Effects of Calcium and Manganese on Sporulation of Bacillus Species Involved in Food Poisoning and Spoilage.

Spores are resistant against many extreme conditions including the disinfection and sterilization methods used in the food industry. Selective prevention of sporulation of Bacillus species is an ongoing challenge for food scientists and fermentation technologists. This study was conducted to evaluate the effects of single and combined supplementation of calcium and manganese on sporulation of common pathogenic and food spoilage Bacillus species: B. cereus, B. licheniformis, B. subtilis and B. coagulans. Sporulation of Bacillus vegetative cells was induced on sporulation media supplemented with diverse concentrations of the minerals. Under the various mineral supplementation conditions, the degree of sporulation was quantified with colonies formed by the Bacillus spores. The results revealed that B. licheniformis and B. cereus displayed the weakest sporulation capabilities on media with minimal supplementation levels of calcium and manganese. The lowest sporulation of B. subtilis and B. coagulans was observed on media supplemented with the highest level of calcium and low levels of manganese. Depending on effect of supplementation on sporulation, the Bacillus species were divided into two distinct groups: B. licheniformis and B. cereus; and B. subtilis and B. coagulans. The information provides valuable insight to selectively reduce sporulation of Bacillus species undesirable in the food industry.

Bacterial Production and Control of Biogenic Amines in Asian Fermented Soybean Foods.

Fermented soybean foods possess significant health-promoting effects and are consumed worldwide, especially within Asia, but less attention has been paid to the safety of the foods. Since fermented soybean foods contain abundant amino acids and biogenic amine-producing microorganisms, it is necessary to understand the presence of biogenic amines in the foods. The amounts of biogenic amines in most products have been reported to be within safe levels. Conversely, certain products contain vasoactive biogenic amines greater than toxic levels. Nonetheless, government legislation regulating biogenic amines in fermented soybean foods is not found throughout the world. Therefore, it is necessary to provide strategies to reduce biogenic amine formation in the foods. Alongside numerous existing intervention methods, the use of Bacillus starter cultures capable of degrading and/or incapable of producing biogenic amines has been proposed as a guaranteed way to reduce biogenic amines in fermented soybean foods, considering that Bacillus species have been known as fermenting microorganisms responsible for biogenic amine formation in the foods. Molecular genetic studies of Bacillus genes involved in the formation and degradation of biogenic amines would be helpful in selecting starter cultures. This review summarizes the presence and control strategies of biogenic amines in fermented soybean foods.

Tyramine reduction by tyrosine decarboxylase inhibitor in Enterococcus faecium for tyramine controlled cheonggukjang.

This study was carried out to find a method to control tyrosine decarboxylase activity (TDC) of a strain of Enterococcus faecium capable of producing high levels of tyramine. To select a TDC inhibitor, enzyme assay was first performed using purified TDC enzyme and 0.1% of TDC inhibiting chemicals. When 0.23% of nicotinic acid was added, tyramine content (363 ug/mL) was lower than that of the control group (873 ug/mL). At the same time, bacterial growth was decreased 1 log cycle from 8.62 to 7.56 log CFU/mL. TDC expression level in E. faecium was measured by using RT-qPCR. Lower expression level (below 0.7) was observed after the addition of 0.23% nicotinic acid (in vitro). When cheonggukjang was manufactured with addition of nicotinic acid, tyramine contents were decreased from 698.67 to 117.27 mg/kg when the concentration of nicotinic acid added was increased from 0.10 to 0.30%. These results suggest that nicotinic acid could be used as an agent (TDC inhibitor) to reduce tyramine content in cheonggukjang.

Potential of a lactic acid bacterial starter culture with gamma-aminobutyric acid (GABA) activity for production of fermented sausage.

The ability of lactic acid bacterial starter cultures to produce gamma-aminobutyric acid (GABA) during sausage fermentation was studied. Among 305 strains of lactic acid bacteria isolated from kimchi samples, 11 strains were selected as starter candidates based on the following criteria: growth speed, pH lowering ability, and biogenic amine productivity including GABA-producing activity. During in vitro tests, the Y8 (Lactobacillus brevis), O52, and KA20 strains produced 39.00 ± 1.36, 49.73 ± 3.80, and 64.59 ± 0.61 mg/kg of GABA, respectively. Interestingly, although isolate Y8 showed low productivity in vitro, the GABA content it produced during in situ tests (61.30 ± 2.61 mg/kg) was similar to that produced by isolate PM3 (L. brevis) used as positive control (69.64 ± 2.20 mg/kg). Therefore, isolate Y8 was selected as the best functional starter culture for the production of fermented sausage because it exhibited rapid growth, safety, and abundant GABA productivity.

Application of Baechu-Kimchi Powder and GABA-Producing Lactic Acid Bacteria for the Production of Functional Fermented Sausages.

This study was carried out to determine the physicochemical, microbiological, and quality characteristics of a new type of fermented sausage manufactured by incorporating Baechu-kimchi powder and gamma-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB). The LAB count was at the maximum level by day nine of ripening in inoculated sausages, accompanied by a rapid decrease in the pH. The addition of kimchi powder decreased the lightness (L*) and increased the redness (a*) and, yellowness (b*) values, while also significantly increasing the hardness and chewiness of the sausage (p<0.05). Moreover, although the thiobarbituric acid reactive substances values increased in all samples during the study period, this increase was lower in the kimchi-treated samples, indicating a reduction in lipid oxidation. Overall, our results show that the addition of Baechu-kimchi powder to sausages reduced the off-flavor properties and improved the taste profile of the fermented sausage in sensory evaluations. The GABA content of all fermented sausages increased from 17.42-25.14 mg/kg on the third day of fermentation to 60.95-61.47 mg/kg on the thirtieth day. These results demonstrate that Baechu-kimchi powder and GABA-producing LAB could be functional materials in fermented sausage to improve quality characteristics.

Quantitative analysis of volatile flavor components in Korean alcoholic beverage and Japanese sake using SPME-GC/MS.

Quantitative analysis of the volatile flavor components in Korean alcoholic beverages (makgeolli and yakju) and Japanese sake was carried out using SPME-GC/MS. Fusel oils (n-butyl alcohol, isobutyl alcohol, isoamyl alcohol, and phenethyl alcohol), ethyl esters (ethyl acetate, isoamyl acetate, ethyl caproate, ethyl caprylate, and ethyl caprate) and aldehydes (furfural and benzaldehyde) were analyzed quantitatively by an 85 µm SPME fiber (carboxen/polydimethylsiloxane) using internal standards (1-pentanol-1-13C and methyl nonanoate). Phenethyl alcohol (85-216 ppm) and isoamyl alcohol (38-115 ppm) constituted the majority of fusel oils in all the samples. Acetic acid was detected in sour makgeolli at a high level (0.02-0.14 ppm) compared with yakju and sake. A very high level of total ethyl esters (ethyl acetate, isoamyl acetate, ethyl caprate, and phenethyl acetate), having fruit and flower flavor, was found in makgeolli. Processing the volatile flavor data by multivariate partial least squares discriminant analysis, makgeolli, yakju, and sake showed cluster separation.

Validation of an HPLC Analytical Method for Determination of Biogenic Amines in Agricultural Products and Monitoring of Biogenic Amines in Korean Fermented Agricultural Products.

An HPLC analytical method was validated for the quantitative determination of biogenic amines in agricultural products. Four agricultural foods, including apple juice, Juk, corn oil and peanut butter, were selected as food matrices based on their water and fat contents (i.e., non-fatty liquid, non-fatty solid, fatty liquid and fatty solid, respectively). The precision, accuracy, recovery, limit of detection (LOD) and quantification (LOQ) were determined to test the validity of an HPLC procedure for the determination of biogenic amines, including tryptamine, ß-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine, in each matrix. The LODs and LOQs for the biogenic amines were within the range of 0.01~0.10 mg/kg and 0.02~0.31 mg/kg, respectively. The relative standard deviation (RSD) of intraday for biogenic amine concentrations ranged from 1.86 to 5.95%, whereas the RSD of interday ranged from 2.08 to 5.96%. Of the matrices spiked with biogenic amines, corn oil with tyramine and Juk with putrescine exhibited the least accuracy of 84.85% and recovery rate of 89.63%, respectively, at the lowest concentration (10 mg/kg). Therefore, the validation results fulfilled AOAC criteria and recommendations. Subsequently, the method was applied to the analysis of biogenic amines in fermented agricultural products for a total dietary survey in Korea. Although the results revealed that Korean traditional soy sauce and Doenjang contained relatively high levels of histamine, the amounts are of no concern if these fermented agricultural products serve as condiments.

Lactic Acid Bacterial Starter Culture with Antioxidant and γ-Aminobutyric Acid Biosynthetic Activities Isolated from Flatfish-Sikhae Fermentation.

The aim of this study is to select a lactic acid bacterial strain as a starter culture for flatfish-Sikhae fermentation and to evaluate its suitability for application in a food system. Four strains of lactic acid bacteria isolated from commercial flatfish-Sikhae were identified and selected as starter culture candidates through investigation of growth rates, salt tolerance, food safety, and functional properties such as antioxidative and antimicrobial activities. The fermentation properties of the starter candidates were also examined in food systems prepared with these strains (candidate batch) in comparison with a spontaneous fermentation process without starter culture (control batch) at 15°C. The results showed that the candidate YG331 batch had better fermentation properties such as viable cell count, pH, and acidity than the other experimental batches, including the control batch. The results are expressed according to selection criteria based on a preliminary sensory evaluation and physiochemical investigation. Also, only a small amount of histamine was detected with the candidate YG331 batch. The radical scavenging activity of the candidate batches was better compared with the control batch, and especially candidate YG331 batch showed the best radical scavenging activity. Also, we isolated another starter candidate (identified as Lactobacillus brevis PM03) with γ-aminobutyric acid (GABA)-producing activity from commercial flatfish-Sikhae products. The sensory scores of the candidate YG331 batch were better than those of the other experimental batches in terms of flavor, color, and overall acceptance. In this study, we established selection criteria for the lactic acid bacterial starter for the flatfish-Sikhae production and finally selected candidate YG331 as the most suitable starter.

Reduction of tyramine by addition of Schizandra chinensis baillon in Cheonggukjang.

This study was performed to examine the microorganisms responsible for the high tyramine content of Cheonggukjang, a traditional Korean fermented soy food, and to establish a technology for controlling the growth of these microorganisms. The tyramine content in 13 collected Cheonggukjang samples averaged 604.9 mg/kg. Since the tyramine content measured from most samples was sufficient to cause harm to the human body, it is necessary to control its production in food. Enterococci were confirmed to be the bacterial species producing most of the tyramine through the microbial examination and were present in high numbers from not detected (<10(1)) to 7.0 × 10(10) colony-forming units (CFU)/g. To control the growth of enterococci, various plant extracts with antimicrobial activity, common salts, and variable temperature conditions were tested. It was found that 4 samples among the 159 plant extracts had a strong antimicrobial activity in Cheonggukjang, especially against Enterococcus faecium, showing viable cell counts of <10(1)-10(3) CFU/g after 24 h of ripening, which were significantly lower values compared to the control (10(9)-10(11) CFU/g). The Cheonggukjang with the addition of the four plant extracts showed ∼83%-95% lower concentrations of tyramine compared to the control. Cheonggukjang prepared with the Schizandra chinensis Baillon extract had the lowest tyramine content without sacrificing the sensory quality. Not only was the bacterial species of E. faecium reduced more remarkably, by up to 10(3) CFU/g compared to the 10(9)-10(11) CFU/g shown in the control, but it also decreased the tyramine content by up to 91%.

Grape seed extract regulates androgen receptor-mediated transcription in prostate cancer cells through potent anti-histone acetyltransferase activity.

Histone acetylation, which is regulated by histone acetyltransferases (HATs) and deacetylases, is an epigenetic mechanism that influences eukaryotic transcription. Significant changes in histone acetylation are associated with cancer; therefore, manipulating the acetylation status of key gene targets is likely crucial for effective cancer therapy. Grape seed extract (GSE) has a known protective effect against prostate cancer. Here, we showed that GSE significantly inhibited HAT activity by 30-80% in vitro (P < .05). Furthermore, we demonstrated significant repression of androgen receptor (AR)-mediated transcription by GSE in prostate cancer cells by measuring luciferase activity using a pGL3-PSA construct bearing the AR element in the human prostate cancer cell line LNCaP (P < .05). GSE treatment also decreased the mRNA level of the AR-regulated genes PSA and NKX 3.1. Finally, GSE inhibited growth of LNCaP cells. These results indicate that GSE potently inhibits HAT, leading to decreased AR-mediated transcription and cancer cell growth, and implicate GSE as a novel candidate for therapeutic activity against prostate cancer.

Protective effects of kaempferol (3,4',5,7-tetrahydroxyflavone) against amyloid beta peptide (Abeta)-induced neurotoxicity in ICR mice.

To determine the effects of kaempferol, rat pheochromocytoma cells (PC12) and Institute of Cancer Research (ICR) mice were utilized as neuronal models. Using in vitro assays, kaempferol was shown to have protective effects against oxidative stress-induced cytotoxicity in PC12 cells. Administration of kaempferol also significantly reversed amyloid beta peptide (Abeta)-induced impaired performance in a Y-maze test. Taken altogether, the results reported here suggest that further investigation is warranted of the influence of kaempferol on pathways related to Alzheimer's disease.

Characterizations of environmental factors in conjugated linoleic acid production by mixed rumen bacteria.

Conjugated linoleic acid (CLA) production by rumen bacteria is closely related to biohydrogenation of linoleic acid (LA) and affected by various environmental factors. Ruminal biohydrogenation and isomerization were characterized in view of incubation conditions using a mixed culture of microorganisms obtained from surgically prepared cows. Free-floating bacteria (FFB) produced more CLA than particle-attached bacteria (PAB). Some major factors affecting the ruminal environment such as diet, concentrations of fat substrates, incubation time, pre-incubation, and the presence of glucose in the medium were found to be important determinants for the ruminal production of CLA and in a close relationship with biohydrogenation. The mixed bacterial culture, which was pre-exposed to LA, produced more CLA than an unexposed control in a medium containing 30% rumen fluid. The rate of conversion of fat substrate (LA) to hydrogenated products (trans-C18:1, C18:0) was negatively correlated with the initial LA concentration. Overall, the present study showed that CLA accumulation can be increased by modification of diet-induced environmental conditions, which affect changes in ruminal microflora.

Inhibitory effect of Poncirus trifoliate on acetylcholinesterase and attenuating activity against trimethyltin-induced learning and memory impairment.

Various native Korean plants were screened to find an effective acetylcholinesterase (AChE) inhibitor for the treatment of Alzheimer's disease (AD). Among these plants, the ethanol extract of Poncirus trifoliate was selected for isolating the AChE inhibitor because it exhibited the highest inhibitory activity (47.31%). To separate the active compound from Poncirus trifoliate, solvent partition, open column chromatography, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) were utilized. The putative chemical structure of the AChE inhibitor was identified as methoxsalen by successive analysis with electron ionization mass spectrometry (EI-MS) and (13)C/(1)H-nuclear magnetic resonance (NMR). To confirm the attenuating effect of the Poncirus trifoliate extract against trimethyltin (TMT)-induced neurotoxicity, in vivo behavior tests were carried out. Our findings suggest that the Poncirus trifoliate extract significantly reversed TMT-induced learning and memory impairment. These results demonstrate that the Poncirus trifoliate extract could possess a wide range of beneficial activities for neurodegenerative disorders, notably AD.

Ameliorative effect of 1,2-benzenedicarboxylic acid dinonyl ester against amyloid beta peptide-induced neurotoxicity.

Amyloid beta peptide (Abeta)-induced oxidative stress may be linked to neurodegenerative disease. Ethanol extracts of Rosa laevigata protected PC12 cells from hydrogen peroxide-induced oxidative stress. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assays revealed a significant increase in cell viability when oxidatively stressed PC12 cells were treated with R. laevigata extract. The effect of R. laevigata on oxidative stress-induced cell death was further investigated by lactate dehydrogenase release assays and trypan blue exclusion assays. Administration of 1,2-benzenedicarboxylic acid dinonyl ester from R. laevigata extract to mice infused with Abeta significantly reversed learning and memory impairment in behavioural tests. After behavioural testing, the mice were sacrificed and brains were collected for the examination of lipid peroxidation, catalase activity and acetylcholinesterase (AchE) activity. These results suggest that 1,2-benzenedicarboxylic acid dinonyl ester from R. laevigata extract may be able to reduce Abeta-induced neurotoxicity, possibly by reducing oxidative stress. Therefore, R. laevigata extract may be useful for the prevention of oxidative stress-induced neurodegenerative disorders.

Paenibacillus tyraminigenes sp. nov. isolated from Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy.

A bacterial strain H3029, a gram-positive, rod-shaped, oxidase-negative, endospore-forming bacterium that characteristically produces tyramine from tyrosine, was isolated from a Myeolchi-jeotgal, a traditional Korean salted and fermented anchovy (Engraulis japonicus). The H3029 strain showed a high ability to produce 4140 microg/ml of tyramine from the culture broth containing 5000 microg/ml tyrosine. On the other hand, the strain produced a relatively low level of other putrefactive amines, at 973 microg/ml of putrescine and 147 microg/ml of cadaverine from the media, with each 5000 microg/ml of ornithine hydrochloride and lysine hydrochloride. The H3029 strain produced no detectable level of histamine (detection limit of 4 microg/ml) from the media containing 5000 microg/ml of histidine hydrochloride. Meanwhile, tyramine, the main product of the strain, showed the antimicrobial activity at the level of over 1 mg/disk against Staphylococcus aureus by agar diffusion test, and the mutagenicity in Ames test at 0.1 mg/plate using Salmonella typhimurium TA98 and TA1535. On the basis of the polyphasic taxonomic study, the H3029(T) strain was assigned a novel species of the genus Paenibacillus as Paenibacillus tyraminigenes sp. nov. The type strain of which is strain H3029(T) (=KCTC 10694BP(T)).

Reduction of soybean oligosaccharides and properties of alpha-D-galactosidase from Lactobacillus curvatus R08 and Leuconostoc mesenteroides [corrected] JK55.

This study was undertaken to investigate the potential for reducing non-digestive oligosaccharides (NDO) in soy foods, as well as the influence of exogenous conditions on intracellular alpha-galactosidase (alpha-Gal) producing lactic acid bacteria. Two strains, Lactobacillus curvatus R08 and Leuconostoc mesenteroides [corrected]JK55, showed the highest levels of raffinose degrading activity at over 40 U mL(-1), and presented maximum activities during the stationary phase in a medium where raffinose was the only carbon source. Raffinose was the most effective inducer, followed by melibiose, and galactose; the enzymes were partially inhibited by fructose and sucrose. On the other hand, limited activity was observed in glucose. The strains displayed optimum activity levels at neutral pH and a 35-37 degrees C temperature range. The alpha-Gal activities of L. curvatus R08 and Leu. mesenteroides [corrected] JK55 were maintained at pH 6.5-10.0. The activity of the alpha-Gal enzyme was stable in a relatively broad range of temperatures from 0 to 40 degrees C for 3h. In soymilk, Leu. mesenteroides [corrected] JK55 and L. curvatus R08 completely hydrolyzed the NDO after 18-24h of fermentation. The abilities of L. curvatus R08 and Leu. mesenteroides [corrected] JK55 to degrade raffinose sugars and, particularly, to produce organic acids from sugar, could contribute to reductions in the anti-nutritional properties of soy, and to the accumulation of compounds with beneficial properties during food processing. Furthermore, this study provides the optimum conditions to induce alpha-Gal from these strains.

Optimization of production of monacolin K from gamma-irradiated Monascus mutant by use of response surface methodology.

Monascus isolate number 711, which is capable of producing monacolin K as an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the key enzyme of cholesterol synthesis, was isolated from Ang-kak, the red yeast rice koji. To increase the monacolin K-producing activity of the strain, spore suspensions of the strain were subjected to gamma-irradiation. One thousand mutants were generated via gamma-irradiation and screened using bioassay and high performance liquid chromatography analysis. Several mutants with higher productivities of monacolin K than that of the parent strain were primarily selected. Mutant KU609 was finally selected because of its characteristics of high monacolin K production and non-citrinin-producing activity under our test conditions. Response surface methodology was used to analyze the effect of culture medium on the production of monacolin K in mixed solid-state cultures. The optimal values of nutritional ingredients for the maximal production were soytone, glucose, MgSO4, and barley at concentrations of 0.5 g, 0.48 g, 0.053 g, and 9 g, respectively. The final monacolin K production of Monascus KU609 was increased almost 100-fold compared to that of the parent strain.

Fungal mycoflora and mycotoxins in Korean polished rice destined for humans.

Rice samples collected from the Republic of Korea were analyzed for fungal mycoflora and mycotoxins: fumonisins, ochratoxin A, trichothecenes, and zearalenone. The potential of the fungi to produce each mycotoxin was also examined, so that the fungal isolates associated with mycotoxins occurring in rice could be verified. Penicillium citrinum and Aspergillus candidus were the most prevalent species infecting the samples, while Fusarium proliferatum was found as the dominant Fusarium species. Ochratoxin A was the most commonly detected mycotoxin analyzed in the present study; moreover, its level in some samples was above the EU tolerable limit (3 ng/g). According to rice culture experiments, it was revealed that in Korea, fumonisins detected in rice were due to F. proliferatum infection, whereas the occurrence of ochratoxin A was caused by Penicillium verrucosum, though there were no symptoms of disease in rice found in any sample. Furthermore, there appears to be an uneven geographical distribution of P. verrucosum as well as ochratoxin A in that most of them are found in the rice samples produced in the northern region of Korea.

Isolation and characterization of anticomplementary beta-glucans from the shoots of bamboo Phyllostachys edulis.

Bamboo, Phyllostachys edulis produces well known edible shoots in Asia. Immunostimulating anticomplementary (complement activating) substances have been recognized as a characteristic biological response modifier (BRM). In the present study, we isolated and characterized three anticomplementary beta-glucans (BS-BGA, BS-BGB, and BS-BGC) from bamboo shoots. Hot-water extraction, DEAE-Toyopearl 650M-column chromatography, amylase digestion and concanavalin A adsorption, and Sephacryl S-100 HR column chromatography were applied to isolate the beta-glucans. The average molecular masses of the beta-glucans were estimated to be from 14,500 to 85,300 Da by HPSEC-MALLS-RI. All three beta-glucans (0.1-1.0 mg/mL) activated the complement system via the alternative pathway, and could cleave human complement C3 under Ca 2+-free gelatin veronal buffered saline. Among them, the largest molecule, BS-BGA was the most potent complement activator. Methylation analysis and NMR spectroscopy were used to achieve their structural characterization. They are all water-soluble and composed mainly of backbone structures of beta-(1-->3)-glucan with beta-(1-->4)-linked side chains varying in degree of branching. BS-BGA consisted of a higher proportion of 3-linked glucopyranosyl residues and a lower degree of branching than BS-BGB and BS-BGC. In particular, BS-BGA contained a small amount of O-acetyl groups at C-6 of the 3-linked glucopyranosyl residues. These data demonstrate that the structural characteristics including molecular size, degree of branching, and O-acetyl substitution are involved, at least in part, in their different anticomplementary activities.