A composite fermentation agent and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a composite fermentation agent of a Staphylococcus carnosus ZF626 and a Pediococcus acidilactici ZF627 and applications thereof. The composite fermentation agent provided by the present invention has high salt tolerance and high ester production capacity, and is used for fermenting fermented black beans. It can significantly increase the content of a variety of key volatile compounds in the fermented black beans, including the total content of flavor substances such as alcohols, esters, phenols, and acids, especially the content of key flavor compounds such as isopentanol, ethyl acetate, γ-caprolactone, and guaiacol. The composite fermentation agent can be used to ferment fermented black beans to significantly improve the flavor, taste, and aroma coordination of the black beans, not only improving the overall aroma and flavor of the black beans, but also making the flavor and taste more rich, complex, layered, and recognizable, significantly improving the overall flavor and quality of the black beans.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a composite fermentation agent of a Staphylococcus carnosus ZF626 and a Pediococcus acidilactici ZF627 and applications 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-mentioned background technology and provide a composite fermentation agent comprising Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627. The fermentation agent has high salt tolerance and high ester production capacity. When used for fermentation of fermented black beans, it can significantly improve the flavor, taste and aroma coordination of the fermented black beans, and improve the overall aroma, flavor and quality of the 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 a first aspect, the present invention provides a composite lactic acid bacteria fermentation agent, which comprises Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627.

[0007] In a second aspect, the present invention provides a composite fermentation method, wherein the bacteria inoculated in the fermentation method include the above-mentioned Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627.

[0008] In a third aspect, the present invention provides the use of the above-mentioned fermentation agent or composite fermentation method 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 fermentation agent during the fermentation process of the fermented black beans for fermentation.

[0011] Furthermore, the fermentation agent is added in the early stage of fermentation.

[0012] Furthermore, the addition amount of the fermentation agent is 10 5 ~10 7 CFU / mL.

[0013] Furthermore, the ratio of Staphylococcus carnosus ZF626 to Pediococcus acidilactici ZF627 in the fermentation bacteria agent is 1:1.

[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 composite fermentation agent provided by the present invention has high salt tolerance and high ester production capacity, and has a strong ability to produce flavor compounds; it can be used in the production of high-salt fermented foods, improving product flavor and thus improving food quality.

[0017] 2. The composite fermentation agent provided by the present invention can significantly increase the content of total volatile flavor substances in fermented black beans when used for fermentation, with an increase rate of 71%, among which the relative content of key aroma substances such as alcohols, esters, phenols, and acids can be increased by 58.4%-132.3%.

[0018] 3. The composite fermentation agent provided by the present invention is used for fermenting fermented black beans. The concentrations of seven key flavor substances in the fermented black beans, including isoamyl alcohol, ethyl acetate, γ-caprolactone, and guaiacol, are significantly increased. Of particular note, γ-caprolactone, which was not detected in the control, was detected in the composite fermented black beans, and the content of guaiacol increased by more than 27 times. In addition, the content of substances such as isoamyl alcohol and ethyl acetate also increased by 48%-140%. The increase in the content of these flavor compounds can significantly improve the flavor, taste, and aroma harmony of the fermented black beans.

[0019] 4. The present invention provides a composite fermentation method for fermented black beans, which has a simple process and can be used for large-scale production. The fermented black beans are significantly superior to naturally fermented black beans and commercially available black beans in terms of overall flavor, taste and aroma coordination. It not only improves the overall aroma and flavor of the black beans, but also makes the flavor and taste more rich, complex, layered and recognizable, 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 Staphylococcus carnosus ZF626;

[0021] Figure 2 This is the colony morphology of Pediococcus acidilactici ZF626;

[0022] The Staphylococcus carnosus ZF626 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: 64878; 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.

[0023] 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

[0024] 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.

[0025] 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.

[0026] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional food-grade reagents, methods and equipment in the art.

[0027] 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.

[0028] 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:

[0029] (1) Sample processing

[0030] 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.

[0031] (2) GC-MS conditions

[0032] 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.

[0033] 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).

[0034] (3) Quantitative analysis

[0035] The internal standard method was used to calculate the relative content of each component. The calculation formula is as follows:

[0036]

[0037] 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.

[0038] Example 1 Screening of target strains

[0039] 1. Processing of raw materials

[0040] 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.

[0041] 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.

[0042] 2. Preliminary screening of bacterial strains

[0043] 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:

[0044] Table 1 Growth of 12 purified strains in different salt media

[0045]

[0046]

[0047] 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.

[0048] 3. Lactic acid bacteria re-screening

[0049] 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.

[0050] Table 2 Results of rescreening of 5 highly salt-tolerant strains

[0051] 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

[0052] 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 ethyl acetate production of strains DC105 and DC111 in the rescreened culture medium was significantly higher than that of other strains, showing a strong ester production ability.

[0053] The DC105 and DC111 strains obtained in the above screening were genetically identified as Pediococcus acidilactici and Staphylococcus carnosus, respectively, which can be used in the food fermentation industry. They were named Pediococcus acidilactici ZF627 and Staphylococcus carnosus ZF626, respectively, and were deposited in the Guangdong Provincial Microbial Culture Collection on July 17, 2024, with the deposit numbers GDMCC NO: 64879 and GDMCC NO: 64878, respectively.

[0054] Example 2 Test of fermented fermented black beans by Pediococcus acidilactici ZF627 and Staphylococcus carnosus ZF626

[0055] (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;

[0056] (2) Steaming: Steam the soaked soybeans at 115°C for 20 min in a high-temperature and high-pressure autoclave;

[0057] (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;

[0058] (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;

[0059] (5) Inoculation: Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627 were cultured to the middle and late stages of logarithmic growth, and the bacterial pellets were collected by centrifugation. The pellets were washed 2-3 times with physiological saline and resuspended to a cell concentration of 5 × 10 7 CFU / mL and then mixed in a ratio of 1:1. When the fermentation time of fermented black beans reached 20 hours, the mixed bacterial liquid was inoculated into the fermented black beans at an inoculum volume of 2% for fermentation; no inoculation and single inoculation of Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627 were used as controls;

[0060] (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.

[0061] (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.

[0062] Table 3 Results of physical and chemical index determination of fermented fermented soybeans by different strains

[0063]

[0064]

[0065] As shown in Table 3, the reducing sugar content of the ZF626 and ZF627 co-fermentation group was significantly higher than that of the other groups, reaching 10.37 g / 100 g, an increase of over 80% compared to the CK group. This suggests that inoculating ZF626 and ZF627 in the early stages of fermentation not only accelerates the conversion of polysaccharides like starch into monosaccharides but also helps generate more reducing sugars, potentially enhancing the sweetness of fermented black beans and enhancing their mellow flavor through the Maillard reaction. Overall, appropriately adjusting the microbial composition can effectively improve the key flavor components of fermented black beans.

[0066] Table 4 Detection of volatile flavor substances in fermented fermented black beans samples

[0067]

[0068] As shown in Figure 4, the total volatile flavor compounds in fermented fermented black beans inoculated with ZF626 and ZF627 were significantly higher than those in the control group, reaching a 71% increase compared to natural fermentation. The relative increases in key aroma compounds, including alcohols, esters, phenols, and acids, reached 58.44%, 132.30%, 73.45%, and 72.48%, respectively. Alcohols are important aroma components in black beans, imparting a rich, mellow aroma. Esters contribute to a more intense fruity and floral aroma. Phenolic compounds are often associated with woody, smoky, and spicy aromas in food. High phenolic content may impart a more complex woody or smoky aroma to black beans, enhancing the depth and layering of their flavor. Acids contribute to the umami and acidity of black beans, making their flavor more distinct.

[0069] Table 5 Different aroma substances in fermented fermented black bean samples

[0070]

[0071] As shown in Table 5, the composite fermentation of fermented black beans by inoculation with ZF626 and ZF627 can significantly increase the contents of seven key flavor substances in the fermented black beans, including isopentanol, ethyl acetate, γ-caprolactone, and guaiacol, and can significantly enhance the overall aroma and flavor of the fermented black beans.

[0072] First of all, γ-caprolactone is a special flavor compound detected in compound fermented fermented black beans, with a content of 1.31 mg / kg. It was not detected in the control group or the content was extremely low. It has herbal aroma, sweet aroma and caramel aroma, which can add a unique aroma to black beans, enrich its flavor level, cover up unpleasant odors, and at the same time make the taste of black beans more mellow and round.

[0073] Secondly, the content of guaiacol in the composite fermented fermented black beans reached 0.86 mg / kg, which was 28.7 times that of the control group. Guaiacol is an important phenolic compound. High guaiacol content can give black beans a more complex woody or smoky aroma, making the aroma of black beans more complex, layered and recognizable. At the same time, it can cooperate with volatile substances such as alcohols, aldehydes, and esters to enhance or regulate the aroma of black beans, making the fragrance of black beans more rich and coordinated.

[0074] Furthermore, the isopentanol content in fermented fermented douchi (fermented ferment ...

[0075] Finally, the content of 1-octen-3-ol, 3-methylbutanal, and 2-methylbutanal in the fermented fermented black beans inoculated with ZF626 and ZF627 was also significantly increased, which was 1.4-1.5 times that of the CK group; among them, 1-octen-3-ol has a mushroom aroma, which can increase the aroma level and recognition of the black beans, 3-methylbutanal has a fruity aroma, and 2-methylbutanal has an almond smell, which can increase the aroma richness and complexity of the black beans.

[0076] Table 6 Sensory evaluation results of different fermented fermented black beans

[0077] 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 5.5 6.2 6.5 6.1 6.1 6.4 Group 3 7.1 6.3 6.5 6.4 6.5 6.9 Group 4 7.5 6.1 7.6 6.9 7.8 7.8

[0078] Note: Each indicator is scored on a 9-point scale, with larger values ​​indicating more prominent indicators.

[0079] The results of the sensory evaluation experiment show that the fermented black beans inoculated with ZF626 and ZF627 performed best in multiple sensory indicators, especially in flavor, taste and aroma coordination, which were significantly better than other groups. This is consistent with the results of volatile compound analysis, indicating that inoculating ZF626 and ZF627 in the early stage of black bean fermentation can significantly improve the flavor and overall quality of black bean.

[0080] In summary, by inoculating Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627 in the early 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, esters, phenols and acids, especially the contents of isopentanol, 1-octen-3-ol, ethyl acetate, γ-caprolactone, 3-methylbutanal, 2-methylbutanal and guaiacol were significantly increased. The increase of these compounds can significantly improve the flavor, taste and aroma coordination of the fermented black beans, which not only improves the overall aroma and flavor of the fermented black beans, but also makes its flavor and taste more rich, complex, layered and recognizable, significantly improving the overall flavor and quality of the fermented black beans.

[0081] 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 composite fermentation agent, characterized in that: The fermentation bacteria agent comprises Staphylococcus carnosus ZF626 with a preservation number of GDMCC NO: 64878 and Pediococcus acidilactici ZF627 with a preservation number of GDMCC NO: 64879.

2. A composite fermentation method, characterized in that: The bacteria inoculated in the fermentation method include Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627 according to claim 1.

3. Use of the fermentation agent according to claim 1 or the composite fermentation method 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 is a method of adding the fermentation agent according to claim 1 during the fermentation process of fermented black beans to carry out fermentation.

7. The preparation method according to claim 6, wherein The fermentation agent is added in the early stage of fermentation.

8. The preparation method according to claim 6, wherein The addition amount of the fermentation agent is 10 5 ~10 7 CFU / mL.

9. The preparation method according to claim 6, wherein The ratio of Staphylococcus carnosus ZF626 to Pediococcus acidilactici ZF627 in the fermentation bacteria agent is 1:

1.

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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