Marine-derived lactobacillus rhamnosus and application thereof

Abstract

The invention provides marine-derived lactobacillus rhamnosus S400M01 and application thereof, and relates to the technical field of heavy metal adsorption and removal, the marine-derived lactobacillus rhamnosus S400M01 is preserved in Guangdong Microbial Culture Collection Center (GDMCC) on November 28, 2022, the preservation address is Guangzhou, and the number is GDMCCNO: 63001. The strain can tolerate cadmium chloride concentration up to 1000 mg / L, can tolerate lead concentration up to 800 mg / L, can adsorb and remove heavy metals cadmium and lead in squid viscera, and reduces accumulation of cadmium and lead in viscera of live crucian in a heavy metal polluted water body.

Description

Technical Field

[0001] The invention relates to the technical field of heavy metal adsorption and removal, and in particular to marine-derived Lactobacillus rhamnosus and application thereof. Background Art

[0002] Harmful heavy metals, especially cadmium and lead, are extremely toxic heavy metal elements to humans, animals and plants. Due to the development of industry and mining and the irrational use of pesticides and fertilizers, toxic heavy metal wastewater has increased day by day, causing dysfunction of various biological organisms. After entering the human body through the food chain, it can cause various diseases. Cadmium and lead can be enriched in the internal organs of animals and plants, enter the human body through the food chain, destroy the molecular structure of protein molecules, and cause acute and chronic poisoning in the human body. For example, cadmium can interfere with the body's absorption of calcium and phosphorus, causing abnormal metabolism of vitamin D, thereby causing kidney and bone lesions.

[0003] In my country, the scale of fish processing is growing day by day. However, with the unsustainable decline of fish resources caused by overfishing, excessive competition in deep-sea fishing, and unstable production due to seasonal and environmental influences, there is an urgent need to rationally utilize and develop existing fish resources to achieve high-value development. At present, the treatment method of fish processing waste in my country is relatively simple. Taking advantage of the easy decay of these wastes, they are directly buried on the spot, and some are dried and made into feed or processed into fish meal. However, due to heavy metal pollution, the heavy metal content in most fish viscera exceeds the standard, so there are certain difficulties in their development and utilization. Therefore, it is particularly important to remove heavy metals from fish. Compared with traditional physical and chemical methods, microbial methods are efficient, economical, simple, and highly feasible, so they have broad application prospects. Probiotics are the dominant flora in the human intestine and are widely used in food processing and production, health product development, disease treatment, and microecological preparations.

[0004] In recent years, more and more scholars have discovered that probiotics have the ability to absorb heavy metals and can effectively absorb heavy metals in food. Therefore, in-depth analysis of the adsorption mechanism of probiotics on heavy metals is of great significance in ensuring food safety and improving the nutritional value of food. Summary of the invention

[0005] The invention aims to provide a marine-derived Lactobacillus rhamnosus, which can survive in an environment with high concentrations of cadmium and lead, prevent heavy metal poisoning, absorb and remove heavy metal cadmium in the viscera of squid, and reduce the accumulation of heavy metal cadmium in the viscera of living crucian carp.

[0006] The present invention provides a marine-derived Lactobacillus rhamnosus S400M01, which is deposited in Guangdong Microbial Culture Collection Center (GDMCC), with a deposit address in Guangzhou, a deposit number of GDMCC No: 63001, a deposit date of November 28, 2022, and a classification name: Lacticaseibacillus rhamnosus.

[0007] Preferably, the base sequence of the Lactobacillus rhamnosus 16S rDNA gene is SEQ ID NO.1.

[0008] Preferably, an application of cadmium-resistant Lactobacillus rhamnosus of marine origin is applied in the adsorption of heavy metals.

[0009] Preferably, a marine-derived Lactobacillus rhamnosus is used, wherein the Lactobacillus rhamnosus is resistant to lead.

[0010] Preferably, the Lactobacillus rhamnosus has a cadmium tolerance concentration of 1000 mg / L and a lead tolerance concentration of 800 mg / L.

[0011] Preferably, the Lactobacillus rhamnosus can simultaneously adsorb and remove heavy metal cadmium and lead in the viscera of squid or crucian carp, and can effectively reduce the accumulation of metal cadmium and lead in the viscera of living squid or crucian carp in heavy metal-contaminated water sources.

[0012] The probiotic bacteria sieved from the ocean water sample and capable of withstanding high concentrations of cadmium and lead is Lactobacillus rhamnosus S400M01, which can tolerate high concentrations of cadmium chloride and lead, and can be used to adsorb and remove heavy metal cadmium in the viscera of squid and reduce the accumulation of heavy metal cadmium and lead in the viscera of living crucian carp.

[0013] The marine-derived probiotic Lactobacillus rhamnosus S400M01 of the present invention can reduce the accumulation of metallic cadmium and lead in the viscera of crucian carp in heavy metal-contaminated water bodies, and experiments have shown that the Lactobacillus rhamnosus has a cadmium tolerance concentration of 1000 mg / L and a lead tolerance concentration of 800 mg / L, and has a cadmium adsorption capacity greater than that of lead. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. The following drawings and tables only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 The colony morphology of Lactobacillus rhamnosus S400M01 on MRS agar plate;

[0016] Figure 2 This is the Gram staining result of Lactobacillus rhamnosus S400M01;

[0017] Figure 3 The phylogenetic tree was constructed based on the 16S rDNA sequence of Lactobacillus rhamnosus S400M01.

[0018] Figure 4 The growth of Lactobacillus rhamnosus S400M01 in different concentrations of cadmium and lead. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0020] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0021] Example 1: Screening of cadmium-resistant and lead-resistant probiotic strains from marine sources

[0022] All the probiotics screened from the ocean water samples were inoculated into MRS liquid culture medium (pH adjusted to 6.8) with cadmium chloride and lead concentrations of 100 mg / L, and cultured at 37°C for 48 hours. The probiotics that can grow when the cadmium chloride and lead concentrations are 100 mg / L were selected and inoculated into 200 mg / L MRS liquid culture medium, and continued to be cultured at 37°C for 48 hours, and so on, until only one probiotic strain can grow at a certain concentration. This probiotic strain is the probiotic strain that is resistant to high concentrations of cadmium and lead. Three replicates were set for each concentration. Finally, the strain Lactobacillus rhamnosus S400M01 was screened when the cadmium chloride and lead concentrations were 600 mg / L, respectively.

[0023] Example 2: Identification of Lactobacillus rhamnosus S400M01

[0024] 1. Identification of Lactobacillus rhamnosus S400M01 culture medium

[0025] Weigh 66.2g of MRS agar medium, heat and dissolve in 1000mL of distilled water, sterilize at 121℃ for 15 minutes, cool to about 50℃, pour into a sterile plate and set aside. Pick a single colony of Lactobacillus rhamnosus S400M01 and divide it into zones on the MRS agar medium plate. Incubate at 37℃ for 24h. Culture results Figure 1 As shown in the figure, the colonies on the MRS agar plate are milky white, round, convex, smooth, with neat edges and a diameter of about 1 mm.

[0026] 2. Gram staining identification of Lactobacillus rhamnosus S400M01

[0027] Pick a single colony of Lactobacillus rhamnosus S400M01 and mix it in 10 μL of sterile saline solution, spread it on a sterile slide, quickly fix it on a flame, cool it down, follow the Gram staining steps, observe it under a microscope with an oil immersion lens, and confirm it is a Gram-positive short rod. Figure 2 shown.

[0028] 3. 16S rDNA biological identification

[0029] The strain obtained in Example 1 was cultured in MRS liquid medium at 37° C. for 18 h, and 1 mL of the culture medium was taken to extract the genome using a rapid microtiter method. The universal primers for the bacterial 16S rDNA gene (27F: AGAGTTTGATCMTGGCTCAG;

[0030] 1492R: GGTTACCTTGTTACGACTT) PCR amplified the 16S rDNA gene of the strain. The PCR amplification program was: 94℃: 3min, 94℃: 1min, 61℃: 1min, 72℃: 1min, 30 cycles, 72℃: 6min, 4℃, ∞. Take 5μL of PCR amplification product and use 1% agarose gel electrophoresis (150V, 100mA, 20min). The remaining PCR product was purified by column and sent to a biotechnology company for sequencing. The sequencing results were compared with the NCBI database, confirming that the bacterium was Lactobacillus rhamnosus.

[0031] Phylogenetic tree constructed based on 16s rDNA sequences Figure 3 As shown, according to Figure 3 As a result, it can be seen that S400M01 is a Lactobacillus rhamnosus strain.

[0032] Example 3: Maximum cadmium tolerance and maximum lead tolerance of Lactobacillus rhamnosus S400M01

[0033] In the above Example 1, Lactobacillus rhamnosus S400M01 was inoculated into 5 mL of MRS liquid culture medium at a 1% inoculum volume and cultured for 16 h. The absorbance was adjusted to 1 (OD 600 ), take 100uL and inoculate into MRS liquid medium (pH adjusted to 6.8) containing 0mg / L, 100mg / L, 200mg / L, 400mg / L, 800mg / L, 1000mg / L of cadmium chloride and lead acetate, respectively, and culture at 37℃ for 48h. OD 600The growth curve was drawn, and the results showed that the growth of Lactobacillus rhamnosus S400M01 was gradually inhibited with the increase of cadmium chloride concentration, and basically reached the minimum at a concentration of 800 mg / L. However, when the cadmium chloride concentration was 1000 mg / L, the OD 600 There is an upward trend, but the time to reach growth is extended by 6h compared with other concentrations. Figure 4 As shown in the growth curve of cadmium in lead, the maximum cadmium concentration that the bacteria can tolerate is 1000 mg / L. The growth of Lactobacillus rhamnosus S400M01 was gradually inhibited with the increase of lead concentration. When the lead concentration reached 1000 mg / L, S400M01 basically did not grow. Figure 4 As shown in the growth curve of lead, the maximum lead concentration that the bacteria can tolerate is 800 mg / L.

[0034] Example 4: Study on the adsorption of cadmium and lead by Lactobacillus rhamnosus S400M01

[0035] 1. Cultivation of Lactobacillus rhamnosus S400M01

[0036] (1) Liquid culture: transfer the Lactobacillus rhamnosus S400M01 mother culture into MRS liquid culture medium, place it on a shaker at 180 r / min, and culture it at 37° C. for 16-24 h to obtain a liquid culture;

[0037] (2) Solid bacterial strain: Under sterile conditions, a single colony on the solid culture medium of the mother culture of Lactobacillus rhamnosus S400M01 was picked up with an inoculation needle and streaked onto MRS solid culture medium, and cultured at 37° C. for 24 h to 48 h to obtain a solid bacterial strain.

[0038] 2. Experimental study on the adsorption of cadmium and lead by Lactobacillus rhamnosus S400M01

[0039] (1) Fermentation medium: The frozen squid by-products (viscera) were thawed and chopped, and then put into a meat grinder to grind into pulp; the fermentation medium was prepared with a ratio of squid viscera pulp to MRS medium of 1:1, the pH of the medium was adjusted to 6.8, and the medium was sterilized at 121°C for 20 min and then cooled for use.

[0040] (2) Adsorption of cadmium and lead in squid viscera by Lactobacillus rhamnosus S400M01

[0041] The above-mentioned Lactobacillus rhamnosus S400M01 liquid strain was inoculated into 5 mL of MRS liquid culture medium at an inoculum volume of 1% and cultured for 16 h, and the absorbance was adjusted to 1 (OD 600), inoculated into the fermentation medium at an inoculum volume of 5% (before inoculation, take 5 mL of the medium, dilute it by appropriate multiples, and determine the cadmium and lead content in the solution by the graphite furnace method), cultured at 37°C for 48 hours, centrifuged at 4000 r / min for 20 minutes, took the supernatant, and diluted it by appropriate multiples, and determined the cadmium and lead content in the solution by the graphite furnace method. The cadmium and lead adsorption rate of Lactobacillus rhamnosus S400M01 was calculated using formula (1):

[0042] Adsorption rate (%) = 100% - (C1 / C0) × 100%

[0043] Where C0 is the initial heavy metal concentration, and C1 is the heavy metal concentration in the final supernatant.

[0044] The results showed that the cadmium content in the fermentation medium was 17.5 mg / kg. After adding Lactobacillus rhamnosus S400M01 for fermentation for 48 hours, the cadmium content in the supernatant was only 2.8 mg / kg, and the adsorption rate of metallic cadmium in squid viscera by this bacterium reached 84%; the lead content in the fermentation medium was 17.5 mg / kg. After adding Lactobacillus rhamnosus S400M01 for fermentation for 48 hours, the lead content in the supernatant was 3.1 mg / kg, and the adsorption rate of metallic lead in squid viscera by this bacterium reached 82.3%.

[0045] Example 5: Study on the accumulation of cadmium and lead in the viscera of crucian carp in cadmium-contaminated water by Lactobacillus rhamnosus S400M01

[0046] 1. Preparation of Lactobacillus rhamnosus S400M01 powder

[0047] The above-mentioned Lactobacillus rhamnosus S400M01 was cultured after expanding the culture volume according to the culture method of the liquid strain of Lactobacillus rhamnosus S400M01 in Example 4, and the obtained culture solution was centrifuged for 15 minutes at 4°C and 4000r / min under sterile conditions to collect the bacteria. The collected bacteria were added with 10% skim milk powder, placed in a freeze dryer, freeze-dried, and then ground into powder, which was stored at -20°C.

[0048] 2. Preparation of crucian carp feed

[0049] The Lactobacillus rhamnosus powder was added to the basic feed formula at an addition rate of 2% to form the experimental group. The basic feed formula was the control (%): imported fish meal: 12, soybean meal 40, wheat flour 20, rice bran 20, calcium hydrogen phosphate 1.5, vitamins and inorganic salts 2, bentonite 1.5. After analysis, it contained 33.3% crude protein, 3.52% crude fat, 12.5% ​​crude ash, and the number of live Lactobacillus rhamnosus in the experimental group was greater than 10 billion CFU / g. Both baits were processed into 2.0mm particles and air-dried for use.

[0050] 3. Crucian carp breeding

[0051] Twenty crucian carp fingerlings weighing about 60 g were purchased from the market. The purchased crucian carp fingerlings were first placed in cadmium-free water for acclimatization for 2 days, and then the experiment was carried out.

[0052] 4. Study on the effect of Lactobacillus rhamnosus S400M01 on reducing the accumulation of cadmium and lead in the viscera of crucian carp

[0053] The experimental group and the control group were placed in water with cadmium and lead concentrations of 50 mg / L with 10 domesticated crucian carp fry. They were fed different feeds at 9:00 and 17:00 every day to achieve satiety. Wastewater was discharged every 2 days. After discharge, the cadmium and lead concentrations in the water environment were readjusted to 50 mg / L. The water temperature was controlled at 28°C, oxygen was supplied in the water throughout the day, and the fish were raised for 20 days.

[0054] If crucian carp is found dead during the experiment, the water is removed immediately, the viscera are removed after being rinsed with deionized water, weighed and stored in a -80℃ refrigerator. After the experiment, the crucian carp is removed, rinsed with deionized water and the viscera are removed, numbered and weighed, and stored in a -80℃ refrigerator. After the experiment is over, the frozen crucian carp viscera are removed from -80℃, thawed, pulped and appropriately diluted, and the cadmium and lead content in the solution is determined by the graphite furnace method.

[0055] Table 1 Cadmium and lead contents in the viscera of crucian carp fry

[0056]

[0057]

[0058] Table 1 shows the effect of Lactobacillus rhamnosus S400M01 on the accumulation of cadmium and lead in the viscera of live crucian carp fry. The results showed that one crucian carp fry died on the 15th day of feeding in the control group, and no deaths were found in the experimental group. The cadmium and lead content in the viscera of crucian carp was determined to be 8-9 mg / kg and 6-7 mg / kg in the viscera of the control group. The cadmium content in the viscera of dead crucian carp was as high as 10 mg / kg and the lead content was 7 mg / kg. In contrast, the cadmium content in the viscera of the experimental group was approximately 1-2 mg / kg, and the lead content was 2-3 mg / kg. It can be seen that feeding feed supplemented with Lactobacillus rhamnosus S400M01 powder can greatly reduce the content of heavy metals in the viscera of crucian carp, and the ability to adsorb cadmium is greater than that of lead.

[0059] The above describes the basic principle, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

[0060] SEQ ID NO.1:

[0061] CATTTTGTCACCTTAGACGGCTCGCTCCCTAAAGGGTTACG

[0062] CCACCGGCTTCGGGTGTTACAAACTCTCATGGTGGTGACGGGCG

[0063] GTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGAT

[0064] CCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAG

[0065] CCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGAC

[0066] CTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGT

[0067] GTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCC

[0068] ACCTTCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAA

[0069] CTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGG

[0070] ACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGC

[0071] ACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCA

[0072] GGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTG

[0073] CTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCG

[0074] TCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCG

[0075] GAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCC

[0076] TCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGG

[0077] TATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCA

[0078] GTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCAT

[0079] ATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTC

[0080] TTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTA

[0081] AGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTC

[0082] GCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTA

[0083] TTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTG

[0084] GATACCGTCACGCCGACACAGTTACTCTGCCGACCATTCTTCTC

[0085] AACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCG

[0086] GGCGTG

Claims

1. A marine-derived Lactobacillus rhamnosus S400M01, characterized in that: It is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), the deposit address is Guangzhou, and the deposit number is GDMCCNo: 63001.

2. The marine-derived Lactobacillus rhamnosus according to claim 1, characterized in that ,, the base sequence of the Lactobacillus rhamnosus 16S rDNA gene is SEQ ID NO.

1.

3. An application of cadmium-resistant Lactobacillus rhamnosus from marine sources, characterized in that: The application of the marine-derived cadmium-resistant Lactobacillus rhamnosus in the adsorption of heavy metals.

4. The use of Lactobacillus rhamnosus from marine sources according to claim 3, characterized in that: The Lactobacillus rhamnosus is resistant to lead.

5. The use of Lactobacillus rhamnosus from marine sources according to claim 4, characterized in that: The Lactobacillus rhamnosus has a cadmium tolerance concentration of 1000 mg / L and a lead tolerance concentration of 800 mg / L.

6. The use of the Lactobacillus rhamnosus from marine sources according to claim 4, characterized in that: The rhamnosus lactobacillus can simultaneously adsorb and remove heavy metal cadmium and lead in the viscera of squid or crucian carp, and can effectively reduce the accumulation of metal cadmium and lead in the viscera of living squid or crucian carp in heavy metal-contaminated water sources.

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