阅读说明：本技术 一种饲料添加剂用复合菌发酵液及其制备方法 (Compound bacterium fermentation liquor for feed additive and preparation method thereof ) 是由 谢璐璐 李克克 朱广跃 李光华 韩亚坤 冯利菲 于 2020-04-03 设计创作，主要内容包括：本发明涉及一种饲料添加剂用复合菌发酵液及其制备方法,属于发酵饲料技术领域。本发明的饲料添加剂用复合菌发酵液,主要由丁酸梭菌发酵离心液和乳酸菌菌株在以下原料的存在下发酵培养得到：糖类碳源；其中,糖类碳源在丁酸梭菌发酵离心液中的质量分数为1％～7％。本发明的饲料添加剂用复合菌发酵液,通过对丁酸梭菌离心液接种乳酸菌菌种,并加入糖类碳源等营养物质同时对丁酸梭菌离心液中的乳酸菌和残留的丁酸梭菌进行发酵,从而达到利用丁酸梭菌离心液发酵乳酸菌的作用效果。在对丁酸梭菌发酵离心液无害化处理的同时又生产出了可应用于动物养殖产业中的含有大量乳酸菌活菌的丁酸梭菌饲料添加剂。(The invention relates to a compound bacterium fermentation liquid for a feed additive and a preparation method thereof, belonging to the technical field of fermented feeds. The composite bacteria fermentation liquor for the feed additive is mainly obtained by fermenting and culturing clostridium butyricum fermentation centrifugate and lactobacillus strains in the presence of the following raw materials: a carbohydrate carbon source; wherein the mass fraction of the carbohydrate carbon source in the clostridium butyricum fermentation centrifugate is 1-7%. According to the composite bacteria fermentation liquid for the feed additive, the lactobacillus strain is inoculated on the clostridium butyricum centrifugal liquid, and nutrient substances such as carbohydrate carbon sources and the like are added to ferment the lactobacillus in the clostridium butyricum centrifugal liquid and residual clostridium butyricum, so that the effect of fermenting the lactobacillus by using the clostridium butyricum centrifugal liquid is achieved. The clostridium butyricum feed additive containing a large amount of live lactobacillus, which can be applied to the animal breeding industry, is produced while carrying out innocent treatment on the clostridium butyricum fermentation centrifugate.)

1. A composite bacteria fermentation liquid for feed additives is characterized by being obtained by fermenting and culturing clostridium butyricum fermentation centrifugate and lactobacillus strains in the presence of the following raw materials: a carbohydrate carbon source;

wherein the mass fraction of the carbohydrate carbon source in the clostridium butyricum fermentation centrifugate is 1-7%.

2. The complex bacterium fermentation liquid for feed additives according to claim 1, wherein the lactic acid bacterium strain exists in the form of a lactic acid bacterium strain solution, the volume of the lactic acid bacterium strain solution is 1-10% of the volume of the clostridium butyricum fermentation centrifugate, and the content of the lactic acid bacterium strain in the lactic acid bacterium strain solution is 2.0 × 10⁹～5×10¹⁰CFU/mL。

3. The complex bacterium fermentation broth for feed additive according to claim 1, wherein the content of Clostridium butyricum in the Clostridium butyricum fermentation centrifugate is 1.0 × 10⁵～5.0×10⁸CFU/mL。

4. The complex bacteria fermentation broth for feed additive of claim 1, wherein the lactic acid bacteria strain is one, two or more of lactobacillus acidophilus, enterococcus faecalis, lactobacillus casei, lactobacillus plantarum, and enterococcus faecium.

5. The composite bacterium fermentation liquid for the feed additive according to claim 4, wherein the lactic acid bacterium strain is a combination of lactobacillus acidophilus, enterococcus faecalis and lactobacillus casei, wherein the concentration ratio of lactobacillus acidophilus, enterococcus faecalis and lactobacillus casei is 2-4: 1-3: 2-4.

6. The complex bacterium fermentation broth for feed additives of claim 1, wherein the carbohydrate carbon source is one or a combination of two or more of glucose, brown sugar and molasses.

7. The compound bacterium fermentation broth for a feed additive according to any one of claims 1 to 6, wherein the raw material further comprises a nitrogen source, and the mass of the nitrogen source is 0.5 to 2% of the mass of the clostridium butyricum fermentation centrifugate.

8. The composite bacteria fermentation broth for feed additive according to any one of claims 1 to 6, wherein the raw material further comprises inorganic salt, and the inorganic salt is MgSO₄、MnSO₄、K₂HPO₄And sodium acetate, of which MgSO₄、MnSO₄、K₂HPO₄And the mass fraction of the sodium acetate in the clostridium butyricum fermentation centrifugate is as follows:

0.025 to 0.075 percent MgSO₄0.015% -0.045% of MnSO₄0.1 to 0.3 percent of K₂HPO₄0.3 to 0.9 percent of sodium acetate.

9. A method for preparing a composite bacteria fermentation broth for a feed additive according to any one of claims 1 to 8, comprising the following steps:

adding the raw materials and lactobacillus strains into clostridium butyricum fermentation centrifugate, and fermenting and culturing at 35-39 ℃ for more than 20 hours to obtain the lactobacillus fermented feed.

10. The method for preparing the composite bacteria fermentation broth for feed additive according to claim 9, comprising the steps of:

(1) adding the raw materials and lactobacillus strains into clostridium butyricum fermentation centrifugate, carrying out temperature-controlled fermentation at 37 ℃ under the stirring condition, and stopping temperature control when the pH is 4.0-4.6;

(2) and then standing for natural fermentation, and obtaining the compound bacterium fermentation liquor for the feed additive when the pH is 4.0-4.3.

Technical Field

The invention relates to a compound bacterium fermentation liquid for a feed additive and a preparation method thereof, belonging to the technical field of fermented feeds.

Background

Clostridium butyricum (Clostridium butyricum), also known as butyric acid bacteria and eurotium, exists in excrement and soil of human and animals in nature, is gram-positive, belongs to Clostridium, is anaerobic, has straight or slightly curved thallus, round or oval spores and endospores, and usually makes the middle part of the thallus expanded to form a fusiform shape; the product has strong resistance to the external environment, is heat-resistant and acid-resistant, and completely survives after heat treatment of heat preservation at the temperature of 80 ℃ for 30min and at the temperature of 90 ℃ for l0 min; heating to 90 deg.C, keeping the temperature for 2min, and survival at 95%; heating to 100 deg.C, keeping the temperature for 5min, keeping 80% of the total viable, keeping the pH at 1.0-5.0, and maintaining the pH at 4.0-9.8 for the growth of the plant. Therefore, the butyric acid bacteria can resist high temperature and high heat in the feed granulation process, and has market potential compared with the existing widely applied non-spore live bacteria preparation. According to the research progress at home and abroad, the clostridium butyricum can adjust the balance of intestinal flora, promote the proliferation of beneficial bacteria in the intestinal tract, enhance the immune function, prevent tumors and generate various nutrient substances, such as vitamin K, B vitamins and the like, in the intestinal tract. At present, clostridium butyricum is mainly applied to livestock breeding and clinical treatment of related diseases caused by imbalance of normal flora in vivo, such as infectious diarrhea, inflammatory bowel disease, irritable bowel syndrome and the like.

Clostridium butyricum fermentations include solid state fermentations and liquid fermentations. Among them, solid state fermentation has the disadvantages of being difficult to control growth, difficult to separate products, and not mature enough method, and is generally less adopted. At present, the clostridium butyricum fermentation mostly adopts a liquid submerged fermentation method, a common clostridium butyricum preparation is subjected to liquid fermentation and then is centrifuged, freeze-dried or spray-dried to prepare bacterial powder, but centrifugation adopted in the preparation process can not remove all thalli and metabolites thereof, partial thalli are remained in a centrifugate, acetic acid, butyric acid, amylase, vitamin K and B vitamins generated in fermentation, unused culture medium components and the like also partially remain in the centrifugate, and the centrifugate is directly discharged to pollute a water body and cause huge waste. Regarding the treatment of fermentation waste liquid, the traditional treatment mode is mainly used for directly treating waste water and discharging the waste water after reaching the standard, and the treatment mode causes certain economic pressure to enterprises.

Disclosure of Invention

The invention aims to provide a composite bacterium fermentation liquid for a feed additive, which changes the clostridium butyricum fermentation centrifugal liquid into valuable, and has better effect by simultaneously containing clostridium butyricum, lactic acid bacteria and metabolites thereof.

The second purpose of the invention is to provide a preparation method of the compound bacterium fermentation liquor for the feed additive.

The clostridium butyricum fermented centrifugate can be directly used as a liquid feed additive, but the clostridium butyricum centrifugate contains nutrient substances such as carbon sources, nitrogen sources, inorganic salts and the like which are not completely utilized, and substances such as butyric acid, lactic acid, small peptides, amino acids, vitamins, digestive enzymes and the like which are generated by the metabolism of the clostridium butyricum, the pH value of the clostridium butyricum centrifugate is close to neutral, the clostridium butyricum centrifugate is easy to cause the growth and the propagation of other mixed bacteria in the storage process, the loss and the pollution of nutrition are caused, and the clostridium butyricum fermented centrifugate is not easy to store for a long time, so the invention provides the following technical scheme for changing the clostridium butyricum fermented centrifugat:

a composite bacteria fermentation liquor for feed additives is mainly obtained by fermenting and culturing clostridium butyricum fermentation centrifugate and lactobacillus strains in the presence of the following raw materials: a carbohydrate carbon source; wherein the mass fraction of the carbohydrate carbon source in the clostridium butyricum fermentation centrifugate is 1-7%.

It should be understood that the fermentation is carried out in a closed fermentor and stirred at 100rpm to ensure uniform distribution of the material and the biomass.

The composite bacteria fermentation liquor for the feed additive is mainly obtained by fermenting clostridium butyricum fermentation centrifugal liquid, lactobacillus strains and a carbohydrate carbon source. The growth of the lactobacillus reduces the pH value of the centrifugate, so that the centrifugate is convenient to store, the types of probiotics and metabolites are enriched, and the prepared composite bacteria fermentation liquid for the feed additive simultaneously contains clostridium butyricum, the lactobacillus and the metabolites thereof. The application effect is the result of the combined action of clostridium butyricum, lactic acid bacteria and metabolites of the clostridium butyricum and the lactic acid bacteria.

According to the composite bacteria fermentation liquid for the feed additive, the lactobacillus strain is inoculated on the clostridium butyricum centrifugal liquid, and nutrient substances such as carbohydrate carbon sources and the like are added to ferment the lactobacillus in the clostridium butyricum centrifugal liquid and residual clostridium butyricum, so that the effect of fermenting the lactobacillus by using the clostridium butyricum centrifugal liquid is achieved. The clostridium butyricum feed additive containing a large amount of live lactobacillus, which can be applied to the animal breeding industry, is produced while the clostridium butyricum fermentation centrifugate is subjected to innocent treatment, so that the technical problems that the clostridium butyricum centrifugate is not easy to treat sewage and residual thalli and rich nutrient substances in the clostridium butyricum centrifugate are recycled are effectively solved.

In the composite bacteria fermentation liquid for the feed additive, the clostridium butyricum fermentation centrifugate can be a clostridium butyricum fermentation centrifugate which is conventional in the field, for example, the clostridium butyricum strain preservation number in the clostridium butyricum fermentation centrifugate is CICC 10390.

Preferably, the lactobacillus strain exists in the form of lactobacillus strain solution, the volume of the lactobacillus strain solution is 1-10% of the volume of clostridium butyricum fermentation centrifugate, and the content of the lactobacillus strain in the lactobacillus strain solution is 2.0 × 10⁹～5×10¹⁰CFU/mL。

In order to obtain a higher content of lactic acid bacteria while controlling costs, it is preferable that the mass of the lactic acid bacteria strain is 5% of the volume of the clostridium butyricum fermentation centrifugate.

Preferably, the content of clostridium butyricum in the clostridium butyricum fermentation centrifugal liquid is 1.0 × 10⁵～5.0×10⁸CFU/mL。

Preferably, the lactobacillus strain is one or a combination of two or more of lactobacillus acidophilus, enterococcus faecalis, lactobacillus casei, lactobacillus plantarum and enterococcus faecium.

The Lactobacillus acidophilus, enterococcus faecalis, Lactobacillus casei, Lactobacillus plantarum, enterococcus faecium can be conventional strains in the art, preferably JBHBS₁The Lactobacillus acidophilus strain has a preservation number of ACCC05489, JBHBSC₁The enterococcus faecalis strain is deposited with the number CICC23658, and the JBHBGL Lactobacillus casei strain is deposited with the number CICC 20995. Lactobacillus plantarum JBHBTS for short, enterococcus faecium JBHBS for short₃。

In order to further improve the fermentation effect, the lactobacillus strain is preferably a combination of lactobacillus acidophilus, enterococcus faecalis and lactobacillus casei, wherein the concentration ratio of the lactobacillus acidophilus to the enterococcus faecalis to the lactobacillus casei is 2-4: 1-3: 2-4. it can be understood that the lactobacillus strain exists in the form of lactobacillus liquid, the lactobacillus liquid can be obtained by mixing lactobacillus acidophilus liquid, enterococcus faecalis liquid and lactobacillus casei liquid according to the volume ratio of 1:1:1, and the content of lactobacillus acidophilus in the lactobacillus acidophilus liquid is 2.0 × 10⁹～4.0×10⁹CFU/mL, the content of enterococcus faecalis in the enterococcus faecalis liquid is 1.0 × 10⁹～3.0×10⁹CFU/mL, content of Lactobacillus casei in Lactobacillus casei liquidThe amount is 2.0 × 10⁹～4.0×10⁹CFU/mL。

Preferably, the carbohydrate carbon source is one or a combination of more than two of glucose, brown sugar and molasses.

In order to further improve the fermentation effect, the content of the final lactic acid bacteria is increased while the pH is reduced, and preferably, the carbohydrate carbon source is a combination of glucose and molasses, wherein the mass of the glucose is 2.0-3.5% of that of the clostridium butyricum fermentation centrifugate, and the mass of the molasses is 1.0-3.5% of that of the clostridium butyricum fermentation centrifugate.

Preferably, the carbohydrate carbon source is a combination of glucose and molasses, wherein the mass of the glucose is 2.0% of the mass of the clostridium butyricum fermentation centrifugate, and the mass of the molasses is 1.5% of the mass of the clostridium butyricum fermentation centrifugate.

In order to further reduce the pH and increase the content of the final lactic acid bacteria, the raw material preferably further comprises a nitrogen source, and the mass of the nitrogen source is 0.5-2% of that of the clostridium butyricum fermentation centrifugate.

Preferably, the nitrogen source is one or a combination of more than two of corn steep liquor, peptone, yeast extract, beef powder, beef extract and peptide powder. These nitrogen sources all have good results.

In order to further control the cost and increase the content of the final lactic acid bacteria while reducing the pH, preferably, the nitrogen source is corn steep liquor, and the mass of the corn steep liquor is 1% of that of the clostridium butyricum fermentation centrifugate.

In order to further reduce the pH and increase the content of the final lactic acid bacteria, the raw material preferably further comprises an inorganic salt, wherein the inorganic salt is MgSO₄、MnSO₄、K₂HPO₄And sodium acetate, of which MgSO₄、MnSO₄、K₂HPO₄And the mass fraction of the sodium acetate in the clostridium butyricum fermentation centrifugate is as follows: 0.025 to 0.075 percent MgSO₄0.015% -0.045% of MnSO₄0.1 to 0.3 percent of K₂HPO₄0.3 to 0.9 percent of sodium acetate.

Preferably, the MgSO₄、MnSO₄、K₂HPO₄And the mass fraction of the sodium acetate in the clostridium butyricum fermentation centrifugate is as follows: 0.025% MgSO₄0.015% of MnSO₄0.1% of K₂HPO₄0.6 percent of sodium acetate.

A preparation method of compound bacteria fermentation liquor for a feed additive comprises the following steps: adding the raw materials and lactobacillus strains into clostridium butyricum fermentation centrifugate, and fermenting and culturing at 35-39 ℃ for more than 20 hours to obtain the lactobacillus fermented feed.

The preparation method of the composite bacteria fermentation liquor for the feed additive only needs to add the raw materials into the clostridium butyricum fermentation centrifugal liquid and then ferment, and the method is simple and convenient to operate and suitable for large-scale production.

In order to further improve the fermentation effect, preferably, the preparation method of the compound bacterium fermentation liquid for the feed additive comprises the following steps:

(1) adding the raw materials and lactobacillus strains into clostridium butyricum fermentation centrifugate, carrying out temperature-controlled fermentation at 37 ℃ under the stirring condition, and stopping temperature control when the pH is 4.0-4.6;

(2) and then standing for natural fermentation, and obtaining the compound bacterium fermentation liquor for the feed additive when the pH is 4.0-4.3.

It is understood that, in the step (2), the standing natural fermentation is fermentation without stirring, and the temperature of the natural fermentation may be 20 to 37 ℃.

It can be understood that the obtained compound bacteria fermentation liquor for the feed additive can be used as a liquid product to be directly used as a feed additive, and can also be used as a seed liquor for solid fermentation.

Preferably, in the step (2), the content of lactic acid bacteria in the compound bacteria fermentation liquor for the feed additive is 5.0 × 10⁹The content of lactobacillus in the compound bacteria fermentation liquid for the feed additive is more than 5.0 × 10⁹Above CFU/mL, a dominant flora is more likely to form in the animal gut.

Preferably, a method for fermenting lactobacillus with clostridium butyricum centrifugate comprises the following steps:

1) pumping a proper amount of clostridium butyricum centrifugate into a sterilized fermentation tank controlled at 37 +/-2 ℃;

2) adding 2.0-3.5% glucose, 1.0-3.5% molasses as carbon source, 0.5-2.0% corn steep liquor as nitrogen source, 0.025-0.075% MgSO₄、0.015％～0.045％MnSO₄、0.1％～0.3％K₂HPO₄0.3 to 0.9 percent of sodium acetate is used as inorganic salt and trace elements required by fermentation;

3) inoculating 1-10% of JBHBS₁:JBHBSC₁JBHBGL is lactobacillus of 1:1:1 as fermentation seed;

4) fermenting at 37 + -2 deg.C and 100rpm for 18-24h, detecting pH, and stopping temperature control when pH is about 4.5.

5) Standing for naturally fermenting for 96 hr, detecting pH every day, and detecting lactobacillus content of 5.0 × 10 or more for 24 hr until pH is reduced to 4.0-4.3⁹CFU/ml, and then filtering to obtain the product.

The composite bacteria fermentation liquor for the feed additive and the composite bacteria fermentation liquor for the feed additive can be used for livestock breeding in a mode of mixing, drinking or drenching, can adjust intestinal microecological balance, inhibit the growth of pathogenic bacteria and prevent piglet diarrhea; the feed intake of the piglets is improved, the utilization rate of the feed is improved, and the daily gain of the piglets is increased; the balance of intestinal flora of the fat pigs is adjusted, the intestinal environment is improved, the planting of beneficial bacteria is promoted, and harmful bacteria are reduced; increase the advantages of the intestinal flora, promote the re-absorption and utilization of the food, improve the utilization rate of the feed, promote the ingestion and improve the growth speed.

The compound bacteria fermentation liquor for the feed additive is applied to aquatic animals such as fish, shrimps, crabs, sea cucumbers and the like, can adjust intestinal flora, and can obviously reduce the occurrence probability of white feces and enteritis after each liter of the compound bacteria fermentation liquor for the feed additive is mixed with 40 kg of feed for one week; 10-20 kg of compound bacteria fermentation liquor mixed material is used for each liter of feed additive, and clostridium butyricum and lactic acid bacteria can adjust the pH of the intestinal tract and promote the intestinal tract of the cultured animals to wriggle, digest and absorb; it can also be used for relieving stress reaction and improving water quality.

The composite bacteria fermentation liquor for the feed additive can also be applied to poultry breeding in a drinking water mode, improves the non-specific immunity of a machine body, improves the feed utilization rate, reduces the death and culling rate, improves the laying rate of laying poultry, reduces the feed-egg ratio, reduces the feed-meat ratio of meat poultry and the like.

The composite bacteria fermentation liquor for the feed additive is applied to laying poultry, and has the following advantages and effects:

1. promote the absorption of yolk of the young poultry and improve the survival rate and the uniformity of brooding.

2. The laying rate of laying poultry is improved, the egg weight is increased, the egg laying peak is prolonged, and the dropping speed of the egg laying peak is reduced.

3. Improve the quality of eggshells, and reduce the content of eggshells, soft shell eggs and broken eggs.

4. Maintaining the integrity of mucosa barrier of organism, and improving nonspecific immunity and disease resistance of organism.

5. Effectively solves the enteritis and overfeeding phenomena, improves the utilization rate of the laying poultry to the feed, and reduces the feed-egg ratio.

6. Improve the breeding environment, reduce the ammonia concentration in the house and reduce the occurrence of respiratory diseases.

Detailed Description

The present invention will be further described with reference to the following embodiments.

In the embodiment of the invention, the clostridium butyricum strain preservation number of the clostridium butyricum centrifugate is CICC 10390.

In the embodiment of the invention, JBHBS₁The lactobacillus acidophilus strain has a preservation number of ACCC 05489.

In the embodiment of the invention, JBHBSC₁The enterococcus faecalis strain is deposited under accession number CICC 23658.

In the embodiment of the invention, the preservation number of the JBHBGL lactobacillus casei strain is CICC 20995.

In the embodiment of the invention, the preparation method of the compound bacteria fermentation liquor for the feed additive is carried out in a micro-aerobic environment.

In the embodiment of the invention, the strain in the lactobacillus liquid is JBHBS₁Lactobacillus acidophilus strain, JBHBSC₁Enterococcus faecalis strain and JBHBGL cheeseThe lactobacillus strain composition contains Lactobacillus acidophilus 1.0 × 10⁹CFU/mL, enterococcus faecalis content of 7.0 × 10⁸CFU/mL, Lactobacillus casei content of 1.0 × 10⁹CFU/mL。

The specific embodiment of the compound bacteria fermentation liquor for the feed additive is as follows: