A strain of Pediococcus acidilactici ZF627 and its application

Abstract

The present invention belongs to the field of microbial technology, and in particular relates to a strain of Pediococcus acidilactici ZF627 and its applications. The present invention provides a strain of Pediococcus acidilactici ZF627, which has high salt tolerance and high ester production. When used in fermenting fermented black beans, it can significantly increase the content of multiple key volatile compounds in the fermented black beans, including the total content of flavor substances such as alcohols, aldehydes, acids, and furans, especially the content of key flavor compounds such as isopentanol, ethyl acetate, and methyl acetate. Using Pediococcus acidilactici ZF627 in fermenting black beans can significantly enhance the flavor, mouthfeel, and aroma harmony of the fermented black beans, not only improving the overall aroma and flavor of the fermented black beans, but also making their flavor and mouthfeel more complex and multi-layered, significantly improving the flavor and overall quality of the fermented black beans.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Pediococcus acidilactici ZF627 strain and an application thereof. Background Art

[0002] Fermented black beans, a traditional fermented soy product beloved by consumers, are renowned for their unique flavor, rich nutritional value, and dual medicinal and edible properties. The fermentation process utilizes enzymes like proteases and cellulases, secreted by microorganisms during koji production, to break down soybean proteins and other macromolecules, producing small peptides, amino acids, and monosaccharides. These flavor precursors undergo a series of biochemical reactions during the subsequent fermentation process, resulting in the color, aroma, and flavor of the fermented black beans. Traditional fermented black beans are typically fermented under natural conditions. Differences in geographical environment and fermenting microorganisms lead to varying quality in the finished product, severely restricting the industry's development. To achieve industrialization, research into pure fermentation processes for black beans is essential. Furthermore, traditional fermented black beans have a unique and rich flavor. However, with the industrialization of black bean production, their flavor has fallen far short of that of naturally fermented black beans. Therefore, improving the flavor of pure fermented black beans is a current research priority.

[0003] Currently, there are many studies on fermentation microorganisms of fermented black beans, but the focus is mainly on molds and yeasts, while there is relatively little research on lactic acid bacteria. CN 104996931A discloses a method for making flavored black beans, which uses Bacillus subtilis and Lactobacillus plantarum to perform step-by-step mixed fermentation combined with low-temperature post-fermentation to produce black beans with good flavor. This method can increase the concentration of diacetyl and 2,3-butanediol in black beans, but the effect on improving the flavor of black beans is limited. Patent CN117678701A discloses a rapid fermentation method for reducing the salinity of black beans and improving the flavor. The black beans are inoculated with Pediococcus acidilactici and Zygosaccharomyces rouxii for enhanced fermentation, and the fermented bean embryos are compounded with spices, white wine extract and licorice extract to obtain a black bean product with a strong sauce and fermented bean flavor. However, this method has a complex production process and high production cost, making it unsuitable for large-scale production. Patent CN117981843A discloses a composite fermentation agent and its use in fermented light fermented black beans. The compound fermentation agent includes brewer's yeast, plant lactic acid bacteria, a Foshan Fengliao-derived light fermented black bean product, Actinidia arguta fruit juice, thiolated Codonopsis pilosula polysaccharide, and sodium citrate. The fermented light fermented black beans exhibit significantly improved flavor, taste, and nutritional activity. However, the compound fermentation agent's complex formulation complicates large-scale production. Summary of the Invention

[0004] Based on the above technical problems, the main purpose of the present invention is to overcome the shortcomings of the above background technology and provide a Pediococcus acidilactici ZF627 strain, which has high salt tolerance and high ester production ability. It is used for fermentation of fermented black beans, which can significantly improve the flavor, taste and aroma coordination of fermented black beans, and improve the overall aroma, flavor and quality of fermented black beans.

[0005] To achieve the above objectives, the inventors conducted in-depth research and, after repeated research and demonstration, completed the present invention, which is as follows:

[0006] In the first aspect, the present invention provides a strain of Pediococcus acidilactici ZF627, which was deposited in the Guangdong Microbiological Culture Collection Center on July 17, 2024, with a deposit number of GDMCC NO: 64879.

[0007] In a second aspect, the present invention provides a fermentation agent, wherein the fermentation agent comprises the above-mentioned Pediococcus acidilactici ZF627.

[0008] In a third aspect, the present invention provides the use of the above-mentioned Pediococcus acidilactici ZF627 or fermentation agent in fermented food processing.

[0009] Furthermore, the fermented food is any one or more types of fermented bean foods, and preferably the fermented bean food is fermented black beans.

[0010] In a fourth aspect, the present invention provides a method for preparing fermented black beans, wherein the method comprises adding the above-mentioned Pediococcus acidilactici ZF627 or a fermentation agent during fermentation of the black beans for fermentation.

[0011] Furthermore, the Pediococcus acidilactici ZF627 is added in the middle of the fermentation period.

[0012] Furthermore, the addition amount of the Pediococcus acidilactici ZF627 is 10 5 ~10 7 CFU / mL.

[0013] Furthermore, the fermentation time is 25-35 days.

[0014] In a fifth aspect, the present invention provides a fermented black beans, which is prepared by the preparation method of the present invention.

[0015] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0016] 1. The Pediococcus acidilactici ZF627 provided by the present invention has high salt tolerance and can grow normally at a salt concentration of 20%. It has a high ester production capacity, and the output of ethyl acetate during the fermentation process can be as high as 201.52 μg / L, and has a strong ability to produce flavor substances. This strain can be used in the production of high-salt fermented foods, improving the product flavor and thus improving food quality.

[0017] 2. The Pediococcus acidilactici ZF627 provided by the present invention can significantly increase the content of total volatile flavor substances in fermented black beans by 51%, among which the relative content of key aroma substances such as alcohols, aldehydes, acids, and furans can be increased by 64%-100%.

[0018] 3. The Pediococcus acidilactici ZF627 provided by the present invention is used for fermentation of fermented black beans. The concentrations of important flavor compounds in the fermented black beans, methyl acetate, ethyl acetate, isopentanol, and 3-octanone, are significantly increased, which are 3.94, 2.20, 2.16, and 1.95 times that of natural fermentation, respectively. This can significantly increase the fruity and mushroom aromas of the fermented black beans, thereby improving the overall flavor of the fermented black beans.

[0019] 4. The fermentation method of fermented black beans provided by the present invention has a simple process and can be used for large-scale production; the overall flavor, taste and aroma coordination of the fermented black beans are significantly better than those of naturally fermented black beans and commercially available black beans, which not only improves the overall aroma and flavor of the black beans, but also makes its flavor and taste more complex and multi-layered, significantly improving the overall flavor and quality of the black beans. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony morphology of Pediococcus acidilactici ZF627;

[0021] The Pediococcus acidilactici ZF627 provided by the present invention has been deposited in the Guangdong Microbial Culture Collection Center on July 17, 2024, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the deposit number is GDMCC NO: 64879; the strain was received and registered by the collection center on July 17, 2024, and was detected as a viable strain by the collection center on July 17, 2024. DETAILED DESCRIPTION

[0022] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0023] Unless defined otherwise, 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 belongs.

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

[0025] The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0026] The headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technique used in the present invention is used to determine the volatile flavor compounds in fermented black beans. The specific method is as follows:

[0027] (1) Sample processing

[0028] Weigh 2 g of the ground sample and place it in a 15 mL headspace bottle. Add 3 g of sodium chloride and an appropriate amount of ultrapure water, then add 15 μL of 2-octanol (concentration 9.7 ppm) internal standard solution and mix evenly. Place it in a constant temperature incubator at 60 °C and shake for 30 min. Insert a DVB / CAR / PDMS solid phase microextraction head for extraction for 30 min and resolve at 250 °C for 1 min.

[0029] (2) GC-MS conditions

[0030] GC conditions: HP-INNOWax capillary column (60 m × 250 μm); splitless injection mode, temperature program: 55 °C for 5 min, then 5 °C / min to 230 °C, and hold for 15 min; helium carrier gas at a flow rate of 1.2 mL / min.

[0031] MS conditions included an EI ion source with ion source stability at 250°C, quadrupole and mass spectrometer interface temperatures of 150°C and 280°C, respectively; an electron energy of 70 eV, full scan mode, and a mass scan range of 29–500 m / z. Mass spectral data of unknown volatile substances were compared with the NIST 2017 standard spectral library, with peak identification determined using a similarity (SI) of >80 (maximum 100).

[0032] (3) Quantitative analysis

[0033] The internal standard method was used to calculate the relative content of each component. The calculation formula is as follows:

[0034]

[0035] Wherein, C represents the relative content of a single component, mg / kg; A1 is the peak area of ​​a single component; A2 is the peak area of ​​the internal standard; C2 is the mass concentration of the internal standard, μg / mL; V2 is the volume of the internal standard, μL; and M is the sample mass, g.

[0036] Example 1 Screening of target strains

[0037] 1. Processing of raw materials

[0038] Take a certain amount of flavored fermented black beans, crush them in a sterile mortar, weigh 10g of the crushed sample and add it to a 250mL conical flask containing 90mL sterile saline. Use a constant temperature shaking incubator to shake and culture at 30℃ and 200rpm for half an hour to allow the microorganisms in the sample to be fully suspended in the saline. Pipette the suspension and add sterile saline to dilute it step by step 10 times to 10 -2 -10 -6 , prepare bacterial dilution liquid; select 2-3 bacterial suspensions with appropriate gradients, draw 200 μL of liquid respectively and add them to LB plates and MRS plates containing 20 mg / L natamycin, evenly spread them with a sterile spreading rod, and culture them upside down in a 37°C incubator for 1-2 days; after clearly visible colonies grow on the LB plates and MRS plates, use a sterile inoculation loop to pick single colonies with inconsistent morphology, inoculate them into corresponding LB or MRS culture media for streak culture at 37°C in an incubator for 1-2 days; repeat this 2-3 times until the morphology of the colonies grown on the plates is basically consistent, and then a pure microbial strain is obtained.

[0039] Through the above-mentioned microbial separation and purification scheme, 12 purified strains were obtained from flavored fermented black beans and conventionally preserved, and were numbered DC101-DC112 respectively.

[0040] 2. Preliminary screening of bacterial strains

[0041] The salt content during fermentation of fermented black beans is between 12-15%. In order to ensure that the target microorganisms can maintain a good growth state during the fermentation of fermented black beans, salt tolerance testing is required. The 12 purified strains isolated were inoculated into the corresponding LB or MRS culture medium for activation, and then a single colony was picked and inoculated into the LB or MRS liquid culture medium to be cultured until the mid-logarithmic growth period; the seed liquid was inoculated into LB liquid culture medium and MRS liquid culture medium with salt contents of 8%, 12%, 16%, and 20% at a 2% inoculation rate, and cultured at 37°C and 200rpm for 3 days. After the fermentation was completed, the absorbance value OD at a wavelength of 600nm was measured. 600 , 3 parallels were set up in each group. The test results are shown in Table 1:

[0042] Table 1 Growth of 12 purified strains in different salt media

[0043]

[0044] As shown in Table 1, the growth rates of all strains decreased with increasing salt content. However, strains DC103, DC104, DC105, DC107, and DC111 exhibited strong salt tolerance and were able to maintain relatively high biomass at salt levels of 12%-20%. Therefore, these five strains can be applied to the fermentation process of fermented black beans. These five strains were selected for rescreening.

[0045] 3. Lactic acid bacteria re-screening

[0046] The five strains initially screened were reactivated and cultured, inoculated into LB or MRS liquid medium, and cultured at 37°C for 24 h. The cells were collected by centrifugation, washed with sterile saline, and diluted to a concentration of 10 9 -10 10 A bacterial suspension containing 100 CFU / mL of culture medium was then inoculated at a 2% inoculum into a rescreening medium (100 mL of sterile fermented black bean extract, 0.5% acetic acid, and 2% ethanol). The culture was incubated at 37°C for 3 days. An uninoculated rescreening medium served as a control. After fermentation, the ethyl acetate content was determined using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results are shown in Table 2.

[0047] Table 2 Results of rescreening of 5 highly salt-tolerant strains

[0048] Group Ethyl acetate (μg / L) control group 13.68±0.54 DC103 fermentation group 60.15±3.11 DC104 fermentation group 70.78±4.21 DC105 fermentation group 201.52±4.68 DC107 fermentation group 50.73±2.32 DC111 fermentation group 174.67±6.17

[0049] As shown in Table 2, the ethyl acetate content in all fermentation groups was higher than that in the control group, indicating that these strains were able to synthesize ester compounds to a certain extent. Among them, the amount of ethyl acetate produced by strain DC105 in the rescreened culture medium was significantly higher than that of other strains, showing a strong ester production ability.

[0050] The DC105 strain obtained by the above screening was genetically identified as Pediococcus acidilactici, which can be used in the food fermentation industry. It was named Pediococcus acidilactici ZF627 and was deposited in the Guangdong Provincial Microbial Culture Collection on July 17, 2024, with the deposit number GDMCC NO: 64879.

[0051] Example 2 Pediococcus acidilactici ZF627 fermentation test of fermented black beans

[0052] (1) Soaking: Soak the screened soybeans at room temperature for 2-3 hours until the beans are swollen and wrinkle-free, feel firm, the skins are not easy to fall off, and there is no large amount of foam on the liquid surface;

[0053] (2) Steaming: Steam the soaked soybeans at 115°C for 20 min in a high-temperature and high-pressure autoclave;

[0054] (3) Koji making: After the soybeans are naturally cooled to 35-40°C, soy sauce koji essence and wheat flour are evenly sprinkled on the soybeans and stirred evenly. The ratio of koji essence to soybean dry weight is 0.1%, and the amount of wheat flour added is 100 times that of koji essence. The koji making temperature is controlled at 32-35°C, the koji making time is 48 hours, and the koji is turned every 12 hours;

[0055] (4) Stack fermentation: 12.5% ​​brine was mixed evenly with the koji material in a ratio of brine to koji material = 8:33, and fermented at 30°C for 30 days, with occasional stirring during the fermentation process;

[0056] (5) Inoculation: Pediococcus acidilactici ZF627 was cultured to the middle and late stages of logarithmic growth, the bacterial pellet was collected by centrifugation, washed 2-3 times with physiological saline, and the bacterial pellet was resuspended to a cell concentration of 4 × 10 7 CFU / mL, when the fermentation time of fermented douchi reached 13 days, Pediococcus acidilactici ZF627 was inoculated into the fermented douchi at an inoculation rate of 2% for fermentation; DC103 strain was not inoculated or inoculated in the same way as a control;

[0057] (6) Detection and analysis: After the fermentation, samples were collected to determine the physical and chemical indicators and volatile flavor compound content of fermented fermented black beans. The results are recorded in Tables 3-4. Based on the orthogonal partial least squares discriminant analysis (OPLS-DA), volatile compounds with variable weight values ​​(VIP) less than 1 and Student's t-test P values ​​less than 0.05 were screened to obtain some key differential aroma compounds, and their concentration measurement results are recorded in Table 5.

[0058] (7) Sensory evaluation: The fermented tempeh described above was compared with a commercially available fermented tempeh for flavor evaluation. Thirty trained professional sensory evaluators were invited to conduct sensory evaluations of the different fermented tempeh based on five dimensions: flavor, color, mouthfeel, appearance, and aroma harmony. The samples were scored and the average score was taken. The sample scores and final sensory evaluation results are recorded in Table 6.

[0059] Table 3 Physical and chemical index determination results of fermented fermented soybeans by Pediococcus acidilactici ZF627

[0060] Group Total free amino acids g / 100g Reducing sugar g / 100g Group 1: No vaccination 4.90±0.08 5.73±0.01 Group 2: DC103 fermentation group 4.38±0.16 6.37±0.00 Group 3: ZF627 fermentation group 5.89±0.09 6.91±0.01

[0061] As shown in Table 3, the ZF627 fermentation group exhibited significant advantages in terms of total free amino acid and reducing sugar content. The ZF627 fermentation group had the highest total free amino acid content, reaching 5.89±0.09 g / 100 g, significantly higher than the other groups. This indicates that inoculating Pediococcus acidilactici ZF627 during the mid-stage fermentation of fermented black beans significantly promoted protein hydrolysis, generating more free amino acids. Free amino acids are important flavor precursors in black beans, generating a variety of aroma compounds through the Maillard reaction, thereby enhancing the flavor of black beans. The ZF627 fermentation group also had the highest reducing sugar content, reaching 6.91±0.01 g / 100 g. This indicates that inoculating strain ZF627 during the mid-stage fermentation of black beans significantly promoted carbohydrate degradation, generating more reducing sugars. Reducing sugars are not only an important source of sweetness in black beans, but also react with free amino acids through the Maillard reaction to form complex aroma compounds, further enhancing the flavor of black beans.

[0062] Table 4 Detection of volatile flavor substances in fermented fermented black beans samples

[0063]

[0064] As shown in Figure 4, the total volatile flavor compounds in fermented fermented black beans inoculated with ZF627 were significantly higher than those in the control group, reaching a 51% increase compared to natural fermentation. Key aroma compounds, such as alcohols, aldehydes, acids, and furans, increased by 66.88%, 75.71%, 100.00%, and 64.06%, respectively. Alcohols are important aroma components in black beans, imparting a rich, mellow aroma. Aldehydes typically have a pungent odor in black beans, but their presence in appropriate amounts can add complexity and depth to the product. Acids contribute to the umami and acidity of black beans, making their flavor more distinct. Furans, which typically impart caramel and roasted aromas, contribute to the complex aroma of black beans.

[0065] Table 5 Different aroma substances in fermented fermented black bean samples

[0066]

[0067]

[0068] As shown in Table 5, fermentation of fermented black beans inoculated with ZF627 significantly increased the content of seven key flavor compounds, including isopentanol, 1-octen-3-ol, ethyl acetate, and methyl acetate. In particular, the levels of isopentanol, ethyl acetate, and methyl acetate were more than double those in the control group. The isopentanol content in fermented black beans inoculated with ZF627 reached 0.80 mg / kg, 2.16 times that of the control group. Isoopentanol, a fruity compound, significantly enhanced the fruity aroma of black beans. The ethyl acetate content in the ZF627-inoculated group was 0.11 mg / kg, 2.2 times that of the control group; the methyl acetate content reached 0.63 mg / kg, 3.94 times that of the control group. These two compounds, known for their fruity aromas, enhance the sweet and fruity aroma of black beans, giving them a richer and fuller flavor. In addition, the content of 1-octen-3-ol, 3-methylbutanal, 2-methylbutanal and 3-octanone in fermented fermented black beans inoculated with ZF627 was also significantly increased, and its content was 1.5-1.9 times that of the CK group; among them, 1-octen-3-ol has a mushroom aroma, which can increase the aroma level of black beans, 3-methylbutanal has a fruity aroma, 2-methylbutanal has an almond smell, which can increase the aroma intensity and complexity of black beans, and 3-octanone has a fruity aroma, which can be integrated with the sauce aroma and ester aroma produced by the fermentation of black beans, and can also improve the taste of black beans, making the flavor of black beans more unique.

[0069] Table 6 Sensory evaluation results of different fermented fermented black beans

[0070] Group Flavor Color Taste Appearance Aroma harmony Comprehensive evaluation Commercially available control 6.1 5.2 6.8 6.5 5.7 6.5 Group 1 5.8 4.9 6.6 6.2 5.4 6.3 Group 2 6.4 5.4 7.0 7.0 6.1 6.8 Group 3 7.4 6.0 7.5 7.0 6.8 7.3

[0071] Note: Each indicator is scored on a 9-point scale, with larger values ​​indicating more prominent indicators.

[0072] The results of the sensory evaluation experiment show that the fermented fermented black beans inoculated with ZF627 performed best in multiple sensory indicators, and its flavor, taste and aroma coordination were significantly better than other groups. This is consistent with the results of the volatile compound analysis, indicating that inoculating lactic acid bacteria ZF627 in the middle stage of fermentation of black beans can significantly improve the overall quality and flavor of black beans.

[0073] In summary, by inoculating Pediococcus acidilactici ZF627 in the middle stage of fermentation of fermented black beans, the contents of various key volatile compounds in the fermented black beans were significantly increased, including the total content of flavor substances such as alcohols, aldehydes, acids and furans, especially the contents of isopentanol, 1-octen-3-ol, ethyl acetate, methyl acetate, 3-methylbutanal, 2-methylbutanal and 3-octanone were significantly increased. The increase of these compounds can significantly improve the flavor, taste and aroma coordination of the fermented black beans, not only improving the overall aroma and flavor of the fermented black beans, but also making its flavor and taste more complex and multi-layered, significantly improving the overall flavor and quality of the fermented black beans.

[0074] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A strain of Pediococcus acidilactici ZF627, characterized in that: The deposit number of the Pediococcus acidilactici ZF627 is GDMCC NO: 64879.

2. A fermentation agent, characterized in that: The fermentation agent comprises the Pediococcus acidilactici ZF627 according to claim 1.

3. Use of the Pediococcus acidilactici ZF627 according to claim 1 or the fermentation agent according to claim 2 in fermented food processing.

4. The use according to claim 3, characterized in that The fermented food is any one or more types of fermented bean foods.

5. The use according to claim 4, characterized in that The fermented bean food is fermented black beans.

6. A method for preparing fermented black beans, characterized in that: The method comprises adding the Pediococcus acidilactici ZF627 according to claim 1 or the fermentation agent according to claim 2 during fermentation of fermented black beans for fermentation.

7. The preparation method according to claim 6, wherein The Pediococcus acidilactici ZF627 is added in the middle of the fermentation process.

8. The preparation method according to claim 6, wherein The addition amount of the lactic acid bacteria ZF627 is 10 5 ~10 7 CFU / mL.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The fermentation time is 25-35 days.

10. A fermented black bean, characterized in that: The fermented black beans are prepared by the preparation method according to any one of claims 6 to 9.

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