Submit

Full Research Article

Asian Journal of Dairy and Food Research, volume 43 issue 2 (june 2024) : 243-252

Technological Interest and Antimicrobial Activity of Lactic Acid Bacteria Isolated from Date Paste of the Ghers Variety (Bechar, South-West of Algeria)

D. Razni1, A. Makhloufi1, L. Mebarki1, E. Benyagoub2,3,*, F. Sahel3, R. Kechnaoui3
1Laboratoire de Valorisation des Ressources Végétales et Sécurité Alimentaire en zones semi-arides dans le Sud-Ouest Algérien, Université Tahri Mohammed de Béchar (08000), Béchar-Algérie.
2Laboratoire d’Architecture et Patrimoine Environnemental (ARCHIPEL), Université Tahri Mohammed de Béchar (08000), Béchar-Algérie.
3Département de Biologie, Faculté SNV, Université Tahri Mohammed de Béchar, (08000), Béchar-Algérie.
Cite article:- Razni D., Makhloufi A., Mebarki L., Benyagoub E., Sahel F., Kechnaoui R. (2024). Technological Interest and Antimicrobial Activity of Lactic Acid Bacteria Isolated from Date Paste of the Ghers Variety (Bechar, South-West of Algeria) . Asian Journal of Dairy and Food Research. 43(2): 243-252. doi: 10.18805/ajdfr.DRF-309.

ABSTRACT

Background: Dates are high nutritional value fruits. They are the basis of many traditional products and compose several oases by-products. Among these products, date paste represents a form of conservation widely practiced in Algeria to valorize common date’s surplus production for commercial purposes. 

Methods: First, microbiological analysis was carried out to isolate lactic acid bacteria (LAB) from date’s paste and microbial contaminants from both date fruit by-products according to standard microbiological methods before investigating the technological characteristics attributed to the isolated LAB. Second, the antimicrobial effect was evaluated through three different techniques.

Result: Nine microbial contaminants were isolated from date paste and date extract, while eight LAB strains were isolated from date paste, which belong to the Streptococci and Leuconostoc strains. The SLM1 strain revealed a good acidifying power going up to 5.13 g/L of lactic acid within 6 hours of incubation compared to the other LAB strains. A moderate to strong antibacterial effect ranging up to a 20 mm zone of inhibition was revealed against bacterial contaminants. In decreasing order, the antifungal effect was significant against Penicillium sp, Aspergillus niger and A. flavus. The isolated lactic strains of streptococci could be good candidates for food preservation by extending the shelf life of foods and improving their nutraceutical and hygienic quality. 
 

INTRODUCTION

The Algerian palm grove is rich with an important range of date varieties that constitute the main source of income for some twenty of these regions and are characterized by very interesting aptitudes for processing and conservation due to the diversification of their morphological and composition characteristics. The date palm sector has experienced remarkable developments in Algeria in recent years with more than 18.7 million palm trees (Laouar et al., 2021). According to the data reported by Fleck (2023), Algeria is the fourth-largest producer of dates in the world, with a total production of 1.2 million tons of dates recorded in 2022. An average of 60000 tons of the total production is made up of waste from palm groves, which undergo a selective sorting where the major part 90% is transformed into date paste using the Ghers as well as other varieties. Dates can be used as a raw material in the development of many by-products, but due to their rich composition and physiochemical properties, various factors can contribute to the contamination of dates (Benyagoub, 2011).
       
Through this nutrient-rich composition, we thought to characterize the microflora it contains by studying the nature of potential interactions between LAB strains and microbial contaminants, as well as the biochemical and technological characteristics of the isolated LAB strains.

MATERIALS AND METHODS

All experiments were carried out at the University of Bechar (Algeria), for six months from February to July 2022.
 
Sampling
 
Two products were analyzed. The first one was a local Ghers variety date paste made without any treatment or additive, except that the dates were pitted and mixed until the paste was obtained. The second one was a date extract prepared at the laboratory scale as described by Benyagoub et al., (2011).
 
Isolation and enumeration of LAB stains and microbial contaminants
 
From a stock solution, a series of dilutions from date paste and date extract was prepared using buffered peptone water (BPW) as a diluent (Benyagoub et al., 2022) to isolate bacterial contaminants from date paste and date extract, as well as LAB strains from date paste.
       
According to Benyagoub (2023a) and Bendada et al., (2022), the microbial isolation was performed as follows:
       
Total and fecal coliforms were isolated on MacConkey agar at 44°C for 24 to 48 hours.
       
Staphylococci were isolated on Baird Parker agar at 37°C for 24 hours.
       
Fungal flora was isolated on acidified Potato Dextrose Agar medium at 25°C for 3 to 5 days.
       
The isolation of LAB strains was carried out after an enrichment step on media suitable for their growth. M17 agar was used for the isolation of mesophilic and thermophilic streptococci at 30 and 45°C, respectively and MSE agar for the isolation of Leuconostoc species in a microbiological jar. For LAB strains, only Gram-positive and catalase-negative bacteria were retained.
 
Microbial identification
 
Microbial contaminants of date paste and date extract
 
Through phenotypic and biochemical tests, bacterial contaminants isolated from date paste and date extract were identified according to the techniques described by Guiraud (2003); Benyagoub et al., (2018). While the isolated molds were identified using the microculture technique (Garcia de Lomas et al., 1981).
 
LAB strains
 
Several tests have made it possible to identify LAB isolated from date paste according to microbiological standards as described by Benyagoub (2022).
 
Antibiotic susceptibility testing for LAB strains and bacterial contaminants
 
The study of antibiotic susceptibility was evaluated using the disk diffusion method on Mueller-Hinton agar. The bacterial inhibition zone was measured and then interpreted based on the recommendations of the CLSI (2018) for bacterial contaminants (Benyagoub et al., 2020; Benyagoub, 2023b) and based on the EFSA (2012) for LAB strains.
 
Technological characteristics of LAB strains
 
In this study, we targeted the following technological properties: Heat resistance test, sugar fermentation test, acidifying power, proteolytic, lipolytic and amylolytic activity (Meyers et al., 1996; Benyagoub, 2022).
 
Antimicrobial effect of LAB strains
 
The antibacterial effect was assessed using the spot test on agar as described by Fleming et al., (1975). However, the antifungal effect was estimated by two methods. First, the mycelial disc method by measuring the radial growth of the mycelial disc expressed in percentage (%). Second, the biomass method in which fungal biomass was weighed and the antifungal effect was assessed based on the fungal biomass rate (%) compared to the control as described by Benyagoub (2022).

RESULTS AND DISCUSSION

Microbial contaminants of date paste and date extract
 
Through microbial isolation, selective culture media, as well as biochemical tests, the obtained results made it possible to isolate and identify the following contaminants: Citrobacter sp, Cronobacter sakazakii, Staphylococcus sp (1 and 2), Enterococcus sp (Table 1, Fig 1).
 

Table 1: Biochemical characteristics of bacterial contaminants.


 

Fig 1: Macro- and microscopic aspects of microbial contaminants.


 
The isolated molds have mainly consisted of three species: Aspergillus niger, Aspergillus flavus and Penicillium sp (Fig 1).
 
Antibiotic susceptibility testing (AST) for bacterial contaminants
 
AST results for bacterial contaminants are presented in Table 2 and Fig 2 below.
 

Table 2: AST results for bacterial contaminants and bacterial reference strains.


 

Fig 2: Sensitivity test results.


 
LAB strains
 
Eight (8) LAB were isolated from the date paste (Table 3). Colonies of LAB strains have a whitish color and smooth textures (Fig 3).
 

Table 3: Microscopic and macroscopic appearance of LAB strains.


 

Fig 3: Macroscopic appearances of LAB strains on M17 agar.


       
The physiological characteristics of LAB strains are presented in Table 4.
 

Table 4: Physiological characteristics of LAB strains.


 
Technological interests of LAB strains
 
Thermoresistance and sugar fermentation tests
 
The obtained results showed that all the isolated LAB strains were heat-resistant, mannitol (+) and were different from one strain to another in the fermentation of sugars (Table 5).
 

Table 5: Sugar fermentation tests.


 
The data reported by Abekhti et al., (2021) confirmed the microbial diversity of ‘Btana’, a product similar to date paste, as follows: Weissella paramesenteroides, W. cibaria, Leuconostoc citreum, L. pseudomesenteroides and Lactobacillus plantarum. The isolated LAB strains have significant potential for sugar assimilation compared to the data reported by Abekhti et al., (2021), which fermented 7 carbohydrates. This could mean that the isolated species are adapted to the high-sugar environment of date paste.
 
Acidifying power
 
Fig 4 shows the evolution of titratable acidity of the culture media inoculated with the isolated LAB strains.
 

Fig 4: Evolution of the titratable acidity of the culture media.


       
The results of the acidifying power study show that the isolated LAB strains have a strong acidifying capacity, in particular, the SLM 1 and SLM 3 strains, while the Leuconostoc strains have less acidifying power. Significant acidification caused by the production of lactic acid is one of the most sought-after technological properties. These metabolites are organic acids that modify the pH of the medium, contributing to the inhibition of microbial contaminants and even pathogenic ones. On the other hand, the production of acidity depends on several parameters such as the incubation temperature, the physiological state of the bacteria, the concentration of the inoculum and the medium composition (Benyagoub, 2023a).
 
Amylolytic, proteolytic and lipolytic activity tests
 
Table 6 and Fig 5 present the results of the amylolytic, proteolytic and lipolytic activity tests of the isolated LAB strains.
 

Table 6: Technological suitability of isolated LAB strains.


 

Fig 5: Technological ability of LAB strains isolated.


       
As part of industrial processes, LAB strains are challenged by various stressful conditions that are likely to affect their metabolic activities, including proteolysis (Savijoki et al., 2006). The obtained results show that the SLM 1 and SLM 3 strains exhibited proteolytic activity; these results were consistent with several studies (Ben Moussa et al., 2008; Essid et al., 2009).
       
The lipolytic power is of great importance in the flavor development and the release of fatty acids but it is not very responsive to LAB compared to other groups of bacteria and this is the case of isolated SLM 2 strains, with an appreciable lipolytic power.
 
Antibiotic susceptibility testing (AST) for LAB strains
 
AST results for LAB strains are presented in Table 7 and Fig 6.
 

Table 7: AST results for LAB strains.


 

Fig 6: Sensitivity test results.


       
The AST study is one of the important selection criteria for probiotics (Zhou et al., 2005). Resistance remains a controversial condition; on the one hand, it is sought for in probiotics so that they are active even after treatment with antibiotics. On the other hand, there are risks that it might be transmitted to other bacteria, which could lead to the development of new pathogenic resistant bacteria (Salminen et al., 1998).
       
The AST results agree with various reports that LAB strains are sensitive to major classes of antibiotics, such as penicillin G, amoxicillin β-lactam, cephalosporins, aminoglycoside, quinolone, imidazole, nitrofurantoin and fluoroquinolone (Halami et al., 2000). According to Amalia et al., (2018), the bacteria isolated from fermented food are widely known to be safe for human consumption due to their safety to human health.
 
Antimicrobial effect of LAB strains
 
The antibacterial effect results of LAB strains against bacterial contaminants are shown in Table 8 and Fig 7.
 

Table 8: Antibacterial effect of LAB strains against bacterial contaminants and bacterial reference strains.


 

Fig 7: Photographic illustration of the antibacterial effect of LAB strains.


       
The antifungal effect of LAB strains against the isolated molds is presented in Fig 8, 9, 10 and 11.
 

Fig 8: Qualitative antifungal effect of LAB strains against the isolated molds.


 

Fig 9: Mycelial disc method.


 

Fig 10: Quantitative antifungal effect of LAB strains against the isolated molds.


 

Fig 11: Fungal biomass method.


       
The obtained results showed a significant rate of inhibition against Penicillium sp compared to the two other fungal species A. niger and A. flavus due to the effect of inhibitory agents secreted by lactic strains of streptococci. No antimicrobial effect of Leuconostoc strains has been detected. It may be related to their weak technological characteristics that do not allow the microorganism to dominate in the environment where it lives.
       
We found that isolated lactic strains of streptococci have a good inhibitory power by synthesis of the inhibitor agents or by modification of the pH of the medium. This antibacterial activity affected both Gram-positive and Gram-negative bacteria, but no effect was revealed against the bacterial reference strains (E. coli and S. aureus), except for the SLM1 strain.
       
These results agree with the findings reported by Dubois et al., (1982); Hadef (2012), Elmoualdi et al., (2008) and Benyagoub (2022) who found that the broad-spectrum antimicrobial activity of LAB strains isolated from different biotopes varies from one microorganism to another. Also, Mameche-Doumandji (2008) proved that lactic acid cocci have a high inhibitory activity on Gram-negative bacteria compared to that obtained against Gram-positive ones. The study carried out by Castellano et al., (2007) showed that substances with antimicrobial effects may have a greater possibility of targeting Gram-negative pathogenic strains if the outer membrane has been destabilized by the presence of another obstacle, such as organic acids, chelating agents, or other agents.
       
The toxic effect of lactic acid bacteria includes a reduction of intracellular pH and dissipation of the membrane’s potential (Kos et al., 2010). On the one hand, LAB strains have less antifungal activity against A. flavus and A. niger and on the other hand, Penicillium sp was sensitive to the effect exhibited by the isolated lactic strains of streptococci. These results are already found by Magnusson et al., (2003) and Sathe et al., (2007). Lactic strains of streptococci include a set of heterogeneous species whose common trait is that they can synthesize antibacterial substances.
       
These results show that dates have native LAB strains able to delay or stop the growth of microbial contaminants.

CONCLUSION

The results of this study suggest that lactic acid bacteria isolated from date paste can play the role of a barrier flora towards microbial contaminants and therefore participate in the preservation of date fruit by-products.

SOURCE OF SUPPORT

Nil.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

REFERENCES


  1. Abekhti, A., Margo, C., Maarten, A. et al. (2021). Comparison of maldi TOF MS profiling and 16s rRNA gene identification of presumptive lactic acid bacteria isolated from the traditional Algerian date product ‘Btana’. Appl Biol Sahar Areas. 3: 27-43.

  2. Amalia, U., Sumardianto, U. and Agustini, T.W. (2018). Characterization of lactic acid bacteria (LAB) isolated from Indonesian shrimp paste (terasi). IOP Conf Ser. 116: 012049. DOI: 10. 1088/1755-1315/116/1/012049.

  3. Bendada, F., Abdellah, M., Benyagoub, E. et al. (2022). Analysis of microbiological risks and effects of refrigeration on the evolution of the bacteriological and physicochemical quality of camel meat from Bechar. South Asian J. Exp Biol. 12: 148-156. 

  4. Ben Moussa, O., Mankai, M., Barbana, C. et al. (2008). Influence of culture conditions on esterase activity of five psychrotrophic Gram-negative strains selected from raw Tunisian milk. Ann Microbiol. 58: 53-59. 

  5. Benyagoub, E. (2011). Place du palmier dattier dans l’ethnonutrition au Sud-Ouest Algérien. Mémoire de Magister, Université de Béchar (Algérie). 100p.

  6. Benyagoub, E., Boulenouar, N. and Cheriti, A. (2011). Palmier dattier et ethnonutrition au sud-ouest Algérien: Analyse d’extrait de datte «Robb». Phytochem Biosub J. 5: 30-37. 

  7. Benyagoub, E., Benchaib, S.M., Zaalan, A. et al. (2018). Prevalence and isolation of pathogenic strains responsible for some infections in Bechar’s community. Bangladesh J. Med Sci. 19: 404-413.

  8. Benyagoub, E., Nabbou, N., Boukhalkhel, S. et al. (2020). The in vitro evaluation of the antimicrobial of Quercus robur L. methanolic and aqueous leaves’ extracts, from the Algerian high plateaus against some uro-pathogenic microbial strains. Phytother. 18: 262-274. 

  9. Benyagoub, E., Ahmed Lali, M. and Lamari, N. (2022). Quality of fresh camel meat (Camelus dromedarius) sold at retail houses in Bechar city: Physicochemical and hygienic approaches. Asian J. Dairy Food Res. 41: 264-271. doi: 10.18805/ajdfr.DRF-262.

  10. Benyagoub, E. (2022). Technological properties and antagonistic effect of lactic acid bacteria isolated from fermented goat milk flavoured with juniper leaves against some microbial contaminants. Nutra Foods. 2: 419-437.

  11. Benyagoub, E. (2023a). Traditional goatskin churn (Chekoua): Quality of fermented goat milk flavoured with leaves of Phoenician juniper stored under ambient conditions. Nutra Foods. 2: 472-484.

  12. Benyagoub, E. (2023b). Methicillin, â-lactams and clindamycin resistance profiles of Staphylococcus aureus strains isolated from patients with UTI in Bechar province (Algeria).  Anti-infect Agents. 21: e220823220161. DOI: 10.2174/ 2211352521666230822104016.   

  13. Castellano, J.J., Shafii, S.M., Francis, K. et al.  (2007). Comparative evaluation of silver-containing antimicrobial dressings and drugs. Int Wound J. 4: 114-122.  

  14. CLSI, (2018). Performance Standards for Antimicrobial Disk Susceptibility Testing M100. 28th Edition, Wayne Pennsylvania 19087- USA. 

  15. Dubois, G., Gharbonneau, W.S. and Gagnon, R.M. (1982). Inhibition de quelques bactéries pathogènes et potentiellement pathogènes par Streptococcus lactis, Streptococcus thermophilus, Lactobacilus acidophilus et Lactobacillus helveticus. Lait. 62: 681-687.

  16. Elmoualdi, L.H.L., Boushama, L., Benzakour, A. et al. (2008). Activité bactéricide d’une souche de Lactococcus lactis subsp. cremoris. Bull Soc Pharm Bordeaux. 147: 7-18.

  17. Essid, I., Medini, M. and Hassouna, M. (2009). Technological and safety properties of Lactobacillus plantarum strains isolated from a Tunisian traditional salted meat. Meat Sci. 8: 203- 208.

  18. EFSA. (2012). Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA Journal. 10: 2740-2749.

  19. Fleck, A. (2023). The world’s biggest producers of dates FAO statistica. 

  20. Fleming, H.P., Etchells, J.L. and Castilow R.N. (1975). Microbial inhibition by an isolate Pediococcus from cucumber bune. Appl Environ Microbiol. 30: 1040-1042.

  21. Garcia de Lomas, J., Marin, F., Altuna, A. et al. (1981). A simple and rapid method for microculture and identification of fungi. Mycopathologia. 76: 119-124.

  22. Guiraud, J.P. (2003). Microbiologie Alimentaire. (Edition DUNOD), Paris. 136-139.

  23. Hadef, S. (2012). Évaluation des aptitudes technologique et probiotiques  des bactéries lactiques locales. Mémoire de Magister, Université d’Ouargla (Algérie). 87p.

  24. Halami, P.M., Chandrashekar, A. and Nand, K. (2000). Lactobacillus farciminis MD, a newer strain with potential for bacteriocin and antibiotic assay. Lett Appl Microbiol. 30: 197-202.

  25. Kos, B., Beganovic, J., Pavunc, A.L. et al. (2010). Antimicrobial activity-the most important property of probiotic and starter lactic acid Bacteria. Food Technol Biotechnol. 48: 296-307.

  26. Laouar, A., Benyagoub, E., Benbelkhir, A. et al. (2021). Valorization of Algerian semi-soft date and traditional preparation of date syrup: Physicochemical and biochemical properties. Indones Food Sci Technol J. 4: 32-36.

  27. Magnusson, J., Ström, K., Roos, S. et al. (2003). Broad and complex antifungal activity among environmental isolates of lactic acid bacteria. FEMS Microbiol Lett. 219: 129-135.

  28. Mameche-Doumandji, A. (2008). Purification et caractérisation de bactériocines produites par des bactéries lactiques autochtones isolées. Thèse de Doctorat, INA-Alger (Algérie). 97p.

  29. Meyers, S.A., Cuppett, S.L. and Hutkins, R.W. (1996). Lipase production by lactic acid bacteria and activity on butter oil. Food Microbiol. 13: 383-389. 

  30. Salminen, S., Von Wright, A., Morelli, L. et al. (1998). Demonstration of safety of probiotics-a review. Int J. Food Microbiol.  44: 93-106.

  31. Sathe, S.J., Nawani, N.N., Dhakephalkar, P.K. et al. (2007). Antifungal lactic acid bacteria with potential to prolong shelf life of fresh vegetables. J. Appl Microbiol. 103: 2622-2628.

  32. Savijoki, K., Ingmer, H. and Varmanen, P. (2006). Proteolytic systems of lactic acid bacteria. Appl Microbiol Biotechnol. 71: 394- 406. 

  33. Zhou, J.S., Pillidge, C.J., Gopal, P.K. et al. (2005). Antibiotic susceptibility profiles of new probiotic Lactobacillus and Bifidobacterium strains. Int J. Food Microbiol. 98: 211- 217.
Reviewed By

Download Article
In this Article
    Contact us
    Follow us

    Editorial Board

    View all (0)