Review Article

Endospores of Bacillus subtilis with probiotic potential in animals of zootechnical interest

0000-0002-4035-8643Grethel Milián^{1  *}  , 0000-0003-3019-1971Ana J. Rondón¹  , 0000-0003-4248-3728Marlen Rodríguez¹  , 0000-0002-8525-6595A. Beruvides¹  , 0000-0002-9473-6507M. L. Pérez² 

¹Universidad de Matanzas. Autopista Varadero km 3 ½. Matanzas, Cuba

²Universidad Estatal Amazónica. Departamento de Ciencias de la Tierra. km 2 ½. Vía a Tena (Paso Lateral). Puyo, Pastaza, Ecuador

ABSTRACT

At present, poultry, pig and cattle production constitute the most important branches of animal production in the world, which currently contributes to fulfill the protein needs of the world population. The use of probiotics, prebiotics and synbiotics is predicted with the purpose of using them as animal growth promoters. Among the species that are most used for the elaboration of these products, are those from Bacillus genus. One of them is Bacillus subtilis, a bacterium able to stimulate the immune system, produce enzymes, resist acid pH gastric barriers, and produce antimicrobial substances that inhibit different pathogenic microorganisms. For these reasons, zootechnical additives, made with Bacillus, are considered an alternative in the current livestock industry. The objective of this review was to show the probiotic potential in pig farming, poultry farming and calf rearing that Bacillus subtilis endospores have.

Key words: alternatives in animal production; calves; chickens; pigs

The World Organization for Animal Health and the Food and Agriculture Organization of the United Nations/World Health Organization (^{FAO/OMS 2001}) are working to introduce new products into animal production systems that counteract the effect of antibiotic growth promoters, without affecting the production of meat, milk and eggs, and in turn not generating adverse effects on the consumer health (^{Vélez et al. 2019}). The expectations of farmers are increasing regarding food additives that guarantee improvements in the growth rate and other production parameters, such as the food absorption and the quality of their products, as well as the protection of health against infections (^{Markowiak and Katarzyna 2019}).

Currently, probiotics are used as animal growth promoters, since they improve the composition of the gastrointestinal microbiota and the efficiency in the use of food, stimulate the immune system and inhibit pathogenic microorganisms, without the use of antibiotics (^{Barros 2018}, ^{Molina 2019} and ^{Rondón et al. 2020a}).

Among the probiotics used in livestock production are those made with of Bacillus spp. endospores (BioPlus 2B^®, Biostart^®, Toyocerin^®, Liqualife^®, Biosporin^®, CenBiot^®, Bactisubtil, Biosubtyl “Dalat” and Clostat^®), with a probiotic effect on a wide category of zootechnical interest animals (^{Kizerwetter and Binek 2016} and ^{Milián et al. 2021}). Due to the aforementioned, the objective of this review was to assess the results of researches on the probiotic potential of Bacillus subtilis endospores and their effect on pig farming, poultry farming and calf rearing.

Probiotic properties of bacillus subtilis endospores

Probiotics allow improvement in physiological indicators and stimulate action mechanisms to avoid side effects on animal products destined to human consumption. In the same way, they favor optimal growth of the animal, which provides good function of the intestinal mucosa, increased digestibility and the synthesis of vitamins; in addition to stimulating motility and the absence of diseases, important elements for production development (^{Sefer et al. 2015}). In addition, they generate the stimulation of the specific immune response of the animal, and this allows the increase in immunoglobulin levels, which is a positive effect on growth and production (^{Kassa 2016} and ^{Molina 2019}). There are several action mechanisms of probiotics made with Bacillus strains, which favor the above.

1) Production of antimicrobial substances. They participate in the destruction of target cells through pore formation or inhibition of cell wall synthesis. In the case of bacteriocins, nisin, for example, forms a complex with the last cell wall precursor, thus inhibiting its biosynthesis in endospore-forming bacilli. Subsequently, it constitutes a complex that adds and incorporates peptides to form a pore in the bacterial membrane and generate cell death (^{Tao et al. 2018}). Figure 1 describes the morphological changes that occur in a bacterial population after exposure to nisin.

Degree of nisin assembly depends on bound densityIncreased density of nisin bound to the membrane surface destabilizes the membrane structure, increases part life and causes pore expansionIncreased dissipation of membrane potential causes accelerated cell divisionLine tension drives pore closure and pushes lipids out of the membrane

Figure 1 Model that represents the interaction of nisin with the bacterial membrane and pores formation (^{Ahumada 2020}). 

2) Production of specific enzymes. Enzymes used as additives in animal feeding are abundant and varied. ^{Pérez-Chabela et al. (2020)} report that among the enzymes produced by Bacillus spp in the vegetative phase are lytic enzymes (proteases, chitinases, cellulase, ß-1,3-glucanases, among others). Its use favors the synergistic action of these proteins on the most complex substrates present in foods. Generally, its use is aimed at improving the availability of polysaccharides, lipids and proteins, which are protected from digestive enzymes by impermeable structures of the plants cell wall, and also to degrade compounds that interfere with digestion, absorption and use of nutrients (^{Aftab and Bedford 2018} and ^{Handique et al. 2018}).

In recent years, the effective use of cellulases, xylanases and β-mannanases, as additives in diets for monogastrics, generated great interest from farmers (^{Alagawany et al. 2018}).

^{Medina-Saavedra et al. (2017)} found that B. subtilis produces xylanase, which has a similar effect to antibiotics on the microbiota in the small intestine. The reduction in viscosity accelerates the intestinal transit speed. These authors reported that in the hydrolysis of arabinoxylans (AX), xylanase reduces the antinutrient effect, and thus increases its nutritional value.

^{Bedford (2018)} added the enzyme xylanase to the diet for laying hens and observed adaptive changes in the microbiome of birds, in addition to verifying that the ability to degrade fibrous sources that are not hydrolyzed in the intestine of this species increased. The inclusion of β-mannanases in soybean-containing diets for broilers at the starter stage increased blood glucose content, anabolic hormone homeostasis, and amino acid digestibility (^{Caldas et al. 2018}). Likewise, the addition of this enzyme in diets composed of corn and soybean increased broilers yield, by reducing the content of galactomannans in the feed (^{Latham et al. 2018}).

The use of enzyme cocktails in laying hens improved nutrient retention and showed a tendency to increase enzyme activity in the intestine (^{Wen et al. 2012}). ^{El-Hack et al. (2017)} pointed out that in this same category, the substitution of soybeans for beans (Vacia faba L.), supplemented with cellulases, xylanases, α-amylases and proteases, improved food efficiency, without affecting the quality and productivity of eggs.

3) Competence to prevent the adhesion of pathogens to epithelial cells. The mechanism of antipathogen competition is described as a process in which a bacterial species rigorously competes for adhesion to receptors in the gastrointestinal tract of an animal (figure 2) (^{Van et al. 2020}).

Figure 2 Mechanism of pathogens adhesion to epithelial cells (^{Ahumada 2020}) 

^{Pérez-Chabela et al. (2020)} reported some of the mechanisms used by Bacillus spp. to prevent the pathogens adhesion: 1) cell depolarization due to the formation of pores in the cell membrane, 2) inhibition of growth due to competition in adhesion sites and 3) inhibition of the expression of virulence genes.

4) Modulation of intestinal immunity. One of the characteristics that distinguish probiotics with Bacillus endospores is the ability to activate the immune system. The implementation of strategies for the care of the microbiota helps the host to maintain normal immune function through the expression of molecular patterns associated with metabolites derived from enzymes and antigens (figure 3). Therefore, the immune system heavily depends on the commensal microbiota for protection against invading pathogens (^{Tao et al. 2018}). In turn, probiotics have an immunomodulatory effect, which stimulates phagocytosis and the proliferation of immune cells (macrophages, monocytes and specialized cells, such as CD3, CD4 and CD8 T), in addition to the formation of antibodies (IgM and IgG) (^{Romero et al. 2013} and ^{Ajuwon 2016}).

Figure 3 Immunomodulatory effect through the interaction of probiotic bacteria with epithelial cells, dendritic cells (DC), monocytes and lymphocytes (^{Ahumada 2020}). 

In Cuba, there is the SUBTILPROBIO^® probiotic product made with the strain of Bacillus subtilis subspecies subtilis (C-31, C-34 and E-44). The use of this product shows its in vitro probiotic properties (^{Milián et al. 2017}).

The studies carried out by ^{Milián (2009)} show that the three strains (C-31, C-34 and E-44) have great capacity for growth and production of endospores, capable of inhibiting Gram-positive and Gram-negative microorganisms; in addition to showing sensitivity to a wide group of antibiotics and producing a group of specific enzymes (alkaline phosphatase, C4 esterase, C8 lipase esterase, C14 lipase, leucine arylamidase, naphthol-A-S-BI phosphohydrolase, α-glucosidase and β-glucosidase). This allows inferring that these strains show favorable ranges for their use as zootechnical additives, which constitute a promising alternative to the use of growth-promoting antibiotics.

5) Effect of Bacillus subtilis endospores in pigs. One of the main objectives of pig production today is to obtain the highest number of weaned piglets per sow in the year, healthy and of good weight. Among the nutritional strategies to improve sow yield, which is generally associated with better efficiency in nutrients use, is the utilization of additives. Among those used in sow feeding are probiotics, which show beneficial results related to milk production and quality (^{Rocha et al. 2018}).

^{Peet et al. (2020)} studied the effects of a probiotic supplement with Bacillus (mixture of spores of Bacillus amyloliquefaciens DSM 25840 and Bacillus subtilis DSM 32324) on the growth and health of fattening pigs. As a result of the study, they managed to improve the conversion rate and the average daily gain. ^{Raudez and García (2020)} evaluated the effectiveness of a probiotic with Lactobacillus lactis, Bacillus subtilis, Lactobacillus acidophilus, Bifidobacterium bifidum (PORCI-BIOTIC COMPLEX) for 28 d. These authors obtained favorable results for the animal health behavior variable, where the control group showed a greater number of respiratory and pulmonary diseases compared to the rest (PORCI-BIOTIC COMPLEX).

^{Rondón et al. (2020b)} evaluated the probiotic effect of the biopreparations PROBIOLACTl^®, SUBTILPROBIO^® and their mixture on productive and health indicators of growing pigs. The evaluated biopreparations produced benefits in the animals, since they improved the eubiosis of the gastrointestinal tract, which contributed to improve (P<0.05) the live weight (27.15 kg/25.59 kg), the average daily gain of animals (408.65 g/445.27 g), weight gain (19.42 kg/16.36 kg) and feed conversion (2.44/2.90). In addition, the incidence of diarrhea decreased (8.57/67.14 %) in the treated animals. The results confirmed the probiotic potential of these biopreparations, when applied to pigs during the growth stage.

6) Effect of Bacillus subtilis endospores on birds. There are several researchers that show the positive response of the inclusion of zootechnical additives with a probiotic effect on the food for the poultry category, from probiotics with Bacillus endospores. ^{Morales et al. (2020)} show this when they evaluated a multienzyme complex (proteases, amylases and xylanases) and a probiotic (Bacillus subtilis) in Bovans White hens. These authors confirmed an increase in egg weight with the addition of enzymes and probiotics. However, for the humoral immunity, cholesterol, LDL and HDL variables, there were not differences (P>0.05) between treatments.

^{Bai et al. (2016)} reported improvements in weight gain and feed conversion ratio in day-old Arbor Acres males when they supplemented basal diets with Bacillus subtilis mbJ (BSfmbJ) at doses of 2, 3, and 4 x 10¹⁰ cfu/kg without using growth promoters.

^{Milián et al. (2019)} evaluated the zootechnical additive SUBTILPROBIO^® in laying hens of Leghorn breed L33. To determine the probiotic effect, they measured: live weight, intake, conversion, total egg production, cracked eggs and total disqualified eggs, as well as mortality, death by pecking or cannibalism and viability. The results showed improvement of the indicators live weight (1640.0 g/1585.0 g), intake (10780 kg DM), conversion (1.92/2.10), egg production (15 540/15 397), craked eggs (1092/1114) and disqualified (69/76) for P<0.01 with respect to the control group. The indicators mortality (1/4), viability (99.6/98.6 %) and death by pecking/cannibalism (1/2) did not show differences between treatments.

^{Milián et al. (2021)}, when evaluating the zootechnical additive SUBTILPROBIO^® E-44 in productive and health indicators in Heavy Pure Breed birds, recently reported that the productive indicators live weight, uniformity and conversion showed significant values for (P<0.001) with respect to the control group and the standard for the breed under study. The indicators mortality (3.8/8) and viability (96.2/92 %) showed differences with respect to the control for P<0.01.

Studies performed by ^{Morales et al. (2020)} showed that when they evaluated a multienzyme complex, composed of amylases, proteases and xylanases, and the probiotic with Bacillus subtilis spores, in sorghum-soybean-rapeseed diets, the results of productive yield showed differences (P<0.05) in egg weight. These authors reported that lower weight was obtained with the treatment (60.0/58.9/59.2), but with the addition of enzymes and probiotics, an increase was shown. For the variables cholesterol (100.2/109.7/122.9 mg/dL), LDL (13.9/17.3/16.2 mg/dL) and HDL (30.0/37.0/35.7 mg/dL) there were not differences (P>0.05) between treatments.

^{Rodríguez et al. (2015)}, when evaluating a probiotic mixture of two zootechnical additives (PROBIOLACTIL^® C65 and SUBTILPROBIO^® E-44) with respect to the standard in birds of Heavy Pure Breed B4 for five weeks, obtained positive results in weight gain from the third week of inclusion of biopreparations (793, 1249 and 1587 g). This result is reaffirmed in the reports by ^{Valdés (2018)} and ^{Rondón et al. (2020b)}, when they refer to the use of mixtures of microorganisms in biopreparations for animal production.

^{Vélez et al. (2019)} evaluated a Bacillus subtilis probiotic in Cobb 500 broiler category. The measured indicators were productive, such as weight gain, feed conversion rate, American efficiency factor, carcass weight, mortality, changes in villi and pathological analysis. The mentioned authors showed that the probiotic Bacillus subtilis has a positive effect on the productive parameters.

Regarding the use of probiotics in poultry farming, there are infinite studies that report and show the effectiveness of biopreparations with Bacillus spp. endospores, a criterion that is refuted by the Engineering your feed solutions (ORFFA). This entity ensures that sporulated probiotics are the most natural option to produce healthy birds, highly effective and very competitive, if used in poultry production, so that their productive yields were optimized (^{ORFFA 2021}).

7) Effect of Bacillus subtilis endospores on calves. In the evaluation studies of the zootechnical additive SUBTILPROBIO^®, in the category of lactating calves from Siboney de Cuba breed, ^{Hernández (2012)} observed that the calves witch intake the zootechnical additive had a live weight higher (probiotic GT 169/Control group 110 kg) upon transfer to the development unit. In this case, the health indicators, incidence of diarrhea (1/7) and pneumonia (-/2), and mortality (-/4) were measured for P≤0.05.

^{Silva (2013)} showed that by supplying the probiotic biopreparation SUBTILPROBIO^® C-31 in lactating calves from Siboney de Cuba breed for 90 days, an increase in the productive weight indicator (74.6/58.3 kg) is achieved with respect to the control for P≤0.05.

Conclusions

Bacillus subtilis is a bacterium with a probiotic effect which is in the digestive tract of zootechnical interest animals and in other environments. The researches carried out to date show the in vitro and in vivo potentialities of this bacterium and its endospores.

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Received: December 12, 2021; Accepted: April 05, 2022

^*Email: [e-mail]

Conflict of interests: The authors declare that there is no conflict between them

Authors contribution: Grethel Milián: Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Writing - original draft. Ana J. Rondón: Investigation, Project administration, Funding acquisition, Supervision. Marlen Rodríguez:Investigation, Project administration, Funding acquisition, Supervision. A. Beruvides: Investigation, Project administration, Funding acquisition, Supervision, Writing - original draft. M. L. Pérez: Investigation, Project administration, Funding acquisition, Supervision.