In Silico Safety Assessment of Bacillus Isolated from Polish Bee Pollen and Bee Bread as Novel Probiotic Candidates.

Bacillus species isolated from Polish bee pollen (BP) and bee bread (BB) were characterized for in silico probiotic and safety attributes. A probiogenomics approach was used, and in-depth genomic analysis was performed using a wide array of bioinformatics tools to investigate the presence of virulence and antibiotic resistance properties, mobile genetic elements, and secondary metabolites. Functional annotation and Carbohydrate-Active enZYmes (CAZYme) profiling revealed the presence of genes and a repertoire of probiotics properties promoting enzymes. The isolates BB10.1, BP20.15 (isolated from bee bread), and PY2.3 (isolated from bee pollen) genome mining revealed the presence of several genes encoding acid, heat, cold, and other stress tolerance mechanisms, adhesion proteins required to survive and colonize harsh gastrointestinal environments, enzymes involved in the metabolism of dietary molecules, antioxidant activity, and genes associated with the synthesis of vitamins. In addition, genes responsible for the production of biogenic amines (BAs) and D-/L-lactate, hemolytic activity, and other toxic compounds were also analyzed. Pan-genome analyses were performed with 180 Bacillus subtilis and 204 Bacillus velezensis genomes to mine for any novel genes present in the genomes of our isolates. Moreover, all three isolates also consisted of gene clusters encoding secondary metabolites.

Probiotics enhance resistance to Streptococcus iniae in Nile tilapia (Oreochromis niloticus) reared in biofloc systems.

Biofloc technology is a rearing technique that maintains desired water quality by manipulating carbon and nitrogen and their inherent mixture of organic matter and microbes. Beneficial microorganisms in biofloc systems produce bioactive metabolites that may deter the growth of pathogenic microbes. As little is known about the interaction of biofloc systems and the addition of probiotics, this study focused on this integration to manipulate the microbial community and its interactions within biofloc systems. The present study evaluated two probiotics (B. velezensis AP193 and BiOWiSH FeedBuilder Syn 3) for use in Nile tilapia (Oreochromis niloticus) culture in a biofloc system. Nine independent 3785 L circular tanks were stocked with 120 juveniles (71.4 ± 4.4 g). Tilapia were fed for 16 weeks and randomly assigned three diets: a commercial control diet or a commercial diet top-coated with either AP193 or BiOWiSH FeedBuilder Syn3. At 14 weeks, the fish were challenged with a low dose of Streptococcus iniae (ARS-98-60, 7.2 × 107 CFU mL-1 , via intraperitoneal injection) in a common garden experimental design. At 16 weeks, the fish were challenged with a high dose of S. iniae (6.6 × 108 CFU mL-1 ) in the same manner. At the end of each challenge trial, cumulative per cent mortality, lysozyme activity and expression of 4 genes (il-1ß, il6, il8 and tnfα) from the spleen were measured. In both challenges, the mortalities of the probiotic-fed groups were significantly lower (p < .05) than in the control diet. Although there were some strong trends, probiotic applications did not result in significant immune gene expression changes related to diet during the pre-trial period and following exposure to S. iniae. Nonetheless, overall il6 expression was lower in fish challenged with a high dose of ARS-98-60, while tnfα expression was lower in fish subjected to a lower pathogen dose. Study findings demonstrate the applicability of probiotics as a dietary supplement for tilapia reared in biofloc systems.

Safety assessment of surfactin-producing Bacillus strains and their lipopeptides extracts in vitro and in vivo.

Beneficial Bacillus strains can be administered to livestock as probiotics to improve animal health. Cyclic lipopeptides produced by Bacillus such as surfactins may be responsible for some of the beneficial effects due to their anti-inflammatory and immunomodulatory activity. The aim of the present study was to isolate and evaluate the biocompatibility of native Bacillus spp. strains and their surfactin-like lipopeptides in vitro and in vivo to determine their potential to be used on animals. Biocompatibility of endospore suspensions (108 UFC/mL), and different dilutions (1:10; 1:50; 1:100; 1:500, and 1:1000) of Bacillus lipopeptide extracts containing surfactin was tested on Caco-2 cells by microculture tetrazolium-based colorimetric assay. Genotoxicity was tested on BALB/c mice (n = 6) administered 0.2 mL of endospore suspensions by the bone marrow erythrocyte micronuclei assay. All the isolates tested produced between 26.96 and 239.97 µg mL- 1 of surfactin. The lipopeptide extract (LPE) from isolate MFF1.11 demonstrated significant cytotoxicity in vitro. In contrast, LPE from MFF 2.2; MFF 2.7, TL1.11, TL 2.5, and TC12 had no cytotoxic effect (V% > 70%) on Caco-2 cells, not affecting cell viability signifficantly in most treatments. Similarly, none of the endospore suspensions affected cell viability (V% > 80%). Likewise, endospores did not cause genotoxicity on BALB/c mice. This study was elementary as a first step for a new line of research, since it allowed us to choose the safest isolates to keep working on the search of new potentially probiotic strains destined to production animals to improve their performance and health.

Mineral-Solubilizing Bacteria-Mediated Enzymatic Regulation and Nutrient Acquisition Benefit Cotton's (Gossypium hirsutum L.) Vegetative and Reproductive Growth.

Many farmers' incomes in developing countries depend on the cultivation of major crops grown in arid and semi-arid regions. The agricultural productivity of arid and semi-arid areas primarily relies on chemical fertilizers. The effectiveness of chemical fertilizers needs to improve by integration with other sources of nutrients. Plant growth-promoting bacteria can solubilize nutrients, increase plant nutrient uptake, and supplement chemical fertilizers. A pot experiment evaluated the promising plant growth-promoting bacterial strain's effectiveness in promoting cotton growth, antioxidant enzymes, yield, and nutrient uptake. Two phosphate solubilizing bacterial strains (Bacillus subtilis IA6 and Paenibacillus polymyxa IA7) and two zinc solubilizing bacterial strains (Bacillus sp. IA7 and Bacillus aryabhattai IA20) were coated on cotton seeds in a single as well as co-inoculation treatments. These treatments were compared with uninoculated controls in the presence and absence of recommended chemical fertilizer doses. The results showed the co-inoculation combination of Paenibacillus polymyxa IA7 and Bacillus aryabhattai IA20 significantly increased the number of bolls, seed cotton yield, lint yield, and antioxidants activities, including superoxide dismutase, guaiacol peroxidase, catalase, and peroxidase. Co-inoculation combination of Bacillus subtilis IA6 and Bacillus sp. IA16 promoted growth attributes, including shoot length, root length, shoot fresh weight, and root fresh weight. This co-inoculation combination also increased soil nutrient content. At the same time, Paenibacillus polymyxa IA7 + Bacillus aryabhattai IA20 increased nutrient uptake by plant shoots and roots compared.

Effects of Bacillus spp. Mixture on Growth, Immune Responses, Expression of Immune-Related Genes, and Resistance of Nile Tilapia Against Streptococcus agalactiae Infection.

The purpose of this study was to evaluate the effect of Bacillus spp. mixture (Bacillus subtilis TISTR001, Bacillus megaterium TISTR067, and Bacillus licheniformis DF001) (1 × 106 CFU/g) on growth, immune parameters, immune-related gene expression, and resistance of Nile tilapia against Streptococcus agalactiae AAHM04. Fish were fed different concentrations of Bacillus spp. 0 (control; T1), 1 (T2), 3 (T3), and 5 (T4) g/kg diets for 120 days. The results showed that weight gain, average daily gain, specific growth rate, feed conversion ratio in T3 diet were significantly higher than the control group and other tested diets (p < 0.05). Immune parameters, such as myeloperoxidase and lysozyme, were significantly higher in the T3 and T4 diets compared to the control group (p < 0.05). Similarly, IL-1ß and TNF-α gene expressions in the spleen of fish fed T2, T3, and T4 diets were significantly higher than the control group (p < 0.05). However, no significant differences in survival rate, hematology, blood chemical indices, malondialdehyde (MDA) levels, body chemical composition, and organosomatic indices (p > 0.05) were noticed in all treatments. No significant differences in survival rate after the challenge test with S. agalactiae AAHM04 were found in fish fed Bacillus spp. mixture diets, except for the T3 diet. These results suggest that Bacillus spp. mixture diet at 3 g/kg diet (T3) could improve growth, immune response, and disease resistance of Nile tilapia.

Bioinoculation using indigenous Bacillus spp. improves growth and yield of Zea mays under the influence of nanozeolite.

Bio-inoculants play an important role for sustainable agriculture. Application of nanocompounds in the agriculture sector provides strength and is reported to enhance crop production but the combined effect of nanocompounds and plant growth-promoting rhizobacteria on plants has not been studied much. Therefore, the present study was planned to observe the effect of two plant growth promotory Bacillus spp. along with nanozeolite on maize under field conditions using a randomized block design. Combined treatment of nanozeolite and bio-inoculants promoted plant height, root length, fresh and dry weight of shoot and root, chlorophyll, carotenoids, total sugar, protein and phenol contents in maize significantly over control. Enhanced level of catalase, peroxidase, superoxide dismutase, phenols, alcohols and acid-esters in treated plants over control showed their role in stress management. An increase of 29.80% in maize productivity over control was reported in the combined treatment of Bacillus sp. and nanozeolite. Our results indicate that the application of bio-inoculants with nanozeolite showed a positive response on the health and productivity of maize plants. Hence, these may be used to enhance the productivity of different crops. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02561-2.

In-vitro Antimicrobial Activity of Essential Oils and Spices Powder of some Medicinal Plants Against Bacillus Species Isolated from Raw and Processed Meat.

BACKGROUND AND AIM: Bacillus species are widely distributed microorganisms in nature that are responsible for outbreaks of food poisoning and a common cause of food spoilage. This study aimed to isolate and identify foodborne Bacillus species from meat and to determine the antimicrobial activities of commercial essential oils and spices powder extracted from certain medicinal plants. METHODS: Sixty meat samples were collected in Assiut city and subdivided into raw meat and processed meat. Bacillus spp were isolated and identified according to their cultural characters, biochemical reactions, serological typing, and 16S rRNA gene sequencing. The antibacterial activity of essential oils and spices powder was measured by using well-diffusion and microbial count techniques. RESULTS: The prevalence of Bacillus spp. in the examined raw meat samples and processed meat samples was 13.34%, and 26.67%, respectively. There was a marked decrease in the total Bacillus species count after treatment of minced beef with essential oils and spices powder compared to the untreated one. Black seed oil was the most potent antibacterial essential oil among the tested oils present in this study. CONCLUSION: Essential oils and spices powder of certain medicinal plants (cumin: Cuminum cyminum, black seeds: Nigella sativa, cloves: Syzygium aromaicum, cinnamon: Cinnamomum zeylanicum, and Marjoram: Origanum majorana) have a potential in vitro antimicrobial activity against Bacillus spp. Furthermore, Nigella sativa oil exhibited the most potent antibacterial activity against Bacillus spp.

Effect-directed screening of Bacillus lipopeptide extracts via hyphenated high-performance thin-layer chromatography.

Bacillus species produce a wide array of biologically active metabolites, including nonribosomaly synthesized lipopeptides (LPs). The high-performance thin-layer chromatography (HPTLC) technique hyphenated with different bioassays and mass spectrometry was demonstrated as a valuable tool for effect-directed analysis of iturins, surfactins, fengycins and kurstakins homologues from complex mixtures of LPs. As proof of this straightforward strategy, the found surfactin and iturin A homologues were characterized and compared with reference substances. This study considered two different extraction methods for LPs produced by five Bacillus strains. The ethyl acetate extraction (Ex-1), and the acidic precipitation followed by methanol extraction (Ex-2) were investigated. Diverse enzyme inhibitions and antimicrobial potentials of LPs were analyzed, and in parallel, high-resolution mass spectra (HRMS) were online recorded from the HPTLC zones of interest. No antimicrobial effect against Gram-positive B. subtilis was evident for iturin, whereas a response was detected for surfactin. The nonpolar kurstakin compounds showed a pronounced B. subtilis antimicrobial activity in Ex-1 of almost all strains, whereas the fengycin homologues were detected in Ex-2 of SS-10.7 and SS-27.2. Iturin had also no activity against Gram-negative Aliivibrio fischeri, while again surfactin showed an enhancing luminescent activity. Contrary, kurstakin compounds caused a decrease in the luminescence in Ex-1 of all strains, particularly for SS-13.1. Both, iturin and surfactin showed a strong acetylcholinesterase (AChE) and α-glucosidase inhibition, but surfactin caused a much stronger inhibition. This was evident in all bacterial strains, except for SS-13.1 in Ex-1 and for SS-38.4 in Ex-2. Although, iturin and surfactin exhibited no DPPH˙ scavenging activity, Ex-1 of all strains contained more intense DPPH˙ scavenging compounds compared to Ex-2, and surfactin methyl esters showed a pronounced DPPH˙ activity, particularly in SS-12.6 in Ex-1. This study pointed to active metabolites of strains that can be used as therapeutics and biocontrol agents with beneficial effects on human health. The straightforward HPTLC profiling served as an excellent bioanalytical tool to control the formed bioactive metabolites. As the fermentation process is very sensitive to external influences, it could be a helpful control tool for standardization of the biotechnological processing.

Effects of dietary isomaltooligosaccharide and Bacillus spp. supplementation during perinatal period on lactational performance, blood metabolites, and milk composition of sows.

BACKGROUND: In this study, we evaluated the effects of isomaltooligosaccharide (IMO) and Bacillus spp. in perinatal diets on lactational performance, blood metabolites, and milk composition of sows. Multiparous gestating sows (N = 130) with similar body conditions were fed diets without containing IMO (control, CON group), IMO (IMO group), IMO and Bacillus subtilis (IMO + S group), IMO and Bacillus licheniformis (IMO + L group), and IMO and both B. subtilis and B. licheniformis (IMO + S + L group). RESULTS: The average daily feed intake of lactating sows in the IMO + S group (P < 0.05) and the average litter gain and total milk yield in the IMO + S + L group (P < 0.01) were significantly higher than those of the sows in the other treatment groups. In addition, the serum alanine aminotransferase and alkaline phosphatase concentrations in all treatment groups were lower than those in the CON group (P < 0.05). Furthermore, the highest proportion of fat in colostrum (0.05 < P < 0.1) and lowest proportion of lactose in milk were observed in the IMO + L group (P < 0.05). The concentrations of growth hormone and immunoglobulins A and G in milk were higher in sows supplemented with IMO, IMO + S, IMO + L, or IMO + S + L (P < 0.05). CONCLUSION: IMO and Bacillus spp. supplementation during the perinatal period improved the sows' lactational performance by affecting their metabolism and milk quality. Sows in the IMO + S and IMO + S + L treatment groups exhibited the best performance. © 2019 Society of Chemical Industry.

Zinc solubilizing Bacillus spp. potential candidates for biofortification in maize.

Bioaugmentation of Zn solubilizing rhizobacteria could be a sustainable intervention to increase bioavailability of Zn in soil which can be helpful in mitigation of yield loss and malnutrition of zinc. In present study, a number of pure rhizobacterial colonies were isolated from maize rhizosphere and screened for their ability to solubilize zinc oxide. These isolates were screened on the basis of zinc and phosphate solubilization, IAA production, protease production, catalase activity and starch hydrolysis. All the selected isolates were also positive for oxidase activity (except ZM22), HCN production (except ZM27) and utilization of citrate. More than 70% of isolates produces ammonia, hydrogen cyanide, siderophores, exopolysaccharides and cellulase. More than half of isolates also showed potential for urease activity and production of lipase. The ZM31 and S10 were the only isolates which showed the chitinase activity. All these isolates were evaluated in a jar trial for their ability to promote growth of maize under axenic conditions. Results revealed that inoculation of selected zinc solubilizing rhizobacterial isolates improved the growth of maize. In comparison, isolates ZM20, ZM31, ZM63 and S10 were best compared to other tested isolates in stimulating the growth attributes of maize like shoot length, root length, plant fresh and dry biomass. These strains were identified as Bacillus sp. (ZM20), Bacillus aryabhattai (ZM31 and S10) and Bacillus subtilis (ZM63) through 16S rRNA sequencing. This study indicated that inoculation of Zn solubilizing strains have potential to promote growth and can be the potential bio-inoculants for biofortification of maize to overcome the problems of malnutrition.

Biodegradation of Diesel, Crude Oil and Spent Lubricating Oil by Soil Isolates of Bacillus spp.

Two species of Bacillus, B. thuringiensis B3 and B. cereus B6, isolated from crude oil-contaminated sites in Ecuador, were tested for their capability in degrading polycyclic aromatic hydrocarbons (PAHs) in diesel (shake-flask), and to remove total petroleum hydrocarbons (TPHs) from crude oil- or spent lubricating oil-polluted soils (plot-scale). TPHs and PAHs were analyzed by Gas chromatography-Flame ionization detector (GC-FID) and High performance liquid chromatography (HPLC), respectively. Degradation percentages of PAHs by strain B6 were in the range of 11-83 after 30 days. A mixed culture of both the strains removed 84% and 28% of TPHs from crude oil- and spent lubricating oil-polluted soils, respectively. Reduction in the abundance of total n-alkane fractions (C8-C40) of spent lubricating oil was 94%, which was 18% higher than the control. Our results clearly indicate that the selected strains have great potential in degrading petroleum hydrocarbons at both laboratory- and field-scales.

Microbial degradation of total petroleum hydrocarbons in crude oil: a field-scale study at the low-land rainforest of Ecuador.

A field-level feasibility study was conducted to determine total petroleum hydrocarbon (TPH)-degrading potential of two bacterial strains, Bacillus thuringiensis B3 and B. cereus B6, and two fungi, Geomyces pannorum HR and Geomyces sp. strain HV, all soil isolates obtained from an oil field located in north-east region of Ecuador. Crude oil-treated soil samples contained in wooden boxes received a mixture of all the four microorganisms and were incubated for 90 days in an open low-land area of Amazon rainforest. The percent removal of TPHs in soil samples that received the mixed microbial inoculum was 87.45, indicating the great potential of the soil isolates in field-scale removal of crude oil. The TPHs-degrading efficiency was verified by determining the toxicity of residues, remained in soil after biodegradation, toward viability of Artemia salina or seed germination and plant growth of cowpea. Our results clearly suggest that the selected soil isolates of bacteria and fungi could be effectively used for large-scale bioremediation of sites contaminated with crude oil.

Probiotic Bacillus spp. in Soy-Curd: Nutritional, Rheological, Sensory, and Antioxidant Properties.

UNLABELLED: The focus of this study was to coculture probiotic Bacillus spp. with dairy starter cultures namely, Streptococcus thermophilus and Lactobacillus bulgaricus for enhanced nutritional properties of soy-curd. Subsequently, rheological, sensory, and antioxidant properties of soy-curd along with mineral as well as fatty acid composition were analyzed. Data revealed an increase in the cell viability of probiotic Bacillus spp. on coculturing rather than as mono-culture. Proximate analysis showed higher nutritional value along with increased trace elements. UFA/SFA ratio, rheology, and sensory properties of probiotic soy-curd were in the acceptable range. Probiotic soy-curd showed higher antioxidant activity as measured by the ability to scavenge free radicals. No significant difference in the overall quality within the probiotic products was observed. However, B. flexus MCC2427 cocultured product displayed slightly better attributes than other samples. In general, the results suggest that soy-curd can be a suitable carrier for probiotic Bacillus spp. and the enhanced nutritional and antioxidant properties could be of additional advantage to combat malnutrition problem. PRACTICAL APPLICATION: In order to supply consumers with intriguing probiotic products for improving health benefits, several criteria including technological and functional properties should be considered as a quality control measures. Further, a meaningful level of probiotics has to be viable to exhibit beneficial effect. Hence, present work has been carried out to improve the quality of soy-curd by supplementation of probiotic Bacillus spp. These Bacillus spp. are well characterized native probiotic cultures with potential functional attributes including antimicrobial, antioxidant, anticholesterol activity (Shobharani and Halami 2014). Hence, the application of these cultures will encourage for development of food product with wider health benefits.