CN116694539A - 直投氏发酵剂植物乳杆菌j26及其制法和应用 - Google Patents
直投氏发酵剂植物乳杆菌j26及其制法和应用 Download PDFInfo
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- CN116694539A CN116694539A CN202310965095.XA CN202310965095A CN116694539A CN 116694539 A CN116694539 A CN 116694539A CN 202310965095 A CN202310965095 A CN 202310965095A CN 116694539 A CN116694539 A CN 116694539A
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- lactobacillus plantarum
- fermentation
- acid
- soybean milk
- starter
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Abstract
本发明公开了一种直投氏发酵剂植物乳杆菌J26及其制法和应用,制法包括以下步骤:将植物乳杆菌J26活化,按4%的接种量接种至100mL优化碳氮源培养基中,在33℃、pH5.6条件下采用5L发酵罐进行高密度发酵24h,在4~24h恒速流加补料,补料速率为15mL/h,补料培养基为优化碳源:优化氮源=5:2;高密度发酵完成后,收获菌液,冻干,得到冻干菌粉。本发明添加复合益生元与直投氏发酵剂植物乳杆菌J26的组合可以促进豆乳发酵,显著提高发酵豆乳的活菌数,减缓发酵乳的后酸化,并改善其持水力和质构。
Description
技术领域
本发明涉及一种直投氏发酵剂植物乳杆菌J26及其制法和应用,尤其涉及一种直投氏发酵剂植物乳杆菌J26、豆乳发酵剂、合生元发酵豆乳及其制法和应用,具体涉及一种直投氏发酵剂植物乳杆菌J26及其高密度发酵工艺与合生元发酵豆乳及其制法,属于微生物技术领域。
背景技术
发酵乳制品在人们日常生活中占有显著的地位,发酵乳制品对机体具有营养作用及良好的益生作用。目前直投式乳酸菌发酵剂具有活菌数含量高等优点,可以直接接种用于生产发酵乳制品,然而由于技术等问题,使得现有的直投式乳酸菌发酵剂发酵乳制品的质量难以保证,严重制约了我国发酵乳制品行业的健康发展。而传统高密度发酵工艺会导致菌体干重少,菌液活菌数低等缺点,因此,提出一种直投氏发酵剂植物乳杆菌J26及其高密度发酵工艺是十分有必要的。
豆乳是中国被大众熟知的传统乳制品,受到消费者的广泛喜爱。目前单一菌种(如鼠李糖乳杆菌)发酵产品仍然存在许多问题,如发酵豆乳的后酸化情况严重,持水力和质构等感官品质差,益生功能较为单一,其中总酚、游离氨基酸含量低,抗氧化活性不好等较为普遍,因此,利用菌种的发酵优势,通过添加益生元等辅助发酵剂,制备一种合生元发酵豆乳,从而改善豆乳品质,进而提高营养价值和生理功能是十分重要的,为后续工业生产提供理论支撑。
发明内容
本发明的目的是为了提供一种直投氏发酵剂植物乳杆菌J26的制法,可获得具有良好发酵特性的直投氏发酵剂。
同时,本发明提供一种直投氏发酵剂植物乳杆菌J26,该直投氏发酵剂可使活菌数由4.35×1010CFU/mL增长为7.82×1011CFU/mL,菌体干重由49g/L增长为62g/L。
同时,本发明提供一种包含直投氏发酵剂植物乳杆菌J26的豆乳发酵剂,该豆乳发酵剂发酵特性好,获得的豆乳总酚含量高、游离氨基酸含量高、体外抗氧化活性好,具有良好质构特性,持水性好,可以防止乳清析出,有利于产品贮藏及风味保留。
同时,本发明提供一种合生元发酵豆乳的制法,该法获得的豆乳活菌数高,感官评价综合得分高。
同时,本发明提供一种高质量的合生元发酵豆乳。
同时,本发明提供一种直投氏发酵剂植物乳杆菌J26在直接接种生产发酵乳制品中的应用。
为解决上述技术问题,本发明采用的技术方案为:
一种直投氏发酵剂植物乳杆菌J26的制法,包括以下步骤:将植物乳杆菌J26活化,按4%的接种量接种至100mL优化碳氮源培养基中,在33℃、pH5.6条件下采用5L发酵罐进行高密度发酵24h,在4~24h恒速流加补料,补料速率为15mL/h,补料培养基为优化碳源:优化氮源=5:2;高密度发酵完成后,收获菌液,冻干,得到冻干菌粉。
冻干菌粉中添加可接受辅料。可接受辅料选自:漂白剂、防腐剂、抗氧化剂、着色剂、甜味剂、酸味剂、增味剂、护色剂等。
优化碳氮源培养基和补料培养基中,优化碳源为25g/L麦芽糖与葡萄糖按照1:1的质量比例混合,优化氮源为10g/L牛肉膏与酵母粉按照1:1的质量比例混合,其余成分、用量与MRS液体培养基中的成分、用量相同(即本发明的优化碳氮源培养基中,用25g/L、1:1的麦芽糖与葡萄糖替换MRS液体培养基中的碳源葡萄糖,用10g/L、1:1的牛肉膏与酵母粉替换MRS液体培养基中的氮源蛋白胨,其余同MRS液体培养基)。
一种直投氏发酵剂植物乳杆菌J26的制法获得的直投氏发酵剂植物乳杆菌J26。
一种包含直投氏发酵剂植物乳杆菌J26的豆乳发酵剂,包括8wt%的复合益生元、1×106CFU/mL的直投氏发酵剂植物乳杆菌J26、6wt%的哈密瓜果酱、2wt%的燕麦和余量的豆奶。
哈密瓜果酱包括哈密瓜原浆74.2%、白砂糖25%、果胶:黄原胶(2:1)的混合物0.3%和柠檬酸0.5%。
复合益生元包括大麦β-葡聚糖:莲子寡糖:菊粉=2:1:1。
一种合生元发酵豆乳的制法,包括以下步骤:
步骤一,在豆奶中加入复合益生元并混匀,再进行超高压杀菌处理,待冷却至室温时添加直投氏发酵剂植物乳杆菌J26,进行发酵豆乳;
步骤二,发酵完成后于4℃后熟24h;
步骤三,4℃完成后,再添加6wt%的哈密瓜果酱和2wt%的燕麦,混匀,即得。
超高压杀菌处理的工艺为:在530MPa下进行10min。
发酵豆乳的工艺为:37℃培养箱发酵,以pH4.5为发酵终点。
一种合生元发酵豆乳的制法获得的合生元发酵豆乳,合生元发酵豆乳中,总酚含量为 94.92μg/g,游离氨基酸含量为0.57mg/mL。
一种直投氏发酵剂植物乳杆菌J26在直接接种生产发酵乳制品中的应用,发酵乳制品包括豆乳、常温酸奶、低温酸奶、搅拌型酸奶、凝固型酸奶、饮用性酸奶、乳酪和乳酸菌饮料。
本发明具有以下有益效果:
本发明提供一种植物乳杆菌J26的高密度发酵工艺,获得的直投氏发酵剂植物乳杆菌J26用于制备冻干菌粉,并且可作为具有良好发酵特性的直投氏发酵剂,用于发酵豆乳,体外消化特性的应用。
本发明的高密度发酵工艺通过Biolog GEN Ⅲ Microstation自动微生物鉴定系统结果分析植物乳杆菌J26对氮源及碳源的利用情况,通过优化培养基中的碳、氮源并在发酵过程以流加补料的方式补充碳、氮源,并对发酵过程中pH、温度、接种量等进行优化,获得的菌液活菌数高,菌体干重显著提高。
本发明公开了植物乳杆菌J26高密度发酵的工艺条件优化及其冻干菌粉作为直投氏发酵剂用于发酵豆乳,植物乳杆菌J26冻干菌粉作为直投氏发酵剂用于豆乳的发酵,通过添加来自天然植物中提取得到益生元(大麦β-葡聚糖、莲子寡糖、菊粉),添加量为8wt%复合益生元(大麦β-葡聚糖:莲子寡糖:菊粉=2:1:1)与植物乳杆菌J26共同发酵一种新型合生元豆乳,对合生元豆乳进行超高压杀菌处理在530MPa下进行10min,超高压杀菌对豆乳品质的保持有显著优势,研究发现合生元豆乳中总酚、游离氨基酸含量显著升高,通过增加总酚含量从而显著提高了体外抗氧化活性(DPPH、ABTS自由基清除率、ACE抑制率),对新型合生元豆乳未来对于继续研究体内抗氧化活性奠定基础,在合生元豆乳中添加6wt%的哈密瓜果酱和2wt%的燕麦,研究发现通过添加哈密瓜果酱可以使豆乳呈哈密瓜果香及特有的浓郁的发酵豆香味,添加燕麦可以丰富豆乳口感,具有发酵豆乳特有的滋味,使哈密瓜燕麦风味合生元豆乳感官评分达95分,对合生元发酵豆乳的质构、持水力、pH、酸度、活菌数分别进行检测,研究发现,与不同复配比例益生元及单种益生元与直投氏发酵剂植物乳杆菌J26发酵豆乳对比,添加复合益生元(大麦β-葡聚糖:莲子寡糖:菊粉=2:1:1)与直投氏发酵剂植物乳杆菌J26的组合可以促进豆乳发酵,显著提高了发酵豆乳的活菌数,减缓了发酵乳的后酸化,并改善了其持水力和质构。主要增加了发酵乳的黏稠度和表面光滑度,发酵出具有良好发酵特性豆乳。研究表明通过添加复合益生元与直投氏发酵剂植物乳杆菌J26制备得新型合生元豆乳可以使总酚、游离氨基酸含量显著升高,从而显著提高了体外抗氧化活性,并且添加哈密瓜果酱及燕麦可以显著提高发酵豆乳风味及口感,发酵特性优于市售豆乳,为新型风味良好的合生元豆乳提供新的研究思路。
附图说明
图1为植物乳杆菌J26生长曲线;
图2为Biolog GEN Ⅲ Microstation自动微生物鉴定系统结果分析植物乳杆菌J26对碳源的利用情况;
图3为Biolog GEN Ⅲ Microstation自动微生物鉴定系统结果分析植物乳杆菌J26对氮源的利用情况;
图4为植物乳杆菌J26最佳碳、氮源利用情况;
其中,A是在不同碳源发酵终点的活菌数,B是在不同氮源发酵终点的活菌数,C是不同浓度最适碳源的活菌数,D是不同浓度最适氮源的活菌数;
图5为植物乳杆菌J26的最适接种量;
图6为植物乳杆菌J26的最适温度;
图7为植物乳杆菌J26的最适pH值;
图8为流加补料对植物乳杆菌J26生长曲线的影响;
图9复合益生元对合生元豆乳总酚含量的影响;
图10复合益生元对合生元豆乳游离氨基酸含量的影响;
图11复合益生元对合生元豆乳pH的影响;
图12复合益生元对合生元豆乳酸度值的影响;
图13复合益生元对合生元豆乳持水力的影响。
具体实施方式
下面结合附图对本发明作更进一步的说明。
本发明中,植物乳杆菌J26即为植物乳杆菌NDC75017,发明人通过菌种保藏中心购买得到。植物乳杆菌NDC75017的分类命名为植物乳杆菌Lactobacillus plantarum,保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),北京,保藏日期为2011年11月08日,保藏编号为CGMCC NO.5448;植物乳杆菌J26简称Lactobacillus plantarumJ26、L. plantarum J26、LJ26或J26。
一种直投氏发酵剂植物乳杆菌J26冻干菌粉的制备:将存放于-20℃冰箱的植物乳杆菌J26活化,按3%的接种量接种至100mL市售MRS液体培养基(蛋白胨10g ,牛肉浸粉 5g,吐温80 1mL,磷酸氢二钾2g,酵母浸粉 4g,硫酸镁0.2g,柠檬酸三铵2g,葡萄糖 20g,硫酸锰0.05g,乙酸钠5g和余量的水)中,在37℃下培养20h。如图1所示,每隔2h测定一次菌液的OD600值,以时间为横坐标,OD600值为纵坐标建立菌株的生长曲线。在此条件下,采用5L发酵罐进行高密度发酵,高密度发酵工艺通过Biolog GEN Ⅲ Microstation自动微生物鉴定系统结果分析植物乳杆菌J26对氮源及碳源的利用情况,通过优化培养基中的碳、氮源并在发酵过程以流加补料的方式补充碳、氮源,并对发酵过程中pH、温度、接种量等进行优化,测定菌液活菌数及菌体干重。收获菌液用真空干燥冻干机进行冻干得到冻干菌粉。
直投氏发酵剂植物乳杆菌J26冻干菌粉发酵豆乳的制备:将豆奶与8wt%不同复配比例益生元及单种益生元混匀,对其进行超高压杀菌处理,在530MPa下进行10min,待冷却至室温时,添加6wt%的哈密瓜果酱和2wt%的燕麦,对添加入不同复配比例益生元及单种益生元与直投氏发酵剂植物乳杆菌J26发酵的合生元豆乳、未添加益生元和市售豆乳(鼠李糖乳杆菌发酵)的总酚含量、游离氨基酸含量、体外抗氧化活性(DPPH、ABTS自由基清除率、ACE抑制率)进行测定。将添加8wt%不同复配比例益生元及单种益生元与直投氏发酵剂植物乳杆菌J26发酵的合生元豆乳、未添加益生元和市售豆乳对比,对不同组发酵豆乳的质构、持水力、pH、酸度、活菌数分别进行检测,比较其发酵特性,优选出植物乳杆菌J26是具有发酵特性良好的直投氏发酵剂。
一、材料与方法
1.植物乳杆菌J26碳、氮源利用:
通过Biolog GEN Ⅲ Microstation自动微生物鉴定系统结果分析植物乳杆菌J26对氮源及碳源的利用情况,用D-甘露醇、D-麦芽糖、蔗糖、葡萄糖依次代替培养基的碳源,并用D-甘露醇、D-麦芽糖、蔗糖、葡萄糖进行碳源混合实验,用酵母粉、牛肉膏、蛋白胨、大豆蛋白、L-半胱氨酸、四氧嘧啶依次代替MRS液体培养基中的主要氮源(蛋白胨),并用酵母粉、牛肉膏、蛋白胨、大豆蛋白、L-半胱氨酸、四氧嘧啶进行氮源混合实验。碳源如下分组实验:(1)按20g/L浓度,添加量:D-甘露醇:D-麦芽糖=1:1添加;(2)按20g/L浓度,添加量:D-甘露醇:蔗糖=1:1添加;(3)按20g/L 浓度,添加量:D-甘露醇:葡萄糖=1:1添加;(4)按20g/L浓度,添加量:D-麦芽糖:蔗糖=1:1添加;(5)按20g/L 浓度,添加量:D-麦芽糖:葡萄糖=1:1添加;(6)按20g/L 浓度,添加量:蔗糖:葡萄糖=1:1添加,按3%接种量接种于含有培养基的250mL的锥形瓶中,初始pH值为6.2,在37℃恒温培养箱中静置发酵24h,然后用发酵罐发酵,测定菌液发酵终点活菌数。筛选出最适碳源后,考察当发酵完成时最适碳源添加量(20g/L、25g/L、30g/L、35g/L、40g/L)对菌液活菌数的影响。
对于氮源,10g/L的酵母粉、牛肉膏、蛋白胨、大豆蛋白、L-半胱氨酸、四氧嘧啶依次代替MRS液体培养基中的主要氮源,进行如下分组实验:(1)按10g/L浓度,添加量:酵母粉:牛肉膏=1:1添加;(2)按10g/L浓度,添加量:酵母粉:蛋白胨=1:1添加;(3)按10g/L浓度,添加量:酵母粉:大豆蛋白=1:1添加;(4)按10g/L浓度,添加量:牛肉膏:蛋白胨=1:1添加;(5)按10g/L浓度,添加量:牛肉膏:大豆蛋白=1:1添加;(6)按10g/L 浓度,添加量:蛋白胨:大豆蛋白=1:1添加,以优化氮源后的MRS培养基作为对照。测定菌液发酵终点活菌数,筛选出最适氮源。在此基础上,考察当发酵完成时最适氮源添加量(10 g/L、15 g/L、20 g/L、25 g/L、30 g/L)对菌液活菌数的影响。
筛选出最适碳氮源后,考察当发酵完成时碳氮源添加量对菌体密度的影响。
2.接种量的影响
选择不同接种量3%、4%、5%,6%接种种子液于发酵培养基中,初始pH5.6置于恒温箱内静置培养 14h,测定培养液的菌落数,初始pH用氨水溶液调节。
3.温度的影响
按最适接种量接种种子液于发酵培养基中,初始pH5.6,分别在30、33、36、40℃静置培养14h,测定其菌落数。
4.pH的影响
选择pH值分别为5.0、5.2、5.4、5.6、5.8、6.0在5L发酵罐内分批发酵,按最佳接种量接种,最佳温度培养,搅拌速度控制在150r/min,每隔2h测定菌落数,选择其最佳pH值。
5.流加补料的影响
补料培养基为麦芽糖与葡萄糖(1:1):牛肉膏与酵母粉(1:1)=5:2的质量比混合添加,通过流加补料的方式即从发酵2h开始补料,在2~24h恒速流加,补料速率为15mL/h;通过与未补料生长曲线对比,观察最适合的补料时间,测定补料及未补料菌体干重及活菌数。
菌体干重测定:取25mL发酵液,于6000rpm离心15min,弃上清。加入同体积的蒸馏水(即25mL蒸馏水),6000rpm离心15min,取沉淀烘干至恒重,称量记录。
6.在豆奶中分别加入不同复配比例益生元及单种益生元并混匀,对其进行超高压杀菌处理,在530MPa下进行10min,待冷却至室温时添加菌体,进行发酵豆乳,对添加不同复配比例益生元、单种益生元与直投氏发酵剂植物乳杆菌J26发酵的合生元豆乳、未添加益生元的直投氏发酵剂植物乳杆菌J26发酵的豆乳和市售豆乳(鼠李糖乳杆菌发酵)的总酚含量、游离氨基酸含量、体外抗氧化活性(DPPH、ABTS自由基清除率、ACE抑制率)进行测定。
7.在豆乳中添加6wt%的哈密瓜果酱和2wt%的燕麦进行感官评价,感官评价由20名接受过专业感官分析培训的食品从业人员分别对样品的色泽(25分)、组织状态(25分)、气味(25分)和滋味(25分)进行评价。
8.对添加不同复配比例益生元、单种益生元与直投氏发酵剂植物乳杆菌J26发酵的合生元豆乳、未添加益生元的直投氏发酵剂植物乳杆菌J26发酵的豆乳和市售豆乳(鼠李糖乳杆菌发酵)的特性进行比较,通过发酵豆乳的质构、持水力、pH、酸度、活菌数分别进行检测,优选出益生元的最佳复配比。
二、实验结果
1.植物乳杆菌J26碳、氮源种类及其添加量的确定
如图2所示,植物乳杆菌J26对碳源底物利用情况:A-1阴性对照孔作为参照,植物乳杆菌J26对D-麦芽糖、D-纤维二糖、蔗糖、D-甘露醇中碳源底物有利用能力,并且利用效果最好;其次,对D-甘露糖、 α-D-葡糖、 N-乙酰-D-葡糖也有较好的利用;同时,对D-果糖、β-甲酰-D-葡糖苷、D-水杨苷表现出一定的可利用性,呈阳性反应;对其他碳源利用能力明显较低。(通过色度的变化来指示微生物对碳源e的利用,其中颜色越深、数值越高的孔表示对碳源利用程度越高),A-10阳性对照孔作为参照,植物乳杆菌J26对化学物较敏感,尤其是乳酸钠化学物质的敏感性最显著;其次,对1% NaCl、万古霉素、四唑紫、四唑蓝、亚碲酸钾有一定的敏感性。
下表1为与图2中代表的各个碳源符号相对应的成分。
表1 Biolog GEN Ⅲ Microstation自动微生物鉴定系统分析的碳源成分表
A1NegativeControl阴性对照 | A2Dexrin糊精 | A3D-Maltose | A4D-TrehaloseD-海藻糖 | A5D-CellobioseD-纤维二塘 | A6Gentiobiose龙胆二糖 | A7Sucrose蔗糖 | A8D-TuranoseD-松二糖 | A9Stachyose水苏糖 | A10PositiveControl阳性对照 | A11pH6 | A12pH5 |
B1D-Raffinose蜜三糖,棉子糖 | B2α-D-Lactoseα-D-乳糖 | B3D-Melibiose蜜二糖 | B4β-Methyl-D-Glucosideβ-甲基-D-葡糖苷 | B5D-SallcinD-水杨苷 | B6N-Acetyl-D-GlucosamineN-乙酰-D-葡糖胺 | B7N-Acetyl-β-DMannosamineN-乙酰-β-D-甘露糖胺 | B8N-Acetyl-D-GalactosamineN-乙酰-D-半乳糖胺 | B9N-AcetylNeuraminicAcidN-乙酰神经氨酸 | B101%NaCl | B114%NaCl | B128%NaCl |
C1a-D-Glucose a-D-葡糖 | C2D-MannoseD-甘露糖 | C3D-Fructose D-果糖 | C4D-Galactose D-半乳糖 | C53-MethylGlucose3-甲酰葡糖 | C6D-Fucose D-岩藻糖 | C7L-FucoseL-岩藻糖 | C8L-Rhamnose L-鼠李糖 | C9Inosine肌苷 | C101%SodiumLactate乳酸钠 | C11FusidicAcid梭链孢酸 | C12D.SerineD-丝氨酸 |
D1D-Sorbitol D-山梨醇 | D2D-MannitolD-甘露醇 | D3D-Arabitol D-阿拉伯醇 | D4myo-Inositol肌醇 | D5Glycerol甘油 | D6D-Glucose6-P04D-葡糖-6-磷酸 | D7D-Fructose-6-P04D-果糖-6-磷酸 | D8D-AsparticAcidD·天冬氨酸 | D9D-Serine D-丝氨酸 | D10Troleandomycin酷竹桃毒素 | D11RifamycinSV利福霉素SV | D12Minocycline二甲胺四环素 |
E1Gelatin明胶 | E2Glycy-L-Proin e氨基乙酰-L-脯氨酸 | E3L-Alanine L-丙氨酸 | E4L-Arginine L-精氨酸 | E5L-AsparticAcidL·天冬氨酸 | E6L-GlutamicAcidL-谷氨酸 | E7L-Histidine L-组胺 | E8L-PyroglutamicAcidL-焦谷氨酸 | E9L-Serine L丝氨酸 | E10Lincomycin林肯霉素,洁霉素 | E11GuanidineHC盐酸胍 | E12Niaproof4硫酸四癸钠 |
F1Pectin果胶 | F2D-GalacturonicAcidD-半乳糖醛酸 | F3L-GalactonicAcidLactone L-半乳糖醛酸内 | F4D-GluconicAcidD-葡糖酸 | F5D-GlucuronicAcidD-葡糖醛酸 | F6Glucuronamide葡糖醛酰胺 | F7Mucic Acid粘酸:粘液酸 | F8QuinicAcid奎宁酸 | F9D-SaccharicAcid糖质酸 | F10Vancomycin万古霉素 | F11TetrazoliumViolet四唑紫 | F12TetrazoiumBlue四唑蓝 |
G1p-Hydroxy.PhenylaceticAcidp-羟基-苯乙酸 | G2MethylPyruvate丙酮酸甲酯 | G3D-LacticAcidMethylEsterD-乳酸甲酯 | G4L-LacticAcidL·乳酸 | G5CitricAcid柠檬酸 | G6a-Keto-GlutaricAcida-酮·戊二酸 | G7D-MalicAcidD-苹果酸 | G8L-MalicAcidL-苹果酸 | G9Bromo-SuccinicAcid澳丁二酸 | G10NalidixicAcid蔡啶酮酸 | G11LithiumChloride氯化锂 | G12PotassiumTellurite亚碲酸钾 |
H1Tween 40吐温40 | H2y-Amino-ButryricAcidV-氨基丁酸 | H3a-Hydroxy-Butyric Acida·羟基·丁酸 | H4B-Hydroxy-D.LButyric AcidB-羟基-D.L丁酸 | H5a-Keto-ButyricAcida-酮·丁酸 | H6AcetoaceticAcid乙酰乙酸 | H7PropionicAcid丙酸 | H8AceticAdid乙酸 | H9FormicAcid甲酸 | H10Aztreonam氨曲南 | H11SodiumButyrate丁酸钠 | H12SodiumBromate溴酸钠 |
如图3所示,植物乳杆菌J26 对氮源底物利用情况:A-1阴性对照孔作为参考,植物乳杆菌J26菌株对L-半胱氨酸、鸟嘌呤、四氧嘧啶有利用这些氮源底物的能力,并且利用效果最好,其次,对D,L-2-氨基辛酸也有较好的利用,呈阳性反应。(通过色度的变化来指示微生物对氮源e的利用,其中颜色越深、数值越高的孔表示对氮源利用程度越高)。
下表2为与图3中代表的各个氮源符号相对应的成分。
表2 Biolog GEN Ⅲ Microstation自动微生物鉴定系统分析的氮源成分表
A1NegativeControl阴性对照 | A2Ammonia氨 | A3Nitrite亚硝酸盐 | A4Nitrate硝酸盐 | A5Urea尿素 | A6Biuret双缩脲 | A7L-AlanineL-丙氨酸 | A8L-ArginineL-精氨酸 | A9L-AsparagineL-天冬酰胺 | A10L-AsparticAcidL-天冬氨酸 | A11L-CysteineL-半胱氨酸 | A12L-GlutamicAcidL-谷氨酸 |
B1L-GlutamineL-谷氨酰胺 | B2Glycine甘氨酸 | B3L-HistidineL-组氨酸 | B4L-IsoleucineL-异亮氨酸 | B5L-LeucineL-亮氨酸 | B6L-LysineL-赖氨酸 | B7L-NethionineL-蛋氨酸 | B8L-PhenylalanineL-苯丙氨酸 | B9L-ProlineL-脯氨酸 | B10L-SerineL-丝氨酸 | B11L-ThreonineL-苏氨酸 | B12L-TryptophanL-色氨酸 |
C1L-TyrosineL-酪氨酸 | C2L-ValineL-缬氨酸 | C3D-AlanineD-丙氨酸 | C4D-AsparagineD-天冬酰胺 | C5D-AsparticAcidD-天冬氨酸 | C6D-GlutamicAcidD-谷氨酸 | C7LysineD-赖氨酸 | C8D-SerineD-丝氨酸 | C9D-ValineD-缬氨酸 | C10L-CitrullineL-瓜氨酸 | C11L-HomoserineL-高丝氨酸 | C12L-OrnithineL-鸟氨酸 |
D1N-Acetyl-D,L-GlutamicAcidN-乙酰-D,L-谷氨酸 | D2N-Phthaloyl-L-GlutamicAcidN-酞酰-L-谷氨酸 | D3L-PyroglutamicAcidL-焦谷氨酸 | D4Hydroxylamine羟胺 | D5Methylamine甲胺 | D6N-AmylamineN-戊胺 | D7N-ButylamineN-丁胺 | D8Ethylamine乙胺 | D9Ethanolamine乙醇胺 | D10Ethylenediamin乙二胺 | D11Putrescine腐胺 | D12Agmatine胍丁胺 |
E1Histamine组胺 | E2β-Phenylethyl-amineβ-苯乙胺 | E3Tyramine酪胺 | E4Acetamide乙酰胺 | E5Formamide甲酰胺 | E6Glucuronamide葡醛酰胺 | E7D,L-LactamideD,L-乳酰胺 | E8D-GlucosamineD-氨基葡萄糖 | E9D-GalactosamineD-半乳糖胺 | E10D-MannosamineD-甘露糖胺 | E11N-Acetyl-D-GlucosamineN-乙酰-D-葡糖胺 | E12N-Acetyi-D-GalactosamineN-乙酰-D-半乳糖胺 |
F1N-Acetyl-D-MannosamineN-乙酰-D-甘露糖胺 | F2Adenine腺嘌呤 | F3Adenosine腺苷 | F4Cytidine胞苷 | F5Cytosine胞嘧啶 | F6Guanine鸟嘌呤 | F7Guanosine鸟苷 | F8Thymine胸腺嘧啶 | F9Thymidine胸苷 | F10Uracil尿嘧啶 | F11Uridine尿苷 | F12inosine肌苷 |
G1Xanthine黄嘌呤 | G2Xanthosine黄原苷 | G3UricAcid尿酸 | G4Alloxan四氧嘧啶 | G5Allantoin尿囊素 | G6ParabanicAcid对羟基苯甲酸 | G7D,L-α-Amino-N-ButyricAcidD,L-α-氨基-N-丁酸 | G8γ-Amino-N-ButyricAcidγ-氨基-N-丁酸 | G9ε-Amino-N-CaproicAcidε-氨基-N-己酸 | G10D,L-α-Amino-CaprylicAcidD,L-α-氨基辛酸 | G11δ-Amino-N-ValericAcidδ-氨基-N-戊二酸 | G12α-Amino-N-ValericAcidα-氨基-N-戊二酸 |
H1Ala-Asp | H2Ala-Gln | H3Ala-Glu | H4Ala-Gly | H5Ala-His | H6Ala-Leu | H7Ala-Thr | H8Gly-Asn | H9Gly-Gln | H10Gly-Glu | H11Gly-Met | H12Met-Ala |
如图4所示,单一碳源与混合碳源中,麦芽糖与葡萄糖相结合为碳源使植物乳杆菌J26菌体活菌数最高,麦芽糖与葡萄糖混合添加发酵效果最好,活菌数达 2.82×1010CFU/mL。并且确定以25g/L 麦芽糖与葡萄糖混合添加量为最适,活菌数达 2.82×1010CFU/mL。
单一氮源与混合氮源中,牛肉膏与酵母粉相结合为氮源使植物乳杆菌J26活菌数最高,牛肉膏与酵母粉混合添加发酵效果最好,活菌数达 2.67×109CFU/mL。且确定以10g/L牛肉膏与酵母粉混合添加量为最适,活菌数达 2.67×109CFU/mL。
2.植物乳杆菌J26最适接种量的确定
如图5所示,当接种量为4%进行发酵时,获得的活菌数最高,当接种量较低时,菌体密度小,当接种量较高时,菌体密度大,消耗营养物质多,营养物质不足导致延缓菌体生长,接种量过高和过低都不利于菌体的生长。
3.植物乳杆菌J26温度的确定
如图6所示,最佳培养温度为33℃,此时菌体生长繁殖快,发酵温度过低会影响菌体自身酶的活性,温度过高会使细胞内蛋白质变性,温度过高和过低会导致菌体生长发育迟缓。
4.植物乳杆菌J26最适pH 值的确定
如图7所示,在pH为5.6时,菌体冻干后存活因子最高,pH过低时不利于菌体保持活力,当pH高于5.6时,菌体存活因子下降,高于或低于最适pH值都不利于菌体的生长。所以最终选择保护剂pH为5.6。
5.对比补料对菌体生长的影响
如图8所示,由对比实验可知,0~4h补充碳、氮源并不能加速菌体生长,而4h以后,补充碳、氮源可以提高菌体生长且效果显著,所以拟定于发酵4h时开始补料,在4~24h恒速流加,补料速率为15mL/h。由表3可见,通过流加补料的方式补充碳、氮源,通过优化发酵条件,可使活菌数由4.35×1010CFU/mL(此时发酵条件:25g/L麦芽糖与葡萄糖混合添加量、10g/L牛肉膏与酵母粉混合添加量、接种量为4wt%、最佳培养温度为33℃、pH为5.6,未流加补料)增长为7.82×1011CFU/mL(与上述工艺的区别仅在于流加补料),菌体干重由49g/L增长为62g/L,说明流加补料的方式补充碳氮源对菌体生长起重要作用。
表3流加补料对菌体生长的影响
6.复合益生元对合生元豆乳总酚含量的影响
由图9可知,通过添加8wt%复合益生元(大麦β-葡聚糖:莲子寡糖:菊粉=2:1:1)与直投氏发酵剂植物乳杆菌J26制备的合生元豆乳总酚含量为 94.92μg/g,显著高于其他不同复配比例益生元及单种益生元与植物乳杆菌J26制备豆乳,而未添加益生元豆乳及市售豆乳(鼠李糖乳杆菌发酵)其总酚含量分别为 59.62μg/g和50.40μg/g,8wt%复合益生元(大麦β-葡聚糖:莲子寡糖:菊粉=2:1:1)的合生元豆乳总酚含量显著高于未添加益生元豆乳及市售豆乳(鼠李糖乳杆菌发酵)总酚含量(P<0.05)。说明复合益生元的添加,可以显著增加豆乳中总酚含量。
7.复合益生元对合生元豆乳游离氨基酸含量的影响
由图10可知,豆乳在发酵过程中游离氨基酸含量升高。通过添加8wt%复合益生元(大麦β-葡聚糖:莲子寡糖:菊粉=2:1:1)与直投氏发酵剂植物乳杆菌J26制备的合生元豆乳游离氨基酸含量为0.57mg/mL,显著高于其他不同复配比例益生元及单种益生元与植物乳杆菌J26制备豆乳,而未添加益生元豆乳及市售豆乳(鼠李糖乳杆菌发酵)其游离氨基酸含量分别为0.27mg/mL和0.24mg/mL,合生元豆乳游离氨基酸含量显著高于未添加益生元豆乳及市售豆乳(鼠李糖乳杆菌发酵)游离氨基酸含量(P<0.05)。说明复合益生元的添加,可以显著增加豆乳中游离氨基酸含量。
8.复合益生元对合生元豆乳抗氧化活性和ACE抑制率的影响
由表4可知,添加8wt%复合益生元(大麦β-葡聚糖:莲子寡糖:菊粉=2:1:1)与直投氏发酵剂植物乳杆菌J26制备的合生元豆乳的DPPH和ABTS自由基清除率分别为88.98±0.22%、50.67±0.34%显著高于未添加益生元豆乳及市售豆乳(鼠李糖乳杆菌发酵)的DPPH的自由基清除率分别为71.55±0.33%、 66.87±0.31%,ABTS自由基清除率分别为38.56±0.22%、32.67±56%(P<0.05),这可能与合生元豆乳中较高的总酚含量有关。研究表明DPPH和ABTS自由基清除率的高低,认为其与抗氧化物质溶出有关,通过对比ACE抑制率发现,合生元豆乳45.67±0.55%的ACE抑制率显著高于未添加益生元豆乳及市售豆乳(鼠李糖乳杆菌发酵)的34.88±0.76%和30.78±0.43%(P<0.05),说明复合益生元的添加,可以显著增加豆乳中DPPH和ABTS自由基清除率和ACE抑制率。
表4 不同益生元对DPPH和ABTS自由基清除率和ACE抑制率的影响
注:不同字母间表示不同组间具有显著性差异,P<0.05。
其中,A:未添加益生元+植物乳杆菌J26;B:大麦β-葡聚糖:莲子寡糖:菊粉=1:1:1+植物乳杆菌J26;C:大麦β-葡聚糖:莲子寡糖:菊粉=4:1:1+植物乳杆菌J26;D:大麦β-葡聚糖:莲子寡糖:菊粉=2:2:1+植物乳杆菌J26;E:大麦β-葡聚糖:莲子寡糖:菊粉=2:1:2+植物乳杆菌J26;F:大麦β-葡聚糖:莲子寡糖:菊粉=2:1:1+植物乳杆菌J26;G:大麦β-葡聚糖:莲子寡糖=2:1+植物乳杆菌J26;H:莲子寡糖:菊粉=1:1+植物乳杆菌J26;I:大麦β-葡聚糖:菊粉=2:1+植物乳杆菌J26;J:大麦β-葡聚糖+植物乳杆菌J26;K:莲子寡糖+植物乳杆菌J26;L:菊粉+植物乳杆菌J26;M:市售豆乳(鼠李糖乳杆菌发酵)。
9.添加哈密瓜果酱及燕麦对合生元豆乳感官评价的影响
发酵结束后,终产品的感官评价结果如表5所示。 综合色泽、组织状态、气味和滋味4个指标,直投氏发酵剂植物乳杆菌J26发酵豆乳产品呈现出良好的品质,感官评价综合得分为95分。
表5 终产品的感官评价结果
10. 复合益生元对合生元豆乳pH与酸度值的影响
如图11~图12所示,由发酵豆乳过程中pH的变化可知,添加复合益生元(大麦β-葡聚糖:水苏糖:低聚果糖=2:1:1)与直投氏发酵剂植物乳杆菌J26作为发酵剂制备的新型合生元豆乳可最快达到发酵终点,添加复合益生元后植物乳杆菌在豆乳中的产酸速度为最快,发酵8h的pH能达到4.35,图11为13种发酵剂在发酵豆乳过程中酸度的变化,在发酵结束时 pH 在4.35左右,添加复合益生元(大麦β-葡聚糖:水苏糖:低聚果糖=2:1:1)与直投氏发酵剂植物乳杆菌J26作为发酵剂制备的合生元豆乳滴定酸度最高达93.98°T,市售发酵剂滴定酸度最高达83.12°T,综上所述,添加复合益生元后植物乳杆菌J26作为发酵剂,发酵速率优于其余组发酵,可证明其有良好的发酵性能。
11.复合益生元对合生元豆乳活菌数的影响
发酵终点活菌数结果证明,由表6可知,与其他组合相比,可知添加8wt%复合益生元(大麦β-葡聚糖:水苏糖:低聚果糖=2:1:1)与直投氏发酵剂植物乳杆菌J26作为发酵剂制备的新型合生元豆乳的活菌数均得到显著提升,植物乳杆菌J26发酵时生长较好,发酵终点的活菌数为3.0×109CFU/mL。高于其他不同复配比例益生元及单种益生元与植物乳杆菌J26制备豆乳在发酵终点时的活菌数,市售发酵剂活菌数为1.15×107CFU/mL,因此添加8wt%复合益生元后植物乳杆J26菌粉更适合发酵豆乳。
表6不同益生元与J26作为发酵剂制备的新型合生元豆乳的活菌数表
12.复合益生元对合生元豆乳持水力的影响
如图13所示,持水力是发酵乳的重要理化指标之一,可知添加复合益生元(大麦β-葡聚糖:水苏糖:低聚果糖=2:1:1)与直投氏发酵剂植物乳杆菌J26作为发酵剂制备的新型合生元豆乳持水力均得到显著提升,与其他组相比具有显著变化。豆乳在发酵过程中持水性呈上升趋势。这可能与豆乳在发酵过程中pH值、黏度、活菌数变化有关。发酵豆乳持水性与其组织状态呈正相关,可以防止乳清析出,有利于产品贮藏及风味保留。
13.复合益生元对合生元豆乳质构的影响
不同发酵剂制备豆乳的质构参数如表7所示,结果表明,添加复合益生元(大麦β-葡聚糖:水苏糖:低聚果糖=2:1:1)与植物乳杆菌J26作为发酵剂制备的新型合生元豆乳的硬度、稠度、黏度指数均显著高于其他组不同复配比例益生元及单种益生元与植物乳杆菌J26制备发酵豆乳(P<0.05),因此,添加复合益生元可发酵具有良好质构特性的合生元豆乳。
表7 不同益生元对合生元豆乳质构的影响
注:不同字母间表示不同组间具有显著性差异,P<0.05。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种直投氏发酵剂植物乳杆菌J26的制法,其特征在于,包括以下步骤:将植物乳杆菌J26活化,按4%的接种量接种至100mL优化碳氮源培养基中,在33℃、pH5.6条件下采用5L发酵罐进行高密度发酵24h,在4~24h恒速流加补料,补料速率为15mL/h,补料培养基为优化碳源:优化氮源=5:2;高密度发酵完成后,收获菌液,冻干,得到冻干菌粉。
2.根据权利要求1所述的一种直投氏发酵剂植物乳杆菌J26的制法,其特征在于,优化碳氮源培养基和补料培养基中,优化碳源为25g/L麦芽糖与葡萄糖按照1:1的质量比例混合,优化氮源为10g/L牛肉膏与酵母粉按照1:1的质量比例混合,其余成分、用量与MRS液体培养基中的成分、用量相同。
3.根据权利要求1或2所述的一种直投氏发酵剂植物乳杆菌J26的制法获得的直投氏发酵剂植物乳杆菌J26。
4.包含权利要求3所述的直投氏发酵剂植物乳杆菌J26的豆乳发酵剂,其特征在于,包括8wt%的复合益生元、1×106CFU/mL的直投氏发酵剂植物乳杆菌J26、6wt%的哈密瓜果酱、2wt%的燕麦和余量的豆奶。
5.根据权利要求4所述的豆乳发酵剂,其特征在于,复合益生元包括大麦β-葡聚糖:莲子寡糖:菊粉=2:1:1。
6.采用权利要求4或5所述的豆乳发酵剂的合生元发酵豆乳的制法,其特征在于,包括以下步骤:
步骤一,在豆奶中加入复合益生元并混匀,再进行超高压杀菌处理,待冷却至室温时添加直投氏发酵剂植物乳杆菌J26,进行发酵豆乳;
步骤二,发酵完成后于4℃后熟24h;
步骤三,4℃完成后,再添加6wt%的哈密瓜果酱和2wt%的燕麦,混匀,即得。
7.根据权利要求6所述的合生元发酵豆乳的制法,其特征在于,超高压杀菌处理的工艺为:在530MPa下进行10min。
8.根据权利要求6所述的合生元发酵豆乳的制法,其特征在于,发酵豆乳的工艺为:37℃培养箱发酵,以pH4.5为发酵终点。
9.根据权利要求6所述合生元发酵豆乳的制法获得的合生元发酵豆乳,其特征在于,合生元发酵豆乳中,总酚含量为 94.92μg/g,游离氨基酸含量为0.57mg/mL。
10.根据权利要求3所述的直投氏发酵剂植物乳杆菌J26在直接接种生产发酵乳制品中的应用,其特征在于,发酵乳制品包括豆乳、常温酸奶、低温酸奶、搅拌型酸奶、凝固型酸奶、饮用性酸奶、乳酪或乳酸菌饮料。
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