Postbiotics: A developing notion to application

Introduction

The concept of "biotics" has recently gained public recognition and includes terms such as probiotics, prebiotics, and synbiotics, which have been popular since the 1990s. The Food and Agriculture Organization of the United Nations and the World Health Organization (FAO-WHO) define probiotics as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." Prebiotics, meanwhile, are non-digestible food ingredients that positively impact the host. The International Scientific Association for Probiotics and Prebiotics (ISAPP) defines prebiotics as "a substrate that is selectively utilized by host microorganisms, conferring a health benefit." Prebiotics, which include dietary fibers, phenolics, phytochemicals, conjugated linoleic acids, polyunsaturated fatty acids, lactulose, inulin, human milk oligosaccharides (HMOs), and various oligosaccharides, can enhance host health by altering the microbiota composition and promoting the growth of beneficial species (Gurunathan S et al., 2023) [14] . Synbiotics, a synergistic combination of prebiotics and probiotics, improve the survival and colonization of live microbes in the gastrointestinal (GI) tract, thereby providing further health benefits (Rasaei N., 2024; Wegh, C.A.M., 2019) [29,39] . However, the effectiveness of probiotics relies on the viability of the microorganisms, prompting researchers to explore alternatives. This led to the development of postbiotics, which, unlike prebiotics and probiotics, do not contain live microorganisms, resulting in a longer shelf life, greater stability, and numerous health benefits. This review provides a concise overview of the concept of postbiotics, their production methods, classification, and their impact on human and health.

Although postbiotics include metabolites, purified metabolites, and vaccines are excluded from this category. Besides the health benefits discussed here the health benefits attributed to postbiotics include immunomodulatory, antimicrobial, and anticancer effects, as well as the reduction of inflammation, oxidative stress, blood pressure, cholesterol levels, and body weight (Gurunathan et al., 2023) [14] . It is believed that the postbiotic content of the body can be enhanced by consuming the foods like buttermilk, cottage cheese, fermented pickles, yogurt, fiberrich oats, garlic, and flaxseeds. However, overconsumption of postbiotics may have adverse effects on immunocompromised individuals. The effectiveness of postbiotics can be influenced by both internal and external factors, as reported in various studies (Rad et al., 2020). Internal factors, such as resident microbiota, enzymes, and food components, can inhibit the function of postbiotic metabolites activity (Rad et al., 2020). Postbiotic dysfunction is mainly associated with proteolytic enzymes (Izuddin et al., 2020) [17] . Proteolytic enzymes that interfere with the activity of postbiotic metabolites include chymotrypsin, pepsin etc., (Rad et al., 2020; Izuddin et al., 2020) [28,17] . The optimal pH required for postbiotic activity is between 4 and 9. Among external factors, hydrogen ions can alter postbiotic activity. Foods like pasteurized milk and ground meat, which have an optimal pH, do not interfere with postbiotic function. (Moradi et al., 2020) [24] . Temperature variation also affects the antimicrobial activity of postbiotics, (Mirnejad et al., 2013) [23] . Therefore, temperature and pH are crucial factors for the production of postbiotics.

Production of postbiotics

The production of postbiotics involves a series of steps. The process begins with the careful selection of specific strains of microorganisms, often those that are known to produce beneficial metabolites during their life cycle or after cell lysis Aguilar-Toalá et al., 2018, Barros et al., 2020) [2,5] . These strains can be probiotics like Lactobacillus, Bifidobacterium, or other beneficial microbes. The selected microorganisms are cultivated under controlled fermentation conditions. This step is critical as the growth environment, including factors such as temperature, pH, and nutrient availability, influences the types and quantities of metabolites produced. After fermentation, the microbial biomass and the culture medium are harvested. The culture medium contains the metabolites and other soluble compounds that are released during the growth or after the lysis of the microbial cells (Barros et al., 2020) [5] . Postbiotic biomass can be initially separated into cell filtrate and inanimate microorganisms by simple filtration techniques (Vinderola et al., 2022, Barros et al., 2020) [38,5] . The most common method for extracting postbiotics involves separating bioactive extracellular metabolites from the cells that produce them through techniques such as centrifugation, sonication, and ultrafiltration. Effective approaches to inactivate live organisms and produce postbiotics include ohmic heating, supercritical CO2 treatment, pH adjustments, pulsed electric fields, and drying (Cuevas-González, 2020; de Almada, C.N. 2016) [9,11] . Emerging technologies like electric fields, ultrasonication, high pressure, ionizing radiation, pulsed light, magnetic field heating, and plasma technology offer potential for inactivating microorganisms by preserving overall quality and nutritional value. Metabolomics can also be utilized to quantify postbiotics (Vinderola et al., 2022) [38] . Figure 1 Depicts the pathways for the preparation of postbiotic cultures comprising of whole cells or their fragments, with and without metabolites or fermentation products. [10,38]

Classification of postbiotics

Postbiotics is an umbrella term that includes a wide range of components such as inanimate microorganisms, cell wall fragments, cell-free supernatants, and metabolites. It includes fermentation products like muropeptides derived from peptidoglycans, exopolysaccharides (EPSs), teichoic acids, surface molecules that protrude out (e.g., fimbriae, pili, or flagella), secreted proteins/peptides, bacteriocins (such as acidophilin, reuterin, and bifidin), organic acids, bioactive peptides, biosurfactants and neurotransmitters. Postbiotics can be classified based on their composition and origin. The main categories include: [43] .

3. Cell-free Supernatants: These are liquids obtained after removing live bacteria from fermented products, containing various bioactive compounds with low or high molecular weights, such as organic acids, diacetylene, carbon dioxide, and bacteriocin-like substances that will help to maintain homeostasis in the body (Kapustian, V et al.; 2018, Siedler, S. et al; 2019) [18, 31] . After incubating the liquid culture, the microbes are centrifuged and removed. The resulting mixture thus obtained in filtered to ensure sterility. Supernatants of different organisms exhibit different activities. Some exhibit anti-oxidant and anti-inflammatory effect on intestinal epithelial cells, macrophages and neutrophiles by reducing the secretion of the pro inflammatory tumour narcosis factor. Many of the cell free supernatants can reduce oxidative stress in vivo and provide direct anti-tumour activity (Amaretti et al., 2013) [3] .

Enzymes and Proteins: Enzymes such as lactase or

antimicrobial proteins produced by microorganisms can contribute to health benefits. Enzymes are categorized into six main groups: transferases, isomerases, hydrolases, oxidoreductases, lyases, and ligases.

Microbial Lysates: Inactivated or dead microbial cells

that release their internal components, which can positively affect the host's health. Characteristics such as oxygen scavenging ability, and modifying host physiology, are retained by nonviable cells. Studies proved that both live and heat-killed cells of Lactobacillus reuteri adhered to caco 2 cell cultures and prevented enteropathogens from adhering to them (Singh, T.P et al., 2017) [32] .

Each type of postbiotic offers distinct health benefits, such as modulating the immune system, improving gut health, or exhibiting antimicrobial properties. Table 1. Shows a classification of postbiotics. [42,26]

Category Description Example

Metabolites Small molecules produced by microbes during fermentation or metabolism.

Short-chain fatty acids (SCFAs), vitamins, amino acids, peptides, exopolysaccharides.

Cell Wall Components

Components of microbial cell walls with immunemodulating and antimicrobial properties

Peptidoglycan derived muropeptides, teichoic acids (TA), lipopolysaccharides (LPS), Surface layer protein, dimethyl acetyl derived plasmalogen, Polysaccharides, cell surface protiens Cell-free Supernatants Liquids containing bioactive compounds after removing live bacteria from fermented products. Organic acids, bacteriocins

Enzymes and Proteins

Microbial enzymes and proteins that contribute to health benefits. Lactase, antimicrobial proteins, exopolysaccharides

Effect of postbiotics on human health Postbiotics as anti-inflammatory agents

Inflammation is considered as the first response of an organism towards infection or inflammatory disorders such as inflammatory bowel disease, atherosclerosis, chronic inflammation etc. Postbiotics influence inflammatory pathways by modulating the transcription of proinflammatory genes and the expression of related proteins. Their effect on the inflammatory response, however, varies based on factors like their source, bacterial species and strains, the use of cell-free supernatant (CFS), and heatkilled bacteria (HKB). Evidence shows that postbiotics can produce both anti-inflammatory and immunostimulatory [12,37] . Bifidobacterium longum, when used as a whole-cell postbiotic, has demonstrated several barrier-protection properties, including antioxidation, anti-inflammation, and the inhibition of bacterial colonization (Ma et al., 2023) [21] . Similarly, certain whole-cell postbiotic products derived from Lactobacillus species have shown anti-inflammatory effects, such as the downregulation of IL-6 and TNF-α and the upregulation of IL-10, along with antioxidative properties like free radical scavenging, in both in vitro and in vivo animal models (Behzadi et al., 2022) [6] . Products from certain Gram-positive bacteria can activate signaling pathways, such as TLR2, leading to anti-inflammatory states and enhancing transepithelial resistance against bacterial invasion. Shortchain fatty acids (SCFAs) are particularly effective in reducing inflammation and inhibiting malignant cell growth, making them therapeutically valuable in treating inflammatory bowel disease (IBD) and colorectal cancer. Additionally, postbiotic metabolites, including amino acids, bacteriocins, SCFAs, peptide glycan-derived muropeptides, and vitamins, contribute to promoting and enhancing antiinflammatory functions in the human gut (Ma et al., 2023) [21] .

Postbiotics as immunomodulators

Postbiotics are widely recognized for their ability to modulate the immune system, including both innate and adaptive responses, while also maintaining intestinal mucosal barrier integrity and countering potential pathogens (Yeşilyurt N et al., 2021; De Marco, S et al., 2018) [41,12] .

Their anti-inflammatory properties help regulate acute inflammatory responses and prevent gut disruption by activating pathways related to specific immune responses. Several bioactive components in postbiotics contribute to these effects. For example, bacterial peptidoglycan (PGN) and lipopolysaccharide (LPS) can stimulate the immune system in a receptor-dependent manner. Similarly, the surface layer (S-layer) of lactobacilli, formed by the selfassembly of glycoprotein subunits, is known to enhance host immune responses. Other microbial metabolites, such as histamine and branched-chain fatty acids, also play a role in stimulating the immune system.Postbiotics offer additional protective benefits against pathogens. Pure teichoic acids isolated from Lactobacillus strains can inhibit biofilm formation by oral and enteric pathogens, including Streptococcus mutans, Staphylococcus aureus, and Enterococcus faecalis. Biosurfactants from Lactobacillus gasseri have demonstrated antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, organic acids and intracellular components like pyrrole derivatives have been found to exhibit oxidative inhibitory activity. In vitro studies have highlighted the antioxidant properties of intracellular contents and cellular fractions from strains such as Lactobacillus casei and Bacillus coagulans (Ma et al., 2023) [21] . Studies also show that postbiotics from Lactobacillus species support immune function by protecting the intestinal barrier and promoting pathogen aggregation through antagonistic activity (Teame et al., 2020) [35] . Interestingly, the immunomodulatory effects of postbiotics produced by inactivating probiotics are often stronger than those of live probiotics. For instance, heat shock proteins (Hsp) generated during the heating process have been found to enhance immunomodulatory activity (De Almada, C.N. et al., 2016) [11] .

Postbiotics against obesity

Consumption of biotics is said to have favourable effect on anthropometric indeces of obesity in the normal population. Major cell wall components that play a crucial role as antiobese factors include muramyl dipeptidase, lipoteichoic acids, exopolysaccharides and S layer proteins. Studies proved that supplementing with muramyl dipeptide, an excellent postbiotic, will assist in lowering adipose inflammation and glucose intolerance in obese mice (Cavallari, et al., 2017) [8] . Another important cell wall component of microorganism is surface layer protein (SLP). SLP generally attach to the host intestinal wall (Nataraj et al., 2020, Fagan, et al., 2014) [25,13] . SLPs act against obesity and metabolic disturbances by improving systemic inflammation, insulin resistance and adipogenesis (Kim et al., 2021) [20] . A recent report demonstrated the fat-reducing effects of lipoteichoic acid (LTA) from Bifidobacterium animalis subsp. lactis BPL1 using the nematode Caenorhabditis elegans as a pre-clinical model. Lipoteichoic acid is a novel lipid modulator which has the ability to reduce fat through insulin-like signalling pathway. Study also suggests that lipotheicoic acid may have potent preventive and therapeutic application in metabolic syndrome and diabetes related diseases. (Balaguer, et al., 2022) [4] . Short chain fatty acids like acetate, propionate, butyrate etc are said to have reduction in cholesterol level, inhibiting weight gain and promoting beige adipogenesis (Lu, et al., 2016, Weitkunat et al., 2017) [19,40] .

Postbiotics as cosmetic

Postbiotics are applied either by incorporating them into topical formulations, such as creams, or by encapsulating them for oral administration (Catic, T et al., 2022; Inoue, Y et al., 2014) [7,16] . Research has shown that cold creams containing postbiotic components from Lactobacillus fermentum ATCC9338, Lactobacillus reuteri ATCC 23272, and Bacillus subtilis can accelerate wound healing (da Silva Vale et al., 2023) [10] . Additionally, postbiotics have been found to enhance UV protection and delay the aging process of skin cells. Oral intake of heat-killed cells from the Lactococcus lactis strain H61 has been shown to improve skin properties, such as reducing dehydration caused by seasonal changes and enhancing skin elasticity on the inner forearms.

Conclusion

Postbiotics represent a promising frontier in microbiome research and therapeutic interventions. They support gut health and immune function to potentially mitigate various diseases. They also play important roles in weight management, skin care and mental health. Through further research, innovation, and collaboration across scientific disciplines, we can unlock the full potential of postbiotics to revolutionize healthcare and enhance the quality of life for individuals worldwide.

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