PROBIOTICS AND PREBIOTICS: MECHANISMS, BENEFITS, AND CHALLENGES – A NARRATIVE REVIEW

PROBIOTICS AND PREBIOTICS: MECHANISMS, BENEFITS, AND CHALLENGES - A NARRATIVE REVIEW

Yara A. Nader, Maya G. Pillai and A. Helen *

Department of Biochemistry, Research Centre, University of Kerala, Karyavattom, Thiruvananthapuram, Kerala, India.

ABSTRACT: The human gut microbiota harbours numerous microbiota which includes archaea, fungi, viruses and bacteria. A balanced and healthy gut microbiota is essential for maintaining optimal physiological functions and thus the overall health and well-being. The state of a balanced gut microbiome is known as eubiosis, while dysbiosis is condition where an imbalances or disruptions in the gut microbiota, occurs. Dysbiosis has been associated with a range of diseases including gastrointestinal disorders, metabolic syndrome, neurological disorders chronic inflammation etc. The term probiotics refers to live microorganisms that, when ingested in adequate amounts, confer health benefits. The emergence of using probiotics therapeutically to repopulate the gut with eubiotic microorganisms and improve health introduces many new concepts.  On the contrary, prebiotics are food components that promote the growth or metabolism of beneficial microorganisms (e.g., bacteria & fungi). They can alter the gut microbiome makeup. Both probiotics and prebiotics have been effective in a variety of diseases. Nevertheless, concerns over standardization and safety dictate rigorous assessment of strains/formulations. New innovative therapeutic strategies are emerging due to recent advances in the understanding of Postbiotics activity and novel concepts such as symbiotics. This review highlights the existing literature regarding the mechanisms of probiotics and prebiotics in general, their health effects on various diseases as evidenced by animal models or intervention studies, also drawing attention to some controversies linked with these agents, which are still under investigation or revisiting after new high-quality evidence emerged mainly related to metabolic features such as weight loss.

Keywords: Gut microbiome, Eubiosis, Dysbiosis, Probiotics, Prebiotics, Postbiotics, Symbiotics

INTRODUCTION: The gut microbiome is a diverse community comprising bacteria, fungi, viruses, and archaea ^{1, 2, 3}. The microbial diversity is shaped by various factors, such as age, gender, and antimicrobial agents leading to differences in bacterial populations ^{4, 5}. The initial colonization of the gut begins during gestation and extends until around 18 months to 3 years after birth. During this period, a distinct bacterial signature forms within the distal small intestine and colon, signifying the development of a mature microbiome. In humans colonization of the gut by Lactobacillus and Bacteroides occurs during early stages of infancy. Subsequently, environmental influences, breastfeeding, psychological factors, medications, and the maternal diet during and after pregnancy play significant roles in shaping the gut microbiota.  By the age of four, these microbial communities typically stabilizes ^{6, 7, 8, 9, 10}.

They spread throughout the body with the digestive system harbouring the largest concentration ⁶. The human body provides a nutrient-rich environment where these microorganisms metabolise nutrients into metabolites ¹¹, interacting with the host immune system and affecting human health.  Dysbiosis is a condition when an imbalance occurs between harmful and beneficial bacteria, and this can lead to many diseases including autoimmune diseases, psychological diseases, and other health issues etc ^{5, 6, 12}. For instance, the rise in BMI is associated with an increase in specific kinds of intestinal bacteria (Firmicutes/Bacteroidetes) and (Prevotella/Bacteroidetes), which are main indicators of gut microbiota composition. Moreover, certain bacteria such as Eubacterium ventriosum are linked with obesity, while others like Oscillospira spp are related to leanness ³. Bacterial population density vary in individuals, with approximately five hundred species in the oral cavity, one thousand and twelve bacteria per gram in the colon, and over hundred per gram in the small intestine and stomach, including Bifidobacterium, Clostridium, Eubacterium, Faecalibacterium, and Peptococcus ^{12, 13}.

Numerous studies have confirmed the interaction of gut bacteria with the different organs via several axes including the axis of microbiota-gut-brain, the axis of microbiota-gut-liver, and the axis of microbiota-gut-lung, the axes of microbiota-gut-immune and other axes ^{14, 15}. They can produce essential vitamins, support the proliferation of epithelial cells, and enhance the metabolism of bile acid, fibres, proteins and fats changed into short-chain fatty acids and simple sugars. Besides this, they influence innate and adaptive immunity. In order to recognize the dynamics of gut microbiota in health management, researchers use a wide range of state-of-the-art research methods. These include DNA-based techniques, which enable the accurate identification and quantification of microbial species. Molecular biology methods such as polymerase chain reaction (PCR) are strain-specific and hence useful in recognizing the expressions of different species.

The 16S rRNA sequencing is a comparatively novel technique of microbial ecology that enables the detection of bacterial communities by examining the expression of the conserved 16S ribosomal RNA gene ¹³. Researchers employ these technologies to define the diversity of microbial flora within the gut and its effect on human health. As we learn more about the different factors that affect gut health, it is necessary to expand our knowledge beyond probiotics and prebiotics.

We have to research more into other dietary supplements like symbiotics and Postbiotics.  All of these supplements have different effects on the composition and functionality of the gut microbiota and may influence overall health in different ways.

Probiotics: Probiotics are defined as living microorganisms that on ingestion in sufficiently large numbers, can have beneficial effects for the host. They are particularly renowned in relation to the promotion of bile production and, therefore, overall intestinal health and well-being ^{12, 13, 14}. They include Saccharomyces boulardii, and strains of Lactobacillus, Bifidobacterium, Streptococcus thermophiles, and Enterococcus faecalis etc ¹⁶.  The biological effect of probiotics is that they can influence the production of cytokines, inhibit the translocation of microorganisms from the gut lumen across the epithelial layer, suppress pathogenic bacteria by competition for resources and change the pH level ^{14, 15, 17, 18}. Prebiotics, on the other hand, are non-digestible food substances, which selectively encourage the growth of the friendly gut bacteria thereby, potentiating the effects of probiotics. They foster the development of constructive genera like Eubacteria, Bifidobacteria and Lactobacilli and suppress pathogenic species like Clostridium by providing the base for the fermentation by the normal flora ^{15, 19, 20}.

The discovery of probiotics initially dependent on identifying beneficial bacteria in a healthy individual that could provide health benefits when administered to those with a health issue.  However, Russian scientist E´lie Metchnikoff pioneered a novel approach by exploring the relationship between fermented foods and their health benefits ²¹. He suggested that Western diets negatively impacted gut health by promoting harmful bacteria, which adversely affected overall health.  Metchnikoff developed the theory that introducing beneficial bacteria could alter the gut microbiota and improve human health ²². This concept led to various definitions of probiotics.

The World Health Organization (WHO) defines probiotics as live microorganisms ^{12, 23} used as nutritional supplements to promote human health and growth by restoring intestinal bacterial balance ^{23, 24, 14}. Parker was the first to use the term “probiotics” in this context, referring to nutritional supplements that support animal growth ²⁵. Several probiotic bacterial strains and genera are known, although the most common are those of the Lactic acid bacteria (LAB) like Lactobacilli, Enterococci, Heterotrophic bacteria, Escherichia, Streptococcus Bacillus and Propionibacterium. However, some fungal strains Saccharomyces (non-pathogenic yeasts) ^{23, 24}, and some non-spore-forming, non-flagellated Coccobacilli ²⁴ have also been used. Related terms have recently appeared that include postbiotics, meaning “non-viable microorganisms or their by-products biological activity”. Another term, pharmabiotics, defines human microbial cells or their products having demonstrated roles in health or disease including psychobiotics and immunobiotics. Of these, next-generation probiotics (NGP) hold a special position.  They are live microorganisms identified by comparative microbiota analyses which when administered in adequate amounts confer a health benefit on the host. Other related terms include Paraprobiotics, Probioceuticals / Probiotaceuticals, and Live biotherapeutic products (LBP) ²⁶.

The recommended daily intake of probiotics for consumers varies depending on the individual’s bacterial environment, typically ranging between(108 and 109 colony-forming units (CFU)) ^{22, 24, 27}. Probiotics are added to many food products such as milk and milk products, cheese, meat products, juices, bread and fruits. The quality and quantity of probiotics, however, may vary during manufacturing and storage.  Also, some probiotic products contain only a single strain or one type of bacteria while others preparations have contain a mixture of numerous strains or types of bacteria ²⁸.

Mechanisms of Action of Probiotics: Understanding how probiotics work is important in order to appreciate their potentials and limitations. Studies have identified many mechanisms of actions based on in vitro or ex vivo, rodent and human examples. However, not all the mechanisms are definitive because probiotic can act on multiple pathways of host physiology simultaneously diversifying the influencing factors and increasing the complexity. Probiotics modulate the gut microbial community by increasing the population of beneficial bacteria, such as Bifidobacteria and Lactobacilli, while reducing pathogenic microbes ²⁹. They can influence the host metabolism and energy balance by the metabolites they produce or providing necessary nutrients for metabolism through cross-feeding and carbohydrate metabolism ^{18, 22, 30, 31}. This section elaborates on the ways through which probiotics exert their effects on the host.

Modulation of Gut Microbiota: One of the fundamental roles of probiotics is the modulation of the gut microbiota composition. It does this by the following mechanism:

Promotion of Beneficial Bacteria: Probiotics enhance the population of beneficial bacteria, such as Lactobacillus and Bifidobacterium. These bacteria ferment dietary fibers to produce short-chain fatty acids (SCFAs) like butyrate, acetate, and propionate.  By lowering the pH of the gut environment SCFAs inhibit pathogenic bacteria and promote the growth of commensal microorganisms ¹⁷.

Inhibition of Pathogenic Bacteria: Probiotics produce antimicrobial substances, including bacteriocins, hydrogen peroxide, and organic acids, which suppress the growth of harmful bacteria.  By competing for nutrients and adhesion sites on the intestinal mucosa, probiotics also prevent pathogens from establishing themselves in the gut ²³.

Enhancement of Gut Barrier Function: Probiotics play a critical role in maintaining and enhancing the integrity of the intestinal barrier:

Tight Junction Integrity: Probiotics enhance the expression of tight junction proteins, such as occludin and zonulin, which are essential for maintaining the tight junctions between epithelial cells. This reduces intestinal permeability and prevents the translocation of harmful substances into the bloodstream.

Mucus Production: Probiotics stimulate the production of mucin, the primary component of mucus, which forms a protective layer on the intestinal lining. This barrier prevents pathogens from coming into direct contact with the epithelial cells. Probiotics interact with host microorganisms through various macromolecules of cell surface. These include protein molecules like LPxTG-binding proteins, and non-protein molecules like peptidoglycan, lipoteichoic, and exopoly-saccharides that affect mucin production, intestinal cells, and dendritic cells. Consequently, probiotics can extend their transit times within the gut and help fortify, the intestinal mucosal barrier ^{14, 18, 32, 33} by enhancing gene expression involved in junction signalling and mucin-glycoprotein production. This support contributes to the repair of the gut barrier following damage. For example, Staphylococcus thermophilus and Lactobacillus acidophilus significantly inhibited the adherence of enter invasive. Escherichia coli to HT29 cells, and increased transepithelial resistance in Caco-2 cells.  Mucins play a crucial role in immune system support. For example, lactobacilli stimulate the secretion of MUC5AC mucin in human intestinal cell lines like HT29 through adhesion to monolayers ^{29, 34, 35}.

Production of Antimicrobial Substances: Probiotic bacteria secrete antimicrobial peptides that serve multiple functions, including signalling to the immune system or microbiota, directly eliminating other microbiota. For instance, certain bacterial strains produce ribosomally synthesized peptides known as bacteriocins.

These are proteinaceous toxins produced by probiotic bacteria that inhibit the growth of similar or closely related bacterial strains.  Bacteriocins can target specific pathogens without disrupting the overall microbiota balance and can act as preservatives in probiotic-treated foods and inhibit both Gram-positive and Gram-negative microorganisms ²². One such antimicrobials peptide, Low Molecular Weight Bacteriocin (LMWB), functions by either disrupting cell wall synthesis or forming pores to lyse target pathogenic cells. Additionally, probiotic bacteria secrete acetic and lactic acids, which acidify the environment and inhibit pathogens like Salmonella sop. Probiotic also release chemicals known as microcins, which penetrate target cells and disrupt cellular functions by binding to iron siderophore receptors. This action leads to the inhibition of enzymes, such as ATP-synthase, RNA polymerase, and DNA gyrase, ultimately resulting in the death of pathogenic organisms ²⁹. Probiotics contribute to the synthesis of various biologically active compounds, including amino acids, small peptides, phenols and lactanes through fermentation of proteins. These compounds play an important role in immunomodulation by promoting the production of antioxidants and inflammatory mediators that target and eliminate harmful organisms. Probiotics also produce vitamins such as vitamin K, also vitamin B12 and propionic acid, with “Propionibacterium shermani” being a notable producer of propionic acid.  Enzymes produced by Probiotic bacterial hydrolyze proteins to create peptides with immune-modulating and anti-inflammatory properties ¹⁵. These peptides stimulate the production of immunoglobulin A (IgA) and interleukin-10 (IL-10), affecting immune responses and inflammatory gene expressions. The production of IgA enhances immune-modulation and the clearance of pathogenic microorganisms by activating dendritic cells, naive T cells, and B cells ^{36, 37}.

Furthermore, probiotics can enhance antioxidant defences by increasing the activities of enzymes like glutathione S-transferase, catalase, and glutathione peroxidase. This enhancement helps reduce oxidative stress and protect cells from damage caused by carcinogens ³⁸. Probiotics produce organic acids, including lactic acid and acetic acid, which lower the pH of the gut. This acidic environment is inhospitable to many pathogenic bacteria, thereby preventing their proliferation ²⁹.

TABLE 1: THE MOST IMPORTANT VITAL COMPOUNDS PRODUCED BY PROBIOTICS

Competitive Exclusion of Pathogens: Probiotics compete with pathogens for resources and adhesion sites in the gut. In the 1970s, research demonstrated that introducing a diverse array of adult intestinal microorganisms could significantly enhance resistance to diseases, such as Salmonella infections, in newly hatched Chicks ⁴². This led to the development of the concept of "competitive exclusion” which involves maintaining a balance of microorganisms in the digestive system. This balance is achieved by reducing the concentration of harmful organisms primarily aerobic bacteria while preserving a greater number of beneficial anaerobic bacteria ^{29, 42}. Competitive exclusion operates through several mechanisms: it reduces harmful bacteria by altering the gut environment to make it less hospitable. This is achieved by decreasing the oxygen levels or adjusting the pH of the intestinal environment ²³. Additionally, competitive exclusion includes nutrients competition and preventing the pathogen adhesion. Probiotics consume available nutrients in the gut, limiting the resources that pathogenic bacteria need to grow and thrive ²⁹.

Probiotics adhere to the intestinal mucosa, occupying the binding sites that pathogens would otherwise use to attach and colonize. This competitive exclusion helps prevent infections and supports a healthy microbiota. Probiotics contribute to these processes by secreting biosurfactants, disrupting receptors through enzymatic activity, and producing receptor analogues, thereby impeding pathogen attachment ^{18, 14, 29, 33}.

Immune system Modulation: Probiotics interact with the host’s immune system to modulate immune responses.

Enhancement of Innate Immunity: Probiotics activate macrophages, dendritic cells, and natural killer cells, enhancing the body's first line of defence against infections. They stimulate the production of immunoglobulins, particularly IgA, which plays a crucial role in mucosal immunity.

Regulation of Inflammatory Responses: Probiotics modulate the production of cytokines, promoting an anti-inflammatory environment. They can decrease the levels of pro-inflammatory cytokines like TNF-α and IL-6 while increasing anti-inflammatory cytokines such as IL-10.

Probiotics enhance both adaptive and innate immunity ⁴². They increase production of antibodies and stimulate macrophages and natural killer (NK) cells activity. This occurs through the interaction of the system humoral immunity with probiotics via toll-like receptors (TLR) ⁶. For example, “Lactobacillus casei shirota” boosts natural killer cells (NK) activity and cytokine IL12 production ⁴³. Probiotics also inhibit the Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway ^{2, 8}, which reduces the activation of B cells, induces T-cell apoptosis and increased levels of anti-inflammatory cytokines such as interleukin-10 (IL-10) while decreasing interleukin 8 (Il-8)¹⁵and tumour necrosis factor alpha (TNF-α) ^{2, 15}. Additionally, Probiotics downregulate TLR expression, which helps to suppress intestinal inflammation ⁴⁴ TLRs, such as TLR2 and TLR 6 are essential in immune regulation processes, and several probiotics, including “L. plantarum ccfm634”, “L. plantarum ccfm734”, “L. Fermentum CCFM381”, “L. acidophilus ccfm137”, and “S. thermophilus ccfm218” ⁴³.

Regulatory T cells are crucial for promoting oral tolerance and immunity to infections and allergies, benefit from new probiotic strains. Immunological effects of probiotics are primarily associated with the initial resident bacteria rather than prolonged probiotic presence.

Research indicates that the immunological effects of probiotics are primarily associated with the initial resident bacteria rather than prolonged probiotic presence ⁸. Among the most significant probiotic species are lactic acid bacteria (LAB) ¹⁹. “Lactobacillus rhamnosus GG”, identified in 1985, was the first probiotic used in  the dairy industry and has been shown to enhance  the immune system by increasing IgA levels and the number of immunoglobulin-releasing cells in the intestinal mucosa ^{23, 19} (LAB) also utilize toll-like receptor (TLR2) to activate Interleukin 6 (IL-6) and Transforming growth factor-beta (TGF-B) from dendritic cells (DC), promote Interleukin 12 (IL-12) synthesis, and enhance antibody formation and cytokine regulation to boost host defence ²⁹.

Metabolic Effects: Probiotics influence host metabolism in several ways. Certain probiotics synthesize essential vitamins, such as B vitamins and vitamin K, which are crucial for various metabolic processes in the host (Poindexter B et al., 2021). By fermenting dietary fibers, probiotics produce SCFAs that serve as an energy source for colonocytes. SCFAs also have systemic effects, including anti-inflammatory properties and the regulation of lipid and glucose metabolism ¹⁷.

Neurological Effects: Emerging research highlights the impact of probiotics on the gut-brain axis neurotransmitter Production: Some probiotics produce neurotransmitters like serotonin and gamma-aminobutyric acid (GABA), which can influence mood and behaviour. These neurotransmitters can affect the central nervous system via the vagus nerve or through systemic circulation.

Modulation of the HPA Axis: Probiotics can influence the hypothalamic-pituitary-adrenal (HPA) axis, which regulates stress responses.  By modulating this axis, probiotics can potentially alleviate stress and anxiety ^{45, 46, 47, 48}.

The multifaceted mechanisms of action of probiotics underline their potential benefits in promoting gut health, enhancing immune function, and even influencing mental well-being.  However, understanding these mechanisms also helps in identifying the limitations and potential side effects of probiotic use. Comprehensive knowledge of how probiotics work is essential for their effective application in clinical practice and for maximizing their health benefits while minimizing risks.

FIG. 1: GENERAL MECHANISMS OF PROBIOTICS ⁴⁹. DCs: Dendritic cells; IECs: Intestinal Epithelial Cells; IL-10: Interleukin 10; TGF-β: Transforming Growth Factor β; Th1: Type 1 T helper; Th2: Type 2 T helper; Th17: T helper 17; Treg: regulatory T cells.

Probiotics for Health and Diseases Management: Probiotics have become an integral part of modern health management due to their wide-ranging benefits. They contribute to maintaining and enhancing health by supporting various bodily systems, including the digestive, immune, and genitourinary systems. They aid in preventing and managing a variety of conditions, from gastrointestinal disorders and infections to mental health issues and many chronic diseases. This section delves into the impact of probiotics on health and disease management, examining their therapeutic potential and the safety standards essential for their effective use. Research has demonstrated that probiotics play a crucial role in protecting the genitourinary system and reducing infants and neonatal mortality, particularly with strains such as "Lactobacillus GG(ATCC53103)" and "Lactobacillus rhamnosus".  By maintaining a balanced vaginal flora, these probiotics may help lower the risk of sexually transmitted infections, a key benefit of lactobacillus species. Additionally, the strain “Banimalis”, has been shown to alleviate the severity of cirrhosis, mucosal candidiasis and to ease gingival pain. Probiotics have also been effective in managing allergy-related conditions like asthma and dermatitis, as well as certain mental health disorders such as depression and anxiety ²³. In severe illnesses like COVID-19, research indicates that diverse beneficial bacteria can mitigate inflammatory symptoms, potentially improving patient outcomes ⁴⁹. Probiotics have been proven effective in reducing symptoms associated with gastrointestinal disorders, cancer, heart disease, constipation, depression, and various autoimmune diseases.

TABLE 2: ROLE OF PROBIOTICS IN DISEASES

Quality and Safety Standards of Probiotics: Selecting the optimal bacterial strain and ensuring safety are critical when using probiotics. Researchers emphasize the need to consider various factors, including bacterial mutations, environmental stability, bile resistance, survivability, adhesion and antimicrobial production and bacterial identity ^{19, 64, 65}.

Studies should address these aspects to select the appropriate strain and assess its qualities and functions, including its survival during storage and transfer ¹⁹. In 2002, the World Health Organisation established guidelines for probiotics use, emphasizing the need for extensive clinical trials, cell lines and animal models testing, adherence to high-quality standards, and understanding in-vivo mechanisms. It is also important to develop probiotics that can be used in protective vaccines and other health applications ^{23, 66}. Key quality standards include maintaining the properties and purity of probiotic products ³¹.

To ensure safety, stool samples are analyzed using selective media or advanced techniques such as 16s rRNA sequencing, Polymerase chain reaction (PCR), optical enumeration and flow cytometry to identify and quantify intestinal bacteria ^{8, 65}. Emerging technologies, including microarray analysis and bioinformatics platforms have led to the development of , Next generation probiotic (NGPs), which include Gram-positive anaerobic bacteria like “Clostridium butyricum”, “Bacteroides ovatus”, “Bacteroides xylanisolvens”, “Bacteroides fragilis”, “Akkermansia muciniphila”, and “Faecalibacterium prausnitzii” ^{27, 67}. Integrating this data provides a comprehensive view of microorganisms and their interactions with the host, guiding effective treatment approaches ²⁷.

Monitoring the presence of these organisms in faeces over several weeks to months helps, assess their ability to proliferate and establish themselves in the gastrointestinal tract ⁶⁸. Probiotic preparations should contain at least (5*10⁹ CFU) and be used for a minimum of five days ²³. Safety standards include testing for bacterial toxicity, potential for systemic infections, and metabolic byproducts ⁶⁴. Probiotic support the immune system, resist bile and hydrochloric acid, reduce permeability and adapt to intestinal conditions ²². Currently, the most common genera of probiotics in the market are lactic acid bacteria, including: Lacyobacilli, Bifidobacteria, and Streptococci ²⁵.

Prebiotics: Prebiotics are useful in synthesizing other beneficial metabolites of the gut microbiota and work to establish a healthy state of the gut microbiome where it contributes to the generation of short-chain fatty acids (SCFAs) ^{69, 70}. This study also demonstrates that probiotics and prebiotics have significant outcomes in disease control and prevention of chronic illnesses such as IBD ⁷¹, CVD ^{61, 72}, and mental illnesses²⁰.The total genome of the gut bacteria is of the least one hundred-fold of the genome size of a human.  These bacteria participate in metabolism through SCFAs, tryptophan metabolites, amino acids and glycans, which are required for bacterial growth in the gut and modulation of the host’s inflammation ¹¹. Research has also shown that host genome may play a role in transmitting other bacterial families for instance Christensenellaceae which may transact with other genetic families and methanogenic archaea ⁴. The concept of "prebiotic" was defined by Gibson and Roberfroid in (1995) as "a selective non-digestible food component".  The more recent definition by the International Scientific Association for Prebiotics and Probiotics (ISAPP) as a "substrate" that is selectively used by gut bacteria only ^{15 ,20}. Prebiotics are food ingredients that must resist acidity in the stomach, remain undigested in the gut, and not hydrolysable by endogenous enzymes. Prebiotics can be classified based on their structure and fermentation properties.  Some of them are carbohydrates ⁷³, others are fibres that a certain degree of polymerization turn into carbohydrates ⁶⁹. Normally, prebiotics are taken in small quantities in diet ⁷⁰.

Based on the level of fermentation, prebiotics can be divided into two major categories, partially digested or non-digested. Thus, they can be either poorly fermented by oral microbiota or well fermented by gut microbiota ⁷⁴. Phytochemicals like polyphenols, carotenoids and organosulfer compounds also act as prebiotics^75,76. Glycomacropeptide (GMP), which contains a variety of amino acids, is another example of a prebiotic ⁷⁷. Resistant starch, whole grains, pectin that forms pectic oligosaccharides or (POS), and fructans, which include inulin and a fructo-oligosaccharide are some of the commonly used prebiotics ^{15, 23, 69}. Other prebiotics include, isomaltooligosaccharides, xilooligosaccharides, lactobionic acid, psyllium and galactomannan ⁷⁰.

Functions of Prebiotics in the Body: Because of the disruption of the immune barrier and metabolic function there may be low-grade endotoxemia, and many inflammatory mediators such as Lipopolysaccharide (LPS) in the gut are transported into the bloodstream leading to various diseases ²⁹. Such conditions can be treated with prebiotics because affect the type and/or number of bacterial species in the large intestinal and its environment by changing the pH that is preferred by acid-sensitive bacteria. Most of the prebiotic fermentation materials like bacteroids are acids ⁵⁰. Chen & Liu, 2018 claimed that the chain length of bacteria may influence the type of fermented prebiotics among the species.  For instance, activities with polysaccharides having DP ≤ 60 can only be conducted by a few species of fructans such as inulin. Inulin decreases the risks associated with oxidative stress that is associated with some inflammatory biomarkers and prevents the effects of lipid peroxidation in the stomach through the use of certain dietary antioxidants ⁶⁹. G protein receptor is found to regulate the immune system, the population density of beneficial bacteria and metabolism which is changed and influenced by prebiotics found in the gut-associated lymphoid tissue (GALT) ⁵.  Cross-feeding is also observed in the functioning of probiotics. A complex prebiotic ferments and produces a by-product that serves as a substrate for another microbe. For instance, Ruminococcusbromii can ferment resistant starches and lactobacilli and bifidobacteria utilize dietary fibers ⁷⁰.

Specific investigations in monoculture have revealed that Bifidobacteria are surpassed by other bacterial species when it comes to assimilation of inulin-type fructans (ITF). It was also established that Bifidobacteria species differ in their versatility towards the different chain lengths of inulin-type fructans (ITF). While Bifidobacteria have a very fast growth rate ⁷⁸ in the presence of Fructo-oligosaccharides (FOS) and gives out lactate and acetate, which is considered to be good sources of energy ⁷⁰. Eubacterium halliu cannot grow in vicinity of Fructo-oligosaccharides (FOS) ⁷⁸. Another major characteristics of prebiotics is specificity for microorganisms, and the ability to enhance the growth of good bacteria to overcome pathogenic ones besides enhancing the formation of fermentation products essential for immunity. The prebiotic fermentation results the in short chain fatty acid (SCAFAs) such as acetate, propionate, and butyrate ⁷⁰;  these change the stomach pH and influence acid-sensitive bacterial forms like Bacteroids, while stimulating Firmicutes to produce butyrate referred to as the butyrogenic effect ⁶⁹.

Other functions of prebiotics include regulation of, cytokines synthesis, which is a way to immune system regulation as well.  Namely, rat mesenteric lymph nodes (LNs) and cell cultures were shown to elevate the synthesis of the anti- inflammatory cytokines interferon gamma (IFN-g) and Interleukin 2 (IL2) and the production of the suppressor of the pro-inflammatory cytokines acetate and propionate. These prebiotics enhance the quantity of lactobacilli in the large intestine; therefore benefiting probiotics by increasing cytokines synthesis of Interleukin 10 (IL10) and Interferon gamma (IFN-g) in relation to intestinal immunity through (DP16, DP8, and DPG4). Inulin or beta-glucan Immunosuppressive polysaccharides can affect the innate immunity of fish in the way that ly50 also increases activity of neutrophil, phagocytosis and lysosome activity ⁴⁸. Studies have established that prebiotics are useful in the moderation of the incidence of allergic atopic dermatitis and urticaria in infants with the long-chain Fructo-oligosaccharides (FOS) and Galactooligosaccharide (GOS) ²⁹. Short chain fatty acids (SCAFs) control hormones and bowel movements prebiotic carbohydrates are responsible for the softening stool because of their water retention property. Studying has further confirmed that a ratio of Galactooligosaccharide (GOS) and fructans of 9:1 lowers respiratory tract infections in new-borns ³². A study done on elderly people taking Galactooligosaccharide (GOS) revealed enhanced phagocytosis, and NKCA and valuable microorganisms ²⁹.

Constant intake of prebiotics can cause accumulation of the mentioned molecules and even the number of metabolites. Molecular dynamism in a diet containing arachidic acid, behenic acid, and oleic acid together with a novel prebiotic Galactooligosaccharide (GOS) was shown to affect approximately 21 receptors in a study.  It was also established that difructose anhydrides (DFA) impacted on the bioavailability of the iron and blood calcium. Moreover, prebiotics increase the permeability of the intestinal membrane according to the molecule size and improve, nutrient absorption in the intestine and, therefore, the proliferation of different kinds of blood cells. From Channastriate fingerlings high percentage of fructo-oligosaccharide and manoligosaccharide enhanced the increase in hemoglobin and serum protein ⁴⁸. It should also be noted that prebiotics enhance feelings of fullness, decrease and prevent constipation and diarrhoea, and are beneficial for cardiovascular health ⁷.

Source of Prebiotics of Vegetable Origin and Their Biochemical Constituents: Many plants contain prebiotics in them because of the carbohydrate they possess. The examples are garlic, onions, wheat, asparagus, bananas, dandelion root, and many tubers. Pectin present in walls of these plants belongs to those Polysaccharides that can synthesize prebiotics. For example, Kiwi has bacterial polysaccharides content, which results into the formation of metabolites and alteration of colonic bacteria. Solutes such as polyphenols found in the food are metabolized with colonic bacteria or their distribution in the intestine.   Some of the phenolic metabolites of low molecular weight can be produced through the metabolic activities of the gut bacteria, such as Lactobacteria and Bifidobacteria ⁷⁸. These interactions synthesise prebiotics; Aspergillus brasiliensis and Aspergillusnidulans are capable of fermenting rice husk carbohydrates into Xylooligosaccharides (XOS) at 37 °C, pH 4. 5–7, for five days. Enzymatic processes also create prebiotics; when cellobiose 2-epimerase is applied on milk, 50 °C for 24 hours lactulose and epilactose are generated ⁷⁹.

TABLE 3: THE SOURCES OF PREBIOTICS

Prebiotics are substances that can develop and enhance their presence in a variety of products such as rice, wheat, oats, barley and aloe vera. Coffee also restores prebiotics derived from roasted, dark, and ground coffee beans (Anacardium occidentalel), which significantly boosts lactobacillus species ⁷. Additionally, biologically active peptides, vitamins and minerals in milk, which contain numerous prebiotics, are major sources of immune-boosting cytokines. Consequently, Prebiotics in milk, particularly in yoghurt, increase Bifidobacteria. Xylooligosaccharides (XOS) have been demonstrated to be more potent growth enhancers of probiotics than Fructo-oligosaccharides (FOS), stimulating the growth of Bifidobacteria and Lactobacillus ⁴⁸.

Metabolic Profiling of Bioactive Compounds in Foods and Supplements: Bioactive compounds in foods and supplements can be directly assessed, and their effect on human health can be understood by measuring metabolic profile.

This helps identify products resulting from the metabolism of short-chain fatty acid (SCFAs) which are produced after fermentation of dietary fibres by gut bacteria ^{12, 78}. Metabolic profiling involves techniques like “hydrogen nuclear magnetic resonance (H-NMR) spectroscopy” and “mass spectrometry”, along with structural analysis experiments to study the composition of the microbiome. Combining metabolic profiling and structural analysis measurements allows fora deeper understanding of metagenomic and metabolic data, leading to a more accurate classification and comprehension of microbiome functions ^{78, 82}.

FIG. 2: ROLE PREBIOTICS IN THE GUT ²⁹. When prebiotics are used selectively in the gut, the microbiota expands to support immunity at many species and strain levels. For a healthy bowel movement, the bacterial cell wall and other biomass boost immunomodulation and faecal bulking. Organic acids and other metabolites lower the luminal pH, which is hazardous to pathogens but helps make minerals like calcium soluble and absorbable. They also have a favourable effect on hormone regulation and epithelial integrity. GLP1: glucagon like peptide1; IL-10: Interleukin 10; M cell: microfold cell; NK: natural killer; PYY: peptide YY; TGFβ: transforming growth factor-β; Th1: type 1 T helper; Th2: type 2 T helper; Treg: regulatory T cells; ZO1: zonula occludens 1.

Effect of Prebiotics on Various Disease: Prebiotics play a very important role in the management of certain health disorders by intervening with gut microbiota and metabolic pathways.  They can manage the raised levels of bile acid by supplementing with good bacteria in to enhance the metabolism of bile acids. These prebiotics inhibit the activity of enzyme that degrade hypoxanthine and short chain fatty acid (SCFAs) such as tryptamine, and thus relieve constipation ⁸². Research has shown that in stool samples of people with obesity, there is generally a lower level of SCFAs. Short-chain fatty acids play an enormous role in the in stimulating the satiety hormone glucagon. Moreover, research has proved that a greater abundance of SCFA-producing bacteria can improve blood sugar level ¹⁶. Vulevic, 2015, proved that when Galactooligosaccharide (GOS) prebiotics were given to the elderly, showed elevated levels of lactate, increased Bifidobacteria and Bacteroids were identified, an indication of an increased colic absorption and reduced excretion ⁷⁸. Changes in faecal metabolites were detected in the stool of irritable bowel syndrome patients ⁸².

Prebiotics were also reported to ameliorate to colon cancer. Butyrate is the main SCFA that affects the proliferation of epithelial cells growth in the colon and serves as an energy source. It also affects the production of anti-inflammatory propionate and acetate in the large intestine which will lower pH that will favour the multiplication, Lactobacilli and Bifidobacteria ³. Butyrate has been shown to suppress Interleukin 12 (IL12), enhance Interleukin 10 (IL10) and Interleukin 14 (IL14) secretion, and inhibit the production of Interferon gamma (IFN-g) and Interleukin 2 (IL 2). Inhibiting colonic immune activation by inducing apoptosis and decreasing inflammatory signals, butyrate affects Interferon (IFN) signalling pathways including, specifically activator of transcription 1 (STAT1) and nitric oxide synthase (INOs) models. The induction of butyrate has shown decreased levels of the pro-inflammatory cytokines (IL6) and tumour necrosis factor-beta (TNF-B) in human studies ⁴⁸. Furthermore, Short chain fatty acid (SCFAs) are ligands for G-protein-coupled receptor 41 (GPR41) and G-protein-coupled receptor 43 (GPR43) on epithelial cells and therefore trigger immune responses to protect against infection by intestinal Escherichia coli ²⁹. Short chain fatty acid (SCFAs) affect the function of the colonic epithelium through the good absorption of various cations such as calcium (Ca+2), magnesium (Mg+2), and iron (Fe+2) ⁷.

Prebiotics appear to have beneficial effects in the management of type 2 diabetes. For example, β-glucans lower glycemic index and cholesterol levels. The intake of carbohydrates has an impact on the insulin and blood sugar responses and thus affects the intestinal microflora. Recently, scientific Studies have established a correlation between microbiome-brain axis with obesity, and diabetes among others ⁴⁸.

In a study in children and women done over a wide span of 60 days or more, it was established that prebiotics extracted from orange juice, other than being fortified with hesperidin and naringen increased, substantially enhanced insulin regulation, lowered glucose levels, triglycerides, and the level of total cholesterol ²⁹. Prebiotics extracted from mushrooms have also demonstrated anticancer activities and activate the immune system ⁷⁹.

TABLE 4: THE ROLE OF PREBIOTICS IN THE DISEASE

AOS, Alginate oligosaccharides; CVD, Cardiovascular; COVID-19, Coronavirus disease 2019; FOS, Fructo-oligosaccharides; GOS, Galacto-oligosaccharides; GALT, Gut-associated lymphoid tissue; NEC, Necrotizing Enterocolitis; SCFA, Short-chain fatty acids.

Prebiotics Safety Levels: Production technologies for prebiotic involve isomerization, fructosyl-transfer, and hydrolysis, and microbial fermentation ⁷⁹ to enhance physical and chemical properties ¹⁸. Though the US Food and Drug Administration does not define prebiotics, the same are regulated and labeled as safe for consumption in countries like Japan, the Netherlands, and Sweden ⁸³. Microbial diversity in the gut varies between developed and underdeveloped regions due to environmental influences on gut flora composition ¹⁹.

Safety levels of prebiotics depend on acidity of the stomach, bile, the site of digestion mainly in the colon ^{15, 20}. Chain length is a vital aspect of prebiotics, and shorter chains can be fermented in the proximal colon, while the longer chain of prebiotics ferments more slowly in the distal colon with a negative influence on health.  Common adverse effects due to excessive intake of prebiotics include osmotic diarrhoea and flatulence and have been reported to cause bacteremia and infections ⁶⁹.

Insights from Ancient Systems of Medicine:

Indian System (Ayurveda): Ayurveda, the ancient Indian system of medicine, emphasizes the importance of gut health through the concept of “Agni” (digestive fire) ⁹¹.  A strong Agni is central to Ayurvedic philosophy, believed to be the cornerstone of good health by aiding in the proper digestion of food, experiences, and emotions.

Traditional Probiotic Preparations in Ayurveda:

Asava and Arishta:  These are traditional fermented medicinal wines. Herbs and other ingredients are soaked in water or fruit juices and allowed to ferment naturally. These preparations are rich in beneficial microorganisms, including lactic acid bacteria and yeasts.

Takra (Buttermilk): Considered a digestive elixir in Ayurveda, takra is made by churning yogurt and adding water.  It contains diverse probiotic strains such as Lactobacillus and Bifidobacterium species, which are known to support gut health.

Kanji: This fermented rice gruel is rich in prebiotics and naturally occurring probiotics. It has been traditionally used to prevent and treat diarrheal diseases.

Shukta: Various vegetables and fruits are fermented to create shukta preparations. These not only preserve food but also enhance its nutritional and medicinal properties ⁹².

Traditional Prebiotic Foods in Ayurveda:

Ginger: Known for its digestive properties, ginger is often used in Ayurvedic preparations to enhance digestion and support gut health.

Turmeric: This powerful anti-inflammatory spice is also considered a prebiotic, promoting the growth of beneficial gut bacteria.

Asafoetida: Commonly used in Indian cooking, asafoetida aids in digestion and helps maintain a healthy balance of gut flora.

Fenugreek: Rich in fiber, fenugreek seeds act as a prebiotic, supporting the growth of beneficial bacteria in the gut ^{93, 94}.

Modern Research and Validation: Modern scientific research has validated many of these traditional practices.  Studies have shown that Ayurvedic fermented foods often contain more probiotic strains than commercial products. Additionally, the use of prebiotic foods like ginger and turmeric has been supported by research for their role in promoting gut health ^{94, 95, 96, 97}.

Chinese System (Traditional Chinese Medicine): Traditional Chinese Medicine (TCM) is one of the most important complementary and alternative therapies, which has played a major role over the years in treating or alleviating various diseases. TCM works to maintain gut health through Chinese plants and herbal compounds that produce prebiotics and some types of good bacteria.

Herbal Combinations in TCM: In one study, three herbs Rheum palmatum, Coptis chinensis, and Scutellaria baicalensis were combined in a traditional herbal formula administered to mice with Type 2 Diabetes Mellitus. This formula was found to improve the disease by modifying the intestinal microflora and producing new bacteria such as Alloprevotella and Barnesiella that contribute to the production of short-chain fatty acids (SCFAs) ¹⁷.

Both Ayurveda and Traditional Chinese Medicine have long recognized the importance of gut health and have utilized probiotics and prebiotics to promote well-being. Ayurveda, with its rich history of fermented foods and digestive spices, offers a holistic approach to maintaining gut health. Traditional Chinese Medicine complements this with its use of herbal compounds to modify gut flora and enhance health. Modern research continues to validate these ancient practices, highlighting their relevance in contemporary health and wellness.  By incorporating these traditional practices, we can support our digestive health and overall well-being, leveraging the wisdom of ancient systems to complement modern scientific understanding.

Synbiotic and Postbiotics: Synbiotics are combinations of probiotics and prebiotics designed to synergistically improve gut health and overall well-being ^{6, 48, 98}. Probiotics are live microorganisms that confer health benefits to the host, while prebiotics are non-digestible substrates that promote the growth and activity of beneficial gut bacteria.  Their combination provides a synergistic effect for better survival of probiotics, increased activity, improved integrity of the gut barrier and digestive health. Research involving humans, fish, and oysters has demonstrated that synbiotics are more effective than pre and probiotics for gastrointestinal health ^{67, 82}.

Postbiotics are metabolic by-products produced by probiotic bacteria. These substances exert effects on the host either directly or indirectly ^{3, 6}, Experimental evidence suggests that postbiotics can modulate the immune system ⁷ and are beneficial for various conditions including Alzheimer’s disease, multiple sclerosis, and SARS-Cov-2 (Severe Acute Respiratory Syndrome Coronavirus). They also aid in managing allergic diseases by restoring the balance between helper T cells (type I and type II) and influencing physical fitness and activity. For example, recent research on rats has identified propionate, a postbiotic derived from lactic acid metabolism by the Veillonella genus, as having potential health benefits ⁶. Additionally, postbiotics may interact with antibiotics such as tetracyclines, quinolones, and macrolides ²⁹.

Challenges and Limitations of Probiotics: Despite the potential benefits, the effectiveness of probiotics remains debated.  Systematic reviews and meta-analyses have highlighted inconsistencies in the results of probiotic studies. For instance, some studies have reported a decrease in C-reactive protein levels, while others have questioned the reliability of these findings due to limitations in study design and the need for more randomized clinical trials ^{99, 100, 101}. Furthermore, genetic factors, dietary differences, and variations in microbial composition can impact individual responses to probiotics, contributing to the variability in study outcomes.  Research involving children, adults, and the elderly has shown mixed results regarding probiotics such as S. boulardii ^{31, 102, 103, 104} and various strains of Lactobacillus, particularly in treating conditions like acute intestinal inflammation and gastroenteritis ³¹.

In neonates, the efficacy of probiotics in treating sepsis and reducing mortality has varied based on factors such as birth weight, milk type, and the use of single versus mixed strains. For instance, probiotics like L. plantarum PP 11-217 and prebiotics such as fructooligosaccharides (FOS) were effective in Indian infants, while Bifidobacteriumbreve BBG-001 showed limited benefits in English infants fed pasteurized milk. Conversely, combining Lactobacillus and Bifidobacteria was effective in treating necrotizing enterocolitis (NEC) but not nosocomial sepsis ^{105, 106, 107, 108, 109, 110, 111}. A comprehensive review by the Southeast Asian Neurogastrointestinal Society (SEAGMA) on the clinical use of probiotics reported differed opinions on their effectiveness. Probiotics were found to be beneficial for treating acute gastroenteritis in children and reducing antibiotic-associated diarrhoea, but less effective for constipation or obesity in children.  They were also noted to have limited efficacy in inducing remission for ulcerative colitis but helpful in maintaining remission for chronic bursitis.  The review emphasized the need clinical trials to validate their efficacy ¹¹².

Future Directions: The development of precision probiotics has numerous challenges. The two types of research studies involved in precision probiotics include observational and mechanistic studies, whereas the utilization of phenotypic screening with target-based discovery is still under development ²¹. The high throughput strategies aim to describe molecular processes and host responses in more detail to design specific probiotic consumption strategies. Newer techniques like multi-omics technologies are helping in gaining better perspective for microbial associated community, bridging the gap in clinical diagnostics and improving the awareness of public regarding prebiotics, probiotics ⁸². These advancements accompanied by the integration of nanotechnology have revealed enhanced product stability, as well as the effectiveness of the chemicals and structure of the probiotics and prebiotic ^{82, 91, 113, 114, 115, 116, 117} and is expected to become a powerful partner alongside drug therapies through advanced research ^{29, 118}.

CONCLUSION: Ongoing research and technological innovations continue to advance the field of probiotics and prebiotics.  Understanding bacterial strains, their mechanisms of action, and adherence to safety standards will be crucial for their integration into global healthcare systems. The growing body of evidence and increasing consumer awareness suggest that probiotics and prebiotics will play a significant role in future therapeutic and preventive health strategies.

ACKNOWLEDGMENTS: We extend our sincere gratitude to the Indian Council for Cultural Relations (ICCR) and the Government of India for their generous funding.  Special thanks also go to the University of Kerala for providing the facilities necessary for this research.

Funding: This work was supported by the Indian Council for Cultural Relations (Grant WC1746326071346 to Yara A Nader) under the Government of India's initiative for foreign students.

CONFLICTS OF INTEREST: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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     Nader YA, Pillai MG and Helen A: Probiotics and prebiotics: mechanisms, benefits, and challenges - a narrative review. Int J Pharmacognosy 2025; 12(9): 697-15. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.12(9).697-15.

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