A strain of Lactobacillus plantarum ZF623 and its application

Abstract

The present invention relates to the field of microbial technology, and specifically discloses a strain of Lactobacillus plantarum ZF623 and an application thereof. The present invention provides a strain of Lactobacillus plantarum ZF623, which is applied to the preparation of fermented food, can rapidly degrade tannic acid in fermented products, and can significantly increase the content of geraniol and isoamyl acetate in the fermented products, making the products have strong floral and fruity aromas, significantly improving the taste of the products, enhancing the aroma and making the taste pure, and significantly improving the quality of the inoculated fermented food.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Lactobacillus plantarum ZF623 and an application thereof. Background Art

[0002] Traditional fermented foods are closely related to people's lives, including alcohol, vinegar, fermented tea, traditional fermented vegetables, fermented beverages, fermented dairy products, fruit vinegar, fermented black beans, fermented bean curd, fermented rice and flour foods, etc. For a long time, the production method of traditional fermented foods has adopted natural fermentation, which has problems such as long fermentation cycle, easy contamination by miscellaneous bacteria, high content of harmful substances and salt, and unstable product quality. Therefore, companies are gradually using artificial inoculation to replace natural fermentation. However, although artificial inoculation takes less time and has uniform product quality, it has the defect that the taste is not as pure and rich as the natural fermentation flavor. The reason is that the inoculation fermentation takes a short time and the strain is single, which leads to the inability to effectively remove some bad flavor substances in the raw materials, and the accumulation of good flavor substances is not rich enough.

[0003] Tannic acid is a secondary metabolite of plants and a polyphenolic compound. It is widely present in many plant raw materials such as fruits and vegetables. It has a bitter taste and is one of the main undesirable flavor substances commonly found in plant raw materials. In addition, tannic acid can combine with proteins, sugars, and metal ions to form complexes that are difficult to digest and absorb, which will reduce the utilization rate of certain nutrients. In addition, tannic acid itself and its metabolites can often have toxic effects on animals, so it is considered an anti-nutritional factor. Selecting appropriate process measures to degrade tannic acid during the processing of plant raw materials is an effective measure to improve product quality. Existing biological methods for degrading tannic acid mainly include enzyme preparation method and microbial method. Among them, the microbial method has the advantages of low cost and simple operation, and has become a research hotspot.

[0004] Geraniol has the sweet smell of roses, which can give fermented foods a floral flavor; isoamyl acetate has a banana smell, which can give fermented foods a fruity flavor. Both of these aromas can promote the balance of the overall flavor of fermented products, and fermented foods with floral and fruity flavors are deeply loved by consumers. Increasing the content of geraniol and isoamyl acetate in fermented foods to enhance their floral and fruity flavors has good application prospects. Lactic acid bacteria are ubiquitous microorganisms in fermented foods, and are very important to the food fermentation process and the quality of the final product. Screening lactic acid strains for fermented foods that can quickly degrade tannins and produce geraniol and isoamyl acetate will help to further improve the quality of inoculated fermented foods. Summary of the invention

[0005] 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 Lactobacillus plantarum ZF623, which is used to prepare fermented foods, can quickly degrade tannic acid in plant raw materials of fermented products, and can significantly increase the content of geraniol and isoamyl acetate in fermented products, so that the products have strong floral and fruity aromas, significantly improve the taste of the products, enhance the aroma and make the taste pure, and significantly improve the quality of fermented foods.

[0006] 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:

[0007] In the first aspect, an embodiment of the present application provides a strain of Lactobacillus plantarum (Lactiplantibacilusplantarum) ZF623, which has been deposited in the Guangdong Microbiological Culture Collection Center on July 25, 2023, with a deposit number of GDMCC No: 63684.

[0008] The colonies of Lactobacillus plantarum ZF623 isolated and screened on MRS agar medium were round and raised, with a smooth and moist surface and neat edges.

[0009] In a second aspect, the present invention provides a fermentation bacterial agent, wherein the fermentation bacterial agent comprises the above-mentioned Lactobacillus plantarum ZF623.

[0010] In a third aspect, the present invention provides the use of the above-mentioned Lactobacillus plantarum ZF623 or fermentation agent in the preparation of fermented food from plant materials, preferably used for any one or more of fermented fruits and vegetables, fermented soy products or fermented tea.

[0011] Optionally, the food is a fermented food containing tannic acid in the raw materials, such as fermented fruits and vegetables, fruit wine, fruit vinegar, fermented black beans, soy sauce, fermented bean curd, fermented tea, etc., and the preparation method of the food includes adding the above-mentioned Lactobacillus plantarum ZF623 or fermentation agent directly as an additive, or replacing the microorganisms in the original fermentation system, or partially replacing the microorganisms in the original fermentation system, to the fermentation process of the food. Optionally, those skilled in the art can make conventional adjustments to the addition method or fermentation process of the above-mentioned Lactobacillus plantarum ZF623 or fermentation agent in combination with common sense.

[0012] In a fourth aspect, the present invention further provides a method for preparing pickled / fermented vegetables, the method comprising adding the above-mentioned Lactobacillus plantarum ZF623 or fermentation agent to the vegetables for fermentation.

[0013] Furthermore, the amount of Lactobacillus plantarum ZF623 added is 10 5 ~107 CFU / mL.

[0014] Furthermore, the fermentation temperature is 28-32°C.

[0015] Furthermore, the fermentation time is 2-4 days.

[0016] In a fifth aspect, the present invention further provides a method for preparing fermented black beans, the method comprising inoculating the above-mentioned Lactobacillus plantarum ZF623 or fermentation agent into fermented black bean paste.

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

[0018] 1. The Lactobacillus plantarum ZF623 provided by the present invention is used to prepare fermented foods, can rapidly degrade tannic acid in plant raw materials of fermented products, significantly improve the taste of fermented foods, and enhance the quality of fermented foods.

[0019] 2. The plant lactobacillus ZF623 provided by the present invention significantly increases the content of geraniol and isoamyl acetate in the fermented product, making the product rich in floral and fruity aroma, significantly improving the aroma of the product and making the taste pure.

[0020] 3. The pickled / fermented vegetables prepared by the Lactobacillus plantarum ZF623 provided by the present invention can maintain good brightness and sensory quality during the shelf life, effectively improving the quality of the pickled / fermented vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a colony morphology diagram of Lactobacillus plantarum ZF623;

[0022] The plant lactobacillus (Lactiplantibacilus plantarum) ZF623 provided by the present invention has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 25, 2023, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and a collection number of GDMCC No: 63684; the strain was received and registered by the collection center on July 25, 2023, and was detected as a surviving strain by the collection center on July 25, 2023. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional food-grade reagents, methods and equipment in the art.

[0026] 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.

[0027] Example 1 Screening of target strains

[0028] 1. Raw material processing

[0029] Use a sterile sampling bag to collect the naturally fermented sour soup sample and bring it back to the laboratory at low temperature. Immediately dilute it to 10% with sterile water. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , take 100 μL of each and spread it on MRS solid culture medium, and then put it in a constant temperature incubator at 37°C for 48 hours. After the culture is completed, pick lactic acid bacteria with typical colony morphology on the solid MRS plate (colony morphology is milky white, convex, round, and smooth), inoculate MRS solid culture medium, and perform plate streaking separation, and repeat this 2 to 3 times until a pure single colony is obtained.

[0030] 2. Preliminary screening of lactic acid bacteria

[0031] (1) Colony picking: 100 single colonies were picked from the MRS plate, and the purified single colony strains were subjected to microscopic examination, Gram staining, and catalase test. Gram-positive, spore-free, and catalase-negative colonies were selected for preservation. A total of 20 strains were obtained, numbered 1# to 20#.

[0032] (2) Radish embryo fermentation: The 20 strains were activated in MRS liquid medium, cultured at 37°C until the logarithmic growth phase, and then 6 The final concentration of CFU / mL was inoculated into radish embryos and placed in a clean kimchi jar for fermentation and cultured at 30°C for 3 days.

[0033] The preparation method of the radish embryo is as follows: wash the radish and cut it into 1.5cm*1.5cm blocks, then add 1.0%-5.0% by weight of salt and 1.0%-5.0% by weight of white sugar per weight of radish, mix well and press until the water output is 20.0%-30.0% of the weight of the radish.

[0034] (3) Detection of physical and chemical indicators: After the fermentation was completed, samples were taken from each jar to detect the content of short-chain fatty acids, tannic acid, geraniol and isoamyl acetate. The detection method was as follows. The detection results are recorded in Table 1.

[0035] High performance liquid chromatography (HPLC) was used to detect the content of short-chain fatty acids: the chromatographic column was C18, 46*250mm, 50cm; the mobile phase was methanol / H3PO4 / water = 5 / 0.05 / 95; the flow rate was 1mL / min; the column temperature was 30°C, and UV 210nm was used for detection; the injection volume was 5uL.

[0036] High performance liquid chromatography (HPLC) was used to detect the tannic acid content: chromatographic column: CAPCELL PAK C18 MG S5 (4.6 mmi.d. 250 mm); mobile phase: A: 1 mM EDTA, 10 mM potassium dihydrogen phosphate (pH adjusted to 2.5 with phosphoric acid) B: methanol (20:80, V:V); flow rate: 0.2 mL / min; column temperature: 30°C; UV detector wavelength: 275 nm; injection volume: 2 μL.

[0037] The content of geraniol and isoamyl acetate was detected by headspace extraction-gas chromatography-mass spectrometry (GC-MS). Chromatographic conditions: 5 g of fermented sample was placed in a 25 mL sample bottle, and the headspace was extracted at 55°C for 30 min, and then the sample was immediately injected for GC-MS analysis. HP-innowax capillary column (60m*0.25mm*0.25μm); carrier gas was helium, flow rate was 1mL / min; injection port temperature was 270°C; non-split injection; temperature program: starting temperature 40°C (retained for 5 min), increased to 85°C at 6°C / min (retained for 1.5 min), then increased to 148°C at 2.5°C / min (retained for 1 min), and finally increased to 280°C at 20°C / min (retained for 1 min). Mass spectrometry conditions: EI ionization source, electron bombardment energy 70 eV; ion source temperature 230°C; quadrupole temperature 150°C; mass scanning range 25-450 amu; auxiliary temperature 280°C.

[0038] Table 1 Detection results of physical and chemical indexes in radish after fermentation of the initial screening strains

[0039]

[0040] As shown in Table 1. Strains 02#, 03#, 16# and 19# with normal fermented radish quality, low tannic acid content, and high short-chain fatty acids, geraniol and isoamyl acetate content were selected for rescreening.

[0041] 3. Lactic acid bacteria re-screening

[0042] (1) Preparation of seed culture medium: Single colonies of the four strains obtained in the initial screening were selected and inoculated into MRS liquid culture medium and cultured at 37° C. for 24 h to obtain seed culture medium.

[0043] (2) Fermentation: The seed culture solution was inoculated into radish embryos at a 1% inoculum amount and cultured at 30°C for 3 days.

[0044] (3) Sensory evaluation: 10 technicians with rich experience in pickled vegetable evaluation conducted blind evaluation on different pickled radish samples, mainly evaluating the taste, color and aroma type. In terms of aroma, the samples were scored from three dimensions: sour aroma, ester aroma, and comprehensive aroma; in terms of taste, the samples were scored from five dimensions: umami, sourness, sweetness, astringency and bitterness, and comprehensive taste; in terms of color, the samples were scored from two dimensions: brightness and comprehensive color. The score ranges from 0 to 10 points, and the higher the score, the better the indicator. The results are the average of the scores of each dimension, and the evaluation results are recorded in Table 2.

[0045] Table 2 Sensory evaluation results of radish after fermentation with rescreened strains

[0046]

[0047] From the results in Table 2, we can see that strain 03# has the best sensory evaluation result. As the target strain for rescreening, the colony morphology of this strain on MRS agar medium is as follows: Figure 1 As shown, the colonies are round and raised with a smooth and moist surface and neat edges.

[0048] The 03# strain obtained by the above screening was subjected to 16S rDNA sequencing and identification. After comparative analysis, the 16S region sequence measured by the strain of the present invention has the highest similarity with the 16s rDNA sequence of Lactiplantibacilus plantarum, and the matching degree reaches 100%. The strain can be determined to be Lactobacillus plantarum, and it is named Lactobacillus plantarum ZF623. It was preserved in Guangdong Provincial Microbiological Culture Collection Center on July 25, 2023, and the preservation number is GDMCC No: 63684.

[0049] Example 2 Verification of the effect of fermenting radish with the target strain

[0050] 1. Seed liquid preparation: The Lactobacillus plantarum ZF623 in Example 1 was activated and inoculated into liquid MRS culture medium, and then cultured in an incubator at 37°C for 24 hours to prepare a fermentation seed liquid having a cell concentration of 1.0×10 8 CFU / mL.

[0051] 2. Radish fermentation: Add the above fermented seed liquid to the radish embryo, and the final concentration of the inoculated bacteria is 1.0×10 6 CFU / mL.

[0052] The same bacterial concentration and inoculum size were used to inoculate plant lactobacillus commonly used in fruit and vegetable fermentation (purchased from the China Industrial Microbiological Culture Collection Management Center, number CICC 22696) as the strain control group, recorded as the CC group; 1% sterile water was added as the blank control group, recorded as the CK group. According to the traditional kimchi fermentation process, the fermentation was continued at 30°C for 3 days.

[0053] 3. Result detection: After the fermentation, samples were taken for physical and chemical index detection and sensory evaluation according to the method of Example 1. The results are recorded in Table 3 and Table 4, respectively.

[0054] Table 3 Determination results of physicochemical indexes in radish after fermentation with target strains

[0055]

[0056] Table 4 Sensory evaluation results of radish fermented with target strains

[0057]

[0058] It can be seen from Tables 3 and 4 above that the strain ZF623 of the present invention is used for the fermentation of fruit and vegetable raw materials, which can quickly and significantly reduce the content of tannic acid, an undesirable substance in the raw materials, while significantly increasing the content of geraniol and isoamyl acetate, significantly improving the taste and aroma of the fermented radish, and improving the quality of the fermented radish.

[0059] Example 3 ZF623 fermented sauerkraut test

[0060] 1. Seed liquid preparation: The Lactobacillus plantarum ZF623 in Example 1 was activated and inoculated into liquid MRS culture medium, and then cultured in an incubator at 37°C for 24 hours to prepare a fermentation seed liquid having a cell concentration of 1.0×10 8 CFU / mL.

[0061] 2. Cabbage fermentation: Add the above fermented seed liquid to the cabbage embryo, and the final concentration of the inoculated bacteria is 1.0×10 6 CFU / mL.

[0062] Preparation method of cabbage embryo: wash the cabbage and cut it into 0.3cm*7.0cm strips, then add 2.0% to 6.0% salt and 1.5% to 5.5% sugar per weight of cabbage, mix thoroughly and press until the water output is 30.0% to 40.0% of the cabbage weight.

[0063] The same bacterial concentration and inoculum size were used to inoculate plant lactobacillus commonly used in fruit and vegetable fermentation (purchased from the China Industrial Microbiological Culture Collection Management Center, number CICC 22696) as the strain control group, recorded as the CC group; 1% sterile water was added as the blank control group, recorded as the CK group. According to the traditional kimchi fermentation process, the fermentation was continued at 30°C for 3 days.

[0064] 3. Result detection: After the fermentation, samples were taken for physical and chemical index detection and sensory evaluation according to the method of Example 1. The results are recorded in Table 5 and Table 6, respectively.

[0065] Table 5 Determination results of physical and chemical indicators in Chinese cabbage after ZF623 fermentation

[0066]

[0067] Table 6 Sensory evaluation results of ZF623 fermented cabbage

[0068]

[0069] As can be seen from Table 5 above, the strain ZF623 of the present invention can be used for fermentation of sauerkraut, which can quickly and significantly reduce the content of tannic acid, an undesirable substance in the raw material, and significantly increase the content of geraniol and isoamyl acetate. According to the sensory evaluation results in Table 6, the sauerkraut obtained by fermentation with the strain ZF623 has a strong fruity and floral aroma, a good sour feeling, and significantly reduced undesirable flavors (a high score for the astringent and bitter taste option indicates that the astringent and bitter taste is not obvious).

[0070] Example 4 Shelf life test of fruit and vegetable fermented by the target strain

[0071] 1. Sterilization treatment: 0.6 g of potassium sorbate antibacterial agent was added to each kg of the fermented pickled radish in Example 2 to effectively inhibit the growth of miscellaneous bacteria in the fermented pickled radish and control the growth of lactic acid bacteria to prevent the pickled radish from becoming too acidic.

[0072] 2. Packaging: Pack the processed sour radish into aluminum foil bags at a material-liquid ratio of 2:1, with a vacuum degree of ≤-0.100MPa, and seal them for pasteurization at 70°C for 30 minutes. The sterilized product is stored at room temperature and away from light for 9 months. The soaking liquid is the clarified liquid obtained by centrifuging the fermentation liquid at 5000rpm for 10 minutes.

[0073] 3. Testing: Take samples monthly for tannic acid and color value testing and conduct sensory evaluation. The results are recorded in Table 7.

[0074] Table 7 Tannin, color value and sensory evaluation results of fermented radish during shelf life

[0075]

[0076] As shown in Table 7, the color difference L* value of the sour radish fermented by strain ZF623 remained at 64 or above during the 9-month shelf life, and the sensory score was 8 or above. It can be seen that the brightness and sensory quality of the sour radish fermented by strain ZF623 were good during the 9-month shelf life.

[0077] Example 5 Experiment on fermentation of fermented black beans by target strain

[0078] 1. Preparation of ZF623 seed solution: The activated Lactobacillus plantarum ZF623 in Example 1 was inoculated into liquid MRS culture medium and cultured in an incubator at 37°C for 24 h to prepare ZF623 seed solution. The seed solution contained a bacterial cell concentration of 1.0×10 8 CFU / mL.

[0079] 2. Soak the washed soybeans in tap water at 28°C for 8 hours at a ratio of 1:3 (soybeans: tap water, W / W). Steam the drained soybeans in a vertical pressure steam autoclave at 121°C for 20 minutes.

[0080] 3. The steamed soybeans were cooled to room temperature, and Aspergillus oryzae koji was inoculated (bean mass / koji mass = 1000 / 1) in a clean and sterile environment. After koji making for 40-48 hours, 9-12% brine was mixed to obtain fermented black bean paste. After natural fermentation for 3 days, ZF623 seed liquid was evenly sprayed on the surface of the fermented black bean paste at an inoculation amount of 1% of the mass of the fermented black bean paste; the fermented black bean paste without inoculation of ZF623 strain was used as the control group, and was placed at 30°C and 70% humidity for 15 days.

[0081] 4. The fermented fermented black beans were subjected to sensory evaluation. Ten technicians with rich experience in evaluation conducted blind evaluation on different fermented black bean samples, mainly evaluating appearance, taste, flavor, and color. The results are recorded in Table 8.

[0082] Table 8 Sensory evaluation results of ZF623 fermented black beans

[0083]

[0084] According to the sensory evaluation results in Table 8, the fermented black beans obtained by strain ZF623 have rich fruity and floral aromas, increased umami and sweetness, less astringent and bitter taste, significantly reduced unpleasant flavors, and significantly improved flavors.

[0085] In summary, the above embodiments show that the Lactobacillus plantarum ZF623 provided by the present invention is used to prepare fermented foods, can rapidly degrade tannic acid in fermented products, and can significantly increase the contents of geraniol and isoamyl acetate in fermented products, so that the products have strong floral and fruity aromas, significantly improve the taste of the products, enhance the aroma and make the taste pure; the pickled / fermented vegetables pickled / fermented with the same have good brightness and sensory quality within a shelf life of 9 months, significantly improving the quality of the pickled / fermented pickled vegetables.

[0086] 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 Lactobacillus plantarum ( Lactiplantibacillus plantarum ) ZF623, characterized in that The deposit number of the Lactobacillus plantarum ZF623 is GDMCC No: 63684.

2. A fermentation agent, characterized in that: The fermentation agent comprises the Lactobacillus plantarum ZF623 as claimed in claim 1.

3. Application of plant lactobacillus ZF623 as claimed in claim 1 or the fermentation agent as claimed in claim 2 in preparing plant raw material fermented food, wherein the food is any one or both of fermented fruits and vegetables and fermented soy products.

4. Use of the plant lactobacillus ZF623 as claimed in claim 1 or the fermentation agent as claimed in claim 2 in degrading tannic acid.

5. A method for preparing pickled / fermented vegetables, characterized in that: The method is a method of adding the plant lactobacillus ZF623 described in claim 1 or the fermentation agent described in claim 2 to vegetables for fermentation.

6. The preparation method according to claim 5, characterized in that: The addition amount of the plant lactobacillus ZF623 is 10 5 ~10 7 CFU / mL.

7. The preparation method according to claim 5, characterized in that: The fermentation temperature is 28-32°C.

8. The preparation method according to claim 5, characterized in that: The fermentation time is 2-4 days.

9. A method for preparing fermented black beans, characterized in that: The method comprises inoculating the plant lactobacillus ZF623 described in claim 1 or the fermentation agent described in claim 2 into fermented black bean paste.

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