CN116836893B - 一株副干酪乳酪杆菌及其后生元产品和应用 - Google Patents
一株副干酪乳酪杆菌及其后生元产品和应用 Download PDFInfo
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Abstract
本发明公开了一株副干酪乳酪杆菌及其后生元产品和应用,涉及微生物技术领域。本发明提供了一株副干酪乳酪杆菌YJS05199,拉丁学名为Lacticaseibacillus paracasei,保藏编号为GDMCC No.63668,于2023年7月19日保藏于广东省微生物菌种保藏中心。本发明还提供了应用该菌制备的后生元,副干酪乳酪杆菌及其制备的后生元具有抑菌作用,可抑制高脂饮食小鼠的体重增加,降低脂肪组织重量和血糖浓度,并能够显著降低小鼠血清TG、TC、LDL‑C的水平,该菌及其制备的后生元在制备预防或治疗肥胖及其相关疾病的产品或制备抑菌的产品中具有较大的应用前景。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一株副干酪乳酪杆菌及其后生元产品和应用。
背景技术
肥胖症是一种由环境、遗传、内分泌等多种因素构成的慢性代谢性疾病,人们生活条件不断改善,饮食中高脂和高糖食物的摄入量增加,饮食的不均衡使得部分人代谢紊乱,肥胖症患病率增加。肥胖症会增加慢性代谢性疾病和心血管疾病发生和发展的风险,所述的慢性代谢性疾病包括糖尿病、高血压、非酒精性脂肪肝疾病、动脉粥样硬化和血脂异常等。
肥胖症的治疗主要通过控制饮食、运动、口服药物等方法,过度超重者会采用抽脂或胃肠道手术。但控制饮食和运动的效果并不明显,需要长期坚持,并且若运动方法不当,容易造成关节损伤。口服西药大多伴有腹泻、呕吐等不良反应,且价格昂贵。抽脂和胃肠道手术存在一定的手术风险,并且抽脂效果难维持,手术的花费较大。
益生菌是调控人体肠道菌群平衡和促进消化的重要微生物,具有安全、无毒副作用等特点。随着对益生菌的研究深入,发现不仅活菌能够发挥益生功能,灭活的菌体细胞、菌体死亡后溶解释放的成分,以及细菌的代谢产物也具有促进健康的作用。
中国专利CN114717146A中公开了一种副干酪乳杆菌制备的缓解脂肪肝与肥胖的后生元及其应用,该专利提供了一种由副干酪乳杆菌CCFM1224制备的后生元,其具有缓解脂肪肝与肥胖的作用,能够显著抑制高脂饮食小鼠的体重增加、肝脏脂肪堆积和病变以及血清TC、HDL-C、CHE的水平;显著改善高脂饮食小鼠的血糖稳态;显著降低白色脂肪细胞大小,维持棕色脂肪的正常形态,促进白色脂肪棕色化;有效降低了高脂饮食小鼠肝脏和附睾白色脂肪组织重量。其制备的后生元在制备预防和/或治疗脂肪肝和肥胖及代谢失调相关的疾病的产品中具有巨大的应用前景。
中国专利CN113322216A中公开了一种副干酪乳杆菌B111H及其在代谢综合征中的应用,所述的副干酪乳杆菌的保藏编号为CGMCC No.22184,该专利中所述的副干酪乳杆菌菌株B111H能够显著抑制干细胞脂质积累,降低体重和血脂水平,进而改善肝脏脂肪变性,对于缓解高脂血症、肥胖和脂肪肝的发生具有重要的意义。
目前,适于治疗或预防肥胖及其相关疾病的肠道微生物仍有待进一步开发,益生菌的后生元具有稳定、保质期长、安全等优势,其加工、储存和运输更便利,在开发新型具有健康功能的产品上具有广阔前景。
发明内容
本发明的目的是提供一株副干酪乳酪杆菌及其后生元产品和应用,本发明的副干酪乳酪杆菌对食源性致病菌具有抑菌作用,并能够有效的降血脂和降血糖,该菌制备的后生元,可有效抑制高脂饮食小鼠的体重增加,降低脂肪重量、血清TC、TG、LDL-C和血糖浓度,同时,本发明开发了副干酪乳酪杆菌及其后生元的新应用。
术语解释:
术语“后生元”是指益生菌经加工处理后的益生菌代谢物成分的统称,包括菌体组成成分与菌体代谢产物。具体为益生菌经由热处理、物理性处理、高静水压处理、冷冻干燥、超音波振荡等方式处理后,仍保持有与其活菌相同活性的菌体成分。所述的菌体组成成分包括肽聚糖、壁磷壁酸、脂磷壁酸、细胞壁多糖、细胞表面相关蛋白。所述的菌体代谢产物包括酶、有机酸、蛋白质、多肽、胞外多糖。
为实现上述发明目的,本发明的技术方案如下:
一方面,本发明提供了一种副干酪乳酪杆菌,拉丁学名为Lacticaseibacillus paracasei,保藏编号为GDMCC No.63668,于2023年7月19日保藏于广东省微生物菌种保藏中心。
具体地,将保藏编号为GDMCC No.63668的副干酪乳酪杆菌命名为副干酪乳酪杆菌(Lacticaseibacillus paracasei)YJS05199。
具体地,所述的副干酪乳酪杆菌YJS05199是从贵州发酵食品中分离获得,该菌株经过测序,将其16S rRNA序列在NCBI中进行核酸序列对比,得到对比结果后鉴定为副干酪乳酪杆菌。
进一步具体地,所述副干酪乳酪杆菌YJS05199在MRS固体培养基上的菌落为白色、光滑、圆形的菌落。其为革兰氏阳性菌,兼性厌氧。
又一方面,本发明提供了应用上述的副干酪乳酪杆菌YJS05199制备的后生元。
又一方面,本发明提供了后生元的制备方法,所述的制备方法包括将副干酪乳酪杆菌YJS05199接种于发酵培养基中培养获得菌液,进行热处理后获得后生元。
优选地,所述的副干酪乳酪杆菌YJS05199的接种量为1-5%。
进一步优选地,所述的副干酪乳酪杆菌YJS05199的接种量为2-3%。
优选地,所述的发酵培养基为液体培养基。
优选地,所述的液体发酵培养基选自MRS培养基、LB培养基中的一种或多种。
进一步优选地,所述的液体发酵培养基为MRS培养基。
优选地,所述的培养的温度为35-37℃,培养的时间为16-24h。
优选地,所述的热处理的条件为巴氏杀菌。
进一步优选地,所述的巴氏杀菌的温度为62-65℃,时间为20-30min。
再进一步优选地,所述的巴氏杀菌的温度为65℃,时间为30min。
优选地,所述后生元可以制备成液态制剂、固态粉剂、膏剂等剂型。
具体地,所述的液态制剂包括但不限于混悬剂、油剂。
具体地,所述的固态制剂包括但不限于粉剂、片剂、胶囊剂、颗粒剂。
具体地,所述固态制剂为将上述后生元经干燥制备而成。
进一步具体地,所述的干燥包括真空冷冻干燥、喷雾干燥、流化床干燥和真空干燥。
又一方面,本发明提供了上述的副干酪乳酪杆菌或上述的后生元在制备预防、治疗或辅助治疗肥胖及其相关疾病的药物中的应用。
具体地,所述的肥胖相关疾病包括肥胖症、糖尿病、高血压、高血脂、内分泌失调、肥胖性心肌病、脂肪肝和动脉硬化。
进一步具体地,所述的肥胖相关疾病为糖尿病。
再进一步具体地,所述的糖尿病包括1型糖尿病和2型糖尿病。
具体地,在所述的药物中含有2×105-2×1014CFU/mL或2×105-2×1014CFU/g的副干酪乳酪杆菌,具体如2×105CFU/mL(CFU/g)、2×106CFU/mL(CFU/g)、2×107CFU/mL(CFU/g)、2×108CFU/mL(CFU/g)、2×109CFU/mL(CFU/g)、2×1010CFU/mL(CFU/g)、2×1011CFU/mL(CFU/g)、2×1012CFU/mL(CFU/g)、2×1013CFU/mL(CFU/g)、2×1014CFU/mL(CFU/g)等,该数值范围内的其他点值均可选择。
具体地,所述的药物中后生元的剂量不低于200mg/kg体重。
具体地,所述药物的作用为降血脂和降血糖。
又一方面,本发明提供了上述的副干酪乳酪杆菌在制备预防或辅助治疗肥胖及其相关疾病的食品、保健品或膳食补充剂中的应用。
优选地,所述的食品包括发酵乳、酸乳酪、奶粉、纯奶、酸奶、布丁、固体饮料、压片糖果、乳酸菌饮料、乳酸菌发酵豆制品和奶酪。
进一步优选地,所述的食品中还可以包括常规食品辅料,所述的食品辅料包括但不限于矫味剂、粘合剂、崩解剂、润滑剂、抗酸剂。
优选地,所述的食品制剂选自固体、乳品、溶液制品、粉末制品和悬浮液制品中的任意一种。
优选地,所述的保健品中含有2×105-2×1014CFU/mL或2×105-2×1014CFU/g的副干酪乳酪杆菌。
进一步优选地,所述的保健品中还包括保健品常规辅料,所述的辅料包括但不限于填充剂、矫味剂、粘合剂、崩解剂、润滑剂、抗酸剂和营养强化剂。
进一步优选地,所述的保健品包括乳液制品、溶液制品、粉末制品和固体制品。
又一方面,本发明提供了上述的副干酪乳酪杆菌或上述的后生元在制备抑菌产品中的应用。
优选地,所述的抑菌为抑制食源性病原菌。
具体地,所述的食源性病原菌包括但不限于沙门氏菌、金黄色葡萄球菌、大肠杆菌、副溶血性弧菌、阪崎肠杆菌、单核细胞增生李斯特氏菌、志贺氏菌。
进一步具体地,所述的沙门氏菌包括但不限于鼠伤寒沙门氏菌。
再进一步具体地,所述的食源性病原菌为鼠伤寒沙门氏菌、金黄色葡萄球菌和大肠杆菌。
又一方面,本发明提供了一种预防、治疗或辅助治疗肥胖及肥胖相关疾病的药品,所述的药品中包括上述的副干酪乳酪杆菌或上述的后生元。
具体地,所述的药品中包含2×105-2×1014CFU/mL或2×105-2×1014CFU/g的副干酪乳酪杆菌。
具体地,所述的药品中还包括药学上可接受的载体。
再进一步具体地,所述的载体选自赋性剂、稳定剂、稀释剂、粘合剂、防腐剂、润滑剂、填充剂中的一种或多种。
具体地,所述赋性剂选自微晶纤维素、乳糖、预胶化淀粉、环糊精、羧甲基纤维素、甘露醇中的至少一种。
具体地,所述稳定剂选自琼脂、藻酸、纤维素醚、羧甲基甲壳酯中的至少一种。
具体地,所述稀释剂选自赤藓糖醇、甘露醇、山梨糖醇、木糖醇、乳糖、蔗糖、玉米淀粉、马铃薯淀粉、磷酸钙、柠檬酸钙、结晶纤维素中的至少一种。
具体地,所述粘合剂选自乙醇、淀粉浆、糖浆、羟丙基甲基纤维素、羧甲基纤维素钠、海藻酸钠、聚乙烯吡咯烷酮中的至少一种。
具体地,所述防腐剂选自羟苯甲酸甲酯、对羟苯甲酸丙酯、尼泊金甲酯、尼泊金乙酯、尼泊金丙酯、三氯叔丁醇、硫柳汞、氧氰化汞、苯氧乙醇、氯己定、苯甲酸、苯甲酸钠、氯甲酚、苯扎溴铵、苯扎氯铵、羟苯乙酯中的至少一种。
具体地,所述润滑剂选自硬脂酸镁、硬脂酸、氯化钠、油酸钠、月桂醇硫酸钠、泊洛沙姆中的至少一种。
具体地,所述的填充剂选自海藻糖、乳糖、壳聚糖、淀粉和糊精中的至少一种。
进一步具体地,所述的药物的剂型选自片剂、胶囊剂、口服液体剂型、颗粒剂、软膏剂、混悬剂、散剂、乳剂、溶液剂、滴丸剂、栓剂、气雾剂中的任意一种。
又一方面,本发明公开了一种预防或辅助治疗肥胖及其相关疾病的食品、保健品或膳食补充剂,所述的食品、保健品或膳食补充剂中包括上述的副干酪乳酪杆菌或上述的后生元。
又一方面,本发明提供了一种抑菌的产品,所述的产品中包括上述的副干酪乳酪杆菌或上述的后生元。
本发明的有益效果为:
本发明提供一株副干酪乳酪杆菌(Lacticaseibacillus paracasei)YJS05199,该菌及其制备的后生元具有的作用包括:
(1)该菌及其后生元对多种食源性致病菌(鼠伤寒沙门氏菌、金黄色葡萄球菌、大肠杆菌)具有抑制作用,可促进宿主肠道健康;
(2)可显著减少高脂饮食小鼠的体重增加,后生元低、高剂量组相比模型组的平均体重增加量分别降低了45.40%和46.48%;
(3)有效降低高脂饮食小鼠脂肪组织重量,低、高剂量的副干酪乳酪杆菌后生元比模型组分别下降了47.21%和53.83%;
(4)显著降低高脂饮食小鼠血清TG、TC、LDL-C的水平,与模型组相比,低、高剂量的副干酪乳酪杆菌后生元分别降低了TC水平27.91%和32.56%;TG水平比模型组分别降低了25.17%和34.83%;LDL-C分别降低了25.69%和36.99%。HDL-C水平分别升高了19.08%和20.09%;
(5)降低高脂饮食小鼠的血糖浓度,改善血糖稳态;
(6)该菌后生元的制备方式简单,适合大批量生产、运输和贮存。
副干酪乳酪杆菌YJS05199及其后生元在制备具有降血脂、降血糖和抑菌功能的产品中,具有巨大的应用前景。
保藏说明
保藏编号:GDMCC No.63668;
分类命名:Lacticaseibacillus paracasei;
保藏时间:2023年7月19日;
保藏单位:广东省微生物菌种保藏中心;
保藏单位简称:GDMCC;
保藏地址:广州市先烈中路100号大院59号楼5楼。
附图说明
图1为副干酪乳酪杆菌YJS05199的菌落形态。
图2为副干酪乳酪杆菌YJS05199的革兰氏染色结果。
图3为副干酪乳酪杆菌发酵上清液及后生元对3种食源性致病菌的抑菌作用。
图4为副干酪乳酪杆菌、发酵上清液、后生元的体外降胆固醇作用。
图5为不同组别小鼠体重增加量(*p<0.05,**p<0.01)。
图6为不同组别小鼠的脂肪组织重量(*p<0.05,**p<0.01)。
图7为不同组别小鼠血清生化指标:TG(甘油三酯)、TC(总胆固醇)、HDL-C(高密度脂蛋白胆固醇)、LDL-C(低密度脂蛋白胆固醇)(*p<0.05,**p<0.01)。
图8为不同组别小鼠口服葡萄糖耐量(*p<0.05,**p<0.01)。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施例,进一步阐明本发明,但下述实施例仅为本发明的优选实施例,并非全部。基于实施方式中的实施例,本领域技术人员在没有做出创造性劳动的前提下所获得其它实施例,都属于本发明的保护范围。下述实施例中,若无特殊说明,所用的操作方法均为常规操作方法,所用设备均为常规设备,各个实施例所用设备材料均相同。
基础实施例:
副干酪乳酪杆菌的分离纯化:
无菌操作称取25 g样品置于225 mL无菌生理盐水中,振匀5 min,制成1:10样液,然后十倍梯度稀释至一定浓度。取200μL稀释液均匀涂布于MRS平板中,37℃静置培养48h。从平板上挑取疑似乳酸菌的单菌落进行划线,划线纯化3次,至菌落形态均一。将革兰氏染色反应阳性、H2O2接触酶阴性的菌株进行分子鉴定。
菌株的16S鉴定:
提取菌株的基因组,采用细菌16S rDNA通用引物进行PCR扩增和测序,将该序列在NCBI中进行比对,结果显示为副干酪乳酪杆菌。该菌在MRS固体培养基上培养24 h后的菌落形态如图1所示,革兰氏染色结果如图2所示。
实施例1副干酪乳酪杆菌YJS05199后生元的制备
将副干酪乳酪杆菌YJS05199种子液按2%接种量接种于MRS液体培养基,37℃培养16h。
将菌液热处理(65℃,30min)后获得后生元,经过浓缩冷冻干燥得到后生元冻干粉备用。
实施例2副干酪乳酪杆菌发酵液上清液及后生元的抑菌作用
采用牛津杯法检测副干酪乳酪杆菌发酵上清液和后生元的抗菌活性
食源性致病菌(鼠伤寒沙门氏菌、金黄色葡萄球菌和大肠杆菌)活化培养后调整菌浓度至107CFU/mL,吸取100 μL菌液到LB固体培养基上,使用无菌涂布棒将致病菌菌液涂布均匀,在固体培养基表面水平放置3个牛津杯。分别吸取200 μL副干酪乳酪杆菌上清液、后生元(液态制剂)于牛津杯孔内,以MRS培养基为阴性对照,平板放置4℃预扩散4 h,后转移到恒温培养箱培养12 h,观察并测量抑菌圈直径,实验平行3次。由图3可知,副干酪乳酪杆菌上清液、后生元对鼠伤寒沙门氏菌、金黄色葡萄球菌、大肠杆菌均具有抑制作用。
实施例3体外降胆固醇作用
测定副干酪乳酪杆菌在胆固醇培养基中培养时的降胆固醇作用,以及发酵上清液、后生元的体外降胆固醇作用,具体方法如下:
(1)副干酪乳酪杆菌在MRS-CHO培养基中培养的降胆固醇作用
将副干酪乳酪杆菌按2%(v/v)接种量接种于MRS-CHO培养基(g/L)(胆固醇0.1g,牛胆盐2g,吐温1mL,冰乙酸1mL,蔗糖八乙酸酯0.12g,充分搅拌均匀,并于60℃超声水浴30min,边加边搅拌加入MRS肉汤中,于121℃灭菌20min)中,37℃培养48 h,培养液于4000×g离心10 min,取0.50 mL上清液中加入3 mL无水乙醇和2 mL 50% KOH,充分混合后于60℃水浴10 min,冷却至室温后加5 mL正己烷,涡旋振荡20 s,加3mL蒸馏水后放置5min左右至分层,取2.50 mL正己烷层(上层)到干净的试管中,60℃水浴,氮吹至溶剂完全挥发,加2 mL显色液,充分混匀后加1mL浓硫酸,冷却后于OD550处测吸光度。
计算公式如下:P0(%) = [(D-C)/D] ×100%;
其中:P0代表胆固醇降低率;C:样品在OD550处的吸光度;D:空白对照(MRS-CHO培养基)在OD550处的吸光度。
(2)发酵上清液、后生元的降胆固醇作用
发酵上清液、后生元分别以1:1的体积比作用于5mL含100μg/mL胆固醇的MRS培养基中,37℃振荡12h后测定其降胆固醇率,具体同测定方法(1)。
结果如图4所示,副干酪乳酪杆菌在胆固醇培养基中培养时具有降胆固醇作用,该菌的发酵上清液、后生元亦具有降胆固醇作用。
实施例4副干酪乳酪杆菌后生元对高脂饮食小鼠体重增加量的影响
取5周龄健康雄性C57BL/6小鼠32只,随机分为4组,每组8只,4组分别为:正常饮食组(对照组)、高脂饲料喂养模型组、副干酪乳酪杆菌YJS05199后生元组低剂量组(H-1组,200mg/kg小鼠体重)和副干酪乳酪杆菌YJS05199后生元组高剂量组(H-2组,600mg/kg小鼠体重)。
小鼠适应性喂养一周后,正常饮食对照组饲喂正常饲料,其余组饲喂高脂饲料(酪蛋白100 g/kg,蔗糖200 g/kg,猪油150 g/kg,大小鼠维持配合饲料522 g/kg,大小鼠预混料4 g/kg,胆固醇12 g/kg,胆酸钠2 g/kg,碳酸氢钙(饲料级1型)6 g/kg,石粉(碳酸钙)4g/kg,基础饲料等购自广东省医学动物实验中心),后生元冻干粉(溶于生理盐水)以0.2mL/只/天的量对小鼠进行灌胃(400 mg/kg BW),正常饮食对照组与模型组灌胃等量生理盐水作为对照。所有组均为自由饮水和摄食。
分别在后生元干预的第1周和第12周对小鼠进行称重,小鼠体重增加量如图5所示。连续喂养高脂饲料使模型组的体重增加量显著高于正常饮食对照组,而后生元低、高剂量组相比模型组的平均体重增加量分别降低了45.40%和46.48%,说明副干酪乳酪杆菌后生元能够缓解高脂饮食小鼠体重增加。
实施例5副干酪乳酪杆菌后生元对高脂饮食小鼠脂肪堆积的影响
上述喂养实验结束后,取血并处死小鼠,收集脂肪组织并称重。由图6可知,模型组的脂肪组织最多,而低、高剂量的副干酪乳酪杆菌后生元均能显著抑制高脂饮食小鼠的脂肪增加,比模型组分别下降了47.21%和53.83%。
实施例6副干酪乳酪杆菌后生元对高脂饮食小鼠血清生化指标的影响
小鼠饲养结束后,取血并处死小鼠,将全血离心(2500 r/min,15min)取血清,采用全自动生化分析仪测定血清相关指标,包括:TG(甘油三酯)、TC(总胆固醇)、HDL-C(高密度脂蛋白胆固醇)、LDL-C(低密度脂蛋白胆固醇)。结果如图7。
副干酪乳酪杆菌后生元在以低、高剂量干预时均降低了高脂饮食小鼠血清TC水平,比模型组分别降低了27.91%和32.56%;低、高剂量干预组的TG水平比模型组分别降低了25.17%和34.83%;低、高剂量干预组的LDL-C水平比模型组分别降低了25.69%和36.99%;低、高剂量后生元的干预对高脂饲料诱导的小鼠血清中HDL-C的下降具有显著的保护作用,低、高剂量组相比于模型组分别升高了19.08%和20.09%。上述结果说明副干酪乳酪杆菌的后生元具有降胆固醇和降血脂作用,有利于缓解肥胖。
实施例7副干酪乳酪杆菌后生元对高脂饮食小鼠血糖稳态的影响
在小鼠喂养实验结束前通过口服糖耐量试验考察小鼠的血糖稳态。小鼠禁食12h后,按照2mg/g体重的剂量灌胃20%葡萄糖溶液,分别在灌胃前(0min)和灌胃后15min、30min、60min、90min、120min尾静脉采血,测定血糖值。
结果如图8所示,灌胃葡萄糖溶液前,模型组的空腹血糖值为9.03mmol/L,副干酪乳酪杆菌后生元低、高剂量干预时的小鼠空腹血糖值分别为7.05mmol/L和6.93mmo1/L,并且2h时内血糖能降低至接近空腹水平。说明本发明的副干酪乳酪杆菌后生元能够维持高脂小鼠的血糖稳态。
对比例1
专利CN114717146A中公开的一株副干酪乳杆菌,保藏编号为GDMCC No.62272。与本申请的效果比对具体如下表:
表1.
从上表中的比对可以看出,本申请的副干酪乳酪杆菌组与模型组相比,体重下降较多,并且本申请的高剂量给药组的给药剂量小于专利CN114717146A(下称为对比例1)中高剂量组的给药剂量,但本申请的空腹血糖较低;对于TC含量,均与模型组相比,本申请的低剂量组TC下降了27.91%,对比例1中下降了24.68%,并且本申请的600mg/kg给药量与对比例1的800mg/kg的给药量TC下降的程度相同;本申请中与模型组相比HDL-C升高了,对比例1中HDL-C下降了,本领域的技术人员公知,HDL-C升高有助于缓解肥胖,上述结果说明本申请的副干酪乳酪杆菌相比于保藏编号为GDMCC No.62272的副干酪乳酪杆菌缓解肥胖的效果较好。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1. 一株副干酪乳酪杆菌(Lacticaseibacillus paracasei)YJS05199,保藏编号为GDMCC No.63668。
2.应用权利要求1所述的副干酪乳酪杆菌制备的后生元。
3.权利要求2所述的后生元的制备方法,其特征在于,所述的制备方法为将副干酪乳酪杆菌接种于发酵培养基中培养获得菌液,进行热处理后获得后生元。
4.根据权利要求3所述的制备方法,其特征在于,所述的副干酪乳酪杆菌的接种量为1-5%。
5.根据权利要求3所述的制备方法,其特征在于,所述培养的温度为35-37℃,培养的时间为16-24h。
6.根据权利要求3所述的制备方法,其特征在于,所述的热处理的条件为巴氏杀菌;所述的巴氏杀菌的温度为62-65℃,巴氏杀菌的时间为20-30min。
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