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2.1. Overview of fermented foods and beverages Fermentation, an ancient practice across cultures worldwide, enriches diets by enhancing food's nutritional value through microorganism-driven synthesis of proteins, vitamins, and amino acids ( Zhang et al. 2016). This process historically provided sustenance for rural populations and remains crucial for food security, especially for marginalized communities (Marshall and Mejia ,2012). Overall, fermentation stands as one of the oldest and cost-effective ways to preserve food safety and quality. Traditional fermentation of food and beverages is a crucial economic activity globally, especially in the informal sector, providing livelihoods for many families and supporting commercial endeavors (Thairu et al., 2014). This indigenous knowledge is often passed down orally, limiting its widespread documentation. Consequently, traditional fermentation practices are primarily found in rural and tribal areas due to factors like cost, accessibility, cultural preferences, and social ties to these products (Paulos et al. 2010). Traditional fermented foods and beverages exhibit unique characteristics influenced by environmental factors, culture, tastes, raw material availability, and technological advancements (Nair et al., 2008).The diversity of microorganisms involved in fermentation shapes these products' qualities (Vogel et al., 2011). These microbes can be native to the food or added intentionally as starter cultures in industrial processes (Stevens et al.,2009).Some of these beneficial organisms produce compounds with food additive potential or enhance fermentation processes naturally (Longo, 2006). Traditional fermented foods and beverages undergo spontaneous fermentation, leading to variability in their microbiological and chemical composition (Bacha et al., 2010). The process relies on microorganisms present in substrates, utensils, or from previous batches, influencing the final product and its quality (Steinkraus, 2002). These microbes biochemically transform the substrates into culturally accepted edible products. Key components of the fermentation ecosystem include organic matter, microbiota (bacteria, yeasts, and molds), the fermentation solution, and the fermenting container (Stolier et al., 2008). 2.2. Types of fermentation Fermentation remains an energy-efficient and cost-effective method crucial for food preservation and safety, extending shelf life (Sadat et ., 2017). Despite modern technologies, fermentation remains fundamental in food production. Different types such as lactic acid, acetic acid, alkaline, and alcoholic fermentation are commonly used, depending on the desired food products (Anal, 2019). Figure 2. 1. Illustrats the schematic representation of types of fermentation, the microorganisms involved, and the resulting end products( Anal, 2019). 2.2.1. Lactic acid fermnation Lactic acid bacteria are crucial for lactic acid fermentation, playing key roles in food preservation and production without requiring much heat, making them cost-effective. The quality and characteristics of these fermented foods depend on microbial actions influenced by factors like water activity, pH, salt, temperature, and food composition (Blandino et al., 2003). While various microorganisms participate in fermentation, specific types significantly impact product quality.The development of microbial communities in food is guided by ecological factors, affecting the physiological behavior of LAB (Park et al., 2018). Although microorganism quantities in food are small, their impact on flavor and other sensory aspects is significant(Okafor et al.,2009). Fermented foods are a significant part of diets worldwide, constituting 25% in developed nations and 60% in many developing countries(Ogunbanwo et al., 2003). Ethiopia, with its diverse cultural backgrounds, has a rich variety of traditional fermented foods and beverages like ergo, ayib, qibe, arrera (Gonfa et al.,2001), kotcho, siljo (Mehari and Ashenafi,1995), awaze, datta (Idris et al. ,2001) and tella, (Derbew et al.,2013), borde and shamita (Bacha et al., 2011). Lactic acid bacteria play a crucial role in food fermentation by rapidly acidifying the environment through the production of lactic acid. Additionally, they generate various metabolites such as acetic acid, ethanol, aromatic compounds, bacteriocins, exopolysaccharides, and enzymes. These compounds not only enhance the texture and sensory attributes of fermented products but also contribute to their extended shelf life and microbiological safety. A wide range of fermented foods and beverages, including dairy products, meat and fish products, fresh vegetables, cereals, and alcoholic/non-alcoholic beverages, are primarily the result of lactic acid fermentation (Rhee et al., 2011). Different species of Lactobacillus are commonly utilized in traditional fermentation practices for items like meat, vegetables, and dairy due to their ability to produce lactic acid. The adoption of these probiotic strains in industrial food technology is justified by their role in improving the conversion process and enhancing the flavor and texture properties of fermented foods (Anal, 2019). Figure 2.2 illustrates the metabolic pathways carried out by lactic acid bacteria. Figure 2. 2. Metabolic pathways of homofermentative and heteroferemntative LAB (Basso et al. 2014). Metabolic pathways of homofermentative (solid line) and heterofermentative (dotted line) LAB: P, phosphate; ADP, adenosine 5'-diphosphate; ATP, adenosine 5'-triphosphate; NAD +, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide (reduced form); (1), lactate dehydrogenase; (2), alcohol dehydrogenase. 2.2.2. Alcoholic Fermentation Alcoholic fermentation, an ancient and crucial food processing method, has been used since ancient times to produce various alcohol-based beverages like beers, wines, and spirits. This process involves microorganisms such as yeast, like Endomycopsis, and bacteria like Zymomonas mobilis fermenting sugars or starch hydrolysates to produce ethanol and carbon dioxide(Anal, 2019). In Africa, sorghum-based beers are extensively are produced, dolo in Burkina Faso and Togo, pito in Ghana and Nigeria (Konlani et al., 1996; Pattison et al., 1998; van der Watt et al., 2018). Ethiopia is known for traditional fermented beverages like; tella (Birhanu Abegaz, 2014), tej(Bahiru et al., 2001), borde (Abegaz, 2007) and shamita ( Bacha et al.,1999). 2.2.3. Acetic Acid Fermentation Fermented foods and beverages often contain acetic acid bacteria (AAB), like Acetobacter, Gluconobacter, Gluconacetobacter, and Komagataeibacter. They oxidize carbohydrates, sugars, alcohols, and ethanol in food substrates to acetic acid. Despite their significance, AAB are not as extensively studied as other industrial bacteria and can spoil alcohol production (De et al., 2018). According to Awulachew (2021), the dominant species in acetic acid fermentation are S. cerevisiae and L. pastorianum. Tella, with a pH of 4.5-4.8 and 2.8-5.0% (v/v) ethanol, is considered best for consumption. However, excessive growth of Acetobacter spp. during aerobic fermentation can make tella too sour to drink (Awulachew, 2021). 2.2.4. Alkanie Fermentation Alkaline fermentation involves Bacillus species and other bacteria to break down proteins into peptides and amino acids, leading to a pH increase to 8-10. This process is crucial in regions where starchy foods are staple but lack diversity in nutrients (Samedi & Charles, 2019). The common Bacillus species found in legume-based fermented foods include B. amyloliquefaciens, B. circulans, B. coagulans, B. firmus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, B. subtilis var. natto, and B. thuringiensis, while B. cereus strains are associated with Prosopis fermentation. Some B. subtilis strains produce polyglutamic acid (PGA), contributing to the sticky texture of Asian fermented soybean dishes (Elshaghabee et al., 2017; Owusu-Kwarteng et al.,2022). 2.3. Function of fermented foods and beverages Further research is essential to ensure the safety and nutritional value of traditional fermented foods and beverages, which are increasingly vital due to population growth and food insecurity. The wide array of fermented foods available globally highlights their significance not just for preservation and safety but also for their valued sensory properties(Holzapfel, 2002). In developing countries, millions rely on fermentation for affordable food preservation (Kalui et al., 2010). Because, the fermentation improves digestibility, flavor, stability, and nutritional value, along with reduced food toxicity, cooking time, energy requirements, and extended shelf life (Parkouda et al., 2009). 2.3.1. Nutritional Quality Fermented products, typically maintained at 22-25°C, encourage microorganism and metabolite activity due to their acidic nature (Mokoena et al.,2016). LAB fermentation not only boosts enzyme activity but also reduces anti-nutrients like phytic acid and tannins in these foods, enhancing the availability of minerals like iron, protein, and simple sugars (Chelule et al.,2010). Compared to unfermented foods, fermented ones are generally more nutritious (Oladeji et al.,2018). Many fermentative microorganisms involved in traditional fermentation processes break down complex compounds and produce complex vitamins and growth factors, elevating the nutritional value of the foods and beverages (Hasan et al.,2014 ; Mahlia et al., 2014). 2.3.2. Enhancement of Sensory Quality Fermentation enhances food and beverage organoleptic features such as flavors, smells, and textures, adding new sensory properties to the diet and making the food more palatable and, ultimately, more popular than unfermented food (Blandino et al.,2003; Chelule et al.,2010). These organoleptic properties made fermented products more popular than unfermented ones in terms of consumer acceptance(Blandino et al., 2003). Thus, fermentation is unique in that it modifies the unfermented food in diverse ways, resulting in new sensory properties in the fermented product (De Vuyst et al., 2007). 2.3.3. Bio-preservation potential The preservative effect of fermented food and beverages is due to several antimicrobial compounds produced by LAB during fermentation process (Santos et al., 2018). These bacteria also produce antimicrobial metabolites, including organic acids, hydrogen peroxides and bacteriocin (Chelule et al.,2010; Santos et al. 2018) which can increase the bio-preservative potential of LAB. In general, many fermented foods and beverages have good keeping qualities and can be kept without refrigeration for longer than fresh, unfermented foods. Fermentation increases the shelf life and decreases the need for refrigeration or other methods of food preservation (Ahn et al., 2002; Aidoo et al., 2011). 2.3.4. Health Benefits Most species of LAB used for the production of fermented foods and beverages are generally recognized as safe (GRAS), thus eliminating any health risk (Endeshaw et al., 2018). Those LAB isolates, largely isolated from traditional fermented foods and beverages are the normal residents of the complex ecosystem of the gastrointestinal tract (GIT) (Tamang,2003). The probiotic effects and lower levels of pathogenic bacteria found in fermented foods and beverages are especially important in developing countries, where fermented foods and beverages have been shown to reduce the severity, duration, and morbidity of diarrhea. According to a study conducted in the Netherlands and Sweden, regular consumption of fermented dairy products has negative effects and lowers the incidence of bladder cancer, cholesterol level, (Keszei et al., 2009) and cardiovascular disease(Sonestedt et al.,2011). list and define all technical teems

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Fermentation: The metabolic process that converts carbohydrates, such as sugars and starches, into alcohol or organic acids using microorganisms like bacteria, yeast, or fungi, under anaerobic conditions.

Microorganism: Living organisms too small to be seen with the naked eye, including bacteria, yeast, molds, and viruses.

Nutritional value: The content of nutrients in food, including proteins, vitamins, minerals, and other essential components required for growth, repair, and maintenance of the body.

Proteins: Large molecules composed of amino acids essential for the structure, function, and regulation of the body's tissues and organs.

Vitamins: Organic compounds required in small amounts for various physiological functions in the body, including metabolism, growth, and immunity.

Amino acids: Building blocks of proteins, essential for synthesizing hormones and neurotransmitters, and for repairing body tissues.

Food security: The condition where all people, at all times, have physical, social, and economic access to sufficient, safe, and nutritious food that meets their dietary needs and food preferences for an active and healthy life.

Informal sector: Economic activities that are not regulated by the government and often involve small-scale or unregistered businesses, including traditional food fermentation practices carried out by families or communities.

Starter cultures: Cultures of known microorganisms used to initiate fermentation processes in food production, ensuring consistent results and desired characteristics.

Spontaneous fermentation: Fermentation that occurs naturally without the addition of starter cultures, relying on microorganisms present in the environment or on food substrates.

Microbiological composition: The types and quantities of microorganisms present in a particular environment or food product.

Chemical composition: The arrangement and proportions of chemical elements or compounds present in a substance, such as nutrients, acids, and other organic and inorganic components.

Shelf life: The length of time a food product can be stored under specified conditions while maintaining its quality and safety for consumption.

Lactic acid fermentation: A type of fermentation process where sugars are converted into lactic acid by lactic acid bacteria, commonly used in the production of dairy products, pickles, and fermented vegetables.

Acetic acid fermentation: Fermentation process where acetic acid bacteria oxidize carbohydrates, sugars, alcohols, and ethanol in food substrates to produce acetic acid, commonly used in the production of vinegar and certain alcoholic beverages.

Alkaline fermentation: A fermentation process where alkaline conditions are created by specific bacteria, such as Bacillus species, to break down proteins into peptides and amino acids, often used in the fermentation of legumes and starchy foods.

Organoleptic: Relating to the sensory properties of food, including taste, smell, texture, and appearance.

Bio-preservation: Preservation of food using natural or controlled microbiota and their antimicrobial metabolites, such as organic acids and bacteriocins, to inhibit the growth of spoilage and pathogenic microorganisms.

Probiotic: Live microorganisms that, when administered in adequate amounts, confer health benefits on the host, often found in fermented foods and beverages.

GRAS: Generally Recognized As Safe, a designation by regulatory agencies indicating that a substance or microorganism is considered safe for consumption under specified conditions.

Pathogenic bacteria: Microorganisms capable of causing disease in humans or animals.

Gastrointestinal tract (GIT): The digestive system, including the stomach, intestines, and associated organs, where food is digested, absorbed, and processed.

Bladder cancer: Cancer that develops in the tissues of the bladder, often associated with environmental and genetic factors.

Cardiovascular disease: A group of disorders of the heart and blood vessels, including coronary artery disease, stroke, and hypertension, often influenced by lifestyle and dietary factors.

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Fermented foods have been part of human diets for over 6,000 years, offering benefits like improved shelf life, safety, digestibility, and nutrition(Ther, 2013).Lactic acid fermentation, where microorganisms convert sugars into lactic acid, is widely used in households and the food industry for preservation and creating various food items (Dorrington & Bowdish, 2013).This natural preservation method is cost-effective and economically vital in developing countries (Moyane et al., 2013). Fermentation of cereals enhances taste, flavor, acidity, and digestibility. In adiition, fermented meats and fish, can be sources of probiotic bacteria if processed correctly (Anihouvi et al., 2012). Fermentation is also a key fish preservation method in coastal African regions, though processing methods and hygiene levels can affect the safety and quality of fermented fish products (Anihouvi et al., 2012). There is a lack of research on the health benefits of fermented foods, the specific bacterial strains utilized, and the potential probiotic microbes in African fermented foods for applications in the food and pharmaceutical sectors. 2.5. Ethiopian traditional fermented foods and beverages Ethiopian traditional fermented foods and beverages, crafted with rural skills and indigenous knowledge, are vital in global diets, particularly in Ethiopia's culturally diverse landscape. Ethiopian traditional fermented foods and beverages that have received attention include ergo (sour milk), ayib (cottage cheese), qibe (traditional butter), arrera (defatted buttermilk) (Gonfa et al., 2001), injerra, kotcho, siljo(Mehari & Ashenafi, 1995), (Idris et al., 2020). Despite their local-scale production, they play a significant role in sustaining communities(Ashenafi, 2006). Extensive research has investigated potential LAB in Ethiopian fermented foods, revealing their ability to combat foodborne pathogens and enhance food shelf life through antimicrobial acitivity (Birhanu Abegaz, 2014; Mamo Nigatu et al., 2015). Furthermore, because of their ability to produce antimicrobial metabolites, they can control food spoilage and improve food shelf life by extending the food and controlling the proliferation and establishment of pathogens in food. 2.5.1. Bukuri Bukuri is one of Ethiopian traditional fermented nonalcoholic beverage which is the most popular among all people of East Wollega zone and it surroundings area which is consumed by large communities of the rural areas. The ingredients for the preparation of Bukuri are cereals like barely (Hordeum vuldare), maize (Zea mays), sorghum (Sorghum bicolor), wheat (Triticum sativum), finger millet (Eleusine coracana) or teff (Eragrostis teff) and it is without gesho. The communities use Bukuri as meal replacement, on holidays, wedding ceremony, and social works and as sources of income of many households. The popularity of traditionally fermented Bukuri is more common among the religious groups because it is considered as low alcoholic beverages. Bukuri is a cereal based Ethiopian traditional fermented beverages prepared from locally available cereals (maize, barley, sorghum, millet, wheat, and teff and malt flours) (Chali et al., 2024). It is considered as alcohol free drink although the preparation follows a lot of fermentation process. The picture depicts the traditional bukuri fermenting process, which is widely practiced in regions consumed in and around East Wollega Zone. This beverage is frequently used in the communities, particularly during holidays, social work, and wedding ceremonies (Figure 2.3 and 2.4). Figure 2. 3. Traditional bukuri preparation and its ingdreindst, in east wollega zone, 2023 (A. Malt preparation, B. Roasted cereal for pourage preparation, C. Bread prepared, malt and cereals D. Ready to drink bukuri). Figure 2. 4. Bukuri preparation flow chart(Chali et al., 2024). 2.5.2. Cabbage-shamita Cabbage-shamita is a thick, traditionally fermented non-alcoholic beverage mostly consumed by women in rural and urban parts of East Wollega zone and its enviroments. Cereals such as barley, maize, cabbage seeds, and spices are used in cabbage-shameta fermentation. Because cabbage-shamita fermentation takes time and energy, it is not for sale, meal replacement, vacations, or social work. Cabbage seed is crushed with a native crusher and pestle and immersed in water for 7-14 days in cabbage-shamita fermentation. The filtered liquid half is boiled with barely or maize flour, chilled, and spices added before being preserved in a pot after 7-14 days. The solid bits that remain are discarded. There is no need for cereal malt in cabbage-shamita fermentation(Chali et al., 2024). Figure 2. 5. Cabbage-shamita prepared at home and ready for consumption, Nekemte Ethiopia, 2023. Figure 2. 6. Ethiopian traditional fermented beverage, Cabbage-shamita preparation flow chart(Chali et al., 2024). 2.5.3. Borde Borde is one of the most commonly prepared and consumed traditional fermented beverages in many part of the county, Nekemte city and its surrounding communities are using borde specially during ceremony, wedding and on holly days’ occasion. Even though, many ingredients such as maize, wheat, teff, sorghum, and finger millet are mostly used for borde preparation, at the study area, mostly borde has been prepared from barley (Hordeum vulgar L), as the information gathered from the traditional practioners. Therefore, it is a popular meal substitute in several sections of the country. Borde is a low-alcohol beverage, and most people believe it helps during lactation. After giving delivery, mothers are recommended to consume large amounts of it. The components for borde fermentation appear to differ between borde-producing villages. The most often utilized indigenous raw materials for borde preparation are maize, wheat, teff, sorghum, and finger millet (Nemo & Bacha, 2020; Tilahun et al., 2018). Barley is necessary for the production of malt borde. Barley is washed to remove dirt and unwanted impurities before steeping in water for roughly a day to make malt. Excess water is drained, and the soaked barley is wrapped in insete eaves for 2-5 days in order to germinate. Germination barley can afterward be sun-dried and crushed finely, result Malt. The steps, procedures and all necessary information required for Borde predation is shown in Figure 2.4. Figure 2. 7. Borde prepared at home and ready for consumption, Nekemte Ethiopia, 2023, A. barley, washed and cleaned carefully; B. Rosted barley, C. germinated barley, D. Barely powder, E. Ready to drink borde. Figure 2. 8. Ethiopian traditional fermented beverage, borde preparation flow chart, from the local borde brewers, during this study 2.5.4. Kotcho Kocho, along with injera, is one of the most popular fermented dishes. It has a high starch content due to the fermentation of pseudostem, pulverized corm, and decorticated pulp (Bosha et al., 2016; Dibaba et al., 2018). The fermentation duration of kocho varied from location to location, depending on their incubation temperature. The period of kocho fermentation is one year (Dibaba et al., 2018; Yemata, 2020). However, fermentation times of traditional fermented kocho take place in a pit for two to three weeks(Dibaba et al. 2018; Karssa et al. 2014) and its fermentation time is varied from region to region based on their indigenous knowledge of people(Karssa et al.,2014; Tsegaye et al., 2015; Weldemichael et al., 2019). Figure 2. 9. Ethiopian Traditional fermented food (Kotcho), prepared at home and sends to market, collected from Jimma Town, 2023 2.5.5. Bulla Bulla is one of the principal enset products, "the best part," acquired from the enset-to-kocho transformation process. To separate the starch from the fiber, the inside part of the pseudostem is scraped using a piece of split bamboo. This pulp is then squeezed with the feet to extract the juice, which is subsequently decanted. This decantation yields the bulla, which is next fermented for many weeks until it is highly starchy and of the highest quality. However, the nutritive value of starchy foods is determined primarily by their nutrient content, starch physicochemical properties, and the presence of nutritional factors and toxic substances (Atlabachew and Chandravanshi ,2008). Bulla is a little amount of water-insoluble starchy substance that can be isolated from kocho by squeezing and decanting the liquid during processing. After decanting, the bulla is let to dry or ferment in the same manner as Kocho. However, instead of being fermented, it is usually dehydrated to make flour that can be stored for long periods or cooked directly without fermentation. It is the highest-quality enset meal, derived primarily from fully matured enset plants (Dibaba et al., 2018; Yemata, 2020). Figure 2. 10. Ethiopian Traditional fermented food (Bulla), prepared at home and send to market, collected from Jimma Town, 2023 2.5.6. Ergo "Ergo" is popular Ethiopian fermented milk similar to yogurt. It is thick, smooth, and white, with a pleasant taste and smell. It can last up to 15-20 days when stored correctly (Figure 2.11). Widely enjoyed across the country, it is made without specific starter cultures, relying on natural milk flora for fermentation. Urban and rural households alike use various methods for fermentation, with temperatures and inoculation methods affecting the final product's consistency (Gonfa et al.,2001). LAB colonize the inside walls of the container and act as starting cultures. Incubation temperature rarely varies much, and the test of the fermented product may be more or less homogeneous in general(Assefa et al., 2008;Temesgen et al., 2020). Figure 2. 11. Traditional fermented dairy prodcuts, ergo, collected from Jimma Town, 2023 list and defines all technical terms

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Here are the technical terms mentioned in the text along with their definitions:

Fermented foods: Foods that have undergone the process of fermentation, where microorganisms such as bacteria, yeast, or fungi metabolize sugars or starches to produce various compounds like alcohol, organic acids, and gases, leading to changes in flavor, texture, and nutritional composition.

Improved shelf life: Extended duration for which a food product remains safe and suitable for consumption without significant deterioration in quality or safety.

Digestibility: The ease with which a food can be broken down and absorbed by the digestive system, often influenced by factors like cooking methods, food processing, and fermentation.

Lactic acid fermentation: A type of fermentation where sugars are converted into lactic acid by lactic acid bacteria, commonly used in food preservation and the production of dairy products, pickles, and fermented vegetables.

Probiotic bacteria: Beneficial bacteria that confer health benefits when consumed in adequate amounts, typically found in fermented foods and known for their positive effects on gut health and immunity.

Hygiene levels: The degree of cleanliness and sanitation maintained during food processing and preparation to prevent contamination and ensure food safety.

Antimicrobial activity: The ability of certain substances or microorganisms to inhibit the growth of pathogenic or spoilage-causing microorganisms, thereby extending the shelf life and safety of food products.

Indigenous knowledge: Traditional knowledge and practices passed down through generations within a particular culture or community, often related to agriculture, food processing, and natural resource management.

Cereal malt: Germinated and dried grains, typically barley, used in brewing and fermentation processes to convert starches into fermentable sugars.

Pseudostem: A false stem formed by tightly packed leaf sheaths in certain plants, such as bananas and enset, used as a source of starch in fermentation processes.

Physicochemical properties: Characteristics of a substance that can be measured or observed, including its physical and chemical attributes, such as texture, color, pH, and molecular structure.

Enset: A genus of flowering plants native to Ethiopia and parts of East Africa, commonly known as "false banana," cultivated for its starchy pseudostem, leaves, and fiber.

Decantation: The process of separating a liquid from solid particles or other liquids by carefully pouring off the liquid portion, leaving the sediment behind.

Homogeneous: Uniform in composition or character throughout, lacking variation or inconsistency.

Incubation temperature: The temperature at which microorganisms are allowed to grow and multiply during fermentation or culturing processes, often optimized for specific microbial activities.

Inoculation methods: Techniques used to introduce microorganisms, such as starter cultures or inoculants, into a substrate to initiate fermentation or other biochemical processes.

Consistency: The degree of firmness, thickness, or uniformity of a food product, often influenced by factors like ingredient proportions, processing methods, and fermentation conditions.

Flora: Microorganisms naturally present in a particular environment or habitat, such as the microbial flora of the digestive tract or the natural flora of fermented foods.

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2.6. Lactic Acid Bacteria Lactic acid bacteria have a long history of preserving foods from spoilage microorganisms since they are extensively utilized in food fermentation and produce numerous metabolites with favorable health effects; consequently, they are "GRAS". Furthermore, because of their ability to produce antibacterial and antifungal compounds, they are one of the most powerful sources of antimicrobial agents that have been used as biological control agents (Corsetti et al., 2004; Magnusson & Schnurer, 2014; Sumi et al., 2015). 2.7. Classification and physiology of lactic acid bacteria The LAB group is currently classified in the phylum Firmicutes and Actinobacter, class Bacilli, and order Atopobium, Bafidobacterium and Latobacillales. LAB are classified based on cellular morphology, mode of glucose fermentation, range of growth temperature, and sugar utilization patterns and sequencing of 16S rRNA (Mokoena, 2017;Yang et al. 2014). Lactobacillus species are often found in human and animal guts, as well as in vegetables and their fermented versions. Leuconostoc species come from meats, clinical sources, plants, fermented dairy, and wines(Goldstein et al., 2015). Pediococcus species are associated with beer spoilage, while Lactococcus is more common in dairy(Parada & Aguilera, 2007). Lactobacillus members are gram-positive, non-motile, non-sporulating, acid-tolerant, facultative anaerobes, and can ferment in different ways (Alkema et al., 2016). Lactic acid bacteria (LAB) play a crucial role in food fermentation by enhancing sensory qualities and ensuring microbiological safety. Their antimicrobial activity, achieved through organic acids, hydrogen peroxide, ethanol, and bacteriocins, has garnered interest for pathogen control in foods. Utilizing LAB and natural components for food preservation may reduce economic losses from microbial degradation and lower foodborne illness occurrences (Dalié et al.,2010; Delesa, 2017). 2.8. Phylogenetic characterization of Lactic acid bacteria The 16S rRNA gene is widely used in bacterial taxonomy due to its ubiquitous presence, stable function, and sufficient size for informatics (Janda and Abbott, 2007). Yet, to propose a new bacterial taxon, additional data like isolation source, phenotypic traits, biochemical profiles, and antibiotic susceptibility are essential. Other genes like 23S rRNA, 16S-23S rRNA ITS, rpoB, and groEL contribute to characterization (Drancourt & Raoult, 2005). For Lactic acid bacteria (LAB), genomic studies are crucial for understanding their metabolism, evolution, and classification (Callan and Westblade 2020). Whole-genome sequences aid in functional genomics, providing permanent scientific resources (Nowrousian et al., 2010). Comparative genomics show LAB genomes are small but diverse, with gene losses reflecting adaptation to nutrient-rich environments (Makarova & Koonin, 2007). Advancements like next-generation sequencing (NGS) and metagenomics offer deeper insights, although pyrosequencing's error rates can affect rare phylotype estimations (Nowrousian et al., 2010) Metagenomics has explored various environments, showcasing its utility in diverse scenarios (Brugger et al., 2012). 2.9. Probiotics property of Lactic acid bacteria Probiotics are live microorganisms and substances that promote gut balance and may benefit health when consumed adequately (Vilander & Dean, 2019). LAB are the most common probiotics, found in nutrient-rich environments and various body parts (Didari et al., 2014). They are considered safe and therapeutic for treating gastrointestinal issues. To qualify as probiotics, LAB must be safe, survive stomach acids, adhere to intestinal walls, combat harmful pathogens, and maintain viability during production and storage (Feng et al., 2017). 2.10. Criteria for Selection of Probiotic Lactic acid bacteria For probiotics, strains must meet safety, functionality, and storage stability criteria. They should survive processing and storage while maintaining their properties. LAB strains for human use must originate from humans, have a non-pathogenic history, and lack transmissible antibiotic resistance genes (Lim & Im, 2009) (Table 2.1). 2.10.1. Acid and bile tolerance A critical step in probiotic selection is ensuring LAB can survive the acidic stomach environment with pH as low as 1.5 (Gotcheva et al., 2002). Studies show probiotic LAB can endure gastric transit at pH 2.0 for short periods (1-2 hours) (Dowarah et al., 2018). Resistance to bile salts is also key, with recommended concentrations of 0.15-0.3% for human usage, matching physiological bile levels of 0.1-0.3% and occasionally up to 0.5% (Mulaw et al., 2020). 2.10.2. Antimicrobial activity Antimicrobial activity is a key factor in selecting probiotic LAB strains. These strains must produce antimicrobial compounds like organic acids, hydrogen peroxide, and bacteriocin to be effective probiotics. Their ability to create these compounds is crucial for developing probiotic supplements and diets (Fugaban et al., 2021). 2.10.3. Adhesion Properties of Probiotics The ability of probiotic strains to adhere to the intestinal surface is crucial for their effectiveness. Strains that adhere well are more likely to persist in the gut, leading to better metabolic and immunomodulatory effects. Adhesion is a critical characteristic as it competitively excludes harmful bacteria from the intestinal epithelium, making it a key selection criterion for probiotic (Bour et al., 2015). Table 2. 1. Selection criteria for potential probiotic lactic acid bacteria The selection criteria to select the potential probiotic Lactic acid bacteria 1) Human or animal origin, these criteria is important for species dependent health effects. 2) Isolated from the gastrointestinal tract of healthy individuals 3) History of safe use, safe for food and clinical use; no invasion and no degradation of intestinal mucus. 4) Absence of data regarding an association with infective disease ,no adverse effect on human 5) Important for oral consumption even if it may not be for other applications for survival through the intestine, maintaining adhesiveness and metabolic activity. 6) Lack of genes liable for antibiotic resistance localized in non-stable elements 7) Much enough to compete with pathogens, this express their ability to survive and maintain the metabolic activity, and to grow in the target site 8) Should resist to low pH in the stomach, bile salts and enzymes 9) Should have to show potential antagonistic activity towards pathogens 10) Resistance to bacteriocin and acids produced by the endogenic intestinal microbiota 11) Adherence and ability to colonize some particular sites within the host organism 12) Easy production of high biomass amounts and high productivity of cultures. 13) Viability and stability of the desired properties of probiotic bacteria during the fixing process, preparation, and distribution of probiotic products 14) High storage survival rate in finished products 15) Guarantee of desired sensory properties of finished products 16) Genetic stability 17) Important to improve immune system, competition with pathogens, maintain metabolic activity, prevent pathogens to adhesion and colonization (Paulin nd Katarzyna -, 2017). 2.11. Mechanisms of Probiotic Action Probiotic LAB employ various mechanisms to defend against pathogens and intestinal diseases. Primarily, they modulate the host's immune response, crucial for preventing infections and addressing gut inflammation. They also compete with pathogens by blocking their attachment to intestinal cells, aiding infection prevention and microbial balance restoration (Figure 2.12). Additionally, probiotics can influence microbial and host products, contributing to their overall beneficial effects (Anees et al., 2020; Plaza-Díaz et al., 2018). Figure 2. 12. Summarizes of the mechanisms of probiotic action, LAB , (Latif et al., 2023). 2.12. Health Benefits of Probiotics Research shows that probiotic bacteria, when consumed with traditional fermented foods and beverages, significantly contribute to maintaining good health, preventing intestinal infections, and improving overall well-being. These benefits include reducing antibiotic-related diarrhea, allergies, lactose intolerance, and colon cancer risk, as well as preventing eczema in children, lowering cholesterol, supporting immune system development, and protecting against gut pathogens (Hong et al. 2010; Rajoka et al. 2019). In addition, probiotic bacteria can enhance immunity by influencing the immune system's function, particularly in boosting natural killer (NK) cell activity. They also regulate inflammatory cytokines, contributing to overall health improvement (Gardiner, 2002). Probiotic LAB exhibit strong antimicrobial activity against various food and human pathogens. Studies by George Kerry et al. (2018) and Tejero-Sariñena et al. (2012), showed that probiotic LAB can inhibit the survival of pathogens like S. Typhimurium and C. difficile. Additionally, research by Figueroa et al., revealed that probiotics produce antimicrobial compounds such as bacteriocin, ethanol, organic acids, and peptides. These probiotics not only directly target infections but also enhance host defense mechanisms against pathogens (Figueroa-GonzálezGonz et al., 2011). Many people experience lactose maldigestion, leading to symptoms like loose stools and bloating. Interestingly, individuals often tolerate lactose in yogurt better than in raw milk. This is due to lactase released from probiotic LAB and yogurt, which breaks down lactose, making it easier to digest and reducing discomfort (Marteau et al., 2002b). Lactic acid bacteria can reduce serum cholesterol, lowering the risk of heart disease. They achieve this by producing bile salt hydrolase (BSH) enzyme, which breaks down bile salts. This process increases bile salt excretion, synthesizes more bile salts from serum cholesterol, and reduces cholesterol uptake from the gut, as shown in studies Nguyen et al. (2013) and Nguyen et al. (2007). 2.12.1. Safety aspects of Probiotics Safety is one the most important criteria and issue should be considered to select potential and good probiotic candidate. Hence, as Marteau et al. (2002), reported that, to be a potential probiotic bacterium the bacteria should not of pathogenicity and toxicity, tolerance to harsh conditions along the digestive tract, adhesion to mucosal surfaces and validated and documented health effects. Moreover, they should not carry transmissible antibiotic resistance genes (Saarela et al., 2002). This is because antibiotic resistant genes, especially those encoded by plasmids could be transferred between microorganisms. The potential strains need to be assayed for their antibiotic resistance to prevent the undesirable transfer of resistance to other endogenous bacteria. Furthermore, the presence of an antibiotic may facilitate the growth of antibiotic resistant mutants (Marteau et al., 2002). 2.13. Lactic Acid Bacteria as Biological food preservatives Bio-preservation involves using living LAB and their metabolites to enhance food safety and prolong shelf life. LAB and their antimicrobial compounds are employed to increase food safety without compromising sensory qualities (Ghafur et al., 2019). These bacteria produce various compounds like organic acids, bacteriocins, hydrogen peroxide, and antifungal agents, which suppress spoilage organisms and food-borne diseases. Their long history in food fermentation and GRAS status make them ideal alternatives to chemical preservatives. LAB's preservative effect is due to the production of active metabolites such as organic acids, ethanol, fatty acids, and antifungal compounds, which lower pH and inhibit microbial growth (Al-Yami et al., 2022; Zapaśnik et al., 2022). 2.14. Antimicrobial activity of Lactic Acid Bacteria The LAB has a number of advantageous qualities. They perform critical functions in the production and storage of fermented foods. Their metabolic products enable them to impart distinct flavors and aromas to foods. The LAB also generates antimicrobial compounds such as organic acids, diacetyl, ethanol, hydrogen peroxide, and bacteriocins, which can impede the growth of food spoilage or harmful bacteria in foods. Furthermore, the LAB can be used as probiotics, which may benefit the host's health. 2.10.1. Organic acids Lactic acid and acetic acid, produced by LAB, are crucial antibacterial compounds in food preservation. The type and quantity of acids produced during fermentation affect microbial activity, improving shelf life and safety (Reis et al.,2012). Lactic acid's solubility and insolubility play key roles in inhibiting pathogenic growth by altering pH gradients and reducing energy for cell growth (Othman et al. 2017). Acetic acid, found in vinegar, is effective against a range of bacteria, yeasts, and fungi, particularly at low pH levels. These acids, along with other preservation methods like temperature control and heat, offer robust protection against foodborne pathogens (Menteş et al.,2007). 2.14.1. Diacetyl, acetaldehyde and acetoin Heterofermentative LABs produce active acetaldehyde, which combines with pyruvate to form a-acetolactate and then diacetyl, converted by enzymes to acetoin. Diacetyl is known for its buttery aroma in fermented dairy, but its high concentration and aroma limit its use as a preservative. Acetaldehyde, though present in lower amounts, works alongside other LAB metabolites to inhibit microbial growth in fermented products (Jyoti et al., 2003). list and define all technical terms

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Here are the technical terms mentioned in the provided text along with their definitions:

Lactic Acid Bacteria (LAB): A group of bacteria that produce lactic acid as a byproduct of carbohydrate fermentation. LAB are commonly used in food fermentation and are known for their antimicrobial properties.

GRAS: Acronym for "Generally Recognized as Safe," a designation by regulatory agencies indicating that a substance is considered safe for its intended use in food or pharmaceuticals.

Antibacterial: Having the ability to inhibit the growth or kill bacteria.

Antifungal: Having the ability to inhibit the growth or kill fungi.

Firmicutes: A phylum of bacteria that includes lactic acid bacteria and other gram-positive bacteria with low G+C content in their DNA.

Actinobacter: A phylum of bacteria that includes lactic acid bacteria and other gram-positive bacteria with high G+C content in their DNA.

Bacilli: A class of bacteria that includes rod-shaped bacteria such as lactobacilli.

Atopobium, Bafidobacterium, and Latobacillales: Orders within the class Bacilli that include various genera of lactic acid bacteria.

16S rRNA: A component of the ribosomal RNA used for phylogenetic analysis and classification of bacteria.

Phylogenetic: Relating to the evolutionary relationships and classification of organisms.

Genomics: The study of the complete set of genes within an organism's genome.

Metabolism: The biochemical processes that occur within an organism to maintain life, including energy production and biosynthesis.

Probiotics: Live microorganisms that confer health benefits when consumed in adequate amounts.

Adhesion: The ability of microorganisms to attach to surfaces, such as the intestinal lining.

Bile salts: Compounds produced by the liver and stored in the gallbladder, involved in the digestion and absorption of fats.

Bacteriocin: Proteinaceous compounds produced by bacteria that inhibit the growth of closely related species.

Immune response: The body's defense mechanism against foreign invaders, including pathogens and toxins.

Colonization: The establishment and growth of microorganisms on a surface or within a host organism.

Bio-preservation: The use of natural or controlled microorganisms and their antimicrobial metabolites to extend the shelf life and improve the safety of foods.

Antimicrobial: Having the ability to inhibit the growth or kill microorganisms, including bacteria, fungi, and viruses.

Organic acids: Acids produced by microorganisms during fermentation, such as lactic acid and acetic acid, which contribute to food preservation.

Diacetyl, Acetaldehyde, and Acetoin: Metabolites produced by LAB that contribute to flavor and aroma in fermented foods and beverages.

These are the technical terms found in the provided text. Let me know if you need further clarification on any of them!

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Lactic acid's solubility and insolubility play key roles in inhibiting pathogenic growth by altering pH gradients and reducing energy for cell growth (Othman et al. 2017)

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In this context, "lactic acid's solubility and insolubility" refer to the ability of lactic acid to dissolve in water. Lactic acid is soluble in water, meaning it can dissolve and form a solution. However, when lactic acid concentration increases beyond its solubility limit, it can become insoluble and precipitate out of solution.

The statement suggests that both the solubility and insolubility of lactic acid play important roles in inhibiting the growth of pathogens. Here's how:

Altering pH gradients: When lactic acid dissolves in water, it releases hydrogen ions (H+), leading to a decrease in pH. This acidic environment created by lactic acid inhibits the growth of many pathogenic bacteria that prefer neutral or slightly alkaline conditions. By altering pH gradients, lactic acid creates an environment that is unfavorable for the growth of pathogens, thereby inhibiting their proliferation.

Reducing energy for cell growth: Pathogenic bacteria require energy to grow and replicate. The presence of lactic acid, especially in its insoluble form, can disrupt the energy metabolism of these bacteria. Insoluble lactic acid may form crystals or precipitates that physically interfere with the bacterial cell membrane or wall, making it more difficult for pathogens to absorb nutrients and generate energy. This reduction in available energy limits the growth and survival of pathogenic bacteria.

Overall, the solubility and insolubility of lactic acid contribute to its antimicrobial properties by creating an acidic environment and reducing the energy available for the growth of pathogenic microorganisms.

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How carbon dioxides produce

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Carbon dioxide (CO2) is produced through various natural and human activities. Here are some common ways in which carbon dioxide is produced:

Respiration: Both animals and humans produce carbon dioxide as a byproduct of cellular respiration. During cellular respiration, organic molecules such as glucose are oxidized to release energy for cellular processes. One of the waste products of this process is carbon dioxide, which is expelled from the body through exhaling.

Combustion: When organic matter such as fossil fuels (coal, oil, and natural gas) or biomass (wood, agricultural residues) is burned for energy production, carbon dioxide is released as a combustion byproduct. This process occurs in power plants, vehicles, industries, and households that rely on fossil fuels for heating and cooking.

Decay and decomposition: Organic matter undergoes decomposition by microorganisms in soil, water, and other environments. During this process, carbon stored in organic compounds is broken down and released into the atmosphere as carbon dioxide and methane (another greenhouse gas).

Volcanic eruptions: Volcanic eruptions release large amounts of carbon dioxide, along with other gases and particulate matter, into the atmosphere. While volcanic activity is a natural process, it can contribute to short-term spikes in atmospheric CO2 levels.

Industrial processes: Various industrial processes, such as cement production, lime manufacturing, and chemical manufacturing, emit carbon dioxide as a byproduct. These emissions can occur directly from industrial sources or indirectly through energy consumption.

Deforestation and land-use changes: When forests are cleared for agriculture, urban development, or logging, the carbon stored in trees and vegetation is released into the atmosphere as carbon dioxide. This process contributes to deforestation-related emissions.

Fermentation: During fermentation processes, microorganisms break down organic compounds such as sugars and produce carbon dioxide as a metabolic byproduct. This occurs in various natural processes (e.g., fermentation of organic matter in wetlands) as well as in human activities such as brewing, winemaking, and bread baking.

These are some of the primary ways in which carbon dioxide is produced naturally and by human activities. It's important to note that while carbon dioxide is a natural component of the Earth's atmosphere and plays a crucial role in the carbon cycle, human activities have significantly increased atmospheric CO2 levels, leading to concerns about climate change and global warming.

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how lactic acid bacteria produce carbon dioxdes prodcude

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Lactic acid bacteria (LAB) do not produce carbon dioxide (CO2) as a primary metabolic byproduct. Instead, they primarily produce lactic acid as a result of fermentative metabolism. However, there are indirect ways in which LAB can contribute to the production of carbon dioxide:

Fermentation: Lactic acid bacteria are known for their ability to ferment sugars, converting them into lactic acid. During this process, certain LAB strains can also produce small amounts of carbon dioxide as a byproduct. This occurs through the decarboxylation of pyruvate, a metabolic intermediate, into carbon dioxide and acetaldehyde, which is then further reduced to ethanol or other end-products.

Leavening in baking: In the context of baking, LAB may contribute to the production of carbon dioxide through fermentation. In sourdough bread-making, for example, naturally occurring LAB in sourdough starter cultures ferment carbohydrates in the flour, producing lactic acid and carbon dioxide. The trapped carbon dioxide gas causes the dough to rise, resulting in a lighter texture in the finished bread.

Yogurt and fermented beverages: During the fermentation of dairy products like yogurt or fermented beverages like kefir, LAB can produce carbon dioxide as a byproduct of fermentation. While the primary metabolic end-products are lactic acid and other compounds, the presence of carbon dioxide may contribute to the effervescence or carbonation in certain fermented beverages.

Overall, while LAB are not significant producers of carbon dioxide compared to other microorganisms like yeast, their metabolic activities can contribute to the generation of small amounts of CO2, particularly in the context of fermentation processes such as baking and dairy fermentation.

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How hydrogen peroxides produced, exter its antmicrobial activity, antioxidant property

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Hydrogen peroxide (H2O2) is produced by lactic acid bacteria (LAB) primarily through the action of certain enzymes, notably pyruvate oxidase. Here's a breakdown of how it's produced, its antimicrobial activity, and its antioxidant properties:

Production of Hydrogen Peroxide by LAB:

Pyruvate Oxidase Activity: Certain LAB strains possess the enzyme pyruvate oxidase, which catalyzes the conversion of pyruvate to acetyl phosphate, releasing hydrogen peroxide in the process.

Antimicrobial Activity of Hydrogen Peroxide:

Oxidative Stress: Hydrogen peroxide acts as an oxidizing agent, generating reactive oxygen species (ROS) within microbial cells. This oxidative stress damages cellular components such as proteins, lipids, and DNA, ultimately leading to cell death.

Disruption of Cellular Functions: Hydrogen peroxide disrupts critical cellular functions, including enzyme activity and membrane integrity, leading to the inhibition of microbial growth.

Broad-Spectrum Activity: Hydrogen peroxide exhibits antimicrobial activity against a wide range of microorganisms, including bacteria, yeasts, and molds. It is effective against both gram-positive and gram-negative bacteria.

Antioxidant Property of Hydrogen Peroxide:

Scavenging Reactive Oxygen Species: Despite being a reactive oxygen species itself, in appropriate concentrations, hydrogen peroxide can act as an antioxidant by scavenging other, more reactive ROS. This can help mitigate oxidative stress and reduce cellular damage.

Regulation of Cellular Signaling: Hydrogen peroxide has been implicated in cellular signaling pathways, where it acts as a signaling molecule. At low concentrations, it can modulate various cellular processes, including gene expression, cell proliferation, and immune responses.

Applications in Food Preservation:

Preservation of Fermented Foods: Hydrogen peroxide produced by LAB during fermentation contributes to the preservation of fermented foods by inhibiting the growth of spoilage microorganisms and foodborne pathogens.

Control of Food Spoilage: In food processing, hydrogen peroxide can be used as a food preservative to extend the shelf life of perishable products by inhibiting microbial growth.

Enhancement of Food Safety: The antimicrobial activity of hydrogen peroxide can help improve the safety of food products by reducing the risk of contamination with pathogenic microorganisms.

Overall, while hydrogen peroxide produced by LAB can exert antimicrobial activity against undesirable microorganisms, its antioxidant properties and regulatory roles in cellular processes underscore its importance in microbial physiology and food preservation.

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Production of Hydrogen Peroxide by LAB

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Hydrogen peroxide (H2O2) is produced by lactic acid bacteria (LAB) primarily through the activity of the enzyme pyruvate oxidase. Here's how the production process typically occurs:

Metabolism of Pyruvate: LAB metabolize glucose or other carbohydrates through the process of glycolysis, generating pyruvate as an intermediate product.

Action of Pyruvate Oxidase: Within LAB cells, the enzyme pyruvate oxidase catalyzes the conversion of pyruvate to acetyl phosphate while simultaneously producing hydrogen peroxide and carbon dioxide as byproducts:Pyruvate+Oxygen→Acetyl phosphate+CO2+H2O2\text{Pyruvate} + \text{Oxygen} \rightarrow \text{Acetyl phosphate} + \text{CO}_2 + \text{H}_2\text{O}_2Pyruvate+Oxygen→Acetyl phosphate+CO2+H2O2This reaction occurs in the presence of molecular oxygen (O2), which serves as the electron acceptor.

Generation of Hydrogen Peroxide: The pyruvate oxidase enzyme facilitates the transfer of electrons from pyruvate to oxygen, resulting in the formation of hydrogen peroxide. This process contributes to the oxidative metabolism of LAB and plays a role in cellular respiration.

Regulation of Production: The production of hydrogen peroxide by LAB is regulated by various factors, including oxygen availability, pH, temperature, and the metabolic state of the cells. Under aerobic conditions, hydrogen peroxide production tends to increase due to the availability of oxygen as an electron acceptor.

Antimicrobial Activity: The hydrogen peroxide produced by LAB exhibits antimicrobial activity against competing microorganisms, including pathogenic bacteria, yeasts, and molds. It contributes to the preservation of fermented foods by inhibiting the growth of spoilage organisms and enhancing food safety.

Overall, the production of hydrogen peroxide by LAB is an essential aspect of their metabolic activity and contributes to their role in food fermentation and preservation.

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Production of Hydrogen Peroxide by LAB if no oxygen

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In the absence of oxygen, lactic acid bacteria (LAB) typically do not produce significant amounts of hydrogen peroxide (H2O2) through the enzymatic action of pyruvate oxidase. Pyruvate oxidase requires oxygen as an electron acceptor to catalyze the conversion of pyruvate to acetyl phosphate, generating hydrogen peroxide as a byproduct, as mentioned earlier.

However, some LAB species possess alternative metabolic pathways that can lead to the production of hydrogen peroxide, even under anaerobic conditions. One such pathway involves the reduction of molecular oxygen by flavoproteins, yielding superoxide radicals (O2^-). These superoxide radicals can then react with hydrogen ions (H+) to form hydrogen peroxide (H2O2) through a process known as dismutation:

2 O2−+2 H+→H2O2+O22 \text{ O}_2^- + 2 \text{ H}^+ \rightarrow \text{H}_2\text{O}_2 + \text{O}_22 O2−+2 H+→H2O2+O2

This reaction occurs spontaneously and does not require the presence of oxygen gas. However, it is generally less efficient than the enzymatic production of hydrogen peroxide by pyruvate oxidase under aerobic conditions.

The production of hydrogen peroxide by LAB in the absence of oxygen is typically minimal compared to aerobic conditions. Consequently, anaerobic fermentation by LAB primarily produces lactic acid as the main end-product of carbohydrate metabolism, rather than hydrogen peroxide.

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list and define all technical terms 2.14. Antimicrobial activity of Lactic Acid Bacteria The LAB has a number of advantageous qualities. They perform critical functions in the production and storage of fermented foods. Their metabolic products enable them to impart distinct flavors and aromas to foods. The LAB also generates antimicrobial compounds such as organic acids, diacetyl, ethanol, hydrogen peroxide, and bacteriocins, which can impede the growth of food spoilage or harmful bacteria in foods. Furthermore, the LAB can be used as probiotics, which may benefit the host's health. 2.10.1. Organic acids Lactic acid and acetic acid, produced by LAB, are crucial antibacterial compounds in food preservation. The type and quantity of acids produced during fermentation affect microbial activity, improving shelf life and safety (Reis et al.,2012). Lactic acid's solubility and insolubility play key roles in inhibiting pathogenic growth by altering pH gradients and reducing energy for cell growth (Othman et al. 2017). Acetic acid, found in vinegar, is effective against a range of bacteria, yeasts, and fungi, particularly at low pH levels. These acids, along with other preservation methods like temperature control and heat, offer robust protection against foodborne pathogens (Menteş et al.,2007). 2.14.1. Diacetyl, acetaldehyde and acetoin Heterofermentative LABs produce active acetaldehyde, which combines with pyruvate to form a-acetolactate and then diacetyl, converted by enzymes to acetoin. Diacetyl is known for its buttery aroma in fermented dairy, but its high concentration and aroma limit its use as a preservative. Acetaldehyde, though present in lower amounts, works alongside other LAB metabolites to inhibit microbial growth in fermented products (Jyoti et al., 2003). 2.14.2. Carbon dioxide Carbon dioxide has two effects on product preservation. Specifically, it creates an anaerobic environment by replacing the existing molecular oxygen, in addition to its own antimicrobial activity, CO2 has antifungal efficacy because it inhibits enzymatic decarboxylation and accumulates in the membrane lipid bilayer, causing defective permeability (Vuyst, 2007). 2.14.3. Hydrogen peroxide (H2O2) According to many research reports, the production of H2O2 by LAB can prevent the growth of foodborne pathogens and can be act as food preservation. At refrigeration temperatures, LAB that produce H2O2 it has been demonstrated to limit the growth of psychrotrophic and pathogenic microbes. In, addition, by increasing the rancidity and coloring of the final product, hydrogen peroxide might interfere with the organoleptic qualities of fermented meat products (Jaroni & Brashears, 2000; Németh et al., 2005). 2.14.4. Bacteriocins Bacteriocins are antibacterial compounds produced by bacteria, known for their amphiphilic, membrane-permeabilizing nature. They can reduce the need for chemical preservatives and intense heat treatment in food processing, leading to naturally preserved foods with enhanced sensory and nutritional qualities (Yang et al. 2012). To maximize their efficacy, careful study of bacteriocin-producing LAB strains is essential, including optimization of production techniques and use of inducible production systems. Heterologous expression of bacteriocin genes can further enhance their properties and production systems (Zhou et al.,2006). 2.14.5. Classification of Bacteriocin Bacteriocin classification depends on various factors like molecular weight, producer strains, structure, and mode of action, biosynthesis, and biological activity. Recent studies categorize LAB bacteriocins into Class I, Class II, and Class III based on these criteria ( Dicks et al., 2018; Soltani et al., 2021). Class I bacteriocins are characterized by unique amino acids and structures resulting from posttranslational modifications, making them small peptides. These modifications include rare amino acids like lanthionine, heterocycles, head-to-tail cyclization, and glycosylation. They also feature a leader peptide crucial for enzyme recognition, transport, and peptide inactivation (Arnison et al., 2012). Genes responsible for lantibiotic biosynthesis are organized in operons, with genes for bacteriocin maturation typically found within the same gene structure (Alvarez-Sieiro et al. 2016). Class II bacteriocins are characterized by a flexible hinge that separates the peptide tract into variable and conserved portions. They feature an N-terminal leader with a C-terminal attached. The cationic N-terminal leader contains two cysteine residues linked by a conserved disulfide bridge, crucial for target engagement and a lethal mechanism of action. In contrast, the C-terminus is less conserved, resulting in reduced specificity in target contact (Johnsen et al., 2005). Class III bacteriocins are larger, heat-labile proteins with unique peptide domains. They encompass non-lytic bacteriocins with diverse modes of action, including inhibition without cell lysis, binding to sugars like mannose or glucose to affect target organisms' sugar absorption, and causing membrane leakage, leading to essential molecule loss (Choyam et a.,2021). 2.14.6. Separation, purification and identification of antimicrobial compounds The first step in bacteriocin isolation involves screening LAB from diverse sources, such as plant material, food products, and human or animal isolates. Researchers like Ogawa et al (2019), found LAB strains in dairy and cheese products, while Rajaram et al (2020) used the well diffusion method on agar plates to detect antagonistic activity. To avoid hydrogen peroxide antagonism, catalase was added to the culture medium, and phosphate buffer was used in the solid medium to prevent organic acid inhibition. Here are some discussions about novel bacteriocin purification methods (Figure 2.12). Table 2. 2. Bacteriocins of LAB and their main characteristics (Yang et al.,2014). Classification/features Bacteriocins Mwt (Da) Producing strain CLASSI The bacteriocins are post-transnationally modified, linear or globular peptides containing lanthionine, β-methyl lanthionine and dehydrated amino acids Nisin A 3352 Lactococcus lactissubsp. lactic Nisin U 3029 Streptococcus uberis Nisin Z 3493 Lactococcus lactissubsp. lactic Mersacidin 1824 Bacillus sp.Y85,54728 Labyrinthopeptin A 21922 Actinomadura sp. subtilosin A 3399 Bacillus subtilis 168 CLASSII Heat stable, unmodified, bacteriocins, heterogeneous class of small peptides Class IIa (pediocinPA-1like bacteriocins) Pediocin PA-1 4629 Pediococcus acidilactici i PAC-1.0 Carnobacteriocin X 3602 Carnobacterium maltaromaticum C2 Class IIb (composed of two peptides) lactacinF 4755 Lactobacillus spp. ABP-11 84096 Lactobacillus salivariussubsp. salivarius UCC118 Class IIc (circular peptide) carnocyclinA 5862 Carnobacterium maltaromaticum UAL307 enterocin AS-4 87149 Enterococcus faecalis Class IId (linear,non-pediocinlike, single-peptide) epidermicin NI01 6074 Staphylococcusepidermidis lactococcin A 5778 Lactococcus lactissubsp. Cremoris CLASSIII Large, heat unstableproteins Caseicin80 ∼42000 Lactobacillus casei B80 Enterolisin A 34501 Enterococcus faecalis LMG2333 HelveticinJ 37511 Lactobacillus helveticus 481 2.14.7. Chromatographic separation and purification The purity of antibacterial compounds from LAB is crucial for structural analysis. Purification typically involves chromatographic techniques or a combination of methods based on different separation principles. For instance, authors have successfully separated and purified reuterin using reversed-phase high-performance liquid chromatography (RP-HPLC) with two C18 columns in series and water as the mobile phase (Pauter et al., 2020). 2.14.8. Ammonium sulfate precipitation Ammonium precipitation is a widely used method for bacteriocin purification, typically as a first step. After culturing bacteriocin producers on MRS agar and recovering the cell-free supernatant through centrifugation, crude bacteriocin is obtained via protein precipitation with ammonium salt saturation. This crude bacteriocin is then further purified through steps like centrifugation, dissolution in water or buffer, and additional purification methods like membrane filtering and gel filtration. While this process, known as partial purification, yields bacteriocin suitable for use as a food preservative, it's not the final purification step (Fugaban et al., 2021). 2.14.9. Identification of antimicrobial compounds Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) can be used to identify antimicrobial substances. The latter is frequently employed to determine molecular mass. Reuterin was identified primarily using proton (1H) and carbon (13C). HPLC-MS and GC-MS analysis of NMR data in deuterium oxide (D2O) and deuterated methanol revealed that the molecular mass of the cyclic dimer form of reuterin was 148 (Ibrahim et al. 2021; Perez et al., 2014; Ye et al. 2021). 2.14.10. Polyacrylamide gel electrophoresis (PAGE) Polyacrylamide gel electrophoresis is used to estimate the molecular weight of bacteriocin after purification, and purified bacteriocin is treated with tricine sodium dodecyl sulfate polyacrylamide electrophoresis. Bacteriocin standard solutions are commonly used as a standard to measure the molecular weight of bacteriocin using gels (stacking solution and separating gel) (Struck et al., 2016; C. Xu et al., 2021; Yson et al., 2013; Zhu et al., 2015). Figure 2. 13. Steps for bacteriocin screening, production and purification 2.15. Application of Bacteriocin as Food Preservative The use of LAB bacteriocin as a unique natural food preservative is based on its health benefits. To be considered a safe and effective natural preservative, it must meet these criteria: (1) not harm consumers' gastrointestinal health; (2) have broad-spectrum activity against various food spoilage microorganisms; (3) be resistant to food enzymes; and (4) show thermal stability across different pH levels, high salt concentrations, and other factors. Failure to meet these criteria would limit bacteriocin's potential as a safe natural food preservative (Ananou et al., 2010). LAB bacteriocin has been explored as a novel natural food preservation method using various approaches, including LAB starter cultures, protective LAB strains, reintroduction of previously fermented foods with bacteriocin-producing LAB during processing, and direct application of purified bacteriocin as a preservative (Ananou et al., 2010). These strategies are adaptable to different food types and specific LAB targets for preservation. Given LAB bacteriocin's "GRAS" status, it's suitable for industrial use, meeting the rising demand for fresh, safe, naturally preserved, and nutritionally enhanced food products with improved sensory qualities (Dhundale et al., 2018). Bacteriocins are commonly used as bio preservatives in foods through three methods: 1) Immunizing food with LAB that produce bacteriocins, crucial for their efficacy; 2) Directly using pure or semi-purified bacteriocins as preservatives; and 3) Incorporating a bacteriocin-producing strain's fermented product into food processing (Dioso, et al., 2022). 2.16. MALDI-TOF MS bacterial identification system MALDI-TOF, or matrix-assisted laser desorption ionization time-of-flight analysis, is a mass spectrometry method used for identifying microorganisms like bacteria, yeast, and molds. A laser breaks down bacteria into protein fragments, which travel through a detection tube. The "time of flight" measures how long these peptides take to reach the tube. By comparing, the mass spectrometry profile with a MALDI-TOF database, bacterial genera and species can be identified. Updating the database can be done by lab personnel or through purchasing an updated version. It relies on detecting the mass-to-charge ratio of ribosomal proteins, providing a unique mass spectrum for bacteria in a short time, and identification is based on the closest match with reference strains' spectra (Ashfaq et al., 2022). 2.16.1. Sample preparation Two main sample preparation methods for MALDI-ToF analysis are direct transfer and protein extraction. Direct transfer involves quickly recovering bacterial cells from growth plates or liquid media, providing reliable and accurate results. Protein extraction, using solvents like ethanol (avoiding toxic ones like formic acid), is recommended for higher spectral quality (Diaz et al., 2019). After preparation, bacterial cells or extracted proteins are placed on a MALDI biotarget plate, covered with a matrix solution (e.g., CHCA, sinapinic acid, DHB), and dried. This plate is then inserted into the MALDI-TOF instrument to generate a spectrum for comparison with the database (Gonzalo et al., 2021).See Figure 2.13 for an overview of the MALDI-ToF mass analysis process. Figure 2. 14. Steps and procedures for bacterial identification using MALDI-TOF MS(Gonzalo et al., 2021; Šedo et al., 2011; Toh-Boyo et al., 2012). 2.16.2. Bacterial identification Online databases like the MALDI Biotyper Library (MBL) compare mass spectra of unknown bacteria to known ones for identification, using software like Biotyper. Other search engines and fingerprint libraries are also available. MALDI-ToF can achieve over 90% accuracy in identifying organisms to the species level, but reproducibility depends on factors like sample culture, preparation, storage, and matrix type (Clark et al., 2013). 2.16.3. Microorganism typing The MALDI-TOF method is widely used for fast and accurate bacterial identification, including subtyping and serotyping. While it is excellent for genus and species identification, its use for subspecies identification is still being explored due to similarities within species. However, some success has been reported with bacteria like Yersinia enterocolitica for subspecies-level typing using MALDI-TOF MS (Sandrin et al., 2013). Different methods exist for identifying strains using MALDI-TOF MS. Comparing mass spectra visually helps distinguish species by specific peaks. The gel view enhances this comparison by highlighting peak presence or absence. Certain peaks, like those around 2000 and 5000 m/z, are often considered biomarkers across many bacteria (Alsayegh et al., 2021)

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Sure, let's define the technical terms mentioned in sections 2.14 and 2.16:

Antimicrobial activity: The ability of a substance to inhibit or kill microorganisms, such as bacteria, viruses, fungi, or parasites.

Lactic Acid Bacteria (LAB): A group of bacteria that produce lactic acid as a byproduct of carbohydrate fermentation. They are commonly used in food fermentation processes and are known for their probiotic properties.

Organic acids: Acidic compounds containing carbon and hydrogen atoms, often produced by microbial fermentation. In the context of LAB, organic acids like lactic acid and acetic acid are crucial antimicrobial compounds in food preservation.

Diacetyl: A volatile compound produced by certain bacteria during fermentation, known for its buttery aroma. It can also exhibit antimicrobial properties, although its high concentration and aroma limit its use as a preservative.

Acetaldehyde: A volatile organic compound produced during fermentation, contributing to the flavor and aroma of fermented foods. It can also exhibit antimicrobial properties at certain concentrations.

Acetoin: A volatile compound produced during fermentation, contributing to the flavor and aroma of fermented foods. It is often formed from the enzymatic conversion of diacetyl.

Carbon Dioxide (CO2): A colorless, odorless gas produced during fermentation by the action of yeast or bacteria on sugars. In food preservation, carbon dioxide creates an anaerobic environment and exhibits antimicrobial activity by inhibiting microbial growth and enzyme activity.

Hydrogen Peroxide (H2O2): A reactive oxygen species with antimicrobial properties. It can be produced by certain bacteria, including LAB, and is used in food preservation to inhibit the growth of spoilage microorganisms.

Bacteriocins: Proteinaceous antimicrobial compounds produced by bacteria, including LAB. Bacteriocins can inhibit the growth of closely related bacteria and are used as natural preservatives in food products.

MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry): A mass spectrometry technique used for the rapid identification of microorganisms based on their mass spectra. It involves ionizing microbial proteins and analyzing their mass-to-charge ratios to generate unique fingerprints for identification.

These terms encompass various aspects of the antimicrobial activity of LAB and the techniques used for their identification and characterization in food preservation and microbiology.

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Bacteriocins are antibacterial compounds produced by bacteria, known for their amphiphilic, membrane-permeabilizing nature. They can reduce the need for chemical preservatives and intense heat treatment in food processing, leading to naturally preserved foods with enhanced sensory and nutritional qualities (Yang et al. 2012). To maximize their efficacy, careful study of bacteriocin-producing LAB strains is essential, including optimization of production techniques and use of inducible production systems. Heterologous expression of bacteriocin genes can further enhance their properties and production systems (Zhou et al.,2006).

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This passage highlights the significance of bacteriocins in food preservation and outlines some strategies for maximizing their efficacy:

Nature of Bacteriocins: Bacteriocins are described as antibacterial compounds produced by bacteria. They are known for their amphiphilic nature, meaning they have both hydrophilic and hydrophobic regions, and their ability to permeabilize bacterial cell membranes. This action disrupts membrane integrity and leads to cell death.

Reduction of Chemical Preservatives and Heat Treatment: Bacteriocins offer an alternative to chemical preservatives and intense heat treatment in food processing. By effectively inhibiting the growth of spoilage and pathogenic bacteria, they help extend the shelf life of food products naturally. This reduction in the use of chemical additives and heat treatment can contribute to the preservation of the sensory and nutritional qualities of foods.

Optimization of Production Techniques: To fully harness the potential of bacteriocins, it is essential to study bacteriocin-producing LAB strains carefully. This involves optimizing production techniques to maximize bacteriocin yield and efficacy. Factors such as culture conditions, fermentation parameters, and nutrient availability can influence bacteriocin production.

Use of Inducible Production Systems: Inducible production systems can be employed to enhance bacteriocin production. These systems involve the controlled activation of bacteriocin production in response to specific environmental cues or inducers. By regulating the expression of bacteriocin genes, inducible systems allow for precise control over bacteriocin production levels.

Heterologous Expression: Heterologous expression involves transferring bacteriocin genes into other bacterial hosts for production. This approach can be used to enhance the properties of bacteriocins or improve their production systems. By introducing bacteriocin genes into genetically tractable hosts, researchers can manipulate expression levels, modify protein structures, or optimize production conditions to enhance bacteriocin efficacy.

Overall, the passage underscores the importance of bacteriocins in food preservation and emphasizes the need for careful study and optimization of production techniques to maximize their effectiveness in ensuring food safety and quality.

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Amphiphilic, membrane-permeabilizing nature

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The terms "amphiphilic" and "membrane-permeabilizing" describe key properties of bacteriocins:

Amphiphilic: This term refers to molecules that have both hydrophilic (water-attracting) and hydrophobic (water-repelling) regions. Bacteriocins typically exhibit amphiphilic properties, meaning they have regions that are attracted to water molecules and regions that repel water. This amphiphilic nature allows bacteriocins to interact with and disrupt biological membranes, such as those found in bacterial cells.

Membrane-permeabilizing: Bacteriocins are known for their ability to disrupt the integrity of bacterial cell membranes. By interacting with the lipid bilayer of the membrane, bacteriocins can cause destabilization and the formation of pores or channels. This permeabilization of the membrane leads to the leakage of cellular contents, loss of membrane potential, and ultimately cell death.

In summary, the amphiphilic nature of bacteriocins enables them to interact with lipid membranes, and their membrane-permeabilizing activity allows them to disrupt bacterial cell membranes, leading to antibacterial effects.

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what is amphiphilic, membrane-permeabilizing nature

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The terms "amphiphilic" and "membrane-permeabilizing" describe important characteristics of molecules, particularly those with biological activity, like bacteriocins:

Amphiphilic: This term describes molecules that have both hydrophilic (water-loving) and hydrophobic (water-repelling) regions. This dual nature allows amphiphilic molecules to interact with both aqueous and lipid environments effectively. In the context of bacteriocins, their amphiphilic nature enables them to interact with the lipid bilayer of biological membranes, such as those surrounding bacterial cells.

Membrane-permeabilizing: Bacteriocins are known for their ability to disrupt the integrity of bacterial cell membranes. This disruption can occur through various mechanisms, but one common mechanism involves the formation of pores or channels in the lipid bilayer. As a result, the membrane becomes permeable, leading to the leakage of cellular contents and eventual cell death. The membrane-permeabilizing nature of bacteriocins contributes to their antimicrobial activity.

In summary, the amphiphilic nature of bacteriocins allows them to interact with lipid membranes effectively, and their membrane-permeabilizing activity enables them to disrupt bacterial cell membranes, ultimately leading to the death of target bacteria.

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what is the advantages for bacteriocins being amphiphilic, membrane-permeabilizing nature

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The advantages of bacteriocins having an amphiphilic, membrane-permeabilizing nature include:

Broad-Spectrum Antimicrobial Activity: Bacteriocins can target a wide range of bacteria due to their ability to interact with and disrupt bacterial cell membranes. This broad-spectrum activity makes them effective against both Gram-positive and Gram-negative bacteria, including antibiotic-resistant strains.

Low Likelihood of Resistance Development: Bacteriocins typically target essential components of bacterial cells, such as the cell membrane. Because of their diverse mechanisms of action and rapid killing of bacteria, the development of resistance is less likely compared to traditional antibiotics.

Selective Toxicity: Bacteriocins can selectively target bacterial cells while sparing host cells. This selective toxicity is often attributed to differences in membrane composition between bacterial and mammalian cells, allowing bacteriocins to specifically interact with bacterial membranes.

Synergistic Effects: Bacteriocins can act synergistically with other antimicrobial agents, such as antibiotics or other bacteriocins, enhancing their overall antimicrobial activity. This synergy can lead to more effective antimicrobial treatments and potentially lower doses of individual agents, reducing the risk of side effects.

Potential for Food Preservation: The membrane-permeabilizing activity of bacteriocins can be utilized in food preservation to inhibit the growth of spoilage and pathogenic bacteria. By disrupting the membranes of these bacteria, bacteriocins can prevent food spoilage and extend the shelf life of perishable foods without the need for chemical preservatives.

Overall, the amphiphilic, membrane-permeabilizing nature of bacteriocins provides several advantages in terms of antimicrobial activity, selectivity, synergy with other antimicrobial agents, and potential applications in food preservation.

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The advantages of bacteriocins having an amphiphilic,

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The advantages of bacteriocins having an amphiphilic nature include:

Enhanced Membrane Interaction: Amphiphilic bacteriocins possess both hydrophilic and hydrophobic regions, allowing them to interact effectively with bacterial cell membranes. This interaction can disrupt the integrity of the membrane, leading to cell lysis and death.

Broad-Spectrum Activity: Due to their ability to target the bacterial cell membrane, amphiphilic bacteriocins often exhibit broad-spectrum antimicrobial activity against a wide range of bacteria, including both Gram-positive and Gram-negative species. This broad activity makes them valuable for combating various bacterial infections.

Low Potential for Resistance Development: The mechanism of action of amphiphilic bacteriocins involves targeting essential components of bacterial cells, such as the membrane. This makes it less likely for bacteria to develop resistance compared to conventional antibiotics, which often target specific cellular processes.

Synergy with Other Antimicrobials: Amphiphilic bacteriocins can act synergistically with other antimicrobial agents, including antibiotics, enhancing their overall effectiveness. This synergy can lead to more potent antimicrobial treatments and help combat antibiotic-resistant bacteria.

Selective Toxicity: Amphiphilic bacteriocins can exhibit selective toxicity towards bacterial cells while sparing host cells. This selective action is attributed to differences in membrane composition between bacterial and mammalian cells, allowing bacteriocins to specifically target bacteria.

Potential Applications in Food Preservation: The amphiphilic nature of bacteriocins makes them suitable for use in food preservation. By disrupting the membranes of spoilage and pathogenic bacteria, bacteriocins can inhibit their growth and extend the shelf life of food products without the need for chemical preservatives.

Overall, the amphiphilic nature of bacteriocins confers several advantages, including broad-spectrum activity, low potential for resistance development, synergy with other antimicrobials, selective toxicity, and applications in food preservation.

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The technical terms mentioned in the passage are:

Bacteriocins: Antibacterial compounds produced by bacteria.

Amphiphilic: Having both hydrophilic (water-attracting) and hydrophobic (water-repelling) regions in the molecular structure.

Membrane-permeabilizing: Capable of disrupting cell membranes, leading to increased permeability.

Chemical preservatives: Substances added to foods to prevent spoilage, typically synthetic compounds.

Inducible production systems: Regulatory mechanisms that allow for controlled expression of genes in response to specific environmental signals or conditions.

Heterologous expression: The expression of a gene in a host organism that is different from the organism in which the gene naturally occurs.

Optimization of production techniques: Refining methods to maximize the yield or efficiency of a particular process.

Enhanced sensory qualities: Improved characteristics related to taste, smell, texture, and appearance of food products.

Enhanced nutritional qualities: Improved nutritional content or bioavailability of nutrients in food products.

These terms are essential for understanding the production, properties, and applications of bacteriocins and their role in food preservation.

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Bacteriocin classification depends on various factors like molecular weight, producer strains, structure, and mode of action, biosynthesis, and biological activity. Recent studies categorize LAB bacteriocins into Class I, Class II, and Class III based on these criteria ( Dicks et al., 2018; Soltani et al., 2021). Class I bacteriocins are characterized by unique amino acids and structures resulting from posttranslational modifications, making them small peptides. These modifications include rare amino acids like lanthionine, heterocycles, head-to-tail cyclization, and glycosylation. They also feature a leader peptide crucial for enzyme recognition, transport, and peptide inactivation (Arnison et al., 2012). Genes responsible for lantibiotic biosynthesis are organized in operons, with genes for bacteriocin maturation typically found within the same gene structure (Alvarez-Sieiro et al. 2016). Class II bacteriocins are characterized by a flexible hinge that separates the peptide tract into variable and conserved portions. They feature an N-terminal leader with a C-terminal attached. The cationic N-terminal leader contains two cysteine residues linked by a conserved disulfide bridge, crucial for target engagement and a lethal mechanism of action. In contrast, the C-terminus is less conserved, resulting in reduced specificity in target contact (Johnsen et al., 2005). Class III bacteriocins are larger, heat-labile proteins with unique peptide domains. They encompass non-lytic bacteriocins with diverse modes of action, including inhibition without cell lysis, binding to sugars like mannose or glucose to affect target organisms' sugar absorption, and causing membrane leakage, leading to essential molecule loss (Choyam et a.,2021). 2.14.6. Separation, purification and identification of antimicrobial compounds The first step in bacteriocin isolation involves screening LAB from diverse sources, such as plant material, food products, and human or animal isolates. Researchers like Ogawa et al (2019), found LAB strains in dairy and cheese products, while Rajaram et al (2020) used the well diffusion method on agar plates to detect antagonistic activity. To avoid hydrogen peroxide antagonism, catalase was added to the culture medium, and phosphate buffer was used in the solid medium to prevent organic acid inhibition. Here are some discussions about novel bacteriocin purification methods (Figure 2.12). Table 2. 2. Bacteriocins of LAB and their main characteristics (Yang et al.,2014). Classification/features Bacteriocins Mwt (Da) Producing strain CLASSI The bacteriocins are post-transnationally modified, linear or globular peptides containing lanthionine, β-methyl lanthionine and dehydrated amino acids Nisin A 3352 Lactococcus lactissubsp. lactic Nisin U 3029 Streptococcus uberis Nisin Z 3493 Lactococcus lactissubsp. lactic Mersacidin 1824 Bacillus sp.Y85,54728 Labyrinthopeptin A 21922 Actinomadura sp. subtilosin A 3399 Bacillus subtilis 168 CLASSII Heat stable, unmodified, bacteriocins, heterogeneous class of small peptides Class IIa (pediocinPA-1like bacteriocins) Pediocin PA-1 4629 Pediococcus acidilactici i PAC-1.0 Carnobacteriocin X 3602 Carnobacterium maltaromaticum C2 Class IIb (composed of two peptides) lactacinF 4755 Lactobacillus spp. ABP-11 84096 Lactobacillus salivariussubsp. salivarius UCC118 Class IIc (circular peptide) carnocyclinA 5862 Carnobacterium maltaromaticum UAL307 enterocin AS-4 87149 Enterococcus faecalis Class IId (linear,non-pediocinlike, single-peptide) epidermicin NI01 6074 Staphylococcusepidermidis lactococcin A 5778 Lactococcus lactissubsp. Cremoris CLASSIII Large, heat unstableproteins Caseicin80 ∼42000 Lactobacillus casei B80 Enterolisin A 34501 Enterococcus faecalis LMG2333 HelveticinJ 37511 Lactobacillus helveticus 481 2.14.7. Chromatographic separation and purification The purity of antibacterial compounds from LAB is crucial for structural analysis. Purification typically involves chromatographic techniques or a combination of methods based on different separation principles. For instance, authors have successfully separated and purified reuterin using reversed-phase high-performance liquid chromatography (RP-HPLC) with two C18 columns in series and water as the mobile phase (Pauter et al., 2020). 2.14.8. Ammonium sulfate precipitation Ammonium precipitation is a widely used method for bacteriocin purification, typically as a first step. After culturing bacteriocin producers on MRS agar and recovering the cell-free supernatant through centrifugation, crude bacteriocin is obtained via protein precipitation with ammonium salt saturation. This crude bacteriocin is then further purified through steps like centrifugation, dissolution in water or buffer, and additional purification methods like membrane filtering and gel filtration. While this process, known as partial purification, yields bacteriocin suitable for use as a food preservative, it's not the final purification step (Fugaban et al., 2021). 2.14.9. Identification of antimicrobial compounds Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) can be used to identify antimicrobial substances. The latter is frequently employed to determine molecular mass. Reuterin was identified primarily using proton (1H) and carbon (13C). HPLC-MS and GC-MS analysis of NMR data in deuterium oxide (D2O) and deuterated methanol revealed that the molecular mass of the cyclic dimer form of reuterin was 148 (Ibrahim et al. 2021; Perez et al., 2014; Ye et al. 2021). 2.14.10. Polyacrylamide gel electrophoresis (PAGE) Polyacrylamide gel electrophoresis is used to estimate the molecular weight of bacteriocin after purification, and purified bacteriocin is treated with tricine sodium dodecyl sulfate polyacrylamide electrophoresis. Bacteriocin standard solutions are commonly used as a standard to measure the molecular weight of bacteriocin using gels (stacking solution and separating gel) (Struck et al., 2016; C. Xu et al., 2021; Yson et al., 2013; Zhu et al., 2015). Figure 2. 13. Steps for bacteriocin screening, production and purification 2.15. Application of Bacteriocin as Food Preservative The use of LAB bacteriocin as a unique natural food preservative is based on its health benefits. To be considered a safe and effective natural preservative, it must meet these criteria: (1) not harm consumers' gastrointestinal health; (2) have broad-spectrum activity against various food spoilage microorganisms; (3) be resistant to food enzymes; and (4) show thermal stability across different pH levels, high salt concentrations, and other factors. Failure to meet these criteria would limit bacteriocin's potential as a safe natural food preservative (Ananou et al., 2010). LAB bacteriocin has been explored as a novel natural food preservation method using various approaches, including LAB starter cultures, protective LAB strains, reintroduction of previously fermented foods with bacteriocin-producing LAB during processing, and direct application of purified bacteriocin as a preservative (Ananou et al., 2010). These strategies are adaptable to different food types and specific LAB targets for preservation. Given LAB bacteriocin's "GRAS" status, it's suitable for industrial use, meeting the rising demand for fresh, safe, naturally preserved, and nutritionally enhanced food products with improved sensory qualities (Dhundale et al., 2018). Bacteriocins are commonly used as bio preservatives in foods through three methods: 1) Immunizing food with LAB that produce bacteriocins, crucial for their efficacy; 2) Directly using pure or semi-purified bacteriocins as preservatives; and 3) Incorporating a bacteriocin-producing strain's fermented product into food processing (Dioso, et al., 2022). 2.16. MALDI-TOF MS bacterial identification system MALDI-TOF, or matrix-assisted laser desorption ionization time-of-flight analysis, is a mass spectrometry method used for identifying microorganisms like bacteria, yeast, and molds. A laser breaks down bacteria into protein fragments, which travel through a detection tube. The "time of flight" measures how long these peptides take to reach the tube. By comparing, the mass spectrometry profile with a MALDI-TOF database, bacterial genera and species can be identified. Updating the database can be done by lab personnel or through purchasing an updated version. It relies on detecting the mass-to-charge ratio of ribosomal proteins, providing a unique mass spectrum for bacteria in a short time, and identification is based on the closest match with reference strains' spectra (Ashfaq et al., 2022). 2.16.1. Sample preparation Two main sample preparation methods for MALDI-ToF analysis are direct transfer and protein extraction. Direct transfer involves quickly recovering bacterial cells from growth plates or liquid media, providing reliable and accurate results. Protein extraction, using solvents like ethanol (avoiding toxic ones like formic acid), is recommended for higher spectral quality (Diaz et al., 2019). After preparation, bacterial cells or extracted proteins are placed on a MALDI biotarget plate, covered with a matrix solution (e.g., CHCA, sinapinic acid, DHB), and dried. This plate is then inserted into the MALDI-TOF instrument to generate a spectrum for comparison with the database (Gonzalo et al., 2021).See Figure 2.13 for an overview of the MALDI-ToF mass analysis process. technical terms

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The technical terms mentioned in the passage are:

Bacteriocins: Antibacterial compounds produced by bacteria.

Amphiphilic: Having both hydrophilic (water-attracting) and hydrophobic (water-repelling) regions in the molecular structure.

Membrane-permeabilizing: Capable of disrupting cell membranes, leading to increased permeability.

Classification: Categorization of bacteriocins into Class I, Class II, and Class III based on various factors like molecular weight, producer strains, structure, mode of action, biosynthesis, and biological activity.

Posttranslational modifications: Changes that occur to a protein after it has been translated from mRNA, such as the addition of lanthionine, β-methyl lanthionine, and dehydrated amino acids in Class I bacteriocins.

Operons: Functional units of genomic DNA containing a cluster of genes under the control of a single promoter, often found in bacteria.

Chromatographic separation and purification: Techniques used to isolate and purify bacteriocins from other components present in a sample.

Ammonium sulfate precipitation: A method for precipitating proteins using ammonium sulfate, commonly used as an initial step in bacteriocin purification.

Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS): Analytical techniques used to identify the structure and composition of antimicrobial substances.

Polyacrylamide gel electrophoresis (PAGE): A method for separating proteins based on their size and charge.

MALDI-TOF MS bacterial identification system: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry used for identifying microorganisms.

Sample preparation: Methods for preparing bacterial samples for MALDI-TOF MS analysis, including direct transfer and protein extraction.

GRAS status: Generally Recognized As Safe status, indicating that a substance is considered safe for consumption by regulatory authorities.

Biosafety: Ensuring that bacteriocins do not harm consumers' gastrointestinal health.

Broad-spectrum activity: Ability to inhibit a wide range of food spoilage microorganisms.

Thermal stability: Ability to maintain activity at different temperatures.

pH stability: Ability to maintain activity at different pH levels.

High salt concentrations: Ability to maintain activity in the presence of high concentrations of salt.

Biomarker: A characteristic feature used for identifying or measuring the presence of a substance or condition.

These terms are essential for understanding the classification, isolation, purification, identification, and application of bacteriocins in food preservation and bacterial identification.

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LAB bacteriocin has been explored as a novel natural food preservation method using various approaches, including LAB starter cultures, protective LAB strains, reintroduction of previously fermented foods with bacteriocin-producing LAB during processing, and direct application of purified bacteriocin as a preservative (Ananou et al., 2010). These strategies are adaptable to different food types and specific LAB targets for preservation. Given LAB bacteriocin's "GRAS" status, it's suitable for industrial use, meeting the rising demand for fresh, safe, naturally preserved, and nutritionally enhanced food products with improved sensory qualities (Dhundale et al., 2018). Bacteriocins are commonly used as bio preservatives in foods through three methods: 1) Immunizing food with LAB that produce bacteriocins, crucial for their efficacy; 2) Directly using pure or semi-purified bacteriocins as preservatives; and 3) Incorporating a bacteriocin-producing strain's fermented product into food processing (Dioso, et al., 2022)., which application is more effective why

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Each of the three methods mentioned for using LAB bacteriocins as bio preservatives in foods has its advantages and effectiveness, depending on the specific context and desired outcome:

Immunizing food with LAB that produce bacteriocins: This method involves adding LAB strains to food products during processing, which then produce bacteriocins that inhibit the growth of harmful bacteria. This approach can be effective because it allows for continuous production of bacteriocins within the food matrix, providing ongoing protection against spoilage organisms. However, the effectiveness may vary depending on factors such as the concentration of LAB and their ability to produce sufficient levels of bacteriocins throughout the shelf life of the product.

Directly using pure or semi-purified bacteriocins as preservatives: This method involves adding purified bacteriocins directly to food products to inhibit the growth of spoilage organisms. Using pure or semi-purified bacteriocins allows for precise control over the concentration and activity of the preservative, potentially leading to more consistent and predictable results. Additionally, this approach may be preferred in situations where the presence of LAB cells is not desirable, such as in certain types of processed foods or where specific sensory characteristics need to be preserved.

Incorporating a bacteriocin-producing strain's fermented product into food processing: This method involves using fermented products containing bacteriocin-producing LAB strains as ingredients in food processing. The bacteriocins produced during fermentation can provide protection against spoilage organisms in the final product. This approach leverages the natural fermentation process to produce bacteriocins and may be particularly suitable for traditional or artisanal food products where fermentation is already a common practice.

The most effective application method may depend on factors such as the specific food product, desired level of preservation, regulatory considerations, and consumer preferences. For example, direct addition of purified bacteriocins may be preferred in certain processed foods where the presence of live LAB cells is undesirable, while immunizing food with LAB strains may be more suitable for products that undergo minimal processing and where the presence of LAB is acceptable or desirable for other reasons, such as flavor development or probiotic benefits. Ultimately, the choice of application method should be based on a thorough understanding of the product requirements and the capabilities of each approach.

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Bacteriocin safety issue,

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Ensuring the safety of bacteriocins for use as food preservatives is paramount, given their direct contact with food products and potential consumption by consumers. Here are some key safety considerations:

Toxicity: Bacteriocins produced by lactic acid bacteria (LAB) are generally considered safe for consumption, especially those derived from strains that are commonly used in food fermentation processes. However, it's essential to assess the toxicity of bacteriocins thoroughly, particularly if they are being used in purified or concentrated forms. Toxicity studies should evaluate potential adverse effects on human health, including acute and chronic toxicity, genotoxicity, and carcinogenicity.

Allergenicity: Some individuals may be allergic to certain proteins or peptides present in bacteriocins. Therefore, it's crucial to assess the allergenic potential of bacteriocins, especially if they are derived from sources known to cause allergies in susceptible populations. Allergenicity testing may involve in vitro assays, animal studies, and clinical trials to evaluate the potential for allergic reactions upon exposure to bacteriocins.

Antimicrobial Resistance: There is concern that prolonged exposure to bacteriocins could lead to the development of antimicrobial resistance in bacteria, potentially reducing the effectiveness of antibiotics or other antimicrobial agents. Therefore, it's essential to monitor for the emergence of resistance mechanisms in target bacteria exposed to bacteriocins. This may involve surveillance studies, genetic analysis, and risk assessments to identify and mitigate potential risks associated with antimicrobial resistance.

Regulatory Compliance: Bacteriocins used as food preservatives must comply with regulatory standards and guidelines established by government agencies responsible for food safety. These regulations may specify permissible levels of bacteriocins in food products, labeling requirements, and documentation of safety and efficacy data. Compliance with regulatory requirements ensures that bacteriocins are used safely and responsibly in food processing.

Consumer Perception: Perception of safety by consumers is critical for the acceptance and adoption of food products preserved with bacteriocins. Clear labeling and transparent communication about the use of bacteriocins in food products can help build consumer trust and confidence. Educating consumers about the safety and benefits of bacteriocins may also alleviate concerns and promote acceptance of these products in the marketplace.

Overall, addressing safety issues associated with bacteriocins requires a comprehensive approach that involves rigorous scientific evaluation, adherence to regulatory standards, and transparent communication with stakeholders, including consumers, food manufacturers, and regulatory agencies. By ensuring the safety of bacteriocins, their potential as natural and effective food preservatives can be fully realized, contributing to the development of safer and more sustainable food systems.

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Possible research question on sample size, sample collections

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Certainly! Here are some possible research questions related to sample size and sample collection methods:

Sample Size Optimization:What is the optimal sample size required to detect significant differences in microbial populations in fermented foods treated with different concentrations of bacteriocins? How does varying sample size affect the accuracy and precision of microbial diversity measurements in soil microbiome studies? What is the minimum sample size needed to reliably assess the efficacy of bacteriocin-producing LAB strains in inhibiting foodborne pathogens in meat products?

Sample Collection Methods:How does the method of sample collection (e.g., swabbing, excision, immersion) influence the recovery and quantification of bacteriocin-producing LAB from fermented dairy products? What are the comparative advantages and limitations of different sample collection techniques (e.g., vacuum filtration, sedimentation) for studying bacteriocin distribution in aquatic environments? How do sample collection methods (e.g., air sampling, surface swabbing) impact the detection and quantification of bacteriocin-producing LAB in food processing facilities, and what are the implications for food safety assessments?

Sample Representativeness:To what extent do sample collection strategies (e.g., random sampling, stratified sampling) influence the representativeness of microbial communities in soil samples for studying the impact of bacteriocin-producing organisms on soil health? How does the spatial distribution of sampling sites (e.g., grid-based sampling, transect sampling) affect the representativeness of bacteriocin-producing LAB populations in agricultural fields, and what are the implications for biocontrol efficacy? What is the impact of sample collection frequency (e.g., daily, weekly, monthly) on the temporal dynamics of bacteriocin production by LAB in fermented food matrices, and how does this influence the stability and shelf-life of the final products?

These research questions can serve as starting points for investigations into the effects of sample size and collection methods on the outcomes of studies involving bacteriocin-producing LAB or other microbial systems. They address key considerations related to experimental design, data quality, and the interpretation of results in various contexts, including food science, environmental microbiology, and biotechnology.

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Possible research question on sample size, sample collections for traditioan feremnted foods

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Absolutely! Here are some research questions focusing specifically on traditional fermented foods:

Optimizing Sample Size for Traditional Fermented Foods:What is the optimal sample size required to capture the microbial diversity and community dynamics in traditional fermented foods from different geographical regions? How does the sample size affect the reliability of detecting potential spoilage microorganisms and beneficial LAB strains in traditional fermented foods like kimchi, sauerkraut, or miso? Can a smaller sample size be used effectively to assess the microbiological safety and quality of traditional fermented foods without compromising the accuracy of microbial analysis?

Comparative Analysis of Sample Collection Methods:What are the differences in microbial community profiles obtained from traditional fermented foods when using different sample collection methods, such as surface swabbing, core sampling, or liquid extraction? How do sample collection techniques impact the recovery and identification of LAB species and their bacteriocin production in traditional fermented foods, such as kefir, sourdough, or tempeh? Are there specific sample collection methods that are more suitable for preserving the indigenous microbiota and sensory attributes of traditional fermented foods during analysis?

Assessing Representativeness of Samples:To what extent do traditional fermentation practices, such as batch size, fermentation vessel type, and fermentation duration, influence the representativeness of samples collected for microbial analysis? How do variations in sampling locations within traditional fermentation facilities (e.g., near the surface, core samples, top vs. bottom layers) affect the characterization of microbial communities and the detection of bacteriocin-producing LAB strains? Can stratified sampling strategies be employed to account for the heterogeneity within large-scale fermentation batches of traditional foods, and how does this approach impact the accuracy of microbial diversity assessments?

These research questions address the unique challenges and opportunities associated with studying traditional fermented foods, considering their diverse microbial ecosystems, cultural significance, and variations in fermentation practices. By investigating sample size optimization and sample collection methods in this context, researchers can enhance our understanding of microbial dynamics in these foods and contribute to their safety, quality, and preservation.

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Why lactic acid react with Calcium carbonate produce clear zone, around the colony, is lactic acid produce by lactic acid bacteria only ?

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The formation of a clear zone around a colony when lactic acid reacts with calcium carbonate is due to a chemical reaction known as acidification. This phenomenon occurs because lactic acid, a weak organic acid produced during fermentation by lactic acid bacteria (LAB), reacts with calcium carbonate, which is a basic compound.

Lactic acid is typically produced as a metabolic byproduct of LAB during the fermentation process. LAB convert carbohydrates, such as sugars, into lactic acid through a series of biochemical reactions. The accumulation of lactic acid in the surrounding environment leads to a decrease in pH, resulting in acidification.

When lactic acid encounters calcium carbonate, which is a basic salt commonly found in many solid culture media, it reacts with it to form calcium lactate, carbon dioxide, and water. This reaction reduces the pH of the medium surrounding the colony, causing a localized decrease in pH. As a result, the calcium carbonate in the immediate vicinity of the colony is dissolved, leaving behind a clear zone or halo.

It's important to note that while lactic acid is primarily produced by LAB, it can also be produced by other microorganisms and even in some chemical processes. However, in the context of microbial cultures, the production of lactic acid and subsequent formation of a clear zone around the colony are often associated with LAB due to their predominant role in lactic acid fermentation.

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gram staiing for lactic acid bacterai, princioles and implication

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Gram staining is a common microbiological technique used to differentiate bacteria into two broad categories based on their cell wall composition: Gram-positive and Gram-negative. While the staining procedure is widely applicable, its interpretation and implications for lactic acid bacteria (LAB) can vary.

Principles of Gram Staining:

Primary Stain (Crystal Violet): Initially, the bacterial smear is stained with crystal violet, which stains all cells purple.

Mordant (Iodine): Iodine is then added, which forms a crystal violet-iodine complex, trapping the dye within the cell wall of Gram-positive bacteria.

Decolorization (Alcohol or Acetone): This step involves washing the slide with alcohol or acetone, which removes the dye from the thinner cell wall of Gram-negative bacteria but not from the thicker peptidoglycan layer of Gram-positive bacteria.

Counterstain (Safranin): Finally, the slide is stained with safranin, which stains the decolorized Gram-negative bacteria pink, contrasting with the purple Gram-positive bacteria.

Implications for Lactic Acid Bacteria (LAB):

Differentiation: Gram staining can help differentiate between LAB and other bacterial groups based on their cell wall composition. LAB are generally Gram-positive, meaning they retain the crystal violet-iodine complex and appear purple under the microscope.

Identification: While Gram staining provides initial information about the cell wall characteristics of LAB, it is usually just one step in the identification process. Additional tests, such as biochemical assays or molecular techniques, may be required for accurate identification to the species or strain level.

Phenotypic Characteristics: Gram staining can offer insights into the phenotypic characteristics of LAB, such as their morphology and cell wall structure. This information can be valuable for understanding their physiology and potential applications, particularly in food fermentation and probiotic development.

Quality Control: In industrial settings, Gram staining may be used as a quality control measure to assess the purity and viability of LAB cultures. Contaminants or other microorganisms present in a culture can be identified based on their Gram stain reaction, helping ensure the integrity of fermentation processes and product quality.

In summary, while Gram staining provides valuable information about the cell wall properties of lactic acid bacteria, its interpretation should be complemented with other microbiological and molecular techniques for accurate identification and characterization.

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steps and princles of gram staiing

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Gram staining is a differential staining technique used to classify bacteria into two groups: Gram-positive and Gram-negative, based on differences in their cell wall composition. Here are the steps and principles involved in Gram staining:

Steps:

Prepare Bacterial Smear:Obtain a bacterial culture and transfer a small amount onto a clean microscope slide. Allow the smear to air dry to fix the bacteria onto the slide.

Primary Stain (Crystal Violet):Flood the bacterial smear with crystal violet solution and let it stand for about 1 minute. Crystal violet stains all cells purple.

Mordant (Iodine):Add iodine solution (Gram's iodine) to the smear and let it stand for about 1 minute. Iodine forms a complex with crystal violet within the cell wall, trapping the dye inside.

Decolorization:Carefully wash the slide with a decolorizing agent, typically alcohol or acetone, for a few seconds. This step removes the crystal violet-iodine complex from Gram-negative bacteria but not from Gram-positive bacteria due to differences in their cell wall structure.

Counterstain (Safranin):Flood the smear with safranin solution and let it stand for about 1 minute. Safranin stains the decolorized Gram-negative bacteria pink.

Wash and Air Dry:Rinse the slide with water to remove excess stain and then blot it dry with bibulous paper. Allow the slide to air dry completely before examination under a microscope.

Principles:

Differential Staining:Gram staining is a differential staining technique that distinguishes between bacteria based on the thickness and composition of their cell walls.

Crystal Violet-Iodine Complex:Gram-positive bacteria have a thick layer of peptidoglycan in their cell walls, which retains the crystal violet-iodine complex, imparting a purple color to their cells.

Decolorization:The decolorizing agent (alcohol or acetone) dehydrates and shrinks the peptidoglycan layer in Gram-negative bacteria, allowing the crystal violet-iodine complex to be washed out, resulting in colorless cells.

Counterstain:The counterstain (safranin) is applied to the decolorized cells, staining Gram-negative bacteria pink, providing a contrast to the purple Gram-positive bacteria.

Microscopic Examination:After staining, the slide is examined under a light microscope using oil immersion to observe the color and morphology of the bacterial cells.

Interpretation:Gram-positive bacteria retain the crystal violet-iodine complex and appear purple, while Gram-negative bacteria are counterstained pink due to the safranin.

Gram staining is a fundamental technique in microbiology, providing valuable information about the cell wall characteristics of bacteria and aiding in their classification and identification.

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The bioactive compounds from LAB isolates were tested for enzyme sensitivity using pepsin, trypsin, proteinase K, and lysozyme at 5 mg/mL concentration (Goa et al., 2022). why important

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Testing bioactive compounds from LAB isolates for enzyme sensitivity is important for several reasons:

Understanding Stability: Enzyme sensitivity testing helps researchers understand how stable the bioactive compounds are in the presence of digestive enzymes. This is crucial for predicting their fate during digestion and absorption in the human gastrointestinal tract.

Bioavailability Prediction: Enzyme sensitivity testing provides insights into the bioavailability of bioactive compounds. Compounds that are resistant to digestion by enzymes like pepsin and trypsin are more likely to reach the intestines intact, where they can exert their beneficial effects.

Optimizing Formulation: Results from enzyme sensitivity tests can guide the formulation of LAB-derived bioactive compounds for oral delivery. For example, if a compound is sensitive to pepsin but resistant to trypsin, encapsulation strategies or protective coatings may be necessary to ensure its survival through the stomach.

Comparative Analysis: Enzyme sensitivity testing allows for the comparison of different bioactive compounds or LAB strains in terms of their susceptibility to digestion. This information can aid in the selection of strains or compounds with superior stability and bioavailability profiles.

Clinical Relevance: Understanding enzyme sensitivity is essential for assessing the potential health benefits of LAB-derived bioactive compounds. Compounds that survive digestion may have greater therapeutic potential for conditions such as gastrointestinal disorders, immune modulation, or metabolic health.

Overall, enzyme sensitivity testing provides valuable information about the fate and bioavailability of bioactive compounds from LAB isolates, facilitating their development as functional ingredients or therapeutic agents.

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Additionally, 10% methanol and ethanol were added to CFS to observe their influence on bioactivity against pathogens. The stability and antagonistic potential of CFS were further evaluated by introducing 1% (w/t) sodium citrate and potassium sorbate as food preservatives and examining their impact on antibacterial activity. The treated CFS was then incubated for 24 hours at 37°C using the disc diffusion method, and residual inhibitory activity was gauged by observing the zone of inhibition (Arena et al., 2016)., why important

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The addition of different compounds such as methanol, ethanol, sodium citrate, and potassium sorbate to cell-free supernatants (CFS) derived from LAB cultures, followed by the evaluation of their impact on antibacterial activity, serves several important purposes:

Stability Assessment: Testing the influence of methanol and ethanol on the bioactivity of CFS provides insights into the stability of bioactive compounds under different solvent conditions. This is crucial for determining the suitability of these compounds for various applications, such as food preservation or therapeutic use.

Preservative Compatibility: Assessing the impact of sodium citrate and potassium sorbate, common food preservatives, on the antibacterial activity of CFS helps evaluate their compatibility for use in combination with LAB-derived bioactive compounds. Understanding whether these preservatives enhance, diminish, or have no effect on the antimicrobial properties of CFS is essential for formulating effective preservation strategies for food products.

Practical Application: The disc diffusion method allows for a qualitative assessment of residual inhibitory activity after treatment with different compounds. This mimics real-world scenarios where LAB-derived products may be exposed to various environmental conditions or additives. By observing the zone of inhibition, researchers can gauge the effectiveness of treated CFS in inhibiting the growth of pathogens.

Optimization of Formulation: Results from these experiments can guide the optimization of formulations for LAB-derived products intended for specific applications. For example, if methanol or ethanol enhances antibacterial activity without compromising stability, they may be incorporated into preservation methods for fermented foods. Similarly, understanding the impact of food preservatives can help tailor formulations to maximize shelf life while retaining bioactivity.

Quality Control: Evaluating the stability and antagonistic potential of CFS under different conditions is essential for ensuring product consistency and quality control. By systematically testing the influence of various additives, researchers can identify optimal formulations that maintain bioactivity over time, thereby enhancing the efficacy and reliability of LAB-derived products.

Overall, these experiments provide valuable information about the compatibility, stability, and effectiveness of LAB-derived bioactive compounds in different environments and applications, facilitating their development for use in food preservation, healthcare, and other industries.

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applications and uses of lyaste I, formic acid, Lysate II, Acetonitrlae, Matric solution A and B on MALDI TOf

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Each of the substances you mentioned plays a specific role in sample preparation for MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry), which is a technique used for identifying microorganisms like bacteria. Here's how each substance is typically used:

Lysate I and Lysate II: Lysates are solutions containing cellular material obtained from microorganisms. They are used to extract proteins or peptides for analysis by MALDI-TOF MS. Lysate I and Lysate II may contain different lysis buffers or other additives optimized for the extraction of proteins from specific types of microorganisms.

Formic Acid: Formic acid is commonly used as a matrix additive in MALDI-TOF MS sample preparation. It helps to ionize proteins and peptides, enhancing their detection by the mass spectrometer. Formic acid is typically added to the matrix solution in small concentrations to improve ionization efficiency.

Acetonitrile: Acetonitrile is often used as a solvent in MALDI-TOF MS sample preparation. It helps to dissolve proteins or peptides extracted from microorganisms, facilitating their analysis by the mass spectrometer. Acetonitrile may also be used as a component of the matrix solution to aid in the crystallization of matrix-analyte complexes.

Matrix Solution A and B: Matrix solutions are mixtures containing a matrix compound (such as α-cyano-4-hydroxycinnamic acid or sinapinic acid) dissolved in a suitable solvent. These solutions are applied to the sample target along with the analyte (extracted proteins or peptides) prior to analysis by MALDI-TOF MS. Matrix Solution A and B may contain different matrix compounds or solvent compositions optimized for specific types of samples or analytical conditions.

In summary, Lysate I and Lysate II are used to extract proteins or peptides from microorganisms, while Formic Acid, Acetonitrile, and Matrix Solutions A and B are components of the sample preparation process for MALDI-TOF MS, aiding in protein solubilization, ionization, and crystallization for accurate mass spectrometric analysis. Each of these substances contributes to the overall effectiveness and reliability of microbial identification using MALDI-TOF MS.

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The isolates' bacteriocin-producing potential was tested using the agar-well diffusion assay against specific pathogens (Yang et al., 2014). To neutralize acids and hydrogen peroxides, 1 NaOH was used to adjust the pH of the cell-free supernatant (CFS) to 7.00, and catalase was added at 5 g/ml. Confirming bacteriocin production involved treating the CFS with acid neutralization and heat, then applying proteinase K (Sigma, St. Louis, USA) and incubating for 2 hours at 37℃. Antimicrobial activity was assessed using the agar well diffusion method (Saad et al., 2015).

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The steps described in the passage outline a method for assessing the bacteriocin-producing potential of microbial isolates and evaluating their antimicrobial activity. Here's a breakdown of the process:

Agar-Well Diffusion Assay: This assay is a common method for testing the antimicrobial activity of substances, including bacteriocins. In this step, wells are punched into an agar plate inoculated with a specific pathogen. The cell-free supernatant (CFS) containing bacteriocins is added to the wells, allowing diffusion into the agar. The formation of clear zones around the wells indicates inhibition of pathogen growth by the bacteriocins.

Neutralization of Acids and Hydrogen Peroxides: To ensure accurate assessment of bacteriocin activity, any acids and hydrogen peroxides present in the CFS that could interfere with the assay are neutralized. Sodium hydroxide (NaOH) is used to adjust the pH of the CFS to a neutral level (pH 7.00), and catalase is added to degrade hydrogen peroxide.

Confirmation of Bacteriocin Production: To confirm that the antimicrobial activity observed in the CFS is due to bacteriocin production, several steps are taken. First, the CFS is treated with acid neutralization and heat, which may help inactivate other antimicrobial compounds that could interfere with the assay. Then, proteinase K is added to degrade any proteins present in the CFS other than bacteriocins. Incubation at 37°C for 2 hours allows sufficient time for enzymatic degradation.

Assessment of Antimicrobial Activity: After confirming bacteriocin production, the antimicrobial activity of the treated CFS is assessed using the agar-well diffusion method. This step helps validate the effectiveness of the bacteriocins against specific pathogens.

Overall, this method provides a systematic approach to evaluating the bacteriocin-producing potential of microbial isolates and determining their antimicrobial activity against target pathogens. It involves multiple steps to ensure accurate and reliable results.

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The in vitro the LAB isolates cell surface hydrophobicity was evaluated by measuring the microbial cell adhesion to hydrocarbons according to the method described by Rokana et al. (2018). The overnight cultures in MRS broth were harvested by centrifugation (at 8,000 rpm at 4°C for 10 min), washed twice with PBS, and re-suspended in PBS buffer followed by absorbance (A0) measurement at 600 nm. A cell suspension of about 3 ml was blended with 1 ml of hydrocarbon (xylene) and incubated at 37°C without shaking for 1 h for separation of the aqueous and organic phases. The aqueous phase (1 ml) was removed carefully and the absorbance (A1) was measured at 600 nm. The percent hydrophobicity was measured by a decrease in absorbance and calculated using the following formula: percentage of cell surface hydrophobicity = (1 − A1/A0) × 100).

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The passage describes a method for evaluating the cell surface hydrophobicity of LAB isolates using a technique based on microbial adhesion to hydrocarbons. Here's a breakdown of the procedure:

Preparation of Overnight Cultures: LAB isolates are cultured overnight in MRS broth to promote cell growth and metabolic activity.

Harvesting of Cells: After incubation, the cultures are harvested by centrifugation at 8,000 rpm and 4°C for 10 minutes. This step helps to separate the bacterial cells from the growth medium.

Washing with PBS: The harvested cells are washed twice with phosphate-buffered saline (PBS) to remove any residual growth medium and debris.

Measurement of Absorbance (A0): The washed cells are resuspended in PBS buffer, and the absorbance of the suspension is measured at 600 nm (A0). This measurement provides a baseline for the initial cell density.

Incubation with Hydrocarbon (Xylene): A portion of the cell suspension (typically 3 ml) is mixed with xylene (1 ml), a hydrophobic solvent. The mixture is then incubated at 37°C without shaking for 1 hour to allow for phase separation between the aqueous and organic phases.

Measurement of Absorbance (A1): After incubation, the aqueous phase is carefully separated from the organic phase, and the absorbance of the aqueous phase is measured at 600 nm (A1). This measurement reflects the residual cell density after exposure to the hydrocarbon.

Calculation of Hydrophobicity: The percentage of cell surface hydrophobicity is calculated using the formula: percentage of cell surface hydrophobicity = (1 − A1/A0) × 100. This calculation compares the initial absorbance (A0) with the absorbance after exposure to the hydrocarbon (A1) to determine the decrease in absorbance, which correlates with the hydrophobicity of the cell surface.

Overall, this method provides a quantitative assessment of the hydrophobicity of LAB isolates' cell surfaces, which can be indicative of their adhesive properties and potential interactions with surfaces in various environments.

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4.2.7.6.Cell Surface Hydrophobicity

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Cell surface hydrophobicity refers to the tendency of microbial cell surfaces to interact with hydrophobic molecules or surfaces. It plays a crucial role in various microbial processes, including adhesion to surfaces, biofilm formation, and interactions with host cells or other microorganisms.

Principle:

The assessment of cell surface hydrophobicity involves measuring the ability of microbial cells to adhere to hydrophobic solvents, typically hydrocarbons like xylene or hexadecane. The principle relies on the fact that hydrophobic interactions between microbial cell surfaces and hydrophobic solvents will result in the attachment of cells to the solvent phase, leading to a decrease in the aqueous phase's turbidity.

Procedure:

Preparation of Overnight Cultures: Microbial cultures are grown overnight in a suitable growth medium to promote cell growth and metabolic activity.

Cell Harvesting: After incubation, the microbial cells are harvested by centrifugation to separate them from the growth medium.

Washing: The harvested cells are washed with a buffer solution, such as phosphate-buffered saline (PBS), to remove any residual medium components.

Measurement of Initial Absorbance (A0): The washed cells are resuspended in buffer, and the absorbance of the suspension is measured at a suitable wavelength, often 600 nm, using a spectrophotometer. This measurement provides a baseline for the initial cell density.

Incubation with Hydrophobic Solvent: A portion of the cell suspension is mixed with a hydrophobic solvent, such as xylene, and incubated under appropriate conditions (e.g., temperature, time) without agitation.

Phase Separation: After incubation, the mixture is allowed to settle, leading to the separation of the aqueous and solvent phases.

Measurement of Final Absorbance (A1): The absorbance of the aqueous phase is measured again after phase separation. This measurement reflects the residual cell density after exposure to the hydrophobic solvent.

Calculation of Hydrophobicity: The percentage of cell surface hydrophobicity is calculated using the formula: Percentage of Hydrophobicity=(1−A1A0)×100\text{Percentage of Hydrophobicity} = \left(1 - \frac{A1}{A0}\right) \times 100Percentage of Hydrophobicity=(1−A0A1)×100This calculation compares the initial absorbance (A0) with the absorbance after exposure to the hydrophobic solvent (A1) to determine the decrease in absorbance, which correlates with the hydrophobicity of the cell surface.

Significance:

Cell surface hydrophobicity is an important microbial characteristic with implications in various fields, including microbiology, biotechnology, and medicine. Understanding the hydrophobic properties of microbial cells can provide insights into their interactions with surfaces, colonization of environments, and pathogenicity. Additionally, it can inform the development of strategies for controlling microbial adhesion, biofilm formation, and microbial contamination.

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A cell suspension of about 3 ml was blended with 1 ml of hydrocarbon (xylene) and incubated at 37°C without shaking for 1 h for separation of the aqueous and organic phases

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In this step of the procedure for assessing cell surface hydrophobicity:

Preparation of Cell Suspension: A cell suspension containing approximately 3 mL of microbial cells, typically harvested from overnight cultures, is prepared.

Addition of Hydrocarbon (Xylene): One milliliter (1 mL) of a hydrophobic solvent, such as xylene, is added to the cell suspension. Xylene is commonly used in hydrophobicity assays due to its low water solubility and ability to interact with hydrophobic cell surfaces.

Incubation: The mixture of cell suspension and xylene is then incubated at a specified temperature, often 37°C, for a designated period, typically 1 hour. During this incubation period, the mixture is left undisturbed without any shaking to allow for the separation of the aqueous and organic phases.

Phase Separation: Over the incubation period, the hydrophobic solvent (xylene) interacts with hydrophobic regions on the microbial cell surfaces, leading to the attachment of cells to the solvent phase. Meanwhile, the aqueous phase contains the remaining cells and any non-interacting components.

Observation and Collection: After incubation, the mixture is allowed to settle, facilitating the separation of the aqueous and organic phases. The phases can then be visually inspected, and the aqueous phase is typically collected for subsequent absorbance measurements to quantify the remaining cells.

This step is crucial for assessing the interaction between microbial cells and hydrophobic solvents, providing insights into the hydrophobicity of microbial cell surfaces.

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why one hour

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The one-hour incubation period is a common duration used in the assessment of cell surface hydrophobicity for several reasons:

Sufficient Time for Interaction: One hour provides adequate time for the hydrophobic solvent (such as xylene) to interact with the microbial cells. This duration allows for the establishment of equilibrium between the cells and the solvent, ensuring that the maximum amount of hydrophobic interaction between the two is achieved.

Consistency and Standardization: Using a standardized incubation time ensures consistency in the experimental procedure across different samples and studies. By employing the same incubation period, researchers can compare results more effectively and draw reliable conclusions about the hydrophobic properties of different microbial strains or conditions.

Optimal Balance of Sensitivity and Efficiency: One hour strikes a balance between sensitivity and efficiency. It is long enough to detect significant differences in hydrophobicity between microbial strains but not excessively prolonged, which could lead to practical limitations or unnecessary delays in the experimental workflow.

Practical Considerations: From a practical standpoint, a one-hour incubation period is manageable and fits well within typical laboratory schedules. Longer incubation times may not offer substantial additional benefits in terms of data quality while potentially increasing the duration and complexity of the experimental procedure.

Overall, the one-hour incubation period is a widely accepted and practical choice for assessing cell surface hydrophobicity, offering a balance between sufficient interaction time and practical considerations.

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Can increasing bacterial concentration in a formulation compensate for inhibition by lactobacillus strain,and if so ,is it right to do so

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Increasing the bacterial concentration in a formulation might help compensate for inhibition by a specific lactobacillus strain to some extent, but it's not always the ideal solution, and there are several factors to consider:

Efficacy of Inhibition: The effectiveness of inhibition depends not only on the concentration of the inhibiting bacteria but also on their metabolic activity, the production of inhibitory compounds (such as bacteriocins), and the sensitivity of the target bacteria to those compounds. Simply increasing the concentration of the inhibiting bacteria may not necessarily overcome inhibition if the inhibitory mechanisms are potent and the target bacteria are highly sensitive.

Impact on Product Quality: Increasing bacterial concentration in a formulation may alter the sensory properties, texture, and other quality attributes of the product. This could affect consumer acceptance and marketability, especially in food and beverage applications where taste, appearance, and mouthfeel are critical factors.

Cost Considerations: Higher bacterial concentrations may increase production costs, including raw materials, labor, and processing expenses. This could impact the economic feasibility of the product, especially if the increase in concentration does not significantly improve inhibition or product quality.

Regulatory Compliance: Regulatory agencies often impose limits on the maximum allowable bacterial concentrations in food and beverage products for safety and quality reasons. Exceeding these limits may lead to regulatory non-compliance and potential legal issues.

Instead of solely relying on increasing bacterial concentration, it's advisable to explore other strategies to address inhibition by lactobacillus strains:

Strain Selection: Choosing alternative strains of bacteria that do not inhibit the desired microbial population or selecting strains with lower inhibitory activity could be a more targeted approach.
Formulation Optimization: Adjusting the formulation ingredients, pH, temperature, or other parameters to create an environment less conducive to inhibition while still meeting product specifications.
Process Modifications: Modifying the production process or conditions to minimize inhibition while maintaining product integrity and safety.
Combination Approaches: Using a combination of strategies, such as incorporating multiple strains with complementary activities or incorporating additional ingredients or additives that mitigate inhibition.

In conclusion, while increasing bacterial concentration may offer a partial solution to inhibition by lactobacillus strains, it should be approached cautiously, considering its potential impacts on efficacy, product quality, cost, and regulatory compliance. Exploring alternative strategies or combination approaches may provide more effective and sustainable solutions in addressing inhibition issues.