Method for promoting activation speed of feed lactic acid bacteria

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for promoting the activation speed of feed lactic acid bacteria. A carbon source and a nitrogen source rapidly activated by lactic acid bacteria are screened, and the adding proportion of the carbon source and the nitrogen source and the fermentation process are determined through a central combination experiment. Finally, the bacterial powder, the brown sugar, the pichia pastoris hydrolysate and the tap water are heated and activated for 12-24 hours in a water bath according to a certain proportion, so that the viable count of the lactic acid bacteria can reach 9.8 E + 08 CFU / m and is increased by 80-100 times compared with other carbon and nitrogen source culture. Therefore, the practical value is high.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for promoting the activation speed of lactic acid bacteria for feeding. Background Art

[0002] Lactic acid bacteria is a general term for a class of Gram-positive bacteria that can use carbohydrates to produce a large amount of lactic acid. Of the 34 microbial feed additives allowed to be added to feed, 22 belong to lactic acid bacteria, including Enterococcus faecium, Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, etc. (Liao Bo, Wang Weiwei, Wang Yongwei, et al. Research Progress on the Application of Feed Lactic Acid Bacteria in Livestock and Poultry Breeding [J]. China Feed, 2019 (19).). The physiological functions of feed lactic acid bacteria mainly include: (1) maintaining the balance of intestinal microecology and preventing pathogenic bacteria from causing harm to animal health; (2) improving animal disease resistance and enhancing animal nonspecific immunity; (3) producing digestive enzymes such as amylase, lipase, and protease during metabolism to promote animal digestion and absorption; (4) producing bacteriocins, which can prevent pathogenic bacteria from adversely affecting the intestinal health of animals after eating. Bacteriocins also have the function of preventing feed from being contaminated by pathogenic bacteria such as Salmonella.

[0003] Feed lactic acid bacteria are mainly stored in the form of powder. The bacteria in the stored state are dormant. When used, they must be activated and expanded for a period of time to obtain a culture with vigorous vitality and sufficient inoculation quantity. Increasing the number of live lactic acid bacteria in the fermentation process of biological feed is not only conducive to the accumulation of lactic acid bacteria metabolites in the feed, but also improves the production performance of animals, prolongs the shelf life of feed, and improves the stability of feed products. However, the price of feed lactic acid bacteria on the market is relatively expensive. If the inoculation amount of live lactic acid bacteria is increased by increasing the inoculation amount of lactic acid bacteria dry powder strains, the production cost of fermented feed will be greatly increased; and although extending the activation time can increase the number of live lactic acid bacteria to a certain extent, the time cost will reduce production efficiency and increase production costs. How to use the readily available carbon and nitrogen source combination to quickly increase the activation speed of lactic acid bacteria and increase the number of live lactic acid bacteria inoculation is a problem that is worth solving.

[0004] Summary of the invention

[0005] The purpose of the present invention is to screen a combination of easily available carbon sources and nitrogen sources for rapid activation of lactic acid bacteria, and to provide a method for rapidly promoting the activation speed of lactic acid bacteria for feeding in a short time, which can reduce the pH value of lactic acid bacteria liquid to below 4.5 and rapidly increase the number of live lactic acid bacteria to 1.0×10 8 -1.0×10 9 Reduce the amount of lactic acid bacteria used by about 100 times, saving the cost of using bacteria in the fermented feed production process.

[0006] The present invention provides a method for promoting the activation speed of lactic acid bacteria for feeding, which comprises the following steps:

[0007] (1) Weigh water, Pichia pastoris hydrolyzate, brown sugar, and lactic acid bacteria powder and dissolve them in a ratio of 100:1-3:1-3:0.5-2 to prepare a bacterial suspension;

[0008] (2) Keep the bacterial suspension at 32-36°C in a water bath for 10-30 hours.

[0009] Preferably, in step (1), water, Pichia pastoris hydrolysate, brown sugar, and lactic acid bacteria powder are weighed and dissolved in a ratio of 100:2:2:1 to prepare a bacterial suspension.

[0010] Preferably, in step (2), the bacterial suspension is kept warm in a well-ventilated environment without direct sunlight.

[0011] More preferably, in step (2), the temperature of the bacterial solution is controlled at 33-35°C and kept in a water bath for 12-24 hours, and more preferably, kept in a water bath at 34°C for 15-20 hours.

[0012] Optionally, the method further includes a step of detecting the number of viable lactic acid bacteria and / or the pH value.

[0013] Specifically, the detection of the number of live lactic acid bacteria is carried out with reference to the method in "GB 4789.35-2016 National Food Safety Standard Food Microbiology Examination Lactic Acid Bacteria Examination"; the pH value detection is carried out with reference to the method in "GB 5009.237-2016".

[0014] The present invention provides a straw silage method, which comprises the following steps:

[0015] S1: obtaining an activated lactic acid bacteria liquid according to the method described;

[0016] S2 uses straw as a substrate and inoculates the lactic acid bacteria liquid to perform anaerobic fermentation.

[0017] Preferably, the lactic acid bacteria liquid is inoculated at 10% (v / w), and the anaerobic fermentation is carried out by culturing in anaerobic bags.

[0018] Specifically, the temperature of the anaerobic fermentation is 32-36°C, preferably 35°C.

[0019] The present invention heats and activates lactic acid bacteria powder, brown sugar, Pichia pastoris hydrolyzate and tap water in a water bath for 12-24 hours according to a certain ratio, and the number of live lactic acid bacteria in the bacterial agent is increased by 80-100 times. The method of the present invention screens and obtains nitrogen source and carbon source, which can reduce production cost; at the same time, the active lactic acid bacteria are fermented for silage, which can increase the sour flavor of silage straw, and greatly increase the quality of silage straw by reducing the content of anti-nutritional factors (crude cellulose) and increasing the content of crude protein, that is, the fermented feed produced by using brown sugar as an activator in the subsequent production process is stable in quality, and the product has a stronger mellow flavor. Therefore, the present invention has significant advantages and promotion significance. DETAILED DESCRIPTION

[0020] Example 1, screening of the best carbon source:

[0021] The lactic acid bacteria agent, carbon source, Pichia pastoris hydrolyzate and tap water were respectively added in a ratio of 1:3:1.5:120, and different carbon sources (grapes, brown sugar, white sugar, sucrose) were added under the same culture temperature and time (cultured at 34℃ for 24h) to screen out the best carbon source activator with the number of lactic acid bacteria live cells and the pH value of the bacterial solution as indicators. The experimental design and results are shown in Table 1:

[0022] Table 1. Screening of the best carbon source activator for lactic acid bacteria agent

[0023]

[0024] Note: Different letters in the same column indicate significant differences (p<0.05).

[0025] As shown in Table 1, when the lactic acid bacteria were activated using four different carbon sources, the number of live lactic acid bacteria in the treatment group with brown sugar added after 12 hours of activation was significantly higher than that in the other treatment groups, and the pH value dropped below 5. When the bacteria were activated for 24 hours, the number of live lactic acid bacteria in the treatment group with brown sugar added was significantly higher than that in the other treatment groups, and the pH value was much lower than that in the other treatment groups. Therefore, brown sugar was selected as the carbon source for strain activation.

[0026] Example 2, screening of the best nitrogen source:

[0027] The lactic acid bacteria agent, brown sugar, nitrogen source and tap water were respectively added in a ratio of 1:3:1.5:120, and different nitrogen sources (Pichia pastoris hydrolysate, soy protein, whey protein, guar bean meal) were added under the same culture temperature and time (cultured at 34℃ for 12h and 24h). The number of viable lactic acid bacteria and the pH value of the bacterial solution were used as indicators to screen out the best nitrogen source activator. The experimental design and results are shown in Table 2:

[0028] Table 2. Screening of the best carbon source activator for lactic acid bacteria agent

[0029]

[0030] Note: Different letters in the same column indicate significant differences (p<0.05).

[0031] As shown in Table 2, when four different nitrogen sources were used to activate lactic acid bacteria, the number of lactic acid bacteria in the treatment group with Pichia pastoris hydrolysate added after 12 hours of bacterial activation was significantly higher than that in the other treatment groups, and the pH value dropped below 5. When the bacterial strain was activated for 24 hours, the number of lactic acid bacteria in the treatment group with Pichia pastoris hydrolysate added was significantly higher than that in the other treatment groups, and the pH value was much lower than that in the other treatment groups. Therefore, Pichia pastoris hydrolysate was selected as the nitrogen source for bacterial strain activation.

[0032] Example 3, orthogonal experiment of optimal fermentation process

[0033] Based on the above single factor experiment on the activation process of lactic acid bacteria, a five-factor four-level orthogonal experiment L16 (4 5 ), the main influencing factors (the amount of Pichia pastoris hydrolysate added, the amount of brown sugar added, the amount of clean water added, and the activation temperature) that affect the activation effect of lactic acid bacteria strains were analyzed by orthogonal experiment. The R value and K value of the orthogonal experiment were used to determine the influence of each factor on the entire lactic acid bacteria activation effect and the combination of the best lactic acid bacteria activation process. The orthogonal experimental design and results of the best activation process scheme of lactic acid bacteria agent and the best viable count of lactic acid bacteria and pH value under these conditions are shown in Table 4, wherein the inoculation amount of lactic acid bacteria agent is 1g, the activation time is 24h, the amount of Pichia pastoris hydrolysate added is 1.5g, 2g, 2.5g, 3g, the amount of brown sugar added is 1.5g, 2g, 2.5g, 3g, the activation temperature is 34°C, 35°C, 36°C, 37°C, and the amount of water added is 80mL, 100mL, 120mL, 140mL. The experimental design and results are shown in Table 3.

[0034] Table 3, L16(4 5 ) Orthogonal experimental design and results of the optimal activation process

[0035]

[0036]

[0037] Note: Numbers 1, 2, 3, and 4 represent factor levels. R1j represents the extreme value of live lactic acid bacteria count; R2j represents the extreme value of pH value of bacterial solution.

[0038] The experimental results in Table 3 show that the order of factors affecting the number of lactic acid bacteria during the activation of lactic acid bacteria is as follows: amount of clean water added > Pichia hydrolysate > brown sugar > activation temperature > blank column (R13 > R11 > R12 > R14 > R15), and the best activation process is: 2g of Pichia hydrolysate, 2g of brown sugar, 100mL of clean water, and 35°C activation temperature. The order of factors affecting the acid production ability of lactic acid bacteria during the activation of lactic acid bacteria is as follows: brown sugar > amount of clean water added > Pichia hydrolysate > temperature > blank column (R22 > R23 > R21 > R24 > R25).

[0039] Example 4: Verification Experiment

[0040] The following verification experiments are carried out based on the above experimental results:

[0041] (1) Confirmation experiment of live lactic acid bacteria count: The average live lactic acid bacteria count of three parallel experiments under the conditions of K21K22K23K24 was 9.8E+08CFU / mL, which was higher than the results of each experiment in Table 3, indicating that K21K22K23K24 is indeed the best lactic acid bacteria agent activation scheme.

[0042] (2) pH verification experiment: The average pH value of the bacterial solution from three parallel experiments under the conditions of K61K82K73K54 was 4.10, which was lower than the results of each experiment in Table 3, indicating that K61K82K73K54 is indeed the best solution for lactic acid bacteria to accumulate acidic metabolites.

[0043] The experimental results are shown in Table 4. When the inoculum was activated for 12 hours, the pH value of the inoculum dropped to 4.76. When the inoculum was activated for 24 hours, the pH value of the experimental group dropped to 4.16. This shows that the addition of brown sugar and Pichia pastoris hydrolyzate to the lactic acid bacteria inoculum is beneficial to the expansion of lactic acid bacteria and the accumulation of metabolites.

[0044] Table 4 Optimal activation effect of lactic acid bacteria agent

[0045]

[0046] Example 5. Experiment on application of activated lactic acid bacteria to straw silage

[0047] With straw as substrate, the lactic acid bacteria cultured under the optimal activation conditions were inoculated at 10% (v / w) and cultured in anaerobic bags at 35°C. After 24 hours, the Ph of the silage straw could be quickly reduced to 3.5, indicating that the number and activity of lactic acid bacteria cultured under the optimal activation agent were optimal. After 15 days of straw silage culture, the results are shown in Table 5.

[0048] Table 5 Results of relevant parameters after 15 days of straw silage cultivation

[0049] Straw index Before silage After silage Ph 4.5 3.0 Crude fiber content (%) 35 27 Crude protein content (%) 7.7 9.2 Number of viable bacteria 9.8E+08 10.3E+09

[0050] The results showed that the Ph of the silage straw was maintained at around 3.0, the crude fiber content was reduced from 35% to 27%, the crude protein content was increased from 7.7% to 9.2%, and the number of viable bacteria increased from the initial 9.8E+08 to 10.3E+09, increasing the sour aroma of the silage straw. The quality of the silage straw was greatly improved by reducing the content of anti-nutritional factors (crude cellulose) and increasing the crude protein content.

Claims

1. A method for promoting the activation speed of lactic acid bacteria for feeding, characterized in that: The steps include: (1) Weigh water, Pichia pastoris hydrolyzate, brown sugar, and lactic acid bacteria powder and dissolve them in a ratio of 100:1-3:1-3:0.5-2 to prepare a bacterial suspension; (2) Keep the bacterial suspension at 32-36°C in a water bath for 10-30 hours.

2. The method according to claim 1, characterized in that In step (1), water, Pichia pastoris hydrolyzate, brown sugar, and lactic acid bacteria powder are weighed and dissolved in a ratio of 100:2:2:1 to prepare a bacterial suspension.

3. The method according to claim 1, characterized in that In step (2), the bacterial suspension is kept warm in a well-ventilated environment without direct sunlight.

4. The method according to claim 1, characterized in that In step (2), the temperature of the bacterial solution is controlled to be 33-35°C and kept in a water bath for 12-24 hours. More preferably, the temperature is kept in a water bath at 34°C for 15-20 hours.

5. The method according to claim 1, characterized in that In step (2), the temperature of the bacterial solution was controlled at 34°C and kept in a water bath for 15-20 hours.

6. The method according to any one of claims 1 to 5, characterized in that: The method also includes the steps of detecting the number of viable lactic acid bacteria and / or the pH value.

7. The method according to claim 6, characterized in that The detection of the number of live lactic acid bacteria is carried out according to the method in "GB 4789.35-2016 National Food Safety Standard Food Microbiology Examination Lactic Acid Bacteria Examination"; the pH value detection is carried out according to the method in "GB 5009.237-2016".

8. A method for straw silage, characterized in that: The steps include: S1 obtaining an activated lactic acid bacteria liquid according to the method according to any one of claims 1 to 7; S2 uses straw as a substrate and inoculates the lactic acid bacteria liquid to perform anaerobic fermentation.

9. The method according to claim 8, characterized in that The lactic acid bacteria liquid is inoculated at 10% (v / w), and the anaerobic fermentation is carried out by placing the culture in an anaerobic bag.

10. The method according to claim 8, characterized in that The temperature of the anaerobic fermentation is 32-36°C, preferably 35°C.

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