CN104996722B - A kind of method of the step combined ferment feed of multi-cultur es two - Google Patents

Abstract

The invention discloses a method for multi-strain two-step joint fermentation of feed, which belongs to the technical field of feed. The present invention uses corn flour, palm meal, soybean meal, bran, and subflour as raw materials, uses Rhizopus niger, Aspergillus oryzae, Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae as fermentation strains, and undergoes aerobic fermentation and anaerobic fermentation. The fermented feed obtained by fermenting two steps. The protein content of the feed fermented by this method is as high as 30% to 34%, which is 45% to 62% higher than that of the raw material. , rich in probiotics and high enzyme activity feed. The method of the invention has strong operability, is suitable for large-scale industrial production, and has good market prospects.

Description

A kind of method of multi-strain two-step combined fermentation feed

technical field

The invention relates to a method for multi-strain two-step joint fermentation of feed, belonging to the technical field of feed.

Background technique

In recent years, fermented feed has become a research hotspot, and the research on various fermented feeds has been developed to a certain extent. Fermented feed is a biologically fermented feed that uses the action of microorganisms to convert feed materials into microbial cell protein, biologically active small peptide amino acids, microbially active probiotics and compound enzyme preparations. Fermented feed can not only make up for the amino acids that are easily lacking in conventional feed, but also degrade the ingredients of roughage raw materials, improve feed conversion rate, and degrade the antigenic protein in soybean meal. In addition, fermented feed can also reduce the diarrhea rate of pigs and significantly reduce pigs Backfat thickness, lactic acid bacteria fermented feed can also reduce the ammonia content of the pig house.

Fermented feed can be divided into silage fermentation, concentrated feed fermentation, agricultural product processing waste fermentation, etc. from the fermentation raw materials. From the physical state of the fermentation substrate, it can be divided into solid fermentation, liquid fermentation and semi-solid fermentation. Compared with fruit and vegetable dregs, soybean whey liquid, canteen swill, silage, etc., the solid fermented feed with corn flour and palm meal as the main raw materials has more stable product quality, is more suitable for storage and transportation, and is conducive to commercialization Large-scale production mainly includes the following production methods: (1) only the fermentation method in the anaerobic fermentation stage, after the strains are mixed with the feed, they are directly sealed in bags for anaerobic cultivation. , the growth of aerobic bacteria is limited, resulting in low hydrolytic enzyme activity in the raw materials, which cannot hydrolyze the macromolecular substances in the feed into small molecular substances, and thus cannot provide sufficient nutrients for the anaerobic fermentation probiotics. The protein content in the finally obtained feed was not significantly increased, the hydrolytic enzyme activity was low, and the total number of probiotic colonies was low. (2) Aerobic and anaerobic two-stage fermentation method, through the solid aerobic and solid anaerobic fermentation methods combined to make the fermented feed produced contain a high proportion of organic acids and lactic acid bacteria Degraded small molecular protein, and lower cellulose content.

Some fermented feeds only use bacteria, yeast and lactic acid bacteria for anaerobic fermentation. This combination requires external addition of enzyme preparations or external addition of simple sugars to provide nutrients for the early growth of the flora. Some only use mold and yeast or Bacillus subtilis for aerobic fermentation without anaerobic fermentation. This kind of fermentation can significantly improve the feed in terms of increasing protein content.

The invention uses palm dregs, soybean dregs, corn, etc. as raw materials, and adopts joint fermentation of mould, yeast, bacteria and lactic acid bacteria. During the aerobic fermentation stage, molds and Bacillus subtilis grow vigorously, produce various biological enzymes such as amylase, protease, cellulase, and hydrolyze macromolecular substances such as starch, protein, and cellulose in raw materials into small molecular nutrients, which are The growth of yeast, bacteria and lactic acid bacteria provides nutrients; in the anaerobic stage, the mold is dissolved, and the hydrolytic enzymes in the cells are released into the feed, and the macromolecular nutrients in the feed are further hydrolyzed into small molecular nutrients. Bacillus subtilis, yeast Bacteria and lactic acid bacteria continue to grow, the generated lactic acid and anaerobic conditions can better inhibit the growth of harmful bacteria, ensure the activity of probiotics, and finally obtain aromatic and palatable feed rich in protein, rich in probiotics and high in enzyme activity.

Contents of the invention

The purpose of the present invention is to provide a method for multi-strain two-step combined fermentation feed, the purpose is to obtain a fragrant and palatable feed rich in protein, rich in probiotics and high in enzyme activity through aerobic and anaerobic two-step fermentation of various microorganisms. feed.

Technical scheme of the present invention mainly comprises the following steps:

(1) activate bacterial classification, and prepare the pure cultures of Rhizopus, Aspergillus oryzae, Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae respectively;

(2) Pretreatment of fermentation raw materials, including pulverizing the raw materials, mixing them with water, and then sterilizing them;

(3) Aerobic fermentation: Inoculate the seed cultures of Rhizopus, Aspergillus oryzae, Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae into the fermentation raw materials, inoculate according to the mass ratio, and inoculate Rhizopus bran for every 100 parts of sterilized materials 0.2-0.7 parts of pure culture of seeds, 0.2-0.7 parts of pure culture of Aspergillus oryzae bran seeds, 0.8-1.2 parts of pure culture of Bacillus subtilis, 0.5-1.5 parts of pure culture of Lactobacillus plantarum, 2 parts of pure culture of Saccharomyces cerevisiae ～8 copies; mix well after inoculation, place in 28～30℃ for 36～48 hours of moisturizing and aerobic culture, after the temperature rises, place in 37℃ constant temperature incubator for ventilation and culture for 24 hours, control the relative humidity of 85%～95%;

(4) Anaerobic fermentation: After the aerobic stage, break up and mix the fermented feed, put it into a one-way air-exhausting sealed bag, and place it at room temperature for anaerobic cultivation for 4-8 days.

In one embodiment of the present invention, the pretreatment of the fermentation raw materials includes: (1) crushing of the fermentation raw materials: taking 10-15 parts of corn, 10-15 parts of bran, 15-25 parts of soybean meal, palm 35 to 45 parts of meal, 10 to 15 parts of secondary flour, 1 to 2 parts of urea, 0.5 to 1 part of glucose, mixed, crushed, and sieved to a fineness of 20 to 40 mesh; The material-to-water ratio is 1:0.6~1.0, mix well, and put on a plate; (3) Sterilization of fermentation raw materials: Sterilize the fermentation raw materials with high-pressure steam, and the sterilization conditions are 0.1Mpa~0.2Mpa, 15~30min.

In one embodiment of the present invention, for every 100 parts of sterilized materials, 0.2 to 0.7 parts of pure culture of Rhizopus bran seeds, 0.2 to 0.7 parts of pure culture of Aspergillus oryzae bran seeds, and pure culture of Bacillus subtilis are inoculated. The inoculum size of Lactobacillus plantarum pure culture is 1 part, and the inoculum size of pure culture of Saccharomyces cerevisiae is 6 parts.

In one embodiment of the present invention, the activation of Rhizopus is to inoculate the spores of Rhizopus on the potato glucose slant test tube medium, cultivate at a constant temperature of 28～30°C for 24～48h, pick the new mycelium, transfer to Potato glucose slant test tube culture medium, 28 ~ 30 ℃ constant temperature culture for 24 ~ 48h.

In one embodiment of the present invention, the activation of Aspergillus oryzae is to inoculate the spores of Aspergillus oryzae on the potato glucose slant test tube medium, cultivate at a constant temperature of 28～30°C for 24～48h, pick the new mycelium, transfer to Potato glucose slant test tube culture medium, 28 ~ 30 ℃ constant temperature culture for 24 ~ 48h.

In one embodiment of the present invention, the activation of Bacillus subtilis is to inoculate Bacillus subtilis on the LB slant test tube culture medium, culture at a constant temperature of 37°C for 20-24h, and then transfer to the LB slant test tube culture medium, 37 Cultivate at constant temperature for 20-24 hours.

In one embodiment of the present invention, the activation of Lactobacillus plantarum is to inoculate Lactobacillus plantarum on MRS liquid medium, culture at a constant temperature of 37°C for 20-24h, and then transfer to MRS liquid medium once, Cultivate at constant temperature for 20-24 hours.

In one embodiment of the present invention, the activation of brewer's yeast is to inoculate brewer's yeast on YPD liquid medium, culture at a constant temperature of 37°C for 20-24h, and then transfer it to YPD liquid medium once, and keep it at a constant temperature of 37°C. Cultivate for 20-24 hours.

In one embodiment of the present invention, the preparation of the pure culture of rhizopus seeds: inoculate the activated rhizopus mycelium on the solid medium of soybean meal bran medium, and culture at a constant temperature of 28-30° C. for 3-5 days.

In one embodiment of the present invention, the preparation of pure culture of Aspergillus oryzae seeds: inoculate the activated Aspergillus oryzae mycelium on the solid medium of soybean meal bran medium, and culture at a constant temperature of 28-30° C. for 3-5 days.

In one embodiment of the present invention, preparation of pure culture of Bacillus subtilis seeds: inoculate activated Bacillus subtilis into 50 ml of LB seed culture solution, and culture at a constant temperature of 37° C. for 10 to 18 hours with shaking.

In one embodiment of the present invention, preparation of pure culture of Lactobacillus plantarum seeds: transfer 1ml-2ml of activated Lactobacillus plantarum liquid culture solution to 50ml MRS seed culture solution, and culture at 37°C for 10-18 hours.

In one embodiment of the present invention, the preparation of pure culture of brewer's yeast seeds: transfer 1ml-2ml of activated brewer's yeast liquid culture solution to 50ml of YPD seed culture solution, and culture at 28-30°C for 12-18 hours.

Compared with traditional manufacturing methods, the present invention has the following advantages:

1. Greatly increased the activity of amylase and protease in the feed. The aerobic culture process of multi-species two-step fermented feed is beneficial to the vigorous growth and reproduction of mold and Bacillus subtilis, and produces abundant hydrolytic enzymes, especially amylase and protease. The problem that external enzyme preparations need to be added when only yeast and bacteria are used for fermentation is solved. During the fermentation process, the activity of α-amylase reaches above 160U/g, and the activity of acid protease reaches above 140U/g; at the end of the anaerobic stage, the activity of α-amylase can still be maintained at 90-110U/g, and the activity of acid protease remains At 110~130U/g. These hydrolytic enzymes can hydrolyze macromolecular substances in raw materials into small molecular nutrients.

2. Significantly increased the protein content of the feed. The protein content of the feed fermented by the method is as high as 30% to 34%, which is 45% to 62% higher than that of raw materials. A variety of hydrolytic enzymes are produced in the process of aerobic culture, and the macromolecular substances in the hydrolyzed raw materials are small molecular nutrients, which continuously provide nutrients for the growth of yeast, Bacillus subtilis, and lactic acid bacteria, and obtain a large amount of bacterial protein. This solves the problem that the protein content does not increase much due to the lack of hydrolytic enzymes in the anaerobic fermentation feed with only bacteria and yeast.

3. The content of probiotics in the feed has been greatly increased. Through the cultivation in the aerobic stage and the anaerobic stage, Aspergillus oryzae, Rhizopus, Saccharomyces cerevisiae, Bacillus subtilis and lactic acid bacteria grow and reproduce in large quantities, and the finally obtained feed contains rich probiotic flora. After fermentation, the lactic acid bacteria are 5.0×10 ¹⁰ to 5.0×10 ¹¹ cfu/g, the Bacillus subtilis is 2.0×10 ⁹ cfu/g to 2.0×10 ¹⁰ cfu/g, and the yeast is 1.5×10 ⁴ to 2.0×10 ³ cfu/g.

The method of the invention has strong operability, is suitable for large-scale industrial production, and has good market prospects.

Description of drawings

Figure 1 The content of crude protein in the feed

Figure 2 α-amylase activity in feed

Figure 3 Acid protease activity in feed

Figure 4 The logarithmic value of the total number of lactic acid bacteria colonies in the feed

Detailed ways

The protein content was determined according to the national standard GB5009.5-2010 method.

The content of α-amylase was determined according to the method of GB/T5521-2008.

The lactic acid bacteria colony count was determined according to the GB4789.35-2010 method.

The solid medium of soybean meal and bran medium consists of 35-45g of soybean meal powder, 30-40g of bran, 40-50mL of water, and the thickness of the material is 1-2cm.

Potato dextrose agar medium was prepared according to the following method: add 200 parts of potatoes, add 500 mL of water and boil for 30 minutes, filter, take the filtrate, boil, add 20 parts of agar strips, stir until completely dissolved, add 20 parts of glucose, replenish water to 1000 parts, sterilized by moist heat at 115-121°C for 20 minutes.

LB medium is prepared according to the following reagents by weight: 3 parts of beef extract, 10 parts of peptone, 5 parts of sodium chloride, 1000 parts of deionized water, and adjust the pH to 7.0-7.2 after mixing the reagents, 115-121 ℃ damp heat sterilization for 20min.

YPD medium is prepared according to the following reagents in parts by weight: 10 parts of yeast powder, 20 parts of peptone, 20 parts of glucose, 1000 parts of deionized water, mixed evenly, and sterilized by damp heat at 115-121° C. for 20 minutes.

MRS broth medium is prepared according to the following reagents by weight: peptone: 10 parts; beef extract: 10 parts; yeast extract: 5 parts; glucose: 20 parts; dipotassium hydrogen phosphate: 2 parts; citric acid Diammonium hydrogen: 2 parts; Anhydrous sodium acetate: 5 parts; Magnesium sulfate: 0.58 parts; Manganese sulfate: 0.25 parts; Tween 80: 1 part; Deionized water: 1000 parts; 6.4, sterilize with moist heat at 115-121°C for 20 minutes.

Embodiment 1 two strains aerobic, anaerobic two-step fermented feed

The specific implementation steps are as follows:

A. Cultivation of seeds

a. Preparation of pure culture of Bacillus subtilis seeds: transfer the activated Bacillus subtilis liquid culture solution to LB seed culture solution, and culture at a constant temperature of 37° C. for 10 to 18 hours with shaking.

b. Preparation of pure culture of Lactobacillus plantarum seeds: transfer activated Lactobacillus plantarum liquid culture solution to MRS seed culture

Nutrient solution, culture at 37°C for 10-18 hours.

B. Crushing of fermentation raw materials

Take 10-15 parts of corn, 10-15 parts of bran, 15-25 parts of soybean meal, 35-45 parts of palm meal, 10-15 parts of subflour, 1-2 parts of urea, 0.5-1 part of glucose, mix and grind , sieved, the fineness is 20-40 mesh.

D. Mixing water for fermentation raw materials

The ratio of material to water is 1:0.6~1.0, mix well and put on a plate.

E. Sterilization of fermentation raw materials

The fermentation raw materials are sterilized by high-pressure steam, and the sterilization conditions are 0.1Mpa-0.2Mpa, 15-30min.

F. Aerobic fermentation

Inoculate the Bacillus subtilis seed culture into the fermentation raw material, the inoculation ratio Bacillus subtilis: The mass ratio of raw material is 1.5～2.5:100, mix well after inoculation, put it in a 28～30℃ incubator for moisturizing and aerobic cultivation, and raise the temperature Afterwards, they were placed in a constant temperature incubator at 37°C for ventilated culture, and the relative humidity was controlled at 85% to 95%.

After 2 days of aerobic fermentation, inoculate the seed culture of Lactobacillus plantarum, the inoculation ratio is Lactobacillus plantarum: The mass ratio of raw materials is 4-6:100, mix well, put in a one-way exhaust sealed bag, and place it anaerobically at room temperature Culture 6d. Determination of protein content, α-amylase content, acid protease content and the number of viable lactic acid bacteria in the feed.

Embodiment 2 multi-strain anaerobic fermentation feed

The specific implementation steps are as follows:

A. Preparation of Seed Solution

a. Preparation of pure culture of Bacillus subtilis seeds: transfer 1ml to 2ml of activated Bacillus subtilis liquid culture solution to 50ml LB seed culture solution, and culture at a constant temperature of 37°C for 10 to 18 hours.

b. Preparation of pure culture of Lactobacillus plantarum seeds: transfer 1ml-2ml of activated Lactobacillus plantarum liquid culture solution to 50ml MRS seed culture solution, and culture at 37°C for 10-18 hours.

c. Preparation of pure culture of brewer's yeast seeds: transfer 1ml-2ml of activated brewer's yeast liquid culture solution to 50ml of YPD seed culture solution, and culture at 28-30° C. for 12-18 hours.

B. Crushing of fermentation raw materials

Take 10-15 parts of corn, 10-15 parts of bran, 15-25 parts of soybean meal, 35-45 parts of palm meal, 10-15 parts of subflour, 1-2 parts of urea, 0.5-1 part of glucose, mix and grind , sieved, the fineness is 20-40 mesh.

C. Mixing water for fermentation raw materials

The ratio of material to water is 1:0.6~1.0, mix well and put on a plate.

D. Sterilization of fermentation raw materials

The fermentation raw materials are sterilized by high-pressure steam, and the sterilization conditions are 0.1Mpa-0.2Mpa, 15-30min.

E. Anaerobic fermentation

Inoculate Bacillus subtilis, Lactobacillus plantarum, and brewer's yeast seed cultures into the fermentation raw materials, and the inoculation mass ratio is as follows, Bacillus subtilis: raw material 0.5～2:100, Lactobacillus plantarum: raw material 0.5～2:100: brewer's yeast: raw material 2-8: 100. Mix well after inoculation, put in a one-way air-tight sealing bag, and carry out anaerobic fermentation. After 6 days of fermentation, the protein content, α-amylase content, acid protease content and the viable count of lactic acid bacteria in the feed were measured.

Embodiment 3 The method for making feed of the present invention

The specific implementation steps are as follows:

A. Activation of strains

a. Activation of Rhizopus: Pick the spores of Rhizopus from the slant of the test tube stored in the refrigerator at 0-4°C, inoculate them on the potato glucose slant test tube medium, cultivate them at a constant temperature of 28-30°C for 24-48 hours, and pick new bacteria The filaments were transferred to the potato glucose slant test tube culture medium, and cultured at a constant temperature of 28-30°C for 24-48h.

b. Activation of Aspergillus oryzae: Pick the spores of Aspergillus oryzae from the slant of the test tube stored in the refrigerator at 0-4°C, inoculate it on the potato glucose slant test tube medium, culture at a constant temperature of 28-30°C for 24-48 hours, and pick new bacteria The filaments were transferred to the potato glucose slant test tube culture medium, and cultured at a constant temperature of 28-30°C for 24-48h.

c. Activation of Bacillus subtilis: Pick a circle of Bacillus subtilis from the slant of the test tube in the refrigerator at 0-4°C, inoculate it on the medium of the LB slant test tube, cultivate it at a constant temperature of 37°C for 20-24 hours, and then transfer it to the LB slant test tube for culture On the base, culture at a constant temperature of 37°C for 20-24h.

d. Activation of Lactobacillus plantarum: pick a ring of Lactobacillus plantarum from the test tube slope of the 0-4°C refrigerator, inoculate it on the MRS liquid medium, cultivate it at a constant temperature of 37°C for 20-24 hours, and then transfer it to the MRS liquid medium once Incubate at 37°C for 20-24 hours.

e. Activation of brewer's yeast: Pick a ring of brewer's yeast from the test tube slope of the refrigerator at 0-4°C, inoculate it on the YPD liquid medium, cultivate it at a constant temperature of 37°C for 20-24 hours, and then transfer it to the YPD liquid medium again. Incubate at a constant temperature of 37°C for 20-24 hours.

B. Preparation of pure culture of strains

a. Preparation of pure culture of rhizopus seeds: inoculate the activated rhizopus mycelium on the solid medium of soybean meal bran medium, and culture at a constant temperature of 28-30° C. for 3-5 days.

b. Preparation of pure culture of Aspergillus oryzae seeds: inoculate the activated Rhizopus mycelium on the solid medium of soybean meal bran medium, and culture at a constant temperature of 28-30° C. for 3-5 days.

c. Preparation of pure culture of Bacillus subtilis seeds: transfer 1ml to 2ml of activated brewer's yeast liquid culture solution to 50ml of LB seed culture solution, and shake at a constant temperature of 37°C for 10 to 18 hours.

d. Preparation of pure culture of Lactobacillus plantarum seeds: transfer 1ml to 2ml of activated beer yeast liquid culture solution to 50ml of MRS seed culture solution, and culture at 37°C for 10 to 18 hours.

e. Preparation of pure culture of brewer's yeast seeds: transfer 1ml-2ml of activated brewer's yeast liquid culture solution to 50ml of YPD seed culture solution, and culture at 28-30°C for 12-18 hours.

C. Crushing of fermentation raw materials

Take 10-15 parts of corn, 10-15 parts of bran, 15-25 parts of soybean meal, 35-45 parts of palm meal, 10-15 parts of subflour, 1-2 parts of urea, 0.5-1 part of glucose, mix and grind , sieved, the fineness is 20-40 mesh.

D. Mixing water for fermentation raw materials

The ratio of material to water is 1:0.6~1.0, mix well and put on a plate.

E. Sterilization of fermentation raw materials

The fermentation raw materials are sterilized by high-pressure steam, and the sterilization conditions are 0.1Mpa-0.2Mpa, 15-30min.

F. Aerobic fermentation

Inoculate the seed cultures of Rhizopus, Aspergillus oryzae, Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae into the fermentation raw materials, and the inoculation mass ratio is as follows, Rhizopus: raw material 0.25～1.5:100, Aspergillus oryzae: raw material 0.25～1.5:100, Bacillus subtilis: raw material 0.5-2:100, Lactobacillus plantarum: raw material 0.5-2:100: brewer's yeast: raw material 2-8:100. Mix well after inoculation, place in a 28-30°C incubator for moisturizing and aerobic cultivation for 48 hours, and then place in a 37°C constant-temperature incubator for ventilated cultivation after the temperature rises, controlling the relative humidity to 85%-95%.

G. Anaerobic fermentation

After the aerobic stage, break up and mix the fermented feed, put it in a one-way air-tight sealed bag, and place it at room temperature for anaerobic cultivation.

After 6 days of anaerobic fermentation, the content of protein, α-amylase, acid protease and the number of viable lactic acid bacteria in the feed were measured.

Embodiment 4 adds enzyme preparation anaerobic fermentation feed method

The specific implementation steps are as follows:

A. Preparation of Seed Solution

a. Preparation of pure culture of Bacillus subtilis seeds: transfer 1ml to 2ml of activated Bacillus subtilis liquid culture solution to 50ml LB seed culture solution, and culture at a constant temperature of 37°C for 10 to 18 hours.

b. Preparation of pure culture of Lactobacillus plantarum seeds: transfer 1ml-2ml of activated Lactobacillus plantarum liquid culture solution to 50ml MRS seed culture solution, and culture at 37°C for 10-18 hours.

c. Preparation of pure culture of brewer's yeast seeds: transfer 1ml-2ml of activated brewer's yeast liquid culture solution to 50ml of YPD seed culture solution, and culture at 28-30° C. for 12-18 hours.

B. Crushing of fermentation raw materials

Take 10-15 parts of corn, 10-15 parts of bran, 15-25 parts of soybean meal, 35-45 parts of palm meal, 10-15 parts of subflour, 1-2 parts of urea, 0.5-1 part of glucose, mix and grind , sieved, the fineness is 20-40 mesh.

C. Mixing water for fermentation raw materials

The ratio of material to water is 1:0.6~1.0, mix well and put on a plate.

D. Sterilization of fermentation raw materials

The fermentation raw materials are sterilized by high-pressure steam, and the sterilization conditions are 0.1Mpa-0.2Mpa, 15-30min.

E. Vaccination

Inoculate Bacillus subtilis, Lactobacillus plantarum, and brewer's yeast seed cultures into the fermentation raw materials, and the inoculation mass ratio is as follows, Bacillus subtilis: raw material 0.5～2:100, Lactobacillus plantarum: raw material 0.5～2:100: brewer's yeast: raw material 2-8: 100.

F. Additional enzyme preparation

The inoculated fermented feed was inoculated with α-amylase 100U/g and acid protease 100U/g, mixed well, put into a one-way air-tight bag, and fermented at room temperature.

After 6 days of fermentation, the content of crude protein, α-amylase, acid protease and the number of viable yeast and lactic acid bacteria in the feed were measured.

The types and proportions of the fermentation raw materials in the above examples are the same, the protein content of the raw materials is 20.11% (calculated as absolute dry material), the content of α-amylase, acid protease and the number of live bacteria of lactic acid bacteria are all 0.

Correlation analysis is carried out to the fermented feed crude protein content, α-amylase, acid protease content, the viable count of lactic acid bacteria in embodiment 1, embodiment 2, embodiment 3, embodiment 4, analysis result is listed in Fig. 1, Figure 2, Figure 3 and Figure 4. As can be seen from Figure 1, among the four implementations, the protein content in the final fermented product of Example 3 is the highest, which is 32.00%, which is 59.12% higher than the protein content in the original feed, and the protein content of the final fermented feed in Example 4 is the second highest , was 27.10%, an increase of 34.26%. It can be seen that the fermentation process of Example 3 is most conducive to the improvement of protein content in the fermentation raw materials. As can be seen from Fig. 2, among the 4 embodiments, the α-amylase activity in the final fermented feed of Example 3 is the highest, which is 10 U/g, the fourth highest in Example 4, which is 70 U/g, and the lowest in Example 2, which is 24U/g. As can be seen from Fig. 3, in 4 kinds of embodiments, the acid protease activity in the final fermented feed of embodiment 3 is the highest, is 123U/g, and embodiment 4 times is high, is 71U/g, and embodiment 2 is the lowest, is 47U/g. g. As can be seen from Figure 4, the number of live lactic acid bacteria in the final fermented feed of Example 3 is the highest, and its logarithmic value is 12.1.

In addition, during the fermentation process, the mold secretes protease and glucoamylase. If the inoculum amount of the mold is too low, it will not be able to provide enough hydrolytic enzymes, and thus cannot obtain enough hydrolyzate, which will affect the growth of yeast and bacteria; Too strong, on the one hand, it will affect the growth of yeast and bacteria due to competition, and on the other hand, it will lead to insufficient oxygen supply.

Lactic acid bacteria, Bacillus subtilis and yeast play different roles in the fermentation process, and the ratio of each strain directly affects the quality of fermented feed, see Table 1 for details.

Table 1 Test results of bacteria and yeast inoculum in fermented feed

It can be seen from Table 1 that the protein content of the fermented feed is the highest when the inoculation amount of Bacillus subtilis is 1%, the inoculation amount of yeast is 6%, and the inoculation amount of lactic acid bacteria is 1%.

The aerobic stage can produce a large number of hydrolytic enzymes, and it is also the most important stage of single-cell protein production. The length of the aerobic phase is also related to temperature. Ferment at 28-30° C. for 36-48 hours, then heat up to 37° C. and continue to ferment for 24 hours. The protein content and enzyme activity of the fermented feed are the highest.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (8)

1. a method for multi-bacteria two-step combined fermentation feed, is characterized in that, mainly comprises the following steps: (1) activate bacterial classification, and prepare the pure cultures of Rhizopus, Aspergillus oryzae, Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae respectively; (2) Pretreatment of fermentation raw materials, including pulverizing the raw materials, mixing them with water, and then sterilizing them; (3) Aerobic fermentation: Inoculate pure cultures of Rhizopus, Aspergillus oryzae, Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae into the fermentation raw materials, mix well after inoculation, and place in 28-30°C for 36-36°C moisturizing and aerobic fermentation. After 48 hours, after the temperature rises, place it in a 37°C constant temperature incubator for ventilation and culture for 24 hours, and control the relative humidity at 85% to 95%; (4) Anaerobic fermentation: After the aerobic stage is over, break up and mix the fermented feed, put it into a one-way air-exhausting sealed bag, and place it at room temperature for anaerobic cultivation for 4-8 days; The step (2) pretreatment of fermented raw materials includes: 1. crushing of fermented raw materials: taking 10 to 15 parts of corn, 10 to 15 parts of bran, 15 to 25 parts of soybean meal, 35 to 45 parts of palm meal, 10 to 15 parts, 1 to 2 parts of urea, 0.5 to 1 part of glucose, mixed, crushed, and sieved to a fineness of 20 to 40 meshes; Mix well and put on a plate; ③Sterilization of fermentation raw materials: Carry out high-pressure steam sterilization on fermentation raw materials, the sterilization conditions are 0.1Mpa～0.2Mpa, 15～30min; The bacterial classification in the described step (3) is inoculated by mass ratio, and every 100 parts of sterilized materials are inoculated with 0.2 to 0.7 parts of the pure culture of Rhizopus bran seeds, and 0.2 to 0.7 parts of the pure culture of Aspergillus oryzae bran seeds, 0.8-1.2 parts of pure culture of Bacillus subtilis, 0.5-1.5 parts of pure culture of Lactobacillus plantarum, and 2-8 parts of pure culture of brewer's yeast. 2. method according to claim 1, is characterized in that, every 100 parts of sterilized materials, inoculate 0.2～0.7 part of rhizopus bran seed pure culture, 0.2～0.7 part of aspergillus oryzae bran seed pure culture, The inoculum size of the pure culture of Bacillus subtilis is 1 part, the inoculum size of the pure culture of Lactobacillus plantarum is 1 part, and the inoculum size of the pure culture of Saccharomyces cerevisiae is 6 parts. 3. method according to claim 1, is characterized in that, the preparation of rhizopus seed pure culture: the rhizopus mycelium after the inoculation activation is on the soybean meal bran culture medium solid medium, 28～30 ℃ constant temperature cultures 3. ~5d. 4. The method according to claim 1, characterized in that, the preparation of the pure culture of Aspergillus oryzae seeds: inoculate the activated Aspergillus oryzae mycelium on the solid medium of soybean meal bran medium, and cultivate at a constant temperature of 28～30°C 3~5d. 5. The method according to claim 1, characterized in that, the preparation of the pure culture of Bacillus subtilis seeds: inoculate the activated Bacillus subtilis on the slant to 50ml LB seed culture solution, and cultivate at 37°C for 10-18h with constant temperature oscillation. 6. The method according to claim 1, characterized in that the preparation of pure culture of Lactobacillus plantarum seeds: transfer 1ml to 2ml of activated Lactobacillus plantarum liquid culture solution to 50ml MRS seed culture solution, and stand at 37°C Cultivate for 10-18 hours. 7. The method according to claim 1, characterized in that the preparation of pure culture of brewer's yeast seeds: transfer 1ml to 2ml of activated brewer's yeast liquid culture solution to 50ml of YPD seed culture solution, and let stand at 28 to 30°C Cultivate for 12-18 hours. 8. The feed prepared by the method according to any one of claims 1-7.