Enzymatic and functional properties of lactic acid bacteria isolated from Algeria fermented milk products.
The role of fermented milk in human nutrition is well documented and the virtues of these products were known to man even during the ancient days of civilization. Fermented milk provides a wide range of important nutrients and contains different components that affect one or a limited number of functions of the body in a positive way. These products represent an important component of functional foods, and intense research efforts are under way to develop dairy products into which probiotic organisms are incorporated to make them more valuable.
Several types of fermented milk products have been reported to exist throughout the world (Stanley, 1998; Tamime, 2002).The most popular of them in North African are Jben, Lben, Klila and Raib, (Mechai et al., 2014; Benkerroum, 2013).
Jben a soft variety cheese is made in the mountainous area of eastern Algeria (souk ahras, Guelma, Tebessa, Khanchla and Batna) of cows' milk, with sometime additions of goats' and sheep's milk from January to March, although some manufacturers who provide a wider market use rennet to coagulate the milk just a few hours after milking. Traditional protocol includes rennet coagulation of raw whole cow's milk; to which a salt was added in the proportion of 10-20 NaCl per litre of milk at room temperature for 2 to 15 d (Benkerroum and Tamime, 2004). Klila a hard variety cheese is made from raw milk by heating whey of curd at 65[degrees]C for 30 minutes without using a starter culture of lactic acid bacteria. Then they obtained curd is sieving through a muslin doth or straw basket to discard whey (Benkerroum, 2013).
Raib and its by-products (Lben and zebda) are traditional fermented dairy products, still widely produced and consumed in Maghreb countries (Morocco, Tunisia, and Algeria) (Benkerroum, 2013; Mechai and Kirane et al., 2008). This traditional fermented dairy product is obtained after spontaneous curdling of raw milk within 24 to 36 h at ambient temperature; however Lben (buttermilk) is obtained by churning spontaneously soured milk to remove butter.
Lactic acid bacteria (LAB), such as lactococci and lactobacilli, play an essential role in the manufacture of cultured dairy products such as cheese. They are primarily responsible for the production of lactic acid from lactose, the degradation of casein and, in some instances, the production of antimicrobial agents. In this way they contribute to optimal curd formation, to the exclusion of undesired spoilage bacteria and to the development of the desired texture and flavour of the cheese (Asmahan, 2010).
LAB are the most commonly used microorganisms in fermented foods. Their crucial importance is associated mainly with their physiological features such as substrate utilization, metabolic capabilities and probiotic properties. Their common occurrence in foods coupled with their long historical use contributes to their acceptance as GRAS (Generally Recognized As Safe) for human consumption (Parmjit, 2011; Silva et al., 2002).
It has been demonstrated that LAB are important in dairy products specially in fermented milk processing because (i) they increase food safety through the release of lactic acid and bacteriocins, (ii) produce aromas and flavor and accelerate the maturation process of cheese via their proteolytic and lipolytic activities, bringing economic advantages to the industry, (iii) bring about desirable food textures via release of polysaccharides that increase the viscosity and firmness, and reduce susceptibility to syneresis, (iv) they may be used to deliver polyunsaturated fatty acids and vitamins, leading to dairy products with increased nutritional value (Parvez et al, 2006).
Proteolytic, caseinolytic, lipolytic, and a-amylase, activities have been detected in various bacteria, but few studies on these enzymatic activities in fermented milk products LAB have been reported (Piraino et al., 2008).
The aim of this study was to evaluate the enzymatic profiles and antimicrobial activity of LAB strains to better understand their role during the fermentation process as well as to identify particular properties that may be relevant to use of the organisms in starter cultures.
MATERIAL AND METHODS
Collection of samples:
Altogether thirty eighth products were used, including a variety of different artisanal fermented milk, it consisted of Sixteen sample of fermented skimmed milk (Raib and Lben) and twenty two samples of Traditional cheeses (Jben and Klila) were sampled in five different regions of north-east of Algeria. For all solid samples (Jben and Klila), 1 g of sample was added to 9 ml of sterile diluent (0.1% peptone, 0.85% NaCl) and homogenized by vortex mixing. For liquid samples (Raib and Lben), 1 ml of sample was added to 9 ml sterile diluent and homogenized by vortex mixing.
Phenotypic characterization of LAB strains:
Cell morphology was observed by using phase contrast microscopy at 1000* magnification and isolates were Gram-stained and catalase activity was deter-mined. Presumptive LAB were tested for growth at different temperatures (10[degrees]C, 15[degrees]C and 45[degrees]C), and at different pH (3.9 and 9.6), as well as the ability to grow in different concentrations of NaCl (6.5, 10 and 18 %) in MRS broth and production of gas from glucose in MRS broth as described by Schillinger and Lucke (1987) and Dykes et al. (1994). The configuration of D (-) and L (+) isomers of lactic acid produced from glucose was determined by enzymatic method (Boehringer Mannheim, 1989). Sugar fermentation patterns of LAB were determined using API 50 CHL test strips (bioMerieux, France) andthe result was obtained using the APILAB PLUS data-base identification software (bioMerieux, France). Strains were tentatively designated to species using APILAB PLUS (Version 3.33, Bio-Merieux) and standard taxonomic descriptions from Wood and Holzapfel (1995), Axelsson (1993), Curk et al. (1996) and Schillinger and Lucke (1987). The isolates were stored at -30[degrees]C in MRS or M17 broth containing 10% glycerol.
Those LAB isolates sharing some of the screened properties were further identified by molecular biology. Total genomic DNA was extracted from overnight culture of bacteria isolates using Bacterial Genomic DNA extraction kit (SpinKlean Genomic DNA Extraction Kit, Canada) (Van Hoorde et al., 2008). Polymerase chain reaction (PCR) amplification of 16S rDNA was carried out using Taq PCR Master Mix Kit (Biomatik, Canada). An approximately 1500-bp fragment of the 16S rDNA was amplified by using the following universal primers 1492R (Bacteria/Archaea-specific) 5'-GGTTACCTTGTTACGACTT-3' and 27F (Bacteria-specific) 5'AGAGTTTGATCCTGGCTCAG-3'. The PCR reaction (Esco Swift MiniPro Thermal Cycler) mixture (20 [micro]l) consisting of 10[micro]l 2x PCR master mix (Biomatik, Canada), 1pl of each primer (2.5[micro]M), 6.5[micro]l nuclease free
water and 1.5 pl template DNA. The thermocycler program was as follows: 94[degrees]C for 1min; 30 cycles of 95[degrees]C for 30 s, 44[degrees]C for 30 s, and 72[degrees]C for 2 min; and a final extension step at 72[degrees]C for 4 min. The nested PCR products were analyzed by electrophoresis on a 1.0 % agarose gel stained with ethidium bromide in 1X TAE buffer at 100 V for 45min. The bands were visualized under UV trans-illuminator by GelCompar II (software Bio1D++).
The acidifying activity of the strains was determined in 10% reconstituted skimmed milk powder inoculated at a level of 1% (v/v) with bacterial suspensions revitalised in MRS broth at 30[degrees]C containing 109 cfu [mL.sup.-1]. After incubation at 30[degrees]C for 6, 12 and 24 h, pH and titratable acidity were measured in accordance with the International Dairy Federation (IDF) standard 306 (IDF, 1995).
The production of extra-cellular proteolytic enzymes was determined on PCA, supplemented with 10% skim milk powder. The plates were incubated at 37[degrees]C for 48 h. The presence of clear zones around the colonies was indicative of proteolysis. Area ([cm.sup.2]) of this zone was measured for each selected strain. Moreover, proteolytic activity of whole cells in milk was determined by using the O-phthaldialdehyde (OPA) spectrophotometric assay (Church et al., 1983). This test is based on the reaction of the free [alpha]-amino groups released by hydrolysis of the casein (after a 24 h period of incubation of the strains in the milk) with Ophthaldialdehyde, in the presence of P-mercaptoethanol, to form a complex which strongly absorbs at 340 nm. The results were calculated from a calibration curve obtained from dilution of glycine in distilled water and were expressed in mM Gly [L.sup.-1] of milk.
The caseinolytic activity of the CFE was determined by measuring absorbance of light at 280 nm at zero time and after 1 h incubation at 30[degrees]C while continuously shaking (Gomez et al., 1988). The reaction mixture consisted of 150mL of enzymatic extract and 1350 mL of substrate solution (Hammersten casein at a level of 2 g [mL.sup.-1]) in 50 mm[Na.sub.2][HPO.sub.4]/NaH2PO4 buffer (pH 7.0), sterilized at 90[degrees]C for 20 min.
One unit of caseinolytic activity was defined as the amount of enzyme required to give an increase in absorbance of 0.01 units at 280 nm after a hydrolysis period of 1 h. Specific activity was expressed as the number of caseinolytic activity units [mg.sup.-1] protein in the CFE.
Production of a-amylase:
To test for a-amylase production, a single streak of a test culture was made on modified MRS agar plates that contained 0.2% soluble starch instead of glucose. The plates were incubated at 30[degrees]C overnight, after which they were flooded with iodine. A colorless area around the growth indicated a positive test. Moreover the [alpha]-Amylase activity was assayed as described by Thapa et al. (2006). Briefly, strain to be tested was grown on broth medium (1.0% soluble starch, 1.0% beef extract, 1.0% peptone, and 0.3% NaCl, pH 7.0) on a rotary shaking incubator at 30[degrees]C at 180 rpm for 48 h. The cultures were immediately centrifuged at 17,000 rpm for 10 min. The enzyme solution was diluted to an appropriate concentration. The enzyme solution and 1.5% soluble starch dissolved in 100 mM Tris-HCl buffer (pH 7.0) were pre-incubated separately at 37[degrees]C for 5 min in waterbath shaker. Then, the reaction mixture was started by adding 1 ml of 1.5% soluble starch to 0.5 ml enzyme solution and incubated at 37[degrees]C for 10 min. Reaction was stopped by addition of 2.5 ml of stop solution (0.5 N acetic acid-0.5 N HCl, 5:1). The 100 ml of the reaction mixture was added to potassium iodide solution, left at room temperature for 20 min and the absorbance at 660 nm of the resulting solution was measured in UV-VIS Spectrophotometer. One unit of a-amylase activity was defined as the amount of a-amylase which produced 10% reduction in the intensity of blue colour at the above condition.
Tested strains were grown overnight at 37[degrees]C in MRS broth. A loopful fresh culture was placed on Tributyrin Agar (Leuschner et al., 1997). Plates were incubated at 37[degrees]C for 4 days and observed daily for halo formation around the colonies. The radius of the halo formation (in mm) at the end of incubation was measured. Moreover lipolytic activity was measured according to Mauriello et al., (2004), 1 ml of an overnight culture of each strain was inoculated in 10 ml of MRS broth supplemented with 4% (w/v) of butter fat in order to pork fat. After incubation at 37[degrees]C for 7 days, the lipolytic activity was measured by titration. The lipid fractions were extracted by adding 10 ml of petroleum ether (Merck) and shaking for 1 min. The free fatty acids of the upper phase (lipid extract) were titrated with 0.1 N NaOH in ethanol using 1% phenolphthalein ethanol as indicator. The lipolytic activity was determined by the following equation:
Lipolytic activity = (a x N x 28.2) / g
Where "a" is ml of NaOH used in the titration, "N" is the normality of NaOH, "28.2" is the percent equivalent weight of oleic acid and "g" the weight of fat in the sample.
Enzymatic profile by API-zym system:
Enzymatic activities of selected LAB strains were assayed using the API- ZYM (bioMerieux, France) galleries as described by the manufacturer. Each strain tested was grown on MRSA plates at 37[degrees]C for 24 h. Colonies were then removed from the media and suspended in 2 ml of sterile distilled water, to produce a dense suspension (corresponding to [10.sup.8] ufc [ml.sup.-1]). The cell suspensions from each strain were used to inoculate the cupules of the API ZYM strips. These latter were then inoculated at 37[degrees]C for 4 h. The reaction was carried out by the addition of the API ZYM reagents (ZYM A and ZYM B). The enzymatic activity was graded from 1 to 5 according to the colour reaction chart. The approximate number of free nmol hydrolyzed substrate may be obtained from the colour intensity, 0: no activity; 1: liberation of 5 nmol; 2:10 nmol; 3: 20 nmol; 4:30 nmol and 5: [greater than or equal to] 40 nmol (Papamanoli et al, 2003).
Screening for antibacterial activity:
Isolated colonies of the assumed LAB were screened for antimicrobial-producing activity essentially using the spot method as described by Spelhaug and Harlender (1989). An overnight culture of the test organism grown in MRS broth supplemented with 2.5% yeast extract (MRSY) was diluted 10-fold in 10 mmol [l.sup.-1] Tris HCl (pH 7.0), and 2 ml aliquots were spotted onto MRS agar. Plates were incubated for approximately 24 h, until growth was evident, and then overlaid with 5 ml Trypticase soft agar (0.7% agar) seeded with 0.1 ml of an overnight culture of L. monocytogenes ATCC7644. Plates were incubated for an additional 18 h, and then checked for clear zones around spots of the putative producers.
Sensitivity to heat, pH, and hydrolytic enzymes:
Samples of crude bacteriocin were used for these tests. Aliquots of the semi-purified bacteriocin were exposed to heat treatments of 65[degrees]C for 40 min, 95[degrees]C for 20 min, 100[degrees]C for 20 min and 121[degrees]C for 20 min, and then were tested for remaining antimicrobial activity. Semipurified preparations of the bacteriocin were adjusted to various pH values in the range of 2 to 12. The pH-adjusted bacteriocin samples were incubated at 37[degrees]C for 20 min and then neutralized to pH 6 and tested for bacteriocin activity.
The following enzymes were tested for their hydrolytic activity on the antimicrobial compounds contained in the supernatants: protease K (2.6 U [mg.sup.-1]), Trypsin (22 U mg-1), pepsin (16U [mg.sup.-1]), catalase (adjusted to a final activity of 2600 U mg-1), lipase (50 U[mg.sup.-1]), and a-amylase (15 U [mg.sup.-1]). The assays were performed at a final concentration of 0.5 mg ml-1 and at pH 6.5, except for pepsin (pH 3.0). Samples with and without enzymes were held at 35[degrees] C for 6 h and the remaining activity was determined by well-diffusion assay as described before using L. monocytogenes ATCC7644 as indicator strain.
RESULATS AND DESCUSSION
108 out of 208 isolates from 36 sample of traditional fermented milk collected in five different regions of north-east of Algeria were identified to LAB based on their Gram reaction, morphology and catalase test.
All strains were recorded as catalase negative and Gram-positive cocci in pairs or long chains, bacilli in pairs or chains and cocobacilli. Of all the isolates, only 12 representative strains were selected randomly from each group on the basis of cell morphology, gas production from glucose and arginine hydrolysis (Table 1) for further phenotypic characterisation including determination of the sugar fermentation pattern, lactate configuration and DAP following the taxonomical keys of Wood and Holzapfel (1995). Based on phenotypic characterisation and interpretation of APILAB PLUS database (table 1) and identification by 16S rDNA sequencing PCR analysis of genomic DNA (figure 1), representative strain JBL102 (isolated from Lben) was identified as L. lactis subsp. cremoris, strains JBL02 and JBL06 (Raib) were identified as L. lactis subsp. lactis biovar. diacetylactis, strain JBLC161 (Klila) showing ovoid cells were identified as Leuconostoc mesenteroides. Strain JBB110 (Klila) and strain JBB102 (Jben) were tentatively identified as Lactobacillus casei subsp. casei, whereas strains JBB71, JBB75 and JBB76 (Jben) were Lactobacillus plantarum. Cocci strains JBL02 (Raib), JBL06 and JBL15 (Lben) were identified as Lactococcus lactis subsp lactis.
Acidification is an important functional logical property in relevance of selection for starter culture among the LAB (de Vuyst, 2000). In the present study, the lactococci strains showed a greater acidifying activity than leuconostoc and lactobacilli, with some strains reaching values around 71-74 g [ml.sup.-1] of lactic acid after 24 h of incubation. (Table 2). After 48 h, the pH of inoculated milk with strain Lactococcus lactis subsp lactis JBL06, decreased to values lower than 4.01. The strains of L. lactis subsp. lactis isolated from fermented milk products showed an acidifying activity after 6h of incubation similar to that detected for lactococci isolated from goats' milk cheeses by Menendez et al. (1998), respectively. L. lactis subsp. lactis var. diacetylactis showed an acidifying capacity similar to that of L. lactis subsp. lactis strains throughout the incubation time. Leuconostoc mesenteroides JBLC161 developed an acidity of 61 g [ml.sup.-1] after 24h incubation. In fact, the capacity of leuconostocs to metabolise lactose is much lower than those of lactococci (Garvie, 1984). Low acid production ability of lactobacilli in milk was also observed for those isolated from traditional Mozzarella cheese production (Morea et al., 1998). Results of acidifying capacity of selected LAB from raw milk agreed with those already observed for the bacteria isolated from traditional dairy products (Cogan et al., 1997).
Proteolytic activity of selected LAB strains, using agar diffusion assay, gave clear zones around spots put on medium supplemented with skim milk. Strains of L. lactis subsp. lactis showed significant differences (p<0,05) with respect to proteolytic activity (Table 3) Strains Lactococcus lactis (strains JBL02, JBL15) and L. lactis subsp. cremoris (strain JBL102) had largest halos surrounding spots. Proteolytic activity evaluated using oPA, methods revealed proteolytic capacity for the 12 selected LAB strains. Strains JBL02, JBL06 of L. lactis subsp. lactis were those showing the highest release of amino groups (expressed as mM Gly [L.sup.-1] of milk), giving values greater than 3.0 mM Gly [L.sup.-1] after 24 h of incubation, which were higher than those reported for lactococci strains by Mayo et al., (1990) and by Centeno et al, (1996). The strains of L. lactis subsp. lactis biovar. diacetylactis showed a proteolytic activity inferior to that of most of the strains of L. lactis subsp. lactis. Concerning the lactobacilli strains, the highest proteolytic activities detected in L.plantarum, and Lb. casei subsp. casei were 1.91, and 0.55 mM Gly [L.sup.-1] respectively. L. mesenteroides subsp. mesenteroides (JBLC161) showed the reduced proteolytic activity (1.30 mM GlyL-1).
Proteolysis is considered one of the most important biochemical processes involved in manufacturing of many fermented dairy products (Fox, 1989). In cheese manufacture, the proteolysis of casein is thought to play a pivotal role because amino acids resulting from proteolysis are the major precursors of specific flavour compounds, such as various alcohols, aldehydes, acids, esters, and sulphur compounds (Herreros et al. (2003). The CFEs of L. lactis subsp. lactis and L. lactis subsp. cremoris showed moderate caseinolytic activities; however, higher values were obtained for the CFE of L. plantarum (JBB76) Lb. casei subsp. casei (JBB110 and JBB102) (Table 3). The results found in this work are in accordance with those reported by Ballesteros et al. (2006).
Preliminary screenings of amylolytic activity of LAB isolates were tested in the starch agar plates. None of the lactic acid bacteria showed the inability to utilize starch by LAB (Table 3).
The [alpha]-amylase activity of all 12 strains of LAB was found in between 1.5 and 5.8 U [ml.sup.-1].
Agar diffusion tests made on mediums supplemented with different fat sources showed that all strains don't have lecithinase activity, neither lipolytic activity. In contrast, Talon and Montel (1994) reported that lipases of lactic acid bacteria had an optimum activity with tributyrin, and also lower with natural lipids. Otherwise, lipolytic activities, expressed by percentage of oleic acid released, detect peak lipolytic capacity for all strains (Table 3). Highest values were obtained for Lb. casei subsp. casei JBB110 releases with 0.62 [+ or -] 0.08% fatty acids from butter fat. Furthermore, value obtained for L. plantarum JBB75 was 0.51 [+ or -] 0.03%. Lactococcus subsp lactis JBL02 was the lowest lipolytic strain (0.14 [+ or -] 0.02%). This property participates in the elaboration of fermented dairy products flavour, although sometimes, it will alter them.
API ZYM system:
The enzymatic activities of the 12 strains of LAB isolated from Algerian fermented milk products, as evaluated by the semiquantitative API-ZYM system, are shown in Table 4. Leucine arylamidase and valine arylamidase detected was strong for all strains tested, However, valine and cystine arylamidase activities were very low or absent in some strains of Lactococcus. These results concur with those of several authors (Tzanetakis and Litopoulou-Tzanetaki, 1989).
Concerning the activities of the enzymes correlated with carbohydrate catabolism, as shown in Table 4, [alpha]-galactosidase exhibited by L. lactis subsp. lactis biovar. diacetylactis (JBB10), Lactobacillus plantarum (JBB71,JBB75 and JBB76), Lactobacillus casei subsp. casei (JBB102), whereas [beta]-galactosidase displayed by the three strains of L. plantarum and Ln. mesenteroides subsp mesenteroides (JBLC 161). Strong N-acetyl- Pglucosaminidase activities were exhibited by all Lactococcus lactis subsp lactis strains. Very weak amannosidase activities were observed for all tested strains. The esterase, esterase-lipase and lipase activities were very low, or not detected, for most strains of lactococci. On the other hand, definite esterase activity was observed for L. mesenteroides subsp. mesenteroides. No lipase, esterase-lipase or esterase activities were detected for the strains of L. plantarum. Strains of L. casei subsp. casei exhibited a definite esterase and esterase-lipase activities. Corroborating with reported results in the literature, alkaline phosphate activity wasn't detected among all tested strains. In contrast, acid phosphatase activity was high for some strains (JBL02, JBB76, JBB110, and JBB102). It has been reported that the commercial API-zym kit is of relevance for selection of strains as potential starter cultures based on superior enzyme profiles, especially peptidases and esterases, for accelerated maturation and flavour development of fermented products (Tamang et al, 2000). In the other hand, absence of proteinases (trypsin and chymotrypsin) and presence of strong peptidase (leucine-, valine-, and cystine-arylamidase) and esterase-lipase (C4 and C8) activities produced by the LAB strains isolated from the Algerian fermented milk products are possible traits of desirable quality for their use in the production of typical flavour. High activity of phosphatase by LAB strains indicates their possible role in phytic acid degradation in fermented vegetable products. It was also shown that strains of L. lactis subsp. lactis biovar. diacetylactis (JBB10), Lactobacillus plantarum (JBB71,JBB75 and JBB76), Lactobacillus casei subsp. casei (JBB102) had moderate to high a-galactosidase activity. This indicated their ability to hydrolyse oligosaccharides of raffinose family (Holzapfel, 2002).
Characterization of substances responsible of antimicrobial activity:
All strain-produced antimicrobial substances were inactivated by the proteolytic enzymes, indicating the proteinaceous nature of the inhibitory substances (Table 5). They were insensitive to lipase and [alpha]-amylase, which eliminates the possibility that synthesized bacteriocins are included in the group of complex biologically active substances containing a lipid or carbohydrate component (Mechai et al., 2014).
The antimicrobial substances of all strains revealed high thermostability, surviving treatments at 95[degrees]C for 20 min. At 100[degrees]C for 20 min, the activity of JBL 102, JBB 71 and JBB76 were entirely retained. At 121[degrees]C for 15 min the activity of three strains slowly decreased (Table 5).
These bacteriocins exhibited inhibitory action within a wide pH range from 2 to 10, retaining full activity in the 5-9 pH range (Table 5). The antimicrobial substances of strains JBL15 and JBB76 remained maximally active within a wide pH range from 3 to 11.
The bacteriocins preserved maximum activity during 90 days of storage at 20[degrees]C. An insignificant reduction in bacteriocin activity was observed during storage at 4[degrees]C. The inhibitory activity of bacteriocin produced by JBL15 was the most stable at 4 and 20[degrees]C for 90 days.
Scientific knowledge on the Arabian Maghreb countries fermented milk is sparse outside the region. From this work, the results confirmed that isolated LAB possess an extensive collection of enzymatic activities, many of which have the potential to influence milk composition and therefore the processing, organoleptic properties, and quality of milk. Regarding their proprieties, we would L. plantarum JBB76 and L. lactis subsp. lactis JBL 02 species had respectively the high caseinolytic and proteolytic activities and, therefore, some suitable strains should be selected because of these properties in order to control the flavour and the texture of fermented milk products. The antagonistic activity possessed by the selected strains might be an additional advantage for the control of unwanted pathogens mainly in dairy products. Further studies should be focused on the mechanisms of action of autochthonous LAB in traditional fermented milk products. Furthermore, genetic engineering of already identified probiotics and those newly discovered to make them more efficacious should be pursued.
Received 2 April 2014
Received in revised form 13 May 2014
Accepted 28 May 2014
Available online 27 June 2014
This study was supported by a grant of the << Ministry of Higher Education>> of Algeria (Project: F02920130004). We gratefully acknowledge Prof (Karim Elmahda) for kindly supplying the proteolytic enzymes and for providing the indicator strain.
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(1,2) Mechai Abdelbasset, (1,2) Debabza Manel, (1) Menasria Taha and (2) Kirane Djamila
(1) Water and Environmental Laboratory, Faculty of Exact Sciences and Natural and Life Sciences, University of Tebessa, 12002 Tebessa, Algeria.
(2) Department of Biochemistry, Microbiology laboratory, Faculty of sciences, Badji-Mokhtar University, Annaba 23000, Algeria.
Corresponding Author: Mechai Abdelbasset, Water and Environmental Laboratory, Faculty of Exact Sciences and Natural and Life Sciences, University of Tebessa, 12002 Tebessa, Algeria.
E-mail: mechai_mabdelbasset@yahoo. fr
Table 1: Grouping of LAB strains isolated from Algerian fermented milk products. Fermented milk product Species Jben (12) (a) Klila (10) Raib (10) L. lactis subsp lactis 15 2 8 L. lactis subsp. lactis 4 0 8 biovar. diacetylactis L. lactis subsp. 0 0 2 cremoris Lb. plantarum 7 1 3 Lb. casei subsp. casei 1 2 12 Ln. mesenteroides 4 2 4 subsp mesenteroide Fermented milk product Species Lben (6) Grouped Cell shape strain L. lactis subsp lactis 7 32 Cocci L. lactis subsp. lactis 2 14 Cocci biovar. diacetylactis L. lactis subsp. 6 8 Cocci cremoris Lb. plantarum 10 21 Rods Lb. casei subsp. casei 2 17 Rods Ln. mesenteroides 6 16 Cocci/ovoid subsp mesenteroide Fermented milk product Species Representative strains (b) L. lactis subsp lactis JBL02, JBL06, JBL15 L. lactis subsp. lactis JBL02, JBL06 biovar. diacetylactis L. lactis subsp. JBL102 cremoris Lb. plantarum JBB71, JBB75, JBB76 Lb. casei subsp. casei JBB110, JBB102 Ln. mesenteroides JBLC161 subsp mesenteroide (a) : Total number of samples in each product are given in brackets. (b) : representative strains of LAB selected from each grouped strains Table 2: Acidifying activity of 12 strains of lactic acid bacteria isolated from Algerian fermented milk products a. Lactic acid bacteria Strain Incubation time 6 h pH Titratable acidity L. lactis subsp lactis JBL02 6.00 28 JBL06 6.02 30 JBL15 5.96 31 L. lactis subsp. lactis biovar. JBB10 5.92 29 diacetylactis JBB41 5.80 18 L. lactis subsp. cremoris JBL102 6.09 22 Lb. plantarum JBB71 5.75 28 JBB75 5.26 30 JBB76 5.42 29 Lb. casei subsp. casei JBB110 5.70 21 JBB102 5.68 20 Ln. mesenteroides subsp JBLC161 5.04 31 mesenteroides Lactic acid bacteria Strain Incubation time 12 h pH Titrable acidity L. lactis subsp lactis JBL02 5.15 51 JBL06 5.12 49 JBL15 5.31 51 L. lactis subsp. lactis biovar. JBB10 5.10 53 diacetylactis JBB41 5.25 45 L. lactis subsp. cremoris JBL102 5.76 38 Lb. plantarum JBB71 5.00 42 JBB75 4.85 49 JBB76 4.90 46 Lb. casei subsp. casei JBB110 4.99 35 JBB102 4.97 34 Ln. mesenteroides subsp JBLC161 4.62 41 mesenteroides Lactic acid bacteria Strain Incubation time 24 h pH Titrable acidity L. lactis subsp lactis JBL02 4.11 74 JBL06 4.01 72 JBL15 4.22 71 L. lactis subsp. lactis biovar. JBB10 4.19 70 diacetylactis JBB41 4.21 68 L. lactis subsp. cremoris JBL102 5.10 40 Lb. plantarum JBB71 4.54 46 JBB75 4.40 55 JBB76 4.44 50 Lb. casei subsp. casei JBB110 4.34 69 JBB102 4.32 65 Ln. mesenteroides subsp JBLC161 4.30 61 mesenteroides (a) : Titratable acidity expressed as g [mL.sup.-1] lactic acid. Standard deviation of acidity values is in the range [[+ or -] 0.06 to [+ or -] 0.12]. Standard deviation of pH values is in the range [[+ or -] 0.01 to [+ or -] 0.04]. Table 3: Enzymatic activity of 12 strains of lactic acid bacteria isolated isolated from Algerian fermented milk products. Lactic acid bacteria Strain Caseinolytic Activity (a) L. lactis subsp lactis JBL02 129 [+ or -] 0.02 JBL06 165 [+ or -] 0.12 JBL15 170 [+ or -] 0.05 L. lactis subsp. lactis JBL02 212 [+ or -] 0.20 biovar. diacetylactis JBL06 207 [+ or -] 0.21 L. lactis subsp. cremoris JBL102 194 [+ or -] 0.17 Lb. plantarum JBB71 422 [+ or -] 0.13 JBB75 485 [+ or -] 0.30 JBB76 504 [+ or -] 0.09 Lb. casei subsp. casei JBB110 678 [+ or -] 0.01 JBB102 734 [+ or -] 0.23 Ln. mesenteroides subsp JBLC161 34 [+ or -] 0.06 mesenteroide Lactic acid bacteria Proteolytic Proteolytic activity activity (c) (PCA agar (at 24h) method (b)) mM Gly [L.sup.-1] L. lactis subsp lactis +++ 3.77 [+ or -] 0.14 ++ 3.59 [+ or -] 0.17 +++ 3.01 [+ or -] 0.07 L. lactis subsp. lactis ++ 1.98 [+ or -] 0.10 biovar. diacetylactis ++ 2.01 [+ or -] 0.12 L. lactis subsp. cremoris +++ 3. 03 [+ or -] 0.02 Lb. plantarum + 1.80 [+ or -] 0.08 + 1.91 [+ or -] 0.13 + 1.05 [+ or -] 0.20 Lb. casei subsp. casei + 0.51 [+ or -] 0.07 + 0.55 [+ or -] 0.11 Ln. mesenteroides subsp ++ 1.30 [+ or -] 0.00 mesenteroide Lactic acid bacteria [alpha]-amylase d Lipolytic (U [ml.sup.-1]) activity (d) (% of oleic acid) L. lactis subsp lactis 6.20 [+ or -] 0.1 0.14 [+ or -] 0.02 5.81 [+ or -] 0.4 0.40 [+ or -] 0.12 5.10 [+ or -] 0.11 0.51 [+ or -] 0.05 L. lactis subsp. lactis 4.21 [+ or -] 0.01 0.20 [+ or -] 0.11 biovar. diacetylactis 4.00 [+ or -] 0.17 0.22 [+ or -] 0.04 L. lactis subsp. cremoris 3.30 [+ or -] 0.09 0.32 [+ or -] 0.14 Lb. plantarum 3.78 [+ or -] 0.15 0.45 [+ or -] 0.02 2.54 [+ or -] 0.20 0.51 [+ or -] 0.03 3.04 [+ or -] 0.12 0.49 [+ or -] 0.15 Lb. casei subsp. casei 3.66 [+ or -] 0.23 0.62 [+ or -] 0.01 4.32 [+ or -] 0.24 0.56 [+ or -] 0.08 Ln. mesenteroides subsp 2.89 [+ or -] 0.33 0.55 [+ or -] 0.14 mesenteroide (a):Caseinolytic activity expressed as units of enzymatic activity [mg.sup.-1]protein. One unit of caseinolytic activity was defines as the amount of enzyme giving an absorbance increase of 0.01 units at 280 nm in 1h. (b): PCA medium. +: < 2 cm2, ++: < 4 cm 2, +++ > 4 [cm.sup.2]. (a): (c): Proteolytic activity measured using the o-pthaldialdehyde (OPA) spectrophoto-metric assay and expressed as mM Gly [L.sup.-1] of milk, after a 24 h period of incubation of the strains in the milk. (d): Strains showing positive hydrolysis test (>2.0 mm) were assayed. Table 4: Enzymatic activity (a) detected using API-ZYM system, of selected LAB isolated from Algerian fermented milk products. Lactic acid bacteria Strain 01 02 03 04 05 L. lactis subsp lactis JBL02 0 1 0 3 1 JBL06 0 0 0 3 1 JBL15 1 0 1 4 0 L. lactis subsp. JBB10 0 3 0 5 1 lactis biovar. JBB41 1 3 0 4 2 diacetylactis L. lactis subsp. JBL102 1 3 0 3 4 cremoris Lb. plantarum JBB71 1 0 0 5 4 JBB75 1 0 0 5 4 JBB76 1 1 0 5 4 Lb. casei subsp. JBB110 0 3 3 4 1 casei JBB102 1 3 3 3 1 Ln. mesenteroides JBLC161 1 1 3 4 1 subsp mesenteroide Lactic acid bacteria Strain 06 07 08 09 10 L. lactis subsp lactis JBL02 1 5 1 2 1 JBL06 2 3 1 2 3 JBL15 1 3 2 3 L. lactis subsp. JBB10 5 2 1 4 3 lactis biovar. JBB41 4 2 2 3 5 diacetylactis L. lactis subsp. JBL102 5 2 2 4 5 cremoris Lb. plantarum JBB71 4 4 1 4 5 JBB75 5 2 1 4 4 JBB76 5 5 1 4 5 Lb. casei subsp. JBB110 4 3 2 3 4 casei JBB102 4 4 2 4 4 Ln. mesenteroides JBLC161 4 3 1 5 5 subsp mesenteroide Lactic acid bacteria Strain 11 12 13 14 15 L. lactis subsp lactis JBL02 1 2 2 5 1 JBL06 2 3 0 5 0 JBL15 3 3 0 5 1 L. lactis subsp. JBB10 3 4 1 2 0 lactis biovar. JBB41 3 3 0 3 1 diacetylactis L. lactis subsp. JBL102 2 3 1 3 1 cremoris Lb. plantarum JBB71 2 3 5 3 1 JBB75 2 4 4 3 0 JBB76 2 4 5 3 1 Lb. casei subsp. JBB110 2 4 1 3 0 casei JBB102 2 3 1 3 1 Ln. mesenteroides JBLC161 1 3 4 3 0 subsp mesenteroide 01:Alcaline phosphatase ; 02: Esterase ; 03: Esterase and lipase ; 04: Leucine arylamidase ; 05: Valine arylamidase ; 06: Cystine arylamidase ; 07: Acid phosphatase ; 08: Naphtol phosphohydrolase ; 09: [alpha]-Galactosidase ;10: [beta]-Galactosidase; 11: [beta]-Glucuronidase; 12: [alpha] - Glucosidase; 13: [beta]-Glucosidase; 14: N-acetyl-[beta]-glucosaminidase; 15: [alpha] -Mannosidase. Table 5: Effect of heat treatment, pH and proteolytic enzymes on the antimicrobial compounds produced in the supernatant by selected lactic acid bacteria isolated from Algerian fermented milk products ab. L. lactis subsp lactis L. lactis L. lactis subsp subssp. lactis cremoris biovar diacetylact Treatments JBL02 JBL06 JBL15 JBB10 JBB41 JBL102 enzymes Trypsin - - - - - - Proteinase K - - - - - - pepsin - - - - - - Lipase +++ +++ +++ +++ +++ +++ [alpha]-Amylase +++ +++ +++ +++ +++ +++ Catalase +++ +++ +++ +++ +++ +++ pH 2 + + - + + - 3 ++ ++ + +++ +++ +++ 5 +++ +++ +++ +++ +++ ++ 7 +++ +++ +++ +++ +++ +++ 9 ++ ++ +++ +++ +++ +++ 11 + - +++ - - + 12 - - - - - ++ Heat Treatment 65[degrees]C/40 ++ ++ +++ +++ +++ +++ min 95[degrees]C/20 ++ ++ ++ +++ +++ +++ min 100[degrees]C/20 ++ ++ ++ + ++ +++ min 121[degrees]C/20 - + + - + + min Storage ++ +++ +++ +++ ++ +++ during 90 days at 20[degrees]C Storage during + + +++ + + + 90 days 4[degrees]C Lb. plantarum Lb. casei subsp. casei Treatments JBB71 JBB75 JBB76 JBB102 JBB110 enzymes Trypsin - - - - - Proteinase K - - - - - pepsin - - - - - Lipase +++ +++ +++ +++ +++ [alpha]-Amylase +++ +++ +++ +++ Catalase +++ +++ +++ +++ +++ pH 2 - + - + - 3 ++ ++ +++ +++ ++ 5 ++ +++ +++ +++ +++ 7 +++ +++ +++ +++ +++ 9 +++ +++ +++ +++ ++ 11 - ++ +++ - - 12 - - + - - Heat Treatment 65[degrees]C/40 +++ +++ +++ +++ ++ min 95[degrees]C/20 +++ ++ +++ ++ ++ min 100[degrees]C/20 +++ ++ +++ ++ ++ min 121[degrees]C/20 - + + - - min Storage +++ ++ +++ +++ ++ during 90 days at 20[degrees]C Storage during ++ ++ + + + 90 days 4[degrees]C Ln. mesenteroides subsp. mesenteroides Treatments JBLC16 1 enzymes Trypsin - Proteinase K - pepsin - Lipase +++ [alpha]-Amylase +++ Catalase +++ pH 2 - 3 ++ 5 +++ 7 +++ 9 +++ 11 - 12 - Heat Treatment 65[degrees]C/40 ++ min 95[degrees]C/20 ++ min 100[degrees]C/20 + min 121[degrees]C/20 - min Storage ++ during 90 days at 20[degrees]C Storage during + 90 days 4[degrees]C (a): All assays were conducted with Listeria monocytogenes ATCC 7644 as indicator strain. (b): -= no inhibition zone, + = inhibition zone up to 5 mm, ++ = inhibition zone up to 10 mm; +++ = inhibition zone over 12 mm.
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|Author:||Abdelbasset, Mechai; Manel, Debabza; Taha, Menasria; Djamila, Kirane|
|Publication:||Advances in Natural and Applied Sciences|
|Date:||Jul 1, 2014|
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