RU2582806C2 - Method for production of bosa using starter - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method for production of Boza. Method of producing Boza comprises addition to sterilised unfermented and filtered Boza prepared from a mixture of grain crops with addition of sugar, a mixture of Lactobacillus plantarum GE223 and Wiessella confusa GEI13, deposited under number LMG P-26178 in Belgian coordinated collections of microorganisms.

EFFECT: invention enables to obtain a product (Boza) having year-round standard microbiological, chemical and sensorial properties, regardless of temperature and ambient flora.

13 cl

Description

The scope of the present invention includes the production method and the bacterial composition of the produced Bose (Boza), which is a microbiologically, physically, chemically and sensory-defined Bose that can exhibit the same characteristics in each production cycle.

The invention encompasses a fermented bose obtained by inoculating Lactobacillus plantarum GE223 and Wiessella confusa GE213 unfermented bosa, prepared in accordance with the method for its preparation.

Physically, chemically, microbiologically, and sensoryly, a standard Bose cannot be obtained due to the fact that the traditional technology of Bose production is carried out as a result of infection of the unfermented Bose with causal microorganisms that were previously present in the environment, or by adding the remains of the fermented Bose from the last production cycle.

Optimal fermentation of the bose depends on the infection of the surrounding microflora and can only be carried out in a certain period of the year. Due to the interruption in production, the bose produced in the next production season cannot be delivered to consumers until the desired microflora in the environment and the desired taste are achieved, resulting in loss of product. Since production is dependent on environmental conditions, it cannot be properly controlled, and unfermented or re-fermented sour bose can be sold in stores.

The novelty of the present invention lies in the proposed solution to these technological problems, which consists in the development of a method for the production of a standardized bose and the use of a composition of bacteria isolated from natural Turkish bose (Turkish Boza). In the present invention, bacteria isolated from Turkish Bose were tested, and suitable bacteria were selected to produce a combination of bacteria that allowed for the production of a Bose standard from a microbiological, chemical and sensory point of view.

The use of baker's yeast (Saccharomyces sp.) Instead of lactic acid bacteria is mentioned in patents BG101548 "Boza composition and method for its production" and BG326 "Composition for making Boza drink", received by Bulgarian authors.

Zorba et al. (2003) describe studies of Bose production using starter cultures in their publication entitled "Use of starter cultures in the fermentation of Boza, a traditional beverage" (process Biochemistry, 2003, 38, 1405 -1411), but although bacteria isolated from the Bose are used for inoculation, due to the use of the unsterilized Bose for inoculation, it contains many other (competing) bacteria, and the possibility of producing a standard Bose on an industrial scale is excluded. Another important point that distinguishes the present invention from others is the use of Wiessella confusa and Lactobacillus plantarum, which are not used in other patents.

In Patent No. BG60703, “Installation for the production of Boza drink and an autoclave for it”, an autoclave was used not to sterilize an unfermented bose, but to heat treat a bose drink. In patent No. BG102205, “Method for preparing a soft drink from cereal crops”, pasteurization or sterilization is used to kill bacteria in a Bose that has already been fermented and ready to eat, to increase shelf life, but not for unfermented bose. The described bose is produced without the use of leaven and does not contain live bacteria in the finished product, unlike our invention.

A feature that distinguishes the bacterial composition of the present invention from commercial starter cultures is that it is isolated from the natural microflora of a commercial market bose, does not contain yeast, and does not contain unnatural genetic changes. For cereal-based products, yeast of the Saccharomyces cerevisea species belonging to the Saccharomyces genus is typically used.

Lactobacillus plantarum GE223 and Wiessella confusa GE213 were isolated from the Bose and did not undergo any genetic manipulations, they were not previously used as the starter cultures of the Bose. Fresh Bose of standard quality can be produced at any time of the year by inoculating with this combination of bacteria a sterilized non-fermented Bose.

A distinctive feature of this starter culture is that it includes a mixture of two lactic acid bacteria with different characteristics. Of these, L. plantarum GE213 is homoenzymatic, produces acidity, acetoin and aromatic compounds, while W. confusa GE213 is heteroenzymatic and produces various organic acids and gives the Bose a distinctive taste. In addition, the selected cultures have a low acid and gas production rate. The Bose obtained as a result of this process is characterized as a lightly drinkable drink with a slightly acidic fruity taste and a viscous consistency.

Cooking boza

Wheat, corn, millet or other crops and their mixtures used as raw material are ground in a mill. Crushed grain is boiled in a 4-5-fold volume of water relative to the grain used until it is completely softened. The cooked mixture is spread on pallets and cooled to approximately 30 ° C until a viscous paste forms. The paste is mixed with 3 volumes of added water, depending on the composition of the mixture and the type of crops. The resulting liquid, in which phase separation is not observed, is subjected to direct filtration through a sieve with a mesh size of 0.2-0.5 mm or it is pre-filtered through a sieve with larger cells to separate large pulp particles and then transferred to smaller sieves. The obtained homogeneous viscous liquid is an unfermented bose. Traditionally, this liquid is fermented by mixing with sugar in a ratio of 10-15% in a vessel containing the remains of the fermented bose from the previous bose production cycle, and after reaching a pH of 5-5.5 it is tasted, cooled and consumed.

In this invention, to allow starter cultures to be used, the filtered liquid Bose is pasteurized (65-85 ° C for 10-30 minutes) or sterilized (121 ° C for 5 minutes) after sugar is added. After that, the leaven is added to the mixture in a ratio of half to half (0.5 + 0.5%) in an amount of 1% of the total volume of the bose, so that it contains 10 ⁶ CFU / ml of the bacterial mixture.

This combination of bacteria is obtained by cultivating two bacteria (GE223 and GE213) separately on MRS agar medium (Man-Rogosa-Sharp) in petri dishes and mixing them after separately adjusting their concentrations with sterile physiological water, and add to the unfermented Bose. Bose is fermented at 25-30 ° C for a maximum of 16 hours until its pH drops to pH 4.5-5, and it will be ready for use after reaching this acidity. After fermentation, the bose is stored at 4-5 ° C. Bosa retains its taste to a pH of 3.9, and many consumers find it sour when it reaches a pH of 3.8.

Properties of selected microorganisms used in the Bose

Cultures designated L. plantarum GE223 and W. confusa GE213 were identified using the Biomerieux® Mini-API 50 CH kit. Tests performed using these kits showed that GE223 utilizes D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, methyl-αD-mannopyranoside, N-acetylglucosamine, amygdalin (amygladin ), arbutin, esculin, salicin, D-cellobiose, D-maltose, D-melibiosis, sucrose, D-trehalose, D-melecitose, D-raffinose, gentiobiosis and does not utilize L-arabinose, D-lactose, D-turanose, D-arabitol and potassium gluconate, and strain G223 was identified as Lactobacillus plantarum 1.

At the same time, GE213 utilizes D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, sucrose, gentiobiosis and potassium gluconate, and does not utilize L-arabinose, D-ribose, methyl-αD-glucopyranoside, D-lactose, D-melibiosis and D-trehalose, and strain G213 is identified as Wiessella confusa.

The ability of these bacteria to produce acetoin and CO ₂ from glucose and to produce dextran from sucrose was studied, survival at 60 ± 1 ° C for 30 minutes and growth in 4% NaCl, 6.5% NaCl at 5 ± 1 ° C, 10 ± 1 ° C, 15 ± 1 ° C and 45 ± 1 ° C, hydrolysis of arginine, hydrolysis of esculin, the formation of a clot in 24 hours in litmus milk, the utilization of carbohydrates and the production of short-chain fatty acids. From isolated Bose cultures, GE223 (Lb. plantarum) produces acetoin, but does not produce CO ₂ from glucose and dextran from sucrose, grows at NaCl concentrations of 4% and 6.5%, does not hydrolyze arginine, but hydrolyzes esculin, does not grow at 5 ° С, 45 ° С, but grows at 10 ° С, 15 ° С.

At the same time, GE213 (W. confusa) does not produce acetoin, produces CO ₂ from glucose and dextran from sucrose, hydrolyzes arginine and esculin. In addition, it grows at 10 ° C, 45 ° C, is resistant to heat treatment at 60 ° C for 30 minutes, and grows at salt concentrations of 4% and 6.5%.

Measuring the antibacterial effect

The inhibitory effect of selected bacteria on the inhibition of growth of pathogens was determined using a Bioscreen®C device. For this, the selected bacteria (L. plantarum and W. confusa) were cultured in liquid MRS at 30 ± 1 ° C for 72 hours and centrifuged at 3000 rpm for 10 minutes, the upper phase was selected (supernatant), which it is believed to have antimicrobial activity, and then microorganisms were removed by passing the supernatant through a 0.45 μm sterile filter. The pH of the filtrate having antimicrobial activity was adjusted to pH 4.6 ± 0.1 so that a low pH of the filtrate does not inhibit the growth of pathogens. The pathogenic bacteria Bacillus cereus, Staphylococcus aureus and Escherichia coli were activated by propagation at 37 ± 1 ° C for 48 hours in cardiac broth (BHI broth).

240 μl of BHI broth, 30 μl of a pathogenic culture diluted with 10 ¹ and 30 μl of a sterile filtrate, which is believed to have antimicrobial activity, were loaded into the wells of a Bioscreen® system tablet. In parallel, as a control, a mixture of antimicrobial-free and glucose-free MRS added to BHI broth containing diluted cultures was loaded. The growth of pathogenic bacteria was determined by turbidometric measurements at 580 nm every 15 minutes after short shaking when compared with wells without antimicrobial solution (control). After an experiment lasting 7 hours, the time required for the growth of pathogenic bacteria to a predetermined optical density of the culture medium was determined (for E. coli and S. aureus: to an optical density of 0.1; for B. cereus: 0.2). Previous studies have shown that the increase in this time was proportional to the logarithm (log) of the decrease in the number of bacteria. Using the previously obtained calibration curves, the percent reduction in the number of pathogenic bacteria was calculated.

When L. plantarum GE223 was added to the E. coli culture, the same optical density was reached in 320 minutes instead of 227, for S. aureus in 255 minutes instead of 245, and in B. cereus culture in 401 minutes instead of 272. Comparison of these coefficients delays with previously determined calibration curves allowed us to calculate that L. plantarum GE223 reduced the number of E. coli by 88%, S. aureus by 8% and B. cereus by 99%.

It was calculated that with the addition of W. confusa GE213 to the E. coli culture, the same optical density was reached in 280 minutes instead of 228, with a 71% decrease, and there was no antimicrobial effect against S. aureus and B. cereus.

Thus, it was determined that when the bacteria of the present invention are used together, they have growth inhibitory activity, especially against E. coli and B. cereus.

In case of contamination of the bose, the bacteria used in this invention will protect the bose due to their antimicrobial effect, the effect of lowering the pH and will also create competition for nutrients.

Determination of shelf life

Bose samples were prepared and stored in a refrigerator at 5 ± 1 ° C for storage shelf life studies. To study the shelf life, two parallel Bose samples were taken on days 1, 7, 14, 21, 28, 35, 42 and 60 and microbiological calculations and determination of pH, acidity, sensory analysis, determination of viscosity, composition of organic acids and sugar distribution were performed.

Since Bose is a starch-based product containing viable bacteria, starch undergoes both degradation and recrystallization throughout its shelf life, and its taste changes continuously under the influence of viable microorganisms. When the Bose is stored in the cold to slow down microbiological growth, its shelf life is reduced because the starch in the Bose undergoes transformations leading to thickening and phase separation. It is not enough to control the souring of Bose by limiting the growth of microorganisms; additional enzymes that degrade starch are needed to prevent starch thickening and phase separation. Thus, as described previously, the prepared bose was placed in a refrigerator at + 5 ° С and souring was prevented, while the ability of the added bacteria to degrade starch prevented thickening of the consistency. Bose observations were made to determine its sensory properties, pH, and microbiological criteria over the shelf life. It was determined that with a properly adjusted bose consistency at the initial time, an increase in the density of the bose or phase separation was not observed due to the ability of the bacteria used in the production of bose to partially degrade starch, and the pH did not fall below an acceptable limit of pH 3.9. The content of acetic acid and ethanol did not exceed the limit values (in accordance with the TS 9778 Bose Standard, volatile acidity should not exceed 0.2%, and alcohol - 2%).

Sensory analysis

According to the results of sensory analysis carried out over the shelf life, the appearance of the bose was defined as transparent, bright from light yellow to cream color, without phase separation, without gas bubbles or cracks and with a specific consistency of a homogeneous viscous liquid. The taste and smell of the bose were defined as slightly acidic, fruity, with a specific pleasant odor and a similar slightly acidic taste, with a characteristic sweet-sour aroma of dried fruit.

Properties of Bose Fermentation Cultures

The biochemical properties of the bacterial cultures referred to in this invention are given in the section entitled "Properties of selected microorganisms used in the Bose."

The most important property of crops for making Bose is that these crops were isolated from Bose and were not genetically modified.

The cultures described in this invention contribute to the production of a fragrant bose by simultaneously producing a wide variety of organic acids and acetoin, which slightly burns the nasal cavity, without exceeding the limits of acetic acid and alcohol, during the fermentation of boza sugar for a short period of time. They increase the safety and shelf life of the bose by showing an antimicrobial effect against E. coli and B. cereus, which may be contaminants that enter the bose from the environment, and may be present in an unfermented bose.

The content of organic acids giving the Bose taste produced by the bacteria mentioned in this invention was analyzed by high performance liquid chromatography (HPLC), with the content of lactic acid remaining at the level of 1.5-2 g / l, acetic acid 0.5-1 g / l of propionic and butyric acids, which create an undesirable taste at high concentrations, remained at the level of trace concentrations over a 2-month period of storage of the bose.

L. plantarum GE223, present in the Bose, can degrade starch, while W. confusa GE113 does not degrade starch, and the problem of thickening of the Bose produced by the classical method when stored in a refrigerator is eliminated due to partial degradation of starch.

By analyzing the sugar composition of unfermented Bose, it was determined that at the beginning the sugar composition included 9888 mg / L sucrose, 156 mg / L fructose, 68 mg / L glucose, with a decrease to 8780 mg / L sucrose, 371 mg / L fructose, 271 mg / l glucose after 2 months of storage of the bose and while maintaining its sweetness.

Bose-forming cultures show growth at 15 ° C and no growth below 10 ° C. This creates the opportunity for fermentation of the bose in cool environmental conditions without heating and increase the shelf life when storing the bose in cold conditions to stop fermentation.

Another property of a given sourdough bose is that probiotic properties can be created by adding cultures (yeast / bacteria) with proven probiotic properties.

The bacteria L. plantarum GE223 and W. confusa GE213 described in this invention are deposited under LMG number P-26178 in the Belgian Coordinated Microorganism Collections (BCCM) / LMG Collections (BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS-BCCM / LMG COLLECTION (international )

Claims (13)

1. A method for producing a Bose, characterized in that the mixture of Lactobacillus plantarum GE223 and Wiessella confusa GEI13, deposited under the number LMG P-26178 in the Belgian coordinated collections of microorganisms (BCCM) / Collections LMG (BELGIAN COORDINATED COLLECTIONS OF MICROORGGISMISMS (MICROORGMISMS) international depository authority), which has not been artificially genetically modified, is added to a sterilized unfermented and filtered Bose with sugar, prepared from a mixture of grain crops. 2. The method according to p. 1, in which the bacterium Lactobacillus plantarum 1 GE223, added to a sterilized unfermented and filtered bose with sugar, has the ability to utilize D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, methyl-αD-mannopyranoside, N-acetylglucosamine, amygdalin (amygladin), arbutin, esculin, salicin, D-cellobiose, D-maltose, D-melibiosis, sucrose, D-trehalose, D-melecitosis, D-raffinose , gentiobiosis and does not utilize L-arabinose, D-lactose, D-turanose, D-arabitol and potassium gluconate. 3. The method according to claim 1, in which the bacterium Wiessella confusa GE113, added to a sterilized unfermented and filtered Bose with sugar, has the ability to utilize D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D -mannitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, sucrose, gentiobiose and potassium gluconate and does not utilize L-arabinose, D-ribose, methyl-αD-glucopyranoside, D-lactose, D-melibiosis and D-trehalose. 4. The method according to claim 1, wherein the bacterial mixture used in the culture of the bose, obtained by mixing the two bacteria in equal amounts, does not contain yeast, but if necessary, a probiotic bose can be obtained by adding yeast / bacteria with known probiotic properties. 5. The method according to p. 1, in which the used mixture of bacteria contains cultures that show growth at 15 ° C and no growth below 10 ° C and, thus, provides the possibility of fermentation of the bose in cool environmental conditions and stops fermentation (souring) in storage conditions at low temperatures to increase shelf life. 6. The method according to p. 1, in which the bacterium Lactobacillus plantarum 1 GE223, added to a sterilized unfermented and filtered bose with sugar, produces the aromatic compound acetoin, quinic, lactic, citric, malic, and a small amount of acetic acid, and Wiessella confusa GE213 not produces the aromatic compound acetoin, but produces quinic, lactic, acetic, citric and malic acids. 7. The method according to p. 1, in which the bacterium Lactobacillus plantarum 1 GE223, added to a sterilized unfermented and filtered bose with sugar, is able to degrade starch, and Wiessella confusa GE213 cannot degrade starch. 8. The method according to p. 1, in which the bacteria used in the method have an antimicrobial effect on the pathogenic bacteria Bacillus cereus and Escherichia coli. 9. The method according to claim 1, in which the bacteria used in the method have the ability to maintain a pH value of the bos above 3.9 for at least 90 days without the use of any preservatives at 4-5 ° C, in a refrigerator. 10. The method according to p. 1, comprising the following stages:
a) Brix Bose is standardized to a value in the range of 10-50% before sterilization;
b) the unfermented filtered bose is sterilized at 65-85 ° C for 10-30 minutes or sterilized at 121 ° C for 5 minutes before fermentation;
c) prepare the bose by adding the culture obtained by mixing equal volumes of Lactobacillus plantarum GE223 and Wiessella confusa GE113, so that it was 1% of the bose, and fermentation at 25-30 ° C until reaching a pH of 5-5.5 for no more than 16 hours ;
d) the resulting fermented bose has a shelf life of at least 3 months when cooled to 4-5 ° C and stored at that temperature. 11. The method according to p. 1, in which the bose obtained by this method, when stored in cold conditions, has a content of ethyl alcohol not exceeding 2% and acetic acid not exceeding 0.2%, in accordance with the limit values established in the standard for the Bose (TS 9778). 12. The method according to p. 1, in which the pH of the bose obtained by this method, in storage at a temperature below 10 ° C, does not decrease below 3.9 ± 0.1 for at least 90 days. 13. The method according to p. 1, in which the bose obtained by this method using cultures partially degrading starch does not lose its fluidity, it does not phase separation and does not contain gas bubbles when stored in a refrigerator at 4-5 ° FROM.