Fusion Weissella sp. HTD7 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a Weissella fusiformis HTD7 and application thereof. The preservation number of the Weissella fusiformis HTD7 is CGMCC No.29775, the Weissella fusiformis HTD7 is good in antibacterial effect and different in color under different pH values, when the Weissella fusiformis HTD7 is applied to fermentation of high-fiber matrix cereals, the content of various beneficial components in a fermentation product can be increased, the product effect can be improved, when the Weissella fusiformis HTD7 is applied to preparation of bread and steamed buns, the product quality can be improved, and the Weissella fusiformis HTD7 can also be used as an edible dye, so that the Weissella fusiformis HTD7 can be used for The method can be widely applied to food preparation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a fusion strain of Weissella HTD7 and its applications. Background Technology

[0002] *Weissella confusa* is a lactic acid bacterium that promotes food fermentation and improves food texture. It is mainly used in the fermentation production of dairy products, pickled vegetables, and meats. However, it still has limitations, including a limited variety of strains, potentially low fermentation efficiency, and poor adaptability.

[0003] Currently, commonly used microbial strains for grain fermentation include yeast and lactic acid bacteria. However, there is still a shortage of microbial strains for plant-based foods and beverages, leading to unsatisfactory fermented products. Combining *Westernobacterium fusion* with whole grain fermentation holds promise for overcoming the shortcomings of existing *Westernobacterium fusion* and grain fermentation methods. The development of novel microbial strains will have a positive impact on improving product quality and inhibiting spoilage, and may create more types of functional foods. This will help expand the application potential of fermentation microorganisms in the food industry and biotechnology fields, and promote the development of the agro-food industry. Summary of the Invention

[0004] One of the objectives of this invention is to provide a novel fusion strain of Weissella suitable for food processing and its applications.

[0005] To achieve this objective, the technical solution of the present invention is as follows:

[0006] This invention provides a strain of Weissella confusa HTD7, with accession number CGMCC No. 29775.

[0007] The present invention also provides a microbial agent containing the above-mentioned Weissella confusa HTD7.

[0008] The bacterial agent of the present invention contains Fusion Weissella HTD7, which can be a solid bacterial agent or a liquid bacterial agent. Those skilled in the art can prepare the bacterial agent according to methods known in the art.

[0009] Preferably, the inoculum comprises the fermentation broth of Weissella confusa HTD7.

[0010] The present invention also provides the application of the above-mentioned fused Weissella confusa HTD7 or bacterial agent in the production of proteases, antibacterial agents or food preservatives.

[0011] This invention has discovered that the fusion of Weissella HTD7 can produce proteases with excellent antibacterial effects, inhibiting Escherichia coli, Salmonella, and molds.

[0012] The present invention also provides the application of the above-mentioned fused Weissella confusa HTD7 or bacterial agent in the preparation of high-fiber matrix cereal beverages or noodle products.

[0013] In the application of this invention, the high-fiber matrix grain is oats.

[0014] Using the fused Weissella HTD7 of this invention for oat fermentation can increase the content of various beneficial components in the product, and the bread prepared using this fermentation product is more ideal in terms of sensory evaluation and texture.

[0015] The present invention also provides the application of the above-mentioned fused Weissella confusa HTD7 or bacterial agent in increasing the content of lactic acid, flavonoids, amino acids, β-glucan, dietary fiber, folic acid and / or reducing sugar in the products after oat fermentation.

[0016] The present invention also provides the application of the above-mentioned fused Weissella confusa HTD7 or bacterial agent in improving the quality of bread or steamed buns or as an edible pigment.

[0017] Using the fused Weissella HTD7 of this invention directly to prepare bread and steamed buns can improve the specific volume, color and sensory evaluation of the products, thereby improving the overall product quality.

[0018] The Fusion Weissella HTD7 of the present invention has different colors at different pH values. Since the bacteria have color and the bacteria are beneficial, it can be used as an edible pigment for food decoration or coloring.

[0019] The present invention also provides an oat beverage or sweetened mash, which is prepared by fermenting oats with the above-mentioned fused Weissella confossa HTD7 or bacterial agent. Preferably, in the preparation of the oat beverage, the oats are first subjected to high-pressure jet homogenization before fermentation to facilitate the increase of the content of active ingredients.

[0020] Compared to other fermentation agents, the sweet mash prepared by fermenting solid oat grains with the fusion of Weissella HTD7 of this invention has a higher content of beneficial components and better product efficacy.

[0021] The beverage prepared from the oat pulp fermented with Fusobacterium fusion HTD7 of the present invention has good stability, is not easy to discolor, and has a high content of beneficial ingredients.

[0022] Preferably, the pressure during high-pressure jet homogenization of oats is 120 MPa, and the infused solution of Weissella HTD7 bacterial culture (concentration of 1×10⁻⁶) is used.6 The inoculum size (CFU / mL) was 0.5% (v / w, mL / g).

[0023] The present invention also provides a bread comprising an oat product fermented with the aforementioned Weissella confusa HTD7 or a bacterial agent, or comprising the aforementioned Weissella confusa HTD7 or a bacterial agent.

[0024] This invention has found that adding sweet mash obtained from oat fermentation using Weissella confusa HTD7 during bread preparation can improve the appearance, taste, and texture of the bread. Directly adding the aforementioned Weissella confusa HTD7 can also improve the bread's specific volume, color, and sensory evaluation.

[0025] The present invention also provides a steamed bun containing the above-mentioned fused Weissella confusa HTD7 or bacterial agent.

[0026] The present invention also provides an edible pigment, food preservative or antibacterial agent, which contains the above-mentioned Weissella confusa HTD7 or bacterial agent.

[0027] The beneficial effects of this invention are at least as follows:

[0028] This invention provides a novel fusion strain of Weissella HTD7, which is a Gram-positive bacterium that is light pink, flesh pink to pink in color. It undergoes heterologous glucose fermentation, is highly acid-resistant, and can grow at pH 2. It can grow and reproduce at 10-50℃, with a rapid growth rate. The color will change when cultured at different pH values ​​for different times.

[0029] The strain of this invention can inhibit the growth of pathogens, is low in cost, safe, reliable, easy to use, and inexpensive. It has a good promoting effect on the fermentation of new whole grain beverages, making up for the current lack of suitable plant-based fermentation strains for whole grains. In addition, this strain can also improve the quality of bread and steamed buns, and can be used as an edible pigment. Attached Figure Description

[0030] Figure 1 For the phylogenetic tree of the family gene.

[0031] Figure 2 To fusion, the colony morphology (left image) and electron micrograph (right image) of *Westernella esculenta* HTD7 grown in MRS medium.

[0032] Figure 3 Growth curves for fusion with Weissella HTD7.

[0033] Figure 4To integrate the growth of Weissella HTD7 at different pH values.

[0034] Figure 5 To integrate the growth of Weissella HTD7 at different temperatures.

[0035] Figure 6 The results show the inhibitory effects of Weissella HTD7 on Escherichia coli (left), Salmonella (middle), and Rhizopus sp. (right).

[0036] Figure 7 This study describes the hydrolysis zone formation observed when *Westernella* HTD7 grows on corn gluten meal medium.

[0037] Figure 8 Photos of soybean paste with added Weissella fusion strain HTD7 (right image) and soybean paste without added Weissella fusion strain HTD7 (left image) after being stored at room temperature for 6 months.

[0038] Figure 9 Photos of sweet mash with added Weissella fusion strain HTD7 (right image) and sweet mash without added Weissella fusion strain HTD7 (left image) after being stored at room temperature for 10 days.

[0039] Figure 10 Photos of whole wheat bread made with sweet mash made with Weissleriane HTD7 (left) and whole wheat bread made without sweet mash (right).

[0040] Figure 11 The images show photos of whole wheat steamed buns and bread made with *Westernella fusion strain HTD7* and whole wheat steamed buns and bread made with the same species control strain CICC 24453, as shown in Example 5. The top left image is a side view of the whole wheat steamed bun, and the top right image is a top view. In both images, the left image shows the whole wheat steamed bun made with the same species control strain CICC 24453, and the right image shows the whole wheat steamed bun made with *Westernella fusion strain HTD7*. The bottom left image is a side view of the bread, and the bottom right image is a top view. In both images, the right image shows the bread made with the same species control strain CICC 24453, and the left image shows the bread made with *Westernella fusion strain HTD7*.

[0041] Figure 12 The results of the Circos genome circle map analysis.

[0042] Figure 13 The results are from the analysis of carbohydrate-active enzymes.

[0043] Figure 14 This is a linear map of gene clusters for the synthesis of secondary metabolites.

[0044] Figure 15This is a colony photograph of the control strain (CICC 24453) of the same genus.

[0045] Figure 16 The color of *Westernella fusion* HTD7 after 24 hours of fermentation in culture media with different pH values.

[0046] Figure 17 The color of *Westernella fusion* HTD7 after 48 hours of fermentation in culture media with different pH values.

[0047] Figure 18 The color of *Westernella fusion* HTD7 after 72 hours of fermentation in culture media with different pH values.

[0048] Figure 19 The color of *Westernella fusion* HTD7 after 96 hours of fermentation in culture media with different pH values.

[0049] Figure 20 Photo of a steamed bun after being coated with HTD7 bacteria of *Westernella vulgaris*.

[0050] Figure 21 A drawing of Fusion Weissella HTD7 cells cultured at different pH values. Detailed Implementation

[0051] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art. Example 1: Isolation and Screening of Fusion Weissella

[0053] In this embodiment, a strain of *Westernella* fusion was obtained through screening, specifically as follows: The bacterium was isolated and cultured, and screened using the dilution plate method: A conical flask containing 500 mL of distilled water, a test tube containing 4.5 mL of distilled water, MRS solid medium, and plates were sterilized. After sterilization, the MRS medium was poured into the plates. Under aseptic conditions, 10 g of traditionally fermented whole-grain sweet mash was mixed evenly, and then 90 mL of sterile distilled water was added and shaken evenly to achieve a final concentration of 10. -1 Then, take 500 μL of this liquid and add it to a test tube containing 4500 μL of sterile distilled water. Shake to mix thoroughly, and then continuously dilute to achieve concentrations of 10 μL and 10 μL respectively. -2 10 -3 10 -4 and 10-5 With a concentration of 10 -3 and 10 -5 Repeatedly streak plate separation with 20 μL of liquid until a single colony is obtained. Inoculate the single colony into a 2 mL cryovial containing 20% ​​glycerol using a sterile inoculation needle and store at -80°C for later use.

[0054] MRS medium consisted of: 10.0 g peptone; 10.0 g beef extract; 5.0 g yeast extract; 20.0 g dextrose; 1.0 g polysorbate 80; 2.0 g ammonium citrate; 5.0 g sodium acetate; 0.1 g magnesium sulfate; 0.05 g manganese sulfate; 2.0 g dipotassium phosphate; and 1000 mL distilled water. The solid medium was then infused with 15 g / L agar and sterilized at 121°C for 15 min.

[0055] Weissella confusa HTD7, a strain with rapid growth rate, strong acid resistance, and wide growth temperature range, was selected for biopreservation. This strain was deposited on January 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and classified as Weissella confusa, with accession number CGMCC No. 29775.

[0056] The genome of *Weissella confusa* HTD7 was extracted, and then the 16S rRNA gene was amplified using PCR primers 27f (5'-AGAGTTTGATCCTGGCTC-3', SEQ ID No. 1) and 1492r (5'-TACGGCTACCTTGTTACGACT-3', SEQ ID No. 2). BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST) was used to identify the species with the highest homology to the gene sequence of the target lactic acid bacteria strain among those of known taxonomic positions. Gene sequences of known lactic acid bacteria strains were selected from GenBank and compared with the 16S rRNA gene sequence of the target lactic acid bacteria strain to determine its similar species and taxonomic position.

[0057] Based on 31 housekeeping genes (dnaG,frr,infC,nusA,pgk,pyrG,rplA,rplB,rplC,rplD,rplE,rplF,rplK,rplL,rplM,rplN,rplP,rplS,rplT,rpmA,rpoB,rpsB,rpsC,rpsE,rpsI,rpsJ,rpsK,rpsM,rpsS,smpB,tsf), 19 strains most closely related at the species level were selected, and a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with MEGA 6.0 software. Results are shown below. Figure 1 .

[0058] Example 2: Culture and observation of *Westernella*

[0059] This embodiment studies the growth characteristics of the fusion Weissella HTD7 obtained in Example 1.

[0060] 1. The *Weissella fusion* HTD7 was cultured in MRS medium for 24 hours, and its colony morphology was observed. The bacterial cells ranged in color from light pink to flesh-pink to pink. Electron microscopy was performed on *Weissella fusion* HTD7; the specific results are shown below. Figure 2 The colonies of the control strain (CICC 24453) were white; see photo below. Figure 15 .

[0061] MRS medium consisted of: 10.0 g peptone; 10.0 g beef extract; 5.0 g yeast extract; 20.0 g dextrose; 1 ml polysorbate 80; 2.0 g ammonium citrate; 5.0 g sodium acetate; 0.2 g magnesium sulfate; 0.05 g manganese sulfate; 2.0 g dipotassium phosphate; 1000 mL distilled water; and 15 g / L agar added to the solid medium. The medium was sterilized at 121°C for 15 min.

[0062] 2. The fused Weissella HTD7 strain was cultured in LB medium (pH 7.0) at 37°C for 60 hours, and the growth was recorded. Specific results (OD) were recorded. 600 )See Figure 3 .

[0063] 3. Under the same culture conditions as in section 2 above, but with the pH values ​​of the LB medium changed to 2, 3, 4, 5, 6, 7, 8, 9, and 10 respectively, *Westernella* HTD7 fusion strains were cultured. Specific results (OD) were obtained. 600 )See Figure 4 .

[0064] 4. Under the same culture conditions as in section 2 above, except that the culture temperature was changed to 10℃, 20℃, 30℃, 40℃, and 50℃ respectively, the fusion strain *Westernella* HTD7 was cultured. Specific results (OD) were obtained. 600 )See Figure 5 .

[0065] 5. The antibacterial activity and protease production of *Westernella fusion-type* HTD7 were detected. Results are shown below. Figure 6 and Figure 7 .

[0066] Detection method: In a sterile operating table, culture 10 μL of bacterial solution (under the same culture conditions as in section 2 above, with a bacterial concentration of 1×10⁻⁶) 6 Inoculate (CFU / mL) onto sterile filter paper discs of LB agar or corn gluten medium evenly coated with the pathogen, and incubate at 30°C for 48 hours. Observe and measure the inhibition zone of the colonies. For Rhizopus, use streak plating with the bacterial suspension, while keeping other conditions the same.

[0067] The inhibitory effects of HTD7 fusion strain on Escherichia coli are shown in the figure. Figure 6 The left image shows a clear zone diameter to colony diameter ratio of 2.2. The inhibitory results of *Westernella fusion* HTD7 on *Salmonella* are shown in [reference needed]. Figure 6 The ratio of the diameter of the transparent zone to the colony diameter in the middle image is 2.5. The inhibition results of *Westernella* fusion HTD7 on *Rhizopus* are shown in [the image / image / etc.]. Figure 6 The right image shows the hydrolysis zone produced by *Westernella fusionis* HTD7 growing on corn gluten meal medium. Figure 7 .

[0068] The same tests were performed using the control strain CICC 24453 of the same genus. CICC 24453 showed a clear zone diameter to colony diameter ratio of 1.2 for Escherichia coli and 1.1 for Salmonella. Under the same conditions, it had no significant inhibitory effect on Rhizopus and the clear zone for protease production could not be clearly observed.

[0069] 6. The utilization and chemical sensitivity of different carbon sources by *Westernella fusionis* HTD7 were tested. Results: After testing with the Biolog method, the carbon sources available were: D-maltose, D-trehalose, sucrose, D-minobiose, α-D-lactose, α-D-glucose, D-mannose, inosine, D-mannitol, glycerol, L-aspartic acid, L-glutamic acid, L-pyroglutamic acid, L-serine, D-gluconic acid, L-malic acid, propionic acid, acetic acid, formic acid, and β-formyl-D-glucosidase.

[0070] Chemically sensitive substances: lincomycin, potassium tellurite, aztreonam, sodium bromate, naphthidone acid.

[0071] Example 3: Using Fusion Weissl bacterium HTD7 for preservation

[0072] Weissella confusa HTD7 bacterial suspension was added to soybean paste at 1% (v / w, mL / g) and stored at room temperature. A control group without the addition of Weissella confusa HTD7 was also included.

[0073] The results showed that the preservative performance was greatly improved after adding Fusion Weissl bacterium HTD7, and the preservation time increased by more than 3 months in the current preservation experiments.

[0074] Adding 1% (v / w, mL / g) of *Westernella fusion-HTD7* bacterial solution to fresh sweet mash, with a control group not containing *Westernella fusion-HTD7*, resulted in a delay of more than 7 days in the appearance of large-scale mold spoilage.

[0075] For specific experimental results, please see [link / details]. Figure 8 , Figure 9 .

[0076] The Fusion Weissl bacterium HTD7 bacterial culture used in this embodiment was obtained by culturing Fusion Weissl bacterium HTD7 in LB medium (pH 7.0) at 37°C for 48 hours, with a bacterial concentration of 1×10⁻⁶. 6 CFU / mL.

[0077] Example 4: Fermentation of oats using Fusion Bacterium HTD7 to produce sweet mash and bread containing it.

[0078] This embodiment utilizes *Weissella confusa* HTD7 to ferment whole-grain oats to produce sweet mash. Specifically, HTD7 bacterial solution (see Example 3) was added to cooked oats at a ratio of 1% (v / w, mL / g), and fermented at 25°C for 48 hours before the nutrient content was analyzed. A control group was prepared by fermentation using the same genus control bacteria CICC 24453 (DZJ) and *Lactobacillus plantarum* L1 (described in: Ren Fei, Liu Yuchun, Wang Chao, et al. Screening and growth characteristics of highly efficient probiotics for degrading corn gluten powder [J]. Grain and Oil Food Science and Technology, 2021, 29(3):183-191, where *Lactobacillus plantarum* L1 was named CGM57). The results are shown in Table 1, where the data represent the average of three tests. It can be seen that the product obtained by fermentation with *Weissella confusa* HTD7 has a better nutrient content.

[0079] Table 1. Nutrient composition of fermented sweet mash samples

[0080]

[0081] In this embodiment, the sweet mash obtained above is further ground into a paste and used for the production of whole wheat sweet mash bread.

[0082] The specific bread recipe by weight includes: 52 parts whole wheat flour, 48 parts all-purpose flour, 4 parts butter, 60 parts distilled water, 5 parts sugar, 1 part yeast, and 20 parts sweetened syrup. Made with a Panasonic SD-P103 bread machine, mode 6.

[0083] A control group of bread was prepared without the addition of sweetened mash. Photos of the prepared bread are shown below. Figure 10 The image shows bread with sweetened syrup on the left and bread without sweetened syrup on the right.

[0084] The specific volume, texture, hardness, and aging rate of the two types of bread were measured, and sensory evaluation was conducted. The results are as follows:

[0085] Specific gravity: Whole wheat bread made with added sweet fermented mash has a specific gravity of 4.57;

[0086] Whole wheat bread made without sweetened mash has a flavor value of 3.31.

[0087] Moisture content (g / 100g): 1.79 for whole wheat sweet fermented bread made with added sweet fermented mash;

[0088] Whole wheat bread made without sweetened mash has a yield of 1.60.

[0089] Ash content (g / 100g): 2.86 for whole wheat bread made with added sweet fermented mash;

[0090] Whole wheat bread made without sweetened mash costs $2.99.

[0091] Starch (g / 100g): 57.55g for whole wheat sweet fermented bread made with added sweet fermented mash;

[0092] Whole wheat bread made without sweetened mash has a weight of 56.31.

[0093] Fat (g / 100g): 3.18g for whole wheat bread made with sweetened fermented mash;

[0094] Whole wheat bread made without sweetened mash has a price of 3.74.

[0095] Egg white (g / 100g): 15.41 for whole wheat sweetened bread made with added sweetened fermented mash;

[0096] Whole wheat bread made without sweetened mash costs 1.80.

[0097] Texture hardness aging rate (7-day interval): 114.79g / day for whole wheat sweetened bread made with added sweetened mash;

[0098] Whole wheat bread made without added sweetened mash contains 160.06g per day.

[0099] Sensory evaluation: 60 points maximum, with higher scores indicating better evaluation. Ten evaluators participated, and the scoring criteria are shown in Table 2.

[0100] Table 2

[0101] Evaluation criteria items Scoring Criteria Full marks / point form Regular shape and good symmetry 10 Color Uniform, yellowish-brown 10 odor Rich aroma 10 taste Delicious, odorless, not sticky, and chewy. 10 Internal structure Smooth, soft and elastic 10 Texture uniformity The pores are fine and uniform with thin walls. 10

[0102] The sensory score of whole wheat sweetened bread made with added sweetened mash was 55±2.3, while the sensory score of whole wheat sweetened mash bread made without added sweetened mash was 45±3.0.

[0103] Example 5: Fermentation of whole wheat steamed buns and bread using Fusion Weissl bacteria HTD7

[0104] Preparing whole wheat steamed buns:

[0105] Whole wheat flour was mixed with water, and then 1% (v / w, mL / g) of a fused *Westernella* HTD7 bacterial solution and a control bacterial solution of the same genus, CICC 24453, were added respectively (see Example 3 for the preparation method of the bacterial solutions). The mixture was kneaded into dough, allowed to rise for 20 minutes, and then steamed for 30 minutes to obtain whole wheat steamed buns weighing 90g. The specific volume and color of the two types of steamed buns were measured, and sensory evaluation was performed.

[0106] Specific volume: 2.13 for whole wheat steamed buns made with HTD7;

[0107] The control group produced whole wheat steamed buns with a yield of 1.66.

[0108] Color saturation (average value taken from 3 different locations):

[0109] HTD7's whole wheat steamed buns are:

[0110] Top: L = 38.79 ± 0.45, a = 6.58 ± 0.12, b = 12.76 ± 0.14;

[0111] Bottom: L = 41.22 ± 0.38, a = 6.23 ± 0.15, b = 13.37 ± 0.16;

[0112] The whole wheat steamed buns made with the control bacteria were:

[0113] Top: L = 34.36 ± 0.43, a = 5.86 ± 0.11, b = 10.88 ± 0.15;

[0114] The bottom L = 37.55 ± 0.25, a = 4.83 ± 0.12, and b = 10.01 ± 0.15.

[0115] The comparison shows that the whole wheat steamed buns made with HTD7 have a better color.

[0116] Sensory evaluation: The higher the score, the better the evaluation. Ten people will evaluate the evaluation, and the scoring criteria are as follows: appearance and shape, structure, elasticity and toughness, viscosity, flavor and color, with scores of 20, 15, 20, 10, 20 and 15 points respectively, for a total score of 100 points.

[0117] The sensory score of whole wheat steamed buns made with HTD7 was 83±1.5, while the sensory score of whole wheat steamed buns made with the control bacteria without HTD7 was 72±3.0.

[0118] Making whole wheat bread:

[0119] The recipe, by weight, includes: 50 parts whole wheat flour, 50 parts all-purpose flour, 3 parts butter, 60 parts distilled water, 5 parts sugar, and 0.5 parts yeast. A 1.5% (v / w, mL / g) solution of *Westernella vulgaris* HTD7 bacterial culture (see Example 3) was added to the total amount of whole wheat flour and all-purpose flour. The control group was prepared using the control strain CICC 24453 instead of HTD7. The bread was made in a Panasonic SD-P103 bread maker, mode 6. The specific volume and color of the two types of bread were measured, and sensory evaluation was performed.

[0120] Specific gravity: 4.25 for whole wheat bread made with HTD7;

[0121] The control strain produced whole wheat bread had a yield of 3.21.

[0122] Color:

[0123] HTD7 makes whole wheat bread as follows:

[0124] Upper crown: L=42.95±0.35, a=9.14±0.14, b=17.20±0.22;

[0125] Bottom: L = 44.41 ± 0.29, a = 7.43 ± 0.15, b = 15.47 ± 0.15;

[0126] Whole wheat bread made with the control bacteria was:

[0127] Upper crown: L = 41.65 ± 0.28, a = 10.95 ± 0.16, b = 18.14 ± 0.19;

[0128] Bottom: L = 39.46 ± 0.25, a = 9.74 ± 0.15, b = 16.01 ± 0.15.

[0129] The comparison shows that whole wheat bread made with HTD7 has a better color.

[0130] Sensory evaluation: 60 points maximum, with higher scores indicating better evaluation. Ten evaluators participated, and the scoring criteria are the same as in Table 2.

[0131] The sensory score of whole wheat bread made with HTD7 was 53±1.5, while the sensory score of whole wheat bread made with the control bacteria without HTD7 was 44±2.0.

[0132] Photos of whole wheat steamed buns and bread made with *Westernella fusionis* HTD7 and whole wheat steamed buns and bread made with the control bacterium CICC 24453 of the same genus can be found below. Figure 11 .

[0133] Example 6: High-pressure jet coupled bacterial fermentation to produce plant-based whole grain beverage

[0134] In this embodiment, roasted oats were mixed with water at 10% w, treated with a high-pressure jet (Beijing Collaborative Innovation Food Technology Co., Ltd., FJ-3037D) at 120 MPa, and then a 0.5% (v / w, mL / g) fusion of *Weissella confusa* HTD7 bacterial solution (the preparation method of the bacterial solution is described in Example 3) was added. The mixture was fermented at 25°C for 8 hours to produce a whole-grain oat beverage. A control strain of the same genus, CICC 24453 (DZJ) and *Lactobacillus plantarum* L1, was used for fermentation as a control. The test results (average of three tests) are shown in Table 3.

[0135] Table 3. Test results of whole grain beverages prepared by high-pressure jet coupling strains.

[0136]

[0137] Whole-grain oat beverages fermented with Weissella confusa HTD7 are high in bioactive substances (dry basis), dietary fiber, β-glucan, and folic acid. Furthermore, stability is high according to the stability clarification index test.

[0138] Example 7: Genomic Analysis

[0139] This embodiment analyzed the genome of Weissella confuse HTD7, and the results are shown in Table 4. Figures 12 to 14 .

[0140] Table 4

[0141]

[0142] The results of the Circos genome mapping analysis are shown below. Figure 12 The results of carbohydrate activity enzyme analysis are shown in [the table below]. Figure 13 The linear map of the gene clusters for secondary metabolite synthesis is shown below. Figure 14 .

[0143] Example 8: Color change of the strain

[0144] In this example, *Weissella confusa* HTD7 was cultured in LB medium at different pH values ​​(2, 3, 4, 5, 6, 6.9, 6.9, 7, 8, 9, 10) at 37°C, and the color of *Weissella confusa* HTD7 during culture was studied. The results are shown below. Figures 16 to 19 .

[0145] Figure 16 (Color after 24 hours): The color of the medium at pH 2.0 and pH 10.0 is brownish-brown, with the former being darker than the latter; the color of the medium at pH 3.0-pH 9.0 changes from pale yellow to golden yellow.

[0146] Figure 17 (Color after 48 hours): The medium at pH 2.0 is brown, and the medium gradually turns red from pH 3.0 to pH 10.0.

[0147] Figure 18 (72-hour color): The color at pH 2.0 is brown; compared to 48 hours, the red color in the pH 3.0-pH 10.0 medium becomes darker.

[0148] Figure 19 (96-hour color): The color of the medium at pH 2.0 is brown; the color of the medium gradually turns reddish-pink from pH 3.0 to pH 8.0, and the color changes from light red to light brown from pH 9.0 to pH 10.0.

[0149] In summary, the color of the fused Weissella HTD7 bacteria varies after culturing at different pH values ​​for different times.

[0150] Example 9

[0151] Fusion Weissella HTD7 is a type of lactic acid bacteria probiotic. Its cells, liquid, and powder have different colors at different pH values. It can be directly applied or used on the surface of food as an edible decorative pigment.

[0152] In this embodiment, the bacterial cells prepared in step 1 of Example 2 are directly applied to the surface of steamed buns. It can be seen that they exhibit a bright color, which can effectively decorate food. See [link to relevant documentation]. Figure 20 .

[0153] In this embodiment, *Westernella* HTD7 is fused and applied to paper to form a flower pattern. The bacterial cells cultured in MRS medium for 72 hours serve as the flower center, and the bacterial suspension cultured in LB medium (pH 9) for 96 hours serve as the petals. See [link to relevant documentation]. Figure 21 .

[0154] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A strain of Weissella confusa HTD7, characterized in that, The accession number is CGMCCNo.29775.

2. A microbial agent, characterized in that, Contains the Weissella confusa HTD7 as described in claim 1; preferably, the inoculum comprises the fermentation broth of Weissella confusa HTD7.

3. The application of the Weissella confusa HTD7 fusion strain as described in claim 1, or the bacterial agent as described in claim 2, in the production of proteases, antibacterial agents, or food preservatives.

4. The application of the Weissella confusa HTD7 strain of claim 1, or the microbial agent of claim 2, in the preparation of high-fiber matrix cereal beverages or noodle products; preferably, the high-fiber matrix cereal is oats.

5. The use of the Weissella confusa HTD7 strain as described in claim 1, or the microbial agent as described in claim 2, in increasing the content of lactic acid, flavonoids, amino acids, β-glucan, dietary fiber, folic acid, and / or reducing sugars in the products after oat fermentation.

6. The use of the Weissella confusa HTD7 as described in claim 1, or the microbial agent as described in claim 2, in improving the quality of bread or steamed buns or as an edible pigment.

7. An oatmeal beverage or sweetened cereal, characterized in that, The oat beverage is prepared by fermenting oats with the fused Weissella confota HTD7 as described in claim 1 or the bacterial agent as described in claim 2. Preferably, in the preparation of the oat beverage, the oats are first homogenized by high-pressure jet jet before fermentation.

8. A type of bread, characterized in that, Oat products fermented with Weissella confusa HTD7 as described in claim 1 or the microbial agent as described in claim 2, or containing Weissella confusa HTD7 as described in claim 1 or the microbial agent as described in claim 2.

9. A type of steamed bun, characterized in that, It contains the Weissella confusa HTD7 strain as described in claim 1 or the bacterial agent as described in claim 2.

10. An edible pigment, food preservative, or antibacterial agent, characterized in that, It contains the Weissella confusa HTD7 strain as described in claim 1 or the bacterial agent as described in claim 2.

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