Pediococcus acidilactici ZF636 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a pediococcus acidilactici ZF636 strain and application of the pediococcus acidilactici ZF636 strain. The pediococcus acidilactici strain ZF636 is obtained by screening, is salt-resistant and acid-resistant, has relatively strong ester production capacity, and particularly has high yield of isoamyl acetate; the strain is used for high-salt food fermentation, the content of ester substances, especially isoamyl acetate, of a product and the total amount of volatile compounds can be remarkably increased, meanwhile, the sauce flavor, the ester flavor, the delicate flavor and the strong flavor of the product are improved, the flavor of the product is more balanced and richer, and the flavor and the overall quality of the product are comprehensively improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Pediococcus lactis ZF636 and its applications. Background Technology

[0002] Soy sauce is a traditional fermented soybean product in my country, highly favored by consumers both domestically and internationally. Its unique and complex flavor is key to its value. The flavor of soy sauce mainly relies on the traditional natural fermentation process, which involves a complex microbial community structure that is easily affected by environmental factors, resulting in poor flavor stability and a long production cycle.

[0003] To improve this situation, existing technologies have attempted to enhance fermentation by adding external microbial agents to increase the content of flavor compounds and improve product flavor and quality. For example, patent CN 202411585296.8 discloses a salt-tolerant Zygomyces rouxii strain that produces high levels of ethanol, which can significantly increase the ethanol content and flavor of soy sauce, enhancing its aroma and taste. Patent CN201910495808.4 discloses a salt-tolerant Pediococcus lactis strain that can grow in high-salt, high-acid environments, producing flavor compounds such as organic acids. This strain can be used as a functional microbial agent in the traditional fermented food industry, such as fermented soybean paste, soy sauce, vinegar, bean paste, sweet bean sauce, and fermented black beans, to optimize production processes, improve product flavor, and enhance product quality. Patent CN 202010661916.7 discloses a method for producing soy sauce by fermentation with highly salt-tolerant lactic acid bacteria, which shortens the fermentation time and improves the flavor and quality of soy sauce.

[0004] The main flavor compounds in soy sauce include alcohols, acids, aldehydes, ketones, and phenols. Among these, esters are crucial for creating a pleasant flavor profile, especially isoamyl acetate. Due to its extremely low odor threshold (only 0.005 mg / L), even trace amounts can significantly contribute to fruity and sweet aromas, making it a core component in the "rich ester aroma" of high-end products and harmonizing the soy sauce flavor. However, in traditional fermentation systems, the biosynthetic pathways of esters are often limited, resulting in generally low overall content of esters in the final product. This has become a key bottleneck restricting further improvement in the flavor and quality of soy sauce.

[0005] Lactic acid bacteria are one of the core microbial communities in the soy sauce fermentation system. Although some studies have attempted to add lactic acid bacteria during fermentation, conventional lactic acid bacteria have limited survival ability in the high-salt, acidic late-stage environment of soy sauce. Their metabolic activity is difficult to sustain and they cannot effectively contribute to the synthesis of volatile flavor compounds. Their role has long been mainly defined as acid production to lower pH and inhibiting other bacteria. Currently, there are no reports of consciously screening and utilizing lactic acid bacteria strains with specific ester-producing aroma capabilities. Discovering a functional microbial strain that can highly adapt to the high-salt, acidic fermentation environment and has the ability to specifically and efficiently synthesize esters, especially isoamyl acetate, is expected to further improve the overall flavor quality of soy sauce products. Summary of the Invention

[0006] Based on the above technical problems, the main objective of this invention is to overcome the shortcomings of the aforementioned background technology and to screen and obtain a strain of *Pediococcus lactis* ZF636. This strain has a strong ester production capacity, especially high production of isoamyl acetate. When applied to the fermentation of high-salt foods, it can significantly increase the content of esters, especially isoamyl acetate, as well as the total amount of volatile compounds. At the same time, it enhances the soy sauce aroma, ester aroma, umami flavor, and richness of the product, thus comprehensively improving the flavor quality of the product.

[0007] To achieve the above objectives, the inventors conducted in-depth research and, through repeated studies and demonstrations, obtained the solution of this invention, as detailed below:

[0008] In a first aspect, the present invention provides a strain of Pediococcus acidilacticii ZF636, the preservation number of which is GDMCC No: 67286.

[0009] The strain was deposited on November 13, 2025 at the Guangdong Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0010] Secondly, the present invention provides a fermentation agent comprising the aforementioned Pediococcus lactis ZF636.

[0011] Thirdly, the present invention provides the application of the above-mentioned Pediococcus lactis ZF636 or fermentation agent in fermented condiments. The condiments mainly refer to fermented condiments that produce flavor by fermentation using microorganisms and their enzymes, such as soy sauce, sauce, vinegar, fermented black beans or fermented bean curd, especially fermented bean products, with soy sauce being preferred.

[0012] Fourthly, the present invention provides a method for preparing soy sauce, the method comprising the step of fermentation using the above-mentioned Pediococcus lactis ZF636 or fermentation agent, preferably the inoculation time of Pediococcus lactis ZF636 or fermentation agent is in the early stage of soy sauce mash fermentation.

[0013] The preparation method described herein can employ fermentation methods commonly used in the field, and the process parameters of the fermentation method can be adjusted according to actual production conditions.

[0014] Furthermore, the amount of *Pediococcus lactis* ZF636 added was 10. 6 -10 7 CFU / g.

[0015] Furthermore, the fermentation time is 90-180 days.

[0016] Fifthly, the present invention provides a soy sauce, which is prepared by any of the preparation methods described in the present invention.

[0017] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0018] 1. This application screened a strain of *Pediococcus lactis* ZF636 from traditional fermented soy sauce mash. It is salt and acid tolerant, and can still slowly proliferate at a salinity of 18%, with little effect on its growth rate at salinity of 12% and below. Moreover, it maintains a good growth rate at a pH of 4.5. It has a strong ester production capacity, especially high production of isoamyl acetate. When applied to the fermentation of high-salt foods, it can significantly increase the content of esters, especially isoamyl acetate, as well as the total amount of volatile compounds. At the same time, it enhances the soy sauce aroma, ester aroma, umami, and richness of the product, thus comprehensively improving the flavor quality of the product.

[0019] 2. The *Pediococcus lactis* strain ZF636 provided by this invention, when used for soy sauce fermentation, increases the number of ester compounds in crude soy sauce from 25 in the control group to 33, with the total content increasing nearly threefold. The levels of several key ester compounds increase by 48.28%-1155.00% compared to the control group, with isoamyl acetate showing the most significant increase at 11.55 times. Simultaneously, the total amount of volatile substances also increases by 30.27%. The resulting soy sauce exhibits superior aroma and ester fragrance compared to the control group, and its umami and rich flavor are also significantly better. The soy sauce has a more balanced and richer flavor, significantly improving its overall taste and quality. Attached Figure Description

[0020] Figure 1 Colony morphology of Pediococcus lactis ZF636;

[0021] The *Pediococcus acidilacticii* ZF636 provided by this invention was deposited at the Guangdong Microbial Culture Collection Center on November 13, 2025, at the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC NO: 67286. This strain was received and registered by the collection center on November 13, 2025, and was confirmed to be a viable strain by the collection center on November 13, 2025. Detailed Implementation

[0022] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional food-grade reagents, methods and equipment in the art.

[0025] The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0026] Example 1: Screening of the target strain

[0027] 1. Strains Isolation and Purification

[0028] 5g of traditionally fermented soy sauce mash was inoculated into 100mL of MRS broth medium and incubated at 37℃ for 48h. The mash was then spread onto MRS screening medium and incubated at 37℃ for 48h. Colonies with typical lactic acid bacteria colony characteristics were picked and isolated and purified by streak plating.

[0029] MRS broth medium: 10g peptone, 5g beef meal, 4g yeast powder, 2g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate and 1000ml distilled water.

[0030] MRS screening medium: 10g peptone, 5g beef meal, 4g yeast powder, 2g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15-20g agar powder, 100g NaCl and 1000ml distilled water.

[0031] 2. Preliminary screening of bacterial strains

[0032] The purified strains were inoculated into MRS broth medium and cultured at 37℃ and 150 r / min for 96 h with shaking to obtain culture solutions. The aromas produced by the fermentation of different purified strains were sensorily evaluated by 10 experienced technicians, with a focus on ester aromas. The intensity was rated on a 0-5 scale: 0 (none), 1 (weak), 2 (relatively weak), 3 (moderate), 4 (relatively strong), 5 (strong). Sterile liquid medium served as a blank control. Based on the sensory results, 10 lactic acid bacteria with strong ester aroma production capabilities were selected (Table 1), and lactic acid bacteria that did not produce aroma or had weak aroma production capabilities were initially eliminated. Among them, RS-005 had the strongest aroma.

[0033] Table 1 Initial screening results of strains

[0034]

[0035] 3. Secondary screening of strains

[0036] Strains with good sensory performance in the initial screening were inoculated into modified MRS broth medium (with 60% soy sauce crude oil added) and cultured at 37℃ and 150 r / min for 96 h with shaking to obtain the culture medium. Modified MRS broth medium (with 60% soy sauce crude oil added) without inoculation was used as a blank control. The content of volatile components in the fermentation broth was detected by solid phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS), and the results are recorded in Table 2.

[0037] Sample preparation: Pipette 5 mL of sample and 25 μL of 3.35 × 10⁻⁶ ppm solution. -3 A 2-octanol solution (internal standard) at a concentration of mg / mL was added to a 20 mL headspace vial and equilibrated at 40 °C for 10 min in an incubator. Then, headspace extraction was performed at 40 °C using a CAR / PDMS solid-phase microextraction head for 30 min. After extraction, the sample was injected at 250 °C for 1 min to complete the extraction.

[0038] GC conditions: HP-INNOWax capillary column (60 m × 250 μm ≠ 0.25 μm); injection port temperature 250℃; temperature program: 40℃ for 5 min, then ramped to 240℃ at 5℃ / min and held for 15 min. Carrier gas: helium, flow rate 1.2 mL / min.

[0039] MS conditions: EI ion source, ion source temperature 250℃, electron energy 70 eV, quadrupole and transfer line temperatures 200℃ and 220℃ respectively, mass scan range 35~500 amu.

[0040] Table 2 Results of strain rescreening (unit: mg / L)

[0041]

[0042] As shown in Table 2, compared with the blank control, the content of esters and the total amount of volatile compounds in the fermentation broth significantly increased after inoculation with 10 strains. Among the 10 lactic acid bacteria strains, RS-005 had the highest content of esters, isoamyl acetate, and total volatile compounds in its fermentation broth. Combined with the results of the initial screening by smell, it can be seen that strain RS-005 can produce pleasant ester flavor compounds, especially with a high production capacity of isoamyl acetate. Therefore, it was selected as the original strain for subsequent mutagenesis.

[0043] 4. Mutagenesis

[0044] The RS-005 strain was inoculated onto MRS broth medium and cultured at 37°C for 48 hours. After centrifugation, the strain suspension was resuspended in sterile physiological saline to dilute it to 2 × 10⁻⁶. 6 CFU / mL. Prepare a sterile 10% (V / V) glycerol mixture and thoroughly mix it with the strain suspension at a 1:1 ratio. Take 10 μL of the mixture and spread it onto a sterile slide, then transfer the slide to a sterile stage.

[0045] ARPT mutagenesis conditions: Helium was used as the working gas, the distance between the slide and the plasma emitter port was 2 mm, the power was 120 W, the gas flow rate was 10 SLM, and the time was set to 70 s.

[0046] 5. Screening of target strains

[0047] After mutagenesis, the strains were inoculated into modified MRS broth medium (with 30% soy sauce crude oil added) and cultured at 37℃ and 150 r / min for 96 h with shaking to obtain the culture solution. Ten experienced technicians conducted sensory evaluation of the aroma produced by the culture solutions of different strains, with the intensity rated from 0 to 5, namely none (0), weak (1), relatively weak (2), medium (3), relatively strong (4), and strong (5). The experiment included a blank control, which was sterilized and uninoculated MRS medium (with 30% soy sauce crude oil added). Based on the sensory results, 10 lactic acid bacteria with strong aroma-producing ability were screened, and lactic acid bacteria with weak aroma-producing ability were initially eliminated.

[0048] Ten strains with the strongest aroma-producing sensory characteristics were selected and inoculated into modified MRS broth medium (with 60% crude soy sauce added). The cultures were then incubated at 37℃ and 150 r / min for 96 h with shaking to obtain the culture broth. The uninoculated modified MRS broth medium (with 60% crude soy sauce added) served as a blank control. The content of volatile flavor compounds in the fermentation broth was measured using GC-MS (the specific method is the same as above). The results are recorded in Table 3.

[0049] Table 3. Results of volatile flavor compounds in the mutagenic strains (unit: mg / L)

[0050]

[0051] As shown in Table 3, the fermentation broth of RS-005-280 contained the highest levels of esters and isoamyl acetate, indicating its strongest ability to produce esters and isoamyl acetate. Strain RS-005-280 was inoculated into MRS medium and cultured at 37℃ for 48 hours. Colony morphology was then observed. Figure 1 As shown, the colonies are round, raised, smooth, with neat edges, and milky white. Sequencing identified the strain as *Pediococcus acidilacticii*, and it was named *Pediococcus acidilacticii* ZF636. It was deposited on November 13, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67286.

[0052] Example 2 Performance Test of Pietrococcus lactis ZF636

[0053] 1. Salt tolerance test

[0054] After activation, 1 mL of the selected *Pediococcus lactis* strain ZF636 was inoculated into 100 mL of MRS broth at different salinities (0%, 3%, 6%, 9%, 12%, 15%, 18%) and cultured at 37°C for 36 h. The culture medium without inoculation was used as a blank control. The absorbance of the bacterial culture medium was measured at 600 nm to determine the salt tolerance of the strain. The results are shown in Table 4. *Pediococcus lactis* ZF636 grew slowly at 18% salinity, but still proliferated; while salinity at 12% and below had little effect on its growth rate.

[0055] 2. Acid resistance test

[0056] After activation, 1 mL of the selected *Pediococcus lactis* strain ZF636 was inoculated into 100 mL of MRS broth at different pH values ​​(3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0) and cultured at 37℃ for 36 h. A culture without inoculation was used as a blank control. The absorbance of the strain culture was measured at 600 nm to determine its acid tolerance. The results are shown in Table 4. *Pediococcus lactis* ZF636 grew slowly at pH 3.5 but still proliferated, and maintained a good growth rate at pH 4.5, fully meeting the requirements for use in soy sauce fermentation environments.

[0057] Table 4. Results of salt and acid resistance tests for Pediococcus lactis ZF636

[0058]

[0059] Example 3: Test on the effect of fermented soy sauce made with Pediococcus lactis ZF636

[0060] 1. Preparation of seed culture medium: The selected strain of Pediococcus lactis ZF636 was inoculated into MRS broth medium with a salinity of 10% and cultured at 37℃ for 48 h to obtain a seed culture medium with high bacterial activity and high bacterial density.

[0061] 2. Koji preparation: Add Aspergillus oryzae Hu Niang 3.042 to wheat bran and incubate at 30℃ for 2-3 days to obtain Aspergillus oryzae Hu Niang 3.042 wheat bran koji. Steam soybeans and roast wheat are mixed together as koji preparation raw materials, cooled, and then the above-mentioned wheat bran koji is added and stirred evenly. The amount of wheat bran koji added is 3%.

[0062] The initial cultivation temperature for koji making is 32℃-33℃. After 11-12 hours of cultivation, when a large amount of koji material has clumped together, the first loosening of the koji is carried out. After the first loosening, the cultivation temperature is maintained at 30℃, and water is sprayed every 2 hours. Depending on the clumping of the koji material, the second loosening is carried out after 17-18 hours. After loosening, the cultivation temperature is maintained at 26-28℃, and the third loosening is carried out after 24-26 hours. After that, the cultivation temperature is kept constant, and the koji is produced after 48 hours.

[0063] 3. Soy sauce fermentation: Mix the starter culture with brine at a ratio of 1:2 (starter culture to brine by weight), with a brine concentration of 18%. Calculate the fermentation process by weight of the soy sauce mash at 10... 7 The experimental group was inoculated with *Pediococcus lactis* ZF636 seed culture at an inoculum concentration of CFU / g into soy sauce mash, while the control group was soy sauce mash without inoculation. Both groups fermented for 3 months using the same soy sauce fermentation process. After fermentation, the crude oil was obtained by filtration through a filter cloth.

[0064] 4. The content of volatile flavor compounds in the crude oil of the experimental group and the control group was measured by GC-MS, and the results are recorded in Table 5.

[0065] Table 5 Comparison of volatile substance content in different soy sauces (unit: mg / L)

[0066]

[0067] As shown in Table 5, the types of esters in the crude soy sauce with added Pediococcus lactis ZF636 increased from 25 in the control group to 33, with the total content increasing nearly threefold. The levels of several key ester compounds increased by 48.28%-1155.00% compared to the control group, with isoamyl acetate showing the most significant increase at 11.55 times. Simultaneously, the total amount of volatile substances also increased by 30.27%. These data fully demonstrate that Pediococcus lactis ZF636 can specifically optimize the flavor of soy sauce, with its core function being to significantly increase the types and content of esters, especially isoamyl acetate.

[0068] 5. Sensory evaluation: Fifteen professional R&D personnel with sensory training were selected to conduct sensory evaluation of the above crude oil. The intensity of soy sauce aroma, ester aroma, umami and richness was rated on a scale of 1 to 5. The scores from 1 to 5 represent the degree of taste sensory intensity as weak (1 point), weak (2 points), medium (3 points), strong (4 points), and strong (5 points), respectively. The arithmetic mean of the scores from the 15 professional R&D personnel was compared and the results are recorded in Table 6.

[0069] Table 6 Sensory Evaluation Results of Fermented Crude Oil

[0070]

[0071] As shown in Table 6, the soy sauce made with the added strain Pediococcus lactis ZF636 was significantly better than the control group in terms of aroma, ester aroma, umami, and rich flavor. In particular, the ester aroma was consistent with the test results of flavor substance content, indicating that Pediococcus lactis ZF636 has a great effect on improving the overall flavor of soy sauce.

[0072] In summary, the addition of Pediococcus lactis strain ZF636 to fermented soy sauce can significantly increase the content of volatile flavor compounds in the fermented product, and significantly increase the types and contents of esters, especially the content of isoamyl acetate. The aroma scores of soy sauce and ester aroma, as well as the umami and rich flavor scores of the finished product are significantly improved, which can comprehensively enhance the flavor quality of soy sauce.

[0073] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A strain of Pediococcus acidilactici (P. acidilactici) ZF636, characterized in that, Pediococcus acidilacticii The preservation number of the Pediococcus acidilactici ZF636 is GDMCC No: 67286. ​ 2. A fermentation inoculant characterized in that, The fermentation agent comprises the Pediococcus acidilactici ZF636 of claim 1.

3. The Pediococcus acidilactici ZF636 of claim 1 or the fermentation agent of claim 2 for use in fermentation of a condiment.

4. Use according to claim 3, wherein the compound is ###0002### The fermented condiment is a legume fermented product.

5. The use of claim 4, wherein the legume fermented product is soy sauce.

6. A method for producing a soy sauce, characterized by, The method comprises a step of fermentation using the Pediococcus acidilactici ZF636 of claim 1 or the fermentation agent of claim 2.

7. The production method according to claim 6, wherein The inoculation time of the Pediococcus acidilactici ZF636 or the fermentation agent is the early stage of soy sauce fermentation.

8. The production method according to claim 6, wherein The amount of Pediococcus acidilactici ZF636 added is 10 6 -10 7 CFU / g.

9. The production method according to any one of claims 6 to 8, characterized by, The fermentation time is 90-180 days.

10. A sauce, characterized by The soy sauce is prepared by the method of any one of claims 6-9.

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