CN113519814A - 含菌丝液体组织培养物上清与食品的组合物及其用途 - Google Patents
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Abstract
本发明涉及含菌丝液体组织培养物上清与食品的组合物及其用途。所述组合物包含食品和从菌丝液体组织培养物分离的上清的组合,其中所述组合苦味减少,不想要的回味减少,和/或涩味减少,其中所述食品包括豌豆蛋白质;以及其中用于培养所述菌丝液体组织培养物的真菌是冬虫夏草Cordyceps sinensis。本发明还提供长菌丝的液体组织培养物分离的上清用于提升食品口味的用途。
Description
本申请是申请日为2015年8月26日发明名称为“菌丝液体组织培养物的生产方法和应用”的申请号为201580045846X(国际申请号为PCT/US2015/047036)的发明专利申请的分案申请。
技术领域
本发明涉及用美味和治疗上更高阶的担子菌及子囊菌的菌丝液体组织培养物通过本发明方法制备的产品和其应用。
背景技术
US2693665讨论了在柑桔汁、梨汁、芦笋汁、“有机材料”、碳水化合物、氮源和任意这些材料组合中培养四孢蘑菇(Agaricus campestris),任选地,补充尿素和/或各种铵盐以生产用作食品的菌丝。
US2761246公开用于人食物的浸没的羊肚菌(Morchella esculenta)和马鞍菌(Helvellaceae spp.)菌丝的生产方法。此文献讨论了各种糖蜜溶液作为有铵盐补充物的培养基的应用。该专利公开了加入的碳酸钙或硫酸钙用作菌丝球成核位点,使生物量产量增加30倍。
US2928210公开了从补充有有机和无机盐的亚硫酸废液介质生产蘑菇菌丝的方法。
US3086320公开了通过在的培养基中培养菌株改善羊肚菌、巨大马鞍菌(Helvellagigas)、鸡腿菇(Coprinus comatus)和四孢蘑菇(Agaricus campestris)浸没的菌丝风味的方法,所述培养基“必须包含溶于水的作为能量源的碳水化合物、氮源和适当矿物质”且包括含牛奶的配方,其声称在适当使用时提高菌丝产量和风味。
US4071973讨论担子菌的培养条件。真菌接种并生长于与50-70g/L蔗糖混合,并以0.2-15g/L“压碎的甘蔗、蔗渣、松树组织和麦麸”细粉补充的氮、磷和钾的无机营养盐。氧控制在培养基的30-90%(v/v),容器压力为0.12-0.5MPa(17.4-72.5psi),供氧为0.1-1.0L/分钟。所用盐包括硝酸铵、磷酸钠、七水硫酸镁、七水硫酸亚铁和磷酸氢二钾。讨论创造性气压循环并用调压器控制。另一工程方案使用背压调节器,用空气接受器槽上的调压器供应空气。
世界各地的机构努力寻找新型苦味阻断剂。仅提交了少量关于苦味阻断剂的专利,且许多是合成化合物或依赖基础分子基序排列,参见例如EP2570035A1、US4154862、US5631292、US6265012、US7939671、US20080226788A1、US20100227039A1、US20020177576、US20110086138和WO2008119197A1。
甜叶菊(Stevia rebaudiana)数千年来被人类社会用作民间医药和甜味剂。今天,许多国家和地区种植该植物,包括韩国、中国台湾、泰国、马来西亚、巴西、哥伦比亚、秘鲁、巴拉圭、乌拉圭和以色列。FDA将莱苞迪苷A和甜菊苷都标为一般认为安全(GRAS),产生一些进入美国市场的甜叶菊提取物食品添加剂。术语“甜叶菊”一般用于指新鲜或干燥的叶和/或植物部分,或甜叶菊的提取物/煎剂/糖浆,其未加工的或进一步纯化成特定糖苷,此文献之后使用的术语“甜叶菊”能指任意这些植物形式。已知负责甜菊(S.rebaudiana)甜味和金属的及苦的回味的化合物是甜菊糖苷。共鉴定了10种,化合物类别标记为苷元二萜甜菊醇的各种糖基化、鼠李糖基化和木糖基化(xylated)形式。
为生成甜菊糖苷,甜叶菊植物进行干燥并接受提取过程。经用各种溶剂如甲醇或乙醇结晶通过柱色谱或过滤能获得不同纯度的各种糖苷。
采用各种方法改变绿茶的口味谱。饮用发酵茶已有数百年,不过这通常用环境植物群进行。茶一般发酵不短于3个月,有时长达50年。
需要生产食品例如甜叶菊或茶的方法,获得美味产品而同时减少口味缺陷。因此,本领域仍需要相对于甜叶菊或茶减少不想要的口味组分的水平和/或增加风味和/或健康促进组分水平的产品,和获得这类产品的方法。本发明涉及克服一个或多个上述问题。
发明内容
在一个实施方式中,本发明包括提升食品口味的方法,所述方法能包括以下步骤:在培养基中培养浸没的菌丝液体组织培养物,收集浸没的菌丝液体组织培养物的上清液;和以足以提升食品口味的量向食品加入所收集的上清液。
用于培养浸没的菌丝组织的真菌可以包括以下物种的至少之一:灵芝(Ganodermalucidum)、树舌灵芝(Ganoderma applanatum)、冬虫夏草(Cordyceps sinensis)、蛹虫草(Cordyceps militaris)、猴头菇(Hericium erinaceus)、香菇(Lentinula edodes)、姬松茸(Agaricus blazei)、灰树花(Grifola frondosa)、黑木耳(Auricularia auricula)、金针菇(Flammulina velutipes)、云芝(Trametes versicolor)、羊肚菌(Morchella spp.)、白桦茸(Inonotus obliquus)、Laricifomes officinalis、木蹄层孔菌(Fomesfomentarius)、药用层孔菌(Fomes officinalis)、Fomes fomitopisis、松口蘑(Tricholoma matsutake)、美味牛肝菌(Boletus edulis)、Clitocybe nuda、Clitocybesaeva、Plearotus spp.、银耳(Tremella fuciformis)、桦孔菌(Piptoporus betulinis)、猪苓(Polyporus umbellatus)、滑菇(Pholiota nameko)、草菇(Volvariella volvacea)、真姬菇(Hypsizygus marmoreus)、大球盖菇(Stropharia rugosoannulata)和硫色绚孔菌(Laetiporus sulfureus)。在一个实施方式中,所述真菌是冬虫夏草。
在一些实施方式中,当与所收集的上清液组合时,食品口味提升。口味提升可采用任何形式,例如减少食品的苦味,减少食品的不想要的回味和减少食品的涩味。
食品能包括食物成分、饮食补充剂、食物添加剂、保健营养品和药物。食品示例包括甜叶菊植物部分、甜菊糖苷、阿斯巴甜、安赛蜜、三氯蔗糖、碳水化合物、罗汉果、可可、可可液、茶、人参、糖醇、咖啡、蔓越橘、葡萄柚、石榴、椰子、葡萄酒、啤酒、酒精饮料和烈酒。
在一个实施方式中,所收集的上清液任选地进行巴氏消毒或灭菌。在任选的巴氏消毒或灭菌步骤之前或之后,还任选地对所收集的上清液进行干燥。
在一些实施方式中,所述培养步骤可以进行约1至约60天。
可对所讨论的实施方式进行各种修改和增补,而不偏离本发明范围。例如,尽管上述实施方式涉及特定特征,本发明的范围还包括有不同特征组合的实施方式和不含所有上述特征的实施方式。
具体实施方式
尽管某些实施方式的各个方面和特征如上概括,以下详细描述进一步阐明一些实施方式以使本领域技术人员能实践这类实施方式。提供所描述的实施例用于阐明目的,而不意在限制发明范围。
以下描述中,出于解释目的,列出许多具体细节以提供对所述实施方式的全面了解。然而,本领域技术人员清楚了解,本发明的其它实施方式可在没有一些这类具体细节的情况下实践。本文描述数个实施方式并要求权利,当多种特征被归于不同实施方式时,应理解涉及一个实施方式所述的特征也可纳入其它实施方式。然而,同样地,任何所述或要求权利的实施方式的一个或多个特征不应视作对本发明每个实施方式必要,因为本发明其它实施方式可省略这些特征。
除非另有说明,本文所用表示量、尺寸等的全部数字应理解为在所有情况中由术语“约”修饰。本申请中,除非另有特别说明,使用单数包括复数,除非另有说明,使用术语“和”以及“或”指“和/或”。此外,使用术语“包括(including)”以及其它形式如“includes”和“included”应视作非排他。同样,除非另有特别说明,术语如“元素”或“组分”涵盖含一个单元的元素和组分以及含多于一个单元的元素和组分。
在一个实施方式中,本发明基于以下发现:真菌培养基(在本文所述任何培养基上)如冬虫夏草、猴头菇或灵芝培养基在适当处理后能直接用作食品,所述处理如食用前进行巴氏消毒或灭菌。培养基能干燥、稀释、浓缩,或以浓缩物、干粉等形式简洁使用。
作为静止菌丝毡(mycelial mat)培养物,真菌代谢物溶液与剩余培养基之间的界面稳定地下沉。界面位移是用于确定培养物健康的方便观察,指示培养物进入静止期或生长期。形成的代谢物池通常具有讨人喜欢的颜色,且不受理论约束的情况下,被认为包含有益的真菌材料如酶、碳水化合物、脂质、小分子等,会使得该材料适合作为食物成分/补充剂/添加剂。发明人发现在一个实施方式中,浸没的菌丝培养物仅需要过滤(如用粗棉布、咖啡滤纸、0.2微米滤器)和巴氏消毒以分离上清液。如果混合,能根据本发明使用漂浮培养。
在一个实施方式中,本发明人发现部分真菌液体组织培养液即上清液(所含菌丝量减少,本文称为“无菌丝部分”)在直接加入食品时,具有改善食品中不想要的口味例如苦味、涩味和/或不想要的回味的能力。提升食品口味包括改善该食品的变甜。风味改良还包括减少与甜叶菊和茶相关的特征性回味,包括但不限于苦味、金属味、甘草味,通常称为回味,其在最初甜味或茶的感觉之后出现。减少这些口味也可称为减少口味缺陷。例如,甜菊糖苷具有剩余的苦味和回味,这影响其质量特征。
通过本发明产品处理的食品的改良的口味可以多种方式测量,如化学分析,证明提高甜味、减少苦味和/或减少口味缺陷。还可进行采用品尝小组的口味测试以提供涉及产品口味改善的定性数据,小组确定所处理产品中是否显示提高甜味和/或减少口味缺陷。
在一个实施方式中,本发明包括使甜叶菊长菌丝,例如用冬虫夏草,从而提供相较未长菌丝对照更美味的甜叶菊水性提取物。然而,发明人也发现向甜叶菊水性提取物样品简单加入冬虫夏草(C.sinensis)完整液体培养物可消除甜叶菊不想要的回味(如令人不适的回味)。例如,60%莱苞迪苷A混合物用冬虫夏草和灵芝(G.lucidum)的完整液体培养物孵育。发明人发现通常与甜菊糖苷混合物相关的回味在和冬虫夏草完整液体培养物混合时孵育6小时后完全消除。用灵芝、猴头菇、灰树花、香菇、松口蘑、羊肚菌、云芝或灵芝没有观察到这种效果。然而,据本发明人理解,改善口味谱的特性可能在其它真菌属中发现,且可能是冬虫夏草真菌其它种,例如线形虫草属(Ophiocordyceps)、Elaphocordyceps或冬虫夏草属(Cordyceps)的任何种,如冬虫夏草(C.sinensis)和蛹虫草(C.militaris)。
特别地,本发明人使用过滤的冬虫夏草液体组织培养物混合甜菊糖苷混合物,孵育6小时。运行研究时程后,本发明人意外发现风味增强作用在向甜菊糖苷混合物加入滤液后立即产生,表明该过程可能是非酶过程。推测过滤的冬虫夏草液体组织培养物具有改善口味和/或苦味阻断剂性质。过滤的冬虫夏草液体组织培养物(滤液)随后与表9的本文所公开其它物质组合并发现具有就这些物质而言普遍的改善口味和/或苦味阻断剂性质。本发明人发现滤液可进一步纯化例如以增加溶解度,且可干燥如喷雾干燥,并与食品组合以改善食品口味谱,包括减少苦味和/或回味。因此,本发明公开了苦味阻断剂,其似乎对一些不同类型食品有效。
在一个实施方式中,本发明包括提升食品口味的方法,所述方法包括以下步骤:在培养基中培养浸没的菌丝液体组织培养物,收集浸培养物的无菌丝部分,和向食品加入无菌丝部分以提升食品口味。
本发明所述食品能包括任何食品,其包含通过口服(经口)服用的任何物质,且包括食品、食物成分、饮食补充剂、食品添加剂、药物、食物、化妆品成分、保健营养品成分、饮食成分和加工助剂。具有或可具有不想要的口味特性如苦味、不想要的回味、涩味等的任何食品能用本发明的苦味阻断剂组合物处理。在一些实施方式中,所述食品包括茶树部分、茶煎剂或茶纯化提取物。在一些实施方式中,所述食品包括甜叶菊植物部分、甜菊糖苷、阿斯巴甜、安赛蜜、三氯蔗糖、碳水化合物、罗汉果、可可、可可液、茶、人参、豌豆蛋白质、糖醇、咖啡、蔓越橘、葡萄柚、石榴、椰子、葡萄酒、啤酒、酒精饮料和烈酒。
食品包括所有谷类、谷物,以下所有种类:小麦、黑麦、糙米、白米、红米、黄金稻(gold rice)、野生稻(wild rice)、稻、大麦、黑小麦、稻、高粱、燕麦、小米、藜麦、荞麦、福尼奥米、苋菜(amaranth)、画眉草(teff)和硬质小麦;苹果和梨、杏子、樱桃、杏仁、桃子、草莓、葡萄干、木薯、可可、香蕉、茜草科物种(咖啡)、柠檬、橙和葡萄柚;番茄、马铃薯、胡椒、茄子、甜胡椒、芒果粉、当归、大茴香(茴芹(Pimpinella anisum))、茴香桃金娘(Aniseed myrtle(Syzygium anisatum))、胭脂树(红木(Bixa orellana))、苹果薄荷(香薄荷(Menthasuaveolens))、艾草、艾蒿、阿魏(阿魏(Ferula assafoetida))、小檗属、香蕉、罗勒(罗勒(Ocimum basilicum))、月桂叶、拳参(拳参(Persicaria bistorta))、黑豆蔻、黑色小茴香、黑醋栗、黑酸橙(Black lime)、墨角藻(囊褐藻(Fucus vesiculosus))、蓝籽类叶牡丹(BlueCohosh)、蓝叶小桉树(多苞叶尤加利(Eucalyptus polybractea))、沼泽拉不拉多茶树(加茶杜香(Rhododendron groenlandicum))、波耳多树(博路都树(Peumus boldus))、玻利维亚香菜(Bolivian Coriander)(Porophyllum ruderale)、琉璃苣(琉璃苣(Boragoofficinalis))、菖蒲、金盏花、Calumba(Jateorhiza calumba)、洋甘菊、大麻、刺山柑(续随子(Capparis spinosa))、葛缕子、小豆蔻、角豆荚、肉桂、木麻黄、猫薄荷、猫爪草、猫耳菊(Catsear)、辣椒、灯油藤(Celastrus paniculatus)、聚合草、香芹盐、芹菜籽、矢车菊、山萝卜(蜡叶峨参(Anthriscus cerefolium))、繁缕、菊苣、红番椒(Chile pepper)、辣椒粉、金鸡纳树、细香葱(北葱(Allium schoenoprasum))、欧洲没药(香没药(Myrrhis odorata))、芫荽叶(参见胡荽)(芫荽(Coriandrum sativum))、桂皮(和肉桂)、肉桂桃金娘(柠檬香桃木(Backhousia myrtifolia))、鼠尾草、猪殃殃、三叶草、丁香、咖啡、款冬、聚合草、芸香、康德郎皮、黄连、胡荽、艾菊(Tanacetum balsamita)、偃麦草、峨参(峨参(Anthriscussylvestris))、黄花九轮草、雪球荚蒾树皮(雪球荚蒾(Viburnum opulus))、水芹、古巴牛至(左手香(Plectranthus amboinicus))、鼠麴草、莳萝、咖喱叶(九里香(Murrayakoenigii))、达米阿那(Turnera aphrodisiaca)、蒲公英(西洋蒲公英(Taraxacumofficinale))、缓和剂、魔鬼爪(南非钩麻(Harpagophytum procumbens))、莳萝子、莳萝(莳萝(Anethum graveolens))、多里戈胡椒(Tasmannia stipitata)、紫锥菊、刺金刚纂(Echinopanax Elatum)、雪绒花、接骨木、接骨木花、土木香、刺五加(Eleutherococcussenticosus)、土荆芥(土荆芥(Chenopodium ambrosioides))、麻黄、刺芫荽(Eryngiumfoetidum)、桉树、茴香(茴香(Foeniculum vulgare))、胡芦巴、小白菊、玄参、五香粉(中国)、何首乌(Fo-ti-tieng)、延胡索、高良姜、葛拉姆马萨拉、独行菜、韭菜、大蒜、姜(生姜(Zingiber officinale))、银杏(Ginkgo biloba)、人参、人参、枞树(刺五加)、山羊豆(山羊豆(Galega officinalis))、Goada masala、一枝黄、北美黄莲、雷公根、摩洛哥豆蔻(非洲豆蔻(Aframomum melegueta))、Grains of Selim(Xylopia aethiopica)、葡萄籽提取物、绿茶、常春藤、瓜柯、吉普赛草、山楂(辽宁山樝(Crataegus sanguinea))、山楂树、大麻、普罗旺斯草、木槿、冬青、水飞蓟、啤酒花、苦薄荷、山葵、马尾(Equisetum telmateia)、牛膝草(神香草(Hyssopus officinalis))、球根牵牛、茉莉、珍珠茉莉(Jasmin pearl)、绞股蓝(七叶胆(Gynostemma pentaphyllum))、Joe Pye weed(Gravelroot)、John the Conqueror、杜松、青柠叶(箭叶橙(Citrus hystrix)、C.papedia)、Kaala masala、紫菀科植物、Kokam、拉不拉多茶、篷子菜、斗篷草、陆生水芹、薰衣草(熏衣草属(Lavandula spp.))、杜香、蜜蜂花(蜜里萨香草(Melissa officinalis))、柠檬罗勒、柠檬香草(柠檬草(Cymbopogoncitratus)、曲序香茅(C.flexuosus)和其它种)、柠檬铁皮木(Eucalyptus staigeriana)、柠檬薄荷、柠檬桃金娘(柠檬巴毫(Backhousia citriodora))、百里香、柠檬马鞭草(柠檬过江藤(Lippia citriodora))、甘草-适应原、酸橙花、紫苏草(Limnophila aromatica)、亚麻籽、甘草、荜拨、独活草(欧当归(Levisticum officinale))、罗汉果、肉豆蔻干皮(Mace)、Mahlab、三条筋树叶(Malabathrum)、满洲荆棘树(Manchurian Thorn Tree)(满洲楤木(Aralia manchurica))、曼德拉草、马郁兰(墨角兰(Origanum majorana))、欧夏至草(Marrubium vulgare)、沼泽拉布拉多茶、药蜀葵、乳香、绣线菊、Mei Yen、天堂椒(非洲豆蔻(Aframomum melegueta))、薄荷、奶蓟草(水飞蓟(Silybum))、佛手柑(管蜂香草(Monardadidyma))、益母草、山地美黄芩(Mountain Skullcap)、毛蕊花(毛蕊花(Verbascumthapsus))、芥末、芥菜籽、Nashia inaguensis、印度楝、假荆芥、荨麻、黑种草(Nigellasativa)、Kolanji、黑页蒿、诺丽、肉豆蔻、肉豆蔻干皮、大麻、月见草(月见草(Oenotherabiennis))、Olida(Eucalyptus olida)、牛至(牛至(Origanum vulgare)、冬牛至(O.heracleoticum))、鸢尾根、香根芹、橄榄叶(用于茶和作为草药补充品)、西洋参(Panaxquinquefolius)、班兰叶、红椒、欧芹(香芹(Petroselinum crispum))、西番莲、广藿香、薄荷油、胡椒(黑胡椒、白胡椒和青胡椒)、胡椒薄荷、杏仁桉(薄荷桉(Eucalyptus dives))、紫苏、车前草、石榴、Ponch phoran、罂粟籽、报春花(报春花)、糖制的花(candied flower)、干茶混合物、车前草、马齿苋、苦木、Quatre epices、熊葱、覆盆子、覆盆子(叶)、蕈类草药(Reishi)、芒柄花、红景天(Rhodiola rosea)、Riberry(Syzygium luehmannii)、芝麻菜/芝麻菜、罗马洋甘菊、路易波士、玫瑰果、迷迭香(迷迭香(Rosmarinus officinalis))、花楸浆果、芸香、红花、藏红花、鼠尾草(鼠尾草(Salvia officinalis))、肉桂、金丝桃、小地榆(小地榆(Sanguisorba minor)或Poterium sanguisorba)、丹参、花椒(山椒)、黄樟、香薄荷(夏季香薄荷(Satureja hortensis)、冬季香薄荷(S.montana))、五味子(五味子(Schisandrachinensis))、哥斯达黎加黄芩(Scutellaria costaricana)、番泻叶(草药)、决明(Sennaobtusifolia)、芝麻籽、羊酸模、荠菜、催涎剂、西伯利亚人参(刺五加(Eleutherococcussenticosus))、罗汉果(Siraitia grosvenorii)(罗汉果)、美黄芩、黑刺李浆果、SmudgeStick、苦苣菜、酸模(酸模(Rumex spp.))、青蒿、绿薄荷、婆婆纳、海葱、八角茴香、甜叶菊、草莓叶、巴西人参(南美苋(Pfaffia paniculata))、漆树、夏季香薄荷、Sutherlandiafrutescens、甜仙草、香根芹(欧洲没药(Myrrhis odorata))、香车叶草、四川胡椒(山椒(Xanthoxylum piperitum))、塔柯胶、罗望子、吞多利粉、艾菊、龙嵩(狭叶青蒿(Artemisiadracunculus))、茶、狭叶香科(Teucrium polium)、泰国罗勒、蓟、百里香、Toor DaIl、直立委陵菜、刺蒺藜(Tribulus terrestris)、圣罗勒(圣罗勒(Ocimum tenuiflorum))、郁金(姜黄(Curcuma longa))、熊果(Uva Ursi)(也称为熊果(Bearberry))、香草(香草(Vanillaplanifolia))、鸭嘴花(Vasaka)、马鞭草、香根草、越南香菜(越南香菜(Persicariaodorata))、芥末(山嵛菜(Wasabia japonica))、豆瓣菜、金合欢籽、细辛、野莴苣、欧百里香、冬香薄荷、金缕梅、西方雪果、水杨梅、马先蒿、车叶草、苦艾、蓍草、加州小薄荷、耶巴马黛茶、育亨宾、Za’atar、蓬莪术,或其溶于水性溶液或半水性溶液的衍生物。
培养浸没的菌丝液体组织培养物的步骤可通过任何本领域已知方法完成。在一个实施方式中,所述培养浸没的菌丝液体组织培养物的方法可参见例如2014年3月15日提交的PCT/US14/29989,2014年3月15日提交的PCT/US14/29998,2014年3月15日提交的U.S.61/953,821,2014年3月15日提交的U.S.61/953,823,2014年8月26日提交的U.S.62/042,071,所有专利都通过引用全文纳入本文。
在一个实施方式中,所述浸没的菌丝液体组织培养物在生物反应器压力容器中完成,所述容器理想上用准球形圆顶、柱体和球冠基底构建,所述基底绕机体夹套,装有磁力驱动混合器,以及通过蜷缩夹套空间的端口以提供含DO探针、pH计、电导仪、热电偶等的设备入口,如本领域已知。这些仪表和探针应记录数据。在一个实施方式中,所述柱形基底具有连接收获线的阀(所述线分支到另一三通的阀,分支到池式地漏并与CIP滑轨同轴),与巴氏消毒滑轨同轴的收获线三通,以及最后的干燥装置如喷雾干燥器、流化床干燥器、锥形干燥器或其它干燥应用。在一个实施方式中,所述加工的菌丝液体组织培养物能从干燥器中立即包装。一个样品应保持为对照且一个合适样品送至有分析供应商证书的第三方质量控制。空气可由连接空气压缩机的120/240V空气接受器槽提供。空气压缩机通过有上游和下游阀的调压器释放空气,紧邻上游阀上游的是三通,分支到通向另一三通的阀,分支到CIP滑轨的阀,与带阀的蒸汽供应同轴,后压调节阀与阀和不锈钢轴承壳中的0.2μm不锈钢滤器(其能在超声波水槽中清洗)同轴,其通向任选的止回阀以使阀在压力容器圆顶上,最终的阀系统任选地在止回阀上游,分支到通向2个类似止回阀的Y型管,阀设置到360°喷雾头。2个喷雾头放置成引起贯穿容器的空气过滤器所呈现的阴影。沿着该设置的压力计可严格放置成监控压力,流量计用于监测空气供给速度。额外气体接受器槽如氧气槽能嵌入置于调压器与滤器之间以矫正任何气体分压。发明人推荐背对背滤筒,尽管这不必需。气体经开启压力低的止回阀耗尽,如门阀或开启压力为2-3psi的弹簧止回阀,到维持容器于5-25psi的背压调节器。背压调节器还能通向疏水阀和池式地漏。在一个实施方式中,所述设置提供0.5-5.0ACH。也可使用本领域技术人员已知的其它工程方案。
所述反应器优选用无菌接种的方式配备。在一个实施方式中,为接种反应器,从冰箱取出真菌的甘油储存物,从其密封包装中移出并结合交叉物,与小室的阀同轴,所述储存物由有带阀的可高温高压消毒(如聚丙烯)容器组成。交叉的十字管在两端都带阀,上游阀连接持有不锈钢0.2μm滤器的不锈钢轴承壳。该管连接带阀的三通(也在上游侧装有阀),与主要的空气供应管同轴。交叉的下游是蒸汽带到池式地漏的阀。运行蒸汽以对甘油储备与小室阀之间的空气进行消毒。一旦消毒和冷却,甘油储存物与小室阀之间的真空被破坏。交叉物任一侧的阀关闭,甘油储备和压力容器上的阀打开以接种培养基。也可使用本领域技术人员已知的其它工程方案。
反应器应配备成装满水。供水系统理想上是WFI系统,在蒸馏器与反应器之间是灭菌管线。应向槽预灭菌加入固体培养基成分,理想上通过真空输送系统。高温灭菌快到足以不对培养基产生有害作用。一旦加入水,槽应温和搅拌并接种。在另一实施方式中,向过滤或蒸馏水加入固体培养基成分且液体培养基在高温灭菌并通过灭菌管线泵入压力容器。在另一实施方式中,所述槽装满过滤或蒸馏水,加入固体培养基成分,培养基通过蒸汽处理夹套、小室或两者来灭菌,同时培养基任选地进行搅拌。
至少一个按比例放大的反应器应在槽体积接近1x105前使用。推荐多至3-4个。发明人推荐级别从1x100 L到1x102 L到1x104 L到1x105-6 L。更丰富的培养基能用于按比例放大的反应器和甘油储存前培养的目的(motif)。
在一个实施方式中,依据简单增殖目的(皮氏培养皿到0.1L-4L锥形摇瓶到50%甘油储存物)制备本文公开的甘油储存物。除了上面就生物反应器目的所述的培养基的变化外,皮氏培养皿能包括25-35g/L琼脂。无菌操作,所选的3-90天任何地方生长的皮氏培养皿能增殖到4L锥形烧瓶(或250-1,000mL Wheaton瓶)以在振动台上孵育。容器越小,摇床应更快。发明人推荐40-160RPM,取决于容器尺寸,摆动半径约为1”。振荡1-10天后,可以将摇瓶等分样品(如10-500mL)倒入无菌、带阀的可高温高压消毒容器,其随后用无菌、室温甘油调至40-60%(v/v)。甘油储存物能用水密封口密封并能置于无菌塑料袋内,密封,放入-20℃冰箱以保存并最终冷冻运输到任何生产基地。冰箱理想上是恒温冰箱。未调整至甘油的液体组织培养物储存物也可使用并在4℃或-20°F保存。还可使用4℃保存的甘油储存物。
本发明运用以下概念:任何人等级培养基能用作生产可食用液体菌丝培养物的培养基配方,排除发明背景中讨论的任何人等级成分,如本领域已知以及他处所公开,例如,2014年3月15日提交的PCT/US14/29989,2014年3月15日提交的PCT/US14/29998,2014年3月15日提交的U.S.61/953,821,2014年3月15日提交的U.S.61/953,823,2014年8月26日提交的U.S.62/042,071,所有专利都通过引用全文纳入本文。优选地,氮盐(若使用)是醋酸铵,因为这是最“天然”的盐。其它补充培养基成分包括糙米糖浆、糖蜜、果泥(芒果、苹果等),浓度等级为1x10-2-1x102 mL/L(或简单作为培养基),短粒糙米粉、营养酵母片、羧甲基纤维素、羧甲基纤维素盐、乳清、酪蛋白、植物和种子蛋白。选择成分以使过敏反应可能性最小和提供高产量。醋酸铵任选地纳入作为批式进料成分。
本发明还可与动物级培养基和动物级食品一起使用。
在一个实施方式中,基本培养基液体组织培养物补充有大量完全培养基,从而利用短log时间和次生代谢。
在一个实施方式中,用于本发明真菌组分的真菌菌株在一个实施方式中是冬虫夏草株WC859,可市售获自宾夕法尼亚州立大学(宾夕法尼亚州立大学蘑菇培养保藏中心,可获自农业科学学院,植物病理与环境微生物学系,117Buckhout实验室,宾夕法尼亚州立大学,美国宾夕法尼亚大学园区,16802)。用于本发明的真菌组分可通过本文所述方法制备。可使用本领域已知的其它方法。
或者,真菌液体组织培养物能包括来自冬虫夏草、线形虫草、大团囊虫草、异虫草(Metacordyceps)属的其它真菌种,例如蛹虫草。许多其它种存在于所述属中,然而,这些种一般不商业培养。但预期例如金龟子虫草(C.scarabaeicola)、高雄山虫草(C.takaomontana)、Ophiocordyceps dipterigena、亚马逊线虫草(Ophiocordycepsamazonica)、C.cylindrica、蜂头虫草(Cordyceps sphecocephala)、Metacordycepsmartialis、Ophiocordyceps melonlonthae、Ophiocordyceps nutans、Ophiocordycepscurculionium、Ophiocordyceps australis、Ophiocordyceps tiputini、Cordycepscaloceroides和Cordyceps variabilis具有与冬虫夏草相同或类似的苦味阻断能力。
或者,适合本发明的真菌包括:灵芝、树舌灵芝、蛹虫草、猴头菇、香菇、姬松茸、灰树花、黑木耳、金针菇、云芝、羊肚菌、白桦茸、苦白蹄提取物、木蹄层孔菌、药用层孔菌、层孔菌、松口蘑、美味牛肝菌、紫丁香蘑、杯伞、平菇、银耳、桦剥管菌、猪苓、滑菇、草菇、真姬菇、大球盖菇、硫色绚孔菌和其组合。
在一个实施方式中,本发明包括在培养后制备浸没的菌丝液体组织培养物的无菌丝部分。无菌丝部分包括菌丝生物分子上清固体、细胞材料和浸没的菌丝液体组织培养物的剩余培养基。
如上文所公开,为制备培养物,所制备的培养基接种入溶于水的无菌人等级培养基的容器,优选通过任何本领域已知方法过滤,如反渗透、去离子作用或蒸馏。在另一实施方式中,所述水未过滤。在另一实施方式中,所述培养基是动物级。如所公开,烧瓶和培养基能通过任何本领域已知方法灭菌,如原位暴露于250°F和23PSI饱和蒸汽,持续适当时间量,如就装有1.5L培养基的4.0L锥形烧瓶而言是2-2.5小时。一旦通过任何本领域已知方法冷却,可接种消毒的烧瓶如通过皮氏培养皿、漂浮或浸没液体培养物、长菌丝的农业物料、甘油储存物等接种。如本领域已知,烧瓶在3–60天合适培养后待用,所述培养是例如在洁净室内130RPM、室温的振动台上培养。可以制备烧瓶中所留下剩余培养物的对照皮氏培养皿以确保烧瓶没有污染。烧瓶还能用于含有相同品质的培养基的规模更大的生物反应器(如5–500L),其能以类似方式使用。
在一些实施方式中,所述真菌液体组织培养物是在液体培养基中生长的冬虫夏草,所述培养基由8g/L有机马铃薯淀粉粉末和0.8g/L有机胡萝卜粉组成。发明人发现该基本培养基是生成前述苦味阻断剂(口味提升食品)的有效培养基配方。用不同培养基,会失去本发明产品的苦味阻断作用/口味提升,如使用加入20g/L有机芒果泥,其在甜菊糖苷水溶液中引入风味缺陷。所得上清液粉末可用作本文所述产品应用的苦味阻断剂。
培养适当时间后,可以从培养物中收集无菌丝部分(如本文所定义),所述时间可由本领域技术人员确定。浸没液体菌丝液体组织培养物的该无菌丝部分任选地用于改善和/或提升食品口味。例如,培养能发生约1-约60天、约2-约50天、约3-约40天、约4-约30天、约5-约25天、约6-约20天、约7-约15天、约8-约12天和约9-约10天。例如,培养时间长度能由以下确定:培养天数的经济方面考量和就特定培养时间观察到的口味提升程度。
用于本发明的培养物可以是含菌丝的任何液体组织培养物,例如浸没或漂浮培养物。浸没培养一般进行搅拌,而漂浮培养最低限度地搅拌,这使得菌丝能以毡样形式生长。本发明所用培养物部分包括任何和所有的培养物部分或区段,包括菌丝、培养物上清或滤液,或其任何比例或分数。在一个实施方式中,所述培养物可在使用前混合(机械或其它),采用完整的混合材料或其一些部分。在一些实施方式中,使用的培养物部分是通常理解为“细胞培养物上清”或“细胞培养物滤液”的培养物部分,即与菌丝细胞分开的培养物液体部分,且包含与菌丝细胞部分相比相对更小或更少量菌丝。因此,应理解此液体组织培养物上清通常也包含菌丝,即使肉眼不可见或甚至在显微镜下容易看到。方便起见,此培养物部分在本文称为“无菌丝”部分,然而,如所述,应理解此部分通常包含一些最少数量的菌丝,即使肉眼不可见。
为制备培养物的无菌丝部分,菌丝能通过任何本领域已知方法取出以分离细胞培养物上清液。例如,培养物可通过任何本领域已知方法过滤以获得滤液,例如0.2μm滤器等。或者,培养物的无菌丝部分可离心收集。所培养浸没的菌丝液体组织培养物的经收集无菌丝部分在本文可称为收集的上清、上清、上清液、冬虫夏草上清、滤液、产物和类似术语如口味提升产品或苦味阻断剂/阻断产品,或苦味阻断剂。
任选地,液体组织培养物能通过本领域已知方法处理以减少或消除活生物体的活力,如巴氏消毒或灭菌。收集的液体组织培养物可通过任何本领域已知方法在分离获得培养物的无菌丝部分之前或之后进行巴氏消毒或灭菌。在一个实施方式中,所述材料在一定条件下灭菌,如约30-50分钟暴露于250°F,23psi的饱和蒸汽。或者,所述材料能如下巴氏消毒:将材料保持于160-170°F热水浴20分钟,2次,在运行之间冷却回室温。
此巴氏消毒或灭菌的液体组织培养物能用作新饮料,或其粉末作为新食物、食物成分、饮食补充剂、膳食成分或食物添加剂,其能在多种产品应用中以0.1-40,000ppm使用。
滤液(收集的上清)可调整其体积或液体组分以生成浓缩物、稀释物或干燥粉末,如本领域技术人员所测。在一个实施方式中,所述滤液任选地通过任何本领域已知方法干燥,包括使用风干、小批量干燥器、真空干燥器、流化床或喷雾干燥器、或冷冻干燥机以使液体干燥成粉末。在一个实施方式中,所述滤液在灭菌/巴氏消毒后干燥。
所得粉末或口味提升产品可用于提高食品口味,且可混合入本文所述任何食物/饮料,浓度根据应用性质为0.1-40,000ppm和甚至更高。确定使用的口味提升产品量可由本领域技术人员实验确定,目标是减少或消除食品中不想要的口味组分和/或提升食品口味,而不引入风味缺陷。
冬虫夏草上清(苦味阻断剂)作为干粉用于多种食品的一般浓度范围示于下表9。根据口味谱确定冬虫夏草上清用于特定产品的最优比例在本领域技术范围内。例如,冬虫夏草上清浓度过高时,风味增强作用会停止或产品会将风味缺陷引入终材料。上清浓度过低时,口味改善程度不足。农业物料的浓度最终确定理想的苦味阻断剂浓度,所述物料如甜菊糖苷混合物通常以200-450ppm使用。例如,连续稀释/浓度能用作确定上清使用上和下限阈值浓度的工具。需要测试以何种初始所需浓度将苦味阻断剂配制到材料中。如果提供所需风味变化,则浓度减半,直到风味变化不足。在起作用和不起作用之间取终浓度,以平均值施用苦味阻断剂。若起作用,则浓度减半,直到其不再起作用,不作用之上的浓度是下限阈值浓度。若不起作用,浓度加倍,直到起作用。下限阈值浓度能无限加倍以达到上限阈值浓度,其中品尝员确定风味改良作用是否最终失去或苦味阻断剂开始引入风味缺陷。
粉末还可再水合,过滤和重新干燥以增加产品溶解度。喷雾干燥产品具有高溶解度且任选地在使用前不再水合,可作为粉末与食品简单混合(尤其是在非营养性甜味剂应用中)。或者,风味提升食品可组合液体形式的食品,食品/风味提升食品任选地一起干燥。上清液粉末还可在流化床中干燥,或在流化产品上喷雾干燥且甚至聚集,如在生成含产品的甜菊糖苷混合物中。
本发明包括由本文所公开方法制备的苦味阻断剂产品。
本发明提供在世界各地培养浸没的菌丝作为人类食物的有效方式,这是通过在生产点呈现液体组织储存物形式的接种体源,所述储存物调至50%(v/v)甘油,其能在-20℃维持。此培养物至少就2种菌株测试(灵芝和冬虫夏草),显示其在-20℃保存越长,依赖于复苏的活力增加,且不需要在增殖前加热。
本发明还提供生产食品的方法,包括在培养基中培养浸没的菌丝液体组织培养物,收集上清的无菌丝部分,和使用培养物的无菌丝部分作为食品。本文公开了使用的合适真菌、合适培养基、收集上清的无菌丝部分的合适方法。培养液(或条件培养基)的无菌丝部分能自身用作食品。无菌丝部分任选地如本文所公开进行浓缩、稀释或干燥,可在使用前组合本文所公开的任何食品。本发明还包括由本文所公开工艺制备的食品。
提供以下实施例仅用于阐明目的,而不意在限制发明范围。
实施例
实施例1
RO过滤的水性提取物制备自1lb.有机/新鲜马铃薯和胡萝卜以及1L有机果汁以在6个4L锥形烧瓶中形成1L培养物。这些培养物用任意0-100%甜叶菊/茶水性提取物制备。烧瓶进行高压灭菌并冷却。一旦冷却,冬虫夏草WC859的对数期皮氏培养皿培养物在烧瓶中增殖并随后搅拌(60RPM,摆动半径为1/2英寸)。在约3-4天中观察到完全发育的液体组织培养物(对数期生长)。将20g甜叶菊叶置于食品级容器,向容器加入约100mL上述对数期液体培养物。容器能在约华氏75度孵育、覆盖约6小时。孵育后,甜叶菊叶进行少许巴氏消毒并干燥。5g经处理甜叶菊叶浸泡入一杯水,过滤并在随机双盲试验中品味,由5名测试人员接受未处理甜叶菊。测试人员发现经处理甜叶菊的甜味相较未处理对照甜叶菊增加并具有减少的苦味/甘草回味。
实施例2
RO过滤水性提取物制备自1lb.有机/新鲜马铃薯和胡萝卜以及1L有机果汁以形成6个4L锥形烧瓶中1L培养物。这些培养物用任意0-100%茶水性提取物制备。烧瓶进行高压灭菌并冷却。一旦冷却,冬虫夏草株WC859的对数期皮氏培养皿培养物在烧瓶中增殖并随后搅拌(60RPM,摆动半径为1/2英寸)。在约3-4天中观察到完全移生的对数期液体组织培养物。将约20g绿茶叶置于食品级容器,向容器加入约100mL上述对数期培养物。容器能在约华氏75度孵育、覆盖约6小时。孵育结束后,根据口味测试,轻轻冲洗绿茶叶,温和巴氏消毒并干燥。5g经处理绿茶叶进行干燥并在一杯水中泡,过滤并在随机双盲试验中品味,由5名测试人员接受未处理对照绿茶叶。测试人员发现经处理绿茶叶的苦味相较对照绿茶叶减少。
实施例3
干净的1.5L带柄玻璃瓶装有1L培养基,所述培养基由17g/L琼脂、8g/L有机马铃薯淀粉、0.8g/L有机胡萝卜粉和20mL/L有机芒果泥组成。带柄玻璃瓶的盖子松松拧上并覆盖有锡箔。发明人推荐使用这些带柄玻璃瓶是因为其手柄使得倾倒更容易。瓶置于高压釜中并以2.33小时液体循环灭菌。一旦循环完成,瓶迅速置于无菌层流净化罩冷却,直至其能触碰,这花费约1.3小时。此时,将瓶的内容物仔细倒入120皮氏培养皿。培养皿在罩内冷却过夜。
一旦冷却,来自存储培养物的真菌用于接种最近倾注的培养皿。这些真菌在相同培养基上生长。真菌用无菌12”竹签转移,所述竹签在梅森玻璃瓶中高温高压灭菌,采用来自前一天的琼脂。一种这类真菌种是猴头菇。制备15个猴头菇培养皿,选择1个用于在增殖后8天到4L锥形烧瓶内增殖。在生长第7天,准备4L锥形烧瓶。该烧瓶包含1.5L培养基,所述培养基由8g/L玉米粉、4g/L有机燕麦粉、2g/L有机芒果泥和2g/L有机马铃薯淀粉粉末组成。烧瓶以60RPM振荡6天,摆动半径为1”。在此培养的第2天,用58L RO水和浓缩物填装100L生物反应器,所述浓缩物含800g有机马铃薯淀粉粉末、80g有机胡萝卜粉、50g混合的有机软白小麦粒和1L有机芒果泥,用RO水调至2L,倒入反应器使体积为60L。反应器无夹套,因而,注入121-122℃并通过连接由本领域技术人员设置的压力容器头部的集合管排入小室。生物反应器以4.5小时液体循环灭菌,由于蒸汽凝结而填充到85L。反应器通过热扩散冷却到室温,持续4天,在该时间点接种。
容器有进气管线的入口,其包括1/4马力,115V,50/60Hz空气压缩机,通过2个嵌入式0.2μm可高温高压灭菌的胶囊滤器,通过止回阀和球阀将空气供应到小室内。完整的胶囊滤器阀设置在生物反应器和培养基消毒前灭菌,以无菌操作组装到生物反应器上。一旦86小时后冷却,运送空气以使容器加压,但不是通过排气支管运送,排气支管关闭且容器头部的压力计立即取出从而产生带正压的喷嘴。本领域技术人员移去浸没猴头菇培养物的盖子,将前5英寸锥形烧瓶在丙烷焰(propane torch)上烧,一旦烧瓶冷却(8秒等待时间),烧瓶通过带正压喷嘴倒入生物反应器。压力计放回反应器且排气支管立即打开。反应器在2-3psi平衡,这是止回阀进口和出口的破裂压力。制备猴头菇接种剂的皮氏培养皿用于QC。
如此供应空气,生物反应器培养13天。培养物似乎在第2天进入对数期,活跃生长,到第9天达0.5cm球体,在该处细胞分裂似乎停止。在第13天,生物反应器的内容物倒入6m2塑料桶,有带边缘的10英寸壁,桶包被有食品级塑料薄片。桶保持约4英尺高度,放置2个风扇以在桶上吹气。4天后,培养物干燥,回收牛肉干样材料并混合产生724g粉末。粉末有很淡的胡萝卜味,主要是极中性的谷物式口味。
实施例4
4L烧瓶装有溶于RO水的1.5L 8g/L有机马铃薯淀粉和0.8g/L有机胡萝卜粉,烧瓶灭菌并从2周龄P1冬虫夏草培养物接种。以60RPM(1”摆动半径)室温培养7天后,培养物经3个堆叠的咖啡滤器过滤,165°F巴氏消毒40分钟并置于140°F的小批量干燥器过夜。第二天收集干燥材料并混合,就总共6.75g而言产量为4.5g/L。将5g收获的材料倒入1L RO水并立即振荡15分钟。从此存储培养物中取53.34mL溶液加入含1kg 97%莱苞迪苷A的另一溶液,莱苞迪苷A溶于1.6L RO水。此溶液充分混合并在小批量干燥器干燥过夜,所得材料混合并包装于干净的自封袋,收集的过滤固体浓度为2,667ppm。向500mL RO水加入150mg此混合物产生300ppm97%莱苞迪苷A溶液,有0.8ppm冬虫夏草上清固体。当针对对照测试口味时,所有3个发明人清楚了解,含冬虫夏草上清固体的甜菊糖苷混合物回味相较于对照即300ppm97%莱苞迪苷A溶液无法检测。
实施例5
4L烧瓶装有溶于RO水的1.5L 8g/L有机马铃薯淀粉和0.8g/L有机胡萝卜粉,烧瓶灭菌并从2周龄P1冬虫夏草培养物接种。以60RPM(1”摆动半径)室温培养15天后,培养物经3个堆叠的咖啡滤器过滤,165°F巴氏消毒40分钟并置于140°F的小批量干燥器过夜。第二天收集干燥材料并混合,就总共6.15g而言产量为4.1g/L。将5g收获的材料倒入1L RO水并立即振荡15分钟。从此存储培养物中取53.34mL溶液加入含1kg 97%莱苞迪苷A的另一溶液,莱苞迪苷A溶于1.6L RO水。此溶液充分混合并在小批量干燥器干燥过夜,所得材料混合并包装于干净的自封袋,收集的过滤固体浓度为2,667ppm。向500mL RO水加入150mg此混合物产生300ppm97%莱苞迪苷A溶液,有0.8ppm冬虫夏草上清固体。当针对对照测试口味时,所有3个发明人清楚了解,含冬虫夏草上清固体的甜菊糖苷混合物回味相较于对照即300ppm97%莱苞迪苷A溶液无法检测。
实施例6
4L烧瓶装有溶于RO水的1.5L 8g/L有机马铃薯淀粉和0.8g/L有机胡萝卜粉,烧瓶灭菌并从2周龄P1冬虫夏草培养物接种。以60RPM(1”摆动半径)室温培养35天后,培养物经3个堆叠的咖啡滤器过滤,165°F巴氏消毒50分钟并置于140°F的小批量干燥器过夜。第二天收集干燥材料并混合,就总共8.25g而言产量为5.5g/L。将5g收获的材料倒入1L RO水并立即振荡,在转向培养基的电热板上加热15分钟。从此存储培养物中取53.34mL溶液加入含1kg 97%莱苞迪苷A的另一溶液,莱苞迪苷A溶于1.6L RO水。此溶液充分混合并在小批量干燥器干燥过夜,所得材料混合并包装于干净的自封袋,收集的过滤固体浓度为2,667ppm。向500mL RO水加入150mg此混合物产生300ppm 97%莱苞迪苷A溶液,有0.8ppm冬虫夏草上清固体。当针对对照测试口味时,所有3个发明人清楚了解,含冬虫夏草上清固体的甜菊糖苷混合物回味相较于对照即300ppm 97%莱苞迪苷A溶液无法检测。
实施例7
4L烧瓶装有溶于RO水的1.5L 8g/L有机马铃薯淀粉和0.8g/L有机胡萝卜粉,烧瓶灭菌并从2周龄P1冬虫夏草培养物接种。以60RPM(1”摆动半径)室温培养7天后,培养物经粗棉布过滤,160°F巴氏消毒50分钟并置于130°F的小批量干燥器过夜。第二天收集干燥材料并混合,就总共6.6g而言产量为4.4g/L。将5g收获的材料倒入1L RO水并立即振荡15分钟。从此存储培养物中取53.34mL溶液加入含1kg 97%莱苞迪苷A的另一溶液,莱苞迪苷A溶于1.6L RO水。此溶液充分混合并在小批量干燥器干燥过夜,所得材料混合并包装于干净的自封袋,收集的过滤固体浓度为2,667ppm。向500mL RO水加入150mg此混合物产生300ppm97%莱苞迪苷A溶液,有0.8ppm冬虫夏草上清固体。当针对对照测试口味时,所有3个发明人清楚了解,含冬虫夏草上清固体的甜菊糖苷混合物回味相较于对照即300ppm 97%莱苞迪苷A溶液无法检测。
实施例8
4L烧瓶装有溶于RO水的1.5L 8g/L有机马铃薯淀粉和0.8g/L有机胡萝卜粉,烧瓶灭菌并从2周龄P1冬虫夏草培养物接种。以60RPM(1”摆动半径)室温培养10天后,培养物经3个堆叠的咖啡滤器过滤,170°F巴氏消毒40分钟并置于140°F的小批量干燥器过夜。第二天收集干燥材料并混合,就总共6.9g而言产量为4.6g/L。将5g收获的材料倒入1L RO水并立即振荡15分钟。从此存储培养物中取40.00mL溶液加入含1kg 97%莱苞迪苷A的另一1.6L溶液。此溶液充分混合并在小批量干燥器干燥过夜,所得材料混合并包装于干净的自封袋,收集的过滤固体浓度为2,000ppm。向500mL RO水加入150mg此混合物产生300ppm 97%莱苞迪苷A溶液,有0.6ppm冬虫夏草上清固体。当针对对照测试口味时,所有3个发明人清楚了解,含冬虫夏草上清固体的甜菊糖苷混合物回味相较于对照即300ppm 97%莱苞迪苷A溶液无法检测。此甜菊糖苷混合物口味与含0.8ppm上清固体的混合物非常相似。
实施例9
4L烧瓶装有溶于RO水的1.5L 8g/L有机马铃薯淀粉和0.8g/L有机胡萝卜粉,烧瓶灭菌并从10天龄P1冬虫夏草培养物接种。以60RPM(1”摆动半径)室温培养4天后,培养物经粗棉布过滤并置于140°F的小批量干燥器过夜。第二天收集干燥材料并混合,就总共6.75g而言产量为4.5g/L。将5g收获的材料倒入1L RO水并立即振荡15分钟。从此存储培养物中取53.34mL溶液加入含1kg 97%莱苞迪苷A的另一溶液,莱苞迪苷A溶于1.6L RO水。此溶液充分混合并在小批量干燥器干燥过夜,所得材料混合并包装于干净的自封袋,收集的过滤固体浓度为2,667ppm。向500mL RO水加入150mg此混合物产生300ppm 97%莱苞迪苷A溶液,有0.8ppm冬虫夏草上清固体。当针对对照测试口味时,所有3个发明人清楚了解,含冬虫夏草上清固体的甜菊糖苷混合物回味相较于对照即300ppm 97%莱苞迪苷A溶液无法检测。
实施例10
4L烧瓶装有溶于RO水的1.5L 8g/L有机马铃薯淀粉和0.8g/L有机胡萝卜粉,烧瓶灭菌并从2周龄P1冬虫夏草培养物接种。以60RPM(1”摆动半径)室温培养7天后,培养物经3个堆叠的咖啡滤器过滤并置于140°F的小批量干燥器过夜。第二天收集干燥材料并混合,就总共6.75g而言产量为4.5g/L。将5g收获的材料倒入1L RO水并立即振荡15分钟。从此存储培养物中取53.34mL溶液加入含1kg 60%莱苞迪苷A的另一溶液,莱苞迪苷A溶于1.6L RO水。此溶液充分混合并在小批量干燥器干燥过夜,所得材料混合并包装于干净的自封袋,收集的过滤固体浓度为2,667ppm。向500mL RO水加入150mg此混合物产生300ppm 60%莱苞迪苷A溶液,有0.8ppm冬虫夏草上清固体。当针对对照测试口味时,所有3个发明人清楚了解,含冬虫夏草上清固体的甜菊糖苷混合物回味相较于对照即300ppm 60%莱苞迪苷A溶液无法检测。
实施例11
4L烧瓶装有溶于RO水的1.5L 8g/L有机马铃薯淀粉和0.8g/L有机胡萝卜粉,烧瓶灭菌并从20天龄P1冬虫夏草培养物接种。以60RPM(1”摆动半径)室温培养7天后,培养物经0.2μm真空滤器过滤并置于150°F的小批量干燥器过夜。第二天收集干燥材料并混合,就总共6.45g而言产量为4.3g/L。将5g收获的材料倒入1L RO水并立即振荡15分钟。从此存储培养物中取53.34mL溶液加入含1kg 60%莱苞迪苷A的另一溶液,莱苞迪苷A溶于1.6L RO水。此溶液充分混合并在小批量干燥器干燥过夜,所得材料混合并包装于干净的自封袋,收集的过滤固体浓度为2,667ppm。向500mL RO水加入150mg此混合物产生300ppm 60%莱苞迪苷A溶液,有0.8ppm冬虫夏草上清固体。当针对对照测试口味时,所有3个发明人清楚了解,含冬虫夏草上清固体的甜菊糖苷混合物回味相较于对照即300ppm 60%莱苞迪苷A溶液无法检测。
实施例12
有16种不同培养基配方用于确定培养基针对60%莱苞迪苷A样品的苦味阻断活性效果,采用实施例4的方法,不同培养基如下所示。下表1显示哪种培养基接受测试和感官反应总结。
表1显示尽管不是每个配方都起作用,但是许多配方可应用于生成苦味阻断剂。发明人推荐本文所述马铃薯/胡萝卜或玉米/燕麦配方。
实施例13
所公开苦味阻断剂的分子组成从2个40L批次的200L冬虫夏草浸没培养物制备的样品中确定,所述培养物生长于8g/L有机马铃薯淀粉和0.8g/L有机胡萝卜粉RO水培养基。培养物在41和48天收获,总共230g粉末苦味阻断剂(产量~2.9g/L),其混合在一起。150g样品用于第三方组成分析。数据在技术上一式二份获取,显示该批苦味阻断剂是86.9%碳水化合物。按浓度降低顺序,所述材料进一步由水、灰分、脂肪和蛋白质组成。此研究中没有检测到对食物供应而言外源的分子。这些数据概括于表2,而更详细的信息示于随后的表。也列出千卡(在食物标签上通常称为‘卡路里’)。苦味阻断剂通常在培养第8-第12天加工,但采用此方法以加强理解产品最浓缩形式,即转化程度最大的培养基。
苦味阻断剂的脂质含量可能引起其一部分疏水性质。苦味阻断剂在水溶液中加热至140–160°F时,溶解更快。所述批次在室温下就0.3g而言花费15分钟溶于500mL,伴有间歇搅拌。脂质含量如表3所示,由10种不同分子组成,有趣的是,其包含2种必需脂肪酸。这些分子和后续表中所有分子的分子结构如附录所示。平均值之和指示这些数据说明99.3%总脂质谱。
脂肪含量如表4所示,提供饱和、多不饱和和单不饱和脂肪分解,以及样品的ω酸分解。
下表5详细说明苦味阻断剂的盐、一些元素小分子和维生素分解。
表6所示苦味阻断剂的稀少氨基酸含量由天冬氨酸、谷氨酸、半胱氨酸和赖氨酸组成。
表7显示苦味阻断剂的碳水化合物含量和分解。β-葡聚糖和几丁质是总真菌生物量的良好指标(如表5所示的麦角固醇和D-甘露醇)。这些数据说明约99.8%的碳水化合物谱。
下表8概括苦味阻断剂的NBST含量。数据表明激活补救途径以生成生长所需的NBST材料。应注意苦味阻断剂NBST含量如何是冬虫夏草粉末NBST含量的精简设置。未保留的NBST必须是胞内的。
GC/MS研究显示苦味阻断剂存在3种可挥发生物分子。这些是棕榈酸甲酯、9-十八酸甲酯和硬脂酸甲酯。一旦实施标准,可确定其浓度。
实施例14
冬虫夏草上清粉末(苦味阻断剂)由实施例4所概括的方法生产并基于ppm用于食品。
呈现多个实施方式的描述用于阐明和描述目的,而不意在穷尽或将发明限于所公开形式。本发明范围仅由下列权利要求范围限制。许多修改和变化对本领域普通技术人员显而易见。选择所述实施方式并描述以解释本发明原理、实际应用和使本领域普通技术人员能理解发明用于多个实施方式和多种修改,适合所考虑特定应用。本文引用的所有参考文献通过引用全文纳入。
Claims (8)
1.一种组合物,包含食品和从菌丝液体组织培养物分离的上清的组合,其中所述组合苦味减少,不想要的回味减少,和/或涩味减少,
其中所述食品包括豌豆蛋白质;以及其中用于培养所述菌丝液体组织培养物的真菌是冬虫夏草Cordyceps sinensis。
2.如权利要求1所述的组合物,其中所述上清是干燥的上清。
3.如权利要求1所述的组合物,其中通过过滤或离心获得从所述菌丝液体组织培养物分离的上清。
4.从长菌丝的液体组织培养物分离的上清用于提升食品口味的用途,
其中所述提升食品口味包括减少食品的苦味,减少食品的不想要的回味,和/或减少食品的涩味,其中所述食品包括豌豆蛋白质;以及其中用于培养所述菌丝液体组织培养物的真菌是冬虫夏草Cordyceps sinensis。
5.如权利要求4所述的用途,其中对所收集的上清液进行灭菌和/或干燥。
6.如权利要求4所述的用途,其中对所述菌丝液体组织培养物进行过滤或离心。
7.如权利要求4所述的用途,其中所述培养步骤进行1-60天。
8.如权利要求5所述的用途,其中所述灭菌是巴氏消毒。
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CN103314782A (zh) * | 2013-06-17 | 2013-09-25 | 吉林农业大学 | 一种制备红汁乳菇菌根的方法 |
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KR102034835B1 (ko) | 2019-10-21 |
JP2017526364A (ja) | 2017-09-14 |
CN106604650A (zh) | 2017-04-26 |
US20160058049A1 (en) | 2016-03-03 |
EP3185700B1 (en) | 2021-07-28 |
KR20170044729A (ko) | 2017-04-25 |
BR112017002819B1 (pt) | 2022-02-22 |
ES2886643T3 (es) | 2021-12-20 |
SG11201701444RA (en) | 2017-03-30 |
WO2016033241A1 (en) | 2016-03-03 |
BR112017002819A2 (pt) | 2017-12-19 |
AU2015308905B2 (en) | 2018-08-09 |
CN113519814B (zh) | 2024-04-02 |
JP6616826B2 (ja) | 2019-12-04 |
US9572363B2 (en) | 2017-02-21 |
EP3185700A4 (en) | 2018-01-10 |
SG10201810374WA (en) | 2018-12-28 |
AU2015308905A1 (en) | 2017-03-09 |
CN106604650B (zh) | 2021-08-24 |
EP3185700A1 (en) | 2017-07-05 |
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