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WO2024056027A1 - 含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用 - Google Patents

含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用 Download PDF

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WO2024056027A1
WO2024056027A1 PCT/CN2023/118810 CN2023118810W WO2024056027A1 WO 2024056027 A1 WO2024056027 A1 WO 2024056027A1 CN 2023118810 W CN2023118810 W CN 2023118810W WO 2024056027 A1 WO2024056027 A1 WO 2024056027A1
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lactobacillus plantarum
ldh gene
enteritis
relieving
lactic acid
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PCT/CN2023/118810
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English (en)
French (fr)
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艾连中
王光强
夏永军
熊智强
张汇
宋馨
杨昳津
刘欣欣
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上海理工大学
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    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02001—Acetate kinase (2.7.2.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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 field of microorganisms, and in particular to the application of Lactobacillus plantarum containing the ldh gene in the preparation of products for relieving enteritis.
  • IBD Inflammatory bowel disease
  • Crohn's disease and ulcerative colitis a chronic, inflammatory gastrointestinal disease, including Crohn's disease and ulcerative colitis. Its symptoms mainly include abdominal pain, blood in the stool, and loss of appetite.
  • Lactobacillus plantarum plays an important role in treating inflammatory diseases, intestinal infections, improving obesity, lowering cholesterol, etc., and is often used in combination with chemotherapy drugs to fight colon inflammation and tumors. .
  • Lactobacillus plantarum belongs to the genus Lactobacillus, which is a type of non-spore-forming, anaerobic or facultative anaerobic Gram-positive bacteria. Lactobacillus plantarum exists in many foods, has a long history of consumption, and is highly safe; it also exists in the human body and is a probiotic group in the human gastrointestinal tract. It metabolizes to produce a variety of natural antibacterial substances, which has great impact on human health. promotion effect. Most Lactobacilli can colonize the intestine and produce many metabolites during the utilization of food. Some short-chain fatty acids and lactic acid are the main metabolites of Lactobacillus plantarum.
  • Acetic acid is a type of short-chain fatty acids (SCFAs), which also has a good effect on preventing cardiovascular diseases. Lactic acid can regulate the body's immune system and also regulate the inflammatory activation of epithelial cells. However, there are few studies on which metabolite has the main anti-inflammatory effect. Although each has its own explanation, it is finally concluded that the lactic acid in Lactobacillus plantarum plays the main alleviation and prevention effect on enteritis, which is the goal of the present invention.
  • SCFAs short-chain fatty acids
  • Lactobacillus plantarum produces many metabolites during the fermentation process, mainly lactic acid and some short-chain fatty acids.
  • the antibacterial effect of lactic acid bacteria is also related to these organic acids.
  • the purpose of the present invention is to overcome the above-mentioned defects in the prior art and provide an application of Lactobacillus plantarum containing the ldh gene in the preparation of products for relieving enteritis.
  • the present invention further provides a screening method for Lactobacillus plantarum for preparing enteritis prevention drugs.
  • Lactobacillus plantarum AR113 knockout acetate kinase (ackA), D-lactate dehydrogenase (D-ldh) and L-lactate dehydrogenase (L-ldh) as the research object
  • the knockout strain induced O157:H7 colitis to explore the important role of lactic acid in alleviating tissue damage caused by O157:H7 and protecting the integrity of intestinal epithelial cells. It can effectively alleviate and prevent different types of enteritis, and screen out lactic acid or lactate dehydrogenase genes as screening markers. After screening bacteria, it was concluded that lactic acid plays the main role in alleviating colitis, laying the foundation for screening probiotics with the function of relieving inflammation.
  • the invention provides an application of Lactobacillus plantarum containing the ldh gene in preparing a product for relieving enteritis.
  • the Lactobacillus plantarum containing the ldh gene has the function of relieving inflammation.
  • inflammation is O157:H7-induced enteritis.
  • enteritis induced by O157:H7 is characterized by inflammation of intestinal tissue, increased intestinal permeability, and severe destruction of colon tissue structure.
  • enteritis induced by O157:H7 is characterized by: increased expression of pro-inflammatory cytokines TNF- ⁇ , IL-1 ⁇ , IL-18 and IL-6, decreased anti-inflammatory cytokine IL-10, and neutral The number of granulocytes increased significantly.
  • Lactobacillus plantarum containing the ldh gene is selected from Lactobacillus plantarum AR113.
  • the preservation number of Lactobacillus plantarum AR113 is CGMCC No. 13909, which was disclosed in the CN111304134A patent.
  • nucleotide sequence of the ldh gene is shown in SEQ ID NO.19 and SEQ ID NO.20.
  • the ldh gene controls the contents of acetic acid and lactic acid in Lactobacillus plantarum and mouse intestines.
  • Lactobacillus plantarum containing the ldh gene controls inflammation as follows: lactic acid content decreases, inflammation levels in intestinal tissues decrease, and intestinal epithelial structural damage is alleviated.
  • lactic acid in controlling inflammation is as follows: the expression of pro-inflammatory cytokines TNF- ⁇ , IL-1 ⁇ , IL-18 and IL-6 decreases, and the expression of anti-inflammatory cytokine IL-10 increases.
  • the product is a functional food.
  • the present invention provides a screening method for Lactobacillus plantarum for preparing enteritis prevention drugs, using lactic acid production or lactate dehydrogenase gene (ldh gene) as screening markers to obtain probiotic strains with high lactic acid production and high lactic acid production.
  • Probiotic strains act as probiotic strains that have the function of preventing and alleviating inflammation.
  • the present invention has the following advantages:
  • Lactobacillus plantarum AR113 provided by the present invention is Lactobacillus plantarum with ldh and ackA genes, and Lactobacillus plantarum with lactic acid as a marker can effectively prevent O157:H7-induced enteritis;
  • Figure 1 shows the animal experiment grouping scheme
  • Figure 2 shows the acetate production content of the knockout strain
  • Figure 3 shows the content of lactic acid produced by the knockout strain
  • Figure 4 shows the antibacterial effect of the knockout strain on enterohemorrhagic E. coli O157:H7;
  • Figure 5 shows the production of SCFAs in the cecum of mice, in which (A) is acetic acid, (B) is propionic acid, and (C) is butyric acid;
  • Figure 6 shows the production of lactic acid in mouse feces
  • Figure 7 shows the physical and chemical indicators of colitis mice, where (A) is the DAI index, (B) is the colon length, and (C) is myeloperoxidase activity;
  • Figure 8 shows HE staining of mouse colon.
  • A is the control group
  • B is the O157:H7 group
  • C is the AR113 group
  • D is the ⁇ 0187 group
  • E is the ⁇ 0273 group
  • F is the ⁇ 1778 ⁇ 0467 group
  • Figure 9 shows the mouse colon tissue damage score
  • Figure 10 shows the immune organ index, where (A) is the thymus index and (B) is the spleen index;
  • Figure 11 shows the transcription levels of colon inflammatory factors in mice in each group
  • Figure 12 shows the expression of intestinal barrier function genes in mice.
  • MRS medium (1L): 10.0g peptone, 5.0g yeast powder, 10.0g beef extract powder, 2.0g diammonium hydrogen citrate, 20.0g glucose, 1.0mL Tween-80, 5.0g sodium acetate, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganese sulfate 0.25g, pH 6.2 ⁇ 6.6, used for activation, culture and viable bacterial count of Lactobacillus, sterilized at 115°C for 20 minutes;
  • LB medium 10.0g tryptone, 5.0g yeast extract powder, 10.0g NaCl, sterilized at 121°C for 15 minutes;
  • N,O-trimethylsilyltrifluoroacetamide N,O-trimethylsilyltrifluoroacetamide, anhydrous sodium sulfate, fecal occult blood reagent, myeloperoxidase (MPO) test kit, Hieff qPCR SYBR Green Master Mix, HiScriptIIIRT SuperMix for qPCR;
  • mice Specific sources of experimental mice:
  • mice used in the laboratory are 5-week-old male SPF grade C57BL/6 mice, weighing 18-20g, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd., in an environment with a constant temperature of 23-25°C and a dark/light cycle of 12 hours. , ad libitum diet, and all mice were housed for at least one week to acclimate before experiments.
  • the acetate kinase (ackA) gene includes 0187 gene and 0273 gene, and the sequence is as follows:
  • the D-lactate dehydrogenase (D-ldh) gene includes 1778 genes, and the 1778 gene sequence is shown in SEQ ID NO.19:
  • the L-lactate dehydrogenase (L-ldh) gene includes the 0467 gene, and the 0467 gene sequence is shown in SEQ ID NO. 20:
  • Lactobacillus plantarum AR113 The preservation information of Lactobacillus plantarum AR113 is as follows: Lactobacillus plantarum AR113
  • the preservation number is CGMCC No. 13909.
  • the preservation unit is the China General Microorganism Collection Center.
  • the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.
  • the preservation date is March 22, 2017. It was disclosed in the CN111304134A patent.
  • Plasmid pHSP01 comes from the article Huang H, Song Epub 2019 May 20.PMID:30927506.
  • DU-800 spectrophotometer Spectra Max i3x multi-function microplate reader, anaerobic incubator, GT200 grinder, Light Cycler real-time fluorescence quantitative PCR instrument, high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC) -MS).
  • HPLC high performance liquid chromatography
  • GC gas chromatography-mass spectrometry
  • the genome of the wild-type strain was used as a template, and the corresponding primers (primer sequences are shown in Table 2) were used for PCR amplification to obtain 0187up, 0187down and 0187gRNA, and then KOD FX was used Neo performed Overlap PCR on the three, and recovered the 0187up-down-gRNA fragment (the operation of 0273 is the same as above), and then seamlessly cloned it with the pHSP01 vector recovered by ApaI and XbaI digestion, and the ligated product was transformed into E. coli Top 10 strain The state cells were spread on erythromycin-resistant plates.
  • the single colonies that grew were verified with primers deleteYZ-F and deleteYZ-R (the primer sequences are shown in Table 2).
  • the correct plasmids obtained were named plcp0187 and plcp0273, the recombinant plasmids plcp0187 and plcp0273 were electroporated into the AR113 (gba) competent state respectively, spread on a resistant plate containing erythromycin and chloramphenicol, and sequenced after verification by PCR to obtain the correct single gene knockout type
  • the strains were named L.plantarum AR113 ⁇ 0187 (abbreviated as ⁇ 0187) and L.plantarum AR113 ⁇ 0273 (abbreviated as ⁇ 0273).
  • mice defecate collect about 50 mg of fecal samples and mix them with 500 ⁇ l of pure water, grind them quickly with a grinder, shake at 1500xg at high speed for 2 minutes at 4°C until a homogenized state is formed, then shake at 300xg at low speed for 30 minutes, and then put it into a refrigerated centrifuge and centrifuge at 13000xg for 30 minutes. . Transfer 100 ⁇ l of the supernatant to a new 0.6 mL centrifuge tube, add 10 ⁇ l of 5M HCl to make the pH value of the stool solution around 2, add 100 ⁇ l of anhydrous ether to the acidified stool homogenate, vortex and mix, and centrifuge at 10000xg for 5 min.
  • Lactobacillus plantarum AR113 and AR113 gene knockout mutant strains were streaked on MRS solid plates and cultured anaerobically until a single colony grew to 1 to 2 mm in size. Single colonies were picked and inoculated into 15 mL MRS liquid culture medium at 37°C. Incubate statically for 12 to 16 hours, then transfer 3% of the inoculum into 500 mL liquid MRS and culture for 16 to 24 hours.
  • Centrifuge at 12000rpm/min for 10min use a 1mL disposable sterile syringe to absorb the supernatant, pass it through a 0.22 ⁇ m aqueous phase filter membrane into the injection bottle, then put it on the machine, use a Sepax carbomix H-NP column, and the mobile phase is 2.5mM H 2 SO 4 , flow rate 0.6mL/min, automatic injection, injection volume 10 ⁇ L, running time 30min, calculate the lactic acid content in the sample.
  • mice in the three groups of experiments were fed 5g/L streptomycin with water from day 1 to day 3 to remove normal intestinal flora and increase susceptibility to enterohemorrhagic Escherichia coli O157:H7.
  • mice in the normal group were orally administered 200 ⁇ L of sterile water every day during the entire subsequent experiment.
  • Prevention model group 200 ⁇ L of sterile water was administered from the 4th to the 10th day, and 200 ⁇ L of O157:H7 sterile water suspension with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 11th to the 20th day.
  • AR113 group 200 ⁇ L of Lactobacillus plantarum AR113 bacterial suspension with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 4th to the 10th day, and 200 ⁇ L with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 11th to the 17th day.
  • O157:H7 sterile aqueous suspension 200 ⁇ L of Lactobacillus plantarum AR113 bacterial suspension with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 4th to the 10th day, and 200 ⁇ L with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 11th to the 17th day.
  • ⁇ 0187 group 200 ⁇ L of ⁇ 0187 bacterial suspension with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 4th to the 10th day, and 200 ⁇ L of O157:H7 with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 11th to the 17th day.
  • Sterile water suspension 200 ⁇ L of ⁇ 0187 bacterial suspension with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 4th to the 10th day, and 200 ⁇ L of O157:H7 with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 11th to the 17th day.
  • ⁇ 0273 group 200 ⁇ L of ⁇ 0273 bacterial suspension with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 4th to the 10th day, and 200 ⁇ L of O157:H7 with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 11th to the 17th day.
  • sterile aqueous suspension 200 ⁇ L of ⁇ 0273 bacterial suspension with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 4th to the 10th day, and 200 ⁇ L of O157:H7 with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 11th to the 17th day.
  • ⁇ 1778 ⁇ 0467 group 200 ⁇ L of ⁇ 1778 ⁇ 0467 bacterial suspension with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 4th to the 10th day, and 200 ⁇ L of O157:H7 with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 11th to the 17th day.
  • Sterile aqueous suspension 200 ⁇ L of ⁇ 1778 ⁇ 0467 bacterial suspension with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 4th to the 10th day, and 200 ⁇ L of O157:H7 with a concentration of 2.5 ⁇ 10 10 CFU/mL was administered from the 11th to the 17th day.
  • GC-MS was used to analyze the acetate production of L. plantarum AR113 and three knockout strains ( ⁇ 0187, ⁇ 0273 and ⁇ 1778 ⁇ 0467). After analysis, it was found that L. plantarum AR113 and the knockout strain only produce acetate, a short-chain fatty acid, so we focused on exploring the changes in acetate. The results are shown in Figure 2.
  • AR113 and ⁇ 0273 have the highest acetic acid content, reaching 50.33 ⁇ 0.87 and 44.34 ⁇ 0.22 ⁇ M respectively.
  • the acetic acid content of ⁇ 0187 and ⁇ 1778 ⁇ 0467 was significantly lower than that of AR113 (p ⁇ 0.05), which were 18.49 ⁇ 0.39 and 10.55 ⁇ 0.88 ⁇ M respectively. There is also a significant difference between ⁇ 0187 and ⁇ 1778 ⁇ 0467 (p ⁇ 0.05).
  • the lactate content of the knockout strain was calculated based on HPLC and the constructed linear equation. The results are shown in Figure 3, which shows that the lactate production of AR113 and ⁇ 0187 and ⁇ 0273 knockout ackA genes has almost no change, which are 30.90 ⁇ 0.08, 30.95 ⁇ 0.21, and 30.66 ⁇ 0.44mg/ml respectively.
  • the acetic acid content produced by ⁇ 1778 ⁇ 0467 is extremely low, with the content being 0.86 ⁇ 0.06mg/ml. This shows that knocking out the ldh gene has a great impact on AR113 lactate production.
  • Lactobacillus plantarum AR113, ⁇ 0273 and ⁇ 0187 all have obvious inhibitory zones, indicating that they all have antibacterial effects on O157:H7, and there is no significant difference between AR113, ⁇ 0273 and ⁇ 0187. (p>0.05). However, the ⁇ 1778 ⁇ 0467 strain had no obvious inhibition zone. In vitro experiments showed that knockout of lactic acid-related genes has an impact on the antibacterial effect of O157:H7.
  • Lactobacillus plantarum knocking out the ldh gene will lead to a decrease in lactic acid and acetate, and from in vitro experiments it was known that Lactobacillus plantarum knocking out the lactic acid-related gene has no effect on E. coli O157:H7 It has significant antibacterial effect, so further in vivo animal antibacterial experiments were carried out to study the inhibitory effect of acetic acid and lactic acid on pathogenic bacteria.
  • the acetic acid content of ⁇ 0187 and ⁇ 1778 ⁇ 0467 increased, but it was significantly different from the AR113 group (2.47 ⁇ 0.31vs.3.34 ⁇ 0.33, 2.23 ⁇ 0.17vs.3.34 ⁇ 0.33mmol/g, p ⁇ 0.05). This may be related to the decrease in the acetate production content of the Lactobacillus plantarum strain itself with the ackA0187 and ldh genes deleted. As shown in Figure 5B, compared with the control group, the propionic acid content in the model group was significantly reduced (p ⁇ 0.05).
  • the propionic acid content of AR113 was significantly different from that of the prevention control group (p ⁇ 0.05), while there was no significant difference between ⁇ 0273, ⁇ 0187 and ⁇ 1778 ⁇ 0467 and AR113 and the model group (p>0.05).
  • the butyric acid content in the model group showed a decrease (p ⁇ 0.05).
  • the propionic acid content of AR113 was significantly increased and was significantly different from the model group (p ⁇ 0.05).
  • the lactate content of the mice's feces on the last day was measured. As shown in Figure 6, compared with the control group, the lactic acid content in the model group increased significantly. The lactic acid content of AR113, ⁇ 0273, ⁇ 0187 and ⁇ 1778 ⁇ 0467 was significantly lower than that of the model group (p ⁇ 0.05). Among them, ⁇ 1778 ⁇ 0467 had the lowest lactic acid content, which was significantly different from the AR113 group (p ⁇ 0.05). Studies have shown that when inflammation is in a rapidly developing stage, the concentration of lactic acid will be too high, affecting the proliferation of immune cells and the production of cytokines.
  • Lactobacillus plantarum may have a preventive effect on the phenomenon of excessive lactic acid.
  • lactic acid can serve as a signaling molecule that mediates the migration of inflammatory cells and cancer cells. Therefore, the reason why Lactobacillus plantarum has a better preventive effect on enteritis may be related to the fact that the lactic acid content in the AR113 group is higher than that in the ⁇ 1778 ⁇ 0467 group.
  • the DAI value of the normal group remained around 0, indicating that the mice did not lose weight, the stool hardness and properties were stable, and there was no loose stool or blood in the stool.
  • the DAI of all groups remained around 0.
  • the DAI values of each group continued to rise, especially in the model group, where the DAI reached the highest value on the 17th day.
  • the DAI value of probiotic AR113 increased to a lower extent, indicating that AR113 may play a role in alleviating enteritis symptoms by maintaining the stability of intestinal flora or regulating the expression of related inflammatory factors.
  • AR113 group The DAI value has the smallest increase, and is significantly different from the model group (p ⁇ 0.05). On day 17, compared with the AR113 group, the DAI values of ⁇ 0273 and ⁇ 0187 were higher, but there was no significant difference (p>0.05), while the DAI value of the ⁇ 1778 ⁇ 0467 group was significantly higher than that of the AR113 group (p ⁇ 0.05). The above shows that the ldh gene plays an important role in AR113 regulating DAI value.
  • the MPO activity in the control group was 0.31 ⁇ 0.02U/g, while the MPO activity in the model group was significantly increased to 0.62 ⁇ 0.04U/g, indicating that O157:H7 induction significantly increased the number of neutrophil cells in the colon.
  • the organizational structure was severely damaged.
  • AR113, ⁇ 0273, ⁇ 0187, and ⁇ 1778 ⁇ 0467 can effectively reduce MPO activity in mouse colon tissue by 0.3 ⁇ 0.03, 0.32 ⁇ 0.03, 0.38 ⁇ 0.02, and 0.42 ⁇ 0.04U/g respectively (p ⁇ 0.05).
  • the ⁇ 1778 ⁇ 0467 group had the highest MPO activity, but there was no significant difference compared with the AR113 group (p>0.05).
  • the colon length of the model group was significantly reduced (7.31 ⁇ 0.17vs.8.6 ⁇ 0.11cm, p ⁇ 0.05).
  • Intragastric administration of AR113 and other knockout strains can significantly improve the colon shortening caused by O157:H7.
  • AR113, ⁇ 0273, ⁇ 0187, and ⁇ 1778 ⁇ 0467 were 8.2 ⁇ 0.77, 8.22 ⁇ 0.74, 7.8 ⁇ 0.67, and 7.91 ⁇ 0.48cm respectively.
  • the colon length of the ⁇ 1778 ⁇ 0467 group was the shortest among the knockout strains, but there was no significant difference compared with the AR113 group (p>0.05).
  • the measurement of various physical and chemical indicators of mice can conclude that intragastric administration of the ldh gene knockout strain cannot alleviate the weight loss and colon damage in mice caused by enteritis.
  • the ldh gene can simultaneously control the acetic acid and lactic acid content in Lactobacillus plantarum and mouse intestines
  • the ackA gene can only effectively reduce the levels of acetic acid production in Lactobacillus plantarum and mouse intestines. Since the ackA gene has no significant effect on relieving colitis, it means that acetic acid does not have a significant effect on relieving enteritis, but lactic acid plays a role in relieving enteritis.
  • the ldh gene has a certain protective effect on intestinal tissue damage, while the ackA gene has no significant protective effect on intestinal tissue.
  • the thymus index of the model group decreased significantly (1.08 ⁇ 0.03vs.2.00 ⁇ 0.04mg/g, p ⁇ 0.05).
  • Oral administration of AR113 can significantly improve the decrease in immune index caused by DSS (1.43 ⁇ 0.04vs.1.08 ⁇ 0.03mg/g, p ⁇ 0.05).
  • the immune organ indices of ⁇ 0273 and ⁇ 0187 are 1.37 ⁇ 0.11 and 1.38 ⁇ 0.43mg/g respectively, and There is no significant difference from AR113 (p>0.05).
  • the thymus index of ⁇ 1778 ⁇ 0467 is significantly different compared to AR113 (1.11 ⁇ 0.03vs.1.43 ⁇ 0.04mg/g, p ⁇ 0.05).
  • the spleen index of the model group increased significantly (4.24 ⁇ 0.09vs.3.31 ⁇ 0.22mg/g, p ⁇ 0.05).
  • Oral administration of AR113 can significantly improve the increase in immune index (3.22 ⁇ 0.07vs.4.24 ⁇ 0.09mg/g, p ⁇ 0.05).
  • the immune organ indices of ⁇ 0273 and ⁇ 0187 were 3.60 ⁇ 0.05 and 3.7 ⁇ 0.44mg/g respectively, and there was no significant difference between them and AR113 (p>0.05). There is a significant difference between ⁇ 1778 ⁇ 0467 and AR113 (4.11 ⁇ 0.08vs.3.22 ⁇ 0.07mg/g, p ⁇ 0.05).
  • the ldh gene has a significant preventive effect on up-regulating the thymus index and down-regulating the spleen index and strengthening immune function.
  • knockout of the ldh gene simultaneously reduced the contents of acetic acid and lactic acid, since there was no significant difference in immune organ index between the ackA gene knockout group and the AR113 group, it was judged that lactic acid played a preventive role in enhancing immune function.
  • the degree of inflammation in mice can be expressed through pro-inflammatory factors.
  • E. coli O157:H7 infection can lead to increased transcription levels of pro-inflammatory cytokines, immune disorder in mice, and increased intestinal inflammation.
  • E. coli O157:H7 caused a significant increase in the mRNA levels of pro-inflammatory factors IL-18, TNF- ⁇ , IL-6, and IL-1 ⁇ .
  • the AR113, ⁇ 0273, and ⁇ 0187 groups were all significantly different from the O157:H7 group (p ⁇ 0.05), and could effectively alleviate the increase in the levels of pro-inflammatory factors.
  • ⁇ 1778 ⁇ 0467 slightly reduced the expression levels of pro-inflammatory factors, there was no difference with the DSS group. Significant difference (p>0.05).
  • the expression levels of IL-1 ⁇ , IL-18, and TNF- ⁇ in the ⁇ 1778 ⁇ 0467 group were significantly different from the AR113 group (p ⁇ 0.05).
  • E. coli O157:H7 induces mice by significantly increasing the expression of pro-inflammatory cytokines TNF- ⁇ , IL-1 ⁇ , IL-18 and IL-6 and reducing the level of anti-inflammatory cytokine IL-10.
  • pro-inflammatory cytokines TNF- ⁇ , IL-1 ⁇ , IL-18 and IL-6
  • IL-10 pro-inflammatory cytokine IL-10.
  • the presence of the ldh gene can significantly prevent the upregulation of pro-inflammatory cytokine expression, while the ackA gene has no significant effect. Since the ackA gene can only reduce the content of acetic acid, it has no significant effect on lactic acid production.
  • lactic acid can effectively adjust the expression of pro-inflammatory cytokines and anti-inflammatory cytokines, effectively reduce the inflammation level in intestinal tissue and alleviate the damage of intestinal epithelial structure, and play an important role in regulating intestinal immunity and alleviating the symptoms of enteritis.
  • ackA gene seems to have no significant effect on regulating the expression levels of ZO-1, Occuldin and Claudin. Therefore, the ldh gene has an intervening effect in preventing the destruction of the intestinal mucosal layer and tight junctions in mice. Since ackA has no significant effect on the destruction of the intestinal mucosa layer of the body, and the ackA gene can only reduce the content of acetic acid, it has no significant effect on lactic acid production. Therefore, it is speculated that it is not acetic acid but lactic acid that enhances the protective effect of the mucosal barrier by increasing colonic tight junction proteins, thereby alleviating the damage to the intestinal mucosal layer.

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Abstract

本发明提供一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，本发明涉及的植物乳杆菌或质粒为含有ldh基因的植物乳杆菌或质粒，通过实验证明，ldh基因具有预防和缓解炎症的功能。本发明以植物乳杆菌AR113敲除乙酸激酶（ackA）、D-乳酸脱氢酶（D-ldh）和L-乳酸脱氢酶（L-ldh）为研究对象，通过敲除菌株对O157:H7诱导的结肠炎来探究乳酸在缓解O157:H7导致的组织损伤和保护肠上皮细胞的完整性具有重要的作用，可以有效缓解和预防不同种类的肠炎，并筛选出乳酸或者乳酸脱氢酶基因作为筛选标记进行筛菌，得出是乳酸对结肠炎起主要缓解作用，为筛选具有缓解炎症功能益生菌的方法奠定基础。

Description

含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用 技术领域
本发明涉及微生物领域，尤其是涉及一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用。
背景技术
炎症性肠病(IBD)是一种慢性、炎症性胃肠道疾病，包括克罗恩病和溃疡性结肠炎，它的症状主要有腹痛、便血、食欲不振等。近几年研究发现，植物乳杆菌(Lactobacillus plantarum)在治疗炎症性疾病、肠道感染、改善肥胖，降胆固醇等方面发挥重要作用，并常和化疗药物联合应用于抗结肠炎症和肿瘤等疾病中。
植物乳杆菌属于乳酸杆菌属，它是一类不产芽孢，厌氧或兼性厌氧的革兰氏阳性菌。植物乳杆菌在存在于许多食品中，食用历史悠久，安全性高；同时也存在于人体中，是人体胃肠道内的益生菌群，代谢产生多种天然抑菌物质，对人体健康具有很大的促进作用。大部分乳杆菌能定殖于肠道中，并在利用食物过程中产生许多代谢产物，部分短链脂肪酸和乳酸是植物乳杆菌主要代谢产物。乙酸是短链脂肪酸(SCFAs)的一种，对防止心血管疾病等也有良好的作用。而乳酸能调节机体免疫系统，还可以调节上皮细胞的炎症激活。但很少有研究具体是哪种代谢物起主要抑炎作用，在各有说辞的情况下，最终得出植物乳杆菌中乳酸对肠炎起主要缓解和预防作用是本发明研究的目标。
植物乳杆菌发酵过程中产生很多代谢物，主要以乳酸为主，还有部分短链脂肪酸。乳酸菌的抑菌作用也与这些有机酸有关。虽然有许多文献对这些有机酸代谢物的抑菌效果做研究，但很少有结合体外和体内动物实验同时探索具体是哪种代谢物有抗炎和抑菌作用。因此需要通过探索代谢产物来建立一种具有缓解炎症功能的益生菌方法。
发明内容
本发明的目的就是为了克服上述现有技术存在的缺陷而提供一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用。本发明进一步提供了制备预防肠炎药物的植物乳杆菌的筛选方法。以植物乳杆菌AR113敲除乙酸激酶(ackA)、D-乳酸脱氢酶(D-ldh)和L-乳酸脱氢酶(L-ldh)为研究对象，通过敲除菌株对O157:H7诱导的结肠炎来探究乳酸在缓解O157:H7导致的组织损伤和保护肠上皮细胞的完整性具有重要的作用，可以有效缓解和预防不同种类的肠炎，并筛选出乳酸或者乳酸脱氢酶基因作为筛选标记进行筛菌，得出是乳酸对结肠炎起主要缓解作用，为筛选具有缓解炎症功能益生菌的方法奠定基础。
本发明的目的可以通过以下技术方案来实现：
本发明提供了一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，含有ldh基因的植物乳杆菌具有缓解炎症功能。
进一步地，所述炎症为O157:H7诱导的肠炎。
上述更进一步地，所述O157:H7诱导的肠炎表现为：肠道组织炎症、肠道通透性增加，结肠组织结构遭到严重破坏。
上述更进一步地，所述O157:H7诱导的肠炎表现为：促炎细胞因子TNF-α、IL-1β、IL-18和IL-6的表达增加，抑炎细胞因子IL-10降低，中性粒的细胞数量显著增加。
进一步地，所述含有ldh基因的植物乳杆菌选自植物乳杆菌AR113，植物乳杆菌AR113保藏编号为CGMCC No.13909，在CN111304134A专利中公开过。
进一步地，所述ldh基因的核苷酸序列如SEQ ID NO.19和SEQ ID NO.20所示。
上述更进一步地，所述ldh基因同时控制植物乳杆菌和小鼠肠道中乙酸和乳酸的含量。
上述更进一步地，所述含有ldh基因的植物乳杆菌控制炎症的表现为：乳酸含量下降，肠道组织中的炎症水平降低，解肠上皮结构损伤缓解。
上述更进一步地，所述乳酸控制炎症的表现为：促炎细胞因子TNF-α、IL-1β、IL-18和IL-6的表达量下降，抑炎细胞因子IL-10的表达量上升。
进一步地，所述产品为功能性食品。
本发明提供一种制备预防肠炎药物的植物乳杆菌的筛选方法，以乳酸产量或者乳酸脱氢酶基因(ldh基因)为筛选标记，获得高产乳酸的益生菌株，高产乳酸的 益生菌株作为具有预防和缓解炎症功能的益生菌株。
与现有技术相比，本发明具有以下优点：
1.本发明提供的植物乳杆菌AR113为具有ldh和ackA基因的植物乳杆菌，筛选出乳酸作为标志物的植物乳杆菌可以有效预防O157:H7诱导的肠炎；
2.以乳酸产量或者乳酸脱氢酶基因为筛选标记，获得高产乳酸的益生菌株，经动物实验验证发现此菌株具有较好的缓解炎症作用，利用此方法可以快速筛选获得缓解炎症功能益生菌。
附图说明
图1为动物实验分组方案；
图2为敲除菌株产乙酸的含量；
图3为敲除菌株产乳酸的含量；
图4为敲除菌株对肠出血大肠杆菌O157:H7的抑菌效果；
图5为小鼠盲肠产SCFAs的情况，其中，(A)为乙酸，(B)为丙酸，(C)为丁酸；
图6为小鼠粪便产乳酸的情况；
图7为结肠炎小鼠的理化指标，其中，(A)为DAI指数，(B)为结肠长度，(C)为髓过氧化物酶活性；
图8为小鼠结肠HE染色，其中，(A)为对照组，(B)为O157:H7组，(C)为AR113组，(D)为Δ0187组，(E)为Δ0273组，(F)为Δ1778Δ0467组；
图9为小鼠结肠组织损伤评分；
图10为免疫器官指数，其中，(A)为胸腺指数，(B)为脾脏指数；
图11为各组小鼠结肠炎症因子的转录水平；
图12为小鼠肠道屏障功能基因的表达。
附图中柱形图横坐标标号说明：(一)为对照组，(二)为O157:H7组，(三)为AR113组，(四)为Δ0187组，(五)为Δ0273组，(六)为Δ1778Δ0467组。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。本实施例以本发明技术方 案为前提进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
以下各实施例中，所需材料如下所示：
MRS培养基(1L)：蛋白胨10.0g，酵母粉5.0g，牛肉浸粉10.0g，柠檬酸氢二铵2.0g，葡萄糖20.0g，吐温-80 1.0mL，乙酸钠5.0g，磷酸氢二钾2.0g，硫酸镁0.58g，硫酸锰0.25g，pH为6.2～6.6，用于乳杆菌的活化、培养和活菌计数，115℃灭菌20min；
LB培养基(1L)：胰蛋白胨10.0g，酵母浸出粉5.0g，NaCl 10.0g，121℃灭菌15min；
试剂：N,O-三甲硅烷基三氟乙酰胺、无水硫酸钠、便隐血试剂、髓过氧化物酶(MPO)测试盒、Hieff qPCR SYBR Green Master Mix、HiScriptⅢRT SuperMix for qPCR；
实验小鼠的具体来源：
实验室所用小鼠为5周龄雄性SPF级C57BL/6小鼠，体重18～20g，购自上海洁思洁实验动物有限公，在恒温23-25℃、暗/光周期为12h的环境下，自由饮食，所有小鼠在实验之前至少安置一周以适应环境。
菌株及质粒来源如表1所示：
表1本实验使用的菌株及质粒
其中，乙酸激酶(ackA)基因包括0187基因和0273基因，序列如下：
0187基因序列，序列如SEQ ID NO.17所示：
0273基因序列，序列如SEQ ID NO.18所示：

D-乳酸脱氢酶(D-ldh)基因包括1778基因，1778基因序列如SEQ ID NO.19所示：

L-乳酸脱氢酶(L-ldh)基因包括0467基因，0467基因序列如SEQ ID NO.20所示：
植物乳杆菌AR113保藏信息如下：植物乳杆菌(Lactobacillus plantarum)AR113 保藏编号为CGMCC No.13909，保藏单位为中国普通微生物保藏中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏日期为2017年03月22日，在CN111304134A专利中公开过。
质粒pHSP01来源于文章Huang H,Song X,Yang S.Development of a RecE/T-Assisted CRISPR-Cas9 Toolbox for Lactobacillus.Biotechnol J.2019 Jul；14(7):e1800690.doi:10.1002/biot.201800690.Epub 2019May 20.PMID:30927506.
以下各实施例中，所需仪器如下所示：
DU-800分光光度计、Spectra Max i3x多功能酶标仪、厌氧培养箱、GT200研磨仪、Light Cycler实时荧光定量PCR仪、高效液相色谱(HPLC)、气相色谱-质谱联用仪(GC-MS)。
其余如无特别说明的原料或处理技术，则表明其均为本领域的常规市售产品或常规处理技术。
实施例
(1)菌株L.plantarum AR113Δ0187和L.plantarum AR113Δ0273的构建
在CRISPR/Cas9编辑质粒pHSP01的基础上进行改造，以野生型菌株的基因组为模板，使用相应引物(引物序列如表2所示)进行PCR扩增，获得0187up、0187down和0187gRNA，然后使用KOD FX Neo将三者进行Overlap PCR，割胶回收获得0187up-down-gRNA片段(0273的操作同上)，再与经ApaI和XbaI酶切回收的pHSP01载体进行无缝克隆，连接产物转化至大肠杆菌Top 10感受态细胞，涂布于红霉素抗性的平板，长出的单菌落用引物deleteYZ-F和deleteYZ-R(引物序列如表2所示)进行验证，获得的正确质粒命名依次命名为plcp0187和plcp0273，将重组质粒plcp0187和plcp0273分别电转于AR113(gba)感受态，涂布在含有红霉素和氯霉素的抗性平板上，经PCR验证后进行测序，获得正确的单基因敲除型菌株命名为L.plantarum AR113Δ0187(简称Δ0187)，L.plantarum AR113Δ0273(简称Δ0273)。
表2质粒plcp0187和plcp0273的引物序列

(2)测定植物乳杆菌及敲除菌株的产酸能力：
小鼠排便后立即收集粪便样本50mg左右与500μl纯水混合，用研磨仪快速研磨，4℃环境1500xg高速震荡2min直至形成匀浆状态，然后300xg低速震荡30min，随后放入冷冻离心机13000xg离心30min。将100μl的上清液转移到一个新的0.6mL离心管中，加入10μl 5M HCl使粪便溶液pH值在2左右，酸化后的粪便匀浆加入100μl无水乙醚，漩涡震荡混匀，10000xg离心5min，取上清转移至含无水Na2SO4的离心管中。重复用无水乙醚萃取两次。将100μl无水乙醚层转移至内插管中，加入5μl的BSTFA，漩涡震荡后保存在气相小瓶中，70℃孵育20min，37℃孵育2h左右，最后将样品上机处理，计算样品中短链脂肪酸的含量；
将植物乳杆菌AR113以及AR113基因敲除突变菌株划线于MRS固体平板后于厌氧培养至长出1～2mm大小的单菌落，分别挑取单菌落接于15mL MRS液体培养基中，37℃静止培养12～16h，按3％接种量转接至500mL液体MRS中，培养16～24h。12000rpm/min离心10min，利用1mL一次性灭菌注射器吸取上清，经过0.22μm水相滤膜进入进样瓶，随后上机，采用Sepax carbomix H-NP柱，流动相为2.5mM H2SO4，流速0.6mL/min，自动进样，进样量10μL，运行时间30min，计算样品中乳酸的含量。
(3)致病菌模型建立及分组给药
动物实验的分组和喂养方案如图1所示，三组实验中所有小鼠在第1天到第3天随水喂食5g/L链霉素，以除去肠道正常菌群，增加肠出血性大肠杆菌O157:H7的易感性，
正常组小鼠在随后的整个实验期间每天灌胃200μL无菌水，
预防模型组：在第4天到第10天灌胃200μL无菌水，第11天到第20天灌胃200μL浓度为2.5×1010CFU/mL的O157:H7无菌水悬液，
AR113组：在第4天到第10天灌胃200μL浓度为2.5×1010CFU/mL植物乳杆菌AR113菌悬液，第11天到第17天灌胃200μL浓度为2.5×1010CFU/mL的O157:H7无菌水悬液，
Δ0187组：在第4天到第10天灌胃200μL浓度为2.5×1010CFU/mLΔ0187菌悬液，第11天到第17天灌胃200μL浓度为2.5×1010CFU/mL的O157:H7无菌水 悬液，
Δ0273组：在第4天到第10天灌胃200μL浓度为2.5×1010CFU/mLΔ0273菌悬液，第11天到第17天灌胃200μL浓度为2.5×1010CFU/mL的O157:H7无菌水悬液，
Δ1778Δ0467组：在第4天到第10天灌胃200μL浓度为2.5×1010CFU/mLΔ1778Δ0467菌悬液，第11天到第17天灌胃200μL浓度为2.5×1010CFU/mL的O157:H7无菌水悬液。
(4)实验结果如下所示：
1野生型AR113及敲除菌株产酸含量
利用GC-MS分析L.plantarum AR113和三株敲除菌株(Δ0187、Δ0273和Δ1778Δ0467)产乙酸的情况。分析后发现L.plantarum AR113及敲除菌株只产乙酸一种短链脂肪酸，因此重点探究乙酸的变化。结果如图2所示，AR113和Δ0273的乙酸含量最多，分别达到50.33±0.87和44.34±0.22μM。而Δ0187和Δ1778Δ0467的乙酸含量与AR113相比显著降低(p<0.05)，分别为18.49±0.39和10.55±0.88μM。其中Δ0187和Δ1778Δ0467之间也存在显著性差异(p<0.05)。
根据HPLC和构建的线性方程计算敲除菌株的乳酸含量。结果如图3，表明AR113以及敲除ackA基因的Δ0187和Δ0273乳酸产量几乎没有变化，分别为30.90±0.08、30.95±0.21、30.66±0.44mg/ml。而Δ1778Δ0467产的乙酸含量极低，含量为0.86±0.06mg/ml。说明敲除ldh基因对AR113乳酸产量有很大的影响。
2敲除菌株对大肠杆菌O157:H7的体外抑菌实验
如图4和表3所示，植物乳杆菌AR113、Δ0273和Δ0187均有明显的抑菌圈，说明其均对O157:H7有抑菌效果，并且AR113、Δ0273和Δ0187三者并没有显著性差异(p>0.05)。而Δ1778Δ0467菌株没有明显的抑菌圈。通过体外实验表明，乳酸相关基因的敲除对O157:H7的抑菌效果是有影响的。
表3抑菌圈的直径
3敲除菌株对大肠杆菌O157:H7肠炎的小鼠肠道产酸能力的影响
从前面实验结果分析得出敲除ldh基因的植物乳杆菌会导致乳酸和乙酸的下降，并从体外实验得知敲除乳酸相关基因的植物乳杆菌对大肠杆菌O157:H7没有 显著的抑菌效果，因此进一步开展体内动物抑菌实验，来研究乙酸和乳酸对致病菌的抑制作用。
为了评估短链脂肪酸对O157:H7致病菌炎症产生的影响，对小鼠解剖后，分别取出小鼠的盲肠内容物对其上GC-MS进行SCFAs含量的测定。如图5A显示，与对照组相比，模型组乙酸含量显著降低(2.02±0.11vs.4.24±0.07mmol/g，p<0.05)。AR113和Δ0273与模型组均有显著性差异(p<0.05)。而Δ0187和Δ1778Δ0467的乙酸含量有所上升，但与AR113组均有显著性差异(2.47±0.31vs.3.34±0.33，2.23±0.17vs.3.34±0.33mmol/g,p<0.05)。这可能与敲除ackA0187和ldh基因的植物乳杆菌菌株本身产乙酸含量下降有关。如图5B显示，与对照组相比，模型组丙酸含量显著降低(p<0.05)。AR113的丙酸含量与预防对照组有显著性差异(p<0.05)，而Δ0273、Δ0187和Δ1778Δ0467与AR113和模型组均没有显著性差异(p>0.05)。如图5C所示，与对照组相比，模型组丁酸含量显示降低(p<0.05)。AR113的丙酸含量明显提高，并与模型组有显著性差异(p<0.05)。与AR113相比，Δ0273和Δ0187并没有显著性差异(p>0.05)，而Δ1778Δ0467与AR113有显著性差异(p>0.05)。
对小鼠最后一天的粪便进行乳酸含量的测定。如图6所示，与对照组相比，模型组的乳酸含量显著上升。AR113、Δ0273、Δ0187和Δ1778Δ0467的乳酸含量与模型组相比都显著降低(p<0.05)，其中Δ1778Δ0467的乳酸含量最低，与AR113组有显著性差异(p<0.05)。有研究表明，当炎症处于快速发展的阶段时，乳酸浓度会因过高而影响免疫细胞的增殖和细胞因子的产生。这与该实验结果吻合，而通过植物乳杆菌的提前干预，可能对乳酸过高的现象有预防作用。而另一方面乳酸又可作为介导炎症细胞和癌细胞迁移的信号分子，因此植物乳杆菌对肠炎的预防效果更好的原因，可能与AR113组的乳酸含量比Δ1778Δ0467组含量高有关。
4大肠杆菌O157:H7诱导肠炎的理化指标
如图7A所示，在实验期间，正常组的DAI值保持在0左右，表明小鼠体重未出现下降，粪便硬度和性状稳定，无稀便及便血现象。并且由于前10天均没有灌胃O157：H7，因此所有组的DAI均保持在0左右。从第11天开始灌胃致病菌开始，各组DAI值持续上升，尤其是模型组，在第17天DAI达到最高值。与模型组相比，益生菌AR113DAI值上升程度更低，表明AR113可能通过维护肠道菌群稳定或调节相关炎症因子的表达从而发挥了缓解肠炎症状的作用。其中AR113组 DAI值上升幅度最小，并与模型组有显著性差异(p<0.05)。第17天，与AR113组相比，Δ0273和Δ0187的DAI值更高，但并无显著性差异(p>0.05)，而Δ1778Δ0467组DAI值显著高于AR113组(p<0.05)。以上表明了ldh基因对于AR113调节DAI值具有重要作用。
由图7B可知，对照组的MPO活性为0.31±0.02U/g，而模型组的MPO活性显著提高至0.62±0.04U/g，说明O157:H7诱导使中性粒的细胞数量显著增加，结肠组织结构遭到严重破坏。而AR113、Δ0273、Δ0187、Δ1778Δ0467可以有效降低小鼠结肠组织中的MPO活性，分别为0.3±0.03、0.32±0.03、0.38±0.02和0.42±0.04U/g(p<0.05)。相比较Δ0273、Δ0187、Δ1778Δ0467三者而言，Δ1778Δ0467组MPO活性最高，但与AR113组相比并没有显著差异(p>0.05)。
由图7C所示，与对照组相比，模型组的结肠长度显著降低(7.31±0.17vs.8.6±0.11cm，p<0.05)。灌胃AR113和其他敲除菌株均可显著改善O157:H7导致的结肠缩短,其中，AR113、Δ0273、Δ0187、Δ1778Δ0467分别为8.2±0.77、8.22±0.74、7.8±0.67、7.91±0.48cm。其中Δ1778Δ0467组在敲除菌株中的结肠长度是最短的，但与AR113组相比没有显著差异(p>0.05)。
综上对小鼠各种理化指标的测定可以得出灌胃敲除ldh基因的菌株不能缓解肠炎导致的体重降低、小鼠结肠损伤。虽然ldh基因可以同时控制植物乳杆菌和小鼠肠道中乙酸和乳酸的含量，但ackA基因仅能有效降低植物乳杆菌和小鼠肠道产乙酸的水平。由于ackA基因对结肠炎的缓解并没有显著效果，说明乙酸对肠炎的缓解并没有显著效果，而是乳酸在缓解肠炎中起作用。
收集所有结肠切片HE染色图像，并对组织病理损伤进行评分。如图8和9所示，与对照组小鼠相比，模型组小鼠绒毛缩短，部分上皮细胞破裂，杯状细胞增多，固有层和黏膜出现炎症细胞浸润的典型特征。O157:H7致病菌诱导小鼠后，模型组则表现出明显的炎症现象，组织学评分为8.14±0.32。相比之下，AR113、Δ0273和Δ0187的治疗可以有效缓解O157:H7造成的结肠损伤，虽然仍有部分炎症细胞浸润，但大部分肠上皮细胞结构完整，其组织学评分分别为3.57±0.30、4.57±0.45和4.14±0.27(p<0.05)。然而，灌胃Δ1778Δ0467菌株结肠组织仍没有缓解肠上皮损伤及隐窝消失等症状(图8F)，组织学评分为7.14±0.88，与AR113组有显著性差异(<0.05)。因此，ldh基因对肠道组织破坏有一定的保护作用，而ackA基因对肠道组织的保护作用不显著。由前面对O157:H7模型中小鼠肠道乳酸和乙酸含量 的分析发现，ldh基因可以同时显著降低肠道中乳酸和乙酸的产量，但ackA基因仅能降低乙酸含量，对乳酸的产量并没有显著影响。因此可以推断出，乳酸对预防肠炎导致的组织损伤和保护肠道上皮细胞的完整性具有重要作用。
由图10A所示，与对照组相比，模型组胸腺指数显著下降(1.08±0.03vs.2.00±0.04mg/g，p<0.05)。灌胃AR113可显著改善DSS导致的免疫指数下降(1.43±0.04vs.1.08±0.03mg/g，p<0.05)，Δ0273和Δ0187免疫器官指数分别为1.37±0.11、1.38±0.43mg/g，且与AR113无显著性差异(p>0.05)。而Δ1778Δ0467的胸腺指数相比AR113有显著性差异(1.11±0.03vs.1.43±0.04mg/g，p<0.05)。
由图10B所示，与对照组相比，模型组脾脏指数显著上升(4.24±0.09vs.3.31±0.22mg/g，p<0.05)。灌胃AR113可显著改善免疫指数的上升(3.22±0.07vs.4.24±0.09mg/g，p<0.05)。Δ0273和Δ0187免疫器官指数分别为3.60±0.05、3.7±0.44mg/g，它们与AR113无显著性差异(p>0.05)。而Δ1778Δ0467与AR113相比有显著性差异(4.11±0.08vs.3.22±0.07mg/g，p<0.05)。
综上说明ldh基因对上调胸腺指数和下调脾脏指数，加强免疫功能有显著的预防效果。虽然ldh基因的敲除同时降低乙酸和乳酸的含量，但由于敲除ackA基因的小组与AR113组的免疫器官指数没有显著差异，因此判断是乳酸对加强免疫功能的效果起预防作用。
5敲除菌株对O157:H7肠炎炎症因子和肠道屏障功能的影响
小鼠体内的炎症程度可以通过促炎因子来表达，大肠杆菌O157:H7感染可导致促炎细胞因子转录水平升高，小鼠免疫混乱，肠道炎症增加。如图11ABCD所示，大肠杆菌O157：H7导致促炎因子IL-18、TNF-α、IL-6、IL-1βmRNA水平均显著升高。而AR113、Δ0273、Δ0187组均与O157:H7组有显著差异(p<0.05)，可以有效缓解促炎因子水平的升高，Δ1778Δ0467虽然略微降低促炎因子的表达水平，但与DSS组并没有显著差异(p>0.05)。其中Δ1778Δ0467组在IL-1β、IL-18、TNF-α表达水平上与AR113组有显著性差异(p<0.05)。
如图11E所示，与对照组相比，DSS组小鼠结肠组织中IL-10的mRNA水平显著降低。而AR113、Δ0273、Δ0187组均与DSS组有显著差异(p<0.05)，可以有效缓解抑炎因子水平的降低，Δ1778Δ0467虽然略微升高抑炎因子的表达水平，但与O157:H7组并没有显著差异(p>0.05)，且Δ1778Δ0467组与AR113组有显著性差异(p<0.05)。
综上，可以得出大肠杆菌O157:H7通过显著增加促炎细胞因子TNF-α、IL-1β、IL-18和IL-6的表达和降低抑炎细胞因子IL-10的水平来诱发小鼠肠道组织炎症、增加肠道通透性，ldh基因的存在可以显著预防促炎细胞因子表达量的上调，而ackA基因没有显著作用。由于ackA基因仅能降低乙酸的含量，对乳酸产量没有显著影响。因此，判断乳酸能有效调整促炎细胞因子和抑炎细胞因子的表达量，有效降低肠道组织中的炎症水平和缓解肠上皮结构的损伤，在调节肠道免疫、缓解肠炎症状发挥重要作用。
如图12A所示，检测各组结肠黏蛋白MUC2转录水平变化，O157:H7处理后的小鼠结肠组织中MUC2的mRNA水平显著降低(p<0.05)，而植物乳杆菌AR113、Δ0273的干预可显著上调MUC2基因的表达(p<0.05)，Δ0187和Δ1778Δ0467菌株对MUC2的mRNA水平与模型组无显著影响(p>0.05)。我们通过RT-qPCR方法检测了各组小鼠结肠组织的紧密连接蛋白ZO-1、Occuldin和Claudin基因的表达水平。如图12BCD所示，经过O157:H7诱导肠炎后，ZO-1、Occuldin和Claudin基因的表达水平均有显著下调(p<0.05)。与模型组相比，AR113、Δ0273、Δ0187组可有效上调ZO-1、Occuldin和Claudin的mRNA水平(p<0.05)，而Δ1778Δ0467组虽然略微提升各基因的表达水平，但与模型组没有显著差异(p>0.05)，且与AR113组均有显著差异(p<0.05)。可以得出，ldh基因可以有效抑制O157:H7导致的紧密连接蛋白相关基因水平的下降，这对于紧密连接的修复十分重要。而ackA基因的存在似乎对调节ZO-1、Occuldin和Claudin的表达水平无显著作用。因此ldh基因对阻止小鼠肠道黏膜层和紧密连接的破坏有干预作用。而由于ackA对机体肠道黏膜层破坏没有显著作用，且ackA基因仅能降低乙酸的含量，对乳酸产量没有显著影响。因此推测出不是乙酸，而是乳酸通过提高结肠紧密连接蛋白来增强黏膜屏障的保护作用，从而缓解肠道黏膜层破坏。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。

Claims (10)

  1. 一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，其特征在于，含有ldh基因的植物乳杆菌具有缓解炎症功能。
  2. 根据权利要求1所述的一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，其特征在于，所述炎症为O157:H7诱导的肠炎。
  3. 根据权利要求2所述的一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，其特征在于，所述O157:H7诱导的肠炎表现为：肠道组织炎症、肠道通透性增加，结肠组织结构遭到严重破坏。
  4. 根据权利要求3所述的一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，其特征在于，所述O157:H7诱导的肠炎表现为：促炎细胞因子TNF-α、IL-1β、IL-18和IL-6的表达增加，抑炎细胞因子IL-10降低，中性粒的细胞数量显著增加。
  5. 根据权利要求1所述的一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，其特征在于，所述含有ldh基因的植物乳杆菌选自植物乳杆菌AR113，植物乳杆菌AR113保藏编号为CGMCC No.13909。
  6. 根据权利要求1所述的一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，其特征在于，所述ldh基因的核苷酸序列如SEQ ID NO.19和SEQ ID NO.20所示。
  7. 根据权利要求6所述的一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，其特征在于，所述ldh基因同时控制植物乳杆菌和小鼠肠道中乙酸和乳酸的含量。
  8. 根据权利要求7所述的一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，其特征在于，所述含有ldh基因的植物乳杆菌控制炎症的表现为：乳酸含量下降，肠道组织中的炎症水平降低，解肠上皮结构损伤缓解。
  9. 根据权利要求8所述的一种含有ldh基因的植物乳杆菌在制备缓解肠炎产品中的应用，其特征在于，所述乳酸控制炎症的表现为：促炎细胞因子TNF-α、IL-1β、IL-18和IL-6的表达量下降，抑炎细胞因子IL-10的表达量上升。
  10. 根据权利要求1所述的一种含有ldh基因的植物乳杆菌在制备缓解肠炎产 品中的应用，其特征在于，所述产品为功能性食品。
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