Lactobacillus paracasei ZF616 and application thereof

Abstract

The present application relates to the technical field of industrial microorganisms, and particularly relates to a lacticaseibacillus paracasei ZF616 and application thereof, and further relates to application of the lacticaseibacillus paracasei in soy sauce production.The lacticaseibacillus paracasei ZF616 is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 62634, and the preservation address is No. 59, Building, Guangzhou, Guangdong Province.The strain of the present application has the advantages of being capable of increasing the content of malic acid in fermented soy sauce, and not affecting other components in fermented soy sauce after being added, and when the strain is applied to a soy sauce fermentation process, the obtained soy sauce not only retains the rich soy sauce flavor of fermented soy sauce, but also increases the pleasant sour taste of the soy sauce, the taste is more sour, the sweet and sour taste is more full and coordinated, and the thickness of the soy sauce is improved.

Description

Technical Field

[0001] This invention relates to the field of industrial microbiology, and more particularly to a strain of Lactobacillus paracasei ZF616 that can increase the malic acid content in soy sauce and its application. Specifically, this invention also relates to the application of the Lactobacillus paracasei in soy sauce production. Background Technology

[0002] Soy sauce is a liquid condiment with a distinctive aroma, made from protein and starch raw materials such as soybeans, wheat, and wheat bran through a series of complex biochemical reactions under the combined action of different enzyme systems secreted by various microorganisms. Lactic acid bacteria are important microorganisms that promote the formation of flavor compounds in soy sauce during the fermentation process.

[0003] Lactic acid bacteria in soy sauce refer to salt-tolerant lactic acid bacteria that grow in the soy sauce mash and undergo lactic acid fermentation. These bacteria originate from the koji-making, mash-making equipment, or the air in the brewery. Studies have shown that the main lactic acid bacteria isolated from soy sauce mash include *Pediococcus halophilus*, *Tetragenocuccus halophilus*, *Weisseria*, and *Tetracocu*. These lactic acid bacteria multiply in soy sauce, producing lactic acid and lowering the pH to below 5.0. This acidity is ideal for the reproduction of soy sauce yeast, which is a crucial role of soy sauce lactic acid bacteria and a key factor in the flavor of soy sauce mash. However, in the later stages of fermentation, when the pH is below 5.0, the growth of lactic acid bacteria such as *Pediococcus halophilus* is inhibited, leading to insufficient accumulation of organic acids in the soy sauce fermentation system, thus affecting the formation of flavor compounds in soy sauce.

[0004] The composition, content, and proportion of organic acids are all important components of soy sauce flavor, mainly including lactic acid, acetic acid, citric acid, and malic acid. This invention found that soy sauce has a high malic acid content, which contributes to a pleasant, mellow, and lasting sour taste, resulting in a more natural and harmonious sweet and sour flavor. Currently, soy sauce brewing commonly uses *Aspergillus oryzae* strain 3.042 or its mutant strains. However, these strains produce very low concentrations of malic acid during soy sauce fermentation, failing to reach the flavor threshold for malic acid. Therefore, the ability of lactic acid bacteria to produce organic acids significantly affects the formation of flavor compounds in soy sauce.

[0005] Therefore, it is essential to find new strains of lactic acid bacteria that can solve the above problems. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a strain of *Lactobacillus paracasei* ZF616 and its applications. The inventors of this application accidentally screened, isolated, and identified a strain of *Lactobacillus paracasei* ZF616 from naturally fermented soy sauce mash. This strain has the advantage of increasing the malic acid content in fermented soy sauce, and its addition does not affect other components in the fermented soy sauce. When applied to the soy sauce fermentation process, the resulting soy sauce retains the rich aroma of fermented soy sauce while adding a pleasant sour taste, resulting in a more refreshing and tangy flavor, a fuller and more harmonious sweet and sour taste, and an enhanced richness.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a strain of Lactobacillus paracasei ZF616, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 62634, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0009] The specific strain preservation information is as follows:

[0010] Name of depositary institution: Guangdong Provincial Center for the Preservation of Microbial Cultures;

[0011] Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province;

[0012] Date of deposit: August 21, 2022;

[0013] Accession number: GDMCC No: 62634.

[0014] The isolated and screened *Lactobacillus paracasei* ZF616 exhibits typical lactic acid bacteria colony morphology: milky white, raised, round colonies with smooth surfaces and regular edges. Microscopic observation revealed that, after screening and staining, the cells were rod-shaped, short rod-shaped, or short chain-shaped, and Gram-positive.

[0015] The *Lactobacillus paracasei* ZF616 strain of this invention exhibits strong acid resistance, maintaining good proliferation ability even below pH 4.5. When inoculated into fermented soy sauce mash, it effectively promotes the accumulation of organic acids in the mash. Soy sauce fermented using *Lactobacillus paracasei* ZF616 has the advantage of high malic acid content, along with a bright color, rich ester aroma, and a more refreshing and tangy taste, resulting in a fuller and more harmonious sweet and sour flavor.

[0016] The present invention also provides the application of the above-mentioned Lactobacillus paracasei ZF616 in the preparation of fermented soy sauce.

[0017] The present invention also provides a screening method for Lacticaseibacillus paracasei ZF616, which includes the following steps:

[0018] 1) Dilute the high-salt soy sauce mash fermented using conventional soy sauce fermentation techniques;

[0019] 2) Spread the diluted solution obtained in 1) onto MRS solid medium for initial screening;

[0020] 3) Inoculate the initially screened strains obtained in 2) and ferment them to obtain fermentation broth;

[0021] 4) The fermentation broth obtained in 3) was subjected to routine index testing and screening to select lactic acid bacteria strains with normal fermentation crude oil quality and the highest content of total nitrogen, amino acid nitrogen and malic acid.

[0022] 5) The lactic acid bacteria strains screened in 4) were identified by morphological, physiological and biochemical, and 16S rDNA sequencing analysis.

[0023] As a preferred embodiment of the application described in this invention, the application of Lactobacillus paracasei ZF616 in increasing malic acid content during soy sauce fermentation.

[0024] The Lactobacillus paracasei ZF616 strain was inoculated onto MRS solid medium for fermentation and passaged for 10 generations. If the malic acid content in the fermentation broth remained within 10% of each generation, it indicates that the strain has good genetic stability.

[0025] This invention also provides a method for preparing soy sauce, comprising the following steps:

[0026] i) Inoculate the fermented sauce mash with the above-mentioned bacterial solution containing Lactobacillus paracasei ZF616;

[0027] ii) Continue fermenting the mash obtained in i) for 7–14 days;

[0028] iii) Press, filter, and sterilize the fermented mash obtained in ii) to obtain crude soy sauce (made according to GB18186-2000 high-salt dilute soy sauce crude oil).

[0029] In a preferred embodiment of the soy sauce preparation method of the present invention, in step i), the concentration of Lactobacillus paracasei ZF616 in the bacterial solution is 1.0 × 10⁻⁶. 7 cfu / mL~1.0×10 8 cfu / mL.

[0030] In a preferred embodiment of the soy sauce preparation method of the present invention, in step i), the inoculation amount of the bacterial solution containing Lactobacillus paracasei ZF616 is 1-3%, preferably, the inoculation amount of the bacterial solution containing Lactobacillus paracasei ZF616 is 1-2%.

[0031] As the amount of *Lactobacillus paracasei* ZF616 added increases, the malic acid content in soy sauce gradually increases. However, when the amount of *Lactobacillus paracasei* ZF616 added increases to a certain extent, the flavor of the soy sauce is affected due to the excessively high malic acid content. Therefore, when using *Lactobacillus paracasei* ZF616 provided in this invention for soy sauce mash fermentation, attention should be paid to the amount of *Lactobacillus paracasei* ZF616 added.

[0032] When the bacterial concentration of Lactobacillus paracasei ZF616 in the bacterial culture is 1.0 × 10⁻⁶ 7 cfu / mL~1.0×10 8 The optimal fermentation mash effect is achieved when the cfu / mL concentration is 2% and the inoculum amount is 2%. This increases the malic acid content, resulting in a more tangy and refreshing taste, a fuller and more harmonious sweet and sour flavor, and enhances the richness of the soy sauce without altering the overall flavor balance due to excessive sourness.

[0033] In a preferred embodiment of the soy sauce preparation method of the present invention, the fermentation time of the fermented soy sauce mash in step i) is 41 to 49 days (e.g., 41 days, 43 days, 45 days, 47 days, 49 days); and the fermentation temperature in step ii) is 30 to 37°C.

[0034] The present invention also provides a soy sauce obtained by the above preparation method.

[0035] The present invention also provides a method for increasing the organic acid content in fermented soy sauce, including adding the above-mentioned Lactobacillus paracasei ZF616 for fermentation.

[0036] In some embodiments, the organic acid includes at least one of malic acid, succinic acid, and lactic acid.

[0037] Organic acids such as lactic acid, succinic acid, and malic acid can, to some extent, generate lipids and alcohols, which can enhance the unique flavor of food and give it a lingering and delicate texture.

[0038] Experimental tests showed that the Lactobacillus paracasei ZF616 of this invention, while producing malic acid, can also produce high levels of succinic acid and lactic acid. Furthermore, the soy sauce produced after fermentation has very low levels of oxalic acid, formic acid, and tartaric acid, which enhances the flavor of the soy sauce without affecting its quality, resulting in excellent sensory evaluation.

[0039] Lactobacillus paracasei ZF616 can significantly increase the malic acid content in soy sauce without affecting other components.

[0040] The present invention also provides a fermentation agent comprising the aforementioned Lactobacillus paracasei ZF616.

[0041] This invention also provides the application of *Lactobacillus paracasei* ZF616 in the production of soy sauce by inhibiting contaminating bacteria through the production of phenyl lactic acid. The contaminating bacteria include at least one of *Bacillus subtilis*, *Micrococcus*, *Enterococcus faecalis*, *Pichia pastoris*, and *Saccharomyces cerevisiae*.

[0042] The *Lactobacillus paracasei* ZF616 of this invention can produce phenyllactic acid to inhibit contamination by other microorganisms and can also enhance the sweet and sour taste and richness of soy sauce. When the phenyllactic acid content produced by *Lactobacillus paracasei* ZF616 is 1.5 g / L, it has an inhibitory effect on *Bacillus subtilis*, *Micrococcus*, and *Saccharomyces cerevisiae*.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The *Lactobacillus paracasei* ZF616 provided by this invention exhibits strong acid resistance and good proliferation ability even below pH 4.5. When applied to soy sauce production, it demonstrates significantly superior malic acid production characteristics compared to other *Lactobacillus paracasei* strains, significantly increasing malic acid content without affecting other components in the fermented soy sauce. Its application in the soy sauce fermentation process results in a soy sauce that retains the rich aroma of fermented soy sauce while adding a pleasant sourness, resulting in a more refreshing and balanced sweet and sour flavor, enhancing the soy sauce's richness. Furthermore, *Lactobacillus paracasei* ZF616 can also produce a high content of phenyllactic acid, effectively inhibiting contamination by other microorganisms during soy sauce production without affecting the flavor. Attached Figure Description

[0045] Figure 1 The colony morphology of Lacticaseibacillus paracasei ZF616 on an MRS plate;

[0046] Figure 2 This is a microscopic image of Lacticaseibacillus paracasei ZF616. Detailed Implementation

[0047] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0048] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0049] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional food-grade reagents, methods and equipment in the art.

[0050] Unless otherwise specified, the experimental conditions used in the embodiments of the present invention are conventional experimental conditions in the art. Unless otherwise specified, all reagents used in the embodiments of the present invention are commercially available.

[0051] Example 1: Isolation and screening of Lactobacillus paracasei ZF616

[0052] 1. Initial screening of strains

[0053] Lactic acid bacteria were isolated and screened from fermented soy sauce mash. High-salt diluted soy sauce was added to physiological saline and serially diluted 10 times. 1 ~10 6 The different dilutions were spread (100 μL each) onto MRS (containing 16% NaCl) solid medium and incubated upside down at 37°C for 48 hours. After incubation, strains with typical lactic acid bacteria colony morphology were selected: milky white, raised, round colonies with smooth surfaces and regular edges. A total of 74 single colonies were selected from the plates for microscopic examination and further screening.

[0054] Under a microscope, Gram-positive strains with rod-shaped, short rod-shaped, or short chain-shaped cells after staining were selected. These strains were then purified by multiple streak purification processes, yielding 23 strains with typical lactic acid bacteria morphology, designated ZF601, ZF602, ZF603, ZF604, ZF605, ZF606, ZF607, ZF608, ZF609, ZF610, ZF611, ZF612, ZF613, ZF614, ZF615, ZF616, ZF617, ZF618, ZF619, ZF620, ZF621, ZF622, and ZF623.

[0055] 2. Secondary screening of strains

[0056] The 23 bacterial strains obtained from the initial screening were activated and inoculated into liquid MRS (containing 16% NaCl) medium, and cultured at 37°C for 24 hours to prepare a bacterial culture containing bacterial cells. During the middle stage of fermentation of the soy sauce mash, i.e., on day 45, the bacterial culture containing bacterial cells was inoculated, with a bacterial cell concentration of 1.0 × 10⁻⁶. 8 The bacterial culture was inoculated at 2% cfu / mL and fermented at 30℃~37℃ for 14 days according to the traditional high-salt dilute state fermentation process. After fermentation, the fermented crude oil was obtained (made according to GB18186-2000 high-salt dilute state soy sauce crude oil, which requires pressing, filtration, sterilization and other processes). The conventional indicators of the fermented crude oil were measured, including total nitrogen, amino acid nitrogen and malic acid content.

[0057] The determination of total nitrogen content refers to the "National Standard for Brewed Soy Sauce GB 18186-2000".

[0058] The determination of amino acid nitrogen content was carried out in accordance with the "National Food Safety Standard GB5009.25-2016 Determination of Amino Acid Nitrogen in Food".

[0059] The determination of malic acid content was carried out in accordance with the "National Food Safety Standard for Determination of Organic Acids in Food GB5009.157-2016".

[0060] The results are shown in Table 1.

[0061] Table 1. Quality Analysis of Fermented Crude Oil

[0062]

[0063]

[0064] As shown in Table 1, strains ZF603, ZF616, ZF608, ZF612, ZF617, and ZF622, which had normal fermentation crude oil quality and high content of total nitrogen, amino acid nitrogen, and malic acid, were selected for sensory evaluation. The standards for sensory evaluation are shown in Table 2.

[0065] Table 2 Sensory Evaluation Scoring Criteria

[0066]

[0067] The sensory evaluation results are shown in Table 3.

[0068] Table 3 Sensory Results of Fermented Crude Oil

[0069] ZF603 92 91 93 92 ZF616 94 95 96 95 ZF608 86 89 87 87 ZF612 88 90 86 88 ZF617 90 90 89 90 ZF622 91 91 90 91

[0070] Note: Each indicator has a maximum score of 100 points. The higher the score, the better the result. The scores for each indicator are the average of the scores given by 10 sensory evaluators.

[0071] Based on the sensory evaluation scores in Table 3, the three strains with the best sensory evaluation results—ZF603, ZF616, and ZF622—were selected for genetic stability testing.

[0072] Genetic stability testing:

[0073] The three selected strains (ZF603, ZF616, and ZF622) were inoculated onto MRS solid medium and passaged for 10 generations. The 1st, 5th, and 10th generation strains were fermented in MRS liquid medium, and the malic acid content in the fermentation broth was detected to determine their genetic stability. If the malic acid content was within 10% of the error between generations, it indicated that the strain had good genetic stability. The genetic stability test results of the three strains are shown in Table 4.

[0074] Table 4. Results of genetic stability testing for the three bacterial strains.

[0075]

[0076] As shown in Table 4, the malic acid content produced by the three strains ZF603, ZF616, and ZF622 at different generations of fermentation had an error range of 10%, indicating that the three strains had good genetic stability. Considering both the evaluation results and the genetic stability results, this invention ultimately selected ZF616 as the final strain. The 16S rDNA sequencing results of strain ZF616 are shown in SEQ ID NO:1:

[0077]

[0078] After identification, strain ZF616 was identified as Lactobacillus paracasei and named Lactobacillus paracasei ZF616.

[0079] This strain was deposited on August 21, 2022, at the Guangdong Microbial Culture Collection Center, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 62634.

[0080] Example 2: Performance testing of Lactobacillus paracasei ZF616

[0081] 1. Acid resistance test of Lactobacillus paracasei ZF616:

[0082] The *Lactobacillus paracasei* ZF616 strain finally screened in Example 1 was passaged twice on MRS solid medium, and then inoculated into MRS liquid medium. The pH of the liquid MRS medium was adjusted to 3.5, 4.0, 4.5, and 5.0 with 1.0 g / L hydrochloric acid. The medium was incubated at 37°C for 12 h, and the counts were performed using the plate dilution method, referring to the "National Food Safety Standard for Microbiological Examination of Food - Lactic Acid Bacteria Examination GB4789.35-2016". After plating and incubation at 37°C for 48 h, viable lactic acid bacteria were counted.

[0083] The acid resistance test results of Lactobacillus paracasei ZF616 are shown in Table 5.

[0084] Table 5 Results of acid resistance test

[0085] 0h <![CDATA[2.1×10 5 ]]> <![CDATA[2.1×10 5 ]]> <![CDATA[2.1×10 5 ]]> <![CDATA[2.1×10 5 ]]> 24h <![CDATA[8.4×10 5 ]]> <![CDATA[3.7×10 6 ]]> <![CDATA[6.8×10 6 ]]> <![CDATA[1.6×10 7 ]]>

[0086] As shown in Table 5, Lactobacillus paracasei ZF616 has strong acid resistance and still has good proliferation ability below pH 4.5.

[0087] 2. Malate production performance and sensory evaluation of Lactobacillus paracasei ZF616:

[0088] This embodiment selected *Lactobacillus paracasei* ZF616 obtained from Example 1 and seven strains of *Lactobacillus paracasei* from the China General Microbiological Culture Collection Center. The names of the seven strains from the China General Microbiological Culture Collection Center are as follows: *Lactobacillus paracasei* CGMCC 1.12731, *Lactobacillus paracasei* CGMCC 1.9089, *Lactobacillus paracasei* CGMCC 1.9088, *Lactobacillus paracasei* CGMCC 1.574, *Lactobacillus paracasei* CGMCC 1.3107, and *Lactobacillus paracasei* CGMCC 1.3107. 1.2744, Lactobacillus paracasei CGMCC 1.2468.

[0089] The seven strains of *Lactobacillus paracasei* and the *Lactobacillus paracasei* ZF616 screened in Example 1 were activated on MRS solid medium. The activated strains were then inoculated into MRS liquid medium (containing 16% NaCl) and cultured at 37°C for 24 hours to prepare a bacterial suspension containing 1.0 × 10⁻⁶ cells. 8 CFU / ml. During the middle stage of fermentation, on day 45, various bacterial solutions were inoculated, with a bacterial concentration of 1.0 × 10⁻⁶. 8 cfu / mL. Following the traditional high-salt, dilute-state fermentation process, the mixture was placed at 30℃~37℃ for 14 days of static fermentation. After fermentation, fermented crude oil was obtained. The total nitrogen, amino acid nitrogen, and malic acid content of the fermented crude oil were determined according to the method described in Example 1. The sensory evaluation of the fermented crude oil was performed with reference to the sensory evaluation scoring criteria in Table 2. The malic acid production test results of the above-mentioned *Lacticaseibacillus paracasei* are shown in Table 6, and the sensory evaluation results are shown in Table 7.

[0090] Table 6. Results of malic acid production by Lacticaseibacillus paracasei

[0091] CGMCC 1.12731 1.08 0.68 0.09 CGMCC 1.9089 1.09 0.79 0.35 CGMCC 1.9088 1.26 0.86 0.43 CGMCC 1.574 0.82 0.52 0.15 CGMCC 1.3107 1.18 0.78 0.21 CGMCC 1.2744 1.16 0.60 0.25 CGMCC 1.2468 1.23 0.83 0.40 ZF616 1.88 1.13 1.65

[0092] Table 7 Sensory evaluation results of Lacticaseibacillus paracasei

[0093]

[0094]

[0095] Note: Each indicator has a maximum score of 100 points. The higher the score, the better the result. The scores for each indicator are the average of the scores given by 10 sensory evaluators.

[0096] As shown in Tables 6-7, the *Lactobacillus paracasei* ZF616 strain screened in this invention exhibits significantly superior malic acid production characteristics compared to other *Lactobacillus paracasei* strains during soy sauce production, resulting in a substantial increase in malic acid content. Compared to other *Lactobacillus paracasei* strains, the *Lactobacillus paracasei* ZF616 strain screened in this invention demonstrates a significant advantage in malic acid production. Furthermore, sensory evaluation reveals that malic acid enhances the soy sauce's flavor, making it more tangy and palatable, with a richer and more robust taste, thus garnering higher praise.

[0097] 3. Performance test of phenyllactic acid production by Lactobacillus paracasei ZF616:

[0098] Lactobacillus paracasei ZF616 from Example 1 was activated and inoculated into MRS liquid medium (containing 16% NaCl). After incubation at 37°C for 48 hours, a bacterial suspension containing bacterial cells was prepared, with a bacterial cell concentration of 1.0 × 10⁻⁶. 8 cfu / mL.

[0099] The above bacterial solution was added to the fermented sauce mash, which had been fermenting for 45 days, with an inoculation amount of 2%. The sauce mash continued to ferment for another 14 days, and the phenyllactic acid content in the sample was tested after fermentation was completed.

[0100] The method for detecting phenyl lactic acid was high performance liquid chromatography (HPLC) using a C18 column, eluting with 0.05% trifluoroacetic acid (TFA) and acetonitrile at a rate of 1 ml / min, with a detection wavelength of 210 nm and a recovery rate of 99.88%–100.08%.

[0101] Phenylated acid is a novel small-molecule antibacterial substance with strong inhibitory effects against many foodborne pathogens and certain filamentous fungi. It is a broad-spectrum antibacterial substance that is non-toxic and harmless to humans and can effectively extend the shelf life of food. To verify whether phenyllactic acid has an inhibitory effect on contaminating bacteria in soy sauce, this invention specifically selected several common contaminating bacteria of soy sauce, including Bacillus subtilis, Micrococcus, Enterococcus faecalis, Pichia pastoris, and Saccharomyces cerevisiae, and tested the inhibitory concentration of phenyllactic acid. The results are shown in Table 8. The initial detection concentration for each contaminating strain was 1×10⁻⁶. 4 cfu / ml.

[0102] Table 8. Detection results of the inhibitory effect of phenyllactic acid on contaminating bacteria in soy sauce.

[0103]

[0104] Note: "-" indicates no antibacterial effect, "+" indicates antibacterial effect.

[0105] The data in Table 8 show that Lactobacillus paracasei ZF616 produces phenyllactic acid. When the content of phenyllactic acid is 1.5 g / L, it has an inhibitory effect on Bacillus subtilis, Micrococcus, and Saccharomyces cerevisiae, indicating that phenyllactic acid has a certain preservative effect on soy sauce contaminants. This demonstrates that Lactobacillus paracasei ZF616 can inhibit contaminants in soy sauce.

[0106] The phenyllactic acid production tests of *Lactobacillus paracasei* ZF616 and *Lactobacillus paracasei* CGMCC 1.12731, 1.9089, 1.9088, 1.574, 1.3107, 1.2744, and 1.2468 screened in Example 1 were performed, and the results are shown in Table 9.

[0107] Table 9

[0108] CGMCC 1.12731 0.72 CGMCC 1.9089 0.03 CGMCC 1.9088 1.84 CGMCC 1.574 2.66 CGMCC 1.3107 1.25 CGMCC 1.2744 0.04 CGMCC 1.2468 0.66 ZF616 1.68

[0109] As shown in Table 9, the *Lactobacillus paracasei* ZF616 screened in Example 1 can also produce phenyllactic acid in soy sauce production. Compared with *Lactobacillus paracasei* CGMCC 1.9088 and *Lactobacillus paracasei* CGMCC 1.574, the phenyllactic acid content is lower. However, among these seven *Lactobacillus paracasei* strains, the phenyllactic acid content of *Lactobacillus paracasei* ZF616 of this invention is still at a relatively high level, and has reached the concentration range that inhibits some soy sauce contamination bacteria. Therefore, *Lactobacillus paracasei* ZF616 of this invention can not only enhance the sweet and sour taste and richness of soy sauce, but also produce phenyllactic acid to inhibit contamination by miscellaneous bacteria without affecting the flavor.

[0110] 4. Performance test of Lactobacillus paracasei ZF616 in producing other organic acids:

[0111] During the fermentation process, soy sauce undergoes complex biochemical changes, producing a variety of organic acids, such as oxalic acid, lactic acid, acetic acid, succinic acid, tartaric acid, formic acid, malic acid, and citric acid. These acids can adjust the soy sauce's acidity, affecting its taste and quality.

[0112] Lactobacillus paracasei ZF616 from Example 1 was activated and inoculated into MRS liquid medium (containing 16% NaCl). After incubation at 37°C for 48 hours, a bacterial suspension containing bacterial cells was prepared, with a bacterial cell concentration of 1.0 × 10⁻⁶. 8 cfu / mL.

[0113] The above bacterial solution was added to the fermented sauce mash, which had been fermenting for 45 days, with an inoculation amount of 2%. The sauce mash continued to ferment for 14 days. After fermentation, the contents of oxalic acid, succinic acid, tartaric acid, and formic acid in the sample were tested. The test results are shown in Table 10.

[0114] Organic acid detection was performed using an Aminex HPX-87H (300 mm × 7.8 mm) organic acid column with a mobile phase of 0.5 mmol / L sulfuric acid solution, a flow rate of 0.6 ml / min, an injection volume of 10 μL, a UV detection wavelength of 210 nm, and a column temperature of 30 °C.

[0115] Table 10. Results of detection of other organic acids produced by Lactobacillus paracasei ZF616

[0116]

[0117]

[0118] Note: "ND" indicates that the substance was not detected.

[0119] During soy sauce fermentation, organic acids such as lactic acid, succinic acid, and malic acid can generate lipids and alcohols, which can enhance the unique flavor of the food and give it a lingering and delicate taste. As shown in Table 10, the *Lactobacillus paracasei* ZF616 screened in Example 1 produces malic acid, as well as high levels of succinic acid and lactic acid. Furthermore, the soy sauce fermented using this invention has very low levels of oxalic acid, formic acid, and tartaric acid. Therefore, *Lactobacillus paracasei* ZF616 can both enhance the tangy and rich flavor without reducing the quality of the soy sauce.

[0120] Example 3

[0121] The *Lactobacillus paracasei* ZF616 strain finally screened in Example 1 was activated and inoculated into MRS liquid medium (containing 16% NaCl). After incubation at 37°C for 48 hours, a bacterial suspension containing bacterial cells was prepared, with a bacterial cell concentration of 1.0 × 10⁻⁶. 8 cfu / mL.

[0122] The above-mentioned bacterial solution was added to the fermented soy sauce mash, which had been fermenting for 45 days at this point, with an inoculation amount of 1%. The mash continued to ferment for another 14 days, and crude soy sauce was obtained after fermentation.

[0123] Example 4

[0124] The *Lactobacillus paracasei* ZF616 strain finally screened in Example 1 was activated and inoculated into MRS liquid medium (containing 16% NaCl). After incubation at 37°C for 48 hours, a bacterial suspension containing bacterial cells was prepared, with a bacterial cell concentration of 1.0 × 10⁻⁶. 8 cfu / mL.

[0125] The above-mentioned bacterial solution was added to the fermented soy sauce mash, which had been fermenting for 45 days at this point, with an inoculation amount of 2%. The mash continued to ferment for another 12 days, and crude soy sauce was obtained after fermentation.

[0126] Example 5

[0127] The *Lactobacillus paracasei* ZF616 strain finally screened in Example 1 was activated and inoculated into MRS liquid medium (containing 16% NaCl). After incubation at 37°C for 48 hours, a bacterial suspension containing bacterial cells was prepared, with a bacterial cell concentration of 1.0 × 10⁻⁶. 8 cfu / mL.

[0128] The above-mentioned bacterial solution was added to the fermented soy sauce mash, which had been fermenting for 45 days at this point, with an inoculation amount of 2%. The soy sauce mash continued to ferment for another 14 days, and crude soy sauce was obtained after fermentation.

[0129] Example 6

[0130] The *Lactobacillus paracasei* ZF616 strain finally screened in Example 1 was activated and inoculated into MRS liquid medium (containing 16% NaCl). After incubation at 37°C for 48 hours, a bacterial suspension containing bacterial cells was prepared, with a bacterial cell concentration of 1.0 × 10⁻⁶. 8 cfu / mL.

[0131] The above-mentioned bacterial solution was added to the fermented soy sauce mash, which had been fermenting for 45 days at this point, with an inoculation amount of 3%. The soy sauce mash continued to ferment for 14 days, and crude soy sauce was obtained after fermentation.

[0132] Comparative Example 1

[0133] The fermentation mash was prepared using a traditional high-salt, low-temperature fermentation process, with *Lactobacillus paracasei* ZF616 not inoculated as a control. Other fermentation conditions were the same as in Example 3. After fermentation, crude soy sauce was obtained.

[0134] The crude soy sauce obtained in Examples 3-6 and Comparative Example 1 was tested for total nitrogen, amino acid nitrogen and malic acid content. For details, please refer to Example 1. The sensory evaluation scoring criteria are shown in Table 2.

[0135] The results of total nitrogen, amino acid nitrogen and malic acid content detection are shown in Table 11; the sensory evaluation results are shown in Table 12.

[0136] Table 11 Results of Crude Oil Quality Analysis for Soy Sauce

[0137] Example 3 1.86 1.13 0.53 Example 4 1.87 1.15 0.68 Example 5 1.87 1.16 0.82 Example 6 1.88 1.16 0.93 Comparative Example 1 1.78 1.09 0.11

[0138] Table 12 Sensory Evaluation Results

[0139] Example 3 94 95 94 94 Example 4 95 96 96 96 Example 5 97 98 98 98 Example 6 97 95 93 95 Comparative Example 1 86 87 86 86

[0140] Note: Each indicator has a maximum score of 100 points. The higher the score, the better the result. The scores for each indicator are the average of the scores given by 10 sensory evaluators.

[0141] As can be seen from the data in Table 11, the total nitrogen and amino acid nitrogen content of the soy sauce crude oil in Examples 3, 4, 5 and 6 were not significantly different from those of the soy sauce crude oil in Comparative Example 1, while the malic acid content was significantly increased compared with Comparative Example 1 due to inoculation with Lactobacillus paracasei ZF616.

[0142] As can be seen from the data in Table 12, the color, aroma, and taste of the crude soy sauce in Examples 3, 4, 5, and 6 are all superior to those in the control example.

[0143] Applying *Lactobacillus paracasei* ZF616, screened in Example 1, to the soy sauce production process significantly increases the malic acid content in soy sauce without affecting other components. In Examples 3 to 6, the malic acid content in soy sauce gradually increased with the increase of *Lactobacillus paracasei* ZF616 addition. However, when the addition of *Lactobacillus paracasei* ZF616 increased to a certain extent, the flavor of the soy sauce was affected due to the excessively high malic acid content. Therefore, when using *Lactobacillus paracasei* ZF616 provided by this invention for soy sauce mash fermentation, attention should be paid to the amount of *Lactobacillus paracasei* ZF616 added.

[0144] The above studies indicate that when the concentration of bacteria (Lactobacillus paracasei) in the bacterial solution is 1.0 × 10⁻⁶, the desired effect is achieved. 8 The optimal fermentation effect of soy sauce mash is achieved when the cfu / mL concentration and the inoculum concentration are 2%. This increases the malic acid content, resulting in a more tangy and refreshing taste, a fuller and more harmonious sweet and sour flavor, and enhances the richness of the soy sauce without altering its overall flavor balance due to excessive sourness.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei ZF616, characterized in that, The Lactobacillus paracasei ( Lacticaseibacillus paracasei ZF616 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 62634, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

2. The *Lactobacillus paracasei* as described in claim 1 ( Lacticaseibacillus paracasei Application of ZF616 in the preparation of fermented soy sauce.

3. The application as described in claim 2, characterized in that, The Lactobacillus paracasei ( Lacticaseibacillus paracasei Application of ZF616 in increasing malic acid content during soy sauce fermentation.

4. A method for preparing soy sauce, comprising the following steps: i) Inoculating the fermented sauce mash with Lactobacillus paracasei as described in claim 1 ( Lacticaseibacillus paracasei ZF616 bacterial culture; ii) Continue fermenting the mash obtained in i) for 7-14 days; iii) Press, filter, and sterilize the fermented mash obtained in ii) to obtain crude soy sauce.

5. The preparation method according to claim 4, characterized in that, In step i), the bacterial solution contains Lactobacillus paracasei ( Lacticaseibacillus paracasei The concentration of ZF616 is 1.0 × 10⁻⁶. 7 cfu / mL ~1.0×10 8 cfu / mL; the inoculum volume of the bacterial suspension is 1-3%.

6. The preparation method according to claim 4, characterized in that, Step i) The fermentation time for the fermented sauce is 41 to 49 days; Step ii) The fermentation temperature is 30 to 37℃.

7. A method for increasing the organic acid content in fermented soy sauce, characterized in that, Includes the addition of Lactobacillus paracasei as described in claim 1 ( Lacticaseibacillus paracasei The organic acid is fermented using ZF616; the organic acid includes at least one of malic acid, succinic acid and lactic acid.

8. The *Lactobacillus paracasei* as described in claim 1 ( Lacticaseibacillus paracasei The application of ZF616 in inhibiting contaminating bacteria during soy sauce production; The contaminating bacteria include at least one of Bacillus subtilis, Micrococcus, Enterococcus faecalis, Pichia pastoris, and Saccharomyces cerevisiae.

Images