Pediococcus acidilactici ZF627 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to pediococcus acidilactici ZF627 and application thereof. The invention provides a Pediococcus acidilactici ZF627, which has high salt resistance and high ester yield, is used for fermented soya bean fermentation, and can significantly increase the content of various key volatile compounds in fermented soya beans, including the total content of flavor substances such as alcohols, aldehydes, acids and furans, especially the content of key flavor compounds such as isoamyl alcohol, ethyl acetate and methyl acetate. The pediococcus acidilactici ZF627 is used for fermenting the fermented soya beans, so that the flavor, taste and aroma coordination of the fermented soya beans can be remarkably improved, the overall aroma and flavor of the fermented soya beans are improved, the flavor and taste of the fermented soya beans are more complex and multi-layered, and the flavor and overall quality of the fermented soya beans are remarkably improved.

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] As a traditional fermented soybean product that is deeply loved by consumers, fermented black beans are famous for their unique flavor, rich nutritional value and the characteristics of being both a medicine and food. The production process of fermented black beans is actually to use various enzyme systems such as proteases and cellulases secreted by microorganisms during the koji making process to decompose macromolecules such as proteins in soybeans to generate small molecular peptides, amino acids and monosaccharides. These flavor precursors undergo a series of biochemical reactions in the later fermentation process to form the color, aroma and taste of fermented black beans. Traditional fermented black beans are generally fermented under natural conditions. The differences in geographical environment and fermentation microorganisms lead to differences in the quality of the finished black beans, which seriously restricts the development of the industry. In order to achieve industrialization, it is necessary to study the pure fermentation process of black beans. In addition, the traditional fermented black beans have a unique and rich flavor. With the industrial production of black beans, its flavor is far inferior to that of naturally fermented black beans. Therefore, the improvement of the flavor of pure fermented black beans is also the focus of current research.

[0003] At present, there are many studies on fermentation microorganisms of fermented black beans, but they are mainly focused on molds and yeasts, and there are relatively few studies on lactic acid bacteria. CN 104996931A discloses a method for making flavored black beans, which uses Bacillus subtilis and Lactobacillus plantarum to mix and ferment in steps and combine 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 of 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 fermented by inoculating Pediococcus acidilactici and Zygosaccharomyces rouxii, and the fermented bean embryos are compounded with spices, liquor extract and licorice extract to obtain a black bean product with a strong sauce and soy sauce flavor. However, this method has a complex production process and high production cost, and is not suitable for large-scale production. Patent CN117981843A discloses a composite fermentation agent and its application in light fermented black beans. The composite fermentation agent includes: brewer's yeast, plant lactic acid bacteria, Foshan Fengliao's light fermented black beans product, aralia kiwifruit juice, thiolated codonopsis polysaccharide, sodium citrate, and the flavor, taste and nutritional activity of the inoculated and fermented light fermented black beans are significantly improved. However, the formula of the composite fermentation agent is relatively complex, which increases the difficulty of 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 strain of Pediococcus acidilactici ZF627, which has high salt tolerance and high ester production ability and is used for fermenting black beans. It can significantly improve the flavor, taste and aroma coordination of black beans, and improve the overall aroma, flavor and quality of black beans.

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

[0006] In a first aspect, the present invention provides a strain of Pediococcus acidilactici ZF627, which has been 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 bacterial agent, wherein the fermentation bacterial 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 fermented black beans for fermentation.

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

[0012] Furthermore, the amount of Pediococcus acidilactici ZF627 added 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 described in the present invention.

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

[0016] 1. The lactic acid bacteria ZF627 provided by the present invention has high salt tolerance and can grow normally at a salt concentration of 20%; it has high ester production ability, and the production 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; the strain can be used for high-salt fermented food production to improve product flavor, thereby improving food quality.

[0017] 2. The lactic acid bacteria 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, furans, etc. can be increased by 64%-100%.

[0018] 3. The lactic acid bacteria 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, such as 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. The fruity aroma, mushroom aroma and other aromas of the fermented black beans can be significantly increased, and the overall flavor of the fermented black beans is improved.

[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 Microbiological Culture Collection Center on July 17, 2024, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and a collection number of 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] In order to facilitate the 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 specification 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 phase mass spectrometry (HS-SPME-GC-MS) technique used in the specification of the present invention is used to determine the volatile flavor substances in fermented black beans. The specific method is as follows:

[0027] (1) Sample processing

[0028] Weigh 2 g of the ground sample, 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 60 °C constant temperature incubator for oscillation 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 (60m×250μm); splitless injection mode, temperature program: 55°C for 5min, then increase to 230°C at 5°C / min and hold for 15min; carrier gas: helium at a flow rate of 1.2mL / min.

[0031] MS conditions: EI ion source; ion source stability of 250°C, quadrupole and mass spectrometer interface temperatures of 150°C and 280°C, respectively; electron energy 70 eV, full scan mode; mass scan range 29-500 m / z. The mass spectrum data of unknown volatile substances were compared and confirmed with the standard spectral library map NIST 2017, and peak identification was performed with similarity (SI) > 80 (the highest value is 100) as the standard.

[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 represents the peak area of ​​a single component; A2 represents the peak area of ​​the internal standard; C2 represents the internal standard mass concentration, μg / mL; V2 represents the internal standard volume, μL; and M represents 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 with a sterile mortar, weigh 10g of the crushed sample and add it to a 250mL conical flask containing 90mL sterile saline, and 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; draw the suspension and add sterile saline to dilute it step by step by 10 times to 10 -2 -10 -6 , make bacterial dilution liquid; select 2-3 bacterial suspensions with appropriate gradients, respectively draw 200 μL of liquid and add them to LB plates and MRS plates containing 20 mg / L natamycin, evenly spread them with a sterile spreading rod, and then invert and culture them 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 up single colonies with inconsistent morphology, inoculate them into corresponding LB or MRS culture media for streak culture at 37°C, and culture them 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 a pure strain of microorganisms is obtained.

[0039] Through the above-mentioned microbial separation and purification scheme, 12 purified strains were obtained from the 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 microorganism can maintain a good growth state during fermentation of fermented black beans, it is necessary to test its salt tolerance. 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 culture until the middle of logarithmic growth; 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 for 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, with the increase of salt content, the growth rate of all strains decreased, but strain DC103, strain DC104, strain DC105, strain DC107 and strain DC111 showed strong salt tolerance and could maintain relatively high biomass at a salt content of 12%-20%, so these 5 strains can be applied to the fermentation process of fermented black beans. These 5 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 culture medium, and cultured at 37°C for 24 h. The cells were collected by centrifugation, washed with sterile saline, and diluted to obtain a concentration of 10 9 -10 10 CFU / mL of bacterial suspension, and then inoculated into the rescreening medium (100mL of sterile fermented black bean extract, 0.5% acetic acid and 2% ethanol) at a 2% inoculation amount, and cultured at 37°C for 3 days, and the uninoculated rescreening medium was used as the control group. After the fermentation was completed, the content of ethyl acetate was detected by 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 ethyl acetate production of strain DC105 in the rescreening 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 Microbiological Culture Collection Center 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 swell without wrinkles, feel firm, the bean skin is 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 and wheat flour are evenly sprinkled on the soybeans and stirred evenly. The ratio of koji to soybean dry weight is 0.1%, and the amount of wheat flour added is 100 times that of koji. 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% ​​of the 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 irregular stirring during the fermentation process;

[0056] (5) Inoculation: Pediococcus acidilactici ZF627 was cultured to the middle and late logarithmic growth stages, the bacterial precipitate was collected by centrifugation, washed 2-3 times with physiological saline, and the bacterial precipitate was resuspended to a cell concentration of 4×10 7 CFU / mL, when the fermentation time of 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 and inoculated in the same way as a control;

[0057] (6) Detection and analysis: After the fermentation was completed, samples were collected to determine the physical and chemical indicators and volatile flavor substance content of the fermented fermented black beans, and the results were recorded in Tables 3-4; and 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 above fermented black beans were used for flavor evaluation with a commercially available fermented black beans. 30 trained professional sensory evaluators were invited to conduct sensory evaluation on the five dimensions of flavor, color, taste, appearance and aroma harmony of different fermented black beans. The samples were scored and the average value 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 black beans 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 showed significant advantages in terms of total free amino acids and reducing sugar content. The total free amino acid content of the ZF627 fermentation group was the highest, reaching 5.89±0.09g / 100g, which was significantly higher than that of other groups. This shows that inoculating lactic acid bacteria ZF627 in the middle stage of fermentation of fermented black beans can significantly promote the hydrolysis of proteins and generate more free amino acids. Free amino acids are important flavor precursors in fermented black beans, and can generate a variety of aroma compounds through the Maillard reaction, thereby enhancing the flavor of fermented black beans. The reducing sugar content of the ZF627 fermentation group was also the highest, reaching 6.91±0.01g / 100g. This shows that inoculating strain ZF627 in the middle stage of fermentation of fermented black beans can significantly promote the degradation of carbohydrates and generate more reducing sugars. Reducing sugars are not only an important source of sweetness for fermented black beans, but also can react with free amino acids to generate complex aroma compounds through the Maillard reaction, further enhancing the flavor of fermented black beans.

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

[0063]

[0064] As shown in Figure 4, the content of total volatile flavor substances in fermented fermented black beans inoculated with ZF627 was significantly higher than that in the control group, with an increase rate of 51% compared to natural fermentation, among which the relative content increases of key aroma substances such as alcohols, aldehydes, acids, and furans reached 66.88%, 75.71%, 100.00%, and 64.06%, respectively. Alcohol compounds are one of the important aroma components in black beans, which can give the product a rich mellow aroma; aldehyde compounds usually have a pungent smell in black beans, but the presence of a moderate amount can increase the complexity and layering of the product; acid compounds help to enhance the umami and acidity of black beans, making their flavor more distinct; furan compounds usually have caramel and roasted aromas, which can increase 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 by inoculation of ZF627 can significantly increase the contents of seven key flavor substances in black beans, including isopentanol, 1-octen-3-ol, ethyl acetate, and methyl acetate, especially isopentanol, ethyl acetate, and methyl acetate, which are more than twice that of the control group. The content of isopentanol in fermented black beans inoculated with ZF627 reached 0.80 mg / kg, which is 2.16 times that of the CK group. Isopentanol is a fruity compound that can significantly enhance the fruity characteristics of black beans. The content of ethyl acetate in the ZF627 fermentation group was 0.11 mg / kg, which was 2.2 times that of the CK group; the content of methyl acetate in the ZF627 fermentation group reached 0.63 mg / kg, which was 3.94 times that of the CK group. Ethyl acetate and methyl acetate have fruity aromas, which can increase the sweetness and fruitiness of black beans, making the taste of black beans richer and fuller. In addition, the contents of 1-octen-3-ol, 3-methylbutanal, 2-methylbutanal and 3-octanone in fermented black beans inoculated with ZF627 were also significantly increased, and their contents were 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, and 2-methylbutanal has an almond smell, which can increase the aroma intensity and complexity of black beans. 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 black beans

[0070] Group Flavor Color Taste Appearance Aroma coordination 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 black beans inoculated with ZF627 performed best in multiple sensory indicators, and its flavor, taste and aroma coordination were significantly better than other groups, which is consistent with the results of the volatile compound analysis, indicating that inoculating lactic acid bacteria ZF627 in the middle stage of black bean fermentation can significantly improve the overall quality and flavor of black bean.

[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 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 fermented black beans, which not only improves the overall aroma and flavor of fermented black beans, but also makes its flavor and taste more complex and multi-layered, significantly improving the overall flavor and quality of fermented black beans.

[0074] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached 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 bacteria 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 is a method for adding the Pediococcus acidilactici ZF627 according to claim 1 or the fermentation agent according to claim 2 during the fermentation process of fermented black beans to carry out fermentation.

7. The preparation method according to claim 6, characterized in that: The adding time of the Pediococcus acidilactici ZF627 is in the middle of the fermentation.

8. The preparation method according to claim 6, characterized in that: The amount of Pediococcus acidilactici ZF627 added 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 described in any one of claims 6-9.

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