Lactic acid bacteria and application thereof in dregs vinegar fermentation

Abstract

The invention relates to the technical field of food fermentation, and discloses a lactic acid bacteria strain and application thereof in dregs vinegar fermentation, the lactic acid bacteria strain involved in the invention is Lactobacillus paracasei ZPC30, is preserved in Guangdong Microbiological Culture Collection Center, has a preservation number of GDMCC NO: 67278, is preserved on November 5, 2025, is obtained by screening dregs vinegar distillers' grains, has a preservation number of GDMCC NO: 67278, has a preservation number of CGMCC NO: 67278, has a preservation number of CGMCC NO: 67278, has a preservation date of CGMCC NO: 67278, and has a preservation number of CGMCC NO: 67278, and has a preservation number of CGMCC NO: 67278. The strain has good growth, acid production and ethanol tolerance. Meanwhile, the invention provides application of the strain in dregs vinegar fermentation and product preparation, saccharified rice is inoculated with the lactic acid bacteria strain and a leavening agent according to a set inoculation sequence and inoculation amount, and dregs vinegar is obtained after standing culture, so that the product quality of the dregs vinegar can be improved by using the strain, and the production cost is reduced. And a new way is provided for development of leavening agents of dregs vinegar or other vinegar products.

Description

Technical Field

[0001] This invention relates to the field of food fermentation technology, and more specifically, to a lactic acid bacterium and its application in the fermentation of fermented grains and vinegar. Background Technology

[0002] Zhaobo vinegar is a unique traditional fermented food from Hainan. Made primarily from rice, it undergoes fermentation using a mixed microbial community including lactic acid bacteria, yeast, and acetic acid bacteria, and is then blended with chili oil and garlic oil. Its flavor is characterized by a core of "sour, spicy, fresh, and fragrant" notes, and it is rich in protein, polypeptides, amino acids, and various organic acids, exhibiting potential health benefits. In recent years, Zhaobo vinegar has emerged as a popular hot pot condiment and is gradually becoming a signature consumer product for Hainan tourism, with significant market demand growth.

[0003] Lactic acid bacteria, as one of the key microorganisms producing the main flavor compounds in the fermentation of fermented grain vinegar, have a significant impact on the final flavor of the product. However, in the traditional natural fermentation mode of fermented grain vinegar, lactic acid bacteria are mainly introduced from raw materials, the environment, and the inoculated lees. Some lactic acid bacteria have poor growth performance and flavor production ability, resulting in low production efficiency, poor fermentation stability, and large fluctuations in product quality. Currently, systematic screening and research on lactic acid bacteria in traditional high-quality fermented grain vinegar are still lacking, which limits the breeding and application of superior strains, and thus restricts further improvement of the quality and optimization of the process of fermented grain vinegar.

[0004] Based on this, the present invention provides a lactic acid bacteria strain isolated and screened from high-quality traditional vinegar lees, which enriches the lactic acid bacteria strain resource library. Furthermore, through enhanced fermentation by this lactic acid bacteria, the total acidity and flavor of the vinegar lees can be improved, which is of great significance for stabilizing and improving the quality of vinegar lees products. Summary of the Invention

[0005] The purpose of this invention is to propose a type of lactic acid bacteria and its application in the fermentation of vinegar made from fermented grains, aiming to solve the problem that there are few lactic acid bacteria resources suitable for the fermentation system of vinegar made from fermented grains in the prior art, and to provide lactic acid bacteria resources for the development of fermentation agents for vinegar made from fermented grains.

[0006] This invention discloses a lactic acid bacteria strain, wherein the lactic acid bacteria strain is Lactobacillus paracasei ZPC30 ( Lacticaseibacillus paracasei The sample has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO:67278 and deposit date of 2025.11.05.

[0007] The present invention also provides a microbial agent comprising the lactic acid bacteria strain described in claim 1.

[0008] The present invention also provides a lactic acid bacteria starter culture, which is obtained by fermentation of the lactic acid bacteria according to claim 1.

[0009] The present invention also provides the application of the lactic acid bacteria described in the above technical solution, the application including: Applications in the fermentation of vinegar made from lees and / or in the preparation and production of vinegar products made from lees.

[0010] Furthermore, the steps for applying the lactic acid bacteria include: (1) After washing, soaking and steaming the rice, inoculate it with Aspergillus rhizogenes for saccharification; (2) Inoculate the lactic acid bacteria-prepared starter and yeast into the saccharified rice according to the set inoculation amount and inoculation order; (3) The saccharified rice after inoculation was left to stand and cultured to obtain the fermentation product, lees vinegar.

[0011] The present invention also provides the application of the microbial agent described in the above technical solution, the application including: Applications in the fermentation of vinegar made from lees and / or in the preparation and production of vinegar products made from lees.

[0012] The present invention also provides the application of the lactic acid bacteria starter culture described in the above technical solution, the application including: Applications in the fermentation of vinegar made from lees and / or in the preparation and production of vinegar products made from lees.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: Most existing technologies for producing vinegar from fermented grains use commercially available bacterial strains from other food production systems, lacking fermentation strains specifically designed for this purpose. This invention provides a high-quality lactic acid bacteria strain isolated from commercially available vinegar mash, exhibiting excellent growth, acid production, and acid tolerance.

[0014] This invention provides the application of a strain of lactic acid bacteria in the enhanced fermentation of fermented grain vinegar. The fermented grain vinegar enhanced by this strain has increased total acidity, total phenol content, antioxidant activity, flavor compounds, and sensory evaluation. Attached Figure Description

[0015] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is a schematic diagram showing the results of the lactic acid bacteria growth capacity test in Example 1 of the present invention; Figure 2 This is a schematic diagram showing the results of the lactic acid bacteria acid production capacity test in Example 1 of the present invention; Figure 3 This refers to the Lactobacillus paracasei ZPC30 lactic acid bacteria strain described in this invention. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0017] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] This invention proposes a lactic acid bacteria strain, wherein the lactic acid bacteria strain is Lactobacillus paracasei ZPC30 ( Lacticaseibacillus paracasei The sample has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO:67278 and deposit date of November 5, 2025.

[0022] The lactic acid bacteria strain was derived from high-quality vinegar lees. To obtain strains with fermentation properties for vinegar lees, the following steps were performed for isolation and screening: Sample pretreatment: Take 1-10 mL of traditional vinegar and wine lees, add 9-99 mL of sterile physiological saline, and dilute 10-100 times in a serial manner; Initial screening: Bacterial solutions of different dilution ratios were spread on MRS agar medium and incubated at 37°C for 24 hours to promote the enrichment and growth of microorganisms that have a fermentation effect on the vinegar of lees on the surface of the medium. Regularly shaped colonies were picked and streaked for purification to obtain single colonies. Growth and acid production screening: The purified strain was inoculated into MRS broth medium and cultured at 37°C for 24 h. pH and OD were then measured. 600 In order to screen strains with strong growth and acid production capabilities; Ethanol tolerance screening: The purified strain was inoculated into MRS broth containing 0-8% (v / v) ethanol and cultured at 37°C for 24 h. Its OD was then measured. 600 To screen for strains with good ethanol tolerance, the desired strains were selected based on a comprehensive assessment of growth, acid production, and ethanol tolerance.

[0023] The present invention also provides a microbial agent comprising the aforementioned lactic acid bacteria strain.

[0024] In this invention, the preparation of the microbial agent includes the following steps: Fermentation culture: Lactobacillus paracasei ZPC30 strain was inoculated into MRS broth medium and anaerobic cultured at 37°C for 18 h until the viable count was ≥10⁻⁶. 9 CFU / mL; Freeze-drying: A preservative was added to the fermentation broth, and after thorough mixing, it was pre-frozen at -40℃ for 2 hours, followed by freeze-drying using a freeze dryer with the following parameters: vacuum degree 10 Pa, sublimation temperature -20℃, desorption temperature 30℃, and drying time 12 hours, to produce freeze-dried bacterial powder with a viable count ≥10⁻⁶. 8 CFU / g, moisture content ≤5%; The present invention also provides a lactic acid bacteria starter culture, which is obtained by fermentation of the lactic acid bacteria strains described above.

[0025] In this invention, the preparation of the lactic acid bacteria starter includes the following steps: The freeze-dried bacterial powder from the bacterial agent preparation process was added to sterile physiological saline for reconstitution to prepare a bacterial suspension; Inoculate the bacterial suspension at a rate of 5% into MRS broth medium and culture anaerobically at 37°C for 24 hours until the pH of the fermentation broth drops to 3.8-4.0. This will produce the lactic acid bacteria starter.

[0026] The viable count of the lactic acid bacteria starter is ≥10. 8 CFU / mL, can be used directly for fermentation, or stored at 4℃ for 3 days.

[0027] In this invention, the lactic acid bacteria strain is obtained by isolating high-quality vinegar lees, screening for growth, acid production, and alcohol tolerance, and then inoculating the lactic acid bacteria into MRS agar medium and culturing at 37°C for 24 hours. Single colonies are then transferred to MRS broth medium and cultured at 37°C for 24 hours to obtain the lactic acid bacteria starter. Growth, acid production, and ethanol tolerance are important indicators for evaluating whether lactic acid bacteria can successfully brew vinegar from vinegar lees.

[0028] The present invention also provides the application of the lactic acid bacteria described in the above technical solution, the application including: Applications in the fermentation of vinegar made from lees and / or in the preparation and production of vinegar products made from lees.

[0029] The steps for applying lactic acid bacteria as described in this invention include: (1) After washing, soaking and steaming the rice, inoculate it with Aspergillus rhizogenes for saccharification; (2) Inoculate the lactic acid bacteria-prepared starter and yeast into the saccharified rice according to the set inoculation amount and inoculation order; (3) The saccharified rice after inoculation was left to stand and cultured to obtain the fermentation product, lees vinegar.

[0030] In this invention, the inoculum amount of the mixed fermentation agent is set at 2%, and the static culture parameters are: temperature 37°C and time 96h.

[0031] In this invention, the preparation steps of the saccharified rice include: Raw material pretreatment: Select commercially available rice, wash the rice to remove impurities, add 3 to 5 times the volume of tap water at a temperature of 25 to 30 degrees Celsius and soak for 4 to 6 hours until the rice grains are no longer hard in the center; Steaming: After soaking, drain the rice and place it in a steamer. Steam at 80-100℃ for 20-30 minutes to ensure the rice grains are soft and do not stick together. Saccharification: Sterile water is added to the cooked rice, with a rice-to-water ratio of 1:2. Aspergillus rhizogenes is added at a rate of 0.2% to 0.5% of the rice mass. The mixture is kept at 28°C for 24 hours until the reducing sugar content is ≥80g / L. The rice is then cooled and stored for later use to obtain the saccharified rice.

[0032] The present invention also provides the application of the microbial agent described in the above technical solution, the application including: Applications in the fermentation of vinegar made from lees and / or in the preparation and production of vinegar products made from lees.

[0033] The present invention also provides the application of the lactic acid bacteria starter culture described in the above technical solution, the application including: Applications in the fermentation of vinegar made from lees and / or in the preparation and production of vinegar products made from lees.

[0034] In this invention, the fermented grain vinegar product prepared using the lactic acid bacteria / lactic acid bacteria starter is... In this invention, the fermented product, vinegar made from fermented grains, contains total acid and total phenols. Total acid and pH are important indicators for evaluating the flavor and stability of the vinegar. Total phenols, ABTS free radical scavenging rate, and DPPH free radical scavenging rate reflect the antioxidant and anti-inflammatory health benefits of the vinegar. Its color, appearance, aroma, and taste are important sensory evaluation indicators for vinegar, reflecting its product quality.

[0035] In this invention, unless otherwise specified, all raw materials required for preparation are commercially available products well known to those skilled in the art.

[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1 I. The lactic acid bacteria strain is derived from commercially available high-quality fermented vinegar mash. To obtain strains with fermentation properties for fermented vinegar mash, the following steps were performed for isolation and screening: Sample pretreatment: Take 10 mL of commercially available traditional fermented vinegar mash, add 90 mL of commercially available sterile saline, and dilute 10 times in a serial gradient. Initial screening: Bacterial solutions of different dilution ratios were spread on MRS agar medium and incubated at 37°C for 24 hours to promote the enrichment and growth of microorganisms that have a fermentation effect on the vinegar of lees on the surface of the medium. Regularly shaped colonies were picked and streaked for purification to obtain single colonies. Growth and acid production screening: The purified strain was inoculated into MRS broth medium and cultured at 37°C for 24 h. pH and OD were then measured. 600 In order to screen strains with strong growth and acid production capabilities; Ethanol tolerance screening: The purified strain was inoculated into MRS broth containing 0-8% (v / v) ethanol and cultured at 37°C for 24 h. Its OD was then measured. 600 To screen for strains with good ethanol tolerance, the desired strains were selected based on a comprehensive assessment of growth, acid production, and ethanol tolerance.

[0038] The results of the lactic acid bacteria performance test are shown in Table 1 below: Table 1: Ethanol Tolerance of Lactic Acid Bacteria

[0039] Based on the results of growth, acid production, and ethanol tolerance experiments, strain No. 10 was selected for molecular biological identification. Genomic DNA was extracted using a genome extraction kit. The full-length 16S rDNA sequence was amplified using bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO:2). Sequencing of the PCR amplification products was performed by the Guangdong Provincial Microbial Culture Collection Center. The obtained 16S rDNA sequence was compared with the NCBI database using BLAST to determine its species. The strain with the highest homology was... Lacticaseibacilus paracasei subsp. tolerans With a similarity of 100%, the strain was named Lacticaseibacilus paracasei ZPC30 .

[0040] II. Preparation of saccharified rice: Raw material pretreatment: Select commercially available rice, wash the rice to remove impurities, add 3 times the volume of tap water at 25℃ and soak for 4 hours until the rice grains are no longer hard in the center. Steaming: After soaking, drain the rice and place it in a steamer. Steam at 100℃ for 20 minutes to ensure that the rice grains are soft and do not stick together. Saccharification: Sterile water is added to the cooked rice, with a rice-to-water ratio of 1:2. The temperature is adjusted to 50°C, and Aspergillus rhizogenes is added at a rate of 0.5% of the rice mass. The mixture is kept warm for 24 hours until the reducing sugar content is ≥80g / L. The rice is then cooled to 37°C for later use to obtain the saccharified rice.

[0041] III. Preparation of Lactic Acid Bacteria Starter Culture: Seed culture: Lactobacillus paracasei ZPC30 lactic acid bacteria strain was inoculated into MRS broth medium and anaerobically cultured at 37°C for 18 h until the viable count was ≥10⁻⁶. 9 CFU / mL was used to obtain the seed culture; Large-scale culture: Using MRS broth medium as the fermentation substrate, the inoculum volume fraction was 3%, the temperature was controlled at 37℃, and the culture was carried out for 24 hours to obtain the fermentation broth; Freeze-drying: Add a protective agent to the fermentation broth, mix well, pre-freeze at -40℃ for 2 hours, and then dry in a freeze dryer with the following parameters: vacuum degree 10Pa, sublimation temperature -20℃, desorption temperature 30℃, and drying time 12 hours to produce freeze-dried bacterial powder. Reconstitution treatment: The freeze-dried bacterial powder from the bacterial agent preparation process is added to sterile physiological saline for reconstitution to prepare a bacterial suspension; Preparation of lactic acid bacteria starter: Inoculate the bacterial suspension into MRS broth medium at an inoculation rate of 5%, and culture anaerobically at 37°C for 24 hours until the pH of the fermentation broth drops to 3.8. This is the lactic acid bacteria starter.

[0042] IV. The specific application steps for lactic acid bacteria strains are as follows: (1) The lactic acid bacteria starter prepared by the lactic acid bacteria strain is mixed with the fermentation agent of lees vinegar wine to obtain a mixed starter; (2) The mixed fermentation agent is inoculated into the saccharified rice at a set inoculation amount of 5%; (3) The inoculated saccharified rice was placed in a constant temperature incubator at 28℃ for 120 hours to obtain the fermentation product.

[0043] Comparative Example 1 I. Preparation of saccharified rice: Raw material pretreatment: Select commercially available rice, wash the rice to remove impurities, add 3 times the volume of tap water at 25℃ and soak for 4 hours until the rice grains are no longer hard in the center. Steaming: After soaking, drain the rice and place it in a steamer. Steam at 100℃ for 20 minutes to ensure that the rice grains are soft and do not stick together. Saccharification: Sterile water is added to the cooked rice, with a rice-to-water ratio of 1:2. Aspergillus rhizogenes is added at a rate of 0.5% of the rice mass. The mixture is saccharified at 28°C for 24 hours until the reducing sugar content is ≥80g / L. The mixture is then cooled to 37°C for later use to obtain the saccharified rice.

[0044] II. Preparation of Distilled Rice Wine: Dissolve the lees vinegar yeast in warm water and inoculate it into saccharified rice at a 2% inoculation rate using a sterile pipette. Saccharified rice is placed in a constant temperature incubator at 28℃ for 120 hours to obtain fermentation product lees vinegar lees, which is then mixed with chili oil and garlic oil to obtain the finished lees vinegar.

[0045] Comparative Example 2 I. Preparation of saccharified rice: Raw material pretreatment: Select commercially available rice, wash the rice to remove impurities, add 3 times the volume of tap water at 25℃ and soak for 4 hours until the rice grains are no longer hard in the center. Steaming: After soaking, drain the rice and place it in a steamer. Steam at 100℃ for 20 minutes to ensure that the rice grains are soft and do not stick together. Saccharification: Sterile water is added to the cooked rice, with a rice-to-water ratio of 1:2. Aspergillus rhizogenes is added at a rate of 0.5% of the rice mass. The mixture is saccharified at 28°C for 24 hours until the reducing sugar content is ≥80g / L. The mixture is then cooled to 37°C for later use to obtain the saccharified rice.

[0046] II. Preparation of Distilled Rice Wine: Only saccharified rice is used as the fermentation substrate; no other substances are inoculated. The fermentation product, lees vinegar, was obtained by static incubation in a 28℃ constant temperature incubator for 120 hours. It was then mixed with chili oil and garlic oil to obtain the finished lees vinegar.

[0047] The fermentation containers used in Example 1 and Comparative Examples 1-2 were all sterile Erlenmeyer flasks of the same model.

[0048] The product detection results of Example 1 and Comparative Examples 1-2 are shown in Table 2 below: Table 2: Detection results of products from Example 1 and Comparative Examples 1-2

[0049] In summary, compared to using only traditional yeast and natural fermentation without inoculation, the fermentation of the fermented grain vinegar using the strain of this invention increases the total acidity, pH, total phenol content, antioxidant activity, and sensory evaluation. Furthermore, the inoculant and fermentation agent prepared by this lactic acid bacteria strain have good stability, can be prepared on a large scale, and the preparation and inoculation amount are easy to control, making it suitable for continuous fermented grain vinegar and industrial production. This provides a new approach for the development of fermentation agents for fermented grain vinegar or other vinegar products.

[0050] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is determined by the appended claims.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A type of lactic acid bacteria, characterized in that, The lactic acid bacteria strain is Lactobacillus paracasei ZPC30 ( Lacticaseibacillus paracasei The sample has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO:67278 and deposit date of 2025.11.

05.

2. A microbial agent, characterized in that, The microbial agent includes the lactic acid bacteria strain described in claim 1.

3. A lactic acid bacteria starter culture, characterized in that, The lactic acid bacteria starter is obtained by fermentation of the lactic acid bacteria as described in claim 1.

4. An application of the lactic acid bacteria according to claim 1, characterized in that, The applications include: Applications in the fermentation of vinegar made from lees and / or in the preparation and production of vinegar products made from lees.

5. The application of the lactic acid bacteria according to claim 4, characterized in that, The steps of the application include: (1) After washing, soaking and steaming the rice, inoculate it with Aspergillus rhizogenes for saccharification; (2) Inoculate the fermentation agent and lactic acid bacteria agent into the saccharified rice according to the set inoculation amount and inoculation sequence; (3) The saccharified rice after inoculation was left to stand and cultured to obtain the fermentation product, lees vinegar.

6. The application of the microbial agent according to claim 2, characterized in that, The applications include: Applications in the fermentation of vinegar made from lees and / or in the preparation and production of vinegar products made from lees.

7. The application of the lactic acid bacteria starter culture according to claim 3, characterized in that, The applications include: Applications in the fermentation of vinegar made from lees and / or in the preparation and production of vinegar products made from lees.

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