CN116731935B - 一种干酪乳杆菌发酵剂的筛选方法 - Google Patents
一种干酪乳杆菌发酵剂的筛选方法 Download PDFInfo
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Abstract
本发明涉及微生物和食品生物技术领域,具体公开了一种干酪乳杆菌发酵剂的筛选方法。该筛选方法是基于lac cluster基因高表达和无β‑果糖苷酶酶活性建立的,用于适合发酵乳饮料干酪乳杆菌发酵剂的筛选方法,具体包括:1)提取干酪乳杆菌总DNA,qPCR检测lac cluster中基因拷贝数;2)制备干酪乳杆菌粗酶液并计算干酪乳杆菌的β‑果糖苷酶酶活性;3)按照lac cluster中基因拷贝数高和无β‑果糖苷酶酶活性定向筛选的干酪乳杆菌发酵剂。本发明的筛选方法可以定向筛选适合发酵乳饮料的干酪乳杆菌发酵剂,具备高效和特异性筛选的特点,且有利于发酵乳饮料品质和稳定性的提高。
Description
技术领域
本发明涉及微生物和食品生物技术领域,具体涉及一种干酪乳杆菌发酵剂的筛选方法。
背景技术
乳饮料是功能性乳制品行业中一个非常活跃的部分,全球乳饮料市场的市值在2021年达139亿美元,其中发酵乳饮料是乳饮料市场中主导产品。发酵乳饮料中含有高水平的蛋白质、钙和维生素B和β-酪蛋白等基本营养物质。发酵乳饮料可以缓解乳糖消化不良症状和缩短轮状病毒引起的腹泻,同时预防便秘等消化系统疾病,增强免疫力和降低感染风险等。
发酵剂菌株赋予发酵乳饮料浓郁风味,略厚的质地和独特的香气。目前发酵乳饮料通常由乳酸菌(Lactic acid bacteria,LAB)种属作为发酵剂驱动发酵,如乳杆菌属(乳杆菌属Lactobacillus)、链球菌属(Streptococcus)和双歧杆菌属(Bifidobacterium)。
在发酵乳饮料中,目前筛选发酵乳饮料发酵剂的方法中,主要是依据单菌株的高产酸速率进行筛选。在干酪乳杆菌中,主要影响干酪乳杆菌产酸速率的因素为碳水化合物的种类和糖的代谢。碳水化合物发酵形成乳酸,乳酸作为主要终产物或与其他有机酸和醇结合。所有干酪乳杆菌都能利用包括葡萄糖、果糖、甘露糖、N-乙酰氨基葡萄糖在内的常见己糖,以及乳糖、海藻糖、纤维二糖。糖代谢中,干酪乳杆菌作为异型发酵菌株,其代谢糖的方式主要由多种途径介导,包括ABC 蛋白依赖性系统、糖透性酶系统、糖特异性磷酸烯醇丙酮酸依赖性磷酸转移酶(PTS)系统等。但是,产酸过快使发酵乳饮料的酸度持续增加,当酸度大于90 °T后,发酵乳饮料的酸甜比失调,出现难以接受的过酸味,导致乳饮料的口感降低,进而降低干酪乳杆菌的存活率并引发发酵乳饮料的稳定性降低,保质期缩短。在干酪乳杆菌中,乳糖通过磷酸烯醇式丙酮酸-乳糖磷酸转移酶系统的gal-lac基因簇进入细胞被水解成葡萄糖与半乳糖,葡萄糖经磷酸果糖激酶和醛缩酶等进入糖酵解途径或磷酸戊糖途径,进一步通过乙醛、乙醇、乳酸脱氢酶等分解成小分子物质;生成的半乳糖在基因簇galR- galKTEM作用下将半乳糖分解成有机酸。laccluster由lacTEGF基因组成,其中lacG 编码磷酸-β-半乳糖苷酶。蔗糖通过磷酸烯醇式丙酮酸-蔗糖磷酸转移酶系统转运至胞内,被磷酸化的蔗糖在细胞内由β-果糖苷酶(EC 3.2.1.26)水解,以产生6-磷酸葡萄糖和果糖,进一步通过果糖激酶将果糖引导至糖酵解途径。
可见,乳糖和蔗糖代谢基因能够调控干酪乳杆菌菌株的产酸能力,但是目前缺乏通过乳糖和蔗糖代谢相关基因筛选优质发酵剂的相关研究。国内应用的干酪乳杆菌发酵剂存在产酸差异大的问题,目前主要依赖国外进口的L. casei Shirota、L. casei Danone和L. casei 01TM等干酪乳杆菌发酵剂。因此,如何筛选出适用于发酵乳饮料的干酪乳杆菌是本领域亟待解决的技术问题。
发明内容
针对现有技术的问题,本发明的目的是提供一种定向、高效筛选发酵乳饮料中干酪乳杆菌发酵剂的方法,具体通过筛选lac cluster基因高表达和无β-果糖苷酶酶活性的干酪乳杆菌菌株来实现。
为了实现该目的,本发明的技术方案如下:
一方面,本发明提供一种定向筛选发酵乳饮料中干酪乳杆菌发酵剂的方法,所述筛选方法包括筛选lac cluster基因高表达且无β-果糖苷酶酶活性的干酪乳杆菌菌株的步骤。具体包括如下步骤:
(1)活化干酪乳杆菌,得到干酪乳杆菌菌液;
优选的,步骤(1)的操作步骤包括:将干酪乳杆菌按照体积比1%接种至MRS液体培养基中,在37 ± 1℃下培养,得到干酪乳杆菌菌液;
更优选的,在37 ± 1℃下培养的时间为8-16h ;
进一步优选的,在37 ± 1℃下培养的时间为12h。
(2)提取干酪乳杆菌总DNA,扩增tuf和lac cluster基因,以tuf基因为对照,采用方程 确定 lac cluster基因的拷贝数;
优选的,所述的laccluster基因为lacG基因。
优选的,步骤(2)中所述的采用方程 确定lacG基因的拷贝数的操作步骤包括:以tuf2-F/R和lacG-F/R为引物, qPCR检测CT值,计算tuf和lacG基因的表达量;通过ΔCT计算基因的拷贝数,所述ΔCT为laccluster和tuf基因扩增产物的荧光信号达到设定阈值所经过的循环数的差值;
其中引物序列为:
tuf2-F/R:tuf2-F:5'- ACTGGTCGTGGTACAGTTGC -3'(SEQ ID NO:1),tuf2-R:5'-CACGAAGCAAGACACCAACG -3'(SEQ ID NO:2);和
lacG-F/R:lacG-F:5'- AGATGGCATTGAGACGACAGATTGG -3'(SEQ ID NO:3),lacG-R:5'- GTCACTGGCACCAACGGATAGTC -3'(SEQ ID NO:4)。
优选的,所述步骤(2)中tuf基因为L. casei基因组上单拷贝基因的延伸因子Tu的基因(GenBank:AJ418937.2)。
(3)制备β-果糖苷酶粗酶液,测定干酪乳杆菌的β-果糖苷酶酶活性;
优选的,具体操作为:取步骤(2)制备得到的所述干酪乳杆菌菌液,按照体积比1%添加量接种于S-MRS液体培养基中,在37 ± 1℃下培养;取发酵液,离心,添加0.85%的生理盐水,混匀,离心,再添加 0.85%生理盐水,按照15s均质3次,对菌株进行破碎,得干酪乳杆菌粗酶液;测定所述干酪乳杆菌粗酶液的β-果糖苷酶酶活性。
更优选的,测定所述干酪乳杆菌的β-果糖苷酶酶活性的方法为以3-3'-5-5’-四甲基联苯胺为底物,在450nm波长下检测粗酶液吸光值;一个酶单位定义为在 37°C、pH = 6.8条件下每分钟水解 1 μmol 3-3'-5-5’-四甲基联苯胺所需的酶量。
(4)以laccluster中基因拷贝数高且无β-果糖苷酶酶活性为标准,定向筛选干酪乳杆菌发酵剂。
第二方面,本发明提供第一方面所述的筛选方法在筛选方法在筛选干酪乳杆菌发酵剂中的应用。
优选的,所述干酪乳杆菌发酵剂用于制备风味发酵酸乳、乳饮料或干酪制品。
与现有筛选方法比较,本发明的有益效果是:
(1)通过laccluster基因的高表达筛选出快速代谢乳糖的干酪乳杆菌菌株预防发酵乳饮料产品存在产酸差异大的问题。
(2)通过筛选无β-果糖苷酶酶活性的干酪乳杆菌,使干酪乳杆菌不代谢蔗糖,进而减弱干酪乳杆菌代谢蔗糖造成的后酸化(图3),降低发酵乳饮料离心沉淀率(图5),避免分层或乳清析出的现象。
(3)与传统筛选方法比较,本发明通过laccluster基因和β-果糖苷酶复合定向筛选发酵乳饮料中干酪乳杆菌发酵剂,具有高效和特异性筛选的特点,避免传统筛选方法中单一性。
附图说明
图1为不同干酪乳杆菌菌株的β-果糖苷酶酶活性对比图。
图2为贮藏期内含不同干酪乳杆菌的发酵乳饮料的酸度对比图。
图3为贮藏期内含不同干酪乳杆菌的发酵乳饮料中微生物数量变化对比图。
图4为贮藏期内含不同干酪乳杆菌的发酵乳饮料中蔗糖和乳糖的含量变化图。
图5为贮藏期内含不同干酪乳杆菌的发酵乳饮料的离心沉淀率对比图。
具体实施方式
本发明的实施并不局限于下面的实施例,对本发明所做的任何形式上的变通和/或改变都将落入本发明保护范围。在本发明中,若非特指,所有的设备和原料等均可从市场购得或是本行业常用的。若无特别指明,实施例采用的方法为本领域通用技术。
对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
实验方法:
基因拷贝数的测定:采用qPCR方法,以干酪乳杆菌基因组上单拷贝基因tuf为参照,确定干酪乳杆菌的lacG基因的拷贝数:采用引物tuf2-F和tuf2-R(tuf2-F:5'-ACTGGTCGTGGTACAGTTGC -3',tuf2-R:5'- CACGAAGCAAGACACCAACG -3')扩增tuf基因,qPCR反应体系(20 µL)如下:9µL TB Green Premix Ex Taq ( Tli RNaseH Plus),1µL 10mmol/L上游引物,1µL 10 mmol/L下游引物,2µL总DNA,7µL超纯水。qPCR反应条件为:95℃,5min;[95℃,20s;60℃,30s;68℃,30s]×40个循环,检测基因的表达量。以单拷贝基因tuf为参照,根据公式计算基因的拷贝数,其中E:扩增效率,E=10-1/K [K=标准曲线斜率];%效率=(E-1)×100%;ΔCT:lacG和tuf基因扩增产物的荧光信号达到设定阈值所经过的循环数的差值。
β-果糖苷酶酶活性的测定:采用江苏酶免实业有限公司的β-果糖苷酶酶联免疫试剂盒。将44 IU/L标准品溶液用标准品稀释液稀释至72、36、18、9和4.5 IU/L;分别设定空白孔、标准孔、待测样品孔。在酶标包被板上添加50μL标准品,待测样品孔中添加40μL样品稀释液,再加10μL待测样品。将样品加入酶标板,轻轻晃动均匀;用封板膜封板后置于37℃温育30min;弃去液体,每孔加满洗涤液,静置30s后弃去,如此重复5次,拍干;每孔加入酶标试剂50μL,空白孔除外;用封板膜封板后置于37℃温育30min;弃去液体,甩干,每孔加满洗涤液,静置30s后弃去,重复5次,拍干;每孔先加入50μL显色剂A,再加入50μL显色剂B,轻柔混匀,37℃避光显色10min;以空白孔调零,450nm波长依次测量各孔的吸光度;3-3'-5-5’-四甲基联苯胺(TMB)作为底物,一个酶单位定义为在 37°C、pH = 6.8 条件下每分钟水解 1 μmol TMB 所需的酶量。
发酵乳饮料的制备:
步骤1. 按照13%(w/v)脱脂乳 + 3%(w/v)葡萄糖 + 5%(w/v)蔗糖混合原料,在50℃溶解30min,经过20MPa的均质,在95℃条件下褐变4小时,冷却至37℃,制备得到发酵基料。按照5×106CFU/g的添加量将干酪乳杆菌接种于发酵基料中,在37℃下培养96h,3个生物学重复。
步骤2. 基料稀释液的制备:按照14%(wt/wt)白砂糖,溶于70℃水中,充分搅拌10min,95℃,5min进行杀菌,配置基料稀释液。
步骤3. 按照基料:基料稀释液= 1:3的比例混合, 20 - 25MPa的条件下均质发酵乳饮料,无菌条件下灌装于无菌包装瓶中。将发酵乳饮料于4℃下储藏28天。
活菌数的检测方法:采用国标GB 4789.35-2016《食品安全国家标准 食品微生物检验 乳酸菌检验》。
酸度检测方法:采用国标GB 5009.239–2016。
乳糖和蔗糖含量的检测:准确称取乳糖和蔗糖标准品分别为0.2500和0.2000于100mL容量瓶中,超纯水定容至100mL;将乳糖的质量浓度分别为2.5、2、1.5、1和0.5mg/mL;蔗糖的质量浓度分别为2、1.8、1.6、1.4、1.2、1、0.8、0.6、0.4和0.2mg/mL;以标准品的浓度为横坐标,以峰面积为纵坐标绘制标准曲线。称取发酵96小时的发酵乳基(发酵基料)5.0g至100mL烧杯中,并添加25.0g去离子水,磁力搅拌器充分溶解;依次添加2.5mL Carrez试剂1和Carrez试剂2,充分溶解30min;上述混合物以5000×g,离心15 min;用蒸馏水将上清液稀释至100 mL,通过0.22 μm滤膜过滤处理;在Bio-Rad Aminex®HPX-87P(300 mm×7.8 mm×9μm)色谱柱和2414型差示折射率检测器(RID)相结合,以5mM硫酸水溶液为流动相,流速为550 μL/min,柱温设定为60℃,进样量为2 μL,采用外标法检测样品中的乳糖和蔗糖。所述Carrez试剂1,其配方为:称取10.60g亚铁氰化钾,去离子水溶解至100mL。所述Carrez试剂2,其配方为:称取21.90g二水乙酸锌,去离子水溶解,添加3mL乙酸溶解,定容至100mL。
离心沉淀率的检测:取50mL离心管(质量记为M),称取发酵乳饮料20mL(质量记为M1)在4070 × g条件下离心15min,离心管在滤纸上倒扣10min,称量底部沉淀物(质量记为M2),按照离心沉淀率=(M2-M)/(M1-M)ⅹ100%计算离心沉淀率。
实施例1 干酪乳杆菌培养及总DNA提取
按照1%(v/v)接种量将干酪乳杆菌于MRS液体培养基中,37 ± 1℃静置培养;当OD600 = 0.8~1.0;取菌液2.5mL,4 ℃,12000 × g离心5 min,去除上清;加入2 mL TES溶液重悬菌体,4 ℃,12000× g离心5 min,去除上清;加入200µL 50mg/mL溶菌酶重悬菌体,置于37 ℃,200 × g条件下消化1.5h;加入1 mL裂解液,轻柔混匀,65 ℃条件下水浴30min;加入400 μL 5 mol/L 氯化钠溶液,轻柔混匀,4 ℃,13000× g离心20 min;加入等体积的Tris饱和酚-氯仿-异戊醇(25:24:1, pH>7.8),充分混匀,4 ℃,12000 × g 离心10min,取1.5mL上层水相溶液;加入2体积的无水乙醇和0.1体积的3 mol/L乙酸钠,混匀后置于-20 ℃沉淀2 h;分装于1.5mL离心管,4 ℃,12000× g离心15 min,除去上清;加入1 mL70%预冷乙醇对沉淀进行洗涤,4 ℃,13000× g离心10 min,除去上清;加入50 µL含有RNase的水溶液溶解DNA,-20 ℃保存。
实施例2 干酪乳杆菌lac cluster基因拷贝数(Plasmid copy number,PCN)检测
采用qPCR方法,以干酪乳杆菌基因组上单拷贝基因tuf为参照,确定干酪乳杆菌的lacG基因的拷贝数:采用引物tuf2-F和tuf2-R扩增tuf基因,qPCR反应体系(20 µL)如下:9µL TB Green Premix Ex Taq ( Tli RNaseH Plus),1µL 10 mmol/L上游引物,1µL 10mmol/L下游引物,2µL总DNA,7µL超纯水。qPCR反应条件为:95℃,5min;[95℃,20s;60℃,30s;68℃,30s]×40个循环,检测基因的表达量。以单拷贝基因tuf为参照,根据公式计算基因的拷贝数,其中E:扩增效率,E=10-1/K[K=标准曲线斜率];%效率=(E-1)×100%;ΔCT:lacG和tuf基因扩增产物的荧光信号达到设定阈值所经过的循环数的差值。
干酪乳杆菌的lacG基因拷贝数见表1,以商业菌株LC_N00为对照,LC_N00为购自丹麦科汉森公司的发酵用干酪乳杆菌。干酪乳杆菌LC_ N111、N07、N51、N50、N02、N72、N32、N00、N33、N87、N23、N114、N12、N29、N35和N110中lacG基因的拷贝数分别为5.10、4.93、4.69、4.31、3.95、3.95、3.71、3.29、2.99、2.51、2.48、2.41、2.31、2.28、2.25和2.20 copy/细胞,15株干酪乳杆菌的拷贝数显著大于其他的10株干酪乳杆菌(p<0.05)。
表1
实施例3 测定干酪乳杆菌的β-果糖苷酶酶活性
干酪乳杆菌按照1%(v/v)添加量接种于MRS液体培养基中,在37 ± 1℃下培养12h;菌株按照1%(v/v)添加量接种于S-MRS液体培养基中,在37 ± 1℃下培养12h;取10mL发酵液,在12000 × g下离心5min;添加10mL 0.85%的生理盐水,充分混匀洗涤菌体,在12000 × g下离心5min;添加1mL 0.85%生理盐水,按照15s均质3次,对菌株进行破碎,制备的粗酶液置于冰上。
将44 IU/L标准品溶液用标准品稀释液稀释至72、36、18、9和4.5 IU/L;分别设定空白孔、标准孔、待测样品孔。在酶标包被板上添加50μL标准品,待测样品孔中添加40μL样品稀释液,再加10μL待测样品。将样品加入酶标板,轻轻晃动均匀;用封板膜封板后置于37℃温育30min;弃去液体,每孔加满洗涤液,静置30s后弃去,如此重复5次,拍干;每孔加入酶标试剂50μL,空白孔除外;用封板膜封板后置于37℃温育30min;弃去液体,甩干,每孔加满洗涤液,静置30s后弃去,重复5次,拍干;每孔先加入50μL显色剂A,再加入50μL显色剂B,轻柔混匀,37℃避光显色10min;以空白孔调零,450nm波长依次测量各孔的吸光度;3-3'-5-5’-四甲基联苯胺(TMB)作为底物,一个酶单位定义为在 37°C、pH = 6.8 条件下每分钟水解 1 μmol TMB 所需的酶量。
干酪乳杆菌的β-果糖苷酶酶活性见图1,干酪乳杆菌LC_N07、N72、N02、N53、N74、N111、N12和N01的β-果糖苷酶酶活力分别为0.271、0.252、0.194、0.183、0.164、0.139、0.122和0.113 U/mL;β-果糖苷酶的酶活性最低的13菌株为N50、N51、N33、N69、N87、N110、N32、N35、N114和LC_N00。
实施例4 测定含干酪乳杆菌的发酵乳饮料的酸度
按照13%(w/v)脱脂乳 + 3%(w/v)葡萄糖 + 5%(w/v)蔗糖混合原料,在50℃溶解30min,经过20MPa的均质,在95℃条件下褐变4小时,冷却至37℃,制备得到发酵基料。按照5×106CFU/g的添加量将干酪乳杆菌接种于发酵基料中,在37℃下培养96h,3个生物学重复。
基料稀释液:按照14%(wt/wt)白砂糖,溶于70℃水中,充分搅拌10min,95℃,5min进行杀菌,配置基料稀释液。
按照基料:基料稀释液= 1:3的比例混合, 20 - 25MPa的条件下均质发酵乳饮料,无菌条件下灌装于无菌包装瓶中。将发酵乳饮料于4℃下储藏28天,检测贮藏期内干酪乳杆菌的酸度变化。贮藏期内含干酪乳杆菌的发酵乳饮料酸度见图2,在第0天,LC_N02酸度最低(47.63 °T),LC_N72、N35和N111在0d的酸度最高(63.69、61.96和61.96 °T);在第28天,LC_N02在28d的酸度最低(57.21 °T),LC_N35、N50和N111在28d的酸度最高(66.10、66.14和66.16 °T)。在后酸化中,贮藏期间LC_N07、N72和N111的酸度大于10°T。
实施例5 测定含干酪乳杆菌的发酵乳饮料的活菌数
其他同实施例4,不同之处在于,检测贮藏期内发酵乳饮料中干酪乳杆菌的活菌数的变化。贮藏期内干酪乳杆菌的活菌数见图3,在第0天,LC_N00和LC_N51活菌数(9.08和9.03)最高,LC_N72和LC_N02活菌数(8.41和7.13)最低;贮藏28天后,LC_N07、N32、N35、N51、N110和N00的活菌数(8.67、8.50、8.67、8.82、8.52和8.49)最高;LC_N02的活菌数(6.21)最低。贮藏期内活菌数变化最小的菌株为LC_N32、N35、N51和N110(0.29、0.13、0.21和0.33)。
实施例6 测定干酪乳杆菌对乳糖和蔗糖的利用情况
取实施例4中的发酵基料,进行乳糖和蔗糖的测定。
准确称取乳糖和蔗糖标准品分别为0.2500和0.2000于100mL容量瓶中,超纯水定容至100mL;将乳糖的质量浓度分别为2.5、2、1.5、1和0.5mg/mL;蔗糖的质量浓度分别为2、1.8、1.6、1.4、1.2、1、0.8、0.6、0.4和0.2mg/mL;以标准品的浓度为横坐标,以峰面积为纵坐标绘制标准曲线。
称取发酵96小时的发酵基料5.0g至100mL烧杯中,并添加25.0g去离子水,磁力搅拌器充分溶解;依次添加2.5mL Carrez试剂1和Carrez试剂2,充分溶解30min;上述混合物以5000×g,离心15 min;用蒸馏水将上清液稀释至100 mL,通过0.22 μm滤膜过滤处理;在Bio-Rad Aminex®HPX-87P(300 mm×7.8 mm×9μm)色谱柱和2414型差示折射率检测器(RID)相结合,以5mM硫酸水溶液为流动相,流速为550 μL/min,柱温设定为60℃,进样量为2μL,采用外标法检测样品中的乳糖和蔗糖。
Carrez试剂1,其配方为:称取10.60g亚铁氰化钾,去离子水溶解至100mL。所述Carrez试剂2,其配方为:称取21.90g二水乙酸锌,去离子水溶解,添加3mL乙酸溶解,定容至100mL。
干酪乳杆菌对乳糖和蔗糖的利用情况见图4,所有干酪乳杆菌对于乳糖的利用大于50%,菌株间无显著性差异;在蔗糖利用中,LC_N02、N07、N72和N111对蔗糖的利用分别为51%、53.6%、49.2%和45%,这四株菌对蔗糖的利用显著大于LC_N32、N33、N35、N35、N51、N69、N87、N110、N114和N00,表明所有菌株对乳糖的代谢能力无显著差异,而NC_N02、N07、N72和N111可快速利用蔗糖,另外9株菌与商业菌株LC_N00不利用蔗糖。
实施例7 测定含干酪乳杆菌的发酵乳饮料的离心沉淀率
其他同实施例4,不同之处在于,检测发酵乳饮料的离心沉淀率。
取50mL离心管(质量记为M),称取发酵乳饮料20mL(质量记为M1)在4070 × g条件下离心15min,离心管在滤纸上倒扣10min,称量底部沉淀物(质量记为M2),按照离心沉淀率=(M2-M)/(M1-M)ⅹ100%计算离心沉淀率。贮藏期内发酵乳饮料的离心沉淀率见图5,在贮藏28天时,LC_N32、N33、N35、N50、N51、N69、N87、N110和N114分别为19.96%、18.66%、19.34%、16.97%、20.34、18.63%、22.21%、19.34%和21.04%,显著低于LC_N02、LC_N72和LC_N111。
依据lacG基因拷贝数和β-果糖苷酶酶活性筛选的多个干酪乳杆菌,其通过检测酸度、活菌数、乳糖蔗糖利用率和发酵饮料离心沉淀率测定,发现依据筛选指标筛选的干酪乳杆菌LC_N32、N33、N35、N50、N51、N69、N87、N110和N114菌株具备上述优良特性。
通过对贮藏期的发酵乳饮料进行酸度、活菌数、乳糖和蔗糖的含量、离心沉淀率进行评价。筛选出的9株菌制备的乳饮料贮藏28天的酸度为 64 °T,微生物数量>7.5 lgCFU/g,离心沉淀率<15.67%,表明基于lacG基因拷贝数和β-果糖苷酶酶活性筛选的菌株,可作为发酵剂进行乳饮料的发酵。
综上所述,基于发酵乳饮料中基于laccluster基因高表达和无β-果糖苷酶酶活性建立干酪乳杆菌发酵剂的筛选方法具有高效和特异性筛选的特点,保证菌株快速代谢乳糖,减弱干酪乳杆菌代谢蔗糖造成的后酸化,以及降低发酵乳饮料离心沉淀率,引起的分层或乳清析出的现象。
Claims (6)
1.一种干酪乳杆菌发酵剂的筛选方法,其特征在于,所述筛选方法包括如下步骤:
(1)活化干酪乳杆菌,得到干酪乳杆菌菌液;
(2)提取干酪乳杆菌总DNA,扩增tuf和lac cluster基因,以tuf基因为对照,采用方程确定 lac cluster基因拷贝数;所述tuf为L. casei基因组上单拷贝基因的延伸因子Tu;
所述Nrelatives为基因拷贝数;
所述E为扩增效率,E=10-1/K,K为标准曲线斜率;
所述-ΔCT为lac cluster和tuf基因扩增产物的荧光信号达到设定阈值所经过的循环数的差值;
所述的tuf基因为干酪乳杆菌基因组上单拷贝基因的延伸因子Tu的基因,所述tuf基因的GenBank编号为AJ418937.2;
所述的lac cluster基因为lacG基因;
(3)制备β-果糖苷酶粗酶液,测定干酪乳杆菌的β-果糖苷酶酶活性;
(4)以lac cluster基因拷贝数和β-果糖苷酶酶活性为标准,定向筛选干酪乳杆菌发酵剂;
步骤(2)中所述的采用方程 确定 lacG基因拷贝数的步骤包括:
以tuf2-F/R: tuf2-F:5'- ACTGGTCGTGGTACAGTTGC -3',tuf2-R:5'-CACGAAGCAAGACACCAACG -3';
和lacG-F/R:lacG-F:5'- AGATGGCATTGAGACGACAGATTGG -3',lacG-R:5'-GTCACTGGCACCAACGGATAGTC -3'为引物,用qPCR检测CT值,计算tuf和lac G基因的表达量;通过ΔCT计算基因拷贝数。
2.根据权利要求1所述的筛选方法,其特征在于,步骤(1)的操作步骤包括:将干酪乳杆菌按照体积比1%接种至MRS液体培养基中,在37 ± 1℃下培养,得到干酪乳杆菌菌液。
3.根据权利要求1所述的筛选方法,其特征在于,步骤(3)的具体操作为:取步骤(2)制备得到的所述干酪乳杆菌菌液,按照体积比1%添加量接种于S-MRS液体培养基中,在37 ±1℃下培养;取发酵液,离心,添加0.85%的生理盐水,混匀,离心,再添加 0.85%生理盐水,均质3次,每次均质15s,对菌株进行破碎,得干酪乳杆菌粗酶液;测定所述干酪乳杆菌粗酶液的β-果糖苷酶酶活性。
4.根据权利要求3所述的筛选方法,其特征在于,测定所述干酪乳杆菌的β-果糖苷酶酶活性的方法为以3-3'-5-5’-四甲基联苯胺为底物,在450nm波长下检测粗酶液吸光值;一个酶单位定义为在 37°C、pH = 6.8 条件下每分钟水解 1 μmol 3-3'-5-5’-四甲基联苯胺所需的酶量。
5.权利要求1-4任一项所述的筛选方法在筛选干酪乳杆菌发酵剂中的应用。
6.如权利要求5所述的应用,其特征在于,所述干酪乳杆菌发酵剂用于制备风味发酵酸乳、乳饮料或干酪制品。
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