CN116426433B - 一种植物乳杆菌及其在制备乳清蛋白降胆固醇肽中的应用 - Google Patents
一种植物乳杆菌及其在制备乳清蛋白降胆固醇肽中的应用 Download PDFInfo
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Abstract
本发明提供了一种植物乳杆菌及其在制备乳清蛋白降胆固醇肽中的应用,涉及微生物技术领域;所述的植物乳杆菌FQR拉丁名为Lactobacillus plantarum FQR,保藏在中国典型培养物保藏中心,保藏编号为:CCTCC NO:M 202128;所述植物乳杆菌FQR发酵复合酸制备乳清蛋白降胆固醇肽的步骤为先将乳清蛋白粉溶液搅拌分散充分溶解,冷藏过夜水合灭菌,再经过所述植物乳杆菌FQR发酵改性,加入苹果酸酸解复合改性,最后冷冻干燥保存;本发明制得的乳清蛋白降胆固醇肽可有效提高乳清蛋白的水解程度,显著提高结合胆酸盐的能力和降胆固醇活性。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一种植物乳杆菌及其在制备乳清蛋白降胆固醇肽中的应用。
背景技术
随着人们生活水平的不断提高,对于健康食品的需求量不断增加,人们日常生活饮食结构也在改变,随之而来的是发病率的升高,如高血压、高血糖、高血脂等慢性疾病,于是对于具有降胆固醇等保健功效的食品成分的研究得到了大家的关注,也成为近年来的研究热点。
现阶段人们主要靠药物来降解血清中的胆固醇,虽然快速有效,但副作用大,价格昂贵。乳清蛋白含有丰富且易被人体消化吸收的必需氨基酸和微量的乳铁蛋白、糖巨肽、生长因子、乳过氧化物酶等,具有丰富的营养价值和独特的保健功能,水解后产生许多具有生物活性的序列,经过释放成为生物活性肽,具有降胆固醇、ACE抑制、抗病毒、免疫调节、神经调节、抑菌等功效。动物试验可以测定乳清蛋白肽在体内对胆固醇水平的影响。有研究证明,对β-乳球蛋白的水解物进行分离纯化,得到了可高效降低胆固醇活性的肽段,并根据动物验证试验,证实所得肽段能够有效降低动物体内血清胆固醇的含量。
传统的乳清蛋白降胆固醇肽的制备方法主要有化学法、酶水解法、超声波法、酸水解法,普遍存在操作复杂、产量及降胆固醇活性低等不足,导致对于肽的提取及利用率不高,很大程度上限制了其在食品和药品领域的运用。因此,对于乳清蛋白降胆固醇肽高活性成品的制备方法的研究尤为重要。
微生物发酵技术是利用微生物的生物转化作用,把大分子的蛋白质转变成具有较高活性的多肽。微生物发酵的原料含有丰富的蛋白质,可以在发酵的过程中通过调节发酵条件生成多种肽类,从而使其能够被人类的消化系统直接吸收。与传统方法相比,微生物发酵法生产的肽类食用安全性更高,发酵过程温度低,操作简单,不需要任何昂贵的设备。值得注意的是,研究表明,乳清蛋白水解物产生过程中的pH条件可能会影响肽的释放,从而影响乳清蛋白水解物的生物活性,酸性条件有利于提高发酵活性,同时更有利于蛋白质水解为疏水性的多肽,而疏水性的多肽有助于提高其结合胆酸盐的能力和降胆固醇活性。有机酸不同于一般的盐酸改性,反应条件更温和,对氨基酸的破坏相对较小,是一种简单而安全的化学方法。
胆汁酸是肝脏胆固醇分解代谢的最终产物。胆固醇转化为胆汁酸可以持续清除人体内相当一部分胆固醇,在调节胆固醇代谢方面发挥关键作用,胆固醇代谢是预防心血管疾病和相关疾病的重要因素。胆汁酸仅由肝脏中的胆固醇合成,在正常人身上,胆汁酸通过一个叫做肠肝循环的闭合回路在体内循环,即从肝脏进入肠道,然后返回肝脏。肝细胞吸收胆汁酸,不到总胆汁酸池的2%溢出到体循环中。正常情况下,超过90%的胆汁酸从肠道被重新吸收进入肠肝循环,只有少数通过粪便排出。研究证明,破坏胆汁酸重吸收会导致肝脏中胆固醇的进一步降解,并降低血液中的胆固醇水平,胆盐的结合能力通常被认为是评价物质降胆固醇效果的一个重要因素。最丰富的胆盐是牛磺胆酸盐、甘氨胆酸盐和脱氧胆酸盐等,这些特殊类型的表面活性剂具有与其生物功能密切相关的性质。乳清分离蛋白(Wheyprotein isolate,WPI)是公认的人体优质蛋白补充剂之一,在食品领域应用广泛。功能性WPI降胆固醇、降血压等作用逐渐受到消费者和食品行业的关注。随着新的改性技术的出现,通过提高改性WPI胆盐结合能力,来抑制胆汁酸的重吸收过程,达到抑制血清中总的胆固醇含量的效果具有重要意义。通过测定胆酸盐的结合能力,能反映改性WPI的降胆固醇活性。
因此,为了有效提高WPI水解物的生物活性,制备出能显著提高结合胆酸盐的能力和降胆固醇活性的WPI水解物,本发明提供了一种植物乳杆菌FQR及其复合酸制备乳清蛋白降胆固醇肽的方法。
发明内容
为了有效提高WPI水解物的生物活性,制备出能提高结合胆酸盐的能力和降胆固醇活性的WPI水解物,本发明提供了一种植物乳杆菌FQR。
同时,本发明还提供了所述植物乳杆菌FQR复合酸制备乳清蛋白降胆固醇肽的方法,所述制备方法包括步骤如下:
A.制备WPI溶液
将乳清蛋白粉配制成WPI溶液,然后在磁力搅拌器下按转速1500~1600 rpm分散2~3小时使乳清蛋白充分溶解,4℃冷藏过夜水合;然后在108℃高压灭菌锅灭菌15 min,冷却得到灭菌的WPI溶液;
B.发酵改性处理
将活化后的植物乳杆菌FQR调节活菌数为≥108 CFU/mL,在无菌操作台接种到灭菌WPI溶液,在 35~37℃培养36~48h。发酵结束后,将培养液在4℃10000~15000r/min 离心10~15min,上清液冷冻干燥后,得到发酵改性处理后的乳清蛋白, 4℃冷藏保存;
C.发酵复合改性处理
发酵改性处理后的乳清蛋白配制成70~90mg/mL的WPI溶液,再加入添加苹果酸,搅拌15~20 min后,用1.0~1.5 mol/L NaOH 终止酸解;
D.冻干保存
将发酵复合改性处理后的WPI溶液,置于冷冻干燥机冻干,得到所述的乳清蛋白降胆固醇肽,4℃冷藏备用;
其中,所述WPI溶液的质量浓度为6.0-9.0%;
其中,所述活化后的植物乳杆菌FQR是在液态MRS培养基中活化,35~37℃恒温静止培养24~28h;
其中,所述植物乳杆菌FQR接种量按体积比为1%~4%;
其中,所述苹果酸添加量为2.0-3.0%。
有益效果
本发明对乳清蛋白降胆固醇肽的制备方法进行改进,选用申请人从传统发酵肉制品筛选出的植物乳杆菌FQR,经过发酵复合苹果酸改性水解来制备乳清蛋白降胆固醇肽,通过测定胆酸钠(Sodium cholate,SC)结合率、牛磺胆酸钠(Sodium taurocholate,STC)结合率以及脱氧胆酸钠(Sodium deoxycholate,SDC)结合率等指标来判断乳清蛋白降胆固醇肽的产量及其降胆固醇活性,结果表明,乳清蛋白降胆固醇肽的胆酸盐结合率显著提高,说明先进行发酵改性处理后使得部分大分子蛋白降解为小分子活性肽,此时加入苹果酸使得水解度加深,更多的蛋白质被水解为疏水性的多肽,使得乳清蛋白具有更高的疏水性氨基酸组成,有助于结合具有疏水性的胆酸盐,从而提高结合胆酸盐的能力和降胆固醇活性。相较于传统工艺的制备方法,此法极大的提高了乳清蛋白的水解程度,水解出更多的疏水性肽,从而显著提高了其与胆酸盐的结合率,有效提高了降胆固醇的活性。发明人的课题组也用普通的乳酸菌做了相同的对照实验,但其制备的乳清蛋白降胆固醇肽的胆酸钠(SC)结合率、牛黄胆酸钠(STC)结合率和脱氧胆酸钠(SDC)结合率均低于本发明保藏的植物乳杆菌FQR,可以看出,本发明保藏的植物乳杆菌FQR对乳清蛋白水解出更多的疏水性肽有一定的作用。
本发明提供的一种植物乳杆菌FQR及其复合酸制备乳清蛋白降胆固醇肽的方法,显著提高了乳清蛋白降胆固醇肽的产量及其降胆固醇活性,整个工艺操作简单,产品功能活性高,易于与食品结合,对于人们的健康生活具有一定的实际意义,具有很好的市场前景。
附图说明
图1:乳清蛋白降胆固醇肽的发酵复合改性制备流程;
图2:各实验组WPI成品热图分析,其中,A-F-WPI组为实施例组, CK组为对照组;
图3:各实验组WPI成品特定肽段热图分析,其中,A-F-WPI组为实施例组, CK组为对照组;
实施方式
一种植物乳杆菌FQR,所述菌株已于2021年3月保藏于中国典型培养物保藏中心,该菌株保藏号为:CCTCC M 2021228,拉丁名Lactobacillus plantarum FQR,地址:湖北省武汉市武汉大学,邮编:430072,电话:027-68754052。
本发明保藏的Lactobacillus plantarum FQR菌落特征为:菌落凸起,呈圆形,表面光滑,细密,色白,偶尔呈浅黄或深黄色,菌体细胞呈圆端直杆状,0.9~1.2μm宽, 3~8μm长。
本发明乳清蛋白(WPI,蛋白含量90%)购自美国Hilmar公司,苹果酸购自安徽雪郎生物科技股份有限公司,NaOH购自南京化学试剂股份有限公司;胆酸钠(SC)、脱氧胆酸钠(SDC)和牛磺胆酸钠(STC)均购自上海 Macklin 生化科技有限公司。
胆酸盐结合能力的测定方法如下:
取9%乳清蛋白降胆固醇肽溶液2ml于离心管中,再加入 4 mL 胆酸盐溶液(0.4mmol/L STC、SD和SDC,PBS 溶液配制),放入37℃培养箱中振荡 2 h 后,于离心机离心20min(4000 r/min)。取2.5 mL 上清液和 7.5 mL H2SO4 溶液(60%,v/v)混匀,70 ℃水浴20min,取出后迅速放入冰水冷却,使用酶标仪测定387 nm处的吸光值,根据标准曲线计算溶液的胆酸盐浓度。计算公式:
式中:C0 为空白对照溶液的胆酸盐浓度mmol/L;C1为样品上清液中的胆酸盐浓度mmol/L。
下面结合具体实施例子进一步阐明本发明,应理解这些实施例仅用于说明本发明,而不用于限制本发明的范围,在阅读了本发明之后,本领域的技术人员对本发明各种等价形式的修改均落于本申请所附权利要求所规定的范围。
实施例
一种植物乳杆菌FQR及其复合酸制备乳清蛋白降胆固醇肽的方法,包括以下步骤:
A.制备WPI溶液
将乳清蛋白粉配制成6.0%(w/v)的WPI溶液,然后在磁力搅拌器下按转速1500 rpm分散3小时使乳清蛋白充分溶解,放置于4℃冰箱冷藏,过夜水合;然后在108℃高压灭菌锅灭菌15 min,并冷却至37℃得到灭菌的WPI溶液;
B.发酵改性处理
将植物乳杆菌FQR在液态MRS培养基中37℃恒温静止培养24h活化,将活化后的植物乳杆菌FQR调节活菌数为≥108 CFU/mL,接种到灭菌的WPI溶液中,在无菌操作台按1%(v/v)接种,在37℃培养36h。发酵结束后,将培养液在4℃10000 r/min 离心10 min,上清液冷冻干燥后,得到发酵改性处理后的乳清蛋白,4℃冷藏保存;
C、发酵复合改性处理
发酵改性处理后的乳清蛋白配制成70 mg/mL的WPI溶液,再加入2.0%苹果酸,搅拌15 min后,用1.0 mol/L NaOH终止酸解;
D、冻干保存
将发酵复合改性处理后的WPI溶液,置于冷冻干燥机冻干,得到所述的乳清蛋白降胆固醇肽,4℃冷藏备用。
实施例
A、制备WPI溶液
将乳清蛋白粉配制成7.5%(w/v)的WPI溶液,然后在磁力搅拌器下按转速1500rpm分散2.5小时使乳清蛋白充分溶解,放置于4℃冰箱冷藏,过夜水合;然后在108℃高压灭菌锅灭菌15 min,并冷却至37℃得到灭菌的WPI溶液;
B、发酵改性处理
将植物乳杆菌FQR在液态MRS培养基中36℃恒温静止培养26h活化,将活化后的植物乳杆菌FQR调节活菌数为≥108 CFU/mL,接种到灭菌的乳清蛋白溶液中,在无菌操作台按2.5%(v/v)接种,在36℃培养42h。发酵结束后,将培养液在4℃12000 r/min 离心12 min,上清液冷冻干燥后,得到发酵改性处理后的乳清蛋白,4℃冷藏保存;
C、发酵复合改性处理
发酵改性处理后的乳清蛋白配制成80mg/mL的WPI溶液,再加入2.5%苹果酸,搅拌18 min后,用1.2 mol/L NaOH 终止酸解;
D、冻干保存
将发酵复合改性处理后的WPI溶液,置于冷冻干燥机冻干,得到所述的乳清蛋白降胆固醇肽,4℃冷藏备用。
实施例
A、制备WPI溶液
将乳清蛋白粉配制成9.0%(w/v)的WPI溶液,然后在磁力搅拌器下按转速1600 rpm分散2小时使乳清蛋白充分溶解,放置于4℃冰箱冷藏,过夜水合;然后在108℃高压灭菌锅灭菌15 min,并冷却至37℃得到灭菌的WPI溶液;
B、发酵改性处理
将植物乳杆菌FQR在液态MRS培养基中35℃恒温静止培养28h活化,将活化后的植物乳杆菌FQR菌株调节活菌数为≥108 CFU/mL,接种到灭菌的WPI溶液中,在无菌操作台按4.0%(v/v)接种,在35℃培养48h。发酵结束后,所有培养液在4℃15000 r/min 离心10 min,上清液冷冻干燥后,得到发酵改性处理后的乳清蛋白,4℃冷藏保存;
C、发酵复合改性处理
发酵改性处理后的乳清蛋白配制成90 mg/mL的WPI溶液,再加入3.0%苹果酸,搅拌20 min后,用1.5 mol/L NaOH 终止酸解;
D、冻干保存
将发酵复合改性处理后的WPI溶液,置于冷冻干燥机冻干,得到所述的乳清蛋白降胆固醇肽,4℃冷藏备用。
对照例
对照组1是植物乳杆菌FQR发酵水解组,接种量为4.0%(v/v);
对照组2是普通乳酸菌结合苹果酸水解组,乳酸菌接种量为4.0%(v/v),苹果酸添加量为3%;
对照组3是空白对照组,乳清蛋白溶液不作任何处理;
以上对照组均以9.0%(w/v)的乳清蛋白溶液为水解溶液,乳清蛋白溶液制备方法均相同,其中,对照组1发酵改性步骤与实施例3相同。通过测定胆酸钠结合率、牛磺胆酸钠结合率以及脱氧胆酸钠结合率等指标来判断乳清蛋白降胆固醇肽的产量及其降胆固醇活性。
结果:
由表1可知,与对照组相比,实施例组的三种胆盐的结合率均有显著的提高,其中,实施例3组的三种胆盐的结合率分别最高可达到胆酸钠52.4%、牛磺胆酸钠 45.3%、脱氧胆酸钠70.1%。说明先进行发酵改性处理后使得部分大分子蛋白降解为小分子活性肽,此时加入苹果酸使得水解度加深,更多的蛋白质被水解为疏水性的多肽,使得乳清蛋白具有更高的疏水性氨基酸组成,有助于结合具有疏水性的胆酸盐,从而提高结合胆酸盐的能力和降胆固醇活性。
表1 乳清蛋白降胆固醇肽水解度和结合胆酸盐的能力测定
组别 | 胆酸钠(SC)结合率 | 牛黄胆酸钠(STC)结合率 | 脱氧胆酸钠(SDC)结合率 |
实施例1 | 51.42% ± 1.35% | 43.17% ± 0.99% | 69.43% ± 1.28% |
实施例2 | 51.32% ± 1.43% | 46.71% ± 1.42% | 69.82% ± 1.13% |
实施例3 | 52.42% ± 1.38% | 45.27% ± 0.26% | 70.13% ± 1.08% |
对照组1 | 41.42% ± 0.79% | 33.17% ± 2.11% | 57.43% ± 1.61% |
对照组2 | 43.76% ± 1.82% | 36.89% ± 1.93% | 52.07% ± 0.61% |
对照组3 | 42.82% ± 1.17% | 32.44% ± 1.35% | 58.98% ± 1.06% |
Claims (7)
1.一种植物乳杆菌FQR,其特征在于:所述的植物乳杆菌FQR拉丁名为Lactobacillusplantarum FQR,保藏在中国典型培养物保藏中心,保藏编号为:CCTCCNO:M 202128。
2.一种权利要求1所述的植物乳杆菌FQR的应用,其特征在于:所述植物乳杆菌FQR复合酸处理用于制备乳清蛋白降胆固醇肽。
3.一种权利要求1所述的植物乳杆菌FQR复合酸制备乳清蛋白降胆固醇肽的方法,其特征在于,所述方法的操作步骤为:
A.制备乳清蛋白溶液:将乳清蛋白粉配制成乳清蛋白溶液,然后按转速1500rpm~1600rpm分散2~3小时使乳清蛋白充分溶解,冷藏过夜水合;然后灭菌并冷却,得到灭菌的乳清蛋白溶液;
B.发酵改性处理:将活化后的植物乳杆菌FQR调节活菌数为≥108CFU/mL,在无菌操作台接种到灭菌的乳清蛋白溶液中,在35~37℃发酵培养36~48h;发酵结束后,将培养液在10000~12000r/min离心10~15min,上清液冷冻干燥后,得到发酵改性处理后的乳清蛋白;
C.发酵复合改性处理:发酵改性处理后的乳清蛋白配制成70~90mg/mL的乳清蛋白溶液,再加入添加苹果酸,搅拌15~20min后,用1.0~1.5mol/L NaOH终止酸解;
D.冻干保存;将发酵复合改性处理后的乳清蛋白溶液冷冻干燥,得到所述的乳清蛋白降胆固醇肽。
4.根据权利要求3所述的方法,其特征在于:所述乳清蛋白溶液的质量浓度为6.0~9.0%。
5.根据权利要求3所述的方法,其特征在于:所述活化后的植物乳杆菌FQR是在液态MRS培养基中活化,35~37℃恒温静止培养24~28h。
6.根据权利要求3所述的方法,其特征在于:按体积比,所述植物乳杆菌FQR接种量为1%~4%。
7.根据权利要求3所述的方法,其特征在于:所述苹果酸添加量为2.0~3.0%。
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