Hyndrixis coagulans and application thereof as a helper starter culture in preparing cheese

Abstract

A strain of H. cohnii and its application as a helper starter in preparing cheese belong to the field of microbial fermentation technology. In view of the problems of poor acid production adaptability, insufficient protein hydrolysis and single flavor of the existing H. cohnii and traditional lactic acid bacteria starter in the system of bovine milk-hammar bean double protein cheese, the present application screens a strain of H. cohnii CCMXJ1007, uses the strain as a helper starter, uses Lactobacillus paracasei as a main starter, and uses bovine milk and hammar bean slurry as protein raw materials to prepare double protein cheese. In the process, the H. cohnii CCMXJ1007 as a helper starter has achieved remarkable beneficial effects in shortening the fermentation period, reducing the pH value, increasing the content of free amino acids and fatty acids, enriching the flavor level, improving the texture and taste, and improving the nutritional value.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a strain of Heinrich's coagulans and its application as an auxiliary fermentation agent in cheese preparation. Background Technology

[0002] Double-protein cheese refers to a dairy product made using both milk protein and plant-based protein as a common protein matrix. Following traditional cheese processing techniques, it undergoes pre-acidification, the addition of a coagulant to co-coagulate the animal and plant proteins, followed by whey removal, pressing, and maturation (or unmatured) processes. The core advantage of double-protein cheese lies in its nutritional balance—the combination of animal and plant proteins achieves complementary amino acid profiles. Simultaneously, replacing some milk protein with plant protein effectively reduces raw material costs, providing nutritional value comparable to or even superior to traditional cheese at a lower price. Therefore, the development of double-protein cheese is of significant practical importance in alleviating milk shortages and enriching the variety of cheese products.

[0003] In the selection of plant protein raw materials, chickpeas (Cicer arietinum) are an ideal ingredient for plant-based cheese due to their excellent nutritional and sensory characteristics. Chickpeas are rich in protein (approximately 20%), dietary fiber, vitamins, and minerals. Compared with commonly used plant protein raw materials such as soybeans and peas, the most prominent sensory advantage of chickpea protein products lies in their neutral and mild flavor, without a noticeable beany taste. Soybean and pea products often produce volatile off-flavor substances such as hexanal, 1-hexanol, and 1-octen-3-ol due to the action of lipoxygenase, forming unpleasant flavors such as raw bean taste, which seriously affects product acceptance; while chickpea protein products have a milder flavor profile and can be directly used in food processing without complex deodorization treatment. This natural advantage allows them to better present the original milky aroma characteristics of dairy products in plant-based cheese production. Existing studies have attempted to partially replace cow's milk with chickpea milk in the production of compound cheeses. The results show that the addition of chickpeas not only reduces production costs and achieves nutritional complementarity between plants and animals, but also effectively improves the flavor of the cheese.

[0004] Traditional cheese production relies primarily on lactic acid bacteria starter cultures, including Lactobacillus bulgaricus (Lactobacillus bulgaricus). Lactobacillus bulgaricus ) and Streptococcus thermophilus ( Streptococcus thermophilusLactic acid bacteria (LAB) are the most commonly used fermentation starter cultures. However, in practical applications, these starter cultures still have several limitations: firstly, the acid production rate of LAB is relatively slow, resulting in a long fermentation cycle; secondly, conventional LAB have a weak ability to hydrolyze proteins, leading to a limited variety and quantity of flavor compounds and flavor precursors (such as amino acids and fatty acids) produced during the maturation process, resulting in a single cheese flavor and insufficient aroma complexity. Therefore, screening and developing novel auxiliary starter cultures with excellent performance to shorten fermentation time and enrich cheese flavor has become an important research direction in the cheese processing field.

[0005] Heinrich's coagulation bacteria ( Heyndrickxia coagulans *Heinzia coagulans* is a Gram-positive, spore-forming bacterium that combines the characteristics of both lactic acid bacteria and Bacillus. It metabolizes various products, including L-lactic acid, bacteriocins, and amylase, and exhibits excellent resistance to stresses such as high temperatures, acid, and bile salts. In recent years, *Heinzia coagulans* has gradually attracted attention due to its unique probiotic functions. Previous studies have used *Heinzia coagulans* as an auxiliary starter in dairy fermentation, showing its potential in shortening fermentation time and improving cheese texture and flavor. However, the metabolic characteristics, enzyme production capacity, and flavor contribution of *Heinzia coagulans* are significantly strain-dependent. Strains obtained from different sources or under different screening conditions show considerable differences in their acid production rate, protease and peptidase activities, and the types and amounts of flavor substances produced. While existing technologies have reported the use of Heinrich's coagulate bacteria in dairy fermentation, the strains used are mostly commercially available general strains or strains isolated from traditional fermented foods. The enzyme production characteristics and fermentation performance of these strains in specific protein matrix environments still need improvement. In particular, in the cow's milk-chickpea milk dual-protein system, the introduction of chickpea protein changes the protein composition and buffering capacity of the system, placing higher demands on the acid production metabolism, protein hydrolysis adaptability, and flavor generation ability of the fermentation strains. Existing strains are still unable to meet the production requirements of high-quality dual-protein cheese.

[0006] Furthermore, current research on dual-protein cheese largely focuses on combinations of cow's milk and soy protein, while research on dual-protein cheese blends of cow's milk and chickpea milk is still in its early stages. The fermentation process still uses traditional lactic acid bacteria starter cultures, and there are no reports of applying *Heinrichia coagulans* to cow's milk-chickpea milk dual-protein cheese systems. In particular, chickpea protein and cow's milk casein differ significantly in molecular structure, gel properties, and buffering capacity. These differences directly affect the acid production kinetics and proteolytic behavior of the strains during fermentation, making it difficult for fermentation strains suitable for pure cow's milk systems to achieve the same excellent fermentation results in dual-protein systems. This further increases the technical difficulty of screening suitable strains and optimizing the fermentation process.

[0007] In summary, current technologies lack a *Hydrilla verticillata* strain and its application technology that can efficiently adapt to the dual-protein matrix and significantly enhance the flavor of milk-chickpea milk dual-protein cheese systems. Therefore, screening for a high-performance *Hydrilla verticillata* strain and applying it to the fermentation of milk-chickpea milk dual-protein cheese is of significant research value and has broad application prospects. This aims to address the technical problems of poor acid production adaptability, monotonous flavor, and incomplete protein hydrolysis in existing starter cultures within dual-protein systems. Summary of the Invention

[0008] To address the issues of poor acid production adaptability, insufficient protein hydrolysis, and monotonous flavor of existing Heinrich's coagulation strains and traditional lactic acid bacteria starter cultures in milk-chickpea dual-protein cheese systems, and the lack of a dedicated Heinrich's coagulation strain that can efficiently adapt to the milk-chickpea dual-protein matrix and significantly improve fermentation performance, this invention provides a Heinrich's coagulation strain and its application as an auxiliary starter culture in milk-chickpea dual-protein cheese fermentation.

[0009] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a strain of Heinrich's coagulation bacteria (Hydrogendra hygroscopicus). Heyndrickxia coagulans The strain CCMXJ1007 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 68293, deposited on May 18, 2026. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology.

[0010] A second objective of this invention is to provide a method for preparing double-protein cheese using the aforementioned *Hydroxella coagulans* CCMXJ1007, the method comprising the following steps: S1. Mix sterilized milk with chickpea milk to prepare raw milk; S2, mix the bacterial solution of coagulated Heinrichia coli CCMXJ1007 with Lactobacillus paracasei ( Lactobacillus paracasei SMN-LBK bacterial culture was inoculated into the raw milk prepared by S1, thoroughly mixed, rennet was added, sheared and stirred, and placed at 40℃ for coagulation fermentation to form curds and whey. S3. After being filtered through hanging bags, molded, vacuum-packed, and then cured, double-protein cheese is obtained.

[0011] In one embodiment of the present invention, the sterilized milk in S1 is obtained by pasteurizing milk.

[0012] In one embodiment of the present invention, the preparation method of the chickpea milk in S1 is as follows: chickpeas are washed, soaked and pretreated, mixed with water and pulped, the pulp is filtered out, and the mixture is heated to a boil and kept for 5-10 minutes.

[0013] In one embodiment of the present invention, the ratio of chickpeas to water is 1 g:(6-10) mL.

[0014] In one embodiment of the present invention, the volume ratio of milk to chickpea milk in S1 is 6:4.

[0015] In one embodiment of the present invention, the bacterial suspensions of *Hydrilla verruca coagulans* CCMXJ1007 and *Lactobacillus paracasei* SMN-LBK, described in S2, both have a concentration of 10. 5 -10 9 CFU / mL.

[0016] In one embodiment of the present invention, the volume ratio of the bacterial suspension of *Hydroxella henryi* CCMXJ1007 and the bacterial suspension of *Lactobacillus paracasei* SMN-LBK in S2 is 1:4, and the total inoculum amount is 5%.

[0017] In one embodiment of the present invention, the amount of rennet added in S2 is 0.02%.

[0018] In one embodiment of the present invention, the fermentation time in S2 is 16 h.

[0019] In one embodiment of the present invention, the hanging bag filtration described in S3 refers to suspending the fermented milk with sterile gauze and filtering the whey in a constant temperature and humidity chamber at 10-15℃ for 5 hours until the whey stops dripping.

[0020] In one embodiment of the present invention, S3 refers to placing the cheese filtered through the hanging bag into a sterilized mold and pressing it to form the basic shape of the cheese.

[0021] In one embodiment of the present invention, S3 refers to the post-ripening of packaged cheese under constant conditions of 10°C and 30% relative humidity for 20-60 days.

[0022] A third objective of this invention is to provide a double-protein cheese prepared by the above method.

[0023] The beneficial effects of this invention are: Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly improves the fermentation performance and physicochemical quality of cheese This invention uses *Hydroxella coagulans* (… Heyndrickxia coagulansCCMXJ1007 was used as an auxiliary fermentation strain in cheese production, exhibiting excellent acid-producing ability during fermentation. Compared with the *Lactobacillus paracasei* and *Bacillus coagulans* BC30 groups, cheese fermented with CCMXJ1007 showed a further decrease in pH, a significantly shorter coagulation time, and effectively improved fermentation efficiency. The lower pH not only helps inhibit the growth of unwanted microorganisms and extend the product's shelf life but also creates a suitable acidic environment for subsequent protein hydrolysis and flavor formation.

[0024] (2) Significantly improves the degree of protein hydrolysis and increases the content of free amino acids. In this invention, the *Heinrichia coagulans* CCMXJ1007 exhibits strong proteolytic activity, effectively decomposing proteins in the cheese matrix and releasing a large amount of free amino acids. Experimental results show that the total amount of free amino acids in cheese fermented with CCMXJ1007 is significantly higher than that in the control group. Specifically, the content of umami-related amino acids—leucine, glutamic acid, and aspartic acid—is significantly increased, while the level of methionine (a sulfur-containing amino acid that can degrade to produce a pungent flavor) is significantly decreased. Free amino acids are not only important flavor substances but also precursors for Maillard reactions and further metabolism into volatile flavor compounds; therefore, increasing their content lays a solid foundation for the multi-layered construction of cheese flavor.

[0025] (3) Significantly enhances lipolysis and increases the content of free fatty acids. The *Hydrilla verruca coagulans* CCMXJ1007 strain in this invention possesses certain lipase activity, which can promote milk fat hydrolysis and significantly increase the content of free fatty acids in cheese. Free fatty acids are not only one of the important flavor compounds in cheese, but also precursors to various volatile flavor compounds such as esters, alcohols, aldehydes, and ketones, providing a sufficient substrate source for the formation of subsequent flavor compounds, thereby endowing cheese with a fuller and more complex flavor profile.

[0026] (4) Synergistically optimize the flavor profile, enrich the aroma layers and reduce unpleasant flavors. The composition of volatile flavor compounds is an important indicator for evaluating cheese quality. The cheese fermented with *Hydrilla verticillata* CCMXJ1007 of this invention exhibits significantly better variety and content of volatile flavor compounds than the control group. Specifically, the cheese shows the highest levels of nonanal (characterized by citrus and beeswax aromas), acetoin, 2-heptanone, and 2-nonanone (characterized by buttery and fruity aromas), and ethyl acetate and butyl acetate (characterized by floral and fruity aromas). Simultaneously, the content of free fatty acids such as butyric acid and hexanoic acid, associated with pungent and rancid odors, is significantly reduced, and sulfides and terpenoids also gradually decrease. Therefore, the addition of *Hydrilla verticillata* CCMXJ1007 effectively enhances beneficial flavor compounds while inhibiting the formation of undesirable flavor compounds, achieving synergistic optimization of the flavor spectrum.

[0027] (5) Synergistically improve textural properties, enhance taste profile and overall acceptability In this invention, *Hydrilla verruca coagulans* CCMXJ1007, through its enzymatic properties, moderately hydrolyzes casein and fat, significantly improving the textural properties of cheese. Compared to the *Lactobacillus paracasei* and *Bacillus coagulans* BC30 groups, cheese fermented with CCMXJ1007 exhibits superior texture: reduced hardness and chewiness, increased elasticity and stickiness, resulting in a finer, silkier texture. In terms of taste, saltiness, acidity, and richness are all enhanced, creating a more complex flavor profile and a richer milky aroma. Sensory evaluation results show that the CCMXJ1007 group of cheese outperforms the control group in flavor pleasantness, saltiness palatability, and flavor persistence. The silky texture helps to moderately release undesirable flavors, significantly improving overall acceptability.

[0028] (6) Simultaneously enhance nutritional functionality and health attributes The cheese fermented with *Heinrichia coagulans* CCMXJ1007 in this invention is rich in free amino acids and free fatty acids, including a variety of essential amino acids and fatty acids, making it easily digestible and absorbable, and significantly enhancing its nutritional value. Furthermore, *Heinrichia coagulans*, a recognized probiotic, provides the cheese with additional gut health regulating functions, giving the product both delicious taste and health benefits.

[0029] In summary, this invention, by employing *Hydrilla verruca coagulans* CCMXJ1007 as an auxiliary starter culture, has achieved significant beneficial effects in shortening the fermentation cycle, lowering the pH value, increasing the content of free amino acids and fatty acids, enriching flavor profiles, improving texture and mouthfeel, and enhancing nutritional value. Compared with conventional lactic acid bacteria starter cultures and known *Hydrilla verruca coagulans* strains in the prior art, the technical solution of this invention achieves synergistic improvements in multiple dimensions, possessing outstanding substantive characteristics and significant progress. It provides a novel solution for the development of high-quality functional cheeses and has broad market application prospects.

[0030] Preservation of biological materials: The coagulating Heinrich's bacterium in this invention ( Heyndrickxia coagulans CCMXJ1007 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 68293, deposited on May 18, 2026. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology. Attached Figure Description

[0031] Figure 1 The images show the morphological identification results of strain CCMXJ1007; where A is a colony morphology image and B is a cell morphology image observed using Gram staining. Figure 2 The graph shows the results of acid production capacity testing for different coagulant Heinrich's bacteria. Figure 3 The graph shows the results of protease production capacity assays for different coagulating Heinrich's bacteria. Figure 4 The graph shows the results of the detection of aminopeptidase production capacity of different coagulant bacteria. Figure 5 The radar chart corresponding to the electronic nose measurement; Figure 6 The radar chart corresponding to the electronic tongue measurement; Figure 7 The radar chart corresponding to the sensory evaluation; Figure 8 The heatmap corresponding to the determination of free amino acids; Figure 9 The heatmap corresponding to the determination of free fatty acids; Figure 10 Thermographs for determining volatile compounds using GC-MS. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0034] The microbial strains involved in this invention originate from the following sources: Heinrich's coagulation bacteria ( Heyndrickxia coagulans CCMXJ1007 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 68293, deposited on May 18, 2026. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology.

[0035] Bacillus coagulans BC30 (also known as Bacillus coagulans GBI-30, 6086, and Bacillus coagulans has now been renamed Heinrich's coagulans), purchased from Shanghai Preservation Microbiology Co., Ltd., with accession number SMHCC D24758.

[0036] Lactobacillus paracasei ( Lactobacillus paracasei SMN-LBK is disclosed in Chinese patent application number 202211496004.4, authorization announcement number CN115517367B, entitled "Application of Lactobacillus paracasei SMN-LBK in the preparation of products promoting intestinal health".

[0037] Example 1: Isolation and identification of Heinrich's coagulans CCMXJ1007 1. Isolation of strain CCMXJ1007 Weigh 10 g of fermented soybean paste and add it to 90 mL of sterile water. Mix the mixture on a shaker at 35℃ and 160 rpm for 1 h. Use a pipette to add 200 μL of the supernatant to 5 mL of MRS liquid culture medium and incubate at 42℃ for 24 h.

[0038] In the prepared MRS liquid medium, 0.5 mL of bacterial suspension was added to 4.5 mL of sterile physiological saline and vortexed to mix. This mixture was then serially diluted. Appropriate dilutions were selected for plating on solid plates. The plated plates were inverted and incubated at 42°C for 48 h.

[0039] Bacteria with inconsistent colony morphology in the solid culture medium were isolated and purified. The selected bacteria were Gram-stained and examined under a microscope. Suspected strains of *Hydrilla verticillata* coagulans were preliminarily screened. These strains were repeatedly streaked on MRS solid medium for purification, and finally, purified *Hydrilla verticillata* coagulans strains were obtained and stored in glycerol tubes at -80°C.

[0040] The formula for MRS medium is as follows: 10 g peptone, 5 g beef extract powder, 5 g yeast powder, 20 g glucose, 5 g anhydrous sodium acetate, 2 g diammonium hydrogen citrate, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, add 1000 mL distilled water, adjust the pH to 6.5, and then autoclave at 120℃ for 30 minutes. For solid MRS medium, an additional 15 g agar powder needs to be added.

[0041] 2. Identification of strain CCMXJ1007 A bacterial strain was obtained using the above isolation and purification method, which was designated CCMXJ1007, and its morphological and molecular biological characteristics were then identified.

[0042] (1) Morphological identification of strain CCMXJ1007 Strain CCMXJ1007 was inoculated onto MRS agar plates using a three-zone streak method and incubated at 37°C for 24 h. The size, shape, and other characteristics of bacterial colonies were observed and recorded. Standard Gram staining was performed on strain CCMXJ1007, and the morphology of the bacteria was observed under an optical microscope after staining.

[0043] Colony observation results as follows Figure 1 As shown in Figure A, strain CCMXJ1007 appears as white spherical pellets on an MRS plate. Gram staining results are as follows... Figure 1 As shown in Figure B, after staining and microscopic examination, strain CCMXJ1007 was found to be a Gram-positive bacterium with a rod-shaped morphology.

[0044] (2) Molecular biological identification of strain CCMXJ1007 The strain CCMXJ1007 was inoculated into a suitable liquid culture medium and cultured. The bacterial cells were collected by centrifugation, and its genomic DNA was extracted for subsequent PCR amplification. Specific primers BCF2 (nucleotide sequence shown in SEQ ID NO.1) and BCR2 (nucleotide sequence shown in SEQ ID NO.2) were used for PCR amplification of the strain's 16S rDNA. The PCR reaction system was as follows: 2 μL upstream primer, 2 μL downstream primer, 2 μL DNA, 25 μL 2×Taq PCR premix reagent II, and ddH2O was added to a total reaction volume of 50 μL. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ repair extension for 5 min. The PCR products were validated by agarose gel electrophoresis. The validated PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequencing. The obtained 16S rDNA sequence was compared with the NCBI database using BLAST to determine its similarity to known species.

[0045] The 16S rDNA sequence of strain CCMXJ1007, obtained by sequencing, is shown in SEQ ID NO.3: SEQ ID NO.1: 5'-TGCCTTCCTCGTCCATCAAAA-3'; SEQ ID NO.2: 5'-AGCAAAAGAATGGCGGAACAC-3'; SEQ ID NO.3:

[0046] The 16S rDNA alignment results showed that the 16S rDNA sequence of strain CCMXJ1007 was similar to that of *Hydroxella coagulans* (…). Heyndrickxia coagulans The highest similarity was found between strain CCMXJ1007 and strain H. coagulans. Therefore, based on the combined morphological and molecular biological identification results, strain CCMXJ1007 was identified as *H. coagulans*. Heyndrickxia coagulans The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 68293, on May 18, 2026. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology.

[0047] Example 2: Detection of acid production, protease production, and aminopeptidase production capabilities of strain CCMXJ1007 This invention compares the acid-producing capacity, protease-producing capacity, and aminopeptidase-producing capacity of strain CCMXJ1007 and other laboratory-preserved Heinrich's coagulans.

[0048] 1. Detection of acid production capacity Each coagulating Heinrich's bacterium was inoculated into 10% sterile skim milk powder medium and cultured at 42°C for 24 h. The pH of the culture system was measured before and after the culture, and the acid production capacity of the strain was expressed as ΔpH.

[0049] Excessive acid production causes the pH to drop too quickly, leading to excessive curd shrinkage, excessive whey discharge, and a hardened, crumbly cheese texture, thus reducing yield. Experimental results show that strains D3, D4, D6, D13, CCMXJ1007, D17, D18, D25, D33, P6, P13, and P22 have an acid production capacity ΔpH ≤ 1.1. Figure 2 These substances will not affect cheese maturation and can be used in subsequent cheese making processes.

[0050] 2. Detection of protease and aminopeptidase production capacity Protease activity assay: The test strain was inoculated into a suitable liquid culture medium and cultured in a shaker at 37°C for 24 h. The fermentation broth was then centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant was collected as the crude enzyme solution. Using 1% (w / v) casein solution (dissolved in glycine-sodium hydroxide buffer at pH 10.0) as the substrate, 1.0 mL of the crude enzyme solution was mixed with 1.0 mL of the substrate solution and reacted in a water bath at 40°C for 15 min. The reaction was then terminated by adding 3.0 mL of Folin-Ciocalteu reagent, and colorimetric analysis was performed. After centrifugation, the absorbance of the reaction solution was measured at 680 nm. The enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute at 40°C and pH 10.0.

[0051] Aminopeptidase activity assay: The test strain was cultured in a suitable medium at 37°C for 24 h, and the cell pellet was collected by centrifugation at 4°C and 8000 r / min for 10 min. The pellet was washed twice with 50 mM phosphate buffer (pH 7.0) and resuspended in the same buffer. The cells were disrupted by sonication (200 W, 5 s on, 5 s off, total time 10 min) under ice bath conditions, and centrifuged at 4°C and 12000 r / min for 20 min. The supernatant was the crude enzyme solution. Following a colorimetric method, using L-leucine-β-naphthamide (L-Leu-β-NA, dissolved in 50 mM Tris-HCl buffer, pH 8.0) as the substrate, 0.5 mL of crude enzyme solution was mixed with 0.5 mL of substrate solution (1 mM). The mixture was incubated in a 37°C water bath for 30 min, then 1.0 mL of 1 M HCl was added to terminate the reaction. Finally, 2.0 mL of 0.1% (w / v) p-dimethylaminocinnamaldehyde (DMACA) ethanol solution was added for color development. The absorbance was measured at 540 nm. Enzyme activity units (U) are defined as the amount of enzyme required to release 1 μmol of L-leucine per minute by hydrolyzing the substrate at 37°C and pH 8.0.

[0052] Proteases degrade casein into polypeptides, which are fundamental to cheese maturation, improving texture and releasing flavor precursors. Aminopeptidases further hydrolyze polypeptides into free amino acids, directly contributing umami and sweetness, while simultaneously degrading bitter peptides, thus reducing bitterness and enhancing umami. Experimental results show that, compared to other *Heinrichia coagulans* strains, strain CCMXJ1007 exhibits the highest levels of both protease and aminopeptidasease activities. Figure 3 and Figure 4This means that this strain can efficiently hydrolyze casein during cheese maturation, releasing a large amount of free amino acids and flavor precursors. It can effectively degrade bitter peptides to improve taste and provide sufficient substrates for the formation of aroma compounds such as fruit and cream. Meanwhile, strain CCMXJ1007 exhibited low acid production characteristics in the initial screening, ensuring that it does not damage the curd structure while enhancing flavor. In summary, *Heinrichia coli* CCMXJ1007 possesses ideal characteristics that balance flavor optimization and texture preservation, making it a high-quality auxiliary fermentation strain. Therefore, *Heinrichia coli* CCMXJ1007 was subsequently selected for auxiliary fermentation of cheese.

[0053] Example 3: Application of Heinrich's coagulans CCMXJ1007 in cheese making 1. Preparation of fermentation agent In this embodiment, different combinations of fermenting agents and their inoculation ratios were used, as shown in Table 1.

[0054] Table 1. Preparation schemes of different fermentation agents

[0055] Note: The inoculation ratio of the starter culture is the percentage of the starter culture volume to the skim milk volume, based on the volume of the raw milk.

[0056] 2. Making chickpea double protein cheese The production process of chickpea double protein cheese is as follows: raw milk → addition of lactic acid bacteria → addition of rennet → curd fermentation → hanging bag filtration → molding → vacuum packaging → post-ripening.

[0057] (1) Raw milk preparation: Milk is pasteurized using pasteurization, and fresh sterilized milk is kept at 65℃ for 30 minutes. Meanwhile, chickpeas are washed, soaked in sufficient water, and then mixed with water at a ratio of 1 g: 8 mL to make a paste. The paste is filtered out using a 200-mesh sieve to obtain chickpea milk. The chickpea milk is then heated to a boil and kept at that temperature for 5-10 minutes to remove the beany taste. After cooling, the chickpea milk is thoroughly mixed with fresh milk at a volume ratio of 40:60.

[0058] (2) Addition of lactic acid bacteria: Inoculate the different combinations of starter cultures in Table 1 above into the sterilized raw milk according to the corresponding inoculation amount, mix thoroughly, and ensure that the starter cultures are evenly distributed in the milk; (3) Adding rennet: Each group was given 0.02% of the raw milk mass of rennet and then sheared and stirred. (4) Curd fermentation: The dairy product is placed at 40°C for 16 h to coagulate and form curds and whey; (5) Hanging bag filtration: Hang the fermented milk with sterile gauze and filter the whey in a constant temperature and humidity chamber at 10-15℃ for 5 hours until the whey stops dripping.

[0059] (6) Molding: The cheese filtered through the hanging bag is placed into a sterilized mold and pressed to form the basic shape of the cheese.

[0060] (7) Vacuum packaging and ripening: The packaged cheese is placed under constant conditions of 10°C and 30% relative humidity for 60 days to ripen.

[0061] 3. Determination of physicochemical properties of cheese Samples of cheese from each group were taken and tested at different maturation times (1 day, 10 days, 20 days, 30 days, 40 days, 50 days, and 60 days). The pH value of each group of cheese was measured using a pH meter. The moisture content of the cheese was determined using the direct drying method in GB5009.3-2016. The fat content of the cheese was determined using the Soxhlet extraction method in GB5009.6-2016. The protein content of the cheese was determined using the Kjeldahl method in GB5009.5-2016. Each group was tested in triplicate. The results of the pH, moisture content, and nutrient content measurements of each group of cheese are shown in Table 2.

[0062] Table 2. Results of pH, moisture content and nutrient content of cheese in each group.

[0063] The physicochemical indicators of cheese are the core basis for evaluating its quality, safety, and process feasibility. They objectively reflect the degree of acidity, protein and fat decomposition during fermentation, and explain differences in texture and flavor. Table 2 shows that the basic physicochemical indicators (protein, fat, and moisture content) of the cheeses in the *Hydrix henryi* CCMXJ1007 group did not differ significantly during the maturation process. Furthermore, the protein content of all cheese samples ranged from 12.5% ​​to 18.5%, the fat content from 1.20 to 2.01 g / 100g, and the moisture content (by weight) from 49.5% to 54.94%. This indicates that the addition of *Hydrix henryi* CCMXJ1007 does not affect the basic characteristics of the cheese and therefore does not alter its nutritional value. As shown in Table 2, compared with the *Lactobacillus paracasei* group and the *Bacillus coagulans* BC30 group, the addition of *Hydrix henryi* CCMXJ1007 resulted in a lower pH. The lower pH inhibits pathogens such as *Listeria*, improves the texture and shelf life of the cheese, promotes curd and whey removal, and helps extend shelf life. Simultaneously, it enhances flavor, accelerates protein hydrolysis, and generates more free amino acids and small peptides, making the umami and richness of the cheese more prominent.

[0064] 4. Determination of cheese texture Each group of cheese samples was cut into cubes of 2 cm × 2 cm × 1 cm, with 3 parallel samples prepared for each batch. The texture analyzer was used for the determination in TPA mode. The measurement temperature was room temperature, and the specific test parameters were: a speed of 5 mm / s before the test, a speed of 1 mm / s during the test, and a speed of 5 mm / s after the test. The probe model was TA36. Each treatment group was measured in triplicate, and the results are shown in Table 3.

[0065] Table 3. Texture analysis results of each group of cheeses

[0066] The results, shown in Table 3, revealed that the addition of *Hydrixerella coagulans* CCMXJ1007 significantly reduced the hardness and chewiness of cheeses fermented with *Lactobacillus paracasei* and those fermented with *Bacillus coagulans* BC30. This improved the cheese's texture, making it softer and smoother, enhancing its sensory appeal and better meeting consumer preferences for a softer texture. Except for the early maturation stage, the addition of *Hydrixerella coagulans* CCMXJ1007 resulted in cheeses exhibiting superior cohesiveness compared to the *Bacillus coagulans* BC30 fermentation group. Enhanced cohesiveness allows the cheese to better maintain its integrity during chewing, preventing it from crumbling or breaking apart, resulting in a fuller, more delicate chewiness, improving the overall texture of the product, and enhancing processing performance. This may be because *Hydrixerella coagulans* CCMXJ1007 accelerates cheese maturation, shortens the maturation period, and thus softens the cheese's texture and reduces its chewiness. In summary, the above results indicate that the addition of Heinrich's coagulating bacteria CCMXJ1007 makes the cheese more soft and silky.

[0067] 5. Electronic nose and electronic tongue measurement of cheese Volatile component profile analysis was performed on 12 cheese samples (3 groups × 4 time periods: early maturity, mid-maturity, late maturity, and late maturity) using the PEN3 electronic nose system from Airsense GmbH, Germany. This system is equipped with 10 metal oxide semiconductor sensors (W1C, W5S, W3C, W6S, W5C, W1S, W1W, W2S, W2W, and W3S) to analyze volatile components including aromatic compounds, nitrogen oxides, ammonia compounds, hydrogen, short-chain alkanes and aromatic compounds, methyl compounds, sulfides and terpenes, alcohols, ethers, aldehydes, ketones, organosulfur compounds and aromatic components, and long-chain alkanes. The results are as follows: Figure 5As shown in the figure. In addition, the electronic tongue taste characteristics of 12 cheese samples were detected and analyzed using the INSENT SA402B intelligent taste analysis system from Japan. This system is equipped with nine artificial lipid membrane sensors (AAE, CT0, CA0, GL1, C00, AE1, BT0, HA0, SW0), corresponding to nine taste attributes: sour, salty, astringent, bitter aftertaste, umami, richness, bitterness, and sweetness. The measurement results are shown in the figure. Figure 6 As shown.

[0068] Radar charts were used to illustrate the changes in flavor of cheese via electronic nose at different groups (Lactobacillus paracasei group, Heinrichia coagulans CCMXJ1007 group, and Bacillus coagulans BC30 group) and at different times (early maturity, middle maturity, late maturity, and late maturity). Figure 5 Overall, methyl compounds, alcohols, aldehydes, ethers, and ketones constitute the main volatile compounds in cheese. Compared with the *Lactobacillus paracasei* and *Bacillus coagulans* BC30 groups, the addition of *Hydrix henryi* CCMXJ1007 significantly increased the levels of methyl compounds and alcohols, ethers, aldehydes, and ketones in the cheese. These compounds are important contributors to the typical flavor of cheese, thus enriching the aroma profile of the cheese. For example, alcohols impart fruity and floral aromas, ketones provide creamy and nutty aromas, while aldehydes contribute nutty and caramel flavors, significantly enhancing the overall complexity and fullness of the aroma, as well as improving aroma release and persistence. The increase in ethers makes the cheese aroma more intense and persistent. Meanwhile, the addition of *Heinrichia coagulans* CCMXJ1007 gradually reduced the sulfides and terpenes in the cheese. Excessive levels of certain sulfides and terpenes can easily lead to unpleasant odors such as sulfur, rotten eggs, and pine resin. Appropriate reduction helps to weaken these pungent odors, making the pleasant flavors such as milk and fruit more prominent. The results indicate that *Heinrichia coagulans* CCMXJ1007 exhibits unique advantages in enhancing cheese flavor and reducing unpleasant odors during cheese fermentation.

[0069] Radar graphs were used to illustrate the changes in the electronic tongue flavor of cheese at different groups and time points (see...). Figure 6The results showed that the cheese samples exhibited the most prominent umami, richness, and saltiness responses. Compared with the *Lactobacillus paracasei* and *Bacillus coagulans* BC30 groups, the addition of *Hydrix hydrilla verticillata* CCMXJ1007 significantly increased Aftertaste-A (richness) in the cheese, which can improve flavor persistence and fullness. Richness reflects the duration and complexity of flavor in the oral cavity. Its increase means that the addition of *Hydrix hydrilla verticillata* CCMXJ1007 allows the cheese aroma and flavor to persist longer after swallowing, resulting in a fuller, rounder overall flavor experience and significantly enhancing the product's taste memory. Furthermore, the cheese with added *Hydrix hydrilla verticillata* CCMXJ1007 exhibited higher acidity and saltiness. Appropriate acidity provides a refreshing sensation and suppresses unpleasant flavors, while saltiness enhances the overall flavor intensity and highlights the aroma. The synergistic effect of both enhances the characteristic flavor of the cheese.

[0070] In summary, compared with the *Lactobacillus paracasei* group and the *Bacillus coagulans* BC30 group, the addition of *Hydrix henryi* CCMXJ1007 significantly increased the levels of methyl compounds, alcohols, aldehydes, ethers, and ketones in the cheese, enriching its aroma layers and enhancing aroma release and persistence. Simultaneously, sulfides and terpenoids gradually decreased, mitigating undesirable flavors. Furthermore, the addition of *Hydrix henryi* CCMXJ1007 increased the cheese's saltiness, acidity, and fullness. These results indicate that *Hydrix henryi* CCMXJ1007-assisted fermentation has a significant impact on the flavor of cheese and may contribute to improving its overall texture and flavor.

[0071] 6. Sensory evaluation of cheese Sensory evaluation of mature cheese samples was conducted by a panel of 10 trained reviewers (5 men and 5 women). Prior to the evaluation, all participants were informed of the compositional information and sensory characteristics of the samples to be tested and signed written informed consent forms. All reviewers had received systematic training in descriptive analytical methods. During the evaluation, a standardized descriptive vocabulary was established using a consensus approach to quantify the color, texture, flavor, aroma, saltiness, bitterness, and overall acceptability of the cheese samples. Each attribute was scored on a scale of 0 to 9 (1-2: very weak; 3-4: weak; 5-6: moderate; 7-8: strong; 9: very strong). During the formal testing, all samples were coded with three random numbers and presented in random order, with each sample measured twice. After evaluating each sample, reviewers rinsed their mouths with purified water to remove any oral residue, ensuring the accuracy of subsequent evaluations. The evaluation was conducted in a standard sensory analysis laboratory under controlled temperature and humidity conditions. Samples were uniformly placed in transparent, covered containers to prevent cross-contamination of odors, and the sample temperature was controlled at approximately 12±2℃ to simulate actual consumption conditions. The presentation amount of each sample was approximately 20 g, and the evaluation interval was 60 seconds to reduce the impact of taste fatigue on the evaluation results.

[0072] Sensory evaluation is one of the most traditional and direct methods for food testing. Figure 7 This study revealed the sensory characteristics of cheese during the early, middle, late, and final stages of maturation. Compared to the *Lactobacillus paracasei* and *Bacillus coagulans* BC30 groups, the addition of *Heinrichia coagulans* CCMXJ1007 resulted in a more pronounced flavor and saltiness in the cheese, along with improved flavor persistence, thus enhancing the overall sensory experience. The more pronounced flavor and saltiness gave the cheese with *Heinrichia coagulans* CCMXJ1007 a stronger taste impact, with distinct characteristics immediately noticeable upon tasting. The enhanced persistence ensured a lingering pleasant sensation in the mouth, resulting in a fuller and more complex tasting experience. This demonstrates the flavor-enhancing potential of *Heinrichia coagulans* CCMXJ1007. These results indicate that the addition of *Heinrichia coagulans* CCMXJ1007 not only strengthens the basic taste but also optimizes the release of cheese flavor, making it a high-quality candidate strain for co-fermentation. On the other hand, the addition of Heinrich's coagulant CCMXJ1007 contributes to the cheese's silky texture, which helps to mitigate unpleasant tastes and flavors. In conclusion, using Heinrich's coagulant CCMXJ1007 to aid fermentation improves the flavor and texture of the cheese.

[0073] 7. Determination of Free Amino Acid Content in Cheese The content of free amino acids in cheese was analyzed using an automated amino acid analyzer. Specifically, 40 mg of each cheese sample was placed in an ampoule, 10 mL of hydrochloric acid (6 mol / L) was added, and the mixture was then sonicated. The ampoule was then sealed with a spray gun and baked in a 110°C oven for 23 hours. 0.3 mL of the filtrate was removed from the cooled ampoule, evaporated to complete dryness at 70°C, and thoroughly mixed with 3 mL of sample diluent. The resulting solution was filtered through a 0.45 μm organic filter membrane and then analyzed. The instrument conditions were as follows: cation exchange column: 4.6 mm × 60 mm; column temperature: 57°C; detector temperature: 135°C; buffer flow rate: 0.45 mL / min; ninhydrin flow rate: 0.25 mL / min; injection volume: 20 μL. The analysis time for each sample was approximately 130 minutes.

[0074] Free amino acids are not only direct flavor contributors to the basic tastes of cheese, such as umami, sweetness, and bitterness, but also important precursors to volatile flavor compounds like alcohols, aldehydes, acids, and esters, playing a crucial role in enriching the aroma profile of cheese. Furthermore, free amino acids are more easily absorbed by the human body than bound amino acids, effectively enhancing the nutritional value of cheese. Therefore, the composition and content of free amino acids directly affect the flavor quality, nutritional characteristics, and consumer acceptance of cheese.

[0075] A total of 15 free amino acids were detected in this study, including aspartic acid, glutamic acid, alanine, leucine, phenylalanine, and arginine. Figure 8 The results showed that, compared with the *Lactobacillus paracasei* group and the *Bacillus coagulans* BC30 group, the leucine content in the cheese with the addition of *Hydrilla xanthii* CCMXJ1007 was significantly increased. Leucine can be converted into 3-methylbutyraldehyde through the Stryker degradation pathway. This compound has a rich malty and cocoa aroma and is an important contributor to the typical aroma of mature cheese. 3-methylbutyraldehyde can be further metabolized into 3-methylbutanol and 3-methylbutyric acid, enriching the fruity and acidic layers of the cheese.

[0076] Meanwhile, compared with the *Lactobacillus paracasei* group and the *Bacillus coagulans* BC30 group, the aspartic acid and glutamic acid content in the cheese of the CCMXJ1007 group was significantly increased. Aspartic acid and glutamic acid can impart umami flavor to cheese and reduce the perception of bitterness. Their metabolites, acetoin and diacetyl, can provide creamy aroma and umami characteristics to fermented cheese. In addition, compared with the control group, the methionine level in the cheese of the CCMXJ1007 group was significantly reduced. Methionine is a key precursor to sulfur-containing volatile compounds (such as methanethiol and dimethyl disulfide), which usually give cheese typical pungent, burnt, fishy, ​​and sulfurous odors. The reduction in its precursor content may inhibit the formation of such unpleasant flavor substances, thereby reducing off-flavors.

[0077] In conclusion, the fermentation-assisted fermentation of Heinrichia coagulans CCMXJ1007 can, to a certain extent, increase the content of aliphatic amino acids and umami amino acids in cheese, enrich the fruit and acid aromas, enhance the creamy aroma and umami flavor, while reducing the level of bitter amino acids and reducing the formation of off-flavors, thus playing a positive role in improving the overall flavor quality of cheese.

[0078] 8. Determination of free fatty acid content in cheese Accurately weigh 10.0 g of cheese sample and place it in a stoppered Erlenmeyer flask. Add 20 mL of a dichloromethane-ethanol mixture (dichloromethane to ethanol volume ratio of 2:3) and extract by shaking at room temperature for 30 min. After filtering the extract through filter paper, collect the filtrate in a round-bottom flask and concentrate it under reduced pressure at 40°C using a rotary evaporator until the residue is approximately 2-3 mL.

[0079] Take approximately 50 mg of the above-mentioned concentrated fat into a stoppered test tube, add 2 mL of 6 mol / L hydrochloric acid solution, and hydrolyze at 100°C for 60 min until the solution becomes clear and transparent. Remove the tube and cool to room temperature (25-30°C). Add 2 mL of n-hexane to the hydrolysate and extract by vortexing (1500 r / min) for 120 s. After standing and separating the layers, collect the upper organic phase, dehydrate and dry it with anhydrous sodium sulfate, and set aside for later use.

[0080] Fatty acid methyl esters were analyzed using gas chromatography-mass spectrometry (GC-MS, Agilent 7890B). A DB-23 capillary column (60 m × 0.25 mm × 0.25 μm) was used, with an injection port temperature of 250°C, a detector (FID) temperature of 250°C, and a split ratio of 1:100. The autosampler injection volume was 1 μL. The total run time was 47 min. Qualitative analysis of fatty acid methyl esters was determined by comparing their retention times with those of the FAME-37 mixed standard. Quantification was performed using peak area normalization or the internal standard method.

[0081] Free fatty acids (FFAs) play a crucial role in catabolism, and their metabolites are important sources of aromatic compounds that contribute to the typical flavor of cheese. In this experiment, 31 FFAs (with carbon chain lengths ranging from C4:0 to C24:1) were detected in three cheese samples. Figure 9 This includes 16 saturated fatty acids (SFA), 8 monounsaturated fatty acids (MUFA), and 7 polyunsaturated fatty acids (PUFA).

[0082] The results showed that, compared with the *Lactobacillus paracasei* group and the *Bacillus coagulans* BC30 group, the saturated fatty acid content in the cheese with added *Hydrix henryi* CCMXJ1007 was significantly increased. Increased saturated fatty acid content typically imparts a denser texture, a fuller creamier feel, and smoothness to cheese, thus improving its chewing characteristics. Furthermore, saturated fatty acids are key precursors to various volatile flavor compounds such as methyl ketones and lactones; their increased content contributes to enriching the aroma layers of cheese and enhancing its typical flavor characteristics.

[0083] Meanwhile, compared with the two control groups, the linoleic acid content in the cheese of the CCMXJ1007 group was significantly increased. Linoleic acid not only has physiological activities such as antioxidation and liver protection, but also serves as a fatty acid precursor, synthesizing a variety of unique aromatic compounds through enzymatic reactions. Furthermore, the content of unsaturated fatty acids (including myristic acid, palmitoleic acid, oleic acid, and erucic acid) in the cheese of the CCMXJ1007 group was significantly higher than that in the two control groups. Unsaturated fatty acids help reduce the hardness of cheese, giving it a softer, smoother texture, indicating that *Heinrichia coagulans* CCMXJ1007 can effectively promote the production or accumulation of unsaturated fatty acids in cheese. The effects of *Lactobacillus paracasei* and *Bacillus coagulans* BC30 groups on the unsaturated fatty acid content were not significant.

[0084] In conclusion, the fermentation-assisted fermentation of Heinrich's coagulated bacteria CCMXJ1007 can, to some extent, increase the fatty acid content in cheese and enrich the aroma layers such as fruit and cream, thus playing a positive role in improving the overall flavor and quality of cheese.

[0085] 9. GC-MS determination of volatile compounds Analysis of volatile compounds was performed using a Shimadzu gas chromatography-mass spectrometry (GC-MS) system. A 3-g cheese sample was placed in a glass vial, and 10 μL of internal standard (2-methyl-3-heptanone, 100 ppm) in methanol was added. The sample was extracted by solid-phase microextraction (SPME) fiber at 40°C for 30 min. A three-phase fiber (2 cm, 50 / 30 μm, Bellafont, USA) was inserted into the vial and held in the injection port of the GC-MS system for 3 min to allow desorption of volatile compounds. Separation was then performed using a capillary column (DB-5MS, 60 m × 0.25 mm × 0.25 mm) with helium as the carrier gas at a flow rate of 1.0 mL / min. Retention rates (RIs) were determined by analyzing alkane sequences under the same conditions as the sample. RIs of volatile compounds were determined by mass spectrometry library analysis. Analysis was performed by comparing the peak area of ​​volatile compounds in the cheese sample with the peak area of ​​the internal standard sample; results are expressed in μg / kg.

[0086] The GC-MS analysis results of volatile compounds in the three cheese samples are as follows: Figure 10As shown in the figure, compared with the *Lactobacillus paracasei* group and the *Bacillus coagulans* BC30 group, the content of off-flavor-related organic acids (such as butyric acid and hexanoic acid) in the *Hydrix henryi* CCMXJ1007 group cheese remained at a lower level, indicating that CCMXJ1007-assisted fermentation helps reduce the generation of undesirable flavors. Meanwhile, the acetic acid content in the CCMXJ1007 group cheese was significantly increased. Acetic acid not only enhances the aroma fullness and richness of handmade cheese, but also inhibits the growth and reproduction of harmful microorganisms to a certain extent, helping to extend the product's shelf life. Regarding aldehydes, the total aldehyde content in the CCMXJ1007 group cheese was significantly higher than that in the two control groups, showing a significant upward trend. Aldehydes (such as benzaldehyde and 3-methylbutyraldehyde) are key contributors to the fruity, nutty, and malty aroma characteristics of cheese, and their increased content helps to enrich the overall aroma profile of the cheese. During the maturation process, a total of nine ketone compounds were monitored. Compared with the two control groups, the total amount of ketone compounds in the CCMXJ1007 group cheese was significantly increased. Ketones, as major contributors to the fruity and creamy aromas of mature cheese, contribute significantly to the richer creamy, sweet, and fruity flavors of CCMXJ1007 fermented cheese, enhancing its overall flavor quality. Furthermore, the ester content in the CCMXJ1007 cheese group was significantly higher than in the two control groups. Esters, characterized by floral and fruity aromas, are important aromatic active substances in cheese, and their increased content positively contributes to the overall complexity and pleasantness of the cheese's aroma.

[0087] In conclusion, the fermentation-assisted fermentation of Heinrich's coagulated bacteria CCMXJ1007 can effectively reduce the formation of undesirable acids in cheese, while significantly increasing the content of key flavor compounds such as acetic acid, aldehydes, ketones, and esters, thereby improving the aroma quality and flavor profile of cheese from multiple dimensions.

[0088] 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. A strain of Hyndrixa coagulans (CCMXJ1007), characterized in that, Heyndrickxia coagulans The accession number is GDMCC No: 68293. ​ 2. A method of making a double protein cheese using the Lactobacillus coryniformis subsp. coryniformis CCMXJ1007 of claim 1, characterized in that, Includes the following steps: S1. Mix sterilized milk with chickpea milk to prepare raw milk; S2, mix the bacterial solution of coagulated Heinrichia coli CCMXJ1007 with Lactobacillus paracasei ( Lactobacillus paracasei SMN-LBK bacterial culture was inoculated into the raw milk prepared by S1, thoroughly mixed, rennet was added, sheared and stirred, and placed at 40℃ for coagulation fermentation to form curds and whey. S3. After being filtered through hanging bags, molded, vacuum-packed, and then cured, double-protein cheese is obtained.

3. The method of claim 2, wherein, The preparation method of the chickpea milk described in S1 is as follows: chickpeas are washed, soaked and pretreated, mixed with water and blended into a paste, the bean pulp is filtered out, and the paste is heated to a boil and kept for 5-10 minutes.

4. The method of claim 2, wherein, The volume ratio of milk to chickpea milk in S1 is 6:

4.

5. The method of claim 2, wherein, S2 The concentration of the bacterial liquid of the coagulase-negative Staphylococcus hyicus CCMXJ1007 and the bacterial liquid of Lactobacillus paracasei SMN-LBK were both 10 5 -10 9 CFU / mL.

6. The method of claim 5, wherein, The volume ratio of the bacterial suspension of *Hydroxytetracycline* CCMXJ1007 and the bacterial suspension of *Lactobacillus paracasei* SMN-LBK described in S2 is 1:4, and the total inoculum amount is 5%.

7. The method of claim 2, wherein, The amount of rennet added in S2 is 0.02%.

8. The method of claim 2, wherein, The fermentation time described in S2 is 16 hours.

9. The method of claim 2, wherein, S3 refers to the post-ripening process where packaged cheese is placed under constant conditions of 10°C and 30% relative humidity for 20-60 days.

10. A double-protein cheese prepared by any one of the methods described in claims 2-9.

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