WO2023226509A1 - 一种快发无糖面用酵母菌株及应用 - Google Patents

一种快发无糖面用酵母菌株及应用 Download PDF

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Publication number
WO2023226509A1
WO2023226509A1 PCT/CN2023/079577 CN2023079577W WO2023226509A1 WO 2023226509 A1 WO2023226509 A1 WO 2023226509A1 CN 2023079577 W CN2023079577 W CN 2023079577W WO 2023226509 A1 WO2023226509 A1 WO 2023226509A1
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Prior art keywords
saccharomyces cerevisiae
strain
dough
amcc31248
yeast
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PCT/CN2023/079577
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English (en)
French (fr)
Inventor
孙雅芳
肖明华
陈莉婷
郭天芬
王龙
匡金宝
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Angel Yeast Co Ltd
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Angel Yeast Co Ltd
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Priority to KR1020247041578A priority Critical patent/KR20250010682A/ko
Priority to US18/870,073 priority patent/US20250331524A1/en
Priority to EP23810585.2A priority patent/EP4530342A4/en
Priority to JP2024569755A priority patent/JP2025518092A/ja
Publication of WO2023226509A1 publication Critical patent/WO2023226509A1/zh
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/047—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with yeasts
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D13/00—Finished or partly finished bakery products
    • A21D13/06—Products with modified nutritive value, e.g. with modified starch content
    • A21D13/062—Products with modified nutritive value, e.g. with modified starch content with modified sugar content; Sugar-free products
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with 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; 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
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces 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
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to the field of microorganisms, and specifically relates to a fast-growing sugar-free pasta yeast strain and its application.
  • the fermentation process of the dough always plays a vital role, not only affecting the softness, taste, nutritional value, etc. of the pasta products, but also determining the speed of the dough.
  • the ambient temperature is often too high, which causes the yeast to rise too quickly during the dough mixing process, resulting in a decline in product quality.
  • low-temperature water is usually added to cool down.
  • yeast cells will enter a state of shock when encountering a low-temperature environment, thus affecting the normal growth of yeast cells. Therefore, there is an urgent need for yeast to withstand cold osmotic shock.
  • the present invention provides a fast-rising yeast strain for sugar-free dough with cold osmotic shock resistance.
  • the present invention provides a Saccharomyces cerevisiae strain, and the Saccharomyces cerevisiae strain is:
  • Saccharomyces cerevisiae AMCC31248 strain (Saccharomyces cerevisiae AMCC31248) is deposited in the China Type Culture Collection Center (CCTCC) with the deposit number CCTCC NO: M 20211686.
  • CTCC China Type Culture Collection Center
  • the present invention provides a fermentation preparation method of Saccharomyces cerevisiae inoculant, which method includes the following steps: cultivating the Saccharomyces cerevisiae strain.
  • the preparation method includes the following steps:
  • step (2) Add the product obtained in step (1) to the liquid culture medium, and ferment and culture it at 26-32°C.
  • the present invention provides a bacterial agent, which contains the Saccharomyces cerevisiae AMCC31248 strain.
  • the bacterial agent is obtained by the fermentation preparation method.
  • the present invention also provides the use of the Saccharomyces cerevisiae strain or the inoculant in fermentation.
  • the present invention also provides the application of the inoculant of the Saccharomyces cerevisiae strain in dough.
  • the present invention provides a dough containing the Saccharomyces cerevisiae strain or the inoculant.
  • the dough contains flour and the Saccharomyces cerevisiae strain in a mass ratio of 100:0.5-5.
  • the present invention also provides a method for preparing the dough, which method includes the following steps: kneading the dough with water at 0-35°C.
  • the 0°C water in the present invention may be 0°C ice or 0°C ice-water mixture or 0°C liquid water.
  • water, flour and Saccharomyces cerevisiae can be added in any order.
  • the flour and yeast are mixed first, and then water at 0-35°C is added to knead the dough.
  • the flour can also be added first. Add 0-35°C water, then add Saccharomyces cerevisiae, or add Saccharomyces cerevisiae to 0-35°C water first, and then add flour.
  • the present invention also provides a noodle product, which is obtained by the dough preparation method.
  • the flour products are steamed buns, steamed buns, bread, biscuits, noodles, pot stickers, etc.
  • Saccharomyces cerevisiae AMCC31248 strain provided by the invention has good fermentation performance in sugar-free dough, can quickly ferment sugar-free dough, and has good cold osmotic shock resistance.
  • the Saccharomyces cerevisiae AMCC31248 strain provided by the invention AMCC31248) was deposited in the China Type Culture Collection Center (CCTCC) on December 29, 2021.
  • the deposit number is CCTCC NO: M 20211686.
  • the Saccharomyces cerevisiae AMCC30010 strain used in the present invention was deposited in the China Type Culture Collection Center (CCTCC) on March 29, 2022.
  • the preservation number is CCTCC NO: M 2022340, and the preservation address is: Wuhan University, China.
  • the Saccharomyces cerevisiae AMCC32101 strain (Saccharomyces cerevisiae AMCC32101) provided by the invention was deposited in the China Type Culture Collection Center (CCTCC) on March 29, 2022.
  • the deposit number is CCTCC NO: M 2022341, and the deposit address is: Wuhan University, China.
  • Figure 1 shows the colony diagram of Saccharomyces cerevisiae AMCC31248 strain
  • Figure 2 shows the sporulation diagram of Saccharomyces cerevisiae strain AMCC31248
  • Figure 3 shows the growth curves of the parent and new strains in wort medium.
  • the present invention uses the Saccharomyces cerevisiae AMCC30010 strain and the Saccharomyces cerevisiae AMCC32101 strain as parents, and obtains the Saccharomyces cerevisiae AMCC31248 strain provided by the invention through a microscopic hybridization method.
  • Saccharomyces cerevisiae AMCC30010 strain is a Saccharomyces cerevisiae strain selected by Angel Yeast Co., Ltd. The original strain was collected from Yichang City, Hubei province. Observed through an optical microscope, the cell shape of this Saccharomyces cerevisiae strain is oval and reproduces through budding. The single colonies grown on the solid plate are spherical with a slightly convex center, milky white, with smooth surface and neat edges. Through morphological observation and molecular biology identification under a high-power microscope, it was determined that it was a strain of Saccharomyces cerevisiae and was a food attribute. It was deposited in the China Type Culture Collection Center (CCTCC) on March 29, 2022, with the preservation number CCTCC NO: M 2022340 (ie CCTCC M 2022340).
  • CTCC China Type Culture Collection Center
  • Saccharomyces cerevisiae AMCC32101 strain is A strain of Saccharomyces cerevisiae selected by Angel Yeast Co., Ltd. The original strain was collected from Ulanqab City, Inner Mongolia Autonomous Region. Observed through an optical microscope, the cell shape of this Saccharomyces cerevisiae strain is oval and reproduces through budding. The single colonies grown on the solid plate are spherical with a slightly convex center, milky white, with smooth surface and neat edges. Through morphological observation and molecular biology identification under a high-power microscope, it was determined that it was a strain of Saccharomyces cerevisiae and was a food attribute. It was deposited in the China Type Culture Collection Center (CCTCC) on March 29, 2022, with the deposit number CCTCC NO: M 2022341 (ie CCTCC M 2022341).
  • the Saccharomyces cerevisiae AMCC31248 strain provided by the invention has good fermentation performance in sugar-free dough and can quickly ferment sugar-free dough.
  • the production of some sugar-free breads, soda crackers, steamed buns, etc. mainly involves the fermentation of unsweetened dough.
  • Most of the flour is starch, which is converted into maltose under the action of amylase in the flour. Therefore, the ability of the yeast to utilize maltose also determines whether The speed of rising of sugar-added dough depends on the maltose utilization enzyme system of yeast including maltose hydrolase and maltose permease. Yeast with high maltose utilization ability is called fast fermentation yeast.
  • Sugar-tolerant yeast refers to a higher tolerance to sucrose in sugar-containing dough, that is, its growth and fermentation performance in sugar-containing bread are higher than that of ordinary yeast.
  • Low-sugar-tolerant yeast is used in dough systems with about 7% sucrose, while high-sugar-tolerant yeast is used in dough systems with higher sucrose concentrations, and the content can be as high as 25%.
  • Sucrose generally cannot be directly utilized by microorganisms, but Saccharomyces cerevisiae contains sucrose hydrolase that can degrade sucrose. It acts on ⁇ -1,2 glycosidic bonds to hydrolyze sucrose into D-glucose and D-fructose, and then glucose and fructose. Entering the glycolytic pathway for utilization by yeast, while the glucose and fructose produced due to the rapid breakdown of sucrose will increase the osmotic pressure around the yeast cells.
  • the yeast cell membrane is a selectively semipermeable biological membrane.
  • the concentration of the outside world will affect the activity of the yeast cells.
  • the cells When the cells are in an environment with high osmotic pressure, the water and protoplasm in the cells will leak out of the cell membrane, causing the cells to become dehydrated and even die. Therefore, the hypertonic environment that Saccharomyces cerevisiae faces in high-sugar dough has an impact on its growth and fermentation performance. Therefore, the gas production ability of sugar-free yeast is determined by the activity of maltose utilization enzyme, and the gas production ability of sugar-tolerant yeast is determined by the activity of sucrase.
  • the formula of the sporulation medium used in the embodiment of the present invention is: 1% potassium acetate, 0.1% yeast extract, 0.05% glucose, and 2% agar.
  • YPD solid medium is used to activate each Saccharomyces cerevisiae strain.
  • the formula of YPD solid medium is: 1% yeast extract powder, 2% peptone, 2% glucose, and 2% agar.
  • YPD liquid medium is used to culture each Saccharomyces cerevisiae strain.
  • the YPD liquid medium formula is: 1% yeast extract powder, 2% peptone, and 2% glucose.
  • the parental strains Saccharomyces cerevisiae AMCC30010 and Saccharomyces cerevisiae AMCC32101 were respectively activated and induced to produce spores. After enzymatic hydrolysis of the cells, single spores were picked using a yeast micromanipulator, and single spores from two different parents were contacted and then placed Cultured at 30°C. Observe the morphology of the spores, and continue culturing at 30°C after the two single spores hybridize successfully. This is a first-generation strain. The obtained first-generation strain was then hybridized with the Saccharomyces cerevisiae strain AMCC30010. After obtaining the second-generation strain, the sporulation test was performed, and the hybrid strain was selected for subsequent screening.
  • the fully automatic growth curve analyzer Bioscreen C was used to measure the growth curve of the new hybrid strains obtained from the cross, and select the top 20 hybrid strains with higher growth efficiency than the parents.
  • a shake flask fermentation test was carried out on the 20 hybrid strains with higher growth efficiency than the parents.
  • the net dry weight of the strains and the fermentation activity of fresh yeast in the 0% sugar dough system were used as screening indicators to select the net dry weight of the yeast milk.
  • a hybrid strain that can reach 95-105% of either parent and 0% sugar dough fermentation activity reaches 95%-150% of either parent.
  • the hybrid strains screened in the above steps were cultured in a 45L system fermentation tank, and the obtained yeast cells were prepared into active dry yeast.
  • the fermentation activity of the active dry yeast in the 0% sugar dough system was measured, and the selected components in the dry yeast preparation process were There are no obvious abnormalities in the new strain, and the 0% sugar dough fermentation activity of active dry yeast can reach 95%-120% of any parent strain.
  • the hybrid strains optimized in the above steps were screened for cold-osmotic shock resistance. Knead the dough with crushed ice at 0°C, prepare 0% sugar dough containing active dry yeast of the hybrid strain, measure the proofing time of the dough, and select the hybrid strain corresponding to the shortest proofing time of the dough as the target strain, thereby screening out the strains with Hybrid strains with cold-osmotic shock resistance properties.
  • Figure 1 shows the colony diagram of the hybrid strain AMCC31248.
  • the obtained hybrid strain AMCC31248 was named Saccharomyces cerevisiae AMCC31248.
  • the Saccharomyces cerevisiae AMCC31248 strain was deposited in the China Type Culture Collection Center (CCTCC) on December 29, 2021, with the preservation number CCTCC NO: M 20211686 (ie CCTCC M 20211686).
  • OD1 OD 600 value corresponding to the strain at t1;
  • OD2 OD 600 value corresponding to the strain at t2;
  • t2 The end time of the logarithmic growth phase.
  • Saccharomyces cerevisiae AMCC31248 The growth curve of Saccharomyces cerevisiae AMCC31248 is shown in Figure 3. It can be seen that this strain can grow rapidly in wort medium. Table 3 below shows the growth efficiency of the parent strain and Saccharomyces cerevisiae AMCC31248 strain.
  • the growth efficiency of the obtained Saccharomyces cerevisiae AMCC31248 strain is significantly higher than that of the parent strains Saccharomyces cerevisiae AMCC30010 and Saccharomyces cerevisiae AMCC32101.
  • the Saccharomyces cerevisiae AMCC31248 obtained in Example 1 was inoculated into a shake flask containing a fermentation medium, and cultured at 30°C.
  • the precipitate collected after centrifugation is the yeast milk.
  • the weight of the yeast milk is measured and the moisture content of the yeast milk is measured.
  • Relative percentage of net dry weight (%) (net dry weight of hybrid new strain/net dry weight of parent strain)*100%
  • the net dry weight of Saccharomyces cerevisiae AMCC31248 strain is 95.7% and 109.9% of the parent strain Saccharomyces cerevisiae AMCC30010 and Saccharomyces cerevisiae AMCC32101, respectively.
  • the 0% sugar dough fermentation of fresh yeast of Saccharomyces cerevisiae AMCC31248 strain The vitality is higher than that of the two parents, with an advantage of about 10%.
  • the Saccharomyces cerevisiae AMCC31248 strain is activated and cultured, then enlarged and cultured in a 45L fermentation tank, and then separated, washed, filtered, and dried to obtain active dry yeast.
  • active dry yeast According to the dough formula shown in Table 7, weigh the flour, salt, water and prepared active dry yeast respectively, and use the SJA method to detect the total carbon dioxide gas produced by 280g of dough prepared by the system shown in Table 7 and fermented by active dry yeast for 1 hour. Quantity, that is, the dough fermentation activity of active dry yeast.

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Abstract

提供一种快发无糖面用酵母菌株及应用。提供的酿酒酵母AMCC31248菌株(Saccharomyces cerevisiae AMCC31248)保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 20211686。提供的酿酒酵母AMCC31248菌株在无糖面团中发酵性能良好，能够快速发酵无糖面团。

Description

一种快发无糖面用酵母菌株及应用 技术领域
本发明涉及微生物领域，具体涉及一种快发无糖面用酵母菌株及应用。
背景技术
从最早的商品面包酵母的发现到酵母生产工艺的正式形成，再到现如今的活性干酵母发展阶段，酵母产品种类日趋多元化且产品质量不断提高，可应用于不同面食的制作。发酵面食作为一种传统主食在百姓膳食结构中占据着重要地位，据统计现我国约一半的人口以面食为主，且多数是馒头、包子等不加糖面团发酵类面食。从健康的角度出发，不加糖或低糖的面食更能受到消费者青睐。
在面食的制作中，面团的发酵过程始终起着至关重要的作用，不仅影响着面食产品的松软度、口感、营养价值等，而且决定了发面的快慢程度。酵母发酵能力越强，面团起发速度就越快，从而缩短发酵周期，加快面食制作进程，提高工业化大生产效率。因此开发出在无糖面团中发酵性能良好的酿酒酵母菌株有着十分重要的现实意义。
发明内容
在面食的制作过程中常会遇到环境温度过高，造成和面过程中酵母起发过快，引起产品品质下降。为了降低面团的温度，通常加入低温的水来降温，但酵母细胞遇到低温的环境会进入休克状态，从而影响酵母细胞的正常生长。所以对酵母的耐冷渗透休克性能有着迫切需求。
所以，针对现有技术中的酿酒酵母发酵无糖面团效率低及耐冷渗透休克性能差的问题，本发明提供一种具有耐冷渗透休克性能的快发无糖面用酵母菌株。
第一方面，本发明提供一种酿酒酵母菌株，所述酿酒酵母菌株为：
酿酒酵母AMCC31248菌株(Saccharomyces cerevisiae AMCC31248)，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M  20211686。
第二方面，本发明提供一种酿酒酵母菌剂的发酵制备方法，所述方法包含如下步骤：培养所述的酿酒酵母菌株。
优选地，所述制备方法包括如下步骤：
(1)将所述的酿酒酵母菌株放大培养；
(2)将步骤(1)得到的产物加入到液体培养基中，在26-32℃条件下发酵培养。
第三方面，本发明提供一种菌剂，所述菌剂中含有所述的酿酒酵母AMCC31248菌株(Saccharomyces cerevisiae AMCC31248)。
优选地，所述菌剂通过所述的发酵制备方法得到。
第四方面，本发明还提供所述的酿酒酵母菌株或所述的菌剂在发酵中的应用。
第五方面，本发明还提供所述的酿酒酵母菌株所述的菌剂在面团中的应用。
第六方面，本发明提供一种面团，所述面团中含有所述的酿酒酵母菌株或所述的菌剂。
优选地，所述面团中含有质量比为100:0.5-5的面粉和所述酿酒酵母菌株。
第七方面，本发明还提供所述的面团的制备方法，所述制备方法包括如下步骤：以0-35℃的水和面。
本发明中0℃的水可以为0℃的冰或0℃的冰水混合物或0℃的液体水。
本发明所述的面团的制备过程中，水、面和酿酒酵母可以为任意的加入顺序，如先将面和酵母混匀，然后加入0-35℃的水进行和面，也可以先将面粉加入0-35℃的水中，再加入酿酒酵母，或者先将酿酒酵母加入到0-35℃的水中，然后再加入面粉。
第八方面，本发明还提供一种面制品，其通过所述的面团制备方法得到。
优选地，所述面制品为馒头、包子、面包、饼干、面条、锅贴等。
本发明提供的酿酒酵母AMCC31248菌株(Saccharomyces cerevisiae AMCC31248)在无糖面团中发酵性能良好，能够快速发酵无糖面团，且具有良好的耐冷渗透休克性能。
菌种保藏信息
本发明提供的酿酒酵母AMCC31248菌株(Saccharomyces cerevisiae  AMCC31248)于2021年12月29日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 20211686，保藏地址：中国.武汉.武汉大学，邮政编码：430072；电话：027-68754052。
本发明所用的酿酒酵母AMCC30010菌株(Saccharomyces cerevisiae AMCC30010)于2022年03月29日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 2022340，保藏地址：中国.武汉.武汉大学，邮政编码：430072；电话：027-68754052。
本发明提供的酿酒酵母AMCC32101菌株(Saccharomyces cerevisiae AMCC32101)于2022年03月29日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 2022341，保藏地址：中国.武汉.武汉大学，邮政编码：430072；电话：027-68754052。
附图说明
图1所示为酿酒酵母AMCC31248菌株的菌落图；
图2所示为酿酒酵母AMCC31248菌株的产孢图；
图3所示为亲本和新菌株在麦芽汁培养基中的生长曲线。
具体实施方式
本发明以酿酒酵母AMCC30010菌株和酿酒酵母AMCC32101菌株为亲本，通过显微杂交的方法获得本发明提供的酿酒酵母AMCC31248菌株。
酿酒酵母AMCC30010(Saccharomyces cerevisiae AMCC30010)菌株是安琪酵母股份有限公司选育的一株酿酒酵母菌种，该原始菌株从湖北省宜昌市采集得到。通过光学显微镜观察，该酿酒酵母菌株细胞形态为椭圆形，出芽生殖，在固体平板上长出的单菌落呈中央略微凸起的圆球状，乳白色，表面光滑，边缘整齐。通过高倍显微镜进行形态观察及分子生物学鉴定，确定其为酿酒酵母种(Saccharomyces cerevisiae)菌株，是食品属性。于2022年03月29日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 2022340(即CCTCC M 2022340)。
酿酒酵母AMCC32101(Saccharomyces cerevisiae AMCC32101)菌株是 安琪酵母股份有限公司选育的一株酿酒酵母菌种，该原始菌株从内蒙古自治区乌兰察布市采集得到。通过光学显微镜观察，该酿酒酵母菌株细胞形态为椭圆形，出芽生殖，在固体平板上长出的单菌落呈中央略微凸起的圆球状，乳白色，表面光滑，边缘整齐。通过高倍显微镜进行形态观察及分子生物学鉴定，确定其为酿酒酵母种(Saccharomyces cerevisiae)菌株，是食品属性。于2022年03月29日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 2022341(即CCTCC M 2022341)。
本发明提供的酿酒酵母AMCC31248菌株在无糖面团中发酵性能良好，能够快速发酵无糖面团。
一些无糖面包、苏打饼干、馒头等的制作主要是不加糖面团发酵，面粉中大部分是淀粉，它在面粉中淀粉酶作用下转化为麦芽糖，因此酵母利用麦芽糖能力的高低也就决定了不加糖面团起发速度的快慢，酵母的麦芽糖利用酶系包括麦芽糖水解酶和麦芽糖透性酶，具有高麦芽糖利用能力的酵母称之为快速发酵酵母。
耐糖酵母是指对含糖面团中的蔗糖具有较高的耐受性，即在含糖面包中的生长和发酵性能较普通酵母都高。
耐低糖酵母是在7％左右蔗糖面团体系中使用，耐高糖酵母是在更高蔗糖浓度面团体系中使用，含量可高达25％。蔗糖一般不能直接被微生物利用，而酿酒酵母中含有可以降解蔗糖的蔗糖水解酶，它作用于β-1,2糖苷键，将蔗糖水解为D-葡糖糖和D-果糖，随后葡萄糖和果糖进入糖酵解途径以供酵母利用，同时由于蔗糖的快速分解生成的葡萄糖和果糖会使酵母细胞周围的渗透压增加。酵母细胞膜是一层选择半透性生物膜，外界的浓度高低会影响酵母细胞的活性，当细胞处于渗透压较高的环境中，细胞内的水分和原生质会渗出细胞膜使细胞脱水甚至死亡。因此，酿酒酵母在高糖面团中面临的高渗的环境，对其生长及发酵性能都有影响。因此，无糖酵母的产气能力是由麦芽糖利用酶活力决定，耐糖酵母的产气能力是由蔗糖酶活力决定。
本发明实施例中所用的试剂及仪器来源信息如下表1、表2所示。
表1试剂信息表

表2仪器信息表
本发明实施例中所用的产孢培养基的配方为：1％乙酸钾，0.1％酵母浸粉，0.05％葡萄糖，2％琼脂。
本发明实施例中用YPD固体培养基对各酿酒酵母菌株进行活化，YPD固体培养基配方为：1％酵母浸粉，2％蛋白胨，2％葡萄糖，2％琼脂。
本发明实施例中用YPD液体培养基培养各酿酒酵母菌株，YPD液体培养基配方为：1％酵母浸粉，2％蛋白胨，2％葡萄糖。
实施例1菌株构建及鉴定
将亲本菌株酿酒酵母AMCC30010菌株和酿酒酵母AMCC32101菌株分别活化并诱导产孢，菌体酶解后利用酵母显微操作仪挑取单孢子，并将来自两个不同亲本的单孢子接触，然后置于30℃条件下培养。观察孢子的形态，待两个单孢子杂交成功后继续置于30℃条件下培养。此为一代菌株。将获得的一代菌株与酿酒酵母AMCC30010菌株再进行杂交，得到二代菌株后进行生孢检验，选取杂合菌株进行后续筛选。
利用全自动生长曲线分析仪Bioscreen C对杂交获得的杂合新菌株进行生长曲线测定，选取生长效率高于亲本且排名前20的杂合菌株。
对得到的生长效率高于亲本的20株杂合菌株进行摇瓶发酵试验，以菌株的净干重及其鲜酵母在0％糖面团体系中的发酵活力为筛选指标，选取酵母乳净干重能达到任一亲本的95-105％且0％糖面团发酵活力达到任一亲本的95％-150％的杂合菌株。
接着对上述步骤筛选得到的杂合菌株进行45L体系发酵罐培养，将获得的酵母细胞分别制备成活性干酵母，测定活性干酵母在0％糖面团体系中的发酵活力，选取在干酵母制备过程中无明显异常情况、活性干酵母的0％糖面团发酵活力能达到任一亲本的95％-120％的新菌株。
最后对上述步骤优选的杂合菌株进行耐冷渗透休克性能筛选。用0℃的碎冰和面，制备含有杂合菌株活性干酵母的0％糖生面团，测定面团的醒发时间，以对应面团醒发时间最短的杂合菌株为目的菌株，从而筛选出具有耐冷渗透休克性能的杂合菌株。
经上述筛选得到一株编号为AMCC31248的杂合菌株，该菌株在0％糖面团中发酵活力较高，且具有良好的耐冷渗透休克性能。对该菌株进行鉴定，鉴定结果为：
光学显微镜下观察到菌株细胞形态为椭圆形，出芽生殖，在固体平板上长出的单菌落呈中央略微凸起的圆球状，乳白色，质地较疏松，易被接种环挑起，表面光滑、较干燥，边缘整齐，图1所示为杂合菌株AMCC31248的菌落图。
杂合菌株AMCC31248的产孢镜检情况如图2所示，在显微镜视野中孢子较多且形态较为饱满，表明其具有产孢能力，即为杂合菌株。
将得到的杂合菌株AMCC31248命名为酿酒酵母AMCC31248菌株(Saccharomyces cerevisiae AMCC31248)。该酿酒酵母AMCC31248菌株于2021年12月29日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 20211686(即CCTCC M 20211686)。
实施例2生长效率测定
将实施例1中获得的酿酒酵母AMCC31248菌株以及亲本菌株酿酒酵母AMCC30010和酿酒酵母AMCC32101接种至麦芽汁培养基(购自海博生物)中，30℃培养48h，通过全自动生长曲线分析仪Bioscreen C实现高通量测定各菌株在不同时刻的OD600值，以时间(h)为横坐标，对应的OD600值为纵坐标，绘制生长曲线，分析试验数据并根据以下公式计算菌株的生长效率。
生长效率＝(OD2-OD1)/(t2-t1)
OD1：t1时菌株对应的OD600值；
OD2：t2时菌株对应的OD600值；
t1：对数生长期阶段的起始时刻；
t2：对数生长期阶段的结束时刻。
酿酒酵母AMCC31248的生长曲线图如图3所示，可以看出，该菌株在麦芽汁培养基中能够快速生长，下表3所示为亲本菌株与酿酒酵母AMCC31248菌株的生长效率。
表3亲本及新菌株的生长效率数据
由上表3可以看出，获得的酿酒酵母AMCC31248菌株的生长效率显著高于亲本菌株酿酒酵母AMCC30010和酿酒酵母AMCC32101。
实施例3鲜酵母发酵活力检测
将实施例1中获得的酿酒酵母AMCC31248接种于装有发酵培养基的摇瓶中，30℃条件下培养。离心后收集的沉淀即酵母乳，称取酵母乳的重量并测定酵母乳的水分。根据以下公式计算得到摇瓶中各菌株的净干重(g/L)：
净干重(g/L)＝酵母乳的重量×(1-水分)
按表4所示的0％糖面团体系制备面团以检测酿酒酵母AMCC31248菌株发酵活力，计算并称取酿酒酵母AMCC31248菌株以及亲本菌株酿酒酵母AMCC30010和酿酒酵母AMCC32101所需添加的酵母乳的质量，并按照所示的面团配方分别称取面粉、盐和水，在和面机中混合均匀制作出生面团，利用SJA发酵仪直接测定由表4所示体系制备得到的280g面团在30℃条件下经酵母发酵1h产生的二氧化碳气体的总体积，即菌株的发酵活力，结果用毫升(mL)数表示。
表4 0％糖面团体系
根据以下公式计算表5中净干重相对百分比：
净干重相对百分比(％)＝(杂合新菌株净干重/亲本菌株净干重)*100％
根据以下公式计算表6中酿酒酵母AMCC31248菌株相比亲本菌株的面团发酵活力相对百分比：
面团发酵活力相对百分比(％)＝杂合新菌株的面团发酵活力/亲本菌株
的面团发酵活力*100％
如表5所示，酿酒酵母AMCC31248菌株的净干重分别为亲本菌株酿酒酵母AMCC30010和酿酒酵母AMCC32101的95.7％、109.9％，如表6所示，酿酒酵母AMCC31248菌株鲜酵母的0％糖面团发酵活力均超过两株亲本，具有10％左右的优势。
表5亲本及新菌株的净干重数据
表6鲜酵母在0％糖面团体系中的发酵活力数据
实施例4活性干酵母发酵活力检测
将酿酒酵母AMCC31248菌株活化培养后进行45L体系的发酵罐放大培养，再经分离、洗涤、压滤、干燥得到活性干酵母。按照表7所示的面团配方分别称取面粉、盐、水和制备的活性干酵母，利用SJA法检测由表7所示体系制备得到的面团280g经活性干酵母发酵1h所产生二氧化碳气体的总量，即活性干酵母的面团发酵活力。
表7 0％糖面团体系
由于亲本菌株AMCC32101在活性干酵母的制备过程中干燥难度较大,无对应的发酵活力数据，所以这里仅以亲本菌株AMCC30010的干酵母活力作为对照。酿酒酵母AMCC31248菌株在干酵母制备过程中无明显异常情况，其活性干酵母的0％糖面团发酵活力检测具体数据见表8。其中，表8中的发酵活力相对百分比的计算方法如下所示：
发酵活力相对百分比＝(杂合新菌株发酵活力/亲本菌株AMCC 30010发
酵活力)*100％
表8活性干酵母在0％糖面团体系中的发酵活力数据
结果显示，相比于亲本酿酒酵母AMCC30010菌株，酿酒酵母AMCC31248菌株的活性干酵母在0％糖条件下的面团发酵活力仍具有11.5％的优势，说明酿酒酵母AMCC31248菌株具有一定的耐干燥性。
实施例5活性干酵母耐冷渗透休克性能检测
按照表9所示的面团配方将0℃的碎冰倒入和面机内，加入活性干酵母低速混合，再加入面粉、盐，继续混合搅拌均匀，制备好面团。将400g的面团整形后置于醒发箱内发酵，控制温度为38±1℃、湿度为85-90％，记录面团发酵达到同一设定高度时所需的时间，即发酵时间。
表9冷渗透休克面团体系
酿酒酵母AMCC31248菌株和亲本酿酒酵母AMCC30010菌株对应面团的发酵时间见表10。其中，表10中发酵时间相对百分比计算方法如下所示：
发酵时间相对百分比＝(杂合新菌株发酵时间/亲本菌株AMCC30010发
酵时间)*100％
表10活性干酵母在冷渗透休克面团体系中的发酵时间数据
结果显示，酿酒酵母AMCC31248菌株的发酵时间仅为亲本酿酒酵母AMCC30010菌株的70.0％，具有30％的优势，说明在同一条件下，酿酒酵母AMCC31248菌株发面更快，具有优良的耐冷渗透休克性能。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (12)

  1. 一种酿酒酵母菌株，其特征在于，所述酿酒酵母菌株为：
    酿酒酵母AMCC31248菌株(Saccharomyces cerevisiae AMCC31248)，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 20211686。
  2. 一种酿酒酵母菌剂的发酵制备方法，其特征在于，所述方法包含如下步骤：培养权利要求1所述的酿酒酵母菌株。
  3. 根据权利要求2所述的制备方法，其特征在于，所述制备方法包括如下步骤：
    (1)将权利要求1所述的酿酒酵母菌株放大培养；
    (2)将步骤(1)得到的产物加入到液体培养基中，在26-32℃条件下发酵培养。
  4. 一种菌剂，其特征在于，所述菌剂中含有权利要求1所述的酿酒酵母AMCC31248菌株(Saccharomyces cerevisiae AMCC31248)。
  5. 根据权利要求4所述的菌剂，其特征在于，所述菌剂通过权利要求2或3所述的发酵制备方法得到。
  6. 权利要求1所述的酿酒酵母菌株或权利要求4或5所述的菌剂在发酵中的应用。
  7. 权利要求1所述的酿酒酵母菌株或权利要求4或5所述的菌剂在面团中的应用。
  8. 一种面团，其特征在于，所述面团中含有权利要求1所述的酿酒酵母菌株或权利要求4或5所述的菌剂。
  9. 根据权利要求8所述的面团，其特征在于，所述面团中含有质量比为100:0.5-5的面粉和所述酿酒酵母菌株。
  10. 权利要求8或9所述的面团的制备方法，其特征在于，所述制备方法包括如下步骤：以0-35℃的水和面。
  11. 一种面制品，其通过权利要求8或9所述的面团制备方法得到。
  12. 权利要求11所述的面制品，其为馒头、包子、面包、饼干、面条、锅贴。
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