WO2019205422A1 - 一种凝结芽孢杆菌xp及其在饲料生产中的应用 - Google Patents

一种凝结芽孢杆菌xp及其在饲料生产中的应用 Download PDF

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WO2019205422A1
WO2019205422A1 PCT/CN2018/105156 CN2018105156W WO2019205422A1 WO 2019205422 A1 WO2019205422 A1 WO 2019205422A1 CN 2018105156 W CN2018105156 W CN 2018105156W WO 2019205422 A1 WO2019205422 A1 WO 2019205422A1
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bacillus coagulans
screening
medium
culture
gys
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PCT/CN2018/105156
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French (fr)
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陈胜杰
金明杰
高翔
巫晓冬
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广东怡和科洁科技有限公司
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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
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus

Definitions

  • the invention belongs to the field of microbiology, in particular to a novel screening and extraction of Bacillus coagulans XP and its application to feed to improve the absorption rate of feed.
  • Bacillus coagulans is a probiotic. Bacillus coagulans is a facultative anaerobic bacterium that grows in both aerobic and anaerobic environments. It can adapt to the hypoxic intestinal environment, has high tolerance to acids and bile, and is capable of lactic acid fermentation. L-lactic acid can lower the intestinal pH, inhibit harmful bacteria, and promote the growth and reproduction of beneficial bacteria such as bifidobacteria.
  • the currently disclosed strains of Bacillus coagulans used in feed also have the effects of improving feed utilization, enhancing animal immunity, and gaining weight.
  • problems such as low activity of the strain, large addition amount, high cost, general antibacterial effect, and low feed utilization rate.
  • the invention provides a Bacillus coagulans XP which is screened and extracted from biomass processing waste, and is used as a feed additive, which has the effects of obviously improving feed utilization rate, reducing diarrhea rate and mortality of animals.
  • the present invention firstly provides a Bacillus coagulans XP which has been deposited with the China Center for Type Culture Collection on December 25, 2017, and has a deposit registration number of CCTCC NO: M2017836.
  • the Bacillus coagulans XP belongs to the genus Bacillus in the classification of bacteria, and is a Gram-positive bacterium. The colonies are single or in pairs, rarely arranged in a straight chain; the cells are rod-shaped and end-shaped. Spores, no flagellum, forming endospores; its individual cell size is 4-6 ⁇ m long and 0.4-0.9 ⁇ m wide.
  • Bacillus coagulans XP Bacillus coagulans XP (Bacillus coagulans XP) of 16S rDNA sequence as follows: gctggctccgtaaaggttacctcaccgacttcgggtgttacaaactctcgtggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcggcatgctgatccgcgattactagcgattccggcttcatgcaggcgggttgcagcctgcaatccgaactgggaatggttttctgggattggcttaacctcgcggtctcgcagcccctttgtaccatccattgtagcacgtgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccccaccttccctc
  • the present invention also provides the use of the Bacillus coagulans XP, which is suitable for use as an animal feed additive, in an amount per gram of feed. Add 5 to 5 million CFU of live bacteria.
  • the present invention also provides a screening and culture method for Bacillus coagulans XP, comprising the following steps:
  • step (1) the obtained bacterial solution is cultured in step (1), and inoculated into liquid MRS medium to be enriched and cultured;
  • the Bacillus coagulans XP strain obtained by the above method can be further inoculated into a triangular flask containing a new sterile liquid GYS medium, and cultured at 50 ⁇ 5° C. for 36 hours; Add 20% of the total mass of the glycerin to the bottle, shake it, and then add it to several 5ml plastic tubes and store in a -20 °C refrigerator.
  • the enrichment culture in the MRS medium was carried out by shaking and culturing in a shaker at a temperature of 45 ° C and a rotation speed of 200 rpm.
  • the biomass-treated waste is a liquid or suspension after the straw fermentation treatment, especially the waste residue waste liquid after the corn straw saccharification and fermentation treatment, especially the upper substance or suspended substance of the waste.
  • the GYS medium is formulated to contain 10 g of yeast powder, 5 g of peptone, 100 g of glucose, and 50 g of calcium carbonate (CaCO 3 ) per 1000 ml of deionized water.
  • the MRS medium is formulated to contain 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, and 2.0 g of diammonium hydrogen citrate [(NH 4 ) 2 HC 6 H 5 O 7 per 1000 ml of deionized water.
  • the beneficial technical effects of the present invention are as follows: (1) A newly screened strain of Bacillus coagulans XP is provided; (2) the screening and extraction cost of the strain is low, and screening can be performed from biomass treatment waste, screening The culture procedure is simple; (3) the strain has the effects of significantly increasing the growth rate of the pigs, meat and poultry, improving the feed utilization rate, and reducing the diarrhea rate and mortality of the animal.
  • Figure 1 is a 400x electron micrograph of Bacillus coagulans XP;
  • Figure 2 is a phylogenetic tree analysis map of Bacillus coagulans XP.
  • step (1) transfer three times repeatedly, and store the bacteria solution at -80 °C after each round of transfer, and then perform DGGE (denaturing gradient gel electrophoresis) to analyze the colony changes;
  • DGGE denaturing gradient gel electrophoresis
  • step (3) Picking up the third round of screening and enrichment of the bacterial liquid obtained in step (2), streaked on a new sterile GYS medium plate, and cultured at 45 ° C for at least 18 hours to select a larger bacterial circle.
  • a single colony having a transparent calcium lysate on the periphery was inoculated on a new sterile GYS medium plate to obtain a pure strain.
  • the GYS medium is formulated to contain 10 g of yeast powder, 5 g of peptone, 100 g of glucose, and 50 g of calcium carbonate (CaCO 3 ) per 1000 ml of deionized water.
  • the formula of the MRS medium is: 10.0 g of peptone per 1 ml of deionized water, 10.0 g of beef extract, 5.0 g of yeast extract, diammonium hydrogen citrate [(NH 4 ) 2 HC 6 H 5 O 7 ] 2.0 g, glucose (C 6 H 12 O 6 ⁇ H 2 O) 20.0 g, Tween 80 1.0 mL, sodium acetate (CH 3 COONa ⁇ 3H 2 O) 5.0 g, dipotassium hydrogen phosphate (K 2 HPO 4 ⁇ 3H 2 O) 2.0 g, magnesium sulfate (MgSO 4 ⁇ 7H 2 O) 0.58 g, manganese sulfate (MnSO 4 ⁇ H 2 O) 0.25 g, agar 18.0 g.
  • the strain is a rod-like cell of Gram-positive bacteria, and the size of the cells is 0.4-0.9 ⁇ 4-6 ⁇ m. They are single, paired, and rarely arranged in a straight chain to form endospores, Gram-positive bacteria, terminal spores. , no flagella.
  • the Bacillus coagulans XP obtained by screening in Example 1 was inoculated into MRS medium (pH 6.5) at a seed inoculation amount of 4%, and cultured at 45 ° C and 200 rpm.
  • the initial concentration of the cells is 0.1% OD600nm, and the cells enter the logarithmic growth phase for 10 to 12 hours, lasting 4 to 6 hours; the cells enter a stable phase in about 15 to 18 hours, and then enter the high-density fermentation period of the cells; At about 60 hours, the absorbance of the fermentation broth at 660 nm began to decrease, at which point the fermentation was stopped.
  • the concentration of the cells in the fermentation broth was determined by the plate counting method, and the final concentration of the cells was 7.8 billion CFU/g.
  • the bacterial fermentation broth obtained by the above method and the active zeolite are mixed at a mass fraction of 1:1 to 2:1, and the activated zeolite is subjected to milling treatment, and the particle fineness is ⁇ 0.06 mm;
  • the heating load, the standing, the filtration separation process (the stirring speed is 100-150 rpm, the temperature is 15 to 65 ° C when the load is applied, the adsorption time is 3 to 45 minutes), and the solid material to be separated is separated.
  • Vacuum freeze-drying (temperature 0 ° C ⁇ -20 ° C, vacuum pressure 1.3 ⁇ 5.5 Pa), the dried solid material is pulverized to a particle fineness ⁇ 0.1 mm, that is, a probiotic solid powder is prepared.
  • the prepared probiotic solid powder can be used as a high-grade animal feed additive, and has high commercial value, and can replace sensitive additives such as antibiotics and hormones.
  • Table 2 shows the results of the bacterial concentration measured after dilution of the probiotic solid powder prepared by mixing the bacterial fermentation broth and the active zeolite in different proportions under different conditions.
  • the probiotic solid powder prepared in Example 2 is added to the feed in an amount of 100-3.5 million CFU of live bacteria per gram of feed, which can increase the growth rate of meat and poultry, etc. by 3.5 to 12.7%, and improve feed utilization. 2.1 to 8.5%, and can significantly reduce the diarrhea rate and mortality of animals.

Abstract

一种凝结芽孢杆菌XP及其在饲料生产中的应用。该凝结芽孢杆菌XP(Bacillus coagulans XP)，其已于2017年12月25日保藏于中国典型培养物保藏中心，其保藏登记号为CCTCC NO:M2017836。该菌属于芽孢杆菌属，革兰氏阳性菌，其菌落呈单个或成对存在；其细胞呈杆状，端生芽孢，无鞭毛，形成内生孢子；其单个菌体大小是，长为4～6μm，宽为0.4～0.9μm。该菌是从生物质处理废渣中筛选提取的，通过GYS培养基筛选、MRS培养基培养。该菌适合用作动物饲料添加剂，具有明显的提高肉猪及肉禽等的生长速度、提高饲料利用率、降低动物的腹泻率和死亡率的作用。

Description

一种凝结芽孢杆菌XP及其在饲料生产中的应用 技术领域
本发明属于微生物学领域，具体地说，涉及一种新型的凝结芽孢杆菌XP的筛选提取及其添加到饲料中可提高饲料被吸收率的应用。
背景技术
人类活动快速发展导致环境污染增加，包括养殖环境的变差，导致养殖过程中在动物饲料中添加抗生素、增强动物抵抗力的添加剂都是必不可少的提高养殖收益的手段。抗生素的添加会导致耐药性的产生、对环境造成污染、对人畜有害。现代养殖中，科研者在不断地研究开发出新的抗生素替代品，其中，益生菌是首选的天然替代物质。大量研究已表明，益生菌添加到动物饲料中能提高饲料的吸收利用率、提高动物的免疫能力、减少环境污染等优势。
凝结芽孢杆菌是一种益生菌。凝结芽孢杆菌是兼性厌氧菌，在有氧及无氧的环境下都可生长，能适应低氧的肠道环境，对酸和胆汁有较高的耐受性，能够进行乳酸发酵，产生的L-乳酸能降低肠道pH值，抑制有害菌，并能促进双歧杆菌等有益菌的生长和繁殖。
目前公开的用于饲料中的凝结芽孢杆菌菌株，也具有提高饲料利用率、增强动物免疫力、增重等作用。但存在菌株活性较低、添加量大、成本高、抗菌效果一般、饲料利用率提高率不高等问题。
为此，有必要筛选提取一种在饲料中添加应用的、具有高效增强营养吸收、提高动物抵抗力及生产性能等作用的凝结芽孢杆菌。
发明内容
本发明提供一种从生物质处理废弃物中筛选提取所得的凝结芽孢杆菌XP(Bacillus coagulans XP)，用作饲料添加剂，具有明显的提高饲料利用率、降低动物的腹泻率和死亡率的作用。
本发明首先提供了一种凝结芽孢杆菌XP(Bacillus coagulans XP)，已于2017年12月25日保藏于中国典型培养物保藏中心，其保藏登记号为CCTCC NO:M2017836。该凝结芽孢杆菌XP(Bacillus coagulans XP)在菌种分类上属于芽孢杆菌属，为革兰氏阳性菌，其菌落呈单个或成对存在，很少成直链排列；其细胞呈杆状，端生芽孢，无鞭毛，形成内生孢子； 其单个菌体大小是，长为4～6μm，宽为0.4～0.9μm。
凝结芽孢杆菌XP(Bacillus coagulans XP)的16S rDNA序列如下：gctggctccgtaaaggttacctcaccgacttcgggtgttacaaactctcgtggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcggcatgctgatccgcgattactagcgattccggcttcatgcaggcgggttgcagcctgcaatccgaactgggaatggttttctgggattggcttaacctcgcggtctcgcagccctttgtaccatccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttcctccggtttgtcaccggcagtcaccttagagtgcccaactgaatgctggcaactaaggtcaagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacaaccatgcaccacctgtcactctgtcccccgaaggggaaggccctgtctccagggaggtcagaggatgtcaagacctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcctttgagtttcagccttgcggccgtactccccaggcggagtgcttaatgcgttagctgcagcactaaagggcggaaaccctctaacacttagcactcatcgtttacggcgtggactaccagggtatctaatcctgtttgctccccacgctttcgcgcctcagcgtcagttacagaccagagagccgccttcgccactggtgttcctccacatctctacgcatttcaccgctacacgtggaattccactctcctcttctgcactcaagcctcccagtttccaatgaccgcttgcggttgagccgcaagatttcacatcagacttaagaagccgcctgcgcgcgctttacgcccaataattccggacaacgcttgccacctacgtattaccgcggctgctggcacgtagttagccgtggctttctggccgggtaccgtcaaggcgccgccctgttcgaacggcacttgttcttccccggcaacagagttttacgacccgaaggccttcttcactcacgcggcgttgctccgtcagactttcgtccattgcggaagattccctactgctgcctcccgtaggagtttgggccgtgtctcagtcccaatgtggccgatcaccctctcaggtcggctacgcatcgttgccttggtgagccgttaccccaccaacaagctaatgcgccgcgggcccatctgtaagtgacagcagaagccgtctttcctttttcctccatgcggaggaaaaaactatccggtattagccccggtttcccggcgttatcccgatcttacaggcaggttgcccacgtgttactcacccgtccgccgctaaccttttaaaagcaagcttttaaaaggtccgcacgacttgc
进一步地，本发明还提供了所述的凝结芽孢杆菌XP(Bacillus coagulans XP)的应用，该凝结芽孢杆菌XP(Bacillus coagulans XP)适合用作动物饲料添加剂，其添加量为在每克饲料中的添加50～500万CFU活菌。
更进一步地，本发明还提供了一种凝结芽孢杆菌XP(Bacillus coagulans XP)的筛选培养方法，包括以下步骤：
(1)筛选：选取生物质处理后的废弃物悬浮分离处理后的上清液，加入到液态GYS培养基中筛选培养，条件是50±5℃培养至少18小时；
(2)富集：取步骤(1)培养所得的菌液，接种到液态MRS培养基中富集培养；
(3)提纯：挑取步骤(2)培养所得的菌液，接种到GYS培养基平板中培养；挑选具有溶钙圈的单菌落，接种到新的GYS培养基平板分离培养；即得纯的凝结芽孢杆菌XP(Bacillus coagulans XP)菌株。
通过上述方法筛选得到的凝结芽孢杆菌XP(Bacillus coagulans XP)菌株，可进一步 地接种到含新的无菌的液态GYS培养基的三角瓶中，50±5℃静置培养36小时；然后往三角瓶中加入其内容物总质量的20％的甘油，摇匀后分装到若干个5ml塑料管放入-20℃冰箱保存，即可。
所述在MRS培养基中富集培养的条件是，温度为45℃、转速为200rpm的摇床中摇匀培养。
所述的生物质处理后的废弃物为秸秆发酵处理后的液体或悬浮液，尤其是玉米秸秆糖化发酵处理后的废渣废液，尤其是废弃物的上部物质或悬浮物质。
其中，所述GYS培养基的配方为：每1000ml去离子水中含有10g酵母粉、5g蛋白胨、100g葡萄糖、50g碳酸钙(CaCO 3)。
其中，所述MRS培养基的配方为：每1000ml去离子水中含有10.0g蛋白胨、10.0g牛肉膏、5.0g酵母膏、2.0g柠檬酸氢二铵[(NH 4) 2HC 6H 5O 7]、20.0g葡萄糖(C 6H 12O 6·H 2O)、1.0mL吐温-80，5.0g乙酸钠(CH 3COONa·3H 2O)、2.0g磷酸氢二钾(K 2HPO 4·3H 2O)、0.58g硫酸镁(MgSO 4·7H 2O)、0.25g硫酸锰(MnSO 4·H 2O)、18.0g琼脂。
本发明的有益技术效果在于：(1)提供了一种新筛选的凝结芽孢杆菌XP(Bacillus coagulans XP)菌株；(2)该菌筛选提取成本低，从生物质处理废渣中筛选即可，筛选培养程序简单；(3)该菌具有明显的提高肉猪及肉禽等的生长速度、提高饲料利用率、降低动物的腹泻率和死亡率的作用。
附图说明
图1为凝结芽孢杆菌XP(Bacillus coagulans XP)的400倍电镜图片；
图2为凝结芽孢杆菌XP(Bacillus coagulans XP)的进化树分析图谱。
具体实施方式
下面列举本发明的优选实施例作为对发明内容的详细阐述，本发明的保护不限于以下的实施例。
实施例1
凝结芽孢杆菌Bacillus coagulans XP的分离筛选及鉴定：
1、分离及筛选：
(1)选取广东怡和科洁科技有限公司试实验室中生物炼制玉米秸秆的废弃物作为菌种，取废弃物的上部残渣及及水样，经过悬浮分离法处理后，取上清液0.5g转接入含有新鲜无 菌的、pH 6.5的100mL GYS培养基的三角瓶中，45℃、200rpm震荡培养18～36小时；至三角瓶内的液体呈浓稠浑浊后，挑取三角瓶内的菌液转接入新的无菌MRS培养基中，45℃、200rpm震荡富集培养；
(2)重复步骤(1)操作，反复转接三次，每轮转接后菌液-80℃保存，后续做DGGE(变性梯度凝胶电泳)分析菌落变化情况；
(3)挑取步骤(2)所得的第三轮筛选富集后的菌液在新的无菌的GYS培养基平板上划线培养，45℃培养至少18小时，挑选出菌圈较大、外周具有透明的溶钙圈的单菌落，接种到新的无菌的GYS培养基平板上划线分离，获得纯菌株。
其中，所述GYS培养基的配方为：每1000ml去离子水中含有10g酵母粉，5g蛋白胨，100g葡萄糖，50g碳酸钙(CaCO 3)。
其中，所述MRS培养基的配方为：每1000ml去离子水中含有蛋白胨10.0g，牛肉膏10.0g，酵母膏5.0g，柠檬酸氢二铵[(NH 4) 2HC 6H 5O 7]2.0g，葡萄糖(C 6H 12O 6·H 2O)20.0g，吐温80 1.0mL，乙酸钠(CH 3COONa·3H 2O)5.0g，磷酸氢二钾(K 2HPO 4·3H 2O)2.0g，硫酸镁(MgSO 4·7H 2O)0.58g，硫酸锰(MnSO 4·H 2O)0.25g，琼脂18.0g。
2、菌株的生理生化鉴定：
从上述方法所得的平板中挑选出3～4株菌圈较大、菌圈圆度较好、外周具有明显的透明的溶钙圈的单菌落菌株进行后续的生理生化鉴定，以及代谢途径的研究。
如图1所示，为以上方法筛选出的菌株的电镜图片。该菌株为革兰氏阳性菌细胞呈杆状，菌体大小0.4～0.9×4～6μm，单个、成对，很少成直链排列，形成内生孢子，革兰氏阳性菌，端生芽孢，无鞭毛。
如图2所示，是使用16S rDNA基因序列分析的结果，该结果显示该菌与凝结芽孢杆菌相似性最高，同时进化树分析结果指出，该菌与凝结芽孢杆菌YSJ进化距离更近。
生理生化鉴定，结果如表1。参考《乳酸细菌分类鉴定及实验方法》《伯杰细菌鉴定手册》与《常见细菌系统鉴定手册》可知，筛选菌为凝结芽孢杆菌(Bacillus coagulans)，命名为凝结芽孢杆菌XP(Bacillus coagulans XP)。该菌株脂肪酸分析的结果同上述一致。
表1生理生化鉴定结果
Figure PCTCN2018105156-appb-000001
Figure PCTCN2018105156-appb-000002
实施例2
凝结芽孢杆菌(Bacillus coagulans)XP在作为动物饲料上的应用：
将实施例1筛选所得的凝结芽孢杆菌(Bacillus coagulans)XP按照4％的接种量接种到MRS培养基(pH 6.5)中，45℃、200rpm情况下培养。菌体的初始浓度OD600nm为0.1，菌体在10～12小时左右进入对数生长期，持续4～6小时；菌体在15～18小时左右进入稳定期，继而进入菌体高密度发酵时期；在60小时左右，发酵液在660nm处吸光值开始下降，此时停止发酵。采用平板计数法，对发酵液菌体进行浓度测定，菌体最终浓度为78亿CFU/g。
将通过以上方法得到的菌体发酵液和活性沸石按质量分数为1:1～2:1比例混合，所述活性沸石为经磨粉处理，其颗粒细度≤0.06mm；搅拌均匀后，送入吸附器中依次进行加温负载、静置、过滤分离处理(搅拌转速为100～150rpm，加温负载时温度为15～65℃，吸附时间为3～45分钟)，将分离得到的固体物料真空冷冻干燥(温度0℃～-20℃，真空压力1.3～5.5帕)，干燥后的固体物料进行粉碎至颗粒细度≤0.1mm，即制得益生菌固体粉剂。
取1g益生菌固体粉剂，用无菌蒸馏水稀释10 -4到10 -5倍，采用平板计数法，对其菌体浓度进行测定，检测为150～200亿CFU/g。
制得的益生菌固体粉剂，可作为高级动物饲料添加剂，具有很高的商业价值，可代替抗生素、激素等敏感添加剂。如表2是菌体发酵液和活性沸石以不同比例混合、在不同参数条件处理下制得的益生菌固体粉剂，稀释后测得的菌体浓度结果。
表2不同配方的益生菌固体粉剂的菌体浓度结果
Figure PCTCN2018105156-appb-000003
实施例3
益生菌固体粉剂在动物饲养中的应用及效果：
在饲料中添加实施例2制备所得的益生菌固体粉剂，添加量为每克饲料中添加100～350万CFU活菌，能提高肉猪及肉禽等的生长速度3.5～12.7％，提高饲料利用率2.1～8.5％，并可明显降低动物的腹泻率和死亡率。
该实施例的以45天龄的肉猪100头为样本，分为五组，添加不同活菌浓度的益生菌固体粉剂，对比其效果如表3：
表3添加不同浓度益生菌固体粉剂到饲料中的饲养效果
Figure PCTCN2018105156-appb-000004
Figure PCTCN2018105156-appb-000005
Figure PCTCN2018105156-appb-000006

Claims (10)

  1. 一种凝结芽孢杆菌XP(Bacillus coagulans XP)，其已于2017年12月25日保藏于中国典型培养物保藏中心，其保藏登记号为CCTCC NO:M2017836。
  2. 如权利要求1所述的一种凝结芽孢杆菌XP(Bacillus coagulans XP)，其特征在于：分类上属于芽孢杆菌属，为革兰氏阳性菌，其菌落呈单个或成对存在；其细胞呈杆状，端生芽孢，无鞭毛，形成内生孢子；其单个菌体大小是，长为4～6μm，宽为0.4～0.9μm。
  3. 一种凝结芽孢杆菌XP(Bacillus coagulans XP)的应用，其特征在于：用作动物饲料添加剂，在每克饲料中的添加量为100～350万CFU活菌。
  4. 一种凝结芽孢杆菌XP(Bacillus coagulans XP)的筛选培养方法，其特征在于，包括以下步骤：
    (1)筛选：选取生物质处理后的废弃物悬浮分离处理后的上清液，加入到液态GYS培养基中筛选培养，条件是50±5℃培养至少18小时；
    (2)富集：取步骤(1)培养所得的菌液，接种到液态MRS培养基中富集培养；
    (3)提纯：挑取步骤(2)培养所得的菌液，接种到GYS培养基平板中培养；挑选具有溶钙圈的单菌落，接种到新的GYS培养基平板分离培养；即得纯的凝结芽孢杆菌XP(Bacillus coagulans XP)菌株。
  5. 如权利要求4所述的一种凝结芽孢杆菌XP(Bacillus coagulans XP)的筛选培养方法，其特征在于，所述GYS培养基的配方为：每1000ml去离子水中含有10g酵母粉、5g蛋白胨、100g葡萄糖、50g碳酸钙(CaCO 3)。
  6. 如权利要求4所述的一种凝结芽孢杆菌XP(Bacillus coagulans XP)的筛选培养方法，其特征在于，所述MRS无机盐培养基的配方为：每1000ml去离子水中含有10.0g蛋白胨、10.0g牛肉膏、5.0g酵母膏、2.0g柠檬酸氢二铵[(NH 4) 2HC 6H 5O 7]、20.0g葡萄糖(C 6H 12O 6·H 2O)、1.0mL吐温-80，5.0g乙酸钠(CH 3COONa·3H 2O)、2.0g磷酸氢二钾(K 2HPO 4·3H 2O)、0.58g硫酸镁(MgSO 4·7H 2O)、0.25g硫酸锰(MnSO 4·H 2O)、18.0g琼脂。
  7. 根据权利要求4所述的一种凝结芽孢杆菌XP(Bacillus coagulans XP)的筛选培养方法，其特征在于：所述步骤(2)富集培养的温度为45℃。
  8. 根据权利要求4所述的一种凝结芽孢杆菌XP(Bacillus coagulans XP)的筛选培养方法，其特征在于：所述富集培养是置于转速为200rpm的摇床中摇匀培养。
  9. 根据权利要求4所述的一种凝结芽孢杆菌XP(Bacillus coagulans XP)的筛选培养方法，其特征在于：所述的生物质处理后的废弃物为秸秆发酵处理后的废渣废液。
  10. 根据权利要求4所述的一种凝结芽孢杆菌XP(Bacillus coagulans XP)的筛选培养方法，其特征在于：所述在步骤(3)的提纯，是先将挑取的步骤(2)所得的菌液用生理盐水进行十倍梯度稀释，再挑取稀释菌液接种到所述GYS培养基平板上。
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