Reduction of cholesterol 2013.pdf

Scholarly Journal of Biological Science Vol.2(3),pp.30-38,March 2013 Available online http:/www.scholarly- ISSN 2315-6147 2013 Scholarly-Journals Full Length Research Paper Reduction of cholesterol by Lactococcus lactis KF147 Wesam A.Hassanein,Nadia M.Awny and Shimaa M.Ibraheim*Department of Botany,Faculty of Science,Zagazig University,Zagazig,44519,Egypt.Accepted 17 January,2013 Lactococcus lactis from yoghurt sample was confirmed based on alignment of 16S rRNA gene sequence in gene bank.The degree of acid and bile tolerance of bacteria was evaluated.L.lactis KF147 was able to maintain viability for 2 h at pH 2 and grew in a medium of 4,000 mg/L bile acids with maximum cholesterol reduction of 66.8%from the culture medium.The fatty acid composition of the cells of L.lactis KF147 was altered by reducing cholesterol from the media.Infrared spectroscopy(IR)analysis and transmission electron microscope were detected in the cholesterol binding activity between L.lactis KF147 and cholesterol.The resting and dead cells of L.lactis KF147 exhibited the cholesterol reducing activity of 32.9 and 29.7%,respectively.Key words:Binding,cholesterol,Lactococcus lactis KF147,reduction.INTRODUCTION Cholesterol is a kind of steroid,found particularly in animal cell membranes(Paniangvait et al.,1995).Cholesterol circulates in blood as a component of lipoproteins(Bansal et al.,2005).Majority of the cholesterol synthesized comes from the liver(Kim et al.,2003).Cholesterol in the intestine comes from diet,bile,and intestinal secretions(Levy et al.,2007).Cholesterol is required for the formation of sex hormones(Young,2001),cell membrane function,formation of vitamin D in the skin(Moser and Savage,2001)and bile acids(Afaf and Ali,2009),precursor for hormones and vitamins(Ikonen,2006),and in addition,plays a vital role in the development,function and stability of synapses(Chattopadhyay and Paila,2007),and pathogenesis of Human Immunodeficiency Virus(HIV)infection and disease progression(Mansfield et al.,2007).Cholesterol reduction by microorganisms takes place by different mechanical bioactivities which include the inhibition of the hydroxymethylglutaryl coenzyme A reductase(Homma,1988),co-precipitation(Klaver and Van der Meer,1993)or uptake and co-precipitation of cholesterol by bacteria(Tahri et al.,1996),production for cholesterol esterase(Young,2001)and precipitation of cholesterol(Saavedra et al.,2004).In addition,*Corresponding author.E-mail:Dr_.cholesterol binding to cell walls of probiotics(Liong and Shah,2005),co-precipitation of cholesterol with deconjugated bile(Liong and Shah,2006),production of short chain fatty acids upon fermentation by probiotics in the presence of prebiotics(De Preter et al.,2007),extracellular cholesterol oxidase enzyme(Mendes et al.,2007),enzymatic deconjugation of bile acids by bile-salt hydrolase of probiotics(Lambert et al.,2008),incorporation of cholesterol into the cellular membranes of probiotics during growth(Lye et al.,2010a)and conversion of cholesterol into coprostanol(Lye et al.,2010b).Some microorganisms assimilated cholesterol grows on cholesterol as a sole carbon source.The non-pathogenic and pathogenic mycobacteria are able to use cholesterol as a sole carbon source(Gay and Sobouti,2000).Kovalenko et al.(2004)observed that,the strains of lactic acid bacteria strain,N 139 belonging to Lactobacillus,Streptococcus,Enterococcus and Leuconostoc genera have used cholesterol as carbon source.Probiotics have been considered to have potential health-promoting benefits as biotherapeutic agents or a biological hypocholesterolemic agent(Park et al.,2007),and have cholesterol lowering effects in aqueous system as liquid media(Saavedra et al.,2004),blood of humans(Park et al.,2007)and rats(Nguyen et al.,2007).Pereira and Gibson(2002b)reported that Bifidobacterium infantis,Streptococcus bovis,Hassanein et al.31 Enterococcus durans,E.gallinarum,E.faecalis,E.faecium,Lactococcus fermentum,L.plantarum,L.reuteri,L.pentosus and L.acidophilus N5,had in vitro cholesterol reduction abilities.The aim of this research is to examine the ability of L.lactis KF147 for bile and acid tolerance and then screening for their cholesterol reduction in the liquid media.Fatty acid profile using IR analysis and transmission electron microscope of L.lactis KF147 with and without cholesterol was studied.MATERIALS AND METHODS Isolation and identification of cholesterol growing bacteria L.lactis was isolated from yoghurt samples on MRS media(De Man et al.,1960)under anaerobic conditions.The isolate was confirmed for the identification based on alignment of 16S rRNA gene sequence available in gene bank.Bile tolerance Strain L.lactis KF147 was evaluated for rapidity of growth in a MRS broth media with and without bile acids according to Pereira and Gibson(2002a),Overnight cultures were inoculated(1%v/v)into mMRS broth both containing 0.2 and 0.4%(w/v)oxgall and incubated anaerobically at 37C for 12 h.Cultures were monitored hourly for growth spectrophotometrically at 650 nm.Comparison of cultures was based on their growth rates in each broth.Acid tolerance Overnight cultures of L.lactis KF147 were inoculated(10%v/v)into MRS broth(Oxoid)previously adjusted to pH 2.0 with HCL according to Pereira and Gibson(2002a).The mixtures were incubated anaerobically at 37C for 2 h.One-milliliter samples were taken at various times(0,15,30,45,60,and 120 min)and serially,10-fold diluted in anaerobic diluent(half-strength peptone water plus 0.5 g of L-cysteine HCL/L,pH 7.0)which was thereafter,plated in triplicate onto MRS agar.The plates were incubated at 37C for 24 h under anaerobic conditions before enumeration.The rate of bile and acid tolerance was calculated according to the method described by Xiao et al.(2003):Tolerance rate=(CFU/ml)/(CFU/ml)control 100 Cholesterol reduction Quantitative determination of cholesterol was measured calorimetrically(Liebrman-Burchard reaction)(Tietz,1987)The residual cholesterol was calculated according to the equation of Agerbaek et al.(1995)as shown:(%cholesterol)/g(dry weight)=wBTB/100 Where B is cholesterol content in the uninoculated control(mg/L),T is cholesterol in the culture media(mg/L),and W is cells dry weight(g)after 12 h of incubation.To measure the cholesterol removed with the cells,pellet cells obtained by centrifugation was resuspended in distilled water to the original volume of the culture and cholesterol determined as earlier mentioned.Cholesterol-binding activity The cholesterol binding activity was estimated according to Pato et al.(2005).Sheep serum was added to MRS broth containing 2%sodium thioglycolate and 0.3%oxgall(MRSO).To obtain a concentration of cholesterol(100 g/ml),5 ml of MRS broth was inoculated with 100 ml of active culture.The cholesterol binding ability was estimated by using the formula:A=100-(B/C)100 Where:A represents the binding of cholesterol(%),B represents cholesterol(g)in the supernatant of the inoculated MRSO broth and C represents cholesterol(g)in the supernatant of MRSO broth without inoculation(control).Bile salt hydrolase activity The Bile Salt Hydrolase(BSH)activity assay was done according to the method as described by Lim et al.(2004).Role of cholesterol as a carbon source Effect of different carbon sources Different carbon sources were introduced separately into basal cholesterol liquid medium.Glucose,fructose,sucrose,maltose,lactose,galactose,sorbitol,mannitol,starch and dextrin were used.All these sources were added to the medium at a final concentration of 1%(w/v)(Kaur et al.,2001).Basal cholesterol medium without additional any carbon source was served as control.Bacterial growth This experiment was carried out to investigate the effect of cholesterol on the growth of L.lactis KF147 according to Liong and Shah(2005a).Scholarly J.Biol.Sci.32 Table 1.Effect of bile salt concentration on the viability of strain L.lactis KF147.Bile concentration(%)Bacterial counts expressed as Log10 cfu/ml incubation time(h)BTR 0 4 8 0.0 9.5 0.50abcde 10.2 0.20abc 10.5 0.40a 100 0.1 9.7 0.70abcd 9.5 0.50abcde 9.2 0.40cdef 87.61 0.3 9.4 0.20bcde 9.0 1.00def 8.7 0.70def 94.56 0.5 9.3 0.30bcde 8.2 0.20f 6.5 0.50g 74.7 Table 2.Effect of acidity on the viability of strain L.lactis KF147.pH value Bacterial counts expressed as Log10 cfu/ml incubation time(h)ATR 0 2 4 6 8 2.0 7.2 0.30gh 7.0 0.50h 6.9 0.60hi 65 0.40ij 6.3 0.20j 84.0 3.0 8.5 0.40d 8.4 0.10d 8.1 0.20de 7.8 0.50ef 7.5 0.40fg 70.09 6.8 9.4 0.20c 9.7 0.30c 10.2 0.10b 10.5 0.30ab 10.7 0.60a 100 Electron microscopy The bacteria with and without cholesterol were prepared for transmission electron microscope according to Lorian(1986)and Ooi and Liong(2010).Effect of cholesterol on cellular fatty acid composition Cellular lipids were extracted using a modified method of Liong and Shah(2005a).The fatty acids were estimated as methyl ester using the Hewlett-Packard of gas chromatograph(HP5890).IR spectroscopy analysis After washing,bacteria with and without cholesterol were prepared for IR analysis according to the method as described by Kleiner et al.(2002).The literature values of standard cholesterol were according to Zhou et al.(1997).Cell state The method used was modified from Marculescu et al.(2005).Cholesterol reduction by growing,resting,and dead cells of L.lactis KF147 was expressed in dry weight to obtain uniformity in all treatments.The following equation was used:Cholesterol assimilation=(C1 C2)/(W2 W1).Where C1 and C2 were the amount of cholesterol present in the fermentation broths at time=0 and 20 h,respectively,and W1 and W2 were the dry weight of the individual culture at time=0 and 24 h,respectively.Statistical analysis Recorded data were subjected to the statically analysis of variance according to Snedecor and Cochran(1980),and means separation was done according to Duncan(1955)at 5%level of significanceRESULTS AND DISCUSSION Hypercholesterolemia is considered as a major risk factor for the development of coronary heart disease and stroke(Aloglu and Oner,2006).The tested organism was confirmed for the identification based on alignment of 16S rRNA gene sequence available in gene bank.BLAST(Basic Local Alignment Search Tool)search indicated that,the tested L.lactis showed 99%identity to L.lactis subsp.lactis KF147 strain(accession number 013656.1).Bile and acid tolerance Table 1 represents the effect of bile salt concentrations on the viability of L.lactis KF147.The results indicated that,the bile tolerance rate at 0.1,0.3 and 0.5%bile concentrations were 87.61,94.56 and 74.7%,respectively,as compared to the control sample(without bile salt).L.lactis KF147 under investigation had the potential to survive under bile and acid environment up to 0.4%bile and pH 2 for 12 and 2 h,respectively.This is in line with the study of Kumer et al.(2010).They recorded that different strains of Lactobacillus plantarum tolerated 2%bile up to 7623 for 3 h.In addition,Table 2 indicates that the acid tolerance rate of L.lactis KF147 at pH 2.0 and 3.0 were 84.0 and 70.0%respectively as compared to the control(pH 6.8).L.lactis KF147 could tolerate bile and acidic conditions Hassanein et al.33 0123456010203040506070Dry weight(g/100 ml)%cholesterol reductionCarbon Source(1%w/v)Lc.lactis KF 147%cholesterol reductionDry weight(g/100 ml)Carbon source(1%W/v)%Cholesterol reduction Dry weight(g/100 ml)Figure 1.Effect of different carbon sources on both biomass and cholesterol reduction by L.lactis KF147.indicated the existence in stomach,intestinal juice and fermented food products.In this relation,Morelli(2007)reported that in the laboratory,acid and bile are added to the media in order to mimic conditions encountered in human gastrointestinal tract.Lactobacillus paraplantarum II 32 showed acid resistance and bile salt tolerance.The results indicated that strain II 32 survived at pH 2.0 after 2 h culture and the living bacterial number could reach 104 CFU/ml.In addition,the growth of strain II 32 was delayed less than 0.5 h in MRS broth with 0.3 to 0.4%bile salt when the absorbance values both increased to 0.6 units(Liu et al.,2009).The time from entrance to release from the stomach has been estimated to be approximately 90 min,with further digestive processes requiring longer residence time.Stresses to organisms begin in the stomach with pH between 1.5 and 3.0,and in the upper intestine that contains bile(Corzo and Gilliland,1999).Survival at pH 3.0 for 2 h and at a bile concentration of 1000 mg/L is considered an optimal acid and bile tolerance for probiotic strains(Usman,1999).Cholesterol reduction Strain L.lactis KF147 screened for the reduction of cholesterol was added in the liquid media and residual and reduced cholesterol as well as percentage of cholesterol reduction was determined.The results indicated that the cholesterol reduction percent was 57.7%(Data not shown)Cholesterol-binding activity The result represents the capability of binding cholesterol by L.lactis KF147 to removing 13%cholesterol from broth(Data not shown).In this relation,Pato et al.(2005)showed that L.lactis subsp.lactis B-4 exhibited the highest cholesterol-binding ability by removing 15%cholesterol from the media broth,while L.lactis subsp.lactis I-2775 showed the lowest activity(5%).Bile salt hydrolase activity The result showed there is no precipitation of the bile salt along with the formation of opague granular white colonies with a silvery shine around them on the medium platesL.lactis KF147 has no bile salt hydrolase activity(Data not shown).This result is in line with the study of Ramasamy et al.(2010)for strain Lactobacillus reuteri.ROLE OF CHOLESTEROL AS A CARBON SOURCE Effect of different carbon sources Figure 1 show that the highest percentage of cholesterol reduction 64.3%was obtained when 1%(w/v)glucose was added as sole carbon source to the broth media of L.lactis KF147,and 61.0%for cholesterol as sole carbon source(control).In this connection,Liong and Shah(2005c)found that FOS was a good specific carbon source in modulating the growth rate of Lactobacillus casei ASCC 292.Kovalenko et al.(2004)reported that the genera Lactobacillus,Streptococcus,Enterococcus and Leuconostoc have been used by cholesterol as a source of carbon.Bacterial growth The culture of L.lactis KF147 on MRS with and without cholesterol for different times is represented in Figure 2.The result indicates that the highest O.D.at 620 nm achieved at 24 h was 1.67 in the presence of cholesterol.Also,the highest O.D.at 620 nm was 1.58 in absence of cholesterol.The growth of strain L.lactis KF147 with cholesterol was higher than the growth without cholesterol.In this relation,(Kimoto et al.2002)found that in the presence of cholesterol,Lactococci N7 reached a higher cell density than in its absence.The dry weight of the cells growing in the absence and presence of cholesterol was 219 and 544 mg/L,respectively.In Scholarly J.Biol.Sci.34 0204060024681012141618202224Time(Hour)%