LITERATURE REVIEW
Overview of Enterococcus spp
Enterococci were first discovered in human fecal flora in 1899 However until 1984, they were still considered part of the genus Streptococci (Murray
Enterococci are gram-positive cocci that typically occur in pairs (diplococci) or short chains, and they are often difficult to differentiate from streptococci based on physical characteristics alone These bacteria are significant human pathogens increasingly resistant to antimicrobial agents, posing challenges in clinical treatment Streptococcus faecalis was first identified in 1906 from a patient with endocarditis, while Streptococcus faecium was discovered in 1919 Based on biochemical and nucleic acid (DNA-rRNA homology and 16S rRNA) studies, serogroup D streptococci were divided into two groups, leading to the reclassification of Streptococcus faecalis and Streptococcus faecium into the genus Enterococcus, emphasizing their distinct genetic and biochemical profiles.
Enterococci are responsible for 5% to 15% of endocarditis cases and are commonly treated with a combination of a cell wall-active agent, such as penicillin or vancomycin, and an aminoglycoside to achieve a synergistic bactericidal effect High-level aminoglycoside resistance, with MICs of 2000 micrograms/ml or greater, negates this effect and has now been documented for all aminoglycosides Besides endocarditis, enterococci can cause urinary tract infections, intraabdominal, pelvic, wound infections, superinfections—especially in patients on expanded-spectrum cephalosporins—and bacteremias, often in coinfection with other organisms They are now the third most common cause of nosocomial infections, with most infections being effectively treated with single-drug therapy using penicillin, ampicillin, or vancomycin.
E faecalis and E faecium are the primary causative agents of human enterococcal infections Since the 1970s, enterococci have become a leading cause of hospital-acquired infections, highlighting their significant role in healthcare-associated microbial infections (Gilmore MS, Lebreton F, van Schaik W).
2013) In the past two decades, E faecium has rapidly evolved as a worldwide nosocomial pathogen by successfully adapting to conditions in a nosocomial setting and acquiring resistance against glycopeptides (Top J and Willems R,
Resistance genes against glycopeptides are organized within van operons positioned on mobile genetic elements (MGEs), facilitating their transfer among bacteria These operons contain regulatory genes that control the expression of ligase genes responsible for conferring resistance, with vanA and vanB being the most prevalent among these genes.
E faecium is a Gram-positive, gamma-hemolytic, or non-hemolytic bacterium in the genus Enterococcus It can be commensal (innocuous, coexisting organism) in the gastrointestinal tract of humans and animals, but it may also be pathogenic, causing diseases such as neonatal meningitis or endocarditis
E faecium has emerged as a major cause of drug-resistant infections in hospitalized patients worldwide, demonstrating an inherent ability to develop resistance to antibiotics and withstand environmental stressors This resilience allows the species to thrive in hospital settings, making it a significant nosocomial pathogen The rise of glycopeptide-resistant strains, such as vancomycin-resistant enterococci (VRE), poses a serious global public health challenge The widespread use of antibiotics in healthcare has been a key factor driving the evolution of E faecium into a highly proficient hospital-associated pathogen, complicating infection control efforts.
E faecium has been reported to produce antimicrobial compounds including bacteriocins Bacteriocin production have be applied to the preservation of a wide range of food products and is now being considered as a probiotic trait (Yang et al., 2014) E faecium probiotics also compete for adhesion sites - areas on the cell surface where harmful cells and other molecules can bind, to establish a protective barrier in the gut Results of animal studies indicate that the probiotic benefits of
E faecium include its ability to boost immunity, improve cell proliferation rate, and increase fat burning
E faecium serves as a valuable probiotic alternative to pharmaceutical antibiotics in animal feed, promoting animal health naturally As with other probiotics, E faecium helps maintain a balanced gut microbiota by competing for nutrients, preventing harmful microorganisms from thriving and improving overall digestive health.
E faecium plays a significant role not only in the livestock industry but also in the biotech food sector This bacteria enhances the taste and aroma of certain foods, thanks to its tolerance to salts and acids, which allows it to thrive in diverse food systems E faecium is actively involved in the fermentation of traditionally made cheese and dry sausages, where it contributes to developing the products’ organoleptic characteristics Its functional properties make it a promising candidate for advancing food fermentation technologies.
Overview of bacteriocins
Bacteriocins are a diverse group of ribosomally synthesized bacterial peptides or proteins with antimicrobial activity against specific bacteria They vary in biochemical properties, molecular weight, mechanism of action, and spectrum of activity, targeting the same or closely related bacterial strains Bacteriocin production is regulated by genes in plasmid or chromosomal DNA that also encode resistance mechanisms, export functions, and posttranslational modification enzymes Both Gram-positive and Gram-negative bacteria produce bacteriocins, which play a key role in microbial competition and can be potential agents in antimicrobial therapies.
(Lactobacillus, Lactococcus, Streptococcus, Enterococcus, Leuconostoc,
Pediococcus, and Propionibacterium) and by Gram-negative bacteria
(Escherichia coli, Shigella, Serratia, Klebsiella, and Pseudomonas) Until now, in the open-access database of BACTIBASE, more than 200 bacteriocins were described (January 2014)
Bacteriocins have significant medical and biotechnological potential, serving as supporting agents or alternatives to chemical food preservatives, antiviral agents, antibiotics, anticancer treatments, and anti-Leishmanial agents Notably, some bacteriocins like nisin A are already approved and widely used in the food industry across various countries These natural antimicrobial peptides are emerging as powerful tools with promising applications in both the food and pharmaceutical sectors.
Classification of bacteriocins
Bacteriocins are highly abundant and exhibit significant diversity across various bacterial species (Cotter, Hill, and Ross, 2005; Cotter, Ross, and Hill, 2013; Yang et al., 2014) Despite their diversity, the classification of bacteriocins remains controversial and not yet clearly defined They are categorized based on criteria such as the producing bacterial family, molecular weight, and amino acid sequence Traditionally, bacteriocins are divided into four classes—Class I, Class II, Class III, and Class IV—according to their sequence, molecular weight, and structural characteristics (Negash & Tsehai, 2020).
Class I bacteriocins (Lantibiotics) are thermostable small molecular weight (< 5 kDa) peptides that act according to cell membrane structures Based on their structural features and mode of action, class I lantibiotics are divided into two subclasses: Ia and Ib
Subclass Ia is flexible screw-shaped molecules with a positive charge of 2–
4 kDa, generally acting by creating holes in the cytoplasmic membrane of susceptible species Nisin belongs to this group
Subclass Ib consists of characteristic peptides weighing 2–3 kDa, which are spherical, inactive, and can be negatively charged or uncharged These peptides primarily function by disrupting vital enzyme molecules in susceptible bacteria, leading to their antibacterial effects Mersacidin is a notable example of this subclass, demonstrating its mechanism of action through targeted enzyme interference.
Bacteriocin Class II, also known as the Non-Lanbiotic class, comprises small antimicrobial peptides with a molecular weight of less than 10 kDa that are thermostable and do not contain lanthionine This class is further subdivided into subclasses IIa, IIb, and IIc, each exhibiting distinct functional and structural characteristics Understanding Bacteriocin Class II is essential for their application as natural preservatives and potential alternatives to antibiotics.
Class IIa peptides are the largest group of anti-Listeria bacteriocins, including pediocin PA-1, Leucocin A, and Sakacin P, which demonstrate potent antimicrobial activity (Marugg, J.D et al., 1992) These bacteriocins are promising for industrial applications due to their strong anti-Listeria effectiveness while sparing beneficial starter cultures, making them more advantageous than class I bacteriocins like nisin that have a broader spectrum of inhibition.
Class IIb bacteria are characterized by complexes of two distinct peptides that are either inactive or active, playing a key role in their antimicrobial activity Notable bacteriocins in this group include Lactococcin G, Plantaricin E and F, as well as Plantaricin J and K, as identified in studies by Anderssen et al (1998) and Diep et al (1996, 1997) These bacteriocins are significant for their potential applications in food preservation and safety.
1996), where two different peptides act together to exert an antibacterial effect
Bacteriocin class IIc consists of small, thermostable peptides known as heterogenous bacteriocins, each with distinct mechanisms of action This subclass includes notable bacteriocins such as Divergicin A and Acidocin B, highlighting their diverse functional properties.
Class III bacteriocins are large peptides with molecular weights exceeding 30 kDa, characterized by their insolubility and thermal instability This group primarily comprises extracellular enzymes that are resistant to bacterial degradation and can mimic the physiological activities of bacteriocins To date, these bacteriocins have only been isolated from members of a specific genus, highlighting their unique biological properties and potential applications in antimicrobial strategies.
Lactobacillus Representing class III is Helvetica J produced by the bacterium L helveticus 481, and lactacin B produced by L acidophilus
Ring-structured bacteriocins represent a unique and less-studied class of antimicrobial peptides, characterized by post-translational modifications that form intramolecular covalent bonds creating a stable ring structure Bacteriocin complexes like Bacteriocin IV often consist of proteins associated with lipid or carbohydrate molecules, enhancing their stability and activity Notable examples include leuconocin S and lactocin 27, which exemplify the diversity of this bacteriocin class (Oscáriz & Pisabarro, 2001).
The mechanism of action of bacteriocins
Bacteriocins are widely recognized for their diverse mechanisms of action, primarily affecting essential cellular functions such as transcription, translation, replication, and cell wall biosynthesis Most bacteriocins inhibit target bacteria by forming membrane channels or pores that disrupt the energy potential of sensitive cells They are particularly effective against Gram-positive bacteria like Lactobacillus, Listeria monocytogenes, and Salmonella typhimurium However, their activity against Gram-negative bacteria is often limited due to the protective outer membrane, which prevents bacteriocins, antibiotics, detergents, and dyes from entering the cytoplasm, reducing their inhibitory effects.
Many bacteriocins can degrade DNA and RNA and attack peptidoglycan, weakening the bacterial cell wall They also exert their antibacterial effects by altering enzyme activity, inhibiting spore germination, and forming pores in bacterial membranes to disrupt ion transport The effectiveness of bacteriocins depends on factors such as their concentration, purity, the physiological state of target cells, and specific experimental conditions.
The mechanisms of action of the bacteriocin groups have been studied:
Class I - Typically nisin: the mechanism of action may be either on the peptidoglycan wall or on the primary membrane, or a combination of both For example, nisin has both mechanisms while lacticin is only able to act on the peptidoglycan wall For the peptidoglycan wall: lipid II plays an important role in the transport of synthetic units to peptidoglycan from the cytoplasm Bacteriocins of this group have the ability to bind to lipids located on the primary membrane, this association is explained by the fact that most bacteriocins are positively charged and membrane lipids are negatively charged, so binding is easily made on the membrane After binding, the bacteriocin will lock the lipids, making them unable to transport the subunits that make up peptidoglycan For the plasma membrane, bacteriocin binds and uses lipids on the primary membrane to carry out the redox process, at which time the lipids act as knives to create holes in the primary membrane These holes cause loss of many ions and substances dissolved in the protoplasm (10 mineral salts, amino acids, nucleic acids ), hydrolysis and loss of ATP, leading to faster cell death
Class II - Typically Sakacin: Because it belongs to the hydrophobic group, after creating a channel in the membrane, the bacteriocins of this group will bind to the lipids in the membrane phospholipid component, they will attach directly to the membrane as a component of the membrane, then perform redox reactions that create holes and act like the group I
Class III - Typical Lysostaphin: Acts directly on the cell wall, dissolving the cell wall and disrupting osmolarity
Class IV- Currently scientists have not yet come to a clear specific conclusion for the mechanism of action of this group According to the scientists' assumption, they think that this group acts mainly on DNA, RNA, and protein synthesis Bacteriocins of this group can form insoluble complexes with DNA, prevent DNA from synthesizing RNA and proteins, bind with DNA to translate abnormal proteins, or it can directly bind and inhibit protein activity
Several bacteriocins demonstrate antibacterial activity through their enzymatic functions For instance, colicin E2 exhibits DNase activity, degrading bacterial DNA, while colicin E3 shows RNase activity that breaks down RNA in target cells Additionally, megacin A-216 possesses phospholipase activity, disrupting the integrity of bacterial cell membranes These enzymatic mechanisms contribute to the potent antimicrobial effects of these bacteriocins against harmful bacteria (Prabhakar et al., 2013).
Biochemical properties of bacteriocins
Recent studies indicate that bacteriocins from different bacteria exhibit varying levels of heat tolerance, with classes I and II demonstrating the highest resistance For instance, the bacteriocins ST28MS and ST26MS from Lactobacillus plantarum maintain their antibacterial activity even after 90 minutes at 100°C or 20 minutes at 121°C The heat stability of bacteriocins may be linked to their molecular structures, and those produced by lactic acid bacteria (LAB) are known for their exceptional heat resistance, making them effective in high-temperature applications.
Certain bacteriocins, such as SR28MS and ST26MS, exhibit stable activity across a wide pH range (pH 2 to 12) for up to two hours, in addition to high thermal stability The stability of bacteriocins under varying pH and temperature conditions depends on their unique structural and chemical compositions These properties are crucial for the effective production and application of bacteriocins, as selecting optimal pH and temperature conditions enhances both production efficiency and antibacterial potency.
Bacteriocins have diverse chemical compositions, making them susceptible to specific enzymatic cleavage that results in the loss of their antibacterial activity This enzymatic inactivation serves as an important indicator to confirm the presence of bacteriocins For example, Plantaricin TF711 from Lactobacillus plantarum TF711, isolated in cheese, was shown by Hernandez et al to be partially inactivated by enzymes like lipase and α-amylase, highlighting the role of enzymatic sensitivity in identifying bacteriocins.
Or Vibriocin AVP10, a bacteriocin produced by Vibrio bacteria strains found in marine fish, is completely inactivated by protease, proteinase K, and trypsin enzymes, while it remains stable in the presence of lipase Its thermal stability, pH stability, and resistance to hydrolytic enzymes have been characterized, with detailed data provided in Table 1.1.
Table 1.1 Heat, pH, and enzymatic hydrolytic stability of some bacteriocins biosynthesized by lactic acid bacteria
Bacteriocin Heat resistance Active pH Bacteriocin hydrolytic enzymes
The application of bacteriocins
For microorganisms, bacteriocin is a way to compete for nutrients and living space between species, helping to balance microbial ecology For humans, bacteriocins play an important role
Bacteriocins are widely used in food preservation, with extensive research focused on their application in the dairy industry, eggs, vegetables, and meat products Their antimicrobial properties help enhance food safety and extend shelf life, making them valuable natural preservatives in various food sectors.
Nisin is approved by the FDA and used in over 48 countries as a natural food protectant, marketed under NisaplinTM It effectively inhibits the growth of a wide range of Gram-positive bacteria, including critical food-borne pathogens like Listeria monocytogenes Primarily applied in canned foods and dairy products, nisin plays a vital role in processed cheese and spreads, safeguarding against heat-resistant spore-forming organisms such as Bacillus and Clostridium Its use is especially crucial for preventing Clostridium botulinum toxin formation, which can have severe health consequences Additionally, other bacteriocins like lacticin 3147 and lacticin 481 are still in development, showing promising potential as natural preservatives and flavor enhancers.
Pediocin PA-1 is a broad-spectrum lactic acid bacteriocin that exhibits particularly strong activity against Listeria monocytogenes and is used as a food preservative
Bacteriocins can enhance food safety through three main methods: incorporating purified or semi-purified bacteriocin preparations directly into food products, adding ingredients that have been pre-fermented with bacteriocin-producing strains, or employing bacteriocin-producing cultures to replace or supplement traditional starter cultures during fermentation, thereby enabling in situ bacteriocin production for improved microbial control.
Bacteriocins enhance food quality and sensory properties by accelerating proteolysis and preventing gas-blowing defects in cheese Additionally, their use in bioactive packaging helps protect food from external contamination, thereby improving food safety and extending shelf life.
Nisin, which effectively inhibits a wide range of gram-positive bacteria, is commonly used to prevent mastitis in cattle Additionally, genetically modified Streptococcus mutans strains producing bacteriocins are incorporated into oral health products like toothpaste and mouthwashes to reduce tooth decay and tartar Bacteriocins present in soaps and cosmetics also play a role in eliminating acne, highlighting their diverse applications across healthcare and personal care.
Bacteriocins are increasingly being applied in the medical field as a promising solution to antibiotic-resistant infections With the rise of microbial resistance, traditional antibiotic therapies are becoming ineffective, highlighting the need for alternative treatments Bacteriocins have the potential to effectively treat localized skin infections and combat multidrug-resistant pathogens Additionally, probiotic and bacteriocin-producing strains offer protective benefits for the gastrointestinal tract, promoting overall gut health.
Scientific and fact-based reports suggest that certain probiotics can help modulate the skin’s microflora, strengthen the lipid barrier, and support the skin’s immune system, promoting skin homeostasis ESL5, a bacteriocin produced by Enterococcus faecalis SL-5, has demonstrated effectiveness as a lotion in reducing inflammatory acne lesions caused by Propionibacterium acnes Clinical studies show that patients treated with ESL5 experience significant reductions in inflammatory lesions and pimples compared to those using a placebo.
In recent years, concerns about food safety and quality have prompted scientists to explore and develop new methods of food preservation, especially bacteriocins However, up to now, the effects of bacteriocins in foods have been mainly experimental This may be due to the strict requirements for a safe bio- preservative product as well as conflicting views on genetically modified starter varieties in food However, as consumers need preserved products and processing equipment, the use of bacteriocins may become more common, as a means of preserving "natural" foods Below are some standards set for bacteriocin products applied in food technology: Regarding safety standards: The production strain must have the form of GRAS (Generally regarded as safe - assessed as safe microorganisms) safety), must be tested for toxicity, with no health risks through the identification of aspects such as cumulative, synergistic, and potential effects Efficacy: Bacteriocin must inhibit the growth of a broad spectrum of pathogens and resistant bacteria such as L monocytogenes and C botulinum with activity against a specific pathogen Bacteriocin must be heat stable and highly active when incorporated into food
Currently, only two bacteriocins are commercially available, nisin from
Lactococcus lactis and pediocin PA-1/AcH from Pediococcus acidilactici, both of which are used in the food industry Nisin and pediocin PA-1/AcH have respective trade names as Nisaplin, and ALTA
Probiotics and gut-friendly foods were among the top food trends of 2019, according to Forbes A variety of probiotic products, including pellets, powders, and enriched yogurts, have become widely available These products typically contain beneficial microorganisms like yeasts and bacteria, especially from the LAB group, such as Lactobacillus acidophilus These bacteria, found naturally in the human and animal gastrointestinal tracts, are added to fermented dairy products like acidophilus yogurt Probiotics from LAB produce bacteriocins that inhibit harmful bacteria, support immune system activity, inactivate carcinogenic compounds, and lower blood cholesterol levels Moreover, LAB-based probiotics are effective in preventing intestinal disorders and managing diarrhea, highlighting their significant health benefits.
Advantages and disadvantages of bacteriocins
Today, the widespread use of bacteriocins in food preservation has brought certain advantages over the use of chemical preservatives such as:
⁃ Safe in food preservation for human consumption, less restrictive than chemical preservatives because the molecules are naturally produced by fermenting microorganisms in traditional fermented foods
⁃ They have no impact on the environment as they degrade quickly
⁃ Bacteriocins are used as a primary food source for pathogens without affecting beneficial bacteria
⁃ Bacteriocin does not change the organoleptic properties of food
⁃ Has a clear spectrum of activity
⁃ Supplement for an antibacterial agent
⁃ Can replace antibiotics in aquaculture without causing toxicity to eukaryotic cells
Pure Bacteriocin is relatively stable with 10% salinity, a relatively narrow spectrum of activity compared to traditional antibiotics, so it will not kill beneficial organisms
With these benefits, bacteriocin is increasingly being used in industry as a safe antibiotic However, they also have some disadvantages as follows:
⁃ Less known than chemical preservatives
⁃ Rapidly degraded by proteolytic enzymes
⁃ High cost in meeting technical features
⁃ Only effective against a certain group of bacteria, there are specific requirements for their use in the pure or semi-pure form.
Effect of medium composition
A wide variety of complex media (CM) are available in today's market for the cultivation of lactic acid bacteria (LAB), including MRS, BHI, NaLa, M17, and TSBYE (C Liu et al., 2002) These media are tailored for specific LAB strains, such as M17 for lactococci and MRS for lactobacilli, ensuring optimal growth conditions.
Optimizing growth medium formulation is crucial for enhancing fermentation processes, requiring a balance of cost-effectiveness, high product yield, shorter fermentation time, and ease of downstream purification (N P Guerra et al., 2005) While highest product yields are desirable, they may not always be the most cost-efficient, making medium selection a strategic trade-off tailored to specific production needs Additionally, formulation aims to not only boost bacteriocin production but also ensure its stability, with components like sodium chloride (NaCl), ethanol, and high carbon source concentrations used to stabilize bacteriocin levels (F Leroy et al., 2003) Medium pH has also been identified as a key factor that significantly influences bacteriocin stability (T Zendo et al., 2005).
Adding sugars, vitamins, and nitrogen sources to the culture medium can enhance bacteriocin production, as these supplements typically promote bacterial growth However, excessive supplementation may inhibit both bacterial proliferation and bacteriocin synthesis, highlighting the importance of optimal nutrient levels Alternatively, designing a tailored, well-adapted culture medium can optimize conditions for maximum bacteriocin yield.
Glucose is the primary carbon source for microorganisms due to its rapid utilization and energy conversion capabilities (S H Al-Zahrani and F S Al-Zahrani, 2006) It is also the preferred sugar for stimulating bacteriocin production, with most research showing higher yields when glucose is present in growth media compared to other monosaccharides (A Delgado et al., 2007) Interestingly, some LAB strains produce higher levels of bacteriocins when other sugars are included alongside glucose, suggesting they possess complex enzymatic systems that enable them to utilize multiple carbohydrates For example, E faecium exhibits variable sugar utilization, with its bacteriocin production differing from glucose-dependent patterns.
A medium rich in yeast extract and protein hydrolysates is essential for optimal growth of lactic acid bacteria (LAB) and effective bacteriocin production Robust cell growth directly supports higher bacteriocin yields, highlighting the symbiotic relationship between bacterial proliferation and antimicrobial peptide synthesis (M Mataragas et al.) Ensuring the availability of nutrient-rich media enhances LAB vitality and their ability to produce bacteriocins, making it a critical factor for successful fermentation processes and probiotic applications.
Semi-synthetic media containing complex peptidic sources like MRS, Tryptone Glucose Yeast Extract (TGYE), or All Purpose Tween (APT) are essential for bacteriocin production Peptone, a key organic nitrogen source, is widely used in microbiological media due to its water-soluble, non-heat coagulable protein hydrolysates, which include peptides, proteoses, and free amino acids For cultivating lactic acid bacteria (LAB), media rich in peptones at high concentrations are recommended, with only a small portion of the peptones consumed during fermentation, ensuring optimal bacterial growth and bacteriocin synthesis.
Effect of cultivation condition
Temperature significantly influences bacteriocin production, with optimal production often occurring at specific temperatures that may differ from those promoting maximum bacterial growth For example, lactocin A, enterocin 1146, lactocin S, nisin Z, plantaricin, and enterocin 1146 show varied optimal temperatures for their biosynthesis Notably, some bacteriocins are produced maximally at suboptimal growth temperatures, as seen with amylovorin L471, where slower bacterial growth at low temperatures enhances energy allocation toward bacteriocin synthesis (L De Vuyst et al., 1996) Similarly, sakacin P demonstrates increased bacteriocin production at lower temperatures due to temperature-dependent metabolic reactions that improve carbon and energy utilization, despite reduced growth rates Elevated temperatures can lead to bacteriocin degradation or inactivation, impacting overall yields Additionally, sakacin A’s production is influenced by a three-component regulatory system sensitive to temperature variations, though its optimal temperature is higher than that of sakacin P (D B Diep and I F Nes, 2002) These findings underscore the complex relationship between temperature and bacteriocin synthesis, highlighting the importance of optimizing environmental conditions for maximum yield.
The optimal incubation temperature for maximizing bacteriocin production varies depending on the specific strain, requiring individual assessment for accurate results Most bacteriocin-producing strains isolated to date typically achieve high yields at temperatures ranging from 30°C to 37°C, highlighting the importance of temperature optimization in enhancing bacteriocin biosynthesis.
Culture pH significantly influences the growth of lactic acid bacteria (LAB) and their production of bacteriocins, affecting cell aggregation, absorption, and proteolytic degradation Bacteriocins are produced within a specific pH range that varies among strains and may differ from the pH at which they remain stable and active (S Abbasiliasi et al., 2016) pH also regulates enzymatic reactions; low pH and lactic acid accumulation can halt cell growth and bacteriocin production due to energy depletion Additionally, the immunity of bacteriocin-producing cells depends on immunity peptides co-transcribed with bacteriocin genes, which decline when bacteriocin production stops.
The optimal pH for bacteriocin production varies depending on the microbial strain, with some producing effectively at pH 5.5 to 6.0, while others do so below pH 5 All these pH levels differ from those ideal for bacterial growth A lower growth rate at suboptimal pH can lead to better energy utilization, enhancing bacteriocin synthesis Since enzymatic reactions involved in energy metabolism are regulated by pH, a reduction in pH may decrease these reactions, lowering bacterial growth but positively influencing bacteriocin production by increasing essential metabolites like ATP.
Our analysis indicates that pH and temperature are the most critical physiochemical factors influencing bacteriocin production by bacteriocinogenic strains Optimizing these conditions is essential for enhancing bacteriocin yield in various applications Proper control of pH and temperature can significantly improve the effectiveness of bacteriocin-producing bacteria in industrial and biomedical settings.
Bacteriocins is produced Enterococcus faecium
Enterococci, a group of lactic acid bacteria (LAB), have gained increased attention in recent years due to their dual roles in human health and disease Unlike lactobacilli, which are well-known for their probiotic benefits, enterococci are increasingly recognized as emerging human pathogens, despite being equally abundant in the gastrointestinal tract Enterococcus faecalis is the predominant species in the gut, followed by E faecium, with other species such as E avium and E hirae also commonly found in human stool samples.
Bacteriocin-producing bacteria are present across all environments, with numerous LAB isolates demonstrating bacteriocin production confirmed through biochemical and genetic studies The bacteriocins produced by enterococci are often similar to those generated by other lactic acid bacteria, indicating a conserved mechanism among these species Additionally, various classes and subclasses of bacteriocins have been isolated and characterized in enterococci, highlighting their diverse antimicrobial capabilities (Cotter, Hill).
Most characterized enterocins belong to Class II bacteriocins, with a few identified as heat-labile lytic enzymes Although these lytic enzymes were formerly categorized as bacteriocins, they are now recognized as a separate class of antimicrobials, highlighting their distinct nature in antimicrobial research (Ross, 2005; Nes et al., 1996; Nes et al., 2007; Cotter, Hill, & Ross, 2005).
Numerous bacteriocin producers have been identified and studied from infection-derived E faecalis and E faecium isolates For instance, bacteriocins such as Bacteriocin 31 and Bacteriocin 41 from E faecalis have been extensively researched, while Bacteriocin 43, Bacteriocin 32, and Bacteriocin 51 have been characterized from E faecium isolates In addition to clinical origins, bacteriocins from enterococci of food sources have also been studied, leading to the identification of several notable enterocins, including enterocin L50A/L50B, enterocin Q, enterocin A, and enterocin P, highlighting the diversity and potential applications of enterococci-derived bacteriocins in food safety and biotechnology.
B (Casaus, Nilsen, Cintas, Nes, Hernández, & Holo, 1997) and others
Numerous enterocins have been characterized across various Enterococcus species, with E faecium and E faecalis being the most thoroughly studied sources These bacteriocins have also been isolated from species such as E muntii, E avium, E hirae, and E durans Enterococci that produce bacteriocins are predominantly found in food, waste, human and animal feces, and gastrointestinal tracts, but can also originate from other sources Fermented foods, human infection specimens, and fecal samples from healthy infants are particularly rich sources for isolating bacteriocin-producing enterococci Most evidence suggests that enterococci primarily originate from the digestive tracts of humans and animals, which aligns with the discovery that identical bacteriocins are present in isolates from diverse mediums, especially those of human origin.
Research status of bacteriocins from Enterococcus faecium
Recent advances in modern methods have led to increased research on bacteriocins worldwide and in Vietnam These studies primarily focus on bacteriocins produced by lactic acid bacteria, highlighting their potential as natural antimicrobial agents The growing interest in bacteriocins underscores their significance in health, food preservation, and biotechnology applications.
In 2013, Dr Nguyen Van Duy Nghien from Nha Trang University conducted comprehensive research on the biological characteristics of marine bacteria that produce bacteriocins, exploring their potential as multi-purpose drugs in aquaculture His study focused on selecting and characterizing a collection of these beneficial bacteria to enhance disease management and promote sustainable aquaculture practices.
Thirty marine bacteria strains from diverse genera, including Enterococcus, have been identified as bacteriocin producers, opening new research opportunities in Vietnam for the development of marine-derived bacteriocins These strains hold significant potential for use in creating novel drugs and probiotics, ultimately contributing to the protection of human and animal health through innovative microbiological applications.
A study conducted by the Vietnam National University of Agriculture focused on selecting and identifying bacteriocin-producing strains from fish intestines as natural alternatives to antibiotics in aquaculture The research also determined the optimal environmental conditions for successful fish culture and identified the most suitable inoculation methods for these beneficial strains, promoting sustainable fish farming practices.
Enterococcus faecium HN1 and Enterococcus faecalis NA1.3 to produce bacteriocin are best liquid MRS medium supplemented with glucose and yeast extract at the rate of 3%
The genus Enterococcus encompasses a diverse group of strains, some of which serve as effective starter cultures or probiotics, while others are associated with spoilage or pathogenicity (Moreno et al., 2006) In 2021, researchers at ProBacLab, Handong Global University in Korea, characterized partially purified bacteriocins produced by Enterococcus faecium strains isolated from soybean paste These bacteriocins demonstrated activity against pathogens such as Listeria monocytogenes, Listeria innocua, and vancomycin-resistant Enterococci, highlighting their potential applications in controlling foodborne pathogens and enhancing food safety (Iyorita Fugaban et al., 2021).
Research by (2007) demonstrated that feeding mice with a bacteriocin-producing Lactobacillus salivarius UCC118 strain can protect against Listeria monocytogenes infection, a benefit not seen with a non-producing mutant strain More recent studies by Kommineni et al (2015) showed that bacteriocin production by intestinal commensal enterococci effectively reduces vancomycin-resistant enterococci to undetectable levels in mice without significantly disrupting the existing microbiota Additionally, the presence of a bacteriocin-encoding plasmid (pPD1) enables E faecalis to successfully colonize the gut, outcompete indigenous enterococci, and replace strains lacking the plasmid, highlighting the potential of bacteriocins in microbiome modulation and pathogen control.
Bacteriocin-producing enterococci hold significant potential as probiotics for preventing and treating intestinal infections To ensure their efficacy, the discovery of new enterococcal strains with high probiotic potential requires thorough assessment of their behavior, survival, and antimicrobial activity within the physiological conditions of the gastrointestinal tract Additionally, evaluating the safety of these strains for human use is essential to harness their full therapeutic benefits.
Two strains of Enterococcus are currently recognized as probiotics and are commercially available, namely, E faecium SF68® (NCIMB 10415, Cerbios
Pharma SA, Barbengo, Switzerland) and E faecalis Symbioflor 1 (SymbioPharm,
Herborn, Germany, highlights the proven efficacy of probiotics such as E faecium SF68® in preventing and treating diarrhea in humans and animals Additionally, E faecalis Symbioflor 1 has been recognized as an immune regulator effective in managing recurrent chronic sinusitis and bronchitis (Franz et al., 2011).
MATERIALS AND METHODS
Research subjects and materials
2.1.1 Location and time of the study
- Time: From July 2022 to December 2022
- Location: Laboratory of Microbiology - Faculty of Biotechnology - Vietnam National University of Agriculture
➢ Enterococcus faecium strain ET02 were provided from the laboratory of the Department of Microbiology - Faculty of Biotechnology - Vietnam
➢ Tested bacterial strains: Aeromonas hydrophila, Staphylococcus aureus were provided from the laboratory of the Department of Microbiology Technology - Department of Biotechnology - Vietnam National University of Agriculture
➢ Equipment: Aseptic culture chamber, cold centrifuge, shaker at 30℃ and 37℃, microbiological cabinet 30℃, aseptic drying oven, autoclave, pH meter, analytical balance, digital balance, refrigerator, micropipette (1àl - 1000àl)
➢ Chemicals: Glucose, meat extract, yeast extract, peptone, tween 80, triamonium citrate, triptone, NaCl, K2HPO4.3H2O, CH3COONa, MgSO4.7H2O, MnSO4.4H2O, agar, distilled water
- MRS medium supplemented with agar (g/l): Glucose 20.0, meat extract 10.0, yeast extract 5.0, peptone 10.0, tween 80 1ml, triamonium citrate 2.0,
K2HPO4.3H2O 2.0, CH3COONa 5.0, MgSO4.7H2O 0.58, MnSO4.4H2O 0.28,
CaCO3 5.0, agar 15.0, distilled water just enough 1 liter, pH = 6.5 ± 0.2; Sterilize at 121℃ for 15 minutes
- MRS liquid medium (g/l): has the same composition as solid MRS but does not add agar and CaCO3
Culture medium of test microorganism strains
- LB medium (Luria - Bertani) (g/l): Yeast extract 5.0, peptone 10.0, NaCl 10.0, agar 20.0, distilled water just enough 1 liter, pH = 7.5, sterilized at 121℃/15 minutes
- NB medium (Nutrient Broth) (g/l): beef broth 3.0, peptone 5.0, sodium chloride 8.0, pH = 6.8, distilled water just enough 1 liter, sterilized at 121℃ for
Methods
2.2.1 Identification and analysis by 16s rRNA gene sequencing
Bacterial strains were identified using molecular biology methods with 16S rRNA primer pairs, ensuring precise classification The 16S rRNA gene fragments of the strains were compared to nucleotide sequences registered in GenBank via the BLAST tool, facilitating accurate identification A phylogenetic tree was constructed using Mega X software, illustrating the evolutionary relationships among the bacterial strains.
An agar plate diffusion test was conducted using Enterococcus faecium strain ET02 The strain was cultured in 30 ml of liquid MRS medium at 30°C for 48 hours Subsequently, 10 ml of the culture was aspirated, centrifuged at 10,000 rpm and 4°C for 10 minutes, and the supernatant was collected The supernatant’s pH was adjusted to 6.5 with NaOH to eliminate the influence of lactic acid, ensuring accurate assessment of the antimicrobial activity.
Microorganism test strains, including Aeromonas hydrophila and Staphylococcus aureus, were cultured in 30 ml of nutrient broth (NB) medium at 30°C for 48 hours A 100 µl aliquot of the culture was uniformly applied to a petri dish containing LB agar medium Wells were then created in the agar using an agar puncher, and 100 µl of the centrifuged culture solution was pipetted into each well to assess antimicrobial activity.
Incubate the plates at 4℃ for 2h then transfer the entire plate to a 30℃ incubator for 12 h (Hernandez et al., 2005)
The antibacterial activity was assessed by measuring the diameter of the clear antibacterial ring surrounding the well, calculated as D – d (mm), where D is the diameter of the antibacterial zone and d is the diameter of the agar well A larger difference between D and d indicates stronger antibacterial efficacy.
2.2.3 Effect of the medium ingredients on bacteriocin production
2.2.3.1 Effects of cacbon sources on bacteriocin production
❖ Effect of different carbon- sources on bacteriocin production
Enterococcus faecium strain ET02 was cultured at 30℃ for 48 hours in four flasks of liquid MRS medium supplemented with different sugar sources—sucrose, fructose, lactose, and glucose—each at 2% w/v, to evaluate the impact of various carbon sources on bacteriocin production Bacteriocin activity was assessed using the agar plate diffusion method, with all experiments conducted in triplicate to ensure accuracy and reproducibility.
❖ Effect of different concentrations of best carbon- source
Enterococcus faecium strain ET02 was cultured at 30°C for 48 hours in liquid MRS medium supplemented with varying concentrations (1%, 2%, 3%, and 4% w/v) of the optimal sugar that yielded the highest bacteriocin activity, to evaluate the effect of different carbon-source concentrations on bacteriocin production Bacteriocin activity was assessed using the agar plate diffusion method, with all experiments conducted in triplicate to ensure reproducibility.
2.2.3.2 Effect of nitrogen sources on bacteriocin production
Enterococcus faecium strain ET02 was cultured in optimal liquid MRS medium supplemented with varying concentrations of Peptone (1%, 2%, 3%, and 4% w/v) to assess the impact of nitrogen sources on bacteriocin production Bacteriocin activity was evaluated using the agar plate diffusion method, with all experiments conducted in triplicate to ensure accuracy and reproducibility.
2.2.3.3 Effect of NaCl concentration on bacteriocin production
Enterococcus faecium strain ET02 was cultured on optimal liquid MRS medium to produce bacteriocin, with experiments conducted to assess the influence of NaCl supplementation at concentrations of 1%, 2%, 3%, and 4% (w/v) The effect of varying NaCl levels on bacteriocin production was evaluated using the agar plate diffusion method, ensuring all experiments were performed in triplicate for accuracy.
2.2.4 Effect of pH on bacteriocin production
Enterococcus faecium strain ET02, capable of producing bacteriocins, was cultured in liquid MRS medium with optimized ingredients, and the pH was adjusted to 4.5, 5.5, 6.5, 7.5, 8.5, and 9.5 using 1M HCl or 1M NaOH The effect of pH on bacteriocin activity was assessed by measuring the antibacterial ring diameters on agar plates, with comparisons made across different pH levels Bacteriocin activity was determined using the agar well diffusion method, and all experiments were conducted in triplicate to ensure accuracy.
2.2.5 Effect of temperature on bacteriocin production
Enterococcus faecium strain ET02, capable of producing bacteriocins, was successfully cultured on liquid MRS medium with optimized ingredients, as determined in experiment 3.2.2 The pH level was further optimized in experiment 3.2.3, with temperature variations of 25°C, 30°C, and 35°C tested over 48 hours Bacteriocin activity was measured using the agar plate diffusion method, ensuring accurate assessment of antimicrobial production All experiments were conducted in triplicate to ensure reliability and reproducibility.
2.2.6 Effect of time on bacteriocin production
Enterococcus faecium strain ET02, capable of producing bacteriocins, was cultivated on optimized liquid MRS medium in experiment 3.2.2 The pH and temperature conditions were fine-tuned in experiment 3.2.3 to maximize bacteriocin production, with time points set at 2, 4, 6, and 8 days Bacteriocin activity was assessed using the agar plate diffusion method, and all experiments were conducted in triplicate to ensure data reliability. -Optimize your research summary effortlessly with Draft Alpha’s expert rewriting tools—[Learn more](https://pollinations.ai/redirect/draftalpha)
RESULTS AND DISCUSSION
Enterococcus faecium strain ET02
Figure 3.1 A: Colony of the Enterococcus faecium strain ET02; B:
Phylogenetic tree of Enterococcus faecium strain ET02
PCR products were sequenced for nucleotides Analyze data with Blast tool on NCBI to compare results on Genbank
Strain ET02 was isolated from the intestines of swallowfish in Hai Phong Phylogenetic analysis using Mega X software revealed that ET02 belongs to a specific bacterial lineage The constructed phylogenetic tree (Figure 3.1B) clearly demonstrates the placement of strain ET02 within the bacterial taxonomy, highlighting its genetic relationship to related species This identification provides important insights into the microbial diversity associated with swallowfish and contributes to understanding its potential probiotic properties.
B addition, the morphological, physiological and biochemical characteristics are similar to strains registered on Genbank Therefore, combining biological and molecular characteristics can conclude that strain ET02 is closely related to
Enterococcus faecium CE 61 Therefore, strain ET02 is named Enterococcus faecium ET02.
Effect of the medium ingredients on bacteriocin production
3.2.1 Effects of cacbon sources on bacteriocin production
❖ Effect of different carbon- sources on bacteriocin production
E faecium strain ET02 was cultured in MRS medium supplemented with various 2% (w/v) carbon sources, including glucose, fructose, lactose, and sucrose, to evaluate its ability to produce bacteriocin The study demonstrated that the type of carbon source significantly influences bacteriocin production, with each carbohydrate affecting the strain's antimicrobial output differently Understanding these effects provides valuable insights into optimizing conditions for maximizing bacteriocin yield from E faecium ET02.
Figure 3.2 The effect of C-source on the antibacterial ability of
Using lactose results in the smallest inhibition zone due to minimal bacteriocin production In contrast, more substantial inhibition zones are observed when saccharose and fructose are used as carbon sources The largest inhibition zone, measuring 12 mm against A., was achieved with glucose, indicating its effectiveness in promoting bacteriocin activity.
Hydrophila, and 11 mm against S Aureus, leading to the best ability to antagonize the test bacteria
Glucose is the preferred carbon source for stimulating bacteriocin production, with numerous studies demonstrating higher yields in the presence of glucose compared to other monosaccharides (S H Al-Zahrani and F S Al-Zahrani, 2006) Therefore, glucose is considered the most suitable carbon source for maximizing bacteriocin synthesis in microbial cultures.
❖ Effect of different concentrations of best carbon- source
Experimental results show that the glucose concentration has a great influence on the bacteriocin activity of strain E faecium strain ET02
Figure 3.3 The effect of different glucose concentrations on the antibacterial ability of Enterococcus faecium strain ET02
Enterococcus faecium ET02 demonstrated maximum antagonistic activity against A hydrophila and S aureus at a 3% glucose concentration, with inhibition zone diameters of 13.33 ± 0.58 mm and 14.67 ± 0.58 mm, respectively The inhibitory effect was optimal at this concentration and diminished gradually as glucose levels increased beyond 3%.
Research by Stefano Schirru et al (2013) demonstrated that Enterococcus faecium SD1 produces the highest levels of bacteriocins at a glucose concentration of 3% Based on these findings, a 3% glucose concentration was selected for subsequent experiments to optimize bacteriocin production.
3.2.2 Effect of nitrogen sources on bacteriocin production
In this experiment, E faecium strain ET02 bacteria were cultured in MRS medium with a modified nitrogen source composition of Peptone The results are shown in figure 3.4
Figure 3.4 The effect of diferent peptone concentrations on the antibacterial ability of Enterococcus faecium strain ET02
Adjusting Peptone concentrations significantly influences the bacteriocin activity of E faecium strain ET02, similar to the effects observed with varying glucose levels The antagonistic activity against A hydrophila increased notably when 3% and 4% (w/v) Peptone were added to the liquid MRS medium The largest inhibition zone was observed at 3% Peptone, indicating optimal bacteriocin production at this concentration.
15 ± 0.58 mm, and S Aureus was 18 ± 0.58 mm This showed that the addition of Peptone provided a nitrogen source for E faecium strain ET02 to increase biomass and synthesize bacteriocin
Research by Paulraj Kanmani et al (2010) demonstrated that Enterococcus faecium MC13 exhibits enhanced bacteriocin production under specific environmental conditions Notably, the strain produced the highest levels of bacteriocin when 3% peptone was added to the growth medium This finding highlights the significance of nutrient optimization, particularly peptone concentration, to maximize bacteriocin yield in E faecium MC13.
3.2.3 Effect of NaCl concentration on bacteriocin production
The addition of NaCl either decreased or had no effect on bacteriocin activity The inhibition zone diameter against A hydrophila was measured at 8 ± 0.58 mm, whereas against S aureus it was 9 ± 0.58 mm, indicating limited antimicrobial effectiveness under these conditions.
Hydrophila was 11 ± 0.58 mm and that of S Aureus 14 ± 0.58 mm
Figure 3.5 The effect of adding NaCl 1%, 2%, 3%, 4% on the antibacterial ability of Enterococcus faecium strain ET02
Petkov et al (2008) found that Enterococcus durans M-3, Enterococcus faecium 3587, and Enterococcus faecalis 3915 produced the highest levels of bacteriocins in salt-free media, indicating that eliminating NaCl enhances bacteriocin biosynthesis Similarly, adding NaCl at higher concentrations did not promote bacteriocin or biomass production in specific media, suggesting that salt-free conditions are optimal for maximizing bacteriocin production in Enterococcus strains, including E faecium ET02.
Effect of pH on bacteriocin production
E faecium strain ET02 demonstrates optimal growth and strong antibacterial activity within a pH range of 4.5 to 8.5 Its antibacterial effectiveness diminishes at pH 9.5, where the bacteria lose their antibacterial properties The highest production of antibacterial agents occurs at pH 6.5, with antibacterial ring diameters of 17.67mm against A hydrophila and 17.33mm against S (species).
Aureus, gave the largest antibacterial ring size in terms of ability to antagonize the two strains of bacteria tested above
Figure 3.6 The effect of pH on the antibacterial ability of Enterococcus faecium strain ET02
The inhibition zone diameter increases linearly within the pH range of 4.5 to 6.5, indicating enhanced antibacterial activity under these conditions For the test strain A hydrophila, the results showed reduced antibacterial activity at pH 5.5 compared to pH 4.5 and 6.5, likely due to experimental error Overall, pH levels significantly influence the antibacterial efficacy of the bacterial strain.
Maintaining a pH between 6.5 and 9.5 is essential for preserving antibacterial activity, as the zone of inhibition gradually decreases beyond this range At higher pH levels, the plasma membranes of microorganisms are disrupted, damaging their cells and impairing their function Additionally, alkaline conditions alter the electrolyte state of nutrient molecules, reducing microbial nutrient uptake These effects collectively lead to decreased bacteriocin production, resulting in smaller antibacterial zones and a loss of microbial inhibitory activity.
Research by J.H Kang et al (2005) demonstrated that Enterococcus faecium GM-1, isolated from an infant, optimally produces bacteriocins at a pH range of 6.0 to 6.5 Similarly, Paulraj Kanmani et al (2010) found that Enterococcus faecium MC13 produces the highest levels of bacteriocins at a pH of 6.5 These studies highlight the significant influence of pH on bacteriocin production in Enterococcus faecium strains.
Thus, the pH value = 6.5 was selected to be used for the next experiments.
Effect of temperature on bacteriocin production
Figure 3.7 The effect of temperature on the antibacterial ability of
Enterococcus faecium strain ET02 demonstrates optimal growth and antibacterial production within a temperature range of 25-35°C, with peak activity observed at 30°C At this optimal temperature, strain ET02 produces the strongest antibacterial effects, creating inhibition zones measuring 16.67 mm against Aeromonas hydrophila and 16.50 mm against Staphylococcus aureus These findings highlight the strain's potential as a powerful source of antibacterial agents for various applications.
Research by Samantha Joy D Valledor et al (2020) indicates that bacteriocin production by E faecium ST10Bz against L monocytogenes increases when cultivated at 25°C and 30°C Similarly, Stefano Schirru et al (2013) found that Enterococcus faecium strains SD3 and SD4 produced the highest levels of bacteriocin at 30°C Based on these findings, 30°C was selected as the optimal temperature for subsequent experiments to maximize bacteriocin production.
Effect of time on bacteriocin production
The antibacterial ability of Enterococcus faecium strain ET02 is significantly influenced by culture duration After four days of shaking culture, ET02 exhibits the strongest antibacterial activity, indicated by the largest zones of inhibition against A hydrophila and S aureus This peak activity correlates with high bacteriocin production, with the antibacterial ring diameters reaching approximately 12.17 ±, highlighting the optimal cultivation period for maximizing antimicrobial effectiveness.
0.29 mm and 18 mm, respectively, then after 8 days of culture, they still kept their activity but there was a decreasing trend
Sequencing results showed that strain ET02 is closely related to
Enterococcus faecium CE 61 Therefore, strain ET02 was named Enterococcus faecium ET02
Enterococcus faecium strain ET02 demonstrated strong antibacterial activity against A hydrophila and S aureus when cultured in MRS medium containing 3% glucose as the carbon source and 3% peptone as the nitrogen source Optimal conditions for this activity were at pH 6.5 and a temperature of 30°C, with the most potent effects observed after 4 days of cultivation These findings highlight the potential of E faecium ET02 as a natural antimicrobial agent under specific culture conditions.
From the research results that have been obtained, I have recommendations and directions for further research:
- To study whether the bacteriocin produced by Enterococcus faecim strain ET02 belongs to which type of bacteriocin
- Determine the bacteriocin-producing mechanism of Enterococcus faecim strain ET02, which gene is bacteriocin produced
- Purify bacteriocin from Enterococcus faecim strain ET02 to apply as probiotic products
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Effect of culture medium composition on bacteriocin
Appendix 1 Effect of culture medium composition on bacteriocin
Effect of cultivation condition
Appendix 3 Effect of culture medium composition on bacteriocin