Lactobacillus coryniformis subsp. coryniformis and its application in improving kimchi quality

Abstract

The invention discloses a coryneform lactobacillus subspecies coryneformis and application thereof in improving the quality of kimchi. The bacteria has excellent fermentation performance, good acid resistance and salt resistance, and can degrade nitrite, thereby improving the food safety of kimchi. Inoculating the bacteria into kimchi for fermentation can greatly improve the success rate of fermentation, improve the kimchi flavor to a certain extent, and effectively improve the quality of kimchi.

Description

(I) Technical field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus coryneformis subspecies with strong fermentation performance and application thereof in improving the quality of pickles. (II) Background technology

[0002] Kimchi is a typical traditional fermented food of the Chinese nation. It is usually made from fresh vegetables such as cabbage, mustard greens, radish, etc., and is fermented by microorganisms such as lactic acid bacteria and yeast. Studies have shown that after microbial fermentation, vegetables retain their original nutrients to a great extent and can also be given a unique fermented flavor. In addition, kimchi is reported to have the effects of being beneficial to the intestinal environment, promoting human growth and metabolism, relieving intestinal inflammation, and lowering human cholesterol. After microbial fermentation, mustard greens will produce a special fresh fragrance, which can not only promote gastrointestinal digestion and increase appetite, but also, because mustard greens contain isothiocyanates, they have a special spicy taste during consumption, and are crispy and refreshing, so they have a relatively high edible value.

[0003] In addition to several common types of kimchi, there is also a special flavor of stinky kimchi in Ningbo, Taizhou and other places in Zhejiang Province - "Qianli Piaoxiang" kimchi. Because of its unique taste and flavor, it is also deeply loved by local consumers. Due to the lack of research on this kind of kimchi, its corresponding fermentation mechanism is not clear, and the specific fermentation flora is not very certain. The success rate of kimchi fermentation in 2 to 3 months of natural fermentation is only 20%. The low fermentation success rate makes it difficult to achieve large-scale production. At the same time, in the process of kimchi processing, because of its long fermentation time, a wide variety of microorganisms, complex colony structure succession, and diverse metabolic pathways, it is difficult to unify the quality and safety of kimchi, resulting in unstable product quality. Inoculation fermentation is to ferment by artificially adding purified and optimized lactic acid bacteria strains, so that it can quickly ferment into a dominant strain and inhibit the growth of spoilage bacteria, thereby solving the drawbacks of natural fermentation and obtaining a relatively stable quality product.

[0004] Microorganisms are the key factors in kimchi fermentation, among which bacteria are the key microorganisms. According to previous studies, the main bacterial phyla found in kimchi are Firmicutes and Proteobacteria, and Lactobacillus, Weissella, Leuconostoc, etc. are the main genera. Among these dominant genera, Lactobacillus has been identified as the main microbial community, which dominates the fermentation process of kimchi. At present, the microbial flora structure and fermentation strains of "Qianli Piaoxiang" kimchi are still unclear. Kimchi fermentation is a high-salt, high-acid environment, and foreign strains may not have good fermentation characteristics. Among them, the original lactic acid bacteria in kimchi have stronger environmental adaptability after long-term natural selection, and are more suitable for inoculation and fermentation of kimchi.

[0005] Therefore, using the successfully fermented Qianli Piaoxiang kimchi as the sample source and screening out lactic acid bacteria with excellent fermentation performance from it has important practical significance for improving the flavor of kimchi and solving the problem of kimchi quality fluctuations caused by differences in spices. (III) Summary of the invention

[0006] The present invention aims to provide a Lactobacillus coryneformis subspecies 523L5 and its application in improving the quality of fermented kimchi. The bacteria have excellent fermentation performance, good acid resistance and salt resistance, and can degrade nitrite to improve the food safety of kimchi. Inoculating the bacteria to ferment kimchi can greatly improve the success rate of fermentation, improve the flavor of kimchi to a certain extent, and effectively improve the quality of kimchi.

[0007] The technical solution adopted by the present invention is:

[0008] The invention provides a coryniform lactobacillus coryniformis subsp. coryniformis 523L5, which is deposited in China General Microbiological Culture Collection Center, with a deposit date of June 24, 2024, a deposit number of CGMCC No.31070, and an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The colony of the Lactobacillus coryneformis subspecies 523L5 of the present invention is round, translucent, white, moist and smooth with neat edges when observed with naked eyes. Under an optical microscope, the Lactobacillus coryneformis appears blue-purple, belongs to Gram-positive bacteria, and is rod-shaped.

[0010] The present invention also provides an application of the Lactobacillus coryneformis subspecies 523L5 in improving the quality of fermented kimchi. The raw materials of the fermented kimchi include mustard stems and Chinese mustard.

[0011] Furthermore, the application method is: wash and drain the raw materials of fresh fermented kimchi, cut into blocks of appropriate size and place in a kimchi jar, add salt water with a mass concentration of 2-4%, mix, inoculate Lactobacillus coryneformis subspecies 523L5, seal, and ferment at 25-35°C for 3 months.

[0012] Furthermore, the volume of the brine added is 1-2 mL / g, preferably 1.2 mL / g, based on the mass of the drained kimchi raw material; and the mass concentration of the brine is preferably 3%.

[0013] Furthermore, the Lactobacillus coryneformis subspecies 523L5 is inoculated in the form of a sterile physiological saline bacterial suspension of wet bacteria, with a wet bacteria concentration of 0.5-5 g / L (preferably 1 g / L), and an inoculation amount of 1-3 mL / 100 g based on the weight of the drained kimchi raw material.

[0014] Furthermore, the wet bacterial cells are prepared as follows: Lactobacillus coryneformis subspecies 523L5 is inoculated on an MRS solid culture medium plate, activated and cultured at 37°C for 48 hours, then inoculated in an MRS liquid culture medium, cultured at 37°C for 16 hours, inoculated into the MRS liquid culture medium at a volume concentration of 2%, cultured on a shaking table at 37°C until OD600nm is 1, centrifuged the culture solution at 4°C and 5000rpm for 10 minutes, removed the supernatant, repeatedly washed the bacterial cells with sterile physiological saline for 3 times, collected the wet bacterial cells, suspended them in the same volume of sterile physiological saline, and prepared a bacterial suspension as a mother starter for kimchi fermentation.

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0016] (1) The strain of the present invention can shorten the fermentation period of kimchi. The strain of the present invention screened out Lactobacillus coryneformis subspecies 523L5 from the "Qianli Piaoxiang" kimchi liquid, which has good growth performance, high acid production capacity, strong salt tolerance and acid tolerance, and can tolerate a salinity of about 7%. It can grow in MRS liquid culture medium with a pH of 4.5 and has good fermentation performance. The success rate of fermenting kimchi by inoculation of Lactobacillus coryneformis subspecies 523L5 is significantly higher than that of naturally fermented kimchi, and can significantly increase the pH value of the kimchi fermentation liquid, reduce the acidity by 42.86%, and can reach a higher degree of maturity in a shorter fermentation period, thereby shortening the fermentation period of kimchi while ensuring the sensory quality.

[0017] (2) The strain of the present invention can improve the umami taste of fermented kimchi. The umami amino acids such as aspartic acid and glutamic acid in the kimchi are increased, and most of the bitter amino acids are decreased, thereby improving the umami taste of the kimchi to a certain extent.

[0018] (3) The strain of the present invention can reduce the pungent sour taste of kimchi. The content of organic acids such as acetic acid and succinic acid in fermented kimchi increases, which can give kimchi a softer sour taste and reduce the pungent sour taste of kimchi to a certain extent.

[0019] (4) The strain of the present invention can improve the flavor of fermented kimchi. There are 51 volatile flavor substances in the kimchi fermented by inoculating Lactobacillus coryneformis subsp. coryneformis, which is 15 more than the volatile flavor substances in the kimchi fermented naturally. At the same time, there is a significant increase in the content of acids and sulfur-containing compounds, among which the content of butanol and n-hexanol occupies a major position in the alcohols. Butyric acid replaces n-hexanoic acid as the acid with the highest content, which can reduce the unpleasant fatty odor of kimchi. The increase in the content of dimethyl disulfide can also increase the onion aroma and vegetable aroma of kimchi, indicating that Lactobacillus coryneformis subsp. coryneformis can improve the flavor quality of kimchi.

[0020] In summary, the kimchi fermented by Lactobacillus coryneformis subspecies 523L5 of the present invention has a higher pH value and lower acidity, and can reach a higher degree of maturity within a shorter fermentation period. In terms of quality and flavor, the kimchi fermented by Lactobacillus coryneformis subspecies 523L5 is not inferior to the naturally fermented kimchi in terms of sensory evaluation, and has higher umami amino acids and lower bitter amino acids, higher contents of acetic acid and succinic acid with softer sourness, and no significant difference in flavor compared with the naturally fermented kimchi. The types and relative contents of volatile flavor substances (especially alcohols and acids) in the kimchi are increased, so the quality of the kimchi can be effectively improved, and the kimchi has good application potential in kimchi fermentation. (IV) Description of the drawings

[0021] Figure 1 , colony (A) and cell morphology (B) of strain 523L5.

[0022] Figure 2 , phylogenetic tree of strain 523L5.

[0023] Figure 3 , growth characteristics of strain 523L5, where (a) is the growth curve and (b) is the acid production rate curve.

[0024] Figure 4 , salt tolerance curve of strain 523L5.

[0025] Figure 5 , acid tolerance curve of strain 523L5. (V) Specific implementation methods

[0026] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0027] MRS liquid culture medium: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 5.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 mL, pH 5.7±0.2, dissolved in 1000 mL of distilled water, sterilized at 118°C for 20 min.

[0028] MRS solid culture medium: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 5.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 mL, agar 14.0 g, pH 6.5±0.2, dissolved in 1000 mL of distilled water, sterilized at 121°C for 20 min.

[0029] Nitrite degradation test medium: Add 2 mL of 5 mg / mL NaNO2 aqueous solution to 50 mL of MRS liquid medium.

[0030] Example 1: Preparation of "Qianli Piaoxiang" pickles

[0031] Wash fresh mustard stems in running tap water, drain, cut into pieces of appropriate size, boil and blanch, wait until cooled, weigh 1.2 kg of mustard stems and put them into a 3L kimchi jar, and add 1% chili and 1% garlic by mass fraction (based on the weight of mustard stems). Add 3% saline solution to cover the mustard stems, seal, and ferment at room temperature (25-30°C) for 3 months. After fermentation, trained panel members will score the color, texture, aroma and taste of the fermented kimchi according to Table 1. The kimchi with a total score greater than 70 is defined as excellent fermented kimchi and used as the sample for the next isolation of bacterial species.

[0032] Table 1 Scoring criteria

[0033]

[0034] Example 2: Screening and identification of Lactobacillus coryneformis subsp. coryneformis 523L5

[0035] 1. Initial screening:

[0036] The "Qianli Piaoxiang" pickle liquid selected in Example 1 was diluted 10 times with sterile physiological saline, and 10 0 (i.e. stock solution), 10 -3 , 10 -4 100 μL of each of the two dilution samples was evenly spread on an MRS solid culture medium plate using a sterile spreader rod. After culturing at 37°C for 48 hours, colonies with calcium-dissolving circles and different morphologies were picked and repeatedly streaked on the MRS solid culture medium plate. Single colonies were finally screened and recorded as strains 523L1, 523L5, and 523L12. The purified strains were frozen in 20% glycerol.

[0037] 2. Rescreening:

[0038] Use a sterile inoculation loop to pick up a loop of the bacterial solution frozen in the glycerol tube in step 1 and streak it on an MRS solid culture medium plate. After culturing at 37°C for 48 hours, pick up a loop of single colonies and put them into a test tube containing MRS liquid culture medium. Mix them evenly and culture them at 37°C for 24 hours. Measure the bacterial density (OD600nm) and pH value of the samples at 12 hours and 24 hours, respectively. The results are shown in Table 2.

[0039] Take 2 mL of activated culture solution (OD600nm=1) and inoculate it into 50 mL of sterilized nitrite degradation test medium. After culturing at 37°C for 70 h, the nitrite content was determined by the naphthylethylenediamine hydrochloride method, and the nitrite degradation rate of each strain was calculated. The results are shown in Table 2.

[0040] The activated strains were respectively spread on MRS solid medium containing different mass concentrations (3%, 4%, 5%) of NaCl and 2% calcium carbonate and cultured at 37°C for 48 h. The appearance of calcium-dissolving rings around the colonies indicated that the strains were salt-tolerant and could tolerate NaCl concentrations. The results are shown in Table 2.

[0041] Table 2 Fermentation performance test results of each strain

[0042]

[0043] Among them, strain 523L5 has good growth ability, strong acid production ability, salt tolerance and strong nitrite degradation ability.

[0044] 3. Identification of strain 523L5

[0045] Morphological identification: 100 μL of the bacterial solution frozen in the glycerol tube was spread on the MRS solid medium plate, and then cultured at 37°C for 48 hours. The colony morphology was observed with the naked eye, and the cell morphology was observed under an optical microscope after Gram staining. Figure 1 As shown in the figure, the colonies are round, translucent, white, moist and smooth with neat edges when observed with the naked eye. Under an optical microscope, the strain appears blue-purple and is a Gram-positive bacterium with a rod shape.

[0046] Molecular identification: The screened strains were inoculated into a test tube containing MRS liquid culture medium, cultured in a shaking incubator at 37°C for 16 hours, and the bacteria were harvested. DNA was extracted using a bacterial genomic DNA extraction kit, and the DNA was used as a template to amplify the full-length sequence of 16SrDNA using bacterial universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'). After the PCR product was tested and qualified, it was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, BLAST was used for homology comparison, and MEGA10.0 was used to construct a phylogenetic tree, such as Figure 2 shown.

[0047] The 16SrDNA sequence (SEQ ID NO.1) was imported into NCBI for BLAST homology comparison, and the results showed that the strain 523L5 had a similarity of 100% with the coryniform lactobacillus coryniformis subsp. coryniformis. Combined with the morphological characteristics, the strain 523L5 was identified as the coryniform lactobacillus coryniformis subsp. Coryniformis 523L5, which was deposited in the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 31070 and a deposit date of June 24, 2024.

[0048] Example 3: Fermentation performance test of strain 523L5

[0049] 1. Growth curve determination

[0050] Growth ability is an important parameter for evaluating fermentation strains, and the growth curve can provide an intuitive understanding of the growth pattern of bacteria.

[0051] The strain 523L5 was inoculated on MRS solid culture medium plates, activated and cultured at 37°C for 48 hours, then inoculated into MRS liquid culture medium, cultured at 37°C for 16 hours, and then inoculated into MRS liquid culture medium at a volume concentration of 2%, and cultured at 37°C in a shaking incubator for 24 hours. During the culture process, samples were taken every 2 hours to measure the bacterial density (OD600nm) of the bacterial solution. The results are shown in Figure 3 In a, we can see that from 0 to 4 hours, strain 523L5 is in the growth lag phase and grows slowly; after 6 hours, it enters the logarithmic growth phase and the number of bacteria increases rapidly; after 14 hours, it grows slowly and begins to enter the stable phase. Therefore, Lactobacillus coryneformis subspecies 523L5 has good growth ability.

[0052] 2. Determination of acid production rate

[0053] Step 1: During the 24-hour incubation at 37°C, take samples every 4 hours to measure the pH value of the bacterial solution. Figure 3 As shown in b, it can be seen that from 0 to 4 hours, the strain 523L5 is in the growth adaptation period, and the pH drops slowly; from 4 to 16 hours, the strain adapts to the culture environment and grows rapidly in the logarithmic growth period, and the pH drops rapidly; after 16 hours, the bacteria grow slowly and begin to enter the stable period, and the pH drops slowly and tends to stabilize.

[0054] 3. Salt tolerance

[0055] The strain 523L5 was inoculated on an MRS solid culture medium plate, activated and cultured at 37°C for 48 hours, then inoculated in an MRS liquid culture medium, cultured at 37°C for 16 hours, and then inoculated into an MRS liquid culture medium with added mass fractions of 0%, 2%, 4%, 6%, 8%, 10%, 12%, and 14% NaCl (i.e., salt concentration) at a volume concentration of 2%, cultured at 37°C for 24 hours, and the OD600nm value of the bacterial solution was measured. The results are as follows: Figure 4 shown.

[0056] Depend on Figure 4 It can be seen that when the salt concentration is 2%-4%, the strain 523L5 is less inhibited; when the salt concentration is increased to 6%, the OD600nm is around 0.8; and when the salt concentration is increased to more than 8%, the strain 523L5 can hardly grow.

[0057] The strain 523L5 was determined to have a certain salt tolerance and could tolerate a salt concentration of about 7%.

[0058] 4. Acid resistance

[0059] The strain 523L5 was inoculated on an MRS solid culture medium plate, activated and cultured at 37°C for 48 hours, then inoculated in an MRS liquid culture medium, cultured at 37°C for 16 hours, and then inoculated into MRS liquid culture medium at pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 at a volume concentration of 2%. After incubation at 37°C in a shaking incubator for 24 hours, the bacterial density (OD600nm) of each culture solution was measured. The results are shown in FIG. Figure 5 shown.

[0060] Depend on Figure 5 It can be seen that when the pH is less than 4.0, strain 523L5 can hardly grow normally; when the pH rises to 4.5, the OD600nm is around 0.6; when the pH rises to 5, strain 523L5 is less inhibited; when the pH rises to above 5.5, strain 523L5 can grow normally.

[0061] Example 4: Wet cells of strain 523L5

[0062] The strain 523L5 was inoculated on an MRS solid culture medium plate, activated and cultured at 37°C for 48 hours, then inoculated in an MRS liquid culture medium, cultured at 37°C for 16 hours, and then inoculated into an MRS liquid culture medium at a volume concentration of 2%, cultured in a shaking incubator at 37°C until the OD600nm was 1, the culture solution was centrifuged at 4°C and 5000rpm for 10 minutes, the supernatant was discarded, the bacteria were repeatedly washed 3 times with sterile saline, and the wet bacteria were collected. Finally, they were suspended in the same volume of sterile saline to prepare a bacterial suspension as a mother starter for kimchi fermentation.

[0063] Example 5: Application of Lactobacillus coryniformis subsp. coryniformis 523L5 in mustard stem fermentation

[0064] 1. Preparation of kimchi: Wash and drain fresh mustard stems, cut into pieces of appropriate size and place in a kimchi jar, add 3% saline solution in an amount of 1 g:1.2 mL, mix, inoculate the wet bacterial suspension of strain 523L5 prepared by the method of Example 4 (wet bacterial concentration 1 g / L) at a ratio of 100 g mustard weight to bacterial suspension volume 2 mL as the test group, and use natural fermentation without inoculation as the blank control group. After sealing, ferment at 25-35° C. for 3 months.

[0065] 2. Determination of the pH value of kimchi: Take the finished kimchi fermentation liquid and measure it directly using a pH meter. The pH value of kimchi inoculated with Lactobacillus coryneformis subsp. coryneformis 523L5 was 5.57 after the fermentation, which is higher than that of naturally fermented kimchi (pH = 4.78). Generally speaking, kimchi is considered mature when the pH value of the fermentation liquid is less than 4.0, but a small amount of kimchi may have a pH greater than 4.0 due to different production processes and fermentation microorganisms. Compared with ordinary kimchi, the pH value of Qianli Piaoxiang kimchi will be higher, and the sour taste will be weaker or even non-existent.

[0066] 3. Determination of total acid in kimchi: Accurately measure 10mL of fermentation liquid and add it to a 100mL volumetric flask, add water to the mark, and mix well. Filter with filter paper or absorbent cotton, and set aside the filtrate. Accurately pipette 25mL of filtrate into a 100mL conical flask, add a few drops of phenolphthalein indicator, and titrate with 0.1mol / L sodium hydroxide aqueous solution until the initial pink color appears. The end point is that the color does not fade for 0.5min, and record the titration volume of sodium hydroxide aqueous solution consumed. The total acid content in the sample is calculated as lactic acid, in grams per liter (g / L).

[0067] The total acid content of kimchi inoculated with Lactobacillus coryneformis subsp. coryneformis 523L5 was 1.84 g / L after fermentation, which was 42.86% lower than that of the natural fermentation group (3.22 g / L).

[0068] 4. Determination of organic acid content: 1 mL of kimchi fermentation liquid was transferred to a centrifuge tube, centrifuged at 10000 r / min for 15 min, filtered through a 0.22 μm membrane, and the filtrate was tested for organic acid content by high performance liquid chromatography. The results are shown in Table 4.

[0069] Chromatographic column: Ultimate AQ-C18 (5μm, 250mm×4.6mm), column temperature: 30℃; mobile phase A is 20mM sodium dihydrogen phosphate aqueous solution (adjusted with phosphoric acid to pH=2.60) and methanol in a volume ratio of 99:1, mobile phase B is methanol, flow rate is 0.7mL / min, injection volume is 10μL, UV detection wavelength is 210nm. The gradient elution program of organic acids is shown in Table 3.

[0070] Table 3 Gradient elution program of organic acids

[0071]

[0072] Table 4 Types and contents of organic acids in kimchi

[0073]

[0074] Sourness is the main taste of kimchi, which mainly comes from the rich organic acids produced by kimchi fermentation. Whether it is inoculated fermented kimchi or naturally fermented kimchi, the main organic acids produced are propionic acid, acetic acid, formic acid and succinic acid, but the content is different. The content of propionic acid and formic acid in naturally fermented kimchi is higher than that in inoculated fermented kimchi, while the content of acetic acid and succinic acid in inoculated fermented kimchi is higher. Propionic acid has a pungent sour taste at room temperature, while the sourness of acetic acid and succinic acid is relatively mild, so it can give kimchi a softer sour taste. Except for the four more organic acids, the content of other organic acids in naturally fermented kimchi is slightly higher than that in inoculated fermented kimchi. Therefore, in terms of organic acids, inoculated fermented kimchi can achieve a sour taste close to that of natural fermentation.

[0075] 5. Determination of amino acid content: 1 mL of fermentation broth was centrifuged at 12000r for 10 min at 4°C, and then filtered with a 0.22 μm filter membrane. The filtrate was analyzed using an automatic amino acid analyzer to determine the free amino acid content. The free amino acid content was expressed in mg / L, and the results are shown in Table 5.

[0076] Table 5 Types and contents of amino acids in kimchi

[0077]

[0078] The types and contents of free amino acids in kimchi directly affect the taste of kimchi. Amino acids are divided into four categories: umami (aspartic acid and glutamic acid), sweet (alanine, threonine, glycine and serine), bitter (arginine, histidine, isoleucine, leucine, methionine, phenylalanine, valine, proline and lysine) and tasteless (cysteine ​​and tyrosine). As shown in Table 5, the content of aspartic acid and glutamic acid in kimchi fermented by Lactobacillus coryneformis subsp. 523L5 is higher than that in naturally fermented kimchi, and aspartic acid and glutamic acid are the main sources of the umami taste of kimchi. Inoculation of Lactobacillus coryneformis subsp. 523L5 helps to improve the umami taste of kimchi. For example, the content of bitter amino acids such as isoleucine, leucine, methionine and valine in kimchi fermented by Lactobacillus coryneformis subsp. 523L5 has decreased, which is also conducive to improving the overall flavor of kimchi.

[0079] 6. Sensory evaluation: A sensory evaluation panel consisting of well-trained panelists from kimchi factories scored the color, texture, aroma and taste of fermented kimchi with different processing according to the sensory scoring criteria in Table 6. Due to the different kimchi production processes and fermentation microorganisms, the sensory scoring criteria are significantly different from those of most kimchi.

[0080] Table 6 Sensory scoring standards

[0081]

[0082]

[0083] The texture and color of kimchi inoculated with Lactobacillus coryniformis subsp. coryniformis 523L5 were not significantly different from those of naturally fermented kimchi, and the taste and aroma scores were slightly lower than those of naturally fermented kimchi, but there was no significant difference. This shows that although Lactobacillus coryniformis subsp. coryniformis 523L5 cannot significantly improve the sensory quality of kimchi, it can also ensure a sensory quality similar to that of naturally fermented kimchi.

[0084] 7. Determination of volatile flavor substances in kimchi: Take 2 g of kimchi liquid and add it into a 20 mL headspace bottle, and add 0.72 g of NaCl, use 0.6 mg / mL methyl octanoate aqueous solution as internal standard, add 100 μL of 0.6 mg / mL methyl octanoate aqueous solution to each sample, and use GC-MS system to detect volatile flavor substances. First, the DVB-PDMS fiber head is adsorbed at 40°C in the headspace bottle for 30 min, and then inserted into the GC-MS injection port for desorption at 250°C for 5 min, split ratio 40:1, carrier gas: He, flow rate: 1 mL / min.

[0085] A GC-MS system equipped with a DB-5MS column (30m*0.25mm*0.25μm) was used to separate and detect volatiles. The temperature program was as follows: 40℃ (1min) at 4℃ / min to 76℃ (hold for 1min), at 2℃ / min to 94℃ (hold for 1min), at 4℃ / min to 114℃ (hold for 1min), at 5℃ / min to 150℃ (hold for 1min), at 10℃ / min to 230℃ (hold for 5min). The injection temperature was 250℃, and the mass spectrometry conditions were full scan acquisition mode, electron impact ion source (EI), electron energy 70eV, ion source temperature 200℃, quadrupole temperature 150℃, and mass scanning range m / z 29-650.

[0086] The results of determination of volatile flavor substances in kimchi fermented by Lactobacillus coryniformis subsp. coryniformis 523L5 are shown in Table 7.

[0087] Table 7 Types and relative contents of volatile components in kimchi

[0088]

[0089]

[0090]

[0091] The results showed that 51 volatile substances were detected in the fermented kimchi inoculated with Lactobacillus coryneformis subsp. 523L5, mainly alcohols, acids and sulfur-containing compounds. There were 36 volatile substances in the naturally fermented kimchi, mainly esters, sulfur-containing compounds and alcohols. After the kimchi was fermented with the inoculated strain Lactobacillus coryneformis subsp. 523L5, the relative content of alcohol substances was lower than that of natural fermentation, and the content of butanol and n-hexanol was relatively high. At the same time, the types of alcohol substances were more abundant, which can give the kimchi aromas such as wine, flowers, and roses. The acid substances in the inoculated fermented kimchi were higher than those in the naturally fermented kimchi, and the types of acids were more abundant, such as pentanoic acid, octanoic acid, heptanoic acid, etc. The main acid substances also changed from n-hexanoic acid to butyric acid, which provided a more complex sour taste for the kimchi while also reducing the unpleasant fat smell of the kimchi. Sulfur compounds play an important role in the flavor of fermented vegetables and are the main source of kimchi flavor. Inoculation fermentation can increase the content of sulfur compounds, especially dimethyl disulfide, which can provide kimchi with onion and vegetable aromas. The content of esters decreased after inoculation fermentation, but many new esters such as hexanoate and octanoate also appeared. It may be because after inoculation fermentation, the species and number of bacteria in the kimchi liquid changed, resulting in the inability of some ester-producing bacteria to grow, and most volatile substances remained on precursor substances such as alcohols and acids.

[0092] Example 6: Application of Lactobacillus coryneformis subspecies 523L5 in Chinese vegetable fermentation

[0093] 1. Preparation of kimchi: First, wash and drain fresh Chinese cabbage, cut into small pieces of 7-8 cm long, boil and blanch for one minute, then remove, add 3% saline solution according to 1g:2mL, add wet bacterial suspension of strain 523L5 prepared by the method of Example 4 according to the inoculation amount of 100g:2mL of Chinese cabbage mass and bacterial suspension volume (wet bacterial concentration 1g / L). Cover and seal with plastic wrap, take natural fermentation without inoculation as blank control group, and ferment naturally for 3 months at room temperature.

[0094] 2. Determination of pH and acidity of kimchi: The pH and acidity were detected using the method of Example 5.

[0095] Table 8 Kimchi pH and acidity measurement results

[0096]

[0097] The pH value of kimchi inoculated with Lactobacillus coryniformis subsp. 523L5 after fermentation was much higher than that of kimchi fermented naturally, while the total acid content was just the opposite. At the same time, the pH value and total acid content of kimchi inoculated with Lactobacillus coryniformis subsp. 523L5 in the first month of fermentation were similar to those of kimchi fermented naturally in the third month, and in the second month of fermentation, the pH and total acid content were similar to those of the excellent "Qianli Piaoxiang" kimchi. It can be seen that inoculation with Lactobacillus coryniformis subsp. 523L5 can greatly accelerate the fermentation process of kimchi and shorten the fermentation cycle.

Claims

1. A coryneform lactobacillus subsp. coryniformis 523L5, deposited in China General Microbiological Culture Collection Center, with a deposit date of June 24, 2024, a deposit number of CGMCC No.31070, and an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Lactobacillus coryniformis subspecies 523L5 described in claim 1 in improving the quality of fermented kimchi.

3. The use according to claim 2, characterized in that The raw materials of the fermented kimchi include mustard stems and Chinese mustard.

4. The use according to claim 2, characterized in that The application method comprises the following steps: washing and draining the raw materials of fresh fermented kimchi, cutting them into blocks of appropriate size and placing them in a kimchi jar, adding salt water with a mass concentration of 2-4%, mixing, inoculating Lactobacillus coryneformis subspecies 523L5, sealing the jar, and fermenting the jar at 25-35°C for 3 months.

5. The use according to claim 4, characterized in that The volume of brine added is 1-2 mL / g based on the mass of the drained kimchi raw material.

6. The use according to claim 4, characterized in that The Lactobacillus coryneformis subspecies 523L5 is inoculated in the form of a sterile physiological saline bacterial suspension of wet bacteria, the wet bacteria concentration is 0.5-5g / L, and the inoculation amount is 1-3mL / 100g based on the weight of the drained kimchi raw material.

7. The use according to claim 6, characterized in that The wet bacterial body is prepared as follows: Lactobacillus coryneformis subspecies 523L5 is inoculated on an MRS solid culture medium plate, activated and cultured at 37°C for 48 hours, then inoculated in an MRS liquid culture medium, cultured at 37°C for 16 hours, inoculated into the MRS liquid culture medium at a volume concentration of 2%, cultured on a shaking table at 37°C until OD600nm is 1, centrifuged the culture solution at 4°C and 5000rpm for 10 minutes, removed the supernatant, repeatedly washed the bacterial body with sterile physiological saline for 3 times, collected the wet bacterial body, and suspended it in the same volume of sterile physiological saline.

Images