Antimicrobial lysozyme-containing starch microgel to target and inhibit amylase-producing microorganisms

The aim of this study is to determine the release of lysozyme from oxidized starch microgels and subsequently test its antimicrobial activity. The gels are made of oxidized potato starch polymers, which are chemically cross-linked by sodium trimetaphosphate (STMP). The microgel is negatively charged and interacts with positively charged lysozyme by electrostatic attraction. Application of the lysozyme-containing starch particles to environments contaminated with microbes, may lead to hydrolysis of the starch by microbial enzymes. As a result, lysozyme is released in the environment where it inhibits microbial growth. In this study, first bacteria were screened for amylase production and lysozyme sensitivity. Then, the bacteria were mixed with empty gel particles (i.e., without lysozyme) in a Nutrient Broth liquid medium to test whether the bacteria that can produce amylase are also able to degrade oxidized starch gel. Subsequently the amylase-producing lysozyme sensitive bacteria, Bacillus licheniformis 7558 and Bacillus subtilis 168, were selected for further quantification of the antimicrobial activity of the gel-lysozyme particles after incubation with these bacteria in Nutrient Broth liquid suspensions. The results prove that the starch microgel has a potential as antimicrobial carrier targeting amylase-producing and lysozyme-sensitive bacteria. The controlled antimicrobial delivery for inactivating undesired microorganisms may find applications in food related systems, where amylase-producing bacteria may be abundantly present.     

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.     

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 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.     

Antibacterial activity of Thai edible plants against gastrointestinal pathogenic bacteria and isolation of a new broad spectrum antibacterial polyisoprenylated benzophenone,chamuangone

Twenty-two edible plant extracts were subjected to evaluation of their antibacterial activity against some gastrointestinal pathogenic bacteria, including Escherichiacoli, Salmonella typhimurium, Salmonella typhi, Shigella sonnei and Helicobacter pylori using the disc diffusion and broth microdilution methods. Sixteen of the plant extracts exhibited antibacterial activity against one or more tested bacteria. Only Garcinia cowa leaf extracts exhibited antibacterial activity against all tested bacteria. Purification of the ethyl acetate extract of G. cowa leaves using an antimicrobial assay-guided isolation afforded a new polyprenylated benzophenone, chamuangone, that exhibited satisfactory antibacterial activity against Streptococcus pyogenes (minimum inhibitory concentration (MIC) 7.8 μg/ml), Streptococcus viridans and H. pylori (MICs 15.6 μg/ml), and Staphylococcus aureus, Bacillus subtilis and Enterococcus sp. (MICs 31.2 μg/ml).     

Isolation of antimicrobial agent from the marine algae Cystoseira hakodatensis

Cystoseira hakodatensis is an unutilised brown algae belonging to family Sargassaceae. A crude methanol extract from the algae showed inhibitory effects on the growths of Bacillus cereus and Bacillus licheniformis. To isolate the major antimicrobial agent, a sequential active‐guided isolation procedure was applied: liquid–liquid extraction, column chromatography and bio‐autography. A marked antimicrobial agent (active α) was isolated in hydrophobic fraction and was determined to phenolics without carbohydrates and proteins by phytochemical test. Regarding the antimicrobial potential, the isolated active α showed better inhibitory effects against B. cereus and B. licheniformis at 2 and 4 times of lower concentrations (62.5 and 31.3 μg mL^?1) in comparison with epigallocatechin gallate. These results showed that C. hakodatensis is a potential source of antimicrobial agent capable of preventing the growth of the two bacteria.     

Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations

Rosmarinus officinalis is widely found in the lands of Aegean and Mediterranean regions of Turkey. The goal of this work was to test the antimicrobial activity of the essential oils and methanolic extracts of R. officinalis collected from three different regions at four different time intervals of the year against Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterococcus feacalis, Escherichia coli, Staphylococcus epidermidis, Bacillus subtilis and Candida albicans. Essential oils were obtained from the aerial parts of the plant by using a Clevenger apparatus, for 4 h. After distillation, the distillates were filtered, air-dried and then extracted by using a Soxhlet apparatus for 9 h to obtain the methanolic extracts. The antimicrobial activities of the methanolic extracts were tested by the disc diffusion technique. The antimicrobial activities of the essential oils obtained from R. officinalis were determined by minimum inhibitory concentration (MIC).The results indicated that the tested bacteria were sensitive to the essential oils and partially to the methanolic extracts. The antimicrobial activities of the essential oils against the tested bacteria differed, depending on location and seasonal variations.     

Isolation and characterization of an antimicrobial substance from Bacillus subtilis BY08 antagonistic to Bacillus cereus and Listeria monocytogenes

A wild-type bacteria producing an antimicrobial substance was isolated from Korean traditional fermented soybean paste, and identified as Bacillus subtilis. The antimicrobial substance purified by TLC, tentatively named UV254-B, displayed a specific antimicrobial activity against pathogenic Bacillus cereus and Listeria monocytogenes, without inhibiting the growth of soybean-fermenting Bacillus species. The antimicrobial substance was susceptible to proteinase K and lipase but resistant to esterase. Antimicrobial activity was observed over a wide range of pH from 3 to 11, with the maximum activity at pH 9, and thermal stability up to 80°C. The minimum inhibitory concentration of the antimicrobial substance was found to be 64 μg/mL for B. cereus and 128 μg/mL for L. monocytogenes. This antimicrobial substance has a putative molecular weight either at 1,133.6 or 1,700.5, which differs from that of other antimicrobial substances described for B. subtilis such as iturin, surfactin, fengycin, and subtilisin.     

Antimicrobial activity of a meat-borne Bacillussubtilis strain against food pathogens

The aim of this study was to test the antibacterial activity of Bacillus subtilis TR50 isolated from a traditional cured sausage against ten food-borne pathogenic bacteria. Most of the pathogens were sensitive to cell-free concentrated supernatant from TR50. The inhibitory activity was retained at pH values between 4 and 8, up to 80 °C for 30 min and after treatment with pepsin and trypsin, whereas it disappeared after chymotrypsin hydrolysis; lipase treatment abolished inhibitory activity only against one target strain. The ammonium sulphate precipitation of TR50 concentrated cell-free culture retained the most residual antibacterial activity unlike the ethyl acetate and acid fractions. All these results suggest that the antimicrobial compound(s) produced by TR50 could be of proteinaceous nature and require a lipid moiety for activity against the pathogens tested. This is the first report on the antimicrobial activity against food-borne pathogens of a B. subtilis strain occurring in a traditional sausage potentially useful for food safety improvement.     

Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus

In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacilluscereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to >10.0), and the substance was thermally stable up to 60 °C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5 μg/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254 nm.     

Chemical Composition and Antioxidative Activity of Echinophora platyloba DC. Essential Oil,and Its Interaction with Natural Antimicrobials against Food‐Borne Pathogens and Spoilage Organisms

Abstract: This study was undertaken to determine the chemical composition and antioxidative capacity of Echinophora platyloba DC. essential oil, and its antimicrobial potency against Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli O157:H7, Pseudomonas aeruginosa, Candida albicans, Candida tropicalis, Rhodotorula rubra, and Rhodotorula mucilaginosa. The essential oil was analyzed by GC and GC‐MS; and evaluated for its antioxidative and antimicrobial (singly or in combination with chitosan, nisin, monolaurin, or amphotericin B) activity. Thirty‐three components were characterized representing 95.69% of the total oil composition in which thymol, trans‐ocimene, carvacrol, and (E)‐sesqui‐lavandulol were the major constituents. The oil exhibited high scavenging (IC₅₀: 49.7 ± 2.3 μg/mL) and relative antioxidative activity (RAA%: 85.21 ± 0.4) in 1,1‐diphenyl‐2‐picrylhydrazyl radicals and β‐carotene/linoleic acid bleaching assays, respectively. The oil showed antimicrobial activity against L. monocytogenes, B. cereus, B. subtilis, S. aureus, S. typhimurium, E. coli O157:H7, P. aeruginosa, C. albicans, C. tropicalis, R. Rubra, and R. mucilaginosa. Moreover, R. mucilaginosa and P. aeruginosa were the most susceptible and most resistant organisms, respectively. Regarding the checkerboard data, 47 fractional inhibitory concentration index (FICIs) (≤0.5) indicated synergistic, whereas 7 FICIs (>0.5 to 1) indicated additive effect. Consequently, E. platyloba DC. essential oil could be used as a recommended natural antioxidant and antimicrobial substance for food preservation.     

Improvement in the yield of lipophilized lysozyme by the combination with Maillard‐type glycosylation

Hen egg white lysozyme was modified using the Maillard‐type glycosylation method prior to the lipophilization with palmitic acid. The yield of lipophilized lysozyme significantly increased by the pre‐glycosylation of the protein. The lipophilized lysozyme derivative was separated into two main fractions with different level of glycosylation. All fractions showed a strong antimicrobial activity against Gram‐negative bacteria,Escherichia coli. The lipophilization of the lysozyme combined with glycosylation is a promising method for potential industrial applications of the lysozyme due to the enhanced antimicrobial activity and the improved yield.     

RAPD-based screening for spore-forming bacterial populations in Uruguayan commercial powdered milk

The occurrence of spore-forming bacteria in powdered milk is of concern to the dairy industry due to potential deleterious effects including those resulting from proteolytic and lipolytic activities. Twenty-two powdered milk samples representative of spring and summer production obtained from Uruguayan retail stores were analyzed for type and number of thermophilic and spore-forming bacterial species. Bacillus licheniformis isolates were found to be the most prominent milk powder contaminant followed by Anoxybacillus flavithermus representing 71.5 to 84% of the total microflora. Geobacillus stearothermophilus, however, was not found. B. licheniformis strains F and G were both found in this study but strain F was the prevalent isolate representing 98.9% of the total isolates of this species. A. flavithermus isolates corresponded to strain C in accordance with 16S rRNA gene sequence analysis, however, in contrast with other reports, the RAPD profiles showed three characteristic bands at approximately 650, 1000 and 1650 bp, but lacking a band at 1250 bp. A third group of isolates was identified corresponding to members of a Bacillus subtilis group and Bacillus megaterium. Isolates designated B. licheniformis, A. flavithermus, B. megaterium and the B. subtilis group represented 89.1 to 93.6% of those analyzed, and depended on previous heat treatment and incubation temperatures of the plates. The remaining isolates were Bacillus pumilus and unidentified spore-formers.     

Evaluation of the Phenolic Content,Antiradical, Antioxidant,and Antimicrobial Activity of Different Floral Sources of Honey

Thirty-five honeys were evaluated for total phenolic content by Folin-Ciocalteu method, for potential antioxidant activity using phosphomolibdenum assay and by the 1,1-diphenyl-2-picrylhydrazyl method for antiradical activity. The antimicrobial activity was studied by the agar diffusion method, using 12 bacteria and 2 yeasts. The means of the total phenolic contents of chestnut, honeydew, multifloral, thyme, and astragalus were 47 ± 18, 24.2 ± 0.6, 14 ± 11, 11 ± 6, and 9 ± 7 mg/100 g honey as gallic acid equivalent, respectively. The lowest antioxidant activity was observed in honeydew 70 ± 5 mg ascorbic acid equivalent/g honey while the highest content was observed in astragalus honey 86 ± 16 mg ascorbic acid equivalent/g honey. Correlation between the phenolic content and antioxidant activity were found to be statistically significant. Chestnut honeys (n = 5) exhibited maximum free radical scavenging activity with an average 68 ± 9%. The honey samples showed the highest antimicrobial activity against some microorganisms, especially Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Proteus mirabilis. On the other side, Aeromonas hydrophila, Bacillus subtilis, and E. coli were the most resistant microorganisms. The results revealed that the Turkish honeys studied proved to be a good source of antioxidant and antimicrobial agents that might serve to protect human health.     

Silver/chitosan-based Janus particles: Synthesis,characterization, and assessment of antimicrobial activity in vivo and vitro

Janus particles containing chitosan and silver were synthesized in an eco-friendly manner and were confirmed using transmission electron microscopy. Based on the data of the antimicrobial activity assessment, this material exhibited a higher antimicrobial activity than virgin chitosan with long–lasting antibacterial effectiveness against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella choleraesuis bacteria, as well as Botrytis cinerea fungi. The results showed that the Janus polymer could completely suppress the growth and germination of B. cinerea at a concentration of 0.02 mg/mL in vitro and in vivo. This Janus polymer is an advanced functional material that combines the suitable properties of both components and could be an alternative new antimicrobial agent due to its unique chemical properties and pronounced antimicrobial activity. This material is a potential candidate for use in the food industry to prevent microbial contamination and to inhibit the growth of microorganisms, enhancing product quality and, extend shelf-life of fresh and processed agri-food products.     

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.     

Antibacterial activity of sourdough lactic acid bacteria: isolation of a bacteriocin-like inhibitory substance fromLactobacillus sanfranciscoC57

A total of 232 strains from nine species ofLactobacillusisolated from sourdoughs were screened for antagonistic activity against sourdough-related micro-organisms. Seventy-seven strains on agar medium and 52 in culture supernatants, re-adjusted to pH 6.5 and catalase-treated, showed antagonistic activity. The activities were species and strain specific showing different spectra of inhibition against sourdough lactobacilli. All the strains were inhibitory toBacillus subtilisbut not against sourdough yeasts and moulds.Lactobacillus sanfranciscoandLactobacillus plantarumstrains had the largest inhibitory spectrum. All the antimicrobial compounds produced from strains of different species contained a protein moiety and were differently sensitive to different proteinases. A bacteriocin-like inhibitory substance, which was heat-stable (100°C for 20 min), insensitive to lipase and α-amylase, of a protein nature, with an inhibitory spectrum centred about lactic acid bacteria and a bactericidal or bacteriolytic mode of action was isolated fromLb. sanfranciscoC57. The antimicrobial substance also inhibitedListeria monocytogenes, and was mainly produced in the stationary phase of growth and at pH 4.0–5.0.Lb. sanfranciscoC57 variants, which did not contain the nativec. 17 kbp plasmid, maintained their antagonistic activity, therefore, the gene encoding for the bacteriocin-like inhibitory substance fromLb. sanfranciscoC57 is chromosomally located.     

Effect of different Bacillus strains on the profile of organic acids in a liquid culture of Daqu

A reversed‐phase high‐performance liquid chromatography method for the analysis of malic, lactic, acetic, citric, succinic, propionic and butyric acids, during the incubation of Bacillus spp., was developed. All samples taken from cultivation were centrifuged (20 min, 11,500g at 5°C) and filtered through a 0.45 µm membrane filter before injection. A programme pumping phosphate buffer at pH 2.50 and acetonitrile was used to separate the compounds on a C₁₈ column. Various parameters affecting analysis were optimized to take less than 30 min with a good linearity (R > 0.999). Bacillus licheniformis or B. subtilis, isolated from Daqu, was inoculated to ferment a liquid culture of Daqu. Growth of bacteria and organic acid production during the fermentation were investigated. Although there were no significant differences in the production of organic acids between B. licheniformis and B. subtilis during the first 8 h, significant differences in the production rates of organic acids, except for citric acid, were observed between B. licheniformis and B. subtilis from 8 to 48 h, with the final concentration of each organic acid varying. Copyright © 2013 The Institute of Brewing & Distilling     

Synthesis,characterisation, antioxidant and antibacterial properties of p-hydroxybenzoic acid-grafted chitosan conjugates

In this study, p-hydroxybenzoic acid-grafted chitosan (PA-g-CS) conjugates with different grafting degrees were synthesised by free radical-regulated grafting approach. The obtained PA-g-CS conjugates were characterised by UV–Vis spectra, fourier transform infrared spectroscopy (FT-IR) spectra, X-ray diffraction, thermogravimetric analysis and scanning electron microscopy. Results revealed that PA was successfully grafted onto the hydroxyl group and the amino group positions of CS. Compared with CS, PA-g-CS showed decreased crystallinity, thermal stability, as well as a loose and porous surface morphology. The antioxidant assays demonstrated that PA-g-CS exhibited remarkably enhanced 2,2-diphenyl-1-pyridohydrazino- and 2,2′-azino-bis(3-ethylbenzothiazoline-6)-sulfonic acid-scavenging activities in vitro. Similarly, the synthesised PA-g-CS also significantly increased the antimicrobial activities towards gram negative and positive bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. Moreover, the products with a relatively higher grafting degree showed stronger biological activities. Our findings suggest that PA-g-CS conjugates have great potential application in the field of active packaging, antioxidants and preservatives.     

The antimicrobial,mechanical, physical and structural properties of chitosan–gallic acid films

Chitosan films incorporated with various concentrations of gallic acid were prepared and investigated for antimicrobial, mechanical, physical and structural properties. Four bacterial strains that commonly contaminate food products were chosen as target bacteria to evaluate the antimicrobial activity of the prepared gallic acid–chitosan films. The incorporation of gallic acid significantly increased the antimicrobial activities of the films against Escherichia coli, Salmonella typhimurium, Listeria innocua and Bacillus subtilis. Chitosan films incorporated with 1.5 g/100 g gallic acid showed the strongest antimicrobial activity. It was also found that tensile strength (TS) of chitosan film was significantly increased when incorporating 0.5 g/100 g gallic acid. Inclusion of 0.5 g/100 g gallic acid also significantly decreased water vapor permeability (WVP) and oxygen permeability (OP). Microstructure of the films was investigated by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) and it was found that gallic acid was dispersed homogenously into the chitosan matrix.