阅读说明：本技术 采用复合益生菌发酵浓香型酒糟生产抗生素代替剂的方法 (Method for producing antibiotic substitute by fermenting Luzhou-flavor vinasse by adopting composite probiotics ) 是由 古绍彬 崔玉明 张献敏 吴影 何昭惠 赵丽娜 李璇 马金亮 于 2021-01-29 设计创作，主要内容包括：本发明涉及采用复合益生菌发酵浓香型酒糟生产抗生素代替剂的方法,属于固体发酵领域,该方法包括如下步骤：S1.原料预处理：将浓香型白酒糟pH调定、烘干,得一定含水率的酒糟；S2.原料发酵前改善：酒糟掺玉米粉,采用尿素和磷酸二氢钾混合处理液喷洒酒糟；S3.将植物乳杆菌、凝结芽孢杆菌、酿酒酵母、枯草芽孢杆菌在各自液体培养基中进行活化扩培；S4.接种发酵：将凝结芽孢杆菌和酿酒酵母溶液接种于发酵物料中,搅拌均匀,控温发酵两天,将植物乳杆菌和枯草芽孢杆菌接种于物料中,搅拌均匀,控温发酵,最终得到抗生素代替剂。本发明工艺简单,通过该方法得到高活菌含量、营养丰富的抗生素代替剂,有助于高效高质利用酒糟。(The invention relates to a method for producing an antibiotic substitute by fermenting Luzhou-flavor vinasse with composite probiotics, belonging to the field of solid fermentation and comprising the following steps: s1, raw material pretreatment: adjusting the pH value of the strong aromatic distiller's grains, and drying to obtain distiller's grains with a certain water content; s2, improving the raw materials before fermentation: mixing distiller's grains with corn flour, and spraying the distiller's grains with mixed treating solution of urea and potassium dihydrogen phosphate; s3, activating and expanding lactobacillus plantarum, bacillus coagulans, saccharomyces cerevisiae and bacillus subtilis in respective liquid culture media; s4, inoculating and fermenting: inoculating the bacillus coagulans and saccharomyces cerevisiae solution into the fermented material, uniformly stirring, fermenting at a controlled temperature for two days, inoculating lactobacillus plantarum and bacillus subtilis into the material, uniformly stirring, fermenting at a controlled temperature, and finally obtaining the antibiotic substitute. The method has simple process, and the antibiotic substitute with high viable bacteria content and rich nutrition is obtained by the method, thereby being beneficial to the high-efficiency and high-quality utilization of the vinasse.)

1. The method for producing the antibiotic substitute by fermenting the Luzhou-flavor vinasse by adopting the composite probiotics is characterized by comprising the following steps of: the method comprises the following steps:

step one, raw material pretreatment: carrying out pH setting and drying pretreatment on the strong aromatic distiller grains to obtain treated distiller grains with the water content controlled within 50 +/-2%;

step two, improving the raw materials before fermentation: adding corn flour into the processed vinasse obtained in the step one, stacking and compacting on plastic cloth to form a vinasse stack, and spraying a processing solution on the vinasse stack to obtain a matrix material; supplementing water to enable the water content of the matrix material to be 60 +/-2%, and then sealing the matrix material; the treatment solution is a mixed solution containing urea and monopotassium phosphate, wherein the urea content in the treatment solution is 0.2-0.3 g/mL, and the monopotassium phosphate content in the treatment solution is 0.05-0.15 g/mL; the spraying amount of the treatment liquid is as follows: spraying 50L of treatment liquid on each ton of vinasse;

step three, strain activation and expanding culture: activating and expanding lactobacillus plantarum HZLp-005 and bacillus coagulans CGMCC9551 in an MRS liquid culture medium; activating and expanding saccharomyces cerevisiae in a YPD liquid culture medium; activating and expanding the bacillus subtilis CGMCC17305 in an LB liquid culture medium;

step four, inoculating and fermenting: inoculating the activated bacillus coagulans CGMCC9551 and the saccharomyces cerevisiae solution obtained in the step three into the substrate material obtained by the step two, uniformly stirring, and fermenting for two days at a controlled temperature to obtain an initial material; inoculating the activated lactobacillus plantarum HZLp-005 and the activated bacillus subtilis CGMCC17305 obtained in the step three into the initial material, uniformly stirring, and performing temperature-controlled fermentation to obtain a fermented material;

step five, post-treatment: controlling the water content of the fermented material obtained in the fourth step to be 50% -55%, freezing, crushing, and vacuumizing and packaging after the detection is qualified to obtain a finished product.

2. The method of claim 1, wherein: in the first step, the pH value of the Luzhou-flavor distiller's grains is adjusted to 7 +/-0.5; the drying temperature is 400 +/-50 ℃.

3. The method of claim 1, wherein: in the second step, the corn flour is added in a total amount of 5 +/-0.5 percent of the weight of the matrix material.

4. The method of claim 1, wherein: in the second step, the urea content in the treatment solution was 0.25g/mL, and the potassium dihydrogen phosphate content was 0.1 g/mL.

5. The method of claim 1, wherein: in the third step, the saccharomyces cerevisiae, the bacillus coagulans CGMCC9551, the lactobacillus plantarum HZLp-005 and the bacillus subtilis CGMCC17305 are activated for 2-3 generations, and the inoculation concentrations are all 10⁸ CFU/g。

6. The method of claim 1, wherein: in the fourth step, the inoculation amounts of the saccharomyces cerevisiae, the bacillus coagulans CGMCC9551, the lactobacillus plantarum HZLp-005 and the bacillus subtilis CGMCC17305 are all 2-3 percent.

7. The method of claim 6, wherein: in the fourth step, after bacillus coagulans CGMCC9551 and saccharomyces cerevisiae are inoculated, fermentation is carried out for 2d at the temperature of 33 +/-2 ℃, and ventilation and stirring are carried out once every 3-4 h in the fermentation period, wherein the ventilation time is 30 min each time; inoculating lactobacillus plantarum HZLp-005 and bacillus subtilis CGMCC17305 after fermenting for 2d, fermenting for 3d at 33 +/-2 ℃, and ventilating and stirring once every 3-4 h during fermentation, wherein the ventilation time is 30 min each time.

8. Use of an antibiotic substitute prepared according to any one of claims 1 to 7 in the preparation of an animal feed, feed additive or digestive aid.

Technical Field

The invention belongs to the technical field of solid fermentation, and particularly relates to a method for producing an antibiotic substitute by fermenting strong aromatic vinasse by adopting composite probiotics.

Background

Due to the pollution of antibiotics to the environment and the threat of drug-resistant bacteria, relevant laws and regulations are continuously released in various countries, and the addition of antibiotics to the feed is forbidden. For this reason, people continuously research technologies for replacing antibiotics in feed formulas and breeding processes and search for effective substitute products, and among numerous potential antibiotic substitute products, the most popular antibiotic substitute products and the most effective feed additives are organic acids, probiotics and enzyme preparations.

Although research and development work on healthy and beneficial antibiotic substitutes is continued all the time, most of the antibiotics are traditional Chinese medicines and extracts thereof, and have the advantages of poor palatability, low nutritional value, small application range, high price, relatively single function and unobvious improvement effect on animal immunity and feed reward.

Meanwhile, China is a country with resource shortage, and a large amount of soybeans and fish meal need to be imported for feed processing and breeding industries every year. And the region of China is wide, and the byproducts of various agricultural products and food after processing are rich, such as vinasse, vinegar residues, sauce residues, fruit residues and the like. Wherein the crude protein and starch in the strong aromatic distiller's grains are respectively more than 13.0% and 15.0%, and the strong aromatic distiller's grains can be used as a high-quality fermentation substrate resource. However, the strong aromatic Chinese spirit vinasse is easy to deteriorate and difficult to transport due to high water content, and the application range of the strong aromatic Chinese spirit vinasse is severely limited.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a method for producing an antibiotic substitute by fermenting Luzhou-flavor vinasse by adopting composite probiotics, the Luzhou-flavor vinasse is used for carrying out composite probiotic fermentation, a large amount of living bacteria are contained in the Luzhou-flavor vinasse, a large amount of organic acid, enzymes and the like are metabolized in the fermentation process, the fermented vinasse can be used as a high-quality antibiotic substitute for being applied to the feed breeding industry, the protein content in the vinasse can be increased through fermentation, the crude fiber content is reduced, the quality of the vinasse is further improved, and the utilization value of vinasse.

In order to achieve the purpose, the invention adopts the specific scheme that:

the method for producing the antibiotic substitute by fermenting the Luzhou-flavor vinasse by adopting the composite probiotics comprises the following steps:

step one, raw material pretreatment: carrying out pH setting and drying pretreatment on the strong aromatic distiller grains to obtain treated distiller grains with the water content controlled within 50 +/-2%;

step two, improving the raw materials before fermentation: adding corn flour into the processed vinasse obtained in the step one, stacking and compacting on plastic cloth to form a vinasse stack, and spraying a processing solution on the vinasse stack to obtain a matrix material; supplementing water to enable the water content of the matrix material to be 60 +/-2%, and then sealing the matrix material; the treatment solution is a mixed solution containing urea and monopotassium phosphate, wherein the urea content in the treatment solution is 0.2-0.3 g/mL, and the monopotassium phosphate content in the treatment solution is 0.05-0.15 g/mL; the spraying amount of the treatment liquid is as follows: spraying 50L of treatment liquid on each ton of vinasse;

step three, strain activation and expanding culture: activating and expanding lactobacillus plantarum HZLp-005 and bacillus coagulans CGMCC9551 in an MRS liquid culture medium; activating and expanding saccharomyces cerevisiae in a YPD liquid culture medium; activating and expanding the bacillus subtilis CGMCC17305 in an LB liquid culture medium;

step four, inoculating and fermenting: inoculating the activated bacillus coagulans CGMCC9551 and the saccharomyces cerevisiae solution obtained in the step three into the substrate material obtained by the step two, uniformly stirring, and fermenting for two days at a controlled temperature to obtain an initial material; inoculating the activated lactobacillus plantarum HZLp-005 and the activated bacillus subtilis CGMCC17305 obtained in the step three into the initial material, uniformly stirring, and performing temperature-controlled fermentation to obtain a fermented material;

step five, post-treatment: controlling the water content of the fermented material obtained in the fourth step to be 50% -55%, freezing, crushing, and vacuumizing and packaging after the detection is qualified to obtain a finished product.

Preferably, in the first step, the pH of the Luzhou-flavor distiller's grains is adjusted to 7 +/-0.5; the drying temperature is 400 +/-50 ℃.

Preferably, in the second step, the corn flour is blended in a total amount of 5 +/-0.5% of the weight of the matrix material.

Preferably, in the second step, the urea content in the treatment solution is 0.25g/mL, and the potassium dihydrogen phosphate content is 0.1 g/mL.

Preferably, in the third step, the saccharomyces cerevisiae, the bacillus coagulans CGMCC9551, the lactobacillus plantarum HZLp-005 and the bacillus subtilis CGMCC17305 are activated for 2-3 generations, and the inoculation concentrations are all 10⁸CFU/g. All the strains are strains which are obtained by screening and have fast growth and strong growth vigor.

Preferably, in the fourth step, the inoculation amounts of the saccharomyces cerevisiae, the bacillus coagulans CGMCC9551, the lactobacillus plantarum HZLp-005 and the bacillus subtilis CGMCC17305 are all 2-3%.

Preferably, in the fourth step, after bacillus coagulans CGMCC9551 and saccharomyces cerevisiae are inoculated, fermentation is carried out for 2d at the temperature of 33 +/-2 ℃, and ventilation and stirring are carried out once every 3-4 h in the fermentation period, wherein the ventilation time is 30 min each time; inoculating lactobacillus plantarum HZLp-005 and bacillus subtilis CGMCC17305 after fermenting for 2d, fermenting for 3d at 33 +/-2 ℃, and ventilating and stirring once every 3-4 h during fermentation, wherein the ventilation time is 30 min each time.

The invention also claims the application of the antibiotic substitute prepared by the method in the preparation of animal feed, feed additives or digestion-aiding medicines.

Has the advantages that:

1. the invention selects strong aromatic vinasse as raw materials, which has larger difference with Maotai-flavor type and fen-flavor type in aspects of brewing process, fermentation container, inoculant and the like, for example, Maotai-flavor type wine raw materials are sorghum (brewing wine) and wheat (making Daqu), the Daqu process is high-temperature Daqu, the raw materials are steamed, the amount of used Daqu is large (1: 1.2), a stacking process is adopted before cellar entry, and a cellar pool is a stone wall mud bottom; the fen-flavor raw material is sorghum, the Daqu is a low-temperature yeast, and the fermentation adopts a ground vat for entering fermentation and has short period; the strong-flavor raw materials are sorghum and wheat, the yeast is medium temperature, the raw materials are subjected to mixed steaming and mixed burning treatment, a repeated ten thousand-year-old vinasse fermentation process is adopted, the yeast consumption is about 20%, and a cellar is a fertilizer mud cellar, so that a good habitat is provided for microorganisms such as caproic acid bacteria and the like. The components of the vinasse with different fragrance types are different, and a large number of practices prove that the finished product prepared by adopting the strong fragrance type vinasse as the raw material and combining the fermentation method disclosed by the invention is better in the aspects of nutrition level, viable count, activity of related enzymes and the like.

2. The invention abandons the method of adopting yeast to treat vinasse by single bacteria for protein enrichment in the prior art, and the selected bacteria are added with lactobacillus plantarum (HZLp-005), bacillus subtilis (CGMCC17305) and bacillus coagulans (CGMCC9551) for combined fermentation besides yeast, wherein the bacillus coagulans is used as the only bacillus capable of producing L-lactic acid, and the bacteria have synergistic effect and very remarkable effect in the aspect of benefiting life.

3. The invention takes the strong aromatic vinasse as the raw material, adopts multi-bacterium combination fermentation, finally obtains high-value viable count, and has higher enzyme activity, and the improvement of protein and amino acid and the degradation of cellulose reach obvious levels. These are critical for the health and nutrient accumulation of livestock.

4. The method firstly dries the raw materials, can kill the mixed bacteria in the vinasse on one hand, and can reduce the water content of the vinasse on the other hand, thereby facilitating the subsequent storage of the raw materials and the spraying of the mixed solution of urea and monopotassium phosphate; by adding corn flour into the vinasse and spraying a mixed solution of urea and monopotassium phosphate, the growth and propagation power of thalli can be increased, the viable bacteria amount of a fermentation product is increased, and the quality of the fermentation product is improved; the metabolite produced by yeast fermentation in the product can improve the solid matrix, and the yeast cell content and the yeast cell wall polysaccharide can also improve the solid matrix; bacillus coagulans (CGMCC9551) in the product can effectively inhibit pathogenic bacteria such as escherichia coli, shigella and the like, can generate a large amount of organic acid in the fermentation process, and improves the fermentation substrate; in the product, a large amount of substances such as organic acid and the like are generated in the fermentation process of lactobacillus plantarum (HZLp-005), so that the fermentation substrate can be effectively improved; during the production and metabolism of bacillus subtilis (CGMCC17305), a large amount of amylase, protease, cellulase and other enzyme systems can be secreted, and the fermentation substrate can be effectively improved; the invention can ferment to obtain the antibiotic substitute with high viable bacteria content, rich enzyme system and organic acid, and is beneficial to high-efficiency and high-quality utilization of the vinasse.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

The experimental procedures for the specific experimental conditions are not noted in the examples below, and conventional experimental conditions are generally followed.

The materials used, reagents and the like, unless otherwise specified, are commercially available reagents and materials.

Example 1

The method for producing the antibiotic substitute by fermenting the Luzhou-flavor vinasse by adopting the composite probiotics comprises the following specific steps:

1. pretreatment of raw materials:

and (3) drying and pretreating the strong aromatic Chinese spirit vinasse with the pH value adjusted to 7 in a roller drying device, controlling the drying temperature to be 400 ℃ to obtain vinasse with the water content of 50%, and after the drying and pretreatment, killing the infectious microbes in the strong aromatic Chinese spirit vinasse, wherein the water content of the vinasse is suitable for adding a mixed solution of corn flour, urea and potassium dihydrogen phosphate in the subsequent process, and controlling to obtain the suitable water content, so that the subsequent fermentation is facilitated.

2. Mixing corn flour with the weight of 5% into one ton of distiller's grains with the water content of 50% obtained in the step (1), spraying 50L of treatment liquid into the distiller's grains to obtain a matrix material, covering and sealing the matrix material by adopting a plastic film, reducing the contact between the matrix material and air, and preventing the mildewing of the distiller's grains and the generation of toxins; the treatment solution is a mixed solution containing urea and monopotassium phosphate, wherein the urea content in the mixed solution is 0.25g/mL, and the monopotassium phosphate content in the mixed solution is 0.1 g/mL. Adding proper water to make the water content of the material be 60%.

3. Activating and expanding Lactobacillus plantarum (HZLp-005) and Bacillus coagulans (CGMCC9551) in MRS liquid culture medium; carrying out activation and expansion culture on saccharomyces cerevisiae in a YPD liquid culture medium; activating and expanding the bacillus subtilis (CGMCC17305) in an LB liquid culture medium; obtaining activated and expanded bacterium liquid;

4. inoculating the activated bacillus coagulans (CGMCC9551) and saccharomyces cerevisiae solution obtained in the step (3) into the substrate material obtained in the step (2) in a manner of spraying by using a metering pump, so that the inoculation amounts of the bacillus coagulans (CGMCC9551) and the saccharomyces cerevisiae are respectively 2.5% of the weight of the material, uniformly stirring, fermenting for 2d at 33 ℃, and ventilating and stirring once every 3-4 h during the fermentation period, wherein the ventilation time is 30 min each time, so as to obtain an initial material;

inoculating the activated lactobacillus plantarum (HZLp-005) and the activated bacillus subtilis (CGMCC17305) obtained in the step (3) into initial materials fermented by bacillus coagulans (CGMCC9551) and saccharomyces cerevisiae for two days in a metering pump spraying manner, so that the inoculation amounts of the lactobacillus plantarum (HZLp-005) and the bacillus subtilis (CGMCC17305) are respectively 2.5% of the weight of the initial materials, uniformly stirring the materials, fermenting the materials for 3 days at 33 ℃, ventilating and stirring once every 3-4 h during fermentation, and ventilating for 30 min every time to obtain fermented materials; and plastic cloth is adopted for covering during fermentation, so that mould pollution can be effectively prevented.

5. Product post-treatment: in order to maintain the probiotics in the material at the highest level as much as possible and keep various beneficial components such as digestive enzyme, acid soluble protein, polysaccharide and the like, the whole treatment and transportation process needs to avoid high temperature; the water content of the fermented material is 50% -55%, the fermented material is frozen and then crushed, and after the fermented material is qualified, the fermented material is vacuumized and packaged to obtain a finished product.

Detecting the content of live bacteria, the content of true protein, the content of fiber and the content of amino acid of the finished product of the probiotic prepared by the embodiment, and the activity of diastase, cellulase and protease of the finished product, and putting the detection data into table 1; wherein the viable bacteria amount is detected by adopting a dilution plate coating method; precipitating the true protein content with copper hydroxide, and detecting with a Kjeldahl azotometer; the crude fiber is detected according to the specification of GB/T6434-1994; amino acids were detected as specified in GB/T18246-2019; saccharifying enzyme and cellulase are determined by 3, 5-dinitrosalicylic acid method, and protease is determined by Folin-phenol method.

Example 2

The present embodiment is basically the same as the steps in embodiment 1, except that in step 1, the pH is adjusted to 6.5, the drying temperature is 350 ℃, and the vinasse with the water content of 50% is obtained; in the step 2, the urea content in the treatment solution is 0.2g/mL, the potassium dihydrogen phosphate content is 0.05g/mL, 50L of treatment solution is added per ton, and proper water is supplemented to ensure that the water content of the material is 58%; in the step 4, the inoculation amounts of bacillus coagulans (CGMCC9551), saccharomyces cerevisiae, lactobacillus plantarum (HZLp-005) and bacillus subtilis (CGMCC17305) are respectively 2%, the stirring is uniform, and the temperature in the fermentation process is 31 ℃.

The content of live probiotic bacteria, the content of true protein, the content of fiber and the content of amino acid in the dry material, and the activities of diastase, cellulase and protease in the finished product prepared in the embodiment are detected, and the detection data are listed in table 1.

Example 3

The present example is basically the same as the step in example 1, except that in step 1, the pH is adjusted to 7.5, the drying temperature is 450 ℃, and the vinasse with the water content of 50% is obtained; in the step 2, the urea content in the treatment solution is 0.3g/mL, the potassium dihydrogen phosphate content is 0.15g/mL, 50L of treatment solution is added per ton, and proper water is supplemented to ensure that the water content of the material is 62%; in the step 4, the inoculation amounts of bacillus coagulans (CGMCC9551), saccharomyces cerevisiae, lactobacillus plantarum (HZLp-005) and bacillus subtilis (CGMCC17305) are respectively 3%, the stirring is uniform, and the temperature in the fermentation process is 35 ℃.

The content of live bacteria, the content of true protein, the content of fiber and the content of amino acid of the finished product prepared by the embodiment are detected, and the detection data are listed in table 1.

Detecting the true protein content, fiber content and amino acid content of the initial fermentation vinasse-based dry material, and the activity of finished saccharifying enzyme, cellulase and protease, and listing the detection data in table 1.

Table 1 composition measurements of final product and initial fermentation base for examples of the invention.

Item	Number of viable bacteria (1010CFU /g)	True egg White (%)	Coarse fiber Vitamin (%)	Met (%）	Thr (%）	Trp (%）	Saccharifying enzyme （u/g）	Cellulose, process for producing the same, and process for producing the same Enzyme (u/g)	Protease enzyme （u/g）
										Example 1	3.2	13.6	26.1	0.41	0.73	0.24	786	68	863
Example 2	5.6	16.2	23.7	0.62	0.87	0.31	846	79	924
										Example 3	4.1	14.8	24.9	0.53	0.79	0.28	804	76	889
Initial matrix	0	8.68	29.2	0.28	0.59	0.18	0	0	0

As shown in Table 1, the fermentation process is optimized, so that a finished product prepared after fermentation has billions of viable bacteria, the true protein content is increased by over 56.7%, the crude fiber content is reduced by over 9.6%, methionine is increased by over 46.4%, threonine is increased by over 23.7%, and tryptophan is increased by over 33.3%, fermented vinasse has stronger glucoamylase, cellulase and protease activities, the content change of vinasse nutrients enables the fermented vinasse to be digested and absorbed by animals more easily, the animal feeding livestock can adjust the intestinal flora of the animals, improve the health level of the animals, improve the immunity of the organism and the digestion and utilization rate of feed, and further improve the production performance, so that the vinasse can be recycled with better quality.

While certain specific embodiments of the present invention have been described in detail by way of illustration, it will be understood by those skilled in the art that the foregoing is illustrative only and is not limiting of the scope of the invention, as various modifications or additions may be made to the specific embodiments described and substituted in a similar manner by those skilled in the art without departing from the scope of the invention as defined in the appending claims. It should be understood by those skilled in the art that any modifications, equivalents, improvements and the like made to the above embodiments in accordance with the technical spirit of the present invention are included in the scope of the present invention.