Cultivable bacterial diversity and amylase production in three typical Daqus of Chinese spirits

Both culture‐dependent method and molecular technique were firstly used to simultaneously investigate the cultivable bacterial diversity and amylase production in three typical Daqus of Chinese spirits. The results showed that both cultivable bacterial diversity and amylase production were obviously different. The species of nine bacteria from Deshan, nine from Baisha and six from Wuling Daqus were identified. The total bacterial strains of 17, 15 and 14, and 9, 16 and 10 could produce α‐amylase and glucoamylase, respectively, from the Daqus, and the enzyme yields were different. Bacillus licheniformis, Bacillus subtilis and Bacillus amyloliquefaciens not only were dominant bacteria in the Daqus, but also possessed high activities of α‐amylase and glucoamylase. By comparison, Bacillus cereus and Bacillus oleronius were found to be another predominant bacterial species and good producers of α‐amylase and glucoamylase in Deshan and Wuling Daqus, respectively.     

Evaluation of in vitro probiotic potential of phytase‐producing bacterial strain as a new probiotic candidate

Sixty‐three phytase‐producing bacterial strains were isolated from natural sources, and their probiotic potential was evaluated. Of these, only fifteen strains were selected on the basis of confirmatory plate assay. Among these, five phytase‐producing strains exhibiting potent probiotic properties were identified as Bacillus cereus P1, Bacillus subtilis P6, B. subtilis P7, Pseudomonas aeruginosa P12 and P. aeruginosa P15 on the basis of morphological, biochemical and molecular characteristics. Maximum phytase activity (2.74 EU mL^?1) with potential probiotic properties, that is more than 70% survivability at pH 2.0, up to 2.0% bile salt tolerance, sporulation efficiency of more than 80% and survival in anaerobic condition (94.31%), was revealed by B. subtilis P6 as compared to the well‐established commercial probiotic strains Lactobacillus sporogenes and Lactobacillus casei. Thus, phytase‐producing B. subtilis P6 with promising probiotic features can be used in food and animal feed applications for betterment of mankind after further validation.     

Effect of thermosonication in a batch system on the survival of spore‐forming bacteria

Thermosonication may help reduce bacteria counts responsible for spoilage in dairy products. Vegetative cells and spores of Geobacillus stearothermophilus, Anoxybacillus flavithermus and Bacillus subtilis (spores only) were treated with either heat alone or thermosonication in a batch system from 0 to 120 s in tryptic soy broth and 2% fat milk at 72 and 73 °C. D‐values for vegetative cells were calculated and were reduced after thermosonication. Maximum reduction in vegetative cells after thermosonication was 1 log after 30–45 s and for spores was ≤0.2 log after 120 s, which may not influence dairy product quality in scale‐up systems.     

Essential oil composition and antibacterial activity of Thymus caramanicus at different phenological stages

Thymus species are well known as medicinal plants because of their biological and pharmacological properties. Thymus caramanicus is an endemic species grown in Iran. Variation in the quantity and quality of the essential oil of wild population of T. caramanicus at different phenological stages including vegetative, floral budding, flowering and seed set are reported. The oils of air-dried samples were obtained by hydrodistillation. The yields of oils (w/w%) at different stages were in the order of: flowering (2.5%), floral budding (2.1%), seed set (2.0%) and vegetative (1.9%). The oils were analyzed by GC and GC–MS. In total 37, 37, 29 and 35 components were identified and quantified in vegetative, floral budding, full flowering and seed set, representing 99.3, 98.6, 99.2 and 97.8% of the oil, respectively. Carvacrol was the major compound in all samples. The ranges of major constituents were as follow: carvacrol (58.9–68.9%), p-cymene (3.0–8.9%), γ-terpinene (4.3–8.0%), thymol (2.4–6.0%) and borneol (2.3–4.0%). Antibacterial activity of the oils and their main compounds were tested against seven Gram-positive and Gram-negative bacteria by disc diffusion method and determining their minimum inhibitory concentration (MIC) values. The inhibition zones (IZ) and MIC values for bacterial strains, which were sensitive to the essential oil of T. caramanicus, were in the range of 15–36 mm and 0.5–15.0 mg/ml, respectively. The oils of various phenological stages showed high activity against all tested bacteria, of which Bacillus subtilis and Pseudomonas aeruginosa were the most sensitive and resistant strains, respectively. Thus, they represent an inexpensive source of natural antibacterial substances that exhibited potential for use in pathogenic systems.     

Antimicrobial Activity of Monoacyl Hexose Coexistent with Lysozyme against Gram-Positive Bacilli

The antimicrobial activities of myristoyl, palmitoyl, or stearoyl hexoses, which were glucose, mannose, and galactose, coexistent with lysozyme against three Gram-positive bacteria, Bacillus coagulans, Bacillus subtilis, and Bacillus licheniformis, were measured in order to investigate the availability of monoacyl hexose as an antimicrobial co-agent. The lysozyme exhibited an antimicrobial activity against Bacillus subtilis and Bacillus licheniformis, but there was no significant difference between the dependencies of the antimicrobial activity against the two bacteria on the lysozyme concentration. However, the antimicrobial activities of the monoacyl hexoses coexistent with the lysozyme were different between those against the two bacteria. The stearoyl hexose coexistent with the lysozyme exhibited the highest antimicrobial activity against two bacteria. It was indicated that the antimicrobial action of the monoacyl hexose would be exerted parallel with the bacterial lysis of lysozyme. Stearoyl hexose, which has a high hydrophobicity, coexistent with the lysozyme could exhibit a higher antimicrobial activity than only the lysozyme.     

High pressure inactivation kinetics of bacillus subtilis cells by a three‐state‐model considering distributed resistance mechanisms

Inactivation of vegetative Bacillus subtilis ATCC 9372 by high hydrostatic pressure treatment (200 ‐ 450 MPa) was tested at 20, 30 and 40°C. Time ‐ inactivation curves of the bacteria suspended in Ringer's solution showed sigmoid asymmetric shapes when plotted in logarithmic scale. Kinetic analysis of the survivor data was performed by fitting a two‐step‐model. It was assumed that during pressure treatment, the bacterial cells pass through a metastable intermediate state which is reached after endogenous homeostatic mechanisms balancing the pressure induced displacements of equilibria can no longer be maintained. Combined pressure‐temperature‐pH effects may target this state and cause lethal cell damage. Modelling this concept, a distributive function describing the initial transition was used in combination with a first‐order reaction which was assumed to govern the irreversible second step. Regressively derived characteristic parameters showed logarithmic‐linear behaviour. Applicability of the model on dynamic treatments with constant rate of pressure increase could be proven.     

Starter culture evaluation for the production of ugba from African oil bean seed Pentaclethra macrophylla

Ugba was prepared in the laboratory by the traditional method from the African oil bean seeds Pentaclethra macrophylla. Microorganisms were isolated from the fermenting slices of the beans at 24 h intervals for 5 days and characterised. The results show that only bacteria were isolated from the bean slices. Fungi and yeasts were not isolated. The bacterial isolates were identified as Bacillus subtilis, Bacillus cereus, Pseudomonas chlororaphis, Micrococcus roseus and Staphylococci saprophyticus. The identified bacterial isolates were evaluated for their ability to ferment the cooked oil bean slices into ugba with its desirable quality characteristics of colour, texture, aroma and overall acceptability. Bacillus subtilis was significantly (P<0·05) more active in fermenting the oil bean than the other isolates. Pseudomonas chlororaphis fermented the oil bean actively but caused greening of the slices probably due to the production of the green chloraphin pigment. Staphylococcus saprophyticus, M roseus and B cereus appeared unimportant in the fermentation and may be contaminants. Bacillus subtilis was therefore selected as the single starter culture for ugba production. © 1998 SCI.     

3‐Hydroxypyridinone‐l‐phenylalanine conjugates with antimicrobial and tyrosinase inhibitory activities as potential shrimp preservatives

To explore the potential of novel tyrosinase inhibitors, 3‐hydroxypyridinone‐l ‐phenylalanine conjugates, as shrimp preservatives, compounds 1 and 2 were evaluated for the antimicrobial activity, and compound 2 was investigated for the shrimp preservative efficacy. It was found that they both possess a stronger antibacterial effect than kojic acid against two Gram‐positive bacteria and three Gram‐negative bacteria. The minimum inhibitory concentrations (MICs) of compound 2 against Escherichia coli, Staphylococcous aureus, Salmonella gallinarum, Vibrio parahaemolyticus and Bacillus subtilis were determined as 18.5, 37, 148, 37 and 295 μg mL^?1, respectively, whereas MICs of kojic acid against the same 5 bacterial strains were determined to be 355, 178, 1420, 1420 and 355 μg mL^?1, respectively. It has also been demonstrated that treatment with compound 2 improves the sensory properties, retards the growth of spoilage bacteria, decreases the total volatile basic nitrogen (TVB‐N) and increases the pH of Penaeus vannamei Boone, thereby extending the shelf life to 10 days. In contrast, the shelf life of shrimp treated with kojic acid and the control group was 7 and 6 days, respectively. Clearly, 3‐hydroxypyridinone‐l ‐phenylalanine conjugates could find application as shrimp preservatives by inhibiting melanosis and by preventing the growth of bacteria during the storage.     

Investigation of the microbial changes during koji‐making process of Douchi by culture‐dependent techniques and PCR‐DGGE

Douchi as a traditional fermentation soyfood with an acquired taste had been appreciated by consumers for thousands of years in China and few Asian countries. Unfortunately, few studies had been conducted on the changes of microbial community during the koji‐making process, and whether the pathogenic microorganisms involving the fermentation process is still unclear. Therefore, the PCR‐denaturing gradient gel electrophoresis (DGGE) targeting the 16S and 18S rRNA genes was applied to characterise the dynamic changes of Bacteria, Bacilli and Fungi during koji‐making process. The results of DGGE showed that eleven species of bacteria, nine species of bacilli and seven species of Fungi had been detected during the koji‐making progress, of which the Bacillus subtilis and Aspergillus oryzae belonged to the dominant microorganisms. Also, eleven strains were isolated from Koji‐making samples and were identified as B. subtilis, Bacillus amyloliquefaciens, A. oryzae, Brachybacterium sp., Aspergillus niger, Staphylococcus saprophyticus, Micrococcus sp., Staphylococcus gallinarum, Absidia corymbifera, Saccharomyces cerevisiae, Malassezia sp.; Our results revealed that pathogenic microorganisms were involved in the koji‐making process, but the probiotic microorganisms occupied the dominant position of community in the koji‐making process.     

Composition, quality control and antimicrobial activity of the essential oil of cumin (Cuminum cyminum L.) seeds from Bulgaria that had been stored for up to 36 years

The essential oil of seeds of cumin (Cuminum cyminum L.) from Bulgaria stored for more than 35 years was analyzed by physicochemical methods, GC, GC‐MS and olfactometry and its antimicrobial activity tested using different strains of microorganisms. More than sixty constituents of this cumin oil could be identified as essential volatiles, responsible for the pleasant fresh, clean, spicy (typical cumin‐like) odour of a high quality product. Cumin aldehyde (36%), β‐pinene (19.3%), p‐cymene (18.4%) and γ‐terpinene (15.3%) were the principal compounds found. Antimicrobial testing showed high activity of the essential C. cyminum oil against the mold Aspergillus niger, the Gram (+) bacteria Bacillus subtilis and Staphylococcus epidermidis as well as the yeast Saccharomyces cerevisiae and Candida albicans.     

Antibacterial activity of chitosans with different degrees of deacetylation and viscosities

Antibacterial activities of six acid‐soluble [two degrees of deacetylation (DD) × three viscosities] and two water‐soluble chitosans (two DD with similar viscosities) were examined against eight gram‐negative (Pseudomonas fluorescens, Proteus vulgaris, Erwinia carotovora, Serratia marcescens, Escherichia coli, Vibrio parahaemolyticus, V. vulnificus, and Salmonella Typhimurium) and six gram‐positive bacteria (Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis, B. cereus, Lactobacillus curvatus, and L. plantarum). Antibacterial activities of chitosans differed depending on the chitosan types and bacteria tested. Water‐soluble chitosans inhibited bacterial growth by one to eight log cycles at 0.1% concentration; however, the effects were much lesser than those observed with 0.05% acid‐soluble chitosans. Minimum inhibitory concentration (MIC) values (0.03% to above 0.1%) of acid‐soluble chitosans were much lower than those (0.05% to above 0.8%) of water‐soluble chitosans. Based on MIC values, the acid‐soluble chitosan with 99% DD and lower viscosity (17.9 mPa s) was most effective in inhibiting bacteria growth among eight chitosans tested.     

Genome Subtyping of Autochthonous Bacillus Species Isolated from Iru,a Fermented Parkia biglobosa Seed

Phylogenetic relationship and strains sub-typing of Bacillus species isolated from iru, a traditional fermented condiment in Africa were studied using polyphasic genomic approaches and the profiles compared with bacilli isolated from similar Asian condiments. The 16S rRNA gene sequencing identified the strains as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, and Brevibacillus formosus. The phylogenetic analysis conducted showed five distinct clusters with genetic relatedness among B. subtilis and B. amyloliquefaciens strains from Africa and Asia. Amplified ribosomal DNA restriction analysis (ARDRA) successfully differentiated species of B. subtilis phylogeny from B. cereus. Combined analyses of ARDRA, internal transcribed spacer-polymerase chain reaction (ITS-PCR), ITS-PCR-restriction fragment length polymorphism (ITS-PCR-RFLP) and randomly amplified polymorphic DNA (RAPD-PCR) further confirmed B. subtilis and B. amyloliquefaciens as the dominant Bacillus species associated with fermentation of iru, and revealed high strains genetic diversity, while multilocus sequence analysis (MLSA) data distinguished B. cereus from B. thuringiensis. This information is essential for selection of starter cultures with desirable functional attributes to guarantee product consistency and safety quality of traditional fermented foods.     

Molecular Identification of Microbial Community in Surface and Undersurface Douchi During Postfermentation

To find the reason for fermentation failure of surface Douchi during postfermentation, the microbial communities in undersurface and surface samples were investigated using cell counting method and denaturing gradient gel electrophoresis (DGGE). The results showed that the microbial biomass in surface Douchi was obviously different from that in undersurface Douchi even sampled from the same fermentation tanks, and a 10‐ to 100‐fold reduction of microbial cell counts in undersurface had been observed. The bacterial DGGE profile and principal component analysis (PCA) results indicated that only Lactococcus lacts subsp. lactis and Bacillus thermoamylovorans were detected from surface Douchi, while Lactococcus lacts subsp. lactis, Staphylococcus lentus and 2 uncultured strains occupied the dominant positions in undersurface Douchi; when amplified using Bacillus‐specific primers, Bacillus thermoamylovorans, Bacillus subtilis, and Enterobacter sp. were found in undersurface Douchi, while only Bacillus thermoamylovorans were detected from surface Douchi; compared to the bacteria and Bacillus, the DGGE profiles and PCA plot of fungi indicated that the fungal community between surface and undersurface Douchi was similar and mainly composed by yeasts. In this study, we detected the microbial biomass and species in postfermentation stage of Douchi, and the various microbial diversity in undersurface and surface samples might be the cause of the fermentation failure in surface fermentation tanks.     

ANTIOXIDATIVE AND ANTIBACTERIAL POTENTIALS OF ESSENTIAL OILS AND EXTRACTS ISOLATED FROM VARIOUS SPICE MATERIALS†

The antioxidant and antibacterial potentials of essential oils and acetone extracts of black pepper, cumin, black cumin and mace were carried out by different techniques. The antioxidative capacity of the essential oils and acetone extracts were evaluated against mustard oil by measuring peroxide and thibarbituric acid values at fixed intervals. In addition, their antioxidant potential was evaluated by 2, 2′‐diphenyl‐1‐picrylhydracyl radical and conjugated diene assays. Their reducing power was determined with standards, which proved the strong antioxidant capacity of essential oils and extracts. The antioxidant activity of essential oils and extracts exerted by all the antioxidant assays can be compared with synthetic antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene. The antibacterial activity was studied by disk diffusion and poison food methods. Black cumin essential oil showed complete zone of inhibition (P < 0.05) against tested bacterial strains of Staphylococcus aureus, Bacillus cereus and Bacillus subtilis at 2 and 6 µL level by disk diffusion method. Black cumin and black pepper extracts showed complete reduction of colonies against tested bacterial strains of S. aureus, B. cereus and B. subtilisat 5 and 10 µL level by poison food method. Poison food method exhibited good results for the tested essential oils and extracts. Essential oils of black pepper, cumin, black cumin and mace may be used to stabilize mustard oil after screening.     

Bacillus subtilis HJ18‐4 from Traditional Fermented Soybean Food Inhibits Bacillus cereus Growth and Toxin‐Related Genes

Bacillus subtilis HJ18‐4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad‐spectrum antimicrobial activity against foodborne pathogens, including Bacillus cereus. In this study, we investigated the antibacterial efficacy and regulation of toxin gene expression in B. cereus by B. subtilis HJ18‐4. Expression of B. cereus toxin–related genes (groEL, nheA, nheC, and entFM) was downregulated by B. subtilis HJ18‐4, which also exhibited strong antibacterial activity against B. cereus. We also found that water extracts of soy product fermented with B. subtilis HJ18‐4 significantly inhibited the growth of B. cereus and toxin expression. These results indicate that B. subtilis HJ18‐4 could be used as an antimicrobial agent to control B. cereus in the fermented soybean food industry. Our findings also provide an opportunity to develop an efficient biological control agent against B. cereus.     

Elimination of the toxic effects of patulin onBacillus subtilis andB.megaterium by irradiation

Summary The bacteriological toxicity of patulin irradiated in aqueous solutions was investigated using two strains of bacteria,Bacillus subtilis andB. megaterium. The results of the plate cultivation test showed that the growth inhibition of both strains decreased in relation to absorbed dose. A dose of 1.8 kGy was sufficient to eliminate the bacteriological toxicity of patulin at an initial concentration of 125 g/ml. There was good correlation between the concentration of patulin in the irradiated solution as measured spectrophotometrically and that calculated from the diameter of the inhibition zone.

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Beseitigung der toxischen Wirkung von Patulin aufBacillus subtilis undB.megaterium durch Bestrahlung

Zusammenfassung Es wurde die Toxizität von Patulin nach der Bestrahlung in wäßrigen Losungen bei der Anwendung von zwei Bakterienstämmen,Bacillus subtilis undB. megaterium, untersucht. Die Ergebnisse des Plattenkultivierungstests haben gezeigt, daß die Wachstumsinhibition der beiden Stämme sich im Verhältnis zur Bestrahlungsdosis vermindert. Die Bestrahlungsdosis von 1,8 kGy war ausreichend zur Eliminierung der bakteriologischen Toxizität von Patulin bei der Anfangskonzentration von 125 g/ml. In den bestrahlten Lösungen wurde eine klare Korrelation zwischen der spektrophotometrisch gemessenen und der aus dem Durchmesser der Inhibitionszonen berechneten Patulinkonzentration festgestellt.

     

Incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia

The purpose of this study was to evaluate the incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia. The distribution of Bacillus species in raw milk, pasteurised milk and UHT milk were 47.5%, 27.5% and 25%, respectively. Seven Bacillus species, including Bacillus pumilus (10%), Bacillus subtilis (12.5%), Brevibacillus brevis (10%), Bacillus cereus (22.5%), Bacillus sphaericus (7.5%), Bacillus licheniformis (12.5%) and Bacillus sporothermodurans (25%) were identified in different milk samples. Bacillus cereus was predominant in raw and pasteurised milk. Although B. sporothermodurans was the predominant sporogenous micro‐organism in UHT milk, B. cereus, B. sphaericus and B. licheniformis were also present. This study showed that there is a high degree of diversity, both phenotypic and genotypic, among Bacillus isolates from Tunisian milk and the persistence of spoilage risk in UHT milk.     

Enzymatic synthesis of glycosylated puerarin using maltogenic amylase from Bacillus stearothermophilus expressed in Bacillus subtilis

BACKGROUND: The maltogenic amylase from Bacillus stearothermophilus (BSMA) is a valuable biocatalyst that has been used to transglycosylate natural glycosides to improve solubility. To ensure safety, BSMA was produced in Bacillus subtilis, using new shuttle vector‐based expression vectors. The transglycosylation of puerarin was also conducted with crude BSMA and analyzed. RESULTS: Two expression systems, each containing one of the promoters from the genes encoding Bacillus licheniformis maltogenic amylase (BLMA) and an α‐amylase from B. subtilis NA64 (amyR2), were constructed. The amyR2 promoter system was chosen as the best system; it yielded 107 mg of pure BSMA from a 2 L culture. In the transglycosylation reactions of puerarin using crude BSMA, relative amounts for maltosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 3)‐puerarin, and puerarin were determined as 26:18:7:49. A two‐step purification process, including gel permeation chromatography, yielded 1.7 g of the transfer products from 3 g of puerarin. CONCLUSION: The crude BSMA produced from a host generally recognized as safe (B. subtilis) can be used to transglycosylate various functional compounds. The expression system developed in this study will be helpful for the production of other food‐grade enzymes by B. subtilis. Copyright © 2010 Society of Chemical Industry     

Evaluation Antibacterial Activity of Quaternary‐Based Chitin/Chitosan Derivatives In Vitro

Abstract: Total of 3 water‐soluble quaternary‐based chitin/chitosan derivatives, which have an identical molecular weight and anion, were synthesized and characterized. Their antibacterial activities against Salmonella cholerae‐suis and Bacillus subtilis were evaluated in vitro. The polysaccharides exhibited the antibacterial efficiency. Their minimum inhibitory concentration (MIC) values vary from 0.02 to 20.48 mg/mL, and their minimum bactericidal concentration (MBC) values vary from 0.08 to 40.96 mg/mL against S. cholerae‐suis and B. subtilis, respectively. Futhermore, the extent of Bacillus subtilis cells damage was examined via transmission electron microscopy (TEM) to show how N,N,N‐trimethylchitosan (TMC) gradually destroyed and killed B. subtilis cells when they were treated with TMC. One of those quaternary polymers, O‐([2‐hydroxy‐3‐trimethylammonium])propyl chitin (OHT‐chitin), which can be directly and easily synthesized from chitin in bulk quantities, also was demonstrated its antibacterial activity. These water soluble quaternary‐based chitin/chitosan derivatives that have antibacterial effect should be potentially used as antimicrobial agents in many fields. Practical Application: The main practical application behind the investigation and evaluation antibacterial activity of 3 water‐soluble quaternary‐based chitin/chitosan derivatives could be potentially used as antimicrobial agents in many fields. These polysaccharides represent a renewable source of natural biodegradable polymers and meet with the emergence of more and more food safe problems.