Lactobacillus plantarum SC6 strain, product prepared from lactobacillus plantarum SC6 strain and application of product

Abstract

The invention discloses a lactobacillus plantarum SC6 strain, a product prepared from the lactobacillus plantarum SC6 strain and application of the lactobacillus plantarum SC6 strain, the lactobacillus plantarum SC6 strain is classified and named as lactobacillus plantarum SC6, the lactobacillus plantarum SC6 strain is preserved in China Center for Type Culture Collection on May 6, 2023, the preservation address is No. 299, Wuchang District, Wuhan City, Hubei Province, the postal code is 430072, the preservation number is CCTCC No: M2023684, and the preservation number is CCTCC No: M2023684. The invention also provides an application of the lactobacillus plantarum SC6 strain in preparation of pleurotus geesteranus pickle species, the content of pathogenic bacteria and coliform bacteria in the finished pleurotus geesteranus pickle products prepared from the lactobacillus plantarum SC6 strain meets the national food safety standard, and compared with natural fermentation, compared with the natural fermentation, the inoculated fermentation has the advantages that the acid production speed is high, the fermentation time is short, and the production cost is low. The total acid content is proper, and the safety is higher; sensory evaluation results show that the score of the inoculated and fermented pleurotus geesteranus pickles is higher than that of naturally fermented pleurotus geesteranus pickles.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a Lactobacillus plantarum SC6 strain and products prepared therefrom and applications thereof. Background Art

[0002] The origins of Chinese kimchi can be traced back to the salt pickling process during the Shang and Zhou dynasties. There are clear descriptions of pickled vegetables in Song Dynasty poetry. At that time, salt pickling was used to preserve vegetables, utilizing the fermentation of microorganisms in the natural environment to extend storage. It was discovered through practice that fermentation dominated by lactic acid bacteria in a low-salt environment not only imparted sourness to vegetables but also inhibited spoilage microorganisms. Kimchi fermentation is primarily carried out by lactic acid bacteria, which convert sugars in vegetables into lactic acid and other metabolites (such as ethanol, CO Summary of the Invention

[0003] In response to the deficiencies of the prior art, the present invention provides a Lactobacillus plantarum SC6 strain and products prepared therefrom and their applications, to enrich the types and sources of lactic acid bacteria and improve the processing rate, added value and nutritional value of edible fungi.

[0004] To achieve the above objectives, the present invention provides a Lactobacillus plantarum SC6 strain, which is classified and named Lactobacillus plantarum SC6 and deposited in the China Center for Type Culture Collection on May 6, 2023, with a deposit address of 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, postal code 430072, and a deposit number of CCTCC No: M2023684.

[0005] The present invention also provides an application of the Lactobacillus plantarum SC6 strain in preparing Pleurotus geesteranus pickle seeds.

[0006] The present invention also provides a Pleurotus geesteranus pickle product, comprising the above strain.

[0007] The morphological characteristics of the Lactobacillus plantarum SC6 strain are: See attached for colony morphology Figure 1 The colonies are round, opaque, white or milky white, with a smooth surface and a protrusion in the middle. The colony diameter is 0.5~1.5mm. Gram staining is positive. Microscopic examination is shown in the attached figure. Figure 2 The bacteria are short rod-shaped and are arranged singly, in pairs or chains, without flagella and spores.

[0008] Molecular biology identification: According to the discussion by Fernando Cobo et al., identification is considered correct when the 16S rRNA sequence is ≥99%. The sequencing results of the strain were analyzed in the EzBioCloud database and showed that its homology with Lactiplantibacillus paraplantarum was greater than 99%. The phylogenetic tree was constructed using the neighbor method in Mega 7.0 software, as shown in the attached figure. Figure 5 As shown, this strain is most similar to Lactiplantibacillus paraplantarum, with a similarity of 99.66%. Combined with the above identification results, this strain was identified as a plant-like Lactobacillus and named Lactiplantibacillus paraplantarum SC6.

[0009] The MRS culture dish of the Lactobacillus plantarum SC6 strain (per liter): peptone 10.0 g, yeast extract 4.0 g, glucose 20.0 g, beef extract 5.0 g, Tween (80) 1.08 g, magnesium sulfate (MgSO The present invention provides a Lactobacillus plantarum SC6 strain and products prepared therefrom and applications thereof, which have the following beneficial effects: The finished product of Pleurotus geesteranus kimchi made using the SC6 strain of Lactobacillus plantarum meets the national food safety standards in terms of pathogenic bacteria and coliform bacteria content. Judging from the results of physical and chemical indicators such as pH and nitrite, compared with natural fermentation, inoculated fermentation produces acid faster, has a suitable total acid content, and is safer; the sensory evaluation results show that the inoculated fermented Pleurotus geesteranus kimchi scored higher than the natural fermentation, with moderate sourness and saltiness and a rich flavor. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of the colony morphology of the Lactobacillus plantarum SC6 strain of the present invention; Figure 2 Schematic diagram of the microscopic morphology of the Lactobacillus plantarum SC6 strain of the present invention; Figure 3 Schematic diagram of the dynamic changes in pH of Pleurotus geesteranus kimchi in different fermentation methods of the present invention; Figure 4 Schematic diagram of a radar chart showing sensory scores of Pleurotus geesteranus kimchi of the present invention in different fermentation methods; Figure 5 This is a schematic diagram of the phylogenetic tree of the Lactobacillus plantarum SC6 strain of the present invention based on the 16SrDNA gene sequence strain. DETAILED DESCRIPTION

[0011] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0012] Example 1 Preparation of a Lactobacillus plantarum SC6 strain ① Sample processing 15 samples of acid bacteria kimchi were collected; fresh Pleurotus pulmonarius: provided by the cooperative enterprise Guizhou Haorun Zhenyuan Food Co., Ltd., the collected samples were placed in a dry environment at 14-17℃ and avoided direct sunlight for later use.

[0013] ②Isolation and inactivation of lactic acid bacteria First, disinfect the clean bench and sterilize the glassware required for the experiment to prevent contamination from exogenous microorganisms. Under aseptic conditions, thoroughly stir and mix the kimchi water sample with a sterile glass rod to ensure uniform distribution of microorganisms in the sample. Then, perform a 10-fold gradient dilution and set aside.

[0014] Take 100 μL of 10 ③Identification of lactic acid bacteria 3.1 Morphological observation Inoculate a single colony of the purified strain onto MRS solid medium and incubate at 36°C for 24 hours. Observe the morphology, size, and color of the colonies. Select strains for Gram staining and microscopic examination, observing microscopic characteristics such as size, morphology, and arrangement under an oil immersion lens.

[0015] 3.2 Molecular Biological Identification Genomic DNA of the selected strain was extracted using a bacterial DNA extraction kit and used as a template for PCR amplification using universal 16S rDNA primers 1492R and 27F. 25 μL of 2× Taq PCR MasterMix, 1.5 μL of upstream and downstream primers, 3 μL of DNA template, and 19 μL of sterile water were added to the mixture, making a total of 50 μL. The DNA was pre-denatured at 95°C for 5 minutes, followed by denaturation for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 90 seconds, followed by 35 cycles, and finally extension at 72°C for 10 minutes. The resulting product was sent to Aoke Biotechnology Co., Ltd. for sequencing. The Identify function in the EzBioCloud database was used to identify the strain's closely related species, and a phylogenetic tree was constructed using the nearest neighbor method in Mega 7.0 software.

[0016] Example 2 Experiment on Pickled Pleurotus geesteranus ① Preparation of fermentation strains The preserved strain was activated and inoculated into MRS liquid medium, cultured at 36°C for 48 h, 2% (v / v) of the bacterial solution was inoculated into MRS liquid medium, cultured at 36°C for 18 h, centrifuged at 4°C (5000 r / min, 20 min), and the precipitate was collected. The precipitate was repeatedly rinsed three times with an appropriate amount of 0.9% sterile saline using a pipette, and the colony count was 1×10 after resuspending. 11 CFU / mL bacterial suspension for later use.

[0017] ② Preparation of naturally fermented Pleurotus geesteranus kimchi Production process of fermented Pleurotus geesteranus kimchi: The first step is to select fresh, intact, smooth-surfaced, and undamaged Pleurotus geesteranus as raw material, remove the inedible parts of the mushroom body, and weigh it; The second step is to wash the selected Pleurotus geesteranus with clean water, remove impurities and cut them into strips, usually about 4mm thick. Step 3: Pour the cut oyster mushrooms into boiling water and blanch for 5 minutes, stirring constantly to ensure that the oyster mushrooms are heated evenly. After blanching, remove them from the water and place them in cold water to quickly cool them down. After cooling, drain the water. Step 4: Select ginger with dry, hard, and heavy skin, garlic with intact, unsprouted, and tightly skinned skin, five spices (Sichuan peppercorns, cumin, cinnamon, star anise, and cloves in a ratio of 2:2:2:1:1), and salt as auxiliary ingredients. Add 500g of Oyster mushrooms, 1% salt (w / w), 1% sugar (w / w), 3% ginger and garlic (w / w), 1% five spices (w / w), and 500ml of cooled boiling water to a jar. The fourth step is to seal the bottle and place it in a constant temperature box at 25°C for natural fermentation (NF) for 7 days.

[0018] ③ Preparation of Pleurotus geesteranus kimchi fermented by lactic acid bacteria The production process of Lactobacillus plantarum inoculation fermentation (Lp F) is different from that of natural fermentation of Pleurotus geesteranus kimchi in 1.1. In the fourth step, before canning, a 1% (v / v) inoculation volume of 1×10 11 10 ml of plant-like Lactobacillus SC6 bacterial suspension with a CFU / ml was added, mixed with the ingredients, canned, and sealed and fermented in a constant temperature box at 25°C for 7 days.

[0019] Example 3 Comparative analysis of the structure of spontaneous fermentation and lactic acid bacteria fermentation ①Microbial indicators Table 1 shows the microbial index test results for the finished product of Pleurotus geesteranus kimchi. The pathogenic bacteria content in both treatments met the limits set forth in the "National Food Safety Standard for Limits of Pathogenic Bacteria in Prepackaged Foods" (GB 29921-2021), and the coliform counts met the limits set forth in the "National Food Safety Standard for Pickled Vegetables" (GB 2714-2015).

[0020] Table 1 Microbial index test results of Pleurotus geesteranus kimchi

[0021] ②Physical and chemical indicators pH can be used as an important indicator to measure the maturity of fermented vegetables. The pH dynamic changes of Pleurotus geesteranus kimchi fermented for 7 days are shown in the attached figure. Figure 2 As shown, the pH values ​​of both treatments dropped below 4, indicating that the Pleurotus geesteranus kimchi was mature and edible by the end of the 7-day fermentation. Furthermore, within one day, the pH value of natural fermentation dropped from 6.7 to 5.68, while that of inoculated fermentation dropped from 5.8 to 4.03. The pH value of inoculated fermentation dropped faster and more significantly than that of natural fermentation, indicating that inoculated fermentation can rapidly and significantly reduce pH.

[0022] Nitrite, a salt containing nitrite anions (NO₂−), is a key indicator of kimchi safety and edibility. Under certain conditions, it can react with amines to form the carcinogenic nitrosamine. Table 2 shows the nitrite content of the two treatments at the end of fermentation. The results show that the nitrite content in the Lp F group was significantly lower than that in the NF group. Furthermore, the nitrite content in the finished kimchi produced by both fermentation methods was well below the 20 mg / kg limit for pickled vegetables specified in GB 2762-2022, "National Food Safety Standard: Limits of Contaminants in Food." This indicates that the nitrite content in the Pleurotus geesteranus kimchi prepared in this study was within a safe range.

[0023] Table 2 Nitrite content of Pleurotus geesteranus kimchi in different fermentation methods

[0024] The total acidity (calculated as lactic acid) of kimchi is an important indicator for evaluating its fermentation status. Studies have shown that kimchi with a total acidity of 6-8 g / kg has the best taste. As shown in Table 3, at the end of the 7-day fermentation, the total acidity of the NF group was lower than that of the Lp F group. Furthermore, the lactic acid content of the inoculated fermentation reached 7.2 g / kg, indicating that inoculated fermented Pleurotus geesteranus kimchi may have a better taste.

[0025] Table 3 Total acid content of Pleurotus geesteranus kimchi in different fermentation methods

[0026] The growth of lactic acid bacteria in the fermentation broth has a significant impact on the taste, texture, and nutritional value of the fermented product. Table 4 shows the total lactic acid bacteria counts in the Lp F and NF groups at the end of the 7-day fermentation period. The total lactic acid bacteria count in the inoculated Pleurotus geesteranus kimchi was higher than that in the naturally fermented group. The number of viable lactic acid bacteria is an important indicator of the nutritional value of kimchi, indicating that the inoculated Pleurotus geesteranus kimchi has a relatively higher nutritional value than the naturally fermented group.

[0027] Table 4 Total number of lactic acid bacteria in Pleurotus geesteranus kimchi fermented by different methods

[0028] ③Sensory evaluation Using human sensory input and a ranking-based weighting method, kimchi products were carefully evaluated for color, shape, aroma, texture, and flavor. Twenty panelists with experience in sensory evaluation were invited to conduct the evaluation. They first placed an appropriate amount of sample on a white porcelain plate or disposable white paper plate. They then observed the sample's appearance, smell, and taste under natural light. The final score was the average of the 20 panelists' scores, with a maximum score of 100. The evaluation criteria are shown in Table 5.

[0029] Table 5 Sensory evaluation criteria for Pleurotus geesteranus kimchi

[0030] The average and total scores of each sensory score of the two groups are shown in Table 6, and their distribution is shown in Figure 4 The sensory score of the inoculated fermented Pleurotus geesteranus kimchi was higher than that of the naturally fermented one. The Pleurotus geesteranus kimchi at this time had the milky white color that the mushroom body should have, a tight and crisp texture, a rich aroma, a delicious taste, and a richer kimchi flavor.

[0031]

[0032] Note: The data in the table are average values.

[0033] The various enzymes produced by lactic acid bacteria during the fermentation process can degrade and transform certain substances in the fermented ingredients, or produce a variety of flavor substances through lactic acid metabolism. Therefore, fermentation gives traditional edible fungi a new taste and nutritional value. The present invention isolates a lactic acid bacterium from the traditional folk pickle - acid bacteria. Through colony and bacterial morphology analysis and 16SrRNA sequence comparison analysis, it is identified as a plant-like lactobacillus and named Lactiplantibacillus paraplantarum SC6; Pleurotus geesteranus is used as raw material and Lactiplantibacillus paraplantarum SC6 is inoculated to ferment Pleurotus geesteranus pickles. The pathogenic bacteria content of the finished product meets the limit of Pathogenic Bacteria in Pre-packaged Foods (GB29921-2021) in the National Food Safety Standard, and the coliform group content meets the coliform group limit in the "National Food Safety Standard for Pickled Vegetables" (GB 2714-2015). Judging from the results of physical and chemical indicators such as pH and nitrite, compared with natural fermentation, inoculated fermentation produces acid faster, has an appropriate total acid content, and is safer; sensory evaluation results show that the inoculated fermented Pleurotus geesteranus pickles scored higher than naturally fermented, has moderate sourness and saltiness, and a strong flavor. It is a safe, edible and nutritious new edible fungus pickle product.

[0034] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A Lactobacillus plantarum SC6 strain, characterized in that: The Lactobacillus plantarum SC6 strain is classified and named Lactobacillus plantarum SC6, and was deposited in the China Center for Type Culture Collection on May 6, 2023. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072, and the collection number is: CCTCCNo: M2023684.

2. Application of a plant lactobacillus SC6 strain according to claim 1 in preparing Pleurotus geesteranus kimchi seeds.

3. A Pleurotus geesteranus kimchi product, characterized in that: The invention comprises the strain as claimed in claim 1.

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