CN111944723B - Technical method for producing bacillus megaterium by using white spirit vinasse - Google Patents
Technical method for producing bacillus megaterium by using white spirit vinasse Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
Abstract
The invention discloses a technical method for producing bacillus megaterium by using white spirit vinasse, which belongs to the technical field of fermentation engineering and comprises the following steps: activating strains, culturing primary seeds, pretreating white spirit vinasse, preparing a fermentation culture medium, and performing solid fermentation to obtain a bacillus megaterium culture; the fermentation process of the invention adopts a method for producing the bacillus megaterium by solid state fermentation, and the whole fermentation process follows the aseptic operation rules, so that the solid state culture production of the bacillus megaterium realizes pure culture and large-scale production. The fermentation culture medium solves the problems of low quantity of live bacteria in fermentation by taking the distiller's grains as the main raw material, easy pollution of mixed bacteria in liquid culture, low nutrient utilization rate and difficult separation of thalli and metabolites, and greatly improves the yield of the bacillus megaterium.
Description
Technical Field
The invention relates to the field of fermentation engineering, in particular to a technical method for producing bacillus megaterium by utilizing white spirit vinasse.
Background
With the development of the white spirit industry, the utilization of the distiller's grains, which is a byproduct of white spirit production, has become the key point of the industry work. The solid vinasse is a main byproduct of the production of the white spirit and also a main pollutant of the production of the white spirit. Because the raw materials such as sorghum, wheat and the like are generally adopted, the vinasse has high acidity and contains abundant proteins, amino acids, vitamins and various trace elements. Practice has proved that the comprehensive utilization degree of the distiller's grains directly affects the development of enterprises, and if the treatment is not good, the pressure on the environment is huge; the level of distiller's grains processed feed is related to the implementation of national feed policy.
For a long time, the distiller's grains are mainly directly used as rural feed and play an important role in promoting the development of rural breeding and the virtuous cycle of the biologic chain. However, the nutrient structure of the fresh distillers ' grains cannot meet the requirements of scientific feeding and the requirements of scale development of the feeding industry, particularly, the fresh distillers ' grains have the water content of over 65 percent, are extremely difficult to store, are not easy to manage and are easy to mildew, and a large amount of distillers ' grains are randomly stacked to seriously pollute the environment, which troubles the development of enterprises.
In the prior art, some patents and other publications disclose that culture is prepared by fermentation of white spirit vinasse and applied to feed, but in the fermentation process, various mixed bacteria are generally adopted: for example, various mixed fermentation cultures of yeast, mould, lactobacillus, bacillus subtilis and the like are complicated and fussy in process, and because the culture conditions of various strains are inconsistent, the final culture can be obtained only by adopting different culture conditions to carry out 2-3 stages of culture; in addition, although a plurality of strains are mixed for fermentation, the content of the final viable bacteria is not high, and analysis can cause death of part of strains due to the fact that the suitable culture conditions of the plurality of strains are different greatly and are not suitable for the environment under different culture conditions, so that the number of the final viable bacteria is not high, and further the application value of the final viable bacteria in the feed needs to be researched; for the reasons mentioned above, incomplete fermentation of the spent grains may also result, and the resulting increase in protein content of the fermentation culture is not very significant. Therefore, there is a need to prepare a fermentation culture based on distiller's grains for efficient application to animal feed for specific fermentation conditions of distiller's grains.
The Bacillus megaterium belongs to Bacillus (Bacillus), is gram-positive bacterium, can form spores, has strong radiation resistance, can grow on glucose and ammonium salt culture media, is aerobic, and is widely distributed in nature. In recent years, researches show that the bacillus megaterium is a multifunctional, safe, efficient, residue-free and toxic-side-effect-free excellent strain, and is widely applied to production of bio-organic fertilizers, production of phosphorus bacterial fertilizers, pesticide degradation, antibiotic synthesis, enzyme industry and the like.
At present, when the bio-organic fertilizer is produced, the bacillus megaterium is only used as one of mixed fermentation floras, and is often used together with bacillus subtilis and other microorganisms, and reports of preparing biological feed by fermenting the bacillus megaterium alone are not seen.
Disclosure of Invention
The invention aims to provide a technical method for producing bacillus megaterium by using distiller's grains so as to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a technical method for producing bacillus megaterium by using white spirit vinasse, which comprises the following steps:
(1) activating strains: streaking the strain preserved at-80 deg.C on solid seed culture medium plate, and culturing at 37-40 deg.C for 20-24 hr; the strain is bacillus megaterium;
(2) first-order seed culture: selecting three to five rings of activated bacillus megaterium by using an inoculating ring, inoculating the bacillus megaterium into a liquid seed culture medium, placing the bacillus megaterium into a shaking table, and performing shake culture for 20-24 hours to prepare a first-level seed bacterium;
(3) pretreating spirit distiller grains: uniformly mixing the distiller's grains with water according to the mass ratio of 1:1-1:2, adding sodium bicarbonate to adjust the pH value to 6.5-7.0, inoculating first-class seed bacteria, fermenting raw materials, and culturing for 3-7 days to obtain pretreated distiller's grains;
(4) preparing a fermentation culture medium;
(5) solid fermentation: inoculating 8-10% of first-class seed bacteria into the fermentation culture medium, uniformly stirring, maintaining the humidity and the temperature, and culturing for 20-24h to obtain second-class seed bacteria;
(6) and (5) further fermenting the secondary seed bacteria according to the step (5) to obtain the bacillus megaterium culture.
Further, in the step (2), the shake culture condition is a condition of a temperature of 35-40 ℃ and 250 rpm.
Further, in the step (3), the raw material fermentation conditions are a draft of 15 to 30% and a temperature of 30 to 35 ℃.
Further, the fermentation medium comprises the following components in parts by weight: 30-35 parts of pretreated distiller's grains, 5-10 parts of cane sugar, 5-9 parts of glucose, 1-3 parts of sodium chloride and KH2PH42-4 parts of MnSO4·H20.2-1.5 parts of O.
Further, the preparation method of the fermentation medium comprises the following steps: mixing the components of the fermentation medium, adding water according to the material-liquid ratio of 1:0.5-0.8, uniformly mixing, and sterilizing at the high temperature of 120-.
Further, in the step (5), the temperature is 35-37 ℃ and the humidity is 80-85%.
Further, the obtained Bacillus megaterium culture was hermetically packaged in a sealed bag and stored at a temperature of 25 ℃ or lower.
The invention also provides a bacillus megaterium microecological preparation, which is prepared by adding the bacillus megaterium culture prepared by the technical method into corn starch with the mass ratio of 10-15 times, uniformly mixing, drying in hot air at 50-70 ℃ for 5-10h, crushing, and sieving with a 40-60-mesh sieve.
The invention also provides the application of the bacillus megaterium prepared by the technical method or the bacillus megaterium microecological preparation in animal feed.
Further, 6-10% of the bacillus megaterium culture is added into animal feed according to the weight of the bacillus megaterium culture, and the mixture is uniformly mixed and used for feeding animals.
The invention discloses the following technical effects:
the invention prepares the bacillus megaterium by fermenting the distiller's grains as the raw material, the distiller's grains are byproducts of the production of the white spirit, the price is low, and the distiller's grains contain rich protein, amino acid, vitamin and various trace elements, which is beneficial to the growth of thalli.
The fermentation process of the invention adopts a method for producing the bacillus megaterium by solid state fermentation, and the whole fermentation process follows the aseptic operation rules, so that the solid state culture production of the bacillus megaterium realizes pure culture and large-scale production. The fermentation culture medium solves the problems of low viable count in fermentation by using the distiller's grains as the main raw material, easy pollution of mixed bacteria in liquid culture, low nutrient utilization rate and difficult separation of thalli and metabolites, greatly improves the yield of the bacillus megaterium, and obtains the fermentation product with the highest viable count reaching 8.4 multiplied by 1012cfu/g, and has high spore rate.
The bacillus megaterium has spores, has the advantages of acid resistance, alkali resistance, 100 ℃ high temperature resistance, extrusion resistance and the like, can keep stability in the granulation process and the acid stomach environment, does not proliferate in intestinal tracts, and only rapidly develops and converts the bacillus megaterium into nutritive cells with the metabolism function in the upper section of the intestinal tracts. The bacillus megaterium can acidify the intestinal tract to facilitate the absorption of iron, calcium, vitamin D and the like, promote the growth of animals, shorten the growth period, obviously improve the activity of protease, lipase and amylase, enhance the available variety of vegetable feeds for the animals, and simultaneously has a certain removing effect on aflatoxin to reduce the damage of aflatoxin accumulation to the animals; moreover, the bacillus megaterium belongs to nonpathogenic bacteria in facultative anaerobes, promotes the growth of anaerobic bacteria such as bifidobacteria, lactobacilli, clostridia and the like by self oxygen consumption, effectively inhibits the growth of aerobic bacteria such as enterobacteria, enterococci and the like in intestinal tracts, promotes the growth of normal flora of host intestinal tracts, keeps micro-ecological balance and improves the immunity of organisms.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The bacillus megaterium strain (Bacillus megaterium) adopted in the embodiment of the invention is purchased from China general microbiological culture Collection center, and the preservation numbers are as follows: CGMCC No. 15119; the bacterial colony characteristics of the bacillus megaterium strain are as follows: the colony has irregular morphology, wrinkled edges, roughness and opacity, and dry surface which is milk white or beige; observed under an optical microscope, the D45 strain thallus is rod-shaped, round-ended and flagellar-like; the gram reaction was positive.
Example 1
A technical method for producing bacillus megaterium by using distiller's grains comprises the following steps:
(1) activating strains: marking the bacillus megaterium strain preserved at the temperature of minus 80 ℃ on a solid seed culture medium plate, and culturing for 22h at the temperature of 38 ℃;
(2) first-order seed culture: selecting three to five rings of activated bacillus coagulans by using an inoculating ring, inoculating the bacillus coagulans into a liquid seed culture medium, placing the bacillus coagulans into a shaking table, and performing shake culture for 23 hours at the temperature of 380 ℃ and the speed of 250rpm to prepare first-class seed bacteria;
(3) pretreating spirit distiller grains: uniformly mixing the distiller's grains with water according to a mass ratio of 1:1.5, adding sodium bicarbonate to adjust the pH value to 6.8, inoculating first-class seed bacteria, keeping the ventilation volume at 23% and the temperature at 33 ℃, fermenting raw materials, and culturing for 5 days to obtain pretreated distiller's grains;
(4) preparing a fermentation medium, wherein the fermentation medium comprises the following components in parts by weight: 33 parts of pretreated distiller's grains, 8 parts of cane sugar, 7 parts of glucose, 2 parts of sodium chloride and KH2PH43 parts of MnSO4·H2O1 parts; mixing the components of the fermentation medium, adding water according to the feed-liquid ratio of 1:0.6, uniformly mixing, and sterilizing at 125 ℃ for 25min under high temperature and high pressure;
(5) solid fermentation: inoculating 9% of first-class seed bacteria into the fermentation culture medium, uniformly stirring, keeping the temperature at 36 ℃ and the humidity at 83%, and culturing for 22h to obtain second-class seed bacteria;
(6) and (5) further fermenting the secondary seed bacteria according to the step (5) to obtain the bacillus megaterium culture.
In the above examples, the total viable count of Bacillus megaterium was determined to be 8.4X 10 by PCA plate assay12cfu/g。
Adding 13 times of corn starch into the bacillus megaterium culture prepared by the method, uniformly mixing, drying in hot air at 60 ℃ for 8 hours, crushing, and sieving with a 50-mesh sieve to obtain the bacillus megaterium microecological preparation.
Example 2
A technical method for producing bacillus megaterium by using distiller's grains comprises the following steps:
(1) activating strains: marking the bacillus megaterium strain preserved at the temperature of minus 80 ℃ on a solid seed culture medium plate, and culturing for 20 hours at the temperature of 40 ℃; the strain is bacillus megaterium;
(2) first-order seed culture: selecting three to five rings of activated bacillus coagulans by using an inoculating ring, inoculating the bacillus coagulans into a liquid seed culture medium, placing the bacillus coagulans into a shaking table, and performing shake culture for 20 hours at the temperature of 40 ℃ and the speed of 250rpm to prepare first-class seed bacteria;
(3) pretreating spirit distiller grains: uniformly mixing the distiller's grains with water according to a mass ratio of 1:2, adding sodium bicarbonate to adjust the pH value to 6.5, inoculating first-class seed bacteria, keeping the ventilation volume at 30% and the temperature at 30 ℃, fermenting raw materials, and culturing for 7 days to obtain pretreated distiller's grains;
(4) fitting for mixingPreparing a fermentation culture medium, wherein the fermentation culture medium comprises the following components in parts by weight: 30 parts of pretreated distiller's grains, 10 parts of cane sugar, 5 parts of glucose, 3 parts of sodium chloride and KH2PH42 parts of MnSO4·H21.5 parts of O; mixing the components of the fermentation medium, adding water according to the feed-liquid ratio of 1:0.5, uniformly mixing, and sterilizing at 130 ℃ for 25min under high temperature and high pressure;
(5) solid fermentation: inoculating 8% of first-class seed bacteria into the fermentation culture medium, uniformly stirring, keeping the temperature at 37 ℃ and the humidity at 80%, and culturing for 24h to obtain second-class seed bacteria;
(6) and (5) further fermenting the secondary seed bacteria according to the step (5) to obtain the bacillus megaterium culture.
In the above examples, the total viable count of Bacillus megaterium was determined to be 6.8X 10 by PCA plate assay12cfu/g。
Adding 10 times of corn starch into the bacillus megaterium culture prepared by the method, uniformly mixing, drying in hot air at 70 ℃ for 5 hours, crushing, and sieving with a 60-mesh sieve to obtain the bacillus megaterium microecological preparation.
Example 3
A technical method for producing bacillus megaterium by using distiller's grains comprises the following steps:
(1) activating strains: marking the bacillus megaterium strain preserved at the temperature of-80 ℃ on a solid seed culture medium plate, and culturing for 24 hours at the temperature of 37 ℃; the strain is bacillus megaterium;
(2) first-order seed culture: selecting three to five rings of activated bacillus coagulans by using an inoculating ring, inoculating the bacillus coagulans into a liquid seed culture medium, placing the bacillus coagulans into a shaking table, and performing shake culture for 24 hours at the temperature of 35 ℃ and the speed of 250rpm to prepare first-class seed bacteria;
(3) pretreating spirit distiller grains: uniformly mixing the distiller's grains with water according to a mass ratio of 1:1, adding sodium bicarbonate to adjust the pH value to 7.0, inoculating first-class seed bacteria, keeping the ventilation quantity at 15% and the temperature at 35 ℃, fermenting raw materials, and culturing for 3 days to obtain pretreated distiller's grains;
(4) preparation of fermentation cultureThe culture medium comprises the following components in parts by weight: 35 parts of pretreated distiller's grains, 5 parts of cane sugar, 9 parts of glucose, 1 part of sodium chloride and KH2PH44 parts of MnSO4·H20.2 part of O; mixing the components of the fermentation medium, adding water according to the feed-liquid ratio of 1:0.8, uniformly mixing, and sterilizing at 120 ℃ for 25min under high temperature and high pressure;
(5) solid fermentation: inoculating 10% of first-class seed bacteria into the fermentation culture medium, uniformly stirring, maintaining the temperature at 35 ℃ and the humidity at 85%, and culturing for 20h to obtain second-class seed bacteria;
(6) and (5) further fermenting the secondary seed bacteria according to the step (5) to obtain the bacillus megaterium culture.
In the above examples, the total viable count of Bacillus megaterium was determined to be 8.3X 10 by the PCA plate assay11cfu/g。
Adding 15 times of corn starch into the bacillus megaterium culture prepared by the method, uniformly mixing, drying in hot air at 50 ℃ for 10 hours, crushing, and sieving with a 40-mesh sieve to obtain the bacillus megaterium microecological preparation.
Experimental example 1
In the experiment, 60 goats with the same weight are selected to perform the experiment in a certain farmer in Baoding City in Hebei province, and are randomly divided into three equal groups, namely group A, group B and group C, common feed purchased in the market is selected, the daily ration adopts the mixture of corn and straw, the common feed is added to the group A on the basis of the daily ration, the common feed of 6% of the bacillus megaterium culture prepared in the embodiment 1 is added to the group B on the basis of the daily ration, the common feed of 10% of the bacillus megaterium culture prepared in the embodiment 1 is added to the group C on the basis of the daily ration, and after the group C is fed for 2 months, compared with the group A, the daily feed intake of the group B and the group C is respectively increased by 0.35kg and 0.32 kg; it was also found that the average daily gain increased 0.23 kg/head, 0.18 kg/head for groups B and C compared to group A.
Experimental example 2
3600 healthy and active AA broilers with similar body weight and age of 19 days are selected, the test broilers are randomly divided into a control group and a test group, each group is provided with 4 replicates, and the test lasts for 37 days. In the feeding test, the feeding condition of each group is consistent with that of the group with free feeding and free drinking. The management of each group is carried out according to the conventional method, the growth activity, health, disease condition and other information of the chickens are observed and recorded every day, and the chickens are weighed on empty stomach and analyzed in the test on the 37 th day (56 days old). Wherein the complete formula feed for the broiler chickens in the experimental group comprises 70% of corn, 25% of soybean meal, 5% of premix and 5% of the complete formula feed in terms of addition of the bacillus megatherium microecological preparation prepared in example 1; the composition of the broiler complete formula feed of the control group is the same as that of the test group, but the bacillus megaterium microecological preparation is not added. The results show that the weight gain of the test group is increased by 8.65 percent (P is less than 0.01), the feed coefficient is increased by 6.24 percent (P is less than 0.05), and the survival rate is increased by 3.75 percent (P is less than 0.05) compared with the control group. The bacillus megaterium microecological preparation prepared by the invention can obviously improve the daily gain, the feed coefficient and the survival rate of the broiler chickens.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (4)
1. A method for producing bacillus megaterium by using distiller's grains is characterized by comprising the following steps of:
(1) activating strains: streaking the strain preserved at-80 deg.C on solid seed culture medium plate, and culturing at 37-40 deg.C for 20-24 hr; the strain is bacillus megaterium;
(2) first-order seed culture: selecting three to five rings of activated bacillus megaterium by using an inoculating ring, inoculating the bacillus megaterium into a liquid seed culture medium, placing the bacillus megaterium into a shaking table, and performing shake culture for 20-24 hours to prepare a first-level seed bacterium;
(3) pretreating spirit distiller grains: uniformly mixing the distiller's grains with water according to the mass ratio of 1:1-1:2, adding sodium bicarbonate to adjust the pH value to 6.5-7.0, inoculating first-class seed bacteria, fermenting raw materials, and culturing for 3-7 days to obtain pretreated distiller's grains;
(4) preparing a fermentation culture medium;
(5) solid fermentation: inoculating 8-10% of first-class seed bacteria into the fermentation culture medium, uniformly stirring, maintaining the humidity and the temperature, and culturing for 20-24h to obtain second-class seed bacteria;
(6) further fermenting the secondary seed bacteria according to the step (5) to obtain a bacillus megaterium culture;
in the step (2), the shake culture condition is a condition of temperature of 35-40 ℃ and 250 rpm;
in the step (3), the raw material fermentation conditions are that the ventilation quantity is 15-30% and the temperature is 30-35 ℃;
the fermentation medium comprises the following components in parts by weight: 30-35 parts of pretreated distiller's grains, 5-10 parts of cane sugar, 5-9 parts of glucose, 1-3 parts of sodium chloride and KH2PH42-4 parts of MnSO4·H20.2-1.5 parts of O;
the preparation method of the fermentation medium comprises the following steps: mixing the components of the fermentation medium, adding water according to the material-liquid ratio of 1:0.5-0.8, uniformly mixing, and sterilizing at the high temperature of 120-;
in the step (5), the temperature is 35-37 ℃, and the humidity is 80-85%;
the obtained Bacillus megaterium culture is sealed and packaged by a sealing bag and stored at the temperature below 25 ℃.
2. A bacillus megaterium microecological preparation is characterized in that a bacillus megaterium culture prepared by the technology of claim 1 is added with corn starch in a mass ratio of 10-15 times, the mixture is uniformly mixed, dried for 5-10 hours in hot air at 50-70 ℃, crushed and sieved by a 40-60-mesh sieve, and the bacillus megaterium microecological preparation is prepared.
3. Use of a Bacillus megaterium culture prepared by the method of claim 1 or a Bacillus megaterium probiotic of claim 2 for the preparation of animal feed.
4. The use as claimed in claim 3, wherein the feed is added to the animal feed in an amount of 6-10% by weight of the Bacillus megaterium culture, and the mixture is used for feeding animals.
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