|Livestock Research for Rural Development 31 (7) 2019||Guide for preparation of papers||LRRD Newsletter||
Citation of this paper
This paper describes the potential probiotic properties of a lactic acid bacterium. The evolution from a spontaneous fermentation to a directed one is realized with selected lactic starters which give many dairy products processing various organoleptic characters. The integration of new lactic acid bacteria strains isolated from diverse ecosystems is now used to increase the duration of bio-preservation of dairy products. Moreover, some lactic acid bacteria probiotic activity is exploited to produce functional food. The aim of this study is to research of the possible probiotic and technological potential with some preventive and therapeutic characteristics of some Lactobacillus species isolated from Algerian Raw Goat Milk. The selected isolate was identified to species level as Lactobacillus plantarum (58) using API 50CH Kits. Microbiological and biotechnological techniques were used to fulfill this work. Results obtained have shown that Lactobacillus plantarum (58) can resist to acidic, basic and enzymatic stresses. So, the former strain can be considered as a probiotic. Moreover, the inhibition activity of the Lactobacillus plantarum (58) against pathogens strains (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25921, Bacillus cereus, Pseudomonas aeruginosa, Vibrio cholerae, Listeria ivanovii ATCC 19119 and Salmonella enterica) is clearly obtained by testing these strains with the direct method. Finally, Raw Goat Milk can be defined as an ecosystem that promotes the development of a microflora with probiotic characteristics.
Keywords: antimicrobial activity, goat milk, Lactobacillus plantarum, pathogen, probiotic
Lactic acid bacteria (LAB) comprise a wide range of genera and include a considerable number of species. These bacteria are the major component of the starters used in fermentation, especially for dairy products, and some of them are also natural components of the gastrointestinal microflora. Lactobacillus is one of the most important genera of LAB (Coeuret et al 2003). During the last fifteen years, the Lactobacillus genus has evolved and contains to date more than 80 species. They are present in raw milk and dairy products such as cheeses, yoghurts and fermented milks (Coeuret et al 2003). Lactobacilli comprise a large and diverse group of gram positive, non- spore forming, catalase negative rod bacteria, able to produce lactic acid as the main end-product of the fermentation of carbohydrates (Pelinescu et al 2009; Malek et al 2013). They are considered as generally recognized as safe (GRAS) organisms and can be safely used as probiotics for medical and veterinary applications (Fuller 1989 ; Hatoum et al 2012 ; Eid et al 2016).
Probiotics, as defined in a FAO/WHO (2002) report, are “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Probiotics are beneficial bacteria in that they favorably alter the intestinal microflora balance, inhibit the growth of harmful bacteria, promote good digestion, boost immune function and increase resistance to infection (Helland et al 2004). Other physiological benefits of probiotics include removal of carcinogens, lowering of cholesterol, immunostimulant and allergy lowering effect, synthesis and enhancing the bioavailability of nutrients, alleviation of lactose intolerance (Parvez et al 2006). In order to exert their beneficial effect, probiotics must survive in the gastrointestinal (GI) tract, persist in the host, and prove safety for consumer (De Vries et al 2006). To survive in the gut, the organisms must be tolerant to low pH and bile toxicity prevalent in the upper digestive tract. Besides, quality assurance programs associated with research, development, production and validation of the health benefits of these bacteria require their relevant characterization and identification.
Over the world, the research of novel probiotic strains is important in order to satisfy the increasing request of the market and to obtain new functional products. These new functional products must contain probiotic cultures more active and with better probiotic characteristics comparing to those already present on the market (Siro et al 2008 ; Hill et al 2017). This work will be focused on the technological, antimicrobial and probiotic properties of Lactobacillus plantarum (58) isolated from raw goat’s milk.
Raw goats' milk samples were obtained from a commercial dairy-goat company. The milk was maintained at 5 °C until used. For comparison, a cows' milk sample (pooled from 60 cows, several breeds) was obtained from a local farm. Samples of raw goat milk were obtained from a local farm located in the town of Es-Senia (Oran, Algeria) (35°39' N ; 0°37' W ; Elevation: 92 m). The milk samples were derived from goats of the Makatia and Arabia breeds (pooled from 40 goats). The samples are realized and performed over a period of four years (2015-2018), under aseptic conditions and then brought to the laboratory for microbiological analysis the same day.
Nine pathogenic strains were used in this study: three strains of Staphylococcus aureus (ATCC 25923, ATCC 29213 and ATCC 43300), Listeria ivanovii ATCC 19119, Escherichia coli ATCC 25921, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter calcoaceticus and Salmonella enterica. These pathogenic strains were provided from Central Medical Laboratory of the University Hospital Centre (Oran, Algeria) (35°41' N ; 0°38' W ; Elevation : 116 m), except the Listeria strains that was provided from the Department of Analytical Chemistry, Nutrition and Food Sciences, University of Santiago de Compostela (Spain) (42°53' N ; 8°32' W ; Elevation : 238 m).
Twelve samples of raw goat milk were incubated at 37 °C until coagulation. Coagulated samples were then activated in MRS broth (De Man et al 1960) at 37 °C for 24h in order to obtain enriched cultures. This culture was streaked on MRS agar medium and incubated (Badis et al 2004 ; Badis et al 2006 ; Moulay et al 2006 ; Moulay et al 2013 ; Bendimerad et al 2012 ; Zarour et al 2013) under anaerobic condition using a candle extinction jar with a moistened filter paper to provide a CO2-enriched, water-vapor saturated atmosphere at 37 °C for 48 h. Single colonies picked off the plates were sub cultured in MRS broth at 37 °C for 24 h before microscopic examination. The cultures of Rod-shaped bacteria were streaked on MRS agar medium for purification (Carr et al 2002 ; Moulay et al 2006 ; Bendimerad et al 2012 ; Moulay et al 2013 ; Benmechernene et al 2013). Purified isolates were stores at −20 °C in sterile MRS broth supplemented with 20% glycerol. Additionally, 0.05% cysteine was added to MRS to improve the specificity of this medium for isolation of Lactobacillus (Hartemink et al 1997 ; Kihal et al 2009). Preliminary identification of the selected isolate.
The purified culture of the selected isolate was identified after investigating morphological and biochemical characters according to standard clinical laboratory methods reported and recommended by Sharpe (1979). The cultures were examined microscopically for gram staining and catalase production (Harrigan and Mac Cance 1976). In addition, all isolates were tested for growth at different temperatures 10°C for 7-10 days and 45 °C for 24-48 hours, growth at different NaCl concentration and CO2 production from glucose, under aerobic and anaerobic conditions. The pathogenic bacteria (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25921, Bacillus cereus) come from the collection of collected from the microbiology laboratory of the university hospital of Oran.
The carbohydrate fermentation profile of purified isolate was determined using API 50 CH system (Biomérieux, France). The API 50 CH (Biomerieux) gallery consists of 50 microtubes for the study of substrate fermentation, belonging to the family of carbohydrates and derivatives (glycosides, polyalcohols, uronic acids). Interpretation of these fermentation profiles were facilitated by systematically comparing all results obtained for the isolates studied with information from the computer-aid database ApiwebTM API 50 CH V5.1 software.
Synthesis gastric juices is prepared as described (Glucose: 3,5 g, NaCl: 2,05 g, KH2PO4: 0,6g, CaCl2: 0,11g, KCl: 0,37 g, Pepsin SIGMA®:13,3 g, Distilled water: sqf 1000 ml) and adjusted to (pH = 2) then conserved under freeze conditions (Fernandez et al 2002).
In order to obtain a rate of concentrations, fresh cultures of Lactobacillus plantarum are cultivated in MRS media (pH = 6.5) and incubated at 37 °C. Each two hours OD600 are evaluated to obtain 0.4, 0.5, 0.6, 0.7 and 0.8 of these cultures. Bacterial count of OD600 is obtained by following 1/100 dilutions and successive plating in MRS media (pH = 6.5) to so we can extrapolate bacterial concentrations on every OD600 (De Souza Oliveira et al 2009).
The antibiotic susceptibility of selected acid and bile tolerant isolates strain (Lb plantarum P6) was determined towards nine antibiotics, namely, penicillin G (6 µg/10 IU), Ampicillin (10 µg), Amoxicillin (20 µg), Erythromycin (15 µg), Tetracycline (30µg), Pristinamycine (15 µg), Rifampine (30 µg), Cefazoline (30µg) and Ciprofloxacine (5 µg). The Strain selection was based on their performance toward acid and bile salts. Antibiotic susceptibility was determined semi- quantitatively using a modification of the agar overlay diffusion methods of the National Committee for Clinical Laboratory Standards NCCLS (1993). Diameters of inhibition zones were measured and results were expressed in terms of resistance (R), intermediate susceptibility (I), and susceptibility (S), according to cut off levels proposed by Prescott et al (1999), NCCLS (2002) and Vlkova et al (2006).
Fresh culture of 18h Lactobacillus plantarum strain are harvested and centrifugated at 7000 rpm during 1min, pellets are inoculated in 500µL of synthesis gastric juices; inoculums are incubated at 37 °C for 30 min, 1h, 1h30 min and 2h (Fernandez et al 2002 ; Kim et al, 2006). Following this test, inoculums are centrifugated and pellets are immediately flooded with 500µL of MRS broth added with 0.5% bile salts and incubated at 37 °C during same times (Shima et al 2009). Afterwards, inoculums are once again harvested and auditioned with 500µL of MRS containing 0,1% of trypsin and α-Chymotrypsine and incubated at 37°C during same times (30, 60, 90 and 120 min) (Kim et al 2006 ; Ruiz-Moyano et al 2008). The final pellets are added to 500 µL of physiological water and diluted until 10-8, plated on MRS pH 7 and at 37 °C for 24h. Colony forming units (CFU) were enumerated after incubation at 37 °C for 24 h (Mami et al 2008 ; Boumehira et al 2011 ; Mami et al 2012).
Gelatinase activity of the most antibiotics sensitive isolates Lactobacillus plantarum (58) strain was investigated as described by Harrigan and McCance (1990). 2 µl of a 6h old culture was spot-inoculated into nutrient gelatin agar. The plates were incubated anaerobically for 48h at 37 °C after which they were flooded with saturated ammonium sulfate solution and observed for clear zones surrounding colonies (positive reaction for gelatin hydrolysis). A strain of Staphylococcus aureus ATCC 25923 was used as positive control.
Haemolysis activity of gelatinase negative isolates Lactobacillus plantarum (58) strain was investigated as described by Gerhardt et al. (1981). 2 µl of a 6 h old culture broth was spot- inoculated into sterile blood agar. The blood agar was prepared by adding 7% sheep-blood, that had been preserved in ethylenediaminetetraacetic acid (EDTA), into sterile blood agar base at 45 °C. Plates were incubated anaerobically at 37 °C for 48h after which they were observed for clear zones surrounding colonies (positive reaction for beta haemolysis). A strain of S. aureus ATCC 25923 was used as positive control.
Antimicrobial activity of the selected probiotic strain (Lb plantarum) was checked by using the agar-spot test (Mami et al., 2008; Mami et al., 2012). Isolates were screened for production of antimicrobial against the collected pathogenic strains; Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25921, Bacillus cereus, Pseudomonas aeruginosa, Vibrio cholerae, Listeria ivanovii ATCC 19119 and Salmonella enterica as the indicator microorganisms. An aliquot of 2 µl of a 6 h old producer isolate culture was spotted on MRS agar and plates were incubated anaerobically at 37 °C for 48 h to allow exhibition of antimicrobial compounds. Cell suspensions of the indicator microorganisms were prepared as follows: each 24 h old culture of the indicator strain on Mueller Hinton Agar Slant was suspended in sterile physiological saline solution (NaCl 0,9%) and the turbidity was compared to 0.5 Mc Farland (corresponding to108 CFU.ml-1).
50 µl of the cell suspension was inoculated in 5 ml of Plate Count Soft Agar and overlaid on colonies of producer isolates. After incubation at 37 °C for 24 h, plates were checked for zones of inhibition surrounding the producer colonies. Inhibition was recorded as positive if the width of the clear zone around the colonies of the producer was 2 mm or larger. The agar well diffusion technique was also used to discriminate antimicrobial activity of the selected probiotic isolates due to organic acid production. The method of Mante et al (2003) was adapted. Isolates were cultured overnight before assay. Bacterial cultures were prepared into cell supernatant (pH = 7). 50 μl sterilized free-cell neutralized supernatant was filled into the well against target microorganisms. After 24 h of incubation time, the diameter of the inhibition zone was measured and scored. The representation of inhibition zone was not included in 6mm diameter of well. The inhibition zone larger than 2 mm was scored positive.
Choice of effective strain and Preliminary identification This specie was chosen because of its capacity to inhibit a broad spectrum of undesirable bacteria. The identification is carried out thanks to the traditional methods established by various authors (Klein et al 1998 ; Kihal et al 1997 ; Carr et al 2002 ; Kihal et al 2009). The latter is identified to species level by microbiological and biochemical methods, as described by Stiles and Holzapfel (1997). The strains retained gave small colonies of approximately 1 mm of diameter, lenticular with a white or milky color, smooth surface and a regular circular circumference were observed on solid medium. The microscopic examination revealed that the tested strains were Gram positive, with a cellular Rod form associated in pairs or in chains (Table 1).
|Table 1. First identification characters of Lactobacilli isolates from raw goat’s milk|
|CO2 from glucose||-|
|CO2 from gluconate||+|
|Growth at 15/45||+/+|
|Group||Group II, Facultative Heterofermentary (Streptobacteria)|
One isolate selected according to their good probiotic potential was identified at phenotypic level as Lactobacillus plantarum (58) using API 50 CHL technique. This isolate was from samples of various origins. Furthermore, a genotypic method was essential to discriminate between strains. The establishment of the percentage of reliability of this strain in comparison with references Lactobacillus plantarum ATCC 14917 (Stiles and Holzapfel 1997 ; Klein et al 1998; Kihal et al 1997 ; Carr et al 2002).
|Table 2. Strain's carbohydrates fermentation patterns on API 50 CHL kits|
|D- Lactose 1||+||+||V|
|MDX : Methyl-βD-Xylopyranoside, MDM : Methyl-αD-Mannopyranoside, MDG : Methyl-αD-Glucopyranoside, NAG: N-AcetylGlucosamine, ESC : Esculine citrate de fer, 2KG : Potassium 2-cetogluconate, 5KG : Potassium 5 cetogluconate, GNT : Potassium Gluconate|
Microbiological characteristics and fermentation profile of the strain Lactobacillus plantarum (58) and of the reference strain Lactobacillus plantarum ATCC 14917 are 100% identical. This similarity coefficient is calculated between (58) strain and the reference strain, what guides the identification of the strain (58) to the species Lactobacillus plantarum. Bile salt tolerance.
After exposure to acidic conditions, one selected acid tolerant Lactobacillus isolate was assayed for bile salt tolerance (Table 2). This isolate demonstrated good capacity to resist bile salts by presenting surviving percentage greater than 50% under exposure to 0.2% bile salts after 24h at 37°C. This isolate was further investigated for their safety properties including sensitivity to antibiotic, hemolysis and gelatinase activity.
Table 3. Surviving percentage of
lactobacilli isolates in MRS broth supplemented
with 0.2% or 0.5% bile salts after 6 and 24 h at 37 °C
|Tested strains||Bile salt concentration|
|6 h||24 h||6 h||24 h|
|Lactobacillus plantarum (58)||87,4||92,8||52,6||90,6|
|Lactobacillus plantarum (ATCC 14917)||85,3||88,1||51,7||85,4|
One potentially probiotic Lactobacilli isolates were subjected to antibiotic susceptibility testing using the agar diffusion method (Table 3). All of them were sensitive to Penicillin, Ampicillin, Amoxicilline, Tetracycline, Pristinamycine, Erythromycin and Rifampine. The isolate Lb. plantarum (P6) demonstrated intermediate resistance to cotrimoxazole. Notable observation is the resistance towards Ciprofloxacin expressed by this isolate.
|Table 4. Susceptibility of potentially probiotic Lactobacilli isolates to antibiotics using the disc diffusion method|
|Isolates||Diameter of inhibition zone in mm|
|Lb. plantarum (P6)||35(S)||33(S)||30(S)||25(S)||26(S)||29(S)||28(S)||0(R)||18(I)|
|Lb. plantarum (ATCC 14917)||28(S)||33(S)||29(S)||21(S)||20(S)||26(S)||22(S)||0(R)||10(I)|
Antibiotics (Disk potency) : P10 :
Penicillin G (10 units) ; Ap10 : Ampicillin (10 µg) ; Am10 :
Amoxicilline (10 µg) ;
T30 : Tetracycline (30 µg) ; PT15 : Pristinamycine (15 µg) ; E15 : Erythromycin (15 µg) ; RA30 : Rifampine (30µg) ;
C5 : Ciprofloxacine (5µg) ; CZ30 : Cefazoline (30 µg). (S) : sensitive ; (R) : resistant ; (I) : intermediate
The results obtained for the aptitude of the strain Lactobacillus plantarum (58) to resist acidic, basic and enzymatic stresses show clearly that this specie can be regarded as probiotic micro-organism (Perea Velez et al 2006). The concentration at the end of the treatments of Lactobacillus plantarum (58) is 8.5 log ufc/ml, knowing that according to Salminen et al (2006), the concentration of administration of a probiotic micro-organism must be 107 or 10s and that according to the definition of FAO/WHO (2002) probiotic must be administrated with a well-defined concentration so that it can achieve these beneficial effects for its host. In general, a probiotic micro-organism is never administrated in the form of bacterial cream, it is added as an adjuvant in agroalimentary products (Champagne and Gardner 2008 ; Russo et al 2017) or in the form of capsules containing these freeze-dried micro-organisms (Corcoran et al 2003). These methods allow, even if the species considered as probiotic resists the gastro-intestinal environment, to increase their chances to arrive in sufficient quantity at the level of intestine (Pan et al 2009).
|Table 5. Susceptibility of potentially probiotic Lactobacilli isolates to antibiotics using the disc diffusion method|
|Lb. plantarum (P6)||OD600||Time (minute)|
|30 min||60 min||90 min||120 min|
|Growth (Log CFU.ml-1)|
The potentially probiotic Lactobacillus isolates was assayed for gelatinase activity and are hemolysis. It showed no activity of gelatinase and positive hemolysis compared to the positive control Staphylococcus aureus ATCC 25923 strains.
Results for antimicrobial activity of Lactobacillus plantarum (58) probiotic was as shown in Table 4. Isolate inhibited the growth of all pathogenic strains when agar spot method was used. It was also noticed that, the neutralized free-cell supernatant from the culture of the Lactobacillus plantarum (58) inhibited the growth of all pathogenic indicators.
Table 6. Inhibitory activity of potentially probiotic lactobacilli isolates
Lb. Plantarum (58)
Lb. Plantarum (58) (mm)
Staphylococcus aureus ATCC 25923
Escherichia coli ATCC 25922
Listeria ivanovii ATCC 19119
+: Diameter of inhibition zone ≥ 2mm; -: No inhibition; MDR: Multi Drug Resistant
Lactobacillus plantarum (58) inhibit the growth of pathogens, displayed BSH activity by providing dosage. Lactobacillus plantarum (ATCC 14917) has an activity of average diameter of BSH demonstrating the precipitation zone of 10 mm and Lactobacillus plantarum (58) shows a strong activity of BSH by expressing the area of diameter greater than 15 mm the zone precipitation around the colonies on the plate.
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Received 20 April 2019; Accepted 24 April 2019; Published 2 July 2019
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