Propionibacterium freudenreichii and application thereof in cereal fermented food

Abstract

The invention relates to propionibacterium freudenreichii and application thereof in cereal fermented food, and belongs to the technical field of food. The invention provides a microbial agent containing propionibacterium freudenreichii, the microbial agent is applied to cereal fermented food (such as bread, steamed buns and steamed stuffed buns), and the preservation performance and sensory quality of the food are remarkably improved by prolonging the shelf life of the food and improving the texture and flavor of the food. The specific method comprises the following steps: adding the microbial agent into a mixed system of water and flour, fermenting to prepare sour dough, mixing the sour dough with other ingredients, and baking. Experimental results show that the microbial inoculum significantly prolongs the shelf life of the bread, inhibits the growth of mould, optimizes the texture parameters of the bread, improves the specific volume and height-diameter ratio of the bread, reduces the baking loss and aging rate of the bread, and improves the overall sensory evaluation.

Description

Technical Field

[0001] The invention discloses a strain of Propionibacterium freudenreichii and application thereof in fermented cereal food, belonging to the technical field of food. Background Art

[0002] Fermented grain foods, including fermented grain foods, steamed bread, dumplings and other high water activity fermented grain foods are good culture media for the growth of fungi, bacteria and other microorganisms. Therefore, they are prone to spoilage during storage, and also provide favorable growth conditions for fungi, bacteria and other microorganisms, resulting in mold, flavor deterioration or potential health risks during storage. Therefore, how to effectively extend the shelf life of fermented grain foods is crucial to ensuring food safety and reducing food waste.

[0003] In order to inhibit the growth of microorganisms, physical preservation methods and the addition of chemical preservatives have traditionally been used. Physical preservation methods such as freezing, improved packaging, high-pressure processing, radiation and magnetic fields have achieved remarkable results in inhibiting the growth of microorganisms, but are often accompanied by high equipment costs, energy consumption and process complexity, making them difficult to promote in large-scale production. Chemical preservatives: Chemical preservatives such as calcium propionate and potassium sorbate also have the advantages of high efficiency and convenient operation in inhibiting mold and bacteria. However, their maximum usage limits are strictly restricted, and excessive use may bring health risks; in addition, with the upgrading of consumer concepts, resistance to "non-natural" attributes has gradually intensified, making it difficult to meet the needs of today's green and healthy consumption trends.

[0004] Against the backdrop of growing demand for natural and safe preservation technologies, new preservation technologies that combine high-efficiency preservation with health properties have become a research hotspot. Sourdough technology can produce a variety of organic acids and antibacterial substances (exopolysaccharides, antimicrobial peptides, bacteriocins, etc.) during the fermentation process, which not only effectively inhibits the growth of microorganisms and prolongs the shelf life of fermented grain foods, but also significantly improves the texture and flavor of fermented grain foods. Because it has the dual advantages of preservation and quality improvement, it is regarded as a potential preservation method with green and safe characteristics.

[0005] As a typical representative of sourdough technology, lactic acid bacteria sourdough has achieved many results in extending the shelf life and improving the quality of fermented cereal foods. This is because lactic acid bacteria can quickly produce acid in a short period of time, enhance antibacterial activity, improve dough structure and water holding capacity, and give fermented cereal foods a unique flavor. However, lactic acid has a low acidity coefficient (smaller pKa) and a high molecular weight, which limits the sustainability of its antiseptic properties.

[0006] In order to overcome the limitations of the inhibitory effect of lactic acid, the introduction of propionic acid bacteria has become a new research direction. Propionic acid bacteria can use lactic acid to generate propionic acid and synthesize metabolites with antibacterial activity such as extracellular polysaccharides and diacetyl, thereby further enhancing the preservative effect of fermented cereal foods. Compared with lactic acid, propionic acid has a smaller molecular weight and a lower acidity coefficient (higher pKa), so it is difficult to ionize and is easier to enter microbial cells, releasing hydrogen ions, leading to the accumulation of intracellular acid, thereby exerting a stronger antibacterial effect. However, if the fermentation time and metabolic rate of propionic acid bacteria are not properly controlled, the excessive propionic acid produced will significantly reduce the pH of the sourdough, inhibit the normal fermentation process of yeast in subsequent cereal fermented foods, lead to a decrease in the bulkiness and texture of the cereal fermented foods, and even affect the overall sensory quality. Therefore, how to balance the fermentation conditions of propionic acid bacteria and yeast and avoid the production of excessive propionic acid is a key challenge in the application of propionic acid bacteria sourdough.

[0007] In order to achieve a comprehensive improvement in the quality and preservative properties of fermented cereal foods, the combination strategy of lactic acid bacteria and propionic acid bacteria has received increasing attention. The advantages of this mechanism mainly include the following two aspects: (1) Persistent and enhanced preservation. Lactic acid bacteria quickly produce acid in sour dough, forming an acidic environment that inhibits the growth of mold and spoilage bacteria. At the same time, lactic acid provides a substrate for the metabolism of propionic acid bacteria, enabling them to synthesize propionic acid with better antibacterial effect, thereby achieving a long-lasting and stable antibacterial effect. In addition, the mixture of multiple metabolites can achieve synergistic antibacterial effect, further enhancing the inhibitory ability against microorganisms such as bacteria and mold. (2) Quality optimization. The combination strategy reasonably controls the fermentation conditions to avoid a sharp drop in pH caused by excessive propionic acid on the one hand; on the other hand, it makes full use of the metabolites of the two strains to jointly improve the texture, flavor and sensory quality of fermented cereal foods. Summary of the invention

[0008] Based on this problem, this study proposed the development of a compound strategy of lactic acid bacteria and propionic acid bacteria. First, lactic acid bacteria are used to quickly produce acid and inhibit foreign bacteria in the early stage of fermentation. Second, the lactic acid metabolized by lactic acid bacteria is used as a substrate for propionic acid bacteria, and propionic acid bacteria continuously produce propionic acid to prolong the antibacterial effect. Third, by coordinating the acid production intensity and fermentation time, excessive acidity accumulation can be avoided, thereby reducing the inhibition of yeast activity while meeting the preservation requirements. Fourth, the texture and flavor of fermented grain foods can be further improved through the interaction of the metabolites of the two strains.

[0009] Based on this, this study focuses on the compound sourdough technology of lactic acid bacteria and propionic acid bacteria, aiming to make full use of the respective advantages and synergistic mechanisms of the two strains, extend the shelf life of cereal fermented foods and improve product quality, and provide feasible solutions and theoretical support for the promotion and application of green preservation technology in the cereal fermented food industry.

[0010] This study introduced a combination strategy of lactic acid bacteria and propionic acid bacteria into sourdough technology, which not only fully utilized the rapid acid production characteristics of lactic acid bacteria to inhibit foreign bacteria, but also strengthened the preservation effect with the help of the long-lasting antibacterial advantages of propionic acid bacteria. By reasonably controlling the acid production intensity and fermentation time, the fermentation quality and preservation requirements of cereal fermented foods were taken into account to the greatest extent. At the same time, the interaction between the metabolites of the two strains is more conducive to improving the texture and flavor of cereal fermented foods. This study provides new ideas and practical paths for the promotion of green preservation technology in the cereal fermented food industry, which not only helps to meet consumers' demand for natural, safe and high-quality cereal fermented foods, but also has important significance for extending the shelf life of cereal fermented foods, reducing food waste and promoting the sustainable development of the industry.

[0011] The present invention provides a strain of Propionibacterium freudenreichii DX0705, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.33491 and a deposit date of January 22, 2025.

[0012] The present invention provides a microbial agent, wherein the microbial agent contains the above-mentioned Propionibacterium freudenreichii.

[0013] In one embodiment of the present invention, the number of Propionibacterium freudenreichii cells in the microbial agent is not less than 1.0×10 6 CFU / g;

[0014] In one embodiment of the present invention, the microbial agent contains living cells of Propionibacterium freudenreichii, dry cells of Propionibacterium freudenreichii obtained by freeze-drying, immobilized cells of Propionibacterium freudenreichii, liquid agent of Propionibacterium freudenreichii, solid agent of Propionibacterium freudenreichii, or Propionibacterium freudenreichii in any other form.

[0015] The present invention provides a microbial agent, wherein the microbial agent contains the above-mentioned Propionibacterium freudenreichii and Lactobacillus sanfranciscensis;

[0016] In one embodiment of the present invention, the Lactobacillus sanfrancisco is Lactobacillus sanfrancisco DSM20451T.

[0017] In one embodiment of the present invention, the amount of Propionibacterium freudenreichii added to the microbial agent is at least: 1.0×10 6 CFU / g; the amount of Lactobacillus sanfrancisco added is at least: 1.0×10 6 CFU / g;

[0018] In one embodiment of the present invention, the microbial agent contains living cells of Propionibacterium freudenreichii or Lactobacillus sanfrancisco, dry cells of Propionibacterium freudenreichii or Lactobacillus sanfrancisco obtained by freeze-drying, immobilized cells of Propionibacterium freudenreichii or Lactobacillus sanfrancisco, liquid agent of Propionibacterium freudenreichii or Lactobacillus sanfrancisco, solid agent of Propionibacterium freudenreichii or Lactobacillus sanfrancisco, or Propionibacterium freudenreichii or Lactobacillus sanfrancisco strains in any other form.

[0019] The present invention also provides the use of the Propionibacterium freudenreichii or the microbial agent or Lactobacillus sanfrancisco in extending the shelf life of fermented cereal foods and / or improving the texture and flavor of fermented cereal foods.

[0020] In one embodiment of the present invention, the Lactobacillus sanfrancisco is Lactobacillus sanfrancisco DSM20451T.

[0021] In one embodiment of the present invention, the fermented cereal food includes bread, steamed buns, and dumplings.

[0022] The present invention also provides a method for making sourdough, which comprises mixing Propionibacterium freudenreichii or the above-mentioned microbial agent or Lactobacillus sanfrancisco with water and flour to prepare the sourdough.

[0023] In one embodiment of the present invention, the Lactobacillus sanfrancisco is Lactobacillus sanfrancisco DSM20451T.

[0024] In one embodiment of the present invention, the method is prepared by adding Propionibacterium freudenreichii or Lactobacillus sanfrancisco to a flour-water system with a weight ratio of water to flour of 0.6-1.5:1, and fermenting at 30°C for 24 hours until the pH of the sourdough drops to 4.0-4.5, and the method comprises mixing the above-mentioned microbial agent with water and flour to prepare the sourdough with antibacterial effect;

[0025] Preferably, the weight ratio of water to flour is 1:1;

[0026] Preferably, the amount of Propionibacterium freudenreichii added is at least: 1.0×10 6 CFU / g;

[0027] Preferably, the amount of Lactobacillus sanfrancisco added is at least: 1.0×10 6 CFU / g;

[0028] Preferably, the amount of the mixed microbial agent added is at least: 1.0×10 6 CFU / g.

[0029] The invention also provides sour dough obtained by the production method.

[0030] The present invention also provides a method for preparing fermented cereal food, which comprises mixing the sour dough with water, flour, yeast and salt, and then stirring, proofing and baking to prepare the sour dough.

[0031] In one embodiment of the present invention, the fermented cereal food includes bread, steamed buns, and dumplings.

[0032] In one embodiment of the present invention, the method comprises: mixing sour dough with appropriate amounts of water, flour, yeast and salt in a certain proportion, and stirring at low and high speeds until gluten is formed; then dividing and shaping the dough, and proofing it under suitable temperature and humidity conditions; and finally baking it in a baking device to obtain the fermented cereal food.

[0033] In one embodiment of the present invention, the sourdough, water, flour, yeast and salt are added in a ratio of: 180-230 g: 180-220 g: 380-420 g: 4-6 g: 4-6 g.

[0034] The present invention also provides a product for extending the shelf life of fermented cereal foods and / or improving the texture and flavor of fermented cereal foods, wherein the product contains the Propionibacterium freudenreichii or the microbial agent or Lactobacillus sanfrancisco.

[0035] In one embodiment of the present invention, the fermented cereal food includes bread, steamed buns, and dumplings.

[0036] Preferably, the amount of Propionibacterium freudenreichii added is at least: 1.0×10 6 CFU / g;

[0037] Preferably, the amount of Lactobacillus sanfrancisco added is at least: 1.0×10 6 CFU / g;

[0038] Preferably, the amount of the mixed microbial agent added is at least: 1.0×10 6 CFU / g.

[0039] In one embodiment of the present invention, the product is a microbial additive or fermentation aid, suitable for baking, fermentation or other food processing processes.

[0040] Specifically, this product can be added directly or indirectly to flour, dough or similar matrices. Through the synergistic effect of probiotic microorganisms such as propionic acid bacteria and lactic acid bacteria, it can improve fermentation efficiency, optimize flavor and significantly extend the shelf life of the product.

[0041] Beneficial Effects

[0042] The present invention achieves a balance between the antiseptic performance and quality improvement of cereal fermented foods, and promotes the widespread application and development of green antiseptic technology in the cereal fermented food industry.

[0043] ① Develop sourdough technology based on the combination of propionic acid bacteria and lactic acid bacteria, make full use of the metabolic advantages of the two strains, extend the shelf life of grain fermented foods, and improve the texture and flavor of grain fermented foods.

[0044] ②Systematically analyze the synergistic effect of propionic acid bacteria and lactic acid bacteria, explore the key metabolites produced by compound sourdough, and their antibacterial advantages and mechanisms.

[0045] This study developed a sourdough made from a combination of lactic acid bacteria and propionic acid bacteria, which provides a new perspective for the development of preservation technology. Taking into account both the fermentation quality and preservation requirements of cereal fermented foods, it provides a new idea and practical path for the promotion of green preservation technology in the cereal fermented food industry.

[0046] Biomaterial Deposit

[0047] A strain of Propionibacterium freudenreichii DX0705, taxonomically named Propionibacterium freudenreichii, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 22, 2025, with the deposit number CGMCC No.33491, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0049] Figure 1 This is the colony morphology of strain DX0705 on the culture medium.

[0050] Figure 2 This is the Gram staining microscopic examination of strain DX0705.

[0051] Figure 3 This is the phylogenetic tree of strain DX0705.

[0052] Figure 4 Texture and sensory results of sourdough fermented by strain DX0705.

[0053] Figure 5The specific volume, high-to-net ratio and baking loss results of sourdough fermented by strain DX0705.

[0054] Figure 6 These are the results of sourdough aging rate fermented by strain DX0705.

[0055] Figure 7 These are the sensory evaluation results of sourdough fermented by strain DX0705.

[0056] Figure 8 The chromatogram is for sourdough without adding DX0705.

[0057] Fig. 9 The chromatogram of sourdough with added DX0705.

[0058] Fig.10 This is a 3D absorption diagram of a mixed organic acid.

[0059] Fig.11 This is the 3D absorption diagram of organic acids in sourdough without adding DX0705.

[0060] Fig.12 3D absorption diagram of organic acids in sourdough with added DX0705. DETAILED DESCRIPTION

[0061] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0062] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0063] The lactic acid bacteria involved in the following embodiments are Lactobacillus sanfrancisco, and further, Lactobacillus sanfrancisco DSM20451T, which are purchased from DSMZ German Collection of Microorganisms.

[0064] The reagents and instrument information involved in the following examples are as follows:

[0065] Reagents: High-gluten wheat flour was purchased from Yihai Kerry Group Co., Ltd. (72% starch, 12.8% protein, 1.6% fat and 13% water); active dry yeast was a product of Angel Yeast Co., Ltd.; salt was purchased from a local supermarket; and the remaining reagents were purchased from Sinopharm Group.

[0066] Main instruments: dough mixer, proofer, oven, slicer, Wuxi Xinmai Machinery Co., Ltd.; large centrifuge, Thermo Fisher, USA; electronic analytical balance, AL204 type, Meltler Toledo, Switzerland; single-sided clean bench, Suzhou Antai Co., Ltd.; constant temperature incubator, Yuejin (Shanghai) Medical Equipment Factory; high pressure sterilizer, Shanghai Shen'an Medical Equipment Factory; pH ​​meter, Meltler Toledo 320pH meter, Switzerland; spectrophotometer, V-1800 type, Shanghai Meipuda Instrument Co., Ltd.; fermentation rheometer, F3 type, Chopin Technology, France; differential scanning calorimeter, X-DSC7000 type, SIINT, Japan; rheometer, AR1000 type, TA, USA; scanning electron microscope, Quanta-200 type, FEI, Netherlands; high performance liquid chromatograph, Agilent, USA; texture analyzer, TA.XTPlus type, SMS, UK.

[0067] The culture medium formula involved in the following examples is as follows:

[0068] Propionic acid bacteria culture medium: Add 5.0 g casein peptone, 5.0 g peptone, 10.0 g yeast extract, 5.0 g beef extract, 5.0 g glucose, and 1.0 mL Tween 80 to 1 L distilled water.

[0069] The solid culture medium for propionic acid bacteria is prepared by adding 1.5% agar powder to the liquid culture medium.

[0070] Lactic acid bacteria culture medium: add 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast extract, 10.0 g of malt extract, 5.0 g of glucose, and 10.0 g of maltose to 1 L of distilled water.

[0071] The solid culture medium for lactic acid bacteria is prepared by adding 1.5% agar powder to the liquid culture medium.

[0072] The Propionibacterium freudenreichii cells involved in the following examples were cultured as follows:

[0073] (1) First, take out the frozen Propionibacterium freudenreichii DX0705 glycerol tube and thaw it. Use an inoculation loop to take a sample from the thawed liquid culture medium of the aforementioned propionibacterium and streak it on a propionibacterium solid culture medium plate. Then, place the plate in an anaerobic sealed bag, add an indicator, seal it, and culture it in an incubator at 30°C for 48 hours.

[0074] (2) Pick a single colony from the surface of the solid culture medium obtained in step (1) and inoculate it into a 50 mL centrifuge tube containing 30 mL of propionic acid bacteria liquid culture medium, and continue to culture at 30° C. for 48 hours. After 48 hours, centrifuge at 5000 r / min for 5 minutes, discard the supernatant, and resuspend the bacteria in 30 mL of sterile water to prepare a propionic acid bacteria suspension.

[0075] The lactic acid bacteria (the lactic acid bacteria is Lactobacillus sanfrancisco DSM20451T) involved in the following embodiments are cultured as follows:

[0076] (1) First, the frozen lactic acid bacteria glycerol strain was taken out and thawed, and streaked on the aforementioned lactic acid bacteria culture plate. The plate was then placed in an anaerobic sealed bag, sealed after adding an anaerobic indicator, and placed in an incubator for anaerobically culturing at 37°C for 48 hours.

[0077] (2) Pick a single colony from the surface of the solid culture medium obtained in step (1), inoculate it into a 50 mL centrifuge tube containing 30 mL mMRS liquid culture medium, and culture it in an incubator at 37°C with anaerobism and shaking for 48 hours. After the culture is completed, centrifuge at 5000 r / min for 5 minutes, discard the supernatant, and resuspend the bacteria in 30 mL of sterile water to prepare a lactic acid bacteria suspension.

[0078] The detection methods involved in the following embodiments are as follows:

[0079] Bread texture determination

[0080] After the baked bread was placed at room temperature for 1 hour, it was cut into 1 cm thick slices using a slicer, and the two middle slices were taken for texture measurement. The texture measurement was carried out using a physical property analyzer according to the AACC 74-09 method. The measurement conditions and parameter settings of the physical property analyzer are as follows: the mode is TPA, using a P / 25 probe; the speed before the test is set to 3.00 mm / s, the test speed is 1.00 mm / s, and the speed after the test is 5.00 mm / s; the probe compression ratio is 40%, and each sample is compressed twice, with a compression interval of 10 seconds.

[0081] Bread specific volume determination

[0082] After the baked bread was placed at room temperature for 1 hour, the mass and volume were measured. The mass was directly measured using an electronic balance (accuracy 0.01g), and the volume was measured using the rapeseed volume displacement method, with specific reference to AACC Method 10-05.01. During the measurement, the bread was placed in a container of known volume, the remaining space was filled with rapeseed, and the displacement volume of the rapeseed was calculated, which was the volume of the bread. The specific volume of bread is expressed as the ratio of volume to mass (mL / g), which is used to evaluate the leavening of bread.

[0083] Determination of bread height-to-diameter ratio

[0084] The height-to-diameter ratio is determined by using a ruler to accurately measure the height and diameter of the bread. Place the baked bread on a horizontal surface and use a ruler to measure the height of the highest point and the diameter of the widest part of the bread (take the average of three times). The height-to-diameter ratio is calculated by the ratio of height to diameter and is used to evaluate the morphological characteristics of the bread.

[0085] Bread baking loss rate

[0086] The baking loss rate is calculated by the weight difference of the dough before and after baking. Before making, use an electronic balance to accurately weigh the dough and record the weight; after baking, cool the bread at room temperature for 2 hours and weigh it again. The baking loss rate is expressed as the percentage of the weight difference before and after making to the weight before making, and the formula is as follows: Baking loss rate (%) = [(raw material weight - weight after baking) / raw material weight] × 100%.

[0087] Determination of color of bread crust and bread crumb

[0088] The colorimeter was used to measure the chromaticity of the cooled bread, and the color difference ΔE was measured by measuring the three indicators of L*, a* and b* of the bread crust and core.

[0089] Bread hardness changes during storage, bread aging rate

[0090] The determination of bread hardness and staling rate was carried out by total texture analysis (TPA) method. First, the bread samples on day 0 and those stored at 4°C for 2, 4, 6, and 8 days were taken out, returned to room temperature, and sliced. Two slices of fresh bread core without obvious dehydration and hardness were selected for testing. The texture was determined by a physical property analyzer according to the AACC 74-09 method. The determination conditions and parameters of the physical property analyzer were set as follows: the mode was TPA, and a P / 25 probe was used; the speed was set to 3.00 mm / s before the test, 1.00 mm / s for the test, and 5.00 mm / s after the test; the probe compression ratio was 40%, and each sample was compressed twice, with a compression interval of 10 seconds. During the test, the hardness value (N) generated by each compression was recorded. Based on the measured hardness data, the aging rate at each storage time point was calculated.

[0091] The formula is: aging rate (N / day) = bread hardness increase (N) / storage time (days).

[0092] Bread sensory evaluation

[0093] The sensory evaluation method was based on the method of Xu Dan et al. and adjusted appropriately. The adjusted scoring criteria are shown in Table 1 below.

[0094] Twenty trained personnel (10 females, 10 males, aged 20 to 35 years) rated the appearance, texture, taste, flavor and overall quality of the bread samples using a nine-point preference method, with a minimum score of 1 (least favorite) and a maximum score of 9 (most favorite), representing the degree of acceptance of the product from low to high. Sensory analysis was performed in the sensory evaluation room of the School of Food Science and Engineering of Jiangnan University at room temperature and white light, and water was provided to clear the taste between samples. Bread samples were stored in glass containers marked with a 3-digit code and presented randomly, providing each panel member with a separate sample. In order to reduce the impact of aging on bread, samples were evaluated within 8 hours after preparation.

[0095] A smooth and symmetrical appearance is rated high, and a milky or creamy white interior is the best. The texture is based on uniform pores and good recovery after pressing. The taste is good when chewing is effortless and does not stick to the teeth. The flavor is required to have a distinct sour taste and no odor.

[0096] Table 1: Scoring criteria

[0097]

[0098] Bread shelf life determination

[0099] The shelf life of bread is determined by evaluating the quality changes and microbial activity of bread under different storage conditions. After the baked bread samples are placed at room temperature for 1 hour, samples are taken at specified time points (such as 1 day, 2 days, 5 days, 7 days, 10 days, etc.) to measure their pH value, texture changes and microbial spoilage. The specific steps include regularly measuring the pH value and texture parameters of the bread, and counting mold to assess the degree of spoilage. The data will be used to compare the differences in the effectiveness of propionic acid sourdough and ordinary sourdough in extending the shelf life of bread.

[0100] Detection and content of organic acids

[0101] 1. Preparation of standard products

[0102] Weigh 0.01g (accurate to 0.0002g) of acetic acid, lactic acid, succinic acid, and propionic acid standards in a 100mL volumetric flask, add water to make up to volume, and prepare a concentrated standard solution with a concentration of 100mg / L. The preparation of the standard series solution is shown in Table 2:

[0103] Table 2: Solutions

[0104] Volume of concentrated standard pipetted, mL 0 0.01 2 5 10 Preparation concentration, mg / L 0 0.1 20 50 100 Volume of volumetric flask, mL 0 10 10 10 10

[0105] Transfer the above volumes of concentrated standard samples respectively, dilute to the scale with water, shake well, and test on the instrument after the instrument is stable.

[0106] 2. Sample preparation

[0107] Weigh a certain mass of sample (0.1 g, accurate to 0.0002 g), put it in a 10 ml volumetric flask, dilute it to the mark with ultrapure water, extract the organic acid with ultrasonic vibration, shake it well, centrifuge it at 4500 r / min, filter it through a 0.45 um microporous filter membrane, and then inject it for analysis.

[0108] 3. Instrument operating conditions

[0109] Liquid chromatography instrument conditions: Chromatographic column: reverse C18 column: 250 mm*4.6 mm*5.0 um ODS; mobile phase: methanol+0.1% phosphoric acid water=5+95 (volume fraction); gradient: constant flow; flow rate: 0.8 ml / min; UV detection wavelength: 210 nm.

[0110] The method for determining the acid resistance of Propionibacterium freudenreichii strains is as follows:

[0111] The pH values ​​of the propionic acid bacteria broth medium were adjusted to 4.0 and 4.5 respectively with 1 mol / L HCl (according to the literature and industrial fermentation experience, most propionic acid bacteria began to be significantly inhibited near pH 4.5). The propionic acid bacteria were inoculated at a 3% (V / V) inoculation amount into the propionic acid bacteria culture medium with different pH values, cultured at 37°C for 4 h, and spread on the propionic acid bacteria agar culture medium after 10-fold gradient dilution. The viable bacteria were counted and the survival rate was calculated as follows:

[0112] Survival rate = (number of viable bacteria after culture / number of viable bacteria before inoculation) × 100%.

[0113] The method for determining the bile salt tolerance of Propionibacterium freudenreichii strains is as follows:

[0114] Prepare propionic acid bacteria culture medium containing 0.3%, 0.4%, and 0.5% (V / V) ox bile salt, inoculate propionic acid bacteria in the propionic acid bacteria culture medium containing different ox bile salts at a 3% (V / V) inoculation amount, culture at 37°C for 4 hours, spread on the propionic acid bacteria agar culture medium mentioned above after 10-fold gradient dilution, count, determine the number of live bacteria, and calculate the survival rate. The calculation method is:

[0115] Survival rate = (number of viable bacteria after culture / number of viable bacteria before inoculation) × 100%.

[0116] Example 1: Isolation, screening, species identification and genetic evolution relationship of propionic acid bacteria strains

[0117] 1. Separation and screening

[0118] Take 10g of sample from traditional handmade cheese in Wuxi, Jiangsu, mix in 90mL sterile NaCl solution (0.85%, W / V) and vortex. Use 10-fold gradient dilution (10^-1~10^-5), take 100μL from each of the 10^-3, 10^-4 and 10^-5 concentration gradient dilutions, evenly spread on modified YPD agar plates (such as the above medium formula), culture anaerobically at 30℃ for 48h, and place in anaerobic bags. After the culture is completed, pick the white colony and streak it for isolation and purification several times until a single colony is obtained, named DX0705.

[0119] 2. Morphological observation of strains

[0120] After the strain DX0705 was cultured at 30°C in an anaerobic environment for 48 hours, the colonies were morphologically observed to be white, moist, with regular, smooth and convex edges ( Figure 1 ). Gram staining and electron microscope observation showed that it was Gram-positive, short rod or rod-shaped bacteria, mainly arranged in single or small aggregates, and short chains ( Figure 2 ). In the liquid culture medium of propionic acid bacteria, the bacterial solution is usually uniformly turbid, and the formation of white precipitate can be observed after standing for a long time. Comprehensive experimental results show that its optimal growth temperature is 30℃, and it needs to grow under anaerobic conditions.

[0121] like Figure 1 , which is a schematic diagram of the colony morphology formed by the Propionibacterium freudenreichii of the present invention after streaking inoculation on a solid culture medium. Light yellow to milky white round or nearly round colonies can be seen on the surface of the culture medium, and the colony distribution changes from dense to sparse along the streaking area. Clearly separated single colonies can be seen in some locations, with a relatively smooth surface and neat edges.

[0122] like Figure 2 As shown, under a scanning electron microscope, Propionibacterium freudenreichii is generally in the shape of a thick, short or slightly curved rod with a blunt end and a smooth surface. It has no flagella and no motility. A few bacteria can be seen to be slightly branched or in a "rod-ball" transitional morphology. They are mainly distributed singly or in pairs, and at a certain magnification, slight aggregation or adhesion between bacteria caused by extracellular polymers can be observed.

[0123] 3. Strain nucleic acid identification

[0124] 16S rDNA gene sequence analysis: Pick a single colony in MRS liquid medium, culture it anaerobically at 30°C overnight, collect the bacteria by centrifugation at 8000 rpm for 1 min, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20 μL. The PCR amplification program was 95℃ pre-denaturation for 5min, 94℃ for 15s, 57℃ for 15s, 72℃ for 1min, 35 cycles; 72℃ extension for 10min; the amplified 16S rDNA consisted of 1510bp. Homology comparison analysis showed that the sequence similarity between DX0705 and Propionibacterium freudenreichii ALA was 99.54%, and the sequence similarity between DX0705 and Propionibacterium freudenreichii DSM_20271T(T) was 99.53%. Phylogenetic tree analysis showed that DX0705 and Propionibacterium freudenreichii were clustered in the same branch, see Figure 3 The sequence of 16S rDNA of Propionibacterium freudenreichii DX0705 is as follows.

[0125] The 16S rDNA sequence of Propionibacterium freudenreichii DX0705 (SEQ ID NO.1) is as follows:

[0126] AGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGGACGG 60

[0127] TAAGGCCCCTTTCGGGGGGTACACGAGTGGCGAACGGGTGAGTAACACGTGAGGAACGTG 120

[0128] CCCTTGACTTCGGTATAGCTCCAGGAAACTGGTGGTAATCCCGAATATGAGCCTGGCCTG 180

[0129] CATGGGTTGGGTTGGAAAGCTATATGCGGTCAGGGATCGTCTCGCGGCCTATCAGCTTGT 240

[0130] TGGTGGGGTAATGGCCTACCAAGGCAGCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCC 300

[0131] ACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCAC 360

[0132] AATGGGCGCAAGCCTGATGCAGCAACGCCGCGTGCGGGATGACGGCCTTCGGGTTGTAAA 420

[0133] CCGCTTTCATCCATGACGAAGCGCAAGTGACGGTAGTGGGAGAAGAAGCACCGGCTAACT 480

[0134] ACGTGCCAGCAGCCGCGGTGATACGTAGGGTGCGAGCGTTGTCCGGAATTATTGGGCGTA 540

[0135] AAGAGCTTGTAGGCGGTTGATCACGTCGGAAGTGAAATTCCAGGGCTTAACTCTGGGCTT 600

[0136] GCTTTCGATACGGGTTGACTTGAGGAAGGTAGGGGAGAATGGAACTCTCGGTGGAGCGGT 660

[0137] GGAATGCGCAGATATCGGGAAGAACACCAGTGGCGAAGGCGGTTCTCTGGACATTTCCTG 720

[0138] ACGCTGAGAAGCGAAAGCGTGGGGAGCAAACAGGCTTAGATACCCTGGTAGTCCACGCCG 780

[0139] TAAACGGTGGGTACTAGGTGTGGGTCCCTTCCACGGGGTCCGTGCCGTAGCTAACGCATT 840

[0140] AAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGCCC 900

[0141] CGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTT 960

[0142] TGACATGTACTGGAAGCGTTCAAAGATGGGCGTGCCTTTTTGGCTGGTACACAGGTGGTG 1020

[0143] CATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACC 1080

[0144] CTCGTCCAATGTTGCCAGCAGTTCGGCTGGGGACTCATTGGAGACCGCCGGGGCCAACTC 1140

[0145] GGAGGAAGGTGGGGATGAGGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACGCATGC 1200

[0146] TACAATGGCCGGTACAAAGAGTTGCGAGCCTGTGAGCGTGAGCGAATCTCAGAAAGCCGG 1260

[0147] TCTCAGTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCA 1320

[0148] GATCAGCAACGCTGCGGTGAATACGTTCCCGGGGCTTGTACACACCGCCCGTCAAGTCAT 1380

[0149] GAAAGTCGGTAACTCCCGAAGCCGGTGGCCCAACCTTTTGGGGGGGAGCCGTCGAAGGTGG 1440

[0150] GACTGGTGATTAGGACTAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCA 1500

[0151] CGCCTCCTTT 1510

[0152] The PCR product was sequenced and compared with the standard sequences published in the GenBank database for homology (BLASTN). The bacterium was identified as a propionibacterium and named Propionibacterium freudenreichii DX0705, which was deposited in the General Microbiology Center of China National Committee for the Collection of Microbiological Cultures.

[0153] 4. Phylogenetic tree analysis of strains

[0154] The sequences obtained by sequencing were compared with the bacterial database sequences in EzTaxon-e (http: / / www.ezbiocloud.net / identify) of the online software EzBioCloud's Identify Service module. Cluster analysis and phylogenetic tree construction were performed using MEGA6.5 software with the NJ method. The sampling was repeated 1000 times, and the stability of the calculated phylogenetic tree topology was tested using the bootstrap method. The developmental tree nodes only displayed values ​​with Bootstrap values ​​greater than 50%, and the superscript "T" indicates the model strain.

[0155] Example 2: Study on the performance of Propionibacterium freudenreichii DX0705

[0156] The acid and bile salt resistance properties of the 12 strains of Propionibacterium freudenreichii (Table 3) obtained by the initial screening using the method of Example 1 and the screened Propionibacterium freudenreichii DX0705 were measured. The results are shown in Tables 3-4.

[0157] Table 3: Survival rate of strains at different pH

[0158]

[0159] Table 4: Survival rate of strains at different bile salt concentrations

[0160]

[0161] From the results in Tables 3 and 4, it can be seen that as the pH value in the environment decreases (from pH = 4.5 to pH = 4.0), the acid resistance of each strain decreases to varying degrees; similarly, as the bile salt concentration in the culture medium gradually increases from 0.3% to 0.5%, the survival rate of each strain also decreases significantly. In terms of comprehensive acid resistance and bile salt resistance, DX0705 performs best: the survival rates at pH = 4.0 and pH = 4.5 can reach 86.10% and 90.70%, respectively; the survival rates at 0.3%, 0.4%, and 0.5% bile salt concentrations reach 45.4%, 31.5%, and 25.7%, respectively. This shows that DX0705 has good tolerance to acidic and high bile salt environments, and is superior to other strains screened from the same Swiss cheese.

[0162] Example 3: Preparation of sourdough

[0163] In this embodiment, the amount of bacteria added to the sourdough is 2%; the lactic acid bacteria involved is Lactobacillus sanfrancisco DSM20451T.

[0164] 1. Sourdough recipe

[0165] The specific steps are as follows:

[0166] Ordinary sourdough: Use about 90-110g sterile water and 90-110g flour.

[0167] Propionibacterium sourdough: 2 g of Propionibacterium freudenreichii suspension (the concentration of the suspension is 1.0 × 10 8 CFU / g), 97-99g sterile water, and 90-110g flour.

[0168] Lactic acid bacteria sourdough: 2g lactic acid bacteria suspension (the concentration of the suspension is 1.0×10 8 CFU / g), 97-99g sterile water, and 90-110g flour.

[0169] Propionic acid bacteria + lactic acid bacteria sourdough: 1g lactic acid bacteria suspension (the concentration of the bacterial suspension is 1.0×10 8 CFU / g) and 1g of Propionibacterium freudenreichii suspension (the bacterial concentration of the bacterial suspension was 1.0×10 8 CFU / g), 97-99g sterile water, and 90-110g flour.

[0170] According to the above formula, sterile water, flour and corresponding bacterial suspension (Propionibacterium freudenreichii, lactic acid bacteria or a combination of the two) are mixed evenly as required, maintained at a constant temperature culture condition of about 30° C.-36° C., and cultured for 18 to 30 hours to complete the fermentation process of the sourdough, thereby obtaining ordinary sourdough (SD), propionic acid bacteria sourdough (PAB group), lactic acid bacteria sourdough or propionic acid bacteria + lactic acid bacteria sourdough.

[0171] 2. Testing the performance of sourdough

[0172] (1) The results of organic acids are shown in Table 5.

[0173] Table 5: Organic acid results of sourdough with and without propionic acid bacteria

[0174]

[0175] The results showed that the addition of propionic acid bacteria significantly increased the contents of propionic acid, succinic acid and acetic acid in sourdough, and significantly enhanced the accumulation of lactic acid, fully demonstrating its important role in prolonging the antibacterial effect and optimizing organic acid metabolism.

[0176] (2) The addition of Propionibacterium freudenreichii significantly increased the organic acid content of fermented sourdough compared with ordinary sourdough without the addition of propionibacterium ( Figure 8 ), sourdough with propionic acid bacteria added ( Fig. 9) showed significant advantages in the production of lactic acid, propionic acid, succinic acid and acetic acid.

[0177] Specifically, the lactic acid content increased from 286.76 mg / kg to 4956.1 mg / kg, an increase of 1628.31%; the propionic acid content increased from 216.75 mg / kg to 1504.41 mg / kg, an increase of 594.08%; the succinic acid content increased from 124.89 mg / kg to 439.21 mg / kg, an increase of 251.68%; the acetic acid content increased from 5.87 mg / kg to 109.14 mg / kg, an increase of 1759.28%; the citric acid content increased from 17.25 mg / kg to 24.62 mg / kg, an increase of 42.72%.

[0178] These metabolites not only significantly enhanced the antibacterial properties of fermented sourdough and prolonged the antibacterial period, but also improved the flavor and sensory quality of sourdough. This shows that Propionibacterium freudenreichii plays an important role in extending the shelf life of dough, improving its antiseptic properties and texture stability by metabolizing lactic acid to produce less volatile propionic acid, as well as synthesizing metabolites such as succinic acid and acetic acid, providing important support for the application of green antiseptic technology in the food industry.

[0179] (3) Fig.10 As shown in the absorption diagram, each organic acid has a corresponding absorption peak within a specific time range, and the absorption intensity distribution is uniform and stable. Fig.10 and Fig.11 It provides a reference for the qualitative and quantitative analysis of organic acids in samples.

[0180] Fig.11 The 3D absorption graph of ordinary sourdough is shown, in which the absorption intensity of sourdough without propionic acid bacteria in the UV-visible range is generally low. The main absorption peaks are concentrated in the generation time range of lactic acid and citric acid, and the maximum absorption intensity is about 121mAU, indicating that the types and contents of organic acids in ordinary sourdough are relatively limited. In particular, in the propionic acid and succinic acid metabolite region corresponding to 7.5-10 minutes, the absorption signal is significantly weaker, indicating that ordinary sourdough has obvious deficiencies in the metabolism of antibacterial related organic acids.

[0181] (4) Fig.12 Shown is a 3D absorption graph of sourdough supplemented with Propionibacterium freudenreichii. Fig.11 , Fig.12The overall absorption intensity was significantly enhanced, especially in the time range corresponding to lactic acid, propionic acid and succinic acid, the absorption peak was significantly higher than that of ordinary sourdough. The maximum absorption intensity reached 37mAU, and the production of propionic acid and succinic acid increased significantly in the time range of 7.5-10 minutes. In addition, the absorption peak of lactic acid was also quite significant, indicating that propionic acid bacteria significantly improved the production efficiency of various organic acids in sourdough through synergistic metabolism.

[0182] The above figure uses 3D absorption diagrams to more clearly show the 3D absorption spectrum effects of ordinary sourdough without adding propionibacterium and sourdough with added Propionibacterium freudenreichii, revealing the metabolic advantages and application potential of propionibacterium in fermenting sourdough.

[0183] In summary, the application of Propionibacterium freudenreichii in fermented sourdough showed significant metabolic advantages. By enhancing the production of propionic acid and other antibacterial metabolites, the strain significantly improved the antibacterial properties and shelf life extension of sourdough, providing a reliable solution and practical basis for green preservation technology in the food industry.

[0184] Example 4: Production of regular bread and sourdough bread

[0185] In this embodiment, the addition amount of all bread recipes is the same, and the specific steps are as follows (the lactic acid bacteria involved are Lactobacillus sanfrancisco DSM20451T):

[0186] 1. Bread recipe (Table 6)

[0187] Ordinary bread: add 280-320g water, 480-520g flour, 4-6g yeast and 4-6g salt.

[0188] Regular sourdough bread: Add 180-230g regular sourdough, 180-220g water, 380-420g flour, 4-6g yeast and 4-6g salt.

[0189] Propionic acid bacteria sourdough bread: 180-230g of propionic acid bacteria sourdough, 180-220g of water, 380-420g of flour, 4-6g of yeast and 4-6g of salt are added and mixed.

[0190] Lactic acid bacteria sour dough bread: 180-230 g of lactic acid bacteria sour dough, 180-220 g of water, 380-420 g of flour, 4-6 g of yeast and 4-6 g of salt are added and mixed.

[0191] Propionic acid bacteria + lactic acid bacteria sour dough bread: 180-230g of propionic acid bacteria + lactic acid bacteria sour dough, 180-220g of water, 380-420g of flour, 4-6g of yeast and 4-6g of salt are added and mixed.

[0192] Table 6: Bread ingredients

[0193]

[0194]

[0195] 2. The process of making bread is as follows:

[0196] The bread making method can be prepared according to a conventional method, and all parameters can refer to the bread preparation parameters in "Study on Quality Improvement of Red Bean Paste Bread" by Wang Lulu et al. The method comprises mixing sour dough with an appropriate amount of water, flour, yeast, and salt in a certain proportion (Table 6), and stirring at a low speed and a high speed until gluten is formed; then dividing and shaping the dough, and proofing it under suitable temperature and humidity conditions; and finally baking it in a baking device to obtain the bread.

[0197] 3. The results of the test in this embodiment:

[0198] (1) Test the texture of each bread separately ( Figure 4 )

[0199] Table 7: Texture results

[0200]

[0201] The sourdough with Propionibacterium freudenreichii and lactic acid bacteria significantly improved the texture and sensory quality of bread compared with lactic acid bacteria sourdough (PAB+LAB). In contrast, the use of Propionibacterium freudenreichii alone inhibited yeast fermentation due to acidity and destroyed the gluten network, resulting in increased bread hardness and reduced chewiness. However, its elasticity and adhesion were not significantly different from those of ordinary sourdough, which may be due to the fact that the extracellular polysaccharides metabolized by propionibacteria partially alleviated the negative effects of the acidic environment.

[0202] Overall, compound sourdough has obvious advantages in improving bread quality and extending shelf life, while also emphasizing the necessity of acid-resistant yeast screening and fermentation process optimization.

[0203] (2) Test the specific volume, height-to-diameter ratio, and baking loss rate of each bread separately ( Figure 5 )

[0204] Specific volume = volume of bread / mass of bread, height-to-diameter ratio (H / D Ratio) = volume of bread / mass of bread

[0205] Table 8: Specific volume, height-to-diameter ratio, and baking loss rate results

[0206] PAB BR SD LAB PAB+LAB Specific volume (mL / g) 3.402±0.114 3.652±0.049 3.725±0.035 3.930±0.031 3.988±0.038 Aspect Ratio 1.020±0.079 1.177±0.057 1.268±0.054 1.365±0.065 1.436±0.038 Roasting loss rate 10.352±0.263 12.017±1.029 9.822±0.171 8.777±0.259 8.485±0.375

[0207] The results show:

[0208] 1) The sourdough mixed with lactic acid bacteria and Propionibacterium freudenreichii showed the highest specific volume and height-to-diameter ratio.

[0209] Lactic acid bacteria sourdough showed similar quality due to its better water holding capacity and fermentation ability. In contrast, the quality of ordinary sourdough was slightly inferior; ordinary bread had a further reduction in specific volume and height-to-diameter ratio due to the lack of sourdough's improvement in texture.

[0210] The excessive drop in pH in the sourdough made from Propionibacterium freudenreichii significantly inhibited yeast activity, resulting in weak fermentation ability, deteriorated pore structure, and the lowest specific volume and height-to-diameter ratio.

[0211] It can be seen that strain synergy and a suitable acidic environment are the key to improving bread quality, while excessive acidity will inhibit yeast activity and deteriorate bread structure. Future research should focus on screening acid-resistant yeast strains to improve fermentation performance and optimize bread quality.

[0212] 2) Baking Loss Rate (%) = [(raw material weight - weight after baking) / raw material weight] × 100%,

[0213] The baking loss rate of bread reflects the water loss during the baking process, and the reasonable range is usually 8% to 15%. Compound sourdough and lactic acid bacteria sourdough performed well in controlling the baking loss rate, and their water holding capacity was significantly enhanced, which may be attributed to the hydrophilic substances such as extracellular polysaccharides produced by lactic acid bacteria metabolism, which effectively reduced water volatilization.

[0214] In contrast, the organic acids produced by the metabolism of Propionibacterium freudenreichii sourdough are highly volatile, resulting in a higher baking loss rate, while ordinary bread has the weakest water-holding capacity and the highest baking loss rate due to the lack of hydrophilic substances produced by microorganisms in the sourdough.

[0215] (3) Detect the color of each bread separately. The brightness (L*) reflects the lightness or darkness of the color. The higher the value, the closer the color is to white, and the lower the value, the darker the color. The red-greenness (a*) indicates whether the color is reddish or greenish, with a positive value indicating reddishness. The yellow-blueness (b*) indicates whether the color is yellowish or blueish, with a positive value indicating yellowishness.

[0216] Table 9: Colors

[0217]

[0218]

[0219] From the data, it can be seen that both the crust and the crumb showed significant color changes at 0 and 8 days of storage.

[0220] After 8 days of storage, the brightness (L*) of each group decreased, while the red-green color (a*) and yellow-blue color (b*) increased significantly, indicating that the browning degree of bread deepened with the extension of storage time.

[0221] The crust of the compound sourdough group (PAB+LAB) had the lowest L value and the highest a and b values ​​on Day 0, indicating that it had a higher degree of Maillard reaction during baking, while the regular bread group (BR) showed the highest L value, the lowest a and b values, and the weakest degree of baking color. After 8 days of storage, the L value of the crust of each group further decreased, and the a and b values ​​increased significantly. The chromaticity analysis of the bread core showed that the compound sourdough group had the highest L value and the lowest a and b values, while the regular bread group had the lowest L value and the highest a and b values. After 8 days of storage, the L value of the bread core also decreased significantly, and the a and b values ​​increased significantly. The Maillard reaction is the main coloring mechanism in the bread baking process, which depends on the carbonyl-amine condensation reaction involving free amino acids and reducing sugars. Compound sourdough can release more free amino acids by hydrolyzing peptide chains, providing sufficient substrate for the Maillard reaction, thereby significantly deepening the color change of the crust and bread core.

[0222] (4) Detect the hardness change of each bread during storage and the staling rate of bread

[0223] During the low-temperature storage of bread, amylopectin is prone to recrystallization, a process called bread aging.

[0224] Bread staling is mainly attributed to the recrystallization of starch and the migration of water, which is often manifested as the bread texture becoming tighter and the inner core becoming harder. The full texture test evaluates the texture of food from an objective perspective by simulating two chewings of food in the mouth. Among them, fresh, palatable, and soft bread should have a lower hardness, which is also the main goal of maintaining quality and delaying staling.

[0225] Figure 6 The hardness of the bread core changes during the 8-day storage period, and the aging rate is from high to low: ordinary bread > bread made from propionic acid bacteria sour dough > bread made from ordinary sour dough > bread made from lactic acid bacteria sour dough > bread made from propionic acid bacteria + bread made from lactic acid bacteria sour dough. The composite sour dough bread has the lowest initial hardness and shows the lowest hardness increase during the entire storage period. Its aging rate is significantly lower than that of other groups, showing a more active role in delaying bread aging than other groups.

[0226] In summary, the sourdough technology of propionic acid bacteria and lactic acid bacteria significantly improved the texture stability of bread during storage and slowed down the aging rate of bread. This may be due to the synergistic effect of propionic acid bacteria and lactic acid bacteria, which improved the moisture retention capacity of bread through substances such as extracellular polysaccharides, reduced water loss and starch recrystallization, and maintained the softness and taste of bread. Therefore, the PAB+LAB group of bread can effectively maintain a low hardness increase during storage, significantly delaying the aging process.

[0227] (5) Sensory evaluation of each bread Figure 7 )

[0228] The results of the sensory evaluation showed that the overall acceptance of the BR group was low, with a total score of 3.5 points, ranking last, indicating that its taste and flavor did not reach the ideal level, especially the lack of obvious sourness in flavor. The PAB and SD groups both scored 5.5 points, among which PAB performed better in acidity, but had poor texture and taste; while the SD group's performance was more balanced and moderate overall. The performance of the LAB group was relatively outstanding, especially in appearance and texture, showing a higher quality standard. The PAB+LAB group performed well in all evaluation indicators, with a total score of 8.25 points, ranking first, showing that it has significant advantages in flavor, taste and texture.

[0229] In summary, although the acidity of the PAB group was higher, its appearance, texture and taste were poor, indicating that although propionic acid bacteria can improve acidity, they have a poor effect on the taste quality of bread. The composite strain (PAB+LAB) can effectively improve these defects and improve the overall quality of bread.

[0230] (6) Test the shelf life of each bread

[0231] Table 10: Statistics of bread mold

[0232]

[0233] - indicates no mold, * indicates mold

[0234] The PAB+LAB group showed no mold within 7 days, and the shelf life of the ordinary dough, ordinary sourdough, and LAB group was extended by 4 days, 2 days, and 1 day, respectively. The synergistic effect of Propionibacterium freudenreichii and lactic acid bacteria significantly improved the antiseptic properties of bread through the metabolism of organic acids and antibacterial substances. Propionibacterium sourdough and compound sourdough effectively extended the shelf life of bread by 48 hours and 4 days, respectively.

[0235] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Propionibacterium freudenreichii, characterized in that It has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.33491 and the deposit date being January 22, 2025.

2. A microbial agent, characterized in that: Contains the Propionibacterium freudenreichii according to claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial inoculum contains living cells of Propionibacterium freudenreichii, dry cells of Propionibacterium freudenreichii obtained by freeze-drying, immobilized cells of Propionibacterium freudenreichii, liquid inoculum of Propionibacterium freudenreichii, solid inoculum of Propionibacterium freudenreichii strains, or Propionibacterium freudenreichii strains in any other form.

4. A microbial agent, characterized in that: The microbial agent contains the Propionibacterium freudenreichii and Lactobacillus sanfanciscensis as claimed in claim 1; Preferably, the amount of Propionibacterium freudenreichii added in the microbial agent is at least: 1.0×10 6 CFU / g, the amount of Lactobacillus sanfrancisco added is at least: 1.0×10 6 CFU / g; Preferably, the microbial agent contains living cells of Propionibacterium freudenreichii or Lactobacillus sanfrancisco, dry cells of Propionibacterium freudenreichii or Lactobacillus sanfrancisco obtained by freeze-drying, immobilized cells of Propionibacterium freudenreichii or Lactobacillus sanfrancisco, liquid agent of Propionibacterium freudenreichii or Lactobacillus sanfrancisco, solid agent of Propionibacterium freudenreichii or Lactobacillus sanfrancisco, or Propionibacterium freudenreichii strains or Lactobacillus sanfrancisco strains in any other form.

5. Use of the Propionibacterium freudenreichii according to claim 1 or the microbial agent according to any one of claims 2 to 4 in extending the shelf life of fermented cereal foods and / or improving the texture and flavor of fermented cereal foods.

6. A method for making sourdough, characterized in that: The method comprises mixing the Propionibacterium freudenreichii described in claim 1 or the microbial agent described in any one of claims 2 to 4 with water and flour and then fermenting the mixture; Preferably, the amount of Propionibacterium freudenreichii added is at least: 1.0×10 6 CFU / g; Preferably, the amount of Lactobacillus sanfrancisco added is at least: 1.0×10 6 CFU / g; Preferably, the amount of the mixed microbial agent added is at least: 1.0×10 6 CFU / g.

7. Sourdough obtained by the preparation method according to claim 6.

8. A method for preparing a fermented cereal food, characterized in that: The method comprises mixing the sour dough of claim 7 with water, flour, sugar, yeast and salt, and stirring until gluten is formed; and then dividing, shaping, proofing and baking the stirred dough to obtain the fermented cereal food.

9. The method according to claim 8, characterized in that The mass ratio of the sour dough, water, flour, sugar, yeast and salt is: (190-210): (190-210): (380-420): (30-40): (4-6): (4-6).

10. A product for extending the shelf life of a fermented cereal food and / or improving the texture and flavor of a fermented cereal food, characterized in that: The product contains the Propionibacterium freudenreichii described in claim 1 or the microbial agent described in any one of claims 2 to 4.

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