CN114032196A - Microbial preparation, preparation method and application thereof - Google Patents

Microbial preparation, preparation method and application thereof Download PDF

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CN114032196A
CN114032196A CN202111344475.9A CN202111344475A CN114032196A CN 114032196 A CN114032196 A CN 114032196A CN 202111344475 A CN202111344475 A CN 202111344475A CN 114032196 A CN114032196 A CN 114032196A
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bacillus
culture medium
follows
preparation
lactobacillus casei
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谢二伟
赵玉山
赵旭栋
赵晓冰
郑瑞娜
周峰
赵艳艳
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Luoyang Espoir Biotechnology Co ltd
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    • 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
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    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/125—Casei
    • 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
    • A23V2400/51—Bifidobacterium
    • A23V2400/533—Longum

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Abstract

The invention relates to a microbial preparation, which comprises bacillus subtilis, bacillus coagulans, candida utilis, lactobacillus casei, bacillus licheniformis and bifidobacterium longum, and the microbial preparation comprises the following components in percentage by dry weight: (10% -50%): (10% -50%): (10% -50%): (10% -50%): (10% -50%): (10% -50%). The removal rate of mycotoxin in the microbial preparation is higher than 90%. The average daily gain of the piglets cultured by using the microbial preparation fermented feed is higher than 400g/d, the feed-meat ratio is lower than 1.4%, the diarrhea rate is lower than 1.5%, and the digestion of the piglets on the feed is good.

Description

Microbial preparation, preparation method and application thereof
Technical Field
The invention belongs to the technical field of microbial preparations, and particularly relates to a preparation for degrading mycotoxin, a preparation method and application.
Background
Mycotoxins are mainly toxic metabolites produced by the mold in food contaminated by the mold, can enter animal bodies through feed, are anti-nutritional factors existing in feed and raw materials, and are mold secondary metabolites with strong toxin. Mycotoxin is produced during the processing, transportation and storage of the feed, and causes acute or chronic toxicity of animals, and damages liver, kidney, nerve tissue, hematopoietic tissue, skin tissue and the like of organisms. According to statistics, more than 300 mycotoxins are known, wherein aflatoxin, zearalenone, T-2 toxin, ochratoxin, vomitoxin and fumonisin have the highest toxicity, the widest distribution and the highest toxin yield.
To date, the methods for removing aflatoxins have been mainly chemical detoxification, physical detoxification and biological detoxification (bioadsorption, biodegradation). Wherein, the chemical detoxification method mainly adopts an oxidant or strong alkali for detoxification. The physical detoxification method mainly comprises an adsorbent water washing method, an adsorption method, a solvent extraction method, a heating detoxification method, an ultraviolet detoxification method, a degerming detoxification method and the like, wherein the adsorbent adsorption method is most commonly used. However, the two methods have the defects of unstable and incomplete detoxification effect, loss of nutrients and trace elements, poor palatability and the like. The biological adsorption method is mainly characterized in that the toxin is adsorbed by microorganisms through cell walls, and the method is a reversible process, although the toxin can be effectively adsorbed, part of the toxin can be released again along with the prolonging of the culture time. Biodegradation detoxification is the breakdown of mycotoxins by secondary metabolites produced by microorganisms, and produces low-or non-toxic degradation products.
Therefore, it is important to develop a microbial preparation for feeding which can effectively remove mycotoxin and is beneficial to digestion and absorption of livestock.
Disclosure of Invention
The invention provides a preparation and a feed for effectively degrading mycotoxin and a preparation method thereof to solve the technical problems.
The invention firstly provides a microbial preparation which comprises bacillus subtilis, bacillus coagulans, candida utilis, lactobacillus casei, bacillus licheniformis and bifidobacterium longum.
Preferably, in the microbial preparation, the viable count of the bacillus subtilis is 5-10 multiplied by 1010CFU/g, the viable count of the bacillus coagulans is 4-10 multiplied by 1010CFU/g, the number of viable candida utilis is 1-10 multiplied by 1010CFU/g, the number of viable candida utilis is 1-10 multiplied by 1010CFU/g, the viable count of the lactobacillus casei is 1-10 multiplied by 1010CFU/g, the viable count of the bacillus licheniformis is 1-10 multiplied by 1010CFU/g, the viable count of the bifidobacterium longum is 1-10 multiplied by 1010CFU/g。
Preferably, the microbial preparation comprises bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight percentage of the bifidobacterium longum is as follows: (10% -50%): (10% -50%): (10% -50%): (10% -50%): (10% -50%): (10% -50%); preferably, bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight percentage of the bifidobacterium longum is as follows: (20% -50%): (20% -50%): (10% -30%): (10% -30%): (10% -30%): (10% -30%).
Preferably, the microbial preparation further comprises at least one of bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, enterococcus lactis, lactobacillus acidophilus, lactobacillus casei, lactobacillus delbrueckii subsp lactis, lactobacillus plantarum, pediococcus acidilactici, pediococcus pentosaceus, saccharomyces cerevisiae, rhodopseudomonas palustris, bifidobacterium infantis, bifidobacterium breve, bifidobacterium adolescentis, streptococcus thermophilus, lactobacillus reuteri, bifidobacterium animalis, aspergillus niger, aspergillus oryzae, bacillus lentus, bacillus pumilus, lactobacillus cellobiosus, lactobacillus fermentum, lactobacillus delbrueckii subsp.
The invention also provides a preparation method of the microbial preparation, which comprises the following steps:
respectively inoculating bacillus subtilis, bacillus coagulans, candida utilis, lactobacillus casei, bacillus licheniformis and bifidobacterium longum on a sterilized plate culture medium, wherein coumarin, ochratoxin and zearalenone are added into the plate culture medium for inoculating the bacillus subtilis;
step two, respectively inoculating bacillus subtilis, bacillus coagulans, candida utilis, lactobacillus casei, bacillus licheniformis and bifidobacterium longum into a sterilized liquid culture medium;
step three, respectively carrying out fermentation tank expanded culture on bacillus subtilis, bacillus coagulans, candida utilis, bacillus licheniformis, lactobacillus casei and bifidobacterium longum;
step four, centrifuging the bacteria obtained in the step three, taking bacteria mud, and performing low-temperature suspension drying or freeze drying to obtain bacteria powder;
step five, respectively and uniformly mixing the bacterial powder obtained in the step four with a physiologically acceptable carrier, wherein the weight ratio of the bacterial powder to the physiologically acceptable carrier is 1:1-1: 10; preferably, the physiologically acceptable carrier is one or more than two of maltodextrin, cyclodextrin, starch, talcum powder and montmorillonite;
step six, mixing the bacterial powder mixture obtained in the step five according to the ratio of bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight percentage of the bifidobacterium longum is as follows: (10% -50%): (10% -50%): (10% -50%): (10% -50%): (10% -50%): (10% -50%); preferably, bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight percentage of the bifidobacterium longum is as follows: (20% -50%): (20% -50%): (10% -30%): (10% -30%): (10% -30%): (10% -30%).
Preferably, in the first step of the preparation method of the microbial preparation, the dosages of coumarin, ochratoxin and zearalenone are respectively 1-20 μ g, 1-20 μ g and 1-20 μ g added in each kilogram of plate culture medium; preferably, the dosages of coumarin, ochratoxin and zearalenone are 5-15 μ g, 5-15 μ g and 5-15 μ g respectively per kilogram of plate culture medium.
Preferably, the preparation method of the microbial preparation comprises the following step two:
the culture conditions of the bacillus subtilis are as follows: culture medium: 5-15g/L of starch, 1-5g/L of beef extract, 5-15g/L of peptone, 1-8g/L of NaCl, 10-200 g/L of seaweed meal and pH 7.0; culturing at 25-40 deg.C for 10-30 h;
the culture conditions of the bacillus coagulans are as follows: culture medium: 10-20 g/L of glucose, 10-20 g/L of peptone, 5-15g/L of yeast powder, 2-10 g/L of magnesium sulfate, 10-200 g/L of seaweed powder and pH 7.5; culturing at 30-40 deg.C for 10-30 h;
the culture conditions of the candida utilis are as follows: culture medium: 10-25 g/L of glucose, 5-15g/L of peptone, 1-10g/L of yeast extract, 10-200 g/L of seaweed meal and pH of 4.5-6.5; culturing at 25-30 deg.C for 10-30 h;
the culture conditions of the lactobacillus casei are as follows: culture medium: 60-120 g/L of skim milk, 10-30 g/L of whey powder, 5-20 g/L of yeast extract, 1-10g/L of diamine hydrogen phosphate, and the pH value is 6.0-7.0; culturing at 30-40 deg.C for 20-50 h;
the culture medium of the bacillus licheniformis comprises: 1-10g/L beef extract, 5-15g/L peptone, 10-10 g/L NaC 11, pH7.0-8.0, 10-200 g/L seaweed meal, culturing at 30-40 deg.C for 20-40 h;
the culture medium of bifidobacterium longum is as follows: 5-15g/L of peptone, 1-10g/L of casein peptone, 1-6 g/L of lactose, 1-10g/L of yeast extract, 1-6 g/L of oligosaccharide, 10-200 g/L of seaweed meal, pH 6-7, and culturing at 30-40 ℃ for 20-40 h;
preferably, the preparation method of the microbial preparation comprises the following steps:
the fermentation conditions of the bacillus subtilis are as follows: culture medium: 15-25g/L of glucose, 5-15g/L of peptone, 1-8g/L of sodium chloride, 10-200 g/L of seaweed meal and pH = 7.2-7.5; culturing at 25-40 deg.C for 20-50 h;
the fermentation conditions of the bacillus coagulans are as follows: culture medium: starch 5-15g/L, corn steep liquor 1-10g/L, peptone 5-15g/L, K2HPO41-5 g/L，MgSO40.1-1 g/L, 10-200 g/L of seaweed powder and pH 7.5; culturing at 30-40 deg.C for 20-50 h;
the fermentation conditions of the candida utilis are as follows: culture medium: 15-25g/L of glucose, 5-15g/L of peptone, 1-10g/L of yeast extract, 10-200 g/L of seaweed meal and pH of 4.5-6.5; culturing at 25-30 deg.C for 20-50 h;
the fermentation conditions of the lactobacillus casei are as follows: culture medium: 60-120 g/L of skim milk, 10-30 g/L of whey powder, 5-20 g/L of yeast extract, 1-10g/L of diamine hydrogen phosphate, and the pH value is 6.3-6.6; culturing at 30-40 deg.C for 40-80 hr;
the fermentation conditions of the bacillus licheniformis are as follows: culture medium: peptone 1-10g/L, yeast extract 5-15g/L, sodium chloride 1-10g/L, starch 1-10g/L, seaweed meal 10-200 g/L, pH7.5; culturing at 30-40 deg.C for 20-40 h;
the fermentation conditions of bifidobacterium longum are as follows: culture medium: 5-15g/L of peptone, 1-10g/L of casein peptone, 1-6 g/L of lactose, 1-10g/L of yeast extract, 1-6 g/L of oligosaccharide, 10-200 g/L of seaweed meal, pH 6-7, and culturing at 30-40 ℃ for 20-40 h.
Preferably, the preparation method of the microbial preparation comprises the following steps: the inoculation amounts of the bacillus subtilis, the bacillus coagulans, the candida utilis, the bacillus licheniformis, the lactobacillus casei and the bifidobacterium longum are respectively 1-10%, 1-10% and 1-10%.
The invention also provides a feed or additive, and the microbial preparation is added into the feed or additive.
The invention provides application of the microbial preparation in removal of mycotoxin, preferably in removal of aflatoxin, zearalenone, T-2 toxin, ochratoxin, vomitoxin and fumonisin.
The invention also provides a method for removing mycotoxin, which comprises the steps of adding the microbial preparation or additive for degrading aflatoxin into a sample to be treated, and fermenting for 10-100h at the temperature of 25-40 ℃ and with the pH value of 6-7.5; the dosage of the microbial preparation or the additive for degrading the aflatoxin is that 0.01-30g of the microbial preparation or the additive for degrading the aflatoxin is added into each kilogram of samples.
The invention adopts the combination of composite probiotics, and the degradation method and the adsorption method are cooperated to remove mycotoxins, in particular aflatoxin, zearalenone, T-2 toxin, ochratoxin, vomitoxin and fumonisin. And can meet the requirements of guaranteeing the culture production in two aspects of promoting the balance of animal intestinal flora and degrading mycotoxin in the feed.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
EXAMPLE 1 preparation of fungal powder
Respectively inoculating bacillus subtilis, bacillus coagulans, candida utilis, lactobacillus casei, bacillus licheniformis and bifidobacterium longum on a sterilized plate culture medium, wherein the dosages of coumarin, ochratoxin and zearalenone added into the plate culture medium for inoculating the bacillus subtilis are respectively 5 mu g, 5 mu g and 5 mu g added into each kilogram of the plate culture medium;
step two, respectively inoculating bacillus subtilis, bacillus coagulans, candida utilis, lactobacillus casei, bacillus licheniformis and bifidobacterium longum into a sterilized liquid culture medium;
the culture conditions of the bacillus subtilis are as follows: culture medium: 10.0g/L of starch, 3.0g/L of beef extract, 10g/L of peptone, 5.0g/L of NaCl, 50g/L of seaweed meal and pH 7.0; culturing at 37 deg.C for 28 h;
the culture conditions of the bacillus coagulans are as follows: culture medium: 15g/L of glucose, 15g/L of peptone, 10g/L of yeast powder, 3.0g/L of magnesium sulfate, 50g/L of seaweed powder and pH 7.5; culturing at 37 deg.C for 28 h;
the culture conditions of the candida utilis are as follows: culture medium: 20g/L of glucose, 10g/L of peptone, 5g/L of yeast extract, 50g/L of seaweed meal and pH 6.0; culturing at 37 deg.C for 28 h;
the culture conditions of the lactobacillus casei are as follows: culture medium: 100 g/L of skim milk, 20g/L of whey powder, 10g/L of yeast extract, 5.0g/L of diamine hydrogen phosphate and pH of 6.0; culturing at 37 deg.C for 37 h;
the culture medium of the bacillus licheniformis comprises: 5.0g/L beef extract, 10.0g/L peptone, 15.0g/L NaC15, 50g/L seaweed meal, pH8.0, culturing at 30 deg.C for 28 h;
the culture medium of bifidobacterium longum is as follows: 10.0g/L of peptone, 5.0g/L of casein peptone, 3.0g/L of lactose, 5.0g/L of yeast extract, 3.0g/L of oligosaccharide, 50g/L of seaweed meal, pH 6.5 and culturing at 37 ℃ for 28 h;
step three, respectively carrying out fermentation tank expanded culture on bacillus subtilis, bacillus coagulans, candida utilis, bacillus licheniformis, lactobacillus casei and bifidobacterium longum;
the inoculation amounts of bacillus subtilis, bacillus coagulans, candida utilis, bacillus licheniformis, lactobacillus casei and bifidobacterium longum are respectively 8%, 6%, 8% and 8%;
the fermentation conditions of the bacillus subtilis are as follows: culture medium: glucose 20.0g/L, peptone 10.0g/L, sodium chloride 4.0g/L, seaweed meal 60g/L, pH = 7.0; culturing at 37 deg.C for 50 h;
the fermentation conditions of the bacillus coagulans are as follows: culture medium: starch 10.0g/L, corn steep liquor 5.0g/L, peptone 10.0g/L, K2HPO43.0 g/L，MgSO40.5 g/L of seaweed powder, 60g/L of seaweed powder, pH7.5, and culturing for 50h at 37 ℃;
the fermentation conditions of the candida utilis are as follows: culture medium: 20.0g/L of glucose, 10.0g/L of peptone, 5.0g/L of yeast extract, 60g/L of seaweed meal and pH of 4.5-6.5; culturing at 25 deg.C for 48 h;
the fermentation conditions of the lactobacillus casei are as follows: culture medium: 100.0 g/L of skim milk, 20.0g/L of whey powder, 10.0g/L of yeast extract, 5.0g/L of diamine hydrogen phosphate and pH of 6.5; culturing at 37 deg.C for 72 h;
the fermentation conditions of the bacillus licheniformis are as follows: culture medium: 10.0g/L of peptone, 10.0g/L of yeast extract, 5.0g/L of sodium chloride, 5.0g/L of starch, 60g/L of seaweed meal and pH 7.5; culturing at 37 deg.C for 25 h;
the fermentation conditions of bifidobacterium longum are as follows: culture medium: 10.0g/L of peptone, 5.0g/L of casein peptone, 3.0g/L of lactose, 5.0g/L of yeast extract, 3.0g/L of oligosaccharide, 60.0g/L of seaweed meal, pH 6.5, and culturing at 37 ℃ for 32 h.
Step four, centrifuging the bacteria obtained in the step three, taking bacteria mud, suspending and drying at low temperature to obtain bacteria powder;
step five, uniformly mixing the bacterial powder obtained in the step four with maltodextrin and cyclodextrin respectively, wherein the weight ratio is as follows: 1:0.5:0.5.
EXAMPLE 2 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 50%: 10%: 10%: 10%: 10%: 10 percent.
EXAMPLE 3 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 10%: 50%: 10%: 10%: 10%: 10 percent.
EXAMPLE 4 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 10%: 10%: 50%: 10%: 10%: 10 percent.
EXAMPLE 5 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 10%: 10%: 10%: 50%: 10%: 10 percent.
EXAMPLE 6 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 10%: 10%: 10%: 10%: 50%: 10 percent.
EXAMPLE 7 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 10%: 10%: 10%: 10%: 10%: 50 percent.
EXAMPLE 8 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 30%: 30%: 10%: 10%: 10%: 10 percent.
EXAMPLE 9 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 30%: 10%: 30%: 10%: 10%: 10 percent.
EXAMPLE 10 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 30%: 10%: 10%: 30%: 10%: 10 percent.
EXAMPLE 11 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 30%: 10%: 10%: 10%: 30%: 10 percent.
EXAMPLE 12 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 30%: 10%: 10%: 10%: 10%: 30 percent.
EXAMPLE 13 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 20%: 20%: 30%: 10%: 10%: 10 percent.
EXAMPLE 14 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 20%: 20%: 10%: 30%: 10%: 10 percent.
EXAMPLE 15 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 20%: 20%: 10%: 10%: 30%: 10 percent.
EXAMPLE 16 preparation of microbial preparation
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 20%: 20%: 10%: 10%: 10%: 30 percent.
Comparative example 1
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 75%: 5%: 5%: 5%: 5%: 5 percent.
Comparative example 2
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 5%: 75%: 5%: 5%: 5%: 5 percent.
Comparative example 3
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 5%: 5%: 75%: 5%: 5%: 5 percent.
Comparative example 4
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 5%: 5%: 5%: 75%: 5%: 5 percent.
Comparative example 5
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 5%: 5%: 5%: 5%: 75%: 5 percent.
Comparative example 6
The bacterial powder obtained in example 1 was mixed, and bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight ratio of the bifidobacterium longum is as follows: 5%: 5%: 5%: 5%: 5%: 75 percent.
Application example 1 removal of mycotoxins
The microbial preparations prepared in examples 2-16 and comparative examples 1-6 (the amounts of the microbial preparations were 2g per 1Kg of medium) were added to the respective media (glucose 15g/L, peptone 15g/L, magnesium sulfate 3.0g/L, beef extract 3.0 g/L);
after aflatoxin B1, zearalenone, vomitoxin, T-2 toxin, fumonisin B1 and ochratoxin A are dissolved respectively, the mixture is added into the culture medium, so that the final concentrations of aflatoxin B1, zearalenone, vomitoxin, ochr T-2 toxin, fumonisin B1 and ochratoxin A are respectively 20 mu g/kg. Fermenting at 37 deg.C for 72h at pH of 7.0, detecting the concentrations of aflatoxin B1, zearalenone, vomitoxin, ochratoxin, T-2 toxin, fumonisin B1, and ochratoxin A in the fermentation broth by high performance liquid chromatography, and calculating mycotoxin removal rate.
Mycotoxin removal rate = (initial concentration of toxin in sample-concentration of toxin in sample after treatment)/initial concentration of toxin in sample x 100% formula.
TABLE 1 mycotoxin removing effect of microbial preparations prepared in examples 2 to 16 and comparative examples 1 to 6
Figure DEST_PATH_IMAGE001
As can be seen from the comparison of the mycotoxins, the microbial preparations prepared in examples 2 to 16 all have a mycotoxin removal rate higher than 90%, and a mycotoxin removal rate higher than that of the microbial preparations prepared in comparative examples 1 to 6.
Application example 2 influence on growth Performance and apparent digestibility of piglets
220 weaned healthy commercial castrated boars of 28 days old and similar weights are selected and divided into 22 groups of 10 pigs. Feeding the basic ration of corn-soybean meal-fish meal for 30 days at regular time three times every day. Wherein the antibiotic group is added with kitasamycin 50 mg/kg-1Oxytetracycline 50 mg/kg-1Chlortetracycline 75 mg/kg-1(ii) a The experimental group added 2g/Kg of the microbial preparations prepared in examples 2-16 and comparative examples 1-6 to corn, soybean meal, whey powder and fish meal in the corn-soybean meal-fish meal type basic ration, respectively, added with appropriate amount of water, fermented at room temperature for 4 hours, and then fed for 30 days at regular time and three times a day after being proportioned according to the trace elements of the antibiotic group.
Detecting the index
1. Growth performance
And (4) measuring the weight and feed intake of the pig, and calculating the average daily gain, the average daily feed intake and the feed conversion ratio.
2. Apparent digestibility test of feed
And (3) measuring the apparent digestibility of the piglets to the main nutrient components of the feed by adopting an endogenous indicator method. The test starts on day 14 and feces are collected for 4 consecutive days, and stored at-20 ℃. After the collection, the mixture is uniformly thawed, dried at 60 ℃, crushed and then detected and analyzed for apparent digestibility of dry matters, crude protein, crude fat and crude fiber.
TABLE 2 Effect on piglet growth Performance
Figure 894894DEST_PATH_IMAGE002
As can be seen from the comparison, the average daily gain weight of piglets fed by the microbial preparation fermented feed prepared in examples 2 to 16 is higher than that of the antibiotic group and that of the microbial preparation group prepared in comparative examples 1 to 6, the feed-meat ratio and the diarrhea rate are lower than those of the antibiotic group and that of the microbial preparation group prepared in comparative examples 1 to 6, and the feed-meat ratio is reduced by 5.8 to 12.8 percent compared with that of the antibiotic group; the average daily food intake was higher than that of the antibiotic group and the microbial preparation groups prepared in comparative examples 1 to 6.
TABLE 3 apparent digestibility test of feeds
Figure DEST_PATH_IMAGE003
As can be seen from the comparison, when piglets fed with the microbial preparation fermented feed prepared in examples 2 to 16 had dry matters, crude proteins, crude fats and crude fibers which were significantly higher than those of the antibiotic group and the microbial preparation group prepared in comparative examples 1 to 6.
It will be apparent to those skilled in the art that modifications and improvements can be made to the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. A microbial preparation is characterized by comprising bacillus subtilis, bacillus coagulans, candida utilis, lactobacillus casei, bacillus licheniformis and bifidobacterium longum.
2. The microbial preparation of claim 1, wherein the viable count of Bacillus subtilis is 5-10 x 1010CFU/g, the viable count of the bacillus coagulans is 4-10 multiplied by 1010CFU/g, the number of viable candida utilis is 1-10 multiplied by 1010CFU/g, the number of viable candida utilis is 1-10 multiplied by 1010CFU/g, the viable count of the lactobacillus casei is 1-10 multiplied by 1010CFU/g, the viable count of the bacillus licheniformis is 1-10 multiplied by 1010CFU/g, the viable count of the bifidobacterium longum is 1-10 multiplied by 1010CFU/g。
3. The microbial preparation of claim 1, wherein the ratio of bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight percentage of the bifidobacterium longum is as follows: (10% -50%): (10% -50%): (10% -50%): (10% -50%): (10% -50%): (10% -50%); preferably, bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight percentage of the bifidobacterium longum is as follows: (20% -50%): (20% -50%): (10% -30%): (10% -30%): (10% -30%): (10% -30%).
4. The microbial preparation of any one of claims 1 to 3, further comprising at least one species of Bifidobacterium bifidum, enterococcus faecalis, enterococcus faecium, enterococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp.
5. A process for the preparation of a microbial preparation according to any one of claims 1 to 4, comprising the steps of:
respectively inoculating bacillus subtilis, bacillus coagulans, candida utilis, lactobacillus casei, bacillus licheniformis and bifidobacterium longum on a sterilized plate culture medium, wherein coumarin, ochratoxin and zearalenone are added into the plate culture medium for inoculating the bacillus subtilis;
step two, respectively inoculating bacillus subtilis, bacillus coagulans, candida utilis, lactobacillus casei, bacillus licheniformis and bifidobacterium longum into a sterilized liquid culture medium;
step three, respectively carrying out fermentation tank expanded culture on bacillus subtilis, bacillus coagulans, candida utilis, bacillus licheniformis, lactobacillus casei and bifidobacterium longum;
step four, centrifuging the bacteria obtained in the step three, taking bacteria mud, and performing low-temperature suspension drying or freeze drying to obtain bacteria powder;
step five, respectively and uniformly mixing the bacterial powder obtained in the step four with a physiologically acceptable carrier, wherein the weight ratio of the bacterial powder to the physiologically acceptable carrier is 1:1-1: 10; preferably, the physiologically acceptable carrier is one or more than two of maltodextrin, cyclodextrin, starch, talcum powder and montmorillonite;
step six, mixing the bacterial powder mixture obtained in the step five according to the ratio of bacillus subtilis: bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight percentage of the bifidobacterium longum is as follows: (10% -50%): (10% -50%): (10% -50%): (10% -50%): (10% -50%): (10% -50%); preferably, bacillus coagulans: candida utilis: lactobacillus casei: b, bacillus licheniformis: the dry weight percentage of the bifidobacterium longum is as follows: (20% -50%): (20% -50%): (10% -30%): (10% -30%): (10% -30%): (10% -30%).
6. The method for producing a microbial preparation according to claim 5,
preferably, in the first step of the preparation method of the microbial preparation, the dosages of coumarin, ochratoxin and zearalenone are respectively 1-20 μ g, 1-20 μ g and 1-20 μ g added in each kilogram of plate culture medium; preferably, the dosages of coumarin, ochratoxin and zearalenone are respectively 5-15 μ g, 5-15 μ g and 5-15 μ g per kilogram of plate culture medium;
preferably, the preparation method of the microbial preparation comprises the following step two:
the culture conditions of the bacillus subtilis are as follows: culture medium: 5-15g/L of starch, 1-5g/L of beef extract, 5-15g/L of peptone, 1-8g/L of NaCl, 10-200 g/L of seaweed meal and pH 7.0; culturing at 25-40 deg.C for 10-30 h;
the culture conditions of the bacillus coagulans are as follows: culture medium: 10-20 g/L of glucose, 10-20 g/L of peptone, 5-15g/L of yeast powder, 2-10 g/L of magnesium sulfate, 10-200 g/L of seaweed powder and pH 7.5; culturing at 30-40 deg.C for 10-30 h;
the culture conditions of the candida utilis are as follows: culture medium: 10-25 g/L of glucose, 5-15g/L of peptone, 1-10g/L of yeast extract, 10-200 g/L of seaweed meal and pH of 4.5-6.5; culturing at 25-30 deg.C for 10-30 h;
the culture conditions of the lactobacillus casei are as follows: culture medium: 60-120 g/L of skim milk, 10-30 g/L of whey powder, 5-20 g/L of yeast extract, 1-10g/L of diamine hydrogen phosphate, and the pH value is 6.0-7.0; culturing at 30-40 deg.C for 20-50 h;
the culture medium of the bacillus licheniformis comprises: 1-10g/L beef extract, 5-15g/L peptone, 10-10 g/L NaC 11, pH7.0-8.0, 10-200 g/L seaweed meal, culturing at 30-40 deg.C for 20-40 h;
the culture medium of bifidobacterium longum is as follows: 5-15g/L of peptone, 1-10g/L of casein peptone, 1-6 g/L of lactose, 1-10g/L of yeast extract, 1-6 g/L of oligosaccharide, 10-200 g/L of seaweed meal, pH 6-7, and culturing at 30-40 ℃ for 20-40 h;
preferably, the preparation method of the microbial preparation comprises the following steps:
the fermentation conditions of the bacillus subtilis are as follows: culture medium: 15-25g/L of glucose, 5-15g/L of peptone, 1-8g/L of sodium chloride, 10-200 g/L of seaweed meal and pH = 7.2-7.5; culturing at 25-40 deg.C for 20-50 h;
the fermentation conditions of the bacillus coagulans are as follows: culture medium: starch 5-15g/L, corn steep liquor 1-10g/L, peptone 5-15g/L, K2HPO41-5 g/L，MgSO40.1-1 g/L, 10-200 g/L of seaweed powder and pH 7.5; culturing at 30-40 deg.C for 20-50 h;
the fermentation conditions of the candida utilis are as follows: culture medium: 15-25g/L of glucose, 5-15g/L of peptone, 1-10g/L of yeast extract, 10-200 g/L of seaweed meal and pH of 4.5-6.5; culturing at 25-30 deg.C for 20-50 h;
the fermentation conditions of the lactobacillus casei are as follows: culture medium: 60-120 g/L of skim milk, 10-30 g/L of whey powder, 5-20 g/L of yeast extract, 1-10g/L of diamine hydrogen phosphate, and the pH value is 6.3-6.6; culturing at 30-40 deg.C for 40-80 hr;
the fermentation conditions of the bacillus licheniformis are as follows: culture medium: peptone 1-10g/L, yeast extract 5-15g/L, sodium chloride 1-10g/L, starch 1-10g/L, seaweed meal 10-200 g/L, pH7.5; culturing at 30-40 deg.C for 20-40 h;
the fermentation conditions of bifidobacterium longum are as follows: culture medium: 5-15g/L of peptone, 1-10g/L of casein peptone, 1-6 g/L of lactose, 1-10g/L of yeast extract, 1-6 g/L of oligosaccharide, 10-200 g/L of seaweed meal, pH 6-7, and culturing at 30-40 ℃ for 20-40 h;
preferably, the preparation method of the microbial preparation comprises the following steps: the inoculation amounts of the bacillus subtilis, the bacillus coagulans, the candida utilis, the bacillus licheniformis, the lactobacillus casei and the bifidobacterium longum are respectively 1-10%, 1-10% and 1-10%.
7. Use of a microbial preparation according to any one of claims 1 to 4 for the removal of mycotoxins.
8. Use according to claim 7, wherein the microbial preparation is used for the preparation of an additive for the removal of mycotoxins.
9. Use according to claim 7, wherein the microbial preparation is used for the preparation of a feed and the microbial preparation is used for the removal of mycotoxins from the feed.
10. The use according to claim 7, wherein the mycotoxins comprise one or more of aflatoxins, zearalenone, T-2 toxin, ochratoxins, vomitoxin, fumonisins.
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