Lactobacillus pentosus JX3 and its application

Abstract

The present invention relates to the field of microbial technology, in particular to Lactobacillus pentosus ( Lactobacillus pentosus ) JX3 and its application. The present invention provides Lactobacillus pentosus ( Lactobacillus pentosus ) JX3, which has strong acid and salt resistance, can efficiently degrade nitrite, has strong antioxidant and antibacterial abilities, and has the ability to produce acid and amino acid nitrogen. The Lactobacillus pentosus ( Lactobacillus pentosus ) JX3 is used in fermentation to increase the content of total acid, amino acid nitrogen and other substances, improve antioxidant capacity, and increase the content of volatile substances such as esters and alcohols with fresh and fruity flavors in fermented products, which is beneficial to improving the quality of fermented products: improving food safety, extending shelf life, increasing nutritional value, and improving flavor.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Lactobacillus pentosus JX3 and applications thereof. Background Art

[0002] Lactic acid bacteria are a type of bacteria that can ferment carbohydrates and produce lactic acid. They have become an important microbial resource in the field of food fermentation. At present, more than 200 species of lactic acid bacteria have been identified, belonging to 18 genera, among which Lactobacillus, Bifidobacterium, Streptococcus and Lactococcus are the most common. In recent years, a large number of studies have shown that lactic acid bacteria play an important role in regulating intestinal flora, anti-oxidation, inhibiting allergies, improving glucose and lipid metabolism, and also show health benefits such as lowering cholesterol and blood pressure. During the food fermentation process, lactic acid bacteria can decompose carbohydrates, proteins, etc. in the raw materials to produce beneficial components such as amino acids, peptides and organic acids. These metabolites not only significantly inhibit the reproduction of spoilage bacteria and pathogenic bacteria, but also improve the nutritional value of food, improve flavor, and extend shelf life. Therefore, lactic acid bacteria show a broad and important application prospect in the field of food fermentation.

[0003] Pepper (Capsicum spp.) is an annual herbaceous plant of the genus Capsicum in the Solanaceae family. Due to its rich nutrients and wide range of uses, it has become one of the vegetables with the largest planting area in my country. The scale of planting is particularly significant in the main pepper producing areas such as Guizhou, Henan, Sichuan and Hunan. Peppers are deeply loved by consumers due to their rich nutrient content and diverse uses. However, due to the high water content of peppers, they are very easy to rot and deteriorate after harvest, and have a short shelf life. This poses severe challenges to the storage and transportation of peppers. In order to effectively extend the shelf life while retaining its nutritional value and flavor characteristics as much as possible, fermentation treatment is considered to be an effective means to solve this problem. Fermentation can not only inhibit the growth of spoilage bacteria, but also further enhance the nutritional value and flavor of peppers, thereby providing technical support for the storage and transportation of peppers and their application in food processing.

[0004] Fermented vegetables are typical representatives of traditional fermented foods. During the processing, the growth and metabolic activities of microorganisms cause a series of physical and chemical changes in the vegetable raw materials, thereby improving their nutritional value and flavor. Traditionally, fermented vegetables are mostly produced by natural fermentation, which mainly relies on the synergistic effect of various microorganisms such as lactic acid bacteria and acetic acid bacteria naturally present on the surface of vegetables to complete the fermentation. However, natural fermentation has some significant defects. For example, the fermentation process involves complex microbial flora, and the process conditions are difficult to accurately control, resulting in food safety hazards, easy contamination by miscellaneous bacteria, and unstable nutritional quality and flavor. These problems have brought significant challenges to the large-scale and industrial production of fermented vegetables. Therefore, it is urgent to use technical means such as exogenous microbial agents to degrade the above problems. Exogenous agents can effectively regulate the microbial composition in the fermentation process, ensure the controllability of fermentation conditions, stabilize and improve the quality of fermented vegetables, including improving food safety, extending shelf life, and improving nutritional value and flavor, thereby promoting the development of fermented vegetables in the modern food industry. Summary of the invention

[0005] In view of this, the present invention provides Lactobacillus pentosus JX3 and its application. The Lactobacillus pentosus JX3 of the present invention has strong acid and salt resistance, can efficiently degrade nitrite, has strong antioxidant and antibacterial abilities, and has the ability to produce acid and amino acid nitrogen, can improve the antioxidant capacity of fermented products, and is beneficial to the accumulation of esters, alcohols and other substances with fresh and fruity flavors in fermented products. The Lactobacillus pentosus JX3 with good fermentation performance provided by the present invention is applied to fermentation, which can significantly increase the content of total acid, amino acid nitrogen and other substances, improve antioxidant capacity, and increase the content of volatile substances such as esters and alcohols, which is beneficial to improving the quality of fermented products, including improving food safety, extending shelf life, improving nutritional value, and improving flavor.

[0006] Compared with natural fermentation, the inoculation of Lactobacillus pentosus JX3 is applied to fermentation, including improving the food safety of fermented products, extending the shelf life, improving the nutritional value, and improving the flavor.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides Lactobacillus pentosus JX3, whose deposit number is CGMCC No.31021.

[0009] In some specific embodiments of the present invention, the Lactobacillus pentosus JX3 is used in the form of one or more of live bacteria, inactivated bacteria, fermentation broth, exosomes or metabolites.

[0010] The present invention also provides a microbial agent, comprising the Lactobacillus pentosus JX3.

[0011] The present invention also provides the use of the Lactobacillus pentosus JX3 or the microbial agent in any of the following items:

[0012] (I) Acid and salt resistance in vegetable fermentation; and / or

[0013] (II) Reducing nitrite in vegetable fermentation; and / or

[0014] (III), preparing antioxidant products; and / or

[0015] (IV) Preparation of antibacterial products; and / or

[0016] (V) Acid production during vegetable fermentation; and / or

[0017] (VI) Amino acid nitrogen produced by vegetable fermentation; and / or

[0018] (VII) increasing the content of esters and alcohols with fresh, fruity flavor in vegetable fermentation;

[0019] The ester substance includes one or more of ethyl 2-hydroxypropionate, isopentyl 3-methylbutyrate-M, ethyl hexanoate-M, 3-methyl-1-butanol acetate-M or hexyl acetate-M;

[0020] The alcohol substance includes one or more of 1-hexanol-M, 1-hexanol-D, 3-methyl-1-butanol-M, 1-heptanol or 2-methyl-1-propanol-M.

[0021] In some specific embodiments of the present invention, the antioxidant activity includes improving the antioxidant capacity of fermented vegetable products.

[0022] In some specific embodiments of the present invention, the anti-oxidation comprises one or more of scavenging DPPH free radicals, scavenging hydroxyl free radicals or scavenging superoxide anions.

[0023] In some specific embodiments of the present invention, the antibacterial activity includes inhibiting the growth of Escherichia coli and / or inhibiting the growth of Staphylococcus aureus.

[0024] The present invention also provides the use of the Lactobacillus pentosus JX3 or the microbial agent in improving the quality of fermented vegetables;

[0025] The method of improving the quality of fermented vegetables includes improving the edible safety of fermented vegetables, extending the shelf life, increasing the nutritional value, and improving the flavor.

[0026] In some specific embodiments of the present invention, improving the quality of vegetable fermentation includes reducing the nitrite content, increasing the total acid and amino acid nitrogen content, improving the antioxidant capacity, and increasing the content of ester and alcohol volatile substances.

[0027] The present invention also provides a vegetable fermented product, which is obtained by fermentation with the Lactobacillus pentosus JX3 or the microbial agent.

[0028] The present invention also provides a method for preparing the vegetable fermented product, comprising taking fresh vegetables, mixing them with pickling liquid, inoculating the Lactobacillus pentosus JX3 or the microbial agent, and fermenting to obtain the vegetable fermented product;

[0029] The pickling liquid includes white sugar, salt, white wine and the like.

[0030] In some specific embodiments of the present invention, the fermentation conditions include fermentation at 10±5° C. for 25 to 30 days.

[0031] In some specific embodiments of the present invention, the mass volume ratio of the fresh vegetables to the pickling liquid is 1:1 to 1:2.

[0032] In some specific embodiments of the present invention, the amount of Lactobacillus pentosus JX3 or the microbial agent added is 2-4%, and the bacterial solution concentration is 1-10×10 8 CFU / g.

[0033] The present invention also provides a method for improving the quality of fermented vegetables, based on any of the following fermentations:

[0034] (I), Lactobacillus pentosus JX3; and / or

[0035] (II) the microbial agent.

[0036] The present invention provides the following beneficial effects:

[0037] The Lactobacillus pentosus JX3 of the present invention has strong acid and salt resistance, can efficiently degrade nitrite, has strong antioxidant and antibacterial abilities, has the ability to produce both acid and amino acid nitrogen, can improve the antioxidant capacity of fermented products, and is conducive to the accumulation of esters, alcohols and other substances with fresh and fruity flavors in fermented products. The Lactobacillus pentosus JX3 with good fermentation performance provided by the present invention is applied to fermentation, which can increase the content of total acid, amino acid nitrogen and other substances, improve antioxidant capacity, and increase the content of volatile substances such as esters and alcohols, which is conducive to improving the quality of fermented products, including improving food safety, extending shelf life, improving nutritional value, and improving flavor.

[0038] Biological Deposit Description

[0039] Biological material: JX3, classified and named Lactobacillus pentosus, was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on June 20, 2024, with the deposit number CGMCCNo.31021; the specific address of the General Microbiology Center of China Culture Collection of Microorganisms is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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 or the description of the prior art are briefly introduced below.

[0041] Figure 1 The colony morphology of JX3, JX4, and JS1 (A) and their morphological characteristics under a microscope (B) are shown (magnification is 100 times);

[0042] Figure 2 The results of 16S rDNA gene sequencing of 3 strains are shown;

[0043] Figure 3 The growth curves (A) and acid production characteristics (B) of the three strains are shown;

[0044] Figure 4 The acid resistance (A), salt resistance (B) and nitrite degradation ability (C) of the three strains are shown; different letters indicate significant differences in acid resistance or salt resistance between different strains (P<0.05);

[0045] Figure 5The scavenging abilities of the three strains on DPPH, hydroxyl radicals and superoxide anions are shown; (A) shows IC, i.e., the free radical scavenging ability of the strain suspension, and (B) shows CFE, i.e., the free radical scavenging ability of the strain extract; different letters indicate significant differences in antioxidant ability between different strains (P<0.05);

[0046] Figure 6 Figure 3 shows the fermentation of peppers using JX3 strain; changes in pH (A), total acid content (B), amino acid nitrogen content (C), and antioxidant capacity (D) during the fermentation of peppers; different letters indicate significant differences in pH, total acid content, amino acid nitrogen content, and antioxidant capacity among different strains at the same fermentation time (P<0.05);

[0047] Figure 7 The fermentation of peppers using JX3 strain is shown; the composition of volatile flavor substances during the fermentation process of peppers; principal component analysis (A), relative abundance analysis of volatile substances (B), and differential fingerprint analysis (C);

[0048] Figure 8 The fermentation of pepper using JX3 strain was shown. DETAILED DESCRIPTION

[0049] The present invention discloses Lactobacillus pentosus JX3 and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0050] The invention provides a Lactobacillus pentosus strain with good fermentation characteristics. The classification name of the strain is Lactobacillus pentosus JX3, namely Lactobacillus pentosus JX3.

[0051] The Lactobacillus pentosus JX3 of the present invention has strong acid and salt resistance, can efficiently degrade nitrite, has strong antioxidant and antibacterial abilities, has the ability to produce acid and amino acid nitrogen, can improve the antioxidant capacity of fermented products, and is beneficial to the accumulation of esters, alcohols and other substances with fresh and fruity flavors in fermented products.

[0052] The present invention also provides the use of the Lactobacillus pentosus in fermenting plants such as peppers.

[0053] The Lactobacillus pentosus JX3 isolated by the present invention is inoculated and applied to fermented peppers, which significantly increases the content of total acid, amino acid nitrogen and other substances in the fermented pepper products, improves the antioxidant capacity, and increases the content of volatile substances such as esters and alcohols, which is beneficial to improving the quality of the fermented products, including improving food safety, extending the shelf life, improving the nutritional value, and improving the flavor.

[0054] The preparation process of the fermented pepper is as follows:

[0055] Fresh peppers were rinsed twice with pure water and spread flat to dry; then pickling liquid (1-2% white sugar, 5-6% salt, 0.1-0.2% liquor) was prepared; the pickling liquid was added to the peppers at a ratio of 1:1.5 (w:v), and then the prepared starter was inoculated at 3% inoculation (bacterial liquid concentration 1-10×10 8 CFU / g), stir evenly and place in a clean and dry transparent glass fermentation tank.

[0056] Furthermore, the inoculated fermentation bacteria are Lactobacillus pentosus JX3 and Weissella greecea JS1.

[0057] Furthermore, the above-mentioned strains Lactobacillus pentosus JX3 and Weissella greecea JS1 were respectively isolated from traditional naturally fermented peppers.

[0058] Beneficial effects:

[0059] The Lactobacillus pentosus JX3 of the present invention has strong acid and salt resistance, can efficiently degrade nitrite, has strong antioxidant and antibacterial abilities, has the ability to produce both acid and amino acid nitrogen, can improve the antioxidant capacity of fermented products, and is conducive to the accumulation of esters, alcohols and other substances with fresh and fruity flavors in fermented products. The Lactobacillus pentosus JX3 with good fermentation performance provided by the present invention is applied to fermentation, which can significantly increase the content of total acid, amino acid nitrogen and other substances, improve antioxidant capacity, and increase the content of volatile substances such as esters and alcohols, which is conducive to improving the quality of fermented products, including improving food safety, extending shelf life, improving nutritional value, and improving flavor.

[0060] If not otherwise specified, Lactobacillus pentosus JX3 is provided by the present invention, and other raw materials and reagents used in the application can be purchased from the market.

[0061] The present invention will be further described below in conjunction with embodiments:

[0062] Example 1 Isolation and purification of JX3 strain and colony morphology characteristics

[0063] (1) Aseptically weigh 10 g of the fermented pepper product, add 90 mL of 0.9% sterile saline, and beat in a homogenizer at a speed of 8.0 / s for 1 min each to make a 1:10 homogenate. Dilute to 10 by the 10-fold dilution method. 7 times; 0.1 mL of the dilution was spread on the surface of MRS solid culture medium and cultured at 37±1℃ for 24-36 h; suspected lactic acid bacteria colonies were picked out and pure strains were isolated by plate streak method; three strains were obtained after separation and numbered as JX3, JX4 and JS1 respectively; the morphological characteristics of the colonies of each strain were observed, and the morphological characteristics of the bacteria after Gram staining were observed under the oil immersion lens of a biological microscope.

[0064] The colony morphology and Gram staining results of the 3 strains are as follows Figure 1 (A) Figure 1 As shown in (B). Figure 1 As shown in the figure, the three strains were white, round, smooth and with neat edges. Figure 1 As shown in (B), Gram staining was positive, and the bacteria were rod-shaped under the microscope, arranged singly, in pairs or in chains. These three strains were preliminarily identified as lactic acid bacteria.

[0065] (2) 16S rDNA identification

[0066] Extraction of genomic DNA: After the strain was activated for two generations, the bacteria were collected; whole genomic DNA was extracted according to the method in the TSINGKE TSP701-50 TreliefBacteria Genomic DNA Kit.

[0067] PCR amplification of 16S rDNA: PCR amplification system: PCR Master mix 12.5 µL + 1 µL of upstream and downstream primers (27F and 1492R for universal bacterial primers) + 2.5 µL of DNA template, add ddH2O to 25 µL and then amplify. PCR reaction conditions: 95℃ pre-denaturation for 5 min; 35 cycles × (94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 50 s); 72℃ extension for 10 min.

[0068] Sequencing and analysis: After PCR amplification, non-specific bands, purity and DNA fragment size of PCR products were checked by agarose gel electrophoresis; PCR amplification products were sequenced; comparison analysis was performed using NCBI's GenBank, and homology searches were performed to find bacteria with high homology and download the gene sequences of these strains; MEGA 11.0 software was used to construct a developmental tree to determine the phylogenetic information of the strains.

[0069] The results of 16S rDNA sequencing of the strain are as follows Figure 2The comparison results with the NCBI database showed that JS1 was significantly different from JX3 and JX4, forming a separate branch and was identified as Weissella greecea; JX3 and JX4 had high sequence similarity and belonged to the same branch, and were both identified as Lactobacillus pentosus.

[0070] Example 2 Physiological and fermentation characteristics of JX3

[0071] (1) Growth curve and acid production capacity determination

[0072] The JX3, JX4, and JS1 strains were inoculated into MRS liquid medium and cultured at 37°C for 18–24 h until the concentration reached 1–10 × 10 8 CFU / g. Then, it was inoculated into a new MRS liquid medium at a ratio of 3% (v / v) and cultured at 37°C. Samples were taken every 2 h, and the absorbance at 600 nm (OD600 value) and pH value were measured; the samples were taken until 16 h, and then the OD600 value was measured again at 26 h; the OD value and pH value were measured three times in parallel. The uninoculated MRS liquid medium was used as the blank control, and the strain growth curve was drawn according to the OD value, and the pH value curve was drawn according to the pH value.

[0073] The results are as follows Figure 3 As shown in Figure A, it can be seen that the lag phase of the three strains is relatively short, and they have entered the logarithmic phase after 2 hours, indicating that the strains have good environmental adaptability; but at the same time, it was found that the growth of JX3 and JX4 was significantly higher than that of JS1. As can be seen from Figure B, the acid production curves of each strain tended to be flat after 12 hours; but at the same time, it was found that the acid production capacity and acid production rate of JX3 and JX4 were significantly greater than JS1, that is, the pH value decrease amplitude and speed were significantly greater than JS1. According to Figures A and B, JX3 and JX4 have good growth and acid production capabilities, which are significantly stronger than JS1.

[0074] (2) Determination of acid and salt resistance

[0075] Acid resistance test: strain activation is the same as (1). Inoculate the activated bacterial solution into MRS liquid culture medium with an initial pH value of 2.0, 3.0, 4.0, 5.0, and 6.0 at a 3% (v / v) inoculation volume. Culture at 37°C for 24 h and measure the OD600 value. Use the uninoculated culture medium as the blank control and draw the strain growth curve.

[0076] Salt tolerance test: strain activation was the same as (1). At a 3% (v / v) inoculation volume, the activated bacterial solution was inoculated into MRS liquid culture medium with NaCl contents of 4% (w / v), 6% (w / v), 8% (w / v), 10% (w / v) and 12% (w / v), respectively. Cultured at 37°C for 24 h, the OD600 value was measured. The uninoculated culture medium was used as the blank control, and the strain growth curve was plotted.

[0077] During the fermentation process, plants continuously produce acid, so the fermentation strains are required to have strong acid resistance. In addition, the salt content of fermented vegetables is usually as high as 5% to 12% (w / v), so the fermentation strains are also required to have strong salt resistance. The growth of the three strains under different initial pH and NaCl concentrations is shown in Figure 2. Figure 4 As shown. Figure 4 As shown in (A), JX3 not only grows well in a weakly acidic environment, but also grows better than other strains in a strong acidic environment such as pH 3.0. Figure 4 As shown in (B), JX3 grew better than other strains at all tested salt concentrations, including high salt concentrations, such as 10% (w / v) and 12% (w / v). Figure 4 Comprehensively judging, JX3 has strong acid and salt resistance.

[0078] (3) Determination of nitrite-reducing ability

[0079] The strain activation was the same as (1). The activated bacterial liquid was inoculated into the MRS liquid medium with an initial nitrite concentration of 200 µg / mL at a 3% (v / v) inoculation volume. The culture was carried out at 37°C, and samples were taken at 24 h, 48 h, and 72 h. The nitrite content was determined according to GB 5009.33-2016 "Determination of Nitrite and Nitrate in Food" and the nitrite degradation ability of each strain was analyzed. The nitrite degradation rate was calculated by combining the nitrite standard curve of y=0.0281x+0.002 (R²=0.9993) and formula (1).

[0080] (1)

[0081] Note: D0 is the nitrite content in the liquid culture medium before inoculation, μg / mL; D1 is the nitrite content in the liquid culture medium after inoculation, μg / mL.

[0082] The degradation trend of nitrite by 3 strains within 24-72 h, such as Figure 4 (C) and Table 1. Within 24 h, the degradation rates of strains JX3 and JX4 reached more than 96%, which were significantly higher than JS1's 7.68%; JX3 had the highest rate, reaching 99.81%. At 48 h and 72 h, JX3 and JX4 both showed significant nitrite degradation capabilities, with degradation rates exceeding 99%.

[0083] Table 1 Analysis of nitrite degradation ability

[0084]

[0085] Note: Different letters indicate significant differences in the degradation rate of nitrite among strains at the same culture time (P<0.05).

[0086] (4) Antioxidant capacity determination

[0087] Preparation of cell suspensions and cell-free extracts:

[0088] After the strain was activated, the cells were cultured in MRS liquid for 18 h and centrifuged at 4°C and 6000 r / min for 15 min to obtain bacterial pellets. The cells were washed three times with sterile phosphate buffer solution (PBS, pH 7.2) and resuspended in PBS to obtain cell suspension (IC).

[0089] The cell suspension was disrupted with an ultrasonic disruptor for 10 min under ice bath conditions; centrifuged at 4°C and 6000 r / min for 15 min; and filtered through a 0.22 µm filter membrane to obtain the extract (CFE).

[0090] DPPH scavenging ability determination:

[0091] Using DPPH mixed with PBS as a control, 1 mL IC (i.e., cell suspension of each strain) or CFE (i.e., extract of each strain) was thoroughly mixed with 1 mL of 0.2 mmol / L DPPH anhydrous ethanol solution and then reacted in a 37°C incubator in the dark for 30 min. The supernatant was collected and the OD517 was measured at a wavelength of 517 nm. The DPPH free radical scavenging rate was calculated according to formula (2):

[0092] DPPH free radical scavenging rate (%) = "OD517 (control) - OD517 (sample)" / "OD517 (control)" × 100% (2)

[0093] Hydroxyl free radical scavenging ability assay:

[0094] 1 mL of 2.5 mmol / L o-phenanthroline solution, 1 mL of PBS, 1 mL of IC (or CFE), and 1 mL of 7.5 mmol / L FeSO4 solution were uniformly mixed (referred to as "mixture A"). 1 mL of 1% H2O2 solution was added to "mixture A", and the mixture was placed in a water bath at 37°C for 1.5 h. OD517 was measured at a wavelength of 536 nm. An equal volume of distilled water was used to replace IC (or CFE) in "mixture A" as control 1, and an equal volume of distilled water was used to replace H2O2 added to "mixture A" as control 2. The hydroxyl radical scavenging capacity was calculated according to formula (3):

[0095] Hydroxyl radical scavenging rate (%) = "A536 (sample) - A536 (control 1)" / "A536 (control 2) - A536 (control 2)" × 100% (3)

[0096] Superoxide anion scavenging ability assay:

[0097] Add 2 mL of 200 mmol / L Tris-HCl buffer (pH=8.2), 1 mL of 1.2 mmol / L pyrogallol solution (prepared with 10 mmol / L HCl solution), and 0.5 mL of IC (or CFE) in sequence, mix thoroughly, react at room temperature for 30 minutes, and measure OD325 at a wavelength of 325 nm. Calculate the superoxide anion scavenging capacity according to formula (4):

[0098] Superoxide anion clearance rate (%) = (1-"A1-A2" / "A3-A4") × 100% (4)

[0099] In the formula: A1 is the absorbance of the sample and pyrogallol; A2 is the absorbance of the sample but not pyrogallol; A3 is the absorbance of the sample but not pyrogallol; A4 is the absorbance of the sample and pyrogallol.

[0100] The results of scavenging rates of DPPH free radical, hydroxyl free radical and superoxide anion free radical by strain (IC) and strain extract (CFE) are as follows: Figure 5 As shown. Figure 5 As shown in (A), the three strains JX3, JX4, and JS1 all have different degrees of in vitro antioxidant capacity. Figure 5 As shown in (B), only the extract of JX3 has the ability to scavenge DPPH free radicals, hydroxyl free radicals, and superoxide anions, while JX4 and JS1 can only scavenge DPPH free radicals and superoxide anions.

[0101] (5) Determination of antibacterial ability

[0102] The activation of strains is the same as (1). The activated strains are made into the test solution. Escherichia coli and Staphylococcus aureus are selected and inoculated into LB liquid culture medium, and cultured at 37°C for 8-12 h to make indicator bacterial solution. Using 0.9% saline as the control, the antibacterial ability of the three strains against Escherichia coli and Staphylococcus aureus is compared by the antibacterial diameter using the agar plate punching method.

[0103] Table 2 shows the antibacterial results of the three strains against Escherichia coli E.coli and Staphylococcus aureus S.aureus. As shown in Table 2, the diameters of the inhibition zones of JX3 and JX4 against Escherichia coli E.coli are both greater than 12 mm, significantly greater than JS1; the antibacterial diameter of JX3 against Staphylococcus aureus S.aureus is greater than 15 mm, significantly greater than JX4 and JS1. As shown in Table 2, both JX3 and JX4 have significant inhibitory effects on Escherichia coli E.coli and Staphylococcus aureus S.aureus; but relatively speaking, JX3 has stronger antibacterial ability, especially against Staphylococcus aureus S.aureus.

[0104] Table 2 Inhibitory effects of three lactic acid bacteria on Escherichia coli and Staphylococcus aureus

[0105]

[0106] Note: Different letters indicate significant differences in the inhibitory effects of the same indicator bacteria on the strains (P<0.05).

[0107] In summary, Lactobacillus pentosus JX3 is a lactic acid bacterium with excellent fermentation performance, strong acid and salt resistance, can efficiently degrade nitrite, has strong antioxidant and antibacterial abilities, has fermentation potential, and is suitable for use in the fermentation industry to prepare fermented products.

[0108] Example 3 Application of JX3 in pepper fermentation

[0109] (1) Preparation of starter culture

[0110] The activated JX3 and JS1 were inoculated into MRS liquid culture medium respectively; after growing to the logarithmic phase, centrifuged at 4°C and 6000r / min for 10 min; the supernatant was discarded to obtain the precipitate; the precipitate was washed repeatedly with 0.9% saline for 2 to 3 times to prepare a bacterial suspension for later use.

[0111] (2) Fermentation preparation

[0112] Select fresh chili peppers of moderate size and without mechanical damage on the surface; wash them with clean water first, then rinse them twice with purified water and lay them flat to dry.

[0113] (3) Pickling and fermentation

[0114] Prepare pickling liquid (1-2% white sugar, 5-6% salt, 0.1-0.2% liquor, etc.); add pickling liquid to peppers at a ratio of 1:1.5 (w:v), and then inoculate the prepared starter at a 3% inoculum amount (bacterial liquid concentration 1-10×10 8CFU / g), stir evenly and place in a clean and dry transparent glass fermenter. Cover the lid and ferment at 10±5℃ for 25-30 days. For natural fermentation samples, samples inoculated with JX3 and JS1 are recorded as D, JX3, and JS1, respectively. Figure 8 shown.

[0115] (4) Changes in pH value and total acid content

[0116] During the fermentation process, 2 g of the fermentation product was weighed, 18 mL of deionized water was added, and the pH value of the supernatant was measured with a pH meter after standing for 30 min.

[0117] During the fermentation process, 2 g of the fermented product was weighed and the total acid content was determined according to GB / T 12345-2008 “Determination of total acid in foods”.

[0118] Changes in pH and total acid content during fermentation Figure 6 (A) Figure 6 (B) shown. Figure 6 (A) shows that compared with the natural fermentation group and the pepper inoculated with JS1, the pH of the pepper inoculated with JX3 decreased the fastest and the largest during the entire fermentation process; at the end of fermentation, the pH value was significantly lower than that of the natural fermentation D. Figure 6 (B) shows that compared with the natural fermentation group D and the JS1 inoculation group, the pepper inoculated with JX3 has the highest total acid content, which shows a continuous increasing trend and the fastest increasing rate; at the end of fermentation, the total acid content is significantly lower than that of the natural fermentation group D and the JS1 fermentation group. The rate of pH decrease and the rate of total acid increase are important indicators for measuring the speed of pickling fermentation, which are crucial to the quality and flavor formation of fermented products; and the appropriate low pH value and high total acid content at the end of fermentation are conducive to the fermented products being in an acidic state, which is beneficial to inhibiting the growth of spoilage bacteria and extending the shelf life of fermented products.

[0119] (5) Changes in amino acid nitrogen content

[0120] During the fermentation process, 2 g of the fermented product was weighed and the amino acid nitrogen content was determined with reference to GB 5009.235-2016 "Determination of Amino Acid Nitrogen in Foods". The change in amino acid nitrogen content during the fermentation of chili peppers in this embodiment is as follows: Figure 6 (C) from Figure 6 (C) It can be seen that the amino acid nitrogen content of the chili peppers inoculated with JX3 and JS1 is higher, significantly higher than that of naturally fermented peppers. The protein in the fermentation raw materials is gradually degraded into amino acids through microbial degradation and hydrolytic enzyme action; the higher the amino acid content, the stronger the umami taste and the higher the nutritional value. The higher the amino acid nitrogen content of the chili peppers inoculated with JX3 and JS1 reflects that the inoculation of these two strains is beneficial to improving the flavor and nutritional value of chili pepper fermented products.

[0121] (6) Changes in antioxidant activity

[0122] During the fermentation process, the fermented juice was taken, and the change law of antioxidant capacity during the fermentation process of chili pepper was determined with reference to Example 2, and the results are shown in 6 (D). As can be seen from 6 (D), both the natural fermentation sample and the JS1 fermentation sample had no scavenging ability for DPPH free radicals. The scavenging rate of the JX3 fermentation sample for DPPH free radicals exceeded 60% as early as the first day of fermentation, and rose to about 80% on the fifth day, and then stabilized at more than 80% during the entire fermentation process. It shows that inoculation of JX3 is beneficial to improving the biological activity and nutritional value of chili pepper. Moreover, this result is consistent with the observation results of the antioxidant capacity of the strain extract in Example 2. Combined with the high acid production capacity of JX3, it is speculated that JX3 has the ability to synthesize or secrete antioxidant substances such as organic acids. Therefore, when JX3 is applied to chili pepper fermentation, it continuously secretes antioxidant substances such as organic acids as the fermentation process progresses, thereby significantly improving the antioxidant capacity of chili pepper fermented products.

[0123] (7) Sensory evaluation

[0124] The control naturally fermented peppers and inoculated fermented peppers were secretly numbered, and 20 professionally trained sensory appraisers (10 men and 10 women, aged 20 to 40 years old) were selected to evaluate the fermented chili peppers based on color, taste, smell, tissue state and impurities. All samples were sensory evaluated twice to prevent errors caused by sensory fatigue.

[0125] The scoring criteria are shown in Table 3, and the evaluation results are shown in Table 4:

[0126] Table 3 Sensory scoring criteria for pickled and fermented peppers

[0127]

[0128] Note: Observe the appearance under natural light, place an appropriate amount of the sample in a clean white porcelain dish, observe the color, texture and impurities, smell it, and taste it.

[0129] Table 4 Sensory evaluation results of pickled and fermented peppers

[0130]

[0131] As shown in Table 4, the pepper samples obtained by inoculation with JX3 had the highest comprehensive score in color, taste and smell in the sensory evaluation, which was higher than the pepper samples of naturally fermented group D and the pepper samples fermented with JS1; this shows that the sensory properties of JX3 fermented peppers are excellent, the flavor is harmonious, and they are more acceptable to consumers.

[0132] (8) Analysis of volatile components of fermented chili pepper

[0133] Headspace-solid phase microextraction-gas chromatography-ion mobility spectrometry (HS-SPME-GC-IMS) combined with multivariate statistical analysis were used to compare the differences in volatile flavor compounds in four types of chili peppers: fresh chili pepper (XLJ), natural fermentation (D), fermentation by inoculation with JX3, and fermentation by inoculation with JS1.

[0134] Sample processing method:

[0135] Accurately weigh 2 g of fresh chili pepper (XLJ), natural fermentation (D), inoculation JX3 fermentation, and inoculation JS1 fermentation samples, place them in 20 mL headspace bottles, incubate at 60°C for 15 min, and then inject the samples. Each sample was measured in triplicate.

[0136] Headspace injection conditions:

[0137] Incubation temperature: 60℃; incubation time: 15 min; injection volume: 500 µL; splitless injection; incubation speed: 500r / min; injection needle temperature: 85℃.

[0138] Gas chromatography (GC) conditions:

[0139] Chromatographic column temperature: 60℃; carrier gas: high-purity nitrogen (purity ≥99.999%); programmed pressure increase: initial flow rate 2.0 mL / min maintained for 2 min, linearly increased to 10.0 mL / min within 8 min, linearly increased to 100.0 mL / min within 10 min, and maintained for 10 min. Chromatographic running time: 30 min; injection port temperature: 80℃.

[0140] IMS conditions:

[0141] Ionization source: tritium source (3H); migration tube length: 53 mm; electric field strength: 500 V / cm; migration tube temperature: 45 ℃; drift gas: high-purity nitrogen (purity ≥99.999%), flow rate: 150.0 mL / min; positive and negative ion modes: positive ion.

[0142] Qualitative analysis:

[0143] Six normal ketones (2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone and 2-nonanone, all of analytical grade) were used to prepare a mixed standard, and a calibration curve of retention time and retention index was established. The retention index of the substance was then calculated based on the retention time of the target substance. The built-in GC retention index database (NIST 2020) and IMS migration time database of the VOCal software were used for retrieval and comparison, and the target substance was qualitatively analyzed.

[0144] Through principal component analysis (PCA), we can visually observe the overall statistical differences between the four groups of samples and the level of parallelism between samples in the same group. The more similar the composition of volatile flavor substances in the samples, the closer their distance in the PCA graph. Figure 7 (A) It can be seen that the distribution of fermented peppers and fresh peppers in the PCA diagram is far apart, indicating that fermentation significantly changes the composition of volatile flavor substances in peppers. At the same time, there is also a certain distance between naturally fermented peppers and inoculated fermented peppers, further indicating that there are differences in the effects of the two fermentation methods on pepper flavor substances. In addition, peppers inoculated with different strains also show a certain distribution distance, reflecting that fermentation with different strains affects the formation of volatile flavor substances.

[0145] The composition of volatile flavor substances among the four groups of samples is as follows Figure 7 (B) As shown. Figure 7 As shown in (B), the volatile flavor substances of pepper mainly include esters, alcohols, ketones, acids, aldehydes, furans, alkenes and other unidentified substances. Among them, the above substances accounted for 33%, 16%, 9%, 1%, 9%, 1%, 3%, 3%, 1% and 14% in fresh peppers, respectively. Compared with fresh peppers, the composition of volatile flavor substances in fermented pepper samples changed significantly. Among them, the relative abundance of esters, acids and aldehydes increased significantly, ketones decreased significantly, and furans disappeared completely. Compared with naturally fermented chili peppers and JS1 fermented chili peppers, JX3 fermented chili peppers were mainly composed of esters and alcohols, and the abundance of these two substances increased more significantly, reaching 44% and 22% respectively; the abundance of ketones also decreased more significantly, and by the end of fermentation, it was only 10%. Esters usually have a strong fruity, floral or vanilla aroma, which is sweet, fresh and pleasant, and is essential for the formation of the flavor of fermented products. Suitable esters can enhance the taste and aroma of fermented products, and can increase the complexity and layering of flavors. The role of alcohols in fermented products is similar to that of esters, and alcohols have high volatility, so they also play a vital role in the formation of the flavor of fermented products. Moreover, alcohols and esters often have a synergistic effect, and the two work together to further enhance the complexity and layering of the aroma of fermented products.

[0146] Further analysis of all volatile substances in pepper was performed, and the results are shown in Figure 7 (C) and Table 5. Figure 7As shown in (C), compared with the naturally fermented chili pepper and the JS1 fermented chili pepper, the contents of 5 esters, 5 alcohols, 7 ketones, 2 aldehydes and 3 unidentified substances in the JX3 fermented chili pepper increased significantly. The 5 esters were ethyl 2-hydroxypropanoate, isopentyl 3-methylbutyrate-M, ethyl caproate-M, 1-Butanol, 3-methyl-, acetate-M and hexyl acetate-M. The five alcohols are 1-hexanol-M, 1-hexanol-D, 1-Butanol, 3-methyl-M, 1-heptanol, 1-Propanol, 2-methyl-M. The seven ketones are 2-methyl-2-hepten-6-one, 2-Butanone, 3-hydroxy-M, 2-Butanone, 3-hydroxy-D, 2-Heptanone-M, 2-Heptanone-D, 1-Octen-3-one, and 2-propanone. The two aldehyde substances are benzaldehyde-M and (E)-2-octenal. The three unidentified substances are substances 1, 4, and 6. The relative contents and odor characteristics of the above substances in JX3 fermented chili peppers are shown in Table 6. As shown in Table 6, most of the above substances have odor characteristics such as freshness and fruity aroma, which give JX3 fermented chili peppers a unique flavor.

[0147] Table 5 Relative contents and differences of volatile flavor substances in different samples

[0148]

[0149] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Lactobacillus pentosus ( Lactobacillus pentosus ) JX3, characterized in that, Its deposit number is CGMCC No.31021.

2. A microbial agent, characterized in that: comprising the Lactobacillus pentosus according to claim 1 ( Lactobacillus pentosus )JX3.

3. The Lactobacillus pentosus according to claim 1 ( Lactobacillus pentosus ) JX3 or the use of the microbial agent as claimed in claim 2 in any of the following items: (I) Reducing nitrite in vegetable fermentation; and / or (II), preparing antioxidant products; and / or (III) Preparation of antibacterial products; and / or (IV) Acid production during vegetable fermentation; and / or (V) Amino acid nitrogen produced by vegetable fermentation; and / or (VI) Increase the content of esters and alcohols with fresh and fruity flavor in vegetable fermentation; The antibacterial action is to inhibit the growth of Escherichia coli and / or inhibit the growth of Staphylococcus aureus; The ester substance is one or more of ethyl 2-hydroxypropionate, isopentyl 3-methylbutyrate-M, ethyl hexanoate-M, 3-methyl-1-butanol acetate-M or hexyl acetate-M; The alcohol substance is one or more of 1-hexanol-M, 1-hexanol-D, 3-methyl-1-butanol-M, 1-heptanol or 2-methyl-1-propanol-M.

4. The use according to claim 3, characterized in that: The anti-oxidation comprises one or more of scavenging DPPH free radicals, scavenging hydroxyl free radicals or scavenging superoxide anions.

5. The Lactobacillus pentosus according to claim 1 ( Lactobacillus pentosus ) Use of JX3 or the microbial agent as claimed in claim 2 in improving the quality of fermented vegetables; The method of improving the quality of fermented vegetables includes improving the edible safety of fermented vegetables, extending the shelf life, increasing the nutritional value, and improving the flavor.

6. Vegetable fermented product, characterized in that: comprising the Lactobacillus pentosus as claimed in claim 1 ( Lactobacillus pentosus )JX3 or obtained by fermentation of the microbial agent as described in claim 2.

7. The method for preparing a vegetable fermented product according to claim 6, characterized in that: The method comprises taking fresh vegetables, mixing them with pickling liquid, and inoculating the Lactobacillus pentosus as claimed in claim 1 ( Lactobacillus pentosus ) JX3 or the microbial agent as claimed in claim 2, fermentation to obtain the vegetable fermented product; The pickling liquid comprises white granulated sugar, salt and white wine.

8. A method for improving the quality of fermented vegetables, characterized in that: Fermented vegetables based on any of the following: (I), the Lactobacillus pentosus according to claim 1 ( Lactobacillus pentosus ) JX3; and / or (II) The microbial agent according to claim 2.

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