A lactic acid bacterium, a fermentation composition containing a lactic acid bacterium and a yeast, and use thereof in the preparation of wholegrain dough or wholegrain bread

Abstract

The present application belongs to the technical field of food processing, and particularly relates to a lactic acid bacteria, a fermentation composition containing lactic acid bacteria and yeast, and application of the fermentation composition in preparation of whole wheat sourdough or whole wheat bread. The whole wheat sourdough is prepared by using whole wheat bread flour as a substrate and through synergistic fermentation of lactic acid bacteria and yeast. In addition, the present application applies the whole wheat sourdough in preparation of whole wheat bread, and through synergistic fermentation of lactic acid bacteria and yeast, the fermentation characteristics and dough processing performance of the sourdough are improved, the specific volume and flavor quality of the whole wheat bread are improved, and the nutritional functional characteristics of the whole wheat bread are improved.

Description

Technical Field

[0001] This invention belongs to the field of food microbiology and fermentation technology, specifically relating to a lactic acid bacteria, a fermentation composition containing lactic acid bacteria and yeast, and its application in the preparation of whole wheat sourdough or whole wheat bread. Background Technology

[0002] Whole wheat bread retains the bran, germ, and endosperm of wheat, and is rich in dietary fiber, vitamins, minerals, and plant-based active ingredients, possessing extremely high nutritional value and meeting the urgent needs of modern consumers for healthy foods. However, the high dietary fiber content and bran particles in whole wheat flour cause significant physical damage to the gluten network, resulting in poor dough extensibility and severely insufficient gas retention. Ultimately, this leads to problems such as small specific volume, coarse and dense texture, poor taste, weak flavor, and easy staling during storage, which greatly limits its widespread application in large-scale industrial production.

[0003] Sourdough fermentation is a traditional biological fermentation technique widely used in the production of whole wheat bread to overcome processing defects. In the sourdough system, lactic acid bacteria and yeast constitute the core fermentation microecology. During fermentation, lactic acid bacteria improve the rheological properties of the dough and extend its shelf life by producing metabolic products such as organic acids, extracellular polysaccharides, and specific enzymes; while yeast mainly contributes to the leavening of the dough and the formation of volatile flavor compounds.

[0004] Studies have shown that the introduction of specific functional fermentation metabolites has significant advantages in addressing the processing defects of whole wheat dough. On the one hand, exopolysaccharides (EPS) secreted by lactic acid bacteria, as a natural biological thickener and water-retaining agent, can effectively compensate for damaged gluten networks, significantly improve the viscoelasticity and mechanical processing properties of dough, thereby increasing the specific volume and softness of finished bread and delaying starch retrogradation. On the other hand, whole wheat contains a large amount of bound phenolic substances, and β-glucosidase secreted by lactic acid bacteria can specifically hydrolyze bound phenolic substances and cellulose oligosaccharides in whole wheat bran, releasing free phenolic compounds with high antioxidant activity. This not only significantly improves the nutrient absorption rate and antioxidant capacity of whole wheat bread, but also further improves its overall flavor.

[0005] Although the fermentation of sourdough using a combination of lactic acid bacteria and yeast has gradually become a research hotspot, existing technologies still have significant shortcomings. First, industrial production often uses single commercial yeast fermentation or relies on the unstable natural fermentation mechanism, resulting in long fermentation cycles, unstable product quality, and uncontrollable flavor. Second, current technologies lack precise screening for specific functions of bacterial strains (such as high production of extracellular polysaccharides or high production of glucosidase), and very few studies have combined functional strains with different advantageous metabolic pathways. Finally, the system for how lactic acid bacteria and brewer's yeast with different characteristics can achieve "synergistic effects" through metabolic complementarity to jointly improve the quality of whole wheat bread remains unclear. Therefore, there is an urgent need to develop a highly efficient starter culture combination with clearly defined functions and a well-defined synergistic mechanism, along with its application process, to meet the standardization and efficiency requirements of high-quality whole wheat bread industrial production. Summary of the Invention

[0006] This invention addresses the problems of poor dough extensibility, insufficient gas retention, small specific volume, coarse texture, thin mouthfeel, and easy aging caused by the high dietary fiber and bran content in existing whole wheat bread. It provides a strain of *Westernella esculenta* HN5 that produces high levels of extracellular polysaccharides and a strain of *Pediococcus pentosaceus* HN14 that produces high levels of glucosidase activity. The two lactic acid bacteria strains have clear fermentation properties and complementary functional characteristics, making them suitable for the production of high-quality sourdough and whole wheat bread.

[0007] Another objective of this invention is to provide a method for preparing whole wheat sourdough and whole wheat bread using the synergistic fermentation of *Westernella esculenta* HN5, *Pediococcus pentosaceus* HN14, and *Saccharomyces cerevisiae* Y1. This invention achieves metabolic complementarity and targeted functional synergy among the microbial strains through a microbial complex fermentation system (improving dough elasticity and enhancing antioxidant nutrition), thereby effectively improving the dough processing performance, specific volume, and flavor of whole wheat bread, and significantly enhancing its nutritional functional properties.

[0008] Firstly, the core lactic acid bacteria strains and their functional characteristics:

[0009] The present invention provides a strain of *Westernella esculenta* HN5 that produces a high amount of extracellular polysaccharides, with the preservation number CGMCC No. 38483. The significant feature of this strain is its excellent biothickening potential: after being cultured in MRS liquid medium at 37°C for 24 h, its extracellular polysaccharide (EPS) yield reaches approximately 53 μg / mL, which can significantly improve the viscoelasticity of dough.

[0010] The present invention provides a Pediococcus pentosaceus strain HN14 with high β-glucosidase activity, which has the preservation number CGMCC No. 38482. The significant feature of this strain is its highly efficient biotransformation enzyme system: after being cultured in MRS liquid medium at 37°C for 24 h, its β-glucosidase activity reaches about 20.5 U / mL, which can specifically destroy the structure of whole wheat bran layer and efficiently hydrolyze bound phenolic substances and cellulose oligosaccharides.

[0011] Secondly, the synergistic fermentation agent composition and its synergistic mechanism:

[0012] This invention provides a fermentation agent composition comprising:

[0013] High-extracellular polysaccharide-producing *Westernella sinensis* HN5, preservation number CGMCC No. 38483;

[0014] Pediococcus pentosaceus HN14, which produces high glucosidase activity, has the accession number CGMCC No. 38482;

[0015] Saccharomyces cerevisiae Y1, preservation number CGMCC No. 38485. This composition can be used to prepare whole-wheat sourdough or whole-wheat bread.

[0016] Preferably, the initial inoculation live cell ratio of *Westernella esculenta* HN5, *Pediococcus pentosus* HN14, and *Saccharomyces cerevisiae* Y1 is 0.5:0.5:1.

[0017] The synergistic effect mechanism of the fermentation composition in the preparation of whole wheat sourdough is manifested in:

[0018] (1) Synergistic effect of physical network structure: The extracellular polysaccharides produced by Weissella tamariscina HN5 can wrap and lubricate whole wheat bran and repair the damaged gluten network; together with the carbon dioxide produced by Saccharomyces cerevisiae Y1, they jointly establish a dough queue with high gas holding capacity and high expansion force.

[0019] (2) Nutritional metabolism complementarity: The high production of β-glucosidase by Pediococcus pentosus HN14 degrades whole wheat cellulose macromolecules, releasing fermentable reducing sugars, providing an additional carbon source for brewer's yeast Y1 to start fermentation; at the same time, the released free phenolic compounds greatly enhance the antioxidant capacity of the dough (such as a significant increase in total phenol content and DPPH free radical scavenging rate).

[0020] (3) Synergistic effect of acidity and flavor: The lactic acid produced by the fermentation of the two lactic acid bacteria rapidly reduces the pH value of the dough to the suitable range of 3.8 to 4.2, which stimulates the activity of endogenous enzymes in the flour while inhibiting miscellaneous bacteria, and reacts with yeast metabolites to generate ester flavor substances, which mask the "raw bran taste" of whole wheat.

[0021] Thirdly, this invention provides a whole wheat sourdough fermented with lactic acid bacteria and yeast. The whole wheat sourdough uses whole wheat bread flour as a base and is obtained through the co-fermentation of lactic acid bacteria and yeast. The whole wheat sourdough of this invention exhibits good fermentation activity and processing adaptability. The whole wheat sourdough has a lower pH, increased total titratable acidity, and high activity. The lactic acid bacteria consist of *Pediococcus pentosaceus* and *Westernella esculenta*.

[0022] The method for preparing the whole wheat sourdough is as follows:

[0023] S1: Preparation of lactic acid bacteria ferment and yeast ferment;

[0024] S2: Mix lactic acid bacteria ferment, yeast ferment, whole wheat bread flour and water evenly and then ferment to obtain whole wheat sourdough.

[0025] In step S1, the lactic acid bacteria are activated and cultured on a solid culture medium, and then inoculated into a liquid culture medium for liquid activation culture to obtain the lactic acid bacteria ferment. The solid culture medium is MRS solid culture medium, and the liquid culture medium is MRS liquid culture medium.

[0026] In step S1, the yeast is activated and cultured on a solid culture medium, and then inoculated into a liquid culture medium for liquid activation culture to obtain yeast fermentation product. The solid culture medium is YPD solid culture medium, and the liquid culture medium is YPD liquid culture medium.

[0027] The activation culture conditions for the above-mentioned lactic acid bacteria on solid culture medium are 30-37 ℃ for 18-24 h, and on liquid culture medium are 30-37 ℃ for 18-24 h; the activation culture conditions for the above-mentioned yeast on solid culture medium are 30-37 ℃ for 40-48 h, and on liquid culture medium are 30-37 ℃ for 40-48 h.

[0028] In step S2, the mass ratio of whole wheat bread flour to water is 100:60, and the initial inoculation amount of both lactic acid bacteria and yeast is 10. 6 ~10 7 CFU / g. The fermentation temperature was 30±1℃, and the fermentation time was 24h.

[0029] Fourthly, the present invention provides the application of the above-mentioned whole wheat sourdough in the preparation of whole wheat bread, as detailed below:

[0030] Whole wheat bread is made by mixing whole wheat sourdough with bread fermentation substrate. After one proofing, degassing and shaping, a second proofing, baking and cooling, whole wheat bread is obtained.

[0031] In a further embodiment, the bread fermentation substrate is composed of wheat bread flour, whole wheat bread flour, purified water, sugar, salt, yeast, and butter, and the mass ratio of the whole wheat sourdough, wheat bread flour, whole wheat bread flour, purified water, sugar, salt, yeast, and butter is 30:50:40:60:6:1.5:1.2:3.

[0032] In a further proposed method, the conditions for the first proofing are: temperature 30°C, relative humidity 85%, and time 90 minutes; and the dough is rolled at 55 minutes and 80 minutes of fermentation to remove air bubbles.

[0033] In a further proposed method, the conditions for the second proofing are: temperature 30℃, relative humidity 35%, and time 45 minutes.

[0034] In a further embodiment, the baking conditions are: temperature 200℃, baking time 20min; after baking, remove immediately and cool to room temperature.

[0035] The present invention also provides a whole wheat bread prepared by the above method.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] ① Significantly improves the rheological properties and gas-holding capacity of dough during fermentation:

[0038] Compared with single-strain fermentation, the mixed-strain fermentation group (Y1+HN5HN14) can effectively prolong the time (Tx) before dough begins to leak gas, with a gas retention coefficient of 89.3%. This indicates that the synergistic system significantly enhances the structural stability and gas retention of the dough.

[0039] ② Formation of a suitable fermentation micro-ecological environment:

[0040] The synergistic metabolism of the three strains promotes the rapid and full formation of organic acids and flavor compounds in the sourdough, moderately lowers the pH and increases the acidity, effectively enhancing the overall fermentation activity of the dough.

[0041] ③ Significantly increases bread specific volume:

[0042] Because compound fermented sourdough greatly improves the expansion performance and gas-holding capacity of dough fermentation, the specific volume of the final baked whole wheat bread is significantly increased, effectively improving the defects of traditional whole wheat bread that are "small in volume and short in height".

[0043] ④ Comprehensive improvement of texture and sensory properties:

[0044] Co-fermentation addresses the shortcomings of traditional whole wheat bread, such as its dense texture and coarse mouthfeel. It results in a more uniform and delicate internal structure after baking, effectively improving elasticity and softness while reducing hardness. At the same time, the flavor is more harmonious, greatly alleviating the bran-like taste and rough texture inherent in whole wheat, leading to better overall sensory appeal.

[0045] ⑤ Enhance product storage stability and extend shelf life:

[0046] The suitable acidity and various metabolites (such as extracellular polysaccharides) produced by the fermentation of complex microorganisms create an internal environment that resists deterioration. This not only helps to inhibit the growth of miscellaneous bacteria, but also effectively delays the aging and quality deterioration of whole wheat dough starch during the preservation and storage process, thereby significantly improving the storage stability and shelf life of the product. Attached Figure Description

[0047] Figure 1 Microscopic images of *Westernella esculenta* HN5, *Pediococcus pentosus* HN14, and *Saccharomyces cerevisiae* (top) and single colony images (bottom);

[0048] Figure 2 Results of extracellular polysaccharide production from lactic acid bacteria derived from sourdough;

[0049] Figure 3 Results of β-glucosidase activity assay of lactic acid bacteria derived from sourdough;

[0050] Figure 4 Results of growth performance assay for candidate yeast strains derived from sourdough;

[0051] Figure 5 The growth kinetics of lactic acid bacteria during the fermentation of sourdough in each group;

[0052] Figure 6 The growth kinetics of yeast during the fermentation of sourdough in each group;

[0053] Figure 7 The pH trend during the fermentation of sourdough in each group;

[0054] Figure 8 The trend of total titratable acidity during the fermentation of each group of sourdough;

[0055] Figure 9 The dynamic rheological properties of each group of whole wheat dough; the first to third images reflect the viscoelasticity of the dough (sweep frequency experiment), and the fourth image reflects the extensibility, strain and recovery ability of the dough (creep experiment).

[0056] Figure 10 Specific volume characteristics of each group of whole wheat bread;

[0057] Figure 11Flavor characteristics of each group of whole wheat bread;

[0058] Figure 12 Total phenol content of whole wheat bread in each group;

[0059] Figure 13 Antioxidant properties of whole wheat bread in each group.

[0060] Preservation Information

[0061] 1. Preserved biological material: Pediococcus pentosaceus HN14;

[0062] Accession number: CGMCC No. 38482;

[0063] Classification and nomenclature: Pediococcus pentosaceus ( Pediococcus pentosaceus );

[0064] Preservation institution: China General Microbiological Culture Collection Center;

[0065] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0066] Preservation date: April 27, 2026.

[0067] 2. Preserved biological material: Weissella esculenta HN5;

[0068] Accession number: CGMCC No. 38483;

[0069] Classification and nomenclature: Weissella esculenta ( Weissella cibaria );

[0070] Preservation institution: China General Microbiological Culture Collection Center;

[0071] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0072] Date of preservation: April 29, 2026.

[0073] 3. Preserved biological material: Saccharomyces cerevisiae Y1;

[0074] Accession number: CGMCC No. 38485;

[0075] Classification and naming: Brewing yeast ( Saccharomyces cerevisiae );

[0076] Preservation institution: China General Microbiological Culture Collection Center;

[0077] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0078] Date of preservation: April 29, 2026. Detailed Implementation

[0079] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0080] The whole wheat ingredient used in the examples is commercial whole wheat bread flour.

[0081] Example 1

[0082] The bacterial strains used in this invention were all obtained through targeted screening of traditional handmade sourdough samples. The specific screening process is as follows:

[0083] I. Screening of functional lactic acid bacteria strains derived from sourdough

[0084] 1. Primary screening and purification of lactic acid bacteria

[0085] Activated traditional sourdough samples were thoroughly dispersed in sterile physiological saline and serially diluted. Suitable dilutions were inoculated into MRS liquid medium and incubated at 37°C for 24 hours. The enriched culture was further serially diluted and plated onto MRS solid agar plates, then incubated at 37°C for 24 hours. After incubation, single colonies with larger sizes, different morphologies, and typical lactic acid bacteria characteristics were selected and purified three times by streak plating to obtain candidate lactic acid bacteria strains HN1–HN30 from the sourdough. The activation method for traditional sourdough was as follows: In a clean, oil-free container, a certain amount of sourdough and flour were mixed (1:2), and then an equal amount of water (1:1) was added, stirring until a uniform, moist dough was formed. The container was sealed with plastic wrap and placed in a warm place (20–25°C) for natural fermentation for 2–3 hours. When the dough volume expanded, small bubbles appeared on the surface, and a distinct sour and alcoholic aroma was detected, the sourdough was successfully activated.

[0086] The selected strains formed round, moist, smooth colonies on MRS solid medium with regular edges, and most were milky white or grayish white, consistent with the typical colony morphology of lactic acid bacteria. Gram staining and microscopic examination showed that the selected strains were morphologically consistent, free from obvious contamination, and all were Gram-positive, preliminarily identifying them as candidate lactic acid bacteria strains.

[0087] 2. Screening of high-extracellular polysaccharide-producing lactic acid bacteria strains

[0088] Candidate lactic acid bacteria strains HN1–HN30 were inoculated into modified MRS liquid medium and cultured at 37°C for 24 h to obtain fermentation broths for each strain. The modified MRS liquid medium was prepared by adding 0.2% glucose and 0.1% yeast extract to MRS liquid medium. The fermentation broth was centrifuged, and the supernatant was collected. After precipitation with anhydrous ethanol, centrifugation, and reconstitution, the extracellular polysaccharide test solution was obtained. The extracellular polysaccharide content was determined using the phenol-sulfuric acid method. A standard curve was plotted using glucose as a standard. The test solution was reacted with 6% phenol solution and concentrated sulfuric acid for color development, and the absorbance was measured at 490 nm. The extracellular polysaccharide yield was calculated based on the standard curve. Three parallel determinations were set up for each strain. High-extracellular polysaccharide-producing lactic acid bacteria candidate strains were screened based on extracellular polysaccharide yield.

[0089] The results of the extracellular polysaccharide production assay of the test strains are shown in the figure. Figure 2 .like Figure 2 As shown, strain HN5 had the highest extracellular polysaccharide production among all candidate lactic acid bacteria strains. 16S rRNA gene sequence analysis of strain HN5 confirmed that it is *Westernella esculenta* (a type of lactic acid bacteria). Figure 1 The 16S rRNA gene sequencing results (column 1) are shown in SEQ ID NO:1.

[0090] 3. Screening of lactic acid bacteria strains with high β-glucosidase activity

[0091] Candidate lactic acid bacteria strains HN1–HN30 were initially screened for β-glucosidase using the esculin-based chromogenic plate method. The candidate strains were inoculated onto MRS solid medium containing 0.5% esculin and 0.025% ferric citrate and incubated at 37°C.

[0092] After 24 hours, strains with larger colonies and obvious black precipitate zones were selected and purified by streak plating to obtain candidate strains producing β-glucosidase. These candidate strains were inoculated into MRS liquid medium and cultured. After centrifugation, the supernatant and bacterial cells were collected separately as samples for extracellular enzyme and intact cell enzyme activity assays. β-glucosidase activity was determined using the pNPG method. A standard curve was plotted using pNP (p-nitrophenol) as a standard, and the absorbance was measured at 405 nm to calculate the enzyme activity of each strain. Based on β-glucosidase activity, candidate lactic acid bacteria strains with high β-glucosidase activity were screened.

[0093] The results of the β-glucosidase activity assay are as follows: Figure 3 As shown. By Figure 3 It can be seen that strain HN14 has the highest β-glucosidase activity among all candidate lactic acid bacteria strains; combined with Figure 1(Column 2) As can be seen, the initial screening plate colonies are large and the black precipitate zone is obvious, indicating that this strain has a strong ability to produce β-glucosidase. 16S rRNA gene sequence analysis identified strain HN14 as *Pediococcus pentosaceus*, and the sequencing results are shown in SEQ ID NO:2.

[0094] II. Screening of Dominant Yeast Strains

[0095] Yeast strains were isolated and screened from traditional sourdough that maintained high and stable fermentation activity after multiple activations and subculturings. Activated sourdough samples were added to sterile physiological saline and vortexed until homogeneous. After serial dilution, the samples were spread onto YPD solid agar plates and incubated at 28–30°C for 48 h. Single colonies of yeast with typical morphology, regular edges, and smooth, moist surfaces were selected and streaked 2–3 times to obtain candidate yeast strains Y1–Y10. Candidate yeast strains Y1–Y10 were inoculated into YPD liquid agar and incubated at 28–30°C for 18–24 h. The bacterial concentration was determined by OD0.05. 600 Further screening was conducted to determine the indicators. The results showed that the colony and microscopic morphology of strain Y1 conformed to typical yeast characteristics. Figure 1 The third column), and the colonies are pure and free from contamination; by Figure 4 It can be seen that the OD of strain Y1 600 The highest value indicates superior growth performance, making it a suitable dominant yeast strain for further research. 18S rRNA gene sequence analysis identified strain Y1 as *Saccharomyces cerevisiae*, and its sequencing results are shown in SEQ ID NO:3.

[0096] Example 2

[0097] I. Preparation of Whole Wheat Sourdough

[0098] (I) Activation of microbial strains and preparation of fermentation products

[0099] The preparation method of lactic acid bacteria (Westernella stolonifera HN5 or Pediococcus pentosus HN14) fermentation product is as follows: The lactic acid bacteria strain preserved in glycerol tubes at -80 ℃ is taken out under aseptic conditions, revived, and streaked onto MRS solid medium. It is then incubated at 37 ℃ for 24 h for plate activation. After colony growth, a single colony is picked and inoculated into MRS liquid medium. It is then incubated at 37 ℃ for 24 h for the first generation of liquid activation. Subsequently, it is transferred to a new MRS liquid medium and the second generation of liquid activation is carried out under the same conditions to obtain the lactic acid bacteria broth for fermentation.

[0100] The above lactic acid bacteria culture was centrifuged at 5000 ×g for 10 min, the supernatant was discarded, the bacterial sludge was collected, and it was washed twice with 0.9% sterile sodium chloride solution and then resuspended in purified water to obtain lactic acid bacteria fermentation product for later use.

[0101] The preparation method of yeast fermentation product is as follows: Saccharomyces cerevisiae Y1 stored in glycerol tubes at -80 ℃ was taken out under aseptic conditions, revived, and streaked onto YPD solid medium. It was then cultured at 37 ℃ for 48 h for plate activation. After colony growth, a single colony was picked and inoculated into YPD liquid medium. It was then cultured at 37 ℃ for 48 h for first-generation liquid activation. Subsequently, it was transferred to a new YPD liquid medium and subjected to second-generation liquid activation under the same conditions to obtain yeast culture for fermentation.

[0102] The above yeast culture was centrifuged at 5000 ×g for 10 min, the supernatant was discarded, the bacterial sludge was collected, and it was washed twice with 0.9% sterile sodium chloride solution and then resuspended in mineral water to obtain the yeast fermentation product for later use.

[0103] (II) Preparation of whole wheat sourdough for each group

[0104] Six groups of whole wheat sourdough were prepared according to the leavening agent combinations shown in Table 1, and were designated as control group 1 to control group 3 and group 1 to group 3.

[0105] Each group prepared sourdough according to the following proportions: when using compound fermentation, the mass ratio of lactic acid bacteria ferment, yeast ferment, commercial whole wheat bread flour, and mineral water was 1:1:100:60; when using single-strain fermentation, the mass ratio of ferment, commercial whole wheat bread flour, and mineral water was 1:100:60. After mixing the ingredients, each group was stirred for 6 minutes to ensure uniform mixing, and then fermented at a constant temperature of 30±1℃ for 24 hours to obtain the corresponding whole wheat sourdough.

[0106] The initial inoculation amount of lactic acid bacteria or yeast in each group of sourdough was controlled to be approximately 10. 6 CFU / g. During multi-strain fermentation, the live counts of lactic acid bacteria and yeast were mixed at a 1:1 ratio, and the total inoculum was consistent with that of the single-strain fermentation group. After fermentation, the live counts of lactic acid bacteria or yeast in the sourdough of each group were greater than 10. 8 CFU / g.

[0107] Table 1. Design of Fermentation Agent Combinations for Each Group

[0108]

[0109] II. Determination of Quality Indicators for Whole Wheat Sourdough

[0110] The core of this invention lies in the biotransformation of whole wheat sourdough. To verify this core step, samples of the whole wheat sourdough obtained in step one were taken after fermentation at 30±1℃ for 0h, 4h, 8h, 12h, and 24h. The microbial growth and acid production characteristics were measured, and the results are shown below. Figures 5-8.

[0111] (a) Microbial community detection

[0112] 1. Determination of total lactic acid bacteria colony count

[0113] The total number of lactic acid bacteria colonies in whole wheat sourdough was determined using the plate count method. The test subjects included control group 2, control group 3, group 1, group 2 and group 3; control group 1 was used as a reference because no lactic acid bacteria were added and the total number of lactic acid bacteria was not measured at 0 h of fermentation (the total number of lactic acid bacteria in control group 1 at 0 h of fermentation was assumed to be 0).

[0114] The specific method is as follows: Accurately weigh 1 g of whole wheat sourdough sample, add 9 mL of 0.9% sterile sodium chloride solution, mix thoroughly on a vortex mixer to obtain a 1:10 sample homogenate; then perform a 10-fold serial dilution, select 1 mL of the sample solution with an appropriate dilution and add it to a sterile Petri dish, pour in MRS solid medium, mix well, and incubate at 37℃ for 48 h, and count the colonies.

[0115] Depend on Figure 5 It can be seen that during the constant temperature fermentation process, the number of viable lactic acid bacteria in each group containing lactic acid bacteria showed a significant increasing trend; when fermentation reached 8 hours, the number of viable lactic acid bacteria had significantly increased and showed significant growth momentum, with the final number of viable lactic acid bacteria reaching as high as 10. 8 CFU / g or higher.

[0116] 2. Determination of total yeast colony count

[0117] The number of viable yeast cells in the fermented sourdough of each group was determined by plate counting. The test subjects included control group 1, group 2, and group 3. Since no yeast was added to control group 2 and control group 3, the total number of yeast cells was not measured at 0 h of fermentation (the total number of yeast cells in control group 2 and control group 3 at 0 h of fermentation was assumed to be 0), and they were used as references.

[0118] The sampling and homogenization methods are the same as those for "Determination of Total Lactic Acid Bacteria Colony Count". Inoculate the appropriately diluted sample solution into a sterile Petri dish, pour in YPD solid medium, mix well, and incubate at 30℃ for 48 h before counting the colonies.

[0119] Depend on Figure 6 It can be seen that the number of viable yeast cells increases rapidly in the early stage of fermentation, and can maintain a high level thereafter in the acidic dough environment; after fermentation, the final number of viable cells in each yeast-containing group is greater than 10. 7 The CFU / g concentration met the fermentation requirements. The overall yeast count in the mixed-culture fermentation group was higher than that in the lactic acid bacteria single-culture fermentation group, indicating a good synergistic growth relationship between lactic acid bacteria and yeast.

[0120] (II) Detection of acid production during fermentation

[0121] 1. pH value measurement

[0122] The pH changes of each group of sourdough samples during fermentation were measured using a digital pH meter. The specific method is as follows: 10 g of sourdough sample was accurately weighed, added to 90 mL of distilled water, mixed well, and then the pH value was measured.

[0123] The test results are shown below. Figure 7 The results showed that compared with yeast-based single-strain fermentation of whole wheat sourdough, the mixed fermentation group introducing lactic acid bacteria significantly improved the acid production rate and rapidly decreased the pH value. After 24 hours of fermentation, the pH values ​​of all samples remained relatively stable within the range of 3.8–4.2. This result confirms a good metabolic synergy between lactic acid bacteria and yeast: the rapid acid production of lactic acid bacteria preempts the microecological advantage to inhibit other bacteria, while the ideal acidic environment created further promotes the various biochemical transformation processes dominated by yeast, effectively improving the overall quality of the fermented product.

[0124] 2. Determination of total titratable acidity

[0125] The total titratable acid content of each group of sourdough samples was determined by acid-base titration, expressed as the volume of 0.1 mol / L NaOH standard solution required to neutralize 100 g of sourdough, with units of mL NaOH / 100 g dough.

[0126] The specific method is as follows: Dissolve the sourdough sample, make up to a certain volume, filter it, accurately pipette 20 mL of the filtrate, titrate it with 0.1 mol / L NaOH standard solution to pH 8.1, and record the volume of NaOH consumed.

[0127] See results Figure 8 The results showed that the total titratable acid value of the sourdough in all groups increased with prolonged fermentation time, with the group containing lactic acid bacteria showing a more significant increase. After fermentation, the total titratable acid value of all groups of sourdough was higher than 5 mL NaOH / 100 g. This result indicates that the participation of lactic acid bacteria in fermentation can significantly enhance the acid-producing capacity of sourdough, which is beneficial for forming a richer grain fermentation flavor.

[0128] III. Preparation of Whole Wheat Dough and Whole Wheat Bread

[0129] The whole wheat sourdoughs obtained from the above groups were mixed with wheat bread flour, whole wheat bread flour, purified water, sugar, salt, yeast, and butter in a mass ratio of 30:50:40:60:6:1.5:1.2:3. The mixtures were then stirred at low speed using a double-helix mixer for 6 minutes until the dough was soft, smooth, and non-sticky. The whole wheat dough was then placed in a 30°C environment with 85% relative humidity for a first proofing of 90 minutes. At 55 minutes and 80 minutes of proofing, the dough was rolled to remove air bubbles. After the first proofing, the dough was degassed and shaped, and then placed in a 30°C environment with 35% relative humidity for a second proofing of 45 minutes. The fully fermented whole wheat dough was then baked in an oven at 200°C (top and bottom heat) for 20 minutes. After baking, the bread was immediately removed and cooled to room temperature to obtain whole wheat bread products of control groups 1-3 and group 1-3, respectively, corresponding to whole wheat bread of groups Y1, HN5, HN14, Y1+HN5, Y1+HN14, and Y1+HN5HN14.

[0130] IV. Determination of Rheological Properties of Whole Wheat Dough

[0131] (I) Determination of the fermentation rheological properties of whole wheat dough

[0132] A Rheofermentometer F3 fermentation rheometer was used to sample and measure the fermentation rheological properties of whole wheat dough prepared by each formula in step three before fermentation. The measured parameters included: maximum fermentation height Hm, final fermentation height h, height loss rate [(Hm-h) / Hm], maximum height of the gas release curve H'm, time to start gas leakage Tx, total gas volume produced, total gas volume retained, and gas retention coefficient.

[0133] The results are shown in Table 2. The results indicate that, compared with the single-strain fermentation group of lactic acid bacteria (especially HN5), the mixed-strain fermentation group performed better in terms of increasing the maximum fermentation height Hm, prolonging the dough's initial gas leakage time Tx, and improving gas retention capacity. This suggests that the combined fermentation of lactic acid bacteria and yeast can effectively improve the fermentation rheological properties of whole wheat dough.

[0134] Table 2. Fermentation rheological properties of whole wheat dough in each group

[0135]

[0136] (II) Analysis of the dynamic rheological properties of whole wheat dough

[0137] The dynamic rheological properties of each group of whole wheat dough samples were determined using a rotational rheometer. Specifically, samples of each group of whole wheat dough were taken after fermentation and placed on the rheometer testing platform (set to a constant temperature of 25℃). Frequency sweep tests were performed within a set frequency range, and the changes in storage modulus (G'), loss modulus (G''), and loss tangent (tan δ) of the dough were recorded. Furthermore, the change in strain (%) of the dough during the application and removal of constant stress was measured using a creep-recovery experiment to evaluate the dough's extensibility and elastic recovery rate.

[0138] The test results are shown below. Figure 9 Dynamic rheological results showed that the synergistic fermentation of the three strains (Y1 + HN5 & HN14) significantly reduced the excessive rigidity of whole wheat dough and greatly improved its extensibility and strain capacity, successfully constructing a "soft yet highly tensile" elastic network. In strain-time tests, the strain value of the three-strain combination group was higher than that of the single-strain fermentation groups, indicating that the dough of this combination group has better deformation adaptability. These results suggest that the combined fermentation of HN5, HN14, and Y1 helps to regulate the viscoelastic properties of whole wheat dough and may improve the continuity and processing adaptability of the dough network in the presence of bran.

[0139] V. Determination of Quality Indicators for Whole Wheat Bread

[0140] The specific volume, flavor, total phenol content, and DPPH free radical scavenging ability of the whole wheat bread obtained in step three were determined and analyzed.

[0141] (a) Specific volume determination

[0142] The volume of each group of whole wheat bread was determined using the volume displacement method, and the mass of each group of bread was weighed. The specific volume of the bread was calculated using the following formula:

[0143] Specific volume (mL / g) = Bread volume (mL) / Bread mass (g).

[0144] The test results are shown below. Figure 10 The results showed that, compared with the single lactic acid bacteria fermentation group, the specific volume of whole wheat bread obtained by the combined fermentation group of lactic acid bacteria and yeast was improved, especially the Y1+HN14 group, which showed the most significant improvement. This indicates that the combined fermentation sourdough can improve the fermentation expansion performance and gas holding capacity of whole wheat dough, thus helping to increase the finished volume of whole wheat bread.

[0145] (ii) Flavor determination

[0146] The volatile flavor compounds in whole wheat bread were determined using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). A suitable amount of bread sample was placed in a headspace vial, sealed, and equilibrated. 10 μL of 4-methyl-2-pentanol (100 mg / L) was added as an internal standard. After solid-phase microextraction, GC-MS analysis was performed, and semi-quantitative analysis of volatile flavor compounds was conducted using a spectral library search.

[0147] The test results are shown below. Figure 11 The results showed that volatile flavor compounds such as alcohols, aldehydes, ketones, esters, and acids were detected in all groups of whole wheat bread. Among them, the group fermented with a combination of lactic acid bacteria and yeast had a richer variety and relative content of flavor compounds, and the fermentation aroma and grain aroma were more harmonious, indicating that the compound fermented sourdough can promote the improvement of the flavor quality of whole wheat bread.

[0148] (III) Determination of total phenol content

[0149] The total phenol content in whole wheat bread samples was determined using the Folin-Ciocalteu method. The bread samples were freeze-dried, pulverized, and extracted with a suitable solvent. After centrifugation, the supernatant was used as the test solution. Folin-Ciocalteu reagent and sodium carbonate solution were added to the test solution, and the reaction was carried out in the dark. The absorbance was measured at a specified wavelength, and a standard curve was plotted using gallic acid as a standard to calculate the total phenol content of the samples.

[0150] The test results are shown below. Figure 12 The results showed that, compared with the yeast Y1 single-strain fermentation group, the total phenolic content in whole wheat bread from the combined fermentation group with added lactic acid bacteria (especially the Y1+HN5HN14 three-strain synergistic group) was significantly increased. This indicates that the synergistic fermentation of lactic acid bacteria and yeast is more conducive to promoting the release and transformation of bound phenolic substances in the whole wheat matrix, thereby significantly increasing the detectable total phenolic content and nutritional value of whole wheat bread.

[0151] (iv) Determination of DPPH free radical scavenging capacity

[0152] The free radical scavenging capacity of whole wheat bread samples in each group was determined using the DPPH method. The bread samples were freeze-dried, pulverized, and extracted with a suitable extraction solvent. After centrifugation, the supernatant was used as the test solution. A certain volume of the test solution was mixed with DPPH ethanol solution, and the absorbance was measured at a specified wavelength after reacting in the dark. A standard curve was plotted with the concentration of the Trolox standard solution as the x-axis (μmol / L) and the measured absorbance value as the y-axis. The antioxidant capacity of the samples was calculated based on the standard curve equation, and the final result was expressed as the Trolox equivalent (mg TE / 100 g dw) per 100 g dry bread.

[0153] The test results are shown below. Figure 13 The results showed that, compared with the yeast Y1 single-strain fermentation group, the whole wheat bread produced by the fermentation group containing Pediococcus pentosaceus HN14 (including the HN14 single-strain group and the Y1+HN14 compound group) exhibited significantly higher DPPH free radical scavenging capacity (p < 0.05). This indicates that Pediococcus pentosaceus HN14 can effectively enhance the antioxidant activity of the whole wheat matrix during fermentation, and through the synergistic effect of lactic acid bacteria and yeast, it helps to further enhance the nutritional and functional properties of whole wheat bread.

[0154] In summary, the whole wheat sourdough prepared by the combined fermentation of lactic acid bacteria and yeast can improve the specific volume, color, texture and flavor of whole wheat bread, while increasing its total phenol content and DPPH free radical scavenging ability. This indicates that the combined fermentation system has a positive effect on improving the quality and nutritional function of whole wheat bread.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of lactic acid bacteria, characterized in that, The lactic acid bacteria mentioned are Pediococcus pentosaceus, classified and named as follows: Pediococcus pentosaceus The accession number is CGMCC No. 38482; Alternatively, the lactic acid bacteria may be *Westernella esculenta*, classified and named as follows: Weissella cibaria The accession number is CGMCCNo.38483.

2. A fermentation agent composition, characterized in that, It includes lactic acid bacteria and yeast, wherein the lactic acid bacteria consist of *Pediococcus pentosaceus* as described in claim 1 and *Westernella esculenta* as described in claim 1, and the yeast is *Saccharomyces cerevisiae*, which is classified and named as follows: Saccharomyces cerevisiae The accession number is CGMCC No. 38485.

3. The fermentation agent composition according to claim 2, characterized in that, In the fermentation agent composition, the ratio of viable viable bacteria of *Westernella esculenta*, *Pediococcus pentosus*, and *Saccharomyces cerevisiae* is 0.5:0.5:

1.

4. A whole wheat sourdough fermented by lactic acid bacteria and yeast, characterized in that, The whole wheat sourdough is obtained by co-fermentation of lactic acid bacteria and yeast using whole wheat bread flour as a base; the lactic acid bacteria are composed of Pediococcus pentosaceus as described in claim 1 and Weissella esculenta as described in claim 1, and the yeast is Saccharomyces cerevisiae.

5. The whole wheat sourdough fermented with lactic acid bacteria and yeast according to claim 4, characterized in that, The method for preparing the whole wheat sourdough is as follows: S1: Preparation of lactic acid bacteria ferment and yeast ferment; S2: Mix lactic acid bacteria ferment, yeast ferment, whole wheat bread flour and water evenly and then ferment to obtain whole wheat sourdough.

6. The whole wheat sourdough fermented with lactic acid bacteria and yeast according to claim 1, characterized in that, In step S1, after activating and culturing lactic acid bacteria or yeast on a solid culture medium, they are then inoculated into a liquid culture medium for liquid activation culture to obtain lactic acid bacteria ferment or yeast ferment.

7. The whole wheat sourdough fermented with lactic acid bacteria and yeast according to claim 1, characterized in that, In step S2, the mass ratio of whole wheat bread flour to water is 100:60, and the initial total inoculum of lactic acid bacteria and yeast is 10. 6 ~10 7 CFU / g. The fermentation temperature was 30±1℃, and the fermentation time was 24h.

8. The application of the whole wheat sourdough according to any one of claims 4 to 7 in the preparation of whole wheat bread, characterized in that, include: Whole wheat bread is made by mixing whole wheat sourdough with bread fermentation substrate. After one proofing, degassing and shaping, a second proofing, baking and cooling, whole wheat bread is obtained.

9. The application according to claim 7, characterized in that, The bread fermentation substrate is composed of wheat bread flour, whole wheat bread flour, purified water, sugar, salt, yeast and butter. The mass ratio of the whole wheat sourdough, wheat bread flour, whole wheat bread flour, purified water, sugar, salt, yeast and butter is 30:50:40:60:6:1.5:1.2:

3.

10. The application according to claim 7, characterized in that, The first proofing conditions are: temperature 30℃, relative humidity 85%, time 90min; and the dough is rolled at 55min and 80min to remove air bubbles; the second proofing conditions are: temperature 30℃, relative humidity 35%, time 45min; the baking conditions are: temperature 200℃, baking time 20min; after baking, the dough is immediately removed and cooled to room temperature.

Images