CN108048353B - Lactobacillus reuteri HI120 highly expressing linoleate isomerase LAI and application thereof - Google Patents
Lactobacillus reuteri HI120 highly expressing linoleate isomerase LAI and application thereof Download PDFInfo
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- CN108048353B CN108048353B CN201711415835.3A CN201711415835A CN108048353B CN 108048353 B CN108048353 B CN 108048353B CN 201711415835 A CN201711415835 A CN 201711415835A CN 108048353 B CN108048353 B CN 108048353B
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Abstract
The invention discloses a lactobacillus reuteri HI120 highly expressing linoleic acid isomerase LAI, which is preserved in Guangdong province microorganism strain preservation center in 2016, 11, 21 days, and the preservation number is GDMCC NO: 60119. the expression level of linoleic acid isomerase of the lactobacillus reuteri HI120 is higher than that of lactobacillus reuteri standard bacteria and other probiotics, and linoleic acid can be converted into conjugated linoleic acid in vivo and in vitro. The invention also discloses a microorganism live bacteria preparation containing the lactobacillus reuteri HI120 and application of the microorganism live bacteria preparation in preparing food additives, foods or medicines with the effects of preventing and/or assisting in treating obesity and diabetes and preventing and/or assisting in treating colorectal cancer.
Description
Technical Field
The invention belongs to the technical field of microecological viable bacteria preparations, and particularly relates to lactobacillus reuteri HI120 highly expressing linoleate isomerase LAI and application thereof.
Background
Diseases caused by abnormal lipid metabolism such as obesity, hyperlipidemia, fatty liver, etc. seriously threaten human health. The long-term abnormal lipid metabolism inevitably causes three high diseases such as hyperlipidemia, hypertension, hyperglycemia and the like, is a main factor for further causing serious diseases such as cardiovascular and cerebrovascular accidents, liver cirrhosis, diabetes, malignant tumors and the like, and has great harm to the health of human bodies.
Most lipid metabolism disorders are due to poor dietary structure and lifestyle habits, and are caused by imbalances in energy intake and consumption. Among them, excessive intake of fatty acids and disorder of fatty acid component ratio are the most important factors. The proper ratio (5-8: 1) of omega-6/omega-3 fatty acids (unsaturated bond at 3/6 th carbon atom on the mesial end) in the diet is a necessary condition for maintaining health. Excessive intake of omega-6 fatty acids, or an imbalance in the ratio of omega-6/omega-3 fatty acids, can lead to abnormal lipid metabolism. Animal oil is mainly saturated fatty acid, and is liable to cause lipid metabolism disorder such as obesity after long-term consumption. Vegetable oils such as peanut oil, rapeseed oil, corn oil and soybean oil, which are edible daily, contain the most omega-6 fatty acids and the least omega-3 fatty acids, although they are mainly unsaturated fatty acids. Wherein the content of omega-6 fatty acid is maximum in Linoleic Acid (LA), accounting for 35-70% of the total weight, and the long-term eating of the food can cause lipid metabolism abnormalities such as obesity and the like. Only a few vegetable oils, such as linseed oil, perilla oil, etc., contain a certain amount of the omega-3 fatty acid alpha-linolenic acid (ALA). However, the vegetable oil has limited sources due to small planting area and low oil yield. Deep sea fish oil and sea algae oil are rich in 20-30% of docosahexaenoic acid (DHA C22:6) and eicosapentaenoic acid (EPA C20:5) and other omega-3 fatty acids. However, with the exhaustion of marine resources, deep sea fish oil has limited sources and too high price, and cannot be used as common edible oil for the general public.
Omega-6 fatty acids are the starting material for the synthesis of mediators of inflammation and cause abnormal lipid metabolism and complications in the body, mainly by causing chronic inflammation for a long period of time. Omega-6 fatty acids are converted in vivo mainly to Arachidonic Acid (AA), which is the raw material for the synthesis of the most potent inflammatory mediators, such as prostaglandin E2(PGE2), thromboxane a2(TXA2) and leukotriene B4(LTB 4). PGE2, LTA4, LTB4, TXA2 and TXB2 are inflammatory mediators with the strongest activity in vivo, participate in pathological changes such as atherosclerosis, thrombosis, islet injury and inflammation generation, and have the effects of stimulating cell proliferation and inducing tumors.
Conjugated Linoleic Acid (CLA) has strong functions of losing weight, reducing blood fat, reducing blood sugar, resisting inflammation and the like. Conjugated Linoleic Acid (CLA) is an isomer of the omega-6 fatty acid Linoleic Acid (LA) and is structurally similar to LA, but functions in a manner that is absolutely opposite to LA. CLA was originally found in milk. Some bacteria in the rumen of ruminants, such as Vibrio cellulolyticus and Megasphaera elsdenii, have higher LA isomerase (LAI) activity. The double bonds in the LA chemical structure are all cis. LAI can allosterize one cis double bond in LA to a trans double bond, forming conjugated double bond linoleic acid, CLA. The most common forms of CLA exist as cis-9, trans-11C18:2(C9t11) and trans-10, cis-12C18:2(t10C 12). Many probiotics, such as lactobacillus reuteri, lactococcus lactis and bifidobacterium breve, have LAI genes in their genomes, but the LAI activity is not high, and the efficiency of CLA as allosteric LA is low.
CLA is eventually converted in vivo to compounds such as PGD3, TXA1/3, LTA5 and prostacyclin I3(PGI3) which are structurally similar to, but act in reverse to, the inflammatory mediators PGE2, TXA2 and LTB4, acting in competition to suppress the inflammatory response. For example, PGI3 has effects of dilating blood vessels, reducing platelet aggregation, inhibiting thrombosis, and reducing risk of cardiovascular and cerebrovascular accidents.
CLA can also be combined with nuclear receptors, and directly enter cell nucleus to regulate the expression of some genes to play the functions of losing weight and reducing fat. CLA binds to a variety of peroxisome proliferator-activated receptors (PPARs), regulating the expression of a range of genes: (1) inhibiting lipoprotein lipase (lipoprotein lipase) activity of fat cells, preventing degradation of apolipoprotein B and A, promoting fat transportation and mobilization, inhibiting fat synthesis, and reducing blood lipid. (2) Down-regulating the expression of Cyclooxygenase-2 (COX-2). COX-2 is a key enzyme catalyzing the conversion of Arachidonic Acid (AA) to PGE 2. Downregulation of COX-2 expression may inhibit the inflammatory response. (3) Up-regulates nitric oxide synthase (iNOS) activity, promotes the production of PGF2 alpha, and competitively inhibits the action of PGE 2. (4) Up-regulating the expression of cancer suppressor genes such as P53, P21, P27 and the like and pro-apoptotic protein Bax, enhancing the activity of caspase 3 and 9, down-regulating the expression of cyclin D, E and anti-apoptotic protein bcl-2, and inhibiting the expression and secretion of insulin-like growth factor II (IGF-II). Therefore, the composition has the effects of inhibiting cancer cell proliferation and promoting apoptosis. Researches show that the CLA can directly inhibit the proliferation of tumor cells in vitro and induce apoptosis.
It has been shown that CLA feeding to Obese (OB) mice significantly up-regulates the expression of adipocyte PPAR γ, mobilizes lipolysis, and improves insulin resistance. Clinical tests show that when 3-4g of mixed CLA is taken every day, the fat content of normal adults, obese patients and middle-aged and elderly people is reduced to a certain extent, the blood sugar of diabetic patients is reduced, and the insulin sensitivity is improved. CLA was approved by the ministry of health care, china, in 2009 as early as being included in the list of new food products. However, chemically synthesized or biosynthesized CLA is expensive and cannot be used as a conventional food or food additive for the general public.
The propionibacterium acnes LAI gene can allosterize LA into t10c12 CLA. Research shows that oral administration of the Propionibacterium acnes LAI gene transformed live lactobacillus can obviously change the composition of fatty acid in mouse adipose tissues and liver, and improve the content of omega-3 fatty acid such as EPA, DHA and the like in intestinal tract and other adipose tissues. However, bacteria such as propionibacterium acnes, vibrio fibrillis, megasphaera elsdenii and the like are conditional pathogenic bacteria, are not listed in the list of edible fungi, and live bacteria cannot be directly eaten. The existing regulations do not allow the recombinant lactic acid bacteria carrying the LAI gene to be marketed as functional foods.
Disclosure of Invention
The invention aims to provide a lactobacillus reuteri HI120 with high expression of linoleic acid isomerase LAI, wherein the expression level of Linoleic Acid Isomerase (LAI) of the lactobacillus reuteri HI120 is higher than that of lactobacillus reuteri standard bacteria and other probiotics, and Linoleic Acid (LA) can be converted into Conjugated Linoleic Acid (CLA) in vivo and in vitro.
The invention also aims to provide a microbial live bacteria preparation, which contains the lactobacillus reuteri HI120, is prepared into live bacteria preparations such as live bacteria suspension, dairy products, live bacteria powder or bacteria tablets and the like, can be proliferated and enriched in intestinal tracts of human bodies after being orally taken, and can convert part of LA from food sources into CLA.
The last purpose of the invention is to provide the application of the live microbial preparation in the preparation of food additives, foods or medicines with the effects of preventing and/or assisting in treating obesity and diabetes and preventing and/or assisting in treating colorectal cancer.
The first purpose of the invention is realized by the following technical scheme: lactobacillus reuteri HI120 highly expressing linoleic acid isomerase LAI is deposited in the Guangdong province culture Collection in 2016, 11, 21 days, with the deposit numbers GDMCC NO: 60119.
the lactobacillus reuteri HI120 is isolated from the feces of healthy infants, has a Linoleic Acid Isomerase (LAI) expression level 6-10 times higher than that of lactobacillus reuteri standard strain DSM20016(DSM) and bifidobacterium longum (as shown in fig. 2), and can convert Linoleic Acid (LA) into Conjugated Linoleic Acid (CLA) in vivo and in vitro.
Specifically, the classification of lactobacillus reuteri HI120 is named as: lactobacillus reuteri, the collection unit is: the Guangdong province microorganism strain preservation center has the preservation addresses as follows: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, of the Zhongluo prefecture, Mieli, Guangzhou city, has the preservation date: 2016, 11/21/2016, with a accession number: GDMCC NO: 60119.
the second purpose of the invention is realized by the following technical scheme: a live microorganism preparation comprises the above Lactobacillus reuteri HI120 highly expressing linoleate isomerase LAI.
The microorganism viable bacteria preparation can be viable bacteria suspension preparation, dairy product, viable bacteria powder, lyophilized powder preparation, spray dried enteric viable bacteria preparation, bacteria tablet, etc., can be proliferated and enriched in intestinal tract of human body after oral administration, and can convert part of food source LA into CLA.
The third purpose of the invention is realized by the following technical scheme: the application of the live microbial preparation in preparing food additives, foods or medicines with the effects of preventing and/or assisting in treating obesity and diabetes and preventing and/or assisting in treating colorectal cancer.
The microorganism live bacteria preparation can be used for preventing and adjuvant treating obesity, diabetes, intestinal malignant tumor, etc.
Compared with the prior art, the invention has the following advantages:
(1) the lactobacillus reuteri HI120 is separated from the feces of healthy infants, the expression level of Linoleic Acid Isomerase (LAI) is 6-10 times higher than that of lactobacillus reuteri standard bacteria (DSM 20016) and other probiotics, and Linoleic Acid (LA) can be converted into Conjugated Linoleic Acid (CLA) in vivo and in vitro;
(2) the lactobacillus reuteri HI120 is prepared into viable bacteria preparations such as viable bacteria suspension, dairy products, viable bacteria powder or bacteria tablets and the like, can be proliferated and enriched in intestinal tracts of human bodies after being orally taken, and can convert LA from partial food sources into CLA;
(3) the microbial live bacteria preparation of the invention is applied to the prevention and auxiliary treatment of diseases such as obesity, hyperlipidemia, diabetes, enteritis, malignant tumor (intestinal tumor), gastrointestinal dysfunction and the like.
Drawings
Fig. 1 is the isolation and colony characteristics of lactobacillus reuteri HI120 in example 1, a: anaerobic lactobacillus colonies cultured in the feces of the infants; b: isolating the purified HI120 bacterial colony; c: morphological structure of HI120 gram stain;
FIG. 2 is a comparison of the in vitro efficiency of the allosterism of LA to CLA by Lactobacillus reuteri HI120 in example 1 with that of a standard strain, BFL: bifidobacterium longum NCC2705, DSM: lactobacillus reuteri DSM20016, HI 120: lactobacillus reuteri HI 120;
FIG. 3 shows BLAST alignment of the 16S rRNA sequence of Lactobacillus reuteri HI120 strain with other 16S rRNA gene sequences of Lactobacillus reuteri in the NCBI database in example 1;
FIG. 4 shows the chemical properties of the HI120 strain in vitro conversion of LA to CLA detected and identified by GC-MS in example 1, A: 19 mixed fatty acid methyl esters; b: HI120 bacteria fermentation product methylation product; c: plot B labels peak mass spectrometry plot;
fig. 5 is the effect of live lactobacillus reuteri HI120 bacteria on food intake, weight and blood lipid in DB diabetes model after oral administration in example 3, a: comparing the food intake of mice in the HI120 strain group and the control strain group; b: comparison of body weights of mice in HI120 strain group and control strain group C: comparison of peripheral blood Triglyceride (TG) and Total Cholesterol (TC) concentrations for body weights of mice in HI120 strain group and control strain group, where BC: blank control; DSM: DSM20016 standard strain; HI 120: HI120 strain, the same as below;
fig. 6 is the effect of live lactobacillus reuteri HI120 bacteria on blood glucose and oral glucose tolerance in DB diabetes model mice in example 3 after oral administration, a: comparison of fasting plasma glucose concentrations of HI120 and control mice, B: comparing the blood sugar change of the mice of the HI120 strain group and the mice of the control strain group after glucose is orally taken;
fig. 7 is a staining of oil red O in example 3 showing the effect of live lactobacillus reuteri HI120 bacteria orally administered on fatty liver in DB diabetes model mice, a: blank Control (BC) group; b: standard bacteria (DSM) treatment group; c: HI120 strain treatment group;
FIG. 8 is the therapeutic effect of live HI120 bacteria orally administered to APC mice with primary colon cancer in example 4, left panel: colon cancer model mice, right panel: HI120 live bacteria has curative effect on preventing and treating colon cancer.
Detailed Description
Example 1 isolation and characterization of Lactobacillus reuteri
1. Isolation of Lactobacillus reuteri strains
Taking 0.5g of fresh excrement of healthy infants, putting the fresh excrement into 9.5mL of MRS culture medium, fully and uniformly mixing, and diluting to 100 times. 0.1mL of the suspension is spread on an MRS solid culture medium and placed in an anaerobic incubator for culturing for 48 hours at 37 ℃. Selecting milky white, middle raised, round and bright colonies, respectively inoculating to MRS liquid culture medium, anaerobically culturing at 37 deg.C for 24 hr, collecting bacteria, performing CLA conversion experiment, screening high-activity linoleate isomerase (LAI) strain, and identifying strains (figure 1).
2. Screening of high Activity Linoleic Acid Isomerase (LAI) Strain
Respectively inoculating the selected colony and Lactobacillus reuteri standard strain DSM20016 into MRS culture medium containing 1mg/ml of tween-80, wherein the bacterial density reaches OD600When 0.8, respectively adding LA to 1mg/mL,the culture was performed for 12 hours with suspension shaking. Collecting bacteria liquid, centrifuging and taking supernatant. Extracting 1mL culture solution, 2mL isopropanol and 1.5mL n-hexane for 3min, centrifuging at 6000r/m for 5min, sucking the upper n-hexane layer, standing for layering, and extracting fatty acid from the upper n-hexane layer. Measuring the absorbance value of a sample at a wavelength of 234nm by using an ultraviolet spectrophotometer, calculating the CLA content in the bacteria liquid according to a standard curve of CLA, and dividing the CLA content in each milliliter of the bacteria liquid by the total amount of LA added in each milliliter of the bacteria liquid to obtain the efficiency of converting each strain into CLA. The standard strain DSM20016 was used as a control.
The results in fig. 2 show that the HI120 strain had an average efficiency of conversion to CLA 6.1 and 9.1 times higher than DSM20016 strain (DSM) and bifidobacterium longum NCC2705(BFL), respectively (fig. 2), and therefore, the HI120 strain had an efficiency of conversion to CLA 6-9 times higher than other bacteria.
3. LAI high-activity strain 16S rRNA gene amplification and sequencing identification
Collecting bacterial liquid, adding lysozyme to crack bacteria and extracting genome DNA. The bacteria 16S rRNA universal primers 7F and 1540R are used as primers, genome DNA is used as a template, PCR amplification 16S RNA is performed by using bacteria liquid as a template, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 4 min: reaction at 94 ℃ for 45s, 55 ℃ for 45s and 72 ℃ for 1min in each cycle, and 30 cycles; extension at 72 ℃ for 10 min. The PCR product is sent to Shanghai Biotech company for determination and identification.
4. Sequence alignment and analysis
Sequence alignment of HI120 strain 16S rRNA gene sequences with other lactobacillus reuteri 16S rRNA gene sequences was performed using BLAST software tools in the NCBI database in the united states.
The results in fig. 3 show that the lactobacillus reuteri HI120 species 16S rRNA gene sequence has up to 99% homology to the lactobacillus reuteri standard strain DSM20016 and other lactobacillus reuteri 16S rRNA gene sequences (fig. 3).
5. Gas chromatography-mass spectrometry (GC-MS) combined instrument for determining main chemical structure of CLA generated by conversion of HI120 bacteria
Taking 1mL of fatty acid extract, adding 1mL of HCL-methanol (10%), mixing well, and water bathing at 80 ℃ for 1 h. After cooling to room temperature, 1mL of a mixed solution of boron trifluoride and methanol (BF3) was added thereto and mixed well. Water bath at 50 deg.c for 10 min. And cooling, adding 3mL of n-hexane, mixing uniformly in a vortex manner, standing overnight at room temperature, and taking the n-hexane layer for detection. Detection conditions of a gas chromatograph and mass spectrometer (GC-MS): chromatographic column DB-5MS elastic quartz capillary column (30m × 0.25mm 0.5 μm); the temperature of a sample inlet is 250 ℃; split-flow sample injection with split-flow ratio of 10.0; flow rate: 1.0 mL/min; the carrier gas is high-purity helium (99.999%); the sample volume is 1 mu L; the solvent was delayed for 3 min. Mass spectrum conditions: the temperature of the four-level bar is 250 ℃; the ionization mode is electron impact ion source (EI); the ion source temperature is 250 ℃; electron energy 70 eV; the mass range is 30-6000 mAu; the scanning interval is 0.2 s/scan; the electron multiplication voltage was 0.90 kV. Gradient temperature-rising procedure: 0-50 min at 60-300 ℃; the speed is 10 ℃/min; the post-operation temperature is 300 ℃; the post run time was 3 min. According to the chromatographic results of the 19 fatty acid methyl ester standards, the elution peaks of the target sample are analyzed by mass spectrometry, and the structural form of CLA in the sample in mass spectrometry data is checked.
The results in fig. 4 show that HI120 strain transformed LA to CLA with the main chemical structure c9t11 (fig. 4).
Example 2 live microbial preparations
1. Culture of viable bacteria solution
Recovering the HI120 strain of Lactobacillus reuteri with MRS culture medium, inoculating into MRS culture medium at a certain ratio (1: 100-.
2. Preparation of viable bacterial suspension
After the bacteria are cultured to a certain density, 5000g-10000g of bacteria are collected by centrifugation for 5-10 minutes, and edible sugar and salt mixed solution or beverage is added for heavy suspension, and the mixture is stored in a refrigerator at 4-8 ℃.
3. Preparation of freeze-dried live bacteria preparation
After bacteria are cultured to a certain density, 5000g-10000g of bacteria are collected by centrifugation for 5-10 minutes, an antifreezing protective solution (15% of skimmed milk powder, 5% of glycerol and 0.9% of NaCl) is added according to a certain proportion for heavy suspension and mixing, the mixture is uniformly dispersed, the mixture is put into an ultra-low temperature refrigerator for pre-freezing until the mixture is completely frozen, and then the mixture is put into a freeze drier for drying under the condition of the vacuum degree of 10.0-12.00Pa until the water content of the bacteria powder is less than 3%. After the freeze drying is finished, taking out the bacterial powder, subpackaging the bacterial powder into a sterile sealed container, and storing at the temperature of minus 20 ℃ for later use.
4. Preparation of spray-dried enteric viable bacteria preparation
After the bacteria are cultured to a certain density, 5000g to 10000g of bacteria are collected by centrifugation for 5 to 10 minutes, the enteric capsule wall material and the protective agent are added for resuspension, and the enteric viable bacteria powder is obtained by spray drying under the conditions of a certain drying temperature of 100-. Storing in a refrigerator at 4-8 deg.C for use.
5. HI120 viable bacteria powder bacterial activity determination
Weighing 0.1g of bacterial powder, resuspending the bacterial powder by using 1.0mL of MRS culture medium, diluting the bacterial powder in a gradient of 1:100, 1:1000 and 1:1000, respectively taking 0.1mL of bacterial liquid, coating the bacterial liquid on an MRS solid culture medium plate, placing the MRS solid culture medium plate in an anaerobic culture box for culturing at 37 ℃ for 48 hours, and calculating the colony forming unit (CFU/g) of each gram of bacterial powder.
Example 3 application of biological viable bacteria preparation in preparation of food additive, food or drug with effects of preventing and/or assisting in treatment of obesity and diabetes
1. Application of HI120 live bacteria in preventing and treating DB obesity diabetes
12 DB mice were randomly divided into 2 groups: DSM20016 strain group (DSM group) and HI120 strain group (HI120 group) were administered orally, respectively. The DSM and HI120 groups were fed 0.1mL (about 6X 109cfu) DSM20016 and HI120 bacterial powders, respectively. Once daily, mice intervene for a total of 4 weeks. Food and body weight of mice were measured every other day, fasting blood glucose was measured weekly, and Oral Glucose Tolerance Test (OGTT) was measured every two weeks. After 4 weeks, mice were fasted for 12h, and after eye ball removal and peripheral blood removal, neck was cut off and sacrificed, and liver, pancreas, intestine and intestinal contents were taken.
2. Fasting blood glucose determination in mice
In the first week, the second week, the third week and the fourth week, 6 mice were taken from each group, fasted for more than 12h, rat tails were cut off, their corresponding fasting blood glucose values were measured with a glucometer and recorded, statistical analysis of the data was performed with SPSS software, and the mean and standard deviation of the data were plotted (fig. 6).
3. General glucose tolerance test in OGTT mice
After the fasting blood glucose of the mice is measured, the weight of the mice is weighed, and each group of the mice is respectively gazed according to the standard of 2g/kg glucose, after 30min, 60min, 90min and 120min, the tail of the mice is cut, the blood glucose value of each group of the mice is measured, and the blood glucose value of the mice is detected and recorded by a glucometer at 0min, 30min, 60min and 120min (figure 6). The area under the curve (AUC) was calculated using SPSS13.0 statistical software to reflect insulin sensitivity.
The results in fig. 6 show that lactobacillus reuteri HI120 significantly reduces fasting plasma glucose and significantly improves glucose tolerance in mice compared to the Blank Control (BC) and the standard strain DSM20016 (DSM).
4. Biochemical detection of plasma
After one month of treatment, the experimental mice were fasted for 12h before treatment, blood was taken after eye ball removal, centrifuged at 2000rpm for 10min, and plasma was stored in a refrigerator at 4 ℃. The biochemical indexes such as plasma Triglyceride (TRIG) and Cholesterol (CHOL) content are detected by a trace full-automatic biochemical detector, and each sample is diluted by 3 times with double distilled water and then is tested on a machine (figure 5).
The results in fig. 5 show that lactobacillus reuteri HI120 significantly reduces the triglyceride TRIG and cholesterol CHOL levels in mice food, body weight and blood compared to the Blank Control (BC) and the standard strain DSM20016 (DSM).
5. Oil red O staining detection of liver
Placing fresh liver tissue or liver tissue frozen at-80 deg.C in liquid nitrogen tank, embedding slices (10 μm slices, adhering and fixing on anti-falling glass slide), and air drying; soaking in distilled water and 60% isopropanol for 2 min; dyeing with oil red O for 10min (37 deg.C incubator); 60% isopropanol 25S toning (color change observed under microscope); washing with distilled water, and counterstaining with hematoxylin for 1.5 min; then flushing the blue with tap water; finally, the film is sealed by gelatin glycerol (before sealing, the water-based sealing agent is heated to be liquid in warm water at 60 ℃ before sealing, and the film can be sealed) and observed and photographed by a conventional optical microscope (figure 7).
The results in fig. 7 show that HI120 significantly reduced fatty liver in mice, decreased liver red fat particles (positive for oil red O staining) and more regular structure compared to the Blank Control (BC) and the standard strain DSM20016 (DSM).
Example 4 use of a live biological preparation for the preparation of a food supplement, food or pharmaceutical product for the prevention and/or adjuvant treatment of colorectal cancer
1. APC for colorectal cancermin/+Model construction
(1) Selection of APCmin/+The male mice and C57BL female mice are combined according to the proportion of 1 male mouse and three female mice per cage. After 2 weeks of delivery, the foot and toe markers are clipped, and a small tail or small ear is clipped. Extracting tissue genome DNA by conventional method, amplifying target DNA fragment by Polymerase Chain Reaction (PCR) method according to Nanjing model animal resource library method, and identifying APCmin/+And (4) heterozygote. Only 700bp bands of PCR products are wild mice; only 300bp bands are mutant homozygote mice; the 300bp and 700bp bands are the APCmin/+Heterozygote mice.
(2) Taking 6-8 weeks APCmin/+And (4) molding male and female, wherein each mouse is injected with 1mg/mL of AOM according to the body weight, and the injection dose is 10 mu L of AOM/g. And then feeding high-fat feed, observing the state and the excrement condition of the mouse, measuring the weight of the mouse every 3 days, and successfully molding when the mouse has hematochezia and even has proctoptosis.
2. HI120 live bacteria oral intestinal tract route for preventing and treating primary mouse colon cancer
APC model mice were randomly divided into 6 groups with a minimum of 6 mice per group. The APC successfully modeled is selected for the treatment experimentmin/+The treatment group was fed 0.1ml (about 6X 10)9cfu) HI120 bacterial powder suspension (HI120 group), fed once daily; the negative control group was fed an equal amount of DSM20016 bacterial powder (DSM group). Prevention group selection of unmolded APCsmin/+Heterozygote mice were colon cancer modeled while feeding HI120 and DSM20016 bacterial powders. The food intake and body weight of the mice were measured every other day, and the state and stool condition of the mice were observed, and the mice were sacrificed when severe rectocele occurred and body weight decreased significantly in the placebo (BC) group of mice. Fasting for 12h before sacrifice, taking eyeball and peripheral blood, neck-breaking, measuring length of intestinal canal, observing size of tumor body in intestinal canal, probing whether mouse mesenteric lymph node, liver and lung have metastasis, cutting intestinal canal tissue and storing intestinal contentReady for use (fig. 8).
The results in fig. 8 show that, left panel: the colon cancer model mouse can show huge colorectal cancer in abdominal cavity, and the metastasis of axillary lymph nodes of four limbs with hepatomegaly and splenomegaly. Right panel: the HI120 bacterium is effective in preventing and treating colon cancer, compared with a Blank Control (BC) and a standard bacterium DSM20016(DSM), the HI120 bacterium remarkably reduces the number and the size of colon cancer tumor bodies, and only one tumor body begins to shrink.
The present invention has been described above by referring to a part of specific embodiments, and it should be noted that the above-mentioned specific embodiments are only used for further description of the present invention and do not represent a limitation to the scope of the present invention. Other insubstantial modifications and adaptations of the present invention can be made without departing from the scope of the present invention.
Sequence listing
<110> Guangzhou Vital Jun Biotechnology GmbH
<120> lactobacillus reuteri HI120 with high linoleic acid isomerase LAI expression and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1521
<212> DNA
<213> Lactobacillus reuteri (Lactobacillus reuteri)
<400> 1
tcctggctca ggatgaacgc cggcggtgtg cctaatacat gcaagtcgta cgcactggcc 60
caactgattg atggtgcttg cacctgattg acgatggatc accagtgagt ggcggacggg 120
tgagtaacac gtaggtaacc tgccccggag cgggggataa catttggaaa cagatgctaa 180
taccgcataa caacaaaagc cgcatggctt ttgtttgaaa gatggctttg gctatcactc 240
tgggatggac ctgcggtgca ttagctagtt ggtaaggtaa cggcttacca aggcgatgat 300
gcatagccga gttgagagac tgatcggcca caatggaact gagacacggt ccatactcct 360
acgggaggca gcagtaggga atcttccaca atgggcgcaa gcctgatgga gcaacaccgc 420
gtgagtgaag aagggtttcg gctcgtaaag ctctgttgtt ggagaagaac gtgcgtgaga 480
gtaactgttc acgcagtgac ggtatccaac cagaaagtca cggctaacta cgtgccagca 540
gccgcggtaa tacgtaggtg gcaagcgtta tccggattta ttgggcgtaa agcgagcgca 600
ggcggttgct taggtctgat gtgaaagcct tcggcttaac cgaagaagtg catcggaaac 660
cgggcgactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg gaatgcgtag 720
atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgcaactga cgctgaggct 780
cgaaagcatg ggtagcgaac aggattagat accctggtag tccatgccgt aaacgatgag 840
tgctaggtgt tggagggttt ccgcccttca gtgccggagc taacgcatta agcactccgc 900
ctggggagta cgaccgcaag gttgaaactc aaaggaattg acgggggccc gcacaagcgg 960
tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt gacatcttgc 1020
gctaacctta gagataaggc gttcccttcg gggacgcaat gacaggtggt gcatggtcgt 1080
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgttact 1140
agttgccagc attgagttgg gcactctagt gagactgccg gtgacaaacc ggaggaaggt 1200
ggggacgacg tcagatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1260
cggtacaacg agtcgcaaac tcgcgagagt aagctaatct cttaaagccg ttctcagttc 1320
ggactgtagg ctgcaactcg cctacacgaa gtcggaatcg ctagtaatcg cggatcagca 1380
tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtttg 1440
taacgcccaa agtcggtggc ctaaccttta tggagggagc cgcctaaggc gggacagatg 1500
actggggtga agtcgtaaca a 1521
Claims (3)
1. Lactobacillus reuteri HI120 highly expressing linoleate isomerase LAI is characterized in that: the strain is preserved in Guangdong province microorganism culture collection center in 2016, 11 and 21 days, and the preservation number is GDMCC NO: 60119.
2. a microorganism live bacteria preparation is characterized in that: lactobacillus reuteri HI120 highly expressing linoleate isomerase LAI as described in claim 1.
3. Use of the live microbial preparation according to claim 2 in the preparation of food additives, foods or medicaments for the prevention and/or adjuvant treatment of obesity, diabetes and colorectal cancer.
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| Title |
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| Probiotic Lactobacillus reuteri biofilms produce antimicrobial and anti-inflammatory factors;Sara E Jones et al.;《BMC Microbiol》;20090211;第1-9页 * |
| 亚油酸异构酶及其性质;熊向峰等;《工业微生物》;20021231;第32卷(第4期);第51-54页 * |
| 产共轭亚油酸植物乳杆菌的筛选、鉴定与诱变;刘佩等;《食品科学》;20100531;第31卷(第9期);第136页第1栏第1段 * |
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Effective date of registration: 20220812 Address after: Room 2714, Building 2, South China International Financial Center, No. 28, Haiwu Road, Guicheng Street, Nanhai District, Foshan City, Guangdong Province, 528200 (Residence declaration) Patentee after: Foshan Bote Biou Microbial Technology Co., Ltd. Address before: Room 714, Building H5, No. 1940 Kaikai Avenue, Huangpu District, Guangzhou City, Guangdong Province, 510515 Patentee before: GUANGZHOU WEISHENGJUN BIOTECHNOLOGY Co.,Ltd. |