Functional Characteristics of Lactobacillus fermentum F1

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1 Curr Microbiol (211) 62:27 31 DOI 1.17/s Functional Characteristics of Lactobacillus fermentum F1 Xiao Qun Zeng Dao Dong Pan Pei Dong Zhou Received: 7 April 21 / Accepted: 3 May 21 / Published online: 19 May 21 Ó Springer Science+Business Media, LLC 21 Abstract In this study, Lactobacillus fermentum (L. fermentum) F1 reduced cholesterol 48.87%. The strain was screened from cattle feces using an API 5 CHL system and the 16S rrna sequence contrasting method. L. fermentum F1 showed acid and bile tolerance, and antimicrobial activity against pathogenic Escherichia coli and Staphylococcus aureus. L. fermentum F1 deconjugated.186 mm of free cholalic acid after it was incubated at 37 C in.2% sodium taurocholate (TCA) broth for 24 h. Heat-killed and resting cells of L. fermentum F1 showed small amounts of cholesterol removal (6.85 and mg/g, respectively, of dry weight) compared with growing cells (33.21 mg/g of dry weight). The supernatant fluid of the broth contained 5.85% of the total cholesterol, while the washing buffer and cell extracts had and 35.39%, respectively. These findings suggest that L. fermentum F1 may remove cholesterol by co-precipitating with deconjugated bile salt, assimilating with cells and by incorporation into cellular membranes. Cholesterol assimilated by cells held 72.% of the reduced cholesterol, while 21.65% of the reduced cholesterol was coprecipitated with deconjugated bile salt and 5.89% was adsorbed into the surface of the cells. X. Q. Zeng D. D. Pan (&) Life Science and Biotechnology College, Ningbo University, Ningbo , People s Republic of China daodongpan@163.com X. Q. Zeng D. D. Pan P. D. Zhou Food Science and Nutrition Department, Nanjing Normal University, Nanjing, Jiangsu, People s Republic of China Introduction Cholesterol plays a major role in human heart health. Cholesterol is needed to insulate nerves, make cell membranes, and produce certain hormones. However, the body makes enough cholesterol for its needs, so dietary cholesterol intake is not required for normal adult metabolism [1]. High serum cholesterol is a leading risk factor for human cardiovascular disease, such as coronary heart disease and stroke [2]. Some research suggests that the consumption of fermented milk products containing certain lactobacilli or bifidobacteria may decrease the concentration of the cholesterol in the human blood stream [3, 4]. Lactobacilli are frequently used in products for human consumption and can be found as probiotics in infant food, cultured milk, and various pharmaceuticals [5]. In vitro and in vivo studies have reported that Lactobacillus strains, such as Lactobacillus acidophilus ATCC43121 [6], Lactobacillus acidophilus RP42 [7], and Lactobacillus casei MUH117 [8] may have a cholesterol-reducing effect. However, so far there has been little study of probiotics and even less research into Lactobacillus fermentum as a probiotic used to lower cholesterol. Different hypotheses have been advanced to explain the hypocholesterolemic effect of lactic acid bacteria (LAB) and bifidobacteria. Gilliland et al. [6] postulated that the effect results from a direct assimilation of cholesterol by L. acidophilus. Klaver and van der Meer [8] claimed that cholesterol removed from a broth medium was coprecipitated with deconjugated bile salts at ph values lower than 5.5. Noh et al. [9] suggested that LAB incorporated some of the cholesterol removed from the medium into its cellular membranes during growth. However, Noh et al. did not discuss the combination of possible mechanisms causing the hypocholesterolemic effect and did not

2 28 X. Q. Zeng et al.: Functional Characteristics of L. fermentum F1 measure the amount of reduced cholesterol caused by any one factor. Therefore, it is unclear which is the most important factor causing the LAB strain s hypocholesterolemic effect and the mechanism by which L. fermentum removes cholesterol remains unknown. In order to further study probiotics in relation to cholesterol removal, a lactobacillus strain isolated from cattle feces was evaluated for its potential probiotic properties, including cholesterol reduction, acid and bile tolerance, antimicrobial activity, and antibiotic sensitivity. The mechanisms by which the strain removed cholesterol were also analyzed and discussed. Materials and Methods Source and Maintenance of Culture Fresh cattle feces samples were collected from Weigang Cattle Farm, Nanjing, Jiangsu, China. LAB was cultured at 37 C in MRS (de Mann, Rogosa, Sharpe) liquid broth. Indicator strains of Escherichia coli ATCC and Staphylococcus aureus ATCC were obtained from the Microbiological Laboratory of the School of Life Science at Nanjing Normal University (Nanjing, China) and were cultured in Luria Bertani (LB) medium at 37 C. Cholesterol broth was MRS supplemented with.2% (w/v) sodium thioglycolate (Sigma, St. Louis, USA),.2% (w/v) TCA (Sigma), and a final concentration of.1 g/l watersoluble cholesterol (Sigma). To perform a curd test, a skimmilk substrate was made from 12.% (w/v) skim milk (Guangming Milk Firm, Shanghai, China). Solid culture media contained 1.5% (w/v) agar in LB or MRS liquid broth. Isolation and Identification of a Strain with Cholesterol- Reducing Effect Fresh cattle feces were diluted and spread onto MRS agar plates for preliminary screening. Preliminary identification of LAB, including colonial morphology, gram staining, microscopic examination, and a curd test, were made for each colony. Each LAB was inoculated in cholesterol broth under microaerobic conditions at 37 C for 24 h. Cells were then centrifuged (Bio-rad centrifuge C16, California, USA) at 12,g and 4 C for 1 min. The cholesterol concentration in the broth was determined by using a modified o-phthalaldehyde colorimetric method as described by Rudel and Morris [1]. Absorbance was read at 55 nm (722 grating spectrophotometer, Shanghai, China) after 1 min. The removal rate of each strain was computed using the formula: the cholesterol-reducing rate = [(A - A)/A ] 9 1% [11], where A is the absorbance of the unfermented broth (55 nm), A the absorbance of the broth fermented for 24 h (55 nm). The strain with the largest cholesterol-reducing rate was selected for further characterization. The strain was identified using gram staining, microscopic examination, and an API 5 CH kit (Biomerieux S.A., La Balme les Grottes, France). The 16S rrna gene was amplified and the sequence submitted to the NCBI Genbank and contrasted with the BLAST sequence database to identify the strain s species. Acid and Bile Tolerance, Antimicrobial Activity, and Drug Sensitivity Acid tolerances were evaluated by growing the F1 strain in MRS broth adjusted to acidic phs of 1., 2., 3., and 6.4 (control) by adding concentrated hydrochloric acid. A cell suspension of F1 strain cultures containing about 1 8 cfu/ml cells were inoculated in each broth and were taken at, 1, 2, 3, and 4 h and then spread on MRS agar plates. MRS broth supplemented with.2 and.3% (w/v) bile salt oxgall were used to mimic bile. MRS broth without bile salt was used as a control. The time required to increase absorbance by.3 units at 62 nm in MRS broth with and without oxgall was measured. The difference in time (h) between the two culture media was considered the lag time by which the bile salt tolerance effect was expressed [12]. The LB agar plates were overlaid with E. coli ATCC or S. aureus ATCC indicator strains comprising 1 6 cfu/ml cells. The metabolites of the F1 strain were obtained by centrifuging and filtering using a bacterial filter with a 22 nm diameter filter membrane. The cell-free broth and culture thalli were inoculated into the wells of Oxford cups (6 mm diameter) at 37 C for 24 h. The diameters of the inhibition zones extending laterally around the wells were measured. The different slips soaked, respectively, with gentamicin, cefazolin, penicillin, trimethoprim/sulfamethoxazole (TMP/SMZ), ampicillin, carbenicillin, erythromycin, norfloxacin, amikacin, or chloramphenicol were obtained from Tianhe Biotec Company (Hangzhou, China). The drug slips were put on MRS agar plates overlaid with active F1 isolates and incubated at 37 C for 24 h. The diameter of each inhibition zone was measured. Drug resistance was estimated following the introduction of a drug sensitivity test slip. Deconjugation of TCA and Cholesterol Removal Strain F1 was inoculated (1.%, v/v) into three equal cholesterol broths at 37 C for 24 h. The amount of free cholic acid liberated by deconjugation of TCA in each culture was measured via the method described by

3 X. Q. Zeng et al.: Functional Characteristics of L. fermentum F1 29 Irvin et al. [13]. After the incubation period, the three cultures were centrifuged and the pellets were washed twice with sterile distilled water. Cholesterol assimilation by the three types of cells (dead, resting, and growing) was measured as described in Ref. [14]. The three fractions of supernatant fluid, the washing fluid, and the cells quassation were collected by the method described by Razin et al. [15]. The three fractions were then separated and used to extract and measure cholesterol levels using o-phthalaldehyde methods [1]. Statistical Analysis Experimental data were presented as the mean ± SD of mean for all groups. Data analysis was conducted using SPSS 1. (SPSS Inc., Chicago, USA). A Student t test was used to perform multiple comparisons between means. All data presented are mean values of two determinations and three replicates. Results Isolation and Identification of F1 Strain with High Cholesterol-Lowering Effects Five strains of bacterium (F1, F2, F3, F4, and F5) were isolated from cattle feces. F1 displayed the highest cholesterol reduction rate of 48.87% after 24 h. Gram staining showed that it was positive and skim milk added to the strain coagulated in 6 h. The thallus of F1 was rod-shaped, amphi-ellipse, single or catenulate (Fig. 1). The results of a API 5 CHL test (data not show) indicated that F1 was L. fermentum. The 16S rrna gene was cloned and sequenced, and the Genbank accession number of HM3612 Fig. 1 Mycelial morphology of isolated L. fermentum F1 (magnification 1) was acquired. It was 1% identical to L. fermentum RE1-1 whose 16S rrna accession number is AB55297 in the NCBI Genbank. Therefore, F1 was identified as a strain of L. fermentum and was named L. fermentum F1. Acid and Bile Tolerance, Antimicrobial Activity, and Antibiotic Sensitivity of L. fermentum F1 As shown in Fig. 2a, the viable count of L. fermentum F1 did not decrease after incubation at ph levels of 6.4 and 3. for 4 h, and there were still 1 4 cfu/ml viable cells after incubation at ph 2. for 4 h. Survival at ph 3. is significant because ingestion of food or dairy products raises stomach ph to 3. or higher [16]. Therefore, L. fermentum F1 exhibited fairly good tolerance to human-stomach ph. Compared with the control of non-bile salt substrate, the F1 in.2 and.3% bile salt substrate displayed a lag time of 1.7 and 2.8 h, respectively (Fig. 2b). They were less than the finding reported for Lactobacillus gasseri SBT173, which was 3.25 h at a.3% oxgall concentration [16]. The a Survivors (mean log cfu/ml) b Absorbance(62nm) Incubating time (h) Incubating time (h) Fig. 2 Survivor curve in different ph (a) and growth curve in different bile salt concentrations (b) of L. fermentum F1. a Survivor curve at (closed circle) ph 6.4 (control), (closed square) ph 3., (closed triangle) ph 2., (filled diamond) ph 1.. b Absorbance at 62 nm in MRS-THIO medium (closed circle) without bile salt (control) and with (closed square).2% and (closed triangle).3% oxgall

4 3 X. Q. Zeng et al.: Functional Characteristics of L. fermentum F1 results showed that L. fermentum F1 had good bile salt tolerance. For L. fermentum F1, the zones of inhibition for E. coli and S. aureus were 18.2 and 19.3 mm, respectively (Fig. 3). A clear zone of 1 mm or more was considered positive inhibition. The thalli and the cell-free filtrate broth of L. fermentum F1 were consistently good at inhibiting enteropathogenic E. coli and S. aureus. Of the 1 common antibiotics, L. fermentum F1 was sensitive to gentamicin, cefazolin, penicillin, trimethoprim/ sulfamethoxazole (TMP/SMZ), ampicillin, carbenicillin, erythromycin, amikacin, and chloramphenicol, but resistant to norfloxacin and amikacin. Deconjugation of TCA and Cholesterol Removal by L. fermentum F1 Sodium taurocholate in the media was deconjugated to free bile salt and free cholalic acid by bile salt hydrolase (BSH) enzymes. The amount of free cholic acid deconjugated by L. fermentum F1 was.186 mm when incubated in MRS- THIO broth with.2% (3.72 mm) TCA, which was 5% of total TCA. The amount of cholesterol removed by dead, resting, and growing L. fermentum F1 cells is illustrated in Fig. 4. Fig. 3 Antimicrobial activity of L. fermentum F1. a Inhibition activity of enteropathogenic Escherichia coli by the thalli (T) and cell-free broth (B) of L. fermentum F1. b Inhibition activity of enteropathogenic Staphylococcus aureus by the thalli (T) and cellfree broth (B) of L. fermentum F1 Cholesterol removed (mg/g of dry weight) Growing cells Resting cells Heat-killed cells Fig. 4 Cholesterol removed by growing, resting, and dead cells of L. fermentum F1 The amount of cholesterol removed by growing cells was significantly higher than the amount removed by resting or dead cells. Heat-killed and resting cells showed a small amount of cholesterol removal (6.85 and mg/g of dry weight, respectively) compared with growing cells (33.21 mg/g of dry weight). The amount of cholesterol in different fractions of supernatant fluid, washing buffers, and cell extract was determined. The supernatant fluid accounted for 5.85% of the total amount of cholesterol added to the broth, while washing buffer and cell extract contributed and 35.39%, respectively. Discussion Lactobacillus fermentum F1 exhibited a high cholesterolreducing rate of 48.87%, which surpassed the rates reported for strains of L. casei NCDC 19 (43.2%) [17] and Lactococcus lactis subsp. lactis LQ-12 (41.83%) [11]. L. fermentum F1 from cattle feces showed good acid and bile salt tolerance, which may be due to the fact that the lactobacillus had to survive amidst the gastric juice and bile salt in the gastrointestinal tract of cattle. An antimicrobial test of L. fermentum F1 demonstrated that its antibacterial properties correlated with metabolites of lactobacilli, including lactic acid, acetic acid, hydrogen peroxide, and bacteriocin, which inhibited enteropathogenic E. coli and S. aureus. One of the safety considerations in probiotic studies is to verify that a potential probiotic strain does not contain transferable resistance genes. Of 1 common antibiotics, L. fermentum F1 was resistant to norfloxacin and amikacin. A recent study reported that most LAB share the same resistance features, which may be a common property of LAB [18]. Considering these data, the antibiotic resistance observed for L. fermentum F1 in this study was considered to be intrinsic or a natural resistance. Conversely, L. fermentum F indicator strains 1 did not contain any transferable or acquired resistances. Lactobacillus fermentum F1 deconjugated 5% of the total TCA into free bile salts. Therefore, some of the cholesterol in the media was reduced by coprecipitation with deconjugated bile salts. Although the heat-killed cells could not take up cholesterol, they were able to remove cholesterol from the media. It seemed that some cholesterol bonded to the cells. Even so, resting cells removed more cholesterol than heat-killed cells. The cholesterol difference may be caused by the BSH of resting cells and coprecipitation with deconjugated bile salts. Cholesterol removed by growing cells was significantly higher than by resting or dead cells, which indicated that the difference in the amount of cholesterol removed between the heat-killed cells and growing cells was attributed to the uptake of

5 X. Q. Zeng et al.: Functional Characteristics of L. fermentum F1 31 cholesterol by L. fermentum F1, and that the degree of bound cholesterol might be dependent on the growth of cells. The results revealed that L. fermentum F1 can remove cholesterol from media by binding cholesterol to dead cells, as well as by uptaking and coprecipitating cholesterol into living cells during growth. Heat-killed cells absorbed 6.85 mg/g of cholesterol, while resting cells absorbed and coprecipitated mg/g, which meant that heat-killed cells accounted for 21.38% and resting cells 78.62% of the cholesterol absorbed and coprecipitated. Pellet cells assimilated 35.39% of the cholesterol in the cell extract. The supernatant fluid accounted for 5.85% of the cholesterol added to the broth. Therefore, L. fermentum F1 reduced 49.15% of the cholesterol added to the broth. Cholesterol assimilated by cells occupied the major part (72%) of the removed cholesterol. Some 21.65% of the reduced cholesterol was coprecipitated with deconjugated bile salt and 5.89% was adsorbed into the surface of the cells. The coprecipitation of cholesterol with deconjugated bile salts only happened at ph values lower than 5.5 [8]; therefore, it could not occur in vivo for the ph values between 6.5 and 7., which occur in the intestine. Cholesterol assimilation by L. fermentum F1 was the most significant factor in the three-part process of cholesterol removal. Therefore, L. fermentum F1 should be able to assimilate cholesterol efficiently in vivo. A microorganism is considered probiotic when it meets the following conditions: human-origin, non-pathogenic, high resistance to passing through the intestine, capable of adhering to mucus and preventing the adherence of other pathogenic microorganisms, and beneficial to the immune system and human health in general [19]. The results of this study indicate that L. fermentum F1 is a probiotic with the potential of assimilating cholesterol, tolerating acid and bile, inhibiting pathogens, and may be able to provide species diversity for lactobacilli in order to develop a functional dairy product. Acknowledgments This work was supported by the State Science and Technology Ministry of the People s Republic of China (programs no. 27AA1Z357, 29C2214, and 26BAD27B9), the Natural Science Ministry of China (program no ) and the K. C. Wong Magna Fund, Ningbo University. 3. Harrison VC, Peat G (1975) Serum cholesterol and bowel flora in the newborn. Am J Clin Nutr 28: Endo T, Nakano M, Shimizu S, Fukushima M, Miyoshi S (1999) Effects of a probiotic on the lipid metabolism of cocks fed on a cholesterol-enriched diet. Biosci Biotechnol Biochem 63: Sanders TA (1999) Food production and food safety. Bmj 318: Gilliland SE, Nelson CR, Maxwell C (1985) Assimilation of cholesterol by Lactobacillus acidophilus. Appl Environ Microbiol 49: Gilliland SE, Walker DK (199) Factors to consider when selecting a culture of Lactobacillus acidophilus as a dietary adjunct to produce a hypocholesterolemic effect in humans. J Dairy Sci 73: Klaver FA, van der Meer R (1993) The assumed assimilation of cholesterol by Lactobacilli and Bifidobacterium bifidum is due to their bile salt-deconjugating activity. Appl Environ Microbiol 59: Noh DO, Kim SH, Gilliland SE (1997) Incorporation of cholesterol into the cellular membrane of Lactobacillus acidophilus ATCC J Dairy Sci 8: Rudel LL, Morris MD (1973) Determination of cholesterol using o-phthalaldehyde. J Lipid Res 14: Pan D, Zhang D (25) Screening of cholesterol reducing lactic acid bacteria and its activity in cholesterol reducing. Food Sci 26: Usman HA (1999) Bile tolerance, taurocholate deconjugation, and binding of cholesterol by Lactobacillus gasseri strains. J Dairy Sci 82: Irvin JL, Johnson CG, Kopalo J (1944) A photometric method of the determination of cholates in bile and blood. J Biol Chem 153: Liong MT, Shah NP (25) Acid and bile tolerance and cholesterol removal ability of lactobacilli strains. J Dairy Sci 88: Razin S, Kutner S, Efrati H, Rottem S (198) Phospholipid and cholesterol uptake by mycoplasma cells and membranes. Biochim Biophys Acta 598: Usman HA (1999) Viability of Lactobacillus gasseri and its cholesterol-binding and antimutagenic activities during subsequent refrigerated storage in nonfermented milk. J Dairy Sci 82: Mishra V, Prasad DN (25) Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics. Int J Food Microbiol 13: Temmerman R, Pot B, Huys G, Swings J (23) Dentification and antibiotic susceptibility of bacterial isolates from probiotic products. Int J Food Microbiol 81: Vanderhoof JA, Whitneyx DB, Antonson DL (1999) Lactobacillus GG in the prevention of antibiotic-associated diarrhea in children. J Pediatr 35: References 1. Ranade VV (1993) Significance of cholesterol in health and disease. Int J Clin Pharmacol Ther Toxicol 31: Tabas I (22) Cholesterol in health and disease. J Clin Invest 11:583 59