lable at ScienceDirect

LWT - Food Science and Technology xxx (2013) 1e5

Contents lists avai

LWT - Food Science and Technology

journal homepage: www.elsevier .com/locate/ lwt

Antibacterial activity and cholesterol assimilation of lactic acidbacteria isolated from traditional Iranian dairy products

Mahdieh Iranmanesh a, Hamid Ezzatpanah a,*, Naheed Mojgani b

aDepartment of Food Science and Technology, Science and Research Branch, Islamic Azad University, P. O. Box 14515-775, Tehran, IranbBiotechnology Department, Razi Vaccine and Serum Research Institute, Karaj, Iran

a r t i c l e i n f o

Article history:Received 18 June 2012Received in revised form29 September 2013Accepted 2 October 2013

Keywords:Bacteriocin like inhibitory substances (BLIS)Cholesterol reductionEwe milkLactic acid bacteriaSour buttermilk

* Corresponding author. Tel.: þ98 21 44865137; faxE-mail addresses: hamidezzatpanah@srbiau.ac.ir, h

(H. Ezzatpanah).

0023-6438/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.lwt.2013.10.005

Please cite this article in press as: Iranmanetraditional Iranian dairy products, LWT - Foo

a b s t r a c t

In this study, lactic acid bacteria were isolated from ewe milk, traditional yoghurt and sour buttermilksamples collected from different areas of Azarbayjan-e-sharqi in Iran. All the isolates were screened fortheir ability to produce bacteriocin like inhibitory substances (BLIS) by studying their inhibitory actionagainst pathogens like Listeria monocytogenes, Salmonella enteritidis and Staphylococcus aureus, aftereliminating the effect of organic acids and hydrogen peroxide. According to results, four of the isolatesidentified as Lactobacillus brevis, Lactobacillus pentosus, Pedoicoccus acidilactici and Lactobacillus paracaseiwere unaffected by the action of pH neutralization and hydrogen peroxide and showed inhibitory actionagainst the tested pathogens. The inhibitory activities demonstrated by these isolates were completelyinhibited in the presence of proteolytic enzymes.

The isolates in study were further characterized for their cholesterol reduction ability. Cholesterolassimilation by both viable and dead cells of these strains was determined in MRS broth containing 0.3 g/100 mL bile salt. According to results, highest level of cholesterol removal was recorded in L. brevis, whileall the other isolates in study were also able to reduce cholesterol to lesser extent. To conclude, the LacticAcid Bacteria isolated from these traditional products might be exploited for their probiotic potential forfuture studies.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Elie Metchinkoff, the Russian physician who proposed theconcept of probiotic, propounded that the longevity of the Bul-garians was in part due to consumption of large quantities of fer-mentedmilk containing lactobacilli (Metchnikoff, 1908). Lactobacilliand Bifidobacter are two kinds of Lactic Acid Bacteria (LAB), whichare found in the gut and are considered as probiotic, because oftheir beneficial effects on health (Mitsuoka, 1998). Later, Fuller(1989) defined probiotics as live, non-pathogenic bacteria thatcontribute to the health and balance of the intestinal tract.

Fermented foods are known throughout the world, and theGram positive LAB have beenwell known for their important role infood industry (Karimi Torshizi, Rahimi, Mojgani, Esmaelkhanian, &Grimes, 2008). These bacteria produced antimicrobial agents suchas acids, hydrogen peroxide and bacteriocins and have great po-tential as food bio-preservatives (Aslim, Yukesekdag, Sarikaya, &Beyatli, 2005; Avonts, Uytven, & Vuyst, 2004). Bacteriocins are

: þ98 21 44865464.amidezzatpanah@gmail.com

All rights reserved.

sh, M., et al., Antibacterial actd Science and Technology (20

ribosomal synthesized proteinaceous substances produced by LAB,containing relatively narrow spectrum of bactericidal activity(Cleveland, Montville, Nes, & Chikindas, 2001).

In recent years, consumption of fermented foods has increaseddue to the reported beneficial health effects of LAB includinglowering of serum cholesterol level. Various studies in vivo haveshown that some Lactobacilli can lower cholesterol (Anderson &Gilliland, 1999). Numerous health benefits of LAB have madethem promising probiotic candidates and being extensively studiedto explore their safety and other desirable properties. The mainobjective of the present study was to investigate and characterizeLactic Acid Bacteria in traditional sour buttermilk made from ewe’smilk, which might provide important information regarding itsprobiotic potential and its utilization in future.

2. Materials and methods

2.1. Bacterial strains and culture conditions

All LAB strains used in this study were grown in MRS broth (deMan, Rogosa Sharpe, HiMedia, Mumbai, India) at 37 �C for 24e48 hin anaerobic jars, pathogenic strains used in this study were grown

ivity and cholesterol assimilation of lactic acid bacteria isolated from13), http://dx.doi.org/10.1016/j.lwt.2013.10.005

M. Iranmanesh et al. / LWT - Food Science and Technology xxx (2013) 1e52

in BHI (Braineheart infusion broth, HiMedia, Mumbai, India) at37 �C for 18e24 h under aerobic condition.

All LAB strains weremaintained at 4 �C and renewed everyweekfor short-term preservation. The long-term conservation of thepurified isolates was carried out in MRS broth with sterile glycerol(15 mL/100 mL) and stored at �70 �C (Badis, Guetarni, Boudjema,Henni, & Kihal, 2004).

2.2. Collection of ewe milk and preparation of buttermilk

A total of 20 samples from ewe milk were analyzed, of which 10samples were collected from Myaneh (15 herds) and 10 samplesobtained from Hashrood (15 herds) two cities in Azarbayjan-e-sharqi (north-west of Iran). In both places, samples were taken at10 different days and collected from bulk milk tank. All sampleswere collected according to EN ISO 707:2001 in sterile bottles of250 mL and transported to the laboratory under refrigeration (4 �C)within 36 h (Anon, 2002).

The traditionally made yoghurt (n: 20) and sour buttermilk (n:20) samples which made from ewe milk, were also collected fromthe same area. Fig.1 shows the preparation operation of yoghurtand sour buttermilk. As indicated, the samples are prepared in atraditional device known as Mashk, which is made from hide(sheepskin) and is used for making butter and buttermilk fromyoghurt. This device is also used widely for preservation of fer-mented dairy products for 10e20 days at temperatures notexceeding 20 �C.

1. Ewe milk (n: 20)

Boiling ewe milk

Cooling to inoculation temperature (42 ˚C)

Addition of 2-3 g traditional

yoghurt 100g-1 ewe milk

Inoculated with traditional yoghurt made earlier as starter culture

(For 3-4 h)

2. Yoghurt (n: 20)

Adding water Sour yoghurt

(Rate of 60:40)

Butter making (using Mashk) *

3. Buttermilk (n: 20) Butter grains

Fig. 1. Preparation of samples including ewe milk, yoghurt and traditional sourbuttermilk. *(Mashk is a traditional device used in this study, which is made from hideand is used for making butter and buttermilk from yoghurt or keeping fermented dairyproducts).

Please cite this article in press as: Iranmanesh, M., et al., Antibacterial acttraditional Iranian dairy products, LWT - Food Science and Technology (20

2.3. Isolation and identification of LAB

One ml of each sample (ewe milk, yoghurt and sour buttermilk)was inoculated in MRS broth under above mentioned conditions(part 2.1) until growthwas observed. The grown samples were thenplated on MRS agar and incubated at 37 �C until appearance ofcolonies. Pure colonies were selected and tested for Gram staining,cell morphology and catalase test (Karimi Torshizi et al., 2008).

2.4. Identification of isolated LAB to species level

Only strains showing maximum inhibitory activity againstothers (4 selected lactic acid bacteria) were selected for identifi-cation to species level. The carbohydrate fermentation profiles ofthe selected strains were investigated using API 50 CHL medium(Bio-Meriux, Marcy I’Etoile, France) according to the manufacture’sinstruction (Iranmanesh et al., 2012).

2.5. Antimicrobial activity of supernatant against pathogens

The antimicrobial effects of selected LAB against Gram positiveand Gram negative pathogens were examined by agar well diffu-sion methods. The pathogens namely Staphylococcus aureus (PTCC1112), Listeria monocytogenes (PTCC 1298), and Salmonella enter-itidis (local isolate) were used as indicator culture in the study. Theculture broths of the producer and indicator strains were adjustedto McFarland Index 3 prior to use. The antimicrobial activity wasrecorded as appearance of clear zone around the wells (Aslim et al.,2005).

2.6. Bacteriocin production

Cell free supernatant of the selected LAB strains was obtained bycentrifuging the culture broth at 10,000 g for 10 min at 4 �C,adjusted to pH 6.5 with 1 mol equi/L NaOH followed by filtrationthrough a 0.22 mm pore size filter. To eliminate the effect ofhydrogen peroxide, catalase was added at a final concentration of1 mg/mL to the supernatant broth and the remaining activitydetermined as described earlier (Ghrairi, Manai, Berjeaud, & Frere,2004).

2.7. Sensitivity of bacteriocin-like substance to enzyme

The supernatant of selected strains was treated with thefollowing enzymes (SigmaeAldrich, St. Louis, Mo, USA) at a finalconcentration of 1 mg/100 mL: lysozyme, pronase, trypsin andproteinase K. The treated samples were incubated at 37 �C for 2 hand the remaining activity determined against the listed indicatorstrains (Bromberg, Moreno, Lopez Zaganini, Regina Delboni, & Deoliveria, 2004).

2.8. Cholesterol removal by viable and dead cell

Water-soluble cholesterol was filter-sterilized and added at afinal concentration of 70 mL/100 mL to MRS broth containing 0.3 g/100 mL oxgall (SigmaeAldrich, St. Louis, Mo, USA). The tube wasinoculated with each selected strains (at 1 mL/100 mL) and incu-bated at 37 �C for 2, 4, 9 and 24 h. After incubation, the cells werecentrifuged at 10,000 g for 10 min.

For preparation of heat-killed cells, the cell pellet of 5 selectedstrains with the highest effect of removing cholesterol was washedtwicewith 10mL of sterile distilledwater and autoclaved for 15minat 121 �C, then diluted with ringer solution and counted by mi-croscope. The heat-killed cells were suspended in MRS broth con-taining 0.3 g/100 mL oxgall and water-soluble cholesterol

ivity and cholesterol assimilation of lactic acid bacteria isolated from13), http://dx.doi.org/10.1016/j.lwt.2013.10.005

Table 2Antagonistic activity cell free supernatant of selected strains against pathogens byagar well diffusion assay.

Strains Pathogens

Listeriamonocytogenes

Staphylococcusaureus

Salmonellaenteritidis

L.b pentosus þ þ þþþL.b brevis þþ þ þþþL.c lactis 1 þþþ þþþ þþþL.c lactis 2 þ þþþ þþþL.b paracasei 1 þ þþþ þþþL.b paracasei 2 þþþ þþþ þþþPed. acidilactici 1 þþþ þ þþþPed. acidilactici 2 þ þþ þþþPed. acidilactici 3 þ þ þþPed. acidilactici 4 þþþ þ þþ

þ: 3 mm < zone.þþ : 3 mm < zone <5 mm.þþþ : 5 mm < zone <7 mm.

Table 3Inhibitory activity of neutralized cell free supernatant of selected lactic acid bacteriaagainst pathogens (agar well diffusion assay).

Strains Pathogens

Strains produced acid against pathogens

M. Iranmanesh et al. / LWT - Food Science and Technology xxx (2013) 1e5 3

concentration. The tubes were incubated for 24 h at 37 �C and thencentrifuged like growing cells (Raghavan et al., 2011).

The residual cholesterol of supernatants for both viable anddead cells was determined by an enzymatic method (Pereira &Gibson, 2002). The cholesterol lowering ability was determinedby measuring the absorbance at 546 nm by using spectrophotom-eter. The ability of selected strains to remove cholesterol frommedia was calculated as percentage from the following equation:

A ¼ ðB� C=BÞ � 100

where A ¼ % removed cholesterol, B ¼ absorbance using blank,C ¼ absorbance using cell supernatant.

2.9. Statistical analysis

Data analysis was carried out with SAS statistical software andthe Duncan’s test was used to detect differences (p< 0.05). All testswere run in triplicate.

3. Results

3.1. Isolation and identification of LAB

Seventy-seven out of 168 isolates from ewe milk, yoghurt andsour buttermilk were identified to LAB based on their Gram reac-tion, morphology and catalase test (data not shown). All strainswere recorded as catalase negative and Gram-positive cocci in pairsor long chains, bacilli in pairs or chains and cocobacilli. Of all theisolates, only ten selected strains showing maximum antibacterialpotential against selected lactic acid bacteria were identified tospecies level as Lactobacillus pentosus, Lactobacillus paracasei (twostrains), Lactobacillus brevis, Pediococcus acidilactici (four strains)and Lactococcus lactis (two strains) (Table 1).

3.2. Antagonistic activity against pathogens

Table 2 shows the results of antagonistic activity of selected 10LAB isolates against three different pathogens. The tested isolatesshowed different level of inhibitory action against the pathogens.However, S. enteritidis appeared to be themost sensitive strain as allthe LAB isolates tested were able to inhibit the growth of thementioned isolate in study.

3.3. Characterization of the inhibitory agent

The activity of the inhibitory agent was tested under conditionswhich eliminates the possible effect of organic acids by adjustingthe pH of the cells free supernatant to pH 6.5. As seen in Table 3 theinhibitory activity demonstrated by L. lactis 1 and P. acidilactici 1, 2and 3 against all pathogens might have been due to acid as theiractivity was completely lost after pH neutralization of their su-pernatant fluid.

Table 1Identification of the selected LAB isolates to species level using standardAPI 50CH identification kit.

Strains Samples

Lactobacillus pentosus MilkLactobacillus brevis MilkLactobacillus paracasei (No.1,2) MilkLactococcus lactis (No.1,2) MilkPediococcus acidilactic (No.1,2) MilkPediococcus acidilactic (No.3,4) Buttermilk

Please cite this article in press as: Iranmanesh, M., et al., Antibacterial acttraditional Iranian dairy products, LWT - Food Science and Technology (20

Effect of enzyme catalase on the inhibitory activity of the iso-lates in study indicated that the antimicrobial activity of L.lactis 2,L.paracasei1 and 2 against S. enteritidis might have been due tohydrogen peroxide as their activities were completely eliminatedafter addition of the mentioned enzyme (Table 4). However, theremaining isolates remained unaffected by this enzyme, whichexcluded inhibition by hydrogen peroxide.

Protease sensitivity assay demonstrated that the antimicrobialsubstances produced by L.pentosus, L.lactis 2, L.paracasei 2 against L.monocytogenes and S. aureus might be due to BLIS, as their anti-microbial activities were completely eliminated after treatmentwith proteolytic enzymes. In addition, Pedicoccus acidilactici 4against all listed pathogens and L. brevis against S. aureus and S.enteritidis could produce BLIS.

3.4. Cholesterol removal by viable and dead cells

The percentage of cholesterol assimilation during different timeperiods revealed wide variations in the mentioned property of thetested isolates as variable levels of cholesterol reduction wereobserved in these isolates. Additionally, different isolates of samespecies also revealed different levels of cholesterol lowering effects.

As depicted in Fig. 2, L. brevis assimilated highest level ofcholesterol in viable cell during 2, 4, and 24 h, while, P.acidilactici 2exhibited the highest cholesterol reduction activity after 9 h.

The degree of cholesterol removal by the heat killed cells of theisolates was also determined. Compared to viable cells cholesterol

Listeriamonocytogenes

Staphylococcusaureus

Salmonellaenteritidis

L.b pentosus þ þ e

L.b brevis e þ þL.c lactis 1 e e e

L.c lactis 2 þ þ þL.b paracasei 1 e e þL.b paracasei 2 þ þ þPed. acidilactici 1 ,2,3 e e e

Ped. acidilactici 4 þ þ þþ: antimicrobial activity was remained.e: antimicrobial activity was inactive and it might be due to acid.

ivity and cholesterol assimilation of lactic acid bacteria isolated from13), http://dx.doi.org/10.1016/j.lwt.2013.10.005

Table 4Inhibitory activity of neutralized cell free supernatant of selected lactic acid bacteriaagainst pathogens after catalase treatment (agar well diffusion assay).

Strains Pathogens

Strains produced hydrogen peroxide against pathogens

Listeriamonocytogenes

Staphylococcusaureus

Salmonellaenteritidis

L.b pentosus þ þL.b brevis þ þL.c lactis 2 þ þ e

L.b paracasei 1 e

L.b paracasei 2 þ þ e

Ped. acidilactici 4 þ þ þþ: antimicrobial activity was remained.e: antimicrobial activity was inactive and it might be due to hydrogen peroxide.Blank: antimicrobial activity was inactive after neutralization.

M. Iranmanesh et al. / LWT - Food Science and Technology xxx (2013) 1e54

reduction activity, the ability of the heat killed cells to reducecholesterol although present was significantly lower (Table 5).

4. Discussion

Bacteriocin producing species have now been reported amongall the genera that comprise the LAB including Lactococcus,

Fig. 2. Cholesterol removal by viable cells of selected lactic acid bacteria in MRS media conafter 2 h incubation. B: Cholesterol lowering after 4 h incubation. C: Cholesterol lowering a

Please cite this article in press as: Iranmanesh, M., et al., Antibacterial acttraditional Iranian dairy products, LWT - Food Science and Technology (20

Streptococcus, Lactobacillus, Leuconstoc and Pediococcus as well asEnterococcus (Jack, Tagg, & Ray, 1995). The growth of S. aureus infoods shows a potential public health risk since this strain produce,enterotoxins that cause food poisoning (Bromberg et al., 2004). Inaddition, food born transmission of L. monocytogenes has beenimplicated of listeriosis in human involving the consumption ofvarious foods (Fleming et al., 1985). In this study all selected LABisolates were not only able to inhibit the growth of Gram-positivebacteria like S. aureus and L. monocytogenes but were also activeagainst Gram-negative bacteria like S. enteritidis.

A number of metabolites such as acid, H2O2 and bacteriocin arebelieved to contribute to antimicrobial activity. Similar to thesereports, the antibacterial activity demonstrated by the LAB isolatesin study and characterized, appeared influenced by catalase andother proteolytic enzymes. The sensitivity of the inhibitory agent tolysozyme, pronase, trypsin and proteinase K indicates of theirproteinaceous nature and thus might be considered as bacteriocin.

There are different possible mechanisms underlying the abilityof LAB to remove cholesterol from media including assimilation(Gilliland, Nelson, & Maxwell, 1985), enzymatic hydrolysis of con-jugated bile salts (Corzo & Gilliland, 1999) and co-precipitation ofcholesterol with deconjugated bile salt (Klaver & Van der Meer,1993). Our results were in agreement with the result of another

taining 0.3 g/100 mL oxgall. Results represent mean � SD, n:3. A: cholesterol loweringfter 9 h incubation. D: Cholesterol lowering after 24 h incubation.

ivity and cholesterol assimilation of lactic acid bacteria isolated from13), http://dx.doi.org/10.1016/j.lwt.2013.10.005

Table 5Cholesterol removal by dead cell in MRS media containing 0.3 g/100 g oxgall after24 h incubation.

Strain No. of cells

% of lowering cholesterol

7 cfu/mL 8 cfu/mL 9 cfu/mL

L.b pentosus 28 � 0.38 44 � 0.29 76 � 0.45L.b brevis 48 � 0.20 59 � 0.35 83 � 0.61L.c lactis 1 60 � 0.33 65 � 0.42 78 � 0.35L.b paracasei 2 36 � 0.66 51 � 0.53 72 � 0.84Ped. acidilactici 4 44 � 0.34 62 � 0.45 69 � 0.66

Data are presented as mean � SD, (n ¼ 3).

M. Iranmanesh et al. / LWT - Food Science and Technology xxx (2013) 1e5 5

study conducted in Iran, which showed significant decrease inserum cholesterol levels by consumption of probiotic yoghurt incomparison with ordinary yoghurt (Ataie-Jafari, Larijani, AlaviMajd, & Tahbaz, 2009). In totally, the highest average of choles-terol removal by viable cells of the selected isolates was achievedwithin 9 h. Additionally, the heat killed cells of all the isolates werealso able to lower cholesterol from the media, which could beattributed to some extent of cholesterol bound to cells. This notionis supported by the observation that cholesterol removal capabilityof dead cells increased with an increase in the number of cells.

5. Conclusion

Due to the varied climate in Iran, there is a wide variety oftraditional dairy products especially in rural areas. Most of theseproducts are made from unpasteurized ewe’s or cow’s milk. Thisstudy proved the presence of LAB micro flora in ewe milk, yoghurtas well as traditional sour buttermilk. The antagonistic activitypossessed by these isolates might be an additional advantage forthe control of unwanted pathogens mainly in dairy products.Moreover, the in vitro cholesterol lowering effect of the selectedisolates in both viable and dead form might indicate their possiblecholesterol lowering effect in vivo.

Overall results of this research suggests that the selected LABstrains isolated appear to possess probiotic potential, and hencecould be exploited further for their use in fermented dairyproducts.

References

Anderson, J. W., & Gilliland, S. E. (1999). Effect of fermented milk (yoghurt) con-taining Lactobacillus acidophilus L1 on serum cholesterol in hypercholesterol-emic humans. Journal of the American College of Nutrition, 18, 43e50.

Please cite this article in press as: Iranmanesh, M., et al., Antibacterial acttraditional Iranian dairy products, LWT - Food Science and Technology (20

Anon, (2002). Official methods of analysis of the Association of Official AnalyticalChemists.

Aslim, B., Yukesekdag, Z. N., Sarikaya, E., & Beyatli, Y. (2005). Determination ofthe bacteriocin-like substances produced by some lactic acid bacteria iso-lated from Turkish dairy products. LWTeFood Science and Technology, 38,691e694.

Ataie-Jafari, A., Larijani, B., Alavi Majd, H., & Tahbaz, F. (2009). Cholesterol-loweringeffect of probiotic yogurt in comparison with ordinary yogurt in mildly tomoderately hypercholesterolemic subjects. Annals of Nutrition and Metabolism,54(1), 22e27.

Avonts, L., Uytven, E. V., & Vuyst, L. D. (2004). Cell growth and bacteriocin pro-duction of probiotic lactobacillus strains in different media. International DairyJournal, 14, 947e955.

Badis, A., Guetarni, D., Boudjema, B. M., Henni, D. E., & Kihal, M. (2004). Identifi-cation and technological properties of lactic acid bacteria isolated from raw goatmilk of four Algerian races. Food Microbiology, 21, 579e588.

Bromberg, R., Moreno, I., Lopez Zaganini, C., Regina Delboni, R., & De oliveria, J.(2004). Isolation of bacteriocin-producing Lactic Acid Bacteria from meat andmeat products and spectrum of inhibitory activity. Brazilian Journal of Micro-biology, 35, 137e144.

Cleveland, J., Montville, T. J., Nes, I. F., & Chikindas, M. L. (2001). Bacteriocins safe,natural antimicrobials for food preservation. International Journal of FoodMicrobiology, 71, 1e20.

Corzo, G., & Gilliland, S. E. (1999). Bile salt hydrolase activity of three strains oflactobacillus acidophilus. Journal Dairy Science, 82, 427e480.

Fleming, D. W., Cochi, S. L., MacDonald, K. L., Brondum, J., Hayes, P. S., Plikaytis, B. D.,et al. (1985). Pasteurized milk as a vehicle of infection in an outbreak of liste-riosis. New England Journal of Medicine, 312, 404e407.

Fuller, R. (1989). A review: probiotics in man and animals. Journal of AppliedBacteriology, 66, 365e378.

Ghrairi, T., Manai, M., Berjeaud, J. M., & Frere, J. (2004). Antilisterial activity of lacticacid bacteria isolated from rigouta, atraditional Tunisian cheese. Journal ofApplied Microbiology, 97, 621e628.

Gilliland, S. E., Nelson, C. R., & Maxwell, C. (1985). Assimilation of cholesterol bylactobacillus acidophilus. Applied and Environmental Microbiology, 49, 377e381.

Iranmanesh, M., Ezzatpanah, H., Mojgani, N., Karimi Torshizi, M. A., Aminafshar, M.,& Mohamad Maohamadi, M. (2012). Isolation of lactic acid bacteria from ewemilk, traditional yoghurt and sour buttermilk in Iran. European Journal of FoodResearch & Review, 2(3), 79e92.

Jack, R. W., Tagg, j.r., & Ray, B. (1995). Bacteriocin of gram-positive bacteria.Microbiology Review, 59, 171e200.

Karimi Torshizi, M. A., Rahimi, S. H., Mojgani, N., Esmaelkhanian, S., & Grimes, J. L.(2008). Screening of indigenous strains of lactic acid bacteria for developmentof a probiotic for poultry. Asian-Australian Journal of Animal Sciences, 21(No. 10),1459e1500.

Klaver, F. A. M., & Van der Meer, R. (1993). The assumed estimation of choles-terol removal by Lactobacilli and Bifidobacterium bifidum is due to their bilesalt deconjugation activity. Applied and Environmental Microbiology, 59, 1120e1124.

Metchnikoff, E. (1908). The prolongation of life. New York: Putnam.Mitsuoka, T. (1998). In B. J. B. Wood (Ed.), The lactic acid bacteria in health and

disease (Vol. 1; pp. 69e114). London: Elsevier Applied Science.Pereira, D., & Gibson, G. R. (2002). Cholesterol assimilation by lactic acid bacteria

and Bifidobacteria isolated from the human gut. Applied and EnvironmentalMicrobiology, 4689e4693.

Raghavan, C. M., Nanda, A., Yuvaraj, R., Mukesh kumar, D. J., Senthil Murugan, A., &Balaji Raja, R. (2011). Assimilation of cholesterol by Lactobacillus species asprobiotic. World Applied Science Journal, 14(4), 552e560.

ivity and cholesterol assimilation of lactic acid bacteria isolated from13), http://dx.doi.org/10.1016/j.lwt.2013.10.005