CN112877262A - 一种植物乳杆菌及其应用 - Google Patents
一种植物乳杆菌及其应用 Download PDFInfo
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- CN112877262A CN112877262A CN202110362228.5A CN202110362228A CN112877262A CN 112877262 A CN112877262 A CN 112877262A CN 202110362228 A CN202110362228 A CN 202110362228A CN 112877262 A CN112877262 A CN 112877262A
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- lactobacillus plantarum
- nkk20
- freeze
- fermentation
- acid
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Abstract
本发明涉及一株植物乳杆菌(Lactobacillus plantarum)NKK20及其应用,属于微生物领域。该菌株分离自健康人体肠道,已保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO:M2020596。该植物乳杆菌菌株具有较强的耐酸、耐胆盐和肠粘膜细胞粘附能力;能够显著抑制肠炎沙门氏菌和金黄色葡萄球菌等致病菌,具有抑制肠胃道病原菌功能;在酸奶发酵中能够显著降低出牛奶中胆固醇含量,可应用于药品、功能性食品及食品添加剂领域,对人体健康具有重要意义与价值。
Description
技术领域
本发明属于微生物领域,特别涉及一种植物乳杆菌,还涉及该菌株的应用。
背景技术
胃肠道疾病主要指一般炎症性胃肠道疾病(急、慢性胃炎,急、慢性阑尾炎等)、消化性溃疡、胃癌、食道癌、大肠癌及肠易激综合征等。现如今,胃肠道疾病发病率高,在我国有"十人九胃病"的说法,其病程较长、治疗较难、反复发作等均是胃肠道疾病的特点。医学专家研究发现,胃肠道疾病产生的原因是胃黏膜的防护因子及攻击因子失衡,造成胃肠道黏膜内的防护因子弱于攻击因子而产生胃肠道疾病。
益生菌可以调节肠胃中的有益菌群含量。植物乳杆菌是广泛存在于发酵食品中,是一种常见的益生菌菌种。益生菌需要顺利通过胃的酸性环境以及十二指肠高胆盐环境,以活菌的状态到达小肠和大肠,并发挥其调节肠道菌群的作用。而益生菌发挥其调节肠道菌群的作用,主要表现在能降低微环境的pH值,能黏附在肠道粘膜上与致病菌竞争黏附位点、与其凝集使致病菌顺便排除体外,还能一定程度上抑制致病菌的生长。
为达到上述效果,植物乳杆菌需具有良好的耐酸、耐胆盐的能力,还要具备凝集黏附、抑制致病菌生长的功能。
植物乳杆菌229v和植物乳杆菌Lp-115均经过美国食品药品监督管理(FDA)公认安全食品添加(Generally recognized as safe,GRAS)认证的两个植物乳杆菌菌株,植物乳杆菌229v和植物乳杆菌Lp-115均具有一定的耐酸、耐胆盐的能力,因此应用广泛,然而,申请人发现,其在耐酸、耐胆盐、抑制致病菌生长的能力有不同,例如,植物乳杆菌NKK20对肠粘膜粘附率显著高于植物乳杆菌229v;再如,植物乳杆菌229v对金黄色葡萄球菌ATCC25923抑制能力较弱;植物乳杆菌LP-115对肠炎沙门氏菌ATCC 14028抑制能力较弱。
因此,寻找具有更高耐酸、耐胆盐、抑制致病菌生长能力的益生菌,以适应更多不同病患,达到更好的治疗效果具有非常积极的意义。
发明内容
发明目的:为了解决植物乳杆菌229v和植物乳杆菌Lp-115耐酸、耐胆盐的能力不同,寻找具有更高耐酸、耐胆盐、抑制致病菌生长能力的益生菌,及确保发酵过程中发酵条件对菌种的稳定性、功能性,本发明提供了一种植物乳杆菌,以及该植物乳杆菌发酵工艺。
技术方案:本发明所采用的技术方案为。
本发明提供了一种植物乳杆菌,其特征在于,命名为植物乳杆菌(Lactobacillus plantarum)NKK20,该菌株已于2020年10月19日保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO:M2020596,保藏地址为中华人民共和国湖北省武汉市武汉大学。
本发明提供的菌株能够耐酸、耐胆盐,代谢生成乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸。
本发明提供的菌株能够粘附肠粘膜,抑制黄色葡萄球菌和肠炎沙门氏菌生长。
本发明还提供了一种菌剂,活性成分中含有上述任一项所述的植物乳杆菌菌株。
优选地,所述菌剂中,植物乳杆菌菌株发酵液体培养基中含有3g/L-10g/L卵磷脂。
优选地,该菌剂在发酵冷冻干燥制备过程中,添加有冷冻干燥保护剂,冷冻干燥保护剂成分为:水60%-80%,脱脂乳粉6%-14%,蔗糖2%-8%,甘露糖醇4%-7%,吐温-80 1%-2.5%,甜菜碱1%-3%,谷氨酸钠0.5%-1.2%,可溶性淀粉2%-8%;优选的水70%,脱脂乳粉10%,蔗糖5%,甘露糖醇5%,吐温-80 2%,甜菜碱2%,谷氨酸钠1%,可溶性淀粉5%。
优选地,所述菌剂在制备过程中经5%-10%壳聚糖溶液与4%-10%海藻酸钠溶液进行铰链包埋保护。
本发明还提供了一种酸奶发酵剂,成分中含有上述任意一项所述的菌剂。本发明还提供了一种组合物,成分中含有上述任一项所述的菌剂。
本发明还提供了上述组合物在制备食品中的应用。
本发明还提供了上述组合物在制备抑制胃肠道致病菌的药物中的应用。
本发明还提供了上述组合物在制备降低胆固醇药物和/或食品中的应用。
本发明还提供了一种植物乳杆菌的冻干粉的制备方法,包括以下步骤。
(1)发酵培养。
将植物乳杆菌NKK20按3%-15%接种量接种于高产培养基中,36℃-39℃发酵培养16-20h;活菌量达到108CFU/ml-1010CFU/ml时,将发酵液降温至20℃以下,离心,收获菌泥;
其中,高产培养基为水溶液,含有:大豆蛋白胨6-10g/L,酵母浸粉3-8g/L,无水葡萄糖15-28g/L,乙酸钠3-6g/L,大豆卵磷脂3g/L-10g/L,磷酸氢二钾1.8-2.2g/L,柠檬酸氢二铵1.5-2.5g/L,硫酸镁0.1-0.3g/L,硫酸锰0.02-0.06g/L,吐温-80 0.5-2.0ml/L,L-半胱氨酸盐酸盐~0.05-0.25g/L,pH值5.5-6.5。
(2)保护剂乳化。
将收获的菌泥稀释至含水量60%-80%,等体积加入菌泥保护剂进行乳化;
其中,菌泥保护剂成分为:水70%,脱脂乳粉10%,蔗糖5%,甘露糖醇5%,吐温-80 2%,甜菜碱2%,谷氨酸钠1%,可溶性淀粉5%。
(3)菌体铰链包埋保护。
乳化液中加入壳聚糖溶液,搅拌均匀,搅拌时间为1-2min,搅拌转速为100-120rpm;再加入与壳聚糖溶液等体积的海藻酸钠溶液,继续搅拌,搅拌时间为1-2min,搅拌转速为60-70rpm。
其中,乳化液:壳聚糖溶液的体积比为4:1;壳聚糖溶液浓度为5%-10%,优选为7.0%;海藻酸钠溶液浓度为4%-10%,优选为6.5%。
(4)真空冷冻干燥,即得植物乳杆菌NKK20冻干粉。
本发明还提供了用于植物乳杆菌(Lactobacillus plantarum)NKK20的培养基,该培养基为水溶液,其成分为:大豆蛋白胨6-10g/L,酵母浸粉3-8g/L,无水葡萄糖15-28g/L,乙酸钠3-6g/L,大豆卵磷脂3g/L-10g/L,磷酸氢二钾1.8-2.2g/L,柠檬酸氢二铵1.5-2.5g/L,硫酸镁0.1-0.3g/L,硫酸锰0.02-0.06g/L,吐温-80 0.5-2.0ml/L,L-半胱氨酸盐酸盐~0.05-0.25g/L;优选地,大豆蛋白胨8g/L,酵母浸粉4g/L,无水葡萄糖25g/L,乙酸钠5g/L,大豆卵磷脂4.5g/L,磷酸氢二钾2g/L,柠檬酸氢二铵2g/L,硫酸镁0.2g/L,硫酸锰0.04g/L,吐温-80 1ml/L,L-半胱氨酸盐酸盐0.2g/L。
本发明还提供了用于植物乳杆菌(Lactobacillus plantarum)NKK20的菌泥保护剂,包括以下组份:水60%-80%,脱脂乳粉6%-14%,蔗糖2%-8%,甘露糖醇4%-7%,吐温-80 1%-2.5%,甜菜碱1%-3%,谷氨酸钠0.5%-1.2%,可溶性淀粉2%-8%;优选地,包括以下组份:水70%,脱脂乳粉10%,蔗糖5%,甘露糖醇5%,吐温-80 2%,甜菜碱2%,谷氨酸钠1%,可溶性淀粉5%。
有益效果:本发明提供的植物乳杆菌同时具有非常优异的高耐酸、耐胆盐、抑制致病菌生长、高产丁酸的能力。
具体而言,申请人发现。
(1)植物乳杆菌NKK20对肠粘膜粘附率很高。
植物乳杆菌NKK20对肠粘膜粘附率与LP-115基本相当,约为81%,显著高于植物乳杆菌229v。
(2)植物乳杆菌NKK20总产酸量,特别是丁酸产量非常高。
植物乳杆菌NKK20总产酸量显著高于植物乳杆菌299v和植物乳杆菌Lp-115,且丁酸产量是植物乳杆菌229v和Lp-115的11倍和7倍;丁酸有助于肠胃粘膜修复起重要作用,因此植物乳杆菌NKK20可有助于消化道健康。
(3)植物乳杆菌NKK20对金黄色葡萄球菌ATCC 25923和肠炎沙门氏菌ATCC14028的抑制性均很强。
植物乳杆菌229v对肠炎沙门氏菌ATCC 14028与植物乳杆菌NKK20相当,而对金黄色葡萄球菌ATCC 25923明显弱于植物乳杆菌NKK20;植物乳杆菌LP-115对金黄色葡萄球菌ATCC 25923与植物乳杆菌NKK20相当,而对肠炎沙门氏菌ATCC 14028明显弱于植物乳杆菌NKK20。
(4)植物乳杆菌NKK20具有非常优异的降胆固醇效果。植物乳杆菌NKK20发酵后的酸奶胆固醇比未发酵前降低了51.23%,对牛奶发酵中降胆固醇的能力明显优于植物乳杆菌229v(39.30%)和Lp-115(25.26%)。
附图说明
图1为植物乳杆菌NKK20扫描电子显微镜(SEM)图。
图2为植物乳杆菌NKK20的生物进化树图。
图3为植物乳杆菌NKK20菌落形态图。
图4为肠粘膜粘附植物乳杆菌NKK20的扫描电子显微镜(SEM)图。
具体实施方式
下面结合附图对本发明做出进一步说明。
实施例1耐酸和胆汁盐植物乳杆菌的筛选
将100µL新鲜采集的健康人体肠道菌群悬浮液以10倍梯度进行稀释后,涂布在MRS琼脂培养基上,装入厌氧培养罐,并用三元混合气(氮气80%、氢气10%和二氧化碳10%)对罐内气体进行置换,使培养罐内最终氧气浓度≤5%,将培养罐在37℃恒温箱中培养48h。挑选独立、色泽光亮、形态饱满的菌落,采用基质辅助激光解吸电离飞行时间质谱(Matrix-assisted laser desorption/ionization time–of-flight massspectrometry, MALDI-TOF MS,简称飞行质谱)技术进行高通量菌种初步鉴定。
对MALDI-TOF MS设备初步鉴定为植物乳杆菌的菌落挑至液体MRS培养基中37℃培养12h后,分别在MRS琼脂培养基和含有0.3%牛胆汁盐的pH=2的MRS琼脂培养基上涂布对比培养,根据在两种琼脂培养基上菌落生长差异情况,筛选出高耐酸和胆汁盐的植物乳杆菌,最后再通过16S rRNA测序进行菌种准确性鉴定,最终从36株植物乳杆菌中筛选出高耐酸和胆汁盐的第20号植物乳杆菌作为目标菌株,并将该菌株命名为植物乳杆菌NKK20。
植物乳杆菌NKK20的扫描电子显微镜(SEM)照片见图1。
植物乳杆菌NKK20的生理活性特征如下:
圆端直杆菌,通常0.9~1.2 µm宽,3~8 µm长,单个、成对或成短链。通常缺乏鞭毛,但能运动。厌氧,表面菌落约3 mm宽,凸起,圆,光滑,细密,白色,偶尔浅黄或深黄色。菌落形态见图3。培养液中的生长物甚至很混浊。通常可发酵L-阿拉伯糖、核酸糖、半乳糖、D-葡萄糖、D-果糖、D-甘露糖、鼠李糖、D-棉子糖、松三糖、β-龙胆二糖、甘露糖醇、山梨糖醇、N-乙酰氨基葡糖、麦芽糖、乳糖、蜜二糖、蔗糖、海藻糖等产生DL-乳酸,不能发酵赤藓糖醇、D-阿拉伯糖、D-木糖、L-木糖、L-山梨糖、肌醇、菊粉、木糖醇等。能够产生胞外多糖。
利用蛋白质组学的方法鉴定植物乳杆菌NKK20有多个表面蛋白,其中富含甘油醛-3-磷酸脱氢酶,一种通常位于细胞内的酶,是糖酵解过程中必不可少的酶,其有助于植物乳杆菌NKK20粘附在肠粘膜上。其他被鉴定的糖酵解蛋白包括磷酸甘油酸激酶、糖-磷酸异构酶、烯醇化酶和磷酸葡萄糖异构酶等,这些酶也被认为在竞争和排斥病原体中发挥重要作用。另外存在的GrpE 和DnaK胁迫蛋白使得植物乳杆菌NKK20在免疫调节中发挥作用。
实施例2 菌株的保存
对筛选出的植物乳杆菌NKK20在MRS琼脂培养基上划线培养48h后,挑选单菌落置于10ml液体培养基中,该液体培养基为含有4.5%大豆卵磷脂和2%碳酸钙的MRS液体培养基。将该培养基置于37℃恒温培养12h-16h,测得培养液OD600值≥1.2时,培养液中按1:1体积加入甘油溶液,该甘油溶液浓度为30%-40%,用吸液器吹打混合均匀后分装至2ml无菌冻存管中,并置于4℃预冷2h,然后置于-20℃预冻4h,最后转至-80℃冰箱或液氮中保存。
实施例3 肠粘膜粘附实验
选择植物乳杆菌NKK20、植物乳杆菌229v、植物乳杆菌LP-115进行肠粘膜粘附实验。
各菌株分别在37℃下培养3h后,扫描电镜分析显示,所有菌株均有粘附肠道黏膜的现象,植物乳杆菌NKK20对肠粘膜粘附率与LP-115基本相当,约为81%,显著高于植物乳杆菌229v,各菌株对肠道黏膜值的粘附率见表2。
肠粘膜粘附植物乳杆菌NKK20的扫描电子显微镜(SEM)图,见图4。
实施例4 植物乳杆菌NKK20抗生素敏感性实验
植物乳杆菌NKK20对临床常用抗生素敏感,但对双氯西林、培氟沙星、甲氧苄啶、头孢他啶耐药(见表2)。观察结果表明,该菌株耐药概率较低,与植物乳杆菌Lp-115耐药性相同,弱于植物乳杆菌229v。虽然对部分抗生素有一定的耐药性,然而,有益菌具备一定耐药性的风险是可控的,且是有益的。
实施例5 植物乳杆菌NKK20有机酸代谢检测
标准品处理:分别称取色谱标准级乙酸91.0mg、丙酸37.0mg、丁酸17.6mg、异丁酸17.6mg、戊酸19.8mg和异戊酸19.8mg,溶于双蒸水中,再各自定容至10mL备用。
样品处理:取发酵16h以上的植物乳杆菌发酵液2ml,10000rpm离心5min,取1mL上清液,加入偏磷酸巴豆酸混合溶液0.2ml,放至-20℃冰箱保存8h-12h,其中偏磷酸巴豆酸混合溶液配置方式为:在10ml的偏磷酸溶液中加入64.64mg的巴豆酸。
GC-14B型气相色谱仪参数设置:色谱柱采用毛细吸管柱,柱温130℃,汽化温度180℃,采用氢离子火焰检测器,检测温度180℃,载气为氮气,压力为60KPa,氢气压力为50KPa,氧气压力50KPa,灵敏度(档)为101,衰减3.0。
气相色谱仪检测:样品解冻后12000rpm离心10min,用1.0μL微量进样器取上清液瞬时注入色谱仪,进样量为0.2μL-1.0μL。与样品处理相同,在1ml标准品中加入0.2mL偏磷酸与巴豆酸的混合样,测出乙酸、丙酸、丁酸、异丁酸、戊酸和异戊酸的保留时间。
有机酸浓度的计算:通过标准样品和内标巴豆酸各自的(或浓度)和峰面积可以计算出乙酸、丙酸及丁酸等有机酸的相对校正因子,然后根据乙酸、丙酸及丁酸的重量(或浓度)与其峰面积成正比计算出各个样品中的乙酸、丙酸及丁酸浓度。
样品某酸浓度(mM)=(样品某酸峰面积值×巴豆酸标样峰面积×某酸标样浓度)/(样品中巴豆酸面积值×某酸标样峰面积)
三株菌检测结果如表3所示,其中植物乳杆菌NKK20总产酸量显著高于植物乳杆菌299v和植物乳杆菌Lp-115,且丁酸产量是植物乳杆菌229v和Lp-115的11倍和7倍。根据相关研究丁酸有助于肠胃粘膜修复起重要作用,因此植物乳杆菌NKK20可有助于消化道健康。
实施例6 植物乳杆菌NKK20对肠道病原菌的抑制
选用肠炎沙门氏菌ATCC14028(Salmonella enteritis ATCC 14028)和金黄色葡萄球菌ATCC25923(Staphylococcus aureus ATCC 25923)两株肠道病原菌标准株进行抑菌性实验。
分别挑取新鲜培养的肠炎沙门氏菌ATCC 14028和金黄色葡萄球菌ATCC 25923单菌落接种于1mLM-肉汤培养基中(青岛海博生物),37℃培养8h,发酵液以120000r/min离心10min收集菌体,菌体用0.9%无菌生理盐水洗涤两次,用0.9%无菌生理盐水制备成OD600为0.5-0.8的菌悬液,4℃保存备用。
将植物乳杆菌NKK20、植物乳杆菌229v、植物乳杆菌LP-115分别接种在50ml MRS液体培养基中,37℃培养18h,测得3株菌的发酵液OD600最小值,用无菌MRS液体培养基将另两株菌OD600值稀释为与之一致,误差在±5%以内。对标准后的发酵液10000r/min离心15min,取上清液测得各菌株pH值后留存备用。分装3份10ml无菌MRS液体,分别调pH至与获得各菌株上清液pH值一致,为对照液。
分别取金黄色葡萄球菌ATCC 25923和肠炎沙门氏菌ATCC 14028菌悬液1mL于100mL灭菌后温度为40℃-42℃的M-肉汤琼脂培养基中,混合均匀,记为样品A和样品B。将样品A及样品B分别倾倒平板冷却,并在琼脂培养基上打直径为0.8cm圆孔后加入3株菌的上清液及对应的MRS对照液100μL,37℃培养16h。测量其抑菌圈大小,以待测液产生的抑菌圈半径大于MRS对照液的为有抑菌活性。结果显示,3株植物乳杆菌对肠炎沙门氏菌ATCC 14028和金黄色葡萄球菌ATCC 25923均有不同程度的抑制作用;其中,植物乳杆菌NKK20对金黄色葡萄球菌ATCC 25923和肠炎沙门氏菌ATCC14028的抑制性均很强;植物乳杆菌229v对肠炎沙门氏菌ATCC 14028与植物乳杆菌NKK20相当,而对金黄色葡萄球菌ATCC 25923明显弱于植物乳杆菌NKK20;植物乳杆菌LP-115对金黄色葡萄球菌ATCC 25923与植物乳杆菌NKK20相当,而对肠炎沙门氏菌ATCC 14028明显弱于植物乳杆菌NKK20;结果如表4所示。
实施例7 植物乳杆菌NKK20耐胆盐培养基优化
(1)大豆卵磷脂对植物乳杆菌NKK20耐胆盐能力的影响
植物乳杆菌除了自身耐胆盐能力外,在培养过程中,不同的营养成分影响其代谢过程,进而对发酵后休眠状态的细胞耐胆盐能力起一定影响。
植物乳杆菌NKK20高产培养基A成分为:大豆蛋白胨8g/L,酵母浸粉4g/L,无水葡萄糖25g/L,乙酸钠5g/L,磷酸氢二钾2g/L,柠檬酸氢二铵2g/L,硫酸镁0.2g/L,硫酸锰0.04g/L,吐温-80 1ml/L,L-半胱氨酸盐酸盐0.2g/L,pH为6.0。
将高产培养基A中添加3g/L-10g/L的卵磷脂,其中卵磷脂来源于蛋黄、花生、大豆、肝脏、牛奶等,基于经济型考虑优选大豆卵磷脂,发酵制备植物乳杆菌NKK20进行耐胆盐实验,当大豆卵磷脂添加量为4.5g/L时发酵产生的植物乳杆菌NKK20耐胆盐能力最强,相比未添加大豆卵磷脂的高产培养基A发酵产生的植物乳杆菌NKK20具有显著耐胆盐特点。
(2)培养基组成对植物乳杆菌NKK20耐胆盐能力的影响
实施例8 植物乳杆菌NKK20的冻干粉制备
步骤1 培养基配置
植物乳杆菌NKK20可选择特定的培养基进行发酵,该培养基为水溶液,按实施例7中高产培养基A+4.5g/L大豆卵磷脂培养基。
步骤2培养基灭菌及接种培养
培养基配置后调节pH值为6.5,121℃灭菌15min,冷却至38℃-39℃时,按3%-15%接种量,优选为12%进行发酵罐接种培养,培养温度为36℃-39℃,pH值为5.5-6.5,发酵过程中发酵液不通气,慢速搅拌。
步骤3 发酵终止及离心
培养16h-20h,活菌量达到108CFU/ml-1010CFU/ml,根据OD600值测得生长曲线进入平台期后快速将发酵液降温至20℃以下,进行离心,收获菌泥。
步骤4 保护剂乳化
收获菌泥稀释为含水量60%-80%,等体积加入菌泥保护剂进行乳化;菌泥保护剂成分为:水70%,脱脂乳粉10%,蔗糖5%,甘露糖醇5%,吐温-80 2%,甜菜碱2%,谷氨酸钠1%,可溶性淀粉5%。
该菌泥保护剂可对细菌冷冻干燥过程形成良好的保护:
(1)可以与细菌表面的自由基结合,避免菌体暴露在介质中;
(2)可以与蛋白质形成氢键以取代水,保证蛋白质的稳定性,防止细胞内外冰晶的形成;
(3)在菌冻结和脱水过程中,可以通过氢键和离子键对水和细胞所产生的的亲和力实现细胞成分结构的稳定。
步骤5 菌体铰链包埋保护
乳化后获得的乳化液中按体积比4:1(乳化液:壳聚糖溶液)加入壳聚糖溶液并快速均匀搅拌,该壳聚糖溶液浓度为5%-10%,优选为7.0%,搅拌时间为1-2min,搅拌转速为100-120rpm。然后加入与壳聚糖溶液等体积的海藻酸钠溶液并缓慢搅拌,该海藻酸钠溶液浓度为4%-10%,优选为6.5%,搅拌时间为1-2min,搅拌转速为60-70rpm。
步骤6 真空冷冻干燥
搅拌均匀后装入真空冷冻干燥机进行冷冻真空干燥,冻干物料厚度≤1cm。冻干机温度曲线为:-50℃保持4h,每隔2h温度提高5℃,到第24h时温度温度为0℃,之后每隔2h温度提高3℃,最终最高温度为30℃保持不变,直至物料干燥,物料干燥的特点为水分含量低于5%,水活度在0.11-0.22aw。
步骤7 粉碎及保存
将最终冻干物料进行粉碎成粉,冻干粉目数≥200目,冻干菌粉活性可达到1011CFU/g-1012CFU/g,即为植物乳杆菌NKK20冻干粉,放至-20℃冷库备用。
通过以上步骤制得的植物乳杆菌NKK20冻干粉,在发酵环节增加了大豆卵磷脂,有助于其耐胆盐,乳化环节已经进行了铰链包埋,有助于其菌粉存放的热稳定性和耐酸性。
实施例9 植物乳杆菌NKK20降低胆固醇
测试植物乳杆菌NKK20作为酸奶发酵剂时对酸奶中胆固醇的影响。
用白砂糖、纯牛奶做酸奶发酵原料,采购蒙牛及伊利的传统发酵酸奶成品各1款作为对照。
将200ml纯牛奶加入10g白砂糖充分溶解后置于85℃水浴锅中杀菌30min,冷却至38℃-40℃时,在无菌环境下加入10ml植物乳杆菌发酵剂,搅拌均匀后分装至50ml无菌杯中,放至37℃恒温箱中发酵培养3h-5h,发酵好的酸奶凝固物流动后置于4℃冷藏保存。
其中,植物乳杆菌(NKK20、229v、Lp-115)发酵剂的制备方式为:经MRS液体培养基活化培养12h后的植物乳杆菌发酵液12000rpm/min离心10分钟后,收集菌泥并用9%生理盐水冲洗后离心获取菌泥,加入发酵液体积50%的灭菌纯牛奶,混合均匀后置于37℃恒温箱中培养4h。其中,灭菌纯牛奶灭菌方式为将纯牛奶在120℃条件下灭菌5min。
采用邻苯二甲醛比色法测得各样品中胆固醇含量,如表8所示。
结果显示:通过植物乳杆菌NKK20发酵后的酸奶胆固醇比未发酵前降低了51.23%,对牛奶发酵中降胆固醇的能力明显优于植物乳杆菌229v(39.30%)和Lp-115(25.26%)。
序列表
<110> 天益健康科学研究院(镇江)有限公司
<120> 一种植物乳杆菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 967
<212> DNA
<213> 植物乳杆菌(Lactobacillus plantarum)
<400> 1
cggcacggtg ggggctgcca aaacatgcaa gtcgaacgaa ctctggtatt gattggtgct 60
tgcatcatga tttacatttg agtgagtggc gaactggtga gtaacacgtg ggaaacctgc 120
ccagaagcgg gggataacac ctggaaacag atgctaatac cgcataacaa cttggaccgc 180
atggtccgag tttgaaagat ggcttcggct atcacttttg gatggtcccg cggcgtatta 240
gctagatggt ggggtaacgg ctcaccatgg caatgatacg tagccgacct gagagggtaa 300
tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca gtaaggaatc 360
ttccacaatg gacgaaagtc tgatggagca acgccgcgtg agtgaagaag ggtttcggct 420
cgtaaaactc tgttgttaaa gaagaacata tctgagagta actgttcagg tattgacggt 480
atttaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca 540
agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggttttttaa gtctgatgtg 600
aaagccttcg gctcaaccga agaagtgcat cggaaactgg gaaacttgag tgcataagag 660
gacagtggaa ctccatgtgt agcggtgaaa tgcgtagata taaggaagaa caccagtggc 720
gaagcggctg tctggtctgt aactgacgct gaggctcgaa agtatgggta gcaacaggat 780
tagataccct ggtagtccat accgtaaacg atgattgcta agtgttggag ggtttccgcc 840
cttcagtgct gcatctaacg cattaagcat tccccctggg gagtacggcc gcaatgctga 900
aacttctagg aattgacggg ggcccgcaca atcggtggac atgtggtttt aattcgtagc 960
tactcta 967
Claims (10)
1.一种植物乳杆菌,其特征在于,命名为植物乳杆菌(Lactobacillus plantarum)NKK20,该菌株已于2020年10月19日保藏在中国典型培养物保藏中心,保藏编号为CCTCCNO:M2020596,保藏地址为中华人民共和国湖北省武汉市武汉大学。
2.根据权利要求1所述的植物乳杆菌菌株,其特征在于,所述菌株能够耐酸、耐胆盐,代谢生成乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸,该菌株能够粘附肠粘膜,抑制黄色葡萄球菌和肠炎沙门氏菌生长。
3.一种菌剂,其特征在于,活性成分中含有权利要求1-2中任一项所述的植物乳杆菌菌株。
4.根据权利要求3所述的菌剂,其特征在于,植物乳杆菌菌株液体发酵培养基中含有3g/L-10g/L卵磷脂。
5.根据权利要求3-4中任一项所述的菌剂,其特征在于,该菌剂在发酵冷冻干燥制备过程中,添加有冷冻干燥保护剂,冷冻干燥保护剂成分为:水60%-80%,脱脂乳粉6%-14%,蔗糖2%-8%,甘露糖醇4%-7%,吐温-80 1%-2.5%,甜菜碱1%-3%,谷氨酸钠0.5%-1.2%,可溶性淀粉2%-8%;优选的,水70%,脱脂乳粉10%,蔗糖5%,甘露糖醇5%,吐温-80 2%,甜菜碱2%,谷氨酸钠1%,可溶性淀粉5%。
6.根据权利要求3-5中任一项所述的菌剂,其特征在于,菌剂在制备过程中经5%-10%壳聚糖溶液与4%-10%海藻酸钠溶液进行铰链包埋保护。
7.一种组合物,其特征在于,成分中含有权利要求3-6中任一项所述的菌剂。
8.一种如权利要求8所述的组合物在制备食品、健康营养品或酸奶发酵剂中的应用。
9.一种如权利要求8所述的组合物在制备抑制胃肠道致病菌的药物中的应用。
10.一种植物乳杆菌的冻干粉的制备方法,其特征在于:包括以下步骤:
(1)发酵培养:
将植物乳杆菌NKK20按3%-15%接种量接种于高产培养基中,36℃-39℃发酵培养16-20h;活菌量达到108CFU/ml-1010CFU/ml时,将发酵液降温至20℃以下,离心,收获菌泥;
其中,高产培养基为水溶液,含有:大豆蛋白胨6-10g/L,酵母浸粉3-8g/L,无水葡萄糖15-28g/L,乙酸钠3-6g/L,大豆卵磷脂3g/L-10g/L,磷酸氢二钾1.8-2.2g/L,柠檬酸氢二铵1.5-2.5g/L,硫酸镁0.1-0.3g/L,硫酸锰0.02-0.06g/L,吐温-80 0.5-2.0ml/L,L-半胱氨酸盐酸盐0.05-0.25g/L,pH值5.5-6.5;
(2)保护剂乳化:
将收获的菌泥稀释至含水量60%-80%,等体积加入菌泥保护剂进行乳化;
其中,菌泥保护剂成分为:水70%,脱脂乳粉10%,蔗糖5%,甘露糖醇5%,吐温-80 2%,甜菜碱2%,谷氨酸钠1%,可溶性淀粉5%;
(3)菌体铰链包埋保护:
乳化液中加入壳聚糖溶液,搅拌均匀,搅拌时间为1-2min,搅拌转速为100-120rpm;再加入与壳聚糖溶液等体积的海藻酸钠溶液,继续搅拌,搅拌时间为1-2min,搅拌转速为60-70rpm;
其中,乳化液:壳聚糖溶液的体积比为4:1;壳聚糖溶液浓度为5%-10%,优选为7.0%;海藻酸钠溶液浓度为4%-10%,优选为6.5%;
(4)真空冷冻干燥,即得植物乳杆菌NKK20冻干粉。
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CN116855423A (zh) * | 2023-08-21 | 2023-10-10 | 广东海洋大学 | 一株植物乳杆菌yj6及其在禽类养殖中的应用 |
CN116855423B (zh) * | 2023-08-21 | 2024-02-09 | 广东海洋大学 | 一株植物乳杆菌yj6及其在禽类养殖中的应用 |
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Application publication date: 20210601 Assignee: Jiangsu Health Home Pharmaceutical Co.,Ltd. Assignor: TIANYI HEALTH SCIENCE RESEARCH INSTITUTE (ZHENJIANG) Co.,Ltd. Contract record no.: X2023320000218 Denomination of invention: A Plant Lactobacillus and Its Application Granted publication date: 20211001 License type: Common License Record date: 20231020 |