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Antibacterial and Enzymatic Activities of Lactic Acid Bacteria From Fermented Foods
*Corresponding author: Prof. Devaraja Gayathri, Department of Studies in Microbiology, Davangere University, Davangere, India. gayathridevaraja@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Chakra PS, Yalagondanahalli CN, Gayathri D. Antibacterial and Enzymatic Potential of Lactic Acid Bacteria From Traditional Fermented Foods. J Health Allied Sci NU. doi: 10.25259/JHS-2024-7-12-R3-(1478)
Abstract
Objectives
The present study aimed to explore the microbial diversity—specifically, the diversity of lactic acid bacteria (LAB)—in traditional fermented foods from rural areas of Davangere, Tumakuru, and Chitradurga districts of Karnataka, India. The objectives included isolating and characterising LAB, evaluating their antibacterial activity against common food-borne pathogens, and screening for the production of the industrially relevant enzyme, alkaline protease.
Material and Methods
Samples were collected from naturally fermented foods and subjected to standard microbial isolation protocols, including serial dilution and plating on MRS agar supplemented with 1% CaCO3. Isolates were physiologically characterised, and antibacterial activity was assessed using the agar overlay method against Salmonella enterica serovar Typhimurium (MTCC 3231), Staphylococcus aureus (MTCC 737), Escherichia coli (MTCC 1687), and Listeria monocytes (MTCC 1143). Isolates with promising antibacterial activity were further screened for alkaline protease production under varying conditions of temperature, pH, and nutrient sources.
Results
A total of 122 isolates were obtained. Among these, isolates GP151, GP338, and GP294 demonstrated significant and broad-spectrum antibacterial activity. Of these, GP338 exhibited optimal alkaline protease production at 37°C, pH 10, with starch and lactose as carbon sources and peptone as the nitrogen source.
Conclusion
This study highlights the presence and diversity of LAB in traditional fermented foods and their potential antibacterial properties. Furthermore, certain LAB isolates, particularly GP338, show promise for industrial applications due to their ability to produce alkaline protease under optimized conditions. These findings suggest potential roles for such isolates in food safety, preservation, and biotechnology, warranting further investigation.
Keywords
Alkaline protease
Antibacterial activity
Lactic acid bacteria
Microbial diversity
Traditional fermentation
INTRODUCTION
Lactic acid bacteria (LAB) are a diverse group of Gram-positive, non-spore-forming microorganisms widely recognised for their ability to ferment sugars into lactic acid. They are commonly found in fermented foods and play a key role in their production and preservation. Their fermentation process not only contributes to the flavour and texture of products but also extends their shelf life, making LAB essential in the food industry.[1,2] Moreover, LAB have been acknowledged for their potential probiotic effects, contributing to gut health, immune modulation, and prevention of gastrointestinal infections.[3]
However, many countries have been practicing food fermentation for ages, which offers significant health and nutritional benefits and has been valued for its simplicity and cost-effectiveness in altering the nutritional profiles of plant and dairy-based foods.[4,5] LAB is involved in the manufacturing of yogurt, cheese, cultured butter, sour cream, sausages, cucumber pickles, olives, sauerkraut, and cocoa, among many other foods. These plant and dairy-based byproducts obtained from fruits, vegetables, and grains consist of nutrients and active ingredients.[6]
However, the primary focus of this study is to explore LAB’s antibacterial properties and their potential in enzyme production, specifically alkaline protease, which has notable industrial applications.[7,8]
In addition to their probiotic properties, LAB has been shown to produce a variety of bioactive compounds, such as organic acids, hydrogen peroxide, and bacteriocins. These compounds exhibit antimicrobial activity against foodborne pathogens, including Salmonella, Staphylococcus aureus, and Escherichia coli, highlighting their crucial role in ensuring food safety. LAB are also of great interest for their enzyme production capabilities, including amylases, proteases, and lipases, which are utilised in pharmaceuticals, textiles, waste management, etc.[9,10]
This research aimed to isolate and characterise LAB from traditional fermented foods, particularly from rural areas of Davangere, Tumakuru, and Chitradurga districts of Karnataka, India, regions known for their long-standing fermentation practices. By examining the microbial diversity in these traditional food systems, this study aims to identify potent LAB strains with dual functions: antibacterial activity and enzyme production, with a particular focus on alkaline protease, an enzyme important for industries like detergent, pharmaceutical, and leather processing.
LAB’s role in producing antimicrobial substances, such as bacteriocins, which inhibit pathogenic microorganisms, further underscores its potential to enhance food safety. Traditional fermented foods are an excellent source of LAB strains due to the rich microbial diversity associated with natural fermentation processes. There is limited research on the industrial enzyme production capabilities of LAB strains isolated from traditional Indian fermented foods. This study aims to fill this gap by investigating the alkaline protease activity of these organisms. This enzyme, known for its application in industries due to its proteolytic nature, can serve as a sustainable and cost-effective alternative to chemical enzymes in various processes.[11]
In conclusion, this study focuses on isolating LAB strains from traditional fermented foods, screening them for antibacterial activity, and evaluating their potential for alkaline protease production. The findings will contribute to both the understanding of LAB’s role in food safety and their potential industrial applications, making them valuable in fields that range from food preservation to enzyme-based industrial processes.
MATERIAL AND METHODS
Collection of samples
The rural areas of Davangere, Tumakuru, and Chitradurga districts of Karnataka, India, were chosen. The naturally fermented curd (mosaru), Idli, and Dosa batter samples were collected in sterile containers in triplicate by following the standard isolation protocol. The samples were taken directly from the fermentation pot, labelled, and brought back to the laboratory under controlled conditions, where they were stored at 4°C until further analysis.[12]
Isolation of the lactic acid bacteria
The curd (mosaru), Idli, and Dosa batter samples were prepared for isolation using the serial dilution technique, using 0.9% saline. One mL of water was added to 9 mL of sterile saline, and then the sample was serially diluted. The 10-5 and 10-6 dilutions were taken and plated on MRS (De Mann Rogosa Sharpe) agar supplemented with 1% CaCO3 using the pour plate technique; then, plates were incubated for 24 to 42 hours at 37°C. The colonies with different morphology and colonies showing clear zones were further selected, inoculated on slants, and stored for further analysis.[13,14]
Physiological characterisation
Further, the stored cultures were subjected to physiological characterisation to choose LAB. Tests like gram staining, catalase test, and endospore staining were performed, and selected cultures were stored at -20°C in 40% glycerol.[15]
Antibacterial activity of LAB cultures
To choose LAB that shows potent antibacterial activity against food-borne pathogens such as Salmonella enterica serovar Typhimurium (MTCC 3231), Staphylococcus aureus (MTCC 737), Escherichia coli (MTCC 1687), and Listeria monocytogenes (MTCC 1143), the agar overlay method was used. The LAB was inoculated on MRS agar, and pathogens were inoculated in Luria-Bertani (LB) agar and overlayed on the MRS agar. The plates were incubated at 37°C for 24 hours, and the zone of inhibition in millimetres was measured.[16,17]
Screening and quantification of alkaline protease
The isolates that showed good antibacterial activity were further screened for the production of alkaline protease. Those isolated were inoculated on the skim milk agar media and incubated at 37°C for 24 hours. After incubation, the plates were observed for clear zones around the colonies.[18] Furthermore, to quantify the crude enzyme produced by GP338, it was inoculated in flasks containing skim milk and incubated in a shaking condition at 120 rpm (revolutions per minute) for 48 hours at 37°C. The crude enzyme was obtained from the supernatant by centrifugation at 2000× g for 20 minutes at 4°C, and the protein was quantified using the standard protein estimation method and the readings were taken at 280 nm in a spectrophotometer.[19]
Determining optimal enzyme production conditions
Furthermore, the isolate exhibiting a zone of clearance was assessed for optimal growth and metabolically suitable conditions for enzyme production by monitoring the increase in the zone of inhibition after inoculating the organism. This was done under varying conditions, including checking for activity at different pH levels (2-10), different temperatures (4-37°C), and with different nitrogen sources such as peptone, yeast extract, beef extract, and casein. Additionally, it was tested with various carbon sources, including sucrose, lactose, starch, and maltose. The zone of inhibition was measured, and the optimal conditions for growth and enzyme production were determined.[20]
Molecular characterisation of proteolytic bacteria
The isolate showing the highest alkaline protease production was identified using the 16S rRNA (16s ribosomal RNA) sequencing method. DNA was isolated using the CTAB (cetyltrimethylammonium bromide) method and then stored in sterile, deionised water. The DNA samples were subjected to PCR (polymerase chain reaction) amplification using the universal 16S rRNA primers 27F (50 AGAGTTTGATCMTGGCTCAG 30) and 1492R (50-GGTTAC CTTGTTACGACTT-30). The PCR mixture with the isolated DNA template was inserted into the thermal cycler at 95°C for 5 minutes, 35 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes, followed by a final extension at 72°C for 10 minutes, then the amplification was confirmed using gel electrophoresis. The DNA was sequenced using an ABI (Applied Biosystem) genetic analyser; the sequence was then assembled and compared to the GenBank database using a BLAST (Basic Local Alignment Search Tool) tool. The phylogenetic analysis was performed by aligning sequences using the MUSCLE (Multiple Sequence Comparison by Log-Expectation) tool within the MEGA X (Molecular Evolutionary Genetics Analytics) software.[21]
Statistical analysis
The study results were tabulated and analysed using descriptive statistics, including standard deviation and standard error, in Microsoft Excel and Python for graphical representation.
RESULTS
Isolation of LAB
Fermented food samples were collected from two locations in Davanagere district in Karnataka, India. The LAB isolates from curd (mosaru) samples prepared from buffalo milk yielded around 39 isolates, and the fermented Dosa batter sample from Baada, Karnataka gave 34 isolates, indicating the diverse population of microorganisms in the fermented samples. Similarly, the Idli batter sample from Lakshmisagara village yielded 44 isolates, and the goat curd (mosaru) sample from Yalagondanahalli and Chitradurga in Karnataka, yielded 5 isolates, respectively. A total of 122 isolates were obtained collectively, representing the culturable LAB diversity of microorganisms present in both dairy and non-dairy products. The difference in isolation counts or the types of isolates may signify the influence and effect of the location and environmental factors influencing the microbial population. Thus, a diverse range of microorganisms is present in the samples [Table 1].
| Sample name | Site with global positioning system coordinates | Nature of sample | pH of the sample | Flavour of the sample | Number of isolated after serial dilution |
|---|---|---|---|---|---|
| S3 |
Mayakonda, Davanagere, Karnataka, India. 14° 17’ 0.77” N 76° 4’ 34.41” E |
Curd sample from Buffalo Milk | 4.8 | Slightly tangy, sweet flavour | GP88-GP126 |
| S9 |
Bada, Davanagere, Karnataka, India. 14° 16’ 44.15” N 76° 0’ 9.56” E |
Dosa batter sample | 4.0 | Tangy and sour flavour | GP127-GP160 |
| S10 |
Lakshmisagara, Tumukur, Karnataka, India. 13° 46’ 31.47” N 76° 49’ 4.41” E |
Idli batter sample | 4.5 | Tangy and sour flavour | GP292-GP335 |
| S11 |
Yalagondanahalli, Chitradurga, Karnataka, India. 14° 3’ 2.73” N 76° 46’ 10.65” E |
Goat curd | 4.4 | Tangy and earthy flavour | GP336-GP340 |
Physiological characterisation of LAB
After isolation, 122 isolates were subjected to screening for LAB from the pool of microorganisms, as seen in Figure 1. The bar chart depicts the distribution of various groups of microorganisms based on the physiological characterisation using the catalase test, gram staining, and endospore staining results. Out of 122 isolates, 34 were catalase-positive, gram-positive, and spore-forming, and 39 were catalase-negative, gram-positive, and non-spore-forming. The latter demonstrated the dominant presence of LAB isolates in the fermented food samples, as this combination is peculiarly characteristic of LAB. Some of the other combinations with fewer isolates were catalase-positive, gram-negative, non-spore-forming isolates (20); catalase-negative, gram-positive, spore-forming isolates (13); and the least common combination, catalase-negative, gram-negative, non-spore-forming isolates (2). The diversity of LAB isolates shown by these combinations highlights the complex microbial diversity in traditionally fermented curd (mosaru), Idli, and Dosa batter from Davangere, Tumakuru, and Chitradurga, indicating a rich potential for identifying unique LAB strains with specific desirable characteristics for food fermentation processes.
-g1.png)
- Bar chart representing the distribution of microorganisms in the sample based on physiological characterisation.
Antibacterial efficiency of LAB
The isolates with physiological characteristics similar to those of LAB were further evaluated for their antibacterial activity against foodborne pathogens. Figure 2(a-d) shows the antibacterial activity of all the isolates against the pathogens. The study uncovered significant variability in efficacy, but the most potent antibacterial activity was shown by isolates GP151 and GP338, which exhibited zones of inhibition against Escherichia coli (MTCC 1687) (15.0 ± 0.57 mm, 12.5 ± 0.16 mm), Salmonella enterica serovar Typhimurium (MTCC 3231) (10.0 ± 0.36 mm, 12±0 mm), and Listeria monocytogenes (MTCC 1143) (14.0 ± 0.27 mm, 14.5 ± 0.27 mm), highlighting their broad-spectrum capabilities. Additionally, GP294 demonstrated notable efficacy against Salmonella enterica serovar Typhimurium (MTCC 3231) and Staphylococcus aureus (MTCC 737) with zones of 10.0 ± 0.36 mm and 14.0 ± 0.47 mm, respectively. Despite these promising findings, many isolates exhibited low to moderate zones of inhibition, and 13 isolates displayed complete resistance to all tested pathogens, highlighting a significant variance in antibacterial properties. This study emphasises the potential of specific LAB isolates in combating key bacterial pathogens while also recognising the need for careful selection and application of these biological agents based on their target-specific activities.
-g2.png)
- Bar charts depicting the antibacterial efficiency of the LAB isolates against pathogens (a) Salmonella enterica serovar Typhimurium (MTCC 3231) (b) Staphylococcus aureus (MTCC 737) (c) Escherichia coli (MTCC 1687) (d) Listeria monocytogenes (MTCC 1143). LAB: Lactic acid bacteria.
Screening and quantification of alkaline protease enzyme
To further screen the isolates producing alkaline protease, potent isolates showing antibacterial activity against pathogens were chosen. Among GP338, GP151, GP294, GP153, and GP155, only GP338 exhibited a zone of clearance around the colony inoculated on skim milk agar, signifying protease production by the isolate. When quantified, there was 43 U/mL of crude enzyme after the fermentation process. This indicates that GP338 can break down the milk protein present in the skim milk agar, making it a good candidate for further investigation into the production of alkaline protease.
Optimisation of enzyme production
The isolate producing alkaline protease was subjected to optimisation of production. Parameters such as temperature, pH, various carbon sources, and protein sources were evaluated for their effects on enzyme production, and the optimal conditions were determined. The optimum conditions were at 37°C and pH 10. Interestingly, two carbon sources, lactose and starch, were shown to cause significant enzyme production. Among nitrogen/protein sources, peptone showed optimum production. The measured zones of inhibition under different conditions are depicted in the accompanying graph, as shown in Figure 3.
-g3.png)
- Graph represents the optimisation parameters: (a) Temperature, (b) pH, (c) Carbon source, and (d) Nitrogen source.
Molecular characterisation
To identify the alkaline protease-producing isolate, it was subjected to 16S rRNA molecular characterisation. It was found to be closely related to Bacillus tropicus, showing a high level of sequence homology when compared with the public database. The sequence was then deposited to the GenBank database, and the accession number allotted by GenBank is “PP993957”. The phylogenetic relationship is depicted in the phylogenetic tree, as shown in Figure 4.
-g4.png)
- Phylogenetic tree of isolate GP338.
DISCUSSION
The present study on the identification of LAB from traditional fermented foods, such as curd (mosaru), Idli, and Dosa, from rural areas of Davangere, Tumakuru, and Chitradurga districts aims to identify the microbial population and its specific purposes in these foods, as well as address safety concerns. Out of the 122 isolated LAB strains, the investigation identified those that possess the features of LAB suitable for use as probiotics. Out of these, GP151, GP338, and GP294 exhibited appreciable antibacterial ability, which indicates that the obtained isolates could be potentially employed as bio-preservatives against the common causes of food-borne ailments including Salmonella enterica serovar Typhimurium (MTCC 3231), Staphylococcus aureus (MTCC 737), Escherichia coli (MTCC 1687), and Listeria monocytogenes (MTCC 1143).
Similarly, a study conducted by Abubakr and Al-Adiwish, LAB-Gr2 and LAB-Bn2 showed a difference in their antibacterial activity. This indicates that LAB-Gr2 was more effective in inhibiting the growth of Salmonella enterica serovar Typhimurium compared to LAB-Bn2.[22] Another study by Adeniyi et al. reported variations in the antimicrobial properties of different LAB isolates against Escherichia coli CB6 and Klebsiella CB2, implying that the LAB possessed strain-specific functions. In particular, Enterococcus hirae CO6M and Weisella confusa CO29M exhibited high levels of antibacterial activity against these pathogens; both can, therefore, be considered prospective candidates for use as bio-preservatives against specific bacteria.[23]
Among the potent isolates, GP338 showed the production of alkaline protease and was identified as Bacillus tropicus. Under optimised conditions, such as temperature, pH, and carbon and nitrogen sources, it showed enhanced enzyme production, highlighting its industrial applicability. A similar study conducted by Chu et al. demonstrated that LAB isolates have the potential to produce alkaline protease and exhibit the ability to degrade organophosphorus pesticides. Their study revealed that 32°C was the optimal temperature, and a pH range of 8 to 9.5 was found to be optimal. This correlates with our study, in which we found 37°C to be the optimum temperature and 10 to be the optimum pH. However, our study showed that lactose, starch, and peptone are suitable carbon and nitrogen sources, which were lacking in the study conducted by Chu et al. Our study adds to the existing knowledge about the alkaline protease produced by LAB strains.[24]
The study sheds light on the variation in antibacterial activity among strains and the production of alkaline protease by lactic acid bacteria (LAB) during the fermentation process. It also explains the influence of environmental factors and the use of traditional practices rather than commercially available strains, which demonstrates the potency of isolating indigenous isolates from traditionally fermented food sources. These isolates are promising candidates for use in the food industry and other applications. Furthermore, molecular research will enhance our understanding of molecular pathophysiology, metabolites, and their impact on enzyme production, which is responsible for conferring these benefits.
CONCLUSION
This research provides valuable insights into the existing body of knowledge on the culturable lactic acid bacteria (LAB) diversity of fermented foods in the Davangere, Tumakuru, and Chitradurga districts. The extensive ability of the isolates to inhibit food-borne pathogens and aid the fermentation process emphasises their pivotal role in potential health benefits conferred to the consumer. Additionally, they can be used for preserving food and as food safety agents. This study also underscores the ability of LAB isolates to produce the industrially important enzyme alkaline protease. Our optimisation protocol provides insights into the carbon and nitrogen sources that can be used to enhance the yield of the enzyme. This enzyme is particularly important in the detergent and leather processing industries. Marking the importance of the dual functionality of LAB, which stems from consuming traditionally fermented foods rather than processed ones, highlights their ability to confer health benefits. It also serves as a cost-effective and scalable method for producing industrially important enzymes. There is further scope and a need for additional research to understand and harness the full potential of these microorganisms in various biotechnological applications.
Ethical approval
Institutional Review Board approval is not required. This study did not involve human participants, animal subjects, or the use of sensitive personal data. The experimental work was limited to in vitro screening and characterization of microbial isolates for their antibacterial and enzyme-producing capabilities. As the research is based solely on microorganisms and standard laboratory procedures, no ethical approval or informed consent is required in accordance with institutional and international guidelines.
Declaration of patient consent
Patient’s consent not required as patients identity is not disclosed or compromised.
Financial support and sponsorship
The authors gratefully acknowledge the generous financial support provided by the Karnataka Science and Technology Promotion Society (KSTePS), Department of Science and Technology, Government of Karnataka, under the Vision Group on Science and Technology (VGST) for sponsoring this project (VGST/GRD-1017/CISEE). Fellowship support was also provided by VGST, KSTePS, the Department of Science and Technology, Government of Karnataka (Award No. DST/KSTePS/Ph.D. Fellowship/LIF-12:2019-20).
Conflicts of interest
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
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