CN117431173A - 一种抗菌高原戊糖片球菌tr-37及其无细胞提取物和应用 - Google Patents
一种抗菌高原戊糖片球菌tr-37及其无细胞提取物和应用 Download PDFInfo
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Abstract
本发明提供了一种抗菌高原戊糖片球菌TR‑37及其无细胞提取物和应用,属于乳酸菌技术领域。本发明的抗菌高原戊糖片球菌(Pediococcus pentosaceus)TR‑37,保藏于中国典型培养物保藏中心,地址:中国.武汉.武汉大学,保藏日期为:2023年09月08日,保藏编号为CCTCC NO:M20231651。本发明的戊糖片球菌TR‑37具有较强的抑菌能力和抗氧化能力,可以用于制备食品发酵剂或食品保鲜剂,所制得的食品功能性更强。
Description
技术领域
本发明涉及乳酸菌技术领域,尤其涉及一种抗菌高原戊糖片球菌TR-37及其无细胞提取物和应用。
背景技术
乳酸菌种类繁多,分布广泛。其作为肠道益生菌的重要组成部分,在人类消化道中大量存在,对人体健康有着重要作用。一方面,某些种类的乳酸菌如乳杆菌属和双歧杆菌属,为抵抗病原体提供了微生物屏障,可以预防或减少病原菌在肠道中的定植,对肠道致病菌有抑制活性,具有干扰或阻断肠道致病菌的能力;另一方面,乳酸菌可以通过与免疫细胞或肠上皮细胞等组织细胞上的特异性受体结合,激活宿主体内的免疫反应。除了对肠道感染、腹泻、结肠癌等肠道疾病有预防和治疗作用外,有研究表明乳酸菌对肥胖、II型糖尿病等代谢问题也有调节作用。乳酸菌的益生作用通常具有特异性,不同种属乃至不同菌株的作用靶点和调节方式往往是不同的。因此对乳酸菌益生作用的开发利用具有广阔的前景。
动物及人类体内细菌感染中最主要的致病菌为金黄色葡萄球菌、大肠杆菌、沙门氏菌等,其均属于最典型的革兰氏阴性菌菌株。分析当前情况,肠道致病菌中,以腐败细菌或者急慢性细菌感染诱发的肠道炎症,已经成为公共卫生研究的关键内容。此外,由于肠道致病菌入侵人体以后,穿过肠道粘膜屏障,侵入组织器官或者血液将形成脏器损伤及败血症。如金黄色葡萄球菌和大肠杆菌一旦动物感染,将产生体温上升、肠道粘膜出血和水样腹泻等表现。另外,消化系统常见致病菌为沙门氏菌,其极易导致食物中毒,引起胃肠道炎症及伤寒。
近年来,抗生素耐药性日益提升。而乳酸菌对病原菌和腐败菌具有良好的抑制活性。首先乳酸菌可以产生大量有机酸(乳酸、乙酸、丙酸、苯乳酸、柠檬酸等),他们可以影响细胞膜表面的酶活,改变细胞膜的通透性和稳定性,破坏细胞内稳态,从而抑制细菌的正常生长和代谢。其次是过氧化氢,低浓度的过氧化氢与乳酸协同作用就可以抑制或杀灭微生物。另外,某些乳酸菌在代谢过程中会产生细菌素,他们具有高效、无毒、耐热、无耐药性等特点,可以抑制多种细菌、真菌甚至病毒。目前,乳酸菌产生的少量细菌素已被批准可以作为安全的添加剂应用在食品当中。
随着人们生活水平的提高,人们对功能性食品的渴求日益增加,不同乳酸菌菌种之间功效各异,所以,近些年针对不同菌种的乳酸菌功效探索也成为研究热点,尤其是乳酸菌功能特性的深入研究,对人类健康和食品领域有巨大的应用价值和开发潜力。
被誉为有“世界屋脊”的青藏高原由于其复杂的地理环境和独特的气候条件,蕴藏着丰富的生物物种资源。尤其是丰富的乳酸菌菌种资源,而当地的特殊环境赋予了乳酸菌的生物学特性及遗传多样性。考虑到高寒地区乳酸菌种质资源的宝贵性和特殊性,保护和开发利用极端环境下优质乳酸菌种质资源,建立极端环境中乳酸菌种质资源数据库,筛选具有明显地域特色和特殊生物学功能的乳酸菌资源具有重要的科学意义。因此十分有必要筛选具有功能特性的高原乳酸菌种。
发明内容
本发明的目的在于提供一种抗菌高原戊糖片球菌TR-37及其无细胞提取物和应用。本发明的戊糖片球菌TR-37具有较强的抑菌能力和抗氧化能力,可以用于制备食品发酵剂或食品保鲜剂,所制得的食品功能性更强。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一株抗菌高原戊糖片球菌(Pediococcuspentosaceus)TR-37,所述高原戊糖片球菌TR-37保藏于中国典型培养物保藏中心,地址:中国.武汉.武汉大学,保藏日期为:2023年09月08日,保藏编号为CCTCCNO:M 20231651。
本发明还提供了一种上述抗菌高原戊糖片球菌TR-37的无细胞提取物,所述无细胞提取物的制备方法为:将所述高原戊糖片球菌TR-37的菌液超声破碎,收集上清液,得到无细胞提取物。
优选的,所述菌液的浓度为(1~9)×109CFU/mL。
优选的,所述超声的频率为300~400W,所述超声的时间为20~40min,超声过程中所述菌液全程置于冰上。
本发明还提供了一种上述抗菌高原戊糖片球菌TR-37或上述无细胞提取物在制备发酵食品或食品保鲜剂中的应用。
优选的,所述发酵食品包括发酵乳制品、泡菜、发酵香肠。
优选的,所述食品保鲜剂包括肉质食品保鲜剂。
本发明提供了一种抗菌高原戊糖片球菌TR-37及其无细胞提取物和应用。本发明的戊糖片球菌TR-37可以通过产生有机酸,过氧化氢或细菌素等抑菌物质对大肠杆菌,金黄色葡萄球菌,沙门氏菌和乙型溶血性链球菌产生较强的抑制作用。
本发明的戊糖片球菌TR-37及其无细胞提取物还具有极强的自由基清除率,相较于现有技术中的乳杆菌,抗氧化能力更强,能够用于食品发酵剂或食品保鲜剂中,使发酵食品具有更强的抗氧化能力,功能性更强,使保鲜食品中菌落数量更少,保鲜期更长。本发明的戊糖片球菌TR-37对于开发高效、安全、稳定的乳酸菌源抑菌剂提供了依据,对于功能食品开发及保藏技术应用具有积极意义。
附图说明
图1为本发明戊糖片球菌TR-37的菌株形态。
图2为本发明戊糖片球菌TR-37的系统进化树。
保藏说明
抗菌高原戊糖片球菌(Pediococcuspentosaceus)TR-37,该菌株保藏于中国典型培养物保藏中心,地址:中国.武汉.武汉大学,保藏日期为:2023年09月08日,保藏编号为CCTCC M 20231651。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
本实施例提供了一种抗菌高原戊糖片球菌(Pediococcus pentosaceusi)TR-37的分离过程,如下:
自青藏高原地区野血牦牛乳头取样,将样本立即放入冰袋冻存,并尽快送回实验室。将样本接种于MRS培养基中,于37℃下富集培养8h后,取部分样品加入1mL无菌生理盐水中,振荡混匀,得到10-1CFU/mL的悬液。取500μL此悬液逐级稀释到1×10-7、1×10-8、1×10- 9CFU/ml。分别吸取3个不同稀释度100μL分别均匀地涂布于MRS平板培养基上,37℃厌氧恒温培养48h,挑选圆形,中等大小,凸起,微白色,湿润,边缘整齐的典型乳酸菌单菌落。重新接种至MRS平板培养基中纯化培养,经过多次纯化后,得到103株菌株,将各菌株用50%的甘油保存,于-80℃下保藏。通过抗氧化的初步筛选,获得了20株乳酸菌,编号1~20#。
将得到的20株乳酸菌进行抗氧化能力验证。过程如下:
(1)DPPH的清除能力测定
测试组:称取0.008g DPPH溶于无水乙醇,定容至100mL,配制成0.2mmol/L DPPH溶液。用MRS培养基将以上20株乳酸菌的菌悬液浓度调整为1×108CFU/mL,并分别吸取1.5mL加入至1.5mL DPPH溶液中,室温下(25℃)避光反应30min,8000g,4℃,离心10min,取上清液,517nm处测定上清液吸光度,为A测试。
对照组:用1.5mL水替换DPPH溶液,其他操作与测试组相同,检测吸光度为A对照。
空白组:用1.5mL水替换菌悬液,其他操作与测试组相同,检测吸光度为A空白。
综合三组的吸光度数据,按照以下公式计算自由基清除率:
DPPH清除率=[A空白-(A测试-A对照)]×100%
(2)OH-离子的清除能力测定
样品组:用MRS培养基将以上20株乳酸菌的菌悬液浓度调整为1×109CFU/mL后,以360W的频率,将样本置于冰上超声30min,收集上清液为无细胞提取物。将以上20株乳酸菌的无细胞提取物分别吸取0.5mL加入到1mL O-菲啰啉(浓度0.1%)中,再加入1mL PBS,1mL2.5mmol/LFeSO4,1mL 20mmol/L H2O2。37℃恒温水浴反应1.5h后,536nm处测吸光值,为A样
空白组:用1mL蒸馏水代替1mL H2O2,其他操作与样品组相同,检测吸光度为A空白。
对照组:用0.5mL蒸馏水代替0.5mL无细胞提取物,其他操作与样品组相同,检测吸光度为A对照。
综合三组的吸光度数据,按照以下公式计算自由基清除率:
OH-清除率%=[(A样-A对照)/(A空白-A对照)]×100%
测定结果如表1所示。
表1乳酸菌抗氧化能力测定
乳酸菌编号 | OH-清除率(%) | DPPH清除率(%) |
1# | 14.426 | 59.199 |
2# | 5.626 | 61.041 |
3# | 12.106 | 60.135 |
4# | 14.014 | 59.925 |
5# | 12.724 | 62.297 |
6# | 13.717 | 61.526 |
7# | 13.045 | 59.394 |
8# | 5.687 | 59.95 |
9# | 9.167 | 62.928 |
10# | 8.717 | 59.795 |
11# | 5.488 | 59.825 |
12# | 7.976 | 61.311 |
13# | 6.366 | 60.135 |
14# | 7.938 | 59.68 |
15# | 11.144 | 61.051 |
16# | 7.457 | 61.151 |
17# | 5.251 | 60.586 |
18# | 9.434 | 59.184 |
19# | 12.297 | 59.214 |
20# | 6.343 | 59.124 |
LGG | 11.220 | 58.534 |
注:对照菌株为鼠李糖杆菌LGG(CICC 6001),目前最为稳定的商业菌株,与其对比能够客观的体现TDM-2菌株的抗氧化性能
从表1可以看出,1#菌株DPPH自由基的清除率可达59%以上,其无细胞提取物对OH-离子的清除率高于其他菌株,并显著高于对照菌株LGG。将1#菌株命名为TR-37,保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 20231651。
实施例2
本实施例对菌株TR-37进行了鉴定,过程如下:
(1)菌落和菌体形态观察以及生理生化鉴定
如图1所示,菌株TR-37的菌落呈乳白色,圆形,凸起,边缘较规则,表面光滑、湿润,易于挑取。将菌株置于电子显微镜下观察,如图1所示,显微镜下呈球状,无芽孢性,为革兰氏阳性菌。经检测,接触酶阴性、氧化酶阴性。
(2)基因测序鉴定
克隆菌株TR-37的16S rDNA序列并对该16S rRNA序列进行测序,把测序结果在Genbank进行BLAST比较,确定了TR-37的系统发育进化地位,系统进化树见图2。菌株TR-37进一步被鉴定为乳酸菌中的戊糖片球菌。
实施例3
本实施例以大肠杆菌、金黄色葡萄球菌、沙门氏菌、乙型溶血性链球菌为致病实验菌株,验证了戊糖片球菌TR-37的抗菌能力。具体过程如下:
大肠杆菌,金黄色葡萄球菌,沙门氏菌和乙型溶血性链球菌均购自北京北纳创联生物技术研究院。
戊糖片球菌TR-37接入已灭菌的MRS培养基中37℃培养过夜,连续传代3次。收集最后一次的传代培养液经8000g离心15min,收集TR-37发酵上清液,放入4℃冰箱备用。采用相同的方法制备鼠李糖杆菌LGG(CICC 6001)发酵上清液。
将大肠杆菌、金黄色葡萄球菌、沙门氏菌分别置于LB培养基上,将乙型溶血性链球菌置于哥伦比亚血培养基上,连续活化三代;挑取单菌落于相应的培养基中,37℃培养12h;用PBS调整活菌数至1×108CFU/mL。
采用琼脂孔扩散法,在相应的指示菌(大肠杆菌、金黄色葡萄球菌、沙门氏菌、乙型溶血性链球菌)培养基平板内均匀的涂布100μL致病菌菌悬液;利用无菌打孔器在平板上按压出直径为7mm的抑菌孔,每个平板打四孔作为平行对照,用镊子小心取出孔内琼脂块,加入200μL TR-37和LGG发酵上清液;室温下静置半小时后,放入37℃培养箱培养24h,通过测定抑菌圈的大小判断乳酸菌的抗菌能力,结果如表2~5所示。
表2戊糖片球菌TR-37对大肠杆菌的抗菌能力测定(mm)
菌种 | 1 | 2 | 3 | 4 | 平均 |
TR-37 | 16.43 | 15.72 | 15.33 | 14.31 | 15.4475 |
LGG | 13.38 | 11.33 | 10.69 | 12.33 | 11.9325 |
表3戊糖片球菌TR-37对金黄色葡萄球菌的抗菌能力测定(mm)
菌种 | 1 | 2 | 3 | 4 | 平均 |
TR-37 | 13.18 | 12.09 | 11.68 | 11.59 | 12.135 |
LGG | 12.34 | 10.95 | 11.44 | 0 | 11.57667 |
表4戊糖片球菌TR-37对沙门氏菌的抗菌能力测定(mm)
菌种 | 1 | 2 | 3 | 4 | 平均 |
TR-37 | 12.16 | 11.28 | 11.14 | 13.70 | 12.07 |
LGG | 11.2 | 8.71 | 11.23 | 0 | 10.38 |
表5戊糖片球菌TR-37对乙型溶血性链球菌的抗菌能力测定(mm)
菌种 | 1 | 2 | 3 | 4 | 平均 |
TR-37 | 12.07 | 12.05 | 11.23 | 12.59 | 11.985 |
LGG | 0 | 0 | 0 | 0 | 0 |
从表2~5可以看出,戊糖片球菌TR-37对四株致病菌的抑制能力均优于LGG,且戊糖片球菌TR-37对大肠杆菌的抑制效果最好,对乙型溶血性链球菌的抑制能力相对最弱。
实施例4
本实施例提供了一种利用戊糖片球菌TR-37制备发酵酸奶的方法,具体过程如下:
实验组:将鲜牛乳水浴加热至70℃,加入质量百分比为7%的蔗糖,混合均匀后将牛乳冷却至45℃,进行均质;随后将牛乳加热至90℃保持10min,进行巴氏杀菌处理;杀菌结束后冷却至40~45℃接种菌株,以3%的接种量将保加利亚乳杆菌,嗜热链球菌和戊糖片球菌TR-37按照1:1:1的比例接种于酸奶杯中。将杀菌冷却后的牛乳与发酵菌种混合均匀后分装,放入37℃培养箱中发酵7h。
对照组:与实验组相比,区别仅在于,不添加戊糖片球菌TR-37。
以上两组酸奶发酵结束后迅速冷却,放入4℃进行储藏。于储藏后1天,7天,14天,21天,28天对酸奶的活菌数、理化特性及抗菌能力(琼脂孔扩散法),抗氧化水平进行测定。检测结果如表6~9所示。并在储藏28天后,对酸奶的感官进行评价,评分标准如表10所示,评分结果如表11所示。
表6不同菌种发酵酸奶储存期活菌数变化
表7不同菌种发酵酸奶储存期酸度变化
表8不同菌种发酵酸奶的抗菌能力(抑菌圈大小:mm)
分组 | 大肠杆菌 | 金黄色葡萄糖球菌 | 沙门氏菌 | 乙型溶血性链球菌 |
实验组 | 14.934 | 12.048 | 12.578 | 11.873 |
对照组 | 11.276 | 0 | 0 | 8.312 |
表9不同菌种发酵酸奶的抗氧化水平
分组 | DPPH清除率 | OH-清除率 |
实验组 | 57.12% | 6.74% |
对照组 | 32.27% | 2.37% |
表10酸奶感官评分标准
表11不同菌种发酵酸奶的感官评分
分组 | 色泽 | 滋味和气味 | 组织状态 | 总分 |
实验组 | 29 | 29 | 30 | 88 |
对照组 | 29 | 28 | 29 | 86 |
从表6~11可以看出,两组酸奶储藏期间的活菌数均呈现先升高后降低的变化趋势,但添加了戊糖片球菌TR-37的酸奶活菌数整体高于未添加戊糖片球菌TR-37的酸奶,表明,本发明的戊糖片球菌TR-37活力高,并能够提高保加利亚乳杆菌,嗜热链球菌的活力。且添加了戊糖片球菌TR-37的酸奶口感更佳,抑菌能力更强,可以延长酸奶货架期;另外添加戊糖片球菌TR-37的酸奶还有增强抗氧化的能力。
实施例5
本实施例提供了一种利用戊糖片球菌TR-37保鲜食品的方法,具体过程如下:
将里脊肉在无菌操作台切分成重25g的肉块,分为两组。处理组在实施例3制备的TR-37发酵上清液中浸泡5min后沥干1min,用保鲜膜包裹后,放入保鲜盒后,将样品分为两组,分别放入4℃冰箱和28℃培养箱。对照组采用实施例3制备的LGG(CICC 6001)发酵上清液做相同处理。
放在4℃的样品分别在1d,2d和3d的时候取样检测菌落总数,每组3个平行;而放在28℃培养箱的样品则分别在0h,4h和8h取样检测菌落总数。通过测定肉制品菌落总数判断戊糖片球菌TR-37的保鲜能力。检测结果如表12~13所示。
表124℃下不同菌种处理后的样品菌落总数(CFU/g)
菌种 | 1d | 2d | 3d |
TR-37 | 4.0×104 | 3.6×105 | 1.9×106 |
LGG | 6.9×104 | 2.5×106 | 5.2×107 |
表1328℃下不同菌种处理后的样品菌落总数(CFU/g)
菌种 | 0h | 4h | 8h |
TR-37 | 9.5×103 | 4.7×103 | 1.8×104 |
LGG | 9.4×103 | 7.3×104 | 2.4×106 |
从表12~13可以看出,与对照菌株LGG相比,采用本发明的戊糖片球菌TR-37发酵上清液处理肉制品后,能够明显减少肉制品中的菌落数量,且在4℃或28℃储藏过程中,也能够抑制肉制品中细菌的繁殖,延长食品的保鲜期。
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 (7)
1.一株抗菌高原戊糖片球菌(Pediococcuspentosaceus)TR-37,所述高原戊糖片球菌TR-37保藏于中国典型培养物保藏中心,地址:中国.武汉.武汉大学,保藏日期为:2023年09月08日,保藏编号为CCTCC NO:M20231651。
2.一种权利要求1所述抗菌高原戊糖片球菌TR-37的无细胞提取物,其特征在于,所述无细胞提取物的制备方法为:将所述高原戊糖片球菌TR-37的菌液超声破碎,收集上清液,得到无细胞提取物。
3.根据权利要求2所述的无细胞提取物,其特征在于,所述菌液的浓度为(1~9)×109CFU/mL。
4.根据权利要求3所述的无细胞提取物,其特征在于,所述超声的频率为300~400W,所述超声的时间为20~40min,超声过程中所述菌液全程置于冰上。
5.一种权利要求1所述的抗菌高原戊糖片球菌TR-37或权利要求2~4任一项所述的无细胞提取物在制备发酵食品或食品保鲜剂中的应用。
6.根据权利要求5所述的应用,其特征在于,所述发酵食品包括发酵乳制品、泡菜、发酵香肠。
7.根据权利要求5所述的应用,其特征在于,所述食品保鲜剂包括肉质食品保鲜剂。
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