INFLUENCE OF EXOGENOUS LIPOLYTIC ENZYMES IN THE PRODUCTION OF ALCOHOL FROM GRAIN RAW

ABSTRACT

The effect of rice bran lipases on wheat lipids was studied. Treatment of milled wheat with lipases increases the content of free fatty acids and this has a positive effect on the formation of secondary products during alcoholic fermentation. It has been established that in this case the content of aldehydes decreases by 2 times, and the content of fusel oils and methanol also decreases.

АННОТАЦИЯ

Изучено действие липаз из рисовой мучки на липиды пшеницы. Обработка пшеничного помола липазами способствует увеличению содержание свободных жирных кислот и это  положительно влияет на образование вторичных продуктов во время спиртового брожения. Установлено, что при этом содержание альдегидов уменьшается в 2 раза, а также снижается содержание сивушных масел и метанола.

Keywords: rice bran, lipase, grain must, lipids, hydrolysis, spiro fermentation, secondary products.

Ключевые слова: рисовая мучка, липаза, зерновое сусло, липиды, гидролиз, спировое брожение, вторичные продукты.

Introduction. Enzymes are biological catalysts used in alcohol technology due to their many benefits in terms of product quality [1].

The use of exogenous enzymes in the production of alcohol is due to numerous technological and economic advantages. Understanding the important role that enzymes play in alcohol technology contributes to the development of strategies for optimizing the production process to improve alcohol [2].

It is known that organic acids are important in carbon metabolism, energy metabolism of microorganisms, synthetic and dissimilation processes. The use of fatty acids as a carbon source depends on the type and race of yeast, the concentration of the acid, the length of its carbon chain, and the degree of electrolytic dissociation. Good substrates are acids with a carbon chain length from C₂ to C₆ (acetic, pyruvic, lactic, butyric, etc.) at a relatively low concentration. Potassium salts of acids containing from 2 to 5 carbon atoms in the molecule stimulate the growth of yeast by 1.4–3.3 times compared to the corresponding acids [3, 4].

Fatty acids with an average length of carbon chain (from C₆ to C₁₀ ) are consumed to a lesser extent by yeast and at very low concentrations in the medium (0.02-0.05%). Higher concentrations inhibit the development of yeast. Fatty acids with 12–17 carbon atoms per molecule are consumed selectively depending on the genus and species of yeast [5–6].

The purpose of this work was to study the effect of lipolytic enzymes on the formation of secondary products during alcoholic fermentation.

Materials and methods

Reagents . We used a lipase preparation obtained by degreasing rice bran with hexane and subsequent extraction with 0.05 M phosphate buffer pH 7.0.

For the preparation of buffer solutions, one- and two-substituted potassium phosphate ( chemically pure " Reakhim " ), rhodamine 6G ( chemically pure " Reakhim " ) , acetic acid ( chemically pure " Reakhim " ) , rectified alcohol (JSC " Biokimyo ") and hexane ( chemically pure " Reachim " ), silica gel KSK ( chemically pure " Reachim " ).

Determination of lipolytic activity

Lipolytic activity was measured by a photocolorimetric method using rhodamine 6G as a chromophore agent [7]. The specific activity was expressed in µmol of fatty acids released in 1 hour per 1 mg of protein.

Refined cottonseed oil emulsified with an equal volume of 1% polyvinyl alcohol was used as a substrate. The reaction mixture with a volume of 2 ml, containing 0.2 ml of the substrate in 0,1 Мphosphate buffer, pH 7.5, and 20 мМ CaCl ₂ , was incubated at 37 ⁰ in the enzyme solution for 1 h. Then 0.25 ml of the reaction mixture was taken and 1 ml of 0.1 M HCl in ethanol was added to stop the reactions. The remaining operations were performed according to the procedure [7].

Enzymatic hydrolysis of lipids of grain milling

A 30% suspension of wheat milling was prepared in 0.1M phosphate buffer pH 6.5, a 0.1% solution of lipases obtained from rice bran was added. The mixture was stirred and kept for a certain time in a thermostat at a temperature of 37^°C, after which 0.25 ml of samples were taken to determine the products of hydrolysis ( released fatty acids), and 1 ml 0,1 М of HCl in ethanol was added to stop the reactions. The content of lipid hydrolysis products was determined from a calibration curve constructed for palmitic acid, according to the procedure [7].

Fermentation of saccharified wort in the presence of lipolytic enzymes. Experiments were carried out at the Yangiyul joint-stock company “Biokhim”. Sugared mash for fermentation was prepared in accordance with the accepted technological scheme. The fermentation process was carried out for 78 hours at a temperature of 30^oC. After the completion of the fermentation process, raw alcohol was isolated from the mash by distillation and analyzed for the content of secondary fermentation products.

Determination of the composition of alcohols.

Raw alcohol samples were analyzed using an Agilent 7890 gas chromatograph connected to a 5975 C spectrophotometer. inert XL EI/CI MSD .

Results and discussion

Figure 1 shows the effect of medium pH and temperature on the specific activity of lipases from rice flour. From the presented data, it can be seen that the maximum lipolytic activity is manifested at a pH value of the medium of 6-7 (Fig. 1, a). The temperature optimum of action was 45-52 ^° C (Fig. 1, b).

a                                  b

Figure 1. Influence of medium pH (a) and temperature (b) on the activity of lipases from rice bran

On fig. 2 shows the kinetics of the formation of free fatty acids during the fermentation of wheat flour slurry in aqueous media. From the presented data, it can be seen that the rate of formation of lipid hydrolysis products depends on the reaction temperature.

It can be seen from the presented data that the initial rate of lipid hydrolysis with rice flour lipases at 30^°C is slow. After 2 hours of fermentation, the content of free fatty acids in the reaction medium was 2.3 μmol/ml. (Figure 2, curve 1.) Increasing the fermentation temperature to 37^oC and 45^oC contributed to an increase in the formation of free fatty acids. For example, at these temperatures, after a 2-hour fermentation, the free fatty acid content was 6.3 and 8.7 µmol/ml, respectively. At 52 ^° C, the initial rate of hydrolysis increases, but due to thermal denaturation, the fatty acid content will eventually be lower than when the reaction is carried out at 45^°C .

Figure 2. The effect of temperature on the hydrolysis of lipids in grain milling
1 - at 30 ^o C, 2 - at 37 ^o C, 3 - at 45 ^o C, 4 - at 52 ^o C

In further experiments, the influence of these enzymes on the formation of secondary products during fermentation was investigated.

It is known that most of the biochemical reactions that occur in the process of obtaining alcohol are catalyzed by enzymes. Acetaldehyde, formed during glyceropyruvine fermentation, is the main source of formation of secondary products of alcoholic fermentation. Acetaldehyde inhibits the fermentation process, so the physiological need for yeast is its transformation into other products, namely, acids, ethyl alcohol, 2,3-butylene glycol, etc. [3, 4].

The effect of lipolytic enzymes on the formation of secondary products during alcoholic fermentation is listed in Table 1.

Table 1.

Influence of lipases from rice bran on the chemical composition of alcohol (product content mg/l)

From the presented data, it can be seen that the use of lipases during the processing of grain wort contributes to a change in the chemical composition of the resulting alcohols.

Lipase from rice flour also contributes to a decrease in the content of aldehydes by almost 1.4 times and it is 10.9 mg/l, esters are reduced by 9%, condensed oils by 10% and isopropanol by 7-8%. At the same time, the content of methanol and an increase of 25%.

The study conducted by us showed that the introduction of lipolytic enzymes from the plant revealed the effect of reducing the content of aldehydes, esters and the content of fusel oils in the mash. This had a positive effect on the fermentation process.

It has been shown that an increase in the yield of ethyl alcohol during the fermentation of wort enriched with lipolysis products is due to a more economical process, namely, an increase in the degree of bioconversion of carbohydrates into ethanol, a reduction in starch losses and the formation of by-products.

Thus, the research results point to the important role of the enzymes of the lipolytic complex in the process of alcoholic fermentation and yeast generation . The effect of lipases on the lipids of grain must increases the efficiency of its hydrolysis, enriches the medium with fatty acids, which ultimately contributes to an increase in the physiological activity of yeast cells, intensification of the fermentation process and an increase in the yield of the target product.

References:

1. Римарева, Л. В. Влияние ферментативных систем на биохимический состав зернового сусла и культуральные свойства осмофильной расы спиртовых дрожжей Saccharomycescerevisiae/Л.В. Римарева, М.Б. Овчеренко, Е.М. Серба, Н.И. Игнатова // Производство спирта и ликероводочных изделий. – 2013. – № 1. – С. 16–18.
2. Поляков, В.А. Научное обеспечение инновационного развития спиртовой отрасли на пути интегрирования в мировую экономику / В.А. Поляков, Л.В. Римарева // Производство спирта и ликероводочных изделий. – 2013. – № 1 –С.4-8 .
3. Алексеева А.А. Зависимость концентрации ароматообразующих компонентов и органических кислот в белом столовом виноматериале от способа технологической обработки // Известия высших учебных заведений. Пищевая технология. 2019. № 5/6 (371-372). С. 27-30.
4. Killian, E. and Ough, C.S. (1979). Fermentation esters – formation and retention as affected by fermentation temperature. American Journal of Enology and Viticulture, 30: 301–30
5. Viegas , C. A., et al. Inhibition of yeast growth by octanoic and decanoic acids produced during ethanolic fermentation. Applied and Environmental Microbiology. 1989, 55(1), pp. 21–28
6. Liu, P., Ivanova-Petropulos, V., Duan, C., and Yan, G. (2021). Effect of unsaturated fatty acids on intrametabolites and aroma compounds of Saccharomyces cerevisiae in wine fermentation. Foods, 10(2): 277.
7. Anderson M.M., McCarty R.E. Rapid and sensitive assay for free fatty acids using rhodamine 6G //Anal.Biochem., 1972, v.45, p.260-270.