WO2022160360A1 - 一种发酵乳杆菌lf-schy34及其应用 - Google Patents

一种发酵乳杆菌lf-schy34及其应用 Download PDF

Info

Publication number
WO2022160360A1
WO2022160360A1 PCT/CN2021/074767 CN2021074767W WO2022160360A1 WO 2022160360 A1 WO2022160360 A1 WO 2022160360A1 CN 2021074767 W CN2021074767 W CN 2021074767W WO 2022160360 A1 WO2022160360 A1 WO 2022160360A1
Authority
WO
WIPO (PCT)
Prior art keywords
schy34
lead
lactobacillus fermentum
liver
kidney
Prior art date
Application number
PCT/CN2021/074767
Other languages
English (en)
French (fr)
Inventor
赵欣
龙兴瑶
Original Assignee
重庆第二师范学院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 重庆第二师范学院 filed Critical 重庆第二师范学院
Priority to KR1020217036127A priority Critical patent/KR102567035B1/ko
Priority to JP2021550088A priority patent/JP2023505400A/ja
Publication of WO2022160360A1 publication Critical patent/WO2022160360A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of microorganisms, in particular to a Lactobacillus fermentum LF-SCHY34 and its application.
  • Heavy metals can be enriched through the food chain, cannot be biodegraded, and may be converted into more toxic metal-organic compounds. Heavy metal pollution has attracted widespread attention in recent years. Heavy metal lead is a kind of heavy metal with affinity and accumulation, and its toxicity is relatively high. With the acceleration of industrialization, lead is widely used in printing, paint, ceramics, alloys, gasoline and other industries. Because of its refractory degradability and strong toxicity, lead has become one of the most serious common pollutants.
  • the human body mainly ingests lead through diet and breathing. After accumulating to a certain amount in the body, it will damage the brain and nerve tissue, and accumulate in the kidney and liver, causing acute or chronic kidney disease and liver disease, and eventually causing various organs of the body. Systemic damage. At present, chelates are mainly used to treat heavy metal poisoning, but the treatment may cause damage to the body at the same time, and there are disadvantages of incomplete detoxification.
  • Oxidative damage is considered to be one of the important mechanisms by which lead exerts its toxic effects. Numerous studies have shown that lead can change the redox state of cells, thereby causing oxidative stress, which can cause excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and reduce the activity of antioxidant enzymes, promoting Tissues and cells produce free radicals. When the body responds to free radical damage, it will form a complex oxidative stress response system, and the body itself can induce a series of protective proteins to alleviate the damage to cells.
  • Nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor in the expression of oxidative stress in response to cellular damage through a large amount of ROS and RNS.
  • Nrf2 Under physiological conditions, most of Nrf2 is coupled to Keap1, which is ubiquitous A protein-mediated degradation system maintains inactive Nrf2 at basal levels in the cytoplasm.
  • protein kinases such as MAPK and phosphatidylinositol 3-kinase (PIK3) can directly phosphorylate Nrf2, prompting the dissociation of the Keap1-Nrf2 coupling body, and Nrf2 is activated, accumulated and transported into the nucleus, where it binds to Maf to form Heterodimer, recognizes and binds to the downstream antioxidant element ARE.
  • ARE is located upstream of the protective protein genes secreted by the body's oxidative stress response system.
  • Nrf2 is an activator of ARE. When Nrf2 binds to ARE, it will mediate the expression of downstream protective antioxidant genes and generate protective proteins such as superoxide. Dismutase (SOD), quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1) and ⁇ -glutamyl cysteine synthase ( ⁇ -GCS), which together play a role in cellular oxidative damage It plays an important inhibitory role and can be used as the first line of defense against oxidative damage in the body.
  • SOD Dismutase
  • NQO1 quinone oxidoreductase 1
  • HO-1 heme oxygenase 1
  • ⁇ -GCS ⁇ -glutamyl cysteine synthase
  • Lactobacillus is a general term for a spore-free, Gram-positive bacteria that utilizes fermentable sugars (carbohydrates) to produce a large amount of lactic acid.
  • lactic acid bacteria also have the functions of enhancing immunity, anti-oxidation, and anti-aging.
  • fermented kimchi mainly uses the lactic acid bacteria contained in the vegetable itself for fermentation, so the types of lactic acid bacteria contained in the fermented kimchi are also varied.
  • Lactobacillus such as Lactobacillus plant arum, Lactobacillus pentosus, Lactobacillus sake, Lactobacillus brevis , Lactobacillus casei (Lactobacillus casei) and Lactobacillus fermentum (Lactobacillus fermentum).
  • Lactic acid bacteria in fermented food can not only degrade nitrite in food, but also have various effects on cholesterol lowering, antioxidant and intestinal health regulation.
  • lactic acid bacteria have good resistance and adsorption performance to heavy metals.
  • the effect of lactic acid bacteria in relieving lead toxicity can also be divided into two ways. The first is that lactic acid bacteria have a good ability to absorb heavy metals; the second is that lactic acid bacteria can play an antioxidant role in the body and resist the damage caused by free radicals to the body.
  • the use of bioremediation of heavy metal poisoning has many advantages, such as wide range of raw materials, low cost, simple operation, environmental protection, etc., and does not produce secondary hazards.
  • the purpose of the present invention is to provide a kind of Lactobacillus fermentum LF-SCHY34 and its application, to solve the problem existing in the above-mentioned prior art.
  • the present invention provides the following scheme: the present invention provides a kind of Lactobacillus fermentum, and the Lactobacillus fermentum is named LF-SCHY34, which is preserved in China General Microorganism Culture Collection and Management Center, and the preservation number is CGMCC No.18795 .
  • the present invention also provides an application of the Lactobacillus fermentum LF-SCHY34 in preparing a product for treating or preventing heavy metal poisoning.
  • the product for treating or preventing heavy metal poisoning is used to adsorb heavy metals.
  • the present invention also provides an application of the Lactobacillus fermentum LF-SCHY34 in preparing a product for treating or preventing diseases caused by heavy metal poisoning.
  • condition caused by heavy metal poisoning is liver damage, kidney damage or brain tissue damage.
  • the heavy metal is heavy metal lead, and the effective dose of the Lactobacillus fermentum is 10 9 CFU/time.
  • the present invention also provides an application of the Lactobacillus fermentum in preparing a product for treating or preventing oxidative damage.
  • the effective dose of the Lactobacillus fermentum is 10 9 CFU/time.
  • the present invention discloses the following technical effects: (1) the lactic acid bacteria LF-SCHY34 disclosed in the present invention can adsorb lead ions in two ways: biological adsorption and accumulation, and is a high-quality lead-adsorbing lactic acid bacteria; (2) the lactic acid bacteria disclosed in the present invention LF-SCHY34 has higher antioxidant capacity in vitro and in vivo, has better adhesion ability, and can be better colonized in the human intestinal tract to play a probiotic effect; (3) The lactic acid bacteria LF-SCHY34 disclosed in the present invention can alleviate the lead ion pair The damage caused by the liver and kidney of SD rats reduces the occurrence of inflammation in the liver and kidney, and protects the integrity of liver and kidney cells; (4) The lactic acid bacteria LF-SCHY34 disclosed in the present invention can enhance the response of the Keap1/Nrf2/ARE signaling pathway , stimulate the expression of more downstream genes to produce HO-1, NQO1 and ⁇ -GCS with antioxidant capacity to alleviate the oxidative stress
  • Figure 1 shows the SEM and TEM images of LF-SCHY34 lactic acid bacteria before and after lead ion adsorption, where a is the SEM image of LF-SCHY34 before adsorption, b is the TEM image of LF-SCHY34 before adsorption, and c is the SEM image of LF-SCHY34 after adsorption Figure; d is the TEM image of LF-SCHY34 after adsorption;
  • Figure 2 shows the detection topography and scanning energy spectrum of LF-SCHY34 lactic acid bacteria before and after lead ion adsorption, where a is the detection topography of LF-SCHY34 before adsorption, and b is the scanning energy spectrum of LF-SCHY34 before adsorption. c is the detected morphology of LF-SCHY34 after adsorption, and d is the scanning energy spectrum of LF-SCHY34 after adsorption;
  • Fig. 3 is a slice of SD rat liver, wherein a is the normal group, b is the lead-induced group, c is the EDTA group, and d is the LF-SCHY34 group;
  • Figure 4 is a slice of SD rat kidney, in which a is the normal group, b is the lead-induced group, c is the EDTA group, and d is the LF-SCHY34 group.
  • lactic acid bacteria a strain of lactic acid bacteria was isolated from kimchi in Chongqing, and the extracted DNA was amplified by PCR.
  • the upstream primer 27F (5'-AGA GTT TGA TCC TGGCTC AG-3') 1 ⁇ L
  • the downstream primer 1495R 5'-CTA CGG CTA CCTTGT TAC GA-3'
  • 2 ⁇ Taq plus Buffer 12.5 ⁇ L 2 ⁇ Taq plus Buffer 12.5 ⁇ L
  • template DNA 1 ⁇ L
  • the template DNA was replaced with sterile ultrapure water as a negative control.
  • the amplification conditions were: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, a total of 29 cycles; and a final extension at 72°C for 5 min.
  • the successfully detected PCR product was sent to Beijing Qingke Biotechnology Co., Ltd.
  • the 16S rDNA sequence was shown in SEQ ID NO: 1, and the sequenced sequence was successfully BLAST compared at NCBI, and the lactic acid bacteria were Lactobacillus, Lactobacillus The genus, the similarity is 99.9%, named LF-SCHY34, and was deposited in the China General Microorganism Culture Collection and Management Center (CGMCC, Beijing) on November 4, 2019, and the preservation number is CGMCC No.18795.
  • CGMCC China General Microorganism Culture Collection and Management Center
  • the artificial gastric juice was mixed with 0.2% NaCl and 0.35% pepsin, adjusted to pH 3.0 with 1 mol/L HCl, and then filtered and sterilized with a 0.22 ⁇ m sterile filter.
  • the LP-KFY04 activated twice in 5ml of MRS liquid medium was centrifuged at 3000rpm for 10min to collect bacterial cells, washed twice with sterile saline and resuspended in 5mL of saline. 1:1 (v/v) mix the bacterial liquid with sterile artificial gastric juice, shake well and place it in a constant temperature incubator for cultivation at 37°C, measure the number of viable bacteria at 0h and 3h respectively, and calculate LF according to formula (1).
  • - Survival of SCHY34 in artificial gastric juice was mixed with 0.2% NaCl and 0.35% pepsin, adjusted to pH 3.0 with 1 mol/L HCl, and then filtered and sterilized with a 0.22 ⁇ m
  • LP-KFY04 activated twice at 2% (v/v) inoculum was inoculated into MRS-THIO medium containing 0.0% and 0.3% porcine bile salts (MRS medium was supplemented with 0.2% sodium thioglycolate and heated at 121°C). Sterilization for 15min), after culturing for 24h in a constant temperature shaker at 37°C, the blank medium (uninoculated MRS-THIO medium) was used as a control, and the blank medium and the inoculated medium were added to the 96-well plate, 200ml per well, measure the absorbance at a wavelength of 600nm, and calculate the growth efficiency according to formula (2).
  • the Lactobacillus fermentum SCHY34 bacterial suspension was centrifuged at 1500 ⁇ g for 10 min to collect the bacterial cells, washed twice with normal saline, and then centrifuged to collect the bacterial cells.
  • the bacterial cells were added to 50 mg/L lead nitrate solution with pH 6.3, the final bacterial concentration was 1 g/L, and the supernatant was collected by centrifugation at 8000 ⁇ g for 20 min after culturing at 37 °C for 24 h, and the lead ions in the original solution were determined by flame atomic absorption method. Concentration C0, lead ion concentration C1 after adsorption, and lead ion adsorption rate is calculated according to formula (3).
  • the Lactobacillus fermentum SCHY34 bacterial suspension was centrifuged at 1500 ⁇ g for 10 min to collect the bacterial cells, washed twice with normal saline, and then centrifuged to collect the bacterial cells.
  • the bacterial cell concentration was adjusted with physiological saline so that the absorbance value at a wavelength of 580 nm was 1.000.
  • Lactobacillus fermentum LF-SCHY34 was prepared as a bacterial suspension sample of 10 9 cfu/mL for later use, and 1 mL of 0.05 mol/L pH7.4 phosphate buffer, 0.5 mL of 6 mmol/L phenanthroline were added to the test tube, After thorough mixing, 0.5 mL of 6 mmol/L FeSO 4 solution was added and mixed immediately.
  • the test tubes are divided into sample tubes, blank tubes and control tubes. Add 0.5mL of bacterial suspension sample solution to the sample tube, add 0.5mL of 0.1 % H2O2 solution to the control tube, mix well, add 0.5mL of 0.1 % H2O2 solution, and finally add the volume to 4mL.
  • the survival rate of LF-SCHY34 in artificial gastric juice was 88.71% ⁇ 0.23%
  • the growth efficiency in artificial bile salt was 85.32% ⁇ 0.41%
  • the surface hydrophobicity rate was 43.78% ⁇ 0.75%
  • the lead ion adsorption rate was 69.58% ⁇ 69.58% ⁇ 0.56%
  • the scavenging rates of hydroxyl radical, superoxide anion and DPPH were 44.15% ⁇ 0.41%, 66.11% ⁇ 0.97% and 79.49% ⁇ 0.87%, respectively
  • the reducing power was 111.66 ⁇ 1.18 ⁇ mol/L.
  • the data of each group showed that LF-SCHY34 had strong antioxidant capacity in vitro and could scavenge free radicals efficiently.
  • Example 2 Scanning electron microscope, scanning energy spectrum analysis and transmission electron microscope analysis of Lactobacillus fermentum LF-SCHY34 before and after adsorption of lead ions
  • Figure 1 is the scanning electron microscope and transmission electron microscope images of LF-SCHY34 lactic acid bacteria before and after the adsorption of lead ions:
  • Figure 1-a is the scanning electron microscope picture of the normal group LF-SCHY34 bacteria before adsorption. Through observation, it can be found that the lactic acid bacteria cells are complete in shape, clear in outline, clean and plump , the surface is smooth, the edge boundary is clear, the surface is free of particles and no adherents;
  • Figure 1-c is the scanning electron microscope picture of LF-SCHY34 bacteria after lead adsorption. , the outline of the cell edge became blurred, and even the phenomenon of adhesion and irregular aggregation appeared. At the same time, it was found that the cell surface was covered with fine particles.
  • Figure 1-b is the transmission electron microscope image of LF-SCHY34 in the normal group before adsorption. It is found that there is no sediment on the section of the normal group strain, the surface is clear, and there is no adhesion;
  • Figure 1-d is the transmission electron microscope image of LF-SCHY34 after adsorption. , Compared with the normal group of bacteria, there are a lot of black deposits on the cut surface of the lead-adsorbed lactic acid bacteria cells, and blank parts appear inside the cells.
  • Figure 2 shows the detection topography and scanning energy spectrum of LF-SCHY34 lactic acid bacteria before and after lead ion adsorption.
  • Table 2 shows the element change table of the scanning energy spectrum of lactic acid bacteria before and after lead ion adsorption.
  • the contents of O and N elements on the surface of the lactic acid bacteria after lead adsorption decreased, while the contents of C, P and Pb elements increased.
  • Table 2 Elemental change table of lactic acid bacteria scanning energy spectrum before and after lead ion adsorption
  • Rats in the EDTA group were injected with EDTA at a concentration of 50 mg/kg every day from the 8th week to the 12th week, and the rats in the LF-SCHY34 group were given 1 ⁇ 10 9 CFU/kg(bw) LF daily from the 1st week to the 12th week. -SCHY34.
  • ad indicates a significant difference (p ⁇ 0.05) between the means of different letters in the same table according to Duncan's new MRT.
  • the lead acetate-induced mice have the highest lead content in their blood, which is much higher than the lead content in tissues and organs.
  • the kidney has the highest lead content, followed by the liver, and finally the brain.
  • liver and kidney tissues of SD rats in each group were taken and fixed in 10% formalin (v/v) for 24 hours.
  • the histological morphology was observed under a microscope (BX43; Olympus, Tokyo, Japan) and photographed.
  • the liver section of SD rats is shown in Figure 4. It can be observed from Figure 4 that the hepatic lobules of the rats in the normal group ( Figure 4a) have an orderly structure, and the central veins and hepatic sinusoids are clear. In rats induced by lead acetate (Fig. 4b), the hepatic lobules were blurred, the arrangement of hepatic cords was disordered, monocytes were aggregated and dispersed in different spaces, focal necrosis of hepatocytes and infiltration of large inflammatory cells, internuclear inclusions and nuclear fragmentation . Lead acetate-induced rats treated with EDTA drugs (Fig. 4c) and LF-SCHY34 (Fig. 4d) had more orderly arrangement of liver cells, less inflammatory cell infiltration, and less damage and necrosis of liver cells.
  • SD rat kidney section is shown in Figure 5. It can be observed from Figure 5 that in the normal group ( Figure 5a), the structure of the glomerulus and renal tubules of the rats is normal, the cells are closely arranged, and the number of cells is normal. In the kidney sections of SD rats induced by lead acetate (Fig. 5b), it was found that renal tubules and glomeruli were enlarged and hypercellular, capillaries were dilated and congested; Cells were disrupted and lymphocytes infiltrated. Compared with the lead-induced group, SD rats in the EDTA group (Fig. 5c) and LF-SCHY34 group (Fig.
  • Organ biochemical indicators of catalase (CAT), reactive oxygen species (ROS), total superoxide dismutase (T-SOD), malondialdehyde (MDA) were determined according to the operation method of the kit (Nanjing Jiancheng Bioengineering Institute, China). and glutathione (GSH) levels.
  • the data of oxidation indexes in serum, liver, kidney and brain tissue of SD rats are shown in Table 3. It can be found from Table 3 that CAT, T-SOD and GSH in the blood, liver, kidney and brain tissue of the normal group were the highest among the four groups, and MDA and ROS were the lowest among the four groups. The trend in the kidney and brain tissues was completely opposite to that in the normal group. The lead-induced group CAT, T-SOD and GSH were the lowest among the four groups, and MDA and ROS were the highest among the four groups. The trend of oxidation index in EDTA group and LF-SCHY34 group was similar to that in normal group, and the oxidation index value of LF-SCHY34 was closer to that of normal group.
  • Table 4 shows the data of inflammatory indicators in the serum, liver and kidney of SD rats. It was found that the levels of IL-1 ⁇ , IL-6, TNF- ⁇ and IFN- ⁇ in the serum, liver and kidney of the normal group were the lowest among the four groups. The -10 level is the highest of the four groups. While the level of IL-10 in the lead-induced group was the lowest among the four groups, and the levels of IL-1 ⁇ , IL-6, TNF- ⁇ and IFN- ⁇ were the highest among the four groups.
  • the levels of IL-1 ⁇ , IL-6, TNF- ⁇ and IFN- ⁇ in the EDTA group and LF-CQPC group all showed a downward trend, and the levels of IL-10 increased compared with the lead-induced group. trend, but the decreasing and increasing trends were more pronounced in the LF-SCHY34 group than in the EDTA group.
  • delta-aminolevulinate dehydratase delta-aminolevulinate dehydratase
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • BUN blood urea nitrogen
  • ALT, AST, ⁇ -ALAD enzyme activities and BUN, CRE contents in serum of SD rats are shown in Table 5.
  • the normal group SD rats had the lowest ATL and AST enzyme activities, the highest ⁇ -ALAD enzyme activities, and the lowest BUN and CRE contents.
  • the enzyme activities of liver-related ATL and AST were the lowest in the lead-induced group, while the enzyme activity of ⁇ -ALAD and the contents of kidney-related BUN and CRE were the highest in the lead-induced group.
  • the three enzyme activity trends and the contents of BUN and CRE in the EDTA group and the LF-SCHY34 group were similar to those in the normal group. From the value of the enzyme activity and the contents of BUN and CRE, the intervention effect of LF-SCHY34 was better than that of EDTA.
  • the synthesized cDNA was subsequently mixed with 10 ⁇ L of SYBR Green PCR Master Mix (Thermo Fisher Scientific), 2 ⁇ L of primers (Table 6) and distilled water, and then placed in a qPCR instrument for processing.
  • qPCR program 95°C for 60s; 95°C for 15s, 55°C for 30s, 72°C for 35s, 40 cycles; 95°C for 30s; 55°C for 35s.
  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene, and the relative mRNA transcript levels were calculated using the 2- ⁇ Ct formula.
  • Keap1, Nrf2, HO-1, SOD, GSH, NQO1 and ⁇ -GCS related to the Keap1/Nrf2ARE pathway in liver and kidney were detected.
  • Table 7 shows the mRNA expression data of Keap1, Nrf2, HO-1, SOD1, SOD2, GSH, NQO1 and ⁇ -GCS in SD liver and kidney.
  • the mRNA expressions of Keap1, Nrf2, HO-1, SOD1, SOD2, GSH, NQO1 and ⁇ -GCS were significantly increased in the EDTA group and the LF-SCHY34 group.
  • the mRNA expression levels of related genes in the LF-SCHY34 group were significantly higher than those in the EDTA group.
  • liver tissue 100 mg was homogenized in 1 mL of RIPA (Thermofisher, Waltham, MA, USA) and 10 ⁇ L of PMSF (Thermofisher) and centrifuged at 12000 xg for 5 min at 4°C. Proteins were quantified using the BCA protein assay kit (Thermofisher). The protein samples were mixed 4:1 with sample buffer (Thermofisher) and heated at 95°C for 5 minutes before spotting the samples into wells of an SDS-PAGE gel and running at 100V.
  • RIPA Thermofisher, Waltham, MA, USA
  • PMSF Thermofisher
  • Biosorption includes extracellular precipitation, surface complexation and ion exchange, mainly the chemical groups on some microbial surface proteins, polysaccharides, lipids and other substances and some ions contained on the cell surface interact with metals to form metal complexes
  • Bioaccumulation includes transmembrane transport, intracellular accumulation, cellular physiological metabolism and self-regulation mechanisms.
  • the adsorption of lead ions by LF-SCHY34 includes biological adsorption and accumulation, and it is a high-quality lead-adsorbing lactic acid bacteria.
  • ROS reactive oxygen species
  • GSH glutathione
  • MDA malondialdehyde
  • ROS reactive oxygen species
  • Lactic acid bacteria can alleviate oxidative damage and lipid peroxidation by removing reactive oxygen radicals around cells, and can further defend against oxidative stress by regulating host cell antioxidant-related signaling pathways.
  • LF-SCHY34 can scavenge free radicals more efficiently and has stronger antioxidant capacity in vitro.
  • the LF-SCHY34 group had higher SOD and CAT activities and GSH content, and lower ROS and MDA levels in serum, liver, kidney and brain tissue compared with the lead-induced group, so it can be inferred that LF-SCHY34 group SCHY34 is a dominant strain with high antioxidant capacity in vitro and in vivo.
  • Hydrophobicity is one of the important indicators for the adhesion of lactic acid bacteria to intestinal epithelial cells. The higher the hydrophobicity, the better the adhesion of lactic acid bacteria. Nadia S.AlKalbani et al. found through experiments that the hydrophobicity of different lactic acid bacteria is 0.5%-44.1%, while the hydrophobicity of LF-SCHY34 in this experiment is 43.78% ⁇ 0.75%, indicating that LF-SCHY34 has better adhesion ability , can better colonize the human intestinal tract to play a probiotic role.
  • the lead in the human body is first excreted through the kidneys.
  • the kidneys reach the maximum amount of lead excretion, the lead is concentrated and deposited in the proximal tubular epithelial cells, affecting cell metabolism and causing damage to the structure and function of the kidneys.
  • the reabsorption function of renal tubules decreases, and creatinine and urea remain in the blood as the reabsorption capacity decreases. Therefore, the concentration of blood creatinine and blood urea nitrogen can reflect whether there is damage to renal function.
  • ⁇ -aminolevulinic acid dehydratase ⁇ -ALAD
  • the liver is the most important detoxification organ in the body. It can convert different toxins that enter the body through the digestive system into low-toxic substances and excrete them under the biochemical reaction. Experiments have shown that lead can cause different degrees of liver lesions, cause severe inflammation, affect the activity of liver-related enzymes, and ultimately cause liver damage. ALT and AST are distributed in liver cells.
  • ALT and AST in the cytoplasm When liver cells are damaged, ALT and AST in the cytoplasm will be released into the blood, so the concentration of ALT and AST in the blood can indicate the degree of liver cell damage. Inflammatory factors in serum and organ tissues can reflect the degree of inflammatory damage in the body. The levels of inflammatory factors in serum, liver and kidney, the detection results of serum ⁇ -ALAD, ALT, AST, CRE and BUN, as well as the case analysis of liver and kidney slices, it can be found that LF-SCHY34 can alleviate the effect of lead ions on SD rats. Damage caused by the liver and kidneys, reduces the occurrence of inflammation in the liver and kidneys, and protects the integrity of liver and kidney cells.
  • Nrf2 Nuclear factor E2-related factor 2
  • Keap1 Kelch-like epichlorohydrin-associated protein 1 binds to Nrf2 in the cytoplasm, causing it to be inactive and gradually degraded by ubiquitination.
  • Nrf2 in the cytoplasm When the body is under oxidative stress, Nrf2 in the cytoplasm is dissociated from Keap1, translocated into the nucleus, and bound to the antioxidant response element (ARE) region upstream of certain genes to initiate the expression of genes ( Figure X), which encode to include NQO1, HO-1 and ⁇ -GCS.
  • ARE antioxidant response element
  • Figure X genes which encode to include NQO1, HO-1 and ⁇ -GCS.
  • the expression of these cytoprotective genes can enhance cell self-defense against harmful stimuli and promote cell survival.
  • HO-1 has antioxidant, anti-inflammatory, anti-apoptotic protective effects on fibroblasts, liver cells, renal epithelial cells, etc.
  • NQO1 is a soluble flavonase ubiquitous in almost all animal species, which is expressed at high levels in adipocytes, vascular endothelial cells, and epithelial cells.
  • NQO1 uses NADPH as a donor to generate stable hydroquinone to avoid one-electron reduction reactions that generate toxic semiquinone radicals and reactive oxygen species, as well as direct reactions of intracellular sulfhydryl groups.
  • NQO1 can detoxify quinones with strong activity and maintain the reduced form of fat-soluble antioxidants, thereby protecting the body from oxidative stress.
  • ⁇ -GCS is also a downstream antioxidant factor of Keap1/Nrf2/ARE signaling pathway. It is the rate-limiting enzyme in GSH biosynthesis. When the expression level increases, it can scavenge a large number of free radicals and reduce the oxidative damage of cells.
  • Nrf2 was significantly positively correlated with the expression levels of Keap1, SOD1, SOD2, GSH, HO-1, NQO1 and ⁇ -GCS in the cytoplasm. Inversely correlated with Keap1 in the nucleus.
  • this experiment by detecting the expression of related genes and proteins in the Keap1/Nrf2/ARE signaling pathway in the liver and kidney, it was found that all lead-induced SD rats would produce oxidative stress, resulting in the activation of Keap1/Nrf2/ARE in vivo , forms a protective mechanism for the body, but the degree of activation of downstream protein expression varies.
  • LF-SCHY34 can enhance the response of Keap1/Nrf2/ARE signaling pathway, stimulate the expression of more downstream genes to produce HO-1, NQO1 and ⁇ -GCS with antioxidant capacity to alleviate oxidative stress induced by lead in SD rats reaction.
  • lactic acid bacteria isolated from naturally fermented kimchi were used to study the adsorption capacity and antioxidant capacity of lead ions in vivo and in vitro. , enhance the body's antioxidant capacity, protect the body from lead-induced oxidative stress damage from two aspects at the same time, and provide theoretical and data support for the later study of food-grade lactic acid bacteria to alleviate lead poisoning.
  • LF-SCHY34 can adsorb lead ions in vivo and in vitro, reduce lead content in blood and organs, and protect liver, kidney and brain tissue.
  • LF-SCHY34 can scavenge free radicals and activate the Keap1/Nrf2/ARE signaling pathway to secrete more antioxidant substances, so as to better alleviate the oxidative damage caused by lead to the body.
  • LF-SCHY34 is an excellent strain with strong lead adsorption capacity and antioxidant capacity.
  • LF-SCHY34 still has more research value, and it has great potential and research value in terms of oxidative stress caused by lead ions in humans and alleviating the toxicity of other heavy metals.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Addiction (AREA)
  • Psychiatry (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供了一种发酵乳杆菌LF-SCHY34及其应用，所述发酵乳杆菌LF-SCHY34保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC No.14957，属于微生物技术领域；发酵乳杆菌LF-SCHY34能够吸附体内和体外的铅离子，减少血液和脏器中的铅含量，保护肝脏、肾脏和脑组织；发酵乳杆菌LF-SCHY34还能清除自由基，激活Keap1/Nrf2/ARE信号通路分泌更多抗氧化物质，以更好地缓解铅对机体造成的氧化损伤。因此，发酵乳杆菌LF-SCHY34是一株具有较强铅吸附能力和抗氧化能力的优良菌株，对人体铅离子造成的氧化应激作用和缓解其他重金属毒性等方面具有较大的潜力和研究价值。

Description

一种发酵乳杆菌LF-SCHY34及其应用 技术领域
本发明涉及微生物技术领域，特别是涉及一种发酵乳杆菌LF-SCHY34及其应用。
背景技术
重金属能通过食物链富集，无法被生物分解，且可能转化为毒性更强的金属有机化合物，近年来重金属污染已引起广泛关注。重金属铅是一种具有亲和性、蓄积性的重金属，其毒性较大。随着工业化进程的加快，铅在印刷、油漆、陶瓷、合金、汽油等行业中广泛使用，因为其难降解性和强毒性，成为最严重的常见污染物之一。人体主要通过饮食和呼吸两大途径摄入铅，在体中蓄积到一定量后，会损伤大脑和神经组织，并积累在肾脏和肝脏，引起急性或慢性肾病和肝病，最终造成机体多种器官系统性损伤。目前主要使用螯合物治疗重金属中毒，但治疗的同时可能会对机体造成损伤，并且存在解毒不完全的弊端。
氧化损伤被认为是铅发挥毒性效应的重要作用机制之一。大量研究表明，铅会使细胞的氧化还原状态发生变化，从而造成氧化应激，氧化应激会引起过量的活性氧(ROS)和活性氮(RNS)生成，并导致抗氧化酶活性降低，促进组织和细胞产生自由基。机体在应对自由基损害时会形成一套复杂的氧化应激应答系统，机体自身能诱导出一系列保护性蛋白，以缓解细胞所受到的损伤。核因子E2相关因子2(Nrf2)是一种通过大量的ROS和RNS来响应细胞损伤的氧化应激表达中的关键转录因子，在生理状态下，大部分Nrf2与Keap1相偶联，Keap1通过泛素介导的蛋白降解系统在细胞质中维持非活性的处于基础水平的Nrf2。当氧化应激刺激机体，MAPK、磷脂酰肌醇3激酶(PIK3)等蛋白激酶均可直接磷酸化Nrf2，促使Keap1-Nrf2耦合体解离，Nrf2活化、蓄积并被转运进入细胞核，结合Maf形成异二聚体，识别并结合下游抗氧化原件ARE。ARE位于机体氧化应激应答系统分泌的保护性蛋白的基因上游，Nrf2是ARE的激活因子，当Nrf2与ARE结合后会介导下游保护性抗氧化基因的表达，生成保护性蛋白如超氧化物歧化酶(SOD)，醌氧化还原酶1(NQO1)、血红素加氧酶1(HO-1)及γ-谷氨酰半胱氨酸合成酶(γ-GCS)，共同在细胞氧化损伤方面发挥着重要的抑制作用，可作为机体内拮抗氧化损伤的第一道防线。Nrf2-ARE通路是迄今为止发现的最为重要的内源性抗氧化应激通路。
乳酸菌是一类利用可发酵糖(碳水化合物)产生大量乳酸的无芽孢、革兰氏染色阳性细菌的总称，具有改善人体肠道菌群、促进肠胃蠕动、提高消化率、抑制腐败菌生长等功能，除此之外，乳酸菌还具有增强免疫力、抗氧化、延缓衰老等功能。传统发酵泡菜主要利用蔬菜本身带有的乳酸菌进行发酵，所以发酵完成的泡菜中所含有的乳酸菌种类也是多种多样。研究表明，中国泡菜中的发酵菌种以乳酸杆菌为主，如植物乳杆菌(Lactobacillus plant arum)、戊糖乳杆菌(Lactobacillus pentosus)、清酒乳杆菌(lactobacillus sake)、短乳杆菌(Lactobacillus brevis)、干酪乳杆菌(Lactobacillus casei)和发酵乳杆菌(Lactobacillus fermentum)等。发酵食品中的乳酸菌不仅可以降解食品中的亚硝酸盐，还具有降胆固醇、抗氧化、调节肠道健康多种作用。
随着对乳酸菌研究的不断深入，有研究发现，一些乳酸菌对重金属有着良好的抗性和吸附性能。乳酸菌缓解铅毒性的作用也可分为两种方式，第一是乳酸菌具有良好的吸附重金属的能力；第二是乳酸菌在机体内能发挥抗氧化作用，抵御自由基对机体造成的伤害。利用生物修复重金属中毒有众多优点，如原料广泛、 成本低、操作简单、环保等，而且不会产生次生危害。但目前的研究中，利用乳酸菌作为生物吸附剂的研究还比较少，主要因为大部分乳酸菌的吸附效果较低，且具有较好吸附能力的乳酸菌难以用于食品生产，因此寻求一株可用于食品且对重金属铅离子有较好吸附能力的乳酸菌成为目前研究的热门。
发明内容
本发明的目的是提供一种发酵乳杆菌LF-SCHY34及其应用，以解决上述现有技术存在的问题。
为实现上述目的，本发明提供了如下方案：本发明提供一种发酵乳杆菌，所述发酵乳杆菌命名为LF-SCHY34，保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC No.18795。
本发明还提供一种根据所述发酵乳杆菌LF-SCHY34在制备治疗或预防重金属中毒的产品中的应用。
进一步的，所述治疗或预防重金属中毒的产品用于吸附重金属。
本发明还提供一种根据所述发酵乳杆菌LF-SCHY34在制备治疗或预防由重金属中毒引起的病症中产品中的应用。
进一步的，所述由重金属中毒引起的病症为肝脏损伤、肾脏损伤或脑组织损伤。
进一步的，所述重金属为重金属铅，所述发酵乳杆菌的有效剂量是10 9CFU/次。
本发明还提供一种根据所述的发酵乳杆菌在制备治疗或预防氧化损伤的产品中的应用。
进一步的，所述发酵乳杆菌的有效剂量是10 9CFU/次。
本发明公开了以下技术效果：(1)本发明公开的乳酸菌LF-SCHY34对铅离子的吸附包括生物吸附和累积两种方式，是一株优质的铅吸附乳酸菌；(2)本发明公开的乳酸菌LF-SCHY34拥有较高体外和体内抗氧化能力，有较好的粘附能力，能更好的定植于人体肠道中发挥益生作用；(3)本发明公开的乳酸菌LF-SCHY34能缓解铅离子对SD大鼠肝脏和肾脏造成的损伤，减少肝脏和肾脏中炎症的发生，保护肝脏和肾脏细胞的完整性；(4)本发明公开的乳酸菌LF-SCHY34能增强Keap1/Nrf2/ARE信号通路的反应，激发更多下游基因表达产生拥有抗氧化能力的HO-1，NQO1和γ-GCS以缓解SD大鼠因铅诱导所导致的氧化应激反应，以更好地缓解铅对机体造成的氧化损伤。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为LF-SCHY34乳酸菌铅离子吸附前后扫描电镜和透射电镜图，其中a为吸附前LF-SCHY34扫描电镜图，b为吸附前LF-SCHY34透射电镜图，c为吸附后LF-SCHY34扫描电镜图；d为吸附后LF-SCHY34透射电镜图；
图2为LF-SCHY34乳酸菌铅离子吸附前后能谱探测形貌图和扫描能谱图，其中a为吸附前LF-SCHY34能谱探测形貌图，b为吸附前LF-SCHY34扫描能谱图，c为吸附后LF-SCHY34能谱探测形貌图，d为吸附后LF-SCHY34的扫描能谱图；
图3为SD大鼠肝脏切片图，其中a为正常组，b为铅诱导组，c为EDTA组，d为LF-SCHY34组；
图4为SD大鼠肾脏切片图，其中a为正常组，b为铅诱导组，c为EDTA组，d为LF-SCHY34组。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图和具体实施方式对本发明作进一步详细的说明。
乳酸菌的分离与鉴定：从重庆地区的泡菜中分离得到一株乳酸菌，提取完的DNA进行PCR扩增，其中上游引物27F(5'－AGA GTT TGA TCC TGGCTC AG－3')1μL、下游引物1495R(5'－CTA CGG CTA CCTTGT TAC GA－3')1μL，2×Taq plus Buffer 12.5μL、模板DNA 1μL，用无菌dd H2O将体系补足至25μL。并以无菌超纯水替代模板DNA作为阴性对照。扩增条件为：94℃5min；94℃30s，55℃30s，72℃1min，共29个循环；最后72℃延伸5min。取5μL扩增产物进行琼脂糖凝胶电泳检测，琼脂糖浓度为1.5％，电泳条件为110V，45min。将检测成功的PCR产物送往北京擎科生物技术有限公司进行测序，16S rDNA序列如SEQ ID NO：1所示，测序成功的序列在NCBI进行BLAST对比，所述乳酸菌为乳杆菌科、乳杆菌属，相似性99.9％，命名为LF-SCHY34，2019年11月4日保藏于中国普通微生物菌种保藏管理中心(CGMCC，北京)，保存号为CGMCC No.18795。
实施例1发酵乳杆菌LF-SCHY34的体外性能测定实验
1.1发酵乳杆菌LF-SCHY34在人工胃液中的存活率测定
人工胃液由0.2％的NaCl，0.35％的胃蛋白酶混合而成，使用1mol/L的HCl调节pH至3.0，随后使用0.22μm的无菌过滤器过滤灭菌。将5ml MRS液体培养基中活化两次后的LP-KFY04于3000rpm离心10min收集菌体，无菌生理盐水清洗2次并重悬于5mL生理盐水。1:1(v/v)将菌液与无菌人工胃液混匀，摇匀后置于恒温培养箱中37℃培养，分别于0h，3h测定活菌数，并按公式(1)计算LF-SCHY34在人工胃液中的存活率。
Figure PCTCN2021074767-appb-000001
1.2发酵乳杆菌LF-SCHY34在胆盐中的生长效率测定
按2％(v/v)接种量取活化两次的LP-KFY04接种于含0.0％和0.3％猪胆盐的MRS-THIO medium中(MRS培养基中添加0.2％的巯基乙酸钠并121℃灭菌15min)，在37℃恒温摇床中培养24h后，以空白培养基(未接种的MRS-THIO medium)为对照，将空白培养基与接种后的培养基添加到96-well板中，每孔200ml，于波长600nm处测量吸光度，并按照公式(2)计算生长效率。
Figure PCTCN2021074767-appb-000002
1.3发酵乳杆菌LF-SCHY34体外铅离子吸附能力测试
发酵乳杆菌SCHY34菌悬液于1500xg离心10min收集菌体，生理盐水洗涤两次后离心收集菌体。将菌体加入50mg/L pH 6.3的硝酸铅溶液中，最终菌体浓度为1g/L，置于37℃下培养24h后8000xg离心20min收集上清液，使用火焰原子吸收法测定原始溶液铅离子浓度C0，吸附后铅离子浓度C1，按照公式(3)计算铅离子吸附率。
Figure PCTCN2021074767-appb-000003
1.4发酵乳杆菌LF-SCHY34表面疏水能力测定
发酵乳杆菌SCHY34菌悬液于1500xg离心10min收集菌体，生理盐水洗涤两次后离心收集菌体。用生理盐水调整菌体浓度，使其在580nm的波长下吸光度值为1.000。取2ml调整好吸光度的菌悬液与2ml二甲苯混合，涡旋120min，室温放置30min，吸取1ml上层水相，以生理盐水为空白对照，在580nm下测定空白对照组吸光度值A0值和样品吸光度值A1，按照公式(4)计算乳酸菌的表面疏水能力。
Figure PCTCN2021074767-appb-000004
1.5发酵乳杆菌LF-SCHY34的体外抗氧化能力测定
1.5.1发酵乳杆菌LF-SCHY34体外羟自由基清除能力测定
将发酵乳杆菌LF-SCHY34配制为10 9cfu/mL的菌悬液样品待用，试管中分别加入1mL 0.05mol/L pH7.4的磷酸缓冲液，0.5mL 6mmol/L的邻二氮菲，充分混匀后，加入0.5mL 6mmol/L的FeSO 4溶液并立即混匀。试管分为样品管，空白管和对照管。向样品管中加入0.5mL菌悬液样品溶液，对照管中加入0.5mL 0.1％的H 2O 2溶液，混匀，加入0.5mL 0.1％的H 2O 2溶液，最后补充体积到4mL，于37℃保温1h，测其在536nm波长处的吸光度分别为Ai和A。空白管中不添加样品溶液和H 2O 2溶液，直接使用磷酸缓冲液补充体积到4mL进行后续实验，最终测得吸光度为A0，按照公式(5)计算羟自由基清除率。
Figure PCTCN2021074767-appb-000005
1.5.2发酵乳杆菌LF-SCHY34体外DPPH基清除能力测定
将1mL发酵乳杆菌LF-SCHY34的菌悬液(10 9cfu/mL)中加入1mL 2mmol/L的DPPH乙醇溶液；1mL发酵乳杆菌SCHY34的菌悬液(10 9cfu/mL)中加入1mL无水乙醇；1mL 2mmol/L的DPPH乙醇溶液中加入1mL无水乙醇分别在室温下静置30min后，在517nm波长处测吸光度分别为Ai，Aj和A0，通过公式(6)计算DPPH自由基清除率。
Figure PCTCN2021074767-appb-000006
1.5.3发酵乳杆菌LF-SCHY34体外超氧阴离子清除能力测定
将0.1mL发酵乳杆菌LF-SCHY34的菌悬液(10 9cfu/mL)加入4.5mL pH8.0的Tris-HCl缓冲液，于25℃水浴预热20min后，加入0.4mL 25mmol/L的邻苯三酚，于25℃水浴反应5min，立即用2滴8mol/L的HCl终止反应，在325nm波长处测吸光度即为A，空白组用0.1mL H 2O代替样品即为A0，通过公式(7)计算超氧阴离子清除率。
Figure PCTCN2021074767-appb-000007
1.5.4发酵乳杆菌LF-SCHY34体外还原力测定
取0.5mL发酵乳杆菌LF-SCHY34菌悬液(10 9cfu/mL)加入0.5mL浓度为0.2mol/L pH7.2的磷酸缓冲溶液及1％铁氰化钾0.5mL，于50℃水浴20min，急速冷却。再加入10％三氯乙酸0.5mL，1500xg离心10min，取上清液1mL，加入1mL蒸馏水及1mL0.1％三氯化铁，混合均匀。10min后，于700nm波长测定其吸 光度。设置不同剂量半胱氨酸组作为标准，将样品组吸光度换算为标准组计量单位(μmol/L)进行比较。
1.6发酵乳杆菌LF-SCHY34的能力测定结果
LF-SCHY34在人工胃液中的存活率为88.71％±0.23％，在人工胆盐中的生长效率为85.32％±0.41％，表面疏水率为43.78％±0.75％，铅离子吸附率为69.58％±0.56％，对羟自由基、超氧阴离子和DPPH的清除率分别为44.15％±0.41％，66.11％±0.97％和79.49％±0.87％，还原力为111.66±1.18μmol/L。各组数据表明，LF-SCHY34体外抗氧化能力较强，能高效的清除自由基。
实施例2发酵乳杆菌LF-SCHY34菌体对铅离子吸附前后的扫描电镜、扫描能谱分析和透射电镜分析
2.1扫描电镜和扫描能谱分析
取不含铅离子溶液的菌体和含50mg/L铅离子吸附后的菌体，于8000xg条件下离心20min，再用灭菌的超纯水冲洗三次后，同上条件离心，随后立即倒入戊二醛固定1.5h，用磷酸缓冲液冲洗3次，然后置于6000xg条件下离心10min，再分别用不同浓度(50％、70％、90％、100％)的乙醇脱水一次，后6000xg条件下离心10min，再用乙醇和叔丁醇混合液(v/v＝1/1)以及纯叔丁醇洗脱一次，后6000xg条件下离心10min，将菌体放入-20℃下冷冻30min，放入冷冻干燥仪中干燥4h，最后用离子溅射镀膜仪在样品表面镀一层厚100-150A的金属膜，放入观察室进行观察，并用于能量色散光谱仪分析元素组成。
2.2透射电镜分析
取不含铅离子溶液的菌体和含50mg/L铅离子吸附后的菌体，于8000xg条件下离心20min，用2.5％的戊二醛溶液4℃固定过夜，倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；用1％的锇酸溶液固定样品1-2h；小心取出锇酸废液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；用梯度浓度(包括30％，50％，70％，80％，90％和95％五中浓度)的乙醇溶液对样品进行脱水处理，每种浓度处理15min，再用100％的乙醇处理20min。用包埋剂与丙酮的混合液(v/v＝1/1)处理样品1h；用包埋剂与丙酮的混合液(v/v＝3/1)处理样品3h；纯包埋剂处理样品过夜；将经过渗透处理的样品包埋起来，70℃加热过夜，即得到包埋好的样品。样品切片后经柠檬酸铅溶液和醋酸双氧铀50％乙醇饱和溶液各染色5-10min，晾干后即可在透射电镜中观察。
2.3结果
图1为LF-SCHY34乳酸菌铅离子吸附前后扫描电镜和透射电镜图：图1-a为吸附前正常组LF-SCHY34菌体扫描电镜图片，通过观察能发现乳酸菌细胞形态完整，轮廓清晰，干净饱满，表面光滑，边缘界限清楚，表面无颗粒物无黏附物；图1-c为铅吸附后LF-SCHY34菌体的扫描电镜图片，发现乳酸菌菌株细胞变形严重，菌体细胞出现凹陷扁塌，外观粗糙，细胞边缘轮廓变得模糊，甚至出现粘连成片和不规则聚集的现象，同时发现细胞便面覆盖有细小颗粒。
图1-b为吸附前正常组LF-SCHY34透射电镜图片，发现正常组菌株细胞切面未出现沉积物，表面清晰，无黏附物；图1-d为吸附后LF-SCHY34菌体的透射电镜图，与正常组菌体对比，经过铅吸附的乳酸菌细胞切面有大量黑色沉积物，细胞内部出现空白部位。
图2为LF-SCHY34乳酸菌铅离子吸附前后能谱探测形貌图和扫描能谱图，表2为铅离子吸附前后乳酸菌扫描能谱元素变化表：通过图2和表1可以发现，与正常组相比，铅吸附后的乳酸菌菌体表面O，N元素含量下降，C，P，Pb元素含 量升高。
表2铅离子吸附前后乳酸菌扫描能谱元素变化表
Figure PCTCN2021074767-appb-000008
实施例3发酵乳杆菌LF-SCHY34对醋酸铅诱导的大鼠氧化应激的缓解作用
以下实验均为平行进行三次血清和组织样品的测量，然后计算平均值。使用SPSS软件(SPSS v.25 for Windows,IBM Software Group,Chicago,IL,USA)对数据进行平均和分析。使用Duncan多范围检验通过单因素方差分析评估各个组平均值之间的差异。p<0.05的差异被认为具有统计学意义。
3.1动物实验
48只6周龄SPF级雄性SD大鼠经过一周适应性喂养后随机分为4组，分别为正常组12只，铅诱导组12只，EDTA(Sigma-Aldrich,St.Louis,MO,USA)组12只和LF-SCHY34组12只。正常组大鼠在整个实验周期中均自由进食AIG-93G饲料以及自由饮用不含醋酸铅的饮用水。其余三组大鼠在自由进食AIG-93G饲料的同时，从第一周到第十二周均自由饮用浓度为200mg/L的醋酸铅溶液代替饮用水。EDTA组大鼠从第8周至第12周，每天注射浓度为50mg/kg的EDTA，LF-SCHY34组大鼠从第1周至第12周每日灌胃1×10 9CFU/kg(b.w)LF-SCHY34。
12周后，所有大鼠禁食12小时后使用乙醚麻醉，经眼眶静脉取血后处死，使用液氮收集大鼠心脏、肝脏、肾脏、脑组织后放入-80℃贮藏待用。本研究按照赫尔辛基宣言进行，方案于2019年6月8日(中国重庆)由重庆市功能食品协同创新中心伦理委员会批准(201906008A)。
3.2 SD大鼠血液、肝脏、肾脏和脑组织中铅含量的测定
精确量取0.0,0.4,0.8,1.2,1.6,2.0mL铅标准溶液置于50mL容量瓶中，再分别加入2mL含12.5％磷酸二氢铵和2.5％硝酸镁的混合溶液，然后用2％的硝酸定容。分别吸取20μL上述不同浓度的标准溶液于石墨炉原子化器中测定吸光度并制作标准曲线。
将收集的血液取500μL和各个组织50mg分别置于四氟乙烯消解罐中，加入5mL硝酸进行消解，冷却后加入1mL含12.5％磷酸二氢铵和2.5％硝酸镁的混合溶液并使用硝酸定容至25mL，加入20μL定容后的溶液于石墨炉原子化器中测定吸光度，通过标准曲线计算血液中铅的含量。
表2 SD大鼠血液、肝脏、肾脏和脑组织中铅含量
Figure PCTCN2021074767-appb-000009
a-d表示根据Duncan的新MRT，在同一表格中不同字母的平均值存在显着差 异(p<0.05)。
通过表2中的数据可以发现，未经过醋酸铅溶液诱导过的正常组大鼠的血液、肝脏、肾脏和脑组织中的铅含量是所有组别中最低的。与之相反，铅诱导组大鼠血液、肝脏、肾脏和脑组织中的铅含量是所有组别中最高的。其中血液和肾脏中的铅含量为正常组大鼠的20倍和17倍。使用EDTA药物和LF-SCHY34干预后的大鼠血液、肝脏、肾脏和脑组织中的铅含量明显降低，特别是LF-SCHY34干预后的大鼠血液和肾脏中铅含量约为正常组的7倍和10倍，干预效果好于EDTA药物。
不仅如此，通过数据的对比还能发现，经过醋酸铅诱导的小鼠，其血液中铅含量是最高的，远高于组织器官中的铅含量。在三个组织器官中，肾脏的铅含量是最高的，其次是肝脏，最后是脑组织。
3.3 SD大鼠肝脏、肾脏组织形态学分析
取各组SD大鼠肝脏和肾脏组织相同部位的组织固定于10％福尔马林(v/v)中24h，经过组织脱水、清除、上蜡、包埋、切片、染色步骤后，于光学显微镜(BX43；Olympus,Tokyo,Japan)下观察组织学形态并拍照。
SD大鼠肝脏切片如图4所示，由图4可以观察到，正常组(图4a)大鼠的肝小叶结构有序，中央静脉和肝血窦清晰。使用醋酸铅诱导的大鼠(图4b)肝小叶模糊，肝索排列紊乱，单核细胞聚集并分散在不同间隙，肝细胞局灶性坏死和大片炎症细胞的浸润，细胞核间包体和细胞核碎裂。使用EDTA药物(图4c)和LF-SCHY34(图4d)干预过的醋酸铅诱导大鼠的肝脏细胞排列更为有序，炎症细胞浸润较少，也较少出现肝脏细胞的破损和坏死。
SD大鼠肾脏切片如图5所示，由图5可以观察到，正常组(图5a)大鼠肾小球和肾小管结构正常，细胞排列紧密，细胞数量正常。在醋酸铅诱导(图5b)的SD大鼠肾脏切片中能发现肾小管、肾小球增大且细胞过多,毛细血管扩张充血；肾小管管腔扩张,出现空泡状，上皮细胞颗粒变性，细胞破碎，淋巴细胞浸润。与铅诱导组相比，EDTA组(图5c)和LF-SCHY34组(图5d)的SD大鼠肾脏切片中的肾小球和肾小管损伤较少，细胞完整度较好，存在较少炎症细胞浸润的现象，未见严重的肾小管扩正和空泡现象。LF-SCHY34干预过的SD大鼠其肝脏和肾脏的形态都与正常组大鼠的形态更为接近。
3.4 SD大鼠血清、肝脏、肾脏和脑组织氧化水平的测定
称取100mg各脏器组织用生理盐水匀浆、血液离心后吸取上清液进行实验。根据试剂盒(Nanjing Jiancheng Bioengineering Institute,China)操作方法进行脏器生化指标过氧化氢酶(CAT),活性氧(ROS),总超氧化物歧化酶(T-SOD),丙二醛(MDA)和谷胱甘肽(GSH)水平的测定。
SD大鼠血清、肝脏、肾脏和脑组织中氧化指标数据见表3。从表3中可以发现，正常组大鼠血液、肝脏、肾脏和脑组织中的CAT、T-SOD和GSH为四组中最高，MDA和ROS为四组中最低，铅诱导组的血液、肝脏、肾脏和脑组织中的趋势与正常组完全相反，铅诱导组CAT、T-SOD和GSH为四组中最低，MDA和ROS为四组中最高。EDTA组和LF-SCHY34组的氧化指标趋势与正常组类似，其中LF-SCHY34的氧化指标数值与正常组氧化指标数值更为接近。
表3 SD大鼠血液、肝脏、肾脏和脑组织中氧化指标的含量
Figure PCTCN2021074767-appb-000010
Figure PCTCN2021074767-appb-000011
3.5 SD大鼠血清、肝脏和肾脏炎症因子水平的测定
取大鼠血清和组织匀浆上清液，按照试剂盒(Shanghai Yaji Biotechnology Co.Ltd.Shanghai,China)操作方法测量血清中IL-6,IL-10,IL-1β,TNF-α,IFN-γ水平。
表4为SD大鼠血清、肝脏和肾脏炎症指标的数据，发现正常组血清、肝脏和肾脏中的IL-1β，IL-6、TNF-α和IFN-γ水平是四组中最低的，IL-10水平是四组中最高的。而铅诱导组IL-10水平是四组中最低的，IL-1β，IL-6、TNF-α和IFN-γ水平是四组中最高的。EDTA组和LF-CQPC组的IL-1β，IL-6、TNF-α和IFN-γ水平与铅诱导组相比都呈下降的趋势，IL-10水平与铅诱导组相比都呈上升的趋势，但LF-SCHY34组的下降和上升趋势比EDTA组更为明显。
表4 SD大鼠血液、肝脏和肾脏中炎症指标的含量
Figure PCTCN2021074767-appb-000012
3.6 SD大鼠血清δ-ALAD,ALT,AST,CRE和BUN水平的测定
取大鼠血清，按照试剂盒(Shanghai Yaji Biotechnology Co.Ltd.，Shanghai,China)操作方法测量血清中δ-氨基酮戊酸脱水酶(δ-ALAD),谷丙转氨酶(ALT),谷草转氨酶(AST),血液肌酸(CRE)和血尿素氮(BUN)水平。
SD大鼠血清中ALT，AST、δ-ALAD酶活性和BUN、CRE含量如表5所示。在四组大鼠中，正常组SD大鼠ATL，AST的酶活性最低，δ-ALAD酶活性最高，BUN、CRE含量最低。而肝脏相关ATL，AST的酶活性在铅诱导组中最低，δ-ALAD酶活性和肾脏相关BUN、CRE含量在铅诱导组中最高。EDTA组和LF-SCHY34组大鼠的三种酶活性趋势和BUN、CRE的含量与正常组相似，从酶活力和BUN，CRE含量的数值上看，LF-SCHY34的干预效果优于EDTA。
表5 SD血清中ALT、AST、BUN、CRE和δ-ALAD的含量
Figure PCTCN2021074767-appb-000013
3.7 SD大鼠肝脏和肾脏组织mRNA分析
使用TRIzol(Invitrogen，Carlsbad，CA，USA)提取肝脏组织中的RNA，并调整RNA的浓度为1μg/μL，使用cDNA逆转录试剂盒(Thermo Fisher Scientific)将RNA合称为cDNA。随后将合成的cDNA与10μL SYBR Green PCR Master Mix(Thermo Fisher Scientific)，2μL引物(表6)和蒸馏水混合，然后放入qPCR仪器中进行处理。qPCR程序：95℃60s；95℃15s,55℃30s,72℃35s，40个循环；95℃30s；55℃35s。使用3-磷酸甘油醛脱氢酶(GAPDH)作为内参基因，并使用2 -ΔΔCt公式计算相对mRNA的转录水平。
表6引物序列
Figure PCTCN2021074767-appb-000014
实验检测了肝脏和肾脏中Keap1/Nrf2ARE通路相关的Keap1、Nrf2、HO-1、SOD、GSH、NQO1和γ-GCS的mRNA表达。表7为SD肝脏和肾脏中Keap1、Nrf2、HO-1、SOD1、SOD2、GSH、NQO1和γ-GCS的mRNA表达量数据。
从表7中能够发现，与正常组相比，除SOD和GSH的表达量以外，其余四组大鼠的Keap1、Nrf2、HO-1、SOD、GSH、NQO1和γ-GCS的mRNA的表达均有不同程度的增加。正常组的SOD和GSH的mRNA表达量为四组中最高的。铅诱导组的Keap1、Nrf2、HO-1、NQO1和γ-GCS的mRNA表达量虽高于正常组，但无显著 性差异(p＜0.05)。与铅诱导组相比，EDTA组和LF-SCHY34组的Keap1、Nrf2、HO-1、SOD1、SOD2、GSH、NQO1和γ-GCS的mRNA表达量均显著升高。LF-SCHY34组在相关基因的mRNA表达量上均显著高于EDTA组的表达量。
Figure PCTCN2021074767-appb-000015
3.8 SD大鼠肝脏和肾脏组织Western blot分析
将100mg肝脏组织在1mL RIPA(Thermofisher，Waltham，MA，USA)和10μL PMSF(Thermofisher)中匀浆，并在4℃下以12000xg离心5分钟。使用BCA蛋白测定试剂盒(Thermofisher)对蛋白质进行定量。将蛋白质样品与样品缓冲液(Thermofisher)以4：1混合，并在95℃加热5分钟，然后将样品点入SDS-PAGE凝胶胶孔中并于100V下进行跑胶。将SDS-PAGE凝胶上的条带转移到PVFD膜上，用5％脱脂奶封闭PVDF膜1h，然后在4℃下与一抗(Thermofisher)孵育过夜，洗膜后加入抗体(Thermofisher)并孵育1小时。使用western ECL substrate(Thermofisher)进行化学发光后于iBright(Thermofisher)上获得图像(图7)。
3.9结果分析
有文献表明10 6CFU的活菌到达肠胃就能发挥较好的益生功效，人体胃液pH值通常为3.0左右，小肠的胆汁盐含量在0.03-0.30％,对酸和胆盐耐受性较低的乳酸菌在此环境则无法存活，能适应此环境的乳酸菌则能消化道中存活。LF-SCHY34在胃酸中的存活能力为88.71％，胆盐中的生长效率为85.32％，这表明，服用10 9CFU的LF-SCHY34能顺利达到肠道发挥益生作用。
乳酸菌吸附重金属一般分为生物吸附和生物积累两个阶段。生物吸附包括胞外沉淀、表面络合及离子交换等作用，主要是一些微生物表面蛋白质、多糖、脂类等物质上的化学基团及细胞表面含有的一些离子与金属相互作用形成金属络合物；而生物积累包括跨膜运输、胞内积累、细胞生理代谢和自身调节机制等。 经过实验发现，LF-SCHY34对铅离子的体外吸附率为74.8％，吸附能力远高于Halttunen等人研究发现的范围在39.70％-69.60％左右铅吸收率的乳酸菌。动物实验结果也清楚显示了LF-SCHY34能降低SD大鼠血液、肝脏、肾脏和脑组织中的铅含量。通过扫描电镜、透射电镜和扫描能谱实验分析发现，LF-SCHY34在参与铅吸附的过程中能较好的吸收溶液中的铅离子，在吸附过程中菌体表面含C，P的基团参与其中。大量吸附Pb离子后，部分乳酸菌菌体形态损坏或内部发生变化，甚至出现破裂，细胞内含O，N元素物质溶出，导致O，N元素含量大量增加。因此可以推断，LF-SCHY34对铅离子的吸附包括生物吸附和累积两种方式，是一株优质的铅吸附乳酸菌。
在另一方面，当人体和动物的组织器官长期处在铅暴露下，活性氧(ROS)含量增加，谷胱甘肽(GSH)含量和一些重要的抗氧化酶如SOD和CAT活性降低甚至失活，丙二醛(MDA)含量升高，加快细胞膜中的脂质过氧化过程。活性氧(ROS)主要包括过氧化物、氧离子和含氧自由基等。乳酸菌能通过清除细胞周围活性氧自由基来缓解氧化损伤和脂质过氧化，还能通过调节宿主细胞抗氧化相关信号通路进一步防御机体的氧化应激。本次的体外数据显示LF-SCHY34对羟自由基、超氧阴离子和DPPH的清除率分别为44.15％±0.41％，66.11％±0.97％和79.49％±0.87％，还原力为111.66±1.18μmol/L。Takashi Kuda等人发现不同乳酸菌的DPPH清除率和超氧阴离子清除率大概为78％-90％和45％-62％，Ramila AZAT等人的实验结果中乳酸菌羟自由基清除率最高为45.79％，Zhai等人测定的乳酸菌的还原能力最高等同于99.41μmol/L的L-半胱氨酸。上述的实验结果与本实验中的数据相比，发现LF-SCHY34能更高效的清除自由基，体外抗氧化能力较强。在动物实验中发现，与铅诱导组相比LF-SCHY34组的血清、肝脏、肾脏和脑组织中的SOD和CAT活性和GSH含量较高，ROS和MDA水平较低，因此可以推断，LF-SCHY34的是一株拥有较高体外和体内抗氧化能力的优势菌种。
疏水性是乳酸菌细胞粘附肠道上皮细胞的重要指标之一，疏水性越高，乳酸菌粘附特性越好。Nadia S.AlKalbani等人通过实验发现，不同乳酸菌的疏水性为0.5％-44.1％，而本实验中LF-SCHY34的疏水性有43.78％±0.75％，说明LF-SCHY34有较好的粘附能力，能更好的定植于人体肠道中发挥益生作用。
人体内的铅最先通过肾脏排出,当肾脏达到排铅最大量时,铅浓缩并沉积于近端小管上皮细胞中,影响细胞代谢,导致肾脏结构和功能的损伤。当细胞损伤或坏死时,肾小管重吸收功能下降，肌酐和尿素随着重吸收能力下降而留在血液中，因此血肌酐浓度和血尿氮素浓度可以反映肾功能是否存在损伤。铅会抑制体内δ-氨基酮戊酸脱水酶(δ-ALAD)活性，使血中的ALA增加，δ-ALA随尿液排出，造成血液中的δ-ALA含量减少。肝脏是机体最重要的解毒器官，可以将经消化系统进入体内的不同毒素在生化反应下，转变为低毒物质排出体外。实验证明，铅会造成肝脏不同程度的病变，造成严重的炎症反应，影响肝脏相关酶活力，最终造成肝损伤。ALT和AST分布在肝细胞中，当肝脏细胞受损时，胞浆中的ALT和AST会释放到血液中，因此血液中ALT和AST的浓度可以表示肝脏细胞受到损伤的程度。血清和脏器组织中炎症因子能够反映机体内的炎症损伤程度。通过血清、肝脏和肾脏中炎症因子水平、血清δ-ALAD,ALT,AST,CRE和BUN的检测结果，以及肝脏、肾脏切片的病例分析都能发现，LF-SCHY34能缓解铅离子对SD大鼠肝脏和肾脏造成的损伤，减少肝脏和肾脏中炎症的发生，保护肝脏和肾脏细胞的完整性。
机体具有抵御环境有害因素的自我防御系统，从而减少其对组织、细胞形态 和功能的有害影响，促进细胞的存活。核因子E2相关因子2(Nrf2)是机体自我防御系统中重要的一员。Nrf2是一个对氧化还原状态敏感的转录因子，它主要参与细胞内抗氧化反应，是机体重要的抗氧化因子。在正常条件下，Kelch样环氧氯丙烷相关蛋白1(Keap1)在胞浆中与Nrf2结合致其处于失活状态，并且逐渐被泛素化降解。当机体受到氧化应激时，胞浆内Nrf2从Keap1上解离，转移入细胞核，与某些基因上游的抗氧化反应原件(ARE)区域结合，启动基因的表达(图X)，这些基因编码了包括NQO1、HO-1和γ-GCS。这些细胞保护作用基因的表达可以增强细胞对有害刺激的自我防御能力，促进细胞存活。HO-1对成纤维细胞、肝脏细胞、肾上皮细胞等具有抗氧化、抗炎、抗凋亡做等保护作用，也是催化血红素的促氧化分解反应的第一步限速酶，最终分解产物为一氧化碳、胆绿素以及离子等，具有抗氧化和抗炎双重作用，并在各种心血管疾病、肝肾功能障碍和中枢神经系统疾病中发挥重要的保护性作用。HO-1蛋白以及mRNA含量增加一般会提高机体内抗氧化应激以及抗细胞损伤的能力。NQO1是一种普遍存在与几乎所有动物物种中的可溶性黄酮酶，其在脂肪细胞、血管内皮细胞和上皮细胞中表达水平较高。NQO1以NADPH为供体，生成稳定的对苯二酚以避免产生有毒的半醌自由基和活性氧的单电子还原反应，以及细胞内巯基的直接反应。NQO1能解毒活性强的醌类物质，保持脂溶性抗氧化剂的还原形态，从而保护机体免受氧化胁迫。γ-GCS也是Keap1/Nrf2/ARE信号通路的下游抗氧化因子，它是GSH生物合成中的限速酶，表达量水平升高时，可以清除大量自由基从而减轻细胞的氧化损伤。Fang Ye等和Miao Long等发现，在由铅诱导的氧化应激模型中，Nrf2与胞浆内Keap1、SOD1、SOD2、GSH、HO-1，NQO1和γ-GCS的表达程度呈显著正相关，与细胞核内Keap1呈负相关。在本实验中，通过检测肝脏、肾脏中Keap1/Nrf2/ARE信号通路上相关基因和蛋白的表达情况，发现所有受到铅诱导的SD大鼠都会产生氧化应激，导致体内Keap1/Nrf2/ARE激活，对机体形成保护机制，但激活下游蛋白表达的程度有所差异。铅诱导组的抗氧化蛋白表达虽有所提升，但与正常组无显著性差别，可能原因是铅诱导的氧化应激首先会使得细胞内SOD和GSH等抗氧化物质减少，虽然机体通过后期自身调节能产生一小部分抗氧化物质，但由于铅离子持续的损伤造成的危害大于机体的修复能力，因此产生的抗氧化物质难以抵御铅对机体造成的氧化损伤。LF-SCHY34能增强Keap1/Nrf2/ARE信号通路的反应，激发更多下游基因表达产生拥有抗氧化能力的HO-1，NQO1和γ-GCS以缓解SD大鼠因铅诱导所导致的氧化应激反应。
本实验利用从天然发酵的泡菜中分离的乳酸菌，研究其对体内和体外铅离子的吸附能力和抗氧化能力，证实了LF-SCHY34一方面能有效地吸附铅离子，另一方面能清除自由基，增强机体抗氧化能力，从两方面同时保护机体免受铅诱导的氧化应激损伤，并为后期研究食品级乳酸菌缓解铅中毒提供理论和数据支持。
综上所述，LF-SCHY34能够吸附体内和体外的铅离子，减少血液和脏器中的铅含量，保护肝脏、肾脏和脑组织。此外，LF-SCHY34能清除自由基，激活Keap1/Nrf2/ARE信号通路分泌更多抗氧化物质，以更好地缓解铅对机体造成的氧化损伤。由此看来，LF-SCHY34是一株具有较强铅吸附能力和抗氧化能力的优良菌株。在未来，LF-SCHY34仍具有更多的研究价值，其对人体铅离子造成的氧化应激作用和缓解其他重金属毒性等方面具有较大的潜力和研究价值。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (8)

  1. 一种发酵乳杆菌，其特征在于：所述发酵乳杆菌命名为LF-SCHY34，保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCC No.18795。
  2. 根据权利要求1所述的发酵乳杆菌在制备治疗或预防重金属中毒的产品中的应用。
  3. 根据权利要求2所述的应用，其特征在于：所述治疗或预防重金属中毒的产品用于吸附重金属。
  4. 根据权利要求1所述的发酵乳杆菌在制备治疗或预防由重金属中毒引起的病症的产品中的应用。
  5. 根据权利要求4所述的应用，其特征在于：所述由重金属中毒引起的病症为肝脏损伤、肾脏损伤或脑组织损伤。
  6. 根据权利要求2-5任一项所述的应用，其特征在于：所述重金属为重金属铅，所述发酵乳杆菌的有效剂量是10 9CFU/次。
  7. 根据权利要求1所述的发酵乳杆菌在制备治疗或预防氧化损伤的产品中的应用。
  8. 根据权利要求7所述的应用，其特征在于：所述发酵乳杆菌的有效剂量是10 9CFU/次。
PCT/CN2021/074767 2021-01-29 2021-02-02 一种发酵乳杆菌lf-schy34及其应用 WO2022160360A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
KR1020217036127A KR102567035B1 (ko) 2021-01-29 2021-02-02 락토바실러스 퍼멘텀 lf-schy34 및 그 응용
JP2021550088A JP2023505400A (ja) 2021-01-29 2021-02-02 ラクトバチルス・ファーメンタム（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｆｅｒｍｅｎｔｕｍ） ＬＦ－ＳＣＨＹ３４とその応用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110123212.9 2021-01-29
CN202110123212.9A CN112708583B (zh) 2021-01-29 2021-01-29 一种发酵乳杆菌lf-schy34及其应用

Publications (1)

Publication Number Publication Date
WO2022160360A1 true WO2022160360A1 (zh) 2022-08-04

Family

ID=75549820

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/074767 WO2022160360A1 (zh) 2021-01-29 2021-02-02 一种发酵乳杆菌lf-schy34及其应用

Country Status (4)

Country Link
JP (1) JP2023505400A (zh)
KR (1) KR102567035B1 (zh)
CN (1) CN112708583B (zh)
WO (1) WO2022160360A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116121108B (zh) * 2022-10-21 2024-05-17 海南大学 发酵粘液乳杆菌hnu312及制备减轻铅脑毒性药品的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000035465A2 (en) * 1998-12-11 2000-06-22 Urex Biotech Inc. Oral administration of lactobacillus for the treatment and prevention of urogenital infection
CN104755090A (zh) * 2012-04-05 2015-07-01 伦敦健康科学中心研究公司 用于除去有毒化合物的食品级细菌
CN111040960A (zh) * 2019-11-19 2020-04-21 西南大学 植物乳杆菌lp33及其在制备用于促进铅排泄的产品中的应用
CN111096459A (zh) * 2019-11-19 2020-05-05 西南大学 植物乳杆菌lp33在制备用于预防铅中毒的产品中的应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102586148B (zh) * 2012-02-28 2013-05-08 江南大学 一种能够缓解铅毒性的植物乳杆菌及其用途
CN103146633A (zh) * 2013-03-08 2013-06-12 青岛农业大学 一株嗜铅菌的克隆与培养
TWI689584B (zh) * 2018-11-17 2020-04-01 原生生物醫學股份有限公司 新穎乳酸菌及其用以治療或預防重金屬相關疾病之用途
CN110628677B (zh) * 2019-09-27 2023-05-02 广东环凯生物科技有限公司 一种吸附重金属铅的干酪乳杆菌syf-08及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000035465A2 (en) * 1998-12-11 2000-06-22 Urex Biotech Inc. Oral administration of lactobacillus for the treatment and prevention of urogenital infection
CN104755090A (zh) * 2012-04-05 2015-07-01 伦敦健康科学中心研究公司 用于除去有毒化合物的食品级细菌
CN111040960A (zh) * 2019-11-19 2020-04-21 西南大学 植物乳杆菌lp33及其在制备用于促进铅排泄的产品中的应用
CN111096459A (zh) * 2019-11-19 2020-05-05 西南大学 植物乳杆菌lp33在制备用于预防铅中毒的产品中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HALTTUNEN TEEMU ET AL.: "Reversible surface binding of cadmium and lead by lactic acid and bifidobacteria", INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, vol. 125, no. 2, 15 July 2008 (2008-07-15), XP022777622, ISSN: 1879-3460, DOI: 10.1016/j.ijfoodmicro.2008.03.041 *
HSIEH PEI-SHAN, CHEN CHING-WEI, KUO YI-WEI, HO HSIEH-HSUN: "Lactobacillus spp. reduces ethanol‑induced liver oxidative stress and inflammation in a mouse model of alcoholic steatohepatitis", EXPERIMENTAL AND THERAPEUTIC MEDICINE, vol. 21, no. 3, 1 March 2021 (2021-03-01), Spandidos Publications, GR, pages 1 - 9, XP055954935, ISSN: 1792-0981, DOI: 10.3892/etm.2021.9619 *
KIRILLOVA ANNA V., DANILUSHKINA ANNA A., IRISOV DENIS S., BRUSLIK NATALIYA L., FAKHRULLIN RAWIL F., ZAKHAROV YURI A., BUKHMIN VLAD: "Assessment of Resistance and Bioremediation Ability of Lactobacillus Strains to Lead and Cadmium", INTERNATIONAL JOURNAL OF MICROBIOLOGY, HINDAWI PUBLISHING CORPORATION, vol. 2017, 4 January 2017 (2017-01-04), pages 1 - 7, XP055954933, ISSN: 1687-918X, DOI: 10.1155/2017/9869145 *

Also Published As

Publication number Publication date
CN112708583B (zh) 2022-05-17
KR102567035B1 (ko) 2023-08-14
CN112708583A (zh) 2021-04-27
JP2023505400A (ja) 2023-02-09
KR20220110663A (ko) 2022-08-09

Similar Documents

Publication Publication Date Title
Long et al. Lactobacillus fermentum CQPC08 protects rats from lead-induced oxidative damage by regulating the Keap1/Nrf2/ARE pathway
CN108094794B (zh) 一种解酒护肝的复合微生物饮品及其制备方法
US20210145903A1 (en) Lactobacillus Plantarum LP33 and Use Thereof in Preparation of Product for Promoting Lead Excretion
CN113234612B (zh) 对结肠炎具有预防效果的Lactobacillus fermentum ZS40
CN114752523B (zh) 一株代谢产生萝卜硫素减轻炎症反应的鼠李糖乳杆菌ccfm1252
CN112438998B (zh) 一种防治幽门螺杆菌感染的益生菌菌剂papch及其制备方法
Long et al. Preventive effect of Limosilactobacillus fermentum SCHY34 on lead acetate-induced neurological damage in SD rats
WO2015146916A1 (ja) 新規ラクトバチラス・パラカゼイ株
Mu et al. Lactobacillus plantarum KFY02 enhances the relieving effect of gardenoside on montmorillonite induced constipation in mice
CN115969957B (zh) 一种炎性肠病复合益生菌制剂及其制备方法
CN114159478A (zh) 一种缓解炎症性结肠炎的菌粉组合物及其制备方法与应用
WO2022160360A1 (zh) 一种发酵乳杆菌lf-schy34及其应用
CN115068629B (zh) 基于姜黄素和氧化铈的载药纳米粒子及其制备方法、应用
CN116445321A (zh) 降核苷降血尿酸罗伊氏乳杆菌a21160及其应用
CN116254190A (zh) 一种副干酪乳杆菌亚种及其应用
Tang et al. Isolation, identification and safety evaluation of OTA-detoxification strain Pediococcus acidilactici NJB421 and its effects on OTA-induced toxicity in mice
CN115992059B (zh) 一株产阿魏酸酯酶的约氏乳杆菌及其缓解溃疡性结肠炎的用途
CN114854623B (zh) 德氏乳杆菌保加利亚亚种、含有该菌的菌剂及应用
CN110628677B (zh) 一种吸附重金属铅的干酪乳杆菌syf-08及其应用
CN116121108B (zh) 发酵粘液乳杆菌hnu312及制备减轻铅脑毒性药品的应用
EP3802785A1 (fr) Nouvelle souche probiotique de lactobacillus brevis
CN117384788B (zh) 一株唾液联合乳杆菌sm4及其在制备美白和降胆固醇食品药品中的应用
CN117025492B (zh) 一种减轻宠物肥胖的嗜热链球菌jyst-81及其应用
CN116676240B (zh) 鼠李糖乳酪杆菌及其在预防或治疗酒精性肝病中的应用
Long et al. The Preventive Effect of Lactobacillus Fermentum SCHY34 Isolated from Traditional Fermented Yak Yogurt on Lead Acetate-Induced Neurological Damage in SD Rats

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 2021550088

Country of ref document: JP

Kind code of ref document: A

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21921960

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21921960

Country of ref document: EP

Kind code of ref document: A1