A composite starter culture and its use in the fermentation of food products

Abstract

The present application belongs to the technical field of microorganism strain screening and application, and particularly relates to a compound fermentation agent and application thereof in food fermentation. Two strains of yeast are screened by a traditional culture method, which are gas-producing Saccharomyces cerevisiae SQJ20 and aroma-enhancing abnormal Wickerhamomyces anomalus GZJ2. The screening source is a traditional sourdough. The application of the two strains of yeast in the fermentation process of flour products improves the flavor and quality of the flour products, especially when the two strains of yeast are mixed as a compound fermentation agent for the fermentation process of flour, the fermentation characteristics are better than those of the commercially available active dry yeast; the prepared product is improved in hardness, chewiness and other indexes, and the flavor and quality of the fermented flour products are greatly improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganism strain screening and application, and particularly relates to a compound leavening agent and application thereof in food fermentation. BACKGROUND

[0002] Fermented flour products are various, and steamed buns and breads are the most representative. As early as the Spring and Autumn and Warring States Period, China has used old flour (sourdough) to make steamed buns, and the steamed buns have gradually become an indispensable traditional staple food in northern China due to their rich nutrition and convenient preparation. The steamed buns are mainly made of wheat flour, a certain amount of water and leavening agent, and are fermented into a dough, then are kneaded, shaped, secondarily fermented and steamed to be cooked, and finally have a soft and elastic texture, a slightly sweet taste, and a fresh wheat flavor. As a staple food, the steamed buns are popular in China, Southeast Asia and other regions, and more than 1.3 billion people often eat steamed buns.

[0003] The leavening agent for making fermented flour products mainly includes two categories, commercial yeast and sourdough. The commercial yeast, also known as active dry yeast, has the advantages of strong fermentation capacity, short time and small amount, and is widely used in the industrial production of steamed buns and breads due to its convenience, but has the disadvantage of single flavor of the finished flour food. The sourdough is a leavening agent prepared by culturing natural microorganisms in fruits, grains, fermented yogurt or wine starter, and is also known as natural yeast. The sourdough is a multi-strain leavening agent containing various yeast and lactic acid bacteria, and a variety of organic acids and unique flavor substances such as alcohols, aldehydes and esters are produced in the fermentation process, which can improve the flavor of the fermented flour product and is popular with consumers. However, the preparation process of the sourdough fermented flour product is complex, time-consuming and unstable, and it is difficult to realize industrial production. SUMMARY

[0004] Therefore, in order to solve one of the above technical problems, the present application provides a compound leavening agent and application thereof in food fermentation. The compound leavening agent provided by the present application includes gas-producing Saccharomyces cerevisiae and abnormal Williopsis sp., and inoculation of the compound leavening agent into a fermentation system helps to improve the aroma and quality of the fermented product, and the prepared food has stable quality and better taste, and is more easily accepted by the public.

[0005] In order to achieve the object of the present application, the present application provides the following technical solutions:

[0006] The present application provides a kind of yeast complex fermenting agent, including two strains of Saccharomyces cerevisiae and Wickerhamomyces anomalus, the Saccharomyces cerevisiae is gas production type Saccharomyces cerevisiae strain, named Saccharomyces cerevisiae SQJ20 (Saccharomyces cerevisiae SQJ20, SQJ20 for short), the preservation agency is China Wuhan Typical Culture Collection Center, and the preservation unit address is: Wuhan University, Wuhan, Hubei Province, Wuchang District, Bayi Road 299;Preservation number is CCTCC NO:M 2023858, and the preservation date is May 29, 2023;The Wickerhamomyces anomalus is aromatic type Wickerhamomyces anomalus strain, named Wickerhamomyces anomalus GZJ2 (Wickerhamomyces anomalus GZJ2, GZJ2 for short), the preservation agency is China Wuhan Typical Culture Collection Center, and the preservation unit address is: Wuhan University, Wuhan, Hubei Province, Wuchang District, Bayi Road 299, preservation number is CCTCC NO:M 2023857, and the preservation date is May 29, 2023.

[0007] Preferably, the number ratio of Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2 in the yeast complex fermenting agent is 1:1-3.

[0008] In the yeast complex fermenting agent, Saccharomyces cerevisiae SQJ20 has gas production characteristics, and Wickerhamomyces anomalus GZJ2 has aromatic characteristics.

[0009] The Saccharomyces cerevisiae and Wickerhamomyces anomalus in the present application are obtained by screening sour dough. Two fermentation strains are obtained by gradient dilution combined with plate coating method from sour dough. They are Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2, and the genetic traits are stable within ten generations of subculture.

[0010] The present application also provides the application of the yeast complex fermenting agent as described above in the field of food fermentation.

[0011] Preferably, the food is a flour product. During the dough fermentation stage, the yeast complex fermenting agent is inoculated into the fermentation system in a proper proportion, which helps to improve the aroma and quality of the flour product.

[0012] Preferably, the addition amount of Saccharomyces cerevisiae SQJ20 in the fermentation system is 1-2 x 10 9 cfu per 100 g of flour.

[0013] The present application provides two yeast strains, one is a gas-producing Saccharomyces cerevisiae strain, named Saccharomyces cerevisiae SQJ20 (abbreviated as SQJ20), and the preservation agency is China Center for Type Culture Collection, Wuhan, Hubei Province, China, the address of the preservation unit is: Wuhan University, 299 Bajiyi Road, Wuchang District, Wuhan, Hubei Province, China, the preservation number is CCTCC NO: M2023858, and the preservation date is May 29, 2023.

[0014] The colony characteristics of the Saccharomyces cerevisiae SQJ20 are as follows: the colony on the YPD medium is milky white, spherical, opaque, with a convex center, smooth surface and regular edge, and easy to pick up; the colony on the WL medium is green, spherical, smooth surface, regular edge, with a convex center, and the color gradually fades from the center to the edge to milky white; the cells are mostly oval or fusiform, showing a budding state.

[0015] The physiological and biochemical characteristics of the Saccharomyces cerevisiae SQJ20 are as follows: the fermentation in Durham tube starts at the 4th hour and ends at the 10th hour, and the gas production capacity of the strain is stronger than that of the commercially available active dry yeast (the fermentation in Durham tube starts at the 4th hour and ends at the 12th hour); when 3 < pH < 7, the growth state of the strain is stable; in the color development test of TTC medium, the colony of the strain shows light red, indicating that it has certain ability to produce metabolites such as alcohol and acid. It can be applied in the fermentation of flour products.

[0016] One strain of Wickerhamomyces anomalus with increased aroma, named Wickerhamomyces anomalus GZJ2 (abbreviated as GZJ2), is preserved in China Center for Type Culture Collection, Wuhan, Hubei Province, China, the address of the preservation unit is: Wuhan University, 299 Bajiyi Road, Wuchang District, Wuhan, Hubei Province, China, the preservation date is May 29, 2023, and the preservation number is CCTCC M 2023857.

[0017] The colony characteristics of the Wickerhamomyces anomalus GZJ2 are as follows: the colony on the YPD medium is milky white, spherical, opaque, without convex, regular edge and easy to pick up; the colony on the WL medium is light blue, spherical, flat, dry surface, regular edge and no convex center; the cells are mostly oval or fusiform, showing a budding state.

[0018] The physiological and biochemical characteristics of the abnormal Wickerhamomyces anomalus GZJ2 are as follows: the Du's tube fermentation starts at the 10th hour and the Du's tube is full at the 21th hour, the strain has a certain degree of gas production capacity, but is obviously lower than that of Saccharomyces cerevisiae strain SQJ20 and commercially available active dry yeast; in the TTC culture medium color test, the colony of the strain presents deep red, the center of the colony has a white circle, and there is white material around the colony, which indicates that the strain has strong alcohol, acid and other metabolite production capacity. The strain can be applied in the fermentation of flour products.

[0019] Compared with the prior art, the beneficial effects of the present application are:

[0020] The present application screens two strains of yeast by a traditional culture method, which are gas-producing Saccharomyces cerevisiae strain SQJ20 and flavor-enhancing abnormal Wickerhamomyces anomalus GZJ2. The screening source is traditional sourdough. The application of the two strains in the fermentation process of flour products improves the flavor or quality, especially the mixture of the two strains as a yeast composite leavening agent in the fermentation process of flour, which has better fermentation characteristics than commercially available active dry yeast fermentation; the prepared finished product is improved in hardness, chewiness and other indicators, and the flavor and quality of the fermented flour product are greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the colony morphology diagram of strain 1 in YPD culture medium; in the diagram, A is the front colony, and B is the side colony;

[0022] Figure 2 is the colony morphology diagram of strain 1 in WL culture medium; in the diagram, A is the front colony, and B is the side colony;

[0023] Figure 3 is the cell morphology diagram of strain 1 under a microscope;

[0024] Figure 4 is the phylogenetic tree picture of strain 1;

[0025] Figure 5 is the colony morphology diagram of strain 2 in YPD culture medium; in the diagram, A is the front colony, and B is the side colony;

[0026] Figure 6 is the colony morphology diagram of strain 2 in WL culture medium; in the diagram, A is the front colony, and B is the side colony;

[0027] Figure 7 is the cell morphology diagram of strain 2 under a microscope;

[0028] Figure 8 is the phylogenetic tree picture of strain 2;

[0029] Figure 9Figure 1 is a gas production capacity graph of Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2;

[0030] Figure 10 Figure 2 is an acid production capacity graph of Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2;

[0031] Figure 11 Figure 3 is a metabolic capacity graph of Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2. In the figure, A is the front of the colony, and B is the back of the colony;

[0032] Figure 12 Figure 4 is a growth curve of Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2;

[0033] Figure 13 Figure 5 is a sensory evaluation score graph of fermented steamed buns;

[0034] Figure 14 Figure 6 is a TPA result graph of fermented steamed buns. In the figure, A is hardness, and B is chewiness;

[0035] Figure 15 Figure 7 is a radar chart of electronic nose detection of fermented steamed buns;

[0036] Figure 16 Figure 8 is a protein content graph of fermented steamed buns. DETAILED DESCRIPTION

[0037] The present application will be further explained in conjunction with the following examples, which are only intended to illustrate the present application and not intended to limit the scope of the present application. Any modification made by those skilled in the art without departing from the content, spirit and scope of the present application shall fall within the protection scope of the present application. The experimental methods not specified in the examples are all according to the conventional conditions; the reagents and biological materials, if not specifically stated, can be obtained from commercial channels.

[0038] Culture medium used in the examples:

[0039] YPD culture medium: 10 g / L of yeast powder, 20 g / L of peptone, 20 g / L of glucose, and distilled water to constant volume. The solid culture medium further contains 20 g / L of agar.

[0040] WL culture medium: purchased from Beijing Solabio Science and Technology Co., Ltd.

[0041] TTC upper culture medium: 0.5 g / L of TTC (triphenyl tetrazolium hydrochloride), 5 g / L of glucose, 15 g / L of agar, and distilled water to constant volume.

[0042] TTC lower layer medium: glucose 10 g / L, proteose peptone 2 g / L, yeast extract 1.5 g / L, potassium phosphate dibasic 1 g / L, MgS04·7H20 4 g / L, agar 20 g / L, distilled water to constant volume.

[0043] Example 1: Screening and isolation and purification of yeast

[0044] 31 pieces of sourdough were collected from all over the country, covering 9 provinces and 17 cities. The sourdough was sealed and transported to the laboratory using an ice box for microbial screening. The specific sourdough source and number are shown in Table 1.

[0045] Table 1 Source and number of sourdough

[0046]

[0047]

[0048] Take 5 g of air-dried sourdough, add 45 mL of sterile normal saline, shake well to make a 10 -1 suspension; 1 mL of the suspension is taken with a sterile gun head and added to 9 mL of sterile normal saline, mixed well to obtain a 10 -2 sample dilution, and sequentially and continuously diluted to 10 -7 . Take 100 μL of sample dilution of 10 -5 , 10 -6 , 10 -7 , respectively, and evenly spread on YPD solid medium, with 3 parallels for each dilution gradient. The coated medium plate is placed in a 30°C constant temperature incubator for 48 h and the colony state is observed. Select the single colonies on the plate as spherical, milky white, and opaque, and perform single-line isolation on YPD solid medium until the morphology is consistent. The isolated yeast is stored as follows:

[0049] (1) Plate or slant preservation: the isolated yeast is inoculated on YPD plate or slant, and after 48 h of incubation in a 30°C constant temperature incubator, it is stored in a 4°C refrigerator for short-term use.

[0050] (2) Glycerol preservation: the isolated yeast is inoculated on YPD plate, incubated at 30°C for 48 h, then inoculated into YPD liquid medium for further incubation for 18-20 h, and then mixed with sterile 30% glycerol at a ratio of 1:1, and stored in a -80°C refrigerator.

[0051] Example 2: Morphological identification of strains

[0052] (1) Colony morphology on YPD medium: the strains obtained after purification in Example 1 were inoculated on YPD solid medium, respectively, and the colony morphology characteristics were observed after 48 h. The colonies with the following characteristics were selected: milky white, spherical, opaque, regular edge, easy to pick up. After screening, strain 1 and strain 2 were obtained; strain 1 was derived from sourdough with sample number SQ1-2, and strain 2 was derived from sourdough with sample number GZ1. Figure 1 is the colony morphology diagram of strain 1 on YPD medium; as shown in Figure 1 , the colony is milky white, spherical, opaque, with a convex center, smooth surface, regular edge, easy to pick up, which belongs to the typical colony morphology of Saccharomyces cerevisiae. Figure 5 is the colony morphology diagram of strain 2 on YPD medium; as shown in Figure 5 , the colony is milky white, spherical, dry and opaque on the surface, no convex in the middle, regular edge, easy to pick up.

[0053] (2) Colony morphology on WL medium: strain 1 obtained was inoculated on WL solid medium, and the colony morphology characteristics were observed after 3-5 days. Figure 2 is the colony morphology diagram of strain 1 on WL medium; as shown in Figure 2 , the colony is green, spherical, smooth surface, regular edge, convex center, and the color gradually fades from the center to the edge to milky white. Strain 2 obtained was inoculated on WL solid medium, and the colony morphology characteristics were observed after 3-5 days. Figure 6 is the colony morphology diagram of strain 2 on WL medium; as shown in Figure 6 , the colony is light green, spherical, dry and opaque on the surface, no convex in the middle, regular edge, easy to pick up.

[0054] (3) Cell morphology under microscope: appropriate amount of pure culture of strain 1 and strain 2 was taken respectively for microscopic examination, Figure 3 is the cell morphology diagram of strain 1 under microscope. As shown in Figure 3 , under 100 times magnification, the cells are mostly oval or fusiform, showing a budding state. Figure 7 is the cell morphology diagram of strain 2 under microscope. As shown in Figure 7 , under 100 times magnification, the cells are mostly oval or fusiform, showing a budding state.

[0055] Example 3: Molecular biological identification of strains

[0056] In order to identify the species of strain 1 and strain 2 obtained in Example 2, molecular biology experiments were carried out respectively. The specific steps include the following:

[0057] (1) Activation of bacterial cells

[0058] Pure cultures of strain 1 and strain 2 were inoculated into YPD solid medium and incubated at 30°C for 48 hours.

[0059] (2) PCR amplification

[0060] Colony PCR was performed on activated strains 1 and 2, respectively. The primers used for identification were yeast 26S rDNA gene D1 / D2 region sequence amplification primers, with the following composition: ① forward primer as shown in Seq_1, i.e., NL1: 5′-GCATATCAATAAGCGGAGGAAAAG-3′; ② reverse primer as shown in Seq_2, i.e., NL4: 5′-GGTCCGTGTTTCAAGACGG-3′.

[0061] The PCR reaction system consisted of: a small amount of strain 1 / strain 2 cells collected from the pipette tip as DNA template, 0.8 μL of forward primer, 0.8 μL of reverse primer, and Premix Taq. TM Add 15 μL of ddH2O to make up the total volume of 30 μL.

[0062] The PCR amplification program was as follows: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 35 s, for a total of 34 cycles, and finally 72℃ extension for 7 min.

[0063] Agarose gel electrophoresis: PCR amplification products were verified by 1% agarose gel electrophoresis.

[0064] (3) Sequence analysis and phylogenetic tree construction

[0065] The PCR amplification products that passed the agarose gel electrophoresis verification were sent to Shanghai Jieli Biotechnology Co., Ltd. for sequencing. The sequencing results were compared and analyzed using the BLAST program in NCBI. Strain 1 (its nucleotide sequence is shown in Seq_3) showed more than 99% similarity to the 26S rDNA D1 / D2 region sequences of several other reported Saccharomyces cerevisiae strains. To further demonstrate the phylogenetic relationship between the sequenced strain and known Saccharomyces cerevisiae, a phylogenetic tree was constructed using MEGA 11 biological software. Figure 4 This is a phylogenetic tree image of strain 1. (Example) Figure 4 As shown, strain 1 was identified as Saccharomyces cerevisiae.

[0066] The strain 1 is named Saccharomyces cerevisiae SQJ20, and the strain is preserved in the China Center for Type Culture Collection, Wuhan, and the preservation number is CCTCC NO: M 2023858, the address of the preservation unit is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, and the preservation date is May 29, 2023.

[0067] The strain 2 (the nucleotide sequence of which is shown in Seq_4) has more than 99% similarity with the 26S rDNA D1 / D2 region sequences of other reported strains of Wickerhamomyces anomalus; in order to further show the genetic relationship between the sequenced strain and the known Wickerhamomyces anomalus, a phylogenetic tree is constructed using MEGA 11 biological software, Figure 8 is a phylogenetic tree picture of the strain 2. As shown in Figure 8 , the strain 2 is identified as Wickerhamomyces anomalus.

[0068] The strain 2 is named Wickerhamomyces anomalus GZJ2, and the strain is preserved in the China Center for Type Culture Collection, Wuhan, and the preservation number is CCTCC NO: M 2023857, the address of the preservation unit is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, and the preservation date is May 29, 2023.

[0069] Example 4: Physiological and biochemical characteristics of Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2

[0070] (1) Gas production capacity

[0071] The Durham tube fermentation method is selected to determine the gas production capacity of Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2, and a commercially available high-activity dry yeast is used as a control. The specific steps are as follows: single colonies are taken from solid culture medium stored at 4°C and streaked on YPD solid medium, and then incubated in a 30°C incubator for 48 h. Single colonies of yeast are then picked from the activated solid medium and inoculated into YPD liquid medium, and incubated at 30°C for 24 h. The fermentation time and Durham tube full time of the yeast strain are determined, and the gas height in the Durham tube is recorded every 1 h after fermentation. Figure 9 is a gas production capacity chart of Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2; as Figure 9As shown, compared with the commercially available high-activity dry yeast, the Saccharomyces cerevisiae SQJ20 has stronger gas production capacity, and the Durham tube is full after 10 hours of fermentation starting from the 4th hour; while the gas production capacity of the Wickerham anomalus GZJ2 is relatively weak, and the Durham tube is full after 21 hours of fermentation starting from the 10th hour. It can be seen that the Saccharomyces cerevisiae SQJ20 has extremely strong gas production capacity, which can ensure that the dough is fully fluffy during the fermentation process, while the Wickerham anomalus GZJ2 has weak gas production capacity and cannot be used alone for dough fermentation.

[0072] (2) Acid resistance

[0073] The acid resistance of the Saccharomyces cerevisiae SQJ20 and the Wickerham anomalus GZJ2 was determined, and the specific steps were as follows: single colonies of the yeasts were picked from the activated solid culture medium and inoculated into YPD liquid medium, and the seed liquid was obtained after 24 hours of culture at 30°C. The seed liquid was inoculated into YPD liquid medium with pH values of 3, 4, 5, and 6 in advance, and the inoculation amount was 2%. The medium was cultured at 30°C for 10 hours, and the OD value of the medium was determined at a wavelength of 600 nm. Figure 10 is a graph of the acid production capacity of the Saccharomyces cerevisiae SQJ20 and the Wickerham anomalus GZJ2. As shown in Figure 10 , the Saccharomyces cerevisiae SQJ20 and the Wickerham anomalus GZJ2 showed different growth trends under different pH media. The Saccharomyces cerevisiae SQJ20 has better stability than the commercially available high-activity dry yeast, while the Wickerham anomalus GZJ2 has poor stability at pH = 3, but has similar stability to the commercially available high-activity dry yeast at 4 < pH < 7. It can be seen that the Saccharomyces cerevisiae SQJ20 has extremely high acid resistance, and the pH of the dough after fermentation is generally in the range of 5-7, so the Wickerham anomalus GZJ2 can ensure stable performance during the dough fermentation process.

[0074] (3) Metabolic capacity

[0075] The metabolic capacity of the Saccharomyces cerevisiae SQJ20 and the Wickerham anomalus GZJ2 was determined, and the specific steps were as follows: single colonies of the yeasts were picked from the activated solid culture medium and inoculated into TTC lower medium by point inoculation method, and the medium was cultured at 30°C for 72 hours. The pre-prepared TTC upper medium was cooled to about 45°C, slowly poured onto the bottom medium to cover the colonies, and then moved to a dark place for color development at a temperature of 30°C. After 2 hours, it was taken out and the color of the colonies was compared quickly. Figure 11 is a graph of the metabolic capacity of the Saccharomyces cerevisiae SQJ20 and the Wickerham anomalus GZJ2. In the graph, A is the front of the colony, and B is the back of the colony; as shown in Figure 11As shown, the Saccharomyces cerevisiae SQJ20 appears light red, while the Wickerhamomyces anomalus GZJ2 appears dark red, and there is white substance in the center and around the colony. Generally, the deeper the color of the colony on the TTC medium, the stronger the ability of the strain to produce metabolites such as alcohol and acid. As can be seen, both the Saccharomyces cerevisiae SQJ20 and the Wickerhamomyces anomalus GZJ2 have high metabolic capacity, and the Wickerhamomyces anomalus GZJ2 has better metabolic capacity.

[0076] (4) Growth curve

[0077] The growth curve is a standard curve describing the lag phase, logarithmic phase and stationary phase under a certain culture condition, and can provide a theoretical basis for subsequent experiments. The specific steps for determining the growth curve are as follows: single colonies were taken from the solid medium stored at 4°C and streaked on YPD solid medium, which was then incubated in a 30°C incubator for 48 h. Single colonies of yeast were then picked from the activated solid medium and inoculated into YPD liquid medium, which was then incubated at 30°C for 24 h as a seed liquid. YPD liquid medium was used as the fermentation medium, and inoculation was performed at a rate of 1%. The medium was incubated at 30°C, and 1 mL of fermentation liquid was taken every 2 h after inoculation. The blank medium was used as a control, and the absorbance at 600 nm was measured.

[0078] The growth curves of the Saccharomyces cerevisiae SQJ20 and the Wickerhamomyces anomalus GZJ2 were plotted using the absorbance values as the vertical coordinates and the culture time as the horizontal coordinates. Figure 12 is the growth curve of the Saccharomyces cerevisiae SQJ20 and the Wickerhamomyces anomalus GZJ2; as shown in Figure 12 As shown, the growth of both strains of yeast showed typical microbial growth characteristics, and the lag phase lasted for 0-4 h. After 4 h, the logarithmic growth phase was entered. The OD values of the Saccharomyces cerevisiae SQJ20 and the Wickerhamomyces anomalus GZJ2 were the largest at 12 h and 24 h, respectively, indicating the highest cell concentration and the strongest activity. Thereafter, the growth entered the stationary phase.

[0079] Example 5: Preparation of a composite starter culture

[0080] (1) Strain activation and seed liquid culture

[0081] Single colonies were taken from the solid medium stored at 4°C and streaked on YPD solid medium, which was then incubated in a 30°C incubator for 48 h. Single colonies of yeast were then picked from the activated solid medium and inoculated into YPD liquid medium, which was then incubated at 30°C for 24 h as a seed liquid.

[0082] (2) Cell amplification and collection

[0083] The seed liquid was inoculated into YPD liquid medium at an inoculation amount of 1% (V / V), and cultured at 30°C to the late logarithmic phase (12h for Saccharomyces cerevisiae SQJ20 and 24h for Wickerhamomyces anomalus GZJ2) as the fermentation liquid. The fermentation liquid in the logarithmic phase was transferred into a sterile centrifuge tube under aseptic conditions, centrifuged at 4000r / min for 15min, and the supernatant was discarded to collect the precipitate. The precipitate obtained by centrifugation of 100mL of fermentation liquid was resuspended with 2mL of sterile normal saline to obtain a bacterial suspension. The bacterial suspension was subjected to viable cell counting, and the viable cell count of Saccharomyces cerevisiae was 3-4x10 8 cfu / mL, and the viable cell count of Wickerhamomyces anomalus was 6-7x10 8 cfu / mL.

[0084] (3) Compound of the strain

[0085] The bacterial suspension was mixed at a ratio of 1:1, 1:2 and 1:3 of the bacterial number of Saccharomyces cerevisiae SQJ20 and Wickerhamomyces anomalus GZJ2 to obtain compound starter 1, compound starter 2 and compound starter 3, respectively, which were used for the subsequent production of steamed buns.

[0086] Example 6: Preparation of compound fermented steamed buns

[0087] The yeast compound starter prepared in Example 5 was used for the production of fermented steamed buns, and commercially available high-activity dry yeast powder was used as control starter 1, and Saccharomyces cerevisiae SQJ20 was used as control starter 2. The amount of Saccharomyces cerevisiae SQJ20 added to the dough was 1-2x10 9 cfu per 100g of flour, and the specific steps were as follows:

[0088] (1) Preparation of dough: an appropriate amount of wheat flour was mixed with 50mL of compound starter-containing warm water at a ratio of 100g of flour to 50mL of water, and the water temperature was controlled at 30-40°C. After simple kneading, the dough was rested for about 10min and then kneaded until smooth.

[0089] (2) Proofing of dough: the dough kneaded until smooth was placed in a fermentation box at 30-40°C for fermentation for 60-90min. The dough was taken out, degassed, and divided into 75g pieces. The dough was manually kneaded and shaped, and then placed in the fermentation box for secondary proofing for 30-60min.

[0090] (3) Steaming of steamed buns: the dough after secondary proofing was placed in a steamer, steamed for 30min after steaming, and then steamed for 10min to obtain compound fermented steamed buns.

[0091] The steamed buns were cooled for 30min, and a professional trained personnel was used for sensory evaluation. The sensory evaluation criteria are shown in Table 2.

[0092] Table 2 Sensory evaluation table

[0093]

[0094]

[0095] Figure 13 is a graph of the sensory evaluation scores of the fermented steamed buns. As shown in Figure 13 the sensory evaluation scores of the steamed buns prepared using the control leavening agent 1 and the control leavening agent 2 were significantly lower than those of the experimental examples. In the experimental examples, the sensory evaluation scores showed a trend of first increasing and then decreasing with the increase of the amount of the abnormal Wickerhamomyces anomalus GZJ2 added, and reached a peak when the Saccharomyces cerevisiae SQJ20: abnormal Wickerhamomyces anomalus GZJ2 = 1:2.

[0096] The steamed buns were cooled for 1 h and then cut into cubes of 2 cm x 1 cm x 1 cm. A food texture analyzer (TA.XT.Plus produced by Stable Micro Systems, UK) was used to perform TPA tests on the bun cubes, and a P / 50 probe was selected. The specific TPA program was as follows: the pre-test speed was 1 mm / s, the test speed was 1 mm / s, the post-test speed was 1 mm / s, the compression ratio was 50%, the interval time was 5 s, and the stress was 5 g. Each sample was tested in triplicate. The TPA test results showed that the elasticity, resilience and cohesiveness indicators had no statistical significance, indicating that the steamed buns of the experimental examples and the control examples had similar internal structures, but the hardness and chewiness of the steamed buns of the experimental examples were significantly lower than those of the control examples, Figure 14 is a graph of the TPA results of the fermented steamed buns. In the graph, A is the hardness and B is the chewiness; as shown in Figure 14 , it is indicated that the steamed buns of the experimental examples were softer and more palatable than those of the control examples, to some extent, indicating that the steamed buns of the experimental examples had better texture properties than those of the control examples.

[0097] The steamed buns were cooled for 1 h, and the bun cores were crushed to about 5 mm x 5 mm in size. 3.0 g of the crushed bun cores were placed in a sealed 30 mL glass sample bottle, and an electronic nose (AIRSENSE electronic nose PEN model 3, Germany) was used for determination. The sensing substances corresponding to each detector of the electronic nose are shown in Table 3.

[0098] Table 3 Sensing substances corresponding to each detector of the electronic nose

[0099]

[0100]

[0101] Figure 15 is a radar chart of the electronic nose detection of the fermented steamed buns. As shown in Figure 15It can be seen that the five detectors of the control example 1, the control example 2 and the embodiment have obvious responses, respectively: W5S (nitrogen-containing compounds), W1S (short-chain alkanes), W1W (inorganic sulfides, terpene compounds), W2S (alcohols, aldehydes and ketones, part of aromatic compounds), W2W (aromatic hydrocarbon compounds, organic compounds of sulfur). It can be seen that the response values of different detectors of each group of the embodiment are significantly higher than those of the control examples, and the response values of each detector are the largest when the Saccharomyces cerevisiae SQJ20: abnormal Wickerhamomyces GZJ2 = 1:2.

[0102] Example 7: Protein content of composite fermented steamed buns

[0103] The Kjeldahl method in GB 5009.5-2016 is used to determine the crude protein content of the prepared steamed buns. Specifically, 0.2-2g of steamed bun freeze-dried powder is weighed, 0.4g of copper sulfate, 6g of potassium sulfate and 20mL of sulfuric acid are added, the sample is carbonized at 200℃, and after the foam stops generating and stabilizes, the temperature is increased to 450℃, heated to liquid boiling, and after the liquid becomes transparent blue-green, continue heating for 1h, and then take out after cooling. After adding water and alkali, distill the liquid, and absorb the escaping ammonia with boric acid. Use the calibrated strong acid standard titration solution, record the acid consumption, and calculate the nitrogen content according to the following formula.

[0104]

[0105] In the formula: w(N) is the mass fraction of nitrogen, unit g / 100g; V a is the volume of sulfuric acid standard titration solution consumed by the sample, unit mL; V b is the volume of sulfuric acid standard titration solution consumed by the blank sample, unit mL; c(1 / 2H2SO4) is the amount-of-substance concentration of the sulfuric acid standard titration solution, unit mol / L; M(N) is the molar mass of nitrogen, i.e. 14.007g / mol; m is the mass of the sample, unit g; 100 is the mass conversion coefficient, unit g / 100g; 1000 is the volume conversion coefficient, unit mL / L. Figure 16 is the protein content diagram of the fermented steamed buns; as Figure 16 shown, compared with the control example 1 and the control example 2, the protein content in the steamed buns of the embodiment is significantly improved (p<0.01) with statistical significance. It can be seen that the yeast composite leavening agent provided by the present application is beneficial to improve the nutritional quality of fermented flour products such as steamed buns, and has higher palatability, and is more easily accepted and loved by consumers.

[0106] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A yeast bacteria composite starter culture, characterized in that, The yeast complex leavening agent comprises two strains of Saccharomyces cerevisiae and Wickerhamomyces anomalus, the Saccharomyces cerevisiae is named Saccharomyces cerevisiae SQJ20, the preservation agency is China Center for Type Culture Collection in Wuhan, the preservation number is CCTCC NO: M2023858, and the preservation date is May 29, 2023; the Wickerhamomyces anomalus is named Wickerhamomyces anomalus GZJ2, the preservation agency is China Center for Type Culture Collection in Wuhan, the preservation number is CCTCC NO: M2023857, and the preservation date is May 29, 2023; the number ratio of the Saccharomyces cerevisiae SQJ20 to the Wickerhamomyces anomalus GZJ2 in the yeast complex leavening agent is 1:1-3.

2. The yeast starter culture according to claim 1, characterized in that, The Saccharomyces cerevisiae SQJ20 in the yeast complex leavening agent has the gas production property, and the Wickerhamomyces anomalus GZJ2 has the flavor enhancement property.

3. Application of the yeast complex leavening agent according to any one of claims 1-2 in the field of fermentation of flour products.

4. The use according to claim 3, characterized in that, In the food fermentation stage, the yeast complex starter culture is mixed and inoculated into the fermentation system to improve the aroma and quality of the flour product; the addition amount of Saccharomyces cerevisiae SQJ20 in the fermentation system is 1~2×10 9 cfu of viable bacteria per 100g of flour, and the food is a flour product.

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