CN116179405B - 一株植物乳植物杆菌zf617及其应用 - Google Patents
一株植物乳植物杆菌zf617及其应用 Download PDFInfo
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Abstract
本发明涉及一株植物乳植物杆菌(Lactiplantibacillus plantarum)ZF617及其应用。本发明植物乳植物杆菌ZF617的保藏编号为GDMCC No:62543。本发明菌株能具有提高果蔬汁中抑菌活性、a‑葡萄糖苷酶抑制率,以及发酵过程中产苯乳酸、γ‑氨基丁酸、苹果酸和抗炎活性的优点,在不需要额外添加防腐剂的条件下延长了青柠汁的货架期,即保留了果蔬的风味,又无安全风险,为果蔬发酵提供丰富的香气物质和滋味。
Description
技术领域
本发明属于酿造微生物技术领域,具体涉及一株植物乳植物杆菌ZF617及其应用。
背景技术
后生元是益生菌经加工处理后的益生菌代谢物成分统称,包括菌体与代谢产物。日本研究证实,经过筛选的后生元,增强免疫能力优于原活菌,即使经由高温作用或肠胃消化液处理,仍保有高度生理活性。证据显示,后生元的代表成分 (例如: 脂壁酸) 是决定后生元对于酸、碱、热耐受性的关键。已知后生元对人体具有多种健康助益。
目前已发现后生元在抗炎、维护肠屏障、肥胖、免疫调节、高血压、心血管疾病、抗菌、保肝、抗氧化以及癌症等方面的作用。在促进益生菌增殖方面,后生元也有着优异表现,丝毫不输益生元。
目前,后生元的研究方向相对较少。专利文件CN114868914A公开一种以嗜酸乳酸菌发酵液中抗菌肽为主要功能性物质的后生元,但内涉及细胞内容物的后元生物质的制备。
专利文件CN202110069179.6公开半固定化益生菌湿热淬灭生元的方法,采用乳酸菌发酵液结合大豆纤维粉混合,然后蒸汽湿热灭活,结合急速冷却制备后生元产品,但此专利并未涉及乳酸菌后生元的功能性和乳酸菌菌体内容物制备后元生。专利文件CN202210415577.3公开一种提高免疫促进生长的后生元、制备方法及应用。专利主要涉及水产养殖饲料领域,采用乳酸菌、酵母菌、凝结芽孢杆菌代谢产物三种混合后生元的功能性,不涉及果蔬汁发酵应用而且没有涉及乳酸菌内容物的后生元制备。
由此可见,目前乳酸菌细胞内容物的后生元制备及其功能性依然有待于开发。乳酸菌发酵代谢物后生元功能性,结合乳酸菌细胞内容物的功能生。不仅可以提高乳酸菌的利用价值,同时增强产品的附加值和竞争力。因此,寻求一种乳酸菌细胞内容物经过工艺加工后具有功能性是本技术领域亟待解决的问题。
发明内容
本发明的目的在于提供一种植物乳植物杆菌(Lactiplantibacillus plantarum)ZF617,该菌细胞内容物经过加工后产生抑菌物质,以及能有效抑制a-葡萄糖苷酶活性。将本发明的植物乳植物杆菌用于果蔬汁的发酵,经过工艺加工后,菌体细胞内容物形成抑菌作用可延长果蔬汁的货架期,产生的后生元防止血糖生高可赋予果蔬汁保健功能。
本发明已将该菌株于2022年6月14日保藏于广东省微生物菌种保藏中心,地址:广东省广州市先烈中路100号大院59号楼5楼,邮编510075,获得保藏编号为GDMCC No:62543,分类学名称为:Lactiplantibacillus plantarum,将其命名为:植物乳植物杆菌Lactiplantibacillus plantarumZF617。
为实现上述目的,发明人进行了深入研究,并经重复多次研究论证而完成获得本发明方案,具体如下:
第一个方面,本发明提供了一株植物乳植物杆菌ZF617,已于2022年6月14日保藏于广东省微生物菌种保藏中心,其保藏编号为GDMCC No:62543。
本发明植物乳植物杆菌ZF617是从发酵酸菜中分离筛选获得,其菌落特征如下:菌落直径达3~5mm,菌落呈现乳白色、菌落凸起、呈圆形,表面光滑,菌落边缘整齐。本发明菌株ZF617所测得的16S rDNA序列与植物乳植物杆菌(Lactiplantibacillus plantarum)相似度达到100%,可确定菌株ZF617为一株植物乳植物杆菌(Lactiplantibacillus plantarum)。
第二个方面,本发明提供了一种发酵菌剂,该菌剂包含本发明所述的植物乳植物杆菌ZF617。
第三个方面,本发明提供了所述植物乳植物杆菌ZF617在食品发酵中的应用。本发明的植物乳植物杆菌ZF617可用于食品发酵,用于提高食品的营养价值以及延长食品的货架期。
在某些实施方案中,所述在食品发酵中的应用为用于果蔬汁的发酵。
第四个方面,本发明提供了一种发酵果蔬汁,其包含果蔬汁和本发明植物乳植物杆菌ZF617。
在某些实施例中,经实检测发现本发明后生元中含有本乳酸、γ-氨基丁酸或苹果酸。本发明实施例有效的证明了,采用植物乳植物杆菌ZF617发酵制备的果蔬汁可提升果蔬汁的色泽、香气,甜味和酸味的口感,明显改善了果蔬汁的品质,并提高了果蔬中苯乳酸、γ-氨基酸丁酸、苹果酸的含量,同时赋予发酵果蔬汁a-葡萄糖苷酶抑制活性、抑菌活性以及抗炎性。
第五个方面,本发明提供了一种发酵果蔬汁的制备方法,所述方法为在果蔬汁中添加本发明植物乳植物杆菌ZF617进行发酵的方法。
在某些实施方案中,所述发酵方法的培养温度为30℃~37℃,发酵时间为6~48h,接种量为1×105~106cfu/mL。
第六个方面,本发明还提供了一种后生元,所述后生元由本发明的植物乳植物杆菌ZF617细胞内容物热处理制备而成,包括菌体与菌体代谢物。
在某些实施例中,经实检测发现本发明后生元中含有本乳酸、γ-氨基丁酸或苹果酸。本发明经实验证明本发明制备的后生元能有效抑制α-葡萄糖苷酶活性,其抑制率可达80%以上,起到有效降血糖的效果;本发明还能改善IL-10/IL-12 比值,起到抗炎的作用;本发明的后生元中还含有高产量的苯乳酸、γ-氨基丁酸或苹果酸。
第七个方面,本发明还提供了所述后生元在制备α-葡萄糖苷酶抑制剂中的应用或制备抗炎制剂中的应用或在制备本乳酸、γ-氨基丁酸或苹果酸中的应用。
第八个方面,本发明还提供一种抑菌制剂,该抑菌制剂由本发明植物乳植物杆菌ZF617的发酵液制备而成。
在某些实施方案中,已证明本发明植物乳植物杆菌ZF617发酵液具有一定的抑菌作用,尤其针对芽孢杆菌中的枯草芽孢杆菌、解淀粉芽孢杆菌、蜡状芽孢杆菌、凝结芽孢杆菌。
与现有技术相比,本发明具备如下有益效果:
本发明植物乳植物杆菌ZF617细胞内容物经过工艺加工后可产生抑制a-葡萄糖苷酶的活性物质,填补了现有技术中未有能对乳酸菌细胞内容物功能性开发的空白,且抑制效果较高。由于本发明菌株ZF617细胞内容物经过加工后可产生抑菌物质,尤其针对芽孢杆菌的抑制作用,使用该菌发酵果蔬汁可延长其货架期,此外其发酵液还产生的γ-氨基丁酸、苯乳酸、苹果酸和抗炎活化。可赋予发酵果汁一定的保健和防腐功能,例如:γ-氨基丁酸改善机体睡眠质量、降血压等生理功效。苹果酸可以用作食品的保色剂提高果蔬汁抗氧化活性。
附图说明
图1为菌株ZF617菌落图
图2为菌株ZF617显微镜观察图
图3为菌株ZF617发酵胡萝卜汁中检测出的苯乳酸与苯乳酸标准品的质谱图
图4为菌株ZF617发酵胡萝卜汁中检测出的γ-氨基丁酸与γ-氨基丁酸标准品的质谱图
图5为菌株ZF617发酵胡萝卜汁的发酵6h(未加热)、12h、24h的抑菌结果示意图(其中1为枯草芽孢杆菌;2为解淀粉芽孢杆菌;3为蜡状芽孢杆菌;4为凝结芽孢杆菌)。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
实施例1 目的菌株的筛选
1、原材料的处理
取传统工艺条件下发酵的酸菜,加工成浆状,加入生理盐水稀释至10-1、10-2、10-3、10-4、10-5、10-6浓度,制成菌体稀释液,分别从各浓度稀释液中取0.1ml均匀涂布于MRS(含1.5%溴甲酚紫)固体培养基平皿上,37℃培养48h,挑选菌落呈现乳白色、菌落凸起、呈圆形,表面光滑,菌落边缘整齐的单菌落,接入MRS液体培养基中,37℃培养24h。
2、菌株初筛
将上一步所培养的单菌落分别在MRS固体培养基上再次划线进行纯化,获得9个单一菌落,分别依次编号为FJ101~FJ109。在胡萝卜浓缩汁(将胡萝卜与水按1:10比例打浆,通过均质机均质20 min,制备而成)中分别接入上述9株活化的菌体,接种终浓度为1×105cfu/mL,37℃条件下,静止发酵12h后,在90℃下处理15min,然后分别取样检测发酵胡萝卜汁苹果酸以及抑菌活性、a-葡萄糖苷酶抑制率、IL10/IL12值,以未加热作为对照。
a.抑菌活性检测方法:采用常规平板打孔法,测定抑菌圈大小。
b.a-葡萄糖苷酶抑制率检测方法:抑制率I(%)的计算:I(%)=(ΔAE-ΔA)/ΔAE×100%,ΔAE=未加抑制剂前固定Α-葡(萄)糖苷酶与4-硝基苯-α-D-吡喃葡萄糖苷反应后的吸光度值(扣除相应空白);ΔA=加入抑制剂后固定化Α-葡(萄)糖苷酶与4-硝基苯-α-D-吡喃葡萄糖苷反应后的吸光度值(扣除相应空白)
c.IL-10/IL12检测:将PBMCs (外周血单个核细胞)细胞分别用含和不含抗生素的1640培养基配成1×106cell/ml,再分别加入适量稀释100倍的灭活后(即热处理)的果蔬发酵液上清液,将细胞在5% CO2培养24h。收集细胞上清液,用ELISA 试剂盒检测细胞因子IL-10和IL-12浓度。
d.苹果酸检测方法:采用高效液相法,色谱柱为:CAPCELL PAK ADME-HRS5(4.6 mm×150 mm),流动相为超纯水(含0.2%磷酸),流速:0.2 mL/min,测检波长为215 nm,上样量为5 μL。
表1 初筛菌株发酵胡萝汁的检测结果
IL-12 能促进细胞免疫,使T 细胞产生IFN-γ,导致巨噬细胞的活化和炎症反应。IL-10 被认为是一个重要的抗炎因子,能逆转慢性炎症,如炎症性肠病等。因此单核巨噬细胞和DC 产生的IL-10/IL-12 比值倍受关注,因为这两种因子的平衡影响了免疫炎症发展的方向,其比值高表示抗炎性高,机体不容易产生炎症,外界比值低表示机体反应敏感,显现炎症反应明显,不利于机体恢复。根据统计学相关与细胞学研究表明,在IL10/IL12大于30%时被认定为具有防护级别。
a-葡萄糖苷酶是生物体糖代谢途径中不可缺少的一种酶,能够直接参与淀粉及糖原的代谢途径,调节人体的糖化学代谢。抑制a-葡萄糖苷酶活性可减缓淀粉分解为葡萄糖的速度,减少和延缓小肠对葡萄糖的吸收,到达降低血糖的效果。因此,a-葡萄糖苷酶抑制率越高表明降糖效果越好。
苹果酸除了赋予果汁特殊愉快的酸味口感,还可以用作食品的保色剂,有保持天然果汁色泽以及本身所具有的抗菌作用。苹果酸可以有效提高机体抗氧化酶的活力及非酶抗氧化物质的水平,降低生物机体活性氧的含量,减轻自由基对机体的损伤,增强机体的抗氧化能力,减少脂质过氧化的发生,起到抗氧化作用。因此,在果蔬汁中添加苹果酸,能提高果蔬汁的抗氧化性,延长产品的货架期。
由表1可知,菌株FJ103、FJ107、FJ108的抑菌活性、a-葡萄糖苷酶抑制率、苹果酸以及IL10/IL12值都较高,因此选取菌株FJ103、FJ107、FJ108进行复筛。
3、菌株复筛
将初筛选出的菌株FJ103、FJ107、FJ108,重新活化培养,将菌液8000g离心10 min,去上清液,用无菌水重悬后。分别添加于胡萝卜汁中,最终接种量为1×105cfu/ml,将发酵胡萝卜汁90℃热处理15 min,取各组胡萝卜汁进行感官评价,结果如表2:
表2 发酵胡萝汁感官评价得分
备注:各项指标满分为100分,得分越高表示结果越好。
根据表2的得分,感官评价结果最好的菌株为FJ107,该菌则为本发明的目的菌株。
菌株FJ107在MRS平板培养基上的菌落呈现乳白色,菌落凸起、呈圆形,表面光滑,菌落边缘整齐,直径为3mm-5mm,菌落形态如图1所示,显微镜观察如图2所示。将菌株FJ107的16S区序列进行鉴定,结果与植物乳植物杆菌(Lactiplantibacillus plantarum)相似度达到100%,则可确定菌株FJ107为一株植物乳植物杆菌。本发明将菌株FJ107保藏于广东省微生物菌种保藏中心,获得的保藏编号为GDMCC No:62543,分类学名称为:Lactiplantibacillus plantarum,本发明将其命名为:植物乳植物杆菌Lactiplantibacillus plantarumZF617。
实施例2目的菌株遗传稳定性检测
将实施例1获得的菌株ZF617在MRS培养基上连续传代10代,分别对第1代、第5代、第10代培养的菌株,离心去上清,用无菌水重悬,接菌于胡萝卜液发酵,然后90℃处理15min,检测其抑菌活性、a-葡萄糖苷酶抑制率、苹果酸含量以及IL10/IL12值,用以判断其传代稳定性。检测方法参照实施例1,结果数据见表3:
表3不同传代菌株ZF617在胡萝卜汁中抑菌活性、a-葡萄糖苷酶抑制率、苹果酸含量以及IL10/IL12值
由表3的结果可以看出,与第1代相比,由第5、10代菌株ZF617在胡萝卜汁中抑菌活性、a-葡萄糖苷酶抑制率、苹果酸和IL-10/IL-12并没有显著的变化,表明本发明菌株ZF617遗传稳定性好,符合生产使用要求。
实施例3目的菌株的功能性物质检测
以实施例1中的菌株FJ106作为对照,将菌株ZF617接种于胡萝卜汁中,终浓度为1×105cfu/mL,发酵24 h,用移液器分别吸取发酵6h、12h、24h发酵胡萝卜汁(非热处理6h作为对照,发酵12h、24h的胡萝卜汁90℃热处理15 min)到琼脂孔内,37℃培养16h~18h后观察抑菌圈大小。实验所用指示菌分别为枯草芽孢杆菌、解淀粉芽孢杆菌、蜡状芽孢杆菌、凝结芽孢杆菌。抑菌结果如表4所示:
表4 胡萝卜发酵液的抑菌圈大小
由表4、图5可以看出,本发明的菌株ZF617对芽孢杆菌有一定的抑制作用,尤其对枯草芽孢杆菌、解淀粉芽孢杆菌、蜡状芽孢杆菌和凝结芽孢杆菌具有显著的抑制效果,可作为这类芽孢杆菌的专用抑菌剂。
本发明采用液质联用(LC-MS/MS)方法检测了胡萝卜汁中的苯乳酸与γ-氨基丁酸的含量。液质联用检测方法具体如下:检测苯乳酸采用负离子模式,以m/z(质核比)165为定性离子,以m/z(质核比)147为定量离子;检测γ-氨基丁酸采用正离子模子,m/z(质核比)104为定性离子,以m/z(质核比)87为定量离子。结果检测出苯乳酸的量含量为210.5±10.2mg/L,γ-氨基丁酸含量为63.6mg/100mL,同时,检测出菌株ZF617在发酵12 h后经过热处理所产生的a-葡萄糖苷酶抑制率为77.14%。
苯乳酸作为天然存在的抑菌化合物和重要有机酸,具有广谱抑菌用途,可以有效抑制真菌、细菌。γ-氨基丁酸是一种重要的中枢神经系统抑制性神经递质,具备改善机体睡眠质量、降血压等生理功效。因此,本发明菌株ZF617发酵后经过热处理所获得后生元中含有苯乳酸、γ-氨基丁酸、苹果酸等功能性物质,使其具有抑菌活性、睡眠质量、降血压、抗炎与抗氧化功能。
实施例4目的菌接种量对发酵胡萝卜汁中抑菌活性和a-葡萄糖苷酶抑制率以及品
质的影响
不同浓度的菌种内容物质热处理后会影响发酵胡萝卜汁的品质。因此设计了热处理不同浓度的菌种内容物。将培养后的菌液,8000g离心,10min,去上清,用无菌水重悬,在胡萝卜汁中终浓度分别稀释至1.0×104 cfu/ml、1.0×105 cfu/ ml、1.0×106 cfu/ml、1.0×107cfu/ml、1.0×108cfu/ml的剂量,接种于胡萝卜汁中发酵后,于90℃,热处理15 min,考察其对抑菌活性、a-葡萄糖苷酶抑制率、苹果酸和IL-10/IL-12以及发酵胡萝卜汁品质的影响。参照实施例1的方法,以不添加菌株ZF617的胡萝卜汁作为空白组,以菌株FJ106作为对照组。结果如表5、表6所示:
表5发酵胡萝卜汁中抑菌活性、a-葡萄糖苷酶抑制率、苹果酸含量和IL-10/IL-12值分析
表6感官评价
注:每项评定满分10分,以0.5为评分间隔,综合评分为各项总和值。
由表5、6可知,用本发明菌株ZF617发酵胡萝卜汁,能够显著提高胡萝卜汁中抑菌活性、a-葡萄糖苷酶抑制率、苹果酸和IL-10/IL-12。实验组1~5显示添加量越多,则胡萝卜汁中功能性物质的含量或活性越高,但是当ZF617添加量增大到一定程度时,例如实验组4、5,使得胡萝卜汁的香气、甜味、酸味因素受到较大影响。因此在使用本发明菌株ZF617发酵时应注意产品的添加量,添加量为1.0×105~1.0×106cfu/ml范围时为最佳添加量,既能维持高抑菌活性和a-葡萄糖苷酶抑制率,又能提高胡萝汁的风味和口感。
实施例5 目的菌株在发酵番茄浆中的应用
将菌株ZF617在液体MRS培养基扩增后,将培养后的菌液,8000g离心,10min,去上清,用无菌水重悬,以终浓度1.0×105cfu/ml接种量接入番茄浆(番茄与水比例为1:5,破碎后,加入均质机中均质20min,备用),温度37℃发酵12h,90℃热处理15min,观察其对发酵的影响,以及检测发酵番茄浆的货架期。以菌株FJ106作为对照组。
采用液质联用(LC-MS/MS)方法检测发酵液中苯乳酸与γ-氨基丁酸的含量,液质联用检测方法具体如下:检测苯乳酸采用负离子模式,以m/z(质核比)165为定性离子,以m/z(质核比)147为定量离子;检测γ-氨基丁酸采用正离子模子,m/z(质核比)104为定性离子,以m/z(质核比)87为定量离子结果如表7所示:
表7发酵番茄浆中苯乳酸、γ-氨基丁酸含量及感官评价
注:每项评定满分10分,以0.5为评分间隔,综合评分为各项总和值。
表8发酵番茄浆货架期中苯乳酸、γ-氨基丁酸含量及感官评价
从表7和表8中苯乳酸、γ-氨基丁酸的含量、色泽、香气、甜味、酸味结果来看,本发明的菌株ZF617可以有效保证番茄浆的防腐效果,延长货架期,而且改善番茄浆的感官水平和功能性,增加番茄的香气,提高番茄浆的风味和口感,番茄香气四溢、风味酸甜可口。表7中番茄浆中抑菌活性保持在20.32mm~23.64 mm之间和a-葡萄糖苷酶抑制率69.43%~72.37%之间。苹果酸含量在1.28~1.39g/L之间,IL-10/IL-12在31.84~32.82%之间,各有效成分在货架期含量相对稳定,能有效保证产品的功能性和质量稳定性。
实施例6目的菌株在发酵青柠汁中的应用
将本发明的植物乳植物杆菌ZF617在液体MRS培养基扩增后,将培养后的菌液,8000g离心,10min,去上清,用无菌水重悬,以终浓度1.0×105cfu/mL接种量接入青柠汁(将青柠与水1:5混合,打成浆状,均质20min 备用)中,温度37℃发酵12h,90℃热处理15min,判断其在青柠汁中发酵影响,以及检测发酵青柠汁的货架期。本实验中以菌株FJ106作为对照组,具体数据见表9、10:
表9发酵青柠汁中功能性物质含量及感官评价
注:每项评定满分10分,以0.5为评分间隔,综合评分为各项总和值。
表10发酵青柠汁货架期中功能性物质含量及感官评价
从表9、表10的苯乳酸、γ-氨基丁酸的含量、色泽、香气、甜味、酸味结果来看,本发明的植物乳植物杆菌ZF617可以有效保证青柠汁的防腐效果,而且改善青柠汁的感官水平和功能性,增加青柠汁香气,提高青柠汁的风味和口感,使青柠汁酸甜可口。表7中番茄浆中抑菌活性保持在22.32 mm~24.58 mm之间和a-葡萄糖苷酶抑制率72.58%~74.26%之间,苹果酸在17.11~17.89g/L之间,IL-10/IL-12在30.08~33.22之间。各有效成分在货架期含量相对稳定,能有效保证产品的功能性和质量稳定性。
本发明提供的植物乳植物杆菌ZF617菌种内容物质热处理(即后生元),具有提高果蔬汁中抑菌活性、a-葡萄糖苷酶抑制率,以及发酵过程中产苯乳酸、γ-氨基丁酸、苹果酸和抗炎活性的优点,并且不影响果蔬加工工艺,在不需要额外添加防腐剂的条件下延长了青柠汁的货架期,即保留了果蔬的风味,又无安全风险,为果蔬发酵提供丰富的香气物质和滋味。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (7)
1.一株植物乳植物杆菌(Lactiplantibacillus plantarum) ZF617,其特征在于,所述植物乳植物杆菌ZF617的保藏编号为GDMCC No:62543。
2.一种发酵菌剂,其特征在于,所述发酵菌剂包含如权利要求1所述的植物乳植物杆菌ZF617。
3.如权利要求1所述的植物乳植物杆菌ZF617在果蔬汁发酵中的应用。
4.一种发酵果蔬汁,其特征在于,所述发酵果蔬汁包含果蔬汁和权利要求1所述的植物乳植物杆菌ZF617。
5.如权利要求4所述的发酵果蔬汁,其特征在于,所述发酵果蔬汁还包括苯乳酸、γ-氨基丁酸或/和苹果酸。
6.一种发酵果蔬汁的制备方法,其特征在于,所述方法为在果蔬汁中添加权利要求1所述的植物乳植物杆菌ZF617进行发酵的方法。
7.如权利要求6所述的制备方法,其特征在于,所述方法的发酵温度为30℃ ~ 37℃,发酵时间为6 ~ 48h,接种量为1× 105 ~106cfu/mL。
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