在高加索山脉地区,牧民们发现储存于羊皮袋中的鲜乳会自然发酵成酸乳,即开菲尔(kefir)。这种发酵作用是由羊皮袋中自然形成的发酵剂开菲尔粒(kefir grain,KG)完成的,之后,世界各游牧区如土耳其、伊朗、新疆、青藏等地也都发展出了自己的KG,在青藏地区,KG又被称为雪莲菌或藏灵菇。现代微生物学研究发现,KG在组成上以乳酸菌和酵母菌为主[1-3],副干酪乳杆菌(Lactobacillus paracasei)、嗜酸乳杆菌(L.acidophilus)、保加利亚乳杆菌(L.bulgaricus)、植物乳植杆菌(Lactiplantibacillus plantarum)、克氏乳杆菌(L.kefiranofaciens)及乳酸乳球菌(Lactococcus lactis)是乳酸菌中的优势种,酵母菌包括马克斯克鲁维酵母(Kluyveromyces marxianus)、酿酒酵母(Saccharomyces cerevisiae)、乳酒假丝酵母(Candida kefyr)、单孢酿酒酵母(Saccharomyces unisporus)、德尔布有孢圆酵母(Torulospora delbrueckii)、发酵毕赤酵母(Pichia fermentans)等,其中,酿酒酵母、单孢酿酒酵母、乳酒假丝酵母和马克斯克鲁维酵母是常见的优势种,间或有醋酸细菌,如巴氏醋酸杆菌(Acetobacter pasteurianus)、可可豆醋杆菌(Acetobacter fabarum)等。
KG分为乳开菲尔粒(milk kefir grain,MKG)和非乳开菲尔粒(水开菲尔粒(water kefir grain,WKG))两类,前者主要适合以鲜乳为底物,包括半脱脂、脱脂和全脂乳,颗粒呈白色至奶油色,后者则主要以还原糖或富含还原糖的植物材料如水果为底物,颗粒呈灰白色、半透明、蜡状,稠度较硬[4]。前者的发酵产物称为乳开菲尔(milk kefir,MK),命名上常冠以底物前缀,如牛乳开菲尔、羊乳开菲尔、骆驼乳开菲尔等,后者的发酵产物称为水开菲尔(water kefir,WK)或非乳开菲尔,命名时也以底物为前缀,如石斛开菲尔、桑葚开菲尔、黑枸杞开菲尔、野樱莓开菲尔等。MK粘稠、不透明,味道微酸,含有少量碳酸和微量乙醇,有乳香味,通常感觉不到酒精味。WK具有较低粘度和半透明外观,并且当底物果蔬含量增大时,透明度会降低。与MK相比,WK含有更高甜度和碳酸与酒精含量,两者饮用时均口感清爽[5]。
益生菌是指摄入足够量时能对人或底物宿主产生有益效应的微生物[6],KG已成为发掘益生菌的重要源泉[2-3,5]。开菲尔因其具有益生菌发酵食品的功能属性,具有优化重塑肠道菌群和调理肠道的功能,可对非传染性慢性病如II型糖尿病(type 2 diabetes mellitus,T2DM)、心血管和神经退行性疾病等产生积极影响[8]。基于此,本文介绍了乳开菲尔(MK)及水开菲尔(WK)的发酵菌株及发酵方式,总结了其对肠道功能紊乱、保肝护肝及代谢综合征疾病的预防与辅助治疗的功效,为开菲尔在益生菌菌株发掘和功能性发酵食品研发提供理论依据。
MK的发酵底物以鲜乳为主,相关乳酸菌包括乳球菌属(Lactococcus)、乳杆菌属(Lactobacillus)和双歧杆菌属(Bifidobacterium)等,其生理生化方面的重要特征是能够利用乳糖,在发酵和风味形成中起着至关重要的作用[9-10]。MKG 所含酵母主要为非酿酒酵母(non-Saccharomyces cerevisiae)[11-12]。 某些地区的MKG由于自然环境条件的影响,含有少量醋酸菌,这种情况下,发酵产物中乙酸含量相对较高[13]。
MK的传统发酵方式是用MKG直接接种液体乳[14]。将原料乳巴氏消毒((70~75) ℃、(20~25)min)后冷却至(20~25)℃,接种MKG(接种量为(1~100)g/L均可,但通常为10 g/L),发酵温度为25 ℃,发酵时间为24 h,之后pH达到4.6[15-16],筛分发酵乳,将过滤分离的KG或添加少量先前发酵液作为接种物,添加新底物可进行新一轮发酵,这种重复发酵方式被称为“后倒法(back-slopping)”,后倒重复次数可以多达50次[17]。MK通常是厌氧发酵,但后倒时引入的空气使得发酵体系的好氧度升高,导致醋酸菌增殖,后倒次数越多,所制得MK中所含乙酸含量越高[18]。发酵过程中产生的乳酸、乙酸、乙醛、二氧化碳和少量乙醇构成了MK独特的感官特性[19]。
除传统发酵方式,还可采用纯菌种复合发酵方式规模化生产MK。 WANG L等[20]将从MKG中分离出的肠膜明串珠菌(Leuconostoc mesenteroides)、开菲尔乳杆菌(Lactobacillus kefiri)、干酪乳杆菌(Lactobacillus casei)和马克斯克鲁维酵母(Kluyveromyces marxianus)成功构建成了能够协同共发酵的纯菌复合发酵剂,所发酵的MK的凝乳时间、酸度、持水量、乙醇含量、菌种活力和感官评分等指标与MKG发酵的MK基本一致,而挥发性香气成分种类明显多于后者。张亦等[21]从源自中国新疆的10种KG中分离获得51株乳酸菌和22株酵母菌,确定了复配组合“乳酸菌∶酵母菌=5∶1”、“肠膜明串珠菌∶开菲尔乳杆菌∶干酪乳杆菌=1∶2∶1”,其发酵所得的MK凝乳时间为6.0 h、酸度为84.65°T、持水力为62.31%、乙醇体积分数为0.53%、乳酸菌活菌数为3.89×109 CFU/mL、酵母菌活菌数为4.61×106 CFU/mL、感官评分为89分,各项指标与MKG自然发酵得到的MK类似,而挥发性风味物质间构成更和谐,主要表现在芳香醇(苯乙醇)和呈味醇(异丁醇)含量更高。后续在更多地分离鉴定KG中菌株并分析其代谢特性的基础上,选择发酵特性优越且益生菌效应显著者,根据其代谢的相容性和互补性进行优化复配,通过纯菌种复合发酵方式生产MK,会更好地提升MK的功能属性。 不依赖于MKG的纯菌种复合发酵方式更适合规模化生产。
WK被视为MK的素食替代品,适用于素食主义者。WKG与MKG所含微生物在种属和生化特性上的重要区别是,其所含酵母主要是酵母属(Saccharomyces)和布鲁塞尔德克酵母(Dekkera bruxellensis),所含乳酸菌的乳糖代谢活力较弱[22-23]。WKG主要代谢还原糖,以红糖或富含还原糖的水果为底物,形成乳酸、乙酸、乙醇、二氧化碳、甘露醇、甘油和一系列风味成分[24],其中某些不同于MK的风味化合物如甲酯等来源于水果等底物[25]。
在WK的传统发酵方式中,红糖溶于水后巴氏消毒,接种WKG后于25 ℃发酵24~72 h[26-29],筛分得到的发酵液即为WK,WKG则干燥低温保存,用于下一次发酵[30]。与MK类似,WK也可采用“后倒法”发酵,并且后倒次数越多,发酵产物中所含乙酸含量也越高。 采用WKG发酵各种植物原料可丰富WK产品种类。BUENO R S等[31]用WKG发酵火龙果/苹果,发酵液中优势酵母菌和细菌分别为布鲁塞尔酒香酵母(Brettanomyces bruxellensis)和红条液体乳杆菌(Liquorilactobacillus satsumensis),所制得的开菲尔具有适宜的酸度(总酸较发酵前分别提高1.8~2.7倍)和显著抗氧化活性。 MENDES R M L等[32]用WKG发酵百香果,制成了滋味良好的益生菌活菌饮料,在体外模拟胃肠道条件下,酵母和乳酸菌的活菌率均超过70%。GÜZEL-SEYDIM Z等[33]用WKG发酵橘子汁和柿子汁,所获得WK中乳酸菌和酵母密度分别在108 CFU/mL和106 CFU/mL以上,并且具有良好的感官接受度。LIMBAD M等[34-35]以椰子为主要原料,WKG自然发酵所制成的WK富含乳酸菌和醋酸菌,密度均在108 CFU/mL以上,适用于乳糖不耐症患者、过敏患者和素食主义者。 SANTOS S A等[36]利用WKG发酵全葡萄汁,在室温静态条件下仅发酵24 h,就将全葡萄汁成功转化成低热量、甜酸比适口、富含以乳酸菌为主的益生菌饮品。其他如橙浆[37]、可可浆[38]、番茄和胡萝卜汁[39]、菠萝汁[40]等均成功地用WKG进行了发酵。目前尚无采用纯菌种复合发酵方式进行WK发酵的报道,但ARRIETA-ECHEVERRI M C等[41]基于对WK发酵体系的多组学分析,建立了涵盖90个菌株的培养集合,主要包括膜醭毕赤酵母(Pichia membranifaciens)、东方醋酸杆菌(Acetobacter orientalis)、希氏乳杆菌(Lentilactobacillus hilgardii)、 副干酪乳杆菌(Lacticaseibacillus paracasei)、果实醋杆菌(Acetobacter pomorum)等,膜醭毕赤酵母(P.membranifaciens)是酵母中的优势菌种,乳杆菌属(Lactobacillus)和醋杆菌属(Acetobacter)是乳酸菌和醋酸菌中的优势种属,且WK的发酵原理与WK类似,根据前述MK的发酵经验,采用纯菌种复合发酵方式实现WK规模化生产可行。
当肠道菌群改变而导致微生态失衡时,会引发便秘、腹泻、应激性肠炎等问题,如果肠道微生态失衡持久不能得到纠正,还会诱发癌症,益生菌是优化肠道菌群组成、纠正微生态失衡的有效手段[42],开菲尔因其富含益生菌及其发酵产物而在预防与辅助治疗肠道功能紊乱方面发挥重要作用。
WANG M C等[43]研究发现,连续3周摄食冻干MK粉(剂量为10 log CFU/小袋), 男性受试者的肠道微生物群分布曲线发生变化,双歧杆菌丰度明显增加,腹痛、腹胀症状减轻,女性受试者胃肠道中总厌氧菌丰度和总细菌丰度明显上升,因此,MK粉能够优化肠道微生态并减轻不适症状。HAMET M F等[44]研究表明,BALB/c小鼠灌胃口服MKG粉后,肠道中乳杆菌数量不变,但双歧杆菌占比上升,显示肠道菌群组成得到了优化。 BOLLA P A等[45]研究表明,来自MK的乳酸菌与酵母组合可以防止福氏志贺氏菌侵入人类肠上皮细胞,具有抗炎作用。 YE Z M等[46]研究表明,WK可有效改善右旋糖酐硫酸钠(dextransulfatesodium,DSS)所致炎症性肠病(inflammatory bowel disease,IBD)小鼠的病症,抑制促炎和抗炎细胞因子的异常表达,灭活DSS诱导的TLR4-MyD88-NF-κB通路。 CHEN Y P等[47]研究表明,给予DSS诱导的结膜炎模型小鼠从MKG中分离出的马乳酒样乳杆菌(Lactobacillus kefiranofaciens)M1后,促炎细胞因子产生减少,抗炎细胞因子IL-10产生增加,表明该菌株能够通过调理免疫而发挥抗结肠炎作用。TURAN I˙等[48]研究表明,符合罗马II标准(Rome II criteria)的慢性便秘患者,摄入高剂量开菲尔可显著减少通便药使用、增加排便频率、提高肠道感受满意度。
综上,开菲尔中的益生菌可以在肠道内驻留,通过提高肠道内有益菌占比、拮抗致病菌、调理免疫功能及相关信号通路等途径,辅助防治肠道功能紊乱、有效改善便秘状况,摄入开菲尔有望发展为肠道生态失调的辅助替代疗法。
肠道微生态失调导致病原菌感染、肠道组织通透性增加而消化吸收不良且毒素入血,是诱发肝脏疾病的重要因素,而益生菌通过调理肠道微生态而有利于肝脏健康[49]。
CUI Y Y等[50]构建了肠道上皮屏障和肠道内稳态破坏而导致肝脏炎症和氧化应激并且胆汁酸(bile acids,BA)代谢失调的酒精性肝病(alcoholic liver disease,ALD)小鼠模型,给予口服开菲尔后发现,肝脏病理改善,丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)及炎症标志物表达水平降低,并且肠道屏障修复,肠道微生态中蓝杆菌属(Blautia)、拟杆菌属(Bacteroides)和副萨特氏菌属(Parasutterella)增加,厚壁菌门/拟杆菌门(Firmicutes/Bacteroidetes,F/B)降低,嗜冷杆菌属(Psychrobacter)、芽孢杆菌属(Bacillus)和单球菌属(Monoglobus)数量减少,总胆汁酸和初级胆汁酸水平降低,次级/初级胆汁酸比例上升,显示开非尔可通过改善肠道屏障、调节肠道生态失调和微生物介导的胆汁酸代谢、降低炎症水平和氧化应激而缓解ALD。 DOS SANTOS F R等[51]构建营养不良小鼠模型,并形成肝功能损伤,给予口服开菲尔4周后,小鼠体质量和生化参数有所改善,与氧化应激抑制相关的基因如超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)的表达水平上升,表明开菲尔可作为膳食补充剂以改善营养状况和修复肝功能损伤。ALIGITA W等[52]研究表明,水开菲尔成分,如富马酸可作用于NF-κB和Nrf2信号通路,这一点被分子对接结果所证实,给予口服开菲尔显著改善了CCl4所致急性肝损伤的大鼠肝损伤状况,血清AST、ALT、肿瘤坏死因子-α(tumor necrosis factor,TNF-α)、转化生长因子-I3(transforming growth factor-I3,TGF-I3)水平降低,肝组织形态改善,表明水开菲尔可通过作用于NF-NF-κB和Nrf2信号通路,降低肝脏组织坏死和纤维化水平而提升肝脏健康。
综上,开菲尔通过优化肠道菌群组成、改善营养状况、调节相关信号通路等方式发挥保肝护肝功效。
肠道微生物通过调节能量吸收、肠道蠕动、食欲、糖脂代谢和肝脏脂肪储存以影响宿主代谢,肠道微生态失调可导致全身性炎症和胰岛素抵抗,进而发生代谢综合征(metabolic syndrome,MS),临床表现为中枢性和腹部肥胖、胰岛素抵抗而致II型糖尿病(T2DM)、动脉粥样硬化性血脂异常、高血压、炎症细胞因子活性升高等,治疗手段包括饮食控制、有效运动和针对“三高”(血脂、血压和血糖)的药物治疗[53-55]。
EBNER J等[56]研究表明,MK的肽谱与生乳完全不同,其发酵过程中产生236种独特肽,大部分具有抗高血压(血管紧张素转换酶抑制剂(angiotensin-converting enzyme inhibitors,ACEI))、抗菌、免疫调节、抗氧化和抗血栓特性。WANG B等[57]采用液相色谱-串联质谱(liquid chromatography tandem mass spectrometry,LC-MS/MS)技术,从MK中鉴定出14个在天然乳中并不存在的具有ACE抑制活性的含脯氨酸肽,并且证实MK的ACE抑制活性经体外模拟胃肠道消化后仍保留63%。
高胆固醇血症引发心血管疾病。开菲尔粉可减少高脂饮食肥胖小鼠脂肪组织和脂肪肝风险,减轻体质量,降低总胆固醇(total cholesterol,TC)、低密度脂蛋白(low-density lipoprotein,LDL)和血清甘油三酯(triglyceride,TG)水平[58-59]。开菲尔降低胆固醇水平的机制与其中的功能性乳酸菌有关,其中嗜酸乳杆菌(L.acidophilus)通过同化作用将胆固醇摄入细胞内降解,而干酪乳杆菌(L.casei)在破坏胆固醇胶束稳定后,再将其与去共轭胆盐共沉淀而从体内排出[60-61]。 KIM D H等[62]从MKG和WKG分离出13株乳酸菌,从中优选出可抑制肥胖和非酒精性脂肪性肝病发展的Lactobacillus kefiri DH5,其作用机制是通过直接代谢消化道内胆固醇和上调脂肪组织ppar-α基因而发挥抗肥胖作用。
T2DM与代谢综合征密切相关,开菲尔对T2DM也显示出辅助防治功效。HOSAINZADEGN H等[63]研究表明,II型糖尿病患者摄入自制MK的3个月后,糖化血红蛋白(hemoglobinA1c,HbA1c)水平明显改善,体质量明显减轻。TALIB N等[64]从马来西亚WKG中分离得到的副干酪乳杆菌(Lactobacillus paracasei)具有优异的益生菌特性和较高的抗氧化活性,其可改变T2DM小鼠的葡萄糖稳态和脂质代谢相关基因表达水平,减轻T2DM病症。SALARI A等[65]研究表明,食用MK和WK的志愿者空腹血糖和胰岛素水平有显著改善。
综上,开菲尔通过提供具有广泛活性的肽、降血压、促进胆固醇代谢、改善心血管功能等机制缓解MS,并对T2DM也具有基于消减胰岛素抵抗的辅助防治功效。
KG分为MKG和WKG两大类,既可通过自然发酵的“后倒法”分别制得MK和WK,也可通过纯菌种复合发酵方式进行开菲尔的规模化生产。 KG及其发酵产物开菲尔能够充分展现益生菌的功能特性,在肠道功能紊乱、保肝护肝、代谢综合征及II型糖尿病等方面的疾病预防与辅助治疗中发挥积极作用,其作为发掘益生菌新菌株的重要源泉和益生菌发酵食品的理想载体,将在健康领域得到更加广泛深入的应用。
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Research progress on fermentation of kefir and its application in disease prevention and adjuvant therapy