CN116814481B - 一株源自酸马奶的益生副干酪乳酪杆菌pc646及其人工智能筛选方法 - Google Patents
一株源自酸马奶的益生副干酪乳酪杆菌pc646及其人工智能筛选方法 Download PDFInfo
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Abstract
本发明提供一株副干酪乳酪杆菌(Lacticaseibacillus paracasei)PC646,其保藏编号为CGMCC No.23515。本发明还涉及所述副干酪乳酪杆菌PC646在食品添加剂中的应用,特别地,所述食品添加剂是乳品发酵剂。本发明还提供一种益生菌快速筛选方法,可实现益生菌的快速初步筛选。本发明的副干酪乳酪杆菌PC646通过胃肠液耐受性和胆盐耐受性测试确认该菌株具有良好的益生菌特性,能够抵御消化道环境的抑制并在肠道内定植。本发明的副干酪乳酪杆菌PC646经过感官评鉴确认具有优秀的风味和口感,可应用于商业化发酵乳制品中。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一株分离自酸马奶的具有益生特性的副干酪乳酪杆菌(Lacticaseibacillusparacasei)PC646,还涉及其在益生菌发酵乳制品中的应用,以及分离该益生菌时使用的筛选方法。
背景技术
酸马奶是我国北方少数民族地区的一种传统乳制品,以新鲜马奶为原料、利用天然发酵剂和传统发酵技术发酵而成,其醇香浓郁,营养价值很高,具有驱寒、活血、舒筋、消食、健胃等功能。
乳酸菌(Lactic acidbacteria,LAB)是一类革兰氏阳性、能发酵糖类产生乳酸的有益微生物的总称。乳酸菌是人类胃肠道共生微生物菌群的主要部分,是公认的食品安全级(General RegardAs Safety,GRAS)微生物,具有增强免疫反应、平衡肠道菌群、降低血清胆固醇等生理功效,经常作为益生菌单独或混合用于食品发酵。其中,益生菌以乳杆菌属和双歧杆菌属最为常见,而乳杆菌属中最常见的有嗜酸乳杆菌(Lactobacillusacidophilus)、干酪乳酪杆菌(Lacticaseibacillus casei)、鼠李糖乳酪杆菌(Lacticaseibacillus rhamnosus)和副干酪乳酪杆菌(Lacticaseibacillusparacasei)等。
副干酪乳酪杆菌(Lacticaseibacillusparacasei)常被作为发酵剂及辅助发酵剂用于乳制品的生产制作,属于兼性厌氧型革兰氏阳性菌,它具有多种益生特性,还具有良好的碳源代谢能力及环境胁迫的耐受能力,因此被广泛应用于医疗、食品及发酵工业等领域。
然而,现有的副干酪乳酪杆菌普遍存在胃肠液耐受性较差或胆盐耐受性较差的特点。例如天原等人在《副干酪乳酪杆菌L1的安全性和益生性评价机器在杂交鸡上的应用》中公开了一株副干酪乳酪杆菌L1,该菌株在0.3g/100mL浓度胆盐下的细菌存活数在0-4h内表现为缓慢下降,表明该菌株活性同样在一定程度上受胆盐抑制;又例如曾维友等人在《泡菜中乳酸菌的分离鉴定及抗性筛选》中公开了一株具有良好的人工胃液耐受性和胆盐耐受性的植物乳杆菌,但没有公开其在模拟肠液中的耐受性,也没有验证该菌株是否适用于制备发酵乳。
发明内容
本发明的目的是通过筛选以期获得一株新的副干酪乳酪杆菌,该如干酪乳杆菌需具备良好的胃肠液耐受性和优秀的胆盐耐受性,并验证该菌株能够适用于制备发酵乳制品,能产生良好的风味而可用于制备发酵剂。
基于此,本发明提供一株分离自酸马奶的副干酪乳酪杆菌(Lacticaseibacillusparacasei)PC646,所述副干酪乳酪杆菌(Lacticaseibacillusparacasei)PC646于2021年9月29日保藏于中国微生物菌种保藏管理委员会普通微生物菌种保藏中心,保藏编号为CGMCC No.23515。
本发明还涉及所述副干酪乳酪杆菌(Lacticaseibacillusparacasei)PC646在食品添加剂中的应用。
在本发明中,所述食品添加剂是乳品发酵剂。
进一步地,所述发酵酸奶或发酵豆乳。
本发明还提供一种益生菌快速筛选方法,所述方法包括对确认为乳杆菌属的菌株进行测序以获得其基因组序列,在人工智能筛选平台iProbiotics进行预测,以所得基因组序列作为输出序列,根据所述平台输出的分数进行判断,当输出分数高于0.5时,判断该菌株为潜在的益生菌。
本发明提供一株分离自中国内蒙古锡林浩特市西乌旗地区的酸马奶样品的副干酪乳酪杆菌(Lacticaseibacillusparacasei)PC646,通过胃肠液耐受性和胆盐耐受性测试确认该菌株具有良好的益生菌特性,特别是在胆盐浓度0.3%环境下培养,OD620nm值增加0.3个单位所需的时间与在不含胆盐的培养基中培养的时间差为0小时,表明0.3%胆盐不会抑制该菌株的活性。因此,该菌株能够抵御消化道环境的抑制并在肠道内定植。
另一方面,本发明的副干酪乳酪杆菌(Lacticaseibacillusparacasei)PC646经过感官评鉴确认具有优秀的风味和口感,且制备成褐色乳饮料后,所得产品在4℃条件下保存21天,每间隔7天测定其酸度和益生菌活菌数,确认其性质变化较小,在贮藏期间始终保持高活菌数,证明本发明的PC646具有良好的益生特性,可应用于商业化发酵乳制品中。
本发明涉及以下微生物:
副干酪乳酪杆菌(Lacticaseibacillusparacasei)PC646,2021年9月29日保藏于中国微生物菌种保藏管理委员会普通微生物菌种保藏中心,保藏号为CGMCC No.23515。
【附图说明】
图1:副干酪乳酪杆菌PC646电镜图(3000倍);
图2:副干酪乳酪杆菌PC646在培养基上的菌落形态;
图3:副干酪乳酪杆菌PC646在模拟人工消化道中的存活率变化情况。
【具体实施方式】
下面将结合实施例对本发明作进一步说明,以下实施例仅为说明性的,本发明并不受这些实例的限制。
在本发明中,如无特殊说明,用于解释浓度的“%”均为重量百分比。
本发明涉及以下培养基:
MRS液体培养基:蛋白胨(动物源)10g、牛肉浸粉8g、酵母浸粉4g、葡萄糖20g、Tween-801ml、磷酸氢二钾2g、乙酸钠5g、柠檬酸三胺2g、硫酸锰0.05g、硫酸镁0.2g,蒸馏水1L,调节pH至6.5±0.2,121℃,15min灭菌。
MRS固体培养基:蛋白胨(动物源)10g、牛肉浸粉8g、酵母浸粉4g、葡萄糖20g、Tween-801ml、磷酸氢二钾2g、乙酸钠5g、柠檬酸三胺2g、硫酸锰0.05g、硫酸镁0.2g,蒸馏水1L,调节pH至6.5±0.2,琼脂15g,121℃、15min灭菌。
实施例1副干酪乳酪杆菌PC646的获取
将从内蒙古锡林浩特市西乌旗地区的酸马奶样品中采集到的样品倍比稀释,得到10-5、10-6、10-7稀释梯度,吸取各梯度酸粥样品200μL均匀涂布于MRS固体培养基平皿内,在37℃厌氧条件下培养48-72h。挑取形态、大小、颜色不同的单克隆接种于MRS液体培养基中,于37℃恒温培养箱培养24h。待菌株生长良好后,进行革兰氏染色、镜检,保存分离株。
本发明筛选得到的副干酪乳酪杆菌(Lacticaseibacillus paracasei)PC646具有如下形态学特征:菌体在显微镜下呈杆状,细胞成单、成对或成链状(见图1),不具鞭毛或纤毛,不会移动。
该副干酪乳酪杆菌(Lacticaseibacillus paracasei)PC646在MRS培养基上形成明显的菌落,菌落直径0.5-1mm,边缘整齐,呈奶油白色的不透明菌落(见图2)。
采用二代测序平台Illumina NovaSeq 6000和三代测序平台PacBio SMRT Sequel对该副干酪乳酪杆菌全基因组进行测定,将测序所得的原始数据进行质量评估(测序原始数据NCBI登录号:SRR16925177),去掉低质量Reads和接头,通过Portal(V2.7)中RS_HGAP_Assembly.3软件对该副干酪乳酪杆菌序列进行质控和菌株基因组组装。使用Circlator(V1.5.5)对组装完成的基因组进行环化。测得其基因组长度为3.05Mb,染色体基因组含有2979个编码基因;质粒基因组长度为88.95kb,编码88个基因。将该副干酪乳酪杆菌基因组作为输入序列,在人工智能筛选平台iProbiotics(http://bioinfor.imu.edu.cn/iprobiotics)进行预测,打分为0.57,通过这个结果判定该菌株为潜在的益生菌,并确认该菌株为副干酪乳酪杆菌。
实施例2耐受性测定
1、菌株胃肠液耐受性
模拟胃肠液的制备方法:将PBS灭菌后,用1mol/L HCl调节pH值至2.5,加入3.0mg/ml胃蛋白酶,用0.22μm微孔滤膜过滤除菌,制成人工模拟胃液;
将PBS灭菌后,用0.1mol/LNaOH调节pH值至8.0,加入0.1%胰蛋白酶和1.8%牛胆盐,用0.22μm微孔滤膜过滤除菌,制成人工模拟胰液。
胃肠液耐受性:将分离纯化得到的菌株活化培养两代,离心洗菌两次,收集菌体,取0.5ml复筛菌体悬液加入到4.5ml pH 2.5模拟人工胃液中,37℃消化3h,同时分别于0h和3h用MRS固体培养基倾注法计数测定活菌数。
然后,取0.5ml已消化3h的人工含菌胃液加入到4.5ml的人工胰液中,继续于37℃条件下水浴培养,分别于4h和8h用MRS琼脂培养基倾注法计数测定活菌数,每个样品做4个平行。
菌株存活率计算公式
N1――菌株处理后活菌数;
N0――菌株初始活菌数。
本发明的副干酪乳酪杆菌PC646在pH2.5的人工胃液中消化3小时(存活率69.05%),之后继续在pH8.0的肠液中消化8小时,其存活率高达75.86%(见图3),耐受性突出,实现最终总存活率为52.4%,显著优于现有技术的其他乳酸菌。结果表明副干酪乳酪杆菌PC646具有良好的胃肠道消化液耐受性,能够以活菌状态进入人体肠道,发挥健康功效。
与PC646一起分离筛选的其他副干酪乳酪杆菌有3株,其余菌株的胃肠液耐受性测定结果见表2。
表2胃肠液耐受性测定结果
可见,本发明的副干酪乳酪杆菌PC646是具有益生菌活性的益生菌,可以合理预期当它以干燥的细胞形态或发酵产物形态用于人或动物的情况下,能够密集停留在肠内的消化管道壁上,从而起到使有害菌不能够停留的作用,并且产生乳酸来降低肠内的pH值,抑制有害细菌的繁殖。并且,所用的活菌体生成细菌素和过氧化物,能够抑制病原菌的繁殖,对负责吸收营养成分的肠绒毛的活动起到帮助作用。
2、菌株胆盐耐受性
副干酪乳酪杆菌PC646活化培养三代后,以1%的接种量分别接种到含0.3%牛胆盐(培养基中添加0.2g/100mL巯基乙酸钠和,0.3g/100mL牛胆盐)的MRS液体培养基中,以不含牛胆盐的MRS培养基为空白对照,置于37℃下厌氧培养。培养期间,每隔一小时取样于620nm测定OD值,每组测定三个平行,直到OD值增加0.3个单位及以上时停止培养。计算菌株在含有胆盐和不含胆盐的MRS培养基中生长,OD值增加0.3个单位所需的时间,二者之差被称为延迟时间(LT)。
PC646在不含胆盐的培养基中培养,OD620nm值增加0.3个单位所需的时间为4.7小时,在含有胆盐的培养基中所需的时间为4.7小时,延迟时间为0小时。根据Gilliland等人的胆盐耐受性实验结论,抗性菌株(延迟时间≤0.25h),耐受性菌株(0.25h<延迟时间≤0.67h),弱耐受性菌株(0.67h<延迟时间<1.00h),敏感菌株(延迟菌株≥1.00h)。因此,菌株PC646的延迟时间为0小时,为抗性菌株,具有突出的胆盐耐受性。
与PC646一起分离筛选的其他副干酪乳酪杆菌3株,其余菌株的胆盐耐受性测定结果见表3。
表3胆盐耐受性测定结果
由于胆盐能破坏细胞膜而使细胞死亡,且在小肠中对益生菌存活性影响最大的因素就是胆盐,所以,对胆盐的耐受性是评价益生菌的重要指标之一。根据现有技术的记载,通常胆盐对乳酸菌活性均存在抑制作用(如表3中其他菌株),而本发明的菌株PC646的延迟时间为0小时,表明浓度0.3%胆盐不会抑制该菌株的活性。因此,副干酪乳酪杆菌PC646具有突出的益生特性。
实施例3用副干酪乳酪杆菌PC646发酵制备褐色乳饮料
褐色乳饮料是以脱脂乳粉和还原糖为原料,水合后经高温热处理、冷却,通过乳酸菌长时间发酵,再经过调配、均质等工艺制得的一种口味香浓、营养丰富、具有调节胃肠道功能的发酵型乳饮料。
将水预热至65℃后加入脱脂乳粉、白砂糖,混料均匀后在55℃下水合30min,均质后在95℃下恒温褐变三小时,然后冷却至35-37℃,接入副干酪乳酪杆菌PC646(1×107CFU/ml)37℃恒温发酵,发酵终点至pH4.50及以下,滴定酸度达到200°T以上,约发酵60小时。置于4℃冷藏备用。另取水预热至65℃后加入白砂糖、苹果汁均匀混合15-20min,95℃杀菌5min,冷却至室温后添加发酵基料搅拌15min,柠檬酸调pH至3.70,然后均质、分装,冷藏于4℃。完成后进行感官评定,每间隔7天测定货架期内酸度和益生菌活菌数变化。结果见表4、表5。
表4发酵褐色乳饮料在4℃贮藏期间酸度和益生菌活菌数的变化
由表4的测定结果可知,随着货架期的延长,褐色乳饮料中活菌数在21d内下降缓慢,到保质期末活菌数存活率为63.5%,酸度变化在10°T以内。酸度变化较缓慢,菌体生长较健壮,货架期内菌体死亡较少,更能适应低酸的环境,证明酸马奶分离源的副干酪乳酪杆菌PC646具有良好的益生特性。
另一方面,感官评鉴得分是一株益生菌是否能够作为商业发酵剂的重要因素之一。尽管不少益生菌都能够作为菌种用于制备发酵饮品,但经常存在滋味或口感评分较低或气味较差的缺陷,使得所得产品风味下降,难以被消费者接受,因此无法应用于商业发酵剂。本发明的菌株的感官评定结果如下:
表5发酵褐乳饮料感官评鉴结果
由表5结果表明,本发明的副干酪乳酪杆菌(Lacticaseibacillus paracasei)PC646使得发酵褐乳饮料的口感酸中带甜,滋气味丰富柔和,具有浓郁的发酵风味与美拉德风味,色泽呈现均匀的浅褐色。
实施例4用副干酪乳酪杆菌PC646发酵制备发酵豆乳饮料
发酵豆乳饮料因其蛋白质含量高,易于消化吸收,能调节肠道功能,克服乳糖不耐症,是一种很有发展前景的发酵型乳饮料。
将4.4%的大豆蛋白粉与2%的葡萄糖按比例混合,加入60℃蒸馏水中搅拌15min,水合30min后于20Mpa压力下进行均质处理,将样品放入水浴锅,升温至90℃,杀菌30min,再水浴冷却至(37±0.5)℃接种副干酪乳酪杆菌PC646(1×107CFU/ml),于37℃恒温发酵至pH3.70以下终止发酵,制得发酵基料。将10%的白砂糖充分溶解于蒸馏水中后进行巴氏杀菌,冷却到室温后与25%的发酵豆乳基料按照比例充分混合后,调节pH值,于18Mpa压力下进行均质处理,制得成品发酵豆乳饮料,并进行感官评定,于温度4℃下贮藏28d。贮藏期间每隔7d测定pH值、滴定酸度以及活菌数,结果见表6、表7。
表6发酵豆乳饮料在4℃贮藏期间酸度和益生菌活菌数的变化
由表6的测定结果可知,在为期28d的贮藏期间,发酵豆乳饮料中的活菌数下降幅度平缓,到贮藏末期活菌数为5.40×108CFU/g,活菌数量表现优异;滴定酸度值变化趋势与pH值变化趋势一致,变化平缓,贮藏结束时pH为3.59,滴定酸度为32°T,说明该菌体活力较强,贮藏稳定性良好。
感官评价是衡量食品品质和风味最直接、有效的方法,也是影响消费者对产品购买意象的关键因素,反映产品的受欢迎情况。本发明菌株制作发酵豆乳饮料的感官评定结果如下:
表7发酵豆乳饮料感官评鉴结果
由表7可知,本发明的副干酪乳酪杆菌(Lacticaseibacillusparacasei)PC646使得发酵豆乳饮料的口感酸甜适中,气味柔和,口感香滑,具有典型的乳饮料香味和豆香味,色泽呈现均匀的乳白色。
Claims (5)
1. 一株副干酪乳酪杆菌(Lacticaseibacillus paracasei)PC646,所述副干酪乳酪杆菌(Lacticaseibacillus paracasei)PC646于2021年9月2保藏于中国微生物菌种保藏管理委员会普通微生物菌种保藏中心,保藏编号为CGMCC No. 23515。
2. 权利要求1的副干酪乳酪杆菌(Lacticaseibacillus paracasei)PC646在食品添加剂中的应用。
3.根据权利要求2所述的应用,其特征在于所述食品添加剂是乳品发酵剂。
4.根据权利要求3所述的应用,其特征在于所述乳品发酵剂用于发酵酸奶或发酵豆乳。
5. 权利要求1的副干酪乳酪杆菌(Lacticaseibacillus paracasei)PC646在制备褐色发酵乳中的应用。
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