Incidence,Antibiotic Susceptibility,and Toxin Profiles of Bacillus cereus sensu lato Isolated from Korean Fermented Soybean Products

Korean fermented soybean products, such as doenjang, kochujang, ssamjang, and cho‐kochujang, can harbor foodborne pathogens such as Bacillus cereus sensu lato (B. cereus sensu lato). The aim of this study was to characterize the toxin gene profiles, biochemical characteristics, and antibiotic resistance patterns of B. cereus sensu lato strains isolated from Korean fermented soybean products. Eighty‐eight samples of Korean fermented soybean products purchased from retails in Seoul were tested. Thirteen of 26 doenjang samples, 13 of 23 kochujang samples, 16 of 30 ssamjang samples, and 5 of 9 cho‐kochujang samples were positive for B. cereus sensu lato strains. The contamination level of all positive samples did not exceed 4 log CFU/g of food (maximum levels of Korea Food Code). Eighty‐seven B. cereus sensu lato strains were isolated from 47 positive samples, and all isolates carried at least one enterotoxin gene. The detection rates of hblCDA, nheABC, cytK, and entFM enterotoxin genes among all isolates were 34.5%, 98.9%, 57.5%, and 100%, respectively. Fifteen strains (17.2%) harbored the emetic toxin gene. Most strains tested positive for salicin fermentation (62.1%), starch hydrolysis (66.7%), hemolysis (98.9%), motility test (100%), and lecithinase production (96.6%). The B. cereus sensu lato strains were highly resistant to β‐lactam antibiotics such as ampicillin, penicillin, cefepime, imipenem, and oxacillin. Although B. cereus sensu lato levels in Korean fermented soybean products did not exceed the maximum levels permitted in South Korea (<10⁴ CFU/g), these results indicate that the bacterial isolates have the potential to cause diarrheal or emetic gastrointestinal diseases.     

Prevalence,Genetic Diversity,and Antibiotic Resistance of Bacillus cereus Isolated from Korean Fermented Soybean Products

Bacillus cereus contamination is a major food safety problem for Korean fermented soybean products, but few studies have assessed its potential to cause foodborne illness. The objectives of this study were to investigate the prevalence and characteristics of B. cereus isolated from Korean fermented soybean products. B. cereus was detected in 110 of 162 (67.9%) samples. The highest B. cereus frequency was observed in deonjang (68 of 93 samples, 73.1%) and cheonggukjang (18 of 25, 72.0%); however, nonhemolytic enterotoxin was detected only in 22 of 162 samples (13.6%). Although the tested B. cereus isolates showed diverse pulsotypes according to repetitive sequence‐PCR banding patterns, they displayed similar antibiotic sensitivity spectra. The low frequency of enterotoxin detection suggests that the potential risk of B. cereus foodborne illness associated with Korean fermented soybean products is lower than generally presumed. However, considering the prevalence of B. cereus and the high content of fermented soybean products in the Korean diet, it is necessary to constantly monitor the level of contamination with B. cereus and its toxins in such Korean food products.     

Enterotoxigenic Profiling of Emetic Toxin‐ and Enterotoxin‐Producing Bacillus cereus,Isolated from Food,Environmental, and Clinical Samples by Multiplex PCR

Bacillus cereus comprises the largest group of endospore‐forming bacteria and can cause emetic and diarrheal food poisoning. A total of 496 B. cereus strains isolated from various sources (food, environmental, clinical) were assessed by a multiplex PCR for the presence of enterotoxin genes. The detection rate of nheA, entFM, hblC, and cytK enterotoxin genes among all B. cereus strains was 92.33%, 77.21%, 59.47%, and 47.58%, respectively. Enterotoxigenic profiles were determined in emetic toxin‐ (8 patterns) and enterotoxin‐producing strains (12 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. Our findings revealed that B. cereus must be considered a serious health hazard and Bacillus thuringiensis should be considered of a greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.     

Development of a Novel PCR Primer to Differentiate and Identify Bacillus subtilis and Closely Related Species Isolated from Thai Fermented Foods

A novel PCR assay based on 16S-23S internal transcribed spacers (ITS) length polymorphism was developed for rapid differentiation and identification of the Bacillus subtilis group, especially B. subtilis, B. licheniformis and B. amyloliquefaciens, the most frequently isolated bacilli from fermented foods. A new group-specific conserved primer pair, CITS-F and CITS-R, was designed for specific amplification of the ITS region in B. subtilis and closely related species. The fingerprints of seven reference species, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus, B. atrophaeus, B. vallismortis and B. mojavensis using CITS-F and CITS-R primers showed the same four signature bands of 227, 400, 542 and 650 bp. They are different from those of other genera and species tested. Therefore, these four signature bands could be used to differentiate and identify the B. subtilis group. Moreover, the sequence of the 227 bp signature band could also be used to distinguish closely related species of the B. subtilis group used in this study. A novel PCR assay based on ITS length polymorphism pattern using CITS-F and CITS-R could be considered as a rapid and easy method for the primary differentiation of the B. subtilis group.     

Development of efficient enzymatic production of theanine by γ‐glutamyltranspeptidase from a newly isolated strain of Bacillus subtilis,SK11.004

BACKGROUND: Theanine, a unique amino acid found almost exclusively in tea plants, has various favourable physiological and pharmacological functions in humans. Gamma‐glutamyltranspeptidase (GGT, EC 2.3.2.2) is considered to be the most effective enzyme for the production of theanine. In fact, GGT can catalyse the transfer of γ‐glutamyl moieties from γ‐glutamyl compounds to water (hydrolysis) or to amino acids and peptides (transpeptidation). RESULTS: A novel strain, SK11.004, which produces GGT with high theanine‐forming ability was isolated from fermented shrimp paste and identified as Bacillus subtilis through its physiological and biochemical properties as well as its 16S rDNA sequence analysis. Theanine (18.9 mmol L^?1) was synthesised by GGT (0.06 U mL^?1) through transfer reaction in the presence of glutamine (20 mmol L^?1) as a donor and ethylamine HCl (50 mmol L^?1) as an acceptor at pH 10 and 37 °C for 4 h, the conversion rate being up to 94%. CONCLUSION: The enzymatic synthesis of theanine using GGT from a newly isolated strain Bacillus subtilis SK11.004 was found to be an efficient method. Moreover, compared with others, the GGT from B. subtilis SK11.004 exhibited the highest ratio of transferring activity to hydrolytic activity using glutamine, suggesting a high potential application in the production of theanine and other functional γ‐glutamyl compounds. Copyright © 2010 Society of Chemical Industry     

Effects of pH and dissolved oxygen on the synthesis of γ‐glutamyltranspeptidase from Bacillus subtilis SK 11.004

BACKGROUND: γ‐Glutamyltranspeptidase (GGT; EC 2.3.2.2) is a widely distributed enzyme that is of interest in the food industry. In this study the effects of pH and dissolved oxygen (DO) on GGT synthesis from Bacillus subtilis SK 11.004 were investigated. RESULTS: GGT production increased to 0.5 U mL^?1 when the pH value was controlled at 6.5. The control of a single DO level revealed that the highest specific growth rate (3.42 h^?1) and GGT production rate (0.40 U g^?1 mL^?1) were obtained at DO levels of 40 and 10% respectively. To satisfy the different oxygen demands at different stages of cell growth and GGT synthesis, a stage DO level control strategy was designed as follows: 40% from 0 to 4 h, 30% from 4 to 6 h and 10% from 6 to 18 h. Furthermore, the maximum biomass (2.27 g L^?1) and GGT production (3.05 U mL^?1) could be obtained using a fermentation strategy combining a constant pH value with stage DO level control. CONCLUSION: The proposed fermentation strategy resulted in a 13.7‐fold increase in GGT production. This finding should be of great importance for the industrial production of GGT. Copyright © 2011 Society of Chemical Industry     

Characterization of a novel protease from Bacillus cereus and evaluation of an eco‐friendly hydrolysis of a brewery byproduct

Proteases and proteolytic enzymes constitute one of the most important groups of enzymes and are attracting worldwide attention in attempts to exploit their physiological and biotechnological applications. In this study, partial purifications and biochemical and antimicrobial characterizations of a protease from Bacillus cereus spp., originally isolated from fermented cabbage, were carried out. The crude extract obtained after purification, involving ammonium sulphate precipitation and dialysis, was designated as a partially purified protease (PPP). The obtained PPP had a specific activity of 0.395–2.539 U/g at 32 °C, with maximum activities for the fractions precipitated at 60 and 80% ammonium sulphate. The PPP activity ranged between 20 and 55 °C, with an optimum temperature at 40 °C. At 60 °C, the PPP retained more than 30% of its activity. The optimum pH for the PPP was achieved at pH 9, indicating the alkaline source of the enzyme. Protease production was specifically dependent on the calcium concentration in the culture medium. Also the robustness of the protease on brewer's spent grain hydrolysis was demonstrated. This suggests a potential eco‐friendly application of the enzyme. Finally, it was found that the PPP inhibited the growth of Escherichia coli O157:H7. This novel property of the PPP liberated by the B. cereus spp. could provide important future benefits to industry. Copyright © 2015 The Institute of Brewing & Distilling     

Enhancing the levels of 4‐vinylguaiacol and 4‐vinylphenol in pilot‐scale top‐fermented wheat beers by response surface methodology

Top‐fermented wheat beers are desired by consumers because of their distinctive clove‐like and phenolic aroma, in which two characteristic flavour substances, 4‐vinylguaiacol (4VG) and 4‐vinylphenol (4VP), are of vital importance. In this paper, the levels of 4VG and 4VP were the subject of an intensive and detailed investigation. The influence of wheat malt proportion, mashing‐in temperature, boiling time and fermentation temperature on 4VG and 4VP was studied by pilot‐scale brewing experiments using a 10 hL mashing vessel and 20 hL fermentation tanks. Response surface methodology (RSM) was used to explore the possibilities for enhancing the concentration of 4VG and 4VP in top‐fermented wheat beers. Statistical analysis of the results showed that for the ranges of wheat malt proportion studied, mashing‐in temperature, boiling time and fermentation temperature all had a significant influence (p <0.05) on 4VG and 4VP production. Based on the response surface plots, the optimal brewing parameters that maximized 4VG and 4VP levels were as follows: (a) wheat malt proportion 40%; (b) mashing‐in temperature 44°C; (c) boiling time 88 min; and (d) fermentation temperature 19.5°C, resulting in significantly increased levels of 4VG and 4VP (2.418 mg/L and 1.402 mg/L, respectively). These brewing and fermentation parameters were concluded to be the optimal conditions to highlight the typical flavour and aroma of top‐fermented wheat beers. Copyright © 2015 The Institute of Brewing & Distilling     

Original article: Modelling the effects of food ingredients and pH on high‐pressure processing inactivation of Bacillus cereus spores: a laboratorial study

The combination of high pressure and heat on Bacillus cereus spores in food matrix was investigated with the purpose of achieving a predictive model of microbial inactivation. The high‐pressure processing (HPP) conditions were fixed at 540 MPa and 71 °C for 16.8 min, which were determined as the optimum conditions considering six‐log‐cycle reductions of B. cereus spores. The effects of soybean protein, sucrose, soybean oil and pH on the inactivation of B. cereus spores by HPP were evaluated, and a quadratic predictive model for the effects of food ingredients and pH on the reductions of B. cereus spores by HPP was built using response surface methodology. The experimental results showed that soybean protein, sucrose and pH significantly affected the reductions of B. cereus spores. The predictive model is significant, because the level of significance was P < 0.0001 and the calculated F‐value is much greater than the tabulated F‐value. The adequacy of the predictive model equation was verified effectively using the experimental test data that were not used in the development of the model.     

Antioxidant Properties of Caffeic acid Phenethyl Ester and 4‐Vinylcatechol in Stripped Soybean Oil

Caffeic acid was used to synthesize 4‐vinylcatechol (4‐VC) by thermal decarboxylation and to prepare caffeic acid phenethyl ester (CAPE) by esterification reaction. The identities of synthesized products were confirmed by ¹H NMR. Antioxidative activities of 4‐VC and CAPE were compared with α‐tocopherol and BHT in stripped soybean oil at 60 °C under the dark. To evaluate the degrees of oxidation at different concentrations and combinations, peroxide value (PV) and ¹H NMR were performed. From the results of PV, the formation of primary oxidation products (i.e., hydroperoxides) in stripped soybean oil containing 200 ppm CAPE was the slowest. The relative oxidation degree of 200 ppm CAPE (9.5%) was lower than other samples on 9 d. Similar results were obtained by ¹H NMR analysis. After 15 d of storage, levels of conjugated diene forms and aldehydes of 200 ppm CAPE sample (57.3 and 0.9 mmol/mol oil) were also lower than other treatments. In addition, 4‐VC and α‐tocopherol were found to have a synergistic antioxidant effect.     

Enhanced production of α‐amylase from Bacillus subtilis subsp. spizizenii in solid state fermentation by response surface methodology and its evaluation in the hydrolysis of raw potato starch

This is an attempt to lower the cost of starch hydrolysis by the discovery of new generation α‐amylase. A natural isolate of Bacillus subtilis subsp. spizizenii was capable of producing appreciable amounts of raw potato starch digesting α‐amylase in solid state fermentation of wheat bran. The enzyme productivity has been substantially enhanced by supplementing various nutrients and statistically studying their interactions by response surface methodology. A central composite design for amylase production system elucidated a wheat bran‐based medium supplemented with soybean meal, threonine, and B‐complex vitamins predicting a yield of 521 391 U/g dry solids. The enzyme preparation could effectively digest 5–15% suspension of insoluble potato starch in 6 h revealing the dextrose equivalent of 32–44. The supplementation of a glucoamylase preparation, thereafter, brought about complete saccharification. The yield achieved in the statistically optimized amylase system may be one of the best to date and its capability in directly liquefying raw potato starch granules makes this study novel.     

Isolation and Characterization of Chitinase‐Producing Bacillus and Paenibacillus Strains from Salted and Fermented Shrimp,Acetes japonicus

Chitinases catalyze the conversion of chitin and are produced by a wide range of bacteria. The biological applications of these enzymes have been exploited in food and pharmaceutical industries. We isolated 2 halophilic chitinase‐producing novel strains of bacteria—SCH‐1 and SCH‐2 from Saeu‐jeot, a traditional Korean salted and fermented food made with shrimp (Acetes japonicus). The isolated strains‐ SCH‐1 and SCH‐2 were Gram‐positive, rod‐shaped, endospore‐forming facultative anaerobes, with strain SCH‐2 showing peritrichous flagella. Molecular characterization of the 16S rRNA gene identified the strains SCH‐1 and SCH‐2 as Bacillus sp. and Paenibacillus sp. respectively. Basic Local Alignment Search Tool and subsequent phylogenetic analysis of strain SCH‐1 showed an identity of 97.83% with Bacillus cereus ATCC 14579 (NR_074540), whereas strain SCH‐2 showed an identity of 99.16% with Paenibacillus lautus JCM 9073 (NR_040882). Furthermore, the SCH‐1 strain could use glucose, N‐acetyl glucosamine, esculin, and maltose as carbon source substrates. Cellular fatty acid analysis showed that iso‐C_15:0 and anteiso‐C_15:0 are the major acids in strain SCH‐1 and SCH‐2, respectively. The SCH‐1 strain showed a higher chitinase activity at 15.71 unit/mg protein compared with SCH‐2 strain. Chitinase isozymes of Bacillus sp. SCH‐1was expressed as 2 bands having sizes of 41 and 50 kDa, and as 4 bands with sizes of 30, 37, 45.7, and 50 kDa in Paenibacillus sp. SCH‐2. The rich chitinase activity with the isozyme profiles of the isolated Bacillus and Paenibacillus strains provide advancement in the study of fermentation and may play putative functions in the chitin bioconversion of sea crustacean foods.     

Angiotensin I‐converting enzyme inhibitory peptides FQPSF and LKYPI identified in Bacillus subtilis A26 hydrolysate of thornback ray muscle

Angiotensin I‐converting enzyme (ACE) inhibitory peptides have been searched in thornback ray (Raja clavata) muscle hydrolysed with Bacillus subtilis A26 proteases until a hydrolysis degree of 18.35%. The hydrolysate showed an IC₅₀ of 0.83 mg mL^?1. To identify peptides responsible for this activity, the extract was eluted through size‐exclusion chromatography and fractions collected. The highest ACE inhibitory activity was found for fractions F2 and F3 which had IC₅₀ of 0.42 and 0.51 mg mL^?1, respectively. These fractions were analysed by nano‐liquid chromatography coupled to tandem mass spectrometry (nLC‐MS/MS). A total of 131 and 108 peptide sequences mainly derived from actin, myosin heavy chain and procollagen alpha 1 chain proteins were identified in fractions F2 and F3, respectively. FQPSF and LKYPI showed the best results with an IC₅₀ of 12.56 and 27.07 μM, respectively. These results prove the potential of thornback ray muscle hydrolysate as a source of ACE inhibitory peptides.     

Culture‐Based and Denaturing Gradient Gel Electrophoresis Analysis of the Bacterial Community from Chungkookjang,a Traditional Korean Fermented Soybean Food

Abstract: The bacterial community of Chungkookjang and raw rice‐straw collected from various areas in South Korea was investigated using both culture‐dependent and culture‐independent methods. Pure cultures were isolated from Chungkookjang and raw rice‐straw on tryptic soy agar plates with 72 to 121 colonies and identified by 16S rDNA gene sequence analysis, respectively. The traditional culture‐based method and denaturing gradient gel electrophoresis analysis of PCR‐amplified 16S rDNA confirmed that Pantoea agglomerans and B. subtilis were identified as predominant in the raw rice‐straw and Chungkookjang, respectively, from Iljuk district of Gyeonggi province, P. ananatis and B. licheniformis were identified as predominant in the raw rice‐straw and Chungkookjang from Wonju district of Gangwon province, and Microbacterium sp. and B. licheniformis were identified as predominant in the raw rice‐straw and Chungkookjang from Sunchang district of Jeolla province. Other strains, such as Bacillus, Enterococcus, Pseudomonas, Rhodococcus, and uncultured bacteria were also present in raw rice‐straw and Chungkookjang. Practical Application: A comprehensive analysis of these microorganisms would provide a more detailed understanding of the biologically active components of Chungkookjang and help improve its quality. Polymerase chain reaction‐denaturing gradient gel electrophoresis analysis can be successfully applied to a fermented food to detect unculturable or more species than the culture‐dependent method. This technique is an effective and convenient culture‐independent method for studying the bacterial community in Chungkookjang. In this study, the bacterial community of Chungkookjang collected from various areas in South Korea was investigated using both culture‐dependent and culture‐independent methods.     

Effect of Various Food Additives on the Levels of 4(5)‐Methylimidazole in a Soy Sauce Model System

In this study, the effect of food additives such as iron sulfate, magnesium sulfate, zinc sulfate, citric acid, gallic acid, and ascorbic acid on the reduction of 4(5)‐methylimidazole (4(5)‐MI) was investigated using a soy sauce model system. The concentration of 4(5)‐MI in the soy sauce model system with 5% (v/v) caramel colorant III was 1404.13 μg/L. The reduction rate of 4(5)‐MI level with the addition of 0.1M additives followed in order: iron sulfate (81%) > zinc sulfate (61%) > citric acid (40%) > gallic acid (38%) > ascorbic acid (24%) > magnesium sulfate (13%). Correlations between 4(5)‐MI levels and the physicochemical properties of soy sauce, including the amount of caramel colorant, pH value, and color differences, were determined. The highest correlations were found between 4(5)‐MI levels and the amount of caramel colorant and pH values (r² = 0.9712, r² = 0.9378). The concentration of caramel colorants in 8 commercial soy sauces were estimated, and ranged from 0.01 to 1.34% (v/v).     

Physicochemical Properties,Fatty Acid Profiles,and Sensory Characteristics of Fermented Beef Sausage by Probiotics Lactobacillus plantarum IIA‐2C12 or Lactobacillus acidophilus IIA‐2B4

Probiotics may be used to enhance the functionality and nutritional values of fermented sausages. This study aims to evaluate the physicochemical and sensory properties of beef sausages fermented by lactic acid bacteria of Lactobacillus plantarum IIA‐2C12 and L. acidophilus IIA‐2B4. These strains were isolated from beef cattle and have shown to display probiotic features. While the nutrient contents were not affected by the probiotics, the pH, texture, and color varied among the sausages. Further analysis on fatty acids showed different profiles of saturated (C14:0, C17:0, and C20:0) and unsaturated (C14:1, C18:1n9c, C18:2n6c, and C22:6n3) fatty acids in sausages with probiotics. Gas chromatography–mass spectrometry further revealed some flavor development compounds including acid, alcohols, aldehydes, aromatic, ketones, sulfur, hydrocarbons and terpenes, varied among the sausages. Hedonic test showed no difference in the preference toward aroma, texture, and color for untrained panelists.