CN114015580B

CN114015580B - Strain and method for remarkably improving content of small peptide in fermented feed

Info

Publication number: CN114015580B
Application number: CN202111198412.7A
Authority: CN
Inventors: 佟毅; 金渭武; 杨鑫; 焦琳; 卢宗梅; 陈影; 李义; 张国峰; 安泰
Original assignee: Cofco Nutrition and Health Research Institute Co Ltd; Cofco Biochemical Anhui Co Ltd; Cofco Jilin Bio Chemical Technology Co Ltd
Current assignee: Cofco Nutrition and Health Research Institute Co Ltd; Cofco Biochemical Anhui Co Ltd; Cofco Jilin Bio Chemical Technology Co Ltd
Priority date: 2021-10-14
Filing date: 2021-10-14
Publication date: 2023-08-08
Anticipated expiration: 2041-10-14
Also published as: CN114015580A

Abstract

The invention relates to the field of microorganisms, and discloses a strain and a method for remarkably improving the content of small peptides in fermented feed. The preservation number of the aspergillus niger is CGMCC NO.22437. The aspergillus niger provided by the invention can produce protease, cellulase, amylase and the like with higher enzyme activity, and is especially suitable for producing small peptides. When the bran koji prepared by the black koji enzyme is used for producing feed by fermentation, the protein content in the feed can be improved, the small peptide content is obviously improved, the crude fiber content is reduced, and the feeding value of the raw materials is improved.

Description

Strain and method for remarkably improving content of small peptide in fermented feed

Technical Field

The invention relates to the field of microorganisms, in particular to aspergillus niger, a microbial agent, a bran koji and a preparation method thereof, and application of the aspergillus niger, the microbial agent and the bran koji in production of feed or microecological preparations.

Background

The microbial fermentation feed has a plurality of advantages, and in the microbial fermentation process, not only partial anti-nutritional factors can be decomposed, but also macromolecular proteins in the raw materials can be decomposed into a large number of small molecular polypeptides which are easy to digest, and meanwhile, special fragrance is generated, so that the feed intake of livestock and poultry is improved, and the microbial fermentation feed is widely applied to livestock and poultry cultivation. The catalogue of feed additive varieties (2013) allows 35 added microorganisms including bacillus, lactobacillus, saccharomycetes and mould to survive in a live bacterial state for a long time, and the probiotics enter the intestinal tracts of livestock and poultry to maintain and regulate the balance of intestinal flora, promote the secretion of immune globulin by the intestinal tracts and further enhance the immune function. At present, the use proportion of microbial fermented feed in countries and regions such as Europe and America is more than 50%, the biological feed feeding rate in pig farms in the Netherlands and Finland scale reaches 60%, the biological feed pig raising proportion in Danish reaches 80%, and the application of China in biological feed enterprises scale, variety, fermentation technology and feed has a certain gap from the international leading level.

Aspergillus niger (Aspergillus niger) is a common species in fungi of the genus Aspergillus, the phylum Deuteromycotina, the order Cellulars, and the family Convergencae. Aspergillus niger has cellulase, amylase, proteinase, lipase, xylanase, etc. and may be used in degrading starch, protein, etc. in fermented substrate into monosaccharide and small peptide for the growth of microbe, and the produced enzyme and the enzyme in gastrointestinal tract act together to degrade the nutrients in diet for easy absorption and utilization by animal. The addition of small peptide in daily ration of ruminant has certain effect of improving the growth performance, milk yield and milk components, and the research shows that the addition of small peptide in daily ration of dairy cow can raise the apparent digestibility of Organic Matter (OM) and acid washing fiber (ADF), raise immunity, raise milk yield and reduce nitrogen discharge. Peptides are used as key factors for influencing the growth of rumen microorganisms to reach maximum efficiency, and are closely related to the synthesis of microbial proteins, the composition of amino acids in small intestines and the like. The small peptide can obviously improve the activity of the fibrinolytic bacteria and the degradation rate of crude fibers, and in addition, the small peptide can not influence the pH of rumen, improve the contents of amino nitrogen and microbial protein and improve the energy utilization efficiency.

The biological feed product with high small peptide content is produced by adding enzyme preparation to decompose macromolecular protein into small peptide and adding functional microbe to ferment to increase small peptide content. The former has high cost, so the production of fermented feed with high small peptide content by using functional microorganisms is the first choice, but the small peptide content of the feed produced by the current industrial strains is generally low.

Disclosure of Invention

The invention aims to provide a strain capable of producing protease at high yield, and relates to aspergillus niger, a microbial inoculum, a bran koji and a preparation method thereof.

The molecular weight of the small peptide is generally below 1000Da, and the small peptide is composed of 2-3 amino acids. The digestive end products in the digestive tract are mostly small peptides, but not free amino acids, which can be completely absorbed into the blood circulation, promoting the digestion and absorption of nutrients. The animal and plant macromolecular proteins can be hydrolyzed into small molecular peptides or amino acids by utilizing the enzymatic reaction of protease, so that the absorption and utilization of the proteins by livestock and poultry animals are effectively promoted, the hydrolysis degree is high, and the flavor is good.

In order to achieve the above object, a first aspect of the present invention provides an aspergillus niger strain having a preservation number of CGMCC No.22437.

In a second aspect the invention provides a microbial agent comprising Aspergillus niger as described above.

In a third aspect, the present invention provides a method for preparing a bran koji, comprising: aspergillus niger as described above was inoculated into a bran koji medium for cultivation.

In a fourth aspect the present invention provides a bran koji prepared according to the method described above.

In a fifth aspect the invention provides the use of an aspergillus niger as described above, a microbial agent as described above, or a bran koji as described above in the manufacture of a feed or a microecological preparation.

The aspergillus niger provided by the invention can produce protease, cellulase, amylase and the like with higher enzyme activity, and is especially suitable for producing small peptides. The strain has simple fermentation process, is suitable for different feed raw materials and combinations thereof, and can effectively reduce the cost of fermented feed.

When the bran koji prepared by the black koji enzyme is used for producing feed by fermentation, the protein content in the feed can be increased, the small peptide content is obviously increased, the crude fiber content is reduced, the faint acid flavor is brought, the palatability of the feed is improved, the ratio of the bran koji to aspergillus is improved, and the feeding value of the raw materials is increased.

Preservation of organisms

Aspergillus niger (Aspergillus niger) of the invention is preserved in China general microbiological culture Collection center (address: north Chen West Lu No. 1, 3 of the area of Korea of Beijing, national institute of sciences of China, postal code: 100101) (preservation unit abbreviated as CGMCC), with preservation number of CGMCC No.22437, at day 27 of 5 of 2021.

Drawings

FIG. 1 is a microscopic morphological view of Aspergillus niger according to the invention.

Detailed Description

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In the invention, the small peptide refers to peptide with molecular weight less than or equal to 1000 daltons, and the content of the small peptide is measured according to the method in national standard GB/T22492-2008 soybean peptide powder. The crude protein has high species specificity macromolecules, is not easy to absorb, and can be absorbed only after being decomposed into amino acids or small peptides in the effect process. Small peptides have advantages over amino acid metabolic patterns. The carrier absorbed by the small peptide is not easy to saturate, the carrier has wide substrate, high speed and high efficiency, and the transport absorption of the small peptide promotes the absorption of amino acid. The small peptide can be directly absorbed and transported into blood circulation and tissue cells by intestinal walls without degradation, and provides raw materials for synthesizing organism proteins for the tissue cells. The small peptide product has obvious growth promoting effect on young prawn, and can promote growth of small intestinal villus of aquatic animal and raise immunity. The small peptide product fed by pigs can promote the synthesis of protein, the absorption and utilization of mineral substances, avoid the absorption competition among amino acids, improve the immunity of organisms and have physiological regulation effect; especially for early weaned pigs, the feed additive can effectively improve intestinal villus height, improve digestion and absorption of nutrient substances, relieve diarrhea and promote growth. Therefore, protease, cellulase and the like generated by fermenting the feed raw material by the aspergillus niger can effectively degrade crude protein and crude fiber, and the content of small peptide in the fermented feed raw material product is improved.

In a first aspect the invention provides an Aspergillus niger strain having a collection number CGMCC No.22437.

As shown in fig. 1, the microscopic examination result of the aspergillus niger shows that the conidium of the aspergillus niger is spherical, black and smooth. The mycelium is developed, the top is provided with a spherical top sac, and the conidium is thrown into a sphere shape, which is similar to chrysanthemum, and the diameter is 700-800 mu m.

Through transparent circle tests, the protease, the cellulase and the amylase secreted by the aspergillus niger have higher activity, and can have better degradation effect on improving the protein and the cellulose in processing byproducts; the strain has higher organic acid yield and is suitable for producing feed products.

The form of the microbial agent may be a form of microbial agent commonly known in the art, such as a solid or liquid microbial agent. Preferably, the microbial agent is present in the form of at least one of a koji, a bran koji, a wheat koji, a mycelium and a spore suspension.

The preparation methods of the Xiaoqu, the bran koji and the wheat koji can be prepared by referring to the preparation methods conventional in the art, and are not described herein.

The preparation method of the mycelium and spore suspension may be a preparation method conventional in the art, and will not be described herein.

The inoculation mode of the aspergillus niger can be a conventional inoculation mode in the field, such as inoculation in the form of spore suspension or inoculation by picking a loop of strain from the inclined surface of the aspergillus niger.

Preferably, the Aspergillus niger is inoculated in the form of a spore suspension in an inoculum size of 10 ⁵ The spores per gram of bran koji medium.

The bran koji culture medium may be a bran koji culture medium conventional in the art, preferably, the bran koji culture medium comprises bran and water.

Preferably, the bran content in the bran koji culture medium is 40-50 wt%.

Preferably, the bran koji culture medium comprises 35-45 parts by weight of bran, 2-8 parts by weight of soybean meal and 5-12 parts by weight of corn husks in 100 parts by weight.

Preferably, 100 parts by weight of the bran koji culture medium further comprises 0.8 to 1.5 parts by weight of ammonium sulfate, 0.05 to 0.1 part by weight of urea, 0.01 to 0.05 part by weight of monopotassium phosphate and 0.01 to 0.1 part by weight of magnesium sulfate.

In a preferred embodiment of the present invention, 100 parts by weight of the bran koji culture medium comprises 35 to 45 parts by weight of bran, 2 to 8 parts by weight of soybean meal, 5 to 12 parts by weight of corn husks, 0.8 to 1.5 parts by weight of ammonium sulfate, 0.05 to 0.1 part by weight of urea, 0.01 to 0.05 part by weight of monopotassium phosphate, 0.01 to 0.1 part by weight of magnesium sulfate and 50 to 75 parts by weight of water. The cellulase can be produced in high yield under the preferred bran koji culture medium.

According to a preferred embodiment of the present invention, the conditions of the culture include: the temperature is 28-30deg.C, and the time is 8-15 days.

During the culturing process, the seed may also be turned over by methods conventional in the art.

Preferably, the number of spores in the bran koji is 1×10 ⁹ CFU/g bran koji.

The present invention will be described in detail by examples.

In the following examples, the culture media used were sterilized at 121℃and 0.1MPa for 30 min.

The bran koji culture medium comprises: 36 parts of bran, 5 parts of soybean meal, 7.5 parts of corn husk, 1 part of ammonium sulfate, 0.075 part of urea, 0.025 part of monopotassium phosphate, 0.05 part of magnesium sulfate and the balance of water. The final moisture content is 50-75 wt.%.

The content of the crude protein in the fermented feed is measured according to the method for measuring the crude protein in the feed of national standard GB/T6432-1994.

The content of the small peptide is measured according to the method in the national standard GB/T22492-2008 soybean peptide powder.

The crude fiber content measurement is carried out according to the method for measuring the crude fiber in the national standard GBT 6434-2006 feed.

The method for measuring the enzyme activity of the protease adopts a transparent ring detection method, 1% (m/v) skim milk (sterilized at 110 ℃ for 10 min) is prepared, 3% (m/v) agar (sterilized at 121 ℃ for 20 min) is prepared, the skim milk and the agar are uniformly mixed in a ratio of 1:1, and the mixture is cooled to 45-50 ℃ to prepare a flat plate. And (3) spotting the cultured bacterial liquid to the middle of a flat plate, standing and culturing for 48 hours, measuring the diameter D of a transparent ring and the size D of a bacterial colony, calculating D/D, and judging the protease activity of the bacterial strain.

The method for measuring the enzyme activity of the cellulase adopts a transparent ring detection method, and dipotassium hydrogen phosphate of 0.5g/l (NH) ₄ ) ₂ SO ₄ 2g/l MgSO4.7H2O 0.25g/l, sodium carboxymethylcellulose (CMC) 1.88g/l, agar 0.2g/l, natural pH value, and sterilizing at 121deg.C for 20min. And (3) spotting the cultured bacterial liquid to the middle of the flat plate, standing and culturing for 48 hours, taking out the flat plate for culturing for a period of time, pouring a proper amount of 1% Congo red solution into the flat plate, then pouring a proper amount of 5.8% NaCl solution, and standing and dyeing for 5 minutes. The solution in the dish was poured off and washed once with distilled water, i.e. the staining was completed. The diameter D of the transparent ring and the colony size D are measured, D/D is calculated, and the cellulase activity of the strain is judged.

The method for measuring the enzyme activity of amylase adopts a transparent ring inspection method, and a flat plate culture medium is prepared: 2% (m/v) of soluble starch, 2% (m/v) of peptone, 1% (m/v) of yeast powder, 2% (m/v) of agar, sterilizing at 121 ℃ for 20min, and cooling to 45-50 ℃ to prepare a flat plate. And (3) spotting the cultured bacterial liquid to the middle of a flat plate, standing and culturing for 48 hours, measuring the diameter D of a transparent ring and the size D of a bacterial colony, calculating D/D, and judging the amylase activity of the bacterial strain.

In the examples below, the starting materials and reagents used were all obtained commercially unless otherwise specified.

Example 1

This example is intended to illustrate a simple evaluation of Aspergillus.

The protease, cellulase and amylase activities of 4 strains of bacteria were detected by the transparent loop method using 3 strains of Aspergillus niger and 1 strain of Aspergillus oryzae, respectively, and the D/D results are shown in Table 1.

TABLE 1

As can be seen from Table 1, the Aspergillus niger BC70113 disclosed by the invention can simultaneously block protease, cellulase and amylase, has higher enzyme activities of the protease, the cellulase and the amylase, and can be used for improving the degradation of protein and cellulose in feed raw materials.

Three strains of Aspergillus niger were evaluated for organic acidity, the results of which are shown in Table 2.

TABLE 2

Wherein, the organic acid yield of the Aspergillus niger BC70113 is higher, partial organic acid can be generated in the process of fermenting the feed in a summarized way, and the palatability and the sensory evaluation effect of the feed product are improved.

The Aspergillus niger BC70113 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22437 in the year 5 and the month 27 of 2021.

Example 2

In this example, the aspergillus niger and aspergillus niger cic 41131 according to the invention are used for preparing the bran koji respectively, and the protease activity and the cellulase activity of the bran koji prepared by the method are compared.

Preparation of bran koji: aspergillus niger is picked into a ring, inoculated into a conical flask containing 100g of bran koji culture medium, cultivated for 24 hours at 28 ℃, turned over once every 12 hours, and cultivated for 10 days to harvest koji. The turning method comprises the following steps: the right hand picks up the bottle, turns over the left palm, prevents the bran koji from caking as much as possible, and spreads the bottle bottom.

Bran koji spore count: 1g of bran koji was weighed and added to 300mL of an aqueous solution containing 0.2% Tween to prepare a spore suspension. Placing in an ultrasonic cleaner, performing ultrasonic treatment for 30min, counting by using a blood cell counting plate,

the invention relates to Aspergillus niger and Aspergillus nigerThe bran koji prepared from CICC 41131 has spore number of about 6X10 respectively ⁹ CFU/g bran koji and about 1.3X10 ⁹ CFU/g bran koji.

The preparation method of the crude enzyme liquid comprises the following steps: 5g of bran koji is weighed and dissolved in 45mL of water, leached in a water bath at 40 ℃ for 1h, and the filtrate is filtered and collected to obtain crude enzyme liquid.

The protease activity (GB/T28715-2012) and the cellulase activity (NY/T912-2004) of the crude enzyme liquid are detected, and the protease activity of the aspergillus niger is 55.38 +/-3.58U/g and the cellulase activity is 485+/-5U/g; the protease activity of the Aspergillus niger CICC 41131 is 10.42+/-1.35U/g, and the cellulase activity is 135+/-2U/g.

Example 3

This example is intended to illustrate the process of single feed material fermentation.

2kg of DDGS, guniting corn husks, rice bran or bean curd residues are respectively weighed, distilled water is correspondingly added according to the water content of the DDGS, guniting corn husks, rice bran or bean curd residues to obtain a single feed raw material with the water content of 30% -50%, the single feed raw material is packaged in a breathing bag according to 700 g/bag, the aspergillus niger bran which is prepared in example 2 is inoculated according to the inoculation amount of 0.3% by weight, and then the aspergillus niger bran is fully beaten and uniformly mixed, 1 part of the aspergillus niger bran is not inoculated, and the aspergillus niger bran is placed in a 28-30 ℃ incubator for standing fermentation for 5-7d.

The content of Crude Protein (CP), small peptide and Crude Fiber (CF) in the material, expressed as weight% before and after fermentation, on a dry basis, was determined and the results are shown in table 3.

TABLE 3 Table 3

As shown in Table 3, after the Aspergillus niger (BC 70113) is fermented, the crude protein content is improved by 10.7-18.9%, the small peptide content is improved by 88.8-165.1%, and the crude fiber content is reduced by about 10%. After fermentation by Aspergillus niger CICC 41131, the crude protein content is improved by 3-5%, the small peptide content is improved by 20-28%, and the crude fiber content is reduced by about 2%.

Therefore, the aspergillus niger disclosed by the invention has better small peptide production performance.

Example 4

This example is intended to illustrate the method of fermenting a mixed feed.

DDGS, rice bran, corn husks and bean curd residues are taken, mixed feed raw materials are prepared according to a formula (unit is g) shown in table 4, and the final moisture content is controlled to be 30% -50%. Packaging 700 g/bag into respiratory bags, inoculating Aspergillus niger bran koji prepared in example 2 according to 0.3 wt% respectively, stirring thoroughly, mixing well in triplicate, wherein 1 part is not inoculated, and standing in 28-30deg.C incubator for 5-7d.

TABLE 4 Table 4

Name of the name	Corn husk	DDGS	Rice bran	Bean curd residue	Sum up
						Formulation 1	800	800	400	0	2000
Formulation 2	600	1000	400	0	2000
						Formulation 3	400	1200	400	0	2000
Formulation 4	400	1200	200	200	2000

The content of Crude Protein (CP), small peptide and Crude Fiber (CF) in the material, expressed as weight% before and after fermentation, on a dry basis, was determined and the results are shown in table 5.

TABLE 5

In conclusion, compared with a control strain, the aspergillus niger fermented feed raw material can improve the protein content, especially the small peptide content, and can reduce the crude fiber content and improve the feeding value of the raw material.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims

1. Aspergillus niger strainAspergillus niger) The method is characterized in that the collection number of the aspergillus niger is CGMCC NO.22437.

2. A microbial agent comprising aspergillus niger according to claim 1.

3. The microbial agent of claim 2, wherein the microbial agent is in the form of at least one of a koji, a bran koji, a wheat koji, a mycelium, and a spore suspension.

4. A method for preparing a bran koji, comprising: inoculating Aspergillus niger of claim 1 into bran koji culture medium for culturing.

5. The method according to claim 4, wherein the Aspergillus niger seeds are inoculated in the form of a spore suspension in an amount of 10 ⁵ The spores per gram of bran koji medium.

6. The method according to claim 4, wherein the bran koji culture medium comprises bran and water;

wherein the content of water in the bran koji culture medium is 50-75 wt%.

7. The method as claimed in claim 6, wherein the bran koji culture medium comprises 35-45 parts by weight of bran, 2-8 parts by weight of bean pulp and 5-12 parts by weight of corn bran in 100 parts by weight.

8. The method as claimed in claim 7, wherein 100 parts by weight of the bran koji culture medium further comprises 0.8 to 1.5 parts by weight of ammonium sulfate, 0.05 to 0.1 part by weight of urea, 0.01 to 0.05 part by weight of monopotassium phosphate and 0.01 to 0.1 part by weight of magnesium sulfate.

9. The method of any one of claims 4-8, wherein the culturing conditions comprise: the temperature is 28-30deg.C, and the time is 8-15 days.

10. A bran koji produced according to the method of any one of claims 4 to 9; wherein the bran koji comprises the aspergillus niger of claim 1.

11. Use of aspergillus niger according to claim 1, a microbial agent according to claim 2 or 3, or a bran koji according to claim 10 for the production of feed or a microecological preparation.