Bacillus clausii: A Review into Story of Its Probiotic Success and Potential Food Applications

Abstract

Probiotics are live microbiota that can confer their hereditary health benefits upon the host. They can positively alter the diversity of the host’s gut microbiota population. Bacillus clausii is a spore-producing potential probiotic. Its application in the food industry has been highly recommended. Clausii spores are greatly resistant to harsh food processing treatment (heat and industrial pressure) and they can maintain their physiological traits (acid, bile salt) inside the human gut. The utilization of spores can enhance the nutritional viability of livestock, as well as the functionality of on-shelf products. This contemporary review covers the great attributes of B. clausii, including physiological characteristics, modes of action, probiotics benefits, a safety assessment, and the bacteria food industry applications for the purposes of producing enhanced functional foods known as probiotic foods. To our knowledge, although B. clausii has been widely applied in food industry, the amount of the literature that is dedicated to its role in sustainable food production and addresses its potential sporogenous probiotic behavior is fewer as compared to non-sporogenous lactic acid bacteria (Lactobacillus spp. and Bifidobacterium spp.). In our review, we also discovered a lack of empirical evidence on the synbiotic and synergistic behavior of clausii in combination with other active nutrients. Therefore, our review suggests that more studies should be conducted on the potential probiotic contribution of B. clausii in sustainable food production and its synergistic and synbiotic behavior in conjunction with other active nutrients.

1. Introduction

Even though for decades, some species of Bacillus probiotics have been used for the optimization of human diets or animal feeds, there have not been as many empirical studies on these species as compared to the lactic acid probiotics (LA). Within B. species, B. subtilis (Bacillus natto), B. cereus (var toyoi), B. coagulants, B. licheniformis, and B. polyfermentans have been extensively examined, nonetheless, there has not been enough focus on their potential probiotic behavior [1,2].

A quick look into the existing literature suggests that lactic acid bacteria (Lactobacillus, Enterococcus and, Streptococcus), alongside fungi species (Aspergillus and Saccharomyces) with probiotic qualities, have been largely covered, yet the potential behavior of genera Bacillus has been widely ignored [3,4]. Indeed, few studies in the literature, like Patron et al. [4], Hoa et al. [5], and Sanders et al. [6], have discussed the safe incorporation of genera Bacillus into animal feeds or its harmless applications in the manufacturing of foods and medicines for human therapeutic purposes.

Most existing products in the market purport that their Bacillus probiotic content is as efficient as LAB probiotics due to the fact that they have overlapping properties including hypo-allergenicity (arthritis treatment), and hyper-durability against salt and acidic environment of the gut. Nevertheless, the number of empirical studies that has elaborated on how or why the potential probiotic behavior of Bacillus could be just as efficient as—or even superior—to the conventional LAB, is not significantly substantial [2,3,6]. There is also the issue of public lack of trust due to the contradiction between insufficient scientific evidence versus the appearance of overwhelming commercials that try to introduce different marketed functional foods containing the genera of Bacillus. Such marketing without enough support from empirical studies has raised concern over the probiotic versus pathogenic behavior of the bacilli, resulting in consumer disorientation [2,6]. Our review posits that the strain of B. clausii is among such concerns, and further studies must be conducted to cover the gap, involving the strain’s potential probiotic benefits and how positively they can benefit food manufacturing processing.

The literature has also referred to Bacillus’ capabilities such as adequate spore production, as well as sustainable spore shell life as great attributes which have qualified the bacteria as a potential economic-based probiotic. Furthermore, other bacillus spores’ qualities, like being highly resistant to harsh environmental treatment, including the harsh acidic climate of the human gut, have made the sporegenous probiotic bacteria enjoy successful germination and propagation inside the gut [3,6].

The characteristics of spores’ enhanced tolerance and high survivability rate have made the species sturdier during rough treatments in food processing as well as during rough transportation and harsh storage conditions. Such characteristics have made the Bacillus’ spores a better candidate for dry-frozen probiotic formulas as compared to other health-promoting microorganisms [3,6]. The bio-therapeutic potential of Bacillus spp. is reflected in its ability to have easy interactions with the internal environment of the host, resulting in the production of a multitude of peptides with assorted antibacterial properties, alongside various enzymatic behaviors. The produced peptides can also manufacture much-needed vitamins, carotenoids, and some small extracellular immune enhancers. Such an ability attests to the species’ possible probiotic characteristics, as well as their great contribution to the sustainability of the food industry [3,6].

Probiotic bacteria that naturally exist inside the human gut predominantly belong to the genera of LAB. Yet, recent discoveries have focused on the isolated species of B. clausii, and B. licheniformis from healthy human stool, and argued that Bacillus spp. that survive in the gut harsh environment and temporarily colonize the gut lumen can also demonstrate probiotic features [7,8]. Such probiotic features have made B. clausii spp. a perfect candidate to be concurrently prescribed with antibiotics in order to alleviate the antibiotic’s adverse effects on the gut system. Such a combination reinforces the argument on the innate antibiotic resistance of clausii and its vibrant probiotic behavior [9,10].

Our literature exploration posits that although the potential probiotic behavior of B. clausii has been widely marketed, probiotics of LAB have been purchased more often due to being less stereotypically pathogenic, better supported by empirical evidence, and more scientifically circulated inside the databases. Therefore, our review aimed to assess the scope of the existing information on B. clausii physiological characteristics, their generic mode of action, their potential probiotics benefits, their safety assessment, and the extent of their food industry applications for the purposes of producing enhanced functional foods.

In this review, we also noticed that there is a lack of substantial literature, for instance, on how the potential probiotic behavior of B. clausii strains can lead to sustainable food or feed production. We noticed there is not enough information on which food manufacturing stages is the best stage to add the spores in order to maintain their great probiotic potential_and their high productivity. These last two are underlying, as they can boost functional food shelf life as well as food composition quality. There is also a lack of information on the production of functional foods with synbiotic or synergistic properties under different methodologies. Furthermore, we discerned that the probiotic safety consumption of the B. clausii strains in contrast to the other Bacillus genera or LAB has not been well investigated. The above-mentioned gaps in our review will provide guidelines for the development of new studies in the near future.

1.1. B. clausii Physiological Characteristics

The natural habitats of B. species are inside the soil, marine environments, dust, air, and the human gut [11,12]. These species are saprophytic Gram-positive bacteria [13] and, besides probiotic health benefits, there are also some strains that are involved in food spoilage, including strains of B. cereus [14]. Compared to other non-spore bacteria (Lactobacillus spp.), spore-producing bacilli display some physiological advantages. These encompass resistance to heat, acidity, and being room-temperature-friendly. Spore-producing bacilli are also gene-driven and demonstrates high viability inside harsh ecosystems [11,12,14]. Such properties have shielded their manufactured products against loss of viability. Among B. strains, fermented strains of B. subtilis, B. licheniformis, B. coagulans, B. toyoi (cereus), B. clausii, and B. poly have been widely endorsed for pharmaceutical and food processing purposes [15,16].

Since 1999, four probiotic strains of B. clausii known as O/C (CNCM I-276), N/R (CNCM I-274), SIN (CNCM I-275), and T (CNCM I-273) have been frequently marketed under the trade-name of Enterogermina as over-the-counter supplements [17,18]. These four strains were primarily extracted from a single penicillin-resistant group, referred to as B. subtilis ATCC 9799 [15,19], and were later reintroduced as B. clausii [9,20].

B. clausii is an aerobic, Gram-positive, rod-shaped, alkaline-tolerant, non-pathogenic bacterium that emanates endospores [21]. Its strains (O/C, N/R, SIN, T) grow in a diverse ecosystem including the human gut [22]. Through an elaborate developmental process, the strains yield resilient spores with the ability to withstand hostile environmental conditions. Such conditions are commonly referred to as starvation, droughts, alkalinity, UV radiation, excessive temperature, and exposure to extreme physical/chemical agents [23]. However, by returning to a customized environmental condition, the spores germinate and turn into vibrant vegetative cells, ready to thrive and propagate [23].

B. spores are metabolically inert, and in comparison to their vegetative cells, they can better endure the acidity of bile salt or gastric HCL [3,15,23]. They also appear to be more resilient to any thermal treatment (during pharmaceutical processing or manufacturing of functional foods), in particular, if they are reinforced by a protectant such as “trehalose sugar” [24].

Among the other physiological benefits of clausii, studies have pointed at the bacteria’s ability to heal the gut’s defective membranes or to restore the gut’s vitamin deficiencies caused by antibiotics or chemotherapeutic agents. Nevertheless, the most remarkable benefit of the bacteria is its probiotic behavior of antibiotic resistance, a unique characteristic that neither can be genetically inherited_nor adopted or transferred to the other pathogens that reside in the same environment

Table 1. Physiological properties of B. clausii. Source: modified version from [20].

Physiological Attributes of B. clausii Spore	Antimicrobial and Immunomodulatory Activity
Acid, heat, bile salt tolerant.	Mucin secretion promoter-gut permeability reducer.
Gut barrier function enhancer.	Lantibiotics producer.
Vitamin synthesizer.	Pro and anti-inflammatory cytokines regulator.
Antibiotic resistant.	Serine protease activity on enterotoxins.
Other biochemical and metabolic properties.	Increasing the level of T cells + CD4, enteropathogens suppressor.

[8,20].

B. clausii has a significant role in the preclusion or treatment of an acute intestinal infection that has specifically been triggered by the consumption of some antibiotic-induced diarrhea [18,25]. Meanwhile, the best clinically celebrated characteristic of the bacteria is the strong physiological ability of its spore that can easily germinate in a complex gut-simulated environment inside the lab and demonstrates a perfect survival mood in that artificial environment [26,27]. The species also has other characteristics, such as being the best regulator in respect to a systemic immunoglobulin reaction [28] and displaying antibiotic properties against Gram-positive pathogens [29].

1.2. B. clausii Modes of Action

1.2.1. Antibiotic Resistance

In recent decades, the rate of infectious disease has declined and is almost overshadowed by the new arrivals of modern diseases such as allergic reactions and/or auto-immune disorders. [30]. Such change has mostly been influenced by meddling effects of factors like lifestyle, hygiene, edible antibiotics, exercise, and fitness. These factors have altered the microbiome profiling of the gut, contributing to the rise in modern diseases [31]. Changes in the biology of microbes have paved the path for the manufacturing of functional foods (probiotics, prebiotics, and synbiotics), with the aim of incorporating them into human diet/animal feeds in order to turn the gut microbiota population back into their previously favorable state of being eubiotic [32].

For example, the application of probiotics in plant-based products (fermented cereals, fermented vegetables, fruits, and soybeans) has been prevalent in Asian countries as compared to dairy sources. Such commonality has been aligned with geographical, economic, and cultural reasons. Asian people stereotypically believe that dairies contain high cholesterol and lactose levels and that can be a serious concern to human health [33,34]. It is also well documented that some intestinal microbiota, commonly referred to as gut indigenous bacteria resemble to the most dairy probiotics of Lactobacillus and Bifidobacterium, which feature favorable effects over metabolism, immunity, and functionality of endocrine glands [11,35].

In 2014, the International Scientific Association (ISA) for Probioticsand Prebiotics defined probiotics as “live microbiota” with the ability to diversify the indigenous population of host intestinal bacteria to enhance the host immunity [36,37]. Probiotics are usually prescribed during or after antibiotic therapy due to the fact that such therapies will inevitably have detrimental effects on the healthy population of gut microbiota. This could be the reason why probiotic supplements of Bacillus species like B. clausii are very much advised to be taken alongside antibiotics due to their antibiotic-resistant quality [17,38]. Furthermore, Hoyles [7] and Lopetuso [8] suggested that probiotic strains of B. clausii and B. licheniformis are naturally extracted from the human gut, and are more bound to have adjusted characteristics with the human gut, hence helping them better endure the acidic climate of the gut.

As previously stated, the antibiotic resistance of B. clausii and its high-quality probiotic effects have led the species to be recommended in parallel with antibiotic therapy under any prescribed dosage [39]. Apparently, B. strains (O/C, SIN, N/R, T) can fully stand up to antibiotics of erythromycin, azithromycin, clarithromycin, spiramycin, clindamycin, macrolides, aminoglycosides (aadD2) lincomycin, and metronidazole [18]; however, in accordance with

Table 2. B. clausii strains of O/C, SIN, N/R, and T-antibiotic resistance profiling [18].

Antibiotic	B. clausii O/C	B. clausii SIN	B. clausii N/R	B. clausii T
Oxacillin	8	0	0	9 ± 1.1
Cefuroxime	10 ± 0.7	0	0	12 ± 0.8
Cefepime	8 ± 1	0	0	11 ± 0.5
Streptomycin	28 ± 0.4	0	26 ± 0.6	30 ± 0.5
Chloramphenicol	0	16 ± 0.6	13	15 ± 0.6
Rifampicin	24 ± 0.5	26 ± 0.5	0	27 ± 0.6
Metronidazole	0	0	0	0

, the strains display slight resistance to other tested agents [40,41].

Few scholars have reported on the least risk transmission ability of B. clausii and suggested that the bacteria cannot transfer its chloramphenicol resistance gene [cat Bd] that features antibiotic resistance to other pathogens [42,43].

The B. clausii strain of N/R with achromosomally encoded lactamase gene (blaBCL-1) can transfer its resistance capability to penicillin antibiotic [44]. On the contrary, the passing of gene_code transmission to pathogens such as E. faecalis JH202, E. faecium HM107, and B. subtilis UCN19 proved to be a failure that sheds light further on the discriminatory gene transferring behavior of B. clausii [45].

Addressing the antibiotic resistance behavior of B. clausii strain T, the literature [46] suggested that the strain has substantial effects in decontamination of tetracycline antibiotics (TCs) inside an aquatic environment. Furthermore, B. clausii strains T and O/C may eliminate the traces of the following antibiotics: cefuroxime, cefotaxime, and cefpirome from the medium culture, followed by the removal of tetracycline, oxytetracycline, and chlortetracycline from an aquatic environment [46,47]. Such behavior simply demonstrates strains’ contribution to sustainability [47].

Some of the literature pointed at the unique property of B. clausii antibiotic resistance and the fact that genetically cannot be mimicked by other pathogens due to its exceptional safety properties [48]. For instance, a study by Elshaghabee et al. [3] pointed at the B. clausii strain _UBBC07 carries intrinsic non-transferrable genes inside its chromosomal DNA, and features antibiotic-resistant properties against three antibiotics of clindamycin, erythromycin, and chloramphenicol.

1.2.2. Gut Barrier and Immunity Function

Research on the gut tolerance of B. clausii spores (exposure time between 0 and 360 min) showed that in contrast to other probiotics, all strains of clausii under a simulated gut climate can have acid/bile_salt resistance within the first 120 min and later 240 min of exposure, without losing their viable cells. Such maintenance in viability is imperative when it comes to having a neat reproduction [49,50]. This could be true for B106 since the strain’s inherited gene displays features of acid resistance as well as riboflavin (B2) production [51,52]. These features can both contribute to the maintenance of healthy gut microbiota and greater immunity function [52].

Sequenced genomes of the UBBC07 strain [53] followed by AKU0647 [54] have also demonstrated strong antimicrobial attributes. For example, the AKU0647 strain releases glycosyl hydrolase enzymes with glycoprotein’s degrading ability. This ability deters other bacterial pathogens from flourishing inside the gut environment [29,52]. Furthermore, genome sequences of O/C, N/R, SIN, and T strains feature antibiotic resistance, bacteriocins, and immuno-modulation [29,50,54].

Concerning probiotics’ modes of action, their intestinal effects are different, and such differences are determined by their involving strains. As

Table 3. Summary of B. clausii modes of action based on the existing literature.

Strain Type of B. clausii	Physiological Effects
O/C, N/R, SIN, and T strains [50].	Antibiotic resistance [50]. Bacteriocins [50]. Immuno-modulation [50].
B106 strain [51,52].	Riboflavin (B2) production [51,52]. Contribution to the maintenance of the healthy gut microbiota [51,52]. Strengthening the immunity function [51,52].
A combination of B. clausii strains [56].	Regulating the gene expression responsible for immunity, cell mitosis, proliferation, apoptosis, and cell signaling transductions in treatment of esophagitis patients [56].
Strain of Pseudoxanthomonas-Streptomyces-Saccharopolyspora-B. clausii [57].	Reforming the microbiome profiling of the gut [57]. Creating a stronger immunity in Pancreas Adenoma [57]. Enhancing health of gut homeostasis [57].
B. clausii SC-109 spores in combination with prebiotic fiber [58].	Creating a synergistic effect [58]. Stimulating production of short-chain fatty acids [58]. Diversifying the colon microbial environment [58].
UBBC07 spores (in a vivo study—on rat-subjected uremia) [59].	Diminishing the symptomatic consequences of acetaminophen therapy such as high levels of serum urea, creatinine, and malondialdehyde [59].
A combination of B. clausii spores [60].	Diminishing high serum urea in patients with chronic renal failure [61].

suggests, a blend of strains (O/C, N/R, SIN, and T) can safeguard the gut enterocytes against rotavirus-induced acute gastroenteritis and reduce complications with respect to trans-epithelial electrical resistance [55]. There is also an increase in the amount of some proteins responsible for the regulation of some mucosal tight junctions that can have a favorable impact on gut barrier functions. Furthermore, a mixture of clausii strains that possess counter-ROS qualities can also protect against rotavirus infection while additionally downregulaing the behavior of Toll-like pro-inflammatory receptor-3 pathways. The literature implies that all the above-mentioned traits can strongly work in favor of gut barriers [54,55].

In the treatment of esophagitis patients, using a combination of B. clausii strains can regulate the gene expression that is responsible for immunity, cell mitosis, proliferation, apoptosis, and cell signaling transductions [56]. With a focus on the health of gut homeostasis, B. clausii strain of Pseudoxanthomonas-Streptomyces-Saccharopolyspora has the potency of reforming the microbiome profiling of the gut, leading to stronger immunity in Pancreas Adenoma [57].

B. clausii SC-109 spores in combination with prebiotic fiber can create a synergistic effect, resulting in the production of short-chain fatty acids. Such fatty acids can diversify the colon’s microbial environment [58]. Also, on rat-subjected uremia, the UBBC07 spores can diminish the symptomatic consequences of acetaminophen therapy, encompassing high level of serum urea, creatinine, and malondialdehyde [59]. Alatriste et al. [61] also highlighted the great probiotic effects of B. clausii against high serum urea in patients with chronic renal failure, as is demonstrated in Table 3.

1.2.3. Antimicrobial Behavior and Immuno-Regulatory Properties

Probiotics that may discharge the antibacterial compound “clausin or lantibiotic” have the potential to cease the growth of harmful bacteria, especially in the gastrointestinal tract. Such behavior is especially beneficial when used in combination with antibiotic therapy [60]. As demonstrated in

Table 4. Summary on the antimicrobial behavior of B. clausii strains, modified from the existing literature.

B. clausii	Antimicrobial Behavior	Targeting Pathogens of …
B. clausii strains [11].	Releasing the anti-microbial substances and the ability to sharpen the host immunity system.	Pathogens of Staphylococcus aureus, Enterococcus faecium, and Clostridium difficile [11].
B. clausii vegetative cells [62].	In whey culture, producing peptides with antibiotic capability.	Salmonella typhimurium, Escherichia coli, Shigella flexneri, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis [62].
B. clausii Sinuberase [62].	In fermented coffee ground beans, releasing probiotic-based antimicrobial peptides.	Salmonella typhimurium, Escherichia coli, Shigella flexneri, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis [62].
Strains of B. clausii—UBBC07 and O/C [29,63].	Releasing of clausin peptide.	Gram-positive bacteria such as Clostridium difficile [29,63]
Strain of O/C strain [11,64].	Producing the clausin peptide.	Cytotoxic effects on pathogens ability of peptidoglycan.

, B. clausii vegetative cells in whey culture produce peptides with antibiotic capability that can hamper the proliferation of the following pathogens: Salmonella typhimurium, Escherichia coli, Shigella flexneri, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis [60,62]. Furthermore, the release of probiotic-based antimicrobial peptides could deter the growth of the same pathogenic species that are developed in the coffee ground culture under B. clausii sinuberase fermentation [60,62].

Due to the release of clausin peptide, the strains of B. clausii UBBCO7 and O/C display antimicrobial activities against certain Gram positive bacteria such as Clostridium difficile [29,63]. In the case of the O/C strain, the produced clausin has cytotoxic effects on pathogens ability to peptidoglycan [35,64].

As [11] suggested, the clinical benefits of probiotic B. clausii are mostly associated with the species’ ability to release anti-microbial substances as well as its ability to sharpen the host immunity system. Such anti-microbial substances can strongly target the Gram positive bacteria, in particular, pathogens of Staphylococcus aureus, Enterococcus faecium, and C. difficile.

Probiotics play a pivotal role in restoring the host’s immune system homeostasis [65]. Regarding the potential immuno-modulatory capability of B. clausii, as is displayed in

Table 5. Summary on B. clausii immuno-regulatory properties, modified from the existing literature.

B. clausii Strains	Immune-Regulatory Properties	Target (In Vivo-In Vitro)
Vegetative cells of B. clausii (O/C, N/R, SIN, and T) [11].	Encouraging nitrite production of murine macrophages. Stimulating formation of pro-inflammatory cytokines. Intensifying proliferation of CD4+T cells inside murine BL/6j spleen cells.	Infection-induced diarrhea; Clinical effectiveness [11].
B. clausii spores in a joint treatment with rehydrate solutions [25].	Regulating intestinal-immune System. Detering symptoms of gut bacterial infection, particularly diarrhea symptoms. Anti-inflammatory properties. Deterring a acute diarrhea as a result of antibiotic consumption or gut ifection.	Clostridium difficile or, Helicobacter pylori pathogens [25].
B. clausii MTCC-8326 [67].	Regulating macrophages’ reactions; both pro-inflammatory and counter-inflammatory agents. Targeting infections caused by inflammatory responses of AW264.7 murine macrophages.	Salmonella typhimurium pathogen [67].
O/C strain of B. clausii [68].	Promoting the release of nitric oxide from RAW 264.7 macrophages.	Immuno-regulatory behavior [68].
A mixture of B. clausii (O/C, N/R, SIN, and T spores) [69].	Diminishing the symptoms of inflammatory colitis. Heightening the population of healthy gutmicrobiota.	Targeting ulcerative colitis in mice [69].
Administration of mixed strains (O/C, N/R, SIN, and T) [69].	Dropping total numbers of worms. Displaying inhibitory effects on pro-inflammatory cytokines of IFN-, TNF-gamma and IL-6. Promoting anti-inflammatory cytokines of IL-10, Treg and Th17 cells, leading to a proper regulatory immune response.	Infested mice by common tropical and sub-tropical parasites (worms) [69]. Targeting irregularity of immune-system [69].
B. clausii, in particular, in joint treatment with other therapies [12,70].	Anti-microbial and anti-inflammatory properties with clinical effects. Improving intestinal infections. Improving antibiotic adverse effects in children. Healing antibiotic adverse effects in adults. Nasal allergies, upper respiratory infections.	No data

, the species bears some healing effects on mice with immune-induced asthma. Such effects ensue because of a decline in the numbers of pro-inflammatory eosinophils, neutrophils, and lymphocytes. Additionally, the application of B. clausii for athematic mice can also decrease the expression of hypoxia-related genes as well as the level of IL-4, and IL-5 interleukins [66].

A look into modulating the effects of B. clausii MTCC-8326 on inflammatory responses of AW264.7 murine macrophages reveals that this strain could act as both a pro-inflammatory and counter-inflammatory agent. Indeed, the MTCC-8326 strain can easily regulate macrophages’ reactions against existing infection that is caused by Salmonella typhimurium pathogen [67].

B. clausii vegetative cells (O/C strain) have clinical effectiveness on both C. difficile- and B. cereus-infection-induced diarrhea [35,64]. Vegetative cells of B. clausii (O/C, N/R, SIN, and T) encourage the nitrite production of murine macrophages and the formation of pro-inflammatory cytokines, while intensifying the proliferation of CD4+T cells inside murine BL/6j spleen cells [35]. There is also an O/C strain of B. clausii that promotes the release of nitric oxide from RAW 264.7 macrophages, thus shedding light on the strain’s precise immuno-regulatory behavior [11,68].

In mice suffering from ulcerative colitis, a mixture of B. clausii (O/C, N/R, SIN, and T spores) within a period of two weeks, can slightly diminish the symptoms of inflammatory colitis, while heightening the population of healthy gut microbiota. In mice infested by common tropical and sub-tropical parasites, administration of mixed strains (O/C, N/R, SIN, and T) can also drop the total numbers of worms, while displaying inhibitory effects on pro-inflammatory cytokines of IFN-, TNF-gamma, and IL-6. Additionally, the mixed strains can promote the anti-inflammatory cytokines of IL-10, Treg and Th17 cells, prompting a proper regulatory immune response [25,69].

B. clausii spores in a joint treatment with rehydrate solutions can also instigate favorable curative effects on an acute diarrhea [25,69]. Such diarrhea, mostly, happens because of antibiotic consumption or due to gut contamination by pathogens of either C. difficile or Helicobacter pylori [25].

There are also the clinical effects of B. clausii, in particular, in joint treatment with other therapies which can benefit nasal allergies, and upper respiratory infections, alongside improvement in intestinal infections and improvement in adverse effects of antibiotic consumption in children [12,70]. The literature has also suggested that the same B. clausii has healing effects on side effects that are caused by H. pylori therapy in adults [39,71,72,73,74]. In conclusion, B. clausii is a commonly applied probiotic. Its anti-microbial and anti-inflammatory properties have supported its administration for the purposes of symptom alleviation or deterrence (particularly, diarrhea symptoms)that are prompted by the gut infectious pathogens.

1.2.4. Bacillus spp. Fermentation Ability

There are plenty of studies on the Gram positive bacteria of spore chain bacilli that can only ferment glucose for acid production purposes, yet they cannot catabolize lactose or maltose [75]. For example, the aromatic fermented products of natto and dawadawa with distinct flavors can be manufactured by hydrolytic capabilities of B. natto (B. subtilis) in a glucose medium culture [76]. There is also, B. subtilis of 168DSABA1, BSP003, and BSJ0232, which in the mixed medium culture of (30 g/L xylose, glucose, and arabinose) can produce lipoprotein of fengycin [76]. Using sugarcane and triticale as fermentable substrates for B. velezensis strains, can also create a palatable strategy for enzyme production [77]. A study by [78] displays that the content of cellulose and hemicellulose in soybean meal can be reduced by fermentation of B. subtilis over a 24-time period. There have also, been other studies such as [79] which point to the optimized emanation of α-glucosidase in soy-based food with the presence of B. Subtills PM NEIST_4. The α-glucosidase enzyme is important, since its role is to hydrolyze disaccharidase and starch-based products to glucose in order to facilitate the carbohydrates’ intestinal absorption. In a study by [80], the use of B. subtilis and S. thermophilus (LAB) caused the degradation of Raffinose Family Oligosaccharides which otherwise are responsible for unpalatable odor and poor digestion inside the gut, and their intestinal fermentation can cause digestive discomfort (e.g., bloating, diarrhea). Indeed, in samples containing B. subtilis strains, the content of oligosaccharides such as; verbascose, stachyose, and raffinose diminished, while melibiose, fructose, and sucrose intensified due to hydrolysis of verbascose, stachyose, raffinose, and sucrose during fermentation. There was also a gradual increment in melibiose content that likely ensued, as the result of the degradation of verbascose, stachyose, and raffinose.

In our review, we could not find enough studies focused on the sugar fermentation capability of B. clausii. However, there was a study by [81] conducted on indigenous B. clausii, and concluded that the bacteria’s total consumption of glucose can result in the emanation of a high level of alkaline protease. Meanwhile, such a lack of evidence on B. clausii sugar fermentation capability implies that there is a void of studies in this domain, which needs to be addressed in-depth by future studies.

2. Compositional Quality and Safety Assessment of B. clausii-Based Products

Since probiotic-based products are most frequently about to be utilized, they must comply with safety protocols and must prove whether or not they are suited for humans or animals consumption [6,82]. Microorganisms are usually profiled, before their innate probiotic characteristics are scientifically unraveled [82]. The source of their origin is the first, safety concern that must be addressed. Such compliance determines the safety and healthy composition of the probiotic, and demonstrates that it has no inherited pathogenic genes. Problematic genes can cause gut discomforts or engineer a clinical condition referred to as antimicrobial gene resistance [83].

The greatest concern, in regard to human consumption of probiotics, is the health composition of the product, and whether or not Goods Manufacturing Practices (GMPs) have fully been addressed in alliance with their safety protocols [6,83]. On the contrary, in regard to animals’ feed, the concern over the use of probiotics pertains to the possibility of inter-species gene transfer that can develop antibiotic-resistant genes [6,83].

In South East Asia, different strains of probiotic bacteria encompassing Bacillus strains in conjunction with Lactobacillus species have formulated some functional products with antibiotic resistance properties. The only safety concern with respect to such formulated products, is the high prospect of transferring antibiotic-resistant genes to gut pathogens that naturally reside inside humans or animals [84,85]. To ensure that probiotic products are safe for both human and cattle consumption, it is necessary to enforce some specific control measures like, strict food and drug regulations in order to ensure probiotic safety concerns of strains. Indeed, it has been clinically proven that some Bacillus strains are serious enteropathogens and exhibit vigorous cytotoxic symptoms that have doomed them to be used as probiotics [86]. Such concern, in particular, amplifies if the recommended probiotic supplement is about to be ingested by the living subject (animal or human). For instance, from the family of Bacillus genus, B. cereus is one of the most controversial food poisoning enteropathogens with the ability to emanate heterogeneous heat-resistant toxins [87]. In addition to B. cereus species, some other Bacillus species (B. subtilis, B. amyloliquefaciens, and B. mojavensis) have also shown cytotoxic abilities that have condemned them to be designated as probiotics [42,87].

Very few members of Bacillus strains have been well investigated by clinical studies and commercially approved as safe for consumption, by both animals and humans [83,87]. Recently, the use of probiotics in livestock manufacturing products has been on the rise and there has been a significant interest in the Bacillus family due to their spore-forming characteristics. These Bacillus strains have been adopted as alternatives to antibiotic growth promotors (AGP) since consumers’ demands for the consumption of free AGP products have been on the rise [88].

Primarily, the commercial preparations of B. clausii probiotics happened because of health reports on their benefits and zero reports on their hazardous or life-threatening administrations [37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90]. Few clinical studies with randomized placebo-controlled design, using meta and/or pooled analytical methods, have supported the species’ efficacy in the cure of antibiotic-caused diarrhea in juveniles, adults and children [73,90]. There has also been a specific case study on the efficacy of B. clausii in relation to the treatment of an acute diarrhea in adults of a certain age [37,91] as well as juveniles [70,92,93].

There are also reports of (>93%) that signify a high tolerance in children who are undergoing treatment with probiotic bacteria. This testifies further that the probiotic has a low hyper-sensitivity. The over-the-counter supplement Enterogermina^® includes spores belong to four strains of B. clausii [OC, NR, SIN and T] that withstand antibiotics of chloramphenicol and tetracycline robustly [9,94]. Thus, the combination of these spores at a ratio of (2 × 10⁹ cfu/mL) with antibiotics consumption can empower the gut microbiota population to fight stronger against infantile diarrhea [9,93].

Results of acute toxicity by the UBBC07 strain on rat models [95] points to an oral tolerance (LD50) of less than 5000 mg/kg (630 billion cfu/kg) that shows no adverse effect level (NOAEL). In [95], the NOAEL is indeed established at 1000 (126 billion cfu) mg/kg _BW/day oral administration in a subacute toxicity. In the same study [95], no toxin genes inside the UBBC07 strain were detected. Apparently, such a lack of toxic genes approved the strain’s full safety consumption and agreed to its edibility as an oral probiotic for humans. Furthermore, there was no significant discrepancy between the safe consumption of OECD443 versus OECD407 strains considering both the control and target groups.

B. clausii strains of (DSM 8716, ATCC 21536, and ATCC 21537) seem to be safe for consumption purposes due to the fact that they are inept to transmit the gene with antibiotic resistance attributes to other pathogenic microorganisms [40,95]. Such a quality has been acquired thanks to the inheritance of a specific gene known as the ERM gene [40,95].

Considering the fact that general safety, possible toxicity, and high quality of probiotics are determined by the type of species in_use, as well as the number of viable cells/spores, it is absolutely pivotal that commercially marketed probiotics precisely meet thesuggesting informative claim on their labels [10]. Probiotic products that are manufactured in different countries may bear inconsistent information in relation to the type of strain, the number of seeds/spores, and the degree of viability. Therefore, such manipulations can clearly influence the efficacy or toxicity of the product, particularly at the time of its administration [10,96]. The compositional quality of Enterogermina^® is highly approved since the product represents the precise mentioned information on its label, contrary to the other commercial probiotics that either have unmentioned bacterial strains or have a poor ratio between reported quantitative numbers on the label versus actual bacterial plate counts [4,10,96].

Among worldwide marketed formulations, products of Biogermin^® and Enterogermina also represent the precise content (quality and quantity) on their products’ labels. On the contrary, other products either contain low viability or accommodate traces of unlabeled bacterial species [10,96]. There have also been incidents in which probiotic products claimed containment of pure B. clausii, yet inside the food quality control lab, traces of other bacteria, some indeed with enteropathogenic behavior, have been detected. These bacteria are: B. cereus, B. licheniformis, B. badius, Brevibacillus choshinensis, Lysinibacillus fusiformis, and Acinetobacter baumannii [10,96].

The bacteria strain type and dosage dependency can impact their probiotic quality effects and their beneficial health outcomes; therefore, patients could benefit from such formulations in case a rigorous quality control test/system has been put in place [97]. Different formulations of B. clausii O/C, N/R, SIN, and T strains in different forms and shapes (solution-based, suspension-based, non-suspension-based, powder-based or orally taken capsules) can have the same analogous effects as long as they are aligned with their kinetic profiling claim. In most cases, kinetic profiling sheds light on spore_viability, longevity, extent of efficacy and resistance inside the human gut [98].

Normally, B. clausii has been administered as a typically safe consumable probiotic formula, yet there have been few clinical studies reporting on a slight number of sepsis cases under very specific conditions [99,100]. Even though few isolated cases have been detected for signs of bacteremia or septicemia, mostly in immuno-sensitive individuals with morbid diarrhea, no life-threatening infection has been reported concerning consumption of commercialized B. clausii products [101]. However, work by Joshi et al. [99] reported on a single-case occurrence in a 4-month-old toddler with congenital heart disease who also had diarrhea, and later, her case was affirmed as an isolated case of B. clausii-induced septicemia. This medical affirmation occurred as the lab results debunked the sepsis diagnosis in favor of Klebsiella pneumonia.

Consecutively, after administration of B. clausii in patients with active ulcers, signs of bacteremia have also been reported. However, in this case, B. clausii was apparently recommended to cease the infectious diarrhea [102]. It is obvious that in this instance, since the administration of B. clausii has been recommended in the long term, the factor of a lack of hygiene should have been taken into diagnostic consideration in order to tackle the real cause of the bacteremia [102].

Commercially available B. clausii products are sealed with lids to determine consumer safety and to conserve the content solution [37,103]. Decades of prescribing numerous Bacillus products without indication of life-threatening side effects can outweigh the small number of cases that lead to infection. Nevertheless, the literature implies that certain conditions or skewed diagnoses can lead to an opportunistic infection [39,103]. For example, acute toxicity studies confirm that the B. clausii strain of UBBC07 is safe enough for human utilization, yet signs of sepsis or bacteremia may be detected due to other influential factors such as lack of hygiene [95,103].

In accordance with the European Food Safety Authority (EFSA), ingestions of different strains of clausii (O/C, N/R, SIN, and T) have been approved as safe, and as a result, such probiotics are listed on the Qualified Presumption of Safety (QPS) [104]. https://doi.org/10.2903/j.efsa.2014.3665 © European Food Safety Authority, 2014. EFSA Journal 2014;12(5):3665. (adopted on: 8 April 2014/accessed on: 5 May 2014).

Furthermore, the USA Food and Drug Administration has separately approved the safety of a different class of B. clausii (088AE) and enlisted the strain on the category of generally regarded as safe (GRAS) (United States Food and Drug Administration. GRN No. 971. Bacillus clausii Strain 088AE Spore Preparation. Available online: https://www.cfsanappsexternal.fda.gov/scripts/fdcc/?set=GRASNotices&id=971&sort=GRN_No&order=DESC&startrow=1&type=basic&search=clausii (accessed on 31 July 2020).

Such a designation has indeed made the probiotic bacteria applicable for specific industrial or commercial usage [20,88,104].

The following

Table 6. B. clausii safety assessment.

Name of the Species	Typical Dosing Form of the Usage Times of Usage	Manufacturer Product Information	Contraindications	Pregnancy/ Lactation	Interactions Toxicology	Adverse Effects
Bacillus clausii	2 × 109 spores administered orally. Capsule/suspension (2/3 times/daily—for 10 days up to 3 months).	Adults: 4 to 6 × 109 spores/daily (2 to 3 times/daily of suspension or 2 to 3 times capsules/day). Children and Toddlers: 2 to 4 × 109 spores/daily (for a short term).	Hypersensitivity to probiotics	According to EFSA, the B. clausii can be used during conception/ pregnancy and breastfeeding infants.	Not well-documented. No data.	None in clinical trials. None on labeled product information.

demonstrates some elements concerning B. clausii’s safety assessment modified from https://doi.org/10.2903/j.efsa.2014.3665. © European Food Safety Authority, 2014. EFSA Journal 2014;12(5):3665. (adopted on: 8 April 2014/accessed on: 5 May 2014).

3. Bacillus spp. Application in the Food Industry

3.1. Poultry Industry and B. clausii

For decades, the application of antibiotics in meat, pig, and poultry production has raised concern. Poultry consumers’ desires have mostly lain on meat-based products that have been cured by other healthy alternatives rather than antibiotic growth promoters (AGPs) due to the concern that AGP consolidates the formation of antibiotic-resistant pathogens, distorting human natural gut microbiota. Such a concern led to an EU ban on the use of AGPs (Regulation (EC) No 1831/2003) [105,106].

Bacillus bacteria have the best quality spores, including spore’s convertible aerobic nature with agile adjustability to an anaerobic climate as harsh as a mammals’ gut which has made the bacteria a potential probiotic and frankly the best existing alternative solution against traditional probiotics (LAB-based) [103,107].

Bacillus species are dubbed as golden probiotics. Their ubiquitous distribution, easy isolation from the living environment, and great survival rate (up to 100%) have qualified their application in the food processing industry [88,108].

Bacillus as feed probiotics can produce high-density spores (as high as 1 × 10¹¹ spores·mL⁻¹). High-density spores are very much in demand in food production due to the advantages of high viability (up to 90%) and a great shelf life of five years (at a concentration of 1 × 10⁹ spores·mL⁻¹). Such advantages have made the poultry industry use Bacillus species preferentially in their marketing products [15,88,108].

Despite the fact that application of Lactobacillus spp. has always been entertained as the optimal standard for the poultry industry, the latest research has suggested that the application of Bacillus spp. may be a better alternative [88,103,108].

In addition to great spore_quality and probiotic effects, Bacillus species have the advantages of not being poultry pathogens. They contain exogenous enzymes that can consolidate the chicken gut microbiota population, leading to better gut functionality [88,103,108]. However, the greatest advantage of Bacillus species is their probiotic detoxification quality which can optimize the functionality of bird organs, leading to fewer ailments and fatalities [109,110].

Since Bacillus probiotics have been approved for animal feed productivity, their increase in yield by up to 5% can result in a sustainable meat production [88,111].

B. clausii, with its probiotic effects, are expected to enhance the histo-morphological architecture of chicken’s intestines. Supplementation with 0.03 ML/L B. claussi reforms the chicken’s intestines’ biological behavior while adding weight to their bodies and helping greater feed conversion ratio (FCR). Furthermore, supplementation by B. clausii enhances the life span of the chicken, causing greater net profit in business and higher poultry economic output [112].

3.2. Pig Industry and B. clausii

Most strains of Bacillus are natural probiotics that can sharpen animals’ health and their performances. Concerning the advantages of bacillus spores, their involvement in all phases of the pig industry (sow herd, nursery and mature pigs) has been worthy of attention [113]. Bacillus spores possess distinctive characteristics that enable them to cope with high concentration, great pressure and immense temperature, thus qualifying them to be used in high-paced food productions. The spore’s most underlying feature is its agreeable morphological structure as it aligns with the host’s morphological organs therefore, it makes it a perfect match for effective sufficient food production, storage, and delivery [113].

3.3. Fermented Products and B. clausii

Various probiotic strains of the Bacillus family, including B. clausii, B. coagulans, B. tequilensis, B. licheniformis, and B. amyloliquefaciens, have been extracted from fermented foods like congee and pickles [114,115]. In India and Sri Lanka, the fermented products idli (fermented rice + black lentils), dosa (fermented ground lentils and rice), appam (fermented rice + coconut milk), dhokla (fermented batter), uttapam (fermented rice + lentils), and selroti (fermented rice flour) have been traditionally popular [116]. In the case of idli, the fermented bacteria are probiotic Bacillus, offering anti-cholesterol, antioxidant, and anti-biofilm health properties. In these countries, idli is known as a reservoir of probiotics thanks to the Bacillus spore’s high heat stability [116].

Much loved throughout Brazil is the processed cheese of “Requeijão cremoso” that undergoes a couple of tedious manufacturing phases. Among such phases, exposure to the high temperatures of 90 °C is the most notorious one. Under such high temperatures, lactic acid probiotics will deform, causing hindrance in the production of probiotic Requeijão cremoso cheese. On the contrary, the Bacillus species endure the high temperature since they have heat resistance spores [116].

When it comes to the manufacturing of probiotic based products, the heat or temperature tolerance of the probiotic species has always been the top concern for the dairy industries [117]. Given this consideration, some strains of Bacillus (B. coagulans MTCC 5856, B. coagulans GBI-30 6086, B. subtilis PXN 21, B. subtilis PB6, and B. flexus HK1) have been tested under survival examination rate [2,116]. For instance, the probiotic fortified cheese under various manufacturing phases (pasteurization, curd, coagulation, and fusion) experience such a test to unravel the survival rate of its added probiotic bacteria known as the starter culture. Furthermore, such tests are conducted to determine the chemical composition, the fatty acid, and the protein stability of in-process products. For example, in cheese manufacturing, the fusion step is the optimal stage for the application of B. coagulans GBI-30 6086. This stage is proven to protect the quality and viability of added probiotics and shields the content of the end_product against recontamination [2].

Soybeans are one of the best protein sources that are driven from plants, and because of their high biological value they have been commonly employed in food processing. The most common example of soybeans is tofu which is approximately composed of 6.0–8.4% protein and 79–87% water and has a neutral PH of 5.2–6.2. Tofu is made from hot soy milk blended with some coagulant inducers commonly known as salts of calcium and magnesium chloride [114].

B. clausii has great probiotic effects with the ability of colonization, immunomodulation, and anti-bacterium [28,91,117,118]. The probiotic bacteria can target symptoms of acute or chronic diarrhea without creating any adverse effects [91,117,118]. Tofu as the carrier of B. clausii probiotics can maintain the viability of the bacteria at the highest possible rate and carry it directly into the intestines. Indeed, the use of tofu as the probiotic carrier can seal the B. clausii against direct contact with the intestinal bile salt, gut acid, and enzymatic degradation. Furthermore, tofu cheese can carry the B. clausii spores throughout the gut as swiftly as possible before spores convert into the active form of vegetative cells [119]. The main purpose of engineering a probiotic tofu is either to have a functional food product that can safeguard against the adverse effects of probiotic pills or to help a particular sensitive demographic (vegan/lactose sensitive elderly) who like to have access to plant based probiotic products [119].

Yogurt is a fermented product in which its taste, texture, and culture can favorably change by probiotic supplementations as well as by fruit flavors. Such supplementation makes yogurt become more resonating with dairy-sensitive consumers [120]. For example, yellow mombin yogurt is a functional dairy product thanks to its high probiotic content. The product is also highly nutritional thanks to its enriched minerals and low-fat content. During 150 days of storage, the product can sustain both its protein content (8.3/100 g) _and fat content (2.6/100 g) [120]. Within the same time frame, its phenolic content, DPPH, and FRAP do not change. Its pH is also acidic and, unlike non-probiotic yogurt, adjusts after 60 days. Moreover, within 30 to 150 days, there can be a steady increase in the quantity of its solid matters (22 to 27) and during this time, its probiotic cells continue to remain as functional as possible with counts of 4.1 × 10 8 CFU/g [120].

Spent coffee grounds are a by-product of coffee beverage preparation and the disposal of the by-product has detrimental effects on the environment. Under the technique of surface response, on one hand, B. clausii probiotic fermentation can amplify the phenolic and flavonoid content (36–13%) of the by-product, while, on the other hand, it can elevate the antioxidant (15%) and antimicrobial level of the by-product. Such development indicates that the enhanced fermented spent coffee grounds can be applied as an antioxidant and antimicrobial supplement_in addition to the fact that it can be used as a functional additive in the food industry, enriching different sorts of food products [62,121]. There is also the protein content of the spent coffee ground, which is highly increased by B. clausii’s good fermentative behavior and its strong protein hydrolysis [62,121]. The peptides in the spent coffee grounds feature profound regulatory/inhibitory attributes against chronic diseases of diabetes, hypertension, and oxidative stress [62,121].

3.4. Confectionary Products and B. clausii

The involvement of B. clasuii in the confectionary industry has also been taken into account with the aim of engineering functional confectionaries. Focusing on probiotic soft jelly candy, containing Bacillus probiotic strains of clausii, coagulans, and subtilis (bacillus natto) has been a new thought in food engineering. Such candy within three months’ storage (90 days with 90% viability) does not exhibit any change in viability or durability of its used probiotics, yet, conversely, the product is believed to show higher total phenolic, and anti-oxidant properties [94].

3.5. Functional Foods with Antioxidant-Probiotic B. clausii Properties

Functional foods with antioxidant-probiotic attributes can also be produced undercertain techniques. One of them is the technique of spray-drying. Using such a technique, a probiotic like B. clausii can favorably influence the antioxidant behavior and cultural functionality of resveratrol. However, such synergy can be enforced by the aid of a carrier capsule composed of inulin and lactose [122]. As a matter of fact, B. clausii bacteria with its probiotic property absorbs the antioxidant of resveratrol to preserve its high quality and its great functionality, creating a palatable synergistic effect [122].

There is also the technique of freeze drying that sporadically has been applied to encapsulate B. clausii and quercetin (antioxidant) into one carrier matrix. In doing so, two matrix compositions can possibly be engineered. The first matrix is composed of inulin (IN), lactose (L) and maltodextrin (MX), while the second one is composed of Arabic (A), guar (G), and xanthan (X) gums [123]. Such chosen composition allows the researchers to surpass the threshold viability that is advised by FAO/WHO-2002 [124].

The subject of functional food, using the technique of spray-drying and co-encapsulation has also led to the imultaneous encapsulation of the probiotic B. clausii and quercetin. To test the synergistic effects, three matrix compositions are hypothetically chosen. Two of them are solo_carriers, composed of either inulin or just a single polymer of maltodextrin, while the third carrier has a blended-micro encapsulated matrix. The blended matrix of the dual carrier possesses greater efficiency than each mono-carrier alone. Indeed, the use of maltodextrin enhances the viability of the B. clausii probiotic, while the use of inulin rectifies the activity of the antioxidant, and the use of inulin and maltodextrin together leads to a highly formed synergy [123].

4. Conclusions and Future Perspectives

Contemporarily, the consumers’ food desires have changed more than ever. A significant shift has been observed towards healthier eating that proposes a marvelous market for the manufacturing of functional foods with nutritious content. Over decades, the spike in consumers’ demand for the consumption of functional foods has rationalized the integration of probiotics into the human or livestock_products.

Among these probiotic bacteria, the spore-producing Bacillus spp., in particular, strains of clausii (which was the concern of this review) have stood out due to their spores’ special characteristics featuring great longevity, tolerance, safety with potential probiotic health advantages. Such characteristics have supported the widespread application of the bacteria in the food industry however, our exploration into the existing literature suggested that there has not been adequate empirical research (both in vitro and in vivo studies) to back up the probiotic benefits of clausii spores. Clearly, in regard to food manufacturing, few studies have so far acknowledged the usage of spore-forming probiotics as better alternatives to traditional lactic acid probiotics or antibiotic growth promoters. In our review, few studies implied that the application of probiotics’ spore-forming clausii in “livestock” can intensify feed productivity, nutrient absorption, growth rate, and economic profitability; regarding “the manufactured products”, few works pointed out that B. clausii’s probiotic potential in relation to its impact in optimization of food functionality, food quality, and functional foods’ shelf life. In general, the literature implies that since B. clausii has greater spore viability and greater spore functionality, it may secure a better shelf life in different foods, undertaking different processing treatments, yet the literature could not provide enough scientific studies on how different methodologies can result in the manufacturing of different functional foods with diverse properties (synbiotic or synergistic-like). Our reviewed literature postulated that the right selection of B. clausii probiotic strains can instigate optimal effects in both feeds and on-shelf food products, butthe majority of the reviewed literature failed to specify on which manufacturing stage should the spore supplementation or fortification happen to meet the optimization purposes.

In this review, some characteristics of B. clausii spores, including physiological characteristics, generic modes of action, potential probiotics benefits, safety assessment and food industry applications for the purposes of manufacturing optimized functional foods were taken into account. Our review concluded that the number of research papers addressing the food industry benefits of (B. clausii) in contrast to LAB probiotics have not been proportionately balanced and suggested that more studies should be conducted, concerning the advantages of incorporating B. clausii into the food industry. Furthermore, in this review, we encountered a lack of empirical evidence addressing more details in relation to the sugar fermentation capability of the B. clausii strains as well as the optimal cultivation condition of the strains. There was also a lack of the literature to support any guidance that fully assesses the toxigenic potential of B. clausii strains that are utilized for both feed and food production.