Method for improving leavening flavor of leavening agent, method for preparing steamed buns by using leavening agent and application

Abstract

The invention discloses a method for improving yeast flavor of a leavening agent, a method for preparing steamed buns by using the leavening agent and application, and belongs to the technical field of food microorganisms. Specifically, the saccharomyces cerevisiae CCFM2101, the lactobacillus plantarum CCFM1472 and the lactobacillus mucus fermentation CCFM1473 are included. The content of flavor substances in the steamed buns prepared by the composite leavening agent is remarkably increased, which is specifically reflected as follows: (1) the flavor substances are richer; (2) the contents of flavor substances esters and alcohols are obviously increased; therefore, the saccharomyces cerevisiae CCFM2101, the plant lactobacillus CCFM1472 and the fermentation mucus lactobacillus CCFM1473 have a huge application prospect in the preparation of the composite leavening agent for the steamed buns.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food microbiology, and particularly relates to a method for improving the flavor of leavening agent and its application in making steamed buns. BACKGROUND

[0002] Steamed buns and other flour products have become an indispensable part of people's daily diet today, and people have higher requirements for the flavor, taste, style and nutrition of bread and Chinese steamed buns. With the rapid development and use of commercial dry yeast, the production of fermented flour products such as steamed buns and bread has become more convenient, and has brought great convenience to people's life. The bread produced by this method often lacks the aroma of steamed buns and the taste is not pure enough. In order to meet the requirements of the public, bakers usually use natural or synthetic food additives to increase the aroma, color, softness, elasticity, water retention and freshness of steamed buns. In addition, the flavor and taste of steamed buns are important sensory qualities, and modern people prefer to consume steamed bun products with traditional sensory qualities. In addition to using traditional leavening agents such as traditional leavening agents and old flour to produce traditional flavor steamed buns, using pure culture yeast strains that can produce specific flavors has incomparable advantages. Not only can it control product fermentation, but also can shorten fermentation time and improve production efficiency, meeting people's needs in consumption and production.

[0003] Multi-strain fermentation technology is to make up for the monotony of single strain through the interaction of two or more strains, so that the flavor of fermented flour products is more full and the quality is more safe. The acid dough microbial flora is composed of yeast, lactic acid bacteria, acetic acid bacteria, mold and other microbial flora. Yeast produces carbon dioxide, ethanol and small molecule flavor substances in the metabolic process, giving steamed buns unique texture and flavor characteristics. Lactic acid bacteria, acetic acid bacteria and other microorganisms in the dough metabolize to produce organic acids (such as lactic acid and acetic acid) and alcohols (such as ethanol) produced by yeast to further react to generate a certain amount of ester compounds. Long-term interaction of multiple strains will also produce a small amount of aldehydes, ketones and other hydroxyl compounds. These substances constitute important flavor volatile and flavor auxiliary substances in acid dough. The exopolysaccharides and organic acids secreted by lactic acid bacteria during fermentation can improve the dough texture, inhibit the growth of microorganisms and prolong the shelf life of fermented products.

[0004] However, the dough fermented by sourdough has unique flavor after long fermentation. During the fermentation process of tens of hours, the endogenous enzyme system of the flour matrix and the metabolic network of microorganisms produce a cascade reaction: lactic acid bacteria produce organic acids and flavor precursors through homo / hetero fermentation pathways; yeast dominates the production of gas and the synthesis of ethanol; at the same time, the directed hydrolysis of glutenin and gliadin occurs under the action of protease. The synergistic effect of these biochemical reactions ultimately gives the product unique sensory properties (such as honeycomb texture, complex flavor profile), nutritional enhancement (phytic acid degradation, bioactive peptide generation), and microbial stability (pH reduction, antibacterial substance accumulation). Flavor, as the core sensory attribute of steamed buns, directly affects the purchasing behavior of consumers. Traditional natural fermented sourdough contains rich microbial resources, and its fermentation products have significant advantages in flavor complexity. Although the industrialization process has led to the popular application of high-activity instant dry yeast, traditional sourdough starters still maintain their vitality due to their unique quality advantages. Market research data shows that yeast fermented flour products perform outstandingly in key indicators such as texture elasticity, flavor complexity, and shelf life: in terms of texture, organic acids and enzymes produced by microbial metabolism synergistically enhance the elasticity of the gluten network; in terms of flavor, multiple metabolic pathways give the product a rich and natural fermentation aroma; in terms of shelf life, natural antibacterial ingredients extend the shelf life. In addition, many traditional workshops in the northern region continue to use old dough starter fermentation processes, and the reason for this is the unique texture characteristics and flavor profile given by the complex microbial community structure of the product. This traditional product has always maintained a high market acceptance. However, traditional sourdough production has a long cycle and is difficult to use for industrial production; and traditional sourdough is generally in a semi-solid state, which is highly sensitive to microbial contamination and difficult to store for a long time. Therefore, separating advantageous strains from traditional sourdough and compounding them with yeast or other strains has become a research hotspot in recent years. Different strains have different fermentation characteristics, so selecting different suitable fermentation strains for mixed fermentation can break through the technical bottlenecks of long fermentation cycle and unstable product texture, which is of great significance for the research and development of new fermented flour products. At present, how to improve the flavor characteristics of fermented flour products through multi-strain fermentation technology and screen and isolate special flavor strains is not only effective but also an effective way to improve the flavor characteristics of fermented flour products. Research and multi-strain fermentation of flour products have great practical significance.

[0005] The purpose of the present application is to provide a method for replacing traditional sourdough fermented steamed buns, which overcomes the problems of inconsistent preparation standards and poor stability of traditional sourdough steamed buns, while retaining the flavor characteristics of traditional sourdough steamed buns and promoting the industrial application of sourdough steamed buns. SUMMARY

[0006] The present application aims to improve the flavor deficiency of the prior art, and provides a new multi-strain composite flour product starter and application.

[0007] To achieve the above-mentioned purpose, the present application provides a starter, which contains Saccharomyces cerevisiae CCFM2101, Lactiplantibacillus plantarum CCFM1472 and Lactobacillus fermentum CCFM1473.

[0008] In an embodiment, the preservation number of the Saccharomyces cerevisiae CCFM2101 is GDMCC No: 65875; the preservation number of the Lactiplantibacillus plantarum CCFM1472 is GDMCC No: 65873; and the preservation number of the Lactobacillus fermentum CCFM1473 is GDMCC No: 65874.

[0009] In an embodiment, the Lactiplantibacillus plantarum, Lactobacillus fermentum and Saccharomyces cerevisiae are all screened from the leaven in Texas area, and are prepared by using any known strain of the corresponding species identified by microbiology through conventional culture and expansion.

[0010] The present application also provides a method for preparing the above-mentioned starter, which comprises the following steps:

[0011] S1. Activating the Saccharomyces cerevisiae CCFM2101, inoculating it in YPD liquid medium for culture to obtain a bacterial liquid; and activating the Lactiplantibacillus plantarum CCFM1472 and Lactobacillus fermentum CCFM1473, inoculating them in MRS liquid medium for culture to obtain a bacterial liquid;

[0012] S2. Inoculating the bacterial liquid in sterilized YPD and MRS liquid medium, and then culturing in a fermentation tank to obtain a culture liquid;

[0013] S3. Centrifuging the culture liquid and collecting the bacterial bodies, and then freeze-drying the bacterial bodies to obtain the starter.

[0014] In some embodiments, in S1, the temperature for culturing the yeast is 30℃, and the time for culturing the yeast is 16-24 hours; the temperature for culturing the lactobacillus is 37℃, and the time for culturing the lactobacillus is 12-16 hours.

[0015] In some embodiments, in S1, the OD value of the bacteria solution at 600 nm is 1.85-2.0.

[0016] In some embodiments, in S2, the temperature for culturing the yeast is 30℃, and the time for culturing the yeast is 16-24 hours; the temperature for culturing the lactobacillus is 37℃, and the time for culturing the lactobacillus is 12-16 hours.

[0017] It should be noted that, in S2, the time for culturing is related to the viable count of the yeast and lactobacillus in the leavening agent. For example, when the viable count is high, the time for culturing is long; when the viable count is low, the time for culturing is short.

[0018] In some embodiments, in S3, the speed for centrifugation is 8000g.

[0019] In some embodiments, in S3, the temperature for freeze-drying is -50--80℃, the time for freeze-drying is 36-48h, and the pressure for freeze-drying is 15-30Pa.

[0020] In one embodiment, in the leavening agent, the number of the cell bodies of Saccharomyces cerevisiae CCFM2101, Lactobacillus plantarum CCFM1472 and Lactobacillus fermentum CCFM1473 is ≥10 9 CFU / mL.

[0021] The application also provides a product containing the leavening agent.

[0022] In one embodiment, the product includes food, medicine or health care product.

[0023] In one embodiment, the product includes fermented bread product, fermented dairy product, fermented condiment or wine.

[0024] The application also provides a method for preparing steamed buns, comprising the following steps:

[0025] (1) preparing sour dough: inoculating Lactobacillus plantarum CCFM1472 and Lactobacillus fermentum CCFM1473 in claim 1 into wheat flour, and fermenting at 33-37℃ for at least 12h to obtain sour dough;

[0026] (2) preparing mixed steamed bun dough: mixing 10-30 parts by weight of sour dough, 40-60 parts by weight of wheat flour and 5-15 parts by weight of water, and then inoculating Saccharomyces cerevisiae CCFM2101 in claim 1 to obtain mixed steamed bun dough;

[0027] (3) Preparation of steamed buns: the mixed steamed bun dough of step (2) is allowed to ferment for 2 h at a temperature of 28-32℃ and a relative humidity of 75%; after shaping, it is allowed to ferment for 20 min at a temperature of 28-32℃ and a relative humidity of 75%; and steamed buns are obtained.

[0028] In an embodiment, the inoculation amount of the Lactobacillus fermentum CCFM1472 and the Lactobacillus mucosae CCFM1473 in the wheat flour of step (1) is 1×10 7 ~ 1×10 9 CFU / g.

[0029] In an embodiment, the inoculation amount of the Saccharomyces cerevisiae CCFM2101 in the mixed steamed bun dough of step (2) is 1×10 5 ~ 1×10 8 CFU / g.

[0030] The application also provides the use of the starter in the preparation of a fermented flour product.

[0031] In an embodiment, the fermented flour product includes but is not limited to steamed buns.

[0032] The application also provides a flour product prepared using the novel multi-strain composite flour product starter described above; preferably, the flour product is a steamed bun or a bread.

[0033] The application has the following advantages:

[0034] The application screens three special-flavor yeast strains CCFM2101, Lactobacillus fermentum CCFM1472 and Lactobacillus mucosae CCFM1473 from Dezhou area leaven, and uses the above strains to prepare steamed buns, which can significantly improve the wheat flavor of steamed buns, and the content of 1-nonanol, acetic acid and ethyl acetate in the volatile flavor substances of steamed buns is 0.0112, 0.4585 and 0.0323 mg / kg respectively, the flavor of the prepared steamed buns is closer to that of traditional sourdough steamed buns, and the defects of high storage condition requirement and unstable product flavor of traditional sourdough steamed buns are overcome, which has a broad application prospect.

[0035] Biological material preservation

[0036] Saccharomyces cerevisiae CCFM2101, preserved in Guangdong Microbial Culture Collection Center (GDMCC), with the accession number GDMCC No: 65875, classified as Saccharomyces cerevisiae, preserved on February 12, 2025, and located at No. 59 Building, 5th Floor, Guangzhou City, 100, Martyrs' Road, Guangzhou City.

[0037] Lactiplantibacillus plantarum CCFM1472, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the accession number of GDMCC No: 65873, classified as Lactiplantibacillus plantarum, deposited on February 12, 2025, and located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou.

[0038] Limosilactobacillus fermentum CCFM1473, deposited in Guangdong Microbial Culture Collection Center (GDMCC), with the accession number of GDMCC No: 65874, classified as Limosilactobacillus fermentum, deposited on February 12, 2025, and located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Morphological characteristics of Saccharomyces cerevisiae (A) strain morphology; (B) cell morphology.

[0040] Figure 2 Morphological characteristics of Lactiplantibacillus plantarum (A) strain morphology; (B) cell morphology.

[0041] Figure 3 Morphological characteristics of Limosilactobacillus fermentum (A) strain morphology; (B) cell morphology.

[0042] Figure 4 Phylogenetic tree of Saccharomyces cerevisiae based on strain gene sketch.

[0043] Figure 5 Phylogenetic tree of Lactiplantibacillus plantarum based on strain gene sketch.

[0044] Figure 6 Phylogenetic tree of Limosilactobacillus fermentum based on strain gene sketch.

[0045] Figure 7 Generation time OD600 of Saccharomyces cerevisiae, Lactiplantibacillus plantarum and Limosilactobacillus fermentum.

[0046] Figure 8 Mixed starter steamed bread flavor substances.

[0047] Figure 9 Sensory evaluation of steamed bread prepared by Comparative Example 3, Comparative Example 4, Comparative Example 2 and Example 4. DETAILED DESCRIPTION

[0048] The application will be further described in detail by the following examples, which are only used to illustrate the application and do not limit the scope of the application.

[0049] The culture medium involved in the following examples is as follows:

[0050] YPD liquid medium (g / L): peptone 20, yeast extract powder 10, glucose 20, natural pH, sterilized at 115℃ for 20min; YPD solid medium is the liquid medium based on which 20g / L agar powder is added.

[0051] MRS liquid medium (g / L): peptone 10, beef extract 10, glucose 20, sodium acetate 2, yeast powder 5, diammonium hydrogen citrate 2, K2HPO4·3H2O 2.6, MgSO4·7H2O 0.1, MnSO4·H2O 0.05, Tween 80 1mL / L; MRS solid medium is the liquid medium based on which 20g / L agar powder is added.

[0052] Sorghum extract medium: the filtrate after high-temperature-resistant amylase and glucoamylase treatment of sorghum powder. It is obtained by the following method: 250g of sorghum powder is crushed, 1L of distilled water is added for 4h, and it is gelatinized at a temperature of 85-90℃ for 1h, 0.5g of high-temperature-resistant amylase is used for liquefaction at 90℃ for 1h, it is cooled to 60℃, 0.2g of glucoamylase is added, and it is saccharified at 60℃ for 2h, it is cooled to room temperature and centrifuged at 1000r / min for 10min, the supernatant is taken, the natural pH is adjusted, and it is sterilized at 115℃ for 20min.

[0053] Among them, the reagents used in the application are common reagents, which can be purchased from conventional reagent production and sales companies.

[0054] The preparation method of the yeast bacterial suspension involved in the following examples is as follows:

[0055] Saccharomyces cerevisiae CCFM2101 is streaked on YPD solid medium and cultured at 30℃ for 48h to obtain single colonies; the single colonies are inoculated in YPD liquid medium and cultured at 30℃ for 18h for activation, and the activation is carried out for two generations in succession to obtain an activation liquid; the activation liquid is inoculated in YPD liquid medium at an inoculation amount of 2% (v / v) and cultured at 30℃ for 18h to obtain a bacterial liquid; the bacterial liquid is centrifuged at 8000g for 10min to obtain a yeast bacterial body; the yeast bacterial body is washed with physiological saline and resuspended in a glycerol solution with a concentration of 200g / L to a bacterial concentration of 1×10 10 CFU / mL to obtain a bacterial suspension, which is stored at -80℃ for use.

[0056] Lactobacillus fermentum CCFM1472 was streaked on MRS solid medium and incubated at 37°C for 48h to obtain single colonies; the single colonies were inoculated in MRS liquid medium and incubated at 37°C for 12h to activate, and the activation was continued for two generations to obtain an activation liquid; the activation liquid was inoculated in MRS liquid medium at an inoculation amount of 2% (v / v) and incubated at 37°C for 12h to obtain a bacterial liquid; the bacterial liquid was centrifuged at 8000g for 10min to obtain a yeast cell mass; the yeast cell mass was washed with physiological saline and resuspended in a glycerol solution with a concentration of 200g / L to a bacterial concentration of 1×10 10 CFU / mL to obtain a bacterial suspension, which was stored at -80°C for later use.

[0057] Lactobacillus fermentum CCFM1473 was streaked on MRS solid medium and incubated at 37°C for 48h to obtain single colonies; the single colonies were inoculated in MRS liquid medium and incubated at 37°C for 12h to activate, and the activation was continued for two generations to obtain an activation liquid; the activation liquid was inoculated in MRS liquid medium at an inoculation amount of 2% (v / v) and incubated at 37°C for 12h to obtain a bacterial liquid; the bacterial liquid was centrifuged at 8000g for 10min to obtain a yeast cell mass; the yeast cell mass was washed with physiological saline and resuspended in a glycerol solution with a concentration of 200g / L to a bacterial concentration of 1×10 10 CFU / mL to obtain a bacterial suspension, which was stored at -80°C for later use.

[0058] Example 1: Isolation and identification of leavening yeast and lactobacillus in Texas region

[0059] 1. Strain isolation

[0060] The sample derived from leavening in Texas region was pretreated and stored in a -80°C refrigerator in 30% glycerol. After thawing, the sample was mixed and 0.2mL of the sample was added to 5mL of YPD medium for enrichment for 8h. The enrichment liquid was gradient diluted with 0.9% physiological saline, and a suitable gradient dilution liquid was coated on YPD solid medium and incubated at 30°C for 48h. Typical colonies were picked and streaked on YPD plates for purification, and single colonies were transferred to liquid YPD medium for enrichment. The strain CCFM2101 was obtained by 30% glycerol preservation. 0.2mL of the sample was added to 5mL of MRS medium for enrichment for 8h. The enrichment liquid was gradient diluted with 0.9% physiological saline, and a suitable gradient dilution liquid was coated on MRS solid medium and incubated at 37°C for 48h. Typical colonies were picked and streaked on MRS plates for purification, and single colonies were transferred to liquid MRS medium for enrichment. The strains CCFM1472 and CCFM1473 were obtained by 30% glycerol preservation.

[0061] 2. Identification

[0062] The genome of the screened strain CCFM2101 was extracted, and the 26S rDNA thereof was amplified and sequenced (completed by Shanghai Eng Bioengineering Co., Ltd.). Through sequencing analysis, the 26S rDNA sequence of the strain is as shown below (SEQ ID No. 1), and the sequence is compared in GenBank, and the homology with the Saccharomyces is 99%; the results show that the strain is Saccharomyces cerevisiae, and is named Saccharomyces cerevisiae CCFM2101.

[0063] SEQ ID No. 1: ACAACGGGGATGCTTAGTACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTCGGTGCCCGAGTTGTAATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGTTCCTTGGAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTGTAAAGTGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATGGTGTTTTGTGCCCTCTGCTCCTTGTGGGTAGGGGAATCTCGCATTTCACTGGGCCAGCATCAGTTTTGGTGGCAGGATAAATCCATAGGAATGTAGCTTGCCTCGGTAAGTATTATAGCCTGTGGGAATACTGCCAGCTGGGACTGAGGACTGCGACGTAAGTCAAGGATGCTGGCATAATGGTTATATGCCGCCCGTCTTG.

[0064] The Saccharomyces cerevisiae CCFM2101 has a milky white colony on YPD solid culture medium, and is glossy, flat, with a neat edge, and round and convex.

[0065] The genome of the screened strain CCFM1472 was extracted, and the 16S rDNA thereof was amplified and sequenced (completed by Shanghai Engy Bioengineering Co., Ltd.). Through sequencing analysis, the 16S rDNA sequence of the strain is as shown below (SEQ ID No. 2). The sequence was compared in GenBank, and the homology with the Lactobacillus was 100%; the results showed that the strain was Lactobacillus plantarum, and was named Lactobacillus plantarum CCFM1472.

[0066]

[0067] The Lactiplantibacillus plantarum CCFM1472 has a colony on MRS solid medium with a milky white color, a round convex shape, a smooth and fine surface, and a neat edge.

[0068] The genome of the screened L. fermentum was extracted, and the 16S rDNA thereof was amplified and sequenced (completed by Shanghai Engy Bioengineering Co., Ltd.). The 16S rDNA sequence of the strain is shown as follows (SEQ ID No. 3). The sequence was compared in GenBank, and the homology with the Lactobacillus genus was 99.81%. The results show that the strain is L. fermentum, which is named L. fermentum CCFM1473.

[0069] SEQ ID No. 3: GGGGTCGGGCGGTTGCTATACATGCAGTCGAACGCGTTGACCCAATTGATTGATGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGATTTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACG.

[0070] The fermentation limosilactobacillus CCFA1473 is milky yellow on the MRS solid medium, round and convex, rough surface, and irregular edge.

[0071] 3. Culture

[0072] The morphology, physiological and biochemical characteristics and molecular biology of Saccharomyces cerevisiae CCFM2101 were observed. After the strain Saccharomyces cerevisiae CCFM2101 was cultured on YPD solid medium at 30℃ for 48h, it grew well, and the colony was milky white, with luster, flat, regular edge, round and convex (A); the cell morphology under microscope was shown in B, which was oval, budding at one end, with pseudohyphae, belonging to the typical characteristics of yeast, so the strain CCFM2101 was yeast. Figure 1 Figure 1 B).

[0073] The morphology, physiological and biochemical characteristics and molecular biology of Lactiplantibacillus CCFM1472 were observed. After the strain Lactiplantibacillus CCFM1472 was cultured on MRS solid medium at 37℃ for 48h, it grew well, and the colony was milky white, round and convex, smooth surface, fine and regular edge (A); the cell morphology under microscope was shown in B, which was oval, budding at one end, with pseudohyphae, belonging to the typical characteristics of lactobacillus, so the strain CCFM1472 was lactobacillus. Figure 2 Figure 2 B).

[0074] The morphology, physiological and biochemical characteristics and molecular biology of Lactiplantibacillus CCFM1473 were observed. After the strain Lactiplantibacillus CCFM1473 was cultured on MRS solid medium at 37℃ for 48h, it grew well, and the colony was milky yellow, round and convex, rough surface, irregular edge (A); the cell morphology under microscope was shown in B, which was oval, budding at one end, with pseudohyphae, belonging to the typical characteristics of lactobacillus, so the strain CCFM1473 was lactobacillus. Figure 3 Figure 3 B).

[0075] After the total DNA of Saccharomyces cerevisiae CCFM2101 was extracted, the 26s rDNA sequence was obtained by universal primer amplification, and after sequencing, sequence alignment was performed in NCBI database, the results showed that the strain had the highest homology with the 26s rDNA sequence of Saccharomyces cerevisiae AA1, and the similarity reached 99.44%. The Neighbor-Joining method in MEGA7.0 software was used to construct the phylogenetic tree of the strain and the strains with high sequence similarity.​​​Figure 4 ).Depend on Figure 4 It can be seen that this yeast is most closely related to *Saccharomyces cerevisiae* AA1. After extracting total DNA from *Lactiplantibacillus plantarum* CCFM1472, its 16S rDNA sequence was amplified using universal primers. Sequencing was performed, and sequence alignment was conducted in the NCBI database. The results showed that this bacterium had the highest homology (99.44%) with the 16S rDNA sequence of *Lactiplantibacillus plantarum* NCU116. A phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 7.0 software for this strain and strains with high sequence similarity. Figure 5 ).Depend on Figure 5 It can be seen that this yeast is most closely related to Lactiplantibacillus plantarum NCU116. After extracting total DNA from fermenting Lactobacillus CCFM1473, its 16S rDNA sequence was amplified using universal primers. Sequencing and sequence alignment in the NCBI database showed that this bacterium had the highest homology (99.44%) with the 16S rDNA sequence of Lactobacillus fermentum EFEL6800. A phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 7.0 software for this strain and strains with high sequence similarity. Figure 6 ).Depend on Figure 6 It can be seen that this yeast is most closely related to Lactobacillus fermentum EFEL6800.

[0076] Example 2: Determination of the growth curve of the strain

[0077] The *Saccharomyces cerevisiae* strain CCFM2101, screened and identified in Example 1, was taken from the preservation tube and inoculated into YPD liquid medium at an inoculum size of 2% (v / v). The culture was activated at 30°C for 16 h, and this activation was repeated twice to obtain activated *Saccharomyces cerevisiae* strain CCFM2101. 100 μL of the bacterial culture was added to 5 mL of YPD liquid medium and incubated at 30°C. Three centrifuge tubes were taken at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 h respectively, and the OD values ​​were measured. 600 The nm value was calculated, and the average value of three parallel experiments at each time point was used to plot the growth curve.

[0078] The plantaricin CCFM1472 and the ferments mucosus CCFM1473 screened and identified in Example 1 were respectively inoculated into MRS liquid medium with an inoculation amount of 2% (v / v) from the preservation tube, activated at 37°C for 12h, and repeated activation for 2 times to obtain the activated plantaricin CCFM1472 and the ferments mucosus CCFM1473; 100μL of the bacterial solution was added into 5mL of MRS liquid medium, and incubated at 37°C, and 3 centrifuge tubes were taken out at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23h respectively, mixed and detected OD 600 nm value, and the average value of 3 parallel experiments at each time point was calculated to draw the growth curve.

[0079] The growth curves of the saccharomyces cerevisiae CCFM2101, the plantaricin CCFM1472 and the ferments mucosus CCFM1473 are shown in Figure 7 The saccharomyces cerevisiae was in logarithmic growth phase at 5-12h, and entered into stationary phase after 14h; the ferments mucosus was in logarithmic growth phase at 1-6h, and entered into stationary phase after 7h; the plantaricin was in logarithmic growth phase at 1-8h, and entered into stationary phase after 10h.

[0080] Example 3: Preparation of sourdough

[0081] (1) The plantaricin CCFM1472 and the ferments mucosus CCFM1473 were respectively inoculated into MRS broth medium from the preservation tube, activated at 37°C for 12h, and repeated activation for 2 times to obtain the activated plantaricin CCFM1472 and the ferments mucosus CCFM1473.

[0082] (2) The bacterial solution of the activated plantaricin CCFM1472 and the ferments mucosus CCFM1473 was centrifuged at 10000g for 10min, and then the bacterial slurry obtained by centrifugation was resuspended in phosphate solution under sterile conditions, and centrifuged again, and the process was repeated for 3 times.

[0083] (3) 100% wheat flour was mixed with 1.5 times weight of water, and the bacterial slurry of the plantaricin CCFM1472 and the ferments mucosus CCFM1473 was added, and the inoculation amount of each bacterium was 1×10 7 CFU / g of wheat flour. The obtained mixture was stirred uniformly, and incubated at 35°C for 12h to obtain the sourdough.

[0084] Example 4: Preparation of sourdough steamed bread by lactic acid bacteria and yeast synergistic fermentation

[0085] (1) Yeast activation: 20 parts by weight of the sour dough prepared in Example 3, 50 parts by weight of wheat flour, and 10 parts by weight of water were stirred in a stirring cylinder to prepare a mixed dough. 1 x 10

[0086] (2) Preparation of mixed steamed bun dough: 20 parts by weight of the sour dough prepared in Example 3, 50 parts by weight of wheat flour, and 10 parts by weight of water were stirred in a stirring cylinder to prepare a mixed dough. 1 x 10 5 CFU / g of Saccharomyces cerevisiae CCFM2101 were added, followed by stirring to obtain a lactic acid bacteria-yeast dough (mixed steamed bun dough).

[0087] (3) Dough proofing: The prepared mixed steamed bun dough was placed in a proofing box and proofed at a temperature of 30°C and a relative humidity of 75% for 2 h. After proofing, the dough was kneaded to uniformity and then cut into a shape, and proofed at a temperature of 30°C and a relative humidity of 75% for 20 min.

[0088] (4) The second proofed dough was placed in a steamer with cold water added, heated and steamed for 25 min to obtain a finished steamed bun product.

[0089] (5) Packaging and preservation: The steamed buns prepared were cooled at 25°C for 50 min, then placed in a self-sealing bag, and stored in a 4°C refrigerator.

[0090] Comparative Example 1: Commercial yeast instead of Saccharomyces cerevisiae CCFM2101

[0091] This comparative example provides a new type of multi-strain composite flour product leavening agent, which is different from Example 4 only in that the Saccharomyces cerevisiae CCFM2101 strain selected from a leavening agent is replaced by Angel instant high-activity dry yeast.

[0092] The preparation method of this comparative example is to first prepare a sour dough, i.e. Example 3. Then 20 parts by weight of the sour dough prepared in Example 3, 50 parts by weight of wheat flour, and 10 parts by weight of water were stirred in a stirring cylinder to prepare a mixed dough. 0.5 g / 100 g of commercially available Angel yeast was added to the mixed dough, followed by stirring to obtain a lactic acid bacteria-yeast dough (mixed steamed bun dough). The prepared mixed steamed bun dough was placed in a proofing box and proofed at a temperature of 30°C and a relative humidity of 75% for 2 h. After proofing, the dough was kneaded to uniformity and then cut into a shape, and proofed at a temperature of 30°C and a relative humidity of 60% for 20 min. The second proofed dough was placed in a steamer with cold water added, heated and steamed for 25 min to obtain a finished steamed bun product.

[0093] Comparative Example 2: Fermentation using only commercially available yeast

[0094] The comparative example uses a commercially available single-strain starter culture, and the difference from Example 4 is only that the Saccharomyces cerevisiae strain CCFM2101 screened from the starter culture is replaced by Angel instant high-activity dry yeast, and no other strains are added.

[0095] In the comparative example, 5 g of commercially available starter culture Angel instant high-activity dry yeast is mixed with 150 mL of Wahaha purified drinking water and melted, and mixed with 300 g of flour in a kitchen machine for 8 min to distribute the microorganisms in the starter culture to 1 x 10 7 CFU / mL of dough. It is placed in a fermentation cabinet at 35°C and 75% relative humidity for 2 h of fermentation. After fermentation is completed, it is kneaded into a uniform dough and cut into shapes, and is fermented at a temperature of 30°C and a relative humidity of 60% for 20 min. The twice-fermented dough is placed in a steamer with cold water added, heated and steamed for 25 min to obtain the finished steamed buns.

[0096] Comparative Example 3: Starter culture fermentation in the Dezhou region

[0097] In this comparative example, steamed buns are directly prepared using a starter culture purchased in the Dezhou region, and the preparation method is the same as in Example 4, except that the sour dough is replaced by the starter culture and no Saccharomyces cerevisiae is added.

[0098] (1) Preparation of mixed steamed bun dough: 20 parts by weight of starter culture, 50 parts by weight of wheat flour, and 10 parts by weight of water are stirred uniformly in a stirring cylinder to prepare a mixed dough;

[0099] (2) Fermentation of the dough: the prepared mixed steamed bun dough is placed in a fermentation box and fermented at a temperature of 30°C and a relative humidity of 75% for 2 h; after fermentation is completed, it is kneaded into a uniform dough and cut into shapes, and is fermented at a temperature of 30°C and a relative humidity of 75% for 20 min;

[0100] (3) The twice-fermented dough is placed in a steamer with cold water added, heated and steamed for 25 min to obtain the finished steamed buns.

[0101] (4) Packaging and preservation: the steamed buns are cooled for 50 min at 25°C, then placed in a self-sealing bag, and stored in a 4°C refrigerator.

[0102] Comparative Example 4: Starter culture fermentation in the Shangqiu region

[0103] In this comparative example, steamed buns are directly prepared using a starter culture purchased in the Shangqiu region, and the preparation method is the same as in Example 4, except that the sour dough is replaced by the starter culture and no Saccharomyces cerevisiae is added.

[0104] (1) Preparation of mixed steamed bun dough: 20 parts by weight of starter culture, 50 parts by weight of wheat flour, and 10 parts by weight of water are stirred uniformly in a stirring cylinder to prepare a mixed dough;

[0105] (2) The dough is placed in a fermentation box and allowed to ferment for 2 h at a temperature of 30°C and a relative humidity of 75%; after fermentation, the dough is kneaded to homogenize and then cut into a shape, and allowed to ferment for 20 min at a temperature of 30°C and a relative humidity of 75%;

[0106] (3) The dough after the second fermentation is placed in a steamer with cold water added, and heated and steamed for 25 min to obtain the finished steamed buns.

[0107] (4) Packaging and preservation: The steamed buns are cooled for 50 min at 25°C, and then placed in a self-sealing bag and stored in a refrigerator at 4°C.

[0108] Comparative Example 5: Fermentation using only Saccharomyces cerevisiae CCFM2101

[0109] The specific implementation is the same as that of Comparative Example 2, except that Saccharomyces cerevisiae CCFM2101 is used instead of Angel instant high-activity dry yeast.

[0110] Example 5: Determination of the texture properties of steamed buns

[0111] A TA-XTPlus texture analyzer is selected to analyze the texture of the steamed buns (TPA), and a P / 36R probe is used, with the following program settings: pre-test speed of 1.00 mm / s; test speed of 1.00 mm / s; post-test speed of 1.00 mm / s; strain of 50%; time of 5.00 s; and trigger force of 5.0 g.

[0112] The results of the texture index of the steamed buns fermented by the lactic acid bacteria and the yeast are shown in Table 1.

[0113] Table 1: Results of the texture index of different steamed bun samples

[0114]

[0115] As shown in Table 1, the hardness of the steamed buns of Example 4 is close to that of Comparative Example 3. Compared with the steamed buns fermented with the commercially available leavening agent (Comparative Example 2), the steamed buns of the present application have increased chewiness and hardness, and reduced cohesiveness, thereby improving the quality of the steamed buns. By balancing the hardness and chewiness, the steamed buns of Example 4 have a comprehensive advantage in multiple dimensions: the hardness avoids the dry and rough feeling caused by the excessive compaction of traditional high-gluten steamed buns (better than Comparative Examples 2 and 5), and the moderate gluten network support maintains the fluffy structure, so that the steamed buns are not prone to collapse after reheating (better than Comparative Example 1 which is too soft); at the same time, the chewiness is controlled to retain the long-lasting and bouncy wheat aroma, while avoiding the defect of easy gum sticking of low-chewiness steamed buns (better than Comparative Example 3).

[0116] Example 6: Determination of the organic acid composition of steamed buns

[0117] The composition of organic acids in the steamed bread sample is determined by using a high performance liquid chromatograph; the specific steps are as follows:

[0118] 1g sample is placed in a 15mL centrifuge tube, 10mL pure water is added, vortex mixing is carried out, and then tissue disruption is carried out, water bath ultrasonic is carried out for 20min, standing is carried out for 2h, centrifugation is carried out at a speed of 5000rpm for 10min, and then the content of organic acids is determined by HPLC through a 0.22μm water filter membrane.

[0119] The results of the organic acid composition of the acid dough steamed bread fermented by the lactic acid bacteria and the yeast are as shown in table 2.

[0120] Table 2: Organic acid composition analysis of different steamed bread samples (g / 100g)

[0121]

[0122] As shown in table 2, the content of organic acids in the steamed bread of the application is more abundant, lactic acid is the dominant organic acid, by regulating the pH of the dough to below 4.6, inducing the dissociation of the gluten subunit and enhancing the swelling capacity thereof; succinic acid as a dicarboxylic acid enhances the binding efficiency of starch granule surface and water molecules by improving the surface potential thereof; and L-malic acid maintains the CO2 release rate at a stable level by adjusting the activity of key enzymes in the tricarboxylic acid cycle. In addition, the organic acids change the secondary structure of the gluten protein, increase the content of β-fold, and form a short-range but high-crosslinking-density reticular matrix, which corresponds to the mechanism of the phenomenon of the hardness value increase in the texture analysis.

[0123] Example 7: Determination of steamed bread flavor substances

[0124] The volatile flavor substances of the steamed bread of the composite ferment are determined by using headspace solid phase microextraction-gas chromatography-mass spectrometry, 5g steamed bread sample is crushed and placed in a 20mL screw cap glass bottle, 5μL methyl heptanoate (5×10 -4 mol / L) is added as an internal standard for semi-quantitative concentration calculation of volatile aroma substances. See Figure 8 .

[0125] Chromatographic conditions: Rt-WAX capillary chromatographic column (30m×0.25mm, 0.25μm); programmed temperature: 35℃ for 5min, temperature is increased to 50℃ at a speed of 5℃ / min, 5min is kept, temperature is increased to 230℃ at a speed of 5.5℃ / min, 5min is kept; the injection port is 230℃; the carrier gas is high-purity helium, the flow rate is 1.0mL / min; no split injection.

[0126] Mass spectrometry conditions: ionization mode is electron impact ion source (EI); electron energy 70eV, ion source temperature 200℃; interface temperature 250℃; scanning range 33-495m / z.

[0127] Data processing: The mass spectrometry data of each component with similarity greater than or equal to 80% in NIST11 and NIST11S standard spectral library was searched by computer, the search results were checked and confirmed, and the relative content of each component was calculated according to the area normalization method, and the results are shown in Table 3.

[0128] Table 3 GC-MS analysis results of volatile aroma components of steamed buns produced by yeast

[0129]

[0130]

[0131] It can be seen from the results in Table 3 that compared with the commercially available leavening agent steamed buns, the volatile flavor substance content of the steamed buns produced by the method of the present application is obviously improved, wherein the content of acid, ester, aldehyde and aromatic heterocyclic substances is higher than that of the commercially available leavening agent steamed buns, and the relative content of alcohol substances in the steamed buns prepared by the present application is significantly lower than that of the commercially available leavening agent steamed buns. It shows that the production method adopted by the present application is helpful to improve the flavor of steamed buns. Using other yeasts instead of Saccharomyces cerevisiae CCFM2101 of the present application, the steamed buns prepared have flavors inferior to the product of the present application, and the steamed buns of the present application have more rich flavors. The cluster analysis of volatile flavor substances of the comparative examples and the examples shows that Example 4 and Comparative Example 3 are clustered into one category, which shows that the flavor characteristics of the steamed buns fermented by the leavening agent of the present application are similar to the flavor characteristics of traditional leavening agent Jiaozi (steamed buns) (. Figure 8

[0132] ​23 volatile flavor substances were detected from the steamed buns of the present application, the main components were acids, alcohols, esters, aldehydes, ketones and aromatic heterocyclics. Among them, ethanol (0.6540 mg / kg) has the unique aroma of alcohol; acetic acid (0.4585 mg / kg) has sour taste; ethyl acetate (0.0323 mg / kg) has strong fruit aroma, similar to the aroma of pear, apple or banana; 3-methyl-1-butanol (0.0336 mg / kg) has a sweet fruit flavor, similar to the aroma of banana or other tropical fruits; 2-pentyl furan (0.0384 mg / kg) has a bean, fruit and vegetable-like aroma; 1-hexanol (0.0223 mg / kg) has a herbaceous or plant-like aroma; 1-octen-3-ol (0.0161 mg / kg) is an alcohol containing a double bond, with a mushroom aroma; 1-nonanol (0.0112 mg / kg) has a fatty and rose flavor; phenylethanol (0.0319 mg / kg) has a rose aroma. These volatile substances have obvious flavor characteristics and should be the main contributing substances of steamed buns. Among the volatile flavor substances, alcohols account for the main component in all steamed buns, esters are formed by the condensation of lower saturated fatty acids and alcohols, and have various fruit flavors. The fermented steamed buns of the present application have the highest content of esters (0.11 mg / kg), which will make the compounded steamed buns have better odor.

[0133] Example 8: Sensory evaluation

[0134] The sensory evaluation of steamed buns was carried out according to the national standard GB / T 23776-2018, the sensory panel consisted of 14 trained personnel (7 males and 7 females), aged 22-26 years old, all were graduate students of Jiangnan University. All subjects were non-smokers, without known diseases, especially diseases related to oral cavity and olfactory organs. Before the sensory evaluation, the panel members were trained, 4 times a week for 1 h during the two periods. Comparative Example 3 and Comparative Example 4 and Example 4 and Comparative Example 2 were assigned random three-digit codes. During the sensory evaluation, the coded samples were displayed simultaneously, and the panel members were required to score them according to different sensory attributes. The evaluation was carried out using a 10-point scale. In order to minimize the interference between samples, the panel members gargled with soda water between two evaluations, and each sample was evaluated three times.

[0135] Figure 9The results show that the sensory evaluation of traditional sourdough fermented steamed buns in different regions presents differences: the color, sweetness and wheat flavor of Comparative Example 3 are significantly better than those of Comparative Example 4, and the overall evaluation score of Comparative Example 3 is better than that of Comparative Example 4 (7.5). In comparison, Example 4 is close to Comparative Example 3 in each index, perfectly replicating the flavor and texture characteristics of traditional sourdough steamed buns. Compared with commercial steamed buns, the samples of Example 4 and Comparative Example 2 present a differentiated sensory profile: Example 4 is comparable to Comparative Example 2 in color, elasticity and toughness, and the sensory evaluation of Example 4 is significantly better than that of Comparative Example 2 in flavor. Compared with Comparative Example 2, the steamed buns of Example 4 retain similar alcohol and nut flavors, have better fruit and sour flavors, less astringency and sweetness, and have a prominent wheat flavor (with a difference of 3 sensory units from Comparative Example 2). In addition, Example 4 performs outstandingly in flavor complexity, with 12 more volatile flavor substances than Comparative Example 2, especially the detection of characteristic components such as phenethyl alcohol (rose flavor) and γ-butyrolactone (coconut flavor), which may be related to the metabolic pathways of the single-strain fermentation system. Overall evaluation shows that Example 4 has more advantages in traditional flavor attributes (wheat flavor intensity, moderate sourness) and visual acceptability. HPLC data show that the types of organic acids in Example 4 are more complex (secondary metabolites such as citric acid and malic acid), which is consistent with the characteristics of traditional old dough fermentation reported in the literature. In comparison, the single commercial strain of Comparative Example 2 may preferentially consume the substrate through efficient glycolysis, resulting in insufficient accumulation of sour substances.

[0136] Example 9: Preparation of a milk beverage rich in the above flavor substances

[0137] Raw cow milk containing 2% fructooligosaccharides and 2% mannanoligosaccharides was heated to 95°C for sterilization for 5 min, cooled to 37°C, and inoculated with Saccharomyces cerevisiae CCFM2101, Lactobacillus fermentum CCFM1473 and Lactobacillus mucosus CCFM1474 at an inoculation amount of 3% (v / v) respectively, and fermented at 37°C for 24 h. The fermentation product was diluted 2-4 times with a mixture of sterilized and cooled water, sugar syrup, emulsion stabilizer, etc. to prepare a directly drinkable lactic acid bacteria beverage. The lactic acid bacteria beverage not only has a sweet and sour taste, but also has good flavor.

[0138] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A leavening agent, characterized in that, The fermenting agent contains Saccharomyces cerevisiae CCFM2101, Lactiplantibacillus plantarum CCFM1472 and Lactobacillus fermentum CCFM1473; The Saccharomyces cerevisiae CCFM2101 has been preserved in the Guangdong Microbial Culture Collection Center on February 12, 2025, with the preservation number of GDMCC No: 65875; The Lactiplantibacillus plantarum CCFM1472 has been preserved in the Guangdong Microbial Culture Collection Center on February 12, 2025, with the preservation number of GDMCC No: 65873; The Lactobacillus fermentum CCFM1473 has been preserved in the Guangdong Microbial Culture Collection Center on February 12, 2025, with the preservation number of GDMCC No: 65874.

3. A product containing the fermenting agent of claim 1 or 2.

2. The fermenting agent according to claim 1, characterized in that, The number of cells of Saccharomyces cerevisiae CCFM2101, Lactobacillus fermentum CCFM1472 and Lactobacillus mucosae CCFM1473 in the fermenting agent is ≥ 1 x 10 9 CFU / mL or 1 x 10 9 CFU / g. The product includes food, medicine or health care products.

4. The product of claim 3, wherein The product includes fermented flour products, fermented dairy products, fermented condiments or wine.

5. The product of claim 3, wherein The steps include:

6. A method of preparing a steamed bun, characterized by, (1) Preparation of sour dough: inoculate the Lactiplantibacillus plantarum CCFM1472 and Lactobacillus fermentum CCFM1473 of claim 1 into flour, and ferment at 33-37℃ for at least 12h to obtain sour dough; (2) Preparation of mixed steamed bun dough: mix 10-30 parts by weight of sour dough, 40-60 parts by weight of wheat flour and 5-15 parts by weight of water, then inoculate the Saccharomyces cerevisiae CCFM2101 of claim 1 to obtain mixed steamed bun dough; (3) Preparation of steamed buns: the mixed steamed bun dough of step (2) is fermented at a temperature of 28-32℃ for at least 2h; after shaping, it is fermented at a temperature of 28-32℃ for at least 20min; and steamed to obtain steamed buns.

9. Use of the fermenting agent of claim 1 or 2 in the preparation of fermented flour products.

7. The method of claim 6, wherein, The inoculation amount of L. plantarum CCFM1472 and L. fermentum CCFM1473 in the wheat flour of step (1) was 1 x 10 7 CFU / g each. 9 CFU / g each.

8. The method of claim 7, wherein, In the mixed steamed bun dough of step (2), the inoculation amount of the Saccharomyces cerevisiae CCFM2101 is 1 x 10 5 CFU / g. 8 CFU / g. The fermented flour products include but are not limited to steamed buns.

10. Use according to claim 9, characterized in that, ​

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