Heavy-sugar-tolerant saccharomyces cerevisiae strain, screening method and application thereof

Abstract

The invention relates to a heavy-sugar-tolerant saccharomyces cerevisiae strain, a screening method and application thereof, the heavy-sugar-tolerant saccharomyces cerevisiae strain is specifically a saccharomyces cerevisiae AMCC 31557 strain, is preserved in China Center for Type Culture Collection (CCTCC), has a preservation number of CCTCC NO: M20231789, has excellent activity in heavy sugars with different concentrations and organic acids and salts thereof with relatively high concentration, and can be used for preparing a heavy-sugar-tolerant saccharomyces cerevisiae strain. The application is wide.

Description

Technical Field

[0001] The present invention relates to the field of microbial screening and food fermentation technology, and in particular to a high-sugar-tolerant brewer's yeast strain, a screening method and applications thereof. Background Art

[0002] Saccharomyces cerevisiae is widely used in bread production as an excellent microbial fermentation agent and leavening agent. Because it is rich in amino acids, vitamins, and trace elements, it is also a bionutrient that can enhance the nutritional value of food. Baker's yeast is generally divided into two categories: low-sugar baker's yeast, suitable for doughs without added sugar or with a sugar content below 7%, and high-sugar baker's yeast, suitable for fermenting high-sugar doughs and commonly used to make baked goods with high sugar content, such as sweet breads, pastries, and cakes. In the production of fermented foods like sweet bread, in addition to flour, yeast, and water, large amounts of sucrose are added. The addition of high sucrose concentrations creates a high osmotic pressure, which can easily cause protoplasm and water in the yeast to leak through the cell membrane, leading to plasmolysis and even death. This significantly limits the fermentation activity of conventional baker's yeast. Therefore, high-sugar yeast with excellent tolerance to high sucrose concentrations is needed to avoid the negative effects of increased yeast usage, such as a strong yeasty flavor and high production costs, while effectively preserving the flavor of the sweet bread. Here, sugar content of 20% and above is defined as heavy sugar.

[0003] In order to expand production scale, reduce costs, and improve the convenience of delivery of baked products, industrial users in the baking industry usually add organic acids and their salts to bread to extend its shelf life, such as propionic acid, dehydroacetic acid, calcium propionate, sodium propionate, sodium dehydroacetate, etc. However, in the 2021 draft for comments on the revised version of the "National Food Safety Standard for the Use of Food Additives", restrictions were placed on the use of dehydroacetic acid and its sodium salts in bread, pastries, and baked goods. As a result, the market demand for yeast strains to be resistant to organic acids and their salts has also changed. At present, calcium propionate, as one of the most widely used mold inhibitors, is often added to foods such as bread and cakes to extend their shelf life. It is worth mentioning that the addition of organic acids and their salts inhibits the growth of bacteria, molds and other fungi, but also affects the proliferation and fermentation activity of yeast to a certain extent. This requires that heavy sugar-tolerant yeast must also have a certain tolerance to organic acids and their salts in order to be suitable for actual application scenarios.

[0004] Therefore, in order to meet the actual application scenario needs of users in the baking industry, it is urgent to develop excellent yeast strains that are tolerant to heavy sugars and high concentrations of organic acids and their salts. Summary of the Invention

[0005] The problem existing in the existing technology is that the existing technology lacks strains that have excellent tolerance to different heavy sugar concentrations and to the addition of higher concentrations of organic acids and their salts under conditions of different heavy sugar concentrations.

[0006] To address the above problems, the present invention has obtained a high-sugar-tolerant Saccharomyces cerevisiae strain through hybrid breeding. The strain has excellent activity in the presence of different concentrations of high sugars and in the presence of relatively high concentrations of organic acids and their salts, and has a wide range of applications. Specifically, the present invention proposes the following technical solutions:

[0007] In a first aspect, the present invention provides a high-sugar-tolerant Saccharomyces cerevisiae strain, characterized in that the strain is Saccharomyces cerevisiae AMCC 31557 strain, which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20231789.

[0008] Preferably, the high-sugar-tolerant Saccharomyces cerevisiae strain is characterized in that the strain has high-sugar tolerance, and / or organic acid tolerance, and / or organic acid salt tolerance;

[0009] Preferably, the organic acid comprises one or both of propionic acid and acetic acid;

[0010] Further preferably, the organic acid salt comprises one or a combination of two or more of calcium propionate, sodium propionate and sodium acetate;

[0011] More preferably, the organic acid salt comprises calcium propionate.

[0012] Preferably, the heavy sugar-tolerant Saccharomyces cerevisiae strain is characterized in that the strain is obtained by hybridizing sexual spores of different parent strains.

[0013] Preferably, the heavy sugar-tolerant Saccharomyces cerevisiae strain is characterized in that the dry weight of yeast milk of the heavy sugar-tolerant Saccharomyces cerevisiae strain is 90-110%, preferably 95-110%, of the dry weight of yeast milk of the parent strain.

[0014] Preferably, the high-sugar-tolerant Saccharomyces cerevisiae strain is characterized in that the fermentation activity of the yeast milk of the high-sugar-tolerant Saccharomyces cerevisiae strain in a dough system containing 20-25% sugar reaches 95-130%, preferably 100-130% of that of the yeast milk of the parent strain; and / or

[0015] The fermentation activity of the yeast milk of the heavy sugar-tolerant Saccharomyces cerevisiae strain in a dough system containing 20-25% sugar and 0.1-1% calcium propionate reaches 95-120%, preferably 100-120%, of the yeast milk of the parent strain.

[0016] Preferably, the high-sugar-tolerant Saccharomyces cerevisiae strain is characterized in that the fermentation activity of the active dry yeast of the high-sugar-tolerant Saccharomyces cerevisiae strain in a dough system containing 16-50% sugar reaches 95-120%, preferably 105-120% of the active dry yeast of the parent strain; and / or

[0017] The active dry yeast of the heavy sugar-tolerant Saccharomyces cerevisiae strain has a fermentation activity in a dough system containing 16-50% sugar and 0-0.6% calcium propionate that reaches 95-120%, preferably 105-120%, of the active dry yeast of the parent strain.

[0018] In a second aspect, the present invention provides a method for screening and obtaining a high-sugar-tolerant Saccharomyces cerevisiae strain, the method comprising: forming sexual spores from a parent Saccharomyces cerevisiae strain and hybridizing single spores from different parents to obtain a hybrid strain, wherein the hybrid strain has:

[0019] The dry weight of the yeast milk of the hybrid bacteria is 90-110% of the dry weight of the yeast milk of the parent Saccharomyces cerevisiae;

[0020] The fermentation activity of the hybrid yeast milk reaches 95-130% of that of the parent yeast milk of saccharomyces cerevisiae in a dough system containing 20-25% sugar.

[0021] Preferably, the method, wherein the hybrid bacteria has:

[0022] The dry weight of the yeast milk of the hybrid bacteria is 95-110% of the dry weight of the yeast milk of the parent Saccharomyces cerevisiae;

[0023] Preferably, the fermentation activity of the hybrid yeast milk in a dough system containing 20-25% sugar reaches 100-130% of that of the parent Saccharomyces cerevisiae yeast milk; and / or

[0024] The hybrid yeast milk has a fermentation activity of 95-120%, preferably 100-120%, of the parent yeast milk of Saccharomyces cerevisiae in a dough system containing 20-25% sugar and 0.1-1% calcium propionate.

[0025] Preferably, the method, wherein the hybrid bacteria has:

[0026] The fermentation activity of the active dry yeast of the hybrid bacteria in a dough system containing 16-50% sugar reaches 95-120%, preferably 105-120% of the active dry yeast of the parent Saccharomyces cerevisiae; and / or

[0027] The fermentation activity of the active dry yeast of the hybrid bacteria in a dough system containing 16-50% sugar and 0-0.6% calcium propionate reaches 95-120%, preferably 105-120%, of the active dry yeast of the parent saccharomyces cerevisiae.

[0028] In a third aspect, the present invention provides a fermentation composition, which comprises the high-sugar-tolerant Saccharomyces cerevisiae strain or the high-sugar-tolerant Saccharomyces cerevisiae strain screened by the method described.

[0029] In a fourth aspect, the present invention provides a bacterial agent, which comprises the high-sugar-tolerant Saccharomyces cerevisiae strain or the high-sugar-tolerant Saccharomyces cerevisiae strain screened by the method described.

[0030] In a fifth aspect, the present invention provides the use of the heavy-sugar-tolerant Saccharomyces cerevisiae strain or the heavy-sugar-tolerant Saccharomyces cerevisiae screened by the method described above, or the fermentation composition or the bacterial agent in food.

[0031] In a sixth aspect, the present invention provides a dough, which is obtained by using the heavy-sugar-tolerant Saccharomyces cerevisiae strain or the heavy-sugar-tolerant Saccharomyces cerevisiae strain screened using the method described.

[0032] The dough is characterized in that it is obtained by fermenting raw materials including the following parts by weight: 100-110 parts of flour, 16-50 parts of sugar, 38-58 parts of water and 1-2 parts of heavy sugar-tolerant brewer's yeast dry yeast; preferably, the raw materials also include 0-1 part of calcium propionate; further preferably, the raw materials also include 1-2 parts of salt; more preferably, the raw materials also include 6-8 parts of butter and 1-2 parts of milk.

[0033] In a seventh aspect, the present invention provides a method for preparing the dough, comprising: fermenting the heavy-sugar-tolerant Saccharomyces cerevisiae strain or the heavy-sugar-tolerant Saccharomyces cerevisiae screened by the method described above to obtain dough.

[0034] In an eighth aspect, the present invention provides a baked product, which is obtained by baking the dough or the dough prepared by the method.

[0035] Preferably, the baked product is one of bread, biscuits, Mala cake and brown sugar cake.

[0036] The beneficial effects of the present invention include:

[0037] The high-sugar-tolerant Saccharomyces cerevisiae AMCC 31557 strain provided by the present invention has excellent activity in high-sugar concentrations of different concentrations and in the presence of organic acids and salts thereof added at relatively high concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shown is the colony morphology of the heavy sugar-tolerant Saccharomyces cerevisiae strain AMCC 31557;

[0039] Figure 2Shown is a microscopic image of the heavy sugar-tolerant Saccharomyces cerevisiae strain AMCC 31557 (microscope model Olympus CX43, magnification 400 times);

[0040] Figure 3 Shown are growth curves of the heavy sugar-tolerant Saccharomyces cerevisiae strain AMCC 31557, the parent Saccharomyces cerevisiae strain AMCC 31248, and the parent Saccharomyces cerevisiae strain AMCC 31195.

[0041] Culture collection information

[0042] The Saccharomyces cerevisiae AMCC 31557 strain provided by the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on September 25, 2023, with a deposit number of CCTCC NO: M 20231789. The deposit address is: Wuhan University, Wuhan, China, Postal Code: 430072; Telephone: 027-68754052.

[0043] The Saccharomyces cerevisiae AMCC 31248 strain provided by the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on December 29, 2021, with the deposit number CCTCC NO: M 20211686, the deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052; and disclosed in the patent application with publication number CN117165460A.

[0044] The Saccharomyces cerevisiae AMCC 31195 strain provided by the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on December 29, 2021, with the deposit number CCTCC NO: M 20211685, the deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052; and disclosed in the patent application with publication number CN117165455A. DETAILED DESCRIPTION

[0045] The heavy sugar-tolerant Saccharomyces cerevisiae AMCC 31557 strain provided by the present invention is based on the Saccharomyces cerevisiae AMCC 31248 strain (deposit number CCTCC NO: M20211686, disclosed in patent application CN117165460A) and the Saccharomyces cerevisiae AMCC 31195 strain (deposit number CCTCC NO: M 20211685, disclosed in patent application CN117165455A) in the strain library of Angel Yeast Co., Ltd. as experimental starting strains. The sexual reproduction characteristics of yeast are utilized to hybridize and select progeny with heterosis, thereby obtaining a new strain with excellent resistance to different concentrations of heavy sugars and relatively high concentrations of organic acids and their salts.

[0046] The heavy sugar tolerance means that the yeast strain has a fermentation activity advantage in a dough system with a sugar content of more than 20%, including a fermentation activity advantage in a dough system with a sugar content of 25%, 30%, 35%, 40%, 45% and 50%.

[0047] First, haploid strains of the parent strains, Saccharomyces cerevisiae AMCC 31248 and Saccharomyces cerevisiae AMCC 31195, were prepared using a yeast micromanipulator. Haploid strains that showed growth advantage in a high-sugar selective medium were selected. After activation and culture, hybridization experiments were performed to obtain heterozygotes. Growth curves of the resulting heterozygous strains were measured using the Bioscreen C automated growth curve analyzer, and those with a clear growth advantage were selected. A shake flask fermentation test is carried out on the preferred hybrid strain, and the net dry weight of the yeast milk of the hybrid strain and the fermentation activity of the yeast milk in various dough systems are used as screening indicators. The specific screening criteria are: the net dry weight of the hybrid strain reaches 90-110%, preferably 95-110%, of that of any parent strain; the fermentation activity of the yeast milk of the hybrid strain with 20% sugar in 2 hours reaches 95-130%, preferably 100-130%, of that of any parent strain; the fermentation activity of the yeast milk of the hybrid strain with 20% sugar + 1% calcium propionate in 2 hours reaches 95-120%, preferably 100-120%, of that of any parent strain; and the fermentation activity of the yeast milk with 25% sugar in 2 hours reaches 95-120%, preferably 100-120%, of that of any parent strain.

[0048] The hybrid strain screened in the above step is then cultured in a 45 L fermentation tank, and the obtained yeast cells are prepared into active dry yeast, and the fermentation activity of the active dry yeast in various dough systems is measured. The different dough systems include 16% sugar, 16% sugar + 0.6% calcium propionate, 20% sugar, 20% sugar + 0.6% calcium propionate, 25% sugar, 25% sugar + 0.6% calcium propionate, 30% sugar, 30% sugar + 0.6% calcium propionate, 40% sugar, 40% sugar + 0.6% calcium propionate, 50% sugar, and 50% sugar + 0.6% calcium propionate. The specific screening criteria are: the hybrid strain has no obvious abnormalities during the active dry yeast preparation process, and the fermentation activity of the active dry yeast in each dough system reaches 95-120%, preferably 105-120%, of that of any parent strain.

[0049] Finally, the hybrid strain selected from the above steps was tested in different formulations, including 20% ​​sugar, 20% sugar + 0.5% calcium propionate, 25% sugar, and 25% sugar + 0.5% calcium propionate. The fermentation time of each strain in dough from these different systems was measured, i.e., the time it took for the dough to rise to the same height. Shorter dough fermentation times indicate faster rise and stronger fermentation capacity. Strains with shorter fermentation times than their parent strains were selected as target strains.

[0050] The above screening yielded a hybrid strain, designated AMCC 31557, which exhibited high fermentation activity in various doughs and demonstrated excellent tolerance to high sugar osmotic pressure and high concentrations of organic acids and their salts. Identification of the strain yielded the following results:

[0051] The cells of the strain were observed to be oval in shape and reproduced by budding under an optical microscope. The single colonies grown on the solid plate were spherical with a slightly raised center, milky white in color, loose in texture, easily picked up by the inoculation loop, with a smooth, dry surface and neat edges (see Figure 1 ).

[0052] The spore production of hybrid strain AMCC 31557 is as follows Figure 2 As shown in the figure, there are many spores in the microscope field and the spores are relatively full, indicating that it has the ability to produce spores, that is, it is a heterozygous strain.

[0053] The obtained hybrid strain AMCC 31557 was named as heavy sugar-tolerant Saccharomyces cerevisiae AMCC 31557 strain.

[0054] The parent yeast strain, Saccharomyces cerevisiae AMCC 31248, was obtained by microhybridization. The specific construction and identification methods for the strain are disclosed in patent application publication number CN117165460A. The strain was identified as follows: Under an optical microscope, the cells of the strain exhibited an oval morphology and budding reproduction. Single colonies grown on solid plates were spherical with a slightly raised center, milky white in color, and had a loose texture that was easily picked up by an inoculating loop. The colonies had a smooth, relatively dry surface and neat edges.

[0055] The parent yeast strain, Saccharomyces cerevisiae AMCC 31195, was obtained by hybridization. The specific construction and identification methods for the strain are disclosed in patent application publication number CN117165455A. The strain was identified as exhibiting a cheese-like colony texture, milky white color, smooth surface, neat edges, an oval microscopic shape, and budding reproduction.

[0056] Unless otherwise specified, the various reagents and instruments used in the examples of the present invention are conventional commercially available products. The source information of the instruments and reagents used in the examples of the present invention is shown in Tables 1 and 2 below.

[0057] Table 1 Instruments used in the examples

[0058]

[0059]

[0060] Table 2 Reagents used in the examples

[0061] Reagents purity Commercial Source Yeast extract powder / Angel Yeast Peptone / Angel Yeast glucose Analytical pure AR Sinopharm Shanghai trial sucrose Analytical pure AR Sinopharm Shanghai trial agar Biochemical reagents BR Huixing Potassium acetate Analytical pure AR Sinopharm Shanghai trial concentrated sulfuric acid Analytical pure AR Xilong Chemical Sodium hydroxide Analytical pure AR Sinopharm 2×PCR Mix / Tiangen

[0062] The x% sugar or x% calcium propionate involved in the dough fermentation system in the embodiment of the present invention means that the mass ratio of sugar to flour or calcium propionate to flour in the dough is X:100.

[0063] The formula of the spore production medium used in the embodiment of the present invention is as follows by mass percentage: 1% potassium acetate, 0.1% yeast extract powder, 0.05% glucose, and 2% agar.

[0064] In the embodiment of the present invention, each strain of Saccharomyces cerevisiae was activated using YPD solid culture medium, the formula of which is as follows by mass percentage: 1% yeast extract powder, 2% peptone, 2% glucose, and 2% agar.

[0065] In the embodiment of the present invention, each strain of Saccharomyces cerevisiae was cultured in a YPD liquid culture medium, wherein the formula of the YPD liquid culture medium is as follows in percentage by mass: 1% yeast extract powder, 2% peptone, and 2% glucose.

[0066] In the embodiment of the present invention, the prepared single spores are screened using a high-sugar screening medium, wherein the formula of the high-sugar screening medium is as follows in percentage by mass: 30% sucrose, 1% yeast extract powder, 2% peptone, and 1% calcium propionate.

[0067] Example 1 Strain Construction

[0068] The present invention uses Saccharomyces cerevisiae strains AMCC 31195 and AMCC 31248 as parents, inducing them to produce spores under the same conditions. Haploids of each parent strain are prepared using a yeast micromanipulator and cultured at 30°C. The number of spores produced is recorded. After colonies grow, individual spores from the AMCC 31248 parent strain are designated dan1-dan25, and individual spores from the AMCC 31195 parent strain are designated dan26-dan60. PCR is then performed on these 60 surviving spores to determine their MATa / α zygosity. The zygosity identification method involves performing colony PCR on the surviving spores using primers MAT-a, MAT-α, and MAT-F. The PCR procedure is as follows: 94°C pre-denaturation for 10 minutes, 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, and 72°C extension for 60 seconds, for 30 cycles, followed by a final extension at 72°C for 10 minutes. The amplified product was detected by 1.5% (1.5g / 100mL) agarose gel electrophoresis. If a band was found at 404bp, the mating type was determined to be α type, and if a band was found at 544bp, the mating type was determined to be a type. The specific results are shown in Table 3. The primer sequences are:

[0069] MAT-a(5'-ACTCCACTTCAAGTAAGAGTTTG-3')(SEQ ID NO.1)

[0070] MAT-α(5'-GCACGGAATATGGGACTACTTCG-3')(SEQ ID NO.2)

[0071] MAT-F (5'-AGTCACATCAAGATCGTTTATGG-3') (SEQ ID NO. 3) survival rate (%) = (number of surviving spores / number of deposited spores) * 100%

[0072] Table 3 Single spore survival rate and zygotic distribution of parent strains

[0073]

[0074] The single spore prepared above was inoculated into a test tube filled with YPD liquid medium, cultured with shaking at 30°C overnight, and then inoculated into a 100-well microplate filled with heavy sugar screening medium. Bioscreen C was used to detect the OD600 value of each strain in real time. The culture temperature was set to 30°C, the culture time was 24 hours, and the measurement wavelength was 600 nm. Data was measured every 30 minutes. A growth curve was drawn with time (h) as the horizontal axis and OD600 value as the vertical axis. The growth efficiency was analyzed based on the growth curve results. The growth efficiency was calculated as follows:

[0075] Growth efficiency = (OD2-OD1) / (t2-t1)

[0076] OD1: OD600 value corresponding to the strain at t1;

[0077] OD2: OD600 value corresponding to the strain at t2;

[0078] t1: the starting time of the logarithmic growth phase;

[0079] t2: The end of the logarithmic growth phase.

[0080] Haploid strains with growth advantages in the heavy sugar screening medium were selected, and the top eight single spores in growth efficiency were selected (the growth efficiency and matching type of each strong spore are shown in Table 4). After activation culture, single spores with different matching types were inoculated into test tubes containing YPD liquid medium according to the hybrid combination (as shown in Table 5). The culture was shaken at 30°C overnight, and the bacterial solution was diluted and spread on YPD solid plates, which were cultured at 30°C. The colony growth was observed, and the 140 largest colonies were selected, marked, and named.

[0081] Table 4 Spore growth efficiency and matching of each strong

[0082]

[0083] Table 5 Hybrid combinations

[0084]

[0085] The marked colonies on the above plates were activated and cultured for typing identification and sporulation test. The screening criteria were: strains with double bands in the electrophoresis results and ascospores observed under a microscope after sporulation culture were judged as heterozygous strains.

[0086] Example 2 Growth efficiency determination

[0087] The constructed hybrid strain was inoculated into a heavy sugar screening medium and cultured at 30°C for 24 h. The OD600 value of each strain at different times was determined by high-throughput measurement using the fully automatic growth curve analyzer Bioscreen C. The growth curve was plotted with time (h) as the horizontal axis and the corresponding OD600 value as the vertical axis. The growth efficiency was analyzed and calculated, and the top 50 hybrid strains in growth efficiency were selected.

[0088] The growth curve of the strain numbered Saccharomyces cerevisiae AMCC 31557 is shown in the figure below. Figure 3 As shown, it can be seen that the strain can grow rapidly in the heavy sugar screening medium. Table 6 shows the growth efficiency of the parent strain and the Saccharomyces cerevisiae AMCC 31557 strain. It can be seen that the growth efficiency of the obtained Saccharomyces cerevisiae AMCC 31557 strain is significantly higher than that of the parent strains Saccharomyces cerevisiae AMCC 31248 strain and Saccharomyces cerevisiae AMCC 31195 strain.

[0089] Table 6 Growth efficiency data of parent and new strains

[0090]

[0091] Example 3 Shake flask yeast milk fermentation activity detection

[0092] The preferred hybrid strain obtained in Example 2 was subjected to a shake flask fermentation test. The net dry weight of the hybrid strain and the fermentation activity of its yeast milk in various dough systems were used as screening indicators. The hybrid strain was inoculated into a shake flask filled with YPD liquid fermentation medium and cultured with shaking at 30°C overnight. Yeast milk was obtained after centrifugation and washing. The mass of the yeast milk was weighed as the biomass, and the moisture content was measured using a rapid moisture meter. The net dry weight (g / L) and the relative percentage (%) of the yeast milk of each strain in the shake flask stage were calculated according to the following formula:

[0093] Net dry weight (g / L) = mass of yeast milk × (1-water %)

[0094] Relative percentage of net dry weight (%) = (net dry weight of hybrid strain / net dry weight of parent strain) * 100%. The sucrase activity of yeast milk of each strain was determined by the DNS (dinitrosalicylic acid) colorimetric method and calculated based on the moisture content of the yeast milk determined above, with the unit being U / g.

[0095] The yeast extracts from the collected strains were tested for fermentation activity in different dough systems: System 1: 20% sugar dough system, System 2: 20% sugar + 1% calcium propionate dough system, and System 3: 25% sugar dough system. The ratios of the raw material components in the different dough systems (based on the weight of flour as 100%) are shown in Table 7. The amount of yeast extract required for each strain was calculated and weighed based on the moisture content of each strain. Flour, salt, sugar, and water were weighed according to the dough recipes shown and mixed uniformly in a dough mixer to produce raw dough. The total volume of carbon dioxide produced by yeast fermentation at 30°C in the doughs prepared using the systems shown in Table 7 was directly measured using an SJA fermentation instrument, representing the fermentation activity of the strains. The results are expressed in milliliters (mL). The total gas production of 70g of dough over 2 hours was measured for both System 1 and System 3, while the total gas production of 70g of dough over 3 hours was measured for System 2.

[0096] Table 7 Ratio of each raw material component in different dough systems (%)

[0097]

[0098] The relative percentage of dough fermentation activity of each strain compared to the parent strain was calculated according to the following formula:

[0099] Relative percentage of dough fermentation activity (%) = (dough fermentation activity of hybrid new strain / dough fermentation activity of parent strain) * 100%

[0100] As shown in Table 8, during the shake flask stage, after overnight cultivation, the dry biomass weight of the Saccharomyces cerevisiae strain, designated AMCC 31557, reached 14.40 g / L, a 5.1% advantage over its parent strain, Saccharomyces cerevisiae AMCC 31195. The sucrase activity of Saccharomyces cerevisiae AMCC 31557 was 304.01 U / g, representing 78% of the activity of its parent strain, Saccharomyces cerevisiae AMCC 31195, and 70% of the activity of its parent strain, Saccharomyces cerevisiae AMCC 31248. Related research has shown that yeast strains with lower sucrase activity exhibit higher fermentation activity in sugar-sweetened dough. However, excessive sucrase activity can lead to a rapid increase in monosaccharide concentration, which, in turn, increases osmotic pressure, leading to a decrease in fermentation activity.

[0101] Table 8 Net dry weight data and sucrase activity of parent and new strains

[0102]

[0103]

[0104] In the shake flask stage, after overnight culture, the net dry weight of the other preferred hybrid strains obtained in Example 2 was between 10.51-14.89 g / L, which was 76.7%-108.7% of that of the parent strain Saccharomyces cerevisiae AMCC 31195; the sucrase enzyme activity was 71.8%-126.4% of that of the parent strain Saccharomyces cerevisiae AMCC 31195.

[0105] As shown in Table 9, using the parent strain AMCC 31195 with higher fermentation activity as the control strain, the CO2 gas volume produced by the Saccharomyces cerevisiae AMCC31557 strain in the 20% sugar dough system and the 25% sugar dough system after 70 g dough fermented for 2 h was 268 mL and 138 mL, respectively, which were 28.2% and 56.8% higher than those of the parent strain AMCC 31195, respectively; the gas production of the Saccharomyces cerevisiae AMCC31557 strain in the 20% sugar + 1% calcium propionate dough system after 70 g dough fermented for 3 h was 145 mL, which was 59.3% higher than that of the parent strain AMCC 31195.

[0106] Table 9 Fermentation activity data of the parent strain and the new strain in each dough system

[0107]

[0108] In the shake flask stage, after overnight culture, the activity of the 20% sugar dough system of the other preferred hybrid strains obtained in Example 2 was 71.8%-129.3% of that of the parent strain Saccharomyces cerevisiae AMCC 31195, the activity of the 25% sugar dough system was 83.0-170.5% of that of the parent strain Saccharomyces cerevisiae AMCC 31195, and the activity of the 20% sugar + 1% calcium propionate dough system was 71.4%-181.3% of that of the parent strain Saccharomyces cerevisiae AMCC 31195.

[0109] Example 4: Detection of fermentation activity of active dry yeast in a small test

[0110] The selected strains screened from shake flask fermentation experiments were scaled up in a 45L fermenter, followed by separation, washing, filter pressing, and drying to produce active dry yeast. According to the dough recipes shown in Table 10, flour, salt, sugar, water, and active dry yeast were weighed to prepare doughs of different systems. The total amount of carbon dioxide produced, i.e., the fermentation activity of the active dry yeast in the corresponding dough system, was measured using the SJA method. The results are expressed in milliliters (mL). The different dough systems include system A1: 16% sugar dough system, the total gas production of 280g dough in 2h is tested; system A2: 16% sugar + 0.6% calcium propionate dough system, the total gas production of 280g dough in 2h is tested; system B1: 20% sugar dough system, the total gas production of 100g dough in 2h is tested; system B2: 20% sugar + 0.6% calcium propionate dough system, the total gas production of 100g dough in 3h is tested; system C1: 25% sugar dough system, the total gas production of 280g dough in 2h is tested; system C2: 25% sugar + 0.6% calcium propionate dough system, the total gas production of 280g dough in 3h is tested. Gas production; System D1: 30% sugar dough system, the total gas production of 280g dough was tested for 2h; System D2: 30% sugar + 0.6% calcium propionate dough system, the total gas production of 280g dough was tested for 3h; System E1: 40% sugar dough system, the total gas production of 280g dough was tested for 2h; System E2: 40% sugar + 0.6% calcium propionate dough system, the total gas production of 280g dough was tested for 3h; System F1: 50% sugar dough system, the total gas production of 280g dough was tested for 3h; System F2: 50% sugar + 0.6% calcium propionate dough system, the total gas production of 280g dough was tested for 4h.

[0111] Table 10 Ratio of each raw material component in different dough systems (%)

[0112]

[0113] The Saccharomyces cerevisiae strain, designated AMCC 31557, exhibited no abnormalities during fermentation and sample preparation, and all physical and chemical indicators were normal. As shown in Table 11, using the parent strain, AMCC 31195, which exhibits more advantageous properties, as a control strain, the active dry yeast prepared from the Saccharomyces cerevisiae strain AMCC 31557 provided by the present invention exhibited significant fermentation activity advantages in various dough systems and exhibited excellent tolerance to high levels of sugar and organic acids and their salts.

[0114] Table 11 Fermentation activity data of dry yeast in various dough systems

[0115]

[0116] Table 12 Relative percentage of fermentation activity of dry yeast in each dough system (%)

[0117]

[0118]

[0119] The fermentation activity of dry yeast of other strains in each dough system was not outstanding, lower than the dough fermentation activity of the parent strain Saccharomyces cerevisiae AMCC 31195, and did not meet the optimization standard.

[0120] Example 5 Application Test

[0121] The superior strains selected during the pilot phase were evaluated in application testing. The formulation used is shown in Table 13. The superior parent strain, AMCC 31195, served as the control strain. Fermentation time was used as an indicator. A shorter fermentation time indicated a faster rise rate in this dough system, indicating a more advantageous yeast strain. The percentage of fermentation time relative to the control strain is shown in Table 14.

[0122] Table 13 Ratio of different raw material components in the application formula (%)

[0123]

[0124] Table 14 Relative percentage data of strain application fermentation time

[0125]

[0126]

[0127] The results showed that compared to the control strain AMCC 31195, the AMCC 31557 strain exhibited superior fermentation times in all of the indicated application recipes, demonstrating that it exhibited superior rise speed in this dough system compared to the control strain. This further demonstrated the AMCC 31557 strain's excellent tolerance to high sugar content and to organic acids and their salts. The resulting hybrid strain, AMCC 31557, was designated the high-sugar-tolerant Saccharomyces cerevisiae AMCC 31557 strain.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A high sugar tolerant Saccharomyces cerevisiae strain, characterized in that: The strain is Saccharomyces cerevisiae AMCC 31557 strain, which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCCNO: M 20231789.

2. The heavy sugar-tolerant Saccharomyces cerevisiae strain according to claim 1, characterized in that The strain has high sugar tolerance, and / or organic acid tolerance, and / or organic acid salt tolerance; Preferably, the organic acid comprises one or both of propionic acid and acetic acid; Further preferably, the organic acid salt comprises one or a combination of two or more of calcium propionate, sodium propionate and sodium acetate; More preferably, the organic acid salt comprises calcium propionate.

3. The heavy sugar-tolerant Saccharomyces cerevisiae strain according to claim 1 or 2, characterized in that The strains are obtained by sexual spore hybridization of different parent strains.

4. The heavy sugar-tolerant Saccharomyces cerevisiae strain according to claim 3, characterized in that The dry weight of the yeast milk of the heavy sugar-tolerant Saccharomyces cerevisiae strain is 90-110%, preferably 95-110%, of the dry weight of the yeast milk of the parent strain.

5. The heavy sugar-tolerant Saccharomyces cerevisiae strain according to claim 3 or 4, characterized in that The fermentation activity of the yeast milk of the heavy sugar-tolerant Saccharomyces cerevisiae strain in a dough system containing 20-25% sugar reaches 95-130%, preferably 100-130%, of the yeast milk of the parent strain; and / or The fermentation activity of the yeast milk of the heavy sugar-tolerant Saccharomyces cerevisiae strain in a dough system containing 20-25% sugar and 0.1-1% calcium propionate reaches 95-120%, preferably 100-120%, of the yeast milk of the parent strain.

6. The heavy sugar-tolerant Saccharomyces cerevisiae strain according to any one of claims 3 to 5, characterized in that The active dry yeast of the heavy sugar-tolerant Saccharomyces cerevisiae strain has a fermentation activity in a dough system containing 16-50% sugar of 95-120%, preferably 105-120%, of the active dry yeast of the parent strain; and / or The active dry yeast of the heavy sugar-tolerant Saccharomyces cerevisiae strain has a fermentation activity in a dough system containing 16-50% sugar and 0-0.6% calcium propionate that reaches 95-120%, preferably 105-120%, of the active dry yeast of the parent strain.

7. A method for screening and obtaining a high-sugar-tolerant Saccharomyces cerevisiae strain, the method comprising: The parent yeast Saccharomyces cerevisiae is formed into sexual spores and single spores of different parents are hybridized to obtain a hybrid bacterium, wherein the hybrid bacterium has: The dry weight of the yeast milk of the hybrid bacteria is 90-110% of the dry weight of the yeast milk of the parent Saccharomyces cerevisiae; The fermentation activity of the hybrid yeast milk reaches 95-130% of that of the parent yeast milk of saccharomyces cerevisiae in a dough system containing 20-25% sugar.

8. The method according to claim 7, wherein: The hybrid bacteria have: The dry weight of the yeast milk of the hybrid bacteria is 95-110% of the dry weight of the yeast milk of the parent Saccharomyces cerevisiae; Preferably, the fermentation activity of the hybrid yeast milk in a dough system containing 20-25% sugar reaches 100-130% of that of the parent Saccharomyces cerevisiae yeast milk; and / or The hybrid yeast milk has a fermentation activity of 95-120%, preferably 100-120%, of the parent yeast milk of Saccharomyces cerevisiae in a dough system containing 20-25% sugar and 0.1-1% calcium propionate.

9. The method according to claim 7 or 8, wherein The hybrid bacteria have: The fermentation activity of the active dry yeast of the hybrid bacteria in a dough system containing 16-50% sugar reaches 95-120%, preferably 105-120% of the active dry yeast of the parent Saccharomyces cerevisiae; and / or The fermentation activity of the active dry yeast of the hybrid bacteria in a dough system containing 16-50% sugar and 0-0.6% calcium propionate reaches 95-120%, preferably 105-120%, of the active dry yeast of the parent saccharomyces cerevisiae.

10. A fermentation composition comprising the high-sugar-tolerant Saccharomyces cerevisiae strain according to any one of claims 1 to 6 or the high-sugar-tolerant Saccharomyces cerevisiae screened by the method according to any one of claims 7 to 9. A bacterial agent comprising the high-sugar-tolerant Saccharomyces cerevisiae strain according to any one of claims 1 to 6 or the high-sugar-tolerant Saccharomyces cerevisiae screened by the method according to any one of claims 7 to 9.

12. Use of the high-sugar-tolerant Saccharomyces cerevisiae strain according to any one of claims 1 to 6, the high-sugar-tolerant Saccharomyces cerevisiae obtained by screening according to any one of claims 7 to 9, the fermentation composition according to claim 10, or the microbial agent according to claim 11 in food.

13. A dough obtained by fermenting flour using the high-sugar-tolerant Saccharomyces cerevisiae strain according to any one of claims 1 to 6 or the high-sugar-tolerant Saccharomyces cerevisiae obtained by screening using the method according to any one of claims 7 to 9.

14. A method for preparing the dough of claim 13, comprising: The high-sugar-tolerant Saccharomyces cerevisiae strain according to any one of claims 1 to 6 or the high-sugar-tolerant Saccharomyces cerevisiae obtained by screening according to any one of claims 7 to 9 is fermented to obtain dough.

15. A baked product, comprising baking the dough according to claim 13 or the dough prepared by the method according to claim 14.

16. The baked product according to claim 15, wherein The baked product is one of bread, biscuits, Mala cake and brown sugar cake.

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