CN103275905B - Lactobacillus rhamnosus CCFM0528 having diabetes preventing effect - Google Patents
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Abstract
The invention relates to lactobacillus rhamnosus CCFM0528 having an inhibitory effect on alpha-glucosidase activity, the bacterial strain is preserved at the China General Microbiological Culture Collection Center (CGMCC), and the accession number is CGMCC No.7317. Cytoplasm and fermentation supernate of the CCFM0528 can respectively exhibit the inhibitory effects on the alpha-glucosidase activity, with the inhibition rates of 29.2% and 14.1% respectively. Studies show that the CCFM0528 exhibits the inhibitory effect not only on the alpha-glucosidase activity, also on glucose transport. The experimental results show that the CCFM0528 can prevent type-2 diabetes, effectively reduce fasting blood glucose and postprandial blood glucose of mice, and improve glucose tolerance of the mice. The principle may be that the CCFM0528 has inhibitory effect on the alpha-glucosidase activity and enzyme expression amount in intestinal tract, and can influence the gene expression level of glucose-transport relating protein SGLT-1 and GLUT-2.
Description
Technical field
The present invention relates to the microorganism field of biological technical field, particularly there is the lactobacillus rhamnosus CCFM0528 of prevent diabetes effect.
Technical background
According to (the World Health Organization of the World Health Organization in 1999, and (the International Diabetes Federation of IDF WHO), the definition of IDF) announcing, it is mainly divided into insulin-dependent diabetes mellitus (1 type is DIDM) and non insulin dependent diabetes (2 types are NIDDM), other type and gestational diabetes Four types, wherein diabetes B patient accounts for more than 90%.In recent years, diabetes are widely current in the whole world, have become the chronic disease of the 3rd serious harm human health after tumour, cardiovascular disorder.China's diabetes prevalence has risen 4 times in 20 years in the past, and within 2007, national diabetes number of patients is 3,980 ten thousand, and being only second to India becomes global diabetic subject's number the second big country.Therefore, be badly in need of preventing and treat diabetes by effective measure.Be extremely important for treatment early diabetes and control postprandial hyperglycemia, wherein controlling the absorption of carbohydrate is after the meal a kind of effective methods for the treatment of that reduces postprandial hyperglycemia.Because small intestine is merely able to absorb and transports monose in blood circulation, and the alpha-glucosidase of being secreted by intestinal epithelial cells can be monose by polysaccharide hydrolysis, so be considered to the important factor of glucose absorption in intestines.Therefore for diabetes B patient, take some hypoglycemic oral medicines that contain alpha-glucosidase inhibitor and can effectively reduce hyperglycemia.These medicines Inhibiting α-glucosidase competitively, suppresses disaccharide and is hydrolyzed into monose, hinders digestion and the absorption of glucose, thereby reduces the blood-sugar content in blood after the meal.The alpha-glucosidase inhibitor kind of finding is at present less, and therefore people are still constantly devoted to development of new alpha-glucosidase inhibitor.
Milk-acid bacteria refer to fermenting carbohydrate primary product be a class of lactic acid without the general name of gemma, gram-positive bacterium, be the probiotic bacterium in a kind of mankind's of being present in body.Probiotic bacterium can be helped digest, and helps the health of human body intestines and internal organs of the body, is therefore often regarded as heath food, is added among Yoghourt.According to recent years research data show, milk-acid bacteria has various health-care as the profitable strain of settling down in enteron aisle: can maintain microecological balance and intestinal tube function 1.; 2. have anti-microbial effect, can suppress spoilage organism, as killed listeria bacteria, staphylococcus and leukonid, to suppress and to eliminate Gram-negative bacteria, catalase-positive organism, intestinal bacteria and Salmonellas etc.; 3. can improve liver function; 4. milk-acid bacteria can make enteron aisle reduce the absorption to cholesterol, and a part of cholesterol can be transformed into cholate and excrete; 5. can strengthen immunologic function and anti-tumor disease.In recent years, studies have reported that milk-acid bacteria has impact energetically to treating diabetes.Milk-acid bacteria can prevent and postpone the generation of dissimilar diabetes.Within 2004, responsible official Kawai Yasuo of Japanese He He milk-acid bacteria institute has found to fall hypoglycemic milk-acid bacteria, and it can be used to the medicine of exploitation prevention and treatment diabetes.Quantity research shows greatly at present, and milk-acid bacteria has the potentiality that reduce onset diabetes rate, but people are still little as the research of Remedies for diabetes for milk-acid bacteria.
Summary of the invention
The present invention relates to a kind of lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM0528 with prevent diabetes effect, it is characterized in that this bacterial strain has the activity of Inhibiting α-glucosidase, at Chinese microorganism strain preservation center, deposit number is CGMCC No.7317.
On the other hand, the invention still further relates to the purposes of lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM0528 in the medicine for the preparation of prevent diabetes, described diabetes are 1 type or diabetes B.
The invention still further relates to the pharmaceutical composition that comprises lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM0528, the bacterial strain comprising in described pharmaceutical composition can be the metabolite of live strain or dry bacterial strain or bacterial strain, in addition pharmaceutical composition also comprises pharmaceutically acceptable carrier, its formulation can be oral preparations, for example tablet, capsule, oral liquid, the oral dosage form that lyophilisate etc. are common.Pharmaceutically the common carrier accepted has vehicle, protective material, solvating agent etc.
The invention still further relates to the food that comprises lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM0528, described food is leavened food, for example soybean milk yoghurt, fermentation jelly, fermented tea beverage or milk preparation, as Yoghourt, yaourt, cheese product, nisin milk powder, Ice cream of egg containing yoghurt etc.
1, according to basic probiotic properties and the restraining effect to alpha-glucosidase activity, set up the prescreening method of a kind of probiotic bacterium with Inhibiting α-glucosidase active function.The inhibiting rate that in the fermentation of CCFM0528, cleer and peaceful tenuigenin is lived to alpha-glucosidase is respectively 29.2% and 14.1%.
2, in Caco-2 cell model, evaluate the influence of CCFM0528 to alpha-glucosidase activity and expression amount.In the fermentation of CCFM0528, cleer and peaceful tenuigenin is respectively 15.7% and 20.0% to the inhibiting rate of alpha-glucosidase activity, and the transhipment inhibiting rate of glucose is respectively to 7.0% and 19.3%; The upper cleer and peaceful tenuigenin of CCFM0528 to alpha-glucosidase (S-I) gene expression dose lower respectively 7.29 times and raise 1.04 times, to sodium-glucose body-1(SGLT-1 that cotransports) gene expression dose lowers respectively 2.5 times and raise 2.2 times, to glucose transporter-2(GLUT-2) gene expression dose lower respectively 1.1 times and 1.2 times.
3, utilize high lipid food to feed and the animal model evaluation CCFM0528 of the STZ injection prophylactic effect to diabetes B.After modeling success, model group fasting blood glucose level is 12.9mmol/L, and CCFM0528 can significantly reduce the fasting blood glucose level of prevention group to 8.13mmol/L(P ﹤ 0.01); Model group 2h-plasma glucose level is 23.0mmol/L, and CCFM0528 can significantly reduce the 2h-plasma glucose level of prevention group to 14.3mmol/L(P ﹤ 0.01); CCFM0528 significantly improves the oral glucose tolerance (OGTT) (P ﹤ 0.01) of prevention group compared with model group.
Brief description of the drawings
The survival rate of Fig. 1 CCFM0528 in pH3.0 simulated gastric fluid;
The survival rate of Fig. 2 CCFM0528 in pH8.0 simulated intestinal fluid;
Fig. 3 CCFM0528 supernatant and the cell-free extract inhibiting rate to rat alpha-glucosidase activity;
Cell monolayer scanning electron microscope (SEM) photograph when Fig. 4 is grown in millipore filtration upper 21 day, A × 1000; B × 10000;
The inhibiting rate of Fig. 5 CCFM0528 to alpha-glucosidase activity in Caco-2 cell;
The restraining effect of Fig. 6 CCFM0528 to glucose transport in Caco-2 cell;
Fig. 7 S-I, the relative expression quantity of SGLT-1 and tri-kinds of genes of GLUT-2, note: * * represents that difference is extremely remarkable compared with control group, P < 0.01;
Fig. 8 respectively organizes body weight change during mouse test;
Each group mouse fasting blood glucose level after Fig. 9 modeling success, note: * * represents that difference is extremely remarkable compared with normal group, P < 0.01;
Each group mouse level of postprandial blood sugar after Figure 10 modeling success, note: * * represents that difference is extremely remarkable compared with normal group, P < 0.01;
The sugared tolerance of each group mouse after Figure 11 modeling success;
Figure 12 modeling is each group mouse AUC comparison after one week, note: * * represents that difference is extremely remarkable compared with normal group, P < 0.01.
Embodiment
1, milk-acid bacteria sample source
CCFM0528 is provided by this culture presevation storehouse, laboratory, be accredited as lactobacillus rhamnosus Lactobacillus rhamnosus CCFM0528 by 16S, this bacterial strain has been deposited in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 15th, 2013, Microbe Inst., Chinese Academy of Sciences), deposit number is CGMCC No.7317.
CCFM0528 thalline is rule in solid MRS, choose single bacterium colony and in liquid nutrient medium, cultivate 18h, go down to posterity after 2 times for follow-up test.
2, the tolerance analysis of CCFM0528 to simulated gastric fluid
CCFM0528 is resuspended in physiological saline, by the liquid-tight degree 10 of bacterium
9cFU/mL is linked into the simulation stomach of pH3.0
In liquid, cultivate 3h for 37 DEG C, respectively at 1h, 2h, 3h calculates the survival rate of milk-acid bacteria, and its survival rate is as shown in Figure 1.CCFM0528 grows after 3h in simulated gastric fluid, and its survival rate can reach 84.2%.
3, CCFM0528 analyzes simulated intestinal fluid tolerance
By the bacterium liquid of cultivating in simulated gastric fluid after 3h, get 1mL and join in 9mL pH8.0 simulated intestinal fluid, cultivate 8h for 37 DEG C, respectively at 2h, 4h, 8h calculates the survival rate of CCFM0528, and its survival rate is as shown in Figure 2.CCFM0528 grows after 8h in simulated intestinal fluid, and its survival rate can reach 85.4%.
4, the bile tolerance capability analysis of CCFM0528
CCFM0528 is joined respectively and is not added the MRS-THIO(MRS of 0.3% cholate according to 2% inoculum size
In substratum, add 0.2% Thioglycolic acid sodium salt) in substratum.Bacterium liquid is cultivated in 37 DEG C, CCFM0528 grows in not adding and being added with bile salt culture-medium, OD value reaches 0.3 o'clock required time and is respectively 3.7 ± 0.1h and 5.2 ± 0.1h, so be 1.5h cholate time of lag of CCFM0528, having reacted this milk-acid bacteria has very strong tolerance to cholate.
5, the adhesive capacity analysis of CCFM0528
CCFM0528 is centrifugal rear with PBS washing 3 times, then adjusts bacterial concentration to 10 with DMEM
9cFU/mL, joins bacteria suspension in cultured Caco-2 monolayer cell, cultivates altogether 2h for 37 DEG C, with gramstaining after the fixing 30min of methyl alcohol.At 100,20 visuals field of electric Microscopic observation cell, the adhesive power that calculates this milk-acid bacteria is 12.0 ± 0.2 each cells.
6, the preparation of cleer and peaceful cell-free extract in CCFM0528 fermentation
To after CCFM0528 activation culture, be inoculated in MRS liquid nutrient medium, cultivate 18h for 37 DEG C, culture is through 6000r/min, 4 DEG C of centrifugal l0min, obtain culture supernatant and bacterial sediment, and bacterial sediment is after twice washing of PBS, by thalline PBS Eddy diffusion, adjusting cell concn is 10
9cFU/mL, supernatant liquor obtains acellular supernatant liquor (CFS) after 0.22 μ m membrane filtration, another group, for the preparation of cell-free extract (CFE), is processed with ultrasonic disruption instrument cell suspending liquid in ice bath, work 3s, interval 8s, ultrasonication 10min, test under microscope does not have complete thalline, then with 4 DEG C, and the centrifugal 20min of 12000 × g, collect supernatant liquor, be cell-free extract through 0.22 μ m membrane filtration.Supernatant and cell-free extract, after 100 DEG C of heating 20min, are made heat-inactivated cleer and peaceful cell-free extract.
The inhibition capability analysis of the alpha-glucosidase activity that 7, CCFM0528 extracts intestine in rats
CCFM0528 suppresses determination of activity to alpha-glucosidase: at 150 μ L PBS(0.1M pH6.8) in add 4-nitrophenol-α-D-pyrans Portugal glucoside (PNPG) solution and the 25 μ L testing samples of 75 μ L20mM, by mixture in 37 DEG C of water-bath 10min.Add the alpha-glucosaccharase enzyme solution (0.17U/mL) that 50 μ L intestine in rats extract to continue reaction 10min.Add 1mL0.1M Na
2cO
3as reaction terminating liquid.And reaction solution is surveyed to its light absorption value in 405nm place, light absorption value is directly proportional to the free amount of 4-nitrophenol (PNP).In reaction system, adopt the 0.1M PBS of pH6.8 as the blank of alpha-glucosaccharase enzyme solution and testing sample, adopt the inhibition activity of following formula calculation sample:
Enzyme inhibition rate (%)=[1-(C-D)/(A-B)] × 100%
Wherein A contains alpha-glucosaccharase enzyme solution but not containing the mensuration light absorption value of sample
B is the mensuration light absorption value that does not contain alpha-glucosaccharase enzyme solution and testing sample
C is the mensuration light absorption value that contains alpha-glucosaccharase enzyme solution and testing sample
D is not containing alpha-glucosaccharase enzyme solution but containing the mensuration light absorption value of testing sample
As shown in Figure 3, the upper cleer and peaceful cell-free extract of CCFM0528 all has restraining effect to alpha-glucosidase activity, the inhibiting rate (29.2%) of supernatant is greater than the inhibiting rate (14.2%) of cell-free extract, and the upper cleer and peaceful cell-free extract after deactivation all reduces (﹤ 8.5%) greatly to the inhibiting rate of enzyme, therefore can infer that some active substance in CCFM0528 has restraining effect to enzymic activity.
8, the foundation of Transwell cell model and assessment
Be between 22～40 generations for the Caco-2 cell that builds Transwell cell model.By cell according to density 2 × 10
5individual/ml is inoculated in (membrane pore size 0.4 μ m growth area 1.12cm on 12 hole Transwell cells
2), transhipment groove enteric cavity side (upper chamber, AP side) adds the cell suspension 500ul that mixes up density, and base side (lower chamber, BL side) adds 1.5ml cell culture fluid, changes liquid every day, for subsequent use in the time that cell grows to 21d.
The assessment of model:
(1) whether scanning electron microscope checking cell forms fine and close individual layer, and result as shown in Figure 4.
From Fig. 4 A, can find out that cell has formed fine and close individual layer at polycarbonate membrane, in Fig. 4 B, can find out that cell surface forms good brush dress edge and iuntercellular and forms tight connection, thereby form cell monolayer closely.
(2) uranine permeability detects: the 21st day time, in 12 hole Transwell plates, the apparent permeability coefficient in each hole is all far smaller than 0.5x10
-6cm
2/ s.Further prove that cell has formed good tight connection individual layer.
(3) alkaline phosphatase AKP polarity detects: the enzyme work of upper strata cell is all the more than 4 times of bottom enzyme work.Cultivate
Alkaline phosphatase activities to its enteric cavity side of the cell monolayers of 21 days is significantly higher than base side enzymic activity, approximately exceeds 4 times more than, illustrate this marker enzyme major part concentrate on brush dress edge one side, cell monolayer has formed polarity.
9, the restraining effect of CCFM0528 to alpha-glucosidase in Transwell cell model
Rinse the Transwell cell top and bottom 3 times of having cultivated 21 days (above add 0.25ml, under add 0.6ml) with PBS, remove glucose.Add maltose (filtration sterilization) that 475ul contains 20mM as substrate, and add the supernatant of CCFM0528 or cell-free extract 25ul(0.5ml altogether) one play mucous layer, 1.5mlPBS joins lower floor, plank is cultivated 2h at 37 DEG C, levels is respectively got 100 μ l solution and detect glucose content in microwell plate, calculates respectively inhibiting rate to enzymic activity and the restraining effect of glucose transport rate with this.Result is shown in respectively Fig. 5 and Fig. 6.
As shown in Figure 5, CCFM0528 cell extract is greater than the inhibiting rate of supernatant to the inhibiting rate of enzyme, and its cell extract to the inhibiting rate of intestine in rats enzymic activity the inhibiting rate higher than supernatant, its size therewith result is inconsistent, illustrate in cellular environment and external environment and still have very large difference, so result is also also not quite identical, but all show, enzymic activity is had to certain restraining effect.
As shown in Figure 6, the upper cleer and peaceful cell extract of CCFM0528 all has certain restraining effect to the glucose transport in Caco-2 cell, and the inhibiting rate of cell extract is higher than the inhibiting rate of supernatant, and this is consistent to the inhibiting rate of enzymic activity with above-mentioned CCFM0528.
10, CCFM0528 is to the S-I in Caco-2 cell, SGLT-1, the impact of GLUT-2 gene expression amount
In the digestion of carbohydrate and absorption process, enzyme related to this and transport vehicle mainly contain alpha-glucosidase (S sucrase-isomaltase, S-I), the sodium glucose carrier 1(sodium glucose cotransponers l that cotransports, SGLT-l) and glucose assist diffusion transport vehicle 2(facilitative glucose transporte2, GLUT-2).CCFM0528 supernatant and cell extract are joined in the Caco-2 monolayer cell of cultivating 21 days and cultivated altogether after 2h with it by 5%, extract total RNA the synthetic cDNA of reverse transcription of Caco-2 with test kit, after carry out quantitative fluorescent PCR, and select β-actin as reference gene.Carry out quantitative fluorescent PCR the primer sequence as shown in table 1.
PCR reaction conditions is: (1) 95 DEG C of for15min; (2) 95 DEG C, 10s, 64 DEG C, 20s, 72 DEG C, 30s, 40 circulations; (3) 72 DEG C, 5min; (4) 95 DEG C, 15s, 60 DEG C, 15s, 95 DEG C, 15s.With 2
-△ △ CTmethod is calculated CCFM0528 to the S-I in Caco-2 cell, SGLT-1, the impact of GLUT-2 gene expression amount.Result as shown in Figure 7.
The oligonucleotide sequence of table 1 quantitative fluorescent PCR
From Fig. 7 A, CCFM0528 supernatant can significantly be lowered the expression amount of S-I gene in Caco-2 cell, and cell extract affects without significance the expression amount of S-I gene.In the supernatant of this milk-acid bacteria, contain a large amount of organic acids, as lactic acid, acetic acid etc.Studies have reported that acetic acid has restraining effect to the enzymic activity of Caco-2 cell, we study some active substance of finding in CCFM0528 supernatant has restraining effect to the alpha-glucosidase activity of intestine in rats extraction, therefore can infer that some active substance and organic acid in the tunning of CCFM0528 have restraining effect to alpha-glucosidase activity, and expression amount that can inhibitory enzyme, thereby the decomposition of effectively alleviating carbohydrate absorbs, and reduces the growing amount of monose from source.
From Fig. 7 B, the supernatant of CCFM0528 can significantly be lowered the expression amount of SGLT-1 in Caco-2 cell, and cell extract can significantly raise the expression amount of SGLT-1 in Caco-2 cell.Result suppresses consistent with CCFM0528 to the Transport Rate of glucose.
From Fig. 7 C, the supernatant of CCFM0528 can significantly be lowered the expression amount of GLUT-2 in Caco-2 cell, and cell extract affects without significance the expression amount of S-I gene.
11, the experimentation on animals of prevent diabetes effect in CCFM0528 body
In body, prevent diabetes test adopts high lipid food to feed and injects the diabetes B model that U-9889 (STZ) causes.Laboratory animal adopts 24 of SPF level C57BL/6 mouse in male 3 week age, is provided by the Shanghai limited public affairs of Si Laike laboratory animal.Mouse is at room temperature raised, and freely drinks water and searches for food, and after 1 week adaptability is raised, is divided into immediately normal group, model group and test group, 8 every group.The normal group basal feed (protein content 25.7%, lipid content 13.8%, carbohydrate 60.5%) of feeding; Model group and test group are with high lipid food feed (protein content 20.0%, lipid content 40.0%, carbohydrate 40.0%); Test group gavage every day milk-acid bacteria 2 × 10
9cFU/mL.Fasting 12h after 3 weeks, add low dosage streptozotocin 100mg/Kg (0.1mol/L citrate buffer solution, pH4.5,0.2 μ m membrane filtration) abdominal injection, after 1 week, to observe: figure is normal for normal group group mouse, behavior is active, hair color light, without depilation, appetite is normal with drinking-water, and stool and urine is normal.Injection STZ modeling group mouse engenders that hair color is unclean, and drinking-water increases, and urine amount increases, mood irritability, and the type II diabetes relevant symptoms such as lose weight.Duration of test is measured weekly body weight once, as shown in Figure 8.After to STZ induction modeling one week, respectively organize mouse fasting and can't help water 12h, measure tail vein sugar value with blood glucose meter, be fasting blood sugar, as shown in Figure 9; After feeding 2h, again measuring tail vein sugar value with blood glucose meter, is 2h-plasma glucose value, as shown in figure 10.After modeling 8 days, respectively organize mouse fasting and can't help water (12h), gavage 2g/kg glucose (50%), and measure respectively 0min, 15min, 30min, 60min, 90min, 120min caudal vein blood glucose value.Use orgin7.5 to draw change of blood sugar curve according to blood glucose value, and calculate area under glucose tolerance curve (Area Under Cerve, AUC) as shown in Figure 11 and Figure 12.
As shown in Figure 8, after the 4th week injection STZ, normal group mouse Normal-weight increases, and it is high that the increase of model group Mouse Weight is not so good as normal group, and CCFM0528 group Mouse Weight also increases slowly.
As shown in Figure 9, injection STZ detects the fasting plasma glucose of each model group and CCFM0528 group mouse all higher than the threshold value 7.0mmol/L of diabetes after mono-week, show diabetes model modeling success.The glucose level of model group is far away higher than normal group, although the glucose level of CCFM0528 group higher than 7.0mmol/L, itself and normal group glucose level there was no significant difference.Show that CCFM0528 can significantly reduce the fasting blood glucose level of diabetic mice.
As shown in Figure 10, injection STZ detects the 2h-plasma glucose of each model group and CCFM0528 group mouse all higher than the threshold value 11.0mmol/L of diabetes after mono-week, show diabetes model modeling success.As can be seen from the figure the level of postprandial blood sugar of model group is the highest, and extremely remarkable with normal group difference.Although the postprandial blood sugar of milk-acid bacteria group is extremely remarkable with normal group difference, its glucose level, well below model group, shows that CCFM0528 can reduce the postprandial blood sugar of diabetic mice.
As shown in Figure 11, after gavage glucose, in 15min to 30min, respectively organize rat blood sugar all in rising trend, normal group changes very mild, the basic normal level that keeps after 2h; Model group and milk-acid bacteria group be significantly rising (p<0.01) in 30min, and reach hyperglycemia, model group still remains on higher level after 2h, illustrate that glucose-tolerant ability significantly reduces (p<0.01), meets the feature of type II diabetes impaired glucose tolerance.Wherein, CCFM0528 group mouse its glucose level in the time of 90min is tending towards normal group mouse blood sugar.
As shown in Figure 12, the AUC value significant difference of modeling each group mouse after a week, the AUC area of model group is significantly greater than normal group, though the AUC area and normal group significant difference of milk-acid bacteria group obviously reduce compared with model group.Sugar tolerance tool the be significantly improved effect of CCFM0528 to prevention group mouse is described.
12, the food or the pharmaceutical composition that contain Lactobacillus rhamnosus CCFM0528
Application Example 1: utilize CCFM0528 to manufacture the soybean milk yoghurt with prevent diabetes function
In raw material soya-bean milk, add 6% sucrose, in 121 DEG C, sterilizing 15min, adds lactobacillus rhamnosus CCFM0528 of the present invention with the amount of 7% volume percent, and fermenting to titration acidity at 37 DEG C is that 0.7%-0.8%(is in lactic acid),
Refrigeration to 4 DEG C and stored refrigerated obtain having the soybean milk yoghurt of prevent diabetes function.
Application Example 2: utilize CCFM0528 to manufacture the lactobacillus drink with prevent diabetes function
Raw dairy (skimmed milk, fresh milk, recovery milk etc.), through 100 DEG C, is cooled to 4 DEG C after sterilizing 10min, adds lactobacillus rhamnosus CCFM0528 of the present invention, make more than its concentration reaches 106cfu/ml, 4 DEG C of stored refrigerated obtain viable bacteria milky-drinks.
Application Example 3: utilize CCFM0528 to manufacture the Yoghourt with prevent diabetes function
By raw dairy (skimmed milk, fresh milk, recovery milk etc.) through 100 DEG C, after sterilizing 10min, be cooled to 4 DEG C, add lactobacillus rhamnosus CCFM0528 of the present invention with the amount of 3%-5% volume percent, and add can symbiosis the business starter of preparing Yoghourt as lactobacillus bulgaricus and thermophilus streptococcus, 37 DEG C of mixed fungus fermentation to titration acidities are that 0.6%-0.7%(is in lactic acid), refrigeration to 4 DEG C and stored refrigerated obtain having the Yoghourt of prevent diabetes function.
Claims (10)
1. one kind has lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM0528 of prevent diabetes effect, this bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 15th, 2013, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Microbe Inst., Chinese Academy of Sciences, deposit number is CGMCC No.7317.
2. comprise the pharmaceutical composition of lactobacillus rhamnosus claimed in claim 1 (Lactobacillus rhamnosus) CCFM0528, it is characterized in that the bacterial strain that wherein comprised is live strain or dry bacterial strain.
3. comprise the pharmaceutical composition of the fermented supernatant fluid obtaining after cultivation and fermentation lactobacillus rhamnosus claimed in claim 1 (Lactobacillus rhamnosus) CCFM0528.
4. according to the pharmaceutical composition that comprises lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM0528 described in claim 2 or 3, it is characterized in that described pharmaceutical composition also comprises pharmaceutically acceptable carrier, its formulation is oral preparations.
5. the pharmaceutical composition that comprises lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM0528 according to claim 4, is characterized in that described oral preparations is tablet, capsule, oral liquid or lyophilisate.
6. comprise the food of lactobacillus rhamnosus claimed in claim 1 (Lactobacillus rhamnosus) CCFM0528 or its fermented liquid.
7. food according to claim 6, is characterized in that described food is leavened food.
8. food according to claim 7, is characterized in that described leavened food is soybean milk yoghurt, milk-product, fermentation jelly or fermented tea beverage.
9. the purposes of the pharmaceutical composition described in lactobacillus rhamnosus according to claim 1 (Lactobacillus rhamnosus) CCFM0528 or claim 2-4 any one in the medicine for the preparation of prevent diabetes.
10. purposes claimed in claim 9, is characterized in that described diabetes are diabetes Bs.
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