Asian Journal of Dairy and Food Research

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Assessment of the Antibacterial Potential and Safety Profile of Lactic Acid Bacteria Strains Isolated from Algerian Raw Milk

Mohamed Amine Hammadeche1,*, Nassim Madi2, Karima Aoues3, Amel Kaced4, Amel Doumandji3
1Biotechnology, Environment and Health Laboratory, Faculty of Nature and Life Sciences, University of Blida-1, 09000, Blida, Algeria.
2Centre de Recherche en Technologies Agroalimentaires (CRTAA), Route de Targa Ouzemmour, Campus Universitaire, Bejaia 06000, Algeria.
3Laboratory of Science, Food Technology and Sustainable Development, Faculty of Nature and Life Sciences, University of Blida-1. 09000, Blida, Algeria.
4Scientific and Technique Research Center for Physicochemical Analyses (CRAPC), 42000, Bouismail, Tipaza, Algeria.

ABSTRACT

Background: Raw milk harbors most lactic acid bacteria. Research in this field is a hot topic with the aim of using them for probiotic and/or technological applications.

Methods: Lactic acid bacteria were isolated on MRS and M17 media. Antibacterial activity was tested by the direct method based on spots against four food-borne pathogens. Isolates demonstrating high antibiosis activity were selected following a statistical analysis (Two-way-ANOVA) and identified by MALDI-TOF MS. The safety profile was assessed by sensitivity to antibiotics, hemolytic activity, the ability to produce gelatinase and DNase and finally, the aptitude of lactic strains to form biogenic amines.

Result: A total of 172 strains were isolated, of which 103 were presumed to be lactic acid bacteria. The results of the antibacterial activity demonstrated that 82 strains exerted an antagonistic effect against all reference strains and 12 of these showed significant antibiosis activity. Selected strains were identified as Lactococcus lactis ssp lactis (50%) and Enterococcus faecium (50%). All strains of Lactococcus lactis ssp lactis (LMMDR33, LMMDR06, LMMDR22, LMMDR26, LMMDR05 and LMMDR23) and strains LMMDR27, LMMDR16 and LVBBR13 of Enterococcus faecium demonstrated a good safety profile. They were retained for valorization.

INTRODUCTION

Lactic acid bacteria (LAB) are a diverse group of microorganisms that play a crucial role in food fermentation and human health. These gram-positive, non-spore-forming bacteria are characterized by their ability to produce lactic acid as a primary metabolic end-product (Ludwig et al., 2015). LAB comprise various genera, including Lactobacillus, Lactococcus, Enterococcus and Streptococcus, among others (Vos et al., 2011). Their ubiquitous presence in fermented foods, particularly dairy products, has made them a subject of intense research in recent years.
       
Raw milk serves as a rich reservoir for diverse LAB species, but their diversity can be influenced by various factors, including animal species, geographical location and farming practices (Tilocca et al., 2020). In Algeria, where traditional dairy products play a significant role in the local diet, the characterization of indigenous LAB from raw milk is of particular interest.
       
The potential applications of LAB extend beyond their traditional role in food fermentation. Recent studies have highlighted their promising probiotic properties and their ability to produce antimicrobial compounds. The antibacterial activity of LAB is primarily attributed to the production of organic acids, hydrogen peroxide and antimicrobial peptides known as bacteriocins (Reis et al., 2012). These compounds can inhibit the growth of various foodborne pathogens, making LAB potential bio-preservatives (Madi and Boushaba, 2017). While the probiotic potential of LAB is well-documented, concerns remain regarding the safety of certain strains, particularly those belonging to the Enterococcus genus (Avci and Tuncer, 2017). Key safety considerations include antibiotic resistance, hemolytic activity and the production of biogenic amines (Sonei et al., 2024).
       
The advent of advanced identification techniques, such as Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), has greatly facilitated the accurate identification of LAB species (Bourassa and Butler-Wu, 2015). This technology allows for rapid and reliable identification, which is essential for characterizing newly isolated strains.
       
In light of these considerations, this study aims to isolate and characterize LAB from Algerian raw milk, with a focus on their antibacterial potential against foodborne pathogens and their safety profile. By employing a combination of classical microbiological techniques and modern identification methods, we seek to identify promising LAB strains that could have potential applications in food preservation or as probiotics. This research contributes to the growing body of knowledge on indigenous LAB strains and their potential utilization in the food industry and healthcare sector.

MATERIALS AND METHODS

The experiment was carried out at the Hygiene Laboratory, the Microbiology department for quality control Laboratory and fraud repression and the Microbiology Laboratory of the public establishment specializing in organ and tissue transplantation in Blida, Algeria, from December 2021 to November 2023.
 
Sampling of raw milk
 
Raw milk was collected from various animals in north and south Algeria (Table 1). The animals’ udders were cleaned, the first three jets discarded and milk collected in sterile glass bottles. Samples were transported to the laboratory in an ice box.

Table 1: Types of animals and geographical distribution of raw milk samples.



Isolation of LAB
 
Raw milk samples were incubated at 37oC and 44oC until coagulation (Fugl et al., 2017). Dilutions (10-5 to 10-9) were inoculated onto MRS (de Man  et al., 1960) and M17 (Terzaghi and Sandine, 1975) media (Table 2). Isolated colonies were purified by subculturing. Gram-positive, non-spore-forming, catalase and oxidase negative bacteria were presumed to be LAB (Ludwig et al., 2015). Short-term storage was on agar slants at 4oC; long-term at -20oC in broth with 30% glycerol.

Table 2: Medium and culture condition for isolation of LAB strains.


 
Screening for anti-bacterial activity
 
Reference strains
 
Four indicator strains were used: two Gram-positive (Listeria monocytogenes ATCC 7644, Staphylococcus aureus ATCC 25923) and two Gram-negative (Escherichia coli ATCC 8739, Salmonella aboney NCTC 6017). Strains were obtained from the Pasteur Institute of Algeria and the Frantz Fanon University Hospital Centre of Blida, Algeria. These represent common food contaminants causing foodborne illnesses.
 
Preparation of inoculum and antibacterial assay
 
Lactic isolates were reactivated in MRS or M17 broth and standardized to 108 CFU/mL in 0.9% NaCl. Indicator strains were cultured in TSB and TSA, then prepared at 106 CFU/mL in semi-solid TSA.
       
The direct spot method was used (Madi and Boushaba, 2017). 5 mL of each lactic isolate was spotted on MRS or M17 agar (5 spots/plate) and incubated at 37 or 44oC for 24 h. Plates were then overlaid with semi-solid TSA containing indicator strains (106 CFU/mL) and incubated at 37oC for 24 h. Inhibitions zones >2 mm were considered positive.
 
Identification by MALDI-TOF MS
 
Isolates with high antibiosis activity were identified using the Extended method on a MALDI-TOF MS (Bruker Daltonics). Results were compared with the BioTyper DB-5989 database. Identification reliability scores are shown in Table 3.

Table 3: Meaning of score values (standard sample).


 
Safety assessment
 
Antibiogram
 
Antibiotic susceptibility was tested using the disk diffusion method (Sakoui et al., 2022) on Mueller Hinton agar. The inoculum (0.5 McF) was swabbed on agar plates before applying antibiotic discs.
       
Antibiotics used for both Lactococcus and Enterococcus: Vancomycin (30 µg), Gentamicin (10 µg), Erythromycin (15 µg), Chloramphenicol (30 µg), Rifampicin (5 µg) and Tetracycline (10 µg).

For Lactococcus only: Amoxicillin (10 µg, 25 µg), Amoxicillin/Clavulanic Acid (20/10 µg).

For Enterococcus only: Ciprofloxacin (5 µg).
       
Incubation was at 37oC for 24 h. Results were interpreted according to Jawan et al., (2021) for Lactococcus and CLSI (2020) for Enterococcus.
 
Hemolysis activity
 
Hemolytic activity was tested using the method of Colombo et al., (2020). Lactic strains were inoculated on TSA agar with 5% defibrinated horse blood and incubated at 37oC for 24 h. Results were interpreted as b (clear halo), a (green halo), or g (no change) hemolysis.
 
Gelatinase production
 
Gelatinase activity was assessed following Sonei et al., (2024). Lactic isolates were spotted on nutrient agar with 3% gelatin, incubated at 37oC for 48 h, then flooded with saturated ammonium sulphate. A clear halo indicated positive gelatinase activity.

DNase test
 
DNase activity was tested as per Barbosa et al., (2010). Lactic strains were spotted on DNase agar with 0.005% methyl green and incubated at 37oC for 48 h. A clear halo indicated DNA degradation.
       
Staphylococcus aureus ATCC 25923 served as a positive control for hemolytic, gelatinase and DNase activities.
 
Biogenic amine formation
 
Biogenic amine production was assessed using the method of Bover-Cid and Holzapfel, (1999). Lactic strains were sub-cultured in MRS broth supplemented with pyridoxal-5-phosphate and amino acids (tyrosine, histidine, lysine). Cultures were then spotted on decarboxylase medium with and without amino acids, incubated at 37oC for 4 days under aerobic and anaerobic conditions. A purple halo indicated positive results.
 
Statistical analysis
 
All tests were performed in duplicate. Data were treated using IBM SPSS Statistics v20. Two-way ANOVA (α = 0.05) followed by Tukey’s test were applied to select isolates with high antibiosis activity.

RESULTS AND DISCUSSION

Isolation and characterization of lactic acid bacteria
 
A total of 172 strains were isolated and purified from raw milk of various animals using MRS and M17 media. Table 4 summarizes the isolates per animal. After eliminating 69 non-lactic isolates based on positive catalase reactions, 103 isolates were presumed to be LAB. Macroscopically, the isolates were lenticular, white and varied in size between 1-4 mm. Microscopically, they were Gram-positive, appearing as cocci, rods, or coccobacilli, with differing groupings among species. All isolates were non-spore-forming, catalase and oxidase negative, consistent with lactic bacteria traits (Ludwig et al., 2015) (Fig 1 and Table 4).

Fig 1: Morphology of lactic acid bacteria strains isolated from raw milk.



Table 4: Number and morphological characteristics of strains isolated from raw milk.


       
Raw milk’s nutrient richness supports the growth of various microorganisms, especially LAB, including genera such as Lactococcus, Lactobacillus, Pediococcus, Leuconostoc, Enterococcus and Streptococcus (Bluma and Ciprovica, 2015). Our isolates aligned morphologically with these genera. Previous studies on LAB in raw milk and dairy products confirm this diversity (Bouchibane et al., 2022; Madi and Boushaba, 2017).
 
Screening for antibiosis activity
 
All 103 isolates were tested against Listeria mono-cytogenes ATCC 7644, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 8739 and Salmonella abony NCTC 6017. Six isolates showed no antibacterial activity against the pathogens (zone of inhibition £2 mm), while 97 isolates demonstrated antagonistic effects against at least one pathogen. Of these, 82 inhibited all pathogens and 15 inhibited up to three (Fig 2). Only isolates with broad-spectrum activity were statistically analyzed. Using two-way ANOVA (a = 0.05) and Tukey’s post hoc test. Inhibition zones ranged from 8.25±0.35 mm (LCBBR04 vs. Listeria) to 27.5±0.71 mm (LMMDR16 vs. Salmonella). 12 isolates were selected for further study, with inhibition zones averaging between 19.75±5.79 mm (LMMDR27) and 17.56 ± 4.59 mm (LVBBR13) (Fig 3).

Fig 2: Distribution of lactic isolates by antibiosis capacity against the pathogens tested.



Fig 3: Graphical representation of inhibition zones for lactic isolates against indicator strains.


       
LAB ability to inhibit pathogens is critical for probiotic selection. Our potent isolates, identified as Lactococcus lactis ssp. lactis and Enterococcus faecium, have demonstrated broad antibacterial effects, consistent with prior studies (Cavicchioli et al., 2015; El-Ghaish et al., 2017). The production of antimicrobial metabolites like organic acids, hydrogen peroxide and bacteriocins likely drives their antagonistic effects, which are valuable in food preservation and combating antibiotic-resistant bacteria (Fernandes and Jobby, 2022; Rajasekhar et al., 2022).

Identification of selected lactic isolates
 
All 12 selected isolates exhibited a similar appearance macroscopically on MRS Agar. Microscopically, they appeared as cocci or coccobacilli, grouped singly, in pairs, or in short chains. Identification using MALDI-TOF MS allowed us to classify these isolates into two distinct species in equal proportions: Lactococcus lactis ssp. lactis (50%) and Enterococcus faecium (50%) (Table 5). The spectral profiles generated for each strain (Fig 4) were compared with those available in the database and score values that varied between species were recorded. Identification reliability was confirmed by a score greater than 2 for most strains. The degree of similarity between the identified strains was illustrated by constructing a dendrogram (Fig 5).

Table 5: MS MALDI-TOF identification results for selected isolates.



Fig 4: Appropriate spectra generated by MS MALDI-TOF for each species of selected strains.



Fig 5: Dendrogram generated by MALDI-TOF MS for Lactococcus lactis ssp lactis (Red) and Enterococcus faecium (Blue) strains.


       
The predominance of Lactococcus lactis ssp lactis and Enterococcus faecium species in raw milk has been demonstrated by Cavicchioli et al., (2015) and Medjahed et al., (2021). In research conducted by Kadri et al., (2021), the Firmicutes phylum was dominated by Lactococcus lactis ssp. lactis and Enterococcus faecium species.
       
The probiotic and technological effect of Enterococcus and Lactococcus strains have been demonstrated in the study of Sonei et al., (2024) and Yerlikaya, (2019) respectively.

Safety aspects of identified strains
 
The results for this section are shown in Table 6.

Table 6: Safety profile for Lactococcus lactis ssp lactis and enterococcus faecium strains.


 
Antibiotic susceptibility
 
The Lactococcus lactis ssp. lactis strains were sensitive to 77.78% of tested antibiotics but resistant to 22. 22%. Rifampicin Resistance of these strains has been documented in a previous study of Jawan et al., (2021).
       
The Enterococcus faecium strains were sensitive to 54.14% of antibiotics but resistant to 14.28%. Intermediate resistance was noted for Erythromycin (83.33%) and Ciprofloxacin (50%). To be useful in applications, Enterococcus strains must be sensitive to Vancomycin (Komprda et al., 2010). Xiao et al., (2024) demonstrated resistance to Rifampicin, while Kim et al., (2018) reported resistance to Erythromycin and Ciprofloxacin at rates of 55.4% and 43.8%, respectively.
 
Hemolytic activity, DNase, Gelatinase and biogenic amines formation
 
All strains exhibited g-hemolysis and tested negative for DNase and gelatinase. Lactococcus lactis ssp. lactis strains were unable to degrade the tested amino acids, indicating the absence of decarboxylases responsible for the formation of tyramine, histamine and cadaverine. In fact, Lactococcus strains can be used to control biogenic amine formation, as demonstrated by Tabanelli et al., (2014).
       
Enterococcus faecium strains, on the other hand, formed tyramine but did not decarboxylate lysine. Histidine decarboxylase activity was observed in strains LMBMR28, LMBMR23 and LVBBR25. Tyramine production by Enterococcus strains has been well documented (Avci and Tuncer, 2017; Bover-Cid and Holzapfel, 1999), as well as histamine production (Komprda et al., 2010). When comparing the safety profiles, LMMDR27, LMMDR16 and LVBBR13 had better safety profiles, with LVBBR13 being the safest.

CONCLUSION

This study has successfully isolated and characterized LAB from Algerian raw milk of various animal origins (camel, cow, goat and sheep), with a focus on their antibacterial potential and safety profile. Our findings contribute significantly to the growing body of knowledge on indigenous LAB strains and their potential applications in food preservation and probiotic development. For this purpose, 12 LAB strains demonstrating high antibiosis activity, were selected and precisely identified by MALDI-TOF MS. After assessing their safety aspect, all six Lactococcus lactis ssp. lactis strains (LMMDR33, LMMDR06, LMMDR22, LMMDR26, LMMDR05 and LMMDR23) and three Enterococcus faecium strains (LMMDR27, LMMDR16 and LVBBR13) were retained to be used for industrial applications (bio-preservatives) or as probiotics after additional tests.

CONFLICT OF INTEREST

There are no conflicts to declare.

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