Use of weissella cibaria strain zuj.1 in the preparation of frozen dough

Abstract

This invention provides the application of *Westernella taurida* strain ZJU.1 in the preparation of frozen dough, belonging to the field of novel applications of functional microorganisms. The *Westernella taurida* strain involved in this invention (… Weissella cibaria The strain ZJU.1 possesses the characteristic of secreting extracellular polysaccharides (EPS). Using this *Westernella esculenta* strain ZJU.1 to prepare frozen dough can alleviate dough texture degradation caused by freeze-thaw cycles, improve dough rheological properties, mitigate starch retrogradation and structural degradation induced by freeze-thaw cycles, reduce mechanical damage to the protein network caused by ice crystals, and decrease water loss during freeze-thaw processes, limiting water migration and preventing the conversion of bound water to free water, thereby improving the stability of the frozen dough. Therefore, this invention not only reduces the structural degradation of frozen dough caused by repeated freeze-thaw cycles but also improves the quality of frozen dough by improving the structure and content of starch, gluten, and water.

Description

Technical Field

[0001] This application belongs to the field of new applications of functional microorganisms, specifically relating to *Westernella esculenta* (…). Weissella cibaria Application of strain ZJU.1 in the preparation of frozen dough. Background Technology

[0002] Frozen dough is a semi-finished product processing method. It is frozen during the production process and then stored for later transportation and use. When needed, it is thawed and further processed. This technology promotes the standardization and large-scale production of dough products through centralized production and expanded distribution. However, due to the immaturity of the cold chain transportation system, frozen dough is prone to temperature fluctuations during storage, leading to freeze-thaw cycles (FTC). Studies have shown that freeze-thaw cycles adversely affect the quality of dough products, an effect attributed to changes in the properties and structure of various components within the dough. Starch, gluten protein, and moisture are widely recognized as major factors influencing the quality of frozen dough in numerous studies.

[0003] Numerous studies have shown that the addition of polysaccharides can improve the water retention of frozen dough, strengthen gluten protein structure, and enhance the stability of dough during frozen storage. For example, guar gum, xanthan gum, κ-carrageenan, and sodium alginate can enhance the stability and rheological properties of dough during fermentation and improve bread characteristics. However, the addition of these polysaccharides to food processing often causes significant side effects in humans, such as bloating, gas production, intestinal irritation, impaired mineral absorption, and disturbance of gut microbiota. Therefore, there is an urgent need for a method that can resist dough structural deformation and deterioration during freeze-thaw cycles and improve the stability of frozen dough. Summary of the Invention

[0004] The purpose of this invention is to provide an application of *Westernella taurida* strain ZJU.1 in the preparation of frozen dough. The extracellular polysaccharides produced by *Westernella taurida* strain ZJU.1 can promote the formation and stability of the gluten network in frozen dough, reduce the contact between starch and water, and convert free water in the dough into bound water that is not frozen, thereby reducing the damage to the quality of frozen dough caused by freezing and thawing.

[0005] This invention provides the application of *Westernella tamariscina* strain ZJU.1 and / or the extracellular polysaccharide produced by said *Westernella tamariscina* strain ZJU.1 in the preparation of frozen dough. The *Westernella tamariscina* strain ZJU.1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 38030.

[0006] Preferably, the *Westernella esculenta* strain ZJU.1 exists in the form of cells or spores.

[0007] Preferably, the *Westernella esculenta* strain ZJU.1 exists in the form of a bacterial suspension; The extracellular polysaccharide produced by the *Westernella esculenta* strain ZJU.1 exists in the form of fermentation supernatant. The method for preparing the fermentation supernatant includes inoculating an activated Weissella esculenta strain ZJU.1 into MRS medium and culturing it. The resulting fermentation broth is then subjected to solid-liquid separation, and the liquid phase is collected to obtain the fermentation supernatant.

[0008] Preferably, the preparation of frozen dough includes resisting structural degradation and / or component loss of the frozen dough caused by freeze-thaw cycles.

[0009] Preferably, the structure of the frozen dough includes textural properties, rheological properties, and micronetwork structure. The frozen dough consists of frozen dough starch, gluten protein, and water.

[0010] This invention provides a method for preparing freeze-thaw resistant frozen dough, comprising the following steps: The *Westernella esculenta* strain ZJU.1 used in this application was mixed with flour, sugar, and water to form a dough, which was then fermented, followed by proofing and freezing.

[0011] Preferably, the amount of *Westernella esculenta* strain ZJU.1 added is 10. 5 ~10 9 CFU / g flour; The amount of sugar added is 0.9% to 8%; The amount of drinking water added is 40% to 70%.

[0012] Preferably, the fermentation temperature is 22~27℃; the fermentation time is 10~14h; and the ambient humidity during fermentation is 80%~90%. The humidity during the proofing process is 70%~90%; the temperature during the proofing process is 25~40℃. The warming time is 20-60 minutes; The freezing temperature is -30℃ to 0℃.

[0013] This invention provides frozen dough prepared by the aforementioned method.

[0014] A preferred food product prepared from the frozen dough.

[0015] This invention provides the application of *Westernella esculenta* strain ZJU.1 and / or the extracellular polysaccharide produced by *Westernella esculenta* strain ZJU.1 in the preparation of frozen dough. *Westernella esculenta* strain ZJU.1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38030. The *Westernella esculenta* strain ZJU.1 isolated by this invention has the characteristic of secreting an extracellular polysaccharide (EPS). Using *Westernella esculenta* strain ZJU.1 to prepare frozen dough can alleviate the textural degradation of dough caused by freeze-thaw cycles and improve the rheological properties of the dough. Simultaneously, the application can reduce the number of holes and cracks in dough, inhibit the exposure of starch granules, and improve the integrity and continuity of the gluten network structure during freeze-thaw cycles; it can alleviate starch aging and structural deterioration caused by freeze-thaw cycles, reduce starch swelling force and gelatinization temperature during freeze-thaw cycles, and inhibit the formation of double helix structures; it can alleviate the mechanical damage of ice crystals to protein networks, protect the covalent bonds (SS) and non-covalent interactions (ionic bonds, hydrogen bonds, hydrophobic interactions) of proteins during freeze-thaw cycles, and inhibit the depolymerization of GMP during freeze-thaw cycles; it can also reduce the water loss rate during freeze-thaw cycles, limit water migration, prevent the conversion of bound water to free water, and thus improve the stability of frozen dough. Therefore, this invention, by incorporating the *Westernella esculenta* strain ZJU.1, which secretes specific EPS, into the preparation of frozen dough, can not only reduce the structural deterioration of frozen dough caused by repeated freeze-thaw cycles, but also improve the quality of frozen dough by improving the structure and content of starch, gluten proteins, and water. Furthermore, the EPS synthesized in situ by *Westernella esculenta* during sourdough fermentation does not need to be listed in the food ingredient list, achieving the goal of clean labeling. Attached Figure Description

[0016] Figure 1 A represents the rheological properties of frozen dough; B represents the changes in the storage modulus of frozen sourdough; C represents the changes in the loss modulus of frozen sourdough; D represents the changes in the loss factor of frozen sourdough. Figure 2 Electron micrograph of the microstructure of frozen dough; Figure 3 Results for the starch and protein network in frozen dough; Figure 4 The results show the determination of the secondary structure of gluten protein; A is the detection diagram of the secondary structure of gluten in frozen dough after different freeze-thaw cycles (FTC), and B is the detection results of four conformations in frozen dough after different freeze-thaw cycles (FTC). Figure 5 The results are for non-covalent bonds in gluten protein; A is 0.1M NaCl, B is 0.6M NaCl, C is 0.6M NaCl + 1.5M urea, and D is 0.6M NaCl + 8M urea. Figure 6The results show the determination of free sulfhydryl groups and disulfide bonds in gluten protein; A is a statistical chart of free sulfhydryl group content, and B is a statistical chart of disulfide bond content. Figure 7 The results show the molecular weight distribution of gluten protein; A is the molecular weight distribution of gluten protein in the experimental group frozen dough as the number of FTC cycles increased, and B is the molecular weight distribution of gluten protein in the control group frozen dough as the number of FTC cycles increased. Figure 8 The results show the effect of freeze-thaw cycles on the moisture content of different frozen sourdoughs. Figure 9 The results show the effect of freeze-thaw cycles on the freezeable water content of different frozen sourdoughs. Figure 10 The results of the T2 distribution curve during the freeze-thaw cycle of frozen dough; Figure 11 Cross-sectional views of steamed buns sliced ​​under different freeze-thaw cycles.

[0017] Information on the preservation of biological materials The Weissella strain that produces extracellular polysaccharides of this invention ( Weissella cibaria Strain ZJU.1 was deposited on April 3, 2026 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 38030. Detailed Implementation

[0018] This invention provides the application of *Westernella tamariscina* strain ZJU.1 and / or the extracellular polysaccharide produced by said *Westernella tamariscina* strain ZJU.1 in the preparation of frozen dough. The *Westernella tamariscina* strain ZJU.1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 38030.

[0019] In this invention, the *Westernella esculenta* strain ZJU.1 was isolated from rice wine yeast and identified as *Westernella esculenta* after whole-genome sequencing. Weissella cibaria The *Westernella stolonifer* strain ZJU.1 preferably exists in the form of cells or spores. The cells are the vegetative form of *Westernella stolonifer* strain ZJU.1. The spores are the dormant form of *Westernella stolonifer* strain ZJU.1. This invention does not have any particular limitation on the source of the cells or spores; methods for preparing cells or spores of *Westernella stolonifer* well-known in the art can be used.

[0020] The *Westernella esculenta* strain ZJU.1 is preferably present in the form of a bacterial suspension. The preparation method of the bacterial suspension involves inoculating activated *Westernella esculenta* strain ZJU.1 into MRS medium and culturing it. The resulting culture is then subjected to solid-liquid separation, the solid phase is collected, and the solid phase is washed to obtain the bacterial cells. The extracellular polysaccharides produced by the *Westernella esculenta* strain ZJU.1 are preferably present in the form of a fermentation supernatant. The preparation method of the fermentation supernatant preferably includes inoculating activated *Westernella esculenta* strain ZJU.1 into MRS medium and culturing it. The resulting fermentation broth is then subjected to solid-liquid separation, the liquid phase is collected, and the fermentation supernatant is obtained.

[0021] In this invention, the *Westernella esculenta* strain ZJU.1 possesses the characteristic of producing EPS, and the EPS can alleviate the textural degradation of dough caused by freeze-thaw cycles and improve the rheological properties of the dough. This is mainly attributed to the hydrophilicity of the EPS, which can bind with water to form hydrophilic colloids, thereby improving the viscoelasticity of the dough. Furthermore, the hydrophilic groups in the EPS may bind with nonpolar amino acids in gluten proteins to form hydrogen bonds, strengthening the gluten network and resisting structural deformation caused by ice recrystallization during freeze-thaw cycles. The *Westernella esculenta* strain ZJU.1 participates in the fermentation of frozen dough, which can reduce the number of holes and cracks in the dough, inhibit the exposure of starch granules, and improve the integrity and continuity of the gluten network structure during freeze-thaw cycles. This may be attributed to the hydrogen bonding ability of the EPS, which interacts with water molecules, inhibiting ice recrystallization and thus maintaining a more continuous and complete network structure. In addition, the highly hydrophilic EPS, when combined with starch, can protect starch granules from damage during freezing, forming a dense wheat starch-EPS complex. The *Westernella esculenta* strain ZJU.1 can alleviate starch retrogradation and structural deterioration caused by freeze-thaw cycles, reduce starch swelling force and gelatinization temperature during freeze-thaw, and inhibit the formation of double helix structures. This may be due to the hydrophilic groups of the EPS, which can competitively bind water, thereby inhibiting starch gelatinization and swelling. Simultaneously, EPS can coat the starch surface, buffering the direct damage of ice crystals to starch granules. The *Westernella esculenta* strain ZJU.1 can also alleviate the mechanical damage of ice crystals to the protein network in frozen dough, protecting the covalent bonds (SS) and non-covalent interactions (ionic bonds, hydrogen bonds, hydrophobic interactions) of proteins during freeze-thaw, and inhibiting the depolymerization of GMPs during freeze-thaw. First, EPS inhibits the mechanical damage of ice crystals to the protein network through steric hindrance. Second, its hydrophilic groups can form hydrogen-bonded complexes with protein molecules, enhancing the stability of non-covalent cross-linking. More importantly, EPS effectively reduces the damage of ice crystals to the protein network by regulating the phase and distribution of water during freeze-thaw, thereby inhibiting the depolymerization of large protein molecules. The method for preparing frozen dough in this invention can also reduce the water loss rate during freeze-thaw processes, limit water migration, and prevent bound water from converting into free water, thereby improving the stability of frozen dough. This may be because EPS can effectively bind and fix water through hydrogen bonds and capillary action, converting more free water into bound water that is less prone to freezing.

[0022] In this invention, the preparation of frozen dough preferably includes resisting structural degradation and / or component loss of the frozen dough caused by freeze-thaw cycles. The structure of the frozen dough preferably includes textural properties, rheological properties, and micronetwork structure. The components of the frozen dough preferably include frozen dough starch, gluten protein, and water.

[0023] This invention provides a method for preparing freeze-thaw resistant frozen dough, comprising the following steps: The *Westernella esculenta* strain ZJU.1 used in this application was mixed with flour, sugar, and water to form a dough, which was then fermented, followed by proofing and freezing.

[0024] In this invention, the flour preferably includes at least one of the following: wheat flour, rye flour, buckwheat flour, or corn flour, etc. The amount of *Wesella esculenta* strain ZJU.1 added is preferably 10... 5 ~10 9 CFU / g flour, can be 10 6 ~10 8 CFU / g flour, or 10 7 CFU / g flour. The amount of added sugar is preferably 0.9%~8%, but can be 1%~5%, 1.5%~4%, or 2%~3%. The sugar preferably includes at least one of the following: sucrose, glucose, and fructose, and can be sucrose. The sugar can serve as a carbon source for *Westernella tamariscina* strain ZJU.1. The amount of added drinking water is preferably 40%~70%, but can be 50%~60%. The drinking water preferably includes distilled water, mineral water, boiled water, etc. This invention does not impose any special limitations on the method of mixing into dough; any mixing method well known in the art can be used, such as hand kneading or machine kneading.

[0025] In this invention, the fermentation temperature is preferably 22-27℃, but can be 23-26℃ or 25℃. The fermentation time is preferably 10-14 hours, but can also be 11-13 hours or 12 hours. The ambient humidity during fermentation is preferably 80%-90%, but can be 82%-88% or 85%. This fermentation promotes the rapid proliferation of *Westernella esculenta* strain ZJU.1 and the rapid secretion of secondary metabolites (EPS) into the dough, thereby improving the dough's cohesiveness.

[0026] In this invention, the humidity during proofing is preferably 70%~90%, but can be 75%~85%, or 80%~85%. The proofing temperature is preferably 25~40℃, but can also be 28~37℃, or 32~35℃. The proofing time is preferably 20~60 minutes, but can be 30~50 minutes, or 40 minutes. Proofing involves kneading the dough to release air, shaping it, and then letting it rest to allow the gluten to relax and regain its expansion capacity.

[0027] In this invention, the freezing temperature is preferably -30℃ to 0℃, more preferably -20℃. The freezing time can be 20 h to 60 days, or 22 h. The purpose of the freezing treatment is to inhibit the growth and metabolism of microorganisms in the proofed dough at low temperatures.

[0028] This invention provides a frozen dough prepared by the aforementioned method. The frozen dough, after undergoing freeze-thaw treatment, helps maintain its original textural and rheological properties, reduces component loss, and prevents ice crystal damage, thus maintaining good dough quality.

[0029] The present invention provides a food product prepared from the frozen dough.

[0030] This invention does not impose any special restrictions on the types of food mentioned, and any types of food well known in the art can be used, such as noodles, steamed buns, dumplings, shumai, dumplings, potstickers, and boxes.

[0031] In this invention, the method for preparing the food preferably includes thawing, fermenting, processing, and cooking the frozen dough.

[0032] In this invention, the thawing temperature can be 4℃-37℃, 10-30℃, or 25℃. The thawing time is preferably 0.5 h-6 h, but can also be 1-5 h, 2-4 h, or even 3 h. The thawing is preferably carried out in a constant temperature and humidity chamber. The fermentation temperature is preferably 25℃-40℃, 30-35℃, or even 37℃. The fermentation time is preferably 30 min-120 min, but can be 40-100 min, 50-80 min, or even 60 min.

[0033] The cooking method preferably includes steaming, boiling, or frying. The steaming temperature is preferably 80℃~120℃, but can be 90~110℃ or 100℃. The steaming time is preferably 20 min~60 min, but can be 30 min~50 min or 35 min.

[0034] The following examples illustrate the application of the *Westernella esculenta* strain ZJU.1 provided by this invention in the preparation of frozen dough, but these examples should not be construed as limiting the scope of protection of this invention.

[0035] Example 1 Methods for the isolation and identification of Weissella strain ZJU.1, which produces extracellular polysaccharides In 2025, the applicant isolated a high-EPS-producing microbial strain ZJU.1 from rice wine starter obtained from Hubei Province, China, using selective isolation (MRS medium containing vancomycin and natamycin). A whole-genome shotgun (WGS) sequencing strategy was employed to construct libraries with different insert fragments. Next-generation sequencing (NGS) and third-generation single-molecule sequencing technologies were used to sequence the libraries. By comparing the genome sequence with a nucleotide sequence database (NT), the species information of the genome was obtained, and the identified species was *Westernella esculenta* (…). Weissella cibaria The library construction and sequencing were specifically outsourced to Shanghai Sangon Biotech Co., Ltd. The assembled genome was compared with NCBI data, and the results were consistent with *Westernella esculenta* (…). Weissella cibaria (Consistent)

[0036] Ingestion of Weissella stomatitis ( Weissella cibaria Strain ZJU.1 was deposited at the China General Microbiological Culture Collection Center on April 3, 2026, with accession number CGMCC No.38030.

[0037] Example 2 I. A method for preparing frozen dough (SW) based on Weissella oryzae producing extracellular polysaccharides and subjecting it to freeze-thaw cycles. 1. Dough preparation: Frozen strain ZJU.1 was cultured in MRS medium for 24 h, and then inoculated into fresh MRS medium at a concentration of 2% (v / v) and cultured for another 24 h. The resulting culture was centrifuged at 6000 g, 4℃ for 10 min, and the cells were collected. The cells were washed twice with 0.8% sodium chloride solution (or sterile water) and then resuspended until the optical density measured at 600 nm was 0.7, at which point the cell density was approximately 102. 7 CFU / mL. Store the bacterial suspension at 4°C before use.

[0038] Mix 100g wheat flour, 50mL sterile water, 1g sucrose, and 10g... 7 The bacterial suspension of CFU / g flour was added to a dough mixer and mixed at low speed for 3 minutes, then at high speed for 3 minutes to form dough. The dough was then fermented in an incubator at 25°C and 80%-90% humidity for 12 hours to obtain SW dough samples.

[0039] Naturally fermented dough (SD) was also used as a control. The preparation method of SD dough is as follows: 1. Preparation of SD dough Add 100g wheat flour, 100g water, and 1g sucrose to a dough mixer and knead at 100-150 rpm for 10 minutes to form a dough. Then, allow it to ferment spontaneously in an incubator at 25℃ and 80%-90% humidity for 12 hours to obtain an SD dough sample.

[0040] 2. Dough freeze-thaw cycle processing The prepared SW dough was proofed for 30 minutes in a constant temperature and humidity chamber at 37°C and 70% humidity, then frozen at -20°C for 22 hours, and then thawed for 2 hours in a constant temperature and humidity chamber at 37°C and 85% humidity. This constitutes one freeze-thaw cycle, and 0, 1, 3, 5, and 7 freeze cycles (FTC) were set accordingly. After freezing, the dough was freeze-dried, pulverized, and sieved, and then stored at 4°C for subsequent processing.

[0041] 3. Determination of textural properties of frozen dough The thawed dough was shaped into cylindrical dough balls with a diameter of 2 cm and a height of 1.5 cm using a mold, and TPA was measured using a texture analyzer. The probe model was P / 36R, the compressive stress was 50%, the interval between two compressions was 5 s, the trigger force was 5 g, the velocity before the test was 1.00 mm / s, the velocity during the test was 1.00 mm / s, and the velocity after the test was 1.00 mm / s.

[0042] 4. Determination of Rheological Properties of Frozen Dough Two g of thawed dough was placed in parallel plates with a diameter of 25 mm and a gap of 1 mm. The test temperature was 25 ± 0.1℃, and frequency scanning was performed within a strain range of 0.1% and a frequency range of 0.1–10 Hz. The storage modulus (G'), loss modulus (G''), and tanδ were defined as functions of the angular frequency (rad / s). Here, tanδ represents the ratio of G'' to G'.

[0043] 5. Electron microscopy observation The dough was freeze-dried for 26 h using a freeze dryer and then stored in a desiccator for later use. A small piece (40 mm × 40 mm × 5 mm) was cut from the center of the freeze-dried sample, wrapped with conductive tape and gold-plated for 2 min, and observed at 500 × and 1000 × magnification with an accelerating voltage of 3.0 kV.

[0044] 6. Laser confocal Frozen-thawed dough samples were cut into 50 μm sections using a cryostat and stained with rhodamine B and fluorescein isothiocyanate (0.025% w / w, 1:1 mixture) for 5 min. After washing with ultrapure water, coverslips were applied and observed under a confocal microscope. Fluorescein isothiocyanate and rhodamine B were excited with 488 nm and 543 nm lasers, respectively, and images were captured for further analysis.

[0045] II. Results A. Texture properties The results are shown in Table 1. With increasing FTC cycles, the hardness, adhesiveness, and chewiness of both the comparative and example samples generally decreased. This is attributed to the mechanical damage to the gluten-starch network structure caused by ice crystal formation during freeze-thaw cycles and the imbalance in the dough system due to moisture redistribution. The SD group showed more significant deterioration; conversely, the SW group exhibited superior stability, with its hardness decreasing by only 27.83% after 7 freeze-thaw cycles, compared to a 39.80% decrease in the SD group. At the same number of FTC cycles, the SW group showed higher elasticity and adhesiveness than the SD group. EPS can bind with moisture to form a hydrophilic colloid, thereby improving the viscoelasticity of the dough.

[0046] Table 1. Texture properties of frozen dough

[0047] Note: Data in the same column with different lowercase superscripts indicate significant differences. p <0.05). SD-FTCi (i=0, 1, 3, 5, 7) represents the SD sourdough in the i-th freeze-thaw cycle; SW-FTCi (i=0, 1, 3, 5, 7) represents the SW sourdough in the i-th freeze-thaw cycle.

[0048] B. Rheological properties See results Figure 1 As the number of freeze-thaw cycles increased, both G' and G'' in the SW and SD groups showed a decreasing trend. This reflects that repeated freeze-thaw cycles damage the integrity of the dough network structure, ultimately causing the dough to become soft and collapse. However, after seven freeze-thaw cycles, the decrease in G' and G'' in the SW group was less than that in the SD group, indicating that Weissella fermentation can alleviate the damage to the dough network structure caused by freeze-thaw cycles.

[0049] C. Electron Microscopy Results The microstructural changes of dough during freeze-thaw cycles were observed using electron microscopy. For example... Figure 2As shown, in the 0th freeze-thaw cycle, both dough groups exhibited a uniform and continuous three-dimensional gluten network morphology, with starch granules evenly dispersed within the network. Neither the SD nor SW doughs showed obvious irregularities, holes, or cracks. With increasing FTC cycles, the number of holes and cracks increased significantly, as starch granules gradually detached from the gluten network structure. At the same FTC cycle, the SW dough had fewer holes and cracks than the SD group. This may be attributed to the hydrogen bonding ability of EPS in the SW sourdough, which interacts with water molecules, inhibiting ice recrystallization and thus maintaining a more continuous and intact network structure. Furthermore, the highly hydrophilic EPS, when combined with starch, can protect starch granules from damage during freezing, forming a dense wheat starch-EPS complex.

[0050] D. Laser confocal results See results Figure 3 Compared to the first FTC (Freezing-Temperature Cycle), after five FTC cycles, the continuity of the gluten protein network in both groups of frozen sourdough decreased, the gluten network distribution became more uneven, larger pores formed, and the starch granule volume also decreased. This is mainly due to the physical damage caused to gluten proteins and starch granules by the formation and development of ice crystals during freeze-thaw cycles, leading to gluten network disintegration and starch granule fragmentation. With only one FTC cycle, the SW group showed a more uniform gluten network distribution, stronger gluten protein continuity, and more uniform pore spacing on the frozen dough, indicating better air retention and higher gluten protein stability. This may be attributed to the hydrogen bonds formed between the hydroxyl groups in EPS and gluten proteins, enhancing the stability of the network connections. After the fifth FTC cycle, the SW group exhibited good continuity and uniform pore distribution, while the SD group showed poorer network continuity and larger pores. In conclusion, EPS promotes gluten network formation, enhances its continuity, and reduces the damage of ice crystals to the quality of frozen dough.

[0051] Example 3 Starch properties analysis of frozen dough 1. Starch separation Thaw the dough and wash approximately 200 g of wheat dough with 600 g of water (added in batches), collecting the starch slurry (washing water). Pass the slurry through a 100-mesh filter cloth. Then, store the starch slurry at 4°C for 6 h, centrifuge at 3500 rpm / min for 20 min, carefully remove the upper pigment layer, wash the precipitate with 300 mL of distilled water and centrifuge twice. Next, resuspend the precipitate in 400 mL of distilled water, adjust the pH to 7.0 with hydrochloric acid, and centrifuge the sample three times. Finally, dry the starch sample at 45°C. All starch samples were stored in PE bags for later use.

[0052] 2. Determination of the swelling power of amylose Add the prepared 1% (w / v) starch solution to the calibration sealed tube, incubate in a 90°C water bath with shaking for 30 min, cool for 10 min, and then centrifuge at 6000 g for 15 min. Discard the supernatant and weigh the precipitate. The swelling force is the ratio of the precipitate mass to the initial starch mass.

[0053] 3. Short-range ordered structure of starch The short-range ordered structure of starch was determined using Fourier transform infrared spectroscopy. The scanning wavelength range was 400-4000 cm⁻¹. -1 Spectral resolution 4 cm -1 A total of 64 scans were performed, and data processing was completed using OMNIC software. The result was 995 / 1022 cm⁻¹. -1 The strength ratio reflects the double helix structure, with 1047 / 1022 cm⁻¹. -1 Peak intensity ratio assesses short-range orderliness.

[0054] 4. Starch gelatinization characteristics Accurately weigh 5 mg of sample and transfer it to a crucible. Add 15 mL of distilled water to the sample. Seal and incubate overnight at 4°C. After equilibration, measure the sample using a thermal analyzer at a rate of 5°C / min within the range of 25°C to 120°C.

[0055] result A. Starch swelling power and gelatinization properties The results are shown in Table 2. The swelling power of starch in both groups increased with increasing FTC cycles. However, the swelling power of the SW group was consistently lower than that of the SD group at different FTC cycles. This is mainly attributed to the hydrophilic groups of EPS produced by *Westernella*, which can competitively bind water, thereby inhibiting starch gelatinization and swelling. Simultaneously, EPS can coat the starch surface, buffering the direct damage to starch granules from ice crystals.

[0056] With increasing FTC cycles, the gelatinization initiation temperature (To), peak temperature (Tp), termination temperature (Tc), and gelatinization hysteresis (ΔH) of both groups of samples showed an increasing trend. However, the SW group exhibited lower gelatinization temperature and ΔH at each freeze-thaw stage. To and ΔH represent the energy required for the dissociation of the double-helix structure in the starch crystalline region, indicating the thermal stability of the ordered molecular structure. On the one hand, the continuous formation and expansion of ice crystals during freeze-thaw cycles exert a mechanical destructive effect on starch granules, disrupting some of their crystalline regions, leading to an increase in the energy required for starch gelatinization, a shift in gelatinization temperature to higher temperatures, and an increase in starch swelling force. On the other hand, in the SW group... W. cibaria The metabolically generated EPS exhibits a lower gelatinization temperature by inhibiting ice crystal growth, protecting the integrity of starch granules, and delaying the hydration and gelatinization process of starch chains through competitive water binding.

[0057] Table 2 Starch swelling power and gelatinization properties

[0058] Note: Data in the same column with different lowercase superscripts indicate significant differences. p <0.05).

[0059] B. Short-range ordered structure of starch The short-range ordered structure of starch is closely related to its single-helix and double-helix arrangement, 1047 cm⁻¹ -1 and 995cm -1 The absorption peaks at 1022 cm⁻¹ characterize the short-range ordered structure and the double-helix conformation, respectively. -1 The absorption peak at that location corresponds to the amorphous region. Both R995 / 1022 and R1047 / 1022 show an increasing trend with increasing FTC cycles, a phenomenon attributed to the repeated formation and growth of ice crystals disrupting the starch structure. During thawing, amylopectin first forms an intramolecular double helix, then rearranges to construct a short-range ordered structure. Furthermore, the increase in the R995 / 1022 ratio may be related to the increased hydroxyl exposure caused by ice crystals disrupting the integrity of starch granules, which is consistent with the swelling force measurement results. The R995 / 1022 and R1047 / 1022 of the SW group were significantly lower than those of the SD group (…). p <0.05), this inhibitory effect is related to the hydrophilicity of EPS. EPS can competitively bind water with starch hydroxyl groups, inhibiting the formation of starch double helix structure. At the same time, the interaction between EPS and water can reduce the contact between starch and water, as well as the helical re-aggregation of starch chain molecules and the ordered structure during the regeneration process of thawing.

[0060] Table 3 Short-range ordered structure of starch

[0061] Note: Data in the same column with different lowercase superscripts indicate significant differences. p <0.05).

[0062] Example 4 Analysis of gluten protein properties of frozen dough 1. Gluten protein isolation The thawed dough was rinsed with a 5% NaCl solution to separate the wheat gluten from the starch and salt-soluble proteins. It was then rinsed again with running deionized water to remove NaCl and water-soluble proteins from the gluten protein. After washing, the wet gluten was freeze-dried, ground, and sieved through a 100-mesh sieve. The freeze-dried gluten protein (50 g) was shaken and soaked in dichloromethane (500 mL) at room temperature for 1 h, then filtered. This process was repeated three times to remove lipids. The resulting defatted gluten was left to stand overnight in a fume hood to remove residual dichloromethane.

[0063] 2. Molecular weight distribution of gluten protein Prepare the gel according to the instructions of the one-step PAGE gel rapid preparation kit, with an upper gel concentration of 4.5% and a lower gel concentration of 10%. First, mix 5 mg of gluten protein sample with 1 mL of protein loading buffer (containing DTT), extract at 25°C for 1 h, then in a 100°C metal bath for 5 min, followed by centrifugation at 12000 rpm for 15 min. Finally, add 10 μL of the supernatant to the lane, set the voltage to 150 V, and continue electrophoresis for approximately 60 min. Then, detach the gel from the apparatus and stain it in Coomassie Brilliant Blue quick staining solution for 20 min. Remove the gel, destain it with distilled water overnight, and then observe it.

[0064] 3. Protein secondary structure analysis Protein samples were mixed with potassium bromide at a mass ratio of 1:100 and ground until homogeneous. The mixture was then pressed into transparent thin films under 20 MPa pressure. After spectral acquisition, baseline correction, smoothing, peak finding, and fitting were performed on the amide I region (1600-1700 cm⁻¹) using OMINC and Peak-fit software. The four conformations were quantified according to the following intervals: β-sheet (1600-1635 cm⁻¹). -1 1685-1695cm -1 ), α-helix (1645-1665cm) -1 ), irregular curls (1635-1645cm) -1 ) and β-turn (1665-1685cm) -1 ).

[0065] 4. SS (disulfide bonds) and -SH (free thiol groups) content The determination of total thiol and free thiol groups was performed according to the instructions of the total thiol assay kit (DTNB method) and the free thiol assay kit (DTNB method), respectively, and the absorbance value at 412 nm was measured. The SS bond content was obtained by subtracting the free SH content from the total SH content and then dividing by 2. The contents of total thiol and free thiol groups were calculated according to Formula I and Formula II: Formula I; Formula II.

[0066] In the formula, A412 is the absorbance value at 412 nm, D is the optical path length of the reaction system (typically 1 cm), and 73.53 is the extinction coefficient (M-1cm). -1 C represents the concentration of Elman's reagent (mg / mL).

[0067] 5. Analysis of non-covalent interactions Four extraction solutions, S1 (0.1 M NaCl), S2 (0.6 M NaCl), S3 (0.6 M NaCl + 1.5 M urea), and S4 (0.6 M NaCl + 8 M urea), were prepared using 0.05 M phosphate buffer (pH 7.0). 100 mg of protein sample was added to 10 mL of each of these solutions, mixed thoroughly, and then 5 mL of Coomassie Brilliant Blue solution was added. The mixture was incubated at room temperature for 1 h. After incubation, the sample was centrifuged at 10,000 rpm for 15 min, and 150 μL of the supernatant was carefully transferred to a microplate. The absorbance of gluten protein in the supernatant was measured at 595 nm using a microplate reader. A standard curve was established using bovine serum albumin standard solution.

[0068] result: A. Secondary structure of gluten protein See results Figure 4 After seven FTC cycles, there was no significant difference in peak shape changes between the two groups. However, protein secondary structure results showed that with increasing FTC cycles, the relative content of α-helices in the SW group decreased from 1.63% to 0.62%, while in the SD group it decreased from 1.11% to 0.43%. The relative content of β-sheets remained unchanged, while the relative content of random coils increased. The mechanical compression of gluten proteins by ice crystals during freeze-thaw cycles disrupts the hydrogen bond stability of proteins, leading to a decrease in the content of ordered structures and an increase in the content of disordered structures. After seven FTC cycles, the relative content of α-helices in the SW group was significantly higher than that in the SD group (p<0.05), indicating that EPS production... W. cibaria Fermentation can enhance the stability of gluten protein.

[0069] B. Non-covalent bond analysis of gluten protein See results Figure 5 Under the same extraction conditions, the absorbance values ​​of the SW group were generally lower than those of the SD group in stages S1-S3, indicating that the protein network mediated by non-covalent forces such as ionic bonds, hydrogen bonds, and hydrophobic interactions in the SW group was more stable. However, under strong denaturation conditions, the absorbance values ​​of the two groups tended to be similar, indicating that the total amount of protein bound by non-covalent bonds was comparable. Furthermore, with the increase of freeze-thaw cycles, the solubility of the proteins in both groups increased, indicating that freeze-thaw cycles disrupt the protein network structure.

[0070] C. Content of free sulfhydryl groups and disulfide bonds in gluten protein See results Figure 6After seven freeze-thaw cycles, -SHfree in the SD group increased from 1.38 mmol / g to 1.56 mmol / g, while SS decreased from 12.33 mmol / g to 3.60 mmol / g. In the SW group, -SHfree increased from 1.37 mmol / g to 1.50 mmol / g, while SS decreased from 12.67 mmol / g to 3.86 mmol / g. Temperature fluctuations and ice crystal growth during freeze-thaw cycles disrupt disulfide bonds in the protein network, while EPS effectively protects the gluten network, reduces SS breakage, and inhibits the increase in -SHfree content. This can be attributed to the hydrophilic and emulsifying effects of EPS.

[0071] D. Molecular weight distribution of gluten protein See results Figure 7 The number of protein bands in both groups of sourdough remained unchanged during freeze-thaw cycles, indicating that no new proteins were formed or depolymerized during the freeze-thaw process. However, with the accumulation of FTC cycles, the subunit bands in the 31.0–50.0 kDa range of the SD group darkened in color. This is presumably because GMP depolymerization occurred during the freeze-thaw process, increasing the content of soluble proteins and thus increasing the content of low molecular weight glutenin subunits in this range. The subunit band color in the SW group did not change significantly during freeze-thaw cycles.

[0072] Example 5 Moisture analysis of frozen dough 1. Total moisture content Dry the aluminum container at 105°C until a stable weight is reached, and label this as M0. Accurately weigh 5 g of the thawed dough and label this as M1. Dry the sample at 101-105°C until a constant weight is reached; record the final mass of the dried dough and beaker as M2. The total moisture content (Wt) is calculated as shown in Formula III: Formula III; 2. Freezeable water content Measurements were performed using DSC. A sample of 15.0 ± 0.5 mg of frozen dough was taken from the center, pressed, and accurately weighed, then placed in a DSC aluminum crucible for measurement. The parameters for measuring the frozen dough were set as follows: nitrogen flow rate was set to 100 cm⁻¹. 3 The initial temperature was 25°C, then cooled to -30°C and held for 5 minutes, followed by heating to 25°C. Both the cooling and heating rates were set to 5°C / min. A blank crucible without a sample was used for zeroing during the test.

[0073] 100% Formula IV; ΔHm: enthalpy of frozen dough (J / g); ΔHi: latent heat of fusion of ice, 335J / g; Tw: moisture content of the sample, determined by vacuum drying at 105℃.

[0074] 3. Moisture distribution and migration LF-NMR analysis was performed. A sample weighing 2.0 ± 0.2 g was carefully removed from the center of the frozen dough and placed in an LF-NMR tube for analysis. The experimental parameters were configured as follows: SW: 200 kHz, TW: 3500 ms, TE: 0.200 ms, NS: 16, DRG1: 3, PRG: 1, NECH: 2000, to obtain T2 relaxation curve data.

[0075] result A. Total moisture content See results Figure 8 During freeze-thaw cycles, the moisture content of both SD and SW frozen sourdough gradually decreased. After seven freeze-thaw cycles, the moisture content of SD dough decreased from 39.15 ± 0.30% to 36.04 ± 0.74%, with a moisture loss rate of 7.94%, significantly higher than that of SW dough (3.72%, p < 0.05). These results indicate that EPS can effectively reduce moisture loss in frozen dough, possibly because EPS can effectively bind and fix moisture through hydrogen bonds and capillary action, converting more free water into bound water that is less prone to freezing.

[0076] B. Freezeable water content See results Figure 9 Compared to the SD group, the freezeable water content of the SW group increased from 33.92% at FTC0 to 39.10% at FTC7, and was significantly lower than that of the SD group at all freeze-thaw time points. This indicates that the synthesis of EPS increased the content of non-freezeable water and reduced the damage of ice crystals to the gluten network. This effect is mainly attributed to the strong hydrophilicity of EPS, i.e., the hydroxyl groups on the molecular chain promote the formation of a strong intermolecular hydrogen bond network. Hydrogen bond interactions can reduce the hydrophobicity of the system and inhibit the generation of free water, thereby enhancing the binding ability between water molecules and the gluten matrix. C Moisture distribution See results Figure 10After five freeze-thaw cycles (FTC), the peak areas corresponding to T21 and T22 in both the SD and SW sourdough decreased, indicating that the FTC process promotes the redistribution of water in the dough, releasing bound water from the gluten network. During freezing, bound water between parallel layers of the gluten structure crystallizes to form ice crystals, a process that damages the gluten network to some extent. During thawing, the ice converts back into water, at which point the damaged gluten network releases bound water. Under the same freeze-thaw conditions, the peak areas corresponding to T21 and T22 in the SD group were significantly lower than those in the SW group, and the relaxation time shifted slightly to the right. This suggests that EPS can restrict the fluidity of water in the dough, preventing the conversion of bound water to free water during freeze-thaw cycles.

[0077] Example 6 Making steamed buns using frozen dough After thawing the frozen dough at room temperature for 2 hours, it was transferred to a fermentation chamber at 37°C and fermented for approximately 60 minutes. The fermented dough was then steamed in boiling water for 25 minutes, and then cooled completely at room temperature for 1 hour. The steamed buns were cut into 15mm thick, uniform slices and photographed for record-keeping.

[0078] See results Figure 11 The exopolysaccharide-producing Weissella bacteria can improve the porosity of steamed buns without the addition of yeast, and this improvement effect weakens with the increase of freeze-thaw cycles.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Weissella esculenta ( Weissella cibaria The application of the extracellular polysaccharide produced by strain ZJU.1 and / or the Weissella esculenta strain ZJU.1 in the preparation of frozen dough, wherein the Weissella esculenta strain ZJU.1 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No.38030.

2. The application according to claim 1, characterized in that, The *Westernella esculenta* strain ZJU.1 exists in the form of cells or spores.

3. The application according to claim 1, characterized in that, The *Westernella esculenta* strain ZJU.1 existed in the form of a bacterial suspension. The extracellular polysaccharide produced by the *Westernella esculenta* strain ZJU.1 exists in the form of fermentation supernatant. The method for preparing the fermentation supernatant includes inoculating an activated Weissella esculenta strain ZJU.1 into MRS medium and culturing it. The resulting fermentation broth is then subjected to solid-liquid separation, and the liquid phase is collected to obtain the fermentation supernatant.

4. The application according to claim 1, characterized in that, The preparation of frozen dough includes resisting the structural degradation and / or component loss of frozen dough caused by freeze-thaw cycles.

5. The application according to claim 1, characterized in that, The structure of the frozen dough includes its textural properties, rheological properties, and microstructure. The frozen dough consists of frozen dough starch, gluten protein, and water.

6. A method for preparing freeze-thaw resistant frozen dough, characterized in that, Includes the following steps: The *Westernella esculenta* strain ZJU.1 used in any one of claims 1 to 5 is mixed with flour, sugar, and edible water to form a dough, fermented, and then proofed and frozen.

7. The preparation method according to claim 6, characterized in that, The amount of the *Westernella esculenta* strain ZJU.1 added was 10. 5 ~10 9 CFU / g flour; The amount of sugar added is 0.9% to 8%; The amount of drinking water added is 40% to 70%.

8. The preparation method according to claim 6, characterized in that, The fermentation temperature is 22~27℃; the fermentation time is 10~14h; and the ambient humidity during fermentation is 80%~90%. The humidity during the proofing process is 70%~90%; the temperature during the proofing process is 25~40℃. The warming time is 20-60 minutes; The freezing temperature is -30℃ to 0℃.

9. Frozen dough prepared by the preparation method according to any one of claims 6 to 8.

10. A food product prepared from the frozen dough of claim 9.

Images