lower than in most popular carbonated soft drinks) [6];
• maintaining and developing the policy of using domestic raw materials, by expanding the range
of domestic products;
• expanding the range of authentic products with new attractive taste and aroma;
• prevention of colds during cold periods of the year.
Objective:
• development of a recipe for kvass based on orange juice with the addition of honey and spices.
Tasks:
• selection of the optimal type of yeast;
• selection of the optimal type of lactic acid bacteria;
• selection of the dose of orange juice and the establishment of the stage of its introduction;
• selection of the dose of honey and spices, the establishment of the stage of their introduction;
• research of quality indicators of kvass samples and selection of the optimal recipe;
• establishing the shelf life of the developed product in terms of physicochemical and organoleptic
parameters.
2. Materials and methods
2.1. Used strains of yeast and LAB
Yeast:
• dry baking yeast of the Lesafr trademark (composition: dry baking yeast Saccharomyces
cerevisiae, emulsifier E 491);
• liquid yeast Saccharomyces cerevisiae, strain San Francisco Ale II-BSG-005A, manufacturer
“Brew Success Group”, St. Petersburg.
Lactic acid bacteria:
• lyophilisate of Lactobacillus acidophillus, strain n.v. 317/402 (NARINE), manufacturer
Narex, Armenia;
• bacterial starter Lactobacillus fermentum, strain Belgian Lactobacillus-BSG-007BL,
manufacturer “Brew Success Group”, St. Petersburg.
2.2. Making kvass in laboratory conditions
This step was based on the fulfilment of two main tasks:
• development of a kvass recipe based on orange juice, honey and spices;
• selection of the optimal combination of yeast and lactic acid bacteria strains, allowing to create
the most balanced organoleptic profile of the finished drink. In order to correctly evaluate this
parameter, the same amount of certain strains of yeast and lactic acid bacteria was introduced
into each of the kvass samples.
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IOP Conf. Series: Earth and Environmental Science 677 (2021) 032044
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doi:10.1088/1755-1315/677/3/032044
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Before starting the tests, the quantities of the main components of kvass and the order of their
introduction were determined. For this, a series of experiments was carried out, during which such
ratios of these components were selected so that the organoleptic balance of the tastes introduced by
each of them was observed. It was also found that when orange juice was introduced at the fermentation
stage, the citrus aroma was not felt in the final product, therefore this component was subsequently
introduced at the stage of blending the drink along with 65% sugar syrup. Thus, the general recipe for
the drink was determined, including:
• Concentrate of kvass wort - 20 g [7];
• Honey - 40 g;
• Water - 1000 g;
• Spices (cloves, ginger, cinnamon, cardamom) - 1 g of each type;
• Dried orange peel - 1.3 g;
• Orange juice - 85 g;
• Sugar syrup (65%) - 45 g;
• Yeast (liquid / dry) - 109 CFU / cm3 (which corresponds to 1 ml / 1 g);
• Lactic acid bacteria (liquid / dry) - 109 CFU / cm3 (which corresponds to 1 ml / 0.2 g).
The technology for making kvass is as follows. Warm drinking water at a temperature of 40 ° C is
poured into laboratory glassware. The calculated amount of kvass wort concentrate, and honey is added
to it. Everything is well mixed until the components are completely dissolved. Then spices and dried
orange peel are added to the wort, the wort is mixed. After that, yeast and lactic acid bacteria are added
to the solution. It should be noted that when adding dry yeast, they must be distributed over the surface
of the wort without further mixing. When all the ingredients have been added, the sample is covered
with a cotton cloth and sent to fermentation at 25-30° C for 24 hours.
At the end of the day, the coarse sediment must be removed from the drink. After that, blending is
carried out, for this, orange juice and sugar syrup are added to it. Then the drink is kept at a temperature
of 2° C for 36 hours in a refrigerator. At the same time, microbiological processes slow down, the final
formation of the organoleptic profile of the drink takes place. It should be noted that in this case only
coarse filtration is carried out without subsequent clarification and pasteurization, therefore, cells of
lactic acid bacteria and yeast remain in the finished drink, which determines its probiotic properties and
allows this product to be identified as unfiltered and unpasteurized [8]. Its shelf life is much shorter
than that of pasteurized drinks, and is no more than 5-7 days, since even when stored in a refrigerator,
alcoholic and lactic acid fermentation continues in the volume of the drink. Thus, the resulting drink is
probiotic.
2.3. Study of the obtained kvass samples
All kvass samples were examined for the following indicators:
2.3.1. Organoleptic (appearance, color, taste and aroma, CO2 saturation). Preparation for testing
consisted in bringing the temperature of drinks to 10-14 ° C. The prepared sample was placed in a clean
dry cylinder with a volume of 250 cm3. Then ten members of the tasting committee visually determined
the appearance and color of the drink, paying special attention to the shade and intensity of kvass color.
With the subsequent organoleptic determination of aroma and taste, which was carried out immediately
after pouring the sample into a tasting glass, the level of compliance of these indicators with the
requirements specified in the standard for this product was revealed [9].
2.3.2. Physical and chemical indicators
• Mass fraction of dry substances [10].
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The determination of dry matter was carried out by the refractometric method using a PTR 46
refractometer by the British company Index Instruments, which is an automated digital measuring
device consisting of an optical system built according to the Abbe scheme. The determination was
carried out in accordance with the specified interstate standard.
• Acidity [11]
A titrimetric method was used, which is based on the titration of all acidic substances with an alkali
solution after the sample has been freed from carbon dioxide, followed by a mathematical calculation
using the formula:
where V is the volume of the alkali solution used for titration, cm3;
К is correction factor for alkali solution;
А is the volume of the drink taken for determination, cm3.
• Volume fraction of alcohol [12]
The method is based on distillation and subsequent calculation of the relative density of the distillate by
the formula:
where m is the mass of the pycnometer with the distillate solution, g;
m1 is the mass of the empty pycnometer, g;
m2 is the mass of the pycnometer with distilled water, g.
The mass fraction of alcohol is found by the calculated relative density using tables from the specified
GOST standard.
• Apparent and actual fermentation [9]
First, the visible extract was determined in kvass using a sugar meter (if alcohol and carbon dioxide
were present in the drink). The apparent degree of fermentation was calculated using the formula:
where m1 is the mass fraction of dry substances in the original wort, %;
m2vid is visible extract, %.
Then the kvass sample was freed from alcohol and CO2. Using a pycnometer, the relative density
was determined, according to which the value of the actual extract was found (using look-up tables).
The actual fermentation rate was calculated using the formula:
where m1 is the mass fraction of dry substances in the original wort, %;
m2d is actual extract, %.
2.4. Determination of shelf life of samples
Since the studied kvass is unfiltered and unpasteurized, after the end of fermentation, no treatment is
carried out that inactivates the activity of yeast and lactic acid bacteria. Therefore, even after filling into
containers, fermentation processes continue in the finished product. Such drinks are stored in cool
places for no more than seven days at a temperature of 2-3 ° C [8]. Thus, in order to confirm the optimal
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IOP Conf. Series: Earth and Environmental Science 677 (2021) 032044
IOP Publishing
doi:10.1088/1755-1315/677/3/032044
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shelf life of the studied samples, in the process of work, changes in their organoleptic properties were
investigated on days 4, 7 and 9 of storage.
3. Results and discussion
3.1 Combinations of strains
As part of the work, 5 samples of kvass were made. The combinations of strains in the samples are
shown in table 1.
Table 1. Combinations of strains in samples.
Yeast Strains /
Lactic acid bacteria strains
Baker's yeast
Saccharomyces cerevisiae
Lesafr (dry)
Brewer's yeast Saccharomyces
cerevisiae strain San Francisco
Ale II-BSG-005A (liquid)
Lactic acid bacteria
Lactobacillus acidophillus,
strain n.v. 317/402 (dry)
Sample II
Sample III
Lactic acid bacteria
Lactobacillus fermentum,
strain Belgian Lactobacillus-
BSG-007BL (liquid)
Sample V
Sample IV
As a control sample (Sample I), dry baker's yeast Saccharomyces cerevisiae was used without the
addition of lactic acid bacteria. This sample will fundamentally determine the degree of influence of
lactic acid bacteria on the sensory profile of the product.
3.2 Physicochemical and organoleptic characteristics of kvass samples
The obtained results of analyses of each of the samples are shown in table 2.
Table 2. Physicochemical and organoleptic characteristics of the samples.
Index
Sample
Normative
indicators po
[8,9,10,11,12]
I
II
III
IV
V
Physical and chemical indicators
Volume fraction of alcohol, %
0.6
0.7
0.3
0.3
0.7
No more 1.2
Acidity, k.ed
2.8
2.7
2.4
2.2
3.8
1.5 to 7.0
Visible fermentation rate, %
18.5
23.9
13.0
13.0
34.8
-
Actual fermentation rate, %
15.4
19.4
10.5
10.5
28.4
-
Mass fraction of dry substances,
%
6.3
6.2
6.9
6.9
5.8
No less 3.5
Organoleptic indicators
Appearance (1-4 s.)
4
4
4
4
4
Foaming opaque
liquid
Colour (1-3 s.)
3
3
3
3
3
Colour from
brown to amber
Taste and aroma (2-12 s.)
8
10
12
9
9
Refreshing,
fermented,
matching raw
materials
Saturation СО2 (2-6 s.)
6
6
4
4
6
Abundant
release of
carbon dioxide
Overall score (5-25 s.)
21
23
23
20
22
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In sensory assessment, the total amount is divided according to the following criteria [8] “Excellent”
- 23 - 25 points; “Good” - 20 - 22 points; “Satisfactory” - 15 - 19 points; “Unsatisfactory” - 14 or less
points.
According to the organoleptic evaluation, samples II and III were chosen as the best. Interestingly,
both samples were made using acidophilic lactic acid bacteria, which are commonly used in the
production of lactic acid products, and not fermented beverages from plant materials. This fact allows
us to consider this strain as potentially very interesting for the production of beverages on a grain and
mixed basis. It should also be noted that all samples received only “good” and “excellent” marks, and
also fully comply with the requirements of international standards. This allows us to conclude about
the successful selection of the recipe and the conduct of the experiment as a whole. The shelf life of the
samples, without loss of organoleptic and physicochemical properties, was six days at a temperature of
2° C.
4. Conclusion
The work done allows us to conclude that the development of technologies for modernized probiotic
drinks based on traditional recipes is one of the key vacant niches in the world market. It should be
noted that kvass, like a non-alcoholic drink, can have a wide range of different tastes, since the bread
aroma is quite versatile and is combined with many other components, including citrus raw materials.
The resulting products meet the standards for physical, chemical and organoleptic properties. The shelf
life of drinks made from mixed grain and citrus raw materials at a temperature of 2° C was established
for six days. The proven possibility of using the lactic acid bacteria Lactobacillus acidophillus in the
production of beverages from plant raw materials is of separate scientific and industrial interest.
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