WO2023087499A1 - 一株高产复合酶同时高效降解霉菌毒素的贝莱斯芽孢杆菌及其应用 - Google Patents

一株高产复合酶同时高效降解霉菌毒素的贝莱斯芽孢杆菌及其应用 Download PDF

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WO2023087499A1
WO2023087499A1 PCT/CN2021/142776 CN2021142776W WO2023087499A1 WO 2023087499 A1 WO2023087499 A1 WO 2023087499A1 CN 2021142776 W CN2021142776 W CN 2021142776W WO 2023087499 A1 WO2023087499 A1 WO 2023087499A1
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feed
raw materials
bacillus velezensis
animal
increase
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French (fr)
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邓雪娟
李冲
蔡辉益
李爽
李淑珍
王腾飞
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天津博菲德科技有限公司
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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
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • 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
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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

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  • the invention relates to the field of agricultural biotechnology, in particular to a strain of Bacillus velezensis and its application, in particular to a strain of Bacillus velezensis LB-Y capable of simultaneously degrading mycotoxins with high yield and high efficiency -1 and its application.
  • Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous and unavoidable contaminants in food and feed. Consumption of mycotoxins by humans and animals can lead to illness and death. Aspergillus, Penicillium and Fusarium species are all capable of producing a variety of mycotoxins. For example, Aspergillus flavus, Aspergillus parasiticus, Aspergillus knossii and Aspergillus pseudoaflatus are the main culprits for the production of a series of highly toxic substances aflatoxin.
  • AFB 1 aflatoxin B 1
  • AFB 1 The existing methods to reduce the impact of AFB 1 mainly include removal, inactivation, transformation or degradation, which can be divided into three types: physical, chemical and biological. Due to unavoidable limitations, most of these methods are inefficient or cost-prohibitive, while biotechnology provides a promising approach for the removal and degradation of AFB 1 in agricultural products and animal feed due to its excellent characteristics such as safety, economy, and stability. An attractive choice. Some strains, such as Stenotrophomonas sp., Mycobacterium fluoranthenivorans sp., etc. have been verified to have good AFB 1 degradation effect. In addition, laccase, horseradish peroxidase, manganese peroxidase Oxidases also have a certain degradation ability.
  • Bacillus velezensis was first isolated in 1999 and officially named in 2005. It has been verified that it can prevent and treat various types of plant pathogen infections, reduce the disease index, and inhibit a variety of pathogenic fungi and bacteria. An excellent biocontrol strain. However, there are few reports on the research of this strain on the degradation of mycotoxins.
  • One object of the present invention is to provide a strain of Bacillus velezensis LB-Y-1.
  • the preservation number of Bacillus velezensis LB-Y-1 provided by the present invention is CGMCC No.21344.
  • the strain has been deposited in the General Microbiology Center of China Committee for Microbial Culture Collection (CGMCC for short) on December 10, 2020; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Zip code: 100101 ).
  • the Bacillus velezensis LB-Y-1 was isolated from the chyme of the digestive tract of healthy animals by the inventor through complex domestication, comparison, screening and other processes. It has the following properties: degrade AFB 1 , High production of amylase, protease and cellulase can improve the structure of animal intestinal flora, improve animal growth performance, and improve the quality of raw materials or feed.
  • Another object of the present invention is to provide a bacterial agent.
  • the active ingredient of the bacterial agent provided by the present invention is the above-mentioned Bacillus velezensis LB-Y-1 or its bacterial suspension or its culture or its culture liquid or its fermentation liquid.
  • the concentration of the bacterial suspension or the culture may be 1.0 ⁇ 10 7 -1.0 ⁇ 10 9 CFU/mL, preferably 1.0 ⁇ 10 8 CFU/mL.
  • Another object of the present invention is to provide the new application of the above-mentioned Bacillus velezensis LB-Y-1 or bacterial agent.
  • the invention provides the application of the above-mentioned Bacillus velezensis LB-Y-1 or bacterial agent in any one of the following 1)-9):
  • a final object of the present invention is to provide a method according to any one of X1)-X6) below:
  • a method for degrading or removing AFB 1 comprising the steps of: using the above-mentioned Bacillus velezensis LB-Y-1 or bacterial agent to ferment and treat raw materials or feed, so as to realize the degradation or removal of AFB 1 ;
  • a method for eliminating or inhibiting AFB 1 contamination in raw materials or feeds comprising the steps of: using the above-mentioned Bacillus velezensis LB-Y-1 or bacterial agent to ferment and treat raw materials or feeds to achieve elimination or inhibition AFB 1 contamination in raw materials or feed;
  • a method for detoxification of raw materials or feed comprising the steps of: using the above-mentioned Bacillus velezensis LB-Y-1 or microbial agent to ferment and treat raw materials or feed, so as to realize detoxification of raw materials or feed;
  • a method for improving the quality of raw materials or feed comprising the steps of: using the above-mentioned Bacillus velezensis LB-Y-1 or microbial agent to ferment and treat the raw materials or feed, so as to realize the improvement of the quality of the raw materials or feed;
  • a method for improving the structure of the intestinal flora of an animal comprising the steps of: feeding the animal with the above-mentioned Bacillus velezensis LB-Y-1 or bacterial agent, so as to realize the improvement of the structure of the intestinal flora of the animal ;
  • a method for improving growth performance of animals comprising the steps of: feeding animals with the above-mentioned Bacillus velezensis LB-Y-1 or bacterial agent, so as to improve the growth performance of animals.
  • the fermentation treatment method includes the following steps: adding the above-mentioned Bacillus velezensis LB-Y-1 or bacterial agent to raw materials or feed.
  • the feeding method comprises the following steps: spraying the above-mentioned Bacillus velezensis LB-Y-1 or bacterial agent in the animal basal diet.
  • the complex enzyme is protease, cellulase and/or amylase.
  • the improvement of the animal intestinal flora structure is embodied in any of the following b1)-b3):
  • the macromolecular substance can be macromolecular protein (such as casein), cellulose (such as sodium carboxymethyl cellulose) or starch (such as soluble starch).
  • macromolecular protein such as casein
  • cellulose such as sodium carboxymethyl cellulose
  • starch such as soluble starch
  • the preparation method of the bacterial suspension comprises the following steps: inoculating the isolated and purified LB-Y-1 bacterial strain in LB solid medium, after solid culture (cultivation time can be 24h), picking Single colonies were placed in LB liquid medium, and cultured at 37°C and 160r/min (cultivation time can be 18-24h), so that the bacterial concentration in the culture system reached 1.0 ⁇ 10 7 ⁇ 1.0 ⁇ 10 9 CFU/ mL, centrifuged (centrifugation condition can be 4000rpm centrifugal 10min), collects thalline, with sterile physiological saline, described thalline is washed successively (washing times can be three times) and resuspended, obtains described bacterial suspension (bacterial suspension The bacterial cell concentration in the solution is 1.0 ⁇ 10 7 ⁇ 1.0 ⁇ 10 9 CFU/mL).
  • the preparation method of the culture comprises the following steps: inoculating the isolated and purified LB-Y-1 strain in LB solid medium, picking a single colony in the logarithmic growth phase into the LB liquid medium, and inoculating at 37°C, Cultivate under the condition of 160rpm/min (cultivation time can be 24h) to obtain seed solution, inoculate the seed solution (inoculum size can be 1%) into LB liquid medium, and carry out under the condition of 37°C and 160rpm/min Cultivate (cultivation time may be 14 hours) to obtain a fermentation liquid, which is the culture (thalline concentration in the culture is 1.0 ⁇ 10 7 -1.0 ⁇ 10 9 CFU/mL).
  • the animal includes but not limited to chicken.
  • the animal is a broiler chicken (such as an AA broiler chicken).
  • the raw material or feed can be agricultural products such as peanuts, corn, rice and cottonseed that are or are not contaminated by AFB 1 .
  • the raw material is peanut meal, specifically peanut meal contaminated with AFB 1 .
  • Fig. 1 is the effect of strain LB-Y-1 of the present invention on the primary screening of degrading casein, sodium carboxymethylcellulose and soluble starch.
  • A is protease producing ability;
  • B is cellulase producing ability;
  • C is amylase producing ability.
  • Figure 2 is the colony characteristics and cell morphology of the bacterial strain LB-Y-1 of the present invention (Gram staining ⁇ 100).
  • A is the characteristics of the colony;
  • B is the shape of the bacteria.
  • Fig. 3 is the analytical result that bacterial strain LB-Y-1 of the present invention adopts API 20NE.
  • Fig. 4 is a phylogenetic tree constructed by the strain LB-Y-1 of the present invention.
  • Fig. 5 is the growth curve of the strain LB-Y-1 of the present invention.
  • Fig. 6 is the effect of the bacterial strain LB-Y-1 of the present invention on the intestinal flora structure of broilers.
  • Figure 7 shows the effect of fermentation time on the degradation of AFB 1 content in naturally moldy peanut meal by strain LB-Y-1.
  • Figure 8 shows the effect of fermentation time on the increase of acid-soluble protein content in naturally moldy peanut meal by strain LB-Y-1.
  • the culture medium involved in the present invention (the solvents are all distilled water, all sterilized at 121°C for 20min before use) are as follows:
  • Nutrient broth (NB) medium (g/L): 10 peptone, 3 beef extract, 5 sodium chloride, pH adjusted to 7.2-7.4, and 2% agar powder added to the solid medium.
  • Hormisch medium (g/L): Dipotassium hydrogen phosphate 0.25, magnesium sulfate heptahydrate 0.25, potassium nitrate 0.5, ammonium sulfate 0.5, calcium chloride 0.005, ferric chloride 0.003, adjust pH to 7.0, add as needed after sterilization 1 ⁇ 3g coumarin, add 2% agar powder to the solid medium.
  • Casein medium (g/L): casein 10, beef extract powder 3, agar powder 15, sodium chloride 5, potassium dihydrogen phosphate 2, adjust the pH to 7.0, add 2% agar powder to the solid medium.
  • Cellulase selection medium g/L: yeast extract 5, sodium chloride 5, tryptone 10, sodium carboxymethylcellulose 10, potassium dihydrogen phosphate 1, pH adjusted to 7.2-7.4, solid medium Add 2% agar powder.
  • Amylase selection medium g/L: soluble starch 10, glucose 5, tryptone 10, beef extract 5, sodium chloride 5, pH adjusted to 7.2-7.4, solid medium added 2% agar powder.
  • LB medium (g/L): Tryptone 10, yeast extract 5, sodium chloride 10, pH adjusted to 7.2-7.4, solid medium added with 2% agar powder.
  • Fermentation medium A (g/L): peptone 10, beef extract 3, sodium chloride 5, dipotassium hydrogen phosphate 1, glucose 1, the pH was adjusted to 6.5, and 2% agar powder was added to the solid medium.
  • Fermentation medium B glucose 5, tryptone 10, yeast extract 5, potassium dihydrogen phosphate 1, sodium chloride 5, magnesium sulfate 0.5, manganese sulfate 0.005, sodium carboxymethylcellulose 5, pH Adjust to 5.5, add 2% agar powder to the solid medium.
  • Fermentation medium C (g/L): soluble starch 5, glucose 5, tryptone 10, yeast extract 5, potassium dihydrogen phosphate 1, sodium chloride 5, magnesium sulfate 0.5, manganese sulfate 0.005, pH adjusted to 5.5, Add 2% agar powder to the solid medium.
  • the detection method involved in the present invention is as follows: the detection of AFB 1 concentration adopts HPLC method; The detection of protease activity adopts Folin-phenol method; The detection of cellulase activity adopts DNS method; The detection of amylase activity adopts DNS method; The protein content was determined according to the method of (GB/T 22492-2008).
  • Example 1 the screening and identification of Bacillus LB-Y-1
  • the culture solution was diluted and spread on NB solid medium (dilution factor from 10 -1 to 10 -6 ), and cultured at 37°C for 24 hours.
  • NB solid medium dialine, aqueous fetal calf serum, fetal calf serum, and fetal calf serum.
  • the purified strains are stored in 50% glycerol and stored at -80°C.
  • Degradation rate calculation formula: AFB 1 degradation rate/% (A-B)/A ⁇ 100%, where A and B represent the control peak area and the peak area after treatment with AFB 1 , respectively.
  • strain LB-Y-1 has the ability to degrade AFB 1 , and the degradation efficiency is 81.56%. It also has the ability to produce protease, cellulase and amylase.
  • the area ratio of the degradation circle to the size of the colony (S/s ) are: 3.67, 4.06, 2.68 respectively.
  • Strain LB-Y-1 was streak-inoculated on LB solid medium, and the growth form of the colony was observed (Figure 2A), which is characterized by: milky white colonies, opaque, and the shape changes gradually from the initial round (full) to irregular ( Folds), the edges are irregular and spread out in the form of clouds, the center of the colony is raised to form a crater, and there is a viscous liquid after being picked apart; the bacteria in the logarithmic phase of growth are taken to smear the plate, fixed and stained with Gram's stain, The morphology was observed and photographed under an oil microscope (Fig. 2B). The bacteria were short rod-shaped and could form spores, and Gram staining was positive.
  • the API 20NE reagent strip was used to analyze the strain LB-Y-1, and the specific results are shown in Figure 3.
  • the carbon source utilization of the strain LB-Y-1 was further analyzed by BIOLOG, and the carbon source of the positive reaction was shown in Table 1.
  • Bacterial Genome DNA Extraction Kit was used to extract the DNA of strain LB-Y-1, and the 16S rDNA and housekeeping gene gyrB were amplified and sequenced. Sequencing results showed that the 16S rDNA PCR product of strain LB-Y-1 obtained a gene fragment with a size of 1476 bp, and its nucleotide sequence was shown in sequence 1.
  • the strain was identified as Bacillus (genus Bacillus), Bacillus velezensis (Bacillus velezensis).
  • the 16S rDNA gene sequence of the strain LB-Y-1 was homologously compared at NCBI, the phylogenetic relationship was analyzed, and a phylogenetic tree was constructed (Fig. 4).
  • strain LB-Y-1 Based on the morphological characteristics of colonies and bacteria, the utilization of BIOLOG carbon sources, the analysis results of API 20NE, and molecular biological identification, the taxa of strain LB-Y-1 were determined to be: Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Bacillus , belonging to Bacillus velezensis.
  • Bacillus velezensis LB-Y-1 has been preserved in the General Microbiology Center of China Committee for Microbial Culture Collection (CGMCC for short) on December 10, 2020; address: Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences; Zip code: 100101), the deposit number is CGMCC No.21344.
  • Embodiment 2 the ability of Veles bacillus LB-Y-1 to produce protease, cellulase and amylase
  • Example 2 Prepare the seed solution according to the method in Example 1, inoculate the seed solution in fermentation medium A, and culture at 37° C. and 160 rpm/min for 36 hours.
  • step 2 After completing step 2, centrifuge at 4°C and 8000 rpm for 15 minutes, and separate the supernatant to obtain the crude enzyme solution.
  • the yield of tyrosine was obtained, and the enzyme activity of the crude enzyme solution was calculated, and the amount of enzyme required to catalyze casein to generate 1 ⁇ g of tyrosine per milliliter of enzyme solution was regarded as 1 enzyme activity as the measurement unit (U/mL).
  • step 2 After completing step 2, centrifuge at 4°C and 8000 rpm for 15 minutes, and separate the supernatant to obtain the crude enzyme solution.
  • the enzyme activity of the crude enzyme solution was calculated, and the amount of enzyme needed to catalyze sodium carboxymethylcellulose to generate 1 ⁇ g of glucose per milliliter of enzyme solution was taken as the measurement unit (U/mL).
  • a maltose standard solution with a concentration of 0.1-0.7 mg/mL. Take 2mL of the above-mentioned standard solution and 2mL DNS reagent and add them to the test tube and mix them thoroughly. After 5 minutes in the boiling water bath, immediately transfer to the ice bath to terminate the reaction. Add 16mL of distilled water to mix well. The relationship curve with maltose concentration.
  • Example 2 Prepare the seed solution according to the method in Example 1, inoculate the seed solution in fermentation medium C, and culture at 37° C. and 160 rpm/min for 36 hours.
  • step 2 After completing step 2, centrifuge at 4°C and 8000 rpm for 15 minutes, and separate the supernatant to obtain the crude enzyme solution.
  • the enzyme activity of the crude enzyme solution was calculated, and the amount of enzyme needed to catalyze starch to produce 1 mg of maltose per milliliter of enzyme solution per 30 min was taken as the unit of measurement (U/mL).
  • Example 3 the effect of Bacillus velei LB-Y-1 on the growth performance of broilers and the diversity of intestinal microorganisms
  • the CON group was fed with basal diet; BV1, 2, and 3 groups were sprayed with Bacillus Veles suspension in the basal diet, so that the bacterial concentration in the basal diet was 1.0 ⁇ 10 7 CFU/kg, 1.0 ⁇ 10 8 CFU/kg, 1.0 ⁇ 10 9 CFU/kg; AGPs group added combination antibiotics (chlortetracycline 100 mg/kg, kitasamycin 20 mg/kg) in the basal diet.
  • the basal ration was a corn-soybean meal ration, and the formula design was based on the "Chicken Feeding Standard" (NY/T 33-2004).
  • the test period was 42 days, and the effects on the growth performance and intestinal flora structure of broilers were analyzed.
  • the preparation method of Bacillus Velez suspension is as follows: inoculate the isolated and purified Bacillus Velez LB-Y-1 in LB solid medium, and after solid culture for 24 hours, pick a single colony and inoculate it into 10 mL LB liquid culture Shake culture at 37°C and 160r/min for 18-24h in culture medium, so that the concentration of bacteria in the culture system reaches 1.0 ⁇ 10 9 CFU/mL, centrifuge at 4000rpm for 10min, collect bacteria, and wash with sterile normal saline first Resuspend three times with sterile normal saline to obtain a bacterial suspension (bacteria concentration: 1.0 ⁇ 10 9 CFU/mL).
  • Embodiment 4 application of Bacillus velei LB-Y-1 to ferment the influence of peanut meal on AFB 1 and acid-soluble protein content
  • Fermentation medium peanut meal (contaminated by AFB 1 ) 50g, sterilized distilled water 50mL, sterilized at 105°C for 15min (AFB 1 content is 103.47 ⁇ g/kg).
  • strain LB-Y-1 on LB solid medium pick a single colony in the logarithmic growth phase into 10mL LB liquid medium, and cultivate it at 37°C and 160rpm/min for 24h as a seed solution.
  • the seed solution was inoculated in LB liquid medium at 1%, and cultured at 37°C and 160rpm/min for 14h, and the logarithmic phase fermentation broth (cell concentration of 1.0 ⁇ 10 9 CFU/mL) was taken for inoculation and fermentation.
  • step 1 Take the fermentation liquid prepared in 2 of step 1, inoculate it into the fermentation medium prepared in 1 of step 1 according to the inoculum amount of 10%, ferment at 37°C for 60 hours at a constant temperature, and ferment at 0h, 12h, 24h, 36h, Samples were taken at 48h and 60h, dried and pulverized at 50°C, and the contents of AFB 1 and acid-soluble protein were determined.
  • the present invention provides a strain of Bacillus velezensis LB-Y-1, which not only can effectively degrade AFB 1 , but also has the ability to produce protease, cellulase and amylase with enzyme activities of 345.47 U/mL, 429.72U/mL, 34.75U/mL.
  • the in vivo test of broiler chickens showed that the strain has potential probiotic properties such as high safety, strong stress resistance, easy intestinal colonization, improvement of intestinal flora structure and growth performance.
  • the bacteria can degrade AFB 1 in mildewed peanut meal by fermentation, increase the content of acid-soluble protein (small peptide + amino acid), and realize biological detoxification and quality improvement of peanut meal.
  • Bacillus velezensis (Bacillus velezensis) LB-Y-1 provided by the invention can be applied to the following aspects: (1) because this bacterium has stronger AFB 1 degradation ability, it can be used for processing AFB 1 polluted raw materials (such as peanut meal); (2) Since the bacterium can degrade macromolecular protein, cellulose and starch, it can be used to prepare fermentation raw materials (such as peanut meal) to improve the quality of raw materials; (3) Since the bacterium can produce compound enzymes, it has high safety , Improve the structure of intestinal flora, and can be directly fed to animals (such as broiler chickens) to improve growth performance.
  • animals such as broiler chickens

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Abstract

一株高产复合酶同时高效降解霉菌毒素的贝莱斯芽孢杆菌及其应用。所述贝莱斯芽孢杆菌为贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1，该菌株不仅可有效降解AFB 1，还具备高产蛋白酶、纤维素酶和淀粉酶的能力。肉鸡在体试验表明该菌株具有安全性高、抗逆性强，易于肠内定植，改善肠道菌群结构和提高生长性能等潜在益生特性。此外，该菌可通过发酵处理降解霉变花生粕中的AFB 1、提高酸溶蛋白含量，实现对花生粕的生物脱毒及品质改善。对于消除和抑制饲料及原料中霉菌毒素污染，降低原料粗纤维含量，提高小肽含量等方面有综合效果。

Description

一株高产复合酶同时高效降解霉菌毒素的贝莱斯芽孢杆菌及其应用 技术领域
本发明涉及农业生物技术领域，具体涉及一株贝莱斯芽孢杆菌(Bacillus velezensis)及其应用，特别涉及一株高产复合酶同时高效降解霉菌毒素的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1及其应用。
背景技术
霉菌毒素是真菌产生的次级代谢物，是食品和饲料中普遍存在且不可避免的污染物，人类和动物食用霉菌毒素会导致疾病和死亡。曲霉属、青霉菌属和镰刀菌属均能够产生多种霉菌毒素。例如，黄曲霉、寄生黄曲霉、诺氏黄曲霉和假黄曲霉是产生一系列剧毒物质黄曲霉毒素的罪魁祸首。自1960年黄曲霉毒素B 1(Aflatoxin B 1，AFB 1)发现以来，已被验证具有高度的致癌、诱变、致畸和遗传毒性，广泛存在于如花生、玉米、大米和棉籽等农产品中。鉴于AFB 1对人类和动物健康的不良影响，寻找安全、实用、廉价和有效的去污染策略是非常必要的。
目前已有的减少AFB 1影响的方法主要包括去除、灭活、转化或降解，方式可分为物理、化学和生物三种。由于不可避免的局限性，这些方法大多低效或成本过高，而生物技术由于其安全性、经济性和稳定性等优良特点，为农业产品和动物饲料中AFB 1的去除和降解提供了一种有吸引力的选择。一些菌株，如寡养单胞菌(Stenotrophomonas sp.)、分枝杆菌(Mycobacterium fluoranthenivorans sp.)等已被验证有很好的AFB 1降解效果，此外漆酶、辣根过氧化物酶、锰过氧化物酶，也有一定降解能力。综上所述，生物手段去除AFB 1似乎是解决局限性的最佳手段。当前已报道的多为单菌株或复合菌对AFB 1的单一降解，如专利CN110804570A所涉及的贝莱斯芽孢杆菌ANSB01E，只公开具有降解霉菌毒素的能力，鲜有发现某一菌株在降解AFB 1的同时兼具其他功能，而饲料原料的品质改善往往是多方面的，如降低粗纤维水平、降解大分子蛋白或抗原蛋白等。
贝莱斯芽孢杆菌(Bacillus velezensis)1999年被首次分离并于2005年正式命名，已经被验证可以防治多种类型的植物病原体感染、降低病情指数、抑制多种致病性真菌和细菌，是一种优良的生防菌株。但目前关于 该菌株在降解霉菌毒素方面的研究鲜有报道。
发明公开
本发明的一个目的是提供一株贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1。
本发明提供的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1的保藏编号为CGMCC No.21344。该菌株已于2020年12月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC；地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；邮编：100101)。该贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1是发明人经过复杂的驯化、比对、筛选等流程，从健康动物消化道食糜中分离得到的，具有如下性能：降解AFB 1，高产淀粉酶、蛋白酶和纤维素酶，改善动物肠道菌群结构，提高动物生长性能，改善原料或饲料品质。
本发明的另一个目的是提供一种菌剂。
本发明提供的菌剂的活性成分为上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或其菌悬液或其培养物或其培养液或其发酵液。
上述菌剂中，所述菌悬液或所述培养物的浓度可为1.0×10 7-1.0×10 9CFU/mL，优选为1.0×10 8CFU/mL。
本发明还有一个目的是提供上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或菌剂的新用途。
本发明提供了上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或菌剂在如下1)-9)中任一种中的应用：
1)降解或脱除AFB 1；
2)产复合酶；
3)降解大分子物质或增加小肽含量；
4)改善动物肠道菌群结构；
5)提高动物生长性能；
6)消除或抑制原料或饲料中AFB 1污染；
7)提高原料或饲料中酸溶蛋白和/或小肽含量；
8)原料或饲料脱毒；
9)改善原料或饲料品质。
本发明的最后一个目的是提供如下X1)-X6)任一所述的方法：
X1)一种降解或脱除AFB 1的方法，包括如下步骤：用上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或菌剂发酵处理原料或饲料，实现AFB 1的降解或脱除；
X2)一种消除或抑制原料或饲料中AFB 1污染的方法，包括如下步骤：用上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或菌剂发酵处理原料或饲料，实现消除或抑制原料或饲料中AFB 1污染；
X3)一种原料或饲料脱毒的方法，包括如下步骤：用上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或菌剂发酵处理原料或饲料，实现原料或饲料脱毒；
X4)一种改善原料或饲料品质的方法，包括如下步骤：用上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或菌剂发酵处理原料或饲料，实现原料或饲料品质的改善；
X5)一种改善动物肠道菌群结构的方法，包括如下步骤：用上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或菌剂饲喂动物，实现动物肠道菌群结构的改善；
X6)一种提高动物生长性能的方法，包括如下步骤：用上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或菌剂饲喂动物，实现动物生长性能的提高。
上述方法中，所述发酵处理的方法包括如下步骤：将上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或菌剂添加至原料或饲料中。
所述饲喂的方法包括如下步骤：在动物基础日粮中喷洒上述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或菌剂。
上述应用或方法中，所述复合酶为蛋白酶、纤维素酶和/或淀粉酶。
上述应用或方法中，所述提高动物生长性能体现在如下a1)-a2)中的任一种：
a1)提高动物体重(如末重、平均日增重)；
a2)降低料重比。
上述应用或方法中，所述改善动物肠道菌群结构体现在如下b1)-b3)中任一种：
b1)增加动物肠道的菌群丰度；
b2)增加动物肠道中有益菌(如Lactobacillus、Alistipes、Lachnospiraceae菌群)的比例；
b3)降低动物肠道中有害菌(如不利于营养物质吸收和降低免疫性能的Escherichia-Shigella菌群)的比例。
上述应用或方法中，所述改善原料或饲料品质体现在如下c1)-c2)中任一种：
c1)降解原料或饲料中的大分子物质；
c2)提高原料或饲料中酸溶蛋白和/或小肽含量。
所述大分子物质可为大分子蛋白(如酪蛋白)、纤维素(如羧甲基纤维素钠)或淀粉(如可溶性淀粉)。
上述应用或方法中，所述菌悬液的制备方法包括如下步骤：将分离纯化后的LB-Y-1菌株接种于LB固体培养基中，固体培养(培养时间可为24h)后，挑取单菌落至LB液体培养基中，在37℃、160r/min条件下进行培养(培养时间可为18-24h)，使培养体系中的菌体浓度达到1.0×10 7～1.0×10 9CFU/mL，离心(离心条件可为4000rpm离心10min)，收集菌体，用无菌生理盐水将所述菌体依次进行洗涤(洗涤次数可为三次)和重悬，得到所述菌悬液(菌悬液中菌体浓度为1.0×10 7～1.0×10 9CFU/mL)。
所述培养物的制备方法包括如下步骤：将分离纯化后的LB-Y-1菌株接种于LB固体培养基中，挑取对数生长期的单菌落至LB液体培养基中，在37℃、160rpm/min条件下进行培养(培养时间可为24h)，得到种子液，将所述种子液接种(接种量可为1％)至LB液体培养基中，在37℃、160rpm/min条件下进行培养(培养时间可为14h)，得到发酵液，即为所述培养物(培养物中菌体浓度为1.0×10 7～1.0×10 9CFU/mL)。
上述应用或方法中，所述动物包括但不限于鸡。在本发明的一个实施例中，所述动物为肉鸡(如AA肉鸡)。
上述应用或方法中，所述原料或饲料可为受或未受AFB 1污染的花生、玉米、大米和棉籽等农产品。在本发明的一个实施例中，所述原料为花生粕，具体为受AFB 1污染的花生粕。
附图说明
图1为本发明菌株LB-Y-1初筛降解酪蛋白、羧甲基纤维素钠和可溶性淀粉的效果。A为产蛋白酶能力；B为产纤维素酶能力；C为产淀粉酶能力。
图2为本发明菌株LB-Y-1的菌落特征及菌体形态(革兰氏染色×100)。A为菌落特征；B为菌体形态。
图3为本发明菌株LB-Y-1采用API 20NE的分析结果。
图4为本发明菌株LB-Y-1构建的系统发育树。
图5为本发明菌株LB-Y-1的生长曲线。
图6为本发明菌株LB-Y-1对肉鸡肠道菌群结构的影响。
图7为发酵时间对菌株LB-Y-1降解自然霉变花生粕中AFB 1含量的影响。
图8为发酵时间对菌株LB-Y-1提高自然霉变花生粕中酸溶蛋白含量的影响。
保藏说明
菌株拉丁名：Bacillus velezensis
菌株编号：LB-Y-1
建议的分类命名：贝莱斯芽孢杆菌
保藏机构：中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称：CGMCC
地址：北京市朝阳区北辰西路1号院3号
保藏日期：2020年12月10日
保藏中心登记入册编号：CGMCC No.21344
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明所涉及到的培养基(溶剂均为蒸馏水，使用前均121℃灭菌20min)如下：
营养肉汤(NB)培养基(g/L)：蛋白胨10，牛肉膏3，氯化钠5，pH调整为7.2～7.4，固体培养基加入2％琼脂粉。
Hormisch培养基(g/L)：磷酸氢二钾0.25，七水硫酸镁0.25，硝酸钾0.5，硫酸铵0.5，氯化钙0.005，氯化铁0.003，pH调整为7.0，灭菌后按需加入1～3g香豆素，固体培养基加入2％琼脂粉。
酪蛋白培养基(g/L)：酪蛋白10，牛肉浸粉3，琼脂粉15，氯化钠5，磷酸二氢钾2，pH调整为7.0，固体培养基加入2％琼脂粉。
纤维素酶选择培养基(g/L)：酵母提取物5，氯化钠5，胰蛋白胨10，羧甲基纤维素钠10，磷酸二氢钾1，pH调整为7.2～7.4，固体培养基加入2％琼脂粉。
淀粉酶选择培养基(g/L)：可溶性淀粉10，葡萄糖5，胰蛋白胨10，牛肉膏5，氯化钠5，pH调整为7.2～7.4，固体培养基加入2％琼脂粉。
LB培养基(g/L)：胰蛋白胨10，酵母提取物5，氯化钠10，pH调整为7.2～7.4，固体培养基加入2％琼脂粉。
发酵培养基A(g/L)：蛋白胨10，牛肉膏3，氯化钠5，磷酸氢二钾1，葡萄糖1，pH调整为6.5，固体培养基加入2％琼脂粉。
发酵培养基B(g/L)：葡萄糖5，胰蛋白胨10，酵母提取物5，磷酸二氢钾1，氯化钠5，硫酸镁0.5，硫酸锰0.005，羧甲基纤维素钠5，pH调整为5.5，固体培养基加入2％琼脂粉。
发酵培养基C(g/L)：可溶性淀粉5，葡萄糖5，胰蛋白胨10，酵母提取物5，磷酸二氢钾1，氯化钠5，硫酸镁0.5，硫酸锰0.005，pH调整为5.5，固体培养基加入2％琼脂粉。
本发明所涉及到的检测方法如下：AFB 1浓度的检测采用HPLC法；蛋白酶活力的检测采用Folin-酚法；纤维素酶活的检测采用DNS法；淀粉酶活力的检测采用DNS法；酸溶蛋白含量参照(GB/T 22492-2008)的方法测定。
下面实施例将对本发明进一步阐述：
实施例1、贝莱斯芽孢杆菌LB-Y-1的筛选及鉴定
一、菌株LB-Y-1的筛选
1、菌株的初筛
收集健康且生长状况良好的若干奶牛瘤胃、鸡盲肠、猪回肠、家兔盲肠食糜样品35例，用于筛选目的菌株。具体方法为：各取上述样品10g，溶于90mL无菌的PBS溶液中，在160rpm条件下恒温震荡20min，吸取500μL转入5.5mL NB液体培养基中，37℃恒温160rpm培养24小时，随后再吸取100μL菌液，接种于5.5mL Hormisch液体培养基中，由1～3mg/mL逐步提高香豆素的含量，在37℃和160rpm条件下培养48h，进行菌株的富集。在5次富集之后，将培养液稀释涂布于NB固体培养基(稀释倍数从10 -1～10 -6)，37℃静置培养24h，通过判断菌落的形态、颜色、边缘光滑度、湿度等方面作为鉴别标准，挑选单菌落在NB固体培养基进行三代纯化，纯化菌株保存于50％甘油中，-80℃保藏。
2、菌株的复筛
取初筛菌株接种于发酵培养基A，37℃培养48h，备以AFB 1标准品，990μL发酵培养液，与10μL AFB 1标准品(浓度为10μg/mL)，在37℃条件下培养48h，反应结束后加入1mL二氯甲烷，重复3次，合并有机相，随后再加入1mL甲醇复溶，过0.22μm滤膜后，HPLC上机，检测条件：C18色谱柱：SB-C18，4.6mm×250mm，5μm；流动相：V(甲醇):V(水)＝60:40，流速0.8mL/min，柱温32℃，运行时间为11min；检测波长365nm，光电二极管阵列(PDA)。降解率计算公式：AFB 1的降解率/％＝(A－B)/A×100％，其中A、B分别表示添加AFB 1的对照峰面积和处理后的峰面积。
挑选AFB 1降解效果较佳的前20株菌进一步筛选；取1μL发酵种子液，接种于酪蛋白固体培养基，培养24h后，测量并计算酪蛋白降解圈面积(S 1)与菌落面积(s 1)的比值，选择降解效率最佳的若干菌株进行下一步纤维素降解能力判定；取上述挑选出的发酵种子液接种于纤维素酶选择固体培养基，培养24h后，通过刚果红染色测量并计算纤维素降解圈面积(S 2)与菌落面积(s 2)的比值，挑选降解效率最佳的若干菌株，进行下一步淀粉降解能力的判定；取上述菌株的发酵种子液接种于淀粉酶选择固体培养基，培养24h后，通过碘液对培养基染色测量并计算淀粉降解圈面积(S 3)与菌落面积(s 3)的比值，判定最佳的降解菌株，降解结果如图1所示。
试验结果表明：菌株LB-Y-1具有降解AFB 1的能力，降解效率为81.56％，同时兼有产蛋白酶、纤维素酶和淀粉酶的能力，降解圈与菌落大小的面积比(S/s)分别为：3.67、4.06、2.68。
二、菌株LB-Y-1的鉴定
1、菌株LB-Y-1的形态观察
将菌株LB-Y-1划线接种于LB固体培养基中，观察菌落生长形态(图2A)，其特征为：乳白色菌落，不透明，形态变化由最初圆形(饱满)逐渐转为不规则(褶皱)，边缘不整齐向四周以云雾状扩散开，菌落中间凸起形成火山口样，挑开后有粘性液体；取生长对数期菌体涂板，固定并用用革兰氏染液染色，在油镜下观察形态拍照(图2B)，菌体为短杆状，可形成芽孢，革兰氏染色阳性。
2、菌株LB-Y-1的生化鉴定
首先采用API 20NE试剂条分析菌株LB-Y-1，具体结果如图3所示，进一步采用BIOLOG分析菌株LB-Y-1的碳源利用情况，阳性反应碳源如表1所示。
表1、BIOLOG GENⅢ分析贝莱斯芽孢杆菌LB-Y-1碳源利用
Figure PCTCN2021142776-appb-000001
Figure PCTCN2021142776-appb-000002
3、菌株LB-Y-1的分子生物学鉴定
采用细菌基因组DNA提取试剂盒提取菌株LB-Y-1 DNA，进行16S rDNA及持家基因gyrB扩增与测序。测序结果表明：菌株LB-Y-1的16S rDNA PCR产物共得到大小为1476bp的基因片段，其核苷酸序列如序列1所示。该菌株被鉴定为Bacillus(杆菌属)，为Bacillus velezensis(贝莱斯芽孢杆菌)。
将菌株LB-Y-1的16S rDNA基因序列在NCBI进行同源性比对，分析亲缘关系，构建系统发育树(图4)。
4、菌株LB-Y-1的生长曲线
将菌株LB-Y-1接种于LB固体培养基，挑取对数生长期的单菌落至 10mL LB液体培养基中，于37℃、160rpm/min的条件下培养24h作为种子液，精确吸取1％接种量的上述种子液，接种于100mLLB液体培养基中，37℃，160rpm/min培养24h，期间每间隔1h取样一次，在紫外可见分光光度计600nm处测其光密度(OD)值，并绘制OD 600与时间的关系曲线图(图5)。
综合菌落及菌体形态特征、BIOLOG碳源利用情况、API 20NE的分析结果、分子生物学鉴定等手段，确定菌株LB-Y-1的分类单元为：Bacteria；Firmicutes；Bacilli；Bacillales；Bacillaceae；Bacillus，属于贝莱斯芽孢杆菌(Bacillus velezensis)。
三、菌株LB-Y-1的保藏
贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1已于2020年12月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC；地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；邮编：100101)，保藏编号为CGMCC No.21344。
实施例2、贝莱斯芽孢杆菌LB-Y-1产蛋白酶、纤维素酶和淀粉酶的能力
一、贝莱斯芽孢杆菌LB-Y-1产蛋白酶的能力
1、绘制L-酪氨酸标准曲线：预先配置好0.1mol/L的HCL溶液作为溶剂，分别以10μg/mL为梯度差，配成浓度为0～70μg/mL的酪氨酸标准溶液。各取1mL的上述标准液与5mL 0.4mol/L的Na 2CO 3溶液和1mL的Folon-Phenol试剂混合，40℃水浴20min后在紫外分光光度计680nm的波长下，测定吸光度，绘制L-酪氨酸OD 680与浓度的关系曲线。
2、按照实施例1中的方法制备种子液，将种子液接种于发酵培养基A，在37℃、160rpm/min的条件下培养36h。
3、完成步骤2之后，4℃、8000rpm条件离心15min，分离上清液即为粗酶液。
4、酶活测定：将1mL步骤3取得的粗酶液1mL，在40℃条件下水浴20min，依次加入1mL底物(1g酪蛋白溶解于pH7.5的缓冲溶液并定容至100mL)和2mL 0.4mol/L三氯乙酸，混合均匀，静置10min，慢速过滤收集 滤液，再将上述滤液1mL转移至新试管，与5mL 0.4mol/L的Na 2CO 3溶液和1mL的Folon-Phenol试剂混合，40℃水浴孵育20min后在紫外分光光度计680nm的波长下，测定吸光度，每次测定均设三个平行，并采用蒸馏水作为空白对照。
计算公式：吸光度＝吸光度(试验组)-吸光度(对照组)。
对照标准曲线，得到酪氨酸产量，计算粗酶液酶活，以每毫升酶液催化酪蛋白生成1μg酪氨酸所需酶量为1个酶活力为计量单位(U/mL)。
计算结果表明：贝莱斯芽孢杆菌LB-Y-1产蛋白酶粗酶液酶活为345.47U/mL。
二、贝莱斯芽孢杆菌LB-Y-1产纤维素酶的能力
1、绘制葡萄糖标准曲线
以蒸馏水为溶剂，分别以0.1mg/mL为梯度差，配成浓度为0.1～0.7mg/mL的葡萄糖标准溶液。各取3mL的上述标准液与1mL DNS试剂加到试管中充分混合，沸水浴5min后，立即转入冰浴终止反应，加16mL蒸馏水充分混合，在紫外分光光度计540nm的波长下测定吸光度，绘制吸光度与葡萄糖浓度的关系曲线。
2、按照实施例1中的方法制备种子液，将种子液接种于发酵培养基B，在37℃、160rpm/min的条件下培养36h。
3、完成步骤2之后，在4℃、8000rpm条件离心15min，分离上清液即为粗酶液。
4、酶活测定：将步骤3得到的粗酶液用蒸馏水适度稀释，得到待测溶液。对照组依次在试管中加入2mL底物(0.5g羧甲基纤维素钠溶于pH7.5的磷酸缓冲液并定容至100mL)、1mL DNS和1mL待测溶液混合均匀，沸水浴5min后冰浴终止反应，加16mL蒸馏水充分混合，在紫外分光光度计540nm的波长下测定吸光度；试验组依次加入2mL底物和1mL待测溶液混合均匀，置于50℃水浴1h，此后加入1mL DNS混合均匀，沸水浴5min后冰浴终止反应，加16mL蒸馏水，在紫外分光光度计540nm的波长下测定吸光度，对照组及试验组每次测定均设三个平行。
计算公式：吸光度＝吸光度(试验组)-吸光度(对照组)。
对照标准曲线，根据葡萄糖的生成量及稀释倍数，计算粗酶液酶活， 以每毫升酶液催化羧甲基纤维素钠生成1μg葡萄糖所需的酶量为计量单位(U/mL)。
计算结果表明：贝莱斯芽孢杆菌LB-Y-1产纤维素酶粗酶液酶活为429.72U/mL。
三、贝莱斯芽孢杆菌LB-Y-1产淀粉酶的能力
1、绘制麦芽糖标准曲线
以蒸馏水为溶剂，分别以0.1mg/mL为梯度差，配成浓度为0.1～0.7mg/mL的麦芽糖标准溶液。各取2mL的上述标准液与2mL DNS试剂加到试管中充分混合，沸水浴5min后立即转入冰浴终止反应，加16mL蒸馏水充分混合，在紫外分光光度计540nm的波长下测定吸光度，绘制吸光度与麦芽糖浓度的关系曲线。
2、按照实施例1中的方法制备种子液，将种子液接种于发酵培养基C，在37℃、160rpm/min的条件下培养36h。
3、完成步骤2之后，4℃、8000rpm条件离心15min，分离上清液即为粗酶液。
4、酶活测定：将步骤3得到的粗酶液用蒸馏水适度稀释，得到待测溶液。对照组依次在试管中加入1mL底物(0.5g可溶性淀粉溶于pH6.5的磷酸缓冲液并定容至100mL)、2mL DNS和1mL待测溶液混合均匀，沸水浴5min后冰浴终止反应，加16mL蒸馏水充分混合，在紫外分光光度计540nm的波长下测定吸光度；试验组依次加入1mL底物、1mL待测溶液混合均匀，置于50℃水浴1h，此后加入2mL DNS混合均匀，沸水浴5min后冰浴终止反应，加16mL蒸馏水，在紫外分光光度计540nm的波长下测定吸光度，对照组及试验组每次测定均设三个平行。
计算公式：吸光度＝吸光度(试验组)-吸光度(对照组)。
对照标准曲线，根据麦芽糖的生成量及稀释倍数，计算粗酶液酶活，以每30min每毫升酶液催化淀粉生成1mg麦芽糖所需的酶量为计量单位(U/mL)。
计算结果表明：贝莱斯芽孢杆菌LB-Y-1产淀粉酶粗酶液酶活为34.75U/mL。
实施例3、贝莱斯芽孢杆菌LB-Y-1对肉鸡生长性能及肠道微生物多样性的影响
一、不同梯度贝莱斯芽孢杆菌LB-Y-1对肉鸡饲养试验
选择300只1日龄的健康艾拔益加肉鸡(AA肉鸡)，随机分为5个处理组，每个处理组6个重复，每个重复10只鸡，各组分别为对照组CON、贝莱斯芽孢杆菌组BV1、BV2、BV3和抗生素AGPs组。CON组饲喂基础日粮；BV1、2、3组分别在基础日粮中喷洒贝莱斯芽孢杆菌悬液，使基础日粮中的菌浓度分别为1.0×10 7CFU/kg、1.0×10 8CFU/kg、1.0×10 9CFU/kg；AGPs组在基础日粮中添加组合抗生素(金霉素100mg/kg、吉他霉素20mg/kg)。基础日粮为玉米-豆粕型日粮，配方设计参考《鸡饲养标准》(NY/T 33-2004)，试验周期为42d，分析对肉鸡生长性能和肠道菌群结构的影响。
贝莱斯芽孢杆菌悬液的制备方法如下：将分离纯化后的贝莱斯芽孢杆菌LB-Y-1接种于LB固体培养基中，固体培养24h后，挑取单菌落接种至10mL LB液体培养基中，在37℃、160r/min条件下振荡培养18-24h，使培养体系中的菌体浓度达到1.0×10 9CFU/mL，4000rpm离心10min，收集菌体，先用无菌生理盐水洗涤3次再用无菌生理盐水重悬得到菌悬液(菌体浓度为1.0×10 9CFU/mL)。
二、贝莱斯芽孢杆菌LB-Y-1对肉鸡生长性能的影响
分别在试验前期(1～21天)和试验后期(22～42天)统计各组肉鸡的如下指标：末重、平均日增重、平均日采食量和料重比。
结果表明：在肉鸡饲养全期，添加1.0×10 8CFU/kg的贝莱斯芽孢杆菌可显著改善肉鸡生长性能(表2)。具体表现为试验前期(1～21天)，BV2组末重及平均日增重均显著高于对照组(P＜0.05)；在试验后期(22～42天)，BV2组末重及饲料转化率(料重比)均显著优于对照组(P＜0.05)，与应用抗生素无显著差异(P＞0.05)。
表2、饲粮添加贝莱斯芽孢杆菌LB-Y-1对肉鸡生长性能的影响
Figure PCTCN2021142776-appb-000003
Figure PCTCN2021142776-appb-000004
注：同行数据肩标不同小写字母表示差异显著(P＜0.05)，相同或无字母表示差异不显著(P＞0.05)。
三、贝莱斯芽孢杆菌LB-Y-1对肠道菌群结构的影响
在肉鸡42日龄时，取回肠食糜，通过16S扩增子测序及生物信息学方法，分析肠道菌群结构。
结果表明：添加1.0×10 8CFU/kg的贝莱斯芽孢杆菌组的肉鸡肠道菌群丰度显著增加，其中有益菌群Lactobacillus、Alistipes、Lachnospiraceae比例增加、不利于营养物质吸收及降低肉鸡免疫性能的菌群Escherichia-Shigella比例显著降低，同时Bacteroides菌群丰度的减少也有利于肉鸡的增重(图6)。
实施例4、应用贝莱斯芽孢杆菌LB-Y-1发酵花生粕对AFB 1及酸溶蛋白含量的影响
一、制备发酵培养基及发酵液
1、发酵培养基：花生粕(受AFB 1污染)50g，灭菌蒸馏水50mL，105℃灭菌15min(AFB 1含量为103.47μg/kg)。
2、将菌株LB-Y-1接种于LB固体培养基，挑取对数生长期的单菌落至10mL LB液体培养基中，于37℃、160rpm/min的条件下培养24h作为种子液，将种子液按照1％接种于LB液体培养基，在37℃、160rpm/min的条件下培养14h，取对数期发酵液(菌体浓度为1.0×10 9CFU/mL)备以接种发酵。
二、贝莱斯芽孢杆菌LB-Y-1发酵花生粕
取步骤一的2中制得的发酵液，按照10％的接种量接种至步骤一的1中 制得的发酵培养基中，37℃恒温发酵60h，分别在发酵0h、12h、24h、36h、48h、60h取样并于50℃烘干粉碎，测定其中AFB 1及酸溶蛋白含量。
结果表明：随着发酵时间的不断增加，AFB 1含量逐渐减少(图7)，酸溶蛋白含量逐渐增加(图8)，在发酵48h即达到较佳水平，其中AFB 1的降解率为60.73％，酸溶蛋白含量由2.89％提高至21.75％。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明提供了一株贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1，该菌株不仅可有效降解AFB 1，还具备高产蛋白酶、纤维素酶和淀粉酶的能力，其酶活分别为345.47U/mL、429.72U/mL、34.75U/mL。肉鸡在体试验表明该菌株具有安全性高、抗逆性强，易于肠内定植，改善肠道菌群结构和提高生长性能等潜在益生特性。此外，该菌可通过发酵处理降解霉变花生粕中的AFB 1，提高酸溶蛋白(小肽+氨基酸)含量，实现对花生粕的生物脱毒及品质改善。对于消除和抑制饲料及原料中霉菌毒素污染，降低原料粗纤维含量，提高小肽含量等方面有综合效果。本发明提供的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1可应用于以下方面：(1)由于该菌具有较强的AFB 1降解能力，可以用于处理AFB 1污染原料(如花生粕)；(2)由于该菌可降解大分子蛋白、纤维素及淀粉，可采用其制备发酵原料(如花生粕)，提高原料品质；(3)由于该菌可产复合酶、安全性高、改善肠道菌群结构，可直接饲喂动物(如肉鸡)改善生长性能。

Claims (23)

  1. 贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1，其保藏编号为CGMCC No.21344。
  2. 根据权利要求1所述的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1，其特征在于：所述贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1具有如下性能：降解AFB 1、产复合酶、改善动物肠道菌群结构、提高动物生长性能、改善原料或饲料品质。
  3. 一种菌剂，其活性成分为权利要求1或2所述的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或其菌悬液或其培养物或其培养液或其发酵液。
  4. 根据权利要求3所述的菌剂，其特征在于：所述菌悬液的浓度为1.0×10 7～1.0×10 9CFU/mL。
  5. 根据权利要求3所述的菌剂，其特征在于：所述培养物的浓度为1.0×10 7～1.0×10 9CFU/mL。
  6. 权利要求1或2所述的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或权利要求3-5任一所述的菌剂在如下1)-9)中任一种中的应用：
    1)降解或脱除AFB 1；
    2)产复合酶；
    3)降解大分子物质或增加小肽含量；
    4)改善动物肠道菌群结构；
    5)提高动物生长性能；
    6)消除或抑制原料或饲料中AFB 1污染；
    7)提高原料或饲料中酸溶蛋白和/或小肽含量；
    8)原料或饲料脱毒；
    9)改善原料或饲料品质。
  7. 根据权利要求6所述的应用，其特征在于：所述复合酶为蛋白酶、纤维素酶和/或淀粉酶。
  8. 根据权利要求6所述的应用，其特征在于：所述提高动物生长性能体现在如下a1)-a2)中的任一种：
    a1)增加动物体重；
    a2)降低料重比。
  9. 根据权利要求6所述的应用，其特征在于：所述改善动物肠道菌群结构体现在如下b1)-b3)中任一种：
    b1)增加动物肠道的菌群丰度；
    b2)增加动物肠道中有益菌的比例；
    b3)降低动物肠道中有害菌的比例。
  10. 根据权利要求6所述的应用，其特征在于：所述改善原料或饲料品质体现在如下c1)-c2)中任一种：
    c1)降解原料或饲料中的大分子物质；
    c2)提高原料或饲料中酸溶蛋白和/或小肽含量。
  11. 根据权利要求6-10任一所述的应用，其特征在于：所述原料或饲料为花生、玉米、大米或棉籽。
  12. 根据权利要求6-10任一所述的应用，其特征在于：所述动物为鸡。
  13. 一种降解或脱除AFB 1的方法，包括如下步骤：用权利要求1或2所述的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或权利要求3-5任一所述的菌剂发酵处理原料或饲料，实现AFB 1的降解或脱除。
  14. 一种消除或抑制原料或饲料中AFB 1污染的方法，包括如下步骤：用权利要求1或2所述的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或权利要求3-5任一所述的菌剂发酵处理原料或饲料，实现消除或抑制原料或饲料中AFB 1污染。
  15. 一种原料或饲料脱毒的方法，包括如下步骤：用权利要求1或2所述的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或权利要求3-5任一所述的菌剂发酵处理原料或饲料，实现原料或饲料脱毒。
  16. 一种改善原料或饲料品质的方法，包括如下步骤：用权利要求1或2所述的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或权利要求3-5任一所述的菌剂发酵处理原料或饲料，实现原料或饲料品质的改善。
  17. 根据权利要求16所述的方法，其特征在于：所述改善原料或饲料品质体现在如下c1)-c2)中任一种：
    c1)降解原料或饲料中的大分子物质；
    c2)提高原料或饲料中酸溶蛋白和/或小肽含量。
  18. 根据权利要求13-17任一所述的方法，其特征在于：所述原料或饲料为花生、玉米、大米或棉籽。
  19. 一种改善动物肠道菌群结构的方法，包括如下步骤：用权利要求1或2所述的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或权利要求3-5任一所述的菌剂饲喂动物，实现动物肠道菌群结构的改善。
  20. 根据权利要求19所述的方法，其特征在于：所述改善动物肠道菌群结构体现在如下b1)-b3)中任一种：
    b1)增加动物肠道的菌群丰度；
    b2)增加动物肠道中有益菌的比例；
    b3)降低动物肠道中有害菌的比例。
  21. 一种提高动物生长性能的方法，包括如下步骤：用权利要求1或2所述的贝莱斯芽孢杆菌(Bacillus velezensis)LB-Y-1或权利要求3-5任一所述的菌剂饲喂动物，实现动物生长性能的提高。
  22. 根据权利要求21所述的方法，其特征在于：所述提高动物生长性能体现在如下a1)-a2)中的任一种：
    a1)增加动物体重；
    a2)降低料重比。
  23. 根据权利要求19-22任一所述的方法，其特征在于：所述动物为鸡。
PCT/CN2021/142776 2021-11-22 2021-12-30 一株高产复合酶同时高效降解霉菌毒素的贝莱斯芽孢杆菌及其应用 WO2023087499A1 (zh)

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