A kind of Lactobacillus pentosus and its application in preparing direct-throw fermented asparagus pickles

Abstract

The present invention discloses a Lactobacillus pentosus and its application in the preparation of direct-fermented fermented asparagus pickles, belonging to the field of microbial fermentation technology. The present invention provides a Lactobacillus pentosus (Lactiplanttibacillus pentosus) JGS34, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on June 18, 2024, with a deposit number of CGMCC NO.30985. The strain screened by the present invention is acid-resistant and bile-resistant, and has good antibacterial and antioxidant activities; fermenting asparagus with the bacteria can improve the comprehensive taste of fermented asparagus, and the product prepared by the bacteria has antioxidant activity, angiotensin-converting enzyme inhibitory activity and xanthine oxidase inhibitory activity, has potential blood pressure and uric acid lowering performance, and has broad application prospects and commercial value.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a Lactobacillus pentosus and an application thereof in preparing direct-throw fermented asparagus pickles. Background Art

[0002] Asparagus is a perennial herb. It is rich in nutrients and contains a variety of bioactive ingredients such as polyphenols, flavonoids, saponins and asparagine. It is known as the "king of vegetables". Domestic asparagus is mainly sold fresh, and its processed food is mainly canned asparagus. The product type is relatively single. In addition, during the asparagus harvest period, a certain proportion of asparagus with poor appearance or incomplete processing is directly discarded, causing serious environmental problems and affecting the sustainable development of the industry. Asparagus is generally picked continuously in summer and autumn, with high yield and extremely poor storage resistance. Therefore, it is often made into pickled asparagus (pickled asparagus) that is easy to store by high-salt pickling or soaking. Its fermentation cycle is relatively long and productivity is low. Only lactic acid bacteria on the surface of vegetables are used for fermentation, and its fermentation cycle is generally more than 7 days. In addition, affected by sanitary conditions, production season and salt consumption, the fermentation quality is unstable, easy to fail, and safety is difficult to guarantee, which makes it difficult to achieve factory production and industrialization. In addition, there are problems such as excessive nitrites and nitrosamines, as well as high salt content.

[0003] Therefore, in view of the problems existing in the above-mentioned prior art, it is of great significance to obtain a strain having good acid and bile resistance, antioxidant activity and antibacterial activity. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a Lactobacillus pentosus and its application in preparing direct-fermented asparagus pickles. The present invention provides a Lactobacillus pentosus with good acid and bile resistance, antioxidant activity and antibacterial activity, and applies it to the preparation of asparagus pickles with high safety, good flavor and short fermentation cycle, and the preparation process is simple and suitable for industrial production.

[0005] Technical Solution 1: A Lactobacillus pentosus (Lactiplanttibacillus pentosus) JGS34, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on June 18, 2024, with the deposit number CGMCCNO.30985.

[0006] Technical solution 2: A bacterial agent, the active ingredient of which includes the Lactobacillus pentosaceus JGS34.

[0007] Furthermore, the dosage form of the bacterial agent includes freeze-dried powder.

[0008] Technical solution three: Use of the Lactobacillus pentosaceus JGS34 or the bacterial agent in the preparation of direct-fermented asparagus pickles.

[0009] Technical solution 4: A method for preparing asparagus pickles, comprising the steps of inoculating and fermenting the Lactobacillus pentosaceus JGS34.

[0010] Furthermore, the method comprises the following steps: fresh asparagus is screened, cleaned and cut into pieces, loaded into a fermentation container, water, edible salt, glucose, white sugar and edible calcium chloride are added and mixed evenly, inoculated and fermented with a lactic acid bacteria direct-injection fermentation agent, packaged and sterilized to obtain a finished product; the lactic acid bacteria direct-injection fermentation agent comprises the Lactobacillus pentosaceus JGS34 described in claim 1.

[0011] Furthermore, the fermentation system includes, by weight, 40-80 parts of fresh asparagus, 40-80 parts of water, 2-5 parts of edible salt, 0.2-0.7 parts of glucose, 0.4-1.0 parts of white sugar, 0.1-0.3 parts of edible calcium chloride and 0.001-0.1 parts of lactic acid bacteria direct-injection starter.

[0012] Furthermore, the effective viable count of the Lactobacillus pentosus JGS34 is 1×10 10 -9×10 10 CFU / g.

[0013] Furthermore, the inoculation fermentation condition is to ferment at 25-37°C for 2-4 days.

[0014] Furthermore, the sterilization condition is pasteurization at 90° C. for 10 minutes.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects:

[0016] The present invention provides a strain of Lactobacillus pentosus, which is classified and named as Lactobacillus pentosus (Lactiplanttibacilluspentosus) JGS34, and is preserved in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee, referred to as CGMCC, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is June 18, 2024, and the preservation number is CGMCC NO.30985. The Lactobacillus pentosus JGS34 provided by the present invention is acid-resistant and bile-resistant, has good antioxidant activity, and has an antibacterial effect on Escherichia coli, Salmonella, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Streptococcus mutans; the strain of the present invention is used to ferment asparagus kimchi, and the comprehensive taste of asparagus kimchi can be improved. According to the above-mentioned activity of the strain of the present invention, the strain of the present invention can also be applied to vegetable fermentation, and can also be combined with plant lactobacillus, thermophilic streptococcus, bulgaricus lactobacillus fermented yogurt and other products, and can also be used as a raw material for meal replacement powder.

[0017] The Lactobacillus pentosus JGS34 screened by the present invention can effectively reduce the content of nitrite and biogenic amine in asparagus kimchi, ensuring the safety of consumption; the Lactobacillus pentosus JGS34 screened by the present invention can effectively increase the content of organic acid and amino acid in asparagus kimchi products, so that asparagus has a soft sour and fresh taste and a pure and rich fermented aroma, and retains the original nutrients of asparagus to the greatest extent, greatly improving the acceptance of consumers; the asparagus kimchi directly fermented by Lactobacillus pentosus JGS34 prepared by the present invention has good performance in DPPH free radical scavenging ability, ABTS free radical scavenging ability and total reducing ability, and has excellent antioxidant ability; the asparagus kimchi directly fermented by Lactobacillus pentosus JGS34 prepared by the present invention has excellent ACE inhibition ability, and its IC 50 The value is 0.24 mg / mL, which has good potential antihypertensive activity; the asparagus pickles directly fermented by Lactobacillus pentosus JGS34 prepared by the present invention have good xanthine oxidase inhibition ability, and its IC 50 The value is 64.25 μg / mL, which has excellent uric acid-lowering potential; the direct fermentation of the Lactobacillus pentosaceus JGS34 starter of the present invention can shorten the pickling time of asparagus kimchi from more than 7 days to less than 3 days, which can greatly improve the production efficiency; the direct fermentation of the Lactobacillus pentosaceus JGS34 starter can reduce the amount of salt used, thereby reducing the production cost, while avoiding the health risks and environmental pressures brought about by high salt intake and wastewater discharge; the production process of the present invention is simple and easy to control, with a high level of standardization, stable and reliable product quality, and is conducive to the realization of large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The morphological staining of the strains;

[0020] Figure 2 The morphological characteristics of the strains under an optical microscope with a magnification of 1000. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The experimental methods in the following examples are all conventional methods unless otherwise specified; the chemical and biological reagents used are all conventional reagents in the art and can be obtained commercially unless otherwise specified.

[0027] Example 1 Isolation, purification and identification of Lactobacillus pentosus JGS34

[0028] 1. Collection of traditional pickled asparagus

[0029] The sampling site is located at Jiangxi Guokang Agricultural Group Co., Ltd., Xingguo County, Ganzhou City. The sampling time is September 30, 2023. The sampling target is traditional pickled asparagus. Use a sterile spoon to draw 10mL of fermentation liquid into a 50mL sterile centrifuge tube, quickly tighten the tube cap and put it into the low-temperature sampling box.

[0030] 2. Isolation and Purification of Strains

[0031] Step 1: Prepare the culture medium, using conventional MRS culture medium, the specific formula is as follows: 10g peptone, 5g beef powder, 4g yeast powder, 2g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g agar powder, 1000mL distilled water. Sterilize the prepared culture medium, cool it to 55-60℃, and pour it into the culture dish (about 15mL per dish, thickness 3-5mm).

[0032] Step 2: Prepare bacterial suspension: After the pickled asparagus samples are brought back to the laboratory, they are first cultured for lactic acid bacteria enrichment. In the clean bench, the sample pickle juice is inoculated into sterile MRS broth at a 5% inoculation rate, and cultured in a constant temperature incubator at 37°C for 18 hours. When the MRS broth becomes turbid and white bacterial bodies are precipitated at the bottom, it indicates that a good enrichment has been achieved, and the next step of separation and purification can be carried out.

[0033] Step 3: Spread the plate and culture at constant temperature: Take 1 mL of the enriched MRS broth suspension and dilute it to 10% with sterile saline under sterile conditions. -3 ~10 -8 . Take 10 respectively -5 ~10 -8 Inoculate 100 μL of the diluted bacterial suspension into the center of the MRS culture medium, spread it evenly on the surface of the culture medium with a sterile spreading stick, and let it stand at room temperature for 5-10 minutes. After the bacterial solution is absorbed into the culture medium, invert the culture dish and place it in a constant temperature incubator at 37°C for 48h-72h.

[0034] Step 4: Plate streak purification: Use an inoculation loop to pick bacteria with different colony morphology on the plate and streak them onto a new MRS medium for streak separation (try to pick out bacteria in the center of a single colony). Repeat this process until a pure single colony with consistent morphology is obtained through separation and purification. Then perform Gram staining to eliminate non-target strains.

[0035] Step 5: Preservation of bacterial strains: After screening, retain the target bacteria, use a pipette to draw 0.5 mL of bacterial suspension and 0.5 mL of 20% sterilized glycerol, mix them evenly in a sterilized bacterial strain preservation tube, and store them in a -80°C refrigerator, named JGS34.

[0036] 3. Morphological identification of strains

[0037] The strain was cultured on the culture medium until the logarithmic growth phase, and then stained according to the instructions of the kit (Solabo Company) and observed under an optical microscope for Gram positive and negative, capsule, flagella and other morphological characteristics of strain JGS34 ( Figure 2 ).

[0038] 4. Molecular Identification of Strains

[0039] DNA extraction and 16S rDNA PCR amplification were performed on the suspected lactic acid bacteria strains. The purified suspected target strains were inoculated into MRS broth and cultured at 37°C for 18-24 hours. The DNA was extracted using a bacterial genomic DNA extraction kit. The extracted DNA was PCR amplified, with 10 μL of upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) 1 μL, 10 μL of downstream primer 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQID NO.2) 1 μL, 2×Taq plus Buffer 12.5 μL, template DNA 1 μL, and the system was supplemented to 25 μL with sterile ddH2O. Sterile ultrapure water was used to replace the template DNA as a negative control. Amplification conditions were: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, for a total of 29 cycles, and finally 72°C for 5 min. The target DNA fragment was recovered and then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing result is shown in SEQ ID NO.3:

[0040]

[0041] 5. Morphology and molecular identification results of strains

[0042] Morphological observation of the suspected lactic acid bacteria colonies purified by plate streaking revealed that the colonies in the culture medium were milky white, smooth, and easy to pick off. Morphological staining of the strain revealed that the strain was a Gram-positive bacterium with a capsule and no flagella (see Figure 1 ). The method of direct amplification of 16S rDNA of bacteria was adopted, and the sequencing results were compared by BLAST. The strain belonged to the genus Lactiplantibacillus. The results of 16S rDNA homology analysis showed that the homology with the known Lactobacillus pentosus in the GeneBank database reached 100%. The isolated Lactobacillus pentosus strain was named JGS34 and preserved. The preservation information is as follows:

[0043] The strain JGS34 was sent to the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) for preservation in June 2024. The address of the preservation unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; preservation date: June 18, 2024; preservation number: CGMCC NO.30985; classification name: Lactiplanttibacillus pentosus.

[0044] Example 2 Analysis of acid and bile resistance of Lactobacillus pentosus

[0045] 1. Acid resistance test

[0046] 1 mL of the culture solution of Lactobacillus pentosus JGS34 was placed in a 1.5 mL centrifuge tube and centrifuged at 8000 × g for 5 min. The collected bacteria were washed twice with sterilized culture medium, and then resuspended in MRS broth culture medium with a pH of 2.5. Finally, the bacteria were placed in a CO2 incubator at 35°C for anaerobically culture, and 1 mL of the bacterial solution was taken at 0 h and 3 h, respectively, and diluted 10 times with 0.9% saline (10 -1 -10 -7 ), then take appropriate concentration and spread it on MRS agar plate, incubate anaerobically at 35℃ for 48-72h, count and determine the number of viable bacteria after the incubation, and calculate its survival rate. The formula for calculating the survival rate of strains is:

[0047]

[0048] N1 is the number of viable bacteria after strain treatment; N0 is the initial number of viable bacteria of the strain.

[0049] After the above Lactobacillus pentosaceus acid was treated for 3 h, N1 was 8.54×10 8 cfu, N0 is 9.88×10 8 cfu, and the survival rate was 86.44%.

[0050] 2. Bile salt tolerance test

[0051] Inoculate 1 mL of the culture solution of Lactobacillus pentosus JGS34 directly into fresh MRS culture medium containing 0.3% bile salts, culture at 35°C, and take samples at 0h and 4h of culture, spread them on MRS agar plates after dilution, and place the spread plates in a CO2 incubator at 35°C for anaerobically cultured for 48-72h. After the culture is completed, count the number of live bacteria on the MRS agar plates and calculate their survival rate. The formula for calculating the survival rate of the strain is:

[0052]

[0053] After the above-mentioned Lactobacillus pentosus bile salt treatment for 3 hours, N1 was 4.85×10 8 cfu, N0 is 9.88×10 8 cfu, and the survival rate was 49.09%.

[0054] The experimental results show that the Lactobacillus pentosus JGS34 screened by the present invention has strong acid and bile salt resistance.

[0055] Example 3 Analysis of the Antioxidant Capacity of Lactobacillus plantarum

[0056] 1. Preparation of bacterial suspension

[0057] When the strain culture concentration reaches 10 9 cfu / mL, centrifuge at 5000g for 5 min to remove the culture medium. Collect the bacteria and resuspend the precipitate with physiological saline to obtain the sample to be tested; 2,2-dipHenyl-1-picrylhydrazyl (DPPH), hydroxyl radical scavenging rate and reducing ability are used to determine the antioxidant capacity of the strain.

[0058] 2. DPPH free radical scavenging experiment

[0059] Mix 2 mL of bacterial suspension with 2 mL of DPPH methanol solution (0.2 mM) and place at room temperature away from light for 30 min. DPPH and saline were used as the control group, and the sample and ethanol were used as the blank group. Measure the absorbance at 536 nm. DPPH free radical scavenging rate (%) = 1-[A536 (sample group)-A536 (blank group) / A517 (control group)]×100%. Sample group: sample + 2 mL DPPH solution; blank group: sample + 2 mL ethanol solution; control group: 2 mL DPPH + saline.

[0060] The results are shown in Table 1: The DPPH free radical scavenging rate of the strain JGS34 of the present invention is 68.25%, which is close to the scavenging rate of Vc (0.8 mg / mL) of 70.12%, which shows that Lactobacillus pentosus JGS34 has a strong ability to scavenge DPPH free radicals and exhibits strong antioxidant activity.

[0061] 3. Hydroxyl free radical scavenging experiment

[0062] The cell-free extract was mixed with 1 mL salicylic acid, 1 mL ferrous sulfate FeSO4 and 1 mL hydrogen peroxide and placed at 37°C for 15 min. The absorbance was measured at 510 nm:

[0063] Hydroxyl radical scavenging rate (%) = 1-[(A1-A2) / A0] × 100%

[0064] Among them: A1 is the sample solution (FeSO4+salicylic acid+sample+hydrogen peroxide), A2 is the blank control of the sample solution (FeSO4+salicylic acid+sample+RO water), and A0 is the control solution (FeSO4+salicylic acid+normal saline+hydrogen peroxide).

[0065] The results are shown in Table 1: The hydroxyl radical scavenging rate of strain JGS34 was 42.93%.

[0066] 4. Determination of reducing ability

[0067] Take 0.5mL of bacterial suspension sample, add 0.2mol / L PBS solution (pH 6.6), 0.5mL of 1% potassium ferricyanide solution, mix well, place in a 50℃ water bath for 20min, cool quickly, add 0.5mL of 10% trichloroacetic acid, centrifuge at 5000g for 5min, take 1mL of supernatant, distilled water, 1mL of 0.1% trichloroacetic acid, mix well, let stand for 10min, and detect at 700nm absorbance. Reducing ability (%) = (A1-A0) / A0×100%. A1 is the absorbance of the sample group; A0 is the absorbance of the sample replaced by PBS buffer.

[0068] The results are shown in Table 1: The reducing capacity of strain JGS34 was 40.84%.

[0069] Table 1 Antioxidant activity of strains

[0070] NO Hydroxyl radical scavenging rate (%) DPPH(%) Reduction performance (%) JGS34 42.93±1.29 68.25±1.08 40.84±1.33 <![CDATA[V C ]]> 45.44±0.23 70.12±0.43 42.22±0.78

[0071] The results of the DPPH free radical scavenging experiment, the hydroxyl free radical scavenging experiment and the reducing ability experiment show that the Lactobacillus pentosus JGS34 of the present invention has acid and bile resistance and strong antioxidant activity, and can be used to prepare foods or health products with anti-aging and antioxidant effects.

[0072] Example 4 Anti-pathogenic bacteria test

[0073] The antibacterial test was carried out using the Oxford cup method. 8 100 μL of LB solution containing CFU / mL Escherichia coli, Salmonella, Listeria, Staphylococcus aureus and Bacillus cereus was spread on a sterilized LB medium. 8 100 μL of BHI solution of CFU / mL mutans streptococci was spread on a sterilized BHI plate medium, an Oxford cup was placed horizontally on it, and 200 μL of the target bacterial solution of the same concentration was added respectively. Three replicates were set up and cultured at 37°C for 24 hours, and the diameter of the inhibition zone was recorded.

[0074] As shown in Table 2, the Lactobacillus pentosus JGS34 of the present invention has significant antibacterial effects on Escherichia coli, Salmonella, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Streptococcus mutans.

[0075] Table 2 Inhibition zone of Lactobacillus pentosus JGS34 (mm)

[0076]

[0077]

[0078] Example 5 Application of Lactobacillus pentosus JGS34 in the preparation of direct-injection fermented asparagus pickles

[0079] Select fresh asparagus without rot and deterioration, wash it with clean water, remove the inedible part and set aside, add 50 parts of asparagus, 50 parts of pure water, 4 parts of edible salt, 0.5 parts of glucose, 1 part of white sugar, and 0.2 parts of edible calcium chloride to the fermentation container, mix well and add 0.01 parts of lactic acid bacteria direct-injection fermentation agent (put the identified Lactobacillus pentosaceus JGS34 with a viable count of 1×1010CFU / g into a test tube containing MRS liquid culture medium for 24 hours and then centrifuge at 8000g to obtain the bacteria, add sterilized skim milk with a mass fraction of 10% (the volume ratio of the bacterial culture medium to the skim milk is 10:1) to the centrifuged bacteria, mix well, put it in a 0℃ refrigerator for 2 hours, and then vacuum freeze-dry it for 24 hours to obtain freeze-dried bacteria powder), mix well and ferment at 37℃ for 3 days. After the fermentation is completed, the asparagus kimchi is vacuum-packed and pasteurized at 90℃ for 10 minutes to obtain the finished product.

[0080] Example 6 Application of Lactobacillus pentosus JGS34 in the preparation of direct-injection fermented asparagus pickles

[0081] Select fresh asparagus without rot and deterioration, wash it with clean water, remove the inedible parts and set aside, add 40 parts of asparagus, 60 parts of pure water, 6 parts of edible salt, 0.75 parts of glucose, 1.5 parts of white sugar, and 0.2 parts of edible calcium chloride to the fermentation container, mix well and add 0.1 parts of lactic acid bacteria direct-injection starter (live bacteria count 1×10 10 CFU / g, Lactobacillus pentosus JGS34), mixed evenly and fermented at 37° C. for 2 days. After fermentation, the asparagus kimchi was vacuum packed and pasteurized at 90° C. for 10 min to obtain a finished product.

[0082] Example 7 Application of Lactobacillus pentosus JGS34 in the preparation of direct-injection fermented asparagus pickles

[0083] Select fresh asparagus without rot and deterioration, wash it with clean water, remove the inedible parts and set aside, add 60 parts of asparagus, 40 parts of pure water, 2 parts of edible salt, 0.25 parts of glucose, 0.5 parts of white sugar, and 0.2 parts of edible calcium chloride to the fermentation container, mix well and add 0.001 parts of lactic acid bacteria direct-injection starter (live bacteria count 1×10 10 CFU / g, Lactobacillus pentosus JGS34), mixed evenly and fermented at 37° C. for 4 days. After fermentation, the asparagus kimchi was vacuum packed and pasteurized at 90° C. for 10 minutes to obtain a finished product.

[0084] Comparative Example 1

[0085] The only difference between this comparative example 1 and example 5 is that no direct-injection fermentation agent is inoculated, and the fermentation is carried out using endophytic bacteria obtained by natural fermentation.

[0086] Comparative Example 2

[0087] The only difference between this comparative example 1 and example 5 is that no additional glucose and white sugar are added.

[0088] Comparative Example 3

[0089] The only difference between this comparative example 1 and Example 5 is that 0.01 parts of lactic acid bacteria direct-injection starter (1×1010 CFU / g, Pediococcus pentosaceus DSM20336 (from German Collection of Microorganisms and Cells) were used for inoculation, and other conditions were the same as those in Example 1.

[0090] Effect analysis

[0091] (1) Detection of volatile flavor compounds

[0092] The volatile flavor components of asparagus kimchi were determined by headspace solid phase microextraction combined with gas chromatography-mass spectrometry using an Agilent triple quadrupole gas chromatography-mass spectrometer tandem time-of-flight mass spectrometer. The specific conditions were as follows: chromatographic column: HP-5MS quartz capillary column (30m×0.25mm, 0.25μm); temperature program: initial temperature 50℃, maintained for 3min, then increased to 100℃ at 2℃ / min and maintained for 2min, then increased to 210℃ at 4℃ / min and maintained for 5min; carrier gas was helium, flow rate 1.0mL / min, pressure 7.6522psi; split ratio 1:50; EI ionization source, ionization energy 70eV, temperature 230℃; injection port temperature 250℃; transfer line 230℃; mass spectrometry scanning range 35-450m / z, 2scans / s. Compound identification: After mass spectrometry data acquisition, Agilent's MassHunter and UnknownAnalysis software were used to perform peak extraction, peak alignment, and baseline filtering on the GC-Q-TOF / MS raw data. The NIST17.0 spectral library was used to compare and identify each compound, and the peak area normalization method was used for quantification. The specific results are shown in Table 3.

[0093] Table 3 Contents of main volatile flavor compounds in direct-throw fermented asparagus of Example 5 and Comparative Examples 1-3

[0094]

[0095]

[0096]

[0097] As shown in Table 3, compared with fermentation without direct-inoculation fermentation agent, fermentation with endophytes, without additional addition of glucose and white sugar, and fermentation with Pediococcus pentosaceus DSM20336, the concentration of esters and alcohols is higher, and the types of volatile aromas are richer, such as ethyl acetate, methyl palmitate, vinyl acetate, ethyl butyrate, ethyl 2-hydroxypropionate, etc. New aroma substances. The sulfide content is low and the types are fewer, especially no methyl mercaptan is produced, and sulfur-containing compounds will give the product a more pungent odor such as sulfur, onion and garlic.

[0098] (2) Free amino acid detection

[0099] The free amino acid detection method is based on GB5009.124-2016 "Determination of amino acids in food", using an amino acid analyzer to determine the type and content of amino acids in the sample. The specific results are shown in Table 4.

[0100] Table 4 Free amino acid content of direct-fermented asparagus in Example 5 and Comparative Examples 1-3

[0101]

[0102] As shown in Table 4, compared with Comparative Examples 1-3, the lactic acid bacteria direct-fermentation asparagus kimchi prepared in Example 5 has higher total amino acid, umami amino acid, and sweet amino acid contents, while the bitter amino acid content is comparable. Therefore, the lactic acid bacteria direct-fermentation fermentation agent corresponding to Example 5 can give asparagus kimchi more umami and sweetness while maintaining the total amino acid level of asparagus kimchi.

[0103] (3) Xanthine oxidase inhibition rate test

[0104] After the fermented asparagus kimchi sample is homogenized, 2.0 g is weighed and added to 30 mL of 70% ethanol. After ultrasonic extraction for 20 minutes, the supernatant is centrifuged, filtered, concentrated by rotary evaporation, and vacuum-freezed. It is reconstituted with 2 mL of PBS solution and set aside. The xanthine oxidase inhibition rate detection method is as follows: the reaction system is 5 mL, and different fermented asparagus kimchi sample extracts 0.2 mL, 2.3 mL of PBS solution (0.2 mmol / L, pH 7.5), and 2.0 mL of xanthine solution (1.2 mmol / L) are added to the sample group respectively. After 20 minutes of constant temperature water bath at 25°C, 50 U / L of xanthine oxidase solution is added, and the reaction is carried out for 10 minutes. The absorbance at 292 nm is measured every 1 minute. In the control group, PBS buffer is used instead of the fermented asparagus kimchi sample extract. The xanthine oxidase inhibition rate of the sample is calculated according to the following formula, and the IC is calculated based on the inhibition rate. 50 Value (inhibition rate %).

[0105]

[0106] Among them, K control is the enzyme reaction rate when no fermented asparagus kimchi sample extract was added (control group), K sample is the enzyme reaction rate when the asparagus kimchi sample extract is added. The specific results are shown in Table 5.

[0107] Table 5 Xanthine oxidase inhibition ability of direct-fermented asparagus kimchi

[0108] Group <![CDATA[Xanthine oxidase inhibition IC 50 (μg / mL)]]> Unfermented 352.05 Example 5 64.25 Comparative Example 1 86.35 Comparative Example 2 125.44 Comparative Example 3 95.63

[0109] As shown in Table 5, the xanthine oxidase inhibition IC values ​​of the lactic acid bacteria direct-fermented asparagus kimchi prepared in Example 5 were 50 The value is much lower than that of Comparative Examples 1-3. This shows that Example 5 has better xanthine oxidase inhibition activity than Comparative Examples 1-3, compared with the non-inoculated direct-throwing fermentation agent, and fermentation with endophytes without additional addition of glucose and white sugar. Moreover, the strain provided by the present invention has better performance in xanthine oxidase inhibition than the Pediococcus pentosaceus in the prior art, and has better uric acid-lowering potential.

[0110] (4) Angiotensin converting enzyme (ACE) inhibition test

[0111] After the fermented asparagus kimchi sample is homogenized, 2.0 g is weighed and added to 30 mL of 70% ethanol by volume. After ultrasonic extraction for 20 minutes, the supernatant is centrifuged and filtered, concentrated by rotary evaporation, and then freeze-dried in vacuum. After reconstitution with 2 mL of PBS solution, it is set aside. Detection method: Take 10 μL of the fermented asparagus kimchi sample extract and 40 μL of 5 mM HHL solution, mix well, preheat in a 37°C water bath for 5 minutes, add 30 μL of 0.02 U / mL ACE, incubate at 37°C for 30 minutes, and shake once every 5 minutes; add 150 μL of 1M HCl solution to terminate the reaction. The content of the product HA (hippuric acid) is detected by high-performance liquid chromatography. Liquid phase conditions: injection volume of 20 μL, chromatographic column Kromasi l100-5-C18 (4.6 x 250 mm), mobile phase of 0.05% formic acid aqueous solution: acetonitrile = 80%: 20%, column oven temperature 30°C, detector is UV detector, detection wavelength 228 nm. ACE inhibition rate is calculated as follows:

[0112]

[0113] In the formula, A a : The peak area of ​​HA in the presence of both ACE and fermented asparagus kimchi extract in the above detection system, A b : The peak area of ​​HA in the absence of the fermented asparagus kimchi extract in the above detection system, Ac: The peak area of ​​HA in the absence of both ACE and the fermented asparagus kimchi extract in the above detection system. The specific results are shown in Table 6.

[0114] Table 6 ACE inhibition ability of direct-fermented asparagus kimchi

[0115]

[0116]

[0117] As shown in Table 6, the ACE inhibition IC values ​​of the lactic acid bacteria direct-fermented asparagus kimchi prepared in Example 5 are as follows: 50 The values ​​are much lower than those of Comparative Examples 1 to 3. This indicates that the lactic acid bacteria direct-fermented asparagus kimchi prepared in Example 5 has excellent blood pressure lowering potential.

[0118] (5) Main physical and chemical indicators of asparagus kimchi fermented by direct-injection fermentation of Lactobacillus pentosus in different embodiments

[0119] Table 7 Main physical and chemical indicators

[0120]

[0121] As shown in Table 7, in Example 5-7, the pH of the asparagus kimchi fermented by direct-throwing Lactobacillus pentosaceus is at a relatively low level, while the total acidity is relatively high, thereby giving the product a strong fermented sour taste. In addition, the fermented asparagus kimchi products prepared by Example 5-7 have a nitrite content of no more than 1.0 mg / kg, which is far lower than the national standard GB2762 for the limit of nitrite content of pickled vegetables (20 mg / kg). In addition, the direct-throwing fermented asparagus kimchi products prepared by different embodiments have a salt content of less than 6%. This not only meets the current health needs of low saltization, but also effectively reduces the environmental pressure caused by the discharge of high-salt wastewater. It is worth mentioning that the lactic acid bacteria direct-throwing fermented asparagus kimchi prepared by Example 5-7 has a high content of vitamin C and protein, which indicates that the nutrients in the lactic acid bacteria direct-throwing fermented asparagus kimchi prepared by it are well retained.

[0122] (6) Biogenic amines from asparagus kimchi fermented by direct injection of Lactobacillus pentosus in different examples

[0123] Biogenic amines are determined by high performance liquid chromatography (HPLC) based on the liquid chromatography method in GB5009.208-2016 National Food Safety Standard Determination of Biogenic Amines in Foods to detect the content of histamine, tyramine, cadaverine and putrescine in samples.

[0124] Table 8 Biogenic amine content

[0125]

[0126]

[0127] Note: “-” means not detected.

[0128] As shown in Table 8, the direct-throw fermented asparagus kimchi prepared in Examples 5-7 has relatively low levels of cadaverine, tyramine, putrescine and histamine. It is particularly noteworthy that the histamine and tyramine contents are lower than the EU standard (histamine is lower than 100 mg / kg, and tyramine is lower than 100-800 mg / kg). The test results fully demonstrate that the fermented asparagus kimchi prepared by the lactic acid bacteria direct-throw fermentation agent corresponding to Examples 5-7 has good safety and is a green and safe fermented product.

[0129] (7) Organic acid content of asparagus kimchi fermented by Lactobacillus sutchusosus in different examples

[0130] Organic acid detection method: The sample was centrifuged at 10000rpm for 10min at 4℃, and 1mL of supernatant was filtered through a 0.22μm water filter for determination. The organic acid content was detected using an Agilent 1260 high performance liquid chromatograph. The chromatographic column used was BIO-RAD HPX-87H ion exchange chromatography column C18 (5μm, 300mm×7.8mm); mobile phase: 6mmol / L dilute sulfuric acid; detector: UV detector, wavelength: 210nm; column temperature: 35℃; injection volume: 20μL; flow rate: 0.5mL / min; organic acid standards were used for external standard quantification.

[0131] Table 9 Organic acid content

[0132]

[0133] Note: “-” means not detected.

[0134] As shown in Table 9, the contents of various organic acids (herbic acid) in the lactic acid bacteria direct-fermented asparagus pickles prepared in Examples 5-7 after fermentation were significantly increased compared to before fermentation. Among them, the content of lactic acid ranks first and occupies the most prominent position. Organic acids such as lactic acid, malic acid and citric acid contribute greatly to the taste of the product, and they mainly give the product a soft and pleasant sour taste. Acetic acid, propionic acid and butyric acid are all important components of short-chain fatty acids. In the physiological functions of the human body, these short-chain fatty acids play an indispensable role and help prevent a series of metabolic syndromes.

[0135] (8) Antioxidant capacity of asparagus kimchi fermented by direct injection of Lactobacillus pentosus in different examples

[0136] DPPH free radical scavenging ability detection method: Asparagus kimchi sample is mixed with ultrapure water in a ratio of 1:1 and homogenized. After ultrasonic extraction for 30 minutes, centrifuge and take the supernatant for use. Add 1.0mL of the extracted sample to 1mL of ethanol DPPH free radical solution (0.1mM), mix well and incubate in a dark environment at room temperature for 30 minutes. After centrifugation at 8000g for 10 minutes, a mixture of 1mL PBS and 1mL ethanol DPPH solution is used as the control group, and 1mL ethanol and 1mL extracted asparagus kimchi sample are used as the background blank, and the absorbance of the resulting solution is measured at 517nm. Trolox is used as the reference standard, and the results are expressed as μM Trolox. DPPH free radical scavenging ability is calculated according to the following formula:

[0137]

[0138] Where: A control is the absorbance of the control group, A sample is the absorbance of the sample treatment group, A blank is the background blank absorbance.

[0139] ABTS free radical scavenging ability test method: The total antioxidant capacity of fermented asparagus kimchi was determined using the ABTS method. The asparagus kimchi sample was mixed with ultrapure water in a ratio of 1:1 and homogenized. After ultrasonic extraction for 30 minutes, the supernatant was centrifuged and used for later use. The ABTS stock solution was mixed with 2.45mmol / L K2S2O8 and reacted in the dark at room temperature for 16 hours to obtain ABTS· + The resulting solution was then diluted with PBS and the absorbance at 734 nm was adjusted to 0.70 ± 0.01. + The solution is prepared and used immediately. Add the extracted sample (10 μL) or Trolox standard solution to 200 μL of fresh ABTS. + The solution was incubated in the dark at room temperature for 6 min and the absorbance of the mixture was measured. Trolox was used as a reference standard and the results were expressed as μM Trolox equivalent antioxidant capacity.

[0140] Total reducing capacity detection method: The total reducing capacity of the fermented asparagus kimchi sample was determined according to the iron reduction / antioxidant capacity (FRAP) method. The asparagus kimchi sample was mixed with ultrapure water in a ratio of 1:1 and homogenized. After ultrasonic extraction for 30 minutes, the supernatant was centrifuged and set aside. The FRAP reagent was prepared according to the instructions of the FRAP total antioxidant capacity assay kit. Fresh FRAP reaction reagent was prepared before the assay and placed in a 37°C water bath. 5 μL of the extracted sample was added to 180 μL of the FRAP reagent and incubated at 37°C in the dark for 5 minutes. The absorbance of the mixture was measured at 593 nm. A FeSO4 standard curve was prepared using 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5 mM FeSO4 solutions. The total antioxidant capacity of the sample was calculated based on the standard curve, and the results were expressed as FeSO4 equivalents (mM).

[0141] The specific results are as follows:

[0142] Table 10 Antioxidant capacity of direct-fermented asparagus kimchi

[0143]

[0144]

[0145] As shown in Table 10, the lactic acid bacteria direct-fermented asparagus kimchi prepared in Example 5-7 has strong DPPH free radical scavenging ability, ABTS free radical scavenging ability and total reducing ability, indicating that the lactic acid bacteria direct-fermented asparagus kimchi prepared in Example 5-7 has excellent antioxidant activity.

[0146] (9) The shelf life of the asparagus pickle products directly fermented by Lactobacillus pentosus prepared by the present invention, i.e., Examples 5-7, was determined by the following sensory quality and microbial indicators

[0147] Table 11 Evaluation of the shelf life of lactic acid bacteria fermented asparagus kimchi (Examples 5-7)

[0148]

[0149] As shown in Table 11, during the 18-month storage period, the sensory indicators such as color, crispness, taste, smell and texture of the asparagus pickles fermented by direct-injection fermentation of Lactobacillus pentosus prepared in Examples 5-7 of the present invention remained unchanged. In addition, the total number of microbial colonies was 0, and no coliform bacteria were detected.

[0150] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of Lactobacillus pentosus JGS34 or its bacterial agent in the preparation of direct-fermented asparagus kimchi, characterized in that: The Lactobacillus pentosus (Lactiplanttibacillus pentosus) JGS34 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on June 18, 2024, with a deposit number of CGMCC NO.30985; the active ingredient of the bacterial agent includes the Lactobacillus pentosus JGS34; the dosage form of the bacterial agent includes freeze-dried powder.

2. A method for preparing asparagus pickles, characterized in that: It includes the steps of inoculating and fermenting Lactobacillus pentosus JGS34; the Lactobacillus pentosus JGS34 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on June 18, 2024, with a deposit number of CGMCC NO.30985.

3. The preparation method according to claim 2, characterized in that: The following steps are involved: Fresh asparagus is screened, cleaned, cut, and put into a fermentation container. Water, edible salt, glucose, white sugar, and edible calcium chloride are added and mixed evenly. A lactic acid bacteria direct-injection fermentation agent is used for inoculation, fermentation, packaging, and sterilization to obtain a finished product. The lactic acid bacteria direct-injection fermentation agent contains the Lactobacillus pentosaceus JGS34.

4. The preparation method according to claim 3, characterized in that: The fermentation system includes, by weight, 40-80 parts of fresh asparagus, 40-80 parts of water, 2-5 parts of edible salt, 0.2-0.7 parts of glucose, 0.4-1.0 parts of white sugar, 0.1-0.3 parts of edible calcium chloride and 0.001-0.1 parts of lactic acid bacteria direct-injection starter.

5. The preparation method according to claim 3, characterized in that: The effective viable count of Lactobacillus pentosus JGS34 is 1×10 10 -9×10 10 CFU / g.

6. The preparation method according to claim 3, characterized in that: The inoculation fermentation conditions are to ferment at 25-37°C for 2-4 days.

7. The preparation method according to claim 3, characterized in that: The sterilization condition is pasteurization at 90°C for 10 minutes.

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