WO2017206146A1 - 假丝酵母菌菌株及其应用，以及加工普洱茶的方法 - Google Patents

假丝酵母菌菌株及其应用，以及加工普洱茶的方法 Download PDF

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WO2017206146A1
WO2017206146A1 PCT/CN2016/084532 CN2016084532W WO2017206146A1 WO 2017206146 A1 WO2017206146 A1 WO 2017206146A1 CN 2016084532 W CN2016084532 W CN 2016084532W WO 2017206146 A1 WO2017206146 A1 WO 2017206146A1
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tea
candida
strain
sun
fermentation
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PCT/CN2016/084532
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English (en)
French (fr)
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高林瑞
唐蜀昆
刘韬
卢开阳
任万增
童一峰
黄华伟
田飞
邹小林
卢晓慧
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勐海茶业有限责任公司
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Priority to PCT/CN2016/084532 priority Critical patent/WO2017206146A1/zh
Publication of WO2017206146A1 publication Critical patent/WO2017206146A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00—Tea; Tea substitutes; Preparations thereof
    • A23F3/06—Treating tea before extraction; Preparations produced thereby
    • A23F3/08—Oxidation; Fermentation
    • A23F3/10—Fermentation with addition of microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida

Definitions

  • the invention belongs to the field of biotechnology, and in particular to a strain of Candida and its application, and a method for processing Pu-erh tea.
  • yeast has been widely used in many aspects of people's lives, such as brewing, fermenting juice, fermenting bread, making dairy products, and making delicious food.
  • Yeast has a natural and rich nutrient system and is a natural, non-polluting, biologically healthy source of nutrients. Yeast is hailed as the most attractive nutrient by the nutrition community. According to reports, yeast contains more than 50 nutrients, proteins, vitamin B groups, minerals, nucleic acids, etc. necessary for human health can be obtained from yeast.
  • the present invention provides a Candida strain and its use, and a method of processing Pu-erh tea.
  • the present invention provides:
  • Candida blankii strain deposited under the accession number CGMCC No. 8682, was deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on January 6, 2014.
  • CGMCC General Microbiology Center
  • the invention also provides a method for processing Pu-erh tea, comprising separating and purifying the silk
  • the yeast Candidadida blankii
  • the yeast is inoculated into the large-leaf sun-dried tea and fermented.
  • the large leaf type sun-green tea is a large leaf type sun-dried tea that has been tidal.
  • the moisture content of the tidal large leaf type sun-dried tea is 20-40% (m/m) based on the total weight of the sun-dried tea before the tidal water.
  • the Candida in the growth index period is inoculated into the sun-dried tea.
  • the inoculum amount of the Candida is 0.5 x 10 4 to 1.0 x 10 6 per gram of the sun-dried tea.
  • the sun-dried tea is sterilized prior to the inoculation.
  • the fermentation culture is fermented for 2-6 weeks at 20-50 °C.
  • the fermentation culture is fermented at 25-45 ° C for 2-4 weeks.
  • the culture is hydrated during the fermentation culture.
  • the culture is turned over during the fermentation culture.
  • the Candida species is a Candida strain deposited under the number CGMCC No. 8682.
  • the invention also provides the application of Candida blankii in the processing of Pu'er tea.
  • the Candida species is a Candida strain deposited under the number CGMCC No. 8682.
  • the invention has the following advantages and positive effects:
  • the present invention separates Candida blankii from the fermentation sample of Pu'er tea, and applies Candida for the first time in the fermentation processing of Pu'er tea.
  • the invention finds that the Candida yeast is inoculated into the large-leaf type sun-dried green tea, and after a period of fermentation, the Pu'er cooked tea with the soup color deep red slightly, the sweetness is obvious, and the scent of the scented flower is high and long-lasting is obtained.
  • the content of theanine in Pu'er tea is 1.5-2.1% (m / m)
  • the content of the tea brown pigment is 7.4-12.4% (m / m)
  • soluble sugar content It is 1.2-2.9% (m/m)
  • the water extract content is 26.3-38.8% (m/m).
  • the use of Candida blankii for the processing of Pu'er tea not only maintains the original quality of Pu'er tea, but also increases the special aroma of the Pu'er tea and the brightness of the soup, and shortens the hair. The cycle of leaven.
  • the present invention further improves the above advantageous effects by further exploring and optimizing the fermentation conditions in which the Candida species is applied to the processing of Pu-erh tea. This has provided an important application basis for the enrichment of Pu'er tea and the development of new products, and has played a positive role in the development of Pu'er tea industry.
  • the present invention provides a Candida blankii strain having the accession number CGMCC No. 8682. This strain is particularly capable of providing the above-described advantageous effects in the fermentation processing of Pu'er tea.
  • Candida blankii TMCC 70011 strain provided by the present invention has been deposited on the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on January 6, 2014, and the deposit address is: Beichen West Road, Chaoyang District, Beijing. No. 3, No. 3, Zip Code: 100101, the deposit number is: CGMCC No.8682.
  • CGMCC General Microbiology Center
  • Candida blankii strain i.e., TMCC 70011 strain.
  • the strain was deposited on January 6, 2014 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), and the deposit number is CGMCC No.8682.
  • CGMCC General Microbiology Center of China Microbial Culture Collection Management Committee
  • Candida blankii of the present invention is a Candida albicans.
  • Candida blankii is collectively referred to as "Candida albicans”.
  • the inventors of the present invention analyzed the fermentation samples of Pu'er tea in different periods by using Roche's high-throughput 454 sequencing technology and Miseq sequencing technology, and found the existence of Candida albicans, and then passed The TMCC 70011 strain was obtained by pure culture technique.
  • the TMCC 70011 strain is obtained by fermenting a sample of a traditional eucalyptus Pu'er tea during the fermentation process of Pu'er tea by means of plate dilution coating, primary screening, rescreening, purification and the like.
  • TMCC 70011 strain can use conventional PDA medium (glucose 20g, potato 200g, agarose 2g, distilled water 1000mL, pH natural (6.8-7.3)), ISP No. 2 medium (peptone 4g, glucose 4g, yeast extract 5g, A variety of media such as 2 g of agarose, 1000 mL of distilled water, and natural pH (6.8-7.3) were used for resuscitation and activation.
  • the morphological characteristics of the TMCC 70011 strain were as follows: after culturing on ISP No. 2 medium for 48 hours, the colonies were neatly creamy, the surface was moist and lustrous, the edges were not wrinkled, and the diameter was about 0.5 mm. Under the microscope, the cells were long oval and had pseudofilaria. It can grow slowly under anaerobic conditions and is a facultative anaerobic bacterium with a growth temperature between 20 and 50 °C.
  • the physical and chemical characteristics of the TMCC 70011 strain are:
  • the TMCC 70011 strain of the invention has a particularly good effect in the fermentation processing of Pu'er tea, which not only can maintain the original quality of Pu'er tea, but also can increase the special aroma of the Pu'er tea and the brightness of the soup color, and obtain a deep red color of the soup.
  • Pu'er cooked tea with obvious sweetness and high-lasting characteristics In addition, the cycle of fermentation can be shortened. This has provided an important application basis for the enrichment of Pu'er tea and the development of new products, and has played a positive role in the development of Pu'er tea industry.
  • Another aspect of the invention provides a method of processing Pu-erh tea, comprising separating The purified Candida blankii is inoculated into the large-leaf sun-dried tea and fermented.
  • the inventors of the present invention found that when Candida albicans is inoculated into the puppling sun-dried green tea of the tide, after a period of fermentation, the soup color can be slightly reddish, the sweetness is obvious, and the scent of the scent is high and lasting. Pu'er cooked tea.
  • the use of Candida albicans in the processing of Pu'er tea not only maintains the original quality of Pu'er tea, but also increases the special aroma of the Pu'er tea and the brightness of the soup color, and shortens the fermentation cycle. Therefore, the present invention applies the purified Candida albicans to the fermentation process of Pu'er tea for the first time.
  • the Candida albicans of the present invention is one of the dominant bacteria in the fermentation of Pu'er tea, and the developed enzyme system plays an important role in the transformation of the contents of Pu'er tea, and the yeast also has abundant nutrients, such as Protein, vitamins, etc., have important significance for the formation of Pu'er tea quality.
  • the large leaf type sun-dried green tea is a tidal large leaf type sun-green tea.
  • the water content of the tidal large leaf type sun-dried tea is preferably 20-40% (m/m) based on the total weight of the sun-dried tea before the tidal water.
  • tanning palm tea refers to the artificial addition of water to a certain amount of water to the tanning palm tea before the plow.
  • tanning palm tea As is known in the art, the large-leaf type sun-dried green tea is produced by the process of drying, killing, rubbing, simmering, and drying the fresh leaves of the big leaf tea tree.
  • the water content is calculated according to the following formula:
  • Water content the quality of the water added / the total mass of the sun-dried tea before adding water
  • the Candida albicans in the growth index phase is inoculated into the sun-dried tea.
  • the growth index period can be determined by depicting the growth curve of the Candida albicans in a conventional manner.
  • the inoculum amount of the Candida albicans is 5.0 x 10 4 to 1.0 x 10 6 per gram of sun-dried tea, preferably 7.0 x 10 4 to 9.0 x 10 5 pieces, more preferably 9.8 x 10 4 to 1.2 ⁇ 10 5 , and most preferably 1.0 ⁇ 10 5 .
  • the sun-dried tea is sterilized prior to the inoculation to further ensure single-fermentation.
  • the fermentation culture is fermented at a temperature of 20 to 50 ° C for 2 to 6 weeks, more preferably at 25 to 45 ° C for 2 to 4 weeks. Further preferably, during the fermentation culture, the culture is hydrated, preferably once a week, and the amount of each hydration may be 6-10% of the total weight of the culture before each hydration (v /m). Preferably, the culture is turned over during the fermentation culture.
  • the medium may be cultured using various media such as a conventional PDA medium, ISP No. 2 medium, and the like, and ISP No. 2 medium is preferably used.
  • the Candida albicans strain is the Candida TMCC 70011 strain described above.
  • Candida blankii in the processing of Pu'er tea.
  • the Candida is preferably the Candida TMCC 70011 strain described above.
  • Example 1 Isolation and purification of Candida sp. TMCC 70011 strain
  • Fresh fermented tea samples were collected from the fermented Pu'er tea pile (collected from Bohai Tea Co., Ltd.), and 150 strains of Candida albicans were isolated by the purification medium described below. After morphological observation and gene sequencing (trusted by Shenzhen Huada Gene Company), TMCC 70011 strain was selected.
  • the prepared medium is sterilized at 121 ° C for 40 minutes, cooled to about 45 ° C, poured into a sterile culture dish, about 20 ml per dish, cooled and solidified for use;
  • Inoculation Inject the bacterial suspensions labeled as 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , and 10 -7 into 500 ⁇ l and inject them into step (1). Petri dishes were evenly coated with a coating bar, three replicates per concentration. After completion, the culture was placed at 35 ° C and observed every 24 hours;
  • TMCC 70011 strain After morphological observation and gene sequencing analysis, TMCC 70011 strain was selected.
  • Example 2 Production and application of Candida TMCC 70011 strain
  • the purified TMCC 70011 strain was inoculated into a ISP No. 2 liquid medium by a single colony by a plate, and cultured in a shaker at 150 rpm, 30 ° C for 7 hours to a growth index period. Adding large-leaf sun-dried green tea (collected from Bohai Tea Industry Co., Ltd.) to water content to 35% (m/m) based on the total weight of sun-dried tea before the tide, and sterilizing at 121 °C for 30 minutes. The TMCC 70011 strain cultured to the growth index phase was inoculated at an inoculation amount of 1.0 ⁇ 10 5 per gram of sun-dried tea, and then placed at 35 ° C for 3 weeks.
  • the water is hydrated and turned over in weeks, and the water is replenished in an amount of 10% (v/m) each time (based on the total weight of the culture before each hydration).
  • TMCC 70011 strain With the proliferation of TMCC 70011 strain, the amount of enzyme secreted by it is also increased. Under the action of enzymes such as protease, cellulase, pectinase and glucoamylase, reducing sugar, amino acid, soluble carbohydrate and water in Pu'er tea. The content of pectin is increasing, which enriches the taste of Pu'er tea.
  • the content of theanine in Pu'er tea is 1.8% (m/m)
  • the content of the tea brown pigment is 12.4% (m/m)
  • the soluble sugar content is 2.9% (m/m)
  • the content of water extract is 36% (m). /m).
  • the determination methods of the content of theanine, the tea brown pigment, the soluble sugar, and the water extract are all tested according to the national standard method.
  • the TMCC 70011 strain was applied to the fermentation of Pu'er tea under different fermentation conditions, and good effects were also obtained. See Table 1 and Table 2 for specific data.
  • Table 1 Test results of the content of TMC 70011 strain fermented Pu'er tea at the same temperature, time and different inoculum: (fermentation conditions: fermentation at 35 °C for 3 weeks; inoculum unit: single cell/gram of green tea)
  • Inoculum size 5.0 ⁇ 10 4 Inoculum size 4.2 ⁇ 10 5 Inoculum size 1.0 ⁇ 10 5 Water extract 34.7% 23.4% 36% Theanine 1.5% 1.6% 1.8% Soluble sugar 2.5% 2.4% 2.9% Tea brown pigment 8.6% 11.3% 12.4%
  • Table 2 Test results of the contents of TMC 70011 strain fermented Pu'er tea at different temperatures and times: (inoculation amount: 1.0 ⁇ 10 5 per gram of sun-dried tea)
  • the Pu'er cooked tea fermented in this example has improved sensory quality such as soup color, aroma and taste compared with Pu'er cooked tea fermented by conventional alfalfa (see Table 4).
  • the application of Candida albicans to the production of Pu'er tea not only maintains the original quality of Pu'er tea, but also increases the special aroma of the Pu'er tea and the brightness of the soup color, and shortens the fermentation cycle. This has provided an important application basis for the enrichment of Pu'er tea and the development of new products, and has played a positive role in the development of Pu'er tea industry.

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Abstract

提供了假丝酵母菌菌株及其应用，以及加工普洱茶的方法。假丝酵母菌（Candida blankii）菌株，其保藏编号为CGMCC No.8682，该菌株可用于普洱茶的加工。加工普洱茶的方法包括将分离纯化的假丝酵母菌（Candida blankii）接种于大叶种晒青毛茶中，进行发酵培养。

Description

[根据细则37.2由ISA制定的发明名称]　假丝酵母菌菌株及其应用，以及加工普洱茶的方法 技术领域
本发明属于生物技术领域，具体而言，涉及一种假丝酵母菌菌株及其应用，以及加工普洱茶的方法。
背景技术
随着现代生物技术的发展，酵母菌已经广泛地应用于人民生活当中的很多方面，如酿酒、发酵果汁、发酵面包、制作乳制品、制造美味等。酵母有着天然丰富的营养体系，是一种纯天然、无污染、生物态的健康营养源。酵母被营养界誉为最有魅力的营养品。据报道，酵母中含有50多种营养成分，人体健康所必须的蛋白质、维生素B群、矿物质、核酸等都可以从酵母中获得。
在传统的普洱茶渥堆发酵中，微生物、湿热作用、氧化及酶促反应等综合作用使普洱茶内含物质发生一系列转化，在6-8周的时间内形成普洱茶所独有的品质特征，这其中微生物发挥着重要的作用。
微生物资源丰富、代谢活跃，普洱茶发酵工艺革新的技术突破需要对微生物资源进行挖掘和开发利用。目前还没有将假丝酵母菌(Candida blankii)用于普洱茶的发酵加工中的报道。
发明内容
本发明提供了一种假丝酵母菌菌株及其应用，以及加工普洱茶的方法。
具体而言，本发明提供了：
一种假丝酵母菌(Candida blankii)菌株，其保藏编号为CGMCC No.8682，已于2014年1月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)。
本发明还提供了一种加工普洱茶的方法，包括将分离纯化的假丝 酵母菌(Candida blankii)接种于大叶种晒青毛茶中，进行发酵培养。
优选地，所述大叶种晒青毛茶为已潮水的大叶种晒青毛茶。
优选地，所述已潮水的大叶种晒青毛茶的含水量为基于潮水之前的晒青毛茶的总重量计为20-40％(m/m)。
优选地，将处于生长指数期的所述假丝酵母菌接种于所述的晒青毛茶中。
优选地，所述假丝酵母菌的接种量为每克所述的晒青毛茶接种5.0×104至1.0×106个。
优选地，在所述接种之前，对所述的晒青毛茶进行灭菌处理。
优选地，所述发酵培养在20-50℃的条件下发酵2-6周。
优选地，所述发酵培养在25-45℃的条件下发酵2-4周。
优选地，在所述发酵培养时，对培养物进行补水。
优选地，在所述发酵培养时，对培养物进行翻堆。
优选地，所述假丝酵母菌为保藏编号为CGMCC No.8682的假丝酵母菌菌株。
本发明还提供了假丝酵母菌(Candida blankii)在普洱茶加工中的应用。
优选地，所述假丝酵母菌为保藏编号为CGMCC No.8682的假丝酵母菌菌株。
本发明与现有技术相比具有以下优点和积极效果：
1.本发明从渥堆普洱茶发酵样品中分离得到假丝酵母菌(Candida blankii)，并且首次将假丝酵母菌应用于普洱茶的发酵加工中。本发明发现，将该假丝酵母菌接种到大叶种晒青毛茶中，经过一段时间的发酵后，可以得到汤色深红略浑、甜感明显、具有槐花香高扬持久特点的普洱熟茶。在20-50℃条件下发酵2-4周，普洱茶中的茶氨酸含量为1.5-2.1％(m/m)，茶褐素含量为7.4-12.4％(m/m)，可溶性糖含量为1.2-2.9％(m/m)，水浸出物含量为26.3-38.8％(m/m)。将假丝酵母菌(Candida blankii)用于普洱茶的加工不仅能保持普洱茶原有的品质，而且能增加普洱茶的特殊香气和汤色的明亮程度，而且缩短了发 酵的周期。此外，本发明通过进一步摸索和优化该假丝酵母菌应用于普洱茶加工时的发酵条件，进一步改善了上述有益效果。这为普洱茶滋味的丰富和新产品的开发提供了重要的应用基础，对普洱茶产业的发展发挥了积极的作用。
2.本发明提供了一种假丝酵母菌(Candida blankii)菌株，其保藏编号为CGMCC No.8682。该菌株在普洱茶的发酵加工中尤其能够提供上述有益效果。
生物材料保藏信息
本发明提供的假丝酵母菌(Candida blankii)TMCC 70011菌株已于2014年1月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址：北京市朝阳区北辰西路1号院3号，邮编：100101，保藏编号为：CGMCC No.8682。
具体实施方式
以下通过具体实施方式的描述并参照附图对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
本发明一个方面提供了一种假丝酵母菌(Candida blankii)菌株(即，TMCC 70011菌株)。该菌株已于2014年1月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC，北京市朝阳区北辰西路1号院3号)，保藏编号为CGMCC No.8682。
本发明所述的假丝酵母菌(Candida blankii)是一种布朗克假丝酵母菌。下文将该假丝酵母菌(Candida blankii)统称为“布朗克假丝酵母菌”。
本发明的发明人通过大量的实验研究，应用罗氏高通量454测序技术和Miseq测序技术对普洱茶不同时期的渥堆发酵样品进行了分析，发现了布朗克假丝酵母菌的存在，之后通过纯培养技术得到了所述TMCC 70011菌株。
所述TMCC 70011菌株在普洱茶的渥堆发酵过程中，利用传统渥堆普洱茶发酵样品，经平板稀释涂布、初筛、复筛、纯化培养等工序而获得。TMCC 70011菌株可使用常规的PDA培养基(葡萄糖20g，土豆200g，琼脂糖2g，蒸馏水1000mL，pH自然(6.8-7.3))、ISP 2号培养基(蛋白胨4g，葡萄糖4g，酵母浸膏5g，琼脂糖2g，蒸馏水1000mL，pH自然(6.8-7.3))等多种培养基进行复苏活化。
所述TMCC 70011菌株的形态学特征为：在ISP 2号培养基上培养48小时后观察，菌落整齐呈奶油色，表面湿润有光泽，边缘无褶皱，直径约0.5mm左右。显微镜下观察，细胞呈长椭圆形，有假丝菌。在无氧条件下也能缓慢生长，为兼性厌氧菌，生长温度在20-50℃之间。
所述TMCC 70011菌株的理化特征为：
(1)对TMCC 70011菌株进行唯一碳源或唯一氮源利用实验呈阳性的物质如下：
Figure PCTCN2016084532-appb-000001
(2)TMCC 70011菌株的酶活性实验阳性结果如下：
序号 测定的酶 酶活性
1 碱性磷酸盐酶 +
2 酯酶(C4) +
3 类酯脂酶(C8) +
4 类酯酶(C14) +
5 白氨酸芳胺酶 +
6 缬氨酸芳胺酶 +
7 胱氨酸芳胺酶 +
8 胰蛋白酶 +
9 α-胰凝乳蛋白酶 +
10 酸性磷酸酶 +
11 萘酚-AS-BI-磷酸水解酶 +
12 α-半乳糖甙酶 +
13 β-半乳糖甙酶 +
15 α-葡萄糖甙酶 +
16 β-葡萄糖甙酶 +
17 N-乙酰-葡萄糖胺酶 +
18 α-甘露糖甙酶 +
通过测序，所述TMCC 70011菌株的18S rRNA基因序列如序列表中的序列1所示，ITS序列如序列表中的序列2所示。
本发明的TMCC 70011菌株在普洱茶的发酵加工中具有特别良好的效果，其不仅能保持普洱茶原有的品质，而且能增加普洱茶的特殊香气和汤色的明亮程度，得到汤色深红略浑、甜感明显、具有槐花香高扬持久特点的普洱熟茶。此外还能缩短发酵的周期。这为普洱茶滋味的丰富和新产品的开发提供了重要的应用基础，对普洱茶产业的发展发挥了积极的作用。
然而，传统的普洱茶发酵需要大概6-8周的时间，且刚出堆的熟茶堆味较重，汤色红褐略混，需要经过一段时间的纯化才能达到较好的品质。
本发明另一个方面提供了一种加工普洱茶的方法，包括将分离 纯化的假丝酵母菌(Candida blankii)接种于大叶种晒青毛茶中，进行发酵培养。
本发明的发明人发现当将布朗克假丝酵母菌接种到已潮水的普洱晒青毛茶中，经过一段时间的发酵后，可以得到汤色深红略浑、甜感明显、具有槐花香高扬持久特点的普洱熟茶。将布朗克假丝酵母菌用于普洱茶的加工不仅能保持普洱茶原有的品质，而且能增加普洱茶的特殊香气和汤色的明亮程度，而且缩短了发酵的周期。因此，本发明首次将纯化的布朗克假丝酵母菌应用到了普洱茶的发酵加工中。
本发明的布朗克假丝酵母菌是普洱茶发酵中的优势菌群之一，其发达的酶系对普洱茶内含物质的转化发挥着重要的作用，同时酵母还有着丰富的营养物质，如蛋白质、维生素等，这对普洱茶品质的形成有着重要的意义。
优选地，在本发明的方法中，所述大叶种晒青毛茶为已潮水的大叶种晒青毛茶。所述已潮水的大叶种晒青毛茶的含水量优选为基于潮水之前的晒青毛茶的总重量计为20-40％(m/m)。
本文所用的术语“潮水”是指在渥堆前对晒青普洱毛茶人为加水至一定含水量。如本领域已知的，大叶种晒青毛茶是由大叶种茶树鲜叶经摊晾、杀青、揉捻、渥黄、干燥等工序制作而成。
在提及已潮水的大叶种晒青毛茶的含水量时，其含水量按照以下公式计算：
含水量＝所加水的质量/加水之前晒青毛茶的总质量
优选地，将处于生长指数期的所述布朗克假丝酵母菌接种于所述的晒青毛茶中。生长指数期可通过用常规的方法描绘所述布朗克假丝酵母菌的生长曲线来确定。
发明人通过大量的试验摸索发现，当接种的菌种浓度过低时，菌种繁殖慢，发酵的时间会延长，时间成本增加；当接种的菌种浓度过高时，菌种生长较快，茶叶内含物质被过度利用，这会造成发酵熟茶的口感淡，对品质影响较大。因此，优选地，所述布朗克假丝酵母菌的接种量为每克晒青毛茶接种5.0×104至1.0×106个，优选7.0×104至9.0×105个，更优选9.8×104个至1.2×105个，最优选1.0×105个。
优选地，在所述接种之前，对所述的晒青毛茶进行灭菌处理，以进一步保证单菌发酵。
发明人还发现，当发酵短于2周时，不能形成熟茶的品质，汤色、口感均接近生茶的特点；当发酵大于6周时，茶叶内含物质被过度利用，内含物质减少较多，这会造成发酵熟茶的口感淡。因此，优选地，所述发酵培养在20-50℃的条件下发酵2-6周，更优选在25-45℃的条件下发酵2-4周。还优选地，在所述发酵培养时，对培养物进行补水，优选每周补水一次，每次补水的量可以为：以每次补水之前的培养物的总重量计为6-10％(v/m)。优选地，在所述发酵培养时，对培养物进行翻堆。
在所述发酵培养过程中，培养基可以使用常规的PDA培养基、ISP 2号培养基等多种培养基进行培养，其中优选使用ISP 2号培养基。
优选地，在本发明的方法中，所述布朗克假丝酵母菌为上文所述的假丝酵母菌TMCC 70011菌株。
本发明的又一个方面提供了假丝酵母菌(Candida blankii)在普洱茶加工中的应用。所述假丝酵母菌优选为上文所述的假丝酵母菌TMCC 70011菌株。
以下通过例子的方式进一步解释或说明本发明的内容，但这些例子不应被理解为对本发明的保护范围的限制。
实施例
实施例1：假丝酵母菌TMCC 70011菌株的分离纯化
从正在发酵的普洱茶渥堆(采集自勐海茶业有限责任公司)中采集新鲜的发酵茶样，通过下文所述的纯化培养基分离得到150株布朗克假丝酵母菌。经过形态观察和基因测序(委托深圳华大基因公司)分析，选取TMCC 70011菌株。
分离纯化方法如下：
(1)纯化培养基准备：
培养基配方：蛋白胨4g，葡萄糖4g，酵母浸膏5g，琼脂糖2g，蒸馏水1000ml，pH为自然状态(6.8-7.3)。
将配制好的培养基121℃灭菌40分钟，冷却至45℃左右，倒入无菌培养皿中，每皿约20ml，冷却凝固后备用；
(2)样品处理：取待分离的普洱茶发酵渥堆茶样，研磨粉碎，取1g，在超净台中加入到装有9ml无菌水的试管中，振荡制成菌悬液，浓度记为100；
(3)稀释：按十倍稀释法逐步稀释，稀释7次为止，分别将浓度记为10-1、10-2、10-3、10-4、10-5、10-6、10-7。十倍稀释法是指下一管的菌悬液浓度是上一管的十分之一；
(4)接种：将标记为10-2、10-3、10-4、10-5、10-6、10-7浓度的菌悬液分别取500μl注入到步骤(1)中已准制好的培养皿中，并用涂布棒均匀涂布，每个浓度三个重复。完成后，放置于35℃下培养，每隔24小时观察一次；
(5)纯化：培养4天后，挑取单菌落转接到新鲜固体PDA培养基中培养，多次划线纯化直至得到纯化菌株；
(6)选取TMCC 70011菌株：经过形态观察和基因测序分析，选取TMCC 70011菌株。
实施例2：假丝酵母菌TMCC 70011菌株的生产应用
将纯化得到的TMCC 70011菌株通过平板挑取单菌落接种于ISP2号液体培养基中，在摇床中以150rpm、30℃培养7小时至生长指数期。将大叶种晒青毛茶(采集自勐海茶业有限责任公司)加水至含水量达到基于潮水之前的晒青毛茶的总重量计为35％(m/m)，121℃灭菌30分钟，按每克晒青毛茶1.0×105个的接种量接种所述培养至生长指数期的TMCC 70011菌株，然后放置于35℃培养3周。以周为单位进行补水并翻堆，每次按照10％(v/m)(以每次补水之前的培养物的总重量计)的量补水。随着TMCC 70011菌株的大量繁殖，其分泌的酶量也增加，在蛋白酶、纤维素酶、果胶酶、糖化酶等酶的作用下，普洱茶中还原性糖、氨基酸、可溶性碳水化合物和水化果胶的含量不断增加，由此丰富了普洱茶的滋味。
经过接种TMCC 70011菌株进行发酵的普洱茶，具有汤色深红略 浑、甜感明显、槐花香高扬持久的特点。普洱茶中茶氨酸含量为1.8％(m/m)，茶褐素含量为12.4％(m/m)，可溶性糖含量为2.9％(m/m)，水浸出物含量为36％(m/m)。其中，茶氨酸、茶褐素、可溶性糖、水浸出物的含量的测定方法均按照国家标准方法检测。
按照上述相同的方法，在不同的发酵条件下将TMCC 70011菌株应用于普洱茶的发酵，同样获得了良好的效果。具体数据请参见表1和表2。
表1：相同温度、时间，不同接种量下，TMCC 70011菌株发酵普洱茶的内含物质检测结果：(发酵条件：35℃发酵3周；接种量单位：个菌体/克晒青毛茶)
  接种量5.0×104 接种量4.2×105 接种量1.0×105
水浸出物 34.7％ 23.4％ 36％
茶氨酸 1.5％ 1.6％ 1.8％
可溶性糖 2.5％ 2.4％ 2.9％
茶褐素 8.6％ 11.3％ 12.4％
表2：不同温度、时间下，TMCC 70011菌株发酵普洱茶的内含物质检测结果：(接种量：每克晒青毛茶1.0×105个)
Figure PCTCN2016084532-appb-000002
表3：传统的渥堆发酵普洱熟茶(渥堆发酵6周)(大益2013年7572普洱熟茶)的品质检测结果：
检测项目 水浸出物 茶氨酸 可溶性糖 茶褐素
含量(m/m) 34.5％ 2.3％ 3.2％ 10.2％
本实施例发酵的普洱熟茶与传统渥堆发酵的普洱熟茶相比，汤色、香气、滋味等感官品质提高(见表4)。
由本发明可知，将布朗克假丝酵母菌应用于普洱茶的生产，不仅能保持普洱茶原有的品质，而且能增加普洱茶的特殊香气和汤色的明亮程度，且缩短了发酵的周期。这为普洱茶滋味的丰富和新产品的开发提供了重要的应用基础，对普洱茶产业的发展发挥了积极的作用。
表4：实施例2发酵的普洱熟茶与传统渥堆发酵的普洱熟茶(渥堆发酵6周，大益2013年7572普洱熟茶)的感官评价比较：(+代表程度)
Figure PCTCN2016084532-appb-000003

Claims (14)

  1. 一种假丝酵母菌(Candida blankii)菌株，其保藏编号为CGMCC No.8682。
  2. 一种加工普洱茶的方法，包括将分离纯化的假丝酵母菌(Candida blankii)接种于大叶种晒青毛茶中，进行发酵培养。
  3. 根据权利要求2所述的方法，其中所述大叶种晒青毛茶为已潮水的大叶种晒青毛茶。
  4. 根据权利要求3所述的方法，其中所述已潮水的大叶种晒青毛茶的含水量为基于潮水之前的晒青毛茶的总重量计为20-40％(m/m)。
  5. 根据权利要求2所述的方法，其中将处于生长指数期的所述假丝酵母菌接种于所述的晒青毛茶中。
  6. 根据权利要求2所述的方法，其中所述假丝酵母菌的接种量为每克所述的晒青毛茶接种5.0×104至1.0×106个。
  7. 根据权利要求2所述的方法，其中在所述接种之前，对所述的晒青毛茶进行灭菌处理。
  8. 根据权利要求2所述的方法，其中所述发酵培养在20-50℃的条件下发酵2-6周。
  9. 根据权利要求2所述的方法，其中所述发酵培养在25-45℃的条件下发酵2-4周。
  10. 根据权利要求2所述的方法，其中在所述发酵培养时，对培养物进行补水。
  11. 根据权利要求2所述的方法，其中在所述发酵培养时，对培养物进行翻堆。
  12. 根据权利要求2所述的方法，其中所述假丝酵母菌为保藏编号为CGMCC No.8682的假丝酵母菌菌株。
  13. 假丝酵母菌(Candida blankii)在普洱茶加工中的应用。
  14. 根据权利要求12所述的应用，其中所述假丝酵母菌为保藏编号为CGMCC No.8682的假丝酵母菌菌株。
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