Compound bacterium high-density culture medium as well as preparation method and application thereof

Abstract

The invention discloses a compound bacterium high-density culture medium as well as a preparation method and application thereof, and relates to the technical field of microbial fermentation. The compound bacterium high-density culture medium comprises soybean meal and bran zymolyte, corn flour, soybean oil, dipotassium phosphate, sodium carbonate, magnesium sulfate heptahydrate and purified water. The soybean meal and bran zymolyte is a product obtained by carrying out enzymolysis on soybean meal and bran through combined enzymes, and the combined enzymes comprise cellulase, pectinase and protease. The preparation method comprises the following steps: mixing the soybean meal and bran zymolyte with the other components, and adjusting the pH value to 5.5-7.5 to obtain the compound bacterium high-density culture medium. The invention further discloses application of the high-density culture medium for the compound bacteria to high-density fermentation in a farm and application of the high-density culture medium for the compound bacteria to high-density fermentation preparation of compound bacteria fermentation liquor. According to the method, the fermentation efficiency and the thallus concentration of the fermentation liquid can be effectively improved, the viable count of the culture liquid can reach 5.8 * 10 < 12 > cfu / mL, the fermentation time is shortened, and high-density fermentation in a farm site is realized.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation, specifically to a high-density culture medium containing compound bacteria, its preparation method, and its application. Background Technology

[0002] Bacillus coagulans, Bacillus subtilis, and lactic acid bacteria are all common probiotic species. They are non-toxic, non-pathogenic, and do not release any toxins inside or outside the body. Sufficient consumption of probiotics can enhance the production of metabolic products, improve immunity, maintain intestinal flora balance, and stimulate growth. They do not have negative antibiotic effects and help maintain the balance of the natural flora in animals or prevent dysbiosis. Furthermore, with the continuous development of biotechnology, they show promising application prospects in the pharmaceutical, food, and livestock industries.

[0003] With the development of high-cell-density cultivation (HCDC), freeze-drying, and other technologies, standardized and highly stable freeze-dried probiotic powders have provided a sufficient guarantee for the development of probiotics. Therefore, how to increase the viable count of probiotics has become a hot topic in probiotic fermentation. The culture medium, as a basic condition for microbial growth, directly affects the proliferation and activity of Bacillus coagulans, Bacillus subtilis, and lactic acid bacteria during the cultivation process, depending on its composition.

[0004] Previous studies have primarily focused on single-strain fermentation, but the relatively simple composition of metabolites produced by single-strain fermentation is insufficient to meet the demands of modern agriculture for microbial agents. With further research, multi-strain mixed fermentation has gained increasing attention. However, obtaining high-concentration fermentation broth from mixed microorganisms through high-density culture technology requires optimizing a series of related process parameters for strict control of the fermentation process. Research indicates that many factors influence mixed microbial fermentation, such as strain selection, culture medium composition, initial pH of the culture medium, temperature, culture time, and inoculum size.

[0005] Therefore, developing a suitable culture medium for compound microbial fermentation is crucial for obtaining high-concentration, high-activity compound microbial fermentation broth. At the same time, the initial compound microbial fermentation will also have a significant impact on the application of the fermentation broth in the farm. Summary of the Invention

[0006] The purpose of this invention is to provide a high-density culture medium for compound bacteria, its preparation method and application, to prepare a high-density culture medium for compound bacteria, and to obtain a high-concentration, high-activity compound bacteria fermentation broth by fermenting a compound bacteria system in the high-density culture medium for compound bacteria, thereby improving the problem of low viable bacteria count in existing probiotic fermentation.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A high-density culture medium for compound bacteria, comprising the following components by weight percentage: 1.5-2.5% soybean meal and bran hydrolysate, 0.8-1.5% corn flour, 0.8-1.5% soybean oil, 0.15-0.25% dipotassium hydrogen phosphate, 0.1-0.2% sodium carbonate, 0.02-0.03% magnesium sulfate heptahydrate, with the balance being water;

[0009] The soybean meal and bran enzymatic hydrolysate is the product obtained by enzymatic hydrolysis of soybean meal and bran using a combination of enzymes, including cellulase, pectinase, and protease.

[0010] Sterile distilled water or reverse osmosis pure water can be used, with a conductivity of ≤5μS / cm. This avoids the inhibition of Bacillus spore germination by chloride ions in ordinary water; it also prevents calcium and magnesium ions from forming precipitates with phosphates, thus reducing nutrient utilization; and it can also reduce microbial contamination.

[0011] This invention does not directly use expensive yeast extracts, peptones, or single soybean meal / bran. Instead, it uses soybean meal (a high-protein nitrogen source) and bran (a high-fiber carbon source, vitamin / mineral source) to produce soybean meal and bran hydrolysates through enzymatic hydrolysis. These two raw materials are widely available and inexpensive. The soybean meal and bran hydrolysates (1.5-2.5%) significantly improve the utilization rate of components such as cellulose, hemicellulose, and protein in the raw materials that are originally difficult for microorganisms to utilize directly. The enzymatic hydrolysis process may produce or release unknown growth factors that promote microbial growth. By setting the hydrolysates as the main organic nutrient component of the culture medium, it replaces the expensive organic nitrogen and carbon sources (such as yeast powder, peptone, glucose, etc.) commonly used in traditional high-density culture media, which can effectively reduce costs and is suitable for large-scale promotion.

[0012] Based on the main organic nutrients provided by soybean meal and bran hydrolysate, corn flour and soybean oil are added. Corn flour provides starch carbon source, which is inexpensive and complements the carbon source of hydrolysate; soybean oil provides lipid carbon source and energy, forming a multi-level carbon source supply to meet the carbon requirements of different strains or different growth stages, and soybean oil is used to suppress foam.

[0013] Dipotassium hydrogen phosphate provides phosphorus, essential for nucleic acid and energy metabolism, and potassium, necessary for maintaining osmotic pressure. An appropriate amount of magnesium sulfate heptahydrate provides magnesium, which is a cofactor for many enzymes, meeting the needs of high-density growth while avoiding excessive inhibition. Sodium carbonate (0.1-0.2%) is mainly used to neutralize acidic substances (such as organic acids) that may be produced in the early stages or during fermentation, maintaining a relatively stable pH within a suitable range; together with phosphate, it forms a buffer system to maintain cell activity and metabolism.

[0014] This invention utilizes a soybean meal and bran complex treated with specific enzymatic hydrolysis (cellulase + pectinase + protease) as the core nutrient source. It combines low-cost, readily available raw materials (enzymatic hydrolysate, corn flour, soybean oil) and optimizes the ratio of key inorganic salts (P, Mg) and buffers (Na) to provide a highly efficient and economical overall nutritional solution based on the deep processing (enzymatic hydrolysis) of inexpensive agricultural by-products. It is specifically designed for high-density culture of compound microorganisms and organically integrates enzymatic hydrolysis technology, raw material combination, nutritional balance, and cost control.

[0015] A preparation method for preparing the aforementioned high-density culture medium containing compound bacteria includes the following steps: mixing soybean meal and wheat bran enzymatic hydrolysate with the remaining components, and adjusting the pH to 5.5–7.5 to obtain the high-density culture medium containing compound bacteria.

[0016] Further, the soybean meal and bran enzymatic hydrolysate, by weight, comprises the following components: 3.5 to 5.5 parts of hydrolysate raw material and 0.95 to 1.2 parts of enzymatic hydrolysate agent, wherein the hydrolysate raw material includes 2 to 3.5 parts of soybean meal and 1.5 to 2 parts of bran; the enzymatic hydrolysate includes 0.2 to 0.25 parts of cellulase, 0.25 to 0.35 parts of pectinase, and 0.5 to 0.6 parts of protease.

[0017] Furthermore, the preparation method of the soybean meal and bran enzymatic hydrolysate includes the following: at a temperature of 25-40°C, the enzymatic hydrolysate raw material is added to deionized water and stirred evenly, while maintaining a humidity of 60-80%, an enzyme preparation is added and mixed evenly to obtain an enzymatic hydrolysate mixture; the enzymatic hydrolysate mixture is placed at room temperature for fermentation, stirred once every 4-6 hours, and fermented for 30-40 hours to obtain the soybean meal and bran enzymatic hydrolysate.

[0018] The preparation method of soybean meal and wheat bran enzymatic hydrolysate employs a specific composite enzymatic hydrolysis process, combining cellulase, pectinase, and protease for effective disruption of plant cell walls (cellulase and pectinase), and thorough hydrolysis of proteins (protease), converting macromolecules into small-molecule peptides, amino acids, fermentable sugars, vitamins, and other nutrients that are easily absorbed and utilized by microorganisms. Through the controlled soybean meal / wheat bran ratio and targeted formulation of the enzymatic hydrolysate, the carbon and nitrogen source forms are upgraded, achieving a small-molecule rate of over 85%. This semi-solid, moisture-limited fermentation system overcomes the problems of high water consumption in traditional liquid enzymatic hydrolysis and low efficiency in solid-state fermentation.

[0019] The application of the aforementioned high-density culture medium for compound bacteria, the application of the high-density culture medium for compound bacteria in high-density fermentation in aquaculture farms, and the application of the high-density culture medium for compound bacteria in the preparation of compound bacteria fermentation broth through high-density fermentation.

[0020] Furthermore, the preparation method of the compound bacterial fermentation broth includes the following steps:

[0021] S100. Two or more strains with synergistic effects are screened using the plate confrontation method to form a compound bacterial system.

[0022] S200. The compound bacterial strain is inoculated into a seed culture medium and cultured to obtain a compound bacterial seed liquid;

[0023] S300. After mixing the compound bacterial seed liquid with the fermentation agent, inoculate it into the compound bacterial high-density culture medium and carry out fermentation until the OD600 of the fermentation liquid is stable, thus obtaining the compound bacterial fermentation liquid.

[0024] Traditional fermentation requires specialized fermentation tanks (≥5 tons) and a sterile workshop. This solution is suitable for existing facilities in farms (1-2 ton mixing tanks), controlling contaminating microorganisms through the composition of the culture medium. The culture medium is prepared directly using the farm's own soybean meal / wheat bran / corn, eliminating transportation costs. Symbiotic microorganisms are screened using the plate confrontation method, and the microbial community is constructed based on the principle of niche competition. OD600 stability is used as the endpoint (not a fixed time) to avoid over-fermentation leading to microbial death, resulting in a viability rate >95%.

[0025] Preparation method of compound bacterial seed liquid:

[0026] Step 1: Inoculate the selected compound bacterial strains into the corresponding liquid culture media:

[0027] Bacillus coagulans and Bacillus subtilis: LB liquid medium, shaken and cultured at 35-37℃ for 12-16 hours;

[0028] Lactic acid bacteria: MRS liquid medium, anaerobic culture at 37℃ for 18-24h;

[0029] Step 2: Mix the culture media of each strain in proportion, collect the bacterial cells by centrifugation, and suspend them in sterile physiological saline to adjust the viable cell concentration to 1×10⁻⁶. 9 ~5×10 9 The concentration of cfu / mL was used to obtain a compound bacterial seed culture.

[0030] This invention integrates four dimensions: culture medium formulation optimization, microbial community construction method, simplified fermentation process, and application scenarios, forming a closed-loop solution for self-made microbial agents in aquaculture farms.

[0031] Further, in step S100, the composite bacterial system includes two or three of Bacillus coagulans, Bacillus subtilis, and lactic acid bacteria, with Bacillus coagulans accounting for 30-55 parts, Bacillus subtilis accounting for 10-30 parts, and lactic acid bacteria accounting for 40-60 parts by weight.

[0032] Furthermore, the fermenting agent, by weight, comprises 30-38 parts molasses, 15-25 parts snowflake powder, and 42-50 parts silkworm pupa protein powder, and the mass ratio of the compound bacterial seed liquid to the fermenting agent is 1:2.3-2.8.

[0033] Furthermore, in step S300, the mass ratio of the high-density culture medium to the seed culture of the compound bacteria is 0.1–0.25:1; the fermentation temperature is 25–40℃.

[0034] Furthermore, in step S300, a fermentation method that removes product repression is adopted: natural fermentation with aeration and stirring is carried out for 0 to 24 hours; the pH of the fermentation broth is monitored for 24 to 40 hours, and when the pH reaches 4.5 to 5.5, 30% to 40% of the volume of fermentation broth is released, and high-density culture medium of compound bacteria is added according to the corresponding volume ratio.

[0035] This invention achieves physical removal of inhibitors and nutrient reset by releasing the fermentation broth and then replenishing the culture medium. A pH threshold (4.5–5.5) trigger mechanism is set, using metabolites as signals. When the pH of the fermentation broth drops to 4.5–5.5 (the critical point for lactic acid / acetic acid accumulation), it indicates an excess of carbon source, byproducts inhibiting enzyme activity (e.g., phosphofructokinase is inhibited by ATP), and a decrease in cell growth rate (product repression effect). At this point, releasing 30–40% of the fermentation broth can remove more than 60% of organic acid inhibitors (measured lactic acid concentration decreased from 35 mM to 15 mM), break the high osmotic pressure environment, and restart logarithmic growth by replenishing the culture medium.

[0036] The fermentation method that removes product repression transforms the continuous feed-in process of industrial fermentation into a semi-continuous replacement process suitable for farms. It achieves the removal of metabolic waste, the restart of nutrient niches, and the generational renewal of microbial communities through the low-cost physical removal process. Using pH, a parameter that can be easily monitored in farms, as a control switch, it finds the golden balance between microbial metabolic regulation and on-site operability.

[0037] Because the bacteria produce a large amount of prebiotics during their growth, such as antimicrobial peptides, bacteriocins, various digestive enzymes (protease, amylase, lipase, and xylanase, etc.), organic acids (lactic / acetic acid, propionic acid, butyric acid), and B vitamins, the released fermentation broth can be directly diluted with water and given to farmed animals for drinking. It has significant effects on improving animal intestinal health, enhancing growth performance, and boosting immunity. In addition, the released fermentation broth, diluted to a certain ratio, can be sprayed into the farming environment, such as pens and manure treatment ponds, to effectively inhibit the growth of pathogens and reduce the incidence of respiratory and skin diseases in farmed animals.

[0038] Compared with the prior art, the beneficial effects of the present invention are:

[0039] This invention adds enzymatic hydrolysates of soybean meal and wheat bran to a high-density culture medium for compound bacteria. The small molecules produced by enzymatic hydrolysis are more suitable for high-density fermentation of compound bacteria. Simultaneously, inexpensive raw materials such as molasses, corn starch, and soybean oil are used as carbon and nitrogen sources for the culture. The raw materials for the high-density culture medium for compound bacteria are widely available, inexpensive, and easy to prepare, allowing for large-scale use. Furthermore, this culture medium can effectively increase the concentration and activity of the compound bacteria in the high-density fermentation broth, reducing fermentation costs and showing promising application prospects.

[0040] The high-density culture medium for compound bacteria of the present invention can be used to prepare compound bacterial fermentation broth through fermentation with compound bacterial strains. The fermentation method is simple and efficient, and can effectively improve fermentation efficiency and cell concentration in the fermentation broth. The viable cell count in the culture broth can reach 5.8 × 10⁻⁶. 12 With a concentration of cfu / mL, a culture medium pH of 3.0–5.0, and a fermentation time of only 30–40 hours, the fermentation time is shortened, enabling high-density fermentation on-site in farms. Detailed Implementation

[0041] Example 1

[0042] The soybean meal and bran enzymatic hydrolysate comprises the following components: 450g of hydrolysate raw material and 105g of enzymatic hydrolysate agent, wherein the hydrolysate raw material includes 280g of soybean meal and 170g of bran; the enzymatic hydrolysate agent includes 22g of cellulase, 30g of pectinase, and 53g of protease.

[0043] The preparation method of the soybean meal and bran enzymatic hydrolysate includes the following: at a temperature of 32°C, the enzymatic hydrolysate raw material is added to deionized water and stirred evenly, while maintaining a humidity of 70%. An enzyme preparation is added and mixed evenly to obtain an enzymatic hydrolysate mixture. The enzymatic hydrolysate mixture is placed at room temperature for fermentation, stirred once every 5 hours, and fermented for 35 hours to obtain the soybean meal and bran enzymatic hydrolysate.

[0044] Example 2

[0045] The soybean meal and wheat bran enzymatic hydrolysate comprises the following components: 350g of hydrolysate raw material and 95g of enzymatic hydrolysate agent, wherein the hydrolysate raw material includes 200g of soybean meal and 150g of wheat bran; the enzymatic hydrolysate agent includes 20g of cellulase, 25g of pectinase, and 50g of protease.

[0046] The preparation method of the soybean meal and bran enzymatic hydrolysate includes the following: at a temperature of 25°C, the enzymatic hydrolysate raw material is added to deionized water and stirred evenly, while maintaining a humidity of 60%. An enzyme preparation is added and mixed evenly to obtain an enzymatic hydrolysate mixture. The enzymatic hydrolysate mixture is placed at room temperature for fermentation, stirred once every 4 hours, and fermented for 30 hours to obtain the soybean meal and bran enzymatic hydrolysate.

[0047] Example 3

[0048] The soybean meal and bran enzymatic hydrolysate comprises the following components: 550g of hydrolysate raw material and 120g of enzymatic hydrolysate agent. The hydrolysate raw material includes 350g of soybean meal and 200g of bran. The enzymatic hydrolysate agent includes 25g of cellulase, 35g of pectinase, and 60g of protease.

[0049] The preparation method of the soybean meal and bran enzymatic hydrolysate includes the following: at a temperature of 40°C, the enzymatic hydrolysate raw material is added to deionized water and stirred evenly, while maintaining a humidity of 80%, an enzyme preparation is added and mixed evenly to obtain an enzymatic hydrolysate mixture; the enzymatic hydrolysate mixture is placed at room temperature for fermentation, stirred once every 6 hours, and fermented for 40 hours to obtain the soybean meal and bran enzymatic hydrolysate.

[0050] The performance of the enzymatic hydrolysates of soybean meal and wheat bran prepared in Examples 1-3 is shown in Table 1.

[0051] Table 1. Performance of the enzymatic hydrolysates of soybean meal and wheat bran prepared in Examples 1-3

[0052]

[0053] As shown in Table 1, the enzymatic hydrolysates of soybean meal and wheat bran prepared in Examples 1-3 can effectively hydrolyze proteins and cellulose. After enzymatic hydrolysis, the amino nitrogen content can reach 41.2 g / kg, and the reducing sugar content can reach 168 g / kg, which can provide sufficient nitrogen and carbon sources for bio-fermentation. The retention rate of B vitamins can reach 91.2%, which enhances the colonization ability of bacteria and promotes the growth rate of Bacillus subtilis by 143%. The proportion of small molecule peptides in Examples 1-3 is all >58%, which proves that the compound enzymatic hydrolysis strategy (cellulase + pectinase + protease) has universality for the efficient degradation of plant proteins.

[0054] Example 4

[0055] A high-density culture medium for compound bacteria comprises the following components: 200g of soybean meal and bran enzymatic hydrolysate, 120g of corn flour, 110g of soybean oil, 20g of dipotassium hydrogen phosphate, 15g of sodium carbonate, 2.5g of magnesium sulfate heptahydrate, and 9532.5g of water; wherein the soybean meal and bran enzymatic hydrolysate is prepared by the method of Example 1.

[0056] A preparation method for preparing the aforementioned high-density culture medium containing compound bacteria includes the following steps: mixing soybean meal and wheat bran enzymatic hydrolysate with the remaining components, and adjusting the pH to 6.5 to obtain the high-density culture medium containing compound bacteria.

[0057] Example 5

[0058] A high-density culture medium for compound bacteria comprises the following components: 150g of soybean meal and bran enzymatic hydrolysate, 80g of corn flour, 80g of soybean oil, 15g of dipotassium hydrogen phosphate, 10g of sodium carbonate, 2g of magnesium sulfate heptahydrate, and 9663g of water; wherein the soybean meal and bran enzymatic hydrolysate is prepared by the method of Example 1.

[0059] A preparation method for preparing the aforementioned high-density culture medium containing compound bacteria includes the following steps: mixing soybean meal and wheat bran enzymatic hydrolysate with the remaining components, and adjusting the pH to 5.5 to obtain the high-density culture medium containing compound bacteria.

[0060] Example 6

[0061] A high-density culture medium for compound bacteria comprises the following components: 250g of soybean meal and bran enzymatic hydrolysate, 150g of corn flour, 150g of soybean oil, 25g of dipotassium hydrogen phosphate, 20g of sodium carbonate, 3g of magnesium sulfate heptahydrate, and 9402g of water; wherein the soybean meal and bran enzymatic hydrolysate is prepared by the method of Example 1.

[0062] A preparation method for preparing the aforementioned high-density culture medium containing compound bacteria includes the following steps: mixing soybean meal and wheat bran enzymatic hydrolysate with the remaining components, and adjusting the pH to 7.5 to obtain the high-density culture medium containing compound bacteria.

[0063] Comparative Example 1

[0064] A high-density culture medium for compound bacteria comprises the following components: 250g of unenzymatically hydrolyzed soybean meal and wheat bran, 150g of corn flour, 150g of soybean oil, 25g of dipotassium hydrogen phosphate, 20g of sodium carbonate, 3g of magnesium sulfate heptahydrate, and 9402g of water; wherein the enzymatic hydrolysate of soybean meal and wheat bran is prepared by the method of Example 1.

[0065] A preparation method for preparing the aforementioned high-density culture medium containing compound bacteria includes the following steps: mixing soybean meal and wheat bran enzymatic hydrolysate with the remaining components, and adjusting the pH to 7.5 to obtain the high-density culture medium containing compound bacteria.

[0066] Comparative Example 2

[0067] A high-density culture medium for compound bacteria comprises the following components: 250g of soybean meal and bran enzymatic hydrolysate, 330g of corn flour, 150g of soybean oil, 25g of dipotassium hydrogen phosphate, 20g of sodium carbonate, 3g of magnesium sulfate heptahydrate, and 9402g of water; wherein the soybean meal and bran enzymatic hydrolysate is prepared by the method of Example 1.

[0068] A preparation method for preparing the aforementioned high-density culture medium containing compound bacteria includes the following steps: mixing soybean meal and wheat bran enzymatic hydrolysate with the remaining components, and adjusting the pH to 7.5 to obtain the high-density culture medium containing compound bacteria.

[0069] Comparative Example 3

[0070] A high-density culture medium for compound bacteria comprises the following components: 180g yeast extract, 200g glucose, 150g soybean oil, 25g dipotassium hydrogen phosphate, 20g sodium carbonate, 3g magnesium sulfate heptahydrate, and 9402g water; wherein the soybean meal and bran enzymatic hydrolysate is prepared by the method of Example 1.

[0071] A preparation method for preparing the aforementioned high-density culture medium containing compound bacteria includes the following steps: mixing soybean meal and wheat bran enzymatic hydrolysate with the remaining components, and adjusting the pH to 7.5 to obtain the high-density culture medium containing compound bacteria.

[0072] Examples 4-6 and Comparative Examples 1-2 all used the same compound bacterial strain (Bacillus subtilis: Lactic acid bacteria: Yeast = 5:3:2), with an initial viable cell density of 3.5 × 10⁻⁶. 8 The fermentation conditions were consistent (35℃, 200rpm, aeration rate 1vvm), and the fermentation performance comparison table is shown in Table 2.

[0073] Table 2 Comparison of fermentation performance of Examples 4-6 and Comparative Examples 1-2 using the same compound microbial strain

[0074] Evaluation indicators Example 4 Example 5 Example 6 Comparative Example 1 Comparative Example 2 Comparative Example 3 Detection method <![CDATA[Maximum OD 600 > 28.5 24.8 32.7 16.2 26.3 30.5 Spectrophotometry viable cell density (cfu / mL) <![CDATA[4.2×10 10 ]]> <![CDATA[3.5×10 10 ]]> <![CDATA[5.1×10 10 ]]> <![CDATA[8.7×10 9 ]]> <![CDATA[3.8×10 9 ]]> <![CDATA[4.8×10 10 ]]> Plate counting method Fermentation cycle (h) 38 42 36 52 45 34 <![CDATA[OD 600 Stability determination Residual sugar content (g / L) 3.2 2.8 3.8 12.5 15.7 4.5 DNS Law Organic acids (mmol / L) 18.3 15.2 20.5 8.4 35.6 22.8 HPLC Cell yield (gDCW / g substrate) 0.48 0.43 0.52 0.21 0.39 0.47 Dry weight method <![CDATA[Viable count per unit cost (×10 13 cfu / yuan)]]> 2.27 2.03 2.58 0.53 2.01 0.58 viable cell count / culture medium cost

[0075] As shown in Table 2, Examples 4-6, which used soybean meal and wheat bran hydrolysate and corn flour, had a maximum OD value of [missing information]. 600 24.8–32.7, viable cell density 3.5–5.1 × 10⁻⁶ 10 The fermentation time was 36–42 h, with a residual sugar content of only 2.8–3.8 g / L, an organic acid production of 15.2–20.5 mmol / L, a cell yield of 0.43–0.52 g DCW / g substrate, and a viable cell count per unit cost of 2.03–2.58 × 10⁻⁶ cells / mL. 13 cfu / yuan.

[0076] Comparative Example 1 used unenzymatically hydrolyzed soybean meal bran and corn flour, and the viable cell density of Comparative Example 1 was only 8.7 × 10⁻⁶. 9 The viable cell density in Example 6 was 5.1 × 10⁻⁶ CFU / mL. 10In Example 6, the viable cell density was increased by 486% (cfu / mL). Example 6 used enzymatic hydrolysate to increase the proportion of small molecule nutrients (amino nitrogen + reducing sugar), thus prolonging the logarithmic growth phase of the cells. The residual sugar content in Comparative Example 1 was 12.5 g / L, while in Example 6 it was only 3.8 g / L, a reduction of 69.6%. In Example 6, the cellulase in the enzymatic hydrolysate pre-decomposed bran, avoiding undegraded fiber residue. The organic acid production in Comparative Example 1 was 8.4 mmol / L, while in Example 6 it was 20.5 mmol / L, an increase of 59%. The enzymatic hydrolysate in Example 6 improved the utilization rate. The viable cell count per unit cost in Comparative Example 1 was 0.53 × 10⁻⁶. 13 CFU / yuan, Example 6: Unit cost viable count 2.58 × 10⁻⁶ 13 CFU / yuan, Example 6 shows a 387% increase in viable bacteria count at the same cost.

[0077] Comparative Example 2 used soybean meal and wheat bran hydrolysate and excess corn flour, resulting in an imbalanced carbon-nitrogen ratio and an organic acid production of up to 35.6 mmol / L. Compared with Example 6, the organic acid accumulation increased by 75%, inhibiting cell growth. The osmotic pressure increased, and the cell yield was 0.39 g DCW / g substrate, which was 25% lower than that of Example 6.

[0078] Comparative Example 3 used yeast extract and glucose, while Comparative Example 2 had the highest OD. 600 30.5, viable cell density, 4.8 × 10⁻⁶ 10 With a CFU / mL fermentation rate of 34 hours, the residual sugar content was only 4.5 g / L, the organic acid production reached 22.8 mmol / L, and the cell yield was 0.47 g DCW / g substrate, all comparable to Example 6; however, the unit cost viable cell count of Comparative Example 2 was 0.58 × 10⁻⁶. 13 CFU / yuan, the unit cost viable count in Example 6 is 2.58 × 10⁻⁶. 13 CFU / yuan, Example 6 shows a 344% increase in viable bacteria count at the same cost.

[0079] This invention demonstrates that the soybean meal and bran enzymatic hydrolysate, in synergy with precisely proportioned inorganic components, achieves a 344% increase in live bacteria yield per unit cost, while maintaining a comparable live bacteria density to that of commercial culture medium (Comparative Example 3), and is also compatible with non-standard equipment in livestock farms.

[0080] Example 7

[0081] Seed liquid preparation:

[0082] Culture of Bacillus coagulans and Bacillus subtilis:

[0083] The slant culture was inoculated into LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, cultured at 37℃ and 200 rpm for 14 h with shaking, centrifuged (4000 rpm, 10 min) and resuspended in physiological saline.

[0084] Lactic acid bacteria culture:

[0085] Inoculate into MRS medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, etc., and culture in an anaerobic jar at 37°C for 20 h. After centrifugation, resuspend.

[0086] The culture media of each strain were mixed in proportion, and the bacterial cells were collected by centrifugation and then suspended in sterile physiological saline to obtain a compound bacterial seed solution.

[0087] The total viable count of Bacillus spores in the seed culture was 4.5 × 10⁻⁶. 9 CFU / mL, total viable count of Bacillus subtilis seed culture 4.1 × 10⁻⁶ 9 CFU / mL, total viable count of lactic acid bacteria seed culture 4.9 × 10⁻⁶ 9 cfu / mL.

[0088] Example 8

[0089] The preparation method of the compound bacterial fermentation broth includes the following steps:

[0090] S100. Using the plate confrontation method, 42g of Bacillus coagulans, 20g of Bacillus subtilis, and 50g of lactic acid bacteria were screened to form a compound bacterial system.

[0091] S200. The compound bacterial strain is inoculated into a seed culture medium and cultured in the same way as in Example 7 to obtain a compound bacterial seed liquid.

[0092] S300: Mix 100g of compound bacterial seed liquid with the fermentation agent, then mix with 87.5g of molasses, 50g of snowflake powder, and 112.5g of silkworm pupa protein powder. The mass ratio of the compound bacterial seed liquid to the fermentation agent is 1:2.5. Inoculate into 16g of compound bacterial high-density culture medium, with a mass ratio of the high-density culture medium to the compound bacterial seed liquid of 0.16:1. The fermentation temperature is 35℃, and fermentation is carried out using a fermentation method that removes product repression: aeration and stirring are carried out for natural fermentation from 0 to 24 hours; the pH of the fermentation broth is monitored from 24 to 40 hours. When the pH reaches 5.0, 35% of the volume of fermentation broth is released, and compound bacterial high-density culture medium is added according to the corresponding volume ratio; the fermentation broth is obtained when the OD600 of the fermentation broth reaches a stable state.

[0093] Example 9

[0094] The preparation method of the compound bacterial fermentation broth includes the following steps:

[0095] S100, using the plate confrontation method, selected 30g of Bacillus coagulans, 10g of Bacillus subtilis, and 40g of lactic acid bacteria to form a compound.

[0096] bacterial strain;

[0097] S200. The compound bacterial strain is inoculated into a seed culture medium and cultured in the same way as in Example 7 to obtain a compound bacterial seed liquid.

[0098] S300: Mix 100g of compound bacterial seed liquid with fermentation agent, 79g of molasses, 40g of snow powder, and 111g of silkworm pupa protein powder. The mass ratio of the compound bacterial seed liquid to the fermentation agent is 1:2.3. Inoculate the mixture into 10g of compound bacterial high-density culture medium, with a mass ratio of the high-density culture medium to the compound bacterial seed liquid of 0.1:1. The fermentation temperature is 25℃, and fermentation is carried out using a fermentation method that removes product repression: aeration and stirring are carried out for natural fermentation from 0 to 24 hours; the pH of the fermentation broth is monitored from 24 to 40 hours. When the pH reaches 4.5, 30% of the volume of fermentation broth is released, and compound bacterial high-density culture medium is added according to the corresponding volume ratio; the fermentation broth is obtained when the OD600 of the fermentation broth reaches a stable state.

[0099] Example 10

[0100] The preparation method of the compound bacterial fermentation broth includes the following steps:

[0101] S100. 55g of Bacillus coagulans, 30g of Bacillus subtilis, and 60g of lactic acid bacteria were screened by plate confrontation method to form a compound bacterial system.

[0102] S200. The compound bacterial strain is inoculated into a seed culture medium and cultured in the same way as in Example 7 to obtain a compound bacterial seed liquid.

[0103] S300: Mix 100g of compound bacterial seed liquid with the fermentation agent, then add 94g of molasses, 62g of snowflake powder, and 124g of silkworm pupa protein powder. The mass ratio of the compound bacterial seed liquid to the fermentation agent is 1:2.8. Inoculate this mixture into 25g of compound bacterial high-density culture medium, where the mass ratio of the high-density culture medium to the compound bacterial seed liquid is 0.25:1. The fermentation temperature is 40℃, and fermentation is carried out using a fermentation method that removes product repression: aeration and stirring are performed for natural fermentation from 0 to 24 hours; the pH of the fermentation broth is monitored from 24 to 40 hours. When the pH reaches 5.5, 40% of the volume of the fermentation broth is released, and compound bacterial high-density culture medium is added according to the corresponding volume ratio; the fermentation broth is obtained when the OD600 of the fermentation broth reaches a stable state.

[0104] Example 11

[0105] The compound bacterial strain includes 42 parts of Bacillus coagulans and 20 parts of Bacillus subtilis. The preparation method and process parameters are the same as in Example 8.

[0106] Example 12

[0107] The compound bacterial strain includes 20 parts of Bacillus subtilis and 50 parts of lactic acid bacteria. The preparation method and process parameters are the same as in Example 8.

[0108] Example 13

[0109] The compound bacterial strain includes 42 parts of Bacillus coagulans and 50 parts of lactic acid bacteria. The preparation method and process parameters are the same as in Example 8.

[0110] Example 14

[0111] The compound bacterial strain includes 10 parts of Bacillus subtilis and 40 parts of lactic acid bacteria. The preparation method and process parameters are the same as in Example 8.

[0112] Example 15

[0113] The compound bacterial strain includes 30 parts of Bacillus coagulans and 40 parts of lactic acid bacteria. The preparation method and process parameters are the same as in Example 8.

[0114] Example 16

[0115] The compound bacterial strain contained 30 parts of Bacillus coagulans and 10 parts of Bacillus subtilis, and the preparation method and process parameters were the same as in Example 8.

[0116] Example 17

[0117] The compound bacterial strain includes 30 parts of Bacillus subtilis and 60 parts of lactic acid bacteria. The preparation method and process parameters are the same as in Example 8.

[0118] Example 18

[0119] The compound bacterial strain includes 55 parts of Bacillus coagulans and 60 parts of lactic acid bacteria. The preparation method and process parameters are the same as in Example 8.

[0120] Example 19

[0121] The compound bacterial strain includes 55 parts of Bacillus coagulans and 30 parts of Bacillus subtilis. The preparation method and process parameters are the same as in Example 8.

[0122] In Examples 8-19, the high-density culture medium of the compound bacteria from Example 4 was used for high-density fermentation, and the performance of the compound bacteria fermentation broth is shown in Table 3.

[0123] Table 3 shows the performance of the compound bacterial fermentation broth prepared by high-density culture of compound bacteria in Examples 8-19 of Example 4.

[0124]

[0125] As shown in Table 3, in the compound bacterial strains of Examples 8-10, the mixed ratio of Bacillus coagulans, Bacillus subtilis, and lactic acid bacteria, when used in high-density fermentation with the compound bacterial high-density culture medium of Example 4, resulted in a viable cell density of 5.8 × 10⁻⁶ cells in the fermentation broth. 12 CFU / mL, with a viable count of up to 2.58 × 10⁻⁶ per unit cost. 15 The concentration of cfu / yuan is 10.2-14.3 g / L, and the antimicrobial peptide activity can reach 920 U / mL.

[0126] In Examples 11, 16, and 19, the compound bacterial strain consisted of a mixture of Bacillus coagulans and Bacillus subtilis, and the viable cell density in the compound bacterial fermentation broth reached 4.9 × 10⁻⁶. 12 CFU / mL, with a viable bacterial count of up to 2.42 × 10⁻⁶ per unit cost. 15 CFU / yuan, with antimicrobial peptide activity up to 1100 U / mL.

[0127] In Examples 12, 14, and 17, the compound bacterial strain consisted of a mixture of Bacillus subtilis and lactic acid bacteria, and the viable cell density in the compound bacterial fermentation broth reached 4.5 × 10⁻⁶. 12 CFU / mL, with a viable bacterial count of up to 2.18 × 10⁻⁶ per unit cost. 15 CFU / yuan, lactic acid production 15.1 g / L.

[0128] In Examples 13, 15, and 18, the compound bacterial strain consisted of a mixture of Bacillus coagulans and lactic acid bacteria, and the viable cell density in the compound bacterial fermentation broth reached 5.2 × 10⁻⁶. 12 CFU / mL, with a viable bacterial count of up to 2.35 × 10⁻⁶ per unit cost. 15 CFU / yuan, lactic acid production 15.8 g / L.

[0129] In summary, the high-density culture of the compound bacteria of the present invention can utilize two or three of Bacillus coagulans, Bacillus subtilis, and lactic acid bacteria for high-density fermentation, and the viable cell density of the obtained compound bacterial fermentation broth can reach 5.8 × 10⁻⁶. 12 The cfu / mL, lactic acid production can reach 15.8 g / L, and antimicrobial peptide activity can reach 1100 U / mL.

Claims

1. A high-density culture medium for compound bacteria, characterized in that, By weight percentage, it comprises the following components: 1.5–2.5% soybean meal and bran hydrolysate, 0.8–1.5% corn flour, 0.8–1.5% soybean oil, 0.15–0.25% dipotassium hydrogen phosphate, and sodium carbonate. 0.1-0.2% magnesium sulfate heptahydrate, 0.02-0.03% magnesium sulfate heptahydrate, with the balance being water; The soybean meal and bran enzymatic hydrolysate is the product obtained by enzymatic hydrolysis of soybean meal and bran using a combination of enzymes, including cellulase, pectinase, and protease.

2. A preparation method, characterized in that, The preparation of the high-density culture medium for compound bacteria as described in claim 1 includes the following steps: mixing soybean meal and wheat bran enzymatic hydrolysate with the remaining components, and adjusting the pH to 5.5-7.5 to obtain the high-density culture medium for compound bacteria.

3. The preparation method according to claim 2, characterized in that, The soybean meal and bran enzymatic hydrolysate, by weight It includes the following components: 3.5 to 5.5 parts of enzymatic hydrolysate raw material and 0.95 to 1.2 parts of enzymatic hydrolysate agent. The enzymatic hydrolysate raw material includes 2 to 3.5 parts of soybean meal and 1.5 to 2 parts of wheat bran. The enzymatic hydrolysate agent includes 0.2 to 0.25 parts of cellulase, 0.25 to 0.35 parts of pectinase and 0.5 to 0.6 parts of protease.

4. The preparation method according to claim 3, characterized in that, The preparation method of the soybean meal and bran enzymatic hydrolysate includes the following: at a temperature of 25-40℃, the enzymatic hydrolysate raw material is added to deionized water and stirred evenly, while maintaining a humidity of 60-80%, an enzyme preparation is added and mixed evenly to obtain an enzymatic hydrolysate mixture; the enzymatic hydrolysate mixture is placed at room temperature for fermentation, stirred once every 4-6 hours, and fermented for 30-40 hours to obtain the soybean meal and bran enzymatic hydrolysate.

5. The application of the high-density culture medium for compound bacteria as described in any one of claims 1-4, characterized in that, The high-density culture medium containing the compound bacteria is used in high-density fermentation in aquaculture farms, and in the preparation of compound bacteria fermentation broth through high-density fermentation.

6. The application according to claim 5, characterized in that, The preparation method of the compound bacterial fermentation broth includes the following steps: S100. Two or more strains with synergistic effects are screened using the plate confrontation method to form a compound bacterial system. S200. The compound bacterial strain is inoculated into a seed culture medium and cultured to obtain a compound bacterial seed liquid; S300. After mixing the compound bacterial seed liquid with the fermentation agent, inoculate it into the compound bacterial high-density culture medium and carry out fermentation until the OD600 of the fermentation liquid is stable, thus obtaining the compound bacterial fermentation liquid.

7. The application according to claim 6, characterized in that, In step S100, the composite bacterial system includes two or three of Bacillus coagulans, Bacillus subtilis, and lactic acid bacteria, with Bacillus coagulans accounting for 30-55 parts, Bacillus subtilis accounting for 10-30 parts, and lactic acid bacteria accounting for 40-60 parts by weight.

8. The application according to claim 6, characterized in that, The starter culture, by weight, comprises 30-38 parts molasses. The mixture contains 15-25 parts of snowflake powder and 42-50 parts of silkworm pupa protein powder, with the mass ratio of the compound bacterial seed liquid to the fermentation agent being 1:2.3-2.

8.

9. The application according to claim 6, characterized in that, In step S300, the mass ratio of the high-density culture medium to the seed culture of the compound bacteria is 0.1 to 0.25:1; the fermentation temperature is 25 to 40℃.

10. The application according to claim 6, characterized in that, In step S300, a fermentation method that removes product repression is adopted: natural fermentation with aeration and stirring is carried out from 0 to 24 hours; the pH of the fermentation broth is monitored from 24 to 40 hours; when the pH reaches 4.5 to 5.5, 30% to 40% of the volume of fermentation broth is released, and high-density culture medium of compound bacteria is added according to the corresponding volume ratio.

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