CN112088211A - 乳酸菌及其用途 - Google Patents
乳酸菌及其用途 Download PDFInfo
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- CN112088211A CN112088211A CN201980030116.0A CN201980030116A CN112088211A CN 112088211 A CN112088211 A CN 112088211A CN 201980030116 A CN201980030116 A CN 201980030116A CN 112088211 A CN112088211 A CN 112088211A
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- lactic acid
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Abstract
本发明的技术问题在于提供一种新型乳酸菌或其处理物。此外,本发明的技术问题还在于提供该乳酸菌或其处理物的增殖方法或其用途。本发明提供乳酸菌耐久肠球菌(E.durans)HS‑08菌株的菌体或其处理物。此外,本发明可起到促进产生IgA的作用、免疫刺激、抗过敏作用、抑制有害菌和/或病原菌增殖的作用、保护粘膜的作用、增强产生短链脂肪酸的作用、增强产生有机酸的作用、及上调GRP43基因表达的作用、调节植物生长的作用、防止植物倒伏的作用、调节植物鲜味的作用、促进动物成长的作用、或提高动物自身免疫的作用。
Description
技术领域
本发明涉及一种新型乳酸菌或其处理物、该乳酸菌或其处理物的增殖方法、及其用途。
背景技术
乳酸菌广泛用于酸奶或腌菜等发酵食品,是一种非常熟悉且安全的菌种。近年来,一些乳酸菌作为益生菌即对人体健康具有积极作用的微生物而受到瞩目,对于这些乳酸菌的各种功效,一直在进行研究。
例如,作为乳酸菌所具备的功能之一,已报道了具有诱导IgA产生的作用的乳酸菌(专利文献1、2)。此外,还已知乳酸菌体或其处理物具有诱导IgA产生的作用,且对于乳酸菌体及其处理物,可期待预防感染、抑制肿瘤、抑制过敏及肠道调节作用等效果,乳酸菌体及其处理物可用于免疫刺激剂或肠道调节剂等(专利文献3)。然而另一方面,众所周知,虽为同一种菌种但菌株不同时,乳酸菌体及其处理物所带来的效果有时会有很大差异。因此,菌株的选择非常重要,一直在尝试分离出功能更强的乳酸菌株。
现有技术文献
专利文献
专利文献1:日本特开2004-305128号公报
专利文献2:日本特开2010-130954号公报
专利文献3:日本特开2008-201708号公报
发明内容
本发明要解决的技术问题
本发明的技术问题在于提供一种新型且优异的乳酸菌或其处理物。此外,本发明的技术问题还在于提供该乳酸菌或其处理物的制备方法或其应用。
解决技术问题的技术手段
本申请的发明人为了解决上述问题反复进行了认真研究,结果成功采集了乳酸菌耐久肠球菌(以下,也称为“E.durans”)HS-08菌株(保藏编号NITE BP-02675),并发现了其多种优异的用途,进而基于该发现进行了更深入的研究,从而完成了本发明。
即,本发明包含以下各发明,以解决上述技术问题。
[1]一种耐久肠球菌HS-08(保藏编号NITE BP-02675)的菌体或其处理物。
[2]一种诱导IgA产生用或免疫刺激用组合物,其含有[1]所述的菌体或其处理物。
[3]一种抗过敏用组合物,其含有[1]所述的菌体或其处理物。
[4]一种抑制有害菌和/或病原菌增殖用组合物,其含有[1]所述的菌体或其处理物。
[5]一种保护粘膜用组合物,其含有[1]所述的菌体或其处理物。
[6]一种组合物,其特征在于,含有[1]所述的菌体或其处理物,且用于选自由增强产生短链脂肪酸、增强产生有机酸、及上调GRP43基因表达组成的组中的至少一种用途。
[7]一种组合物,其特征在于,含有[1]所述的菌体或其处理物,且用于选自由调节植物生长、防止植物倒伏、及调节植物鲜味(umami)组成的组中的至少一种用途。
[8]一种组合物,其特征在于,含有[1]所述的菌体或其处理物,且用于促进动物成长和/或提高动物自身免疫的用途。
[9]一种植物用肥料,其特征在于,含有[1]所述的菌体或其处理物、或[7]所述的组合物。
[10]一种动物用饲料,其特征在于,含有[1]所述的菌体或其处理物、或[8]所述的组合物。
[11]一种饮食品、化妆品或药物,其特征在于,含有[1]所述的菌体或其处理物、或[2]~[8]中任一所述的组合物。
[12]一种混凝土构件,其特征在于,含有[1]所述的菌体或其处理物、或[2]~[8]中任一所述的组合物。
[13]一种组合物,其含有[1]所述的菌体或其处理物、以及选自由包括其他乳酸菌、双歧杆菌、或酵母菌的有益菌、及霉菌组成的组中的至少一种或它们的处理物。
[14]一种耐久肠球菌HS-08菌体的增殖方法,其特征在于,培养耐久肠球菌HS-08(保藏编号NITE BP-02675)的菌体。
发明效果
本发明提供一种乳酸菌耐久肠球菌(Enterococcus durans;以下也称为“E.durans”)HS-08菌株(保藏编号NITE BP-02675)或其处理物。
本发明的乳酸菌可促进下述作用:显著诱导作为一种免疫抗体的IgA产生的作用、及由此带来的生物体内的免疫刺激作用。
此外,本发明的乳酸菌表现出抗过敏作用,更令人惊讶的是表现出抑制有害菌和/或病原菌增殖的作用、保护粘膜的作用、增强产生短链脂肪酸的作用、增强产生有机酸的作用、上调GRP43基因表达的作用、调节植物生长、抗倒伏或调节鲜味的作用、或促进动物成长或提高自身免疫的作用。
附图说明
图1为示出小鼠粪便中的IgA量(μg/day)的图表。
图2为示出小鼠血液中的IgA浓度(μg/ml)的图表。
图3为示出小鼠体重(g)的图表。
图4为示出小鼠血液中的CRE浓度(μg/ml)的图表。
图5为示出小鼠血液中的ALT浓度(iU/L)的图表。
图6为示出小鼠血液中的AST浓度(iU/L)的图表。
图7为示出参与IgA产生的各种基因的表达量的图表。
图8为示出小鼠粪便中的有机酸及短链脂肪酸浓度(μmol/g)的图表。
图9为示出小鼠粪便中的pH的图表。
图10为示出GRP43基因的表达量的图表。
具体实施方式
E.durans HS-08菌株(保藏编号NITE BP-02675)已作为依照布达佩斯条约的国际保藏而在2018年4月10日被独立行政法人制品评价技术基盘机构生物技术中心专利微生物保藏中心(NPMD)(地址:邮政编码292-0818日本千叶县木更津市上总镰足2-5-8)接受。
该菌可通过向上述保藏中心进行申请而获得,但其也可以为下述菌:例如将形态特征(例如,菌落的形状、细胞的形状等)、生理、生化特性(例如,糖的同化性、生长温度、最适Ph值等)、化学分类学的特性(菌体脂肪酸组成等)等特性与E.durans HS-08菌株(保藏编号NITE BP-02675)进行比较,根据该特性的比较结果而确定的菌,也可以为根据16S rRNA基因的碱基序列的分析而确定的菌。
E.durans HS-08菌株优选通过例如下述方法进行培养。
(i)培养液(培养基):包括MRS(de Man,Rogosa and Sharpe)培养基、MRS琼脂培养基、LBS培养基、改性LBS琼脂培养基、Rogosa培养基、其他公知的乳酸菌用培养基或培养液等。(ⅱ)pH值为4~9左右,优选为5~8左右、进一步优选为5.5~7.5左右。(ⅲ)培养方法可以为静置培养,也可以为搅拌培养。搅拌培养时的搅拌次数可以为每1分钟100~250次左右,优选为每1分钟120~180次左右,进一步优选为每1分钟140~160次左右。(ⅳ)培养时间通常为1~72小时左右,优选为3~48小时左右,进一步优选为6~24小时左右。(ⅴ)培养温度通常为20~40℃左右,优选为25~39℃左右、进一步优选为30~38℃左右。(ⅵ)其他,MRS培养基、MRS琼脂培养基、LBS培养基、改性LBS琼脂培养基、Rogosa培养基、其他公知的乳酸菌用培养基或培养液中,也可以含有其他添加剂。
作为这些培养基中所含有的成分或添加剂,并不仅限于上述,可列举出来源于酵母的成分,来源于大豆的成分,来源于玉米的成分,动植物蛋白质或其提取物以及分解产物;选自由硝酸铵、硫酸铵、氯化铵、乙酸铵等铵盐,氨、硝酸钠、硝酸钾、谷氨酸钠、尿素、氨基酸、谷蛋白、酪蛋白、蛋白胨等组成的组中的一种以上的氮源;选自由葡萄糖、肌醇、麦芽糖、木糖、甘露糖、果糖、淀粉、乳糖、甘油(glycerol)、阿拉伯糖、核糖、半乳糖、果糖、肌醇、甘露醇、山梨糖醇、葡萄糖胺、纤维二糖、蔗糖、海藻糖、木糖醇、醇、淀粉糖浆、淀粉、糖蜜、甘油(glycerin)、有机酸、有机酸盐、及烃组成的组中的一种以上的碳源。此外,还可列举出维生素A、B、C、D、E、K等维生素类或其衍生物、或它们的盐类、锌、铁、镁、钾、钙、磷等矿物质、pH缓冲剂、表面活性剂、抗生素、稳定剂等,但并不仅限于此。作为其他成分或添加剂,可列举出蔗糖、碳酸盐、碳酸氢盐、HEPES、白蛋白、胰岛素、氨基酸、细胞因子、生长因子、激素等,但并不仅限于此。添加剂可以单独使用一种,也可以组合使用两种以上。另外,对于培养时的厌氧或有氧条件,可依照本领域公知的方法。
本发明的乳酸菌可以通过冷冻干燥、低温干燥、喷雾干燥或L-干燥等或这些干燥方式的组合而被制成粉末状,从而进行使用,此外,也可以直接以通过上述方法而制得后的状态(糊状、液状)等进行使用。将乳酸菌制成粉末状进行使用时,活菌数优选为107~108cfu/g,更优选为109~1012cfu/g,但并不仅限于此。将乳酸菌制成液状进行使用时,活菌数优选为107~108cfu/mL,更优选为109~1012cfu/mL,但并不仅限于此。活菌数的测定因菌体的不同而不同,但可依据例如日本药局方外医药品规格中所记载的各种菌体的定量方法而容易地测定。
本发明的乳酸菌的“处理物”或其他乳酸菌的“处理物”优选为对该乳酸菌进行加工后的物质或该乳酸菌的培养物,但并不仅限于此。此外,菌可以是活菌,也可以是死菌。在本发明中,可以直接使用这些处理物,也可以通过冷冻干燥、低温干燥、喷雾干燥或L-干燥等或这些干燥方式的组合而将这些处理物制成粉末状而进行使用。此外,这些处理物(培养物)可以用适宜的溶剂(水、醇、有机溶剂等)稀释而进行使用,也可以添加适宜的添加剂并制成凝胶或固体剂而进行使用。
本发明的乳酸菌或其处理物可以与除其以外的有益菌(例如属于乳酸杆菌属、明串珠菌属、链球菌属的乳酸菌;双歧杆菌;酵母菌或霉菌等)或其处理物同时使用。作为其他乳酸菌,例如可列举出嗜酸乳杆菌(Lactobacillus acidophillus)、德氏乳杆菌(L.delbrueckii)、干酪乳杆菌(L.casei)、赖氏乳杆菌(L.leichmannii)、食果糖乳杆菌(L.fructivorans)、瑞士乳杆菌(L.helveticus)、希氏乳杆菌(L.hilgardii)、高加索酸奶乳杆菌(L.kefiri)、马乳酒样乳杆菌(L.kefiranofaciens)、高加索酸奶粒乳杆菌(L.kefirgranum)、类高加索酸奶乳杆菌(L.parakefir)、戊糖醋酸乳杆菌(L.pentoaceticus)、caret乳杆菌(L.caret)、保加利亚乳杆菌(L.bulgaricus)、乳酸乳杆菌(L.lactis)、植物乳杆菌(L.plantarum)、类干酪乳杆菌(L.paracasei)、纤维二糖乳杆菌(L.cellobiosus)、融合乳杆菌(L.confusus)、嗜粪乳杆菌(L.coprophilus)、发酵乳杆菌(L.fermentum)、旧金山乳杆菌(L.sanfrancisco)、嗜热乳杆菌(L.thermophilus)、巴伐利亚乳杆菌(L.bavaricus)、短乳杆菌(L.brevis)、布氏乳杆菌(L.buchneri)、棒状乳杆菌(L.coryniformis)、弯曲乳杆菌(L.curvatus)、清酒乳杆菌(L.sake)等乳杆菌属;乳脂明串珠菌(Leuconostoc cremoris)、肠膜明串珠菌(Leuc.mesenteroides)等明串珠菌属,乳链球菌(Streptococcus lactis)、唾液链球菌(Streptococcus salivarius)、嗜热链球菌(S.thermophilis)等链球菌属等,但并不仅限于此。这些有益菌可以通过现有公知的方法培养获得,此外,还可以通过例如ATCC(注册商标)或IFO等机构或财团法人、日本双歧杆菌中心、独立行政法人制品评价技术基盘机构专利微生物保藏中心等而容易地获得。此外,也可以适当使用市售产品。
对于能够与本发明的乳酸菌或其处理物同时使用的双歧杆菌的实例,例如可列举出两歧双歧杆菌(Bifidobacterium bifidum)、角双歧杆菌(B.angulatum)、青春双歧杆菌(B.adolescentis)、短双歧杆菌(B.breve)、链状双歧杆菌(B.catenulatum)、齿双歧杆菌(B.dentium)、高卢双歧杆菌(B.gallicum)、纤细双歧杆菌(B.subtile)、星状双歧杆菌(B.asteroides)、牛双歧杆菌(B.boum)、豚双歧杆菌(B.choerinum)、棒状双歧杆菌(B.coryneforme)、串孔双歧杆菌(B.cuniculi)、鸡胚双歧杆菌(B.gallinarum)、印度双歧杆菌(B.indicum)、大双歧杆菌(B.magnum)、瘤胃双歧杆菌(B.merycicum)、最小双歧杆菌(B.minimum)、嗜冷双歧杆菌(B.psychraerophilum)、小鸡双歧杆菌(B.pullorum)、反刍双歧杆菌(B.ruminantium)、史卡杜维双歧杆菌(B.scardovii)、嗜热双歧杆菌(B.thermophilum)、婴儿双歧杆菌(B.infantis)、摩恩格里艾恩斯双歧杆菌(B.mongoliense)、假链状双歧杆菌(B.pseudocatenulatum)、嗜热嗜酸双歧杆菌猪亚种(B.thermacidophilum subsp.porcinum)、动物双歧杆菌乳亚种(B.animalissubsp.lactis)、动物双歧杆菌动物亚种(B.animalis subsp.animalis)、长双歧杆菌长亚种(B.longum subsp.longum)、假长双歧杆菌假长亚种(B.pseudolongumsubsp.pseudolongum)、假长双歧杆菌球形亚种(B.pseudolongum subsp.globosum)等双歧杆菌属或其亚种等,但并不仅限于此。这些双歧杆菌可以通过现有公知的方法培养获得,此外,还可以通过例如与上述乳酸菌相同的机构或中心等而容易地获得。此外,也可以适当使用市售产品。
对于能够与本发明的乳酸菌或其处理物同时使用的酵母菌的实例,可列举出酿酒酵母(Saccharomyces cerevisiae(也以出芽酵母、上面发酵酵母而为人所知))、开菲尔酵母(S.kefir)、乳酸酵母(S.lactis)、嘉士伯酵母(S.carlsbergensis(也以下面发酵酵母而为人所知))、单孢酵母(S.unisporus)、巴氏酵母(S.pastrianus)、贝酵母(S.bayanus)、嘉士伯酵母(S.carlsbergensis)、德氏酵母(S.delbrueckii)、大连酵母(S.dairensis)、糖化酵母(S.diataticus)、少孢酵母(S.exiguus)、克鲁弗酵母(S.kluyveri)、鲁氏酵母(S.rouxii)、葡萄汁酵母(S.uvarum)、罗斯酵母(S.rosei)等酵母属;粟酒裂殖酵母(Schizosaccharomyces pombe(也以裂变酵母而为人所知))、鲁氏裂殖酵母(Schizosaccharomyces rouxii)等裂殖酵母属;产朊假丝酵母(Candida utilis(也以圆酵母而为人所知))、热带假丝酵母(C.tropicalis)、梅林假丝酵母(C.milleri)、克鲁斯假丝酵母(C.krusei)、葡萄牙假丝酵母(C.lusitaniae)、阿赛假丝酵母(C.aaseri)、季也蒙假丝酵母(C.guilliermondii)、土生假丝酵母(C.humicola)、光滑假丝酵母(C.glabrata)、郎比可假丝酵母(C.lambica)、解脂假丝酵母(C.lipolytica)、近平滑假丝酵母(C.parapsilosis)、热带假丝酵母(C.tropicalis)、副热带假丝酵母(C.paratropicalis)、拟热带假丝酵母(C.pseudotropicalis)、褶皱假丝酵母(C.rugosa)、类星形假丝酵母(C.stellatoidea)、涎沫假丝酵母(C.zeylanoides)等假丝酵母属;德尔布有孢圆酵母(Torulaspora delbrueckii)等有孢圆酵母属;celluculosa球拟酵母(Torulopsiscelluculosa)、白色球拟酵母(Torulopsis candida)等球拟酵母属;开菲尔圆酵母(Torulakefir)等圆酵母属;保加利亚克鲁维酵母(Kluyveromyces bulgaricus)、脆壁克鲁维酵母(K.fragilis)、耐热克鲁维酵母(K.thermotolerans)、乳酸克鲁维酵母(K.lactis)、马克斯克鲁维酵母(K.marxianus)、脆壁克鲁维酵母(K.fragilis)等克鲁维酵母属;膜醭毕赤酵母(P.membranaefaciens)、树干毕赤酵母(P.stipitis)、异常毕赤酵母(P.anomala)、斋藤毕赤酵母(P.saitoi)等毕赤酵母属;异常汉逊酵母(Hansenula anomala)等汉逊酵母(Hansenula)属;汉逊德巴利酵母(Debariomyces hansenii)等德巴利酵母属等,但并不仅限于此。这些酵母菌可以通过现有公知的方法培养获得,此外,还可以通过例如与上述乳酸菌相同的机构或中心等而容易地获得。此外,也可以适当使用市售产品。
对于能够与本发明的乳酸菌或其处理物同时使用的其他有益菌的实例,例如可列举出包括醋化醋杆菌(Acetobacter aceti)、东方醋杆菌(Acetobacter orientalis)、巴氏醋杆菌(Acetobacter pasteurianus)、木醋杆菌(Acetobacter xylinum)等醋杆菌属的醋酸菌;包括丁酸梭菌(Clostridium butyricum)等的丁酸菌、有害片球菌(Pediococcusdamnosus)、戊糖片球菌(Pediococcus pentosaceus)、嗜盐片球菌(Pediococcushalophilus)等片球菌属,丝状真菌(曲霉菌)、纳豆枯草芽孢杆菌(Bacillus subtilisnatto)等枯草芽孢杆菌(Bacillus subtilis)、放线菌、光合成细菌、氨基酸生产菌、纤维素分解菌(例如梭菌、木霉菌等)、根瘤菌、菌根菌、VA菌、硝化菌等,但并不仅限于此。这些酵母菌可以通过现有公知的方法培养获得,此外,还可以通过例如与上述乳酸菌相同的机构或中心等而容易地获得。此外,也可以适当使用市售产品。
对于能够与本发明的乳酸菌或其处理物同时使用的霉菌的实例,可列举出米曲霉(Aspergillus oryzae)、酱油曲霉(Aspergillus sojae)、毛霉曲霉(Aspergillus Mucor)、黑曲霉(Aspergillus niger)、构巢曲霉(Aspergillus nidulans)等曲霉属;娄地青霉(Penicillium roqueforti)、根霉青霉(Penicilliun Rhizopus)等青霉属等,但并不仅限于此。这些霉菌可以通过现有公知的方法培养获得,此外,还可以通过例如与上述乳酸菌相同的机构或中心等而容易地获得。此外,也可以适当使用市售产品。
本发明的乳酸菌或其处理物、或含有它们的组合物可以直接播散在植物(叶、茎、果实等)上或播散(给予)在土壤中。或者也可以混入植物的肥料中,在旱田或稻田的情况下,可以设置在取水口处等,也可以作为取水口的混凝土原料或混凝土结构物的材料而混入。此外,当本发明的乳酸菌或其处理物、或含有它们的组合物适用于畜牧领域时,可以混合在动物的饲料或饮用水中而给与,也可以作为牛舍或鸡舍等的墙壁或地板等的混凝土原料或混凝土结构物的材料等而混入。
本发明提供一种含有本发明的乳酸菌的组合物。本发明的组合物中可以使用本领域通常使用的公知的添加剂,例如可列举出水、溶剂、pH调节剂、保湿剂、着香剂、甜味剂、增稠剂、调味剂、胶凝剂、溶解剂、着色剂、防腐剂、表面活性剂、悬浮剂、乳化剂及稳定剂等,但并不仅限于此。
例如,作为可以在本发明中使用的pH调节剂,只要为可将本发明的组合物的pH值调节至所需的值的调节剂即可,可用于调节至pH约2.5~10.0、更优选调节至pH约5.0~8.5的范围。作为优选的pH调节剂,可列举出碱金属氢氧化物、碱土金属氢氧化物、碱土金属氧化物、碱金属氧化物、碳酸盐、硼酸盐、硅酸盐、磷酸盐、有机酸及有机碱等。具体而言,可列举出磷酸或其盐、安息香酸或其盐、水杨酸或其盐、氢氧化钠、氢氧化钾、碳酸钠、碳酸钾、碳酸镁等碱金属碳酸盐;氢氧化镁、氢氧化钙碱土金属碳酸盐、碳酸氢钾、碳酸氢钠等碱金属碳酸氢盐、碳酸氢镁、碳酸氢钙等碱土金属碳酸氢盐、硼酸、柠檬酸、富马酸盐、苹果酸、酒石酸、琥珀酸、马来酸或它们的盐、乳酸、咪唑、三乙醇胺、二乙醇胺、氨丁三醇、葡甲胺、利多卡因及它们的盐等,但并不仅限于此。
作为可在本发明中使用的优选的保湿剂,例如可列举出山梨糖醇、木糖醇、甘油、丁二醇、聚乙二醇及丙二醇等多元醇,但并不仅限于此。
作为可在本发明中使用的着香剂,例如可列举出椒样薄荷油、留兰香油、薄荷油、薄荷脑、茴香脑、鼠尾草、柠檬油、橙油、肉桂、香兰素、百里酚、芳樟醇等,但并不仅限于此。
作为可在本发明中使用的甜味剂,例如可列举出蔗糖、葡萄糖、糖精、甘草酸、葡萄糖、果糖、乳糖、甘露醇、山梨糖醇、果糖、麦芽糖、木糖醇、蜂蜜、淀粉糖浆、糖精、阿斯巴甜、D-色氨酸、安赛蜜、环拉酸及它们的盐等,但并不仅限于此。
作为可在本发明中使用的调味剂,例如可列举出抗坏血酸、柠檬酸、甘草酸、谷氨酸、琥珀酸、酒石酸、富马酸、苹果酸、牛磺酸、肌氨酸、甘草酸或它们的盐、或者赤藓糖醇、乳糖醇、还原帕拉金糖、氯化钠、氯化镁、橙油、藏红花油、山椒油、紫苏油、罗勒油、薄荷油、柠檬油、柠檬草油、玫瑰油、迷迭香油、可可、焦糖、甘草、樟脑、肉桂油、藏红花、水杨酸甲酯、芍药提取物、生姜、肉桂醛、甜叶菊提取物、日本当药、山梨醇、环糊精、大豆油、大枣提取物、牛磺酸、单宁酸、丁香油、本岛云杉提取物、苦木提取物、梅肉提取物、蜂蜜、薄荷水、薄荷油、薄荷脑、聚维酮、冰片、桉树油、柠檬油、玫瑰油等,但并不仅限于此。
可在本发明中使用的表面活性剂可以为非离子型、两性离子型、阴离子型或阳离子型。作为非离子表面活性剂,可以使用任意的非离子表面活性剂,例如可列举出聚山梨醇酯20、聚山梨醇酯40、聚山梨醇酯60、聚山梨醇酯80等聚氧乙烯山梨糖醇酐脂肪酸酯;聚氧乙烯10氢化蓖麻油、聚氧乙烯40氢化蓖麻油、聚氧乙烯50氢化蓖麻油、聚氧乙烯60氢化蓖麻油等聚氧乙烯氢化蓖麻油;聚氧乙烯聚氧丙二醇醚,聚乙二醇单月桂酸酯、乙二醇单硬脂酸酯、聚氧乙烯40硬脂酸酯等聚乙二醇单硬脂酸酯;聚乙二醇单油酸酯、乙二醇单硬脂酸酯等聚乙二醇脂肪酸酯等,但并不仅限于此。作为阴离子表面活性剂,例如可列举出烷基苯磺酸钠、十二烷基苯磺酸铵等,但并不仅限于此。作为两性离子表面活性剂,例如可列举出含有羧基、磺酸基、硫酸酯基的仲胺或叔胺的脂肪族衍生物、或杂环仲胺或叔胺的脂肪族衍生物,例如(C8-20烷基)甜菜碱及(C8-20烷基)酰胺(C6-8烷基)甜菜碱、及它们的混合物等,但并不仅限于此。作为阳离子表面活性剂,可以列举出季铵盐、胺盐或胺等,但并不仅限于此。
此外,作为可在本发明中使用的乳化剂及悬浮剂,可以适宜地使用上述的表面活性剂,其他,还可以使用大豆卵磷脂、蛋黄卵磷脂、氢化卵磷脂、酶解卵磷脂等卵磷脂类或鲸蜡醇、月桂醇、硬脂醇及羊毛脂醇等高级醇等,但并不仅限于此。
作为可在本发明中使用的稳定剂,可列举出聚丙烯酸钠、己二酸、抗坏血酸、亚硫酸钠、亚硫酸氢钠、二丁基羟基甲苯、丁基羟基苯甲醚、乙二胺四乙酸钠、氯化钠、柠檬酸、环糊精、半胱氨酸等,但并不仅限于此。
本发明的乳酸菌或其处理物、或含有它们的组合物,可给予至包括人的动物。本发明的乳酸菌或其处理物、或含有它们的组合物可以直接作为饲料(农业物料)、药物、食品而给予,或者也可以通过与饲料(农业物资)、药物、食品的原料混合而给予,也可以自身直接用作药物、化妆品等而涂布,或者也可以作为药物、化妆品等的原料而涂布。
此外,给予本发明组合物的动物没有特殊限定,例如可以是人、或除人以外的动物等。作为除人以外的动物,没有特殊限定,例如可以列举出牛、马、猪、羊等家畜类;狗、猫、兔子、仓鼠、松鼠、豚鼠、鼠等宠物或实验动物;沙丁鱼、金枪鱼、鲨鱼、翻车鱼、鳐鱼、鲹、远东哲罗鱼、鰤鱼、香鱼、鲤鱼、鲷鱼、鲭鱼、牙鲆鱼、鲽、鳗鱼、鯙、鮟鱇、河豚、丝背细鳞鲀、热带鱼等鱼类;虾、蟹、墨鱼、虾夷扇贝、牡蛎、蝾螺等海鲜类;鸡、鹌鹑、火鸡、鸭、家鹅、鹦哥、鹦鹉等鸟类;独角仙、锹甲、菜粉蝶、燕尾蝶、螳螂、蝗虫、铃虫、鼠妇等虫类;壁虎、变色龙、鬣蜥、蜥蜴等爬行类;蝾螈、蛙、山椒鱼等两栖类动物等。其中,特别优选给予至人、牛、猪、鸡、鱼类、虾。
此外,本发明的给予方式没有特别限定,可列举出口服给予、非口服给予(静脉内给予、经皮给予等)。作为本发明的给予剂型,为口服剂时,可列举出片剂、胶囊剂、颗粒剂、散剂等,为非口服剂时,可列举出注射剂、软膏、插入剂(intercalator)等。
此外,本发明的剂型例如为片剂、胶囊剂、颗粒剂、散剂等时,没有特别限定,可以根据需要适当选择并使用下述试剂进行制备:例如,甘露醇、磷酸钙、淀粉、蔗糖、乳糖、葡萄糖等赋形剂;羧甲基纤维素、淀粉、预胶化淀粉、羧甲基纤维素钙、交联羧甲基纤维素钠、交联聚维酮、低取代羟丙基纤维素等崩解剂;羟丙基纤维素,乙基纤维素,阿拉伯胶,淀粉、部分预胶化淀粉,聚乙烯吡咯烷酮,聚乙烯醇等粘合剂;硬脂酸镁、硬脂酸钙、滑石粉、水合二氧化硅、固化油等润滑剂;聚乙烯吡咯烷酮、糖、羟丙基甲基纤维素、羟丙基纤维素、甲基纤维素、乙基纤维素等包衣剂等。
静脉内给予本发明的制剂时,例如当剂型为注射剂时,可以根据需要适当选择并使用下述试剂进行制备:例如,氯化钠等的等渗调节剂;磷酸钠等缓冲剂;聚氧乙烯脱水山梨醇单油酸酯等表面活性剂;甲基纤维素等增稠剂等,但并不仅限于此。
眼睛局部给予本发明的制剂时,例如当剂型为滴眼剂等时,可以根据需要适当选择并使用下述试剂进行制备:例如,氯化钠、浓甘油等的等渗调节剂;磷酸钠、醋酸钠等缓冲剂;聚氧乙烯脱水山梨醇单油酸酯、聚氧乙烯(40)单硬脂酸、聚氧乙烯氢化蓖麻油等表面活性剂;柠檬酸钠、依地酸钠等稳定剂;苯扎氯铵,对羟基苯甲酸酯等防腐剂等,但并不仅限于此。其pH值只要在眼科用制剂允许的范围内就没有问题,对于pH调节剂,例如可以使用以下所述的pH调节剂。
本发明的制剂为软膏、透皮剂、贴剂等时,可以使用下述试剂进行制备:例如,凡士林、角鲨烷、石蜡、液体石蜡、月桂酸、肉豆蔻酸、硬脂酸、异硬脂酸、油酸等高级脂肪酸;蜂蜡、羊毛脂等蜡类等油脂类等的常用基质。
本发明的制剂为插入剂时,可以使用下述生物可降解聚合物进行制备:例如,羟丙基纤维素、羟丙基甲基纤维素、羧基乙烯基聚合物、聚丙烯酸等,也可以根据需要适当选择并使用上述稳定剂、pH调节剂等。
本发明的给予量可根据剂型、患者的症状、年龄、体重等适当进行选择,没有特别限定。例如,在口服给予时,每1kg体重每天给予0.05~5000mg、优选给予0.1~2000mg、特别优选给予1~1000mg,且可以每天1次或分多次给予。为注射剂时,每1kg体重每天给予0.0001~2000mg、优选给予0.001~1500mg、特别优选给予0.01~500mg,且可以每天1次或分多次给予。
[饮食品、食品添加物]
本发明提供一种含有本发明的乳酸菌或其处理物、或含有它们的组合物的食品。食品包括健康食品、功能性表示食品、特定保健用食品、患者用食品。食品的形态没有特别限定。作为具体实例,例如可列举出作为所谓的营养辅助食品或补剂的片剂、颗粒剂、散剂、健康饮品等。除此之外,例如可列举出清凉饮料、营养饮料、碳酸饮料、果肉饮料、乳酸饮料、茶饮料等饮料;荞麦面、乌冬面、中华面、方便面等面类;巧克力、零食点心、糖果、口香糖、饼干、果冻、果酱、奶油等点心类;面包类;香肠、鱼糕、火腿等水产加工食品;加工乳、发酵乳(包括酸奶)等乳制品;人造黄油、蛋黄酱、起酥油、打发鲜奶油、调味料、调味汁、色拉油、天妇罗油等油脂或油脂加工食品;酱油、沙司等调味料;咖喱、盖饭、粥、菜粥、炖菜等蒸煮袋食品;冰淇淋、冰糕(sherbet)等冰制点心等,但并不仅限于此。相对于食品整体的质量,食品中E.durans HS-08菌株的比例优选为0.1~10%,更优选为1~10%。
本发明提供一种含有本发明的乳酸菌或其处理物、或含有它们的组合物的食品添加物。作为食品添加物的用途,例如可列举出pH调节剂、保存剂、杀菌剂、抗氧化剂、防霉剂、着色剂、发色剂、漂白剂、光亮剂、香料、香辛料提取物、软化剂、营养强化剂、甜味剂、酸味剂、调味剂、苦味剂、乳化剂、增稠剂、稳定剂、胶凝剂、淀粉胶粘剂、膨松剂、生产用溶剂、酶、胶基、酵母食物或它们的制剂等,但并不仅限于此。另外,也可以同样使用上述例示的添加剂等。相对于食品添加物整体的质量,食品添加物中E.durans HS-08菌株的比例优选为0.1~10%,更优选为1~10%。
[化妆品]
本发明提供一种含有本发明的乳酸菌或其处理物、或含有它们的组合物的化妆品。化妆品包括所谓的药用化妆品(医药部外品)。作为化妆品,例如可列举出清洁剂、洗发水、护发素、生发液、发乳、须后水、身体乳、化妆水、洗面奶、按摩霜、润肤霜、气溶胶产品、脱臭剂、芳香剂、除臭剂或沐浴剂等,但并不仅限于此。除了本发明的卵磷脂以外,本发明的化妆品还可以根据需要适当掺合化妆品中通常所使用的成分,例如表面活性剂、保湿剂、来自于动植物的油脂、来自于微生物的油脂、有机硅类、高级醇、低级醇、来自于动植物的提取物、来自于微生物的提取物、紫外线吸收剂、消炎剂、金属螯合剂、维生素、抗氧化剂、增稠剂、防腐剂、杀菌剂、pH调节剂、着色剂、各种香料等。这些同样可以使用上述例示的成分。相对于化妆品整体的质量,化妆品中E.durans HS-08菌株的比例优选为0.1~10%,更优选为1~10%。
[药物]
本发明提供一种含有本发明的乳酸菌或其处理物、或含有它们的组合物的药物。除了含有本发明的乳酸菌之外,药物还可以含有有效成分,以及根据需要适当掺合药学上允许的载体、添加剂,从而进行制备。具体而言,可将药物制成片剂、包衣片剂、丸剂、散剂、颗粒剂、胶囊剂、液体制剂、混悬剂、乳剂、注射剂、输液剂、栓剂、软膏、喷雾剂、软膏剂、霜剂、胶凝剂、贴剂等,但并不仅限于此。关于载体或添加剂的掺合比例,只需基于药物领域中通常采用的范围适当设定即可。可掺合的载体或添加剂没有特别限定,例如可列举出水、生理盐水、其他水溶性溶剂、水溶性或油性基质等各种载体、赋形剂、粘合剂、pH调节剂、崩解剂、吸收促进剂、润滑剂、着色剂、矫味剂、香料等各种添加剂。除此之外,也可优选使用上述例示的成分。相对于药物整体,药物中E.durans HS-08菌株的比例优选为0.1~10%,更优选为1~10%。
[动物用饲料]
本发明提供一种含有本发明的乳酸菌或其处理物、或含有它们的组合物的动物用饲料。作为饲料,例如可列举出牛、马、猪、羊等家畜类用饲料;狗、猫、兔子、仓鼠、松鼠、豚鼠、鼠等宠物或实验动物类用饲料;沙丁鱼、金枪鱼、鲨鱼、翻车鱼、鳐鱼、鲹、远东哲罗鱼、鰤鱼、香鱼、鲤鱼、鲷鱼、鲭鱼、牙鲆鱼、鲽、鳗鱼、鯙、鮟鱇、河豚、丝背细鳞鲀、热带鱼等鱼类饲料;虾、蟹、墨鱼、虾夷扇贝、牡蛎、蝾螺等海鲜类用饲料;鸡、鹌鹑、火鸡、鸭、家鹅、鹦哥、鹦鹉等鸟类用饲料;独角仙、锹甲、菜粉蝶、燕尾蝶、螳螂、蝗虫、铃虫、鼠妇等虫类用饲料;壁虎、变色龙、鬣蜥、蜥蜴等爬行类用饲料;蝾螈、蛙、山椒鱼等两栖类动物用饲料等,但并不仅限于此。除了在饲料中添加本发明的乳酸菌之外,可以使用通常的饲料制备方法制备本发明的饲料。相对于饲料整体,饲料中E.durans HS-08菌株的比例优选为0.1~10质量%,更优选为1~10质量%。该动物用饲料可促进动物成长或提高其自身免疫,缩短给与饲料的动物(尤其是家畜或家禽)的养殖周期,促进早期出栏。
[植物用肥料]
本发明提供一种含有本发明的乳酸菌或其处理物、或含有它们的组合物的植物用肥料。此处,作为肥料,例如可列举出蔬菜、花、花卉苗木或水果等的肥料,但并不仅限于此。此外,作为蔬菜或水果,可列举出萝卜、牛蒡、胡萝卜、芜菁、莲藕等根菜类;土豆、番薯、中国薯蓣、野芋、芋头、魔芋、日本薯蓣等芋类;毛豆、蚕豆、豌豆、菜豆、兵豆、赤豆、芝麻等豆类;卷心菜、生菜、白菜、菠菜、野泽菜、雪菜、三叶芹、小菘菜、茼蒿等叶类蔬菜;蜜柑、夏橙、文旦、椪柑、伊予柑、八朔、柠檬、酸橙、葡萄柚等柑橘类;洋葱、大葱、葱白等葱类;黄瓜、金针菇、杏鲍菇、黑木耳、蟹味菇(shimeji)、滑子菇、松茸、椎茸、舞茸等蘑菇类;西蓝花、花椰菜、菜花、洋蓟等花菜类;黄瓜、西瓜、南瓜、西葫芦、葫芦、丝瓜、冬瓜、铁炮瓜、瓠瓜、苦瓜、苦瓜、甜瓜等瓜类;水稻、黑麦、薏苡、黍、粟、稗、高粱等禾谷类;小麦、大麦等麦类;蕨、紫萁等野菜;其他还可列举出苏铁、银杏、松、竹子、桃子、栗子、柿子、草莓、苹果、梨、葡萄、麝香葡萄、番茄、茄子、芥末、芹菜、笋、大蒜等。此外,作为花或花卉苗木等,可列举出蓟、溪荪、满天星、酸浆草、蒲公英、豌豆、凤仙花、康乃馨、洋甘菊、樱草花、百合、一串红、牡丹、油菜花、菊花,毛茛、彼岸花、三色堇、堇菜,向日葵、风信子、郁金香、水仙花、芙蓉、香豌豆、万寿菊、铃兰、石竹、非洲菊、天竺葵、百日菊、桔梗、波斯菊、大丽花、牵牛花、旋花、番红花、青葙、绣球花等花;玫瑰、梅、樱花、桃、木瓜、李、杏等蔷薇科;金木樨、茉莉、刺桂、丁香、连翘、迎春花等木犀科;山茱萸等山茱萸科;瑞香科;紫藤、胡枝子等豆科;山茶、茶梅等山茶科;皋月杜鹃、映山红、石南花、马醉木、蓝莓等杜鹃花科;其他还可列举出锦葵科,槭树科,瑞香科,安息香科,七叶树科,漆树科,忍冬科等花卉苗木,但并不仅限于此。除了在肥料中添加本发明的乳酸菌之外,可以按照通常的肥料制备方法制备本发明的肥料。相对于肥料整体,肥料中E.durans HS-08菌株的比例优选为0.1~10质量%,更优选为1~10质量%。此外,该植物用肥料可具有调节植物生长、防止植物倒伏、或调节植物鲜味等作用。该植物用肥料可以促进例如植物的根、茎、枝、叶、果实或花的生长。此外,该植物用肥料可以增强例如植物的根、茎、枝、叶、果实或花的鲜味。
[工业产品]
本发明提供一种含有本发明的乳酸菌或其处理物、或含有它们的组合物的工业产品(例如混凝土构件等)。作为除混凝土构件以外的工业产品,例如可列举出石油产品、树脂产品、磁性产品、皮革产品、纺织品等,但并不仅限于此。相对于工业产品,工业产品中E.durans HS-08菌株的比例优选为0.1~10质量%,更优选为1~10质量%。
该工业产品由于含有本发明的乳酸菌或其处理物、或含有它们的组合物,而本发明的乳酸菌或其处理物、或含有它们的组合物具有促进动物成长、提高动物自身免疫、调节植物生长、防止植物倒伏、或调节植物鲜味等作用,因此当该工业产品为用于动物饲养的工业产品(例如饲养箱、围栏等)、用于植物生长的工业产品(例如盆栽、喷壶、水桶、软管、取水口的混凝土等)时,具有促进动物成长、提高动物自身免疫、调节植物生长、防止植物倒伏、或调节植物鲜味等功能。
[套装(kit)]
本发明提供一种含有本发明的乳酸菌或其处理物、或含有它们的组合物的套装。套装优选由像分装瓶或箔包装等可另外含有组合物的容器构成,但也可以由单一的容器容纳组合物。作为优选套装的实例,可列举出用于片剂、胶囊等包装的泡罩包装,或填装药液的注射筒及容器等,但并不仅限于此。另外,也可以对套装进行射线、高压釜等公知的灭菌处理。
[免疫刺激作用]
本发明的乳酸菌优选具有免疫刺激作用。关于免疫刺激作用,例如,当与非给予组相比,本发明的乳酸菌或含乳酸菌的组合物的给予组的粪便中或血液中的IgA浓度高且有显著性差异或呈显著性趋势时,可以判断具有免疫刺激作用。此外,例如将本发明的组合物混入饲料中并给予至动物时,能够缓解免疫性疾病(例如乳腺炎、大肠杆菌病、腹泻等)的症状。由此,也可以减少抗生素的用量。
病原体或病毒的感染是由于它们粘附于上皮细胞表面而引起的,派尔集合淋巴结产生的IgA会阻止这样的粘附,在免疫系统中发挥重要作用。派尔集合淋巴结位于肠道内,由产生T细胞、B细胞及IgA的浆细胞等构成。
此外,关于粘膜组织的免疫刺激作用,已知乙酸通过诱导视黄酸从而诱导IgA,此外还已知派尔集合淋巴结的树突细胞分泌的IL-6、RALDH2、APRIL、BAFF等也会诱导产生IgA。因此,当与非给予组相比,本发明的乳酸菌或含乳酸菌的组合物的给予组的乙酸的浓度或RALDH2、IL-6、APRIL、BAFF的基因表达量高且有显著性差异或呈显著性趋势时,可以判断具有刺激粘膜组织的免疫、预防感染症的效果。此处,优选粘膜组织为例如消化系统、呼吸系统、泌尿系统等,进一步优选激活肠道的粘膜组织的免疫系统。
[保护粘膜效果]
本发明的乳酸菌优选具有保护粘膜的作用。肠道菌群产生的乳酸或乙酸等有机酸有助于包括人在内的动物的各种生化反应,这些有机酸被粘膜组织吸收,成为粘膜上皮细胞的能量源,具有修复上皮细胞、保护粘膜的作用。因此,例如,当与非给予组相比,本发明的乳酸菌或含乳酸菌的组合物的给予组的粘膜组织中的乳酸或乙酸量高且有显著性差异或呈显著性趋势时,可以判断具有粘膜保护效果。
[抗过敏作用]
本发明的乳酸菌优选具有抗过敏作用。例如,当与非给予组相比,本发明的含乳酸菌的组合物的给予组的血液中或粪便中的IgA的浓度高且有显著性差异或呈显著性趋势时,可以判断具有抗过敏作用。此外,例如,当对人给予本发明的组合物时,可以缓解花粉症等的症状。
[增强产生短链脂肪酸及有机酸的效果]
本发明的乳酸菌优选具有增强产生短链脂肪酸或有机酸的作用。已知乳酸菌等肠道菌群产生的短链脂肪酸或有机酸有助于IgA的产生,因此,例如,当与非给予组相比,本发明的乳酸菌或含乳酸菌的组合物的给予组的短链脂肪酸或有机酸的浓度高且有显著性差异或呈显著性趋势时,可以判断具有增强产生短链脂肪酸或有机酸的作用。
作为通过本发明的乳酸菌增强产生的短链脂肪酸或有机酸,可列举出乳酸、乙酸等,但并不仅限于此。已知短链脂肪酸通过IgA发挥免疫刺激作用或抗过敏作用,被认为会给人或动物的生命体带来各种积极影响。
[上调GRP43基因表达的效果]
本发明的乳酸菌优选具有上调乙酸受体GRP43基因表达的作用。已知GRP43是与短链脂肪酸、特别是乙酸的亲和性高的受体,伴随着肠道内短链脂肪酸量的增加,GRP43也会增加。即,认为若GRP43的基因表达量增加,则肠道内短链脂肪酸(乙酸等)也会增加。
此外,GRP43基因表达的上调具有各种积极作用:通过调节胰岛素信号预防糖尿病的效果、通过诱导参与保持体内稳态的调节性T细胞所带来的抑制结肠炎的效果等(NatureCommunications volume 4,Article number:1829(2013))。因此,例如,与非给予组相比,本发明的乳酸菌或含乳酸菌的组合物的给予组的GRP43基因表达量高且有显著性差异或呈显著性趋势时,可以判断具有预防糖尿病的效果或抑制结肠炎的效果。
[抑制有害菌和/或病原菌增殖的效果]
本发明的乳酸菌优选具有抑制有害菌和/或病原菌增殖的效果。已知人或小鼠等动物的有害菌或病原菌的增殖在酸性环境下会被抑制,因此认为若乳酸或乙酸等有机酸增加,则肠道内环境酸性化,pH值下降,肠道内的有害菌或病原菌的增殖被抑制。例如,当与非给予组相比,本发明的乳酸菌或含乳酸菌的组合物的给予组的肠道内的pH值低且有显著性差异或呈显著性趋势时,可以判断具有抑制有害菌和/或病原菌增殖的作用。
另外,作为本发明中增殖被抑制的有害菌或病原菌,可列举出产气荚膜梭菌、大肠杆菌等,但并不仅限于此。
[调节植物生长的作用、防止植物倒伏的作用及调节植物鲜味的作用]
本发明的乳酸菌优选具有调节植物生长的作用。例如,当与非给予组相比,本发明的乳酸菌或含乳酸菌的组合物的给予组在根、叶、枝、果实或花的大小方面生长显著时,例如优选当其大小生长了10~100%左右、进一步优选当其大小生长了30~50%左右时,可以判断具有调节植物生长的作用。由此,也可以减少用于植物生长的肥料的用量。
本发明的乳酸菌优选具有防止植物倒伏的作用。倒伏的程度例如可以以倒伏植物的倾斜度大小而表示为无(0)~严重(5)这6个等级。例如,优选本发明的乳酸菌或含乳酸菌组合物的给予组相对于非给予组的倒伏率,可防止1~5等级的倒伏,进一步优选可防止2~4等级的倒伏,但并不仅限于此。植物可列举出上述例示的植物,特别优选米或水稻,但并不仅限于此。此外,组合物还可以含有钙、锌、铁、镁、钾、磷等矿物质。通过同时使用矿物质,可以进一步提高防止植物倒伏的效果。认为这是由于提高植物自身免疫的作用而引起的,由此,可以减少肥料的用量。
本发明的乳酸菌优选具有调节植物鲜味(糖度)的作用。例如,相对于原糖度,优选增加1%~70%左右的糖度,进一步优选增加20%~50%左右的糖度,但并不仅限于此。植物可列举出上述所示的植物,特别优选米、甜瓜、桔子、柠檬、番茄、白菜等,但并不仅限于此。
[促进动物成长的作用及提高动物自身免疫的作用]
本发明的乳酸菌优选具有促进动物成长的作用。例如,本发明的乳酸菌或含有乳酸菌的组合物的给予组相对于非给予组的体重,优选增加10~200质量%左右的体重、更优选增加30~100质量%左右的体重,或者相对于非给予组的体长,优选增加10~200%左右的体长,更优选增加30~100%左右的体长,但并不仅限于此。由此,可减少饲料的用量。作为本公开文本中的动物,可列举出如上述的动物,但并不仅限于此。
本发明的乳酸菌优选具有提高动物自身免疫的作用。例如,当与非给予组相比,本发明的乳酸菌或含乳酸菌的组合物的给予组的粪便中或血液中的IgA浓度高且有显著性差异或呈显著性趋势时,可以判断具有提高自身免疫的作用。此外,例如,当将本发明的组合物混入饲料中并给予至动物时,可缓解自身免疫性疾病(例如花粉症、过敏等)的症状。作为本公开文本中的动物,可列举出上述的动物,但并不仅限于此。
实施例
以下,通过实施例对本发明进行详细说明,但本发明并不仅限于此。
[实施例1]E.durans HS-08菌株的诱导产生IgA的活性
(1)供试小鼠为BALB/cA、交付时的周龄为6周龄,性别为雄性。另外,刚交付时的小鼠有可能会因环境变化所带来的压力的原因而对试验结果造成影响,因此将适应期设定为17天,适应期仅摄取精饲料(AIN-76A、Research Diets,New Brunswick,NJ),然后设定下述给予饲料的时期及观察时期(约60天)。
(2)以使体重及粪便中的IgA尽量相同的方式将结束适应的小鼠分为4组,并设为无E.durans HS-08菌株的分组(以下为对照组),饲料中E.durans HS-08菌株的比例为0.38%的分组(以下为0.38%组)这两组,供试数均为每组5只(表1)。
[表1]
(3)用于掺合在小鼠摄取的饲料中的乳酸菌粉末是以MRS培养基(DifcoLaboratories,Detroit,MI,USA)培养的E.durans HS-08菌株。具体而言,首先将E.duransHS-08菌株的菌落接种到5ml的MRS培养液中,在密闭状态下于37度静置培养24小时,得到培养液。然后,将该培养液加入到2000ml的MRS培养液中,在密闭状态下于37度静置培养24小时。然后,通过离心分离收集细菌,加入菌体2倍量的10%蔗糖溶液作为赋形剂,然后进行冷冻干燥,得到本发明的乳酸菌粉末。另外,乳酸菌粉末的活菌数为1.1×1012cfu/g。
(4)各分组的饲料配比如图2所示。
[表2]
(5)在摄取乳酸菌粉末后的第0天、第14天、第28天、第42天及第60天收取小鼠1整天的粪便。将该粪便与加入有蛋白酶抑制剂的磷酸生理盐水混合,在冰上静置30分钟后,将混合液离心分离,用小鼠IgA ELISA试剂盒测定其上清液中IgA的浓度。
(6)在摄取乳酸菌粉末后的第60天采集血液。将该血液与肝素钠混合,进行离心分离,用小鼠IgA ELISA试剂盒测定其上清液(以下也称为“实施例1中获得的血浆”)中IgA的浓度。
如图1所示,关于IgA量的经时变化,与对照组相比,0.38%组在第28天时IgA量具有增加趋势,在第42天时IgA量显著增多。在第60天时,与第42天时相比IgA量虽减少,但具有多于对照组的趋势。另外,在用两因素重复测量方差法对粪便中IgA量的数据进行分析后,通过多重比较检验进行统计分析。其他数据通过多重比较检验进行统计分析。
此外,如表3及图2所示,与对照组相比,0.38%组的血液中IgA浓度显著增高。
[表3]
平均值±标准误差(n=5)
*p<0.05vs对照组
确认到摄取乳酸菌粉末的组(0.38%组)的粪便中IgA量比对照组高。由此可以确认E.durans HS-08菌株可以刺激肠道免疫系统。
此外,摄取乳酸菌粉末的组(0.38%组)的血液中IgA浓度比对照组高。可以说血液中IgA浓度提高,则全身免疫力提高,因此可以确认E.durans HS-08菌株可以刺激全身免疫系统。
[实施例2]E.durans HS-08菌株的安全性试验
(1)度过适应期后,在摄取乳酸菌粉末后的第0天、第7天、第14天、第21天、第28天、第35天、第42天、第49天、第56天及第60天测量体重。
(2)用肌酐定量检测试剂盒(LabAssayCreatinine)测定上述实施例1中获得的血浆的肌酐(以下为CRE)浓度。若肾脏发生异常,则血液中肌酐(CRE)浓度增加。本试验的目的是比较对照组和乳酸菌粉末摄入组的CRE浓度来确认对小鼠肾功能的安全性。
(3)此外,用转氨酶CII试剂盒测定上述实施例1中获得的血浆的谷丙转氨酶(ALT)浓度、及谷草转氨酶(AST)浓度。若肝脏发生异常,则血液中ALT及AST增加。本试验的目的是比较对照组和乳酸菌粉末摄入组的ALT浓度及AST浓度来确认对小鼠肝功能的安全性。
(4)进一步,使用小鼠IgE ELISA试剂盒测定上述实施例1中获得的血浆的IgE浓度。本试验的目的是比较对照组和乳酸菌粉末摄入组的IgE浓度来确认小鼠对乳酸菌不过敏。
关于体重,对对照组和乳酸菌粉末摄入组进行了比较,结果无显著性差异(图3)。此外,关于CRE浓度,对对照组和乳酸菌粉末摄入组进行了比较,结果无显著性差异(表4及图4)。进一步,关于ALT浓度及AST浓度,对对照组和乳酸菌粉末摄入组进行了比较,结果无显著性差异(表5及图5~6)。此外,关于IgE,对照组和乳酸菌粉末摄入组中均未检测到IgE。
[表4]
平均值±标准误差(n=5)
[表5]
平均值±标准误差(n=5)
与对照组相比,乳酸菌粉末摄入组的体重、CRE浓度、ALT浓度及AST浓度无显著性差异。由此可认为即使使用E.durans HS-08菌株,也不会对小鼠的体重、肾功能及肝功能产生不良影响。
此外,对照组、乳酸菌粉末摄入组这两个分组中均未检测到IgE。由此可以认为小鼠对E.durans HS-08菌株不过敏,即,E.durans HS-08菌株不是过敏源。
进一步,也进行了耐药性试验和鲎试验等,但均可确认本发明乳酸菌的安全性。归纳上述结果并将其示于下述表6。
[表6]
平均值±标准误差(n=5)
[实施例3]派尔集合淋巴结细胞的基因分析
在摄取乳酸菌粉末后第60天,通过颈椎脱臼的方式使实施例1的(1)~(4)中获得的小鼠安乐死,采集小鼠的派尔集合淋巴结细胞,用illustra RNAspin Mini RNA Icolation试剂盒(GE Healthcare,Piscataway,NJ,USA)提取RNA。称取0.15μg RNA,通过寡核苷酸引物(Invitrogen,Carlsbad,CA)、Super ScriptIII逆转录酶(Invitrogen)对该RNA进行反转录。然后,使用PCR纯化试剂盒(Qiagen,Cambrige,MA,USA),由反转录的样品纯化cDNA,通过基于SYBR Green(Life Technologies)的实时PCR法,进行肠道内免疫诱导因子的基因表达分析。
如图7所示,乳酸菌粉末摄入组的RALDH2、APRIL、BAFF、IL-6的基因表达量显著高于对照组,或者具有高的趋势。由受刺激的树突细胞分泌的IL-6、BAFF、APRIL促进来自B细胞的IgA的产生,由树突细胞的RALDH2产生的视黄酸促进产生IgA的B细胞归巢至粘膜。因此,由图7的结果明确可知本发明的乳酸菌在基因表达水平方面也表现出了对肠粘膜中IgA的增强。
另外,上述分析的基因为视黄醛脱氢酶2(RALDH2)、增殖诱导配体(APRIL)、B细胞活化因子(BAFF)、白介素-6(IL-6),本实施例中使用的内参基因为β-肌动蛋白。此外,基因分析用引物为下述表7所记载的引物。
[表7]
[实施例4]短链脂肪酸或有机酸及pH值测定
对于实施例1的(1)~(4)中获得的小鼠,在摄取乳酸菌粉末后第60天收取小鼠1整天的粪便,将该粪便冷冻干燥,以超纯水混悬。然后于4℃静置1小时后离心,过滤其上清液。以短链脂肪酸分析用标记试剂(YMC CO,Kyoto,Japan)处理过滤后的上清液,然后以HPLC测定有机酸的量(乳酸、乙酸、丙酸、丁酸)。并且,测定过滤后的上清液的pH值。
如图8所示,与对照组相比,乳酸菌粉末摄入组的小鼠粪便中的乳酸及乙酸的量显著增加(乳酸:p<0.01、乙酸:p<0.05)。
此外,如图9所示,与对照组相比,乳酸菌粉末摄入组的小鼠粪便中的pH值显著下降(p<0.05)。
[实施例5]GRP43的基因表达分析
对于实施例1的(1)~(4)中获得的小鼠,在摄取乳酸菌粉末后第60天,通过颈椎脱臼的方式使小鼠安乐死,采集小鼠的派尔集合淋巴结细胞,用illustra RNAspin Mini RNAIcolation试剂盒(GE Healthcare,Piscataway,NJ,USA)提取RNA。称取0.15μg RNA,通过寡核苷酸引物(Invitrogen,Carlsbad,CA)、Super ScriptIII逆转录酶(Invitrogen)对其进行反转录。然后,使用PCR纯化试剂盒(Qiagen,Cambrige,MA,USA)由反转录的样品纯化cDNA,通过基于SYBR Green(Life Technologies)的实时PCR法,进行肠道内DRP43的基因表达分析。本实施例中使用的内参基因与实施例4相同,为β-肌动蛋白。此外,基因分析用引物使用了下述引物。
[表8]
如图10所示,乳酸菌粉末摄入组中的GRP43的基因表达量具有高于对照组的趋势(P=0.42)。使用Excel统计(SSRI、Tokyo、Japan)进行统计分析。此外,数据通过多重比较检验(Dunnett’s test)进行统计分析。
[实施例6]
对患有腹泻的牛(奶牛、肉牛)、猪、鸡(肉鸡、蛋鸡)等动物给予本发明的乳酸菌或其处理物,结果可以止泻、使液状的粪便固化等。此外,可以减少通常的饲养过程中所需的药物的饲料添加量,对于奶牛或鸡,可以抑制免疫系统疾病(例如乳腺炎、大肠杆菌病等)的发生。此外,观察到肉鸡的死亡率降低。即,确认到了本发明的乳酸菌的免疫刺激作用。
[实施例7]
对有花粉症的健康成人给予本发明的乳酸菌或其处理物,结果抑制了花粉症的症状。即,确认到了本发明的乳酸菌的抗过敏作用。
[实施例8]
对米、甜瓜、番茄或白菜给予本发明的乳酸菌或其处理物,结果米、甜瓜、番茄的果实部分以及白菜的叶子变大,赋予了这些植物活力。即,确认到了本发明的乳酸菌的调节植物生长的作用。
[实施例9]
将本发明的乳酸菌或其处理物混入牛(肉牛)、猪、鸡(肉鸡)的动物饲料中并进行给予,结果与给予不含本发明的乳酸菌或其处理物的饲料相比,确认到了饲料需求率减少、体型结实等变化。即,确认到了本发明的乳酸菌的促进动物成长的作用。
[实施例10]
将本发明的乳酸菌或其处理物混入水稻的肥料中,结果与给予不含本发明的乳酸菌或其处理物的肥料相比,发挥了植物的自身免疫作用,整体不易倒伏。由此,可认为本发明的乳酸菌具有防止植物倒伏的作用。
[实施例11]
将本发明的乳酸菌或其处理物混入米的肥料中,结果与给予不含本发明的乳酸菌或其处理物的肥料相比,米的鲜味增加。由此,可认为本发明的乳酸菌具有调节植物鲜味的作用。
[实施例12]
按照公知的方法,确定本发明的耐久肠球菌(E.durans HS-08菌株)碱基序列,结果如下所示(序列号13)。
[表9]
由以上结果可知,本发明的乳酸菌在例如给予至动物或混入饲料中时是有用的,原因在于具有下述效果:(1)由于可促进动物(家畜)的肠道调节的作用、预防传染病等,因此有助于减少抗生素等的用量或降低死亡率;(2)动物(家畜)的适口性高、成长促进率良好,可缩短出栏天数;(3)由粪便导致的臭味减少,因此降低由畜舍环境引起的压力,具有提高肉质的效果。
此外,本发明的乳酸菌在例如散布于植物或混入肥料中时是有用的,原因在于具有下述效果:(1)由于能够赋予植物活力,因此具有可调节植物生长的效果;(2)由于可发挥植物自身免疫作用,因此具有防止植物倒伏的效果;(3)具有增加植物鲜味的效果。
工业实用性
本发明提供一种新型菌株乳酸菌耐久肠球菌(E.durans HS-08菌株)或其处理物及其增殖方法,具有工业实用性。
序列表
<110> 株式会社好固工
<120> 乳酸菌及其用途
<130> KHP202112617.2
<150> JP 2018-104321
<151> 2018-05-31
<160> 13
<170> PatentIn version 3.1
<210> 1
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 1
gacttgtagc agctgtcttc act 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 2
tcacccattt ctctcccatt tcc 23
<210> 3
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 3
tcacaatggg tcaggtggta tc 22
<210> 4
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 4
tgtaaatgaa agacacctgc actgt 25
<210> 5
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 5
tgctatgggt catgtcatcc a 21
<210> 6
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 6
ggcagtgttt tgggcatatt c 21
<210> 7
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 7
aatagtcctt cctaccccaa tttc 24
<210> 8
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 8
atttcaagat gaattggatg gtct 24
<210> 9
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 9
catccgtaaa gacctctatg ccaac 25
<210> 10
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 10
atggagccac cgatccaca 19
<210> 11
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 11
ggcttctaca gcagcatcta 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 12
aagcacacca ggaaattaag 20
<210> 13
<211> 1483
<212> DNA
<213> 耐久肠球菌
<400> 13
gacgaacgct ggcggcgtgc ctaatacatg caagtcgtac gcttcttttt ccaccggagc 60
ttgctccacc ggaaaaagaa gagtggcgaa cgggtgagta acacgtgggt aacctgccca 120
tcagaagggg ataacacttg gaaacaggtg ctaataccgt ataacaatcg aaaccgcatg 180
gttttgattt gaaaggcgct ttcgggtgtc gctgatggat ggacccgcgg tgcattagct 240
agttggtgag gtaacggctc accaaggcca cgatgcatag ccgacctgag agggtgatcg 300
gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc 360
ggcaatggac gaaagtctga ccgagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt 420
aaaactctgt tgttagagaa gaacaaggat gagagtaact gttcatccct tgacggtatc 480
taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc 540
gttgtccgga tttattgggc gtaaagcgag cgcaggcggt ttcttaagtc tgatgtgaaa 600
gcccccggct caaccgggga gggtcattgg aaactgggag acttgagtgc agaagaggag 660
agtggaattc catgtgtagc ggtgaaatgc gtagatatat ggaggaacac cagtggcgaa 720
ggcggctctc tggtctgtaa ctgacgctga ggctcgaaag cgtggggagc aaacaggatt 780
agataccctg gtagtccacg ccgtaaacga tgagtgctaa gtgttggagg gtttccgccc 840
ttcagtgctg cagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa 900
actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca 960
acgcgaagaa ccttaccagg tcttgacatc ctttgaccac tctagagata gagcttcccc 1020
ttcgggggca aagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080
gttaagtccc gcaacgagcg caacccttat tgttagttgc catcattcag ttgggcactc 1140
tagcaagact gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc 1200
cttatgacct gggctacaca cgtgctacaa tgggaagtac aacgagttgc gaagtcgcga 1260
ggctaagcta atctcttaaa gcttctctca gttcggattg caggctgcaa ctcgcctgca 1320
tgaagccgga atcgctagta atcgcggatc agcacgccgc ggtgaatacg ttcccgggcc 1380
ttgtacacac cgcccgtcac accacgagag tttgtaacac ccgaagtcgg tgaggtaacc 1440
tttttggagc cagccgccta aggtgggata gatgattggg gtg 1483
Claims (14)
1.一种耐久肠球菌HS-08(保藏编号NITE BP-02675)的菌体或其处理物。
2.一种诱导产生IgA用或免疫刺激用组合物,其含有权利要求1所述的菌体或其处理物。
3.一种抗过敏用组合物,其含有权利要求1所述的菌体或其处理物。
4.一种抑制有害菌和/或病原菌增殖用组合物,其含有权利要求1所述的菌体或其处理物。
5.一种保护粘膜用组合物,其含有权利要求1所述的菌体或其处理物。
6.一种组合物,其特征在于,含有权利要求1所述的菌体或其处理物,且用于选自由增强产生短链脂肪酸、增强产生有机酸、及上调GRP43基因表达组成的组中的至少一种用途。
7.一种组合物,其特征在于,含有权利要求1所述的菌体或其处理物,且用于选自由调节植物生长、防止植物倒伏、及调节植物鲜味组成的组中的至少一种用途。
8.一种组合物,其特征在于,含有权利要求1所述的菌体或其处理物,且用于促进动物成长和/或提高动物自身免疫的用途。
9.一种植物用肥料,其特征在于,含有权利要求1所述的菌体或其处理物、或权利要求7所述的组合物。
10.一种动物用饲料,其特征在于,含有权利要求1所述的菌体或其处理物、或权利要求8所述的组合物。
11.一种饮食品、化妆品或药物,其特征在于,含有权利要求1所述的菌体或其处理物、或权利要求2~8中任一项所述的组合物。
12.一种混凝土构件,其特征在于,含有权利要求1所述的菌体或其处理物、或权利要求2~8中任一项所述的组合物。
13.一种组合物,其含有权利要求1所述的菌体或其处理物、以及选自由包括其他乳酸菌、双歧杆菌、或酵母菌的有益菌、及霉菌组成的组中的至少一种或它们的处理物。
14.一种耐久肠球菌HS-08菌体的增殖方法,其特征在于,培养耐久肠球菌HS-08菌株(保藏编号NITE BP-02675)的菌体。
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US20210205378A1 (en) | 2021-07-08 |
KR20210005671A (ko) | 2021-01-14 |
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