A Lactobacillus plantarum with the functions of enriching selenium and degrading nitrite and its application

Abstract

The present invention discloses a Lactobacillus plantarum with the functions of selenium enrichment and nitrite degradation, named Lactobacillus plantarum h32, and its preservation number is: CGMCC No. 29092. Lactobacillus plantarum h32 has high nitrite degradation and selenium enrichment capabilities. Under certain conditions, it can not only degrade harmful substances and reduce food safety risks, but also convert inorganic selenium into organic selenium, improving the nutritional quality of food. The present invention also discloses the application of the Lactobacillus plantarum with the functions of selenium enrichment and nitrite degradation in pickles. By applying Lactobacillus plantarum h32 to produce selenium-enriched pickles, the obtained pickles have a lower nitrite content, and their selenium content, color, smell, taste, texture and tissue structure are all higher than those of naturally fermented pickles.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and more particularly, relates to a Lactobacillus plantarum with the functions of selenium enrichment and nitrite degradation and its application. Background Art

[0002] Selenium is a non-metallic element and one of the essential mineral elements for humans and animals, having physiological functions such as enhancing immunity, anti-cancer, and antioxidant properties.

[0003] The valence states of selenium are generally elemental selenium (0), selenide (Ⅱ), selenite (Ⅳ), and selenate (Ⅵ), and its chemical form affects the exertion of functions. Selenium exists in two forms in nature, inorganic selenium and organic selenium. Inorganic selenium has high toxicity and is not easily absorbed by the human body, while organic selenium has low toxicity, high safety, and is easily absorbed by the human body. Only by converting inorganic selenium into organic selenium can it be used to regulate the physiological functions of the human body.

[0004] Lactic acid bacteria (LAB) are Gram-positive heterotrophic bacterial probiotics present in the human body, which can ferment carbohydrates into lactic acid. Those beneficial to human health are called probiotics, represented by lactic acid bacteria and Bifidobacterium. Moreover, as probiotics in the human body, lactic acid bacteria have been confirmed by many studies to have the ability to enrich and transform selenium, and their selenium-enriched metabolites have various biological activities and physiological functions such as antioxidant, antibacterial, anti-tumor, improving immunity, delaying aging, and promoting growth.

[0005] Nitrite is a common food additive. Excessive intake of nitrite may cause acute nitrite poisoning, leading to symptoms such as headache, vomiting, and abdominal pain, affecting the food safety of food. The degradation of nitrite in food can be carried out through different ways such as physical, chemical, and microbial methods. Physical degradation is to degrade nitrite in food through physical means such as high temperature and ultraviolet radiation. However, physical degradation may cause loss of nutrients in food, affecting the quality, taste, and flavor of food. Chemical degradation is to convert nitrite into other compounds through chemical reactions to achieve the degradation effect. However, chemical degradation may generate harmful substances such as nitrosamines, bringing potential risks to food safety. In addition, chemical degradation may require the addition of chemical reagents, which may arouse consumers' concerns about food safety. Using biological methods, especially using lactic acid bacteria fermentation to degrade nitrite, is considered a safe and feasible method and is deeply favored by consumers.

[0006] During the production process of pickled vegetables, nitrite is inevitably produced. Excessive intake of nitrite will endanger human health. Therefore, screening out a strain of lactic acid bacteria with the functions of selenium enrichment and simultaneous nitrite degradation for the development of selenium-enriched pickled vegetables is a problem to be solved. Summary of the Invention

[0007] To address the above deficiencies, the present invention provides a Lactobacillus plantarum with the functions of enriching selenium and degrading nitrite, named Lactobacillus plantarum h32. Lactobacillus plantarum h32 is deposited in the General Microbiological Center of the China National Culture Collection of Microorganisms. The deposit date is November 21, 2023, and its deposit number is: CGMCC No. 29092;

[0008] Absorb 25 mL of pickled vegetable juice into 50 mL of MRS broth, perform enrichment culture at 37°C for 24 h, then perform gradient dilution with physiological saline, dilute 7 gradients (select gradients 3 - 6 for culture), culture the diluted bacterial solution on MRS solid medium containing 0.1% CaCO3 (screening lactic acid bacteria by the calcium solubilization circle method), determine the colonies of lactic acid bacteria (the discolored colonies), and perform culture at 37°C for 48 h to complete the isolation step of strain h32;

[0009] Pick single colonies with obvious calcium solubilization circles and a moist surface on the above solid medium, repeatedly streak and purify, and perform culture at 37°C for 48 h. Then, perform Gram staining on the purified strain, and store the Gram-positive strain on a 4°C slant and in a -80°C glycerol tube to complete the purification step of strain h32.

[0010] Furthermore, this strain was identified as Lactobacillus plantarum by colony morphology, biochemistry, and 16S rRNA sequencing analysis, and named Lactobacillus plantarum h32.

[0011] Furthermore, the object of the present invention lies in the application of the Lactobacillus plantarum with the functions of enriching selenium and degrading nitrite in pickled vegetables.

[0012] Furthermore, the object of the present invention also lies in a method for preparing pickled vegetables. First, perform selenium enrichment treatment on Lactobacillus plantarum h32, then wash, drain, and package fresh vegetables and inject water, inoculate the above selenium-enriched Lactobacillus plantarum h32, and finally perform anaerobic fermentation.

[0013] Furthermore, the growth amount of Lactobacillus plantarum h32 is 10 7 cfu / mL, and the inoculation amount is 3%, and the unit of the inoculation amount is mL / L. Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. By screening multiple strains of bacteria, Lactobacillus plantarum h32 was obtained. Lactobacillus plantarum strain h32 has a high nitrite degradation ability, can achieve efficient degradation under relatively mild conditions, and has a certain selectivity when degrading nitrite, which can avoid the production of harmful degradation products and reduce food safety risks;

[0015] 2. Applying Lactobacillus plantarum strain h32 to the production of selenium-enriched pickles, the color, smell, taste, texture and tissue structure of the obtained pickles are all better than those of naturally fermented pickles. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a morphological photograph of Lactobacillus plantarum strain h32 under Gram staining under a microscope.

[0017] Figure 2 It is a phylogenetic tree of Lactobacillus plantarum strain h32.

[0018] Figure 3 It is the change of pH value and total acid during the cultivation of Lactobacillus plantarum strain h32.

[0019] Figure 4 It is a schematic diagram of the change of nitrite degradation rate during the cultivation of Lactobacillus plantarum strain h32.

[0020] Figure 5 It is a schematic diagram of the change of nitrite content during the process of making pickles using Lactobacillus plantarum strain h32.

[0021] Figure 6 It is a standard curve graph in the determination of selenium enrichment ability. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] To facilitate the understanding of the present invention, the device of the present invention will be described more comprehensively below with reference to the relevant drawings. Embodiments of the device are shown in the drawings. However, the device can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0023] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installed", "connected", "set" should be understood in a broad sense. For example, it can be fixedly connected, set, or detachably connected, set, or integrally connected, set. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0024] Example 1

[0025] This example is the isolation and purification of Lactobacillus plantarum strain h32:

[0026] 1. Isolation of Lactobacillus plantarum strain h32: Absorb 25 mL of the self - made Chinese cabbage pickle juice from Luoyang farmers into 50 mL of MRS broth, and perform enrichment culture at 37 °C for 24 h. Then, perform gradient dilution with normal saline for 7 gradients (select gradients 3 - 6 for culture). Culture the diluted bacterial solution on MRS solid medium containing 0.1% CaCO3 (screening lactic acid bacteria by the calcium - dissolving circle method), determine the colonies of lactic acid bacteria (the discolored colonies), and culture them in an environment at 37 °C for 48 h.

[0027] 2. Purification of Lactobacillus plantarum strain h32: Pick the single colonies with obvious calcium - dissolving circles and moist surfaces on the above - mentioned solid medium, repeatedly streak and purify, and culture them in an environment at 37 °C for 48 h. Then, perform Gram staining on the purified strains, and store the Gram - positive strains on a slant at 4 °C and in a glycerol tube at - 80 °C for subsequent experiments.

[0028] Example 2

[0029] This example is for screening strains:

[0030] Inoculate the strain isolated in Example 1 into MRS broth medium for activation twice, and then inoculate it into MRS broth containing 150 mg / L NaNO2 at an inoculation amount of 2% (volume fraction), culture at 37 °C for 48 h, and measure the degradation rate of nitrite by the strain within 48 h according to the following method to screen out the strain h32 with good nitrite - degrading effect:

[0031] Determine nitrite by the N - (1 - naphthyl) ethylenediamine dihydrochloride method in GB / T5009.33—2016, and draw a standard curve: Accurately weigh 0, 1, 2, 3, 4, 5, 7.5, 10, 12.5 μg of NaNO2 respectively, add 40 mL of water and 2 mL of sulfanilic acid solution, mix well, stand in the dark for 5 min, then add 1 mL of N - (1 - naphthyl) ethylenediamine dihydrochloride solution, mix well and make up the volume to 50 mL, stand in the dark for 15 min, measure the absorbance at 538 nm, and obtain the equation of the standard curve through linear regression analysis, and obtain the nitrite content according to the standard curve.

[0032] The nitrite degradation rate is calculated according to the following formula:

[0033] R / %=(C0 - C1) / C0;

[0034] In the above formula: R represents the nitrite degradation rate; C0 represents the initial content of NaNO2 in the medium, with the unit of mg / L, and C1 represents the content of NaNO2 in the medium at different culture times, with the unit of mg / L.

[0035] Sample treatment: Add 12.5 mL of saturated borax solution to 5.0 mL of fermentation broth, then add 40 mL of distilled water, heat in a boiling water bath for 15 min, cool to room temperature, add 5 mL of potassium ferrocyanide solution, then add 5 mL of zinc acetate solution and mix well. Let stand for 30 min to precipitate proteins. Dilute the above liquid to 100 mL, filter, discard the first 5 mL of the filtrate, and collect the remaining filtrate for use.

[0036] Sample determination: Take 2 mL of the above-treated solution, add 40 mL of water and 2 mL of sulfanilic acid solution, mix well, let stand in the dark for 5 min, then add 1 mL of hydrochloric acid naphthyl ethylenediamine solution, dilute to 50 mL, let stand in the dark for 15 min, measure the absorbance at 538 nm, and use the same amount of distilled water as a blank control at the same time.

[0037] Example 3

[0038] Identification steps for the strains screened in 2:

[0039] 1. Physiological and biochemical identification:

[0040] Observe the morphological structure of the strains, and preliminarily determine the strain types in combination with the "Common Bacterial System Identification Manual";

[0041] 2. Extraction of lactic acid bacteria genomic DNA:

[0042] Extract the genomic DNA of lactic acid bacteria using a DNA extraction kit;

[0043] 3. 16S rDNA identification:

[0044] After amplifying the genome extracted in the previous step with universal primers, submit the amplification results to a biological engineering institution for 16S rDNA sequencing, and compare the obtained results with the genomic sequences of related strains in GenBank;

[0045] 4. Construction of a phylogenetic evolution tree:

[0046] After performing a Blast alignment on the NCBI website based on the full 16S rRNA sequence obtained by sequencing, select the lactic acid bacteria strain sequences with higher homology (preferably greater than 97%) to the target sequence and construct a phylogenetic evolution tree;

[0047] 5. Determination of the growth curve:

[0048] Inoculate the strains into MRS broth medium for activation twice, and inoculate them into MRS broth at an inoculation amount of 2% by volume, culture in an environment of 37 °C, and measure the OD 600nm absorbance every 2 h;

[0049] 6. Determination of acid production ability:

[0050] The strain was inoculated into MRS broth medium for activation twice, and then inoculated into MRS broth at an inoculum volume fraction of 2%, and cultured at 37 °C for 72 h. The pH value and total acid content of the fermentation broth were measured at 0, 4, 8, 12, 24, 36, 48, 60, and 72 h respectively. As Figure 2 shown, the results showed that the acid production of the strain during fermentation could reduce the pH value of the medium to about 3.65, which was beneficial to the fermentation and acidification of pickles and the inhibition of miscellaneous bacteria. The total acid content was determined by the potentiometric titration method of pH meter in GB / T 12456—2021;

[0051] 7. Determination of the rate of nitrite degradation:

[0052] The strain was inoculated into MRS broth medium for activation twice, and then inoculated into MRS broth containing 150 mg / L NaNO2 at an inoculum volume fraction of 2%, and cultured at 37 °C for 72 h. The nitrite degradation rate at 0, 4, 8, 12, 24, 36, 48, 60, and 72 h was obtained. As Figure 3 shown, at 60 h, the degradation efficiency was 100%;

[0053] 8. Determination of selenium enrichment ability:

[0054] Accurately pipette 0, 1, 2, 3, 4, and 5 mL of 10 μg / mL sodium selenite standard solution into 50 mL beakers respectively, add water to 35 mL, then add 1 mL of 5% EDTA-2Na solution, shake well, and adjust the pH value to about 2.5 with hydrochloric acid with a volume ratio of 1:1. Add 4 mL of 0.5% DAB solution to each, shake well, place in the dark for reaction for 30 min, then adjust the pH value to neutral with 5% NaOH solution, add to a separatory funnel, and add 10 mL of xylene and shake for 2 min. Let it stand for layering, remove the aqueous layer, collect the organic layer in a cuvette, and measure the absorbance value at 420 nm. As Figure 5 shown, draw a standard curve.

[0055] Centrifuge the fermentation broth at 8000 r / min for 15 min, take 2 mL of the supernatant into a 50 mL beaker, add water to 35 mL. The following steps are the same as those for drawing the standard curve, and at the same time, use the non-selenium-enriched group as a blank control. Zero with the blank control, measure the absorbance value of the sample, and find the corresponding selenium content from the standard curve for the obtained data. This is the residual inorganic selenium content. The result shows that when the inoculum volume is 2%, the selenium enrichment rate reaches the maximum value of 78.39%.

[0056] Example 4

[0057] This example is the application of strain h32 in pickles:

[0058] The strain h32 screened in Example 2 was inoculated into pickles, and the process was as follows:

[0059] Preparation of selenium - enriched lactic acid bacteria: Sodium selenite (final concentration 10 μg / mL) was added to 100 mL of fresh MRS medium. After sterilization, it was cooled to about 40 °C. Lactobacillus plantarum h32 that had been activated was inoculated at a ratio of 1% (V / V) and stirred evenly. Fermentation was carried out at 37 °C for 12 h. Subsequently, centrifugation was performed (4000 g, 5 min). The cells were washed 3 times with sterile water and then dissolved in 50 mL of sterile water to obtain selenium - enriched lactic acid bacteria.

[0060] Select fresh vegetables (such as Chinese cabbage, radish, etc.) → Wash → Drain → Package → Inject water (6% sterilized saline, cooled) → Inoculate the selenium - enriched Lactobacillus plantarum h32 at a ratio of 3% (V / V) → Anaerobic fermentation (at 28 °C).

[0061] The sensory scores of pickles fermented by natural fermentation and by strain h32 are as follows in the table:

[0062]

[0063] As Figure 4 shown, the nitrite content and pH value in the two kinds of pickles were measured every 12 h. After fermentation, the selenium content was measured. When measuring nitrite, only 5 mL of the fermentation broth needed to be replaced with 5 g (accurate to 0.0001 g) of pickles, and the other steps remained unchanged. The results showed that after adding Lactobacillus plantarum strain h32, the degradation rate of nitrite was effectively accelerated, and the organic selenium content reached 0.54 μg / g.

[0064] It should be noted that the instruments and equipment used in the above - mentioned embodiments include but are not limited to: microscope, ultraviolet analyzer, centrifuge, DNA gene amplification instrument, ultra - clean workbench, high - pressure sterilizer, analytical balance, electro - thermostatic incubator;

[0065] The materials and reagents used for screening strains include but are not limited to: distilled water, physiological saline, CaCO3 reagent, 95% ethanol solution, borax, zinc acetate, potassium ferrocyanide, sodium chloride, NaNO2, sulfanilic acid solution, hydrochloric acid naphthyl ethylenediamine solution, MRS broth, agar, bacterial genomic DNA extraction kit.

[0066] It should be noted that the structure described in the present invention can be implemented in many different forms and is not limited to the embodiments. Any equivalent transformation made by those of ordinary skill in the art using the description and drawings of the present invention, or directly or indirectly applied in other related technical fields, such as the loading and unloading of other articles, is included in the protection scope of the present invention.

Claims

1. A Lactobacillus plantarum with the functions of being rich in selenium and degrading nitrite, characterized in that: It is named Lactobacillus plantarum h32, which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit date is November 21, 2023, and the deposit number is: CGMCC No. 29092.

2. Application of the Lactobacillus plantarum with the functions of selenium enrichment and nitrite degradation as described in claim 1 in fermented pickles.

3. Use of Lactobacillus plantarum according to claim 2 in the fermentation of pickled vegetables, characterized in that: A method for preparing pickles, which comprises washing fresh vegetables, draining them, packing them separately, injecting water, inoculating the Lactobacillus plantarum h32 as described in claim 1, and finally performing anaerobic fermentation.

4. Use of Lactobacillus plantarum as described in claim 3 in fermented pickles, characterized in that: The inoculation amount of the Lactobacillus plantarum h32 is 3% V / V.

Images