CN109517750A - 制备发酵羽毛粉的枯草杆菌菌株及其用途 - Google Patents
制备发酵羽毛粉的枯草杆菌菌株及其用途 Download PDFInfo
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- CN109517750A CN109517750A CN201810036061.1A CN201810036061A CN109517750A CN 109517750 A CN109517750 A CN 109517750A CN 201810036061 A CN201810036061 A CN 201810036061A CN 109517750 A CN109517750 A CN 109517750A
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- 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
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- 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
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- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K10/20—Animal feeding-stuffs from material of animal origin
- A23K10/26—Animal feeding-stuffs from material of animal origin from waste material, e.g. feathers, bones or skin
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F1/00—Fertilisers made from animal corpses, or parts thereof
- C05F1/005—Fertilisers made from animal corpses, or parts thereof from meat-wastes or from other wastes of animal origin, e.g. skins, hair, hoofs, feathers, blood
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- 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
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- C12N1/20—Bacteria; Culture media therefor
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- 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
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- 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
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- 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
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
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- Mycology (AREA)
- Sustainable Development (AREA)
- Environmental & Geological Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明涉及制备发酵羽毛粉的枯草杆菌菌株及其用途,其目的有二,第一为协助改善水解羽毛粉的缺点,利用枯草杆菌加入水解的羽毛粉,经一日的作用后,提高其羽毛粉胃蛋白酶消化率。第二目的为本菌株另具有蛋白分解酶及角蛋白分解酶,可供动物饲料的添加物,增加营养价值。本发明的枯草杆菌(Bacillus subtilis)531‑7是由堆肥中筛检出来,再经羽毛分解力及酶活性比对,确认有商业价值。本发明的新颖枯草杆菌(Bacillus subtilis)菌株,经16S rDNA基因序列分析及gyrB基因序列分析比对鉴定确认。
Description
技术领域
本发明有关于一种自堆肥中分离的新颖枯草杆菌菌株,用于产生羽毛粉及蛋白酶包含角蛋白酶活性的菌株。本发明另涉及一种水解羽毛粉再加工的制备方法。
背景技术
羽毛中主要蛋白是由不可溶的结构蛋白质-角蛋白(keratin)构成。角蛋白含有高量的疏水性双硫键和氢键键结,不易被一般蛋白质水解酶分解,而动物体本身缺乏此种蛋白质分解酶,所以对禽畜而言,角蛋白不能直接利用。
现有技术中水解羽毛粉的方法主要为利用蒸气水解羽毛来实现。具体而言如图7所示,将湿羽毛去水后(步骤301),将羽毛输入精炼炉内,注入蒸气后将羽毛均匀加温并搅拌。当羽毛及水在蒸煮机中,加入10巴(bar)蒸气,并将温度控制在187℃的条件下;此时蒸煮机中的水化为水蒸气,水蒸气体积涨大产生压力,帮助羽毛的水解作用;而后,将蒸煮机维持在3bar、145℃的环境下持续蒸煮50分钟,将羽毛水解而保存可利用的蛋白质。高压水解后的羽毛成糊状(步骤302),再经真空干燥后,抽出含水量在12%以下的干燥羽毛粉(步骤303)。待获得干燥羽毛粉后,将其放入收集槽散热以降低温度,等干燥羽毛粉降温后再输送至震动筛网并将粗块或废弃物筛出;而后再输送至粉碎机、装袋及称量即为水解羽毛粉(步骤304)。
然而,根据以往水解羽毛粉的方法,蒸气压与时间的控制会影响水解羽毛粉质量。过度水解会降解大量氨基酸,使养分流失;但水解不足反而会使双硫键未完全分解,致使蛋白质的质量不良。
现有技术指出,水浴加热法虽可一定程度地增加营养成分,但会损失必需氨基酸[如赖氨酸(lysine)、甲硫氨酸(methionine)和色氨酸(tryptonphan)等]的含量,并导致赖氨丙氨酸(lysinoalanine)和羊毛硫氨酸(lanthionine)等非必需氨基酸含量的增加。即便是利用水浴加热法水解羽毛,若过度水解也会存在破坏氨基酸、降低蛋白质质量、或所得的羽毛粉消化率较低等问题。
再者,不同来源或不同批次生产的水解羽毛粉质量可能不同。现有饲料公司福寿实业提供8种不同来源或不同批次的水解羽毛粉商品,经分析水解羽毛粉的胃蛋白酶消化率约在23.5%至92.8%,粗蛋白含量约在79.7%至93.3%。饲料业者对于胃蛋白酶消化率非常重视,消化率低常会导致家禽或家畜生病。一般政府要求水解羽毛粉商品中胃蛋白酶消化率需高于70%或75%。国产或进口的水解羽毛粉粗蛋白含量在79.7%至93.3%,但消化率则在23.5%至92.8%之间,显示不能以粗蛋白含量来判断水解羽毛粉的消化率。对于水解羽毛粉中的氨基酸成分,饲料业者认为可通过后续添加调整,因此水解羽毛粉中的氨基酸成分对家禽或家畜的重要性不如胃蛋白酶消化率。
近年由于饲料鱼粉价格高升,废弃羽毛转制的羽毛粉即为产业锁定的替代品之一,但目前水解羽毛粉质量不稳定,造成以其调配的饲料消化率也不稳定,对于从业者而言是相当大的困扰。此外,如羽毛通过蒸气水解得到的产物,动物不易消化,因此寻求生物分解以改善消化的困难性。但生物分解需一定时日,而屠宰场的废弃羽毛需立即处理避免发臭。
有鉴于此,现有技术有需改善的必要。提供快速制备稳定优质的羽毛粉质量,同时避免产生羽毛堆积的情形仍待发展。
发明内容
为了克服现有技术的缺点,本发明的目的有二,第一为协助改善水解羽毛粉的缺点,利用枯草杆菌加入水解的羽毛粉,经一日的作用后,提高其羽毛粉胃蛋白酶消化率。第二目的为利用本菌株生产蛋白分解酶及角蛋白分解酶,可供动物饲料的添加物,增加营养价值。本发明的枯草杆菌(Bacillus subtilis)确有商业价值。
为达到上述的发明目的,本发明提供一种枯草杆菌531-7(Bacillus subtilis531-7) 菌株,其寄存于中国的台湾财团法人新竹食品工业发展研究所,寄存编号为BCRC910793,寄存日期为2017年08月09日;亦寄存于中国典型培养物保藏中心,保藏号为 CCTCCNO:M 2017495,保藏日期为2017年09月06日。
本发明另提供一种微生物制剂,其包含如前述的枯草杆菌531-7菌株的培养物做为有效成分。
本发明另提供一种如上述的枯草杆菌531-7菌株作为饲料或饲料添加剂的用途;较佳的,所述饲料为动物饲料或鱼饲料。
本发明另提供一种如上述的枯草杆菌531-7菌株作为肥料或肥料添加剂的用途。本发明的枯草杆菌531-7菌株供添加至有机堆肥,可以促进堆肥肥效。
本发明另提供一种制备细菌发酵羽毛粉的方法,其包含以下步骤:(1)备齐羽毛(如白肉鸡羽毛,黄土鸡羽毛,乌骨鸡羽毛),将羽毛剪碎后置于培养基,所述培养基包含但不限于基础盐类培养基或地下水(地下水pH值7.72,电导度为657μS/cm,盐度 325ppm,总溶解固体(TDS)为456mg/L),获得重量体积比为1%(w/v)至20%(w/v)羽毛培养基;(2)将如前所述的枯草杆菌531-7菌株添加于羽毛培养基,使枯草杆菌531-7 菌株浓度成为每毫升105至107菌落单位(CFU/mL)进行培养3天至8天,温度介于20℃至40℃;(3)将培养后的含枯草杆菌531-7菌株的羽毛培养基灭菌后进行干燥,再以球磨机研磨至适当大小,获得细菌发酵羽毛粉;较佳的,前述的适当大小为小于0.15mm。此外,本发明当所述步骤(1)培养基为地下水时,亦能达成相似的效果。
较佳的,所述的步骤(1)中,重量体积比为5%(w/v)至6.5%(w/v)羽毛培养基。较佳的,羽毛培养基的羽毛包含,但不限于鸡羽毛;更佳的,鸡羽毛包含,但不限于白肉鸡羽毛。
较佳的,所述的步骤(2)中,将枯草杆菌531-7菌株添加于羽毛培养基培养1天至8天,更佳为6天。
较佳的,所述的步骤(2)中,培养温度是37℃。
本发明提供一种细菌发酵羽毛粉,其为上述的方法所制备而得。其中胃蛋白酶消化率为86.3%、粗蛋白94.7%、氨基酸总含量99.1%;其中水解氨基酸含量值(氨基酸除以粗蛋白,a.a.mg/CP mg,%)为:甲硫氨酸(Met)0.3%、离氨酸(Lys)1.6%、组氨酸 (His)0.5%、色氨酸(Trp)0.5%、苏氨酸(Thr)4.6%、缬氨酸(Val)6.3%、胱氨酸(Cys) 6.7%、异白氨酸(Ile)4.4%、白氨酸(Leu)8.1%、酪氨酸(Tyr)3.3%、苯丙氨酸(Phe)5.4%、精氨酸(Arg)6.4%、丙氨酸(Ala)4.5%、天门冬氨酸(Asp)6.6%、麸氨酸(Glu)10.4%、甘氨酸(Gly)7.6%、脯氨酸(Pro)11.6%、丝氨酸(Ser)10.5%。
本发明另提供一种如前述的细菌发酵羽毛粉作为饲料或饲料添加剂的用途。
本发明另提供一种如前述的细菌发酵羽毛粉作为肥料或肥料添加剂的用途。
本发明另提供一种加工发酵羽毛粉的制备方法,其包含以下步骤:(1)备齐水解羽毛粉,将水解羽毛粉置于培养基,所述培养基包含但不限于基础盐类培养基或地下水,获得重量体积比为1%(w/v)至20%(w/v)水解羽毛粉培养基;(2)将如前所述的枯草杆菌531-7菌株添加于水解羽毛粉培养基,使枯草杆菌531-7菌株浓度成为每毫升105至107菌落单位(CFU/mL)进行培养1天至3天(37℃);(3)培养1天至3天后,将含有枯草杆菌531-7菌株的水解羽毛粉培养基经灭菌干燥后研磨,获得加工发酵羽毛粉。此外,本发明当所述步骤(1)培养基为地下水时,亦能达成相似的效果。
较佳的,所述水解羽毛粉包含,但不限于市售水解羽毛粉。
本发明所述“市售水解羽毛粉”,是指未经本发明枯草杆菌531-7菌株培养的羽毛粉,将羽毛以高压水解成水解羽毛粉。
较佳的,所述的步骤(2)中,培养时间为72小时。低度水解羽毛粉培养72小时后,其消化率由23.5%改善至83.4%。如培养24小时消化率改善至62.8%。高度水解羽毛粉培养72小时后,其消化率由78.9%改善至84.9%。
本发明另提供一种加工发酵羽毛粉,其是上述的方法所制备而得。
本发明另提供一种酶的制备方法,其包含以下步骤:(1)备齐原料,将原料置于培养基,获得重量体积比为1%(w/v)至10%(w/v)原料培养基;(2)将上述枯草杆菌 531-7菌株添加于原料培养基,使枯草杆菌531-7菌株浓度成为每毫升105至107菌落单位(CFU/mL)进行培养。(3)将含有枯草杆菌531-7菌株的原料培养基进行干燥,获得酶。
较佳的,其中于步骤(1)中,原料为羽毛;其中于步骤(1)至(3)中,原料培养基为羽毛培养基;其中于步骤(3)中,酶包含角蛋白酶。
较佳的,其中于步骤(1)中,原料为黄豆;其中于步骤(1)至(3)中,原料培养基为黄豆培养基;其中于步骤(3)中,酶包含酪蛋白酶。
本发明的优点在于:
1.本发明的枯草杆菌531-7菌株,为具备蛋白酶,主要包括角蛋白酶与酪蛋白酶等两种功能活性的菌种,通过菌株生产蛋白酶做为肥料或动物饲料;此外,前述菌株分解的羽毛粉质量稳定(如下表4),如一致的胃蛋白酶消化率(如下表4)。基于上述特征本菌可开发为羽毛废弃物处理、动物饲料添加、肥料等的用途。
2.本发明可利用制备细菌发酵羽毛粉的方法进行酶的收集。细菌加入基础盐类培养基中(含2%羽毛),培养6天(37℃)后,过滤后取滤液得到粗酶,不经灭菌处理,获得角蛋白酶。蛋白酶的收集,以下列方法进行:细菌加入基础盐类培养基中(含1%豆粉),培养2天(37℃)后,过滤后取滤液得到粗酶,不经灭菌处理,获得蛋白酶。通过本发明所获得的蛋白酶(主要包括角蛋白酶与酪蛋白酶)可做为动物饲料的添加物增加营养价值,亦可做为肥料使用。
3.本发明的加工发酵羽毛粉的制备方法,通过前述菌株加入低度水解的羽毛粉,作用后提高其羽毛粉质量(如胃蛋白酶消化率、粗蛋白含量),改善市售水解羽毛粉的缺点,使加工发酵羽毛粉做为动物饲料的添加物更具营养价值且易消化,亦可做为肥料使用。
附图说明
图1为本发明的枯草杆菌531-7菌株培基后在各个时间点的菌液O.D.600值及菌落形成单位(CFU)与时间关系的折线图。
图2为本发明的枯草杆菌531-7菌株与8株公开菌以16S rDNA的亲缘演化树状图。
图3为本发明的枯草杆菌531-7菌株与8株公开菌以gyrB基因的亲源演化树状图。
图4为本发明的枯草杆菌531-7菌株的蛋白质电泳染色图。
图5为发酵羽毛粉的制备流程图;S101至S103代表步骤101至步骤103。
图6为加工羽毛粉的制备流程图;S201至S203代表步骤201至步骤203。
图7为现有技术水解羽毛粉的制备流程图;S301至S304代表步骤301至步骤304。
用于专利程序的微生物保存:
本发明的菌种531-7;
保藏日期:2017年09月06日;
保藏单位:中国典型培养物保藏中心(CCTCC);
保藏单位地址:中国武汉武汉大学邮编:430072;
保藏编号:CCTCC NO:M 2017495;
分类命名:枯草杆菌(Bacillus subtilis)。
具体实施方式
以下配合图及本发明的较佳实施例,进一步阐述本发明为达成预定发明目的所采取的技术手段。
制备例1细菌菌株的来源与分离纯化
本案新颖菌株代号为枯草杆菌531-7菌株(Bacillus subtilis 531-7)是分离自堆肥,再经羽毛分解效力比对,并寄存于中国台湾新竹的财团法人食品工业发展研究所生物资源保存及研究中心,编号为BCRC 910793,寄存日期为2017年08月09日;亦寄存于中国典型培养物保藏中心,保藏号为CCTCC NO:M 2017495,保藏日期为2017年09 月06日。以下为菌株的三阶段筛选方法:
第一阶段:酪蛋白分解力比较
取堆肥1克(g)加入9毫升(mL)无菌水,以80℃、20分钟水浴处理或是直接(室温)稀释成0.01倍和0.001倍后涂在具有1%酪蛋白的基础盐培养基(basal salt medium)[内含 0.7g/L磷酸二氢钾(KH2PO4)、1.4g/L磷酸氢二钾(K2HPO4)、0.5g/L氯化钠(NaCl)、0.1 g/L硫酸镁(MgSO4)、10g/L酪蛋白(casein)、pH 7.2、15g/L洋菜胶(agar)]上,选择有透明环的单一菌落以LA培养基[LB培养基(Lysogeny broth)加上青霉素(ampicillin)]活化 20小时后,以灭菌牙签沾取单一菌落,点于具有1%酪蛋白的基础盐培养基上,于37℃培养24小时后,观察菌落周围是否出现透明环,拍照并记录菌落及透明环大小,选择透明环与菌落比较大者进入第2阶段。
第二阶段:单根羽毛分解力目视比较
配制羽毛磷酸缓冲液(phosphate buffered saline,PBS):将PBS分装10mL于螺旋试管中,并添加一根完整的鸡羽毛单根重约为0.05克,进行高温高压灭菌备用。将供试菌株于LA上活化并以5mL LB液培20小时后,调整菌量为OD600=0.3(108cfu/mL)并吸取100微升(μl)加入试管中,于37℃下培养6天后观查羽毛的完整性,并以不添加供试菌株者作为空白组(blank)。取分解力三级或四级的菌株进行下一个阶段4克羽毛的细菌分解率比较。分解力共分五级,零级:无明显分解情形。一级:有部分分解,只有少许羽毛碎屑悬浮。二级:羽毛明显分解,且碎屑沉淀量多。三级:羽毛呈粉碎状,只剩羽轴。四级,羽毛全分解,外观无残留。
第三阶段:4克羽毛的细菌分解率(%)比较
配制200mL基础盐培养基(0.7g/L KH2PO4,1.4g/L K2HPO4,0.5g/L NaCl,0.1g/LMgSO4,pH 7.2),并添加4克白肉鸡羽毛于500mL三角瓶中成为2%羽毛培养基,高温灭菌后,加入已活化增量于LB中24小时的供试菌株,菌液为OD600=0.3(108CFU/ml),使菌液于羽毛培养基的体积百分比为2%(v/v)。将三角瓶于37℃、150rpm下培养6天后,滤纸[ADVANTECNo.1]进行过滤(收集的滤液为粗酶),将截留的羽毛残留物放入65℃烘箱烘干后,称重并计算羽毛分解率(%),分解率大于80%以上的菌株,再进行羽毛粉的制作及两种粗酶活性试验(蛋白酶包含角蛋白酶),以筛检出具商品价值的具分解羽毛的菌株及酶。
制备例2菌株基本生理特性测试
1.菌株的基本特性:
枯草杆菌531-7菌株的外观:为革兰氏阳性菌,单个细胞大小约为0.4μm至0.6μm×1.0μm至1.6μm,细胞顶端为半圆形且单个或呈短链。在细胞顶端部位形成芽孢,芽孢为椭圆形或圆形,大小约为0.6μm至0.7μm×1.0μm至1.6μm。枯草杆菌531-7菌株的电子显微镜相片可看到前孢子。本发明枯草杆菌531-7菌株在LA培养基生长时,初期(24小时)菌落形狀为圆形,白色,表面形成***的菌落,边缘不规则的菌落或边缘光滑整齐;在3至4天后菌落表面干燥皱褶。
2.pH值测定
将3天及6天的羽毛培养基取过滤后的羽毛分解滤液测pH值。结果显示羽毛分解液在3天后pH值7.6至7.7,6天后pH值为8.5至8.7。
3.生长曲线测定-菌体混浊度(OD)值及菌落数(CFU)
以接种环沾取单一完整菌落于含6mL LB培养液于15mL离心管中,在37℃下振荡培养(转速为150rpm)24小时。调整菌液OD600<0.06。取调整好的1mL菌液(OD600< 0.06)置50mLLB培养液中(OD600<0.01),于37℃下振荡培养(转速为150rpm)再分别于0、1、2、4、6、8、24、26、28、30及32小时(视生长曲线调整后端时间点)取样计数生菌数及测OD值。每个时间点取2mL测其OD值(校正液为LB培养液)。并另取0.1 mL计数生菌数(37℃,24小时)(如图1所示)。菌液在培养2小时至6小时为对数生长期,在培养6小时之后进入迟滞期。
制备例3菌株鉴定方法
1.BIOLOG菌种鉴定与碳源利用能力测定
(1)以BIOLOG鉴定***测定本发明菌株的菌种,将所获得的数值与BIOLOG公司资料库比对,若该菌株71种碳源与23种化学敏感试验的OD595值与资料库的菌株数值相似度达到0.50以上便给予该测定菌株的接近鉴别ID。以BIOLOG公司提供的 MicroStationTM***软件分析的结果,得知其表现型与Bacillus subtilis菌株的相似度最高,其相似度为0.64,可能机率为0.77,因此将菌株命名为枯草杆菌531-7菌株(Bacillus subtilis 531-7)。
(2)枯草杆菌531-7菌株可利用的碳源及化学敏感试验:可利用的碳源为23种。分别为葡萄糖(α-D-Glucose)、糊精(Dextrin)、甘露糖(D-Mannose)、麦芽糖(D-Maltose)、海藻糖(D-Trehalose)、纤维二糖(D-Celloblose)、龙胆二糖(Gentioblose)、蔗糖(Sucrose)、苹果酸(L-Malic Acid)、葡萄糖酸(D-Gluconic Acid)、乳酸(L-Lactic Acid)、柠檬酸(Citric Acid)、丙氨酸(L-Alanine)、天门冬氨酸(L-Aspartic Acid)、麸氨酸(L-GlutamicAcid)、山梨糖醇(D-Sorbitol)、甘露醇(D-Mannitol)、丙三醇(Glycerol)、肌醇(myo-inositol)、半乳糖酸内酯(L-Galactonic Acid Lactone)、水杨苷(D-Sallcin)、甲基葡萄糖苷 (β-methyl-D-Glucoside)及葡萄糖二酸(D-Saccharic acid)共23种。
化学敏感试验结果为耐盐度1、4、8%NaCl,耐酸碱度pH 5及pH 6,对1%乳酸钠(Sodium Lactate),安达菌素(Aztreonam),氯化锂(Lithium Chloride),丁酸钠(SodiumButyrate),亚碲酸钾(Potassium tellurite)有化学敏感性。碳源利用能力及化学敏感试验结果可供枯草杆菌531-7菌株增量培养的参考。
2.API-50CHB菌种鉴定与碳源利用能力测定
(1)以API-50CHB套组(bioMerieux,Inc.公司)测定本发明菌株的菌种及碳水化合物利用能力。在API 50CHB strip的各个试验孔中,含有49种不同的碳水化合物,可测知细菌对碳源的利用情形;在接种细菌之后,细菌在其中生长会造成培养基的pH 值的改变,可由培养基的颜色变化来判定细菌的种类。依照bioMerieux,Inc., Hazelwood,MO公司提供的说明书所述的方法进行判读,结果显示,531-7菌株经API 50CHB鉴定为Bacilluslicheniformis(99.9%)。然而,API 50CHB主要用于分析不同碳水化合物的利用性,菌种鉴定的准确性已知不如DNA序列鉴定的准确。
(2)碳源利用能力:本发明筛选的531-7菌株由API 50CHB分析结果可知本菌在49种碳源中可利用34种,分别为甘油、L-***糖、D-核糖、D-木糖、L-木糖、D-核糖醇、D-半乳糖、D-葡萄糖、D-果糖、D-甘露糖、L-鼠李糖、半乳糖醇、肌醇、D-甘露醇、D-山梨糖醇、甲基α-D-吡喃葡萄糖苷、N-乙酰葡萄糖胺、扁桃苷、熊果素、栗糖/柠檬酸铁、水杨苷、D-纤维二糖、D-麦芽糖、D-蜜二糖、D-蔗糖、D-海藻糖、菊糖、D-棉子糖、淀粉、肝糖、龙胆二糖、D-松二糖、D-塔格糖、葡萄糖酸钾。
3.细菌菌株16S核糖体DNA(rDNA)鉴定法
萃取菌株总(total)DNA,并以细菌16S rDNA通用引子对:正向引子(F:SEQ IDNO.1)及反向引子(R:SEQ ID NO.2)进行聚合酶连锁反应(PCR),产物长度为1481碱基对(base pair,bp)。将增殖后的DNA产物进行核苷酸定序,并以NCBI BLAST的程序进行在线基因库(GenBank)的查询与比对,以进行菌属鉴定。分离的菌株经以细菌通用引子对进行16SrDNA基因进行增幅后,结果可发现分离株可增幅到16S rDNA基因片段,定序为Bacillussubtilis(1504bp,SEQ ID NO.3,相似度达99%)。
4.细菌菌株gyrB基因鉴定法
萃取菌株DNA,并以细菌gyrB基因引子对:正向引子(F:SEQ ID NO.4)及反向引子(F:SEQ ID NO.5)针对Bacillus属菌种进行种的鉴定。增殖后的DNA产物进行核苷酸定序与比对,确认此菌株片段序列为枯草杆菌菌种,大小约为1.3kb,定序结果为 Bacillussubtilis(1259bp,SEQ ID NO.6,相似度为99%)。
将菌株以16S rDNA(图2)与gyrB基因比对亲缘性(图3):菌株包括枯草杆菌531-7菌株与8株寄存于美国典型培养物保藏中心(american type culture collection,ATCC)不同芽孢杆菌,以由Neighbor-joining(NJ)方法分析亲缘演化树;枝条上的数字代表自展值(bootstrap值)。
实施例1酶活性试验
(1)经羽毛培养基所获得的蛋白酶中角蛋白酶活性分析
取经制备例3筛选出的枯草杆菌531-7菌株,培养于LB并将菌液调整为OD600=0.3(108CFU/mL),取4mL菌液至2%羽毛培养基(4克羽毛)共200mL,使枯草杆菌531-7 菌株于羽毛培养基的浓度成为2×106CFU/mL,再于37℃、150rpm下培养3天后,培养液置于冰上,再以布氏漏斗过滤(ADVANTEC 1号滤纸),于冰上收集液体即为第一粗酶液(约180mL),并立即取新鲜的第一粗酶液1mL分析角蛋白酶活性(U/mL)及蛋白质含量,换算为比活性(U/mg);其中,枯草杆菌531-7菌株经羽毛培养基以所培养出的蛋白酶种类繁多,在此以主要的蛋白酶-角蛋白酶做为活性分析。其余的第一粗酶液除菌后进行冷冻干燥成为冷冻干燥的第一粗酶。除菌方式为滤液除菌:以4℃、 8500×g离心后,分次取上清液(约1mL至2mL)在冰上以0.45μm过滤膜(mixed cellulose ester,)抽气过滤(全程需置于冰上)。
表1、枯草杆菌531-7菌株第一粗酶与市售商品角蛋白酶比活性
*市售商品取自中国台湾永信药品工业股份有限公司帮美国BioResourceInternational(BRI)代工生产的福硕酶600散(600,1kg;使用对象:家禽、家畜;用途:改善饲料效率,促进动物生长及增加家禽产蛋率)。
由上表1可知本发明的枯草杆菌531-7菌株的第一粗酶液因冷冻干燥成浓缩状态而增加角蛋白酶比活性,且枯草杆菌531-7菌株的第一粗酶液无论是新鲜或是经冷冻干燥,角蛋白酶比活性皆比市售商品来得更佳,约高出3至5倍。
(2)经黄豆培养液所获得的蛋白酶中酪蛋白酶活性分析:
取经制备例3筛选出的枯草杆菌531-7菌株,培养于LB并将菌液调整为OD600=0.3(108CFU/mL),取4mL菌液至1%黄豆培养液(2克黄豆粉加入200mL的基础盐类培养基),使枯草杆菌531-7菌株于黄豆培养基的浓度成为2×106CFU/mL,于37℃、150rpm 下培养2天后,再以滤膜(ADVANTEC 1号滤纸)过滤收集液体即为第二粗酶液(约180 mL),并取第二粗酶液分析酪蛋白酶活性(U/mL)及蛋白质含量,换算为比活性(U/mg);其中,枯草杆菌531-7菌株经黄豆培养液所培养出的蛋白酶种类繁多,在此以主要的蛋白酶-酪蛋白酶做为活性分析。其余的第二粗酶液除菌后进行冷冻干燥成为冷冻干燥的第二粗酶。
表2、枯草杆菌531-7菌株第二粗酶与市售商品酪蛋白酶比活性
*市售商品取自中国台湾永信药品工业股份有限公司帮美国BioResourceInternational(BRI)代工生产的福硕酶600散(600,1kg;使用对象:家禽、家畜;用途:改善饲料效率,促进动物生长及增加家禽产蛋率)。
由上表1可知本发明的枯草杆菌531-7菌株的第一粗酶液不会因为冷冻干燥而响影角蛋白酶比活性,且枯草杆菌531-7菌株的第一粗酶液无论是新鲜或是经冷冻干燥,角蛋白酶比活性皆比市售商品来得更佳。由上表2可知本发明的枯草杆菌531-7菌株的第二粗酶液不会因为冷冻干燥而响影酪蛋白酶比活性,且枯草杆菌531-7菌株的第二粗酶液无论是新鲜或是经冷冻干燥,酪蛋白酶比活性皆比市售商品来得更佳,约高出 2倍。
(3)粗酶的浓缩与蛋白分子量分析。
实验分为3组:
11594粗酶:将BCRC 11594(地衣芽孢杆菌,Bacillus licheniformis)同实施例1(1) 的方式获得11594粗酶。
531-7粗酶:取自实施例1(1)的第一粗酶液。
531-7沉淀酶:将未经稀释的新鲜的第一粗酶液[取自实施例1(1),其中菌液经2%或10%羽毛培养基培养]50mL置于冰浴中,缓慢添加固体硫酸铵40%至80%饱和度(约增加10mL),待溶解后静置20分钟,离心9000×g、20分钟后去除上清液,将沉淀物溶解于10mL0.1M PBS(pH 7.2),获得531-7沉淀酶。
并将以上3组进行聚丙烯酰胺胶体电泳(SDS-PAGE),以考马斯蓝(coomassieblue) 染剂进行胶片染色。
请参阅图4所示,第1行及第8行为蛋白标准品(Protein Ladder,Thermo, #26616LCS)。第2行和第4行为531-7粗酶(2%羽毛培养基)经0.2倍稀释的粗酶液,条带不明显。第3行和第5行为531-7沉淀酶(2%羽毛培养基)经5倍稀释,约有4个明显条带;其中有1个条带介于55-70kDa之间,有1个条带介于55-40kDa之间,有2个条带介于 35-25kDa之间。第6行为531-7沉淀酶(10%羽毛培养基)经5倍稀释,约有7个条带;其中有1个条带位于130kDa,有1个条带介于55-70kDa之间,有1个条带位于35kDa,有2个条带介于35-25kDa之间,有2个条带介于15-25kDa之间。第7行为11594粗酶经0.2倍稀释,约有5个条带;其中有1个条带介于55-40kDa之间,有1个条带位于40 kDa,有1个条带介于35-25kDa之间,有1个条带位于15kDa,有1个条带位于10kDa。大部分的文献指出角蛋白酶分子量在30-40kDa之间,因此枯草杆菌531-7菌株的角蛋白酶分子量约在25-35kDa之间,为碱性角蛋白酶(pH值8.9)。SDS PAGE分析显示羽毛分解物中含有大量的低分子量多肽(小于13kDa)。
实施例2细菌发酵羽毛粉制备
(1)请参阅图5所示,取经过清洗灭菌干燥的鸡羽毛剪碎后25克加入500mL基础盐类培养基(成为5%至6.5%(w/v)羽毛培养基)(步骤101),将含有制备例3的枯草杆菌 531-7菌株的菌液调整为OD600=0.3(108CFU/mL),并添加5mL菌液于5%羽毛培养基500mL中,使枯草杆菌531-7菌株于羽毛培养基的浓度成为106CFU/mL,再于37℃、 150rpm下培养发酵1至6天后(1天开始分解,3天后分解力约有60%,6天后分解力大于 86%)(步骤102),将此培养液灭菌15分钟后(121℃,每平方英寸15psi),再以80℃烘干 48小时,收集干燥物进行研磨至小于0.15mm,即获得细菌发酵羽毛粉(步骤103)。其质量分析如下表3:
表3、531-7细菌发酵羽毛粉消化率、粗蛋白及氨基酸分析
表4、市售水解羽毛粉与发明菌株产制的细菌发酵羽毛粉质量比较
*市售水解羽毛粉9批次的平均值。统计方式为平均值±标准偏差(mean±SD)。
细菌发酵羽毛粉的品质(表3),较市售水解羽毛粉佳(表4),且由上表4可知,市售水解羽毛粉的标准偏差较大,表示每批次的质量相当不稳定;相较于本发明的细菌发酵羽毛粉标准偏差小,表示质量稳定。最重要的是改善胃蛋白消化率,由65.6%提高至86.3%。且粗蛋白含量也提高,而氨基酸总含量略高于市售水解羽毛粉。
(2)以实施例2(1)步骤,其中菌液于2%或10%羽毛培养基培养发酵6天后的分解液进行静置分两层,并过滤上层液与下层液。其中分解液、上层液与下层液含大量氨基酸(表5),可用于有机肥料的添加物。
表5、以2%或10%羽毛培养基培养获得分解液的氨基酸分析
实施例3加工发酵羽毛粉制备
请参阅图6所示,取市售低度水解羽毛粉10克加入200mL基础盐类培养基(5%水解羽毛粉培养基)(步骤201),将含有制备例3的枯草杆菌531-7菌株的菌液调整为 OD600=0.3(108CFU/mL),并取10mL菌液至5%水解羽毛粉培养基200mL中,使枯草杆菌531-7菌株于水解羽毛粉培养基的浓度成为5×106CFU/mL,再于37℃、150rpm下培养发酵1天后(步骤202),将此培养液灭菌15分钟后以80℃烘干至少48小时,收集干燥物进行研磨即为加工发酵羽毛粉(步骤203)。将市售低度水解羽毛粉(代号L5、L6、 M3)(取自中国台湾福寿实业),与加工发酵羽毛粉进行胃蛋白酶消化率比较。胃蛋白酶分解试验如下:取0.3g羽毛粉加入50mL新鲜配制且已预热45℃的0.2%胃蛋白酶 (活性1:10000)的0.075N盐酸(HCl)中,混合后于45℃、150rpm震荡反应16小时,以 Whatmen#541滤纸过滤,将分解后的剩余不溶物以80℃烘干称重,并将剩余不溶物进行蛋白含量分析,及胃蛋白酶消化率。
如下表6所示,经比较胃蛋白酶的消化率,市售水解羽毛粉L5、L6、M3的消化率分别为23.5、29.2及68.3%,经加工后获得的加工发酵羽毛粉消化率分别提高至62.8、 67.4及79.2%,消化率约提升2.3、2.7及1.2倍。
表6、市售水解羽毛粉与经加工发酵羽毛粉对消化率的比较
如下表7所示,加工发酵羽毛粉其粗蛋白含量较加工前水解羽毛粉,粗蛋白含量增加约3.5%至13.8%。
表7、市售水解羽毛粉与经加工发酵羽毛粉对粗蛋白的影响
以上所述仅是本发明的较佳实施例而已,并非对本发明做任何形式上的限制,虽然本发明已以较佳实施例揭露如上,然而并非用以限定本发明,任何本领域的技术人员,在不脱离本发明技术方案的范围内,当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例,但凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (15)
1.一种枯草杆菌531-7(Bacillus subtilis 531-7)菌株,其特征在于,所述菌株寄存于中国典型培养物保藏中心,保藏号为CCTCC NO:M 2017495。
2.一种微生物制剂,其特征在于,所述微生物制剂包含根据权利要求1所述的枯草杆菌531-7菌株的培养物做为有效成分。
3.一种菌株作为饲料或饲料添加剂的用途,其特征在于,所述菌株为根据权利要求1所述的枯草杆菌531-7菌株。
4.一种菌株作为肥料或肥料添加剂的用途,其特征在于,所述菌株为根据权利要求1所述的枯草杆菌531-7菌株。
5.一种制备细菌发酵羽毛粉的方法,其特征在于,所述方法包含以下步骤:
(1)备齐羽毛,将羽毛剪碎后置于培养基,获得重量体积比为1%(w/v)至20%(w/v)羽毛培养基;
(2)将根据权利要求1所述的枯草杆菌531-7菌株添加于羽毛培养基,使枯草杆菌531-7菌株浓度成为每毫升105至107菌落单位(CFU/mL)进行培养;以及
(3)将含有枯草杆菌531-7菌株的羽毛培养基进行干燥研磨,获得细菌发酵羽毛粉。
6.根据权利要求5所述的方法,其特征在于,所述步骤(2)中,将枯草杆菌531-7菌株添加于羽毛培养基培养1天至8天。
7.一种细菌发酵羽毛粉,其特征在于,所述细菌发酵羽毛粉为根据权利要求5或6所述的方法所制备而得。
8.一种细菌发酵羽毛粉作为饲料、肥料或其添加剂的用途,其特征在于,所述的细菌发酵羽毛粉为根据权利要求7所述的细菌发酵羽毛粉。
9.一种加工发酵羽毛粉的制备方法,其特征在于,所述方法包含以下步骤:
(1)备齐水解羽毛粉,将水解羽毛粉置于培养基,获得重量体积比为1%(w/v)至20%(w/v)水解羽毛粉培养基;
(2)将根据权利要求1所述的枯草杆菌531-7菌株添加于水解羽毛粉培养基,使枯草杆菌531-7菌株浓度成为每毫升105至107菌落单位(CFU/mL)进行培养;以及
(3)将含有枯草杆菌531-7菌株的水解羽毛粉培养基进行干燥研磨,获得加工发酵羽毛粉。
10.根据权利要求9所述的方法,其特征在于,所述步骤(2)中,将该枯草杆菌531-7菌株添加于水解羽毛粉培养基培养24小时至72小时。
11.一种加工发酵羽毛粉,其特征在于,所述加工发酵羽毛粉是根据权利要求9或10所述的方法所制备而得。
12.一种加工发酵羽毛粉作为饲料、肥料或其添加剂的用途,其特征在于,所述的加工发酵羽毛粉为根据权利要求11所述的加工发酵羽毛粉。
13.一种酶的制备方法,其特征在于,所述方法包含以下步骤:
(1)备齐原料,将原料置于培养基,获得重量体积比为1%(w/v)至10%(w/v)原料培养基;
(2)将根据权利要求1所述的枯草杆菌531-7菌株添加于原料培养基,使枯草杆菌531-7菌株浓度成为每毫升105至107菌落单位(CFU/mL)进行培养;以及
(3)将含有枯草杆菌531-7菌株的原料培养基进行干燥,获得酶。
14.根据权利要求13所述的方法,其特征在于,所述步骤(1)中,原料为羽毛;其中于步骤(1)至(3)中,原料培养基为羽毛培养基;其中于步骤(3)中,酶包含角蛋白酶。
15.根据权利要求13所述的方法,其特征在于,所述步骤(1)中,原料为黄豆;其中于步骤(1)至(3)中,原料培养基为黄豆培养基;其中于步骤(3)中,酶包含酪蛋白酶。
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CN114214241B (zh) * | 2021-12-24 | 2023-08-22 | 内蒙古科为博生物科技有限公司 | 一种枯草芽孢杆菌及其应用和产品 |
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