Technological Characterization of Lactic Acid Bacteria Isolated from Different Sheep’s Milk

Milk samples, isolation and identification of LAB
 
50 strains of LAB were isolated from different samples sheep’s milk collected in 3 regions of North-Western of Algeria, namely, three samples sheep’s milk from Naâma (BNF1, BNF2 and BNF3), two samples from Mecheria (BME1 and BME2) and two samples from Mascara (BMA1 and BMA2).
        
All strains were identified using phenotypic tests and only 18 strains were performed using the MALDI-TOF MS Biotyper (Bruker Daltonik GmbH, Bremen, Germany) (Data not shown). All experiments were performed in the Laboratory of Sciences and Technics of Animal Production, Faculty of Nature and Life Sciences, Abdelhamid Ibn Badis University, Mostaganem, Algeria.
 
Biotechnological characterization
EPS production
 
Production of EPS was screened in MSE medium (sucrose (10%) after incubation at 30°C for 24 h. EPS production was assessed based on the presence of mucoïd strains and are characterized by the formation of large, slimy and sticky colonies (Fguiri et al., 2016).

Diacetyl production
 
Diacetyl production was detected by the Voges Proskauer (VP) reaction. Strains were seeded on MRS medium, incubated at 30°C for 24 h. Then,0.5 mL of 1% (wt/vol) α-naphthol solution plus 0.5 mL of 16% (wt/ vol) sodium hydroxide solution was added to 1 mL of microbial culture. After 10 minutes, positive result was visualized by a red ring at the top of the culture (Ribeiro et al., 2014).
 
Evaluation of proteolytic activity
 
Proteolytic activity was determined as by De-Almeida et al., (2015). LAB strains were spotted on surface of MRS agar supplemented with 10% skimmed milk (wt/vol) and incubated at 30°C for 72 h. Positive result was indicated by a clear zone around the spots.
 
Evaluation of lipolytic activity
 
Lipolytic activity was determined on solid MRS medium (at pH 7) supplemented with different lipid substrates. This activity was detected on MRS devoid of Tween 80 and supplemented with 3% olive oil. For artificial source, Tween 80 (3%) was used. Plates were inoculated with the corresponding culture in spots and incubated at 30°C for 72 h. Lipolytic activity was detected by clear zones surrounding the spots (Hantsis-Zacharov and Halpern, 2007).
 
Acidification activity
 
200 mL of skim milk (10%) were inoculated (1% v/v) with each strain from 16 selected LAB and incubated at 30°C. Titratable acidity was determined by titrimetric method as described by AOAC (2000) and measured at 0, 2, 4, 6, 8, 10, 12 and 24 h. For most samples, 1% phenolphthalein (1g phenolphthalein in 100 mL ethyl alcohol 95°) was added (0.5 mL) and the sample was titrated with N/9 sodium hydroxide to the first permanent (30 s) color change to pink.
 
pH was measured using a pH meter (PHSJ-3F). Acidity in dairy products is expressed in Dornic degree (1°D = 0.1 g/L of lactic acid).
 
Antibacterial activity
Agar spot test method
 
Selected microorganisms were screened using the agar-spot-test method against two pathogen bacteria of test microorganisms Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). LAB strains were spotted on the surface of MRS or BHI containing 1% agar, seeded with one of the test microorganisms (10⁸ UFC/mL), and incubated for 24 h at 37°C (Fleming et al., 1975). Results were determined by measuring clear zones of the inhibition.
 
Agar well diffusion method
 
Inhibitory activity was determined, according to Harris et al. (1989). indicator strains: E. coli and P. aeruginosa were grown in Tryptone Soya Agar (TSA, Himedia) supplemented with 0.6% Yeast extract for 24 h at 37°C. Each pathogen was suspended in 4 mL of sterile water and standardized to approximately 10⁸ CFU/ mL and 1 mL was spread on the surface of plate with MRS agar and allowed to absorb. After, 70 µL of cell free supernatant obtained by centrifugation (Sigma 4-16 KS N°136689) (6000 rpm /10 min at 4°C) from each isolate of LAB in exponential growth phase were placed into wells bored of 5 mm in diameter in agar plates.
 
After diffusion of supernatants for 18 h at 4°C, plates were incubated for 24 at 37°C. Inhibitory activity was asset by measuring the dimension of the clear halos.
        
All experiments of technological properties were performed in triplicate.
 
Statistical studies
 
The data collected was subjected to an analysis of variance (SAS Institute, 2008). A one-way ANOVA analysis was applied to the results obtained from the activities, using the Student-Newman-Keuls test for comparison of the mean values (P<0.05).

Technological properties
 
According to phenotypic identification, Enterococcus genus was the most group frequently isolated from sheep’s milk with a predominance of 70%, followed by Leuconostoc with 16% and Lactobacillus with 14%. The proportions rate of the different bacterial groups are eminently variable from milk to another, in relation to the variety of operating environments (Dahou et al., 2020).
 
EPS production
 
EPS production was observed in only 10 strains isolated mainly from the two milk samples collected in the region of Mascara, BMA1 and BMA2. All strains belonging to the genus Leuconostoc (Ln) and 2 strains belonging to the genus Enterococcus (En) were found to produce EPS with large and sticky colonies (Table 1). Our results corroborate with those described by Maina et al., (2008), who showed that several strains of Leuconostoc (Ln. mesenteroïdes and Ln. citreum) are known to produce exopolysaccharides, which are mainly dextran, which is the case of the three species Ln. mesenteroïdes ssp dextranicum (BMA1.A5, BMA2.B1 and BMA2.E2). Also, Patil et al., (2015), showed that LAB isolated from different milk’s produced EPS and played important role in improving of flavor and texture of fermented dairy products.
 

 
Diacetyl production
 
In our study, 56% of the isolates produced diacetyl (Table 1). 71.42% of the Lactobacillus (Lb) produce diacetyl. In agreement with our results, Nikolic et al., (2008) also observed that a high proportion of Lactobacillus paracasei isolated from goat cheeses had the capacity to produce diacetyl. 60% of the isolates of Enterococcus produce diacetyl. 3 strains were considered as high-level diacetyl producers, Lb plantarum (BNF1.A8) and two strains of Enterococcus (BNF2.B3 and BNF3.B5). However, only 25% of the Leuconostoc genus strains produce diacetyl. Moreover, Garabal et al., (2008) showed that optionally heterofermentative Lactobacillus produce the highest amounts of diacetyl-acetoin in milk, while Leuconostoc produce the lowest amounts of diacetyl-acetoin in acidified milk. This corroborates the results obtained in our study.
 
Proteolytic activity
 
Proteolysis is considered to be the most important biochemical traits during cheese maturation. The isolates studied, hydrolyzed the proteins present in the medium with the exception of strains BNF1.A7, BNF2.B3 and BNF2.f1 (Table 1). All Leuconostocs were positive for the proteolytic activity. Lactobacillus revealed significant proteolytic activity by 6/7 positive results. Our results corroborate with the data reported by Madrau et al., (2006) who showed that Lactobacillus isolated from traditional Pecorino Sardo cheese and goat’s milk had significant proteolytic activity. 33/35 of Enterococcal strains are proteolysis positive.
 
Lipolytic activity
 
Lipolytic activity with Tween 80 and olive oil, is pronounced in 56% and 60% of the isolates respectively (Table 1). 85% of the Lactobacillus strains degrade olive oil and only 28% degrade Tween 80. Nieto-Arribas et al., (2010) showed that Lactobacillus isolated from Tenerife cheese and Manchego cheese have no lipolytic activity on tributyrin agar. For Enterococcus genus, 62% showed positive lipolytic activity with olive oil and 60% with Tween 80. Enterococcus are the main genus contributing to the lipolysis of cheese (Giraffa, 2003). For Leuconostoc genus, 87% strains degraded tween 80 and only 14% degrade olive oil.
 
Acidifying power
 
The results of acidification activity of 16 strains studied (10 Enterococcus, 3 Lactobacillus and 3 Leuconostoc) revealed differences in acidifying power after 24 h of incubation (Fig 1).
 

        
LAB strains have their ability to ferment lactose into acid lactic (Saha et al., 2017). For Enterococcus genus, all the strains tested gave acidification values between 8.6 g/L (BME2.D4. En durans) and 4 g/L (BME2.C2. Enterococcus). pH changes by our Enterococcus strains ranged between 5.78 and 5.01 of incubation (Fig 1).
 
Lactic acid production by strains belonging to Lactobacillus strains was low except for the Lb plantarum (BNF1.A3) which showed moderate acidifying activity, i.e., 4.9 g/L compared to the BNF2.A6 (Lactobacillus) strain where the amount of lactic acid produced was 3.5 g/L. Lactobacillus strains slowly metabolize lactose (Carafa et al., 2015).
        
The production of lactic acid by the Leuconostoc strains was significantly lower than the other two genus studied. The acidification capacity was of the order of 5.6 g/L (BMA1.E1. Leuconostoc) and 2.9 g/L (BMA2.E2 Ln. mesenteroïdes ssp dextranicum) and pH values reached with these strains vary between 6.13 and 5.60 after 24 h of incubation.
 
Antimicrobial activity
 
We classified the inhibitions observed in our results in 3 types depending on the dimension of the halos in low (+: 1-2 mm) to moderate (++: 2-4 mm) and high (+++: >4 mm) (Table 1). Lactobacillus (Lb plantarum) and Enterococcus (En. durans) strains were found to have a higher antagonistic activity against pathogens. The inhibitory effect is at least weakest for Leuconostoc strains.
        
For Lactobacillus genus, all strains isolates showed inhibitory activity against both pathogens tested, however no supernatant showed inhibitory activity except Lactobacillus plantarum species which showed moderate inhibition against E. coli.
        
Leuconostoc genus had the lowest antimicrobial activity against E. coli and P. aeruginosa with the exception of the three Ln. mesenteroïdes ssp dextranicum species (BMA1.A5, BMA2.B1 and BMA2.E1) which did not inhibit the indicator strains.
        
Most strains belonging to the Enterococcus genus have shown their inhibitory effect against both pathogens. En. durans species strongly inhibited E. coli with an inhibition zone exceeding 4 mm. No supernatant was able to inhibit P. aeruginosa, but 6 strains of Enterococcus inhibited E. coli. Thus, these data demonstrate that the antagonistic activity shown against indicator pathogens allows the application of the LAB studied as bioconservatives in the production of dairy products, increasing their shelf life.

The 50 strains isolated from sheep milk showed efficient technological activities. Our LAB strains have high potential for probiotic application, with elevated production of EPS by Leuconostoc genus, high diacetyl production and also lipolytic and proteolytic activity by Lactobacillus which confers them to have a potential to be used as adjunct cultures in cheese-making. Enterococcus genus showed the most efficient antimicrobial activity against E. coli and P. aeruginosa.
        
High variability in technologically revealing traits was found among isolates and could be the basis for the selection of strains specifically to be used as a supplement culture in the production of dairy products and other biotechnological applications.