乳酸菌降胆固醇

APPLIED MICROBIAL AND CELL PHYSIOLOGYEffects of Lactobacillus plantarum MA2isolated from Tibet kefir on lipid metabolism and intestinal microflora of rats fed on high-cholesterol dietYanping Wang &Nv Xu &Aodeng Xi &Zaheer Ahmed &Bin Zhang &Xiaojia BaiReceived:15March 2009/Revised:13April 2009/Accepted:15April 2009#Springer-Verlag 2009Abstract The objective of this study was to evaluate the effects of Lactobacillus plantarum MA2,an isolate from Chinese traditional Tibet kefir,on cholesterol-lowering and microflora of rat in vivo.Rats were fed on cholesterol-enriched experimental diet,supplemented with lyophilized L.plantarum MA2powder,with a dose of 1011cells/day per mice.The results showed that L.plantarum MA2feeding significantly lowered serum total cholesterol,low-density lipoprotein cholesterol,and triglycerides level,while there was no change in high-density lipoprotein cholesterol.In addition,liver total cholesterol and trigly-cerides was also decreased.However,fecal cholesterol and triglycerides was increased significantly (P <0.05)in comparison with the control.Also,L.plantarum MA2increased the population of lactic acid bacteria and bifidobacteria in the fecal,but it did not change the number of Escherichia coli as compared to control.Moreover,pH,moisture,and organic acids in the fecal were also measured.The present results indicate the probiotic potential of the L.plantarum MA2strain in hypocholesterolemic effect and also increasing the probiotic count in the intestine.Keywords Lactobacillus plantarum MA2.Kefir .Cholesterol-lowering effectIntroductionElevated serum cholesterol is generally a risky factor correlated with the development of coronary artery diseasesfor humans.However,the current drug therapy has the disadvantage owing to its cost and side effects,so there is an increasing interest in alternative therapies to lower cholesterol.Since Mann and Spoerry (1974)firstly ob-served a hypocholesterolemic effect of milk fermented by wild-type starters in Maasai tribesmen,the ingestion of probiotic lactic acid bacteria might be a more natural way to decrease serum cholesterol in humans (Bazzare et al.1983).There are evidences showing the effects of fermented dairy products by lactic acid bacteria on serum cholesterol levels in animal models (Rao et al.1981;Gilliland et al.1985;Nakajima et al.1992;Akalin et al.1997;Beena and Prasad 1997)and humans (Hepner et al.1979;Steinmetz et al.1994;Agerbaeck et al.1995;Richelsen et al.1996;Schaafsma et al.1998).Although contradictory results have been obtained,there is still reason to believe that probiotic lactic acid bacteria have hypocholesterolemic properties.The mechanisms of the hypocholesterolemic activity of lactic acid bacteria have been proposed to involve inhibition of exogenous cholesterol absorption from the small intestine by binding and incorporation of cholesterol with bacterial cells,assimilation of cholesterol,as well as suppressing bile acid resorption by deconjugation as a function of the bacterial bile salt hydrolase activity (Gilliland et al.1985;Danielson et al.1989;De Smet et al.1998).Kefir has been widely recommended in Soviet and European countries for consumption by healthy people in order to lower the risk of chronic diseases and has also been provided to some patients for the clinical treatment of a number of gastrointestinal and metabolic diseases,hyper-tension,and allergy (St-Onge et al.2002).Also,kefir culture was reported to possess the ability to assimilate cholesterol in milk (Vujicic et al.1992).Furthermore,Liu et al.(2006)proved the hypocholesterolemic effect of kefirAppl Microbiol BiotechnolDOI10.1007/s00253-009-2012-xY .Wang (*):N.Xu :A.Xi :Z.Ahmed :B.Zhang :X.Bai Key Laboratory of Food Nutrition and Safety,Ministry of Education,Tianjin University of Science &Technology,Tianjin 300457,Chinae-mail:ypwang40@milk in male hamsters fed with a cholesterol-enriched diet. However,St-Onge et al.(2002)obtained a contrary result and reported that kefir consumption did not result in lowering plasma lipid concentrations,even though kefir resulted in increasing fecal isobutyric,isovaleric,and propionic acids as well as the total amount of fecal short chain fatty acids.In our previous studies,we isolated a cholesterol-lowering bacteria MA2from Chinese traditional Tibet kefir grains,and it was identified as Lactobacillus plantarum based on the pattern of carbohydrate fermentation and16S ribosomal DNA(rDNA)sequencing results.The vitro experiment showed that the strain has an effective cholesterol-lowering activity.Also,the strain possesses a strong acid and bile salt tolerance(unpublished data)and could successfully pass through the artificial gastric and intestine juice.Based on the above,the present study was conducted to demonstrate the effect of L.plantarum MA2 on cholesterol-lowering and intestinal microflora in rats. Moreover pH,moisture,and organic acids in fecal was also investigated.Materials and methodsBacterial strains The strain of MA2was isolated from Chinese traditional Tibet kefir grains and showed good survival at low pH,tolerance to high bile concentration,and ability to reduce serum cholesterol in vitro in our previous studies.Strain MA2was identified as L.plantarum based on the pattern of carbohydrate fermentation and16S rDNA (GenBank accession no.FJ785723)and was deposited at China General Microbiological Culture Collection Center with accession number CGMCC3005.Preparation of lyophilized strain powder L.plantarum MA2was grown in MRS broth at37°C for18h.The cells were harvested by centrifugation at3,000×g for15min, washed twice with sterile distilled water,and resuspended in threefold volumes of freeze-drying protective solution (including10%skim milk,1.5%fucose,0.5%glycerol,2% sorbitol,1%malt dextrin,[wt/vol])to improve bacterial survival when frozen.Viable cell number of the lyophilized strain powder was2.0×1011cfu/g.Animal feeding and grouping Twenty rats(male Sprague–Dawley),CL grade(Clean animal)and4weeks of age, were obtained from the National Laboratory Animal Breeding and Research Center,Beijing,China.The rats were fed a commercial chow(Kangqiao Inc.,Beijing, China;which includes32%protein,5%fat,2%fiber,Ca 1.8%,P1.2%,and59%nitrogen-free extract)for1week. After this adaptation period,rats were divided into two groups of ten each:group A fed with high-cholesterol diet only and group B fed with high-cholesterol diet and lyophilized L.plantarum MA2powder(lyophilized powder was dissolved in physiological saline and administrated by gavage with a dose of1011cells/day per mice).A high-cholesterol diet includes1%cholesterol(Aoboxing Biotech Co.,Ltd.,Beijing,China),10%lard,5%sucrose,0.3% sodium cholate(Aoboxing Biotech Co.,Ltd.,Beijing, China),0.2%propylthiouracil(Aoboxing Biotech Co., Ltd.,Beijing,China),and78.5%chow.Rats were individ-ually housed in metal cages with controlled temperature (23±2°C)and humidity(55±5%)and in a cycle of12h of light and12h of dark.The animals were fed for5weeks, and during this period,blood was obtained at1-week interval for serum cholesterol and triglycerides analysis. Also,the fecals were collected at4-day interval for bacteria, pH,water content,and organic acid analysis.Simulta-neously,body weight and food intake were also recorded daily.After the feeding period,the rats were euthanized with ether and the viscera of the rats were collected for other tests.Assay for serum lipids Blood(1mL)was obtained from the retro-orbital sinus into a sterile tube at the end of every week following food deprivation for14h.Serum total cholesterol(TC),high-density lipoprotein cholesterol (HDL-C),low-density lipoprotein cholesterol(LDL-C), and triglycerides(TG)were measured enzymatically with a commercial kit(Biosino Biotech&Sci,Inc,Beijing, China).Assay for liver TC and TG After an animal had been killed, the viscera was opened and the liver was removed,rinsed with physiological saline solution,blotted dry with filter paper,and weighed.Liver cholesterol(TC)and triglyceride (TG)content was determined according to the method of Chiu et al.(2006).The same method was used for determination of fecal cholesterol and TG content.Assay for fecal microflora Fecal samples for microbial analyses were collected at an interval of4days in separate sterile tubes and analyzed within1h.Each sample was homogenized using sterile physiological saline diluents. Subsequent tenfold serial dilutions of each sample were plated in triplicate.EMB agar(CM105)was used for E. coli,whereas LBS agar(CM1504)was used for lactic acid bacteria and BSM agar(CM1508)for bifidobacteria.All enumeration media were obtained from Luqiao Tech,Inc, Beijing,China.Assay for fecal organic acid The concentration of organic acid was determined using gas chromatography,which was carried out on a GC-7890II(Tian Mei,Shanghai,China)Appl Microbiol Biotechnolequipped with a FID detector and GDX 401packed column.The following operating conditions were used:The carrier nitrogen gas flow was adjusted to 40mL/min,hydrogen gas to 40mL/min,and air to 400mL/min.The injector and detector temperatures were maintained at 240°C,and the column temperature was held at 200°C and the sample volume was1μL.Measurement of fecal pH and water content The moisture in fecal was determined as a difference between the wet mass and the dry mass of the samples after drying at 80°C (Tianyu Test Instrument,Co,Ltd.,Tianjin,China)until a constant weight was achieved.The pH of the samples was measured with a pH meter (Delta-320,Mettler Toledo,Switzerland).Statistical analysis Experimental data are presented as the mean and standard errors of the mean.Paired t tests were conducted using Microsoft Excel and SPSS 11.5(SPSS Inc.,Chicago,IL,USA).ResultsWeight and food intake All rats were generally healthy throughout the feeding trial period.Both groups of rats showed no significant differences (P >0.05)in body weight gain,food intake,and food efficiency.This indicates that the animals supplemented with L.plantarum MA2grew in similar patterns compared to the control (Table 1).Blood lipid analysis Changes of serum TC,TG,HDL-C,and LDL-C levels of group A and group B are depicted in Table 2.A gradient increase in TC and LDL-C in both groups was observed across the whole experiment time,and the TG level of both groups was significantly higher (P <0.05)at the end of fourth week as compared to the starting 3weeks and was kept stable in the fifth week.HDL-C,Table 1Body weight gain,total food intake,and food efficiency of rats fed on high-cholesterol dietGroup AGroup B Initial body weight (g)140.25(4.50)140.54(4.30)Final body weight (g)217.66(13.92)221.31(20.32)Body weight gain (g/40days)77.41(8.43)80.77(6.47)Food intake (g/40days)713.12(23.00)732.45(14.50)Food efficiency (%)10.86(0.50)11.03(0.45)Group A,high-cholesterol diet only;group B,high-cholesterol diet +Lactobacillus plantarum MA2;food efficiency (%)=(body weight gain/food intake)×100.Results are shown as means (standard deviations;n =10)T a b l e 2S e r u m T C ,H D L -C ,L D L -C ,a n d T G c o n t e n t s o f r a t s f e d o n h i g h -c h o l e s t e r o l d i e tG r o u p AG r o u p BT o t a l c h o l e s t e r o l (m g /d L )H D L c h o l e s t e r o l (m g /d L )L D L c h o l e s t e r o l (m g /d L )T G (m g /d L )T o t a l c h o l e s t e r o l (m g /d L )H D L c h o l e s t e r o l (m g /d L )L D L c h o l e s t e r o l (m g /d L )T G (m g /d L )W e e k 1184.66(19.45)43.74(7.88)120.55(11.52)48.76(5.69)175.62(14.23)45.37(5.72)113.19(14.38)50.13(9.84)W e e k 2264.51(16.29)60.4(15.34)177.79(15.05)41.13(8.56)239.48(19.98)*59.83(11.17)150.01(14.10)**67.06(10.97)**W e e k 3346.69(22.36)61.58(6.96)241.25(14.03)44.45(9.43)276.80(17.34)**58.89(7.74)178.88(10.47)**40.6(8.34)W e e k 4451.51(33.43)70.85(6.61)289.94(21.62)119.63(14.64)338.47(24.76)**60.85(7.34)*221.89(19.68)**99.08(11.98)*W e e k 5490.34(26.25)62.16(9.11)300.88(31.91)124.38(15.93)388.54(8.9)**58.27(4.28)240.56(14.45)**93.11(15.01)*G r o u p A ,h i g h -c h o l e s t e r o l d i e t o n l y ;g r o u p B ,h i g h -c h o l e s t e r o l d i e t +L a c t o b a c i l l u s p l a n t a r u m M A 2.R e s u l t s a r e s h o w n a s m e a n s (s t a n d a r d d e v i a t i o n s ;n =10)*P <0.05;**P <0.01Appl Microbiol Biotechnolhowever,did not show much difference in both group across the feeding trial period.Group B expressed significant lower total cholesterol levels compared with group A,with a reduction of9.46%at the end of week2, 20.16%at week3,25.03%at week4,and20.76%at week5, respectively.Also,the difference of LDL-C between the two groups was significant:group B had the biggest reduction rate of25.85%at the end of the third week,from 241.25to178.88mg/dL.In contrast,the levels of HDL-C in group B decreased slightly as compared to control,but the highest reduction rate of14.10%was in the fourth week.TG had not so much difference between the two groups during the earlier3weeks until the experiment reached up to the fourth week when the difference became greater,and finally the reduction rate reached25.14%at the end of the feeding period.Liver and fecal lipid analysis Table3shows variation in weight and lipid content of the liver and fecal.Liver weight showed a little change among the two groups.Group B had a great reduction in liver cholesterol and TG content as compared to group A.Also,the fecal weight and cholesterol level of the L.plantarum MA2-treated group increased significantly.Fecal TG of the L.plantarum MA2-treated group exhibit a higher level than that of the control group,but the difference was not significant(P<0.05). Microbial populations Figure1depicted the effect of L.plantarum MA2on fecal microflora of rats fed on a high-cholesterol diet.Total E.coli content did not show much change in both two groups and remained8–9cfu log10/g of fresh wet fecal samples across the whole experiment time.A significant increase in total lactic acid bacteria was observed in fecal samples of rats supplemented with L. plantarum MA2,averaged from9.34to11.53cfu log10/g of fresh wet fecal sample;however,the control group showed a consistent population of lactic acid bacteria during the whole feeding period.Also,bifidobacterial content of groupB showed a significant increase from the 24th day and finally contributed a higher count of9.78cfu log10as compared to the control group(8.84cfu log10). Content of fecal organic acids The content of organic acids in the fecals of rats is depicted in Table4.The concentration of acetic acid and propionic acid showed a gradient increase in group B,which averaged from5.53to 15.63mg/g and from2.80to12.15mg/g,respectively,but butyric acid did not show much change and was just from 0.55to0.75mg/g.In group A,only the content of acetic acid increased from8.70to12.85mg/g;however,propionic acid and butyric acid had not changed so much across the whole feeding period.The supplementation with L.planta-rum MA2in high-cholesterol diet caused an increase in the concentration of propionic acid as compared to the control, and the increase was significant(P<0.05).pH and moisture content of fecal The pH of fecal was not significantly different(P>0.05)between both groups (Fig.2).Fecal water content can be used as an index of fecal elimination.Both groups showed a significant decrease in fecal water content across the whole experimental span,Table3Liver and fecal lipid of rats fed on high-cholesterol dietGroup A Group BLiverLiver weight(g)7.26(0.08) 6.95(0.10) Liver cholesterol(mg/g)20.6(0.22)16.23(0.89)* Liver TG(mg/g)32.1(0.34)27.4(9.85)* FecalFecal weight(g/day) 4.07(0.02) 4.25(0.03)* Fecal cholesterol(mg/g)11.75(0.23)15.6(0.40)* Fecal TG(mg/g)8.54(0.03)10.45(0.3)Group A,high-cholesterol diet only;group B,high-cholesterol diet+ Lactobacillus plantarum MA2.Results are shown as means(standard deviations;n=10)*P<0.05Fig.1Population of Escherichia coli,lactic acid bacteria,and bifidobacteria from fecal of rats fed on high-cholesterol diet.Group A,high-cholesterol diet only(triangles);group B,high-cholesterol diet+Lactobacillus plantarum MA2(circles).a Count of Escherichia coli.b Count of lactic acid bacteria.c Count of bifidobacteria colony in fecal.Results are shown as means(n=10)Appl Microbiol Biotechnolranging from 75%to 50%in group A and 80%to 57%in group B,respectively,and L.plantarum MA2caused an increase in fecal water but was not significant,and the difference just ranged between 3%and 7%.DiscussionHigh concentrations of TC and LDL-C are highly associated with an increased risk of coronary heart disease.Reduction in TC and LDL-C in hypercholesterolemic men can reduce the incidence of cardiovascular disease (Probstfield and Rifkind 1991).Our results showed that supplementation of L.plantarum MA2to the high-cholesterol diets did not significantly affect the body weight,food intake,and feed efficiency of rats.The results obtained here correlated withthe findings of Bernardeau et al.(2002)and Liong and Shah (2006),who used Lactobacillus acidophilus and Lactobacil-lus casei ASCC 292,fructooligosaccharide,and maltodextrin (LFM)as supplementation to a high-cholesterol diet,respectively,and found a little change in body weight gain and food intake.The present study showed that L.plantarum MA2resulted in a reduction of serum TC,LDL-C,and TG levels of rats fed high-cholesterol diets.These findings were in agreement with previous reports (Danielson et al.1989;Gilliland et al.1985;Grunewald 1982;Harrison and Peat 1975;Usman 2000).In addition,the great reduction in liver cholesterol and TG content of the L.plantarum MA2-treated group proved that the cholesterol was reduced,not re-distributed between the blood and liver.However,some researchers (Grunewald and Mitchell 1983;Thompson et al.1982;St-Onge et al.2002)did not observe hypocholes-terolemic effect from lactic acid bacteria consumed by mice and humans.Akalin et al.(1997)and Taranto et al.(1998)suggested that these conflicting results may be due to the different properties of cultures used (e.g.,acid,bile tolerance,different mechanisms of lowering cholesterol in vitro).Other important factors included bacterial ingestion dosage,cholesterol content in diet,animal used,and length of the feeding period.HDL-C did not show much difference in the experiment.Similar results in rats and humans had been reported by Ibrahim et al.(2005),Fukushima and Nakano (1996),and St-Onge et al.(2002).However,Chiu et al.(2006)reported a reduction in HDL cholesterol in hamsters fed on high-cholesterol diets.Also,Keim et al.(1981)and Rossouw et al.(1981)got similar results in humans.Otherwise,in a study performed by Hashimoto et al.(1999),a diet containing L.casei TMC 0409was found to raise the concentration of HDL-C in the blood,and similar results were also shown in other findings (Akalin et al.1997;Danielson et al.1989;De Smet et al.1998).Table 4Concentration of organic acids of fecal from rats fed on high-cholesterol dietGroup A Group B Acetic acid (mg/g)Propionic acid (mg/g)Butyric acid (mg/g)Acetic acid (mg/g)Propionic acid (mg/g)Butyric acid (mg/g)Week18.70(0.26) 2.35(0.1))0.63(0.03) 5.53(0.06) 2.80(0.07)0.55(0.01)Week212.65(0.06) 4.70(0.07)0.65(0.01)10.65(0.09) 6.45(0.15)0.63(0.003)Week312.7(0.21) 3.50(0.04)0.75(0.02)11.40(0.10)7.53(0.15)**0.65(0.03)Week412.8(0.13) 3.65(0.05)0.80(0.01)13.08(0.08)8.55(0.04)**0.60(0.02)Week512.85(0.34)4.00(0.20)0.88(0.03)15.63(0.05)12.15(0.12)**0.75(0.01)Group A,high-cholesterol diet only;group B,high-cholesterol diet +Lactobacillus plantarum MA2.Results are shown as means (standard deviations;n =10)**P<0.01Fig.2pH and water content of fecal from rats fed on high-cholesterol diet,group A,high-cholesterol diet only (filled triangle );group B,high-cholesterol diet +Lactobacillus plantarum MA2(circles ).a pH value of fecal.b Water content in fecal.Results are shown as means (n =10)Appl Microbiol BiotechnolLactic acid bacteria may alter serum cholesterol by three proposed mechanisms:(a)directly binding,absorb-ing cholesterol into the cell and assimilation before cholesterol can be absorbed into the body(Gilliland et al.1985;Noh et al.1997);(b)deconjugating bile acids and produce free bile acids,which are more likely to be excreted from the body,and drain the cholesterol pool as more bile acids are synthesized(Corzo and Gilliland1999; Dietschy1966;Chikai et al.1987);and(c)inhibiting HMG-CoA reductase by some metabolites of lactic acid bacteria like propionic acid(Fukushima and Nakano 1996).In our experiment,more fecal cholesterol was detected in the L.plantarum MA2supplementation group, and this phenomenon was similar to the results of Mott et al.(1973)and Park et al.(2008).Based on our previous vitro studies,we presume that cholesterol was tightly bound,incorporated into the cell and excreted from feces, which resulted in an inhibition of cholesterol resorption in intestine(Gilliland et al.1985;Hosono and Tono-oka 1995).The exact vivo mechanism of reducing cholesterol for L.plantarum MA2should be studied further to verify this presumption.Many studies have shown that lactic acid bacteria inhibit the proliferation of pathogenic bacteria,improve intestinal probiotics,reduce the risk of diseases,and promote the health of the host(Fuller1989;Van Winsen et al.2002; Mikkelsen and Jensen1998).In our test,the number of fecal lactobacilli and Bifidobacterium increased in the L. plantarum MA2group compared with the control.This indicated that L.plantarum MA2could successfully tolerate gastric acid and bile salt and play biological effects. Moreover,Donnet-Hughes et al.(1999)suggested that survival in the feces following oral administration reflects large bowel colonization and proliferation.However,in both groups,fecal E.coli showed a constant level throughout the feeding period,and these results indicated that growth of E.coli was not inhibited by L.plantarum MA2in vivo.It must be noted that the concentration of fecal propionic acid in rats supplemented by L.plantarum MA2was significantly increased,almost three times higher than the control.An increase in propionic acid concentration may have altered the cholesterol synthesis pathways and leads the cholesterol concentration to decrease(Liong and Shah 2006).However,the acetate content was not changed too much in both groups.Acetate is a lipogenic substrate (Delzenne and Kok2001),so a decrease in the concentration of acetic acid may lead to a decreased lipogenesis(Liong and Shah2006).Also,the concentration of butyric acid showed no significant change,and it was contrary to the findings of Liong and Shah(2006),who reported that the supplemen-tation of LFM significantly decreased the concentration of butyric acid in rats fed with a cholesterol-rich diet.Our results showed that the L.plantarum MA2did not change fecal pH too much,and many reports indicate that a decreased intestinal pH can inhibit the binding of entero-pathogenic E.coli to intestinal cells(Bernet et al.1994; Swanson et al.2002).There was a higher fecal moisture in L.plantarum MA2group,but the difference was very small,just7%.However,Chiu et al.(2006)reported a20% variation in fecal water content between the L.acidophilus-treated group and control.Our results here indicated that L. plantarum MA2had laxative potential,but the effect is very minute and there is a need to combine it with prebiotics to facilitate fecal elimination.The results of this study indicate that L.plantarum MA2 is a potential probiotic to reduce serum cholesterol,low-density lipoprotein cholesterol,and triglyceride levels. 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