Antioxidant lactobacillus pentosus and application thereof

Abstract

The invention discloses antioxidant lactobacillus pentosus and application thereof, the antioxidant lactobacillus pentosus Lpe.C1 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.33427. Gram staining of the lactobacillus pentosus Lpe.C1 is positive, catalase contact test is negative, a 16S rDNA sequence is shown as SEQ ID NO: 1, the hydroxyl free radical scavenging capacity of the lactobacillus pentosus Lpe.C1 is larger than 65%, the DPPH free radical scavenging capacity of the lactobacillus pentosus Lpe.C1 is larger than 45%, and the lactobacillus pentosus Lpe.C1 grows well under the salt concentration of 4% or above. The application of the antioxidant lactobacillus pentosus comprises the step of preparing antioxidant pickled cabbage type brassica campestris and pickled Chinese cabbage type brassica campestris by utilizing the separated and purified antioxidant lactobacillus pentosus Lpe.C1. According to the pickled brassica campestris prepared by applying the antioxidant lactobacillus pentosus Lpe.C1 disclosed by the invention, the oxidation resistance of a product is remarkably improved, the vitamin C content, the delicate flavor, the sweet amino acid content and the chewiness of the product are higher, and the quality, the mouth feel and the flavor of the product are better.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and particularly relates to an antioxidant Lactobacillus pentosus and its application. Background Art

[0002] As a major oil crop in the world, rapeseed is widely used in edible oil, feed, medicine, vegetables, industry, and ornamental fields. Among them, vegetable rapeseed can pick the main or side shoots as vegetables (rapeseed shoots) during the budding stage. Rapeseed shoots not only taste fragrant and delicious, but also are rich in nutrients such as vitamins, calcium, iron, and potassium. However, as a seasonal vegetable, fresh rapeseed shoots have the problem of poor storage tolerance, a short shelf life, and it is difficult to meet the market demand. Pickling and processing is an effective way to solve this problem, but there are problems such as too long fermentation cycle and easy oxidation and blackening during the pickling process.

[0003] The extended fermentation cycle will prolong the production time, increase costs, reduce efficiency, and may even lead to unstable supply chains, affecting the timely supply of products and making it difficult to meet the market demand. The oxidation and blackening of pickled rapeseed shoots will seriously affect the appearance quality of rapeseed shoots and reduce consumers' desire to buy; at the same time, the oxidation process may also cause the loss of some nutrients (such as antioxidant substances such as vitamin C), reducing the nutritional value of the product.

[0004] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention, and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0005] The purpose of the present invention is to provide an antioxidant Lactobacillus pentosus and its application, which can solve the technical problems raised in the above background art.

[0006] In order to achieve the above purpose, the technical solution provided by a specific embodiment of the present invention is as follows:

[0007] An antioxidant Lactobacillus pentosus, the Lactobacillus pentosus Lpe.C1 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 33427.

[0008] In order to achieve the above purpose, the technical solution provided by a specific embodiment of the present invention is as follows:

[0009] An application of an antioxidant Lactobacillus pentosus for preparing highly antioxidant rapeseed shoots. The preparation method of the highly antioxidant rapeseed shoots includes the following steps:

[0010] S1. Prepare several portions of antioxidant Lactobacillus pentosus Lpe.C1 with the preservation number of CGMCC No. 33427;

[0011] S2. Preparation of rape shoots: Pick a portion of rape shoots and wash them.

[0012] S3. Pretreatment of rape shoots: Add a portion of edible salt to a portion of rape shoots, knead the rape shoots until water appears on the surface of the rape shoots to obtain pretreated rape shoots.

[0013] S4. Pickling of rape shoots: Place the pretreated rape shoots in a pickling jar, level and compact them, add a portion of brine that has been boiled and cooled to room temperature to the pickling jar, and make the brine submerge the rape shoots to obtain pickled rape shoots.

[0014] S5. Inoculation with Lactobacillus pentosus with high antioxidant activity: Add a portion of Lactobacillus pentosus agent Lpe.C1 with high antioxidant activity to the pickled rape shoots, and seal the container again.

[0015] S6. Seal and pickle the rape shoots after step S5 for 50 - 70 days to obtain rape shoots with high antioxidant activity.

[0016] In one or more embodiments of the present invention, in step S5, the preparation method of the antioxidant Lactobacillus pentosus is as follows:

[0017] Activate Lactobacillus pentosus Lpe.C1 separated, purified and stored at -80°C to the logarithmic growth phase, centrifuge and precipitate the bacterial cells at 4°C, wash them 1 - 2 times with sterile physiological saline, and then resuspend them with an equal volume of physiological saline to obtain an antioxidant Lactobacillus pentosus agent.

[0018] In one or more embodiments of the present invention, the Gram staining of the antioxidant Lactobacillus pentosus Lpe.C1 is positive.

[0019] In one or more embodiments of the present invention, the catalase contact test of the antioxidant Lactobacillus pentosus Lpe.C1 is negative.

[0020] In one or more embodiments of the present invention, the 16S rDNA sequence of the antioxidant Lactobacillus pentosus Lpe.C1 is as shown in SEQ ID NO:1.

[0021] In one or more embodiments of the present invention, the hydroxyl radical scavenging ability of the antioxidant Lactobacillus pentosus Lpe.C1 is greater than 65%, and the DPPH· radical scavenging ability of the antioxidant Lactobacillus pentosus Lpe.C1 is greater than 45%.

[0022] In one or more embodiments of the present invention, the weight ratio of a portion of the rape shoots to the edible salt is (500:20) - (500:50).

[0023] In one or more embodiments of the present invention, the mass of salt in the brine accounts for 5% - 8% of the mass of the brine.

[0024] Compared with the prior art, an antioxidant Lactobacillus pentosus of the present invention and its application shorten the pickling period of rape bolts to 50 - 70 days by introducing Lactobacillus pentosus Lpe.C1, improve the production efficiency and reduce the cost. In addition, Lactobacillus pentosus Lpe.C1 inhibits the oxidation reaction, avoids the blackening of rape bolts, maintains the appearance quality and nutritional components such as vitamin C, and solves the key problems in pickling. At the same time, the pickled rape bolt products inoculated with antioxidant Lactobacillus pentosus Lpe.C1 have higher contents of umami and sweet amino acids and better chewiness, enhancing the market competitiveness of the products and the satisfaction of consumers, and providing technical support for the development of vegetable-type rape. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0026] Figure 1 It is a flowchart of the application of an antioxidant Lactobacillus pentosus in an embodiment of the present invention;

[0027] Figure 2 It is a screening diagram of antioxidant lactic acid bacteria in an embodiment of the present invention;

[0028] Figure 3 It is a morphological diagram of antioxidant Lactobacillus pentosus in an embodiment of the present invention;

[0029] Figure 4 It is a pH value change diagram during the fermentation process of pickled rape bolts inoculated with antioxidant Lactobacillus pentosus and naturally fermented rape bolts in an embodiment of the present invention;

[0030] Figure 5 It is a comparison diagram of the hydroxyl radical scavenging ability during the fermentation process of pickled rape bolts inoculated with antioxidant Lactobacillus pentosus and naturally fermented rape bolts in an embodiment of the present invention;

[0031] Figure 6 It is a chewiness comparison diagram of pickled rape bolts inoculated with antioxidant Lactobacillus pentosus and naturally fermented rape bolts in an embodiment of the present invention;

[0032] Figure 7 It is a vitamin C content comparison diagram of pickled rape bolts inoculated with antioxidant Lactobacillus pentosus and naturally fermented rape bolts in an embodiment of the present invention;

[0033] Figure 8 It is a colony diagram of antioxidant Lactobacillus pentosus in an embodiment of the present invention;

[0034] Figure 9 This is the serial number of Lactobacillus pentosus with antioxidant property in an embodiment of the present invention. Detailed implementation manners

[0035] To enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0036] An application of Lactobacillus pentosus with antioxidant property in an embodiment of the present invention. The present invention relates to a Lactobacillus pentosus with antioxidant property and its application. Among them, the Lactobacillus pentosus used was deposited in the Institute of Microbiology, Chinese Academy of Sciences (CGMCC) on January 15, 2025, with the deposit number CGMCC No. 33427, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0037] As Figures 1 to 6 shown, an application of Lactobacillus pentosus with antioxidant property in an embodiment of the present invention is used for preparing Brassica parachinensis with high antioxidant property, and specifically includes the following steps:

[0038] S1. Take 1 mL of pickled fermentation broth, add 9 mL of sterilized normal saline for dilution, and after fully mixing, perform gradient dilution with bacterial normal saline in sequence. The dilution degrees are 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , and 10 -7 dilution degrees. Select the latter 4 dilution degrees and respectively pipette 500 μ μL of the dilution solution and spread it on the MRS-CaCO3 agar plate. Invert the plate and culture it in an incubator at 37 °C for 48 h. Isolate according to the presence or absence of calcium circles, size, color, shape, and edge characteristics of the colonies.

[0039] S2. Purify the isolated strain on the MRS solid medium by repeating the streaking operation until a single and pure colony is observed on the medium.

[0040] S3. Perform Gram staining, catalase contact test, 16S rDNA, and antioxidant ability identification on the single strain screened and isolated.

[0041] As Figures 2 to 3As shown, according to a method for processing rapeseed with high antioxidant capacity of the present invention, by measuring the hydroxyl radical scavenging ability of Lactobacillus pentosus, we successfully screened out the Lactobacillus pentosus strain Lpe.C1 ( Figure 2 ), after Gram staining, its morphology was observed under a microscope to be rod-shaped ( Figure 3 ).

[0042] like Figure 1 As shown, a method for processing high antioxidant rapeseed stalks according to one embodiment of the present invention comprises the following steps:

[0043] S1. Isolation, purification and identification of the antioxidant Lactobacillus pentosus;

[0044] S2. Preparation of rapeseed stalks: Pick the upper part of the rapeseed stalks and wash them clean;

[0045] S3, pretreatment of rapeseed stalks: adding one portion of edible salt to one portion of rapeseed stalks, kneading the rapeseed stalks until water comes out of the surface of the rapeseed stalks, and obtaining pretreated rapeseed stalks;

[0046] S4, pickling rape stalks: placing the pre-treated rape stalks in a pickle jar, flattening and compacting them, and adding a portion of brine that has been boiled and cooled to room temperature into the pickle jar until the rape stalks are submerged;

[0047] S5, inoculating with high antioxidant Lactobacillus pentosus: adding a portion of Lactobacillus pentosus Lpe.C1 bacterial agent that has been pre-screened and activated to the logarithmic growth phase and has a strong antioxidant degradation ability to the pickled rapeseed stalks in S4, and sealing the container again;

[0048] S6. Sealing the cauliflower in the kimchi jar and pickling it for about 60 days to obtain a cauliflower product with good antioxidant capacity.

[0049] According to a processing method of high antioxidant rapeseed stalks of the present invention, the flavor of the rapeseed stalks becomes more intense as the fermentation time is prolonged. When pickling the rapeseed stalks, it is necessary to ensure that the utensils used are completely oil-free to ensure the pickling quality. Before eating, the taken rapeseed stalks should be washed and soaked with clean water. According to personal preference, it can be chopped or not chopped, and then used to make a variety of delicacies such as stir-fry, stew or soup.

[0050] like Figures 4 to 7 As shown, a is a Brassica oleracea type rapeseed stalk, and b is a Brassica rapa type rapeseed stalk.

[0051] The use of this specific strain to pickle rapeseed stems has shortened the fermentation cycle and simplified the production process while maintaining product quality. The ability to scavenge hydroxyl free radicals in rapeseed stems fermented with Lactobacillus pentosus has been significantly improved; the vitamin C content has been increased, and the nutritional value has been higher; the umami and sweet amino acid content has been increased, and the flavor has been better; the chewiness has been significantly improved, and the taste has been better, providing consumers with safer and healthier pickled vegetable products, which have broad promotion value.

[0052] The application of the antioxidant Lactobacillus pentosus significantly improves the pickling effect and product quality of rapeseed stalks by introducing the antioxidant Lactobacillus pentosus. First, the addition of the antioxidant Lactobacillus pentosus effectively shortens the fermentation cycle, improves production efficiency, reduces production costs, and ensures the stability of the supply chain, which can better meet market demand. In addition, the introduction of the antioxidant Lactobacillus pentosus effectively inhibits the oxidation reaction of rapeseed stalks during the pickling process, avoids the problem of easy oxidation and blackening of cabbage-type rapeseed stalks, maintains the appearance quality of the product, and enhances consumers' desire to buy. At the same time, the protective effect of the antioxidant Lactobacillus pentosus also reduces the loss of antioxidant substances such as vitamin C, retains the nutritional value of rapeseed stalks to the greatest extent, and makes it reach a high level in taste, nutrition and appearance. Secondly, the method significantly reduces the generation of nitrite during the pickling process through the action of Lactobacillus pentosus, reduces the risk of consumption, and makes the product safer and more reliable. In summary, this technical solution not only solves the key problems in the process of pickling rapeseed stems, but also significantly improves the market competitiveness and consumer satisfaction of the product, providing important technical support for the multifunctional development of vegetable rapeseed.

[0053] An antioxidant Lactobacillus pentosus in one embodiment of the present invention has a preservation number of CGMCC No. 33427. The preparation steps include:

[0054] Step 1: Isolation and purification of lactic acid bacteria: Take 1 mL of fermentation broth and add it to 9 mL of sterile saline for dilution. After fully mixing, dilute it with sterile saline in a gradient manner. -3 , 10 -4 , 10 -5 For three gradients, take 500 μL of dilutions for each, spread them on the modified MRS-CaCO3 agar plate, and cover them with sterile agar. Place the plate in a vacuum bag, invert it in an incubator under vacuum sealing conditions, and culture it at 37°C for 24 to 48 hours. Select strains with good growth and large calcification circles, streak them on MRS solid culture medium, culture them at 37°C for 24 to 48 hours, purify them for three generations, and store them for later use. All operations are performed in a clean bench.

[0055] Step 2. Identification of lactic acid bacteria. The identification of lactic acid bacteria includes morphological identification, catalase test, Gram staining test, and molecular biology identification. As Figure 8 shown, in terms of colony morphology in morphological identification, the color, shape, surface smoothness, vertical concavity and convexity of the colony are mainly observed and recorded. For the catalase test, a single colony needs to be picked and placed on a clean glass slide or in a small test tube, and 1-2 drops of 3% hydrogen peroxide are added, and then whether bubbles are generated is immediately observed. If bubbles are generated, it is judged as positive, otherwise it is negative.

[0056] Among them, the Gram staining test includes the following steps: A drop of sterile normal saline is dropped in the center of a clean glass slide. Using an inoculation loop, perform aseptic operation, pick a single colony, mix it evenly with normal saline, and smear it into a thin layer. After smearing, slightly warm it on an alcohol lamp to make it dry quickly. Hold one end of the glass slide with the sample facing up, and quickly move it back and forth on the outer side of the flame of the alcohol lamp. The temperature should not be too high to prevent the denaturation of the bacterial protein. Wait until it cools down before staining. After dropping crystal violet staining solution on the fixed sample and staining for 1 min, rinse off the staining solution with pure water and blot the excess water with lens paper. Drop iodine solution and cover the glass slide. After staining for 1 min, wash with water and blot the excess water with lens paper. Drop decolorizing solution ethanol and decolorize for 30 s, then immediately rinse off the decolorizing solution with pure water and blot the excess water with lens paper. Drop safranin staining solution and stain for 1 min, then rinse off the staining solution with pure water and air dry. Observe the color, morphology and arrangement of the strain cells under an oil immersion objective of an optical microscope and take pictures for recording.

[0057] For molecular biology identification, after mixing the enriched liquid culture medium, the genomic DNA of each strain is extracted using a Shengong bacterial genomic DNA extraction kit (B518255-0055). Using universal primers for identifying bacterial 16S rDNA (forward primer 27F: 5'-GAGAGTTTGATCCTGGCTCAG-3', reverse primer 1492R: 5'-TACGGCTACCTTGTTACGAC-3') for PCR amplification, and 2×Rapid Taq MasterMix is used for PCR amplification. The PCR reaction system is 10 μL: 1 μL of DNA template, 0.5 μL of each of primers 27F and 1495R, 5 μL of 2×Master Mix, and ddH2O is added to make up to 10 μL. The PCR reaction program is: pre-denaturation, 95 °C, 5 min; denaturation, 95 °C, 30 s, annealing, 55 °C, 30 s, extension, 72 °C, 1 min (30 cycles); post-extension, 72 °C, 10 min. After the reaction is completed, the high-quality PCR products are sent for Sanger sequencing.

[0058] The obtained sequences were blasted in the NCBI database, and the strain sequences with similarity higher than 99% were selected to construct a phylogenetic tree. The NJ (Neighbor-Joining) phylogenetic tree was constructed using MEGAN11 (Molecular Evolutionary Genetics Analysis), and the Bootstrap value was set to 1000.

[0059] Lactic acid bacteria include the following fermentation characteristics:

[0060] I. Growth curve: The activated strains were inoculated into MRS liquid medium at an inoculum volume fraction of 2%, and cultured with shaking at 37 °C for 24 h. Samples were taken every 2 h under sterile conditions, and the OD value of the bacterial solution at a wavelength of 600 nm was measured using an ultraviolet spectrophotometer. Each group was subjected to 3 replicate experiments. With the culture time (x) as the abscissa and the OD 600nm value (y) as the ordinate, a growth curve was plotted.

[0061] II. Acid production characteristics: The activated strains were inoculated into MRS liquid medium at an inoculum volume fraction of 2%, and cultured with shaking at 37 °C for 24 h. Samples were taken every 2 h under sterile conditions, and the pH value was measured using a pH meter. Each group was subjected to 3 replicate experiments. With the culture time (x) as the abscissa and the pH value (y) as the ordinate, a dynamic pH curve was plotted.

[0062] III. Salt tolerance: The activated strains were inoculated into sterilized NaCl-containing MRS liquid medium at an inoculum volume fraction of 2%. The final concentrations of NaCl were 0, 6%, 8%, and 10% respectively. Tubes without inoculation were used as blank controls. The cultures were shaken at 37 °C for 24 h, and the OD value of the bacterial solution at a wavelength of 600 nm was measured. Each group was subjected to 3 replicate experiments.

[0063] IV. Screening of lactic acid bacteria strains with in vitro antioxidant ability: The activated strains were inoculated into MRS liquid medium at an inoculum volume fraction of 2%, and cultured with shaking at 37 °C for 24 h to obtain the culture solution of lactic acid strains. The culture solution was centrifuged at 12000 rpm for 5 min at 4 °C. After the supernatant was centrifuged at 10000 rpm for 5 min, it was filtered through a 0.22 μm microporous membrane filter to obtain a cell-free fermentation supernatant (CFS). The cells were washed 2 - 3 times with sterile water, and the cell density OD600 was adjusted to 1.0 (about 1×10 9 CFU / mL), which was used as intact cells (IC) for the determination of antioxidant indexes.

[0064] V. Determination of DPPH radical scavenging rate: The micro-method for determining the DPPH radical scavenging ability of lactic acid bacteria was carried out using the DPPH radical scavenging ability detection kit (BC4755). The specific operation was referred to the kit instruction manual, and the calculation was carried out using the following formula:

[0065] DPPH radical scavenging (%) = [[A blank - (A determination - A control)] ÷ A blank] × 100%

[0066] VI. Determination of hydroxyl radical scavenging rate: The micro-method for determining the hydroxyl radical scavenging ability of lactic acid bacteria was carried out with reference to the hydroxyl radical scavenging ability detection kit (BC1325). The specific operation was referred to the kit instruction manual, and the calculation was carried out using the following formula:

[0067] Hydroxyl radical scavenging rate (%) = (A measurement - A control) ÷ (A blank - A control) × 100%

[0068] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claims involved.

[0069] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An antioxidant Lactobacillus pentosus, characterized in that, The Lactobacillus pentosus is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC No. 33427.

2. Application of Lactobacillus pentosus with antioxidant property, characterized in that, For the preparation of highly antioxidant Brassica parachinensis, the preparation method of the highly antioxidant Brassica parachinensis comprises the following steps: S1. Prepare several portions of antioxidant Lactobacillus pentosus Lpe.C1 with the preservation number of CGMCC No. 33427; S2. Preparation of Brassica parachinensis: Pick one portion of Brassica parachinensis shoot and wash it; S3. Pretreatment of Brassica parachinensis: Add one portion of edible salt to one portion of Brassica parachinensis shoot, knead the Brassica parachinensis shoot until water appears on the surface of the Brassica parachinensis shoot to obtain pretreated Brassica parachinensis shoot; S4. Pickling of Brassica parachinensis: Place the pretreated Brassica parachinensis shoot in a pickling jar, level and compact it, add one portion of 5% - 8% brine that has been boiled and cooled to room temperature to the pickling jar to submerge the Brassica parachinensis shoot to obtain pickled Brassica parachinensis shoot; S5. Inoculation with highly antioxidant Lactobacillus pentosus: Add one portion of antioxidant Lactobacillus pentosus Lpe.C1 inoculant to the pickled Brassica parachinensis shoot, and seal the container again; S6. Seal and pickle the Brassica parachinensis shoot obtained in step S5 for 50 - 70 days to obtain highly antioxidant Brassica parachinensis.

3. Use of an antioxidant Lactobacillus pentosus according to claim 2, characterized in that, In the step S5, the preparation method of the antioxidant Lactobacillus pentosus is as follows: Activate the Lactobacillus pentosus Lpe.C1 separated, purified and stored at -80°C to the logarithmic growth phase, centrifuge and precipitate the bacterial cells at 4°C, wash them with sterilized normal saline for 1 - 2 times, and then resuspend them with the same volume of normal saline. After detecting the density and activity of the bacteria, obtain the antioxidant Lactobacillus pentosus Lpe.C1 inoculant.

4. Use of an antioxidant Lactobacillus pentosus according to claim 2, characterized in that, The antioxidant Lactobacillus pentosus is Gram-positive, negative in catalase contact test, and its 16S rDNA sequence is as shown in SEQ ID NO:

1.

5. The application of Lactobacillus pentosus with antioxidant property according to claim 2, characterized in that, The hydroxyl radical scavenging ability of the antioxidant Lactobacillus pentosus is greater than 65%, and the DPPH· radical scavenging ability of the antioxidant Lactobacillus pentosus is greater than 45%.

6. Use of an antioxidant Lactobacillus pentosus according to any one of claims 2 to 5, characterized in that, The weight - volume ratio of one portion of the Brassica parachinensis shoot to the antioxidant Lactobacillus pentosus is (500:8) - (500:24).

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