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Journal of Oleo ScienceCopyright ©2020 by Japan Oil Chemists’ Societydoi : 10.5650/jos.ess20224J. Oleo Sci. 69, (12) 1579-1584 (2020)

Probiotic Properties of a Lactobacillus fermentum Isolated from New-born FaecesSamet Kocabay1 and Serap Çetinkaya2＊

1 Department of Molecular Biology and Genetics, Faculty of Science and Art, Inonu University Malatya, TURKEY2 Department of Molecular Biology and Genetics, Science Faculty, Sivas Cumhuriyet University, Sivas, TURKEY

1 IntroductionProbiotics are described by Food and Agriculture Orga-

nization of the United Nations/World Health Organization as “living microorganisms that when consumed with ade-quate amounts positively contribute to the health of their host through some adjustments in the microbiota of large intestine1, 2）. Lactic acid bacteria（LAB）are one of the most important group of probiotic bacteria, including Lactoba-cillus, Lactococcus, Carnobacterium, Enterococcus, Streptococcus, Pediococcus, Propionibacterium, and Leuconostoc3, 4）. Their applications can be traced through-out the history5）. They have been used to produce fer-mented food products such as bread, yogurt, cheese6）. They also play a crucial role in the establishment of a healthy gut microflora by fighting the pathogens7－9）.

LABs are Gram positive, facultative-anaerobe, catalase-negative, and motile organisms10）. They play an important role in food preservation through the production of antimi-crobial agents such as lactic acid, diacetyl, hydrogen per-oxide, and bacteriocins11）.

The marketing size of the probiotics has reached USD 46.55 billion in 2020 and is estimated to reach USD 64.02 billion by 202212）. Further expansion of the market size depends on the increasing of the global health awareness on probiotic consumption12）. It is recommended that 100g/d of Lactobacillus acidophilus, Bifidobacterium anima-lis ssp. lactis, Lactobacillus casei should be consumed per day13, 14）. Aging and the use of antibiotics are two fun-damental factors that reduce the number of probiotics in the large intestine10）.

＊Correspondence to: Serap Çetinkaya, Department of Molecular Biology and Genetics, Science Faculty, Sivas Cumhuriyet University, Sivas, TURKEYE-mail: serapcetinkaya2012@gmail.comAccepted August 26, 2020 (received for review August 12, 2020)Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 onlinehttp://www.jstage.jst.go.jp/browse/jos/　　http://mc.manusriptcentral.com/jjocs

LABs also exert some therapeutic properties against some types of cancer15）. They have been the centre of in-terest due to their ability to modulate cancer cell’s prolifer-ation and apoptosis. These features can be summed up as bacteriotherapy which has serious implications in the de-velopment of alternative therapies to replace harmful chemo- or radiotherapy16）. Other bacteria also seem to have therapeutic potential against cancer17）.

This work indicated that the Lactobacillus fermentum isolate can be a good probiotic candidate and that faeces of the breast-fed new-borns could be a rich source for the iso-lation of good probiotics.

2 Materials and MethodsMRS culture medium, agar（Sigma-Aldrich）. Pepsin

（Sigma-Aldrich）, bile salts（Sigma-Aldrich）, trypsin（Sigma-Aldrich）, antibiotic discs（Sigma-Aldrich）. The other all chemicals are analytical grade.

2.1 Isolation and identi�cation of L. fermentumL. fermentum was isolated from a faecal sample which

had been collected for a previous research18）and kept at －80℃. An overnight cell culture was obtained by inocula-tion an aliquot of the sample into 10 mL MRS（1％ peptone, 1％ meat extract, 0.5％ yeast extract, 2％ glucose, 0.2％ K2HPO4, 0.5％ sodium acetate, 0.2％ tri-ammonium citrate, 0.02％ MgSO4・7H2O, 0.005％ MnSO4・4H2O, pH 6.3）, and incubated at 37℃. Single bacterial colonies were grown

Abstract: Lactic acid bacteria (LAB) have been demonstrated to have roles in many applications, ranging from lowering of cholesterol to immunological development. In this study, Lactobacillus fermentum was isolated from a new-born’s faeces and its genotypic and probiotic characterizations were performed. Our results showed that the survival rate of isolated Lactobacillus fermentum was 39.39% at pH 2 and 81.34% in the stimulated gastric juice at pH 3. It also digested bile salts. Its surface hydrophobicity was found to be 57.59% in n-hexane. These findings indicated that the isolate can be a good probiotic candidate.

Key words: faecal sample, isolate, Lactobacillus fermentum, new-born, probiotic

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from the overnight culture by using the pour-plate method. One hundred of the colonies were then transferred onto MRS-agar plates, containing 0.5％ starch. The colonies producing clear zones around them were retransferred into fresh starch-agar media until the isolates were purified. The pure cultures with amylolytic activity were stored at －80℃. For the molecular characterisation, DNA was pre-pared and used for the amplification of 16S rRNA gene by using the method described in Bulut et al.（2005）19）. The sequences obtained from an automatic DNA sequencer were subjected to BLAST analysis and similarities were de-termined using the National Center of Biotechnology Infor-mation databases（http://www.ncbi.nlm.nih.gov）20）.

2.2 Survivals of bacterial strain at in low pHAccording to our previously published articles21, 22）the

Lactobacillus isolates were grown overnight in 10 mL sterile MRS broth（1％ peptone, 1％ meat extract, 0.5％ yeast extract, 2％ glucose, 0.2％ K2HPO4, 0.5％ sodium acetate, 0.2％ tri-ammonium citrate, 0.02％ MgSO4・7H2O, 0.005％ MnSO4・4H2O, pH 6.3）with shaking at 110 rpm at 37℃. Ten milliliters of the bacteria were centrifuged for 10 min at 4,000 rpm at ＋4℃. The pellet was suspended in fresh MRS broth（pH 6.3, pH 4, pH 3, pH 2）. Bacterial sur-vival at pH 6.3, pH 4, pH 3, pH 2 was determined. Sequen-tial dilution（1012-fold）was performed in sterile 4.5 mL NaCl（0.85％）. One hundred microliters of the last two dilutions were inoculated by the pour-plate method. The cells were then incubated overnight at 37℃. The colonies grown were counted and CFU/mL was counted and plotted. Survival rate was calculated by using the following formula:

Survival rate ％＝（log CFU N1/log CFU N0）×100％

（N1＝Total number of the cells survived after each of the pH treatments, N0＝Total number of alive cells before the treatment）.

2.3 Tolerance to stimulated gastric acid juiceThe isolate was treated with stimulated gastric juice23）.

Fresh stimulated gastric juice solutions were prepared, in-cluding 3 g/L pepsin in 1x PBS at differing pH points（pH 2, 3, and 4）. The solutions were filter-sterilized. Cell pellets of a 10 mL bacterial culture were suspended in each of these solutions. Treatment lasted for 5 h. After this, 0.5 mL of the samples were spread on agar plates after a serial dilu-tion step, and cell survival rate was estimated by the number of colonies, which were obtained the following day:

Survival rate ％＝（（log CFUN1/ log CFUN0）×100

（N1＝Total bacterial number in stimulated gastric juice N0＝Total bacterial number at first time in stimulated gastric juice）.

2.4 Tolerance to stimulated intestinal juiceThe isolate was were examined in stimulated intestinal

juice24）. Fresh stimulated intestinal juice was prepared 1x PBS, pH 8, and supplemented with 1 g/L trypsin. The solu-tion was sterilized using the 0.20 nm filters. Bacterial sur-vival rate was monitored for 24 h with 5 h intervals. Surviv-al rate was estimated by using the formula below:

Survival rate ％＝（log CFUN1/ log CFUN0）×100

（N1＝Total bacterial number after 24 h in stimulated in-testinal juice, N0＝Total bacterial number after 5 h in stim-ulated gastric juice）23）.

2.5 Tolerance to bile saltsThe bile tolerance of the isolate was assessed as de-

scribed25）. Ox-bile salt solutions（0.5％, 1％, and 2％）were prepared in MRS broth, pH 6.3. The survival rate was moni-tored for 4 h with 1 h intervals, and assessed as described above.

2.6 Antibiotic sensitivityDisc diffusion method was used26）. Fresh agar（1.5％）

medium was prepared and 100 μL bacterial culture was taken and spread on the agar plates. Antibiotic discs in-cluded kanamycin K 30, ampicillin AM 10, streptomycin S 10, tetracycline TE 30, gentamicin CN 30, chloramphenicol C 30, penicillin P 2 units, erythromycin E 15, rifampin RA 5, neomycin N 30, vancomycin VA 30, and a negative control 00. The antibiotic discs were placed on the agar surface. After overnight incubation under the optimum growth conditions, dimeters of inhibition zones were mea-sured with a ruler and the measurements were recorded.

2.7 HydrophobicitySurface hydrophobicity was investigated as described27）.

The isolate was grown overnight under the optimum condi-tions and the culture was divided into 3 mL aliquots in sterile falcon tubes. Cells were pelleted by centrifugation for 10 min at 4000 rpm at ＋4℃. The supernatant was removed and 5 mL of phosphate-urea-magnesium sulphate buffer, pH 6.5, were added. The pellet was re-suspended and the centrifugation step was repeated for 3 times. The initial cell densities were set to 1 at 450 nm and then 0.6 mL of n-hexadecane, n-hexane, xylene were gently added onto the cell suspensions（3 mL）. The mixed solution was placed in a water bath at 37℃ and incubated for 15 min. The samples were then vortexed for 2 min and left on the bench for 25 min or until a hydrocarbon layer formed. Ab-sorbance values were recorded at 450 nm. Percent hydro-phobicity was calculated by using the formula below:

Hydrophobicity ％＝ （（OD450nm N0－OD450nm N1）/ OD450nm N0）×100

（OD450nm N1: the absorbance value for final bacteria con-

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centration, after the experiment, OD450nm N0: the absor-bance value for initial bacteria concentration before the experiment）.

2.8 Degradation of Sodium SaltsThe isolate was grown on MRS agar overnight at 37℃.

Sodium salts, 0.005 g/mL,（sodium glycocholate hydrate, sodium taurodeoxycholate, sodium taurocholic acid）were prepared in MRS agar, pH 6.3. The colonies formed were immersed in the sodium salt solution and left for overnight incubation at 37℃. Salt break-down was checked and the results were recorded28）.

3 Results 3.1 Molecular identi�cation

A phylogenetic analysis based on 16S ribosomal RNA（rRNA）gene sequence comparison showed that the isolate belonged to Lactobacillus fermentum. The sequence was submitted to GenBank（Accession Number: MT796490）（Fig. 1）.

3.2 Survival rate at low pHSurvival rate up to pH 3 was the same as that of at pH

6.3. However, it decreased（39.39％）dramatically at pH 2 in a time dependent manner（Fig. 2）.

3.3 Survival rate in arti�cial gastric juiceSurvival rate was 16.57 log（CFU/mL）at pH 2. After the

3rd hour, it decreased to 5.55 log（CFU/mL）. In stimulated intestinal juices the survival rate was 1.6 log（CFU/mL）after 24 h at pH 8（Fig. 3）.

3.4 Bile toleranceThe survival rate was 21.9 ％ at 0.5％ bile concentration

after 4 h. Higher concentrations, especially 2％ led to the death of all the cells within one hour（Fig. 4）.

3.5 Antibiotic sensitivityThe isolate showed the relatively high susceptibility to

Fig. 1　Dendrogram produced by 16S rRNA gene sequence homology（http://www.ncbi.nlm.nih.gov）.

Fig. 2　Cell survival at low pH points.

Fig. 3　 Graphical representations of the survival rates in gastric juice.

Fig. 4　Survival rates in oxbile salts.

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chloramphenicol（C30）, erythromycin（E15）, rifampin（RA5）, vancomycin（VA30）, and tetracycline（30）（Fig. 5）.

3.6 Surface hydrophobicityThe isolate displayed 23.96％, 57.59％, and 63.94％ in n-

hexadecane, n-hexane, and in xylene, respectively（Table 1）.

3.7 Sodium salt degradationThe isolate decomposed three of the four sodium salts

（Table 2）.

4 DiscussionThe isolate 14 showed good survival rates at pH 6.3, pH

4, and pH 3. Similar results could be found in the litera-ture29）. These data suggested that LABs could be influ-enced negatively by high hydrogen concentration. The presence of abundant protons might interfere with the glucose fermentation and thus with the ATP synthesis30）.

As probiotics are generally consumed orally, they must be resistant low pH in order to survive through the stomach. Therefore, high survival rates have generally

been accepted as the primary criterion. The survival rates in intestinal environment were 28.82％, 58.14％, 30.91％ at pH 2, pH 3, and pH 4, respectively. These results were also compatible with those of the literature31）. The isolate dis-played 21.9 ％ survival rate at 0.5％ bile concentration after 4 h. In the literature only 0.1％ bile concentration have been used and this made the comparison difficult.

Antibiotic sensitivity is another important feature of pro-biotics as resistance is the negative aspects of all the mi-croorganisms. Horizontal transfer of the resistance genes in the gut flora to the pathogenic bacteria is possible. The tested microorganism showed differing amounts of resis-tance to the antibiotics used. Lactic acid bacteria are gen-erally known to be sensitive to chloramphenicol31－34）and to vancomycin34－36）.

Surface hydrophobicity is another important property which determines the capacity for cellular attachment between bacteria and to the mucosal cells of the intes-tine37）. Attachment to the epitel cells of the gastrointestinal track renders a survival advantage to the microorganisms38,

39）. The highest hydrophobicity percentage was obtained in xylene. In other studies, hydrophobicity values have varied greatly, ranging from 16.90 to 96.62％40, 41）. The surface hy-drophobicity is also important in the decomposition of milk lipids42）in yoghurt industry.

Degradation of sodium salts by probiotics is one of the key ways of controlling the cholesterols levels in the host organism. The tested isolate degraded sodium glycolcho-late hydrate, sodium taurodeoxycholate, and sodium tauro-cholic acid salts. These data were also compatible with those of the literature43, 44）.

5 ConclusionThe tested isolate of Lactobacillus fermentum ap-

peared the possess many of the desired characteristics of a good probiotic strain. This isolate might deserve in vivo studies with laboratory animals in the near future.

Fig. 5　 Histogram representation of antibiotic sensitivity results.

Table 1　 Surface hydrophobicity percentages of isolate X in three organic solvents.

Surface Hydrophobicity of Lactobacillus fermentum

Nonpolar solvents n-Hexadecane n-Hexane Xylene Hydrophobicity (%) 23.96 57.59 63.94

Table 2　Degradation capacity for sodium salts.

Degradation of Sodium Salts by Lactobacillus fermentum

Sodium SaltsSodium

glycocholate hydrate

Sodium tauro- deoxycholate

Sodium taurocholic acid

Sodium tauroglycocholate

Isolate 14 ＋ ＋ ＋ －

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AcknowledgementThis work（F-498）was supported by CUBAP, Sivas Cum-

huriyet University.

Conflicts of InterestThe authors declare that there are no conflicts of inter-

est.

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