Fermentation carbon source, preparation method and application thereof

Abstract

This invention relates to the field of fermentation carbon source preparation technology, and particularly to a fermentation carbon source, its preparation method, and its application, comprising the following components by weight: 10-13 parts solid raw material and 100-130 parts purified water; the fermentation carbon source further includes a high-concentration bacterial solution; the volume of the high-concentration bacterial solution is 0.01-0.03 times the total mass of the solid raw material and purified water. The fermentation carbon source of this invention can persistently and efficiently alter the COD, TP, and TN concentrations of aquaculture water.

Description

Technical Field

[0001] This invention relates to the field of fermentation carbon source preparation technology, and in particular to a fermentation carbon source, its preparation method and application. Background Technology

[0002] Fermentation carbon sources, as a key component in the microbial fermentation process, play a crucial role. They are not only essential nutrients for providing carbon to microbial cells and metabolic products, but also directly influence the efficiency of the fermentation process and the quality of the products. In microbial cells, carbon accounts for approximately 50% of the dry matter, forming the cornerstone of cell structure and maintaining life activities. Fermentation carbon sources are diverse, mainly including sugars (such as glucose, sucrose, and lactose), lipids, organic acids, and some hydrocarbons. During fermentation, these substances not only provide the energy needed for microbial growth but also participate in cell construction and the synthesis of metabolic products. For example, glucose is degraded by microorganisms through the glycolysis pathway (EMP) and the pentose phosphate pathway (HMP), producing ATP and intermediate metabolites, which provide the driving force for microbial growth and metabolic activities. In specific fermentation processes, selecting a suitable fermentation carbon source is crucial for improving product yield and quality. Rapidly utilized carbon sources, such as sugars, can quickly participate in metabolism and promote cell growth, but may produce catabolic metabolites that are detrimental to product synthesis. On the other hand, slowly utilized carbon sources, such as lipids, are beneficial for extending the synthesis time of metabolites and improving product stability.

[0003] Fermentation carbon sources have a wide range of applications, including biopharmaceuticals, food industry, bioenergy, and environmental protection. In the biopharmaceutical field, fermentation carbon sources are used to produce antibiotics, vaccines, and other biological drugs. In the food industry, fermentation carbon sources are used to produce yeast, enzyme preparations, organic acids, and alcoholic beverages. In addition, fermentation carbon sources also play an important role in bioenergy production, such as the manufacture of bioethanol and biodiesel.

[0004] In recent years, the application of fermented carbon sources in aquaculture has become increasingly widespread. Fermented carbon sources play an irreplaceable role in key ecological events such as "bacterial-algal ecological balance" and "nitrogen cycle." They not only promote the growth of beneficial bacteria and maintain the bacterial-algal balance in ponds, but also effectively promote the nitrogen cycle, reduce ammonia nitrogen and nitrite levels in the water, and improve water quality. For example, using fermented corn liquor or fermented wheat bran as a carbon source can be adjusted according to different seasons and water quality conditions to meet the needs of aquaculture.

[0005] For example, CN116119840A discloses "a fermented carbon source for in-situ treatment of aquaculture wastewater and its preparation and use method", which is prepared using fermentation concentrate, citric acid solution, Rhodopseudomonas photosynthetic bacteria, Lactobacillus, Bacillus subtilis and rare earth lanthanum as the main raw materials. This fermented carbon source effectively improves the treatment efficiency of carbon source for wastewater.

[0006] With the continuous development and progress of society, people are paying more and more attention to health, which poses a greater challenge to the aquaculture industry. The quality of water directly affects the quality and yield of aquatic products, and there is still room for improvement in the use of fermented carbon sources in the aquaculture industry.

[0007] Therefore, it is urgent to develop a fermentation carbon source that can sustainably and efficiently alter the concentrations of COD, TP, and TN in aquaculture water. Summary of the Invention

[0008] The main objective of this invention is to provide a fermentation carbon source and its preparation method, which can persistently and efficiently alter the COD, TP, and TN concentrations in aquaculture water.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] The present invention provides a fermentation carbon source comprising the following components by weight: 10-13 parts solid raw material and 100-130 parts purified water; the fermentation carbon source further comprises a high-concentration bacterial solution; the volume of the high-concentration bacterial solution is 0.01-0.03 times the total mass of the solid raw material and purified water.

[0011] The fermentation carbon source of this application uses simple and readily available raw materials with minimal environmental pollution. The raw materials can be agricultural crops, which further improves the utilization rate of resources. The fermentation carbon source obtained can be applied to the field of aquaculture and can persistently and efficiently change the concentration of COD, TP, and TN in aquaculture water, thereby improving the aquaculture efficiency of aquatic products.

[0012] In some embodiments, the high-concentration bacterial solution is a Lactobacillus acidophilus bacterial solution.

[0013] In some embodiments, the method for preparing the high-concentration bacterial solution includes the following steps:

[0014] A1. Solid culture: Lactobacillus acidophilus strain is inoculated onto MRS solid medium and cultured at 36-38℃ for 2-3 days to obtain activated Lactobacillus acidophilus;

[0015] A2. Liquid culture: The activated Lactobacillus acidophilus from step A1 is inoculated into MRS liquid culture medium and cultured at 26-28℃ for 22-26 hours to obtain Lactobacillus acidophilus bacterial solution;

[0016] A3. Expanded culture: Inoculate the Lactobacillus acidophilus culture from step A2 into a sterilized seed tank containing MRS liquid culture medium, and ferment at 35-37℃ for 30-32 hours. When the fermentation time reaches 10-12 hours, add an antifoaming agent and continue fermentation until the end, thus obtaining a high-concentration culture.

[0017] In some embodiments, the Lactobacillus acidophilus is registered under the accession number CICC 20248.

[0018] This application selects a specific Lactobacillus acidophilus as the fermentation strain, and uses corn and wheat bran as raw materials to prepare a fermentation carbon source, so that the prepared fermentation carbon source can persistently and efficiently change the COD, TP and TN concentrations of aquaculture water.

[0019] In some embodiments, in step A3, the inoculation amount of the Lactobacillus acidophilus culture is 1 to 3% of the volume of the MRS liquid culture medium.

[0020] This application enables the fermentation carbon source to efficiently alter the COD, TP, and TN concentrations in aquaculture water by adjusting the inoculation amount of Lactobacillus acidophilus.

[0021] This application can also prevent the yield of fermentation products from decreasing by controlling the inoculation amount of Lactobacillus acidophilus. The reason may be that Lactobacillus acidophilus will accelerate the consumption rate of the culture medium and the cells will quickly enter the stable and death phase.

[0022] In some embodiments, the concentration of the high-concentration bacterial solution is (9-11)×10⁻⁶. 9 CFU / mL.

[0023] This application enables the fermentation carbon source obtained by adjusting the concentration of the bacterial solution to efficiently change the COD, TP, and TN concentrations of aquaculture water.

[0024] In some embodiments, the amount of the defoamer added is 0.1 to 0.3 wt% of the MRS liquid culture medium.

[0025] In some embodiments, the preparation method of the defoamer includes the following steps: mixing tetraethyl orthosilicate with polyether polyol, adding triethylamine, heating to 72-76°C and stirring at a constant temperature for 1-3 hours, then adding dimethylsilane and heating to 90-100°C and stirring at a constant temperature for 1-3 hours, followed by adding 2-ethyl-1-butanol, continuing to stir for 20-40 minutes, and drying under reduced pressure to obtain the defoamer.

[0026] During microbial fermentation, a large amount of foam is generated due to factors such as aeration and stirring, nutrient composition of the culture medium, and microbial metabolism. This foam not only affects fermentation efficiency but may also cause various problems in the production process, such as overflow, equipment damage, and impaired dissolved oxygen transfer efficiency. Therefore, using defoamers to control foam generation is an important aspect of the fermentation industry.

[0027] Conventional defoamers have poor defoaming effects and cannot be used in the microbial fermentation process of this application. Therefore, it is necessary to find a defoamer suitable for defoaming in the microbial fermentation process of this application. This application uses tetraethyl orthosilicate, polyether polyol and dimethyl silicone oil as the main raw materials, and uses 2-ethyl-1-butanol for end-capping to obtain a defoamer with good defoaming effect, which is suitable for the microbial fermentation process of this application and improves fermentation efficiency.

[0028] In some embodiments, the molar ratio of tetraethyl orthosilicate to polyether polyol is 1:(0.3 to 0.5).

[0029] In some embodiments, each portion of the solid raw material comprises, by weight percentage, 23-26% wheat bran and 74-77% corn or starch.

[0030] This application uses specific solid raw materials as fermentation raw materials, which enables the fermented carbon source to persistently and efficiently change the COD, TP, and TN concentrations of aquaculture water.

[0031] Another aspect of the present invention provides a method for preparing a fermentation carbon source, comprising the following steps:

[0032] S1. Crush the solid raw material and pass it through a 15-20 mesh sieve to obtain solid raw material powder;

[0033] S2. Mix the solid raw material powder from step S1 with purified water to obtain a raw material slurry;

[0034] S3. Pump the high-concentration bacterial solution and the raw material slurry from step S2 into a fermenter for fermentation. After fermentation, filter by squeezing and collect the filtrate as the fermentation carbon source.

[0035] In some embodiments, the fermentation temperature in step S3 is 35-38°C, and the fermentation time is 70-74 hours.

[0036] Another aspect of the present invention provides an application of fermented carbon source in aquaculture.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The present invention uses solid raw materials, purified water and bacterial liquid as the main raw materials to ferment carbon source. The raw materials used for fermentation carbon source are simple and easy to obtain, and have less environmental pollution. The raw materials can be agricultural crops, which further improves the utilization rate of resources. The fermented carbon source can be applied to the field of aquaculture and can change the COD, TP and TN concentration of aquaculture water in a sustained and efficient manner, thereby improving the aquaculture efficiency of aquatic products.

[0039] (2) The present invention uses Lactobacillus acidophilus with preservation number CICC 20248 as a fermentation strain, which can promote the fermentation of bran and corn or starch, and the fermentation carbon source produced can persistently and efficiently change the COD, TP and TN concentrations of aquaculture water.

[0040] (3) The present invention uses a specific ratio of Lactobacillus acidophilus inoculation amount, which enables the fermentation carbon source to efficiently change the COD, TP and TN concentrations of aquaculture water, while also preventing the yield of fermentation products from decreasing. The reason may be that Lactobacillus acidophilus will accelerate the consumption rate of the culture medium and the bacteria will quickly enter the stable and death phase. Detailed Implementation

[0041] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] In the following examples, MRS solid culture medium and MRS liquid culture medium were purchased from Beijing Aoboxing Biotechnology Co., Ltd.; polyether polyol was purchased from Shanghai Kaiyin Chemical Co., Ltd.; and dimethyl silicone oil was purchased from Zhejiang Rongli High-Tech Materials Co., Ltd.

[0043] Preparation Example 1

[0044] The preparation method of the defoamer includes the following steps: 1 mol of tetraethyl orthosilicate and 0.4 mol of polyether polyol are mixed, 3 mol of triethylamine is added, the mixture is heated to 74℃ and stirred at a constant temperature for 2 hours, then 2 mol of dimethyl silicone oil is added and the mixture is heated to 95℃ and stirred at a constant temperature for 2 hours, followed by the addition of 0.5 mol of 2-ethyl-1-butanol, and the mixture is stirred for another 30 minutes. The mixture is then dried under reduced pressure to obtain the defoamer.

[0045] Preparation Example 2

[0046] The preparation method of the defoamer includes the following steps: 1 mol of tetraethyl orthosilicate and 0.7 mol of polyether polyol are mixed, 3 mol of triethylamine is added, the mixture is heated to 74℃ and stirred at a constant temperature for 2 hours, then 2 mol of dimethyl silicone oil is added and the mixture is heated to 95℃ and stirred at a constant temperature for 2 hours, followed by the addition of 0.5 mol of 2-ethyl-1-butanol, and the mixture is stirred for another 30 minutes. The mixture is then dried under reduced pressure to obtain the defoamer.

[0047] Preparation Example 3

[0048] The method for preparing high-concentration bacterial solutions includes the following steps:

[0049] A1. Solid culture: The Lactobacillus acidophilus strain with preservation number CICC 20248 was inoculated onto MRS solid medium and cultured at 37℃ for 2 days to obtain activated Lactobacillus acidophilus.

[0050] A2. Liquid culture: The activated Lactobacillus acidophilus from step A1 was inoculated into MRS liquid culture medium at an inoculation rate of 2% of the volume of MRS liquid culture medium and cultured at 27℃ for 24 hours to obtain Lactobacillus acidophilus bacterial solution.

[0051] A3. Scale-up culture: Inoculate the Lactobacillus acidophilus culture from step A2 into a sterilized seed tank containing MRS liquid culture medium. Ferment at 36°C for 31 hours. When the fermentation time reaches 11 hours, add 0.2 wt% of antifoaming agent from the MRS liquid culture medium and continue fermentation until completion, thus obtaining a concentration of 10 × 10⁻⁶. 9 High concentration of bacterial solution (CFU / mL).

[0052] The defoamer was prepared in Preparation Example 1.

[0053] Preparation Example 4

[0054] The preparation method of high-concentration bacterial solution is the same as that in Preparation Example 1, except that the preservation number of Lactobacillus acidophilus is CICC 6089.

[0055] Preparation Example 5

[0056] The preparation method of high-concentration bacterial solution is the same as that in Preparation Example 1, except that Bacillus subtilis is used instead of Lactobacillus acidophilus. The preservation number of Bacillus subtilis is CICC 10732.

[0057] Preparation Example 6

[0058] The method for preparing high-concentration bacterial solution is the same as in Preparation Example 1, except that the fermentation time in step A3 is 26 hours, resulting in a concentration of 7 × 10⁻⁶. 9 High concentration of bacterial solution (CFU / mL).

[0059] Preparation Example 7

[0060] The preparation method of high-concentration bacterial solution is the same as that in Preparation Example 1, except that the inoculum amount of Lactobacillus acidophilus is 6%.

[0061] Preparation Example 8

[0062] The preparation method of high-concentration bacterial solution is the same as that in Preparation Example 1, except that the defoamer is prepared in Preparation Example 2.

[0063] Example 1

[0064] A fermentation carbon source comprises the following components by weight: 12 parts solid raw material and 120 parts purified water; the fermentation carbon source further comprises a high-concentration bacterial solution; the volume of the high-concentration bacterial solution is 0.02 times the total mass of the solid raw material and purified water.

[0065] Each portion of solid raw material, by mass percentage, comprises 25% wheat bran and 75% corn, and the high-concentration bacterial solution was prepared in Preparation Example 3.

[0066] The method for preparing a fermentation carbon source includes the following steps:

[0067] S1. Crush the solid raw material and pass it through a 17-mesh sieve to obtain solid raw material powder;

[0068] S2. Mix the solid raw material powder from step S1 with purified water to obtain a raw material slurry;

[0069] S3. Pump the high-concentration bacterial solution and the raw material slurry from step S2 into a fermenter and ferment at 37°C for 72 hours. After fermentation, filter by squeezing and collect the filtrate as the fermentation carbon source.

[0070] Example 2

[0071] A fermentation carbon source comprises the following components by weight: 10 parts solid raw material and 100 parts purified water; the fermentation carbon source further comprises a high-concentration bacterial solution; the volume of the high-concentration bacterial solution is 0.01 times the total mass of the solid raw material and purified water.

[0072] In this case, each portion of solid raw material, by mass percentage, includes 23% wheat bran and 77% corn, and the high-concentration bacterial solution was prepared by Preparation Example 3.

[0073] The method for preparing a fermentation carbon source includes the following steps:

[0074] S1. Crush the solid raw material and pass it through a 17-mesh sieve to obtain solid raw material powder;

[0075] S2. Mix the solid raw material powder from step S1 with purified water to obtain a raw material slurry;

[0076] S3. Pump the high-concentration bacterial solution and the raw material slurry from step S2 into a fermenter and ferment at 35°C for 74 hours. After fermentation, filter by squeezing and collect the filtrate as the fermentation carbon source.

[0077] Example 3

[0078] A fermentation carbon source comprises the following components by weight: 13 parts solid raw material and 130 parts purified water; the fermentation carbon source further comprises a high-concentration bacterial solution; the volume of the high-concentration bacterial solution is 0.03 times the total mass of the solid raw material and purified water.

[0079] Each portion of solid raw material, by mass percentage, comprises 26% wheat bran and 74% corn, and the high-concentration bacterial solution was prepared in Preparation Example 3.

[0080] The method for preparing a fermentation carbon source includes the following steps:

[0081] S1. Crush the solid raw material and pass it through a 17-mesh sieve to obtain solid raw material powder;

[0082] S2. Mix the solid raw material powder from step S1 with purified water to obtain a raw material slurry;

[0083] S3. Pump the high-concentration bacterial solution and the raw material slurry from step S2 into a fermenter and ferment at 38°C for 70 hours. After fermentation, filter by squeezing and collect the filtrate as the fermentation carbon source.

[0084] Example 4

[0085] A fermentation carbon source and its preparation method are described. The specific implementation method is the same as in Example 1, except that the high-concentration bacterial solution is prepared in Example 4.

[0086] Example 5

[0087] A fermentation carbon source and its preparation method are described. The specific implementation method is the same as in Example 1, except that the high-concentration bacterial solution is prepared by Example 5.

[0088] Example 6

[0089] A fermentation carbon source and its preparation method are described. The specific implementation method is the same as in Example 1, except that the high-concentration bacterial solution is prepared by Example 6.

[0090] Example 7

[0091] A fermentation carbon source and its preparation method are described. The specific implementation method is the same as in Example 1, except that the high-concentration bacterial solution is prepared in Example 7.

[0092] Example 8

[0093] A fermentation carbon source and its preparation method are described. The specific implementation method is the same as in Example 1, except that the high-concentration bacterial solution is prepared in Example 8.

[0094] Example 9

[0095] A fermentation carbon source and its preparation method are described. The specific implementation method is the same as in Example 1, except that peanut shells are used instead of corn.

[0096] Example 10

[0097] A fermentation carbon source and its preparation method are described, with the specific implementation method being the same as in Example 1, except that bran is not added.

[0098] Performance testing:

[0099] (1) COD: Tested according to GB / T 11892 "Determination of Permanganate Index in Water" standard;

[0100] (2) TP content: Tested according to GB / T 11893 "Determination of total phosphorus in water by ammonium molybdate spectrophotometric method";

[0101] (3) TN content: Tested according to HJ 636 "Determination of total nitrogen in total nitrogen" standard;

[0102] (4) Durability test: COD, TN and TP levels were tested again 15 days after using the fermentation carbon source of each of the above embodiments.

[0103] The fermented carbon source prepared in each embodiment was sprinkled into water at a ratio of 1g:7L, and the test was carried out according to the above test method. In addition, a control group was set up to test the COD, TP and TN content in the initial water quality. The test results are shown in Table 1.

[0104] Table 1

[0105]

[0106] According to the experimental data in Table 1, the fermented carbon sources prepared in Examples 1-3 can persistently and efficiently change the COD, TP, and TN values ​​of aquaculture water, making them more suitable for aquaculture. In Example 4, the use of different preservation numbers of *Lactobacillus acidophilus* resulted in a decrease in the ability of the fermented carbon source to reduce COD, TP, and TN values ​​in aquaculture water. In Example 5, the use of *Bacillus subtilis* instead of *Lactobacillus acidophilus* led to a decrease in the ability of the fermented carbon source to reduce COD, TP, and TN values ​​in aquaculture water, and also a decrease in its persistence. In Example 6, the change in the concentration of the bacterial solution resulted in a decrease in the ability of the fermented carbon source to reduce COD, TP, and TN values ​​in aquaculture water. In Example 7, the change in the concentration of the bacterial solution further reduced the ability of the fermented carbon source to reduce COD, TP, and TN values. The amount of Lactobacillus inoculation resulted in a decrease in the ability of the fermentation carbon source to reduce COD, TP, and TN values ​​in the aquaculture water. In Example 8, the change in the molar ratio of tetraethyl orthosilicate to polyether polyol weakened the surface activity of the defoamer, thereby reducing the concentration of the high-concentration bacterial solution and further decreasing the ability of the fermentation carbon source to reduce COD, TP, and TN values ​​in the aquaculture water. In Example 9, the use of peanut shells instead of corn reduced the ability of the fermentation carbon source to reduce COD, TP, and TN values ​​in the aquaculture water, and also reduced its persistence. In Example 10, the absence of wheat bran reduced the ability of the fermentation carbon source to reduce COD, TP, and TN values ​​in the aquaculture water, and also reduced its persistence.

[0107] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A fermentation carbon source, characterized in that, It comprises the following components by weight: 10-13 parts solid raw material and 100-130 parts purified water; the fermentation carbon source also includes a high-concentration bacterial solution; the volume of the high-concentration bacterial solution is 0.01-0.03 times the total mass of the solid raw material and purified water.

2. The fermentation carbon source according to claim 1, characterized in that, The method for preparing the high-concentration bacterial solution includes the following steps: A1. Solid culture: Lactobacillus acidophilus strain is inoculated onto MRS solid medium and cultured at 36-38℃ for 2-3 days to obtain activated Lactobacillus acidophilus; A2. Liquid culture: The activated Lactobacillus acidophilus from step A1 is inoculated into MRS liquid culture medium and cultured at 26-28℃ for 22-26 hours to obtain Lactobacillus acidophilus bacterial solution; A3. Expanded culture: Inoculate the Lactobacillus acidophilus culture from step A2 into a sterilized seed tank containing MRS liquid culture medium, and ferment at 35-37℃ for 30-32 hours. When the fermentation time reaches 10-12 hours, add an antifoaming agent and continue fermentation until the end, thus obtaining a high-concentration culture.

3. The fermentation carbon source according to claim 2, characterized in that, The Lactobacillus acidophilus has the accession number CICC20248.

4. The fermentation carbon source according to claim 2, characterized in that, In step A3, the inoculation volume of the Lactobacillus acidophilus culture solution is 1-3% of the volume of the MRS liquid culture medium.

5. The fermentation carbon source according to claim 2, characterized in that, The concentration of the high-concentration bacterial solution is (9-11)×10⁻⁶. 9 CFU / mL.

6. The fermentation carbon source according to claim 2, characterized in that, The preparation method of the defoamer includes the following steps: mixing tetraethyl orthosilicate with polyether polyol, adding triethylamine, heating to 72-76°C and stirring at a constant temperature for 1-3 hours, then adding dimethyl silicone oil and heating to 90-100°C and stirring at a constant temperature for 1-3 hours, followed by adding 2-ethyl-1-butanol, continuing to stir for 20-40 minutes, and drying under reduced pressure to obtain the defoamer.

7. The fermentation carbon source according to claim 6, characterized in that, The molar ratio of tetraethyl orthosilicate to polyether polyol is 1:(0.3-0.5).

8. The fermentation carbon source according to claim 1, characterized in that, Each portion of the solid raw material comprises, by weight percentage (100%), 23-26% wheat bran and 74-77% corn or starch.

9. A method for preparing a fermentation carbon source according to any one of claims 1 to 8, characterized in that, Includes the following steps: S1. Crush the solid raw material and pass it through a 15-20 mesh sieve to obtain solid raw material powder; S2. Mix the solid raw material powder from step S1 with purified water to obtain a raw material slurry; S3. Pump the high-concentration bacterial solution and the raw material slurry from step S2 into a fermenter and ferment at 35-38°C for 70-74 hours. After fermentation, filter by squeezing and collect the filtrate as the fermentation carbon source.

10. The application of the fermentation carbon source according to any one of claims 1 to 8 in aquaculture.

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