CN111480729B - 一种含菜粕的菌酶协同发酵方法 - Google Patents
一种含菜粕的菌酶协同发酵方法 Download PDFInfo
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- CN111480729B CN111480729B CN202010278169.9A CN202010278169A CN111480729B CN 111480729 B CN111480729 B CN 111480729B CN 202010278169 A CN202010278169 A CN 202010278169A CN 111480729 B CN111480729 B CN 111480729B
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- rapeseed meal
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- lactobacillus plantarum
- jun
- meal
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
本发明公开了一种含菜粕的菌酶协同发酵方法,属于发酵工程技术领域。本发明是以植物乳杆菌JUN‑DY‑6、中性蛋白酶、纤维素酶为发酵剂,以菜粕为底物,菌酶发酵后的菜粕有机酸的产量提高,适口性增加,硫苷含量降低,可用于制备饲料添加剂。且本发明的植物乳杆菌还能抑制禽畜类肠道中有害病菌如大肠杆菌、金黄色葡萄球菌、沙门氏菌的生长,有助于肠道健康。该方法还提高了菜粕的附加值,有利于废弃物再利用。
Description
技术领域
本发明涉及一种含菜粕的菌酶协同发酵方法,属于发酵工程技术领域。
背景技术
乳酸菌(Lactic acid bacteria,LAB)归类于乳酸菌科。乳酸菌是革兰氏染色阳性、无芽孢(个别属除外)、不运动或少运动、耐酸的球菌或杆菌,其能利用可发酵糖产大量乳酸。乳酸菌广泛存在于人和动物肠道及许多食品中。乳酸菌不但可以提升食品的营养价值,改善食品风味,帮助改善食品的保藏性,还可以调节人体胃肠道正常菌群、保持微生态平衡,对人和动物健康都有益。乳酸菌通过发酵碳水化合物,可以分泌大量有机酸、乳酸菌素、过氧化氢等抑菌物质来抑制腐败菌的生长来改善食品的风味和品质。近年来,在豆粕发酵过程中常常人工添加发酵剂以获得更好的产品风味和质量,而乳酸菌因其优异的性能在食品生产方面被认为是良好的发酵剂且可取代添加剂。
菜籽粕是生产菜籽油中产生的一种副产物,含有粗蛋白、纤维素、碳水化合物等营养因子,但由于含有硫苷等抗营养因子,限制了菜籽粕的饲用价值,其中硫苷的存在可能造成动物的甲状腺功能障碍,导致畜禽出现中毒。此外,由于菜籽粕中小肽含量较少,不利于牲畜的吸收利用。因此,有效降低菜籽粕中硫苷含量,并且提升小肽以及各种有机酸含量,对提高菜粕的营养价值和解决原料的供给具有重大的意义。
随着对菜籽粕脱毒方法的深入研究,已经提出物理、化学、生物等脱毒方法,但是物理与化学法脱毒效果不理想,并且存在着安全性问题,而使用微生物发酵法脱毒不仅环境友好,而且脱毒率较高。在微生物发酵中产出的蛋白酶的量较少,会导致营养物质丰富度较低。酶解法虽可以提升菜籽粕中肽类物质的含量,但是产物中存在部分苦味肽,既影响了饲料口味,又增加了处理的成本。为了降低菜籽粕中硫苷,提高营养物质,将微生物发酵法与酶解法相结合,通过乳酸菌、酵母菌、枯草芽孢杆菌等微生物发酵生产独特的香味物质遮盖苦味物质,并且加入蛋白酶克服了微生物产酶不足的问题,对提升菜籽粕的饲用价值具有重大意义。
发明内容
本发明的第一个目的是提供一种制备发酵菜粕的方法,是将植物乳杆菌JUN-DY-6、中性蛋白酶、纤维素酶和菜粕混合,进行酶解和发酵。
在一种实施方式中,调节所述菜粕的水分含量为30-50%(按质量计),所述纤维素酶的用量为300-400U/g菜粕,所述中性蛋白酶的用量为800-1500U/g菜粕。
在一种实施方式中,所述植物乳杆菌JUN-DY-6为植物乳杆菌(Lactobacillusplantarum)JUN-DY-6,保藏编号为CCTCC NO:M 2017138,已公开于公开号为CN107446852A的专利申请文件中。
在本发明的一种实施方式中,所述植物乳杆菌JUN-DY-6的菌体浓度为105-108CFU/g菜粕。
在一种实施方式中,所述中性蛋白酶的用量为1350-1500U/g菜粕。
在本发明的一种实施方式中,所述发酵的条件为在35℃-37℃发酵40-60h。
在本发明的一种实施方式中,所述水分的含量为50%(m/m),所述纤维素酶的含量为300U/g底物,所述中性蛋白酶的含量为1500U/g底物,所述植物乳杆菌JUN-DY-6的接种量为5%。
在本发明的一种实施方式中,所述水分的含量为40%(m/m),所述纤维素酶的含量为400U/g底物,所述中性蛋白酶的含量为1250U/g底物,所述植物乳杆菌JUN-DY-6的接种量为4%。
在本发明的一种实施方式中,所述水分的含量为30%(m/m),所述纤维素酶的含量为300U/g底物,所述中性蛋白酶的含量为1000U/g底物,所述植物乳杆菌JUN-DY-6的接种量为4%。
本发明的第二个目的是提供一种用于制备低硫苷含量发酵菜粕的发酵剂,包括植物乳杆菌JUN-DY-6、中性蛋白酶和纤维素酶;所述发酵剂中纤维素酶与中性蛋白酶的酶活之比为(3-4):(12-15);所述植物乳杆菌JUN-DY-6的菌浓≥107CFU/g或≥107CFU/mL。
本发明的第三个目的是提供上述发酵剂或上述方法在生物饲料防腐方面的应用。
在本发明的一种实施方式中,所述应用是先利用上述的降解菜粕的方法进行发酵,再将发酵菜粕添加到动物饲料中抑制金黄色葡萄球菌、沙门氏菌和/或大肠杆菌的繁殖。
本发明的第四个目的是提供上述发酵剂或上述方法在制备饲料中的应用。
在本发明的一种实施方式中,所述应用用于提高饲料的有机酸含量并降低硫苷含量。
在本发明的一种实施方式中,所述有机酸包括乳酸、柠檬酸、苹果酸的一种或多种。
在一种实施方式中,所述饲料以所述发酵菜粕为组分。
本发明的有益效果:
(1)本发明提供的植物乳杆菌菌酶协同发酵菜粕方法,不仅大大提高了菜粕的抑菌能力和有机酸(尤其是乳酸)的含量,还有效地减少了菜粕中硫苷含量,提高适口性,这让菜粕成为饲料添加剂变成了可能;
(2)本发明制备的发酵菜粕中的乳酸含量较未发酵之前提高了4.2倍,发酵剂产酸能力适中,不会导致菜粕的过分酸化;
(3)本发明制备的发酵菜粕中的硫苷含量与菜粕原料相比,降低了38.25%,大大降低了菜粕的毒性;
(4)本发明的方法还提高了发酵菜粕的有机酸含量和小肽含量,提高了菜粕的附加值,有利于废弃物再利用。
附图说明
图1是植物乳杆菌JUN-DY-6在平板培养基形态。
图2是植物乳杆菌JUN-DY-6在MRS培养基中的生长曲线。
图3是植物乳杆菌JUN-DY-1~JUN-DY-6抑菌实验抑菌圈。
具体实施方式
(一)菌株抑菌圈直径的测定方法
指示菌菌液的制备:将大肠杆菌、沙门氏菌和金黄葡萄球菌三种指示菌接种于LB液体培养基,37℃培养24h。
采用牛津杯法:取直径约90mm的平板,分别倾注加热融化的营养琼脂培养基15-20mL,使其在平板内均匀摊布,放置水平台面上使凝固,作为底层。另取半固体营养琼脂培养基(琼脂含量为1%)适量加热融化后,放冷至48-50℃,每50-100mL培养基加入指示菌的菌悬液0.1-0.2mL,在每个平板中分别加5mL,使其在底层上均匀摊布,作为菌层。在每个平板中以等距离均匀安置牛津杯4-5个备用,每个双层平板中的牛津杯中分别滴装200μL乳酸菌上清液,37℃培养10-13h后,测量各抑菌圈直径以作出评价。
(二)有机酸含量测定:用紫外法测定发酵上清液中有机酸的含量,有机酸标样1g/L,有机酸柱(Aninex Hpx-87H ion exchange column)柱温30℃,流动相5mmol/L H2SO4溶液,流速0.6mL/min,进样量20μL,标样及样品跑14min。输出图谱进行分析。根据出峰时间和峰面积和标准品比对,计算样品中各种有机酸的含量。
(三)硫苷含量测定:准确称取菜粕100mg,移入10mL具塞刻度试管中,沸水浴中干蒸10min,加沸蒸馏水6~8ml,再蒸煮30min。取出冷却后定容至10mL,过滤。取滤液2mL于10mL比色管,加4mL0.15的羧甲基纤维素钠,摇匀后再加2mL8mmol/L的氯化钯显色溶液,在22±3℃放置2h,在分光光度计540mm处,以氯化钯-羧甲基纤维素钠空白溶液作参比溶液,测其消光度。
(四)小肽含量测定:将待测样品过80目筛,等体积加入10%三氯乙酸(TCA),在160r/min下30min,而后4000r/min离心15min,取上清液做适当稀释后用Folin-酚法测定蛋白质总量。
实施例1:菌株的筛选
从实验室保藏的15株乳酸菌中筛选出抑菌和降低硫苷效果好的革兰氏阳性菌株。筛选方法如下所述:
1、配制MRS培养基:蛋白胨10.0g,牛肉膏8.0g,酵母粉4.0g,葡萄糖20.0g,磷酸氢二钾2.0g,柠檬酸三铵2.0g,乙酸钠5.0g,七水硫酸镁0.58g,四水硫酸锰0.25g,吐温801mL,蒸馏水1L,115℃灭菌20分钟;
2、将15株乳酸菌分别接种于MRS培养基中,37℃,200r·min-1培养24h,8000r·min-1离心15min,测定发酵上清液的抑菌活性。
3、根据菌株抑菌活性,一共获得5株抑菌效果较好的乳酸菌,由表1可以看出JUN-DY-6菌株能有效抑制金黄色葡萄球菌、大肠杆菌、沙门氏菌,抗菌谱广,且在五株乳酸菌中对大肠杆菌和金黄色葡萄球菌抑制效果最佳,对沙门氏菌的抑菌效果仅次于DY1菌株。
4、将待筛选的菌株接种于MRS培养基中,培养至OD=1,按照相同的量转接于含硫苷的菜粕中培养48h,测定菌株发酵前后菜粕中的硫苷含量,计算硫苷降解能力,结果如表1所示。植物乳杆菌JUN-DY-6表现出较强的对硫苷的降解能力和对致病菌的抑制能力,硫苷的降解率达50.23%,对大肠杆菌、沙门氏菌和金黄色葡萄球菌的抑菌圈直径达13.18~16.08mm。
表1不同乳酸菌的抑菌结果
注:所述植物乳杆菌(Lactobacillus plantarum)JUN-DY-6(简写为植物乳杆菌DY6),已于2017年6月5日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M2017138,并公开于公开号为CN107446852A的专利申请中。
实施例2发酵剂的制备
制备植物乳杆菌JUN-DY-6菌液:将植物乳杆菌JUN-DY-6接种于MRS培养基中,37℃,200r·min-1培养24h,获得植物乳杆菌菌液。可选地,向菌液中加入适量保护剂,进行冷冻干燥制备菌粉。
将植物乳杆菌JUN-DY-6、中性蛋白酶和纤维素酶混合,制备发酵剂,使发酵剂中纤维素酶与中性蛋白酶的酶活之比为(3-4):(12-15);使植物乳杆菌JUN-DY-6的菌浓≥107CFU/g或≥107CFU/mL。
发酵剂还含有辅料,所述的辅料可选用本领域常规的辅料,较佳地包括水、乳糖、蔗糖、麦芽糊精、谷氨酸钠、明胶、甘油、山梨醇、海藻糖、酵母膏和β-环糊精中的一种或多种。
实施例3菌酶协同发酵菜粕
(1)将水、植物乳杆菌JUN-DY-6、纤维素酶、蛋白酶添加到菜粕中进行发酵;使菜粕中的水分的含量为50%(m/m),所述纤维素酶的含量为400U/g底物,所述蛋白酶为中性蛋白酶,所述蛋白酶含量为1500U/g底物,所述植物乳杆菌JUN-DY-6按照实施例2的方法培养,并按照5%(v/m,mL/g)的接种量将菌液加入至底物中,使菌浓达1×106CFU/g底物。
将加入蛋白酶并接种植物乳杆菌后的菜粕在37℃下发酵48h。测定发酵菜粕中小肽含量为85.75mg/g、硫苷含量15.98μmol·g-1、总酸含量5.35%,乳酸含量2.16g·L-1,小肽含量提高41.59%,硫苷含量降低53.27%,总酸含量提高19.58倍,乳酸含量提高3.24倍。
实施例4菌酶协同发酵菜粕
将水、植物乳杆菌JUN-DY-6、纤维素酶、蛋白酶添加到菜粕中进行发酵;所述水分的含量为50%(m/m),所述纤维素酶的含量为400U/g底物,所述蛋白酶为碱性蛋白酶,所述蛋白酶含量为1200U/g底物,所述植物乳杆菌JUN-DY-6按照实施例2的方法培养,并按照3%(v/m,mL/g)的接种量将菌液加入至底物中,使菌浓达1×106CFU/g底物。
将加入蛋白酶并接种植物乳杆菌的菜粕在37℃下发酵48h。测定发酵菜粕中小肽含量为80.48mg/g、硫苷含量15.98μmol·g-1、总酸含量4.85%,乳酸含量1.95g·L-1(表2),小肽含量提高32.89%,硫苷含量降低43.80%,总酸含量提高17.63倍,乳酸含量提高2.82倍。
实施例5菌酶协同发酵菜粕
将水、植物乳杆菌JUN-DY-6、纤维素酶、蛋白酶添加到菜粕中进行发酵;所述水分的含量为50%(m/m),所述纤维素酶的含量为300U/g底物,所述蛋白酶为中性蛋白酶,所述蛋白酶含量为1350U/g底物,所述植物乳杆菌JUN-DY-6按照实施例2的方法培养,并按照3%(v/m,mL/g)的接种量将菌液加入至底物中,使菌浓达1×106CFU/g底物。
将加入蛋白酶并接种植物乳杆菌的菜粕在37℃下发酵60h。测定发酵菜粕中小肽含量为85.88mg/g、硫苷含量15.10μmol·g-1、总酸含量5.45%,乳酸含量2.31g·L-1(表2),小肽含量提高41.80%,硫苷含量降低55.84%,总酸含量提高19.96倍,乳酸含量提高3.53倍。
实施例6菌酶协同发酵菜粕
将水、植物乳杆菌JUN-DY-6、纤维素酶、蛋白酶添加到菜粕中进行发酵;所述水分的含量为50%(m/m),所述纤维素酶的含量为350U/g底物,所述蛋白酶为中性蛋白酶,所述蛋白酶含量为1350U/g底物,所述植物乳杆菌JUN-DY-6按照实施例2的方法培养,并按照3%(v/m,mL/g)的接种量将菌液加入至底物中,使菌浓达1×106CFU/g底物。
将加入蛋白酶并接种植物乳杆菌的菜粕在40℃下发酵48h。测定发酵菜粕中小肽含量为113.21mg/g、硫苷含量26.32μmol·g-1、总酸含量2.29%,乳酸含量1.12g·L-1(表2),小肽含量提高86.94%,硫苷含量降低23.04%,总酸含量提高10.26倍,乳酸含量提高1.20倍。
实施例7不同条件制备的发酵菜粕成分比较
(1)发酵前后总酸和有机酸含量的变化
酸化剂可以降低饲料pH值,使胃内pH值下降,提高消化酶的活性。酸化剂在构建禽畜健康的肠道菌群的能力不如有机酸,为了禽畜的抗病性能,常常在饲料中添加过量的酸化剂,而这一方面影响了饲料的适口性,一方面增加了成本。
本发明用菌酶协同发酵菜粕后得到的发酵产物乳酸代替酸化剂能很好地弥补酸化剂在构建健康肠道菌群能力方面的不足。测定实施例2中所得发酵上清液中的总酸和有机酸含量,发现总酸和有机酸含量显著提高(见表2)。其中总酸含量最高可提高19.96倍,乳酸含量最高可提高3.5倍。
(2)菌酶协同发酵前后硫苷含量的变化
硫苷是菜粕的主要抗营养因子,限制了菜粕的可饲用性,而微生物发酵法能降低菜粕中硫苷含量,降低菜粕的毒性,测定实施案例2中发酵菜粕上清液中硫苷含量可知,通过菌酶协同发酵后菜粕中硫苷含量显著降低(表2),可降低55.84%。
(3)菌酶协同发酵前后小肽含量的变化
小肽含量的提高主要源于蛋白酶对菜粕中大分子蛋白质的酶解,通过不同的蛋白酶降解效果对比,测定发酵菜粕中的小肽含量,实施案例2(4)最高可提高86.94%。
表2发酵前后菜粕中各种物质含量的变化
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (5)
1.一种制备低硫苷发酵菜粕的方法,其特征在于,将植物乳杆菌JUN-DY-6、中性蛋白酶、纤维素酶和菜粕混合,同步进行酶解和发酵;所述菜粕的水分含量为30-50%,所述纤维素酶的用量为300-400 U/g菜粕,所述中性蛋白酶的用量为1350-1500 U/g菜粕;所述植物乳杆菌JUN-DY-6的保藏编号为CCTCC NO:M 2017138,菌体浓度为105-108 CFU/g菜粕;发酵的条件为:在35℃-37℃发酵40-60 h。
2.一种低硫苷菜粕发酵剂,其特征在于,由保藏编号为CCTCC NO:M 2017138的植物乳杆菌JUN-DY-6、中性蛋白酶和纤维素酶组成;所述发酵剂中纤维素酶与中性蛋白酶的酶活之比为(3-4):(12-15);所述植物乳杆菌JUN-DY-6的菌体浓度≥107 CFU/g或≥107 CFU/mL。
3.权利要求1所述方法制备的低硫苷含量的发酵菜粕。
4.权利要求2所述的发酵剂或权利要求1所述的方法在制备饲料中的应用。
5.一种饲料,其特征在于,以权利要求3所述的发酵菜粕为组分。
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