CN115093998B

CN115093998B - Bacillus belicus for fermenting soybean meal

Info

Publication number: CN115093998B
Application number: CN202210725418.3A
Authority: CN
Inventors: 束刚; 江青艳; 朱灿俊; 曾宇贤; 李金成; 王雨君; 朱云龙
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2023-10-17
Anticipated expiration: 2042-06-24
Also published as: CN115093998A

Abstract

The application discloses bacillus belicus for fermenting soybean meal, which is preserved in the Guangdong province microorganism strain collection at the 10 th month 26 of 2020, and the preservation number is GDMCC No.61197. The bacillus beljalis (Bacillus velezensis) SG.X-1 provided by the application has antibacterial capability, acid production and low drug resistance, is used for fermenting soybean meal, and is applied to soybean meal fermentation and livestock and poultry feed for improving the feed intake and the body weight gain of animals and improving the oxidative damage of intestinal tracts.

Description

Bacillus belicus for fermenting soybean meal

Technical Field

The application relates to the technical field of biology, in particular to bacillus subtilis for fermenting soybean meal.

Background

The protein feed in China is deficient in raw material resources, and especially the external dependence of high-quality protein resources such as fish meal and the like is always high. The soybean meal is a high-quality protein source, is a byproduct of soybean oil extraction, and has high nutritive value and good amino acid balance. After the soybean meal is fermented, macromolecular substances can be degraded into small peptides, amino acids and various digestive enzymes, so that the soybean meal is suitable for feeding livestock and poultry to promote the growth and development of the livestock and poultry and improve the feed return level. Fermented soybean meal is commonly used in aquaculture to replace high-price fish meal so as to play a role in promoting growth; in poultry production, for improving poultry growth performance, carcass quality and intestinal microecology; in pig raising production, the production performance of pig groups is improved, the intestinal health of piglets is protected, the immunity is improved, the reproductive performance of sows is improved, and the like.

The effect of fermented soybean meal is directly dependent on its production process. The existing fermented soybean meal has uneven quality, incomplete elimination of anti-nutritional factors and macromolecular protein degradation, and is easy to be polluted by harmful miscellaneous bacteria. The quality of fermented soybean meal is affected by a number of factors. The quality of the biological fermentation feed is closely related to the selection of strains (single strain or multiple strains of mixed strains), the ratio of culture medium (liquid state and solid state) to fermentation substrate (material-water ratio) and the fermentation process parameters (temperature and humidity, oxygen, water content and the like), wherein the strains are key factors influencing the quality of fermented soybean meal. A variety of microorganisms exist, such as Lactobacillus (Lactobacillus plantarum and Bifidobacterium lactis), bacillus (Bacillus subtilis and Bacillus licheniformis), aspergillus (Aspergillus oryzae and Aspergillus niger), and yeast, etc., can be used to ferment the soybean meal. However, the different strains have different secretion proteases and metabolic characteristics, so that the nutritional ingredients and effects of the strain applied to the fermented feed are greatly different. Therefore, screening of suitable strains is an important break for improving the effect of fermented soybean meal.

Intestinal microorganisms are a complex microflora, large in number and of a wide variety. Studies have shown that the structure of the intestinal microbiota is closely related to host health. The livestock and poultry intestinal tract is a system with regional diversity, has a plurality of intestinal microorganisms and provides specific functions, and each region has a microbial community which is specially used for gene expression and functions and regulating complex and diverse digestive, immune, metabolic and endocrine processes. Many specific diseases or conditions are reported to be closely related to intestinal microorganisms, such as inflammatory bowel disease, obesity, and hypertension. In addition, as the protein raw material of the livestock and poultry feed takes the bean pulp as a main material, the intestinal microorganisms of the livestock and poultry feed have good substrate adaptability, and various digestive enzymes related to degrading the bean pulp can be produced. Therefore, how to obtain a strain which can ferment bean pulp and adapt to the intestinal environment has great significance.

Disclosure of Invention

The present application is directed to solving, at least to some extent, one of the problems in the related art. Therefore, the application aims to apply bacillus beijerinckii (Bacillus velezensis) SG.X-1 in soybean meal fermentation and livestock and poultry feed for improving the feed intake and the weight gain of animals and improving the oxidative damage of intestinal tracts.

In order to achieve the above purpose, the present application adopts the following technical scheme: the application provides bacillus beleidsi (Bacillus velezensis) SG.X-1, which is obtained by separating from the intestinal tract of a mouse, performing induced culture by a soybean meal culture medium, and then screening. The strain SG.X-1 was Bacillus bailii (Bacillus velezensis) by 16S rRNA gene sequence analysis. The strain has been deposited in the Guangdong province microorganism strain collection (GDMCC for short, address: no. 59 building 5 of the 100 th university of Mitsui, guangzhou City, ministry of microorganisms, post code 510070) on day 10 and 14 in 2020, and is classified and named as Bacillus belicus Bacillus velezensis, with deposit number GDMCC No.61197.

The microbiological characteristics of Bacillus belicus (Bacillus velezensis) SG.X-1 are: gram positive bacteria, the cell shape is rod-shaped, the diameter is not more than 2 mu m, the single colony is 3-5mm in size, the color is milky white, the bacterial colony is opaque, the surface of the bacterial colony is wrinkled, and the edge is irregular. The thalli can grow in an acidic environment with the pH value of more than 3.0, and has strong bile salt resistance.

An application of bacillus beleiensis in fermentation of soybean meal, which uses bacillus beleiensis SG.X-1 in fermentation of soybean meal.

Further, bacillus bailii strain SG.X-1 was inoculated into soybean meal in an amount of 1X 10 for fermentation ⁵ CFU/g。

Further, the condition parameters of the fermentation are set as follows: the fermentation temperature is 32-40deg.C, the fermentation time is 11-13d, and the total bacterial count of Bacillus bailii SG.X-1 after fermentation is 1×10 ¹⁰ CFU/g～1×10 ¹³ CFU/g。

Further, the soybean meal fermentation process comprises the following steps: inoculating bacillus bailii SG.X-1 into an MRS broth culture medium for expansion culture; preparing soybean meal with the moisture content of 60%, and sterilizing at high temperature; the bacillus beijerinus SG.X-1 after the expansion culture is processed according to the method of 1X 10 ⁵ Inoculating CFU/g strain into soybean meal; anaerobic fermentation is carried out for 11-13d in a bacterial incubator at the temperature of 32-40 ℃; the total bacterial count of bacillus belgium SG.X-1 in the finally obtained soybean meal is 1 multiplied by 10 ¹⁰ CFU/g～1×10 ¹³ CFU/g。

Furthermore, the soybean meal also comprises corn and bran, and the mass relation of the corn, the bran and the soybean meal is as follows: bean pulp, corn, bran=8:1:1.

The application also provides livestock and poultry feed, and the prepared soybean meal fermentation product is added into the livestock and poultry feed.

Further, the addition amount of the soybean meal fermentation product in the livestock and poultry feed is more than 5000ppm.

Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages: (1) The bacillus bailii (Bacillus velezensis) SG.X-1 provided by the application can resist acid production and bile salts, can resist the internal environment of the gastrointestinal tract, and has the performance of probiotics. (2) According to the bacillus belicus (Bacillus velezensis) SG.X-1 disclosed by the application, soybean meal can be fermented rapidly, the content of small peptide is increased, the levels of D-type and L-type amino acids are increased, and simultaneously organic acids such as lactic acid, acetic acid and the like are produced through metabolism. (3) According to the application, the fermented soybean meal of bacillus belicus (Bacillus velezensis) SG.X-1 is added into livestock and poultry feed, so that the feed intake and the weight gain of animals can be obviously improved, and the oxidative damage of intestinal tracts can be improved.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the application and together with the description, serve to explain the principles of the application.

In order to more clearly illustrate the embodiments of the application or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to a person skilled in the art that other drawings can be obtained from these drawings without inventive effort.

In the accompanying drawings:

FIG. 1 is a colony morphology of Bacillus belicus SG.X-1 in example 1;

FIG. 2 is a gram stain of Bacillus belicus SG.X-1 of example 1;

FIG. 3 is a spore stain of Bacillus bailii SG.X-1 of example 1;

FIG. 4 is an acid-producing acid chart of Bacillus belicus SG.X-1 in example 2;

FIG. 5 is a graph showing the results of bacteriostasis of Bacillus belicus SG.X-1 in example 2;

FIG. 6 is a graph showing the effect of Bacillus belicus SG.X-1 fermented soybean meal in example 3;

FIG. 7 is a graph showing the effect of adding Bacillus bailii SG.X-1 fermented soybean meal in example 4 on the productivity and intestinal health of white-feather broilers.

Detailed Description

The following examples are illustrative of the application and are not intended to limit the scope of the application. Modifications and substitutions to methods, procedures, or conditions of the present application may be made without departing from the spirit and nature of the application and are intended to be within the scope of the present application. Unless otherwise indicated, the experimental materials, reagents, instruments, etc. used in the examples of the present application are commercially available; all technical means in the embodiments of the present application are conventional means well known to those skilled in the art unless specifically indicated.

The MRS medium used in the following examples of the present application was formulated as follows: 10.0g of casein enzyme digest, 10.0g of beef extract powder, 4.0g of yeast extract powder, 2.0g of triammonium citrate, 5.0g of sodium acetate, 0.2g of MgSO4.7H2O, 0.05g of MnSO4.4H2O, 2.0g of dipotassium hydrogen phosphate, 20.0g of glucose, 1.08g of Tween 80, 15g of agar, and adjusting the pH to 5.7+/-0.2 by using distilled water to fix the volume to 1L. Placing into a sterilizing pot at 121deg.C, and sterilizing for 20min.

EXAMPLE 1 isolation and characterization of Bacillus bailii SG.X-1

1. Isolation of strain SG.X-1, comprising the steps of:

s1: isolation culture of strains

Taking 1g of mouse intestinal chyme sample, putting the sample into a test tube with 9mL of physiological saline, shaking and uniformly mixing by a vortex device to obtain 1:10 diluent, then taking the diluent for 10-time incremental dilution, and then selecting 3 diluents with proper gradients to be coated on MRS culture medium in 1mL each. Anaerobic culture is carried out for 48-72h at 37 ℃, colony morphology is observed and recorded, single colony with good growth vigor is picked up, streaked, separated and purified.

S2: gram staining of strains, specifically including:

s21: wiping the slide with alcohol, drying on an alcohol lamp, and then picking a loop of sterilized deionized water or physiological saline onto the slide by using an inoculating loop, and picking single colony to be smeared on the water drops until the colony is uniformly dispersed, wherein the colony morphology is shown in figure 1. And inactivating and fixing the smear on the flame of the alcohol lamp.

S22: 1-2 drops of crystal violet staining solution are dripped for 1min, and the solution is lightly washed by deionized water.

S23: after drying, gram iodine solution is dripped to act for 1min, and deionized water is used for lightly washing.

S24: the color was eluted with 95% alcohol drops and the slide was shaken from time to time until no purple fall off, which was continued for about 20-30 seconds, with deionized water.

S25: and (5) after drying, adding a sallow counterstain solution dropwise for counterstaining for 30s. Washing with deionized water, drying, and microscopic examination.

S26: and (5) oil microscopic examination. The purple bacteria are gram-positive bacteria; the red person is gram negative bacteria. The results are shown in FIG. 2.

S3: spore staining of the strain, specifically comprising:

s31: preparing bacterial liquid: adding 1-2 drops of sterile water into a small test tube, picking 2-3 rings of thalli from the inclined plane by using an inoculating loop, fully stirring the thalli in the test tube, and preparing thick bacterial liquid.

S32: adding a staining solution: adding 2-3% of malachite green water solution into a small test tube, and stirring with an inoculating loop to thoroughly mix the dye with the bacterial liquid.

S33: heating: immersing the test tube in boiling water bath, and heating for 15-20min.

S34: smearing: and (4) picking the ringworm liquid from the bottom of the test tube by using an inoculating loop, coating a slice on a clean glass slide, and airing.

S35: fixing: the smear was passed through an alcohol burner flame 3 times.

S36: decoloring: washing with water until no sparrow green color is present in the effluent.

S37: counterstaining: after 5min of dyeing with the red aqueous solution, the dyeing solution was decanted and directly blotted with absorbent paper without washing with water.

hS38: and (5) microscopic examination: first low power, then high power, and finally observing with an oil lens. Results: the spores are dark, and the spores and thalli are light. The results are shown in FIG. 3.

And (3) separating and screening in the step (1-3) to finally obtain a gram-positive bacterial strain with spores. This strain was numbered SG.X-1.

2. Identification of Strain SG.X-1

1. Morphological identification

The strain SG.X-1 in the logarithmic growth phase and stable in colony size was subjected to single colony status description, which mainly includes the colony size, color, transparency, colony surface status and colony edge status. The obtained single colony has a size of 3-5mm, and is milky white, opaque, wrinkled on the surface of the colony, and irregular in edge.

Then, the strain SG.X-1 in the logarithmic growth phase was stained, and the cell morphology was observed by an optical microscope. The strain has positive gram staining, rod shape cell morphology, diameter not more than 2 μm, and spore formation.

2. 16S rRNA sequence homology analysis

The total bacterial DNA is extracted by using a bacterial DNA extraction kit of Guangzhou Meiyu Biotechnology Co. The extracted samples were sent to Shanghai Bioengineering Co.Ltd for sequencing. BLAST homology alignment was performed on the results of the measurement in GenBank database, and strain type was determined to be Bacillus bailii (Bacillus velezensis).

The above test results show that the strain SG.X-1 is Bacillus bailii (Bacillus velezensis). The strain has been deposited at the Guangdong province microorganism strain collection (GDMCC for short, address: no. 59 building 5 of the 100 th university of Mitsui, guangzhou City, ministry of microorganisms, post code 510070) on day 10 and 14 in 2020, and is classified and named as Bacillus bailii Bacillus velezensis, with the deposit number of GDMCC No.61197.

EXAMPLE 2 stress resistance test of Bacillus bailii SG.X-1

1. Acid production resistance identification

Bacillus bailii SG.X-1 was inoculated into MRS medium at pH 6.2, and changes in pH were detected at 1d, 2d, 3d, 4d, 5d, 6d, 7d, respectively, using a hand-held pH meter, as shown in FIG. 4. When the bacillus belicus SG.X-1 is in an acidic environment, namely the pH is 3-6, the bacillus belicus SG.X-1 can still grow normally, the pH value is continuously reduced, and when the pH is about 3.5, the bacillus belicus SG.X-1 does not continuously produce acid. The results indicate that the strain has strong acid tolerance and can tolerate the influence of gastric acid.

2. Antibiotic resistance detection

S1: the developer red tetrazolium and MRS broth medium was prepared at 0.4% with deionized water and autoclaved. Preparing bile salt and antibiotics, and filtering and sterilizing for later use.

S2: 100. Mu.L of medium (20. Mu.L TTC and 80. Mu.L of blank broth) was added to each well of a 96-well plate using a row gun.

S3: 100 mu L of different antibiotics with initial concentration of 1000 mu g/mL are added to each row of first holes, 200 mu L is added, 100 mu L of liquid which is uniformly mixed in the first holes is sucked into the second holes by a row gun, double dilution is continued, 100 mu L is sucked into the third holes, blow mixing is continued, and 100 mu L in the last hole is discarded after the last hole.

S4: finally, 100uL of diluted SG.X-1 bacterial liquid is added into each hole, and the concentration of each row is about: 250. 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, 0.24,0.12 (unit: μg/mL).

S5: a row of positive controls with no antibiotics added to the broth and a row of negative controls with no antibiotics added to the blank broth alone were made on the plates.

S6: the 96-well plate was placed in an anaerobic incubator at 37℃for 24 hours, and the results were observed. TTC will appear red if there is bacterial growth; TTC does not change color if no bacteria grow. The concentration corresponding to the last hole which does not turn red on the horizontal row is the minimum antibacterial concentration of the medicine. The results are shown in Table 1 below.

TABLE 1 susceptibility results of Bacillus bailii SG.X-1 to different antibiotics

Name of the name	Minimum inhibitory concentration μg/mL	Sensitivity to
			Gentamicin	＞250	Extremely sensitive
Ciprofloxacin	62.5	Extremely sensitive
			Cefotaxime	7.81	Mesosensitization
Polymyxin	＞250	Extremely sensitive
			Trimethoprim	31.25	Extremely sensitive
Tigecycline	7.81	Mesosensitization
			Meropenem	7.81	Mesosensitization
Florfenicol	0.98	Hyposensitization
			Ampicillin (ampicillin)	128	Extremely sensitive

The experimental result shows that bacillus belicus SG.X-1 does not have good drug resistance, so that the bacillus belicus SG.X-1 is safe and reliable to use as a feed probiotic.

3. Bacteriostasis experiment of Bacillus bailii SG.X-1 on Escherichia coli and Salmonella

Bacillus bailii SG.X-1 was inoculated into MRS medium and cultured anaerobically at 37℃for 5d. Centrifuge at 12000rpm/min for 10min, collect supernatant and filter with bacterial filter for use. Salmonella and Staphylococcus aureus were inoculated onto LB-containing agar plates, punched, and the supernatants were added separately. After 1 day of culture, the results of drug sensitivity experiments were observed. The diameters of the inhibition zones of salmonella and staphylococcus aureus are respectively 9mm and 10mm, and the results are shown in figure 5; referring to the antibacterial drug efficacy test judgment standard, the bacillus beijerinus SG.X-1 strain is judged to be low sensitive to salmonella and staphylococcus aureus.

Example 3 Bacillus bailii SG.X-1 fermented soybean meal and detection

The soybean meal is fermented by bacillus belicus SG.X-1, and the fermentation process is as follows:

bacillus beleiensis sg.x-1 was inoculated into MRS broth for expansion culture. The ratio of the fermented materials is soybean meal, corn, bran=8:1:1, the moisture content is 60%, and the ratio is 1×10 after high-temperature sterilization ⁵ cfu/g strain is inoculated into the fermentation material, and anaerobic fermentation is carried out for 12 days in a bacterial incubator at 37 ℃. The total bacterial count of bacillus bailii SG.X-1 in the finally obtained fermentation material is 1 multiplied by 10 ¹⁰ CFU/g～1×10 ¹³ CFU/g。

After solid state fermentation of soybean meal for 15 days continuously by using bacillus bailii SG.X-1, sampling and detecting the content change of organic acid and amino acid in the fermented material every 3 days, and monitoring the fermentation acid production effect. As a result, as shown in FIG. 6, the soybean meal was fermented with Bacillus bailii SG.X-1 for 15 days, and it was found that Bacillus bailii SG.X-1 fermented soybean meal was metabolized to produce organic acids such as lactic acid, acetic acid and citric acid, with lactic acid content as high as 200mg/mL. In addition, the contents of D-glutamic acid, L-glutamic acid, D-alanine, L-alanine, D-aspartic acid and L-aspartic acid are obviously improved to reach the highest value after 3 days of fermentation. The bacillus belicus SG.X-1 is suggested to have better fermentation acid-producing capability.

Example 4 Effect of Bacillus bailii SG.X-1 fermented soybean meal addition on white-feathered broiler production performance and intestinal health

As shown in FIG. 7, 5kg/t corresponds to 5000ppm; the bacillus belicus SG.X-1 is used for fermenting soybean meal, and the soybean meal is dried, crushed and added into medium-sized chicken basic ration for production test, so that compared with a control group, the feed intake of chickens can be obviously improved by 0.2kg/t, 2kg/t and 5kg/t of fermented materials; in addition, the ferment material of 5kg/t obviously improves the weight gain of chickens; all three groups have no obvious influence on the material-weight ratio. Meanwhile, the mucous membrane detection of the ileum shows that the contents of superoxide dismutase, glutathione peroxidase and malondialdehyde in 5kg/t group broiler chickens are obviously reduced. In conclusion, 5kg/t of fermented soybean meal material is suggested to be capable of improving the growth performance of broilers and relieving the ileum oxidation injury.

It is to be understood that the above examples only represent preferred embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the application; it should be noted that, for a person skilled in the art, the above technical features can be freely combined, and several variations and modifications can be made without departing from the scope of the application; therefore, all changes and modifications that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

1. The application of bacillus beleiensis in the fermentation of soybean meal is characterized in that bacillus beleiensis (Bacillus velezensis) SG.X-1 is applied to the fermentation of soybean meal, and the bacillus beleiensis (Bacillus velezensis) SG.X-1 is deposited at the microorganism strain collection in Guangdong province at 10 months and 14 days in 2020, and the deposit number is GDMCC NO.61197.

2. The use according to claim 1, wherein bacillus beziacillus (Bacillus velezensis) sg.x-1 is inoculated in soybean meal for fermentation, wherein the inoculation amount is 1 x 10 ⁵ CFU/g。

3. The use according to claim 2, characterized in that the condition parameters of the fermentation are set as: the fermentation temperature is 32-40deg.C, the fermentation time is 11-13d, and the total bacterial count of Bacillus bailii (Bacillus velezensis) SG.X-1 after fermentation is 1×10 ¹⁰ CFU/g～1×10 ¹³ CFU/g。

4. The use according to claim 2, characterized in that the soybean meal fermentation process comprises the steps of: inoculating bacillus bailii (Bacillus velezensis) SG.X-1 into MRS broth culture medium for expansion culture; preparing soybean meal with the moisture content of 60%, and sterilizing at high temperature; amplifying the cultured Bacillus bailii (Bacillus velezensis) SG.X-1 according to 1X 10 ⁵ Inoculating CFU/g strain into soybean meal; anaerobic fermentation is carried out for 11-13d in a bacterial incubator at the temperature of 32-40 ℃; the total bacterial count of the Bacillus belicus (Bacillus velezensis) SG.X-1 in the finally obtained soybean meal is 1 multiplied by 10 ¹⁰ CFU/g～1×10 ¹³ CFU/g。

5. The use according to claim 4, wherein the soybean meal further comprises corn and bran, and the mass relationships of the corn and the bran are: bean pulp, corn, bran=8:1:1.

6. A livestock feed characterized in that the soybean meal fermentation product prepared in claim 2 is added into the livestock feed.

7. The livestock feed of claim 6, wherein the soybean meal fermentation product is added to the livestock feed in an amount of more than 5000ppm.