CN110447765B - Method for preparing bacillus natto culture by using distiller's grains and application of bacillus natto culture - Google Patents
Method for preparing bacillus natto culture by using distiller's grains and application of bacillus natto culture Download PDFInfo
- Publication number
- CN110447765B CN110447765B CN201910785245.2A CN201910785245A CN110447765B CN 110447765 B CN110447765 B CN 110447765B CN 201910785245 A CN201910785245 A CN 201910785245A CN 110447765 B CN110447765 B CN 110447765B
- Authority
- CN
- China
- Prior art keywords
- culture
- bacillus natto
- fermentation
- distiller
- grains
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
- A23K10/38—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material from distillers' or brewers' waste
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- 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
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21062—Subtilisin (3.4.21.62)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Biomedical Technology (AREA)
- Animal Husbandry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Physiology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Sustainable Development (AREA)
- Botany (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing a bacillus natto culture by using white spirit vinasse and application thereof, belonging to the technical field of fermentation engineering and comprising 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 natto culture; the fermentation process of the invention adopts a method for producing the bacillus natto by solid state fermentation, and the whole fermentation process follows the aseptic operation rules, so that the solid state culture production of the bacillus natto realizes pure culture and can realize 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 natto.
Description
Technical Field
The invention relates to the technical field of fermentation engineering, in particular to a method for preparing a bacillus natto culture by utilizing white spirit vinasse and application thereof.
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 shows that the final viable bacteria is not high in quantity because the suitable culture conditions of the plurality of strains are different greatly and part of the strains are dead due to the fact that the strains are not suitable for the environment under different culture conditions, so that the application value of the feed is still to be studied; 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.
Currently, the application effect and action mechanism of bacillus natto kinase as a novel microecological preparation in livestock and aquaculture are attracting wide attention of many scholars. In the high-density liquid fermentation industry of bacillus natto, sucrose, yeast extract, tryptone, sodium chloride and the like are generally used as basic culture media to ferment the bacillus natto, and the high-density liquid fermentation industry has the defects of low concentration of the quantity of bacteria generated in a unit culture medium, high cost of bacteria liquid separation and the like from the production aspect; meanwhile, the purification of the thallus after the liquid fermentation generally adopts a freeze drying or spray drying method to ensure high viable count, but the liquid fermentation contains much moisture, so a complex link of pre-dewatering is needed to carry out the freeze drying or spray drying process, which results in extremely high cost. The research and application of solid fermentation in the fermentation of bacillus natto at present are immature, and related reports of a relatively stable process capable of producing high-quality bacillus natto products do not exist, and the solid fermentation in an industrial fermentation method has the defects of strict requirements on production environment, difficulty and complexity in disinfection of production materials, easiness in contaminating mixed bacteria in the production process, low nutrient utilization rate, difficulty in separating thalli and metabolites and the like.
Furthermore, due to the complexity of microbial fermentation conditions, the fermentation conditions of different types of microbial bacteria are also very different, and cannot be used for reference, so a fermentation method and conditions specially for bacillus natto, which can utilize distiller's grains and can obtain bacillus natto with high viable count, are urgently needed.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a method for preparing a bacillus natto culture by using distiller's grains and application thereof.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for preparing a bacillus natto culture 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 natto;
(2) first-order seed culture: selecting three to five rings of activated bacillus natto by using inoculating rings, inoculating the bacillus natto into a liquid seed culture medium, placing the bacillus natto into a shaking table, and performing shake culture for 20-24 hours to prepare primary seed bacteria;
(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 natto culture.
Further, the bacillus natto has a strain name of TK-1 and a classification name of Bacillus subtilis, and the preservation number is as follows: CGMCC No.4731, preservation date: 2011, 4 months and 2 days, the preservation address is as follows: beijing, sunward area, Beicheng Weolu No. 1, storage unit: china general microbiological culture Collection center.
Further, in the step (2), the shake culture conditions are conditions of a temperature of 37-40 ℃ and 250 rpm.
Further, in the step (3), the raw material fermentation conditions are a draft of 15 to 30% and a temperature of 37 to 40 ℃.
Further, the fermentation medium comprises the following components in parts by weight: 25-65 parts of pretreated distiller's grains, 5-10 parts of sucrose, 3-9 parts of glucose, 3-5 parts of sodium chloride and KH2PO42-4 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 1:0.3-0.8, uniformly mixing, and sterilizing at the high temperature of 120-.
Further, in the step (5), the temperature is 37-40 ℃, and the humidity is 50-70%.
Further, the above steps further include: sealing and packaging the obtained Bacillus natto culture with a sealing bag, and storing at below 25 deg.C.
The invention also provides a bacillus natto microecological preparation, which is prepared by adding the bacillus natto culture prepared by the method into corn starch with the weight ratio of 5-9 times, uniformly mixing, drying for 5-13h in hot air with the temperature of 50-80 ℃, crushing, and sieving with a 40-60-mesh sieve.
The invention also provides application of the bacillus natto culture in animal feed, wherein the bacillus natto culture prepared by the method is added into the animal feed according to the weight ratio of 6-10%, and the bacillus natto culture is used for feeding animals after being uniformly mixed.
The invention discloses the following technical effects:
the bacillus natto takes the distiller's grains as raw materials to prepare the nattokinase by fermentation, the distiller's grains are byproducts of the production of white spirit, the price is low, and the distiller's grains contain rich protein, amino acid, vitamin and various trace elements, thereby being beneficial to the growth of thalli.
The fermentation process of the invention adopts a method for producing the bacillus natto by solid state fermentation, and the whole fermentation process follows the aseptic operation rules, so that the solid state culture production of the bacillus natto realizes pure culture and can realize 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 natto, and obtains the fermentation product with the highest viable count reaching 7.8 multiplied by 1012cfu/g, and has high spore rate, and the enzyme activity of the natto kinase can reach 9450FU/g dry basis at most.
The biological fermentation product rich in the bacillus natto and the nattokinase produced in the fermentation process is an ideal raw material for preparing a microecological preparation, and the bacillus natto has spores, is acid-resistant, alkali-resistant, 100 ℃ high-temperature-resistant and extrusion-resistant, can maintain stability in the granulation process and the acid stomach environment, does not proliferate in intestinal tracts, and only rapidly develops and is transformed into nutritive cells with metabolism in the upper section of the intestinal tracts. The bacillus natto can acidify the intestinal tract, is beneficial to the absorption of iron, calcium, vitamin D and the like, promotes the growth of animals, shortens the growth period, can obviously improve the activity of protease, lipase and amylase and enhances the available variety of plant feed for the animals; and the bacillus natto belongs to a non-pathogenic bacterium in aerobic bacteria, 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
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The bacillus natto adopted in the embodiment of the invention is purchased from the common microorganism center of China Committee for culture Collection of microorganisms. The strain name is TK-1, the classification name is Bacillus subtilis, and the preservation number is as follows: CGMCC No.4731, preservation date: 2011, 4 months and 2 days, the preservation address is as follows: beijing, sunward area, Beicheng Weolu No. 1, storage unit: china general microbiological culture Collection center.
Example 1
A method for preparing Bacillus natto culture from distiller's grains comprises the following steps:
(1) activating strains: streaking the strain preserved at-80 deg.C on a solid seed culture medium plate, and culturing at 38 deg.C for 22 h; the strain is bacillus natto;
(2) first-order seed culture: selecting three to five rings of activated bacillus natto by using inoculating rings, inoculating the bacillus natto into a liquid seed culture medium, placing the bacillus natto into a shaking table, and performing shake culture for 22 hours at the temperature of 38 ℃ and the speed of 250rpm to prepare first-grade 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 amount at 25% and the temperature at 38 ℃, fermenting raw materials, and culturing for 5 days to obtain pretreated distiller's grains;
(4) preparing a fermentation medium: the fermentation medium comprises the following components in parts by weight: 50 parts of pretreated white spirit vinasse, 6 parts of cane sugar, 7 parts of glucose, 4 parts of sodium chloride and KH2PO43 parts of MnSO4·H20.9 part of O; the preparation method comprises the following steps: mixing the components of the fermentation medium, adding water according to the feed-liquid ratio of 1:0.5, uniformly mixing, and sterilizing at 121 ℃ for 20min under high temperature and high pressure;
(5) solid fermentation: inoculating 9% of first-class seed bacteria into the fermentation culture medium, uniformly stirring, maintaining the humidity of 60% and the temperature of 39 ℃, and culturing for 23h to obtain second-class seed bacteria;
(6) and (5) further fermenting the secondary seed bacteria according to the step (5) to obtain the bacillus natto culture.
In the above examples, the total viable count of Bacillus natto was determined to be 7.8X 10 by PCA plate assay12cfu/g, the highest enzyme activity of the nattokinase reaches 9450FU/g dry basis.
Adding 6 times of corn starch into the bacillus natto culture prepared by the method, uniformly mixing, drying in hot air at 65 ℃ for 8 hours, crushing, and sieving with a 50-mesh sieve to obtain the bacillus natto microecological preparation.
Example 2
A method for preparing Bacillus natto culture from distiller's grains comprises the following steps:
(1) activating strains: streaking the strain preserved at-80 deg.C on a solid seed culture medium plate, and culturing at 37 deg.C for 24 hr; the strain is bacillus natto;
(2) first-order seed culture: selecting three to five rings of activated bacillus natto by using inoculating rings, inoculating the bacillus natto into a liquid seed culture medium, placing the bacillus natto into a shaking table, and performing shake culture for 24 hours at the temperature of 37 ℃ and the speed of 250rpm to prepare first-grade 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 40 ℃, fermenting raw materials, and culturing for 3 days to obtain pretreated distiller's grains;
(4) preparing a fermentation medium: the fermentation medium comprises the following components in parts by weight: 65 parts of pretreated distiller's grains, 5 parts of cane sugar, 9 parts of glucose, 3 parts of sodium chloride and KH2PO44 parts of MnSO4·H20.2 part of O; the preparation method comprises the following steps: mixing the components of the fermentation medium, adding water according to the feed-liquid ratio of 1:0.3, uniformly mixing, and sterilizing at 120 ℃ for 20min 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 humidity of 50% and the temperature of 40 ℃, 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 natto culture.
In the above examples, the total viable count of Bacillus natto was determined to be 6.5X 10 by PCA plate assay12cfu/g, the highest enzyme activity of the nattokinase reaches 8560FU/g dry basis.
Adding 9 times of corn starch into the bacillus natto culture prepared by the method, uniformly mixing, drying in hot air at 50 ℃ for 13h, crushing, and sieving with a 40-mesh sieve to obtain the bacillus natto microecological preparation.
Example 3
A method for preparing Bacillus natto culture from distiller's grains comprises the following steps:
(1) activating strains: streaking the strain preserved at-80 deg.C on a solid seed culture medium plate, and culturing at 40 deg.C for 20 hr; the strain is bacillus natto;
(2) first-order seed culture: selecting three to five rings of activated bacillus natto by using inoculating rings, inoculating the bacillus natto into a liquid seed culture medium, placing the bacillus natto into a shaking table, and performing shake culture for 20 hours at the temperature of 40 ℃ and the speed of 250rpm to prepare first-grade 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 37 ℃, fermenting raw materials, and culturing for 7 days to obtain pretreated distiller's grains;
(4) preparing a fermentation medium: the fermentation medium comprises the following components in parts by weight: 25 parts of pretreated distiller's grains, 10 parts of cane sugar, 3 parts of glucose, 5 parts of sodium chloride and KH2PO42 parts of MnSO4·H21.5 parts of O; the preparation method comprises the following steps: mixing the components of the fermentation medium, adding water according to the feed-liquid ratio of 1:0.8, uniformly mixing, and sterilizing at 130 ℃ for 20min under high temperature and high pressure;
(5) solid fermentation: inoculating 8% of first-class seed bacteria into the fermentation culture medium, uniformly stirring, maintaining the humidity of 70% and the temperature of 37 ℃, 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 natto culture.
In the above examples, the total viable count of Bacillus natto was determined to be 8.9X 10 by PCA plate assay11cfu/g, the highest enzyme activity of the nattokinase reaches 8279FU/g dry basis.
Adding corn starch in an amount which is 5 times of the weight of the bacillus natto culture prepared by the method, uniformly mixing, drying for 5 hours in hot air at 80 ℃, crushing, and sieving by a 60-mesh sieve to obtain the bacillus natto microecological preparation.
Example 4
A method for preparing Bacillus natto culture from distiller's grains comprises the following steps:
(1) activating strains: streaking the strain preserved at-80 deg.C on a solid seed culture medium plate, and culturing at 39 deg.C for 21 hr; the strain is bacillus natto;
(2) first-order seed culture: selecting three to five rings of activated bacillus natto by using inoculating rings, inoculating the bacillus natto into a liquid seed culture medium, placing the bacillus natto into a shaking table, and performing shake culture for 21 hours at the temperature of 39 ℃ and the speed of 250rpm to obtain first-grade seed bacteria;
(3) pretreating spirit distiller grains: uniformly mixing the distiller's grains with water according to the mass ratio of 1:1.3, adding sodium bicarbonate to adjust the pH value to 6.6, inoculating first-class seed bacteria, keeping the ventilation amount at 18% and the temperature at 38 ℃, fermenting raw materials, and culturing for 34 days to obtain pretreated distiller's grains;
(4) preparing a fermentation medium: the fermentation medium comprises the following components in parts by weight: 35 parts of pretreated distiller's grains, 7 parts of cane sugar, 5 parts of glucose, 4 parts of sodium chloride and KH2PO43 parts of MnSO4·H20.5 part of O; the preparation method comprises the following steps: mixing the components of the fermentation medium, adding water according to the feed-liquid ratio of 1:0.6, uniformly mixing, and sterilizing at the high temperature and the high pressure of 126 ℃ for 20 min;
(5) solid fermentation: inoculating 8.5% of first-class seed bacteria into the fermentation culture medium, uniformly stirring, maintaining the humidity of 55% and the temperature of 37 ℃, 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 natto culture.
In the above examples, the total viable count of Bacillus natto was determined to be 8.3X 10 by PCA plate assay11cfu/g, the highest enzyme activity of the nattokinase reaches 8294FU/g dry basis.
Adding 6 times of corn starch into the bacillus natto culture prepared by the method, uniformly mixing, drying for 7 hours in hot air at 75 ℃, crushing, and sieving with a 60-mesh sieve to obtain the bacillus natto microecological preparation.
Experimental example 1
In the experiment, 30 goats with the same weight are selected as an experiment for a certain farmer in Chengde city of Hebei province and are randomly divided into three equal groups, namely, group A, group B and group C, and common feed purchased in the market is selected, wherein the daily ration adopts a 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 the bacillus natto culture prepared in 6% is added to the group B on the basis of the daily ration, the common feed of the bacillus natto culture prepared in 10% 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 0.32kg and 0.28 kg; it was also found that the average daily gain increased 0.18 kg/head, 0.15 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 amount of the bacillus natto microecological preparation prepared in the addition 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 natto microecologics are not added. The results show that the weight gain of the test group is improved by 7.21 percent (P is less than 0.01), the feed coefficient is improved by 5.84 percent (P is less than 0.05) and the survival rate is improved by 3.24 percent (P is less than 0.05) compared with the control group. The bacillus natto microecological preparation prepared by the invention can obviously improve the daily gain, the feed coefficient and the survival rate of the broiler chicken.
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 (5)
1. A method for preparing a bacillus natto culture by using distiller's grains is characterized by comprising 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 natto;
(2) first-order seed culture: selecting three to five rings of activated bacillus natto by using inoculating rings, inoculating the bacillus natto into a liquid seed culture medium, placing the bacillus natto into a shaking table, and performing shake culture for 20-24 hours to prepare primary seed bacteria;
(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 medium: the fermentation medium comprises the following components in parts by weight: 25-65 parts of pretreated distiller's grains, 5-10 parts of sucrose, 3-9 parts of glucose, 3-5 parts of sodium chloride and KH2PO42-4 parts of MnSO4·H20.2-1.5 parts of O;
(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 natto culture;
in the step (2), the shake culture condition is a condition of 37-40 ℃ and 250 rpm;
in the step (3), the raw material fermentation conditions are that the ventilation quantity is 15-30% and the temperature is 37-40 ℃;
in the step (5), the temperature is 37-40 ℃, and the humidity is 50-70%.
2. The method for preparing a bacillus natto culture using distiller's grains according to claim 1, wherein the fermentation medium is prepared by: mixing the components of the fermentation medium, adding water according to the material-liquid ratio of 1:0.3-0.8, uniformly mixing, and sterilizing at the high temperature of 120-.
3. The method for preparing a culture of bacillus natto using distiller's grain according to claim 1, further comprising: sealing and packaging the obtained Bacillus natto culture with a sealing bag, and storing at below 25 deg.C.
4. A bacillus natto microecological preparation is characterized in that a bacillus natto culture prepared by the method of any one of claims 1 to 3 is added with corn starch with the weight ratio of 5 to 9 times, the mixture is uniformly mixed, dried for 5 to 13 hours in hot air with the temperature of 50 to 80 ℃, crushed and sieved by a sieve with 40 to 60 meshes, and the bacillus natto microecological preparation is prepared.
5. An application of the natto bacillus culture in animal feed is characterized in that the natto bacillus culture prepared by the method of any one of claims 1 to 3 is added into the animal feed according to the weight ratio of 6 to 10 percent and is used for feeding animals after being uniformly mixed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910785245.2A CN110447765B (en) | 2019-08-23 | 2019-08-23 | Method for preparing bacillus natto culture by using distiller's grains and application of bacillus natto culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910785245.2A CN110447765B (en) | 2019-08-23 | 2019-08-23 | Method for preparing bacillus natto culture by using distiller's grains and application of bacillus natto culture |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110447765A CN110447765A (en) | 2019-11-15 |
| CN110447765B true CN110447765B (en) | 2021-03-23 |
Family
ID=68488857
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910785245.2A Active CN110447765B (en) | 2019-08-23 | 2019-08-23 | Method for preparing bacillus natto culture by using distiller's grains and application of bacillus natto culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110447765B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111019861B (en) * | 2019-12-25 | 2020-08-14 | 烟台富康生物科技有限公司 | Bacillus natto and application thereof |
| CN111518727A (en) * | 2020-05-09 | 2020-08-11 | 李晟雪 | High-strength and high-density fermentation medium for bacillus subtilis and method for preparing microbial inoculum by fermenting bacillus subtilis |
| CN111793589B (en) * | 2020-08-21 | 2022-03-11 | 四川润格生物科技有限公司 | Technical method for producing bacillus mucilaginosus by utilizing white spirit vinasse |
| CN111944723B (en) * | 2020-08-21 | 2022-03-11 | 四川润格生物科技有限公司 | Technical method for producing bacillus megaterium by using white spirit vinasse |
| CN111748504B (en) * | 2020-08-21 | 2023-06-06 | 四川润格生物科技有限公司 | Technical method for producing enterococcus faecium by using white spirit lees |
| CN111938020A (en) * | 2020-08-21 | 2020-11-17 | 四川润格生物科技有限公司 | Technical method for producing enterococcus faecalis by using distiller's grains |
| CN113621674B (en) * | 2021-08-27 | 2022-06-21 | 泸州老窖股份有限公司 | Method for producing L-lactic acid by using liquor distiller grains |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001352975A (en) * | 2000-06-15 | 2001-12-25 | Hiroyuki Sumi | Physiologically active substance derived from bacillus natto |
| CN101215535A (en) * | 2007-12-28 | 2008-07-09 | 无锡市高宝特生物工程技术有限公司 | Solid fermentation process for preparing bacillus natto microecological preparation |
| CN101869906A (en) * | 2010-06-24 | 2010-10-27 | 天津北洋百川生物技术有限公司 | Biological in-situ pretreatment method of solid organic wastes |
| CN102715342A (en) * | 2012-06-01 | 2012-10-10 | 陈华友 | Method for processing microbiological feed based on spirit vinasse and miscellaneous meal |
| CN102783557A (en) * | 2012-08-06 | 2012-11-21 | 贵州大学 | Method for producing thallus feed by white spirit distiller's grain |
| CN103005147A (en) * | 2012-10-29 | 2013-04-03 | 重庆百奥帝克微生态科技有限公司 | Biological feed based on vinasse |
| CN103141666A (en) * | 2013-03-28 | 2013-06-12 | 山东轻工业学院 | Method for producing microbe feed probiotics by using white spirit vinasse |
| CN103289910A (en) * | 2012-10-23 | 2013-09-11 | 广州格拉姆生物科技有限公司 | Solid fermentation production method of bacillus coagulans |
| CN103289916A (en) * | 2013-03-20 | 2013-09-11 | 广州格拉姆生物科技有限公司 | A solid fermentation production method for Bacillus Subtilis natto |
| CN104905013A (en) * | 2015-06-30 | 2015-09-16 | 河南双成生物科技有限公司 | Method for producing protein feed raw materials by carrying out enzymolysis and fermentation on white spirit vinasse |
| CN107594082A (en) * | 2017-09-11 | 2018-01-19 | 辽宁爱普罗斯饲料有限公司 | A kind of liquid raw material fermentation wine brewing lees feedses preparation method and feeding method |
-
2019
- 2019-08-23 CN CN201910785245.2A patent/CN110447765B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001352975A (en) * | 2000-06-15 | 2001-12-25 | Hiroyuki Sumi | Physiologically active substance derived from bacillus natto |
| CN101215535A (en) * | 2007-12-28 | 2008-07-09 | 无锡市高宝特生物工程技术有限公司 | Solid fermentation process for preparing bacillus natto microecological preparation |
| CN101869906A (en) * | 2010-06-24 | 2010-10-27 | 天津北洋百川生物技术有限公司 | Biological in-situ pretreatment method of solid organic wastes |
| CN102715342A (en) * | 2012-06-01 | 2012-10-10 | 陈华友 | Method for processing microbiological feed based on spirit vinasse and miscellaneous meal |
| CN102783557A (en) * | 2012-08-06 | 2012-11-21 | 贵州大学 | Method for producing thallus feed by white spirit distiller's grain |
| CN103289910A (en) * | 2012-10-23 | 2013-09-11 | 广州格拉姆生物科技有限公司 | Solid fermentation production method of bacillus coagulans |
| CN103005147A (en) * | 2012-10-29 | 2013-04-03 | 重庆百奥帝克微生态科技有限公司 | Biological feed based on vinasse |
| CN103289916A (en) * | 2013-03-20 | 2013-09-11 | 广州格拉姆生物科技有限公司 | A solid fermentation production method for Bacillus Subtilis natto |
| CN103141666A (en) * | 2013-03-28 | 2013-06-12 | 山东轻工业学院 | Method for producing microbe feed probiotics by using white spirit vinasse |
| CN104905013A (en) * | 2015-06-30 | 2015-09-16 | 河南双成生物科技有限公司 | Method for producing protein feed raw materials by carrying out enzymolysis and fermentation on white spirit vinasse |
| CN107594082A (en) * | 2017-09-11 | 2018-01-19 | 辽宁爱普罗斯饲料有限公司 | A kind of liquid raw material fermentation wine brewing lees feedses preparation method and feeding method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110447765A (en) | 2019-11-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110447765B (en) | Method for preparing bacillus natto culture by using distiller's grains and application of bacillus natto culture | |
| CN101139560B (en) | Process for producing feeding micro-ecological preparation by solid fermentation of brewery mash with various bacterium and fermentation culture medium thereof | |
| CN108208335B (en) | Process for improving feeding nutritive value of cottonseed meal by microbial fermentation method | |
| CN107535671B (en) | Microbial fermentation yellow wine lees feed for improving rumen protein utilization rate and preparation method thereof | |
| CN104186957A (en) | Method for improving forage nutritional value of cottonseed meal through microbial fermentation | |
| CN106260504B (en) | Method for producing microbial fermentation wet feed by using beer yeast paste | |
| CN101816371B (en) | High-protein straw feed and preparation method thereof | |
| CN102334611A (en) | Solid-state fermentation method for bacillus natto-saccharomycete composite viable bacteria preparation with rice bran as matrix | |
| CN103087964A (en) | Bacillus subtilis, microecological preparation and application of bacillus subtilis in animal feed | |
| CN103468594A (en) | Candidautilis strain and application thereof | |
| CN114107073B (en) | Method for producing hypha protein by utilizing molasses | |
| CN101214006B (en) | Method for using EM original liquid fermentation apple pomace to produce protein feed | |
| CN111944723B (en) | Technical method for producing bacillus megaterium by using white spirit vinasse | |
| CN103184174A (en) | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium | |
| CN102406068A (en) | Method for preparing microbial fermented feed capable of keeping intestinal health of pigs | |
| CN102178036B (en) | Method for producing high-methionine and high-lysine feed by using composite bacteria fermented corn protein powder | |
| CN102178038B (en) | Method for preparing fermented high-lysine high-protein feed | |
| CN114698752A (en) | Agricultural waste resource utilization method based on biological fermentation and insect transformation | |
| CN109486724A (en) | A kind of synchronization preparation process and its feed applications mixing probiotics leaven | |
| CN1151834A (en) | Process for producing thallus protein feed by fermentation culture of spirit stillage | |
| CN113647518B (en) | Special biological fermentation feed for fattening cattle in northern Jiang region and preparation method thereof | |
| RU2243678C1 (en) | Method for preparing protein-vitamin fodder | |
| RU2391859C2 (en) | Method of protein-vitamin fodder production | |
| CN108424872A (en) | A kind of preparation method of aquaculture composite bacteria agent | |
| KR102135554B1 (en) | Method for manufacturing probiotic materials for livestock |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |