CN107365718B

CN107365718B - Bacillus megaterium MYB3 and application thereof in straw fermented feed

Info

Publication number: CN107365718B
Application number: CN201710298708.3A
Authority: CN
Inventors: 李光春; 白冰
Original assignee: Yanbian University
Current assignee: Yanbian University
Priority date: 2017-04-27
Filing date: 2017-04-27
Publication date: 2021-02-02
Anticipated expiration: 2037-04-27
Also published as: CN107365718A

Abstract

The invention relates to Bacillus megaterium (Bacillus megaterium) MYB3 and application thereof in straw fermented feed. The preservation number of the bacillus megaterium MYB3 is CGMCC No.13429, and the bacillus megaterium MYB3 has cellulose and hemicellulose degradation capability. The bacillus megatherium MYB3 can be used as a microbial agent to be added into the process of producing feed by fermenting straws, and can be used for producing vitamin B12. The invention overcomes the defect that the prior leaven for various micro-storage feeds almost has no key bacteria related to the degradation of cellulose and hemicellulose, and is more suitable for the production of straw fermented feeds.

Description

Bacillus megaterium MYB3 and application thereof in straw fermented feed

Technical Field

The invention belongs to the technical field of agricultural microorganisms, and relates to bacillus megaterium MYB3 and application thereof in straw fermented feed.

Background

China is a big agricultural country, a large amount of straws are produced along with the improvement of crop yield, except that one part of the straws is applied to biogas fermentation and mushroom cultivation, most of the surplus straws are burned in the open air. The annual yield of the straws in China exceeds 7 hundred million tons, the straws are ranked first in the world, the crop straws contain rich nutrient elements such as nitrogen (N), phosphorus (P), potassium (K) and the like, the straws which are used as organic fertilizer resources account for 12-19% of the total amount of the organic fertilizer resources in China are not effectively utilized, the straws are burnt in the open air in the field to cause serious pollution to the surrounding environment such as air, soil and the like, and precious biological resources are wasted. With the development of animal husbandry and industry, the utilization of straws is more and more paid attention. However, the utilization mode of the straws in China is still at a lower level at present, and a great gap is still left compared with the developed countries.

The crop straw utilization approaches of Jilin province are mainly four, including passing through abdomen and returning to field, making fertilizer and returning to field, rural domestic energy and industrial raw materials, and are used for manufacturing industrial paper pulp, novel building material plates and the like. Among them, it is widely used to return straws to fields by incineration. At present, most residents in rural areas in Jilin province still use crop straws, grass and trees and the like as cooking and heating energy. The biomass energy resources comprise crop straws, firewood and various organic wastes, are main energy sources in rural areas, have the utilization amount of about 5280 ten thousand tons of standard coal and account for about 70 percent of energy consumption in rural areas. At present, biomass energy is mainly used for direct combustion, and the utilization rate is low. The comprehensive utilization of the straws not only can drive the traditional agriculture to change to the modern agriculture and promote the sustainable development of agricultural production, but also can provide a large amount of cheap raw materials for livestock raising, edible fungi, energy sources, processing industry and the like and promote the development of the livestock raising, the edible fungi, the energy sources, the processing industry and the like to scale, commercialization and industrialization, thereby driving the change of agricultural structures and even the whole rural structures and forming a new growth point of rural economy. Aiming at the current situation of straw utilization in China, the straw resources are more needed to be utilized according to local conditions and by means of advanced modern technologies, the utilization rate of the straws is improved, the utilization approach of the straws is expanded, the additional value of the straws is increased, and therefore the development requirements of the society and the economy are effectively met.

The straw softening method by utilizing the microbial preparation can improve the straw softening mode, obtain higher economic benefit, achieve the effects of not polluting the environment and keeping ecological balance, and accord with the principle of sustainable development. At present, most of the microbial preparations for straw fermentation sold in the market are composite microorganisms mainly comprising lactic acid bacteria, bacillus subtilis and saccharomycetes, or microbial preparations for composting, wherein mould and other bacteria with cellulose degradation capability are used as straw fermentation agents, and few descriptions are provided for the degradation capability of cellulose and hemicellulose in straws and the bacillus megaterium fermented into straw feed as main fermentation agents.

Although the microbial preparation contains various types of microorganisms and is complete in variety, most of straw fermentation inoculants sold in the market at present, such as lactic acid bacteria, saccharomycetes, bacillus subtilis and the like, are developed around the storage property, palatability and the like of the straws, and correspond to the theories and concepts of ensiling, yellow storage (micro storage) and the like of the straws, a quality firming method of ensiling and the like. In straw fermentation such as yellow corn silage and the like, microbial agents lack decomposition and conversion effects on cellulose, hemicellulose and the like, and the conversion rate of useful components of the straws is influenced by using the microbial agent products to ferment the straws.

Disclosure of Invention

The invention is made in view of the problems in the prior art, and the invention aims to provide bacillus megaterium MYB3 and application thereof in straw fermented feed, wherein the bacillus megaterium MYB3 has cellulose and hemicellulose degradation capacity.

In order to achieve the purpose, the Bacillus megaterium strain MYB3 is preserved in China general microbiological culture Collection center (CGMCC) in 2016, 12 and 07 days, the preservation number is CGMCC No.13429, and the preservation address is as follows: no. 3 of Xilu No.1 of Beijing, Chaoyang, and institute of microbiology of Chinese academy of sciences, and the Bacillus megaterium MYB3 has cellulose and hemicellulose degrading ability.

Further, the growth pH value of the bacillus megaterium MYB3 is 3-12.

Further, the growth temperature of the bacillus megaterium MYB3 is 20-45 ℃.

In order to achieve another purpose, the application of the bacillus megaterium MYB3 in the straw fermented feed is realized by the technical scheme that the bacillus megaterium MYB3 is added into the process of producing the feed by straw fermentation as a microbial agent.

Further, the application of the bacillus megaterium MYB3 in the straw fermented feed can simultaneously produce vitamin B12 in the process of producing the feed through straw fermentation.

Has the advantages that:

1. the Bacillus megaterium strain MYB3 has high-efficiency cellulose and hemicellulose degradation capability, is different from other micro-storage fermentation preparations, and is suitable for being used as a straw fermentation microbial agent to prepare micro-storage feed, particularly yellow storage feed.

2. The Bacillus megaterium strain MYB3 has wide acid-base resistance range, and the growth pH range is 3-12.

3. The viable thallus number of the Bacillus megaterium strain MYB3 cultured for 24 hours at 20-45 ℃ is 6.34-6.61(Log, CFU/mL), the viable thallus number of the thallus cultured for 24 hours at 35 ℃ reaches 6.8(Log, CFU/mL), and the strain is suitable for fermentation in different temperature ranges and survival under the temperature condition in animal intestinal tracts.

4. The vitamin B12 can be produced in the process of producing the feed by fermenting the straws, and the vitamin B12 additive is not used or is added in a small amount as the feed, so that the feed ingredients are saved, and the nutritional value of the feed is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention, the drawings used in the description of the embodiments section below are briefly described.

The reference numerals are explained below:

FIG. 1 is a photograph of the colony morphology of Bacillus megaterium (MYB 3) isolated in the first embodiment of the present invention on LB medium and observed under a microscope after Gram staining;

FIG. 2 shows the results of 16S rDNA analysis of Bacillus megaterium strain MYB3 isolated in the first embodiment of the invention;

FIG. 3 shows the viable cell count of Bacillus megaterium strain MYB3 at different temperatures according to a second embodiment of the present invention;

FIG. 4 shows the viable cell count of Bacillus megaterium strain MYB3 under different pH conditions according to a second embodiment of the present invention;

FIG. 5 shows the variation of the decomposition rate of cellulose and hemicellulose in the Bacillus megaterium strain MYB3 in the straw fermentation process for producing feed according to the third embodiment of the invention;

FIG. 6 shows the content variation of vitamin B12 in a straw fermented feed according to a third embodiment of the present invention;

FIG. 7 shows the activity change of the decomposition enzyme in straw fermentation according to the fourth embodiment of the present invention;

FIG. 8 shows the cellulose decomposition rates of different experimental groups of a fifth embodiment of the present invention;

fig. 9 shows the hemicellulose decomposition rates of different experimental groups of the fifth embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

Isolation and characterization of the first example species

Collecting rotten wood and humus soil around the rotten wood in hat mountains in Yanji city as samples, adding 195mL of sterile water into each sample, shaking uniformly at the rotating speed of 120rpm for 30min, standing, taking 2mL of supernatant, inoculating into a 300mL triangular flask containing 100mL of enrichment medium (containing filter paper strips), adding 3g of pretreated corn straw fragments (2-2.5 cm of yellow storage feed standard) into the triangular flask, and culturing at the rotating speed of 30 ℃ and 120 rpm. After 5d, the filter paper is disintegrated, and yellow flocculated substances appear in the sample liquid, namely straw is decomposed. Inoculating the sample into an enrichment culture medium with the same requirement for subculture, repeating the process for ten times, stabilizing straw decomposition bacteria, inoculating domesticated bacteria into an LB liquid culture medium for culture, screening strains on a solid culture medium after 24 hours, separating different strains one by one, circulating the strain on the LB solid culture medium for 5 times, and screening out a strain MYB3 when no mixed bacteria exist in the culture of the strains. As shown in fig. 1-2, the strain MYB3 was identified as bacillus megaterium by colony morphology on LB medium, observation under microscope after Gram staining and 16SrDNA identification analysis, and the bacillus megaterium MYB3 was deposited in china general microbiological culture collection center (CGMCC) at 2016, 12 and 07 months, with the deposit number of CGMCC No.13429, and the deposit address: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

Enrichment Medium K₂HPO₄ 2g，(NH₄)₂SO₄ 1.4g，MgSO₄·7H₂O 0.3g，CoCl₂·2mg，CaCl₂0.3g，FeSO₄·7H₂O 5mg,ZnSO₄ 1.7mg，MnSO₄·H₂O1.6 mg, filter paper (qualitative filter paper), distilled water 1L.

LB culture medium, yeast extract 5g, peptone 10g, NaCl 10g, distilled water 1L.

Growth characteristics of the second example species under different conditions

Quantitative variation of strain MYB3 at different temperatures: accurately sucking 0.1mL of bacterial liquid to be detected in an aseptic operation table, and inoculating the bacterial liquid to be detected into a centrifuge tube filled with 0.9mL of distilled water after sterilization, wherein the centrifuge tube is the bacterial liquid diluted by 10 times. Then accurately sucking 0.1mL of the diluted 10-fold centrifugal tube and connecting the diluted 10-fold centrifugal tube to the next centrifugal tube filled with 0.9mL of distilled water, namely bacterial liquid diluted 100-fold, and continuing the method until the diluted 10-fold centrifugal tube is obtained after connection⁸A bacterial liquid of the double. Respectively sucking 1mL of bacterial liquids with different dilutions, placing the bacterial liquids on a culture dish filled with LB solid culture medium after sterilization, and carrying out a plate counting method experiment. The culture dish is put into an incubator with the temperature of 20 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃ respectively for 24 hours, and then the colony number in the culture dish is calculated.

Quantitative variation of strain MYB3 under different pH conditions: inoculating the strain in solid culture medium into LB liquid culture medium, culturing at 30 deg.C for 1d, sucking 0.1mL, transferring into 10mL phosphate buffer solution with pH of 2, 3,4,5,6,7,8,9,10,11, 12, culturing at 30 deg.C for 4h, diluting bacterial liquid at each pH by 10^-1-10^-8Respectively coating the bacterial cells on a flat plate, culturing at 30 ℃ for 24h, and then carrying out a flat plate counting method experiment to calculate the viable count.

As shown in FIG. 3, the number of viable cells of MYB3 cultured at 20 ℃ for 24 hours was 6.34(Log, CFU/mL), and the number of viable cells cultured at 45 ℃ for 24 hours was 6.61(Log, CFU/mL). The strain has the highest viable count under the condition of 35 ℃, and the number of the strains reaches 6.8(Log, CFU/mL), namely the optimal growth temperature of the MYB3 strain is 35 ℃. The test range is 20-45 ℃, the temperature condition during ordinary straw fermentation is met, and the temperature is suitable for survival in animal intestinal tracts.

As shown in fig. 4, MYB3 strain was viable at pH range 3-12. The number of viable bacteria was 2.48(Log, CFU/mL) at pH 3, grew rapidly at pH 6, was 3.87(Log, CFU/mL), grew most vigorously at pH 8, reached 5.4(Log, CFU/mL), then began to decrease as the pH increased, and decreased to 4.39(Log, CFU/mL) at pH 12.

Application of third embodiment strain MYB3 in production of feed through straw fermentation

Chopping the straw (preferably corn straw) to length of 2-5cm, adjusting water content to 70% with distilled water (or underground water), adjusting pH to 3 with lactic acid, sealing at room temperature for 1-2 days, and sterilizing (after sampling, culturing in LB solid culture medium, and no bacteria can be observed). After sterilization, the strain MYB3 separated in the first embodiment is uniformly inoculated into straws (preferably corn straws) to serve as a microbial agent for straw fermentation, and the straws are bagged, sealed and fermented in a 30 ℃ constant temperature incubator. The viable count of the strain MYB3 is kept to 6.8 multiplied by 10 after fermenting for 10 days⁵cfu/mL。

As shown in FIG. 5, after the straw (preferably corn straw) is fermented for 10 days, the cellulose and hemicellulose decomposition rates reach 41% and 28%, respectively. The pH value of the fermented straw is 4.8, and the straw meets the requirements of yellow storage feed.

The fermented product was analyzed for vitamin B12 by taking samples at 3,5,10 and 20 days after fermentation of the straw (preferably corn straw), as shown in FIG. 6, the content thereof increased with the increase of the fermentation time, and the concentration reached 13.5mg/L at 20 days.

Fourth example measurement of Activity of lytic enzymes in straw liquid fermentation Process

And (3) cellulase activity determination: taking 0.5mL of fermented bacterial liquid, adding 1mL of CMC solution (prepared by 0.1mol/L HAC-NaAC with pH 4.8) with the mass fraction of 1%, preserving the temperature for 30min at 50 ℃, adding 3mL of DNS reagent, carrying out boiling water bath for 5min, cooling, diluting to 25mL, and measuring reducing sugar under the absorbance of 540 nm.

And (3) determination of hemicellulase activity: 0.5mL of the appropriately fermented bacterial solution was taken, 1.5mL of a 1.0g/100mL xylan solution (prepared using 0.1mol/L HAC-NaAC having a pH of 4.8) was added thereto, the mixture was incubated at 50 ℃ for 30min, 3mL of DNS reagent was added thereto, the mixture was cooled in a boiling water bath for 5min, and then diluted to 25mL, and the reducing sugar was measured at 520nm absorbance.

And (3) amylase activity determination: taking 0.5mL of fermented bacterial liquid, adding 0.5mL of 0.1mol/L citric acid buffer solution (pH is 5.6), preserving heat in 40 ℃ water bath for 15min, adding 1mL of 1% soluble starch preheated at 40 ℃, preserving heat in a 40 ℃ water bath kettle for 5min, immediately adding 0.4mol/L NaOH 2mL, and stopping enzyme activity. 2mL of the solution is taken and put into a 25mL volumetric flask, then 3mL of DNS reagent is added, boiling water bath is carried out for 5min, the solution is diluted to 25mL after cooling, and reducing sugar is measured under 540nm absorbance.

As shown in FIG. 7, the enzyme activity was in the state of an inverted U-shaped curve as a whole. After 1 day of fermentation, the activity of the cellulolytic enzyme was 4.672(U/mL) and gradually increased, reaching a peak at day 3, the activity was 19.279(U/mL), and the activity began to decrease as the straw continued to be decomposed, and was only 5.818(U/mL) at day 5. After 1 day of fermentation, the activity of the hemicellulase-degrading enzyme was 3.95(U/mL), then gradually increased, reaching a peak at day 2, and was 6.668(U/mL), and as the straw continued to be degraded, the activity began to decrease, with only 4.65(U/mL) at day 5. The amylase activity was 4.1(U/mL) after 1 day of fermentation, then gradually increased, reaching a peak at day 3, the enzyme activity reached 9.844(U/mL), and then the activity began to decline, with an activity of 7.045(U/mL) at day 5.

Fifth example straw fermented feed Properties

The MYB3 strain separated in the first embodiment is used as a microbial agent to ferment a mixture of corn straws and corn flour in different proportions, water is added into the corn straws in a control group, and the cellulose content and the hemicellulose content are respectively measured after 3 days and 5 days.

As shown in FIG. 8, on day 15, the cellulose decomposition rate of corn stover (30:0) reached 43.18%, and the cellulose decomposition rate of the corn stover-corn meal mixture (30:6) reached 77.26% compared to the control group (straw plus water), and the experimental results showed that adding corn meal increased the cellulose decomposition rate in straw fermentation.

As shown in FIG. 9, at day 15, the hemicellulose decomposition rate of corn stover (30:0) reached 27.91%, and the hemicellulose decomposition rate of the corn stover and corn flour mixture (30:6) reached 32.58% compared to the control (straw plus water), and the experimental results showed that adding corn flour increased the hemicellulose decomposition rate in straw fermentation, but did not increase much in comparison to the cellulose decomposition rate.

The Bacillus megaterium strain MYB3 has high-efficiency cellulose and hemicellulose degradation capability, is different from other micro-storage fermentation preparations, and is suitable for being used as a straw fermentation microbial agent to prepare micro-storage feed, particularly yellow-storage feed; the Bacillus megaterium strain MYB3 has wide acid-base resistance range, and the growth pH range is 3-12; the viable thallus number of the Bacillus megaterium strain MYB3 cultured for 24 hours at the temperature of 20-45 ℃ is 6.34-6.61(Log, CFU/mL), the viable thallus number of the thallus in 24 hours cultured at the temperature of 35 ℃ reaches 6.8(Log, CFU/mL), and the Bacillus megaterium strain is suitable for fermentation in different temperature ranges and survival under the temperature condition in animal intestinal tracts; the vitamin B12 can be produced in the process of producing the feed by fermenting the straws, and the vitamin B12 additive is not used or is added in a small amount as the feed, so that the feed ingredients are saved, and the nutritional value of the feed is improved.

The above-described embodiments are only intended to specifically illustrate the spirit of the present invention, and the scope of the present invention is not limited thereto, and it is a matter of course that other embodiments can be easily made by those skilled in the art by means of alterations, substitutions or modifications based on the technical contents disclosed in the present specification, and these other embodiments should be covered by the scope of the present invention.

Claims

1. The Bacillus megaterium MYB3 is characterized in that the preservation number of the Bacillus megaterium MYB3 is CGMCC No.13429, the Bacillus megaterium MYB is collected from rotten wood and humus soil around the rotten wood in hat mountains of Yangji city, Jilin province, and the Bacillus megaterium MYB3 has cellulose and hemicellulose degradation capacity.

2. The Bacillus megaterium MYB3 of claim 1, wherein the Bacillus megaterium MYB3 has a growth pH of 3-12.

3. Bacillus megaterium MYB3 as in claim 1 or 2, wherein the growth temperature of the Bacillus megaterium MYB3 is 20-45 ℃.

4. The use of bacillus megaterium MYB3 in straw fermented feed according to any one of claims 1-2, wherein bacillus megaterium MYB3 is added as a microbial agent to the process of straw fermented feed production.

5. The use of bacillus megaterium MYB3 in straw fermented feed as claimed in claim 3, wherein the bacillus megaterium MYB3 is added as a microbial agent to the process of producing feed by straw fermentation.

6. The use of bacillus megaterium MYB3 in straw fermented feed according to claim 4, wherein vitamin B12 is produced simultaneously during the process of producing feed by straw fermentation.

7. The use of bacillus megaterium MYB3 in straw fermented feed according to claim 5, wherein vitamin B12 is produced simultaneously during the process of producing feed by straw fermentation.