Lactobacillus plantarum for effectively enriching and removing cadmium under high salt and low pH and application of lactobacillus plantarum

Abstract

The embodiment of the invention belongs to the field of food microorganisms, and particularly relates to lactobacillus plantarum capable of effectively enriching and removing cadmium under high salt and low pH and application of the lactobacillus plantarum. The lactobacillus plantarum is lactobacillus plantarum FJS005, the lactobacillus plantarum is preserved in the China General Microbiological Culture Collection Center on December 26, 2022, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.26254. The lactobacillus plantarum can be used for preparing the lactobacillus plantarum. According to the lactobacillus plantarum and the application thereof provided by the embodiment of the invention, lactobacillus strains are preliminarily separated and screened from fermented food through plate coating, and an excellent strain FJS005 is obtained by determining the enrichment rate of heavy metal cadmium and the total accumulation amount of heavy metal cadmium in unit bacteria; the accumulation amount of heavy metal cadmium in cells and the accumulation amount of heavy metal cadmium on cell surfaces are further determined, oxidative damage of high NaCl and low pH to the strain is determined, and the enrichment mechanism of the strain to heavy metal cadmium is clarified.

Description

Technical Field

[0001] The invention belongs to the field of food microorganisms, and in particular relates to a plant lactobacillus strain capable of effectively enriching and removing cadmium under high salt and low pH conditions and application thereof. Background Art

[0002] The most important environmental pollution is heavy metal pollution. This kind of environmental pollution has been common in recent years. With the rapid development of industry and the intensification of human activities, the content of heavy metals in the atmosphere, soil and water has increased, exceeding the standard range, such as heavy metals cadmium, mercury, lead, etc., which directly lead to the decline of environmental quality. In addition, heavy metals cannot be degraded in the natural environment and organisms. They will flow along the food chain, be absorbed by animals and plants, and gradually accumulate, and eventually flow to humans. Animals and plants are eaten by humans, causing heavy metal pollutants to continue to amplify and accumulate in the food chain. They cannot be decomposed by organisms and endanger human health. The main impact is that heavy metals accumulate in the liver and kidneys of the human body, causing renal failure; replace calcium in the bones, soften bones; cause gastric dysfunction; and cause high blood pressure. Heavy metal cadmium (Cd) is widely distributed in media such as water, atmosphere, soil and food. It is highly toxic, has carcinogenic, mutagenic, genotoxic and immunotoxic effects, and cadmium is non-biodegradable, posing a serious threat to human health. The International Agency for Research on Cancer (IARC) has classified cadmium as a Class I carcinogen, and the World Health Organization (WHO) has classified cadmium as a priority food contaminant. Cadmium is one of the most concerning heavy metals in the soil-plant contact pathway. After cadmium enters the human body, its half-life is 10 to 30 years. Cadmium can cause damage to the kidneys, liver, lungs, bones, etc., and may even cause cancer.

[0003] Because cadmium cannot be biodegraded, before eating food contaminated with heavy metal cadmium, we should first consider removing the heavy metal cadmium. Traditional heavy metal cadmium removal technologies are roughly physical, chemical, and biological. The physical method refers to removing it without changing the chemical structure, such as solidification technology, ion exchange technology, and membrane separation technology. The chemical method is to change its state in water, sludge or soil to remove it, such as chemical precipitation and oxidation-reduction methods. The biological method uses animals, plants or microorganisms to adsorb, transform, enrich metal ions, etc. to reduce the metal concentration in them.

[0004] On the one hand, food is a special type of substance. It contains a large amount of organic compounds and a low content of heavy metal cadmium. Traditional heavy metal removal methods are mostly ineffective for food. Therefore, the technology of microbial enrichment of heavy metal cadmium came into being. On the other hand, in the food industry and polluted water, high NaCl and low pH environments are extremely common. In actual operations, high NaCl and low pH will affect the growth of microorganisms and hinder the enrichment of heavy metals by microorganisms. Therefore, finding salt-resistant and acid-resistant microorganisms is of great significance to actual production operations.

[0005] Microbial adsorption is to use the structure of microorganisms to adsorb heavy metal ions on the cell surface, or to absorb necessary nutrients to absorb heavy metal ions autonomously, and to enrich heavy metal ions on the cell surface or inside. Compared with traditional technologies, microbial adsorption has several advantages: First, microbial adsorption can enrich heavy metal ions without introducing new heavy metal ions and generating no secondary pollution. Second, biological adsorption can treat low-content heavy metal pollutants, reaching mg / L level, and has extremely high adsorption efficiency. Third, biological adsorption technology has low operating costs and requires little capital.

[0006] There are many conditions for microorganisms to enrich heavy metal cadmium ions, and there are roughly several factors: First, pH value is an important factor affecting microbial enrichment. Secondly, inorganic salt concentration is another important factor affecting microbial enrichment. In some environments, the inorganic salt content is extremely high, and the performance of microorganisms in removing heavy metals is different.

[0007] Therefore, it is of great significance to provide a plant lactobacillus that has the ability to enrich and remove heavy metal cadmium in the presence of high salt and low pH. Summary of the invention

[0008] To solve the above problems, the purpose of the present invention is to provide a Lactobacillus plantarum strain that can effectively enrich and remove cadmium under high salt and low pH conditions and its application.

[0009] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions: According to the first aspect of an embodiment of the present invention, a strain of Lactobacillus plantarum that effectively enriches and removes cadmium under high salt and low pH is provided, wherein the Lactobacillus plantarum is Lactobacillus plantarum FJS005 (Lactobacillus plantarum), and the Lactobacillus plantarum was deposited in the General Microbiology Center of China National Culture Collection Administration on December 26, 2022, with a storage address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a storage number of CGMCC NO.26254.

[0010] Furthermore, the 16S rDNA of the Lactobacillus plantarum is shown as SEQ ID NO.1.

[0011] According to a second aspect of an embodiment of the present invention, a fermentation method for preparing Lactobacillus plantarum is provided, comprising the following steps: S1, amplifying and culturing the Lactobacillus plantarum and collecting the product; S2. The product obtained in step S1 is subjected to freeze centrifugation, spraying or freeze drying to obtain the Lactobacillus plantarum.

[0012] Furthermore, the method for amplifying the culture of Lactobacillus plantarum includes inoculating the Lactobacillus plantarum into an MRS liquid culture medium at an inoculation rate of 1% to 5% for culture.

[0013] Furthermore, the culture temperature is 37° C. and the culture time is 24-48 hours.

[0014] According to a third aspect of the embodiments of the present invention, a bacterial agent is provided, wherein the bacterial agent contains the above-mentioned Lactobacillus plantarum.

[0015] Furthermore, the bacterial agent includes a fermentation agent.

[0016] According to a fourth aspect of an embodiment of the present invention, there is provided a method for preparing a microbial preparation enriched in heavy metal cadmium, comprising compounding any one of the above-mentioned Lactobacillus plantarum with a prebiotic to obtain the microbial preparation enriched in heavy metal cadmium.

[0017] According to a fifth aspect of an embodiment of the present invention, there is provided a method for preparing a postbiotic product enriched with heavy metal cadmium, comprising sterilizing the fermentation broth after fermentation by any of the above-mentioned Lactobacillus plantarum at 63° C. for 30 minutes, and spraying or freeze-drying to obtain the postbiotic product.

[0018] According to a sixth aspect of the embodiments of the present invention, there is provided a significant enrichment effect of Lactobacillus plantarum on heavy metal cadmium under high NaCl and low pH conditions.

[0019] Compared with the prior art, the advantages of the embodiments of the present invention are: The embodiment of the present invention provides a plant lactobacillus that can effectively enrich and remove cadmium under high salt and low pH conditions and an application thereof. Lactic acid strains are preliminarily separated and screened from fermented foods by plate coating, and an excellent strain FJS005 is obtained by determining the heavy metal cadmium enrichment rate and the total accumulation of heavy metal cadmium per unit cell. The accumulation of heavy metal cadmium in cells and on the cell surface is further determined, and the oxidative damage to the bacteria caused by high NaCl and low pH is simultaneously determined, so as to clarify the enrichment mechanism of heavy metal cadmium. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings: Figure 1 is a phylogenetic tree constructed in a typical embodiment of the present invention; Figure 2 This is the effect of high concentration of NaCl on the MDA content in Lactobacillus plantarum when enriching heavy metal cadmium; Figure 3This is the effect of high concentration of NaCl on SOD when Lactobacillus plantarum enriches heavy metal cadmium; Figure 4 This is the effect of high concentration of NaCl on the POD activity of the bacteria when enriching heavy metal cadmium; Figure 5 This is the effect of low pH on the MDA content in the cells of the bacteria when enriching heavy metal cadmium; Figure 6 This is the effect of low pH on the SOD of the bacteria when enriching heavy metal cadmium; Figure 7 This is the effect of low pH on the POD activity of the bacteria when enriching heavy metal cadmium; Figure 8 It is the antagonism diagram between Lactobacillus casei and Lactobacillus pentosus; Fig. 9 This is a diagram of the antagonistic experiment between Lactobacillus casei and Lactobacillus plantarum; Fig.10 This is a diagram of the antagonism experiment between Lactobacillus pentosus and Lactobacillus plantarum. DETAILED DESCRIPTION

[0021] In view of the deficiencies in the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution, its implementation process and principle will be further explained as follows.

[0022] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0023] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0024] The embodiment of the present invention provides a plant lactobacillus that effectively enriches and removes cadmium under high salt and low pH conditions, wherein the plant lactobacillus is plant lactobacillus FJS005, and the plant lactobacillus was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 26, 2022, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCCNO.26254. The 16S rDNA of the plant lactobacillus is shown in SEQ ID NO.1.

[0025] In order to better understand the technical solution of the present invention, it is described in detail below in conjunction with specific embodiments.

[0026] Example 1 Isolation and screening of lactic acid bacteria that accumulate heavy metal cadmium (1) Sample collection: Fermented fish, fish sauce, shrimp paste, yogurt, and kimchi were collected from food companies. The batches with better quality and longer shelf life were selected compared with other batches, and then the samples from these batches were screened for lactic acid strains.

[0027] (2) Sample processing: Take 25 g of fermented solid aquatic products under sterile conditions, cut them into pieces, add them to 225 mL of sterile saline and homogenize them with a slapping homogenizer; take 25 mL of sauerkraut juice and 25 mL of yogurt and add them to 225 mL of sterile saline and homogenize them. Then perform a 10-fold serial dilution, select the sample solution with the appropriate dilution and apply it to the MRS containing calcium carbonate to isolate the colonies with calcium-soluble rings, and purify them repeatedly to obtain pure strains of lactic acid bacteria as the initial screening strains.

[0028] (3) Inoculate the primary screening strains stored at 4°C onto MRS slants and culture at 37°C for 24 h. Scrape a small amount of bacteria and inoculate into MRS liquid and culture at 37°C for 24 h.

[0029] (4) Isolation of lactic acid bacteria that accumulate heavy metal cadmium High salt conditions: 100 ml of MRS broth was prepared into a 0.1 mmol / L heavy metal solution, and then NaCl (0, 10, 20, 40, 60 g / L) was prepared in each 150 ml conical flask. Under low pH conditions, prepare 0.1 mmol / L heavy metal solution with 100 mL of MRS broth, and then prepare pH (2, 3, 4, 5, 6) in each 150 mL conical flask.

[0030] Inoculate the activated lactic acid bacteria in high-salt and low-pH MRS broth to make the initial bacterial concentration reach 0.1 g / L, and culture at 37 °C for 48 h. Centrifuge for 15 min at 8000-10000 r / min, wash the lower sediment twice with sterile water, weigh the wet weight, and calculate the biomass M (g / L). Take the supernatant and add a certain volume of mixed acid (HNO3 and HClO4 volume ratio of 4:1), heat and digest thoroughly, and dilute appropriately to measure the Cd content using an atomic absorption spectrophotometer. 2+ Concentration. The heavy metal cadmium enrichment rate R (%) in the solution is calculated according to the following formula: R=(1- Ct / C0)×100% C0: Heavy metal cadmium concentration in the initial MRS liquid culture medium (mmol / L) Ct: Ending concentration of heavy metal cadmium in MRS liquid culture medium (mmol / L) Finally, the strain FJS005 with the best enrichment effect for heavy metal cadmium in the presence of high salt (at a salt concentration of 20 g / L, the enrichment rate of cadmium was as high as 74%) and low pH (at pH 5, the enrichment rate of cadmium was as high as 70%) was screened out as an excellent strain.

[0031] Example 2 Identification of excellent lactic acid strains that are enriched in heavy metal cadmium (1) Physiological and biochemical identification of strains The physiological and biochemical identification of FJS005 was carried out using a microbiochemical kit. It was inoculated into biochemical identification tubes of esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, and raffinose, respectively, and cultured at 37°C for 24 hours to identify the results. The identification results are shown in Table 1.

[0032] Table 1 Physiological and biochemical reaction results of strain FJS005

[0033] The identification results were consistent with the biochemical reaction characteristics of Lactobacillus plantarum.

[0034] (2) Molecular biological identification of strains The template DNA of the strain was extracted using a genome extraction kit, and 1 μL of the template solution was added to 50 μL of the PCR mixture to amplify the 16S r DNA of the strain. 6 μL of the PCR product was subjected to agarose gel electrophoresis at 120 V / min for 30 min, and the electrophoresis bands were observed using a gel imaging analysis system. The PCR product was sequenced and analyzed by Shanghai Shenggong Biotechnology Co., Ltd. After sequencing, the 16S rDNA sequence of the strain was obtained, with a length of 1450 kb. The sequencing results were searched using BLAST on NCBI, and the gene sequences were compared with the sequences in the GenBank database for homology, and a phylogenetic tree was constructed as shown in Figure 2. Figure 1 As shown, the bacteria was classified as Lactobacillus plantarum.

[0035] The 16S rDNA sequence of the strain is shown in SEQ ID NO.1: Example 3 Effect of high concentration of NaCl on cadmium resistance and cadmium accumulation of Lactobacillus plantarum FJS005 Prepare 100 ml of MRS broth into a 0.1 mmol / L heavy metal solution, and then prepare NaCl (0, 10, 20, 40, 60 g / L) in each 150 ml conical flask. Inoculate strain FJS005 in the MRS broth to make the initial bacterial concentration reach 0.1 g / L, culture at 37 °C for 48 h, and centrifuge at 8000-10000 r / min for 15 min. Separate the supernatant and the lower precipitate, wash the lower precipitate twice with sterile water, weigh the wet weight, and calculate the biomass M (g / L). Take the supernatant and add a certain volume of mixed acid (HNO3 and HClO4 volume ratio of 4:1), heat and digest thoroughly, and dilute appropriately to measure the Cd content using an atomic absorption spectrophotometer. 2+ Concentration. The total cadmium accumulation per unit cell Q (μmol / g) was calculated according to the following formula: Q = (C0-Ct) / M C0: Heavy metal cadmium concentration in the initial MRS liquid culture medium (mmol / L) Ct: Ending concentration of heavy metal cadmium in MRS liquid culture medium (mmol / L) M: Biomass under different cultivation conditions (g) After the bacteria were weighed, they were resuspended in a 20 mmol / L EDTA solution and shaken to desorb the heavy metal cadmium accumulated on the cell surface. The EDTA solution was centrifuged (8000-10000 r / min, 15 min) to separate the upper solution from the lower precipitate. The upper solution was diluted and the Cd content was measured using an atomic absorption spectrophotometer. 2+ The amount is the amount of heavy metal accumulation on the cell surface; after adding mixed acid (nitric acid and perchloric acid) to the lower sediment for high temperature wet digestion, the Cd in it is measured by atomic absorption spectrophotometer after dilution. 2+ The concentration is the amount of heavy metal accumulation in cells.

[0036] Table 2 Effects of high concentration of NaCl on cadmium resistance and cadmium accumulation of Lactobacillus plantarum FJS005

[0037] Note: M---biomass, R---heavy metal cadmium enrichment rate, Q---total cadmium accumulation per unit cell, I---heavy metal cadmium accumulation in cells, C---heavy metal accumulation on the cell surface. Different lowercase letters abcde indicate significant differences between the values ​​(P<0.05).

[0038] As can be seen from Table 2, when the culture medium contains 0.1 mmol / L cadmium ions, the biomass of Lactobacillus plantarum FJS005 first remains unchanged and then gradually decreases with the increase of NaCl concentration. This shows that a certain concentration of NaCl (10 g / L - 20 g / L) has little effect on the cadmium resistance of the bacteria, and only excessively high concentrations of NaCl (40 g / L - 60 g / L) will reduce the cadmium resistance of the bacteria, but the NaCl in general food and environment will not exceed 20 g / L.

[0039] For Lactobacillus plantarum FJS005, within a relatively low concentration of NaCl (<20 g / L), as the NaCl concentration increased, the total accumulation of cadmium per bacterial cell and the accumulation of heavy metal cadmium on the cell surface increased significantly, and there was no significant change in the accumulation of heavy metal cadmium in the cell. This indicates that the total accumulation of cadmium per bacterial cell and the accumulation of heavy metal cadmium on the cell surface of Lactobacillus plantarum FJS005 are both promoted by NaCl within a certain range.

[0040] Within a certain range of NaCl concentration (<20 g / L), Lactobacillus plantarum FJS005 is a good strain for enriching heavy metal cadmium. The enrichment of heavy metal cadmium by this bacterium is mainly on the cell surface, and the accumulation of heavy metal cadmium inside the cell is relatively small.

[0041] Example 4 Effect of low pH on cadmium resistance and cadmium accumulation of Lactobacillus plantarum FJS005 Prepare 100 ml of MRS broth to make a 0.1 mmol / L heavy metal solution, and then prepare a solution containing pH (2, 3, 4, 5, 6) in each 150 ml conical flask. Inoculate strain FJS005 in the MRS broth to make the initial bacterial concentration reach 0.1 g / L, culture at 37 °C for 48 h, and centrifuge at 8000-10000 r / min for 15 min. Separate the supernatant and the lower precipitate, wash the lower precipitate twice with sterile water, weigh the wet weight, and calculate the biomass M (g / L). Take the supernatant and add a certain volume of mixed acid (HNO3 and HClO4 volume ratio of 4:1), heat and digest thoroughly, and dilute appropriately to measure the Cd content using an atomic absorption spectrophotometer. 2+ Concentration. The total cadmium accumulation per unit cell Q (μmol / g) was calculated according to the following formula: Q = (C0-Ct) / M C0: Heavy metal cadmium concentration in the initial MRS liquid culture medium (mmol / L) Ct: Ending concentration of heavy metal cadmium in MRS liquid culture medium (mmol / L) M: Biomass under different cultivation conditions (g) After weighing the bacteria, resuspend them in a 20 mmol / L EDTA solution and shake them to desorb the heavy metal cadmium accumulated on the cell surface. Use a centrifuge (8000~10000 r / min, 15 min) to separate the upper solution from the lower precipitate. After diluting the upper solution, use an atomic absorption spectrophotometer to measure the amount of Cd2+ in it, which is the amount of heavy metal accumulation on the cell surface; add mixed acid (nitric acid and perchloric acid) to the lower precipitate for high-temperature wet digestion, and then dilute it and use an atomic absorption spectrophotometer to measure the Cd 2+ The concentration is the amount of heavy metal accumulation in cells.

[0042] Table 3 Effects of low pH on cadmium resistance and cadmium accumulation of Lactobacillus plantarum FJS005

[0043] Note: M---biomass, R---heavy metal cadmium enrichment rate, Q---total cadmium accumulation per unit cell, I---heavy metal cadmium accumulation in cells, C---heavy metal accumulation on the cell surface. Different lowercase letters abcde indicate significant differences between the values ​​(P<0.05).

[0044] As can be seen from Table 3, when the medium contains 0.1 mmol / L cadmium, in the low pH range (2-6), the biomass of Lactobacillus plantarum FJS005 increases significantly with the increase of pH, indicating that in the low pH range (2-6), the relatively high pH (3-6) improves the cadmium resistance of the bacteria. The pH of most acidic foods and environments is 4-6, so this bacterium is a good strain for enriching heavy metal cadmium in food and environment.

[0045] In the low pH range (2-6), as the pH increased, the total accumulation of cadmium per unit cell, and the accumulation of heavy metal cadmium in cells and on the cell surface of Lactobacillus plantarum FJS005 increased significantly, indicating that in the low pH range (2-6), the relatively high pH (3-6) improved the enrichment effect of the bacteria on cadmium. It may be that the relatively high pH increased the cadmium resistance of Lactobacillus plantarum FJS005, promoted the increase of total biomass, and enhanced the enrichment effect.

[0046] Example 5 Effect of high salt on oxidative damage of Lactobacillus plantarum FJS005 100 mL of MRS broth was prepared into a 0.1 mmol / L heavy metal solution, and then NaCl (0, 10, 20, 40, 60 g / L) was prepared in each 150 mL conical flask. The strain FJS005 was inoculated in the MRS broth to make the initial bacterial concentration reach 0.1 g / L, and cultured at 37 °C for 48 h. Centrifuge (8000-10000 r / min, 15 min). The precipitate was washed twice with PBS solution, resuspended in PBS solution, and the cells were broken by ice-bath ultrasonication (400 W, 5 s, interval 5 s, 15 min), and centrifuged at 4 °C, 10000 r / min for 20 min, and the supernatant was aspirated. The supernatant was collected and the intracellular MDA, SOD, and POD contents were determined using malondialdehyde, total superoxide dismutase, and peroxidase detection kits.

[0047] Cells produce oxygen free radicals through non-enzymatic and enzymatic systems. The former can destroy polyunsaturated fatty acids in biological membranes, leading to lipid peroxidation and the formation of lipid peroxides such as malondialdehyde (MDA). Lipid peroxidation not only converts reactive oxygen into active chemical agents, but also amplifies reactive oxygen through chain or chain branching reactions, which may lead to cell metabolism and dysfunction, and in severe cases, death. Therefore, malondialdehyde (MDA) is often used as a marker to evaluate the degree of lipid oxidative damage in cells, thereby indirectly reflecting the degree of cell damage. Figure 2 It can be seen that when the culture medium contains 0.1mmol / L cadmium ions, within the range of NaCl content (10-40 g / L), the MDA content of the bacteria has an upward trend compared with no NaCl. When the NaCl concentration increases to 60 g / L, the MDA content decreases significantly. This indicates that as the NaCl concentration increases, the oxidation and damage of lipids are first promoted and then inhibited.

[0048] In order to reduce oxidative damage to cells, cells themselves form a relatively complex antioxidant defense system. One is non-enzymatic substances, such as uric acid, glutathione, and vitamin E; the other is antioxidant enzymes, such as SOD, CAT, and POD. SOD can remove superoxide anion free radicals (O 2- ), to avoid damage. Therefore, the activity of SOD is also regarded as the antioxidant capacity of cells.

[0049] from Figure 3It can be seen that when the culture medium contains 0.1mmol / L cadmium ions, with the increase of NaCl concentration, the influence of different concentrations of NaCl is significantly different (P<0.05), and the SOD content of the bacteria remains unchanged at first, and then significantly decreases. This shows that when the NaCl concentration is lower than 10 g / L, NaCl has no obvious inhibitory effect on the production of SOD of the bacteria, which can prevent the cells from being damaged by cadmium. When the NaCl concentration is higher than 10g / L, the production of SOD of the bacteria begins to decrease. Therefore, under a certain concentration of NaCl (<10g / L), the bacteria has a greater resistance to cadmium and is a good strain for enriching heavy metal cadmium.

[0050] The content of peroxidase (POD) can reflect the antioxidant capacity of cells, remove superoxide in cells, and thus protect cells. Figure 4 It can be seen that when the culture medium contains 0.1 mmol / L cadmium ions, as the NaCl concentration increases, the effects of different concentrations of NaCl are significantly different (P < 0.05), and the POD content of the bacteria first increases and then decreases, but is higher than when no NaCl is added. This shows that NaCl promotes the production of POD of the bacteria, avoids cadmium damage, and increases the resistance of the bacteria to cadmium. Therefore, the bacteria is a good strain for enriching heavy metal cadmium under high salt conditions.

[0051] Example 6 Effect of low pH on oxidative damage of Lactobacillus plantarum FJS005 100 ml of MRS broth was prepared into a 0.1 mmol / L heavy metal solution, and then pH (2, 3, 4, 5, 6) was prepared in each 150 ml conical flask. The strain FJS005 was inoculated into the MRS broth to make the initial bacterial concentration reach 0.1 g / L, cultured at 37 °C for 48 h, and centrifuged in a centrifuge (8000-10000 r / min, 15 min). The precipitate was washed twice with PBS solution, resuspended in PBS solution, and the cells were broken by ice-bath ultrasonication (400 W, 5 s, interval 5 s, 15 min), and centrifuged at 4 °C, 10000 r / min for 20 min, and the supernatant was aspirated. The supernatant was collected and the intracellular MDA, SOD, and POD contents were determined using malondialdehyde, total superoxide dismutase, and peroxidase detection kits.

[0052] from Figure 5It can be seen that when the culture medium contains 0.1mmol / L cadmium ions and the pH is 2, the MDA content is the lowest, below 0.5nmol / mgprot. This indicates that pH 2 inhibits the production of MDA in the bacteria, inhibits the oxidative damage of cadmium to lipids in cells, and improves the resistance of the bacteria to cadmium. However, when the pH is 3-6, the production of MDA in the cells increases significantly, but the MDA value is still very low, all below 2nmol / mgprot. Therefore, under low pH conditions, the bacteria is a good strain for enriching heavy metal cadmium.

[0053] from Figure 6 It can be seen that when the culture medium contains 0.1 mmol / L cadmium ions and the pH is 2, 3, and 5, the bacteria still has a strong antioxidant capacity, and the SOD content is above 8U / mL. When the pH is 4 and 6, the SOD content increases significantly, reaching a maximum of 26.45U / mL. Therefore, pH 4 and 6 enhance the bacteria's resistance to cadmium.

[0054] from Figure 7 It can be seen that when the culture medium contains 0.1 mmol / L cadmium ions, the POD content of the bacteria does not change significantly as the pH decreases. When the pH continues to decrease to 3, the POD content increases significantly. Therefore, when the pH is 3, the resistance to cadmium is significantly improved, and the damage to the cells caused by cadmium is reduced. Therefore, under low pH conditions, especially when the pH is around 3, the bacteria is a good strain for enriching heavy metal cadmium.

[0055] Example 7 Preparation of plant lactobacillus FJS005 powder and microecological preparation of the present invention Lactobacillus plantarum FJS005 was inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours. Then, 1% inoculation amount was inoculated into MRS liquid culture medium for expansion culture at 37°C for 12-24 hours. The obtained Lactobacillus plantarum FJS005 expansion culture solution was subjected to freeze centrifugation, spraying or freeze drying to obtain Lactobacillus rhamnosus powder. Lactobacillus plantarum powder was compounded with prebiotic inulin, oligoxylose and oligomaltose to prepare a microbial preparation enriched with heavy metal cadmium, and the number of viable bacteria in the microbial preparation was 10-1000 billion / g.

[0056] Example 8 Preparation of postbiotics of Lactobacillus plantarum FJS005 of the present invention Lactobacillus plantarum FJS005 was inoculated into MRS liquid culture medium at a 1% inoculum rate and cultured at 37°C for 24-48 h. The obtained Lactobacillus plantarum FJS005 culture solution was sterilized at 63°C for 30 min, and then sprayed or freeze-dried to obtain the strain FJS005 that can be used as a postbiotic for enriching heavy metal cadmium.

[0057] Example 9 Antagonistic study between Lactobacillus plantarum FJS005 and Lactobacillus casei and Lactobacillus pentosus The fermented strains were inoculated into the culture medium and cultured on a shaker for 16 h. The OD600 of each bacterial solution was measured and the number of viable bacteria was calculated. First, 100 μL of the bacterial solution was evenly spread on the MRS solid culture medium. Three sterilized circular filter papers with a diameter of 6 mm were placed on the surface of the culture medium. 4 μL of the indicator bacterial solution was dripped on the filter paper. The culture was kept at 37 °C for 48 h. The antagonism was measured. The antagonism results were as follows: Figure 8-10 As shown, there is no antagonism between the three bacteria, and they can be used together for composite bacterial fermentation. Figure 8 It is the antagonism diagram between Lactobacillus casei and Lactobacillus pentosus; Fig. 9 This is a diagram of the antagonistic experiment between Lactobacillus casei and Lactobacillus plantarum; Fig.10 This is a diagram of the antagonistic experiment between Lactobacillus pentosus and Lactobacillus plantarum. Among them, Lactobacillus casei and Lactobacillus pentosus were purchased from the China Industrial Microbiological Culture Collection Center.

[0058] Example 10: Brewing low-cadmium fruit and vegetable beverages using the composite strains of Lactobacillus plantarum FJS005 of the present invention in combination with Lactobacillus casei and Lactobacillus pentosus Cadmium is one of the most concerned heavy metals in the soil-plant contact pathway, and its content in fruits and vegetables is relatively high. The plant lactobacillus FJS005 of the present invention is compounded with Lactobacillus casei and Lactobacillus pentosus to brew low-cadmium fruit and vegetable beverages using composite strains. In this study, fresh fruits and vegetables are washed and juiced, and 70% fruit and vegetable juice, 25% water, and 5% white sugar are added according to the mass fraction ratio, followed by high-temperature instant sterilization. After high-temperature heat sterilization at 140°C for 2 seconds, the temperature is immediately cooled to about 37°C, and strains (plant lactobacillus FJS005: Lactobacillus pentosus: Lactobacillus casei = 1:1:1, inoculation amount 8%) are added, and the temperature is kept constant at 37°C for 48 hours, and then the mixture is allowed to stand and blended (5% white sugar, 0.2% citric acid, 0.2% stabilizer (CMC)), the precipitate is filtered out, and the mixture is sterilized again at 85°C for 15 min. Cool down, can seal, and label.

[0059] Example 11 Study on processing low-cadmium kimchi using Lactobacillus plantarum FJS005 of the present invention The invention uses Lactobacillus plantarum FJS005 and Lactobacillus casei and Lactobacillus pentosus to brew low-cadmium kimchi using composite bacteria. Edible salt is dissolved in cold boiled water, allowed to stand and precipitate, the salt water concentration is 3%, filtered and poured into a kimchi jar, 2% white sugar is added, and appropriate amounts of aged vinegar and cooking wine are dissolved to make kimchi water, which is covered and stored for standby use. The vegetables and auxiliary materials are washed with clean water, fished out, drained, dried, and cut. The cut vegetables and auxiliary materials are placed in a kimchi jar in order, kimchi water is added, inoculated (Lactobacillus plantarum FJS005 Lactobacillus casei: Lactobacillus pentosus = 1:1:1, inoculation amount 6%), and fermented at room temperature for 7-10 days. The kimchi water is required to overflow the vegetables, and then sealed with a plastic film or the like.

[0060] Example 12 Research and production of low-cadmium fruit and vegetable enzyme yogurt using Lactobacillus plantarum FJS005 of the present invention The plant lactobacillus FJS005 of the present invention is compounded with thermophilic streptococcus: Lactobacillus bulgaricus to brew low-cadmium fruit and vegetable enzyme yogurt using composite bacteria. Fruit and vegetable enzyme refers to a fermentation product containing biologically active substances obtained by reasonably selecting food materials and fermentation processes to make various fruits undergo deep fermentation. Enzymes have a very large effect on the human body. They can improve the dynamic balance of intestinal microorganisms in the body, promote the metabolism of the digestive system, inhibit the invasion of harmful bacteria and toxins, further assist various nutrients to be absorbed by the human body, and create an excellent environment for the reproduction of probiotics in the body. Using the plant lactobacillus FJS005 of the present invention to study and manufacture probiotic low-cadmium fruit and vegetable enzyme yogurt can improve the health and safety of yogurt, and at the same time is of great significance for developing new varieties of dairy products, expanding the dairy product market, and improving the people's tools.

[0061] (1) Preparation of fruit and vegetable enzymes: Wash fresh fruits and vegetables with purified water, drain and cut into pieces. Fruits and vegetables are added to white sugar in a sterilized fermentation container at a mass ratio of about 2:1. A layer of fruits and vegetables and a layer of sugar are placed in the fermentation container. Lemons are placed on the bottom and last layer to fill the container to 80% of its capacity. Ferment at a constant temperature of 25°C for 2-3 months. During the fermentation period, pay attention to degassing. When the fruits and vegetables sink completely, the fermentation liquid is clear, no gas is generated, and the pH is about 3-4, the fermentation is over, and the fruit enzyme is obtained by filtration.

[0062] (2) Preparation of fruit and vegetable enzyme yogurt Standardize the raw milk, add 8% white sugar, heat to 65℃, stir and dissolve thoroughly, homogenize (homogenization pressure is 20-25 MPa), sterilize (95℃ sterilization for 5-10min), immediately cool to 42℃, add bacteria (Lactobacillus plantarum FJS005: Streptococcus thermophilus: Lactobacillus bulgaricus = 1:1:1, inoculation amount 6%), about 3%-4% enzyme, ferment at 42℃ for about 6 hours, refrigerate at 4℃ overnight after complete solidification, and make low-cadmium fruit and vegetable enzyme yogurt containing FJS005.

[0063] Example 13 Research and production of low-cadmium fruit and vegetable soluble beans using Lactobacillus plantarum FJS005 of the present invention The plant lactobacillus FJS005 of the present invention is compounded with thermophilic streptococcus and bulgaric lactobacillus to brew low-cadmium fruit and vegetable soluble beans using composite strains. The raw milk is standardized, 8% white sugar is added, the temperature is raised to 65°C, fully stirred and dissolved, homogenized (homogenization pressure is 20-25 MPa), sterilized (95°C sterilization for 5-10min), immediately cooled to 42°C, strains (plant lactobacillus FJS005: thermophilic streptococcus: bulgaric lactobacillus = 1:1:1, inoculation amount 6%) are added, fermented at 42°C for about 6 hours, and refrigerated at 4°C overnight after complete solidification to prepare yogurt containing FJS005. The fruits and vegetables are washed, peeled, chopped, steamed, pre-frozen at -20°C for 48 hours, and then transferred to a -80°C freeze dryer for freeze drying for 72 hours, taken out, ground, and sieved to obtain a finer freeze-dried powder. The fruit and vegetable powder was stirred into a paste (fruit and vegetable powder: water = 1:3). Using sensory score as an indicator, single factor analysis was performed on the amount of yogurt added (20, 30, 40, 50, 60 g), the amount of light cream added (6, 10, 14, 18, 22 g), and the amount of milk powder added (1, 2, 3, 4, 5 g). The result showed that 40.00 g of yogurt, 14.00 g of light cream, and 4 g of milk powder were the best.

[0064] Response surface optimization: The addition amount of yogurt (35, 40, 45g), light cream (12, 14, 16g), and milk powder (3, 4, 5g) were selected for response surface, and the sensory score was used as an indicator for response surface optimization. The validation of the significant difference of the response surface quadratic regression equation is shown in Table 4, and the variance analysis of the response surface quadratic regression equation is shown in Table 5. Among them, thermophilic Streptococcus and Bulgarian Lactobacillus were purchased from the China Industrial Microbiological Culture Collection Center.

[0065] Table 4 Verification of significant difference of response surface quadratic regression equation

[0066] Note: A is the amount of yogurt added; B is the amount of light cream added; C is the amount of milk powder added.

[0067] Depend on Figure 3 and4 It can be seen that the F value of the quadratic regression equation is 37.94, which means that the probability due to error is only 0.01%, and the model is significant. "Probability greater than F value" is less than 0.0500, indicating that the model item is significant.

[0068] The F value of the lack of fit term is 1.28, indicating that the lack of fit is not significant relative to the pure error. The p value of the lack of fit term is 0.3954, which indicates that the model fits the data well and there is no significant lack of fit.

[0069] Table 5 Analysis of variance of response surface quadratic regression equation

[0070] Table 5 shows that the R-square value predicted by the model is 0.8266, and the adjusted R-square value is 0.9541. Adeq Precision is used to measure the signal-to-noise ratio. Adeq Precision greater than 4 is ideal. The Adeq Precision of this model is 24.6478, indicating sufficient signal. In summary, this regression equation can be used to determine the basic formula of soy bean dissolving. The optimal formula is 44.98g of yogurt, 13.49g of whipped cream, and 3.98g of milk powder. The response surface score is 90.66.

[0071] After three verifications, the product was finally made: 45.00g of yogurt, 13.50g of light cream, 4.00g of milk powder, and 10.00g of fruit and vegetable pulp (pumpkin pulp, kiwi pulp and blueberry pulp, etc.) were added respectively. After sufficient stirring, they were injected into the mold, pre-frozen at -20℃ for 48h, demolded, placed in a container for secondary freeze-drying, and taken out after 72h at -80℃ to obtain the final product.

[0072] The embodiments described above are only for illustrating the preferred implementation of the present invention. It should be pointed out that, for ordinary technicians in this field, several improvements can be made without departing from the scope of protection of this patent, and these improvements should also be regarded as within the scope of protection of this patent.

Claims

1. A plant lactobacillus that effectively enriches and removes cadmium under high salt and low pH conditions, characterized in that: The plant lactobacillus is plant lactobacillus FJS005, which was deposited in the General Microbiology Center of China National Culture Collection Administration on December 26, 2022. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.26254.

2. Plant Lactobacillus according to claim 1, characterized in that: The 16S rDNA sequence of the Lactobacillus plantarum is shown in SEQ ID NO.

1.

3. A fermentation method for preparing Lactobacillus plantarum as claimed in claim 2, characterized in that: The following steps are involved: S1, amplifying and culturing the Lactobacillus plantarum according to claim 2, and collecting the product; S2. The product obtained in step S1 is subjected to freeze centrifugation, spraying or freeze drying to obtain the Lactobacillus plantarum.

4. The fermentation preparation method according to claim 3, characterized in that: The method for amplifying the cultivation of Lactobacillus plantarum comprises inoculating the Lactobacillus plantarum into an MRS liquid culture medium at an inoculation rate of 1% to 5% for cultivation.

5. The fermentation preparation method according to claim 4, characterized in that: The culture temperature is 37° C. and the culture time is 24-48 hours.

6. A bacterial agent, characterized in that: The bacterial agent contains the plant lactobacillus according to any one of claims 1 to 2.

7. The bacterial agent according to claim 6, characterized in that: The bacterial agent includes a fermentation agent.

8. A method for preparing a microbial preparation enriched with heavy metal cadmium, characterized in that: The method comprises compounding the plant lactobacillus described in any one of claims 1 to 2 with prebiotics to prepare the microbial preparation enriched with heavy metal cadmium.

9. A method for preparing a postbiotic product enriched with heavy metal cadmium, characterized in that: The method comprises sterilizing the fermentation liquid after fermentation of the plant lactobacillus according to any one of claims 1 to 2 at 63° C. for 30 minutes, and spraying or freeze-drying to obtain the postbiotic product.

10. A significant enrichment effect of Lactobacillus plantarum on heavy metal cadmium under high NaCl and low pH conditions as described in any one of claims 1 to 2.

Images