A strain of Lactobacillus plantarum C-17 and its application

Abstract

This invention discloses a strain of Lactobacillus plantarum ( Lactobacillus plantarum This invention relates to Lactobacillus plantarum C-17 and its applications, belonging to the field of microbial and fermentation technology. This Lactobacillus plantarum strain was deposited on December 30, 2024, at the China Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33230. The Lactobacillus plantarum C-17 provided by this invention can be used for the fermentation of shallots, and the nutritional indicators of the fermented shallots are significantly improved. This strain can be used to prepare fermentation agents for the pickling and fermentation of pickled vegetables, with particularly significant effects in the pickling and fermentation of shallots.

Description

Technical Field

[0001] This invention relates to the field of microbiology and fermentation technology, specifically to a strain of *Lactobacillus plantarum*. Lactobacillus plantarum C-17 and its applications. Background Technology

[0002] Mongolian leeks ( Allium mongolicum Regel (AMR) is a perennial herbaceous plant belonging to the genus Allium in the family Liliaceae, also known as sand onion. It grows in deserts, sandy areas, or arid slopes at altitudes of 800-2800 meters. Wild AMR is most widely distributed in Inner Mongolia, China. Both the leaves and flowers of AMR are edible and it is a traditional wild edible plant of the Mongolian people in China. AMR contains volatile sulfur compounds, steroidal compounds, saponins, flavonoids, polysaccharides, and other substances with anti-tumor, antibacterial, anti-inflammatory, antiviral, anti-allergic, antioxidant, and cardiovascular disease prevention effects. AMR has a high content of unsaturated fatty acids. Its protein is a complete protein, with the ratio of essential amino acids to total amino acids conforming to the ideal protein model proposed by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO). AMR contains essential minerals, with high potassium and iron content; its iron content is 7.7 times that of celery and 5.0 times that of spinach (iron-rich vegetables), respectively. Compared with 80 other vegetables, AMR has relatively high levels of Ca, Mg, and P. Because AMR contains a complete range of nutrients, including higher levels of biominerals, essential trace elements, and amino acids than ordinary vegetables, it has extremely high nutritional value.

[0003] In recent years, Mongolian leeks, as a green wild vegetable with both medicinal and edible uses, have been favored by Chinese researchers. According to academic literature, existing research on sand leeks is based on the identification of their flavor, nutritional components, and bioactive components. For example, Wang Junkui (Wang Junkui, Developing Sand Leek Sauce Products by Combining Sensory Evaluation and Fingerprinting Technology) conducted a relatively detailed preliminary study on the aroma of Mongolian leeks and detected 24 volatile compounds using headspace solid-phase microextraction technology. Quantitative descriptive analysis of homemade sand leek sauce products revealed that saltiness, shrimp-like flavor, and umami flavor are important research directions for the production of sand leeks as condiments. Siqinbatuer (Siqinbatuer, Liu Xinmin, Nutritional Components and Ethnobotany of Mongolian Leeks) analyzed the amino acid content and mineral nutrition of Mongolian leeks. Hou Yanru (Hou Yanru, Genetic Diversity and Flavor Quality Analysis of Mongolian Leeks from Different Populations) conducted a comparative study on the total free amino acids, sweet free amino acids, umami free amino acids, and volatile substances of Mongolian leeks from four different populations. On the other hand, functional identification of the bioactive components in Mongolian leeks is being conducted. Especially in recent years, numerous scholars have gained a deeper understanding of the bioactive components of Mongolian leeks. For example, Mongolian leek flavonoids have been gradually proven to improve gastrointestinal function, alleviating constipation by maintaining colonic water content and increasing intestinal transit capacity. Water extracts of Mongolian leeks, with high phenolic and flavonoid content, possess high antioxidant capacity and good inhibitory potential against important enzymes related to obesity and hypertension, thus potentially serving as functional or nutritional foods for the prevention and treatment of obesity and hypertension. Furthermore, many scholars have confirmed that polysaccharides from wild onions have immunomodulatory, antioxidant, and antitumor effects. However, no research on lactic acid bacteria fermentation of wild onions has been reported domestically or internationally.

[0004] Freshly picked Mongolian leeks are difficult to store, typically wilting and becoming sticky within 48 hours. Many local herders and residents usually freeze them. However, commercially available pickled leeks or leek sauces often contain large amounts of salt, spices, and various additives, posing a potential threat to consumer health. Based on my observations of the lifestyle of Mongolian residents in Inner Mongolia over the past few years, I've found that many locals enjoy eating leeks pickled in "Shari-Usu" (Mongolian for the sour pulp left over from making yogurt or brewing milk wine). This type of pickled leeks has a unique flavor and is considered a high-class dish for entertaining guests, as it is also resistant to spoilage and has a long shelf life. It is well known that lactic acid bacteria are the dominant microorganisms in vegetable fermentation, and they are generally recognized as safe (GRAS) microorganisms worldwide, thus being widely used in food fermentation globally. In addition, lactic acid bacteria inoculation fermentation can significantly shorten fermentation time, quickly bring the fermentation environment into a low-acid environment, inhibit the growth of miscellaneous bacteria, reduce the risk of fermentation failure, and reduce the generation of harmful substances (nitrite, bacterial toxins, and fungal toxins, etc.), thereby improving product quality. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Lactobacillus plantarum that can be used for fermenting shallots and to improve the nutritional value of fermented shallots.

[0006] To achieve the above objectives, the present invention provides a strain of *Lactobacillus plantarum* (…). Lactobacillus plantarum Lactobacillus plantarum C-17 was deposited at the China Culture Collection Center for Microbial Cultures on December 30, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33230. This Lactobacillus plantarum C-17 can be used in fermentation, especially for the fermentation of shallots.

[0007] The Lactobacillus plantarum C-17 provided by this invention can be used in the field of shallot fermentation.

[0008] The Lactobacillus plantarum C-17 provided by this invention can extend the shelf life of fermented shallots by increasing the citric acid content; and can improve the flavor of fermented shallots by increasing the glutamic acid content.

[0009] The *Lactobacillus plantarum* C-17 provided by this invention can be used to increase the content of tyrosine and kynurenine in fermented shallots, and to increase the content of beneficial fatty acids in fermented shallots, including propionic acid, butyric acid and valeric acid.

[0010] The present invention also provides a fermentation agent comprising the above-mentioned Lactobacillus plantarum C-17.

[0011] Preferably, the concentration of Lactobacillus plantarum C-17 in the above-mentioned fermentation agent is 1×10⁻⁶. 7 CFU / mL.

[0012] The fermentation agent provided by this invention can be used in the field of fermented shallots, including increasing citric acid in fermented shallots to extend their shelf life; increasing glutamic acid in fermented shallots to improve their flavor; increasing the content of tyrosine and kynurenine in fermented shallots; and / or increasing the content of beneficial fatty acids in fermented shallots.

[0013] This invention also provides a pickling method to improve the nutritional value of sand onions, comprising the following steps:

[0014] 1) Culterate the above-mentioned Lactobacillus plantarum C-17 to obtain a bacterial suspension;

[0015] 2) Inoculate the bacterial suspension into a container containing sand onions;

[0016] 3) Seal and ferment to obtain fermented shallots;

[0017] Its nutritional value is reflected in the beneficial indicators of wild onions, which include tyrosine, kynurenine, glutamic acid, propionic acid, butyric acid, and valeric acid.

[0018] Preferably, in the pickling method provided by the present invention, the bacterial cell concentration of the bacterial suspension is 1×10⁻⁶. 7 CFU / mL; the inoculum size was 1% of the weight of the shallots; sealed fermentation was carried out at room temperature for 7 days; the salt concentration in the sealed fermentation system was 3%.

[0019] The present invention has the following advantages:

[0020] This invention screened and obtained a strain of *Lactobacillus plantarum* C-17. Experimental comparisons showed that adding strain C-17 to the fermentation of wild onions significantly increased the content of glutamic acid, tyrosine, kynurenine, tartaric acid, phenyllactic acid, gluconic acid, and citric acid in the fermented onions. Compared with natural onion fermentation without fermentation bacteria, fermentation with C-17 at a 3% salt concentration particularly increased the content of tyrosine, citric acid, and kynurenine. Simultaneously, after fermentation with C-17, a large amount of glutamic acid was generated in the fermentation system, significantly increasing the glutamic acid content. After fermentation with this strain, the content of beneficial indicators in wild onions was significantly increased, indicating that this strain has significant application prospects in onion fermentation. Attached Figure Description

[0021] Figure 1 The results show the index determination of sand onion before and after fermentation with different lactic acid bacteria in a saline environment in this invention.

[0022] Figure 2 The results show the index determination of *Lactobacillus plantarum* C-17 before and after fermentation of *Allium mongolicum* at different salt concentrations in this invention.

[0023] Figure 3 A phylogenetic tree constructed based on the 16S rDNA sequence of C-17.

[0024] Figure 4 Image showing C-17 cultured on an MRS plate.

[0025] Figure 5 This is a microscopic morphology diagram of C-17 on an MRS plate. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0028] Example 1: Isolation and Identification of Strains

[0029] 1. Strains Isolation

[0030] ① Sample Collection. Samples were collected from fermented foods such as kimchi, rice wine, sauerkraut, dried plum vegetables, wine, fermented bean curd, and fermented black beans. Approximately 5.0g of sample was placed directly into a sterile sampling tube using a sterile spoon, sealed with sealing film, and labeled with a sample number. The collected samples were kept at a low temperature and quickly brought back to the laboratory for isolation and preservation of lactic acid bacteria as soon as possible.

[0031] ② Viable count of lactic acid bacteria in the samples. The lactic acid bacteria in the samples were counted using the pour plate method. The samples were diluted 10-fold with 0.85% sterile physiological saline. 1 mL of each dilution was placed in a sterile petri dish, sterile MRS medium was poured in and mixed thoroughly, and the dishes were incubated anaerobically at 37°C for 48-72 hours before counting.

[0032] ③ Isolation and purification of lactic acid bacteria in the samples. The sample dilutions of the same dilution factor as in the previous step were thoroughly mixed using a shaker. Then, 200 mL of the dilution was pipetted onto sterile solid MRS and M17 agar plates containing bacteriostatic agents and 1.5% CaCO3, respectively, and evenly spread. The plates were then anaerobically incubated at 37°C for 48 hours. Single colonies with different characteristics were streaked 2-3 times onto the corresponding solid MRS or M17 agar plates using a sterile inoculation needle for purification. Finally, Gram-positive, catalase-negative, non-spore-forming cocci, rods, and streptococci were tentatively identified as lactic acid bacteria.

[0033] ④ Preservation of Lactic Acid Bacteria. After activating the pure culture tentatively identified as lactic acid bacteria, incubate at 37℃ for 24 hours. Centrifuge at 3000g for 10 minutes to precipitate the bacteria. Discard the supernatant and add 5 mL of sterile PBS buffer (0.02% KH₂PO₄, 0.115% Na₂HPO₄, 0.8% NaCl) to the bacterial precipitate and wash thoroughly. Repeat the centrifugation, washing, and freezing process twice. Then, add an appropriate amount of lactic acid bacteria preservative (10 g skim milk powder, 90 mL distilled water, 0.1 g monosodium glutamate, sterilized at 121℃ for 7 minutes, rapidly cooled at 98℃) to the bacterial precipitate. After mixing thoroughly, aliquot evenly into 1.5 mL sterile cryovials using a sterile Pasteur needle and store at -80℃.

[0034] ⑤ Identification of lactic acid bacteria isolates. DNA was extracted from the isolated single colonies (see Table 1) and the 16S rRNA gene sequence was determined. The sequencing results were then analyzed using BLAST on the NCBI website to match the strains with the highest similarity, thus preliminarily determining the classification of all screened strains.

[0035] 2. Initial screening of lactic acid bacteria for pickled shallots

[0036] The isolated bacterial colonies were inoculated into sand onions pickled using the above method, with pH as the screening index. Since the whey solution is acidic and the initial pH varies between batches, the pH of the whey solution was adjusted to above 4.5 with baking soda before pickling the sand onions, and the whey solution was sterilized at 95℃ for 5 minutes. The pH results of the sand onions after pickling for each bacterial strain are shown in Table 1 below:

[0037] Table 1. pH changes after pickling sand onions with different strains

[0038]

[0039] As can be seen from the results in Table 1 above, Lactobacillus pentosus C-1 Lactobacillus plantarum C-4 Lactobacillus plantarum C-17 Lactobacillus pentosus J-2, Lactobacilluscasei J-16 produced acid rapidly in the sand onion environment and was identified as the initial screening strain. Lactobacillus plantarum PS-8 and Lactobacilluscasei Although Zhang's pH did not decrease significantly, due to Lactobacillus plantarum PS-8 and Lactobacilluscasei Zhang refers to two commercially available probiotic strains whose probiotic effects have been scientifically proven, and therefore they were also adopted in the experiment of pickling sand onions.

[0040] 3. Making fermented Mongolian leeks

[0041] (1) Traditional herders' method of pickling sand onions

[0042] The wild onions and whey used in this study were sourced from herder families in Saihantala Town, Sunite Right Banner, Xilingol League, Inner Mongolia Autonomous Region. Following local herders' traditional methods, fresh wild onions were washed and drained. A layer of onions was then layered in a container, covered with a thin layer of salt, until the container was nearly full. The contents were then compacted, and whey was poured in. The container was sealed and left to marinate at room temperature for at least 7 days before consumption.

[0043] (2) A new method for pickling with lactic acid bacteria

[0044] The selected lactic acid bacteria were inoculated into MRS liquid medium and incubated statically at 37°C for 24 h. After centrifugation at 5000 r / min for 2 min, the bacterial cells were washed 2-3 times with sterile physiological saline. Then, sterile physiological saline was added to adjust the bacterial concentration to 1×10⁻⁶. 7 CFU / mL was added to Mongolian chives using the same process as the natural fermentation group (the whey had been pre-sterilized at 95℃ for 5 minutes), and fermented at room temperature in a sealed container for 7 days. Wild Mongolian chives (non-fermented), commercially available lactic acid bacteria agents, and natural fermentation without added lactic acid bacteria were used as controls.

[0045] 4. Results

[0046] The samples selected during the above process Lactobacillus plantarum C-1 Lactobacillus plantarum C-4 Lactobacillus plantarum C-17 Lactobacillus pentosus J-2, Lactobacilluscasei J-6, along with commercially available probiotics PS-8 and Zhang, were added to Mongolian leeks prepared using traditional herding methods. The indicators before and after lactic acid bacteria fermentation were measured and compared, including indicators such as Zn, propionic acid, butyric acid, valeric acid, oxalic acid, acetylpropionic acid, kynurenine, itaconic acid, and citric acid. The results show that, compared with fresh shallots, fermented shallots have higher levels of zinc, short-chain fatty acids, glutamic acid and tyrosine, natural antibacterial and antioxidant substances such as phenyllactic acid, tartaric acid and citric acid, vitamins B2 and B6, acetylpropionic acid (biomass raw material), gluconic acid (a natural high-value organic acid that reduces the chance of chemical carcinogenesis, lowers serum cholesterol, and polymer synthesis, and is considered "one of the most valuable biorefining products"), kynurenine (related to mood and nerve regulation), and lower oxalic acid content (oxalic acid can reduce the bioavailability of mineral elements and easily forms calcium oxalate in the human body, leading to kidney stones; therefore, oxalic acid is often considered an antagonist to the absorption and utilization of mineral elements), as well as lower salt content. The research and development of this fermented product yielded a large amount of experimental data and accumulated extensive experimental experience, providing a theoretical basis for subsequent applications.

[0047] Considering that the traditional fermentation environment for wild onions by herders is whey, which is not conducive to commercial application, the whey in the fermentation system was replaced with sterilized 3% saline solution. Lactobacillus plantarum C-1 Lactobacillus plantarum C-4 Lactobacillus plantarum C-17 Lactobacillus pentosus J-2, J-6 repeated the above experiment, using the natural fermentation treatment without the addition of lactic acid bacteria as a blank control. This further verified whether the changes in the above beneficial indicators were solely caused by lactic acid bacteria or influenced by whey. The results showed that, disregarding the influence of whey, Lactobacilluscasei Strain C-17 significantly increased the content of glutamic acid, tyrosine, kynurenine, tartaric acid, phenyllactic acid, gluconic acid, and citric acid in fermented shallots. Compared with natural shallot fermentation without fermentation bacteria, C-17 fermentation at a 3% salt concentration particularly increased the content of tyrosine, citric acid, and kynurenine. Furthermore, C-17 fermentation generated a large amount of glutamic acid in the fermentation system, significantly increasing the glutamic acid content. Specific measurement results can be found in [link to specific results]. Lactobacillus plantarum As shown in the figure, Figures A, B, and C represent the measurement comparison results of different indicators.

[0048] Commercially available pickled shallots typically contain around 6% salt. However, because lactic acid bacteria produce natural antibacterial substances such as phenyllactic acid, tartaric acid, and citric acid, the salt content of lactic acid bacteria-pickled shallots is significantly lower compared to commercially available products. This study further investigates... Figure 1 The salt concentrations (1%, 2%, 3%, 4%, and 5%) for C-17 pickled shallots were validated, with results showing that 3% salt concentration yielded the best results, followed by 4%. It can be seen that under a 3% salt concentration fermentation system, the content of tyrosine, citric acid, and kynurenine in the fermentation system of shallots after C-17 fermentation was significantly increased, and glutamic acid was also generated during fermentation. Simultaneously, C-17 fermentation also increased the content of beneficial short-chain fatty acids, including propionic acid, butyric acid, and valeric acid, with a particularly significant increase in propionic acid. This indicates that shallots fermented with C-17 have higher nutritional value and health benefits. Furthermore, the significant increase in glutamic acid and citric acid content not only improves the flavor of fermented shallots but also effectively extends their shelf life. The results of multiple indicators after fermentation at a 3% salt concentration are shown below. Lactobacillus plantarum As shown in the figure, Figures A, B, and C represent the measurement comparison results of different indicators.

[0049] Based on the above results, select Figure 2 C-17 is considered the optimal lactic acid bacteria for fermenting shallots.

[0050] The physiological and biochemical indicators of lactic acid bacteria C-17 were detected, and the specific results are shown in Table 2 below.

[0051] Table 2. Results of physiological and biochemical index determination of lactic acid bacteria C-17

[0052]

[0053] The symbols are explained as follows: "+" indicates a positive result; "-" indicates a negative result; and "w" indicates a weak positive result.

[0054] Strain C-17 was sent to the company for testing. The results showed that the hemolytic test of strain C-17 was negative; no antibiotic resistance-related genes were detected; no virulence-related genes were detected; and pathogenicity analysis indicated that the strain was not a potential human pathogen.

[0055] Further identification of this lactic acid bacterium was performed. DNA was extracted from a single colony of C-17, and the 16S rDNA gene sequence was determined. Homology comparisons were performed between the sequencing data and the 16S rDNA gene sequences of archived type strains in the NCBI database using software, and a phylogenetic tree was constructed. The results are shown below. Lactobacillus plantarum As shown. Using the bacterial pheS housekeeping gene identification method, C-17 was ultimately identified as *Lactobacillus plantarum* (…). Figure 3 Meanwhile, the whole genome of this strain was also sequenced. See the image of C-17 cultured on an MRS plate. Lactobacillus plantarum As shown, its micromorphology is... Figure 4 Figure 5 As shown. This strain was deposited at the China Culture Collection Center for Microbial Cultures on December 30, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33230.

[0056] This application focuses on traditional plant-derived fermented products, isolating, purifying, and identifying major lactic acid bacteria, and screening out those with excellent fermentation performance and health benefits after fermentation. Using these strains as fermentation starters, fermentation agents can be prepared for the production of fermented shallot products. Optimal fermentation conditions are determined through single-factor experiments and response surface methodology. Finally, the quality of fermented shallot products is analyzed using sensory evaluation, pH, nutritional indicators, and flavor compounds. This research is expected to fill a gap in the field of lactic acid bacteria fermented shallots. Using the *Lactobacillus plantarum* C-17 provided by this invention, or by preparing a fermentation agent from this strain, fermenting shallots can significantly improve various nutritional value indicators, enhance flavor, and extend shelf life. This research has promising applications in the field of shallot fermentation and can also promote the development of the shallot fermentation industry in Inner Mongolia.

[0057] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A strain of Lactobacillus plantarum ( Lactobacillus plantarum C-17, characterized in that, The Lactobacillus plantarum C-17 was deposited at the China Culture Collection Center for Microbial Cultures on December 30, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33230.

2. The application of Lactobacillus plantarum C-17 as described in claim 1 in the field of shallot fermentation.

3. The application according to claim 2, characterized in that, The applications include extending the shelf life of fermented shallots and / or improving the flavor of fermented shallots.

4. The application according to claim 3, characterized in that, The shelf life of fermented shallots can be extended by increasing the citric acid content; the flavor of fermented shallots can be improved by increasing the glutamic acid content.

5. The application according to claim 2, characterized in that, The application also includes increasing the content of tyrosine and kynurenine in fermented shallots; increasing the content of beneficial fatty acids in fermented shallots, including propionic acid, butyric acid and valeric acid.

6. A fermentation agent comprising Lactobacillus plantarum C-17 as described in claim 1.

7. The fermentation agent according to claim 4, characterized in that, The concentration of *Lactobacillus plantarum* C-17 in the fermentation agent is 1 × 10⁻⁶. 7 CFU / mL.

8. The application of the fermentation agent as described in any one of claims 6-7 in the field of shallot fermentation, characterized in that, The applications include extending the shelf life of fermented shallots, improving the flavor of fermented shallots, increasing the content of tyrosine and kynurenine in fermented shallots, and / or increasing the content of beneficial fatty acids in fermented shallots.

9. A method for pickling sand onions to improve their nutritional value, characterized in that, It includes the following steps: 1) Cultivate the bacteria as described in claim 1 to obtain a bacterial suspension; 2) Inoculate the bacterial suspension into a container containing sand onions; 3) Seal and ferment to obtain fermented shallots; The nutritional value mentioned refers to beneficial indicators in sand onions, including tyrosine, kynurenine, glutamic acid, propionic acid, butyric acid, and valeric acid.

10. The pickling method according to claim 9, characterized in that, The bacterial suspension has a bacterial cell concentration of 1×10⁻⁶. 7 CFU / mL; the inoculation amount is 1% of the weight of the shallots; the sealed fermentation is carried out at room temperature for 7 days; the salt concentration in the sealed fermentation system is 3%.

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