Wageningen Academic P u b l i s h e r s
Beneficial Microbes, 2015; 6(1): 119-128
Characterisation and preliminary lipid-lowering evaluation of Lactobacillus isolated http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0018 - Saturday, January 20, 2018 2:14:49 AM - University of Guelph IP Address:131.104.97.86
from a traditional Serbian dairy product G. Zavišić1, S. Ristić1, S. Petričević1, J. Novaković Jovanović1, Ž. Radulović1, B. Janać Petković2, I. Strahinić3 and V. Piperski1* 1Galenika
a.d., R&D Institute, Batajnički drum bb, 11080 Belgrade, Serbia; 2Institute for Biological Research ‘Siniša Stanković’, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia; 3Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010 Belgrade, Serbia; [email protected] Received: 12 February 2014 / Accepted: 18 April 2014 © 2014 Wageningen Academic Publishers
RESEARCH ARTICLE Abstract We investigated the potential probiotic properties of indigenous lactic acid bacteria (LAB) isolated from Serbian homemade cheese. Seventeen LAB strains were isolated and characterised using standard protocols. One of the strains showed several probiotic properties: survival at low pH and in bile salts solution, antimicrobial activity, susceptibility to antibiotics and adhesion to hexodecane. DNA analysis identified the isolate as Lactobacillus casei, hereafter named L. casei 5s. The lipid lowering effect of L. casei 5s was evaluated in vivo using a hyperlipidemic rat model. Orally administered L. casei 5s significantly decreased the elevated total serum cholesterol and triglycerides, and attenuated macro vesicular steatosis in the liver. Moreover, L. casei 5s improved the intestinal microbial balance in favour of lactobacilli, while decreasing the number of Escherichia coli cells. The bacteria were re-isolated and identified from the surface of the intestinal mucosa and from the faecal samples of treated animals, indicating adhesiveness and colonisation ability. The results of an acute oral toxicity study in mice and the absence of translocation to other organs demonstrated the safety of the strain. In conclusion, L. casei 5s demonstrated promising probiotic potential and might be a good candidate for more detailed investigations. Keywords: probiotic, Lactobacillus casei, lipid-lowering effect, safety
1. Introduction Some lactic acid bacteria (LAB), especially lactobacilli, are probiotics that provide numerous health benefits: they enhance immune responses (Maroof et al., 2012), exert antimutagenic and anticarcinogenic activities (Kandasamy et al., 2011), and protect against gastrointestinal diseases (Dicks and Botes, 2010; Katsumi et al., 2012). In addition, one very important health benefit of LAB is the reduction of serum cholesterol. Elevated serum cholesterol is recognised as a major risk factor for the development of cardiovascular disease, the leading cause of death in many countries (Law et al., 1994). Many drugs can effectively reduce cholesterol levels, however, they are expensive and are known to have severe side effects (Bliznakov, 2002). For that reason it would be significant to develop new ways of lowering serum
cholesterol, such as diet modification by supplementation with dairy products containing probiotics. The possibility of cholesterol reduction by LAB has been extensively studied and documented in numerous animal and human studies (Homayouni et al., 2012; Nagpal et al., 2007). Nevertheless, the search for a better and more efficient probiotic strain continues. To survive in the gastrointestinal tract (GIT) and exert their beneficial effects, potential probiotic bacteria must be resistant to the high acidity of the stomach and high concentration of bile salts in the proximal intestine (Faye et al., 2012). Also, the probiotics must have the ability to adhere to the gut epithelium in order to colonise the GIT. These characteristics may be observed in vitro and can be used for the selection of strains (Marteau et al., 1997; Ocana et al., 1999).
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0018119
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G. Zavišić et al.
Antimicrobial activity against different pathogens is another important property of probiotics. Lactobacilli are associated with the alteration of the intestinal microflora population and have been used to suppress the growth of pathogens. This is accomplished by the production of antimicrobial compounds, such as organic acids, hydrogen peroxide, diacetyl and bacteriocins, and also by their competition for nutrients (Isolauri et al., 2002). Probiotic bacteria such as Lactobacillus are generally regarded as safe (GRAS) for consumption. However, there have been reports indicating that probiotic bacteria are capable of translocating from the intestine to other organs. Namely, Lactobacillus casei, Lactobacillus plantarum and Lactococcus lactis were isolated in cases of bacterial enterocarditis, while Bifidobacterium adolescentis was isolated from blood stream infections (Gasser, 1994; Graf and Sarasin, 2007). Such translocations are difficult to induce in healthy individuals, but they could occur in the case of injury of the intestinal mucosa, immunodeficiency of the host, or abnormal intestinal bacterial flora (Ishibashi and Yamazaki, 2001). Regarding probiotic safety, the antibiotic resistance of probiotics is an area of increasing concern, because of the possibility of transferring resistance to GIT pathogens (Mathur and Singh, 2005), thus the absence of antibiotic resistance can be considered a positive feature for probiotic bacteria. In this study, we identified and characterised a Lactobacillus strain from traditional Serbian homemade cheese, estimated its potential probiotic properties, including preliminary lipid lowering effects, and evaluated its safety.
2. Materials and methods In vitro experiments Strain characterisation and identification Our laboratory isolate, from a cheese sample, traditionally homemade in the mountain region of Rajac, Serbia, was preliminarily identified using standard protocols, according to the methods and criteria described by Sharpe (1979) and Kandler and Weiss (1986): Gram-staining, cell morphology, catalase reaction, growth at different temperatures (15, 30, 37 and 45 °C) in De Man Rogosa and Sharpe broth (MRS; Merck GmbH, Darmstadt, Germany) for 5 days; growth in MRS broth with 2, 4 and 6.5% (w/v) NaCl for 5 days; CO2 production from glucose in tube containing inverted Durham’s tubes; L-arginine hydrolysis; liquefaction of gelatine and growth in 10% (w/v) skimmed milk medium (AD Mlekara, Subotica, Serbia). The pattern of carbohydrate fermentation and enzymatic activity of the bacteria were determined by using the API 50CH and API ZYM systems (Gas pack vessel, BioMerieux, France), respectively. All tests were conducted in triplicates. 120
Resistance to artificial gastric juice and bile salts solution The test of bacterial survival in artificial gastric juice (AGJ) and bile salts solution (BSS) was performed according to Graciela and Maria (2001) with modifications, as follows: 10 ml of MRS medium was innoculated at 1% (v/v) with the bacteria and incubated at 37 °C for 18 h. After washing of the bacterial cells, 10 ml of cell suspension containing 108 cfu/ ml was added to 90 ml of AGJ (0.03 M NaCl, 0.32% pepsin at pH 2.0 adjusted with 10 M HCl) and incubated with gentle agitation (58 rpm) to simulate peristaltics. Bacterial aliquots were taken for determination of viable cells at 0, 60, and 120 min. The effect of BSS on bacterial survival was studied on cells which survived in AGJ conditions after 2 h incubation. The cells were transferred to MRS medium containing 0.5% bovine bile salts (oxgall) and incubated at 37 °C for up to 2 h with gentle agitation (58 rpm). The samples for total viable counts were taken at 0, 60 and 120 min. Pepsin, HCl and oxgall were obtained from Sigma (Sigma-Aldrich, St. Louis, MO, USA). All tests were conducted in duplicate.
Microbial adhesion to hexadecane In order to estimate the adhesion potential of the bacterial isolates in vitro, the test of microbial adhesion to hexadecane (MATH) was employed (Sigma Aldrich). The adhesion of microorganisms to hexadecane is a criterion of the hydrophobicity of the bacterial surface due to the absence of electrostatic interaction (caused by a large quantity of electrolytes in 0.1 M KNO3). The percentage of adhered cells was calculated using the formula (1 – A1/ A0) × 100. The values 0-35% indicate low hydrophobicity, 36-70% intermediate hydrophobicity and 71-100% high hydrophobicity (Ocana et al., 1999). All tests were conducted in duplicate.
Detection of antimicrobial activity The antimicrobial activity of the bacterial isolates was determined using the agar diffusion method. The overnight cultures of the indicator strains (Staphylococcus aureus ATCC 6538-P, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Salmonella abony NTCC 6017, Candida albicans ATCC 10231) and of the sporulation cultures (Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778 and Clostridium sporogenes ATCC 19404) were mixed at 1% (106 cfu/ml) with melted nutrient agar, transferred to sterile Petri dishes and allowed to solidify. A 6-mm wide well was cut in the agar across the centre of the dish. Aliquots (100 µl) of the lactobacilli cultures, cell-free filtrate and cell-free filtrate after neutralisation were poured in the wells. The plates were first incubated at 4 °C for 2 h to allow the test material to diffuse in the agar and then incubated for 18 h at 37 °C for bacteria, and 48 h at 25 °C for C. albicans. After incubation, the diameter of the inhibition zone was measured. The test was conducted in duplicate. Beneficial Microbes 6(1)
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Probiotic properties and safety of L. casei isolated from Serbian cheese
DNA isolation and manipulations
In vivo experiments in rats
The lactobacilli’s genomic DNA was isolated using the QIA DNA Mini Kit (Qiagen GmbH, Hilden, Germany). PCR amplicons were generated using Taq polymerase (Pharmacia, Vienna, Austria), according to the supplier’s instructions. PCR products were analysed on 1% agarose gels and purified using the QIAquick gel extraction kit (Qiagen GmbH). Species determination was done by PCR, using primers complementary to 16S rDNA: UNI16SF (5’-GAG AGT TTG ATC CTG GC-3) and UNI16SR (5’AGG AGG TGA TCC AGC CG-3’). PCR amplifications were performed by using the GeneAmp PCR System 2700 thermal cycler (Applied Biosystems, Foster City, CA, USA) and Taq polymerase. PCR amplification conditions were as follows: 5 min at 96 °C; 30 cycles of 30 s at 96 °C, 30 s at 55 °C, and 30 s at 72 °C, and an additional extension step of 5 min at 72 °C. The resulting PCR amplicons were purified with QIAGENE PCR Purification Kit (QIAGEN GmbH), following the manufacturer’s instruction. Sequencing was done in Central Service of Macrogen (Macrogen, Seoul, South Korea) by using the dideoxynucleotide DNA chain termination method. The BLAST algorithm (http:// www.ncbi.nlm.nih.gov/BLAST; RID: 1138633900-27581131272740575. BLASTQ4) was used to determine the most related sequence relatives in the NCBI nucleotide sequence database.
Experimental animals and diets
Sensitivity to antibiotics The susceptibility of isolate 5s was determined towards 15 antibiotics: chloramphenicol (30 µg), penicillin (6 µg), gentamicin (30 µg), ampicillin (10 µg), streptomycin (30 µg), vancomycin (30 µg), rifampicin (5 µg), neomycin (30 µg), kanamycin (30 µg), erythromycin (15 µg), tetracycline (30 µg), lincomycin (15 µg), novobiocin (5 µg), using the standard disc diffusion method (Bauer et al., 1966). Discs infiltrated with antibiotics (Torlak, Belgrade, Serbia) were placed on Petri dishes with MRS agar innoculated with isolate 5s and incubated for 24 h at 37 °C. The diameters of inhibition zones were measured and results were expressed as sensitive, S (≥21 mm); intermediate, I (16-20 mm) and resistant, R (≤15 mm), (Liasi et al., 2009).
Preparation of probiotic culture for in vivo trial L. casei 5s was lyophilised and stored at -20 °C. After recovery at 37 °C for 30 min, the bacteria were innoculated into MRS liquid medium and incubated at 37 °C for 18 h. The bacteria were harvested by centrifugation at 5,000 rpm for 20 min at 4 °C, washed and resuspended at 1×109 cfu/ml in sterile physiological solution (0.9% w/v NaCl).
Beneficial Microbes 6(1)
Eight weeks old male Wistar rats, with an initial body weight of 180-200 g, (obtained from the Vivarium of Galenika a.d., Belgrade, Serbia) were used for in vivo investigation of the hypolipidemic effect of L. casei 5s. The rats were housed in groups of four per standard cage, in a room with a 12 h light-dark cycle and at a temperature of 22±2 °C. The animals were maintained in accordance with the principles enunciated in the Guide for the Care and Use of Laboratory Animals, (NIH publication No. 85-23). To create a model of hyperlipidemia, 12 animals were fed with high-fat high-cholesterol feed (consisting of standard rodent pelleted feed with addition of 2.0% cholesterol, 20% sunflower oil and 0.5% sodium cholate) during 8 weeks. After 8 weeks, blood was collected from tail vein and serum cholesterol and triglycerides were determined to confirm hyperlipidemia. The animals were then divided randomly into 3 experimental groups, each group containing 4 rats (Table 1). The first group (model control group) continued to receive only high-fat highcholesterol feed; the second group (experimental group) was fed high-fat high-cholesterol feed with the addition of L. casei 5s (1×109 cfu/ml); the third group (positive control group) was fed high-fat high-cholesterol feed and treated with the antilipidemic drug Simvastatin (10 mg; Zokor, Sandoz, Holzkirchen, Germany). This regime was maintained for 4 weeks. The animals were treated with L. casei 5s or with Simvastatin every day, by gastric gavage. The amount of bacteria administered was established based on the dose most frequently consumed by humans during probiotic treatment. The Simvastatin dose was also determined according to the human daily dose. The fourth group consisted of 4 animals which were fed standard pellet rodent feed during the entire experimental period, and served as a blank control. The rats were allowed free access Table 1. Experimental groups and diets/treatments.1 Group
Diet/treatment
Model control Experimental
High-fat high-cholesterol diet High-fat high-cholesterol diet + Lactobacillus casei 5s High-fat high-cholesterol diet + Simvastatin Standard rodent feed diet
Positive control Blank control 1
High-fat high-cholesterol diet consisted of standard rat pelleted feed with addition of 20% sunflower oil, 2% cholesterol and 0.5% sodium cholate. L. casei 5s (1×109 cfu/ml) and antilipemic drug Simvastatin (10 mg) were administered daily by gastric gavage, for 28 days. Each group contained 4 rats.
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to experimental food and water and their body weights were measured weekly. The activity, behavior and general health of the rats were monitored daily. At the end of the experiment, the animals were briefly exposed to ether and blood was obtained by cardiac puncture. After cervical dislocation, the viscera were examined macroscopically, and liver samples were taken and processed for histology.
euthanising the animals by cervical dislocation (subsequent to brief exposure to ether) 1 cm fragments of the small intestine were placed on MRS agar plates in order to transfer adherent lactobacilli (with other intestinal bacteria). After 6 h the intestinal fragments were removed and the plates incubated in anaerobic conditions at 37 °C for 48 h. The obtained material was isolated on MRS agar and individual colonies were identified using biochemical and enzymatic tests (API CH 50 and API ZYM).
Biochemical analysis of serum cholesterol and triglycerides
Safety assessment in mice
Blood samples were transferred directly into centrifuge tubes, allowed to clot at room temperature for 20 min, and centrifuged for 10 min at 3,500 rpm. The supernatant obtained was transferred into test tubes for lipid analysis. The total serum cholesterol and triglycerides were determined by the appropriate enzymatic colorimetric methods, using commercial Randox kits (Randox Laboratories Ltd., Crumlin, UK).
Acute toxicity test of L. casei 5s
Liver histology
An acute oral toxicity study was performed according to OECD (1995) guidelines, on 10 NMRI Hann mice, six weeks old, weighing from 18 to 22 g. L. casei 5s in dose of 1×107 cfu/ml was administered by gastric intubation to each animal daily throughout the experimental period of 10 days. The control group of 5 mice received 1 ml of saline by gastric intubation. Feed and water were freely available, and the housing conditions were the same as for rats during the in vivo test of hypolipidemic effect.
The liver tissue samples were fixed in 4% buffered formaline and embedded in parafine wax. Sections 5 µm thick were made using the Reichert microtome (Depew, NY, USA), stained with haematoxyllin and eosin (HE) and analysed with a light microscope (Olympus BX60, Olympus,Tokyo, Japan). Representative photomicrographs were taken using the Promicra Quickphoto Camera 2.3 system (Prague, Czech Republic) for digital photomicrography, image editing and measurements.
All tested animals were observed daily during the experimental period, and examined for general toxicity signs: changes in the skin and fur, eyes and mucous membranes, excreta, behavioral pattern, and feed and water consumption. At the end of experiment, all mice were sacrificed by cervical dislocation (after brief exposure to ether), and macroscopic examination of internal organs was performed.
Microbial analysis of faecal samples
Bacterial translocation
Faecal samples were collected before the beginning and after the end of treatment with L. casei 5s and analysed the same day for the number of lactobacilli and E. coli. The total number of lactobacilli was determined by plate count on MRS agar, after anaerobic incubation at 37 °C for 48 h. For determining the total number of viable E. coli MacConkey agar (MCA; Merck) was used, and aerobic incubation at 35 °C. Individual colonies from MCA were transferred into MacConkey broth (MCB; Merck) and incubated at 43 °C for 24 h. Growth of red, non-mucoid colonies of gram-negative rods, subcultured on plates for 24 h at 35 °C, indicated the possible presence of E. coli, which was confirmed by the indole production test and API E system (European Pharmacopeia, 2011).
In the same animals, translocation of bacteria to blood and tissues was assessed according to Zhou et al. (2000). Briefly, one drop of blood (50 µl) was placed onto the surface of MRS agar plates and incubated at 37 °C anaerobically to test for bacteriemia. The excised spleen, liver and kidney were homogenised in 5 ml of sterile saline solution, and 100 µl of tissue suspensions plated on MRS agar plates. The plates were then incubated anaerobically at 37 °C for 48 h.
Adhesiveness to intestinal mucosa The adhesiveness of L. casei 5s cells to the intestinal mucosa was investigated in another group of 4 male Wistar rats, body mass of 180-220 g, which were treated intragastrically with L. casei 5s (1×109 cfu/ml) once daily for 7 days. After 122
Statistical analysis The levels of cholesterol and triglycerides in the serum, as well as faecal bacterial counts are presented as mean ± standard deviation for all groups. The effect of L. casei 5s was evaluated by two-way analysis of variance (ANOVA) with group as the between-subject factor and time (before and after treatment) as the within-subject (repeated measure) factor, followed where appropriate by post hoc Fisher’s least significant difference (LSD) test. Before statistical analysis, normal distribution of data was assessed using Kolmogorov-Smirnov test. Beneficial Microbes 6(1)
Probiotic properties and safety of L. casei isolated from Serbian cheese
3. Results
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Characterisation and provisional identification Our laboratory isolate, denoted as 5s, was Gram-positive, non-sporulative, catalaze negative, gelatinase-negative, non-haemolitic bacilli growing on MRS agar, in anaerobic, microaerofilic and aerobic conditions, indicating that it belongs to the Lactobacillus genus. According to its fermentative characteristics, the isolate showed greatest similarity to bacteria belonging to L. casei/ Lactobacillus paracasei: glycerol–, erytrol–, D-arabinose–, L-arabinose–, ribose+, D-xylose–, L-xylose–, D-adonitol–, metil-β D-xylopyranoside–, galactose+, glucose+, fructose+, manose+, sorbose–, rhamnose–, dulcitol–, inositol–, manitol+, sorbitol+, metil-α D-manopyranoside–, metil-α D-glucopyranoside–, N-acetylglucosamine+, amygdaline+, arbutine+, esculin+, salicin+, D-celobiose+, D-maltose+, D-lactose+, D-melibiose–, D-saccharose+, D-trehalose+, inuline+, D-melecitose+, D-rafinose–, amidon–, glycogen–, xylitol–, gentiobiose+, D-turanose+, D-lyxose–, D-tagatose±, D-fucose–, L-fucose–, D-arabitol–, L-arabitol–, potasium gluconate–, 2-keto-gluconate–, 5-keto-gluconate–. Subsequently, isolate 5s was more accurately identified to the species level by partial sequencing of 16S rRNA genes. According to the nucleotide sequence, showing 97% similarity, the isolate was identified as L. casei, and marked among our laboratory isolates as L. casei 5s.
Acid and bile salt tolerance of L. casei 5s To explore the potential probiotic properties of L. casei 5s, we tested bacterial viability in artificial gastric and intestinal juice, adhesion to hexadecane, antimicrobial activity and
susceptibility to antibiotics. L. casei 5s showed a high degree of survival in AGJ and in the solution containing 0.5% bovine bile salts. After 120 min exposure to AGJ, the count of viable cells decreased by 1.99 log units (from 6.96×109 to 7.11×107 cfu/ml). The cells that survived AGJ conditions were then exposed to the bovine bile salt solution; after 120 min exposure, the number of viable cells decreased by 0.86 log units (from 7.11×107 to 9.68×106 cfu/ml).
MATH test The percentage of adherent L. casei 5s cells to the hydrophobic solution of hexadecane was 47.26±2.3, indicating intermediate hydrophobicity of the cells.
Antimicrobial activity The antimicrobial activity of L. casei 5s is shown in Table 2. Both complete culture and cell free filtrate of L. casei 5s were able to inhibit the growth of S. aureus ATCC 6538-P, E. coli ATCC 8739, P. aeruginosa ATCC 9027, S. abony NTCC 6017 and C. sporogenes ATCC 19404, but failed to inhibit B. cereus ATCC 11778 and C. albicans ATCC 10231. The strongest antimicrobial activity was against S. aureus. After neutralisation of overnight cell-free filtrate, no zone of inhibition was detected, indicating that antimicrobial activity of L. casei 5s is related to organic acids, mainly lactic acid (data not shown).
Sensitivity to antibiotics L. casei 5s showed sensitivity (S) to chloramphenicol, ampicillin, streptomycin, rifampicin, neomycin, kanamycin, erythromycin, novobiocin (zone of inhibition ≥21 mm); intermediate (I) sensitivity to tetracyclin, lincomycin, penicillin and gentamicin (zone of inhibition 16-20 mm); it was resistant (R) only to vancomycin (no zone of inhibition).
Table 2. Antimicrobial activity of Lactobacillus casei 5s.1 Indicator strain
Staphylococcus aureus ATCC 6538-P Bacillus subtilis ATCC 6633 Bacillus cereus ATCC 11778 Escherichia coli ATCC 8739 Pseudomonas aeruginosa ATCC 9027 Salmonella abony NTCC 6017 Candida albicans ATCC 10231 Clostridium sporogenes ATCC19404 1
Diameter of growth inhibition zone (mm) Overnight culture
Cell free filtrate before neutralisation
Cell free filtrate after neutralisation
18 12 0 12 12 12 0 12
18 12 / 10 12 12 / 12
0 0 / 0 0 0 / /
Mean values from two experiments are presented. 0: no zone of inhibition; /: not tested.
Beneficial Microbes 6(1)
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In vivo hypolipidemic effect of L. casei 5s The effect of assigned diets on serum cholesterol and triglyceride levels are presented in Table 3. For both parameters, two-way ANOVA revealed a significant main effect of group (cholesterol: F(3,12)=108.86, P
Beneficial Microbes, 2015; 6(1): 119-128
Characterisation and preliminary lipid-lowering evaluation of Lactobacillus isolated http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0018 - Saturday, January 20, 2018 2:14:49 AM - University of Guelph IP Address:131.104.97.86
from a traditional Serbian dairy product G. Zavišić1, S. Ristić1, S. Petričević1, J. Novaković Jovanović1, Ž. Radulović1, B. Janać Petković2, I. Strahinić3 and V. Piperski1* 1Galenika
a.d., R&D Institute, Batajnički drum bb, 11080 Belgrade, Serbia; 2Institute for Biological Research ‘Siniša Stanković’, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia; 3Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010 Belgrade, Serbia; [email protected] Received: 12 February 2014 / Accepted: 18 April 2014 © 2014 Wageningen Academic Publishers
RESEARCH ARTICLE Abstract We investigated the potential probiotic properties of indigenous lactic acid bacteria (LAB) isolated from Serbian homemade cheese. Seventeen LAB strains were isolated and characterised using standard protocols. One of the strains showed several probiotic properties: survival at low pH and in bile salts solution, antimicrobial activity, susceptibility to antibiotics and adhesion to hexodecane. DNA analysis identified the isolate as Lactobacillus casei, hereafter named L. casei 5s. The lipid lowering effect of L. casei 5s was evaluated in vivo using a hyperlipidemic rat model. Orally administered L. casei 5s significantly decreased the elevated total serum cholesterol and triglycerides, and attenuated macro vesicular steatosis in the liver. Moreover, L. casei 5s improved the intestinal microbial balance in favour of lactobacilli, while decreasing the number of Escherichia coli cells. The bacteria were re-isolated and identified from the surface of the intestinal mucosa and from the faecal samples of treated animals, indicating adhesiveness and colonisation ability. The results of an acute oral toxicity study in mice and the absence of translocation to other organs demonstrated the safety of the strain. In conclusion, L. casei 5s demonstrated promising probiotic potential and might be a good candidate for more detailed investigations. Keywords: probiotic, Lactobacillus casei, lipid-lowering effect, safety
1. Introduction Some lactic acid bacteria (LAB), especially lactobacilli, are probiotics that provide numerous health benefits: they enhance immune responses (Maroof et al., 2012), exert antimutagenic and anticarcinogenic activities (Kandasamy et al., 2011), and protect against gastrointestinal diseases (Dicks and Botes, 2010; Katsumi et al., 2012). In addition, one very important health benefit of LAB is the reduction of serum cholesterol. Elevated serum cholesterol is recognised as a major risk factor for the development of cardiovascular disease, the leading cause of death in many countries (Law et al., 1994). Many drugs can effectively reduce cholesterol levels, however, they are expensive and are known to have severe side effects (Bliznakov, 2002). For that reason it would be significant to develop new ways of lowering serum
cholesterol, such as diet modification by supplementation with dairy products containing probiotics. The possibility of cholesterol reduction by LAB has been extensively studied and documented in numerous animal and human studies (Homayouni et al., 2012; Nagpal et al., 2007). Nevertheless, the search for a better and more efficient probiotic strain continues. To survive in the gastrointestinal tract (GIT) and exert their beneficial effects, potential probiotic bacteria must be resistant to the high acidity of the stomach and high concentration of bile salts in the proximal intestine (Faye et al., 2012). Also, the probiotics must have the ability to adhere to the gut epithelium in order to colonise the GIT. These characteristics may be observed in vitro and can be used for the selection of strains (Marteau et al., 1997; Ocana et al., 1999).
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0018119
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0018 - Saturday, January 20, 2018 2:14:49 AM - University of Guelph IP Address:131.104.97.86
G. Zavišić et al.
Antimicrobial activity against different pathogens is another important property of probiotics. Lactobacilli are associated with the alteration of the intestinal microflora population and have been used to suppress the growth of pathogens. This is accomplished by the production of antimicrobial compounds, such as organic acids, hydrogen peroxide, diacetyl and bacteriocins, and also by their competition for nutrients (Isolauri et al., 2002). Probiotic bacteria such as Lactobacillus are generally regarded as safe (GRAS) for consumption. However, there have been reports indicating that probiotic bacteria are capable of translocating from the intestine to other organs. Namely, Lactobacillus casei, Lactobacillus plantarum and Lactococcus lactis were isolated in cases of bacterial enterocarditis, while Bifidobacterium adolescentis was isolated from blood stream infections (Gasser, 1994; Graf and Sarasin, 2007). Such translocations are difficult to induce in healthy individuals, but they could occur in the case of injury of the intestinal mucosa, immunodeficiency of the host, or abnormal intestinal bacterial flora (Ishibashi and Yamazaki, 2001). Regarding probiotic safety, the antibiotic resistance of probiotics is an area of increasing concern, because of the possibility of transferring resistance to GIT pathogens (Mathur and Singh, 2005), thus the absence of antibiotic resistance can be considered a positive feature for probiotic bacteria. In this study, we identified and characterised a Lactobacillus strain from traditional Serbian homemade cheese, estimated its potential probiotic properties, including preliminary lipid lowering effects, and evaluated its safety.
2. Materials and methods In vitro experiments Strain characterisation and identification Our laboratory isolate, from a cheese sample, traditionally homemade in the mountain region of Rajac, Serbia, was preliminarily identified using standard protocols, according to the methods and criteria described by Sharpe (1979) and Kandler and Weiss (1986): Gram-staining, cell morphology, catalase reaction, growth at different temperatures (15, 30, 37 and 45 °C) in De Man Rogosa and Sharpe broth (MRS; Merck GmbH, Darmstadt, Germany) for 5 days; growth in MRS broth with 2, 4 and 6.5% (w/v) NaCl for 5 days; CO2 production from glucose in tube containing inverted Durham’s tubes; L-arginine hydrolysis; liquefaction of gelatine and growth in 10% (w/v) skimmed milk medium (AD Mlekara, Subotica, Serbia). The pattern of carbohydrate fermentation and enzymatic activity of the bacteria were determined by using the API 50CH and API ZYM systems (Gas pack vessel, BioMerieux, France), respectively. All tests were conducted in triplicates. 120
Resistance to artificial gastric juice and bile salts solution The test of bacterial survival in artificial gastric juice (AGJ) and bile salts solution (BSS) was performed according to Graciela and Maria (2001) with modifications, as follows: 10 ml of MRS medium was innoculated at 1% (v/v) with the bacteria and incubated at 37 °C for 18 h. After washing of the bacterial cells, 10 ml of cell suspension containing 108 cfu/ ml was added to 90 ml of AGJ (0.03 M NaCl, 0.32% pepsin at pH 2.0 adjusted with 10 M HCl) and incubated with gentle agitation (58 rpm) to simulate peristaltics. Bacterial aliquots were taken for determination of viable cells at 0, 60, and 120 min. The effect of BSS on bacterial survival was studied on cells which survived in AGJ conditions after 2 h incubation. The cells were transferred to MRS medium containing 0.5% bovine bile salts (oxgall) and incubated at 37 °C for up to 2 h with gentle agitation (58 rpm). The samples for total viable counts were taken at 0, 60 and 120 min. Pepsin, HCl and oxgall were obtained from Sigma (Sigma-Aldrich, St. Louis, MO, USA). All tests were conducted in duplicate.
Microbial adhesion to hexadecane In order to estimate the adhesion potential of the bacterial isolates in vitro, the test of microbial adhesion to hexadecane (MATH) was employed (Sigma Aldrich). The adhesion of microorganisms to hexadecane is a criterion of the hydrophobicity of the bacterial surface due to the absence of electrostatic interaction (caused by a large quantity of electrolytes in 0.1 M KNO3). The percentage of adhered cells was calculated using the formula (1 – A1/ A0) × 100. The values 0-35% indicate low hydrophobicity, 36-70% intermediate hydrophobicity and 71-100% high hydrophobicity (Ocana et al., 1999). All tests were conducted in duplicate.
Detection of antimicrobial activity The antimicrobial activity of the bacterial isolates was determined using the agar diffusion method. The overnight cultures of the indicator strains (Staphylococcus aureus ATCC 6538-P, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Salmonella abony NTCC 6017, Candida albicans ATCC 10231) and of the sporulation cultures (Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778 and Clostridium sporogenes ATCC 19404) were mixed at 1% (106 cfu/ml) with melted nutrient agar, transferred to sterile Petri dishes and allowed to solidify. A 6-mm wide well was cut in the agar across the centre of the dish. Aliquots (100 µl) of the lactobacilli cultures, cell-free filtrate and cell-free filtrate after neutralisation were poured in the wells. The plates were first incubated at 4 °C for 2 h to allow the test material to diffuse in the agar and then incubated for 18 h at 37 °C for bacteria, and 48 h at 25 °C for C. albicans. After incubation, the diameter of the inhibition zone was measured. The test was conducted in duplicate. Beneficial Microbes 6(1)
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Probiotic properties and safety of L. casei isolated from Serbian cheese
DNA isolation and manipulations
In vivo experiments in rats
The lactobacilli’s genomic DNA was isolated using the QIA DNA Mini Kit (Qiagen GmbH, Hilden, Germany). PCR amplicons were generated using Taq polymerase (Pharmacia, Vienna, Austria), according to the supplier’s instructions. PCR products were analysed on 1% agarose gels and purified using the QIAquick gel extraction kit (Qiagen GmbH). Species determination was done by PCR, using primers complementary to 16S rDNA: UNI16SF (5’-GAG AGT TTG ATC CTG GC-3) and UNI16SR (5’AGG AGG TGA TCC AGC CG-3’). PCR amplifications were performed by using the GeneAmp PCR System 2700 thermal cycler (Applied Biosystems, Foster City, CA, USA) and Taq polymerase. PCR amplification conditions were as follows: 5 min at 96 °C; 30 cycles of 30 s at 96 °C, 30 s at 55 °C, and 30 s at 72 °C, and an additional extension step of 5 min at 72 °C. The resulting PCR amplicons were purified with QIAGENE PCR Purification Kit (QIAGEN GmbH), following the manufacturer’s instruction. Sequencing was done in Central Service of Macrogen (Macrogen, Seoul, South Korea) by using the dideoxynucleotide DNA chain termination method. The BLAST algorithm (http:// www.ncbi.nlm.nih.gov/BLAST; RID: 1138633900-27581131272740575. BLASTQ4) was used to determine the most related sequence relatives in the NCBI nucleotide sequence database.
Experimental animals and diets
Sensitivity to antibiotics The susceptibility of isolate 5s was determined towards 15 antibiotics: chloramphenicol (30 µg), penicillin (6 µg), gentamicin (30 µg), ampicillin (10 µg), streptomycin (30 µg), vancomycin (30 µg), rifampicin (5 µg), neomycin (30 µg), kanamycin (30 µg), erythromycin (15 µg), tetracycline (30 µg), lincomycin (15 µg), novobiocin (5 µg), using the standard disc diffusion method (Bauer et al., 1966). Discs infiltrated with antibiotics (Torlak, Belgrade, Serbia) were placed on Petri dishes with MRS agar innoculated with isolate 5s and incubated for 24 h at 37 °C. The diameters of inhibition zones were measured and results were expressed as sensitive, S (≥21 mm); intermediate, I (16-20 mm) and resistant, R (≤15 mm), (Liasi et al., 2009).
Preparation of probiotic culture for in vivo trial L. casei 5s was lyophilised and stored at -20 °C. After recovery at 37 °C for 30 min, the bacteria were innoculated into MRS liquid medium and incubated at 37 °C for 18 h. The bacteria were harvested by centrifugation at 5,000 rpm for 20 min at 4 °C, washed and resuspended at 1×109 cfu/ml in sterile physiological solution (0.9% w/v NaCl).
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Eight weeks old male Wistar rats, with an initial body weight of 180-200 g, (obtained from the Vivarium of Galenika a.d., Belgrade, Serbia) were used for in vivo investigation of the hypolipidemic effect of L. casei 5s. The rats were housed in groups of four per standard cage, in a room with a 12 h light-dark cycle and at a temperature of 22±2 °C. The animals were maintained in accordance with the principles enunciated in the Guide for the Care and Use of Laboratory Animals, (NIH publication No. 85-23). To create a model of hyperlipidemia, 12 animals were fed with high-fat high-cholesterol feed (consisting of standard rodent pelleted feed with addition of 2.0% cholesterol, 20% sunflower oil and 0.5% sodium cholate) during 8 weeks. After 8 weeks, blood was collected from tail vein and serum cholesterol and triglycerides were determined to confirm hyperlipidemia. The animals were then divided randomly into 3 experimental groups, each group containing 4 rats (Table 1). The first group (model control group) continued to receive only high-fat highcholesterol feed; the second group (experimental group) was fed high-fat high-cholesterol feed with the addition of L. casei 5s (1×109 cfu/ml); the third group (positive control group) was fed high-fat high-cholesterol feed and treated with the antilipidemic drug Simvastatin (10 mg; Zokor, Sandoz, Holzkirchen, Germany). This regime was maintained for 4 weeks. The animals were treated with L. casei 5s or with Simvastatin every day, by gastric gavage. The amount of bacteria administered was established based on the dose most frequently consumed by humans during probiotic treatment. The Simvastatin dose was also determined according to the human daily dose. The fourth group consisted of 4 animals which were fed standard pellet rodent feed during the entire experimental period, and served as a blank control. The rats were allowed free access Table 1. Experimental groups and diets/treatments.1 Group
Diet/treatment
Model control Experimental
High-fat high-cholesterol diet High-fat high-cholesterol diet + Lactobacillus casei 5s High-fat high-cholesterol diet + Simvastatin Standard rodent feed diet
Positive control Blank control 1
High-fat high-cholesterol diet consisted of standard rat pelleted feed with addition of 20% sunflower oil, 2% cholesterol and 0.5% sodium cholate. L. casei 5s (1×109 cfu/ml) and antilipemic drug Simvastatin (10 mg) were administered daily by gastric gavage, for 28 days. Each group contained 4 rats.
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to experimental food and water and their body weights were measured weekly. The activity, behavior and general health of the rats were monitored daily. At the end of the experiment, the animals were briefly exposed to ether and blood was obtained by cardiac puncture. After cervical dislocation, the viscera were examined macroscopically, and liver samples were taken and processed for histology.
euthanising the animals by cervical dislocation (subsequent to brief exposure to ether) 1 cm fragments of the small intestine were placed on MRS agar plates in order to transfer adherent lactobacilli (with other intestinal bacteria). After 6 h the intestinal fragments were removed and the plates incubated in anaerobic conditions at 37 °C for 48 h. The obtained material was isolated on MRS agar and individual colonies were identified using biochemical and enzymatic tests (API CH 50 and API ZYM).
Biochemical analysis of serum cholesterol and triglycerides
Safety assessment in mice
Blood samples were transferred directly into centrifuge tubes, allowed to clot at room temperature for 20 min, and centrifuged for 10 min at 3,500 rpm. The supernatant obtained was transferred into test tubes for lipid analysis. The total serum cholesterol and triglycerides were determined by the appropriate enzymatic colorimetric methods, using commercial Randox kits (Randox Laboratories Ltd., Crumlin, UK).
Acute toxicity test of L. casei 5s
Liver histology
An acute oral toxicity study was performed according to OECD (1995) guidelines, on 10 NMRI Hann mice, six weeks old, weighing from 18 to 22 g. L. casei 5s in dose of 1×107 cfu/ml was administered by gastric intubation to each animal daily throughout the experimental period of 10 days. The control group of 5 mice received 1 ml of saline by gastric intubation. Feed and water were freely available, and the housing conditions were the same as for rats during the in vivo test of hypolipidemic effect.
The liver tissue samples were fixed in 4% buffered formaline and embedded in parafine wax. Sections 5 µm thick were made using the Reichert microtome (Depew, NY, USA), stained with haematoxyllin and eosin (HE) and analysed with a light microscope (Olympus BX60, Olympus,Tokyo, Japan). Representative photomicrographs were taken using the Promicra Quickphoto Camera 2.3 system (Prague, Czech Republic) for digital photomicrography, image editing and measurements.
All tested animals were observed daily during the experimental period, and examined for general toxicity signs: changes in the skin and fur, eyes and mucous membranes, excreta, behavioral pattern, and feed and water consumption. At the end of experiment, all mice were sacrificed by cervical dislocation (after brief exposure to ether), and macroscopic examination of internal organs was performed.
Microbial analysis of faecal samples
Bacterial translocation
Faecal samples were collected before the beginning and after the end of treatment with L. casei 5s and analysed the same day for the number of lactobacilli and E. coli. The total number of lactobacilli was determined by plate count on MRS agar, after anaerobic incubation at 37 °C for 48 h. For determining the total number of viable E. coli MacConkey agar (MCA; Merck) was used, and aerobic incubation at 35 °C. Individual colonies from MCA were transferred into MacConkey broth (MCB; Merck) and incubated at 43 °C for 24 h. Growth of red, non-mucoid colonies of gram-negative rods, subcultured on plates for 24 h at 35 °C, indicated the possible presence of E. coli, which was confirmed by the indole production test and API E system (European Pharmacopeia, 2011).
In the same animals, translocation of bacteria to blood and tissues was assessed according to Zhou et al. (2000). Briefly, one drop of blood (50 µl) was placed onto the surface of MRS agar plates and incubated at 37 °C anaerobically to test for bacteriemia. The excised spleen, liver and kidney were homogenised in 5 ml of sterile saline solution, and 100 µl of tissue suspensions plated on MRS agar plates. The plates were then incubated anaerobically at 37 °C for 48 h.
Adhesiveness to intestinal mucosa The adhesiveness of L. casei 5s cells to the intestinal mucosa was investigated in another group of 4 male Wistar rats, body mass of 180-220 g, which were treated intragastrically with L. casei 5s (1×109 cfu/ml) once daily for 7 days. After 122
Statistical analysis The levels of cholesterol and triglycerides in the serum, as well as faecal bacterial counts are presented as mean ± standard deviation for all groups. The effect of L. casei 5s was evaluated by two-way analysis of variance (ANOVA) with group as the between-subject factor and time (before and after treatment) as the within-subject (repeated measure) factor, followed where appropriate by post hoc Fisher’s least significant difference (LSD) test. Before statistical analysis, normal distribution of data was assessed using Kolmogorov-Smirnov test. Beneficial Microbes 6(1)
Probiotic properties and safety of L. casei isolated from Serbian cheese
3. Results
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Characterisation and provisional identification Our laboratory isolate, denoted as 5s, was Gram-positive, non-sporulative, catalaze negative, gelatinase-negative, non-haemolitic bacilli growing on MRS agar, in anaerobic, microaerofilic and aerobic conditions, indicating that it belongs to the Lactobacillus genus. According to its fermentative characteristics, the isolate showed greatest similarity to bacteria belonging to L. casei/ Lactobacillus paracasei: glycerol–, erytrol–, D-arabinose–, L-arabinose–, ribose+, D-xylose–, L-xylose–, D-adonitol–, metil-β D-xylopyranoside–, galactose+, glucose+, fructose+, manose+, sorbose–, rhamnose–, dulcitol–, inositol–, manitol+, sorbitol+, metil-α D-manopyranoside–, metil-α D-glucopyranoside–, N-acetylglucosamine+, amygdaline+, arbutine+, esculin+, salicin+, D-celobiose+, D-maltose+, D-lactose+, D-melibiose–, D-saccharose+, D-trehalose+, inuline+, D-melecitose+, D-rafinose–, amidon–, glycogen–, xylitol–, gentiobiose+, D-turanose+, D-lyxose–, D-tagatose±, D-fucose–, L-fucose–, D-arabitol–, L-arabitol–, potasium gluconate–, 2-keto-gluconate–, 5-keto-gluconate–. Subsequently, isolate 5s was more accurately identified to the species level by partial sequencing of 16S rRNA genes. According to the nucleotide sequence, showing 97% similarity, the isolate was identified as L. casei, and marked among our laboratory isolates as L. casei 5s.
Acid and bile salt tolerance of L. casei 5s To explore the potential probiotic properties of L. casei 5s, we tested bacterial viability in artificial gastric and intestinal juice, adhesion to hexadecane, antimicrobial activity and
susceptibility to antibiotics. L. casei 5s showed a high degree of survival in AGJ and in the solution containing 0.5% bovine bile salts. After 120 min exposure to AGJ, the count of viable cells decreased by 1.99 log units (from 6.96×109 to 7.11×107 cfu/ml). The cells that survived AGJ conditions were then exposed to the bovine bile salt solution; after 120 min exposure, the number of viable cells decreased by 0.86 log units (from 7.11×107 to 9.68×106 cfu/ml).
MATH test The percentage of adherent L. casei 5s cells to the hydrophobic solution of hexadecane was 47.26±2.3, indicating intermediate hydrophobicity of the cells.
Antimicrobial activity The antimicrobial activity of L. casei 5s is shown in Table 2. Both complete culture and cell free filtrate of L. casei 5s were able to inhibit the growth of S. aureus ATCC 6538-P, E. coli ATCC 8739, P. aeruginosa ATCC 9027, S. abony NTCC 6017 and C. sporogenes ATCC 19404, but failed to inhibit B. cereus ATCC 11778 and C. albicans ATCC 10231. The strongest antimicrobial activity was against S. aureus. After neutralisation of overnight cell-free filtrate, no zone of inhibition was detected, indicating that antimicrobial activity of L. casei 5s is related to organic acids, mainly lactic acid (data not shown).
Sensitivity to antibiotics L. casei 5s showed sensitivity (S) to chloramphenicol, ampicillin, streptomycin, rifampicin, neomycin, kanamycin, erythromycin, novobiocin (zone of inhibition ≥21 mm); intermediate (I) sensitivity to tetracyclin, lincomycin, penicillin and gentamicin (zone of inhibition 16-20 mm); it was resistant (R) only to vancomycin (no zone of inhibition).
Table 2. Antimicrobial activity of Lactobacillus casei 5s.1 Indicator strain
Staphylococcus aureus ATCC 6538-P Bacillus subtilis ATCC 6633 Bacillus cereus ATCC 11778 Escherichia coli ATCC 8739 Pseudomonas aeruginosa ATCC 9027 Salmonella abony NTCC 6017 Candida albicans ATCC 10231 Clostridium sporogenes ATCC19404 1
Diameter of growth inhibition zone (mm) Overnight culture
Cell free filtrate before neutralisation
Cell free filtrate after neutralisation
18 12 0 12 12 12 0 12
18 12 / 10 12 12 / 12
0 0 / 0 0 0 / /
Mean values from two experiments are presented. 0: no zone of inhibition; /: not tested.
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In vivo hypolipidemic effect of L. casei 5s The effect of assigned diets on serum cholesterol and triglyceride levels are presented in Table 3. For both parameters, two-way ANOVA revealed a significant main effect of group (cholesterol: F(3,12)=108.86, P
