CN115299527A - Method for degrading vomitoxin in DDGS feed and DDGS feed - Google Patents

Method for degrading vomitoxin in DDGS feed and DDGS feed Download PDF

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CN115299527A
CN115299527A CN202110502537.8A CN202110502537A CN115299527A CN 115299527 A CN115299527 A CN 115299527A CN 202110502537 A CN202110502537 A CN 202110502537A CN 115299527 A CN115299527 A CN 115299527A
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fermentation
vomitoxin
feed
culture
detoxified
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胡世洋
惠继星
屈海峰
岳军
宁艳春
徐友海
王继艳
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Petrochina Co Ltd
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    • 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
    • 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
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • 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
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • 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
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A23K20/26—Compounds containing phosphorus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
    • 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
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum
    • 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

Abstract

The invention provides a method for degrading vomitoxin in DDGS feed and the DDGS feed. The method comprises the following steps: respectively inoculating bacillus subtilis, bacillus licheniformis and lactobacillus plantarum into the wet grains and the distiller's grains concentrated solution for fermentation treatment to obtain detoxified wet grains and detoxified distiller's grains concentrated solution; drying to obtain DDGS feed capable of degrading vomitoxin; wherein the bacterial liquid concentrations of the bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum are respectively and independently 2 multiplied by 1010 CFU/mL-4 multiplied by 1010CFU/mL. The three mixed strains are used for respectively degrading the vomitoxin in the wet grains and the concentrated liquor of the vinasse, so that the content of the vomitoxin in the feed is lower, the problem that the vomitoxin residue in the existing DDGS feed harms animals and human health is solved, the content of probiotics in the feed is increased, and the application value of the feed is increased.

Description

Method for degrading vomitoxin in DDGS feed and DDGS feed
Technical Field
The invention relates to the field of microorganisms, and in particular relates to a method for degrading vomitoxin in DDGS feed and the DDGS feed.
Background
With the general attention of consumers to food hygiene and safety and the continuous and intensive research on mycotoxins by researchers, the contamination of raw materials and feed with mycotoxins has attracted increasing attention from the animal nutrition and feed industries. Investigation shows that the exceeding proportion of the pollution of Chinese feed and raw material mycotoxin is 60-70%, wherein the exceeding proportion of vomitoxin (a common mycotoxin in food or feed, also called deoxynivalenol DON) is close to 70%. Wang Rejun (survey report of mycotoxin pollution of Chinese feed and feed raw materials, feed industry, 2003, 24 (7): 53-54) on 194 parts of feed and raw materials of corn and wheat bran in 28 provinces and municipalities in China shows that the pollution rates of vomitoxin (B type trichothecene mycotoxin), T-2 toxin (A type trichothecene mycotoxin) and zearalenone are 50.0%, 18.1% and 26% respectively. Wang Jinyong and the like (report of mycotoxin detection of Chinese feeds and raw materials in 2012, journal of livestock raising in China, 4 th stage in 2013, 29-34) detect 841 feed and raw material samples in every part of China in 2012, and find that the mycotoxins mainly existing in the feeds and the raw materials at present are vomitoxin, zearalenone, fumonisin and aflatoxin. The positive detection rate and the average value of vomitoxin are highest.
Vomitoxin (vomitoxin), also called Deoxynivalenol (DON), is a type B trichothecene compound with molecular formula C 15 H 20 O 6 The molecular weight is 296, the chemical name is 3a,7a, 15-trixylyl-12,13-epoxy single-ended claspin-9-ene-8-ketone (3,7,15-trihydroxy-12,13-epoxyytrichotec-9-en-8-one), the crystal is a colorless needle crystal, the melting point is 151-153 ℃, and no absorption peak is produced by ultraviolet spectrum scanning. The grain detergent is easy to dissolve in solvents such as water, methanol, ethanol, ethyl acetate, acetone, chloroform and the like, is insoluble in butanol, petroleum ether and normal hexane, has stable property and strong heat resistance, is damaged only a little after being heated at the temperature of 121 ℃ for 25min under high pressure, has strong acid resistance, but has reduced toxicity of DON under alkaline conditions, and reports that grains polluted by DON can remove 70% of toxicity in sodium carbonate solution and can be almost completely removed after long-time action. Mainly comprises toxic secondary metabolites produced by Fusarium graminearum and Fusarium culmorum in Fusarium, and vomitoxin widely exists in grain and feed raw materials such as barley, wheat, corn, oat and the like. Humans and animals can produce a wide range of toxic effects after eating food grains contaminated with the toxin by mistake, with clinical symptoms: unstable standing, sluggish response, piloerection, decreased appetite, vomiting, etc., with pigs being most sensitive to vomitoxin and weaned piglets being particularly sensitive. The trace toxin of 0.3 mg/kg-0.5 mg/kg in the feed can cause the symptoms of food refusal, growth reduction and resistance reduction to infectious diseases of pigs, and the symptoms of food refusal, lethargy, serious growth obstruction, body weight gain slowing, immunologic hypofunction, muscle coordination loss, emesis and the like of the pigs can be caused when the feed contains more than 1mg/kg of vomitoxin. In addition, vomitoxin can accumulate in human and animal bodies, can induce acute or chronic diseases, and has teratogenicity, neurotoxicity, embryotoxicity and immunosuppressive effects.
The United states Food and Drug Administration (FDA) requires that the content of vomitoxin in beef cattle and chicken feed for more than 4 months cannot be more than 10mg/kg, and the content of vomitoxin in other animal feed cannot be more than 5mg/kg; the concentration of vomitoxin in the grain products such as wheat bran, flour, germ and the like for human use must be lower than 1mg/kg. The European Union stipulates that the maximum content of vomitoxin in cereals and cereal by-products, corn by-products is 8mg/kg and 12mg/kg, respectively. The content of vomitoxin in the feed of pig, sheep and other animals is not more than 0.9mg/kg,2mg/kg and 5mg/kg respectively. China stipulates that the quantity of vomitoxin in wheat, flour and corn is 1mg/kg in the national food safety Standard (GB 2715-2016).
At present, raw materials used by fuel ethanol production enterprises are mainly corns, and the corns inevitably mildew in the storage and transportation process, so that the corn raw materials contain mycotoxins (vomitoxin, zearalenone, fumonisin and aflatoxin). When corn containing mycotoxins is used for fermentation to produce fuel ethanol, most of the mycotoxins are concentrated and enriched in the byproduct distillers grains (DDGS feed) during the fermentation production process. However, the mycotoxins (vomitoxin, zearalenone and fumonisin) content in DDGS feed can not be utilized when it exceeds the national limit standards. DDGS feed is also called vinasse protein, and is popular in feed industry due to the advantages of high protein, high available phosphorus, low phytate phosphorus, high vitamin and the like. DDGS is used as an important substitute of conventional protein feed, the current situation of shortage of conventional protein resources in China is greatly relieved by using the DDGS, the production cost is effectively reduced, the content of mycotoxin exceeds standard seriously, the content of mycotoxin in DDGS almost reaches 3 times of that of fermented grain raw materials, according to the 2010 DDGS market survey, the detection rate of zearalenone is 73%, and the detection rate of vomitoxin is 98%.
The microbial degradation and conversion can efficiently degrade and convert the mycotoxin into a nontoxic product, is environment-friendly and safe, and the biological enzyme catalysis method has strong specificity and high conversion efficiency. Has become the most promising treatment technology approach in the current mycotoxin reduction technology. The toxic groups of mycotoxins are broken down by microbial utilization to produce secondary metabolites or by intracellular and extracellular enzymes secreted by the microbes, while producing non-toxic degradation products. For microorganisms, the method has the characteristics of quick growth and propagation, easy culture and the like, is easy for large-scale production, and is very suitable for popularization and use.
The microbial degradation and conversion of pollutants mainly achieves the effects of conversion, degradation, mineralization and the like of pollutants through one or more physiological and biochemical reactions such as oxidation reaction (beta-oxidation, epoxidation, nitrogen oxidation, sulfur oxidation, methyl oxidation and the like), reduction (sulfate reduction, double bond reduction, triple bond reduction) reaction, hydrolysis reaction, de-radical reaction (dehalogenation, deamination, decarboxylation), hydroxylation reaction, esterification reaction, metabolism (ammonia metabolism, oxime metabolism, nitrilo-amine metabolism) and the like, so that the molecular structure of pollutants is changed, and the toxicity of the pollutants is reduced or removed. The mycotoxin in the corn vinasse is decomposed and converted by utilizing the characteristics of the microorganisms, so that the content of the mycotoxin in the DDGS feed is reduced.
The microbial bacteria can degrade the mycotoxin into nontoxic or low-toxicity substances under mild conditions, and have little influence on the sensory properties, palatability and the like of raw materials, so the microbial bacteria becomes a hotspot for researching the biodegradation of the mycotoxin. There are three main ways of microbial degradation of vomitoxin: (1) ring-opening oxidation; (2) C3-OH oxidation, glycosylation and isomerization; and (3) hydration. The target for degrading vomitoxin is shown as formula 1.
Figure BDA0003056954330000031
Microorganisms which have been found to degrade vomitoxin are, at present, agrobacterium (Agrobacterium), rhizobium (Rhizobium), rhodotorula glutinis (Rhodotorula glutinis), geotrichum fermentum (Geotrichum fermentum), metschnikowia pulcherima, kluyveromyces marxianus (Kluyveromyces marxianus), rhodotorula rubra (Rhodotorula ubra) and Acetobacter bacteria (Eubacterium BBSH 797), microorganisms in bovine rumen, soil microorganisms, fish viscera microorganisms and chicken intestinal microorganisms. Some progress has been made in the study of the microbial degradation of vomitoxin (as shown in table 1).
TABLE 1 Elimination of vomitoxin by microorganisms
Figure BDA0003056954330000032
Soil microorganisms are a general term for bacteria, fungi, actinomycetes and algae living in soil. The individual is tiny, usually measured in microns or nanometers, and usually 10 in 1 gram of soil 6 To 10 9 The variety and the number of the plants vary with the soil environment and the depth of the soil layer. The organic fertilizer can be oxidized, nitrified, ammoniated, nitrogen-fixed, sulfurized and the like in soil to promote the decomposition of soil organic matters and the conversion of nutrients. Soil microorganisms generally have the largest number of bacteria, and beneficial bacteria include azotobacter, nitrobacteria and saprophytic bacteria; the harmful bacteria include denitrifying bacteria. The application of organic fertilizer is beneficial to the growth and reproduction of microorganisms.
Chinese patent No. CN 102485883B discloses the use of clostridium (clostridium sp.) microorganisms to break down vomitoxin. The use of bacillus cereus to decompose vomitoxin has been disclosed in the literature on screening and identification of a strain of bacillus cereus that degrades vomitoxin in journal food science, but none of the microorganisms used by them is listed as safe microorganisms that can be used for feed addition.
Although several schemes for degrading emetic toxin are proposed in the prior art, in order to further reduce the content of emetic toxin in the feed, there is still a need to improve the existing scheme for degrading emetic toxin in the current feed.
Disclosure of Invention
The invention mainly aims to provide a method for degrading vomitoxin in DDGS feed and DDGS feed, so as to solve the problem of high content of vomitoxin in the existing feed.
To achieve the above object, according to one aspect of the present invention, there is provided a method for degrading vomitoxin in DDGS feed, comprising: step S1, respectively inoculating bacillus subtilis, bacillus licheniformis and lactobacillus plantarum in wet grains and distiller' S grains concentrated solutionFermenting to obtain detoxicated wet grains and concentrated liquid of detoxicated grains; and step S2, mixing and drying the detoxified wet grains and the detoxified distilled grain concentrated solution to obtain the DDGS feed with degraded vomitoxin; wherein the bacterial liquid concentrations of the bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum are respectively and independently 2 multiplied by 10 10 CFU/mL～4×10 10 CFU/mL。
Furthermore, the inoculation volume proportion of the bacterial liquid of the bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum is respectively and independently 2-5%.
Further, the fermentation conditions in the wet grains are: the rotating speed of the fermentation tank is 200 r/min-400 r/min, the oxygen ventilation amount is 1.0 vvm-1.5 vvm, the fermentation temperature is 28-37 ℃, and the fermentation time is 18-24 h; the fermentation conditions in the distiller's grains concentrated solution are as follows: the rotating speed of the fermentation tank is 200 r/min-400 r/min, the oxygen ventilation amount is 1.0 vvm-1.5 vvm, the fermentation temperature is 28-37 ℃, and the fermentation time is 24-48 h.
Further, prior to inoculating the bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum, the method further comprises: the concentration of the cultured and obtained bacterial liquid is respectively and independently 2 multiplied by 10 10 CFU/mL～4×10 10 CFU/mL of Bacillus subtilis, bacillus licheniformis and Lactobacillus plantarum; preferably, respective thalli of bacillus subtilis, bacillus licheniformis and lactobacillus plantarum are inoculated into a shake flask containing 100 ml-200 ml of culture medium for primary seed culture to obtain primary seeds; inoculating the first seed into a fermentation tank, and culturing to obtain the product with concentration of 2 × 10 10 CFU/mL～4×10 10 CFU/mL bacterial liquid; wherein, the culture conditions of the fermentation culture in the shake flask are as follows: the fermentation temperature is 28-37 ℃, the fermentation time is 18-24 h, the rotation speed of the shake flask is 150-180 r/min, and the pH value of the culture medium is 6.0-7.4; the culture conditions of the fermentation tank culture are as follows: the fermentation temperature is 28-37 ℃, the fermentation time is 36-48 h, the rotating speed of the fermentation tank is 200-400 r/min, the oxygen ventilation amount is 1.0-1.5 vvm, and the pH value of the culture medium is 6.0-7.0; preferably, the first-class seeds are inoculated in a fermentation tank according to the volume proportion of 1-10% for fermentation tank culture; preferably, the fermentation culture is carried out in a shake flaskThe medium of (a) and the medium of the fermenter culture are each independently: 4-8 g of peptone, 4-8 g of yeast extract, 10-20 g of glucose, 5-10 g of monopotassium phosphate, 0.5-1 g of magnesium sulfate heptahydrate, 5-10 g of sodium chloride, adding distilled water to 1000-1400 mL, and adjusting the pH value to 6.0-7.4.
Further, during the fermentation treatment of the wet grains and/or during the fermentation treatment of the vinasse concentrated solution, the method also comprises the step of adding nutrient salt into a fermentation system; preferably, the addition amount of the nutrient salt is 0.1-0.3 g/L of ammonium sulfate, 0.1-0.3 g/L of urea and 0.1-0.3 g/L of diammonium hydrogen phosphate in terms of final concentration respectively.
Further, step S2 includes: mixing the detoxified wet grains and the detoxified concentrated liquid of the lees according to the mass ratio of 4-6:1, drying the detoxified wet grain mixture, and controlling the content of dry matters in the mixture to be less than 15 percent to obtain the DDGS feed degraded with the vomitoxin.
Furthermore, the mass concentration of the detoxified distiller's grains dry matter is 62-65%, and the mass concentration of the distiller's grains concentrated solution dry matter is 27-30%.
Furthermore, the drying temperature is 80-200 ℃, and the drying time is 4-48 h.
According to another aspect of the present invention, there is provided DDGS feed degraded by any one of the above methods for degrading emetic toxin in DDGS feed, wherein the content of emetic toxin in DDGS feed is below 1mg/kg.
By applying the technical scheme of the invention, the three mixed strains are utilized to respectively degrade the vomitoxin in the wet grains and the concentrated liquor of the vinasse, so that the content of the vomitoxin in the feed is lower, the method for degrading the toxin has no pollution to the feed and the environment, and the problems of the traditional detoxification method are effectively solved. And the microorganisms used in the method meet the requirements of microbial feed additives, so that the problem that the vomitoxin residue in the existing DDGS feed harms animal and human health is solved, the content of probiotics in the feed is increased, and the application value of the feed is increased.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
As mentioned in the background, the present feed has the highest content of emetic toxins and the degradation efficiency is still relatively low. In order to further reduce the content of vomitoxin in the feed and improve the feed quality, the inventor of the application improves the scheme of degrading the vomitoxin by utilizing the existing microbial strains capable of degrading the vomitoxin, and provides a scheme with stronger degradation capability, which is concretely as follows.
Three safe microorganisms which are definitely used for feed additive use are utilized to degrade vomitoxin in the application, namely bacillus subtilis, bacillus licheniformis and lactobacillus plantarum. The inventor finds that the three strains can obtain higher reproductive capacity and stronger degradation capability on vomitoxin by mixed culture under the same culture condition, so that the degradation efficiency on the vomitoxin is higher.
Based on the above findings, in one exemplary embodiment of the present application, there is provided a method for degrading emetic toxin in DDGS feed, the method comprising: s1, respectively inoculating bacillus subtilis, bacillus licheniformis and lactobacillus plantarum into wet grains and distiller 'S grain concentrated solution for fermentation treatment to obtain detoxified wet grains and detoxified distiller' S grain concentrated solution; and step S2, mixing and drying the detoxified wet grains and the detoxified distilled grain concentrated solution to obtain the DDGS feed with degraded vomitoxin; wherein the bacterial liquid concentration of the bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum is respectively and independently 2 multiplied by 10 10 CFU/mL～4×10 10 CFU/mL。
According to the method for degrading the vomitoxin in the DDGS feed, the vomitoxin in the wet lees and the lees concentrated solution is degraded by using the three mixed strains, so that the content of the vomitoxin in the feed is lower, the method for degrading the vomitoxin does not pollute the feed and the environment, and the problems of the traditional detoxification method are effectively solved. And the microorganisms used in the method meet the requirements of the microbial feed additives allowed to be added in the feed additive catalog (2013) published by Ministry of agriculture of the people's republic of China 2045, so that the problems that the vomitoxin residue in the existing DDGS feed harms the animal and human health are solved, the content of probiotics in the feed is increased, and the application value of the feed is increased.
According to the method for degrading the vomitoxin in the DDGS feed, a large amount of vinasse supernatant (supernatant obtained by centrifuging fermented liquor) is generated in the production process of fuel ethanol, most of vinasse concentrate is generated after evaporation and concentration, and vinasse protein feed (DDGS feed) is generated after wet vinasse and vinasse concentrate are mixed, evaporated and dried. Therefore, the method for degrading the vomitoxin in the DDGS feed can achieve the aim of degrading the vomitoxin in the DDGS feed only by the synergistic effect of two ways. One approach is to add high-density mixed bacteria liquid into wet grains for fermentation treatment to achieve the purpose of degrading water-soluble vomitoxin dissolved in the wet grains. The other way is to add high-density mixed bacteria liquid into the distiller's grains concentrated solution for fermentation treatment to achieve the purpose of degrading the water-insoluble vomitoxin in the distiller's grains concentrated solution. After the two approaches are adopted for treatment, the wet grains and the concentrated liquor of the vinasse are dried together to produce the vinasse protein feed, and the purpose of degrading the vomitoxin in the DDGS feed can be achieved.
The bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum used in the method belong to probiotics, and the bacillus subtilis can promote animals to grow and synthesize various enzyme substances and various B vitamins in the animals, so that the digestion, absorption and utilization rate of the feed is improved. The bacillus licheniformis can produce various active enzymes, such as protease, amylase, lipase, pectinase, glucanase, cellulase and the like, and can also produce various enzymatic factors to enhance the activity of digestive enzymes of animals. The lactobacillus plantarum has wide probiotic effect, and can improve the growth speed of animals and the immunity of organisms, reduce the content of cholesterol in the organisms, synthesize unsaturated fatty acid and polysaccharide, reduce oxidative stress and the like. At 2X 10 10 CFU/mL～4×10 10 Mixture in the form of bacterial liquid in high concentration range of CFU/mLIn the bacterial liquid, the bacterial biomass is higher, and the degradation treatment capacity and efficiency of toxins in the feed are higher.
The bacillus subtilis belongs to a soil microorganism belonging to bacillus, and has a single cell of 0.7-0.8 μm multiplied by 2-3 μm and uniform coloring. Without capsule, the perigenic flagellum can move. Gram-positive bacteria, spores of 0.6-0.9 microns multiplied by 1.0-1.5 microns, oval to columnar shape, located in the center of the bacteria or slightly deviated, and the bacteria do not expand after the spores are formed. The colony surface is rough and opaque, and is dirty white or yellowish, and when the colony grows in a liquid culture medium, the skin becomes always formed. Aerobic bacteria. Tryptophan can be decomposed to form indole by using protein, various sugars and starch. The method is widely applied to genetic research, and the synthesis path and the regulation mechanism of purine nucleotide of the bacterium are clearly researched. Widely distributed in soil and putrefactive organic substances, and is easy to reproduce in the Sucus subtilis extract.
The vomitoxin is degraded by the composite strain containing the bacillus subtilis, and the method has the following beneficial effects that 1) the animal growth is promoted to synthesize various enzyme substances and various B vitamins in the animal body, so that the digestion, absorption and utilization rate of the feed is improved. 2) The compound preparation has the advantages of inhibiting pathogenic bacteria, and various antibacterial active substances such as bacillus subtilis, polymyxin, nystatin, gramicidin and the like generated by thalli, and has obvious inhibiting effect on pathogenic bacteria in an animal body or conditional pathogenic bacteria of endogenous infection. 3) Improving immunity, stimulating growth and development of animal immune organs, increasing immunoglobulin and antibody level, and improving immunity. 4) The stress is slowed down, and the stress response of animals caused by various reasons is slowed down. 5) Regulating intestinal tract balance, establishing advantageous flora in intestinal tract, and regulating microecological balance in digestive tract. 6) Improve the feed environment and reduce the odor of excrement and harmful gas emission.
The bacillus licheniformis is gram positive bacillus belonging to soil microorganism, single cell is 0.8 Mum multiplied by 1.5 Mum-3.5 Mum, the cell shape and arrangement is rod-shaped and single, and the near-midlife elliptic spore is generated, and the cyst is slightly expanded. The colonies on the gravy culture medium were flat, irregular edges, white, rough and wrinkled on the surface, and the diameter of the colonies after 24h was 3mm.
The bacillus licheniformis can generate antibacterial active substances and kill pathogenic bacteria, has a unique biological oxygen-deprivation action mechanism, can inhibit the growth and reproduction of the pathogenic bacteria, and has a growth promoting effect on beneficial bacteria. The bacillus licheniformis can produce various active enzymes, such as protease, amylase, lipase, pectinase, glucanase, cellulase and the like, and can also produce various enzymatic factors to enhance the activity of digestive enzymes of animals. The bacillus licheniformis can generate stress-resistant endospores under certain conditions, can generate various antibiotics such as lipopeptides, peptides, phospholipids, polyene and amino acids, and has good inhibition effect on various pathogenic bacteria.
The bacillus licheniformis can simultaneously generate a plurality of nutrient substances such as vitamins, amino acids, organic acids, growth promoting factors and the like, participate in the metabolism of animal organisms and provide nutrient substances for the organisms. The bacillus licheniformis can adjust the balance of intestinal flora of animals, improve the micro-ecological environment of intestinal tracts, promote the growth of animals, reduce the occurrence of intestinal diseases of the animals and improve the disease resistance of animal organisms. Meanwhile, it can also take effect in many aspects such as immunosuppression, competitive adsorption and synthesis of antibacterial substances. The Bacillus licheniformis can stimulate growth and development of animal immune organs, activate lymphocytes, increase immunoglobulin and antibody levels, enhance cellular immunity and humoral immunity, and improve immunity. In addition, the feeding bacillus licheniformis has the effects of antagonizing pathogenic bacteria of intestinal tracts, maintaining and regulating the microecological balance of the intestinal tracts, enhancing the disease resistance and immunity of animal organisms, effectively improving the conversion rate of feed and increasing the production of livestock and poultry products.
Lactobacillus plantarum belongs to the genus Lactobacillus, rectus rotundus, and is usually 0.9 μm to 1.2. Mu. M.times.3.0 μm to 8.0. Mu.m, single, paired or short-chained. Usually lacking flagella, but able to move. Gram positive, non-sporulating. Facultative anaerobe, surface colony diameter about 3mm, convex, round, smooth surface, fine, white, occasionally light yellow or dark yellow. Belongs to chemoheterotrophic bacteria, can grow at 15 ℃, and usually has the optimal growth temperature of 30-35 ℃.
The lactobacillus plantarum can promote various functions such as nutrient absorption and the like by generating various antibacterial substances such as organic acid, bacteriocin, hydrogen peroxide, diacetyl and the like, improve and regulate the balance of intestinal microbial flora and enhance the immunity of organisms. The lactobacillus plantarum has wide probiotic effects, such as increasing the growth speed of animals and the immunity of organisms, reducing the cholesterol content of the organisms, synthesizing unsaturated fatty acid and polysaccharide, reducing oxidative stress and the like. Is widely applied to the fields of food fermentation, microecological preparation and the like, and is one of microbial feed additives allowed to be added in the feed additive variety catalogue (2013) published by Ministry of agriculture of the people's republic of China No. 2045. The French scientist Niderkorn respectively detected the removal effect of 29 strains of bacteria on mycotoxin, and found that all lactic acid bacteria have the capability of removing vomitoxin. The realization of the function of degrading the vomitoxin by the lactic acid bacteria requires that the number of the viable bacteria reaches a certain number.
The mixed bacterial liquid formed by the three strains is adopted in the method, so that the synergistic promotion effect on the degradation of the vomitoxin in the feed is achieved, other possible toxins in the feed can be degraded, the toxin content in the treated feed is lower, and the strains belong to probiotics beneficial to human and animals, so that the nutritive value of the obtained feed is higher.
In the method, the inoculation proportion of each bacterium in the wet grains and the concentrated solution of the vinasse is preferably 2-5% in volume ratio.
In the method, the specific conditions for fermenting and culturing the wet grains and the distiller's grains concentrated solution to degrade the vomitoxin are the propagation conditions of the mixed microbial strains, and all the conditions which are favorable for the maximum propagation of the mixed microbial strains are all favorable for realizing the high-efficiency degradation of the vomitoxin.
In a preferred embodiment of the present application, the fermentation conditions in the wet spent grain are: the rotating speed of the fermentation tank is 200 r/min-400 r/min, the oxygen ventilation amount is 1.0 vvm-1.5 vvm, the fermentation temperature is 28-37 ℃, and the fermentation time is 18-24 h; preferably, the fermentation conditions of the distiller's grains concentrated solution are as follows: the rotating speed of the fermentation tank is 200 r/min-400 r/min, the oxygen ventilation amount is 1.0 vvm-1.5 vvm, the fermentation temperature is 28-37 ℃, and the fermentation time is 24-48 h; more preferably, the method further comprises the step of adding nutrient salt to the fermentation tank during the fermentation of the wet grains and/or during the fermentation of the distiller's grains concentrated solution, and more preferably, the nutrient salt is 0.1-0.3 g/L of ammonium sulfate, 0.1-0.3 g/L of urea and 0.1-0.3 g/L of diammonium hydrogen phosphate.
The wet grains and the vinasse concentrated solution are fermented respectively under the preferable conditions, so that the mixed strains can be propagated to the maximum, and then the vomitoxin in the wet grains and the vinasse concentrated solution is efficiently degraded, the content of the vomitoxin in the treated feed is greatly reduced, the content of probiotics in the feed is correspondingly increased, and the feed has higher application value.
The method specifically comprises the following steps: the fermentation treatment steps in the wet grains are as follows: taking the bacterial liquid with the concentration of 2 multiplied by 10 10 CFU/mL～4×10 10 The CFU/mL bacillus subtilis, the Bacillus licheniformis and the Lactobacillus plantarum are inoculated in a wet grain fermentation tank for fermentation culture according to the proportion of 2-5 percent, and the fermentation conditions are as follows: the rotation speed is 200 r/min-400 r/min, the ventilation volume is 1.0 vvm-1.5 vvm, the natural pH value, the fermentation temperature is 28-37 ℃, the fermentation culture is 18-24 h, and a certain amount of nutrient salt (preferably, the addition amount of the nutrient salt is 0.1-0.3 g/L of ammonium sulfate, 0.1-0.3 g/L of urea and 0.1-0.3 g/L of diammonium hydrogen phosphate respectively according to the final concentration) is required to be added in the fermentation process for promoting the growth of microorganisms.
The fermentation treatment steps in the vinasse concentrated solution are as follows: taking the bacterial liquid with the concentration of 2 multiplied by 10 10 CFU/mL～4×10 10 The CFU/mL bacillus subtilis, the Bacillus licheniformis and the Lactobacillus plantarum are inoculated in a vinasse concentrated solution fermentation tank for fermentation culture according to the proportion of 5-10 percent, and the fermentation conditions are as follows: the rotating speed is 200 r/min-400 r/min, the ventilation volume is 1.0 vvm-1.5 vvm, the natural pH value, the fermentation temperature is 28-37 ℃, the fermentation culture is 24-48 h, and a certain amount of nutrient salt is required to be added for promoting the growth of microorganisms in the fermentation process. Wherein the addition amount of the nutrient salt is 0.1-0.3 g/L of ammonium sulfate, 0.1-0.3 g/L of urea and 0.1-0.3 g/L of diammonium hydrogen phosphate.
In order to further prolong the storage time of the feed after the microbial agent degrades the vomitoxin and improve the storage and transportation conditions of the feed, in a preferred embodiment of the present application, the step S2 comprises: mixing the detoxified wet grains and the detoxified concentrated solution of the lees according to the mass ratio of 4-6:1; and drying the detoxified wet grain mixture to reduce the water content to below 15 percent to obtain the DDGS feed capable of degrading the vomitoxin. Preferably, the dry matter content in the detoxified wet grains is 62-65 wt%, and the dry matter content in the detoxified concentrated solution of distillers' grains is 27-30 wt%. Preferably, the drying temperature is 80-200 ℃, and the drying time is 4-48 h.
In a second exemplary embodiment of the present application, there is provided a DDGS feed degraded by the above degradation method, wherein the content of vomitoxin in the DDGS feed is less than 1mg/kg. Compared with the DDGS feed in the prior art, the content of vomitoxin in the DDGS feed is greatly reduced, and the edible safety of the feed is improved.
It should be noted that, before the three kinds of strains are mixed to degrade the vomitoxin, high-density culture is required to be carried out separately or in a mixture to obtain the high concentration. The specific steps can be obtained by optimizing and adjusting each culture condition on the basis of the existing bacillus culture condition. In a preferred embodiment of the present application, the above concentration of 2X 10 is obtained by high density culture 10 CFU/mL～4×10 10 The CFU/mL bacterial liquid comprises the following steps: inoculating the thallus into a shake flask containing 100-200 ml of culture medium to culture first-stage seeds, and inoculating the first-stage seeds into a fermentation tank to culture in the fermentation tank to obtain 2X 10 10 CFU/mL～4×10 10 CFU/mL bacterial liquid; wherein, the culture conditions of the fermentation culture in the shake flask are as follows: the fermentation temperature is 28-37 ℃, the fermentation time is 18-24 h, the rotation speed of the shake flask is 150-180 r/min, and the pH value of the culture medium is 6.0-7.4; the culture conditions of the fermentation tank culture are as follows: the fermentation temperature is 28-37 ℃, the fermentation time is 36-48 h, the rotation speed of the fermentation tank is 200-400 r/min, the oxygen ventilation amount is 1.0-1.5 vvm, and the pH value of the culture medium is 6.0-7.0.
By performing the fermentation tank culture under the preferable culture conditions, the bacterial liquid with the high density can be obtained, so that the degradation efficiency of the toxin in the feed is high under the same bacterial liquid dosage condition, and the nutritional value of the treated feed is high.
In the step of inoculating the first-stage seeds into the fermentation tank for culture, the specific inoculation proportion can be reasonably adjusted according to the bacterial liquid concentration of the actual first-stage seeds. In a preferred embodiment of the present application, the first seed is inoculated into a fermentation tank at a volume ratio of 1% to 10% for fermentation tank culture to obtain the microbial agent. The inoculation according to the volume proportion of 1 to 10 percent can ensure that the fermentation speed in the fermentation tank is high and the fermentation efficiency is high.
In the steps of primary seed culture and fermentation tank culture of the mixed strain, the mixed strain can be obtained by reasonably adding or adjusting a carbon source, a nitrogen source and other nutrient elements on the basis of a culture medium for culturing a single strain. In a preferred embodiment of the present application, the medium for the fermentation culture in the shake flask and the medium for the fermentation culture in the fermenter are each independently: 4-8 g of peptone, 4-8 g of yeast extract, 10-20 g of glucose, 5-10 g of monopotassium phosphate, 0.5-1 g of magnesium sulfate heptahydrate, 5-10 g of sodium chloride, adding distilled water to 1000-1400 mL, and adjusting the pH value to 6.0-7.4.
In a most preferred embodiment of the present application, the method for culturing bacillus subtilis, bacillus licheniformis and lactobacillus plantarum in high density specifically comprises: under the aseptic condition, respectively selecting 3-ring to 5-ring thalli by utilizing inoculating loops, inoculating the thalli into 100ml to 200ml of culture medium, and carrying out shake flask fermentation culture (first-stage seed culture) under the culture conditions that: the fermentation temperature is 28-37 ℃, the fermentation time is 18-24 h, the rotation speed is 150-180 r/min, and the pH value is 6.0-7.4. Inoculating the cultured first-stage seeds into a fermentation tank according to the proportion of 1-10 percent for high-density culture of bacillus subtilis, bacillus licheniformis and lactobacillus plantarum, wherein the culture conditions are as follows: the fermentation temperature is 28-37 ℃, the fermentation time is 36-48 h, the rotating speed is 200-400 r/min, the ventilation volume is 1.0-1.5 vvm, the pH value is 6.0-7.0, and finally the concentration of each bacterial liquid reaches 2 multiplied by 10 10 CFU/mL～4×10 10 CFU/mL. The fermentation seed culture medium consists of the following components: 4-8 g of peptone, 4-8 g of yeast extract, 10-20 g of glucose and diphosphoric acid5-10 g of potassium hydride, 0.5-1 g of magnesium sulfate heptahydrate, 5-10 g of sodium chloride, 1000-1400 mL of distilled water and 6.0-7.4 of pH value. The culture medium of the fermentation tank consists of the following components: 4-8 g of peptone, 4-8 g of yeast extract, 10-20 g of glucose, 5-10 g of monopotassium phosphate, 0.5-1 g of magnesium sulfate heptahydrate, 5-10 g of sodium chloride, adding distilled water until the volume is 1000-1400 mL, and adjusting the pH value to be 6.0-7.4.
The advantageous effects of the present application will be further described with reference to specific examples. The strains used in the following examples are all commercial strains purchased from the China center for Industrial culture Collection of microorganisms (CICC). The specific information is as follows:
b, bacillus subtilis: (Bacillus subtilis) with the collection numbers of CICC 10089 and CICC 20445.
B, bacillus licheniformis: (Bacillus licheniformis), accession numbers CICC20446 and CICC20514.
Lactobacillus plantarum: (Lactobacillus plantarum) with the deposit number CICC 6002.
It should be noted that, in the following embodiments, the detection method of vomitoxin content is to use a vomitoxin (DON) ELISA rapid detection kit (this kit uses an indirect competitive ELISA method, vomitoxin antigen is pre-coated on a micropore strip of an ELISA plate, vomitoxin in a sample and antigen pre-coated on the micropore strip compete for an anti-vomitoxin antibody (anti-reagent), and the vomitoxin antibody is combined with an enzyme-labeled secondary antibody (enzyme-labeled substance), and the sample absorbance value is negatively correlated with the quantity of vomitoxin contained in the sample, and the absorbance value is compared with a standard curve and multiplied by the corresponding dilution multiple to obtain the content of vomitoxin in the sample.
EXAMPLE 1 high-Density culture method of Bacillus subtilis
Under aseptic conditions, two kinds of bacillus subtilis with 5 rings are selected by an inoculating ring and inoculated in 100ml of culture medium (300 ml of triangular flask) for shake flask fermentation culture (first-stage seed culture), and the culture conditions are as follows: the fermentation temperature is 37 ℃, the fermentation time is 24h, the rotating speed is 180r/min, and the pH value is 7.0. Culturing the above-mentioned oneThe grade seeds are inoculated in a fermentation tank according to the proportion of 10 percent for high-density culture of the bacillus subtilis, and the culture conditions are as follows: the fermentation temperature is 37 ℃, the fermentation time is 48h, the rotating speed is 200r/min, the ventilation volume is 1.5vvm, the pH value is 7.0, and after the fermentation is finished, the fermentation liquid is stored in a refrigerator at 4 ℃ for later use. Adopts a plate counting method, and 1mL of fermentation liquor is diluted by 10 -9 After doubling, the number of colonies growing on the plate after 24h culture at 37 ℃ is respectively as follows: CICC 10089 reaches 3.8 multiplied by 10 10 CFU/mL and CICC 20445 reach 3.5 x 10 10 CFU/mL。
Wherein the shake flask fermentation medium consists of the following components: 4g of peptone, 4g of yeast extract powder, 10g of glucose, 5g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate and 5g of sodium chloride, distilled water is added until the volume is 1000mL, and the pH value is 7.0. The culture medium of the fermentation tank consists of the following components: 16g of peptone, 16g of yeast extract powder, 40g of glucose, 20g of sodium chloride, 20g of monopotassium phosphate, 2g of magnesium sulfate heptahydrate and 2000mL of distilled water, and the pH value is 7.0.
EXAMPLE 2 high Density culture method of Bacillus licheniformis of the present invention
Under aseptic conditions, two kinds of bacillus licheniformis with 5 rings are picked by utilizing an inoculating ring and inoculated in 100ml of culture medium (300 ml of triangular flask) for shake flask fermentation culture (first-stage seed culture), and the culture conditions are as follows: the fermentation temperature is 37 ℃, the fermentation time is 24h, the rotating speed is 180r/min, and the pH value is 7.0. Inoculating the cultured first-stage seeds into a fermentation tank according to the proportion of 10 percent for high-density culture of the bacillus licheniformis, wherein the culture conditions are as follows: the fermentation temperature is 37 ℃, the fermentation time is 48h, the rotating speed is 400r/min, the ventilation volume is 1.5vvm, the pH value is 7.0, and after the fermentation is finished, the fermentation liquid is stored in a refrigerator at 4 ℃ for later use. Adopts a plate counting method, and 1mL of fermentation liquor is diluted by 10 -9 After doubling, the number of colonies growing on the plate is counted after 24h culture at 37 ℃, and the CICC20446 reaches 2.7 multiplied by 10 10 CFU/mL, CICC20514 reaches 2.8 x 10 10 CFU/mL。
Wherein the shake flask fermentation medium consists of the following components: 4g of peptone, 4g of yeast extract powder, 10g of glucose, 5g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate and 5g of sodium chloride, adding distilled water to 1000mL, and adjusting the pH value to 7.0. The culture medium of the fermentation tank consists of the following components: 16g of peptone, 16g of yeast extract powder, 40g of glucose, 20g of sodium chloride, 20g of monopotassium phosphate, 2g of magnesium sulfate heptahydrate and 2000mL of distilled water, wherein the pH value is 7.0.
EXAMPLE 3 high-Density culture method of Lactobacillus plantarum in accordance with the present invention
Under aseptic conditions, inoculating 5-ring lactobacillus plantarum into 100ml of a culture medium (300 ml of a triangular flask) by utilizing an inoculating ring, and performing shake flask fermentation culture (first-stage seed culture) under the culture conditions that: the fermentation temperature is 30 ℃, the fermentation time is 24h, the rotating speed is 180r/min, and the pH value is 6.2. Inoculating the cultured first-stage seeds into a fermentation tank according to a proportion of 10% for high-density culture of lactobacillus plantarum, wherein the culture conditions are as follows: the fermentation temperature is 30 ℃, the fermentation time is 48h, the rotating speed is 200r/min, the ventilation volume is 1.5vvm, the pH value is 6.2, and after the fermentation is finished, the fermentation liquid is stored in a refrigerator at 4 ℃ for later use. Adopts a plate counting method, and 1mL of fermentation liquor is diluted by 10 -9 After doubling, the number of colonies growing on the plate after 24h culture at 30 ℃ reaches 2.6 multiplied by 10 10 CFU/mL。
Wherein the shake flask fermentation medium consists of the following components: 4g of peptone, 4g of yeast extract powder, 10g of glucose, 5g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate and 5g of sodium chloride, adding distilled water to 1000mL, and adjusting the pH value to 6.2. The fermentation tank culture medium consists of the following components: 16g of peptone, 16g of yeast extract powder, 40g of glucose, 20g of sodium chloride, 20g of monopotassium phosphate, 2g of magnesium sulfate heptahydrate and 2000mL of distilled water, wherein the pH value is 6.2.
Example 4 the method of the present invention for degrading emetic toxin in DDGS feed
And (3) fermentation treatment of the mixed microbial liquid of the wet grains: taking Bacillus subtilis liquid (CICC 10089) with concentration of 3.8 × 10 10 CFU/mL, the concentration of Bacillus licheniformis liquid (CICC 20446) is 2.7 × 10 10 CFU/mL, lactobacillus plantarum solution concentration of 2.6X 10 10 CFU/mL, respectively inoculating in a wet grain fermentation tank together according to the proportion of 2%, 2% and 5% for fermentation culture, wherein the fermentation conditions are as follows: the rotation speed is 200r/min, the ventilation volume is 1.0vvm, the natural pH value and the fermentation temperature are 37 ℃, the fermentation culture is carried out for 24 hours, and after the fermentation is finished, the fermentation liquor is stored in a refrigerator at 4 ℃ for standby. Wherein, fermentingThe nutrient salt added in the process consists of the following components: 0.2g/L of ammonium sulfate, 0.1g/L of urea and 0.3g/L of diammonium hydrogen phosphate.
Fermentation treatment of mixed microbial liquid of the vinasse concentrated solution: taking Bacillus subtilis liquid (CICC 10089) with concentration of 3.8 × 10 10 CFU/mL, the concentration of Bacillus licheniformis liquid (CICC 20446) is 2.7 × 10 10 CFU/mL, lactobacillus plantarum solution concentration of 2.6X 10 10 CFU/mL, respectively inoculating in the distiller's grains concentrated solution fermentation tank according to the proportion of 5%, 5% and 10% for fermentation culture, wherein the fermentation conditions are as follows: the rotating speed is 400r/min, the ventilation volume is 1.5vvm, the natural pH value and the fermentation temperature are 37 ℃, the fermentation culture is carried out for 48 hours, and after the fermentation is finished, the fermentation liquor is stored in a refrigerator at 4 ℃ for standby. Wherein the nutrient salt added in the fermentation process consists of the following components: 0.1g/L ammonium sulfate, 0.2g/L urea and 0.1g/L diammonium hydrogen phosphate.
Preparing a DDGS feed for degrading vomitoxin: after the two ways of treatment, mixing the detoxified wet grains and the detoxified distilled grain concentrated solution according to the mass ratio of 4:1, wherein the dry matter concentration in the detoxified wet grains is 62-65%, and the dry matter content in the detoxified distilled grain concentrated solution is 27-30%. And then drying for 24 hours in an oven at 100 ℃ to reduce the water content to below 15 percent, thus obtaining the DDGS feed with degraded vomitoxin, and realizing that the vomitoxin content in the DDGS feed is below 1mg/kg.
Example 5 the method of the present invention for degrading emetic toxin in DDGS feed
And (3) fermentation treatment of the mixed microbial liquid of the wet grains: taking Bacillus subtilis liquid (CICC 20445) with concentration of 3.5 × 10 10 CFU/mL, bacillus licheniformis liquid (CICC 20514) concentration of 2.8 × 10 10 CFU/mL, lactobacillus plantarum solution (CICC 6002) concentration 2.6X 10 10 CFU/mL, respectively inoculating in a wet grain fermentation tank according to the proportion of 2%, 2% and 5% for fermentation culture, wherein the fermentation conditions are as follows: the rotation speed is 200r/min, the ventilation volume is 1.0vvm, the natural pH value and the fermentation temperature are 37 ℃, the fermentation culture is carried out for 24 hours, and after the fermentation is finished, the fermentation liquor is stored in a refrigerator at 4 ℃ for standby. Wherein the nutrient salt added in the fermentation process consists of the following components: 0.2g/L ammonium sulfate, 0.1g/L ureaAnd 0.3g/L diammonium hydrogen phosphate.
Fermentation treatment of mixed microbial liquid of the vinasse concentrated solution: taking Bacillus subtilis liquid (CICC 20445) with concentration of 3.5 × 10 10 CFU/mL, bacillus licheniformis liquid (CICC 20514) concentration of 2.8 × 10 10 CFU/mL, lactobacillus plantarum solution (CICC 6002) concentration 2.6X 10 10 And (3) the CFU/mL is respectively inoculated in a vinasse concentrated solution fermentation tank together according to the proportion of 5%, 5% and 10% for fermentation culture, and the fermentation conditions are as follows: the rotating speed is 400r/min, the ventilation volume is 1.5vvm, the natural pH value and the fermentation temperature are 37 ℃, the fermentation culture is carried out for 48 hours, and after the fermentation is finished, the fermentation liquor is stored in a refrigerator at 4 ℃ for standby. Wherein the nutrient salt added in the fermentation process consists of the following components: 0.1g/L ammonium sulfate, 0.2g/L urea and 0.1g/L diammonium hydrogen phosphate.
Preparing DDGS feed for degrading vomitoxin: after the two ways of treatment, mixing the detoxified wet grains and the detoxified distilled grain concentrated solution according to the mass ratio of 6:1, wherein the dry matter content in the detoxified wet grains is 62-65%, and the dry matter content in the detoxified distilled grain concentrated solution is 27-30%. And then drying for 48 hours in an oven at 80 ℃ to reduce the water content to below 15 percent, thus obtaining the DDGS feed with degraded vomitoxin, and realizing that the vomitoxin content in the DDGS feed is below 1mg/kg.
Comparative example 1
The difference from example 5 is that: in the raw materials of the wet lees and the concentrated lees solution which are artificially added with 1.2mg/kg of vomitoxin, bacillus subtilis liquid (CICC 20445) with the concentration of 1.5 multiplied by 10 is taken 7 CFU/mL, bacillus licheniformis liquid (CICC 20514) concentration of 1.8 × 10 7 CFU/mL, lactobacillus plantarum solution concentration 1.6X 10 7 And inoculating the mixture in a CFU/mL mode, and fermenting the wet grains and the concentrated liquor of the vinasse respectively.
Through detection, the content of vomitoxin in the DDGS feed is 0.8mg/kg.
Comparative example 2
The difference from example 5 is that: inoculating two kinds of bacteria in wet grains and concentrated lees solution containing 1.2mg/kg vomitoxin, respectively collecting Bacillus subtilis solution (CICC 20445) with concentration of 3.5 × 10 10 CFU/mL, bacillus licheniformis liquid (CICC 20514) 2.8X 10 10 CFU/mL was inoculated.
Through detection, the content of vomitoxin in the DDGS feed is 0.6mg/kg.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects:
(1) The bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum used in the application are all proved to be microorganisms capable of degrading vomitoxin, and the microorganisms meet the requirements of microbial feed additives allowed to be added in the feed additive variety catalog (2013) published by the ministry of agriculture of the people's republic of China No. 2045.
(2) The bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum used in the application belong to probiotics, and the bacillus subtilis can promote animals to grow and synthesize various enzyme substances and various B vitamins in the bodies of the animals, so that the digestion, absorption and utilization rate of the feed is improved. The bacillus licheniformis can produce various active enzymes, such as protease, amylase, lipase, pectinase, glucanase, cellulase and the like, and can also produce various enzymatic factors to enhance the activity of digestive enzymes of animals. The lactobacillus plantarum has wide probiotic effect, and can improve the growth speed of animals and the immunity of organisms, reduce the content of cholesterol in the organisms, synthesize unsaturated fatty acid and polysaccharide, reduce oxidative stress and the like.
(3) The method for degrading vomitoxin by using bacillus subtilis, bacillus licheniformis and lactobacillus plantarum is simple to operate, does not pollute feed and the environment, and effectively solves the problems existing in the traditional detoxification method.
(4) The bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum used in the application have high biological activity, and the concentration of each bacterium liquid can reach 2 multiplied by 10 after high-density culture 10 CFU/mL～4×10 10 CFU/mL, the purpose of efficiently degrading the vomitoxin in the DDGS feed is achieved, and the content of the vomitoxin in the DDGS feed is below 1mg/kg.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (10)

1. A method of degrading emetic toxin in DDGS feed, comprising:
s1, respectively inoculating bacillus subtilis, bacillus licheniformis and lactobacillus plantarum into the wet grains and the distiller 'S grains concentrated solution for fermentation treatment to obtain detoxified wet grains and detoxified distiller' S grains concentrated solution; and
s2, mixing and drying the detoxified wet grains and the detoxified distilled grain concentrated solution to obtain the DDGS feed degraded with vomitoxin;
wherein the bacterial liquid concentrations of the bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum are respectively and independently 2 multiplied by 10 10 CFU/mL～4×10 10 CFU/mL。
2. The method according to claim 1, wherein the inoculation volume ratio of the bacterial liquid of the bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum is 2-5% respectively and independently.
3. The method of claim 1, wherein the fermentation conditions in the wet stillage are: the rotating speed of the fermentation tank is 200 r/min-400 r/min, the oxygen ventilation amount is 1.0 vvm-1.5 vvm, the fermentation temperature is 28-37 ℃, and the fermentation time is 18-24 h;
the fermentation conditions in the vinasse concentrated solution are as follows: the rotating speed of the fermentation tank is 200 r/min-400 r/min, the oxygen ventilation amount is 1.0 vvm-1.5 vvm, the fermentation temperature is 28 ℃ -37 ℃, and the fermentation time is 24 h-48 h.
4. The method of claim 1, wherein prior to inoculating the bacillus subtilis, the bacillus licheniformis, and the lactobacillus plantarum, the method further comprises: culturing and obtaining bacterial liquid concentrationDegree is each independently 2X 10 10 CFU/mL～4×10 10 CFU/mL of the Bacillus subtilis, bacillus licheniformis, and Lactobacillus plantarum;
preferably, the respective thalli of the bacillus subtilis, the bacillus licheniformis and the lactobacillus plantarum are inoculated into a shake flask containing 100 ml-200 ml of culture medium for primary seed culture to obtain primary seeds; and
inoculating the first-stage seeds into a fermentation tank for fermentation tank culture to obtain the seed with the concentration of 2 multiplied by 10 10 CFU/mL～4×10 10 CFU/mL bacterial liquid;
wherein the culture conditions of the fermentation culture in the shake flask are as follows: the fermentation temperature is 28-37 ℃, the fermentation time is 18-24 h, the rotation speed of the shake flask is 150-180 r/min, and the pH value of the culture medium is 6.0-7.4; the culture conditions of the fermentation tank culture are as follows: the fermentation temperature is 28-37 ℃, the fermentation time is 36-48 h, the rotating speed of the fermentation tank is 200-400 r/min, the oxygen ventilation amount is 1.0-1.5 vvm, and the pH value of the culture medium is 6.0-7.0.
5. The method of claim 4, wherein the primary seeds are inoculated into a fermentor for the fermentor culture at a volume ratio of 1% to 10%.
6. The method according to claim 4, wherein the medium of the fermentation culture in the shake flask and the medium of the fermenter culture are each independently: 4-8 g of peptone, 4-8 g of yeast extract, 10-20 g of glucose, 5-10 g of monopotassium phosphate, 0.5-1 g of magnesium sulfate heptahydrate, 5-10 g of sodium chloride, adding distilled water to 1000-1400 mL, and adjusting the pH value to 6.0-7.4.
7. The method of claim 3, wherein during the fermentation treatment of the wet stillage and/or during the fermentation treatment of the stillage concentrate, the method further comprises the step of adding nutrient salts to the fermentation system;
preferably, the addition amounts of the nutrient salts are 0.1-0.3 g/L of ammonium sulfate, 0.1-0.3 g/L of urea and 0.1-0.3 g/L of diammonium hydrogen phosphate in terms of final concentration respectively.
8. The method according to claim 1, wherein the step S2 comprises:
mixing the detoxified wet grains and the detoxified concentrated solution of the lees according to the mass ratio of 4-6:1 to obtain a detoxified wet grain mixture;
and drying the detoxified wet grain mixture to reduce the water content to below 15 percent to obtain the DDGS feed with degraded vomitoxin.
9. The method of claim 8, wherein the dry matter content of the detoxified wet grains is 62-65 wt% and the dry matter content of the detoxified distillers' grains concentrate is 27-30 wt%.
10. The DDGS feed degraded by the method for degrading the vomitoxin in the DDGS feed according to any one of claims 1 to 9, wherein the content of the vomitoxin in the DDGS feed is less than 1mg/kg.
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