CN115232768B - 一种副干酪乳杆菌jn-8及其应用 - Google Patents
一种副干酪乳杆菌jn-8及其应用 Download PDFInfo
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Abstract
本发明公开了一种副干酪乳杆菌JN‑8及其应用,本发明的副干酪乳杆菌(Lactobacillus paracasei)JN‑8,于2021年6月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保存地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.22746。本发明的副干酪乳杆菌JN‑8在胃肠液中模拟消化存活率达到95.2%,且耐胆盐特性强,在胆盐浓度达到0.6%时,菌株生物量0.058,此外,本发明的副干酪乳杆菌JN‑8发酵酸奶后具有强的DPPH自由基抑制活性和总抗氧化活性。
Description
技术领域
本发明涉及一种副干酪乳杆菌JN-8及其应用,属于微生物技术领域。
背景技术
氧化应激是指体内氧化与抗氧化作用失衡,导致中性粒细胞炎性浸润,产生大量对机体有害的产物。机体内氧化应激被认为是多种疾病并发生的一个重要因素,并与机体的老化和日常饮食生活习惯有着密切的关系。
乳酸菌是一种益生菌。利用乳酸菌发酵不仅可以改善风味,还能提供多种功能成分。此外,益生菌发酵后可产生多种益生元,益生元具有抗氧化,抗炎的功能,在多种疾病中显示出许多有益的作用。此外,增加机体内有益细菌的数量,以及由于竞争作用而减少病原菌的数量,可降低炎症因子的级联反应,减轻炎症反应,从而改善疾病。
虽然目前有较多的能够耐受胃酸的乳酸杆菌,但是目前的乳酸杆菌对于胃肠道消化系统,尤其是肠道消化系统的抗性较弱,以及对胆盐的耐受能力较弱,到达肠道后,对肠道中的炎症的治疗效果较差,往往需要复配不同的菌株进行治疗,但是不同菌株之间的复配较为复杂,且受到不同菌株活性效果的影响,导致实际治疗效果并不理想。
发明内容
为解决上述技术问题,本发明提供一种副干酪乳杆菌JN-8及其应用,本发明的副干酪乳杆菌JN-8具有强的胃肠道消化系统的抗性及对胆盐耐受能力,能够应用于预防和治疗自发性肠炎。
本发明的第一个目的是提供一种副干酪乳杆菌,命名为副干酪乳杆菌(Lactobacillus paracasei)JN-8,于2021年6月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保存地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCCNo.22746。
进一步地,所述的副干酪乳杆菌在胃肠液中模拟消化存活率达到95.2%。
进一步地,所述的副干酪乳杆菌具有较强的胆盐耐受特性。
进一步地,所述的副干酪乳杆菌发酵乳制品具有强的抗氧化能力。
本发明的第二个目的是提供一种所述的副干酪乳杆菌在功能酸奶发酵产品中的应用。
进一步地,所述的应用是将所述的副干酪乳杆菌制备成含有所述的副干酪乳杆菌的药品。
进一步地,所述的药品为口服制剂。
进一步地,所述的产品为含有所述的副干酪乳杆菌的冻干菌粉。
进一步地,所述的冻干菌粉中,副干酪乳杆菌的活菌数不低于1×109CFU/g。
进一步地,所述的冻干菌粉通过如下方法制备得到:
(1)菌种的活化:将所述的副干酪乳杆菌JN-8接种量接种于MRS固体培养基中,36~38℃培养36~48h,获得菌株单菌落;
(2)初级种子液制备:挑取固体培养基中的副干酪乳杆菌JN-8单菌落,接种于MRS液体培养基中,36~38℃厌氧培养20~24h,得到初级种子液;
(3)菌种的扩大培养:将初级种子液以4~5%的接种量接种于液体MRS培养基中,36~38℃厌氧培养20~24h,得到发酵液;
(4)菌体收集:将得到的发酵液3000~5000rpm/min离心10~15min,收集并洗涤菌体,得到副干酪乳杆菌JN-8菌体;
(5)菌体冻干:向收集到的副干酪乳杆菌JN-8菌体进行冷冻干燥,得到所述的冻干菌粉。
进一步地,MRS固体培养基:胰蛋白胨8~12g/L;酵母粉4~6g/L;牛肉膏8~12g/L;葡萄糖16~25g/L;柠檬酸氢二胺1~3g/L;乙酸钠4~6g/L;K2HPO4·3H2O 2.6~2.7g/L;MgSO4·7H2O 0.1~0.3g/L;MnSO4·4H2O 0.04~0.06g/L;吐温80 0.8~1.2mL/L;1.6~2%的琼脂粉。
进一步地,MRS液体培养基:胰蛋白胨8~12g/L;酵母粉4~6g/L;牛肉膏8~12g/L;葡萄糖16~25g/L;柠檬酸氢二胺1~3g/L;乙酸钠4~6g/L;K2HPO4·3H2O 2.6~2.7g/L;MgSO4·7H2O 0.1~0.3g/L;MnSO4·4H2O 0.04~0.06g/L;吐温80 0.8~1.2mL/L。
本发明的第三个目的是提供所述的副干酪乳杆菌发酵制备得到的酸乳品。
进一步地,所述的酸乳品为酸奶。
本发明的第四个目的是提供一种含有所述的副干酪乳杆菌的菌剂。
进一步地,所述的菌剂为固态菌剂。
进一步地,所述的菌剂为液态菌剂。
本发明的有益效果是:
本发明的副干酪乳杆菌(Lactobacillus paracasei)JN-8,于2021年6月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保存地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.22746。本发明的副干酪乳杆菌JN-8在胃肠液中模拟消化存活率达到95.2%,且耐胆盐特性强,在胆盐浓度达到0.6%时,生物量达到0.038,此外,本发明的副干酪乳杆菌JN-8进行酸奶发酵,表现出强的抗氧化作用。本发明的副干酪乳杆菌JN-8具有强的胃肠道消化系统的抗性及对胆盐耐受能力,能够应用于生产功能性酸乳品,具有功能和风味俱佳的效果。
生物材料保藏:
副干酪乳杆菌(Lactobacillus paracasei)JN-8,于2021年6月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保存地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.22746。
附图说明:
图1不同菌株发酵乳品的抗氧化活性比较,***与正常组比较P<0.001;**与正常组相比较,P<0.01;
具体实施方式
下面结合具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
MRS固体培养基:胰蛋白胨10g;酵母粉5g;牛肉膏10g;葡萄糖20g;柠檬酸氢二胺2g;乙酸钠5g;K2HPO4·3H2O 2.6g;MgSO4·7H2O 0.2g;MnSO4·4H2O 0.05g;吐温80 1mL;1.6~2%的琼脂粉。pH调节6.8±0.2;定容至1L。
MRS液体培养基:胰蛋白胨10g;酵母粉5g;牛肉膏10g;葡萄糖20g;柠檬酸氢二胺2g;乙酸钠5g;K2HPO4·3H2O 2.6g;MgSO4·7H2O 0.2g;MnSO4·4H2O 0.05g;吐温80 1mL。pH调节6.8±0.2;定容至1L。
LB培养基的配置:胰蛋白胨10.0g,酵母提取物5.0g,氯化钠10.0g,超纯1.0L,调节pH至7.4。121℃灭菌15min后备用。
实施例1:菌株的分离与鉴定
1、乳酸菌分离自宁夏银川地区发酵企业周围环境,取样后进行标号,冷藏盒保存。
2、采用稀释梯度涂布平板方法,将样品稀释并涂布MRS平板上,每个浓度涂三个平行,置于37℃的厌氧培养。
3、挑取符合乳酸菌菌落特征的单菌落接入MRS液体培养基进行富集培养。获得的菌株保存于甘油管中并保藏于-80℃的低温环境中。
4、菌种鉴定:取1mL富集好的乳酸菌菌液,进行DNA的提取,获得的DNA利用通用引物27F(5’-AGAGTTTGATCMTGGCTCAG-3’),1492R(5’-GGTTACCTTGTTACGAC TT-3’)进行PCR扩增,得到未纯化的DNA序列。对序列进行鉴定,鉴定单位为天霖生物科技(无锡)有限公司。
5、鉴定结果
该菌株被鉴定为副干酪乳杆菌(lactobacillus paracei),并命名为副干酪乳杆菌JN-8,已于2021年6月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保存地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.22746。
实施例2:菌株的人工胃肠液抗性实验
1、模拟唾液、胃液和肠液的制备:
(1)胃肠储备液的制备(见表1)
表1模拟唾液,胃液和肠液制备
药品 | 储备液浓度(mol/L) | 胃液盐浓度(mmol/L) | 肠液盐浓度(mmol/L) |
KCl | 0.5 | 6.9 | 6.8 |
<![CDATA[KH<sub>2</sub>PO<sub>4</sub>]]> | 0.5 | 0.9 | 0.8 |
<![CDATA[NaHCO<sub>3</sub>]]> | 1 | 25 | 85 |
NaCl | 2 | 47.2 | 38.4 |
<![CDATA[MgCl<sub>2</sub>(H<sub>2</sub>O)<sub>6</sub>]]> | 0.15 | 0.12 | 0.33 |
<![CDATA[(NH<sub>4</sub>)<sub>2</sub>CO<sub>3</sub>]]> | 0.5 | 0.5 | - |
HCl | 6 | 15.6 | 8.4 |
<![CDATA[CaCl<sub>2</sub>(H2O)2<sup>a</sup>]]> | 0.3 | 0.15 | 0.6 |
a在使用前添加CaCl2(H2O)2
(2)调节pH:利用NaOH溶液或HCl溶液调节SGF溶液pH至3;SIF溶液pH调节至7。
(3)SGF溶液中添加胃蛋白酶溶液和胃脂肪酶溶液,最终混合物的活性分别达到2,000U/mL和60-75U/mL;SIF溶液中添加胰蛋白酶,最终混合物的活性分别达到100U/mL。
(4)在实验过程中保持37℃的温度,不断搅拌,可以用来模拟胃肠的蠕动。
2、胃肠液抗性实验操作
乳酸菌菌液以10%的接种量接种到SGF中,置37℃厌氧培养,分别在0h和3h时,取样测定OD600计算各菌种的存活率(%)。吸取经人工胃液处理3h后的菌液,接种于无菌的SIF中,静置于37℃厌氧培养,在0h,3h取样测定OD600。存活率=A1/A0×100%
3、按照以上操作步骤对植物乳杆菌植物亚种CICC 20263,副干酪乳杆菌CICC20262,副干酪乳杆菌CICC 20273,副干酪乳杆菌JN-8和嗜酸乳杆菌CICC 22162进行胃肠液抗性实验。结果见表2,菌株JN-8的存活率大大高于其他菌株,显示出更强的胃肠液抗性。
表2乳酸菌落对人工胃肠液的耐受性结果
实施例3:菌株的对胆盐的耐受性实验
1、含胆盐的MRS培养基配置:胰蛋白胨10g;酵母粉5g;牛肉膏10g;葡萄糖20g;柠檬酸氢二胺2g;乙酸钠5g;K2HPO4·3H2O 2.6g;MgSO4·7H2O 0.2g;MnSO4·4H2O 0.05g;吐温801mL,定容至1L。胆盐分别添加0.1%,0.2%,0.3%,0.4%,0.5%和0.6%。
2、耐胆盐实验步骤:将乳酸菌按2%的接种量分别接种于含不同浓度胆盐的MRS液体培养基中,放置于37℃厌氧培养,24h分别测定OD600。
3、耐胆盐实验结果见表3,与其他菌株相比,JN-8展现出更强的耐胆盐特性见表3,与其他菌株相比,JN-8展现出更强的耐胆盐特性。
表3乳酸菌样品在含有不同浓度胆盐的培养基中培养16h的生物量
实施例4:副干酪乳杆菌JN-8应用产品制备
1、副干酪乳杆菌JN-8于2021年6月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保存地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.22746。
2、副干酪乳杆菌JN-8冻干菌粉中副干酪乳杆菌的活菌数为1×109CFU/g。
3、副干酪乳杆菌JN-8冻干菌粉的制备方法,包括如下步骤:
(1)菌种的活化:将副干酪乳杆菌JN-8接种量接种于MRS固体培养基中,37℃培养48h,获得菌株单菌落;
(2)初级种子液制备:挑取固体MRS培养基中的副干酪乳杆菌JN-8单菌落,接种于MRS液体培养基中,37℃厌氧培养24h,得到初级种子液;
(3)菌种的扩大培养:将初级种子液以4%的接种量接种于液体MRS培养基中,37℃厌氧培养24h,得到发酵液;
(4)菌体收集:将得到的发酵液4500rpm/min离心15min,收集菌体,用PBS缓冲液洗涤菌体,重悬菌体,离心去上清,重复一次洗涤,得到副干酪乳杆菌JN-8的菌泥;
(5)菌体冻干:向收集到的菌体进行冷冻干燥,得到冻干菌粉。
实施例5:副干酪乳杆菌JN-8应用产品制备
1、副干酪乳杆菌JN-8于2021年6月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保存地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC No.22746。
2、副干酪乳杆菌JN-8冻干菌粉中副干酪乳杆菌的活菌数为1×1010CFU/g。
3、副干酪乳杆菌JN-8冻干菌粉的制备方法,包括如下步骤:
(1)菌种的活化:将副干酪乳杆菌JN-8接种量接种于MRS固体培养基中,37℃培养48h,获得菌株单菌落;
(2)初级种子液制备:挑取固体MRS培养基中的副干酪乳杆菌JN-8单菌落,接种于MRS液体培养基中,37℃厌氧培养22h,得到初级种子液;
(3)菌种的扩大培养:将初级种子液以5%的接种量接种于液体MRS培养基中,37℃厌氧培养24h,得到发酵液;
(4)菌体收集:将得到的发酵液4500rpm/min离心15min,收集菌体,用PBS缓冲液洗涤菌体,重悬菌体,离心去上清,重复一次洗涤,得到副干酪乳杆菌JN-8的菌泥;
(5)菌体冻干:向收集到的菌体进行冷冻干燥,得到冻干菌粉。
实施例6:副干酪乳杆菌JN-8应用产品制备
利用植物乳杆菌植物亚种CICC 20263,副干酪乳杆菌CICC 20262,副干酪乳杆菌CICC20273,副干酪乳杆菌JN-8和嗜酸乳杆菌CICC 22162进行酸奶的制作,具体操作为向发酵袋中加入全脂牛奶、白砂糖,充分混匀,在40-50℃进行加热搅拌,巴氏灭菌法对其进行灭菌处理,控制温度不超过80℃,灭菌30min.待温度冷却到40-45℃之后,向其中加入发酵剂。将样品放置在42℃的酸奶机内发酵6.5h,后熟24h获得发酵酸乳产品。称取各菌株发酵的酸奶及超市购买酸奶5.0g,5500rpm,离心10min,取上清液用于抗氧化活性的检测。超市购买酸奶作为对照组。
1.总抗氧化还原能力测定
用铁氰化钾法检测总抗氧化还原能力,吸光值越高,表明抗氧化还原能力越强.取酸奶上清液0.25mL,先后加入0.5mL的磷酸缓冲液(pH=6.6),0.5mL1%铁氰化钾溶液,混匀,置于50℃水浴20min后流水冷却.再加入0.5mL10%三氯乙酸溶液(TCA),充分混匀,离心(5000rpm,20min)后取上清液90μL于96孔板中,加入90μL纯化水以及20μL0.1%氯化铁溶液,混匀静置10min,测定其OD700,以超纯水为空白组,以维生素C为对照组.每个样品设三组平行。
2.DPPH自由基清除率测定
取各菌株的发酵酸奶上清液20μL,向其中加入180μL 0.2mmol/L DPPH乙醇溶液,避光室温反应30min后测定其OD517,记为AS;取20μL超纯水,向其中加入180μL DPPH乙醇溶液,测定其OD517记为A0;另取各酸奶上清液20μL,向其中加入180μL乙醇,其吸光度值记为AC,每个样品设三组平行。
结果如图1所示,与对照组相比,经过不同菌株发酵的乳品其DPPH清除率都有所增加。经过菌株CICC20263,CICC20262和CICC20273发酵乳品的DPPH清除率与对照组不具有显著性,菌株CICC22162和JN-8发酵的乳品DPPH清除率显著高于对照组(p<0.05,p<0.001),其中JN-8菌株发酵的乳品DPPH清除率最强。此外,对不同菌株发酵乳品的总抗氧化能力也进行了测定。结果显示,菌株CICC20262,CICC20273和JN-8发酵乳品的总抗氧化活性与对照组具有显著性(p<0.05,p<0.01,p<0.001),其中JN-8发酵乳品的总抗氧化活性最高。以上结果表明菌株JN-8发酵增强乳品的抗氧化能力,可产出强的抗氧化能力的酸乳品。
综上可知,副干酪乳杆菌JN-8在胃肠消化后具有强的存活率,达到95.2%。耐胆盐实验中,胆盐浓度达到0.6%时,副干酪乳杆菌JN-8培养16h的生物量达到0.058。此外,副干酪乳杆菌JN-8冻干粉发酵酸奶能够显著提高酸奶的抗氧化性能。其能增强对DPPH自由基的清除能力,增强总抗氧化能力,可产出抗氧化活性强的酸乳品。以上结果说明副干酪乳杆菌JN-8冻干菌粉可以作为一种增强抗氧化性的产品。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (10)
1.一种副干酪乳杆菌,其特征在于,所述的副干酪乳杆菌命名为副干酪乳杆菌(Lactobacillus paracasei)JN-8,于2021年6月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保存地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCCNo.22746。
2.一种权利要求1所述的副干酪乳杆菌在制备抗氧化产品中的应用。
3.根据权利要求2所述的应用,其特征在于,所述的应用是将所述的副干酪乳杆菌制备成含有所述的副干酪乳杆菌的药品。
4.根据权利要求2所述的应用,其特征在于,所述的产品为含有所述的副干酪乳杆菌的冻干菌粉。
5.根据权利要求4所述的应用,其特征在于,所述的冻干菌粉中,副干酪乳杆菌的活菌数不低于1×109CFU/g。
6.根据权利要求5所述的应用,其特征在于,所述的冻干菌粉通过如下方法制备得到:
(1)将所述的副干酪乳杆菌JN-8接种量接种于MRS固体培养基中,36~38℃培养36~48h,获得菌株单菌落;
(2)挑取固体培养基中的副干酪乳杆菌JN-8单菌落,接种于MRS液体培养基中,36~38℃厌氧培养20~24h,得到初级种子液;
(3)将初级种子液以4~5%的接种量接种于液体MRS培养基中,36~38℃厌氧培养20~24h,得到发酵液;
(4)将得到的发酵液3000~5000rpm/min离心10~15min,收集并洗涤菌体,得到副干酪乳杆菌JN-8菌体;
(5)向收集到的副干酪乳杆菌JN-8菌体进行冷冻干燥,得到所述的冻干菌粉。
7.一种采用权利要求1所述的副干酪乳杆菌发酵制备得到的酸乳品。
8.一种含有权利要求1所述的副干酪乳杆菌的菌剂。
9.根据权利要求8所述的菌剂,其特征在于,所述的菌剂为固态菌剂。
10.根据权利要求8所述的菌剂,其特征在于,所述的菌剂为液态菌剂。
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