Acid bacteria lactic acid bacteria

LAB have been used in food preservation for many years because they produce different compounds as a result of their metabolism, such as organic acids, hydrogen peroxide and bacteriocins. Likewise, their role as probiotics has also been studied (^{Agudelo et al., 2015}; ^{Gorbeña and Sáenz, 2008}).

LAB are a diverse group of Gram-positive microorganisms (cocci or bacilli), with a length (depending on the genus) of 0.5 - 0.8 µm (^{Carr et al., 2002}; ^{Ibrahim and Raman, 2021}), that are catalase negative (although in some cases some pseudo catalase can be found), and oxidase and benzidine negative (^{Vázquez et al., 2009}). They are facultative anaerobes, do not form spores, lack cytochromes, are not toxigenic or pathogenic, and are tolerant to acidity, although some can grow at a pH of 9.6. They also grow at different temperatures and can be classified as mesophilic (optimum growth temperature of 20 to 25 °C) and thermophilic (temperature of 40 to 45 °C) (^{Parra Huertas, 2010}). Some have the ability to be aero-tolerant and grow in the presence of oxygen (^{McKee and McKee, 2016}). LAB synthesize ATP through carbohydrate fermentation, and are divided into two large homofermentative and heterofermentative groups based on their biochemical characteristics. Among the metabolites produced by LAB are lactic acid, as well as other acids in smaller proportions such as propionic, acetic, citric, succinic, and formic acids, in addition to hydrogen peroxide, diacetyl, acetaldehyde, reuterin, and bacteriocins (^{Heredia et al., 2017}). Some LAB produces carboxylic acids (^{Kaiting, 2021}).

Criteria for considering a probiotic strain

Among the main criteria for considering a LAB strain to be probiotic are growth at a pH below 4.0, exert control over pathogenic bacteria, survive in the gastrointestinal tract, tolerate bile salts, an ability to adhere to intestinal mucus and epithelial cells, and display co-aggregation and self-aggregation (^{Ramírez-Chavarín et al., 2013}), in addition to not having antibiotic resistance genes, immunostimulation without proinflammatory effect, displaying resistance to phages, and having antimutagenic and anticarcinogenic properties (^{Rondon et al., 2015}).

Beneficial effect of probiotics

Probiotics are responsible for mediating a wide variety of health problems, as they are involved in regulating intestinal homeostasis, suppressing pro-carcinogenic enzymatic activities, interfering with the ability of pathogenic bacteria to infect the intestinal mucosa, improving nutrient bioavailability, reducing symptoms of lactose intolerance, positively influencing the urogenital flora, reducing infections in the gastrointestinal tract, and improving the regulation of intestinal motility (^{Cunningham et al., 2021}; ^{Veiga et al., 2020}). Probiotics establish competition for both nutrients and for adherence sites of digestive tract cells with other (pathogenic) bacteria and stimulate the local and systemic immune system by activating macrophages and elevating immunoglobulin concentrations (^{Prats Capote, 2007}).

Mechanisms of action involved in probiotics

The mechanisms of action that give probiotics the ability to provide these benefits to humans are still largely unknown, but may involve modification of intestinal pH (^{Rondon et al., 2015}), antagonism of pathogens through the production of antimicrobial compounds, competition for binding sites and receptors, competition for available nutrients and growth factors by stimulating immunomodulatory cells, increased erythrocyte regeneration, and production of short-chain fatty acids and regulation of gastrointestinal disorder (^{Cunningham et al., 2021}; Figure 1).

Adapted from ^{Plaza-Diaz et al., 2019}

Figure 1 Mechanism of action of traditional probiotics. 

Patents on traditional probiotics

There are several patents on the use of traditional probiotics (Table 1).

Characteristics of new probiotics

For a probiotic to be considered new, it must meet certain characteristics, which are listed in Table 2.

Classification of new probiotics

Among the new probiotics we can mention immunobiotics, psychobiotics, oncobiotics, pharmabiotics, paraprobiotics, postbiotics, probioceuticals and pseudoprobiotics (^{Martin and Langella, 2019}). The characteristics of each of these are described below.

Immunobiotics

Defined as probiotic microorganisms with favorable immunological action for the organism. Immunobiotics increase the humoral immune response and promote better functioning of the intestinal barrier, stimulate resistance to pathogenic organisms and decrease the hypersensitivity reaction (^{Aviña et al., 2006}). The activity on non-specific immune modulation allows immunobiotics to increase the host immune response and facilitate the elimination of pathogenic germs in the intestine by releasing proinflammatory cytokines such as Tumor Necrosis Factor-alpha (TNF-α). It has been reported that immunobiotics can stimulate macrophages and increase phagocytosis by early activation of the inflammatory response prior to antibody production (^{Isolauri et al., 2001}; ^{Molina et al., 2016}).

Psychobiotics

Defined as a special class of probiotics that when ingested exert mental health benefits through an interaction with the gut microbiota, stimulating the production of short-chain fatty acids, enteroendocrine hormones, anti-inflammatory cytokines and neurotransmitters such as gamma amino butyric acid, serotonin, acetylcholine, catecholamines and norepinephrine, which helps regulate mood, cognitive, memory and learning functions (^{Sharma and Shukla, 2016}; ^{Zendeboodi et al., 2020}). An ability to modulate the expression of neurochemical receptors such as endocannabinoids are also attributed to psychobiotics. Table 3 lists the most commonly used psychobiotics in neurological conditions.

Oncobiotics

Oncobiotics are probiotics that enhance the response to cancer immunotherapy, helping the immune system to eliminate tumor cells through cytotoxicity stimulated by biological agents aimed at preventing apoptosis or lymphocyte inhibition (^{Zitvogel et al., 2016}). There are studies that suggest that oncobiotic probiotics play an important role in the immune response after chemotherapy and radiotherapy treatment, thus helping the host to avoid future relapses (^{Gopalakrishnan et al., 2018}).

Pharmabiotics

Pharmabiotics are probiotics that can function as pharmacological active ingredients with preventive and curative potential. Pharmabiotics are used for the adjunctive treatment of various diseases such as acute diarrhea and irritable bowel syndrome. Studies have determined that specific pharmabiotics can improve many lower gastrointestinal tract symptoms in adults (^{Belkaid and Hand, 2014}; ^{Navarro et al., 2021}).

Paraprobiotics or phantom probiotics

^{Taverniti and Guglielmetti (2011)} described paraprobiotics as inactive, dead, and non-viable cells, or cell fractions, from intact or broken probiotics that provide health benefits to the recipient. Other authors have shown that cells from probiotics, even when dead or inactivated, continue to have a beneficial effect on the host, such as decreasing the inflammatory response, as well as preventing the adhesion of pathogenic bacteria (^{Zendeboodi et al., 2020}). They are also attributed with attenuation of colitis, stimulation of the intestinal immune system, anti-adhesion capacity against several pathogens in CaCo-2 experimental models (^{Zhang et al., 2005}; ^{Nataraj et al., 2020}). Some paraprobiotics produce compounds such as teichoic acid, peptidoglycan-derived muropeptides, exopolysaccharides and biosurfactants bound to their cell wall (^{Nataraj et al., 2020}).

Postbiotics

Defined as a complex mixture of molecules that are part of the products or by-products derived from probiotic metabolism and must be cell-free (^{Aguilar-Toalá et al., 2018}; ^{Salminen et al., 2021}). Postbiotic molecules have a known chemical formula, prolonged storage stability, as well as an ability to trigger various treatment mechanisms for diseases such as inflammation, adhesion of pathogens to the gastrointestinal tract, obesity, coronary artery disease, cancer, and oxidative stress (^{Aguilar-Toalá et al., 2018}). Some examples of postbiotic molecules are enzymes, peptides, proteins, exopolysaccharides, organic acids, serine protease inhibitors (serpin), short-chain fatty acids, and cell wall components such as teichoic acid, peptidoglycan, hydrogen peroxide, reuterin, diacetyl, bacteriocins, phenol, amino acids, benzoic acid, phenylactic acid, and vitamins (^{Jastrząb et al., 2021}). There is scientific evidence attributing different antimicrobial, antioxidant and immunomodulatory functions to postbiotics, properties that could positively affect the homeostasis of the gut microbiota, as well as signaling pathways and metabolic pathways of the host, thus having effect on specific physiological, immunological, neuronal, hormonal, biological, regulatory and metabolic reactions (^{Sharma and Shukla, 2016}; ^{Salminen et al., 2021}).

Probioceuticals

Probioceuticals are biologically active substances derived from the metabolism of probiotics that are attributed with health benefits, as well as the treatment and prevention of diseases. An example of probioceutical compounds are exopolysaccharides which are long-chain polymers of sugars with antioxidant and immunomodulatory properties (^{Kumar et al., 2020}; ^{Adebayo et al., 2018}).

Pseudoprobiotics

Pseudoprobiotics are viable, but not active, probiotic cells that were inactivated by environmental factors such as high or low temperatures, extreme pH, lack of nutrients, low water activity, so they have low or no growth rates. Based on these characteristics they cannot be classified as traditional or true probiotics since they need to be viable and active cells. The advantages that these pseudoprobiotics have is that they can function in an inactive manner as paraprobiotics or be activated and used as traditional probiotics, with the advantages that both classifications give them (^{Zendeboodi et al., 2020}; ^{Blinkova et al., 2014}).

Candidate microorganisms for new probiotics

Among the species that have been reported as new probiotics are Akkermansia miciniphila (^{Castañeda-Guillot, 2019}), Ruminococcus bromii (^{Kumari et al., 2021}), Roseburia intestinalis (Kasahara et al., 2018), Anaerobutyricum hallii (^{Kumari et al., 2021}), Faecalibacterium prausnitzii (^{Castañeda-Guillot, 2019}; ^{Kumari et al., 2021}), Oscillospira (^{Yang et al., 2021}) and Christensenella minuta (^{Castañeda-Guillot, 2019}). The main characteristic of these microorganisms is that they are isolated from the human gastrointestinal tract. Therefore, they must be resistant to a low pH in the intestinal tract, have no resistance to antibiotics and exercise pathogen control, although they may be sensitive to oxygen.

Patents for new probiotics

Table 4 shows some of the patents for new probiotics.

Studies on probiotics

Studies using traditional probiotics

Several authors have studied the role of probiotic LAB. ^{Ramírez-Chavarín et al. (2013)} evaluated the probiotic potential of ten thermotolerant LAB strains, which were studied for tolerance to low pH and bile salts, co-aggregation and self-aggregation, adhesion to epithelial cells, concluding that the thermotolerant LAB studied are promising for their probiotic potential. ^{Sanchez and Tromps (2014)} characterized 72 LAB strains by growth at different pH, temperature and tolerance to high sodium chloride concentrations, selecting 14 strains as probiotic candidates which were tested for hydrophobicity, self-aggregation and microbial antagonism. The results showed that strains such as Lactobacillus spp., Leuconostoc spp., Lactococcus spp., Streptococcus spp. and Pediococcus spp. have microbial antagonism to Bacillus cereus and Staphylococcus aureus at an inhibition rate of 63.6%, to Salmonella typhimurium at 75.7% and to Pseudomonas aeruginosa at 72.7%. ^{Jurado and Fajardo (2017)} evaluated the susceptibility of two strains of lactic acid bacteria to different antibiotics, the inhibition effect of Lactobacillus gasseri and its supernatant on Staphylococcus epidermidis, the growth of the lactic acid strain at different pH and different temperatures, resistance to bile salts and bovine bile, established fermentation kinetics and determined the count of viable microorganisms for each condition. They concluded that both strains have resistance to the antibiotics gentamicin and dicloxacillin, with L. gasseri showing growth inhibition of S. epidermidis, reaching the maximum growth peak at 12 h, at pH 4.2, and a lactic acid percentage of 1.26. ^{Zachary et al. (2020)} evaluated the mechanisms of adhesion to intestinal mucus of different probiotic strains, for which they immobilized the mucus on the surface of the plate and tested the use of different cell concentrations, finding that surface proteins and cellular components influence mucoadhesion and were heterologously expressed or altered in Lactococcus lactis and Escherichia coli, concluding that adhesion to mucus depends on the type of strain, as well as on the concentration of cells.

Studies with new probiotics

In recent years, studies on new probiotics have been developed. ^{Molina et al. (2016)} evaluated the use of the immunobiotic Lactobacillus rhamnosus and its influence on the immunological alterations that occur naturally in aging mice, demonstrating that this strain is capable of improving the phagocytic activity of macrophages. Therefore, they concluded that L. rhamnosus may have application as an immunomodulator given its positive influence on aging and help in reinforcing intestinal and systemic immunity.

^{O'Toole et al. (2017)} sought a sustainable route to the delivery of novel probiotics from a pharmaceutical perspective, where this relatively new concept overlaps with live biotherapeutic products. Through the development of improved cultivation methodologies, accessibility to genome and metagenome sequencing will allow these probiotics to be targeted.

^{Nishida et al. (2017)} analyzed the relationship that the parapsychobiotic Lactobacillus gasseri has with stress and sleep quality. A group of 21 men and eleven women were orally administered the parapsychobiotic for 5 weeks, finding that sleep quality can improve with the administration of L. gasseri, particularly in men, shortening sleep latency and increasing the sleep duration. A decrease in the growth of strains such as Bacteroides vulgatus was observed, responsible for intestinal inflammation, suggesting that the L. gasseri strain is a good ally against stress and its side effects, as well as improving sleep.

^{Salva and Alvarez (2017)} studied the role of microbiota and immunobiotics in granulopoiesis (the process of renewal of granulocytic cells circulating in the blood that are part of the body's defense system), which occurs in the bone marrow of immunocompromised hosts. The authors determined that dietary supplementation with immunobiotics is an interesting alternative to improve granulopoiesis in stationary and emergency states, improving the respiratory innate immune response and resistance against respiratory pathogens in immunocompromised hosts.

Chang et al. (2019) conducted a review of existing information on novel probiotics and how they influence most diseases such as chronic intestinal inflammation, colitis, obesity, metabolic syndromes, diabetes mellitus, liver disease, cardiovascular diseases, cancer, neurodegenerative diseases, using new strains such as Prevotella copri, Christensenella minuta, Parabacteroides goldsteinii, Akkermansia muciniphila, Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii and Bacteroides fragilis.

^{Kumar et al. (2020)} studied the probioceutical produced by Pediococcus acidilactis, in its exopolysaccharide form and its influence on human health. They analyzed the Pediococcus acidilactis genome and identified ten genes responsible for the production of exopolysaccharides, which are linear homopolysaccharides (α-glucans) with few α-(1→3) branches, which have antioxidant activity, reducing power, and anticancer activity, suggesting this probioceutical can be used as an antioxidant and anticancer agent.

^{Akter et al. (2020)} suggested the use of paraprobiotics in the treatment for people with weak immune systems as live probiotics could do more harm than good regarding host health, highlighting their long shelf life, modulation of immune responses, biological response modification, anti-inflammatory and antiproliferative properties.

Different methods of extraction of paraprobiotics and postbiotics produced by bacilli have been studied, such as heat treatment, enzymatic treatments, solvent extraction, radiation, sonication, supercritical CO₂, chromatography, centrifugation, dialysis and lyophilization (^{Teame et al., 2020}).

^{Moradi et al. (2020)} analyzed the applications of postbiotics in food preservation, packaging and biofilm control, as well as their use as a biodegradant against chemical compounds in food (e.g., biogenic amines), concluding that further studies on food biosafety are needed to develop international standards and regulations on the use and applications of postbiotics.

^{Amirí et al. (2021)} proposed the use of postbiotics to produce linoleic acid, exopolysaccharides, and bacteriocins using Bifidobacterium lactis, showing how the effect of yeast extract had a beneficial effect on the production yield of postbiotic metabolites.

Recent studies have determined that complementary probiotic therapy can reduce the duration and severity of intestinal diseases, since using the pharmabiotic Lactobacillus acidophilus LB can reduce intestinal pH and inhibit the growth of pathogenic organisms (^{Navarro et al., 2021}).

^{Michels et al. (2022)} investigated the molecular patterns of the immune response using Bifidobacterium lactis, Lactobacillus casei, L. gasseri, L. paracasei and Streptococcus thermophilus. Different doses of these strains on macrophage cell lines stimulated with polysaccharides was examined; the immune response was analyzed after application of these strains as paraprobiotics, concluding that the paraprobiotics had an effect on the production of interleukin in addition to inhibiting free radicals, increasing cell viability, making it likely that these paraprobiotics contribute to improve intestinal homeostasis, immunomodulation and host metabolism. The identification of microbial strains of intestinal origin for the development of new probiotics requires extensive knowledge about the cultivation of these microorganisms, because unlike traditional probiotics these strains are very sensitive to oxygen (^{De Filippis et al., 2022}).