Engineering Bacillus subtilis J46 for efficient utilization of galactose through adaptive laboratory evolution.

Efficient utilization of galactose by microorganisms can lead to the production of valuable bio-products and improved metabolic processes. While Bacillus subtilis has inherent pathways for galactose metabolism, there is potential for enhancement via evolutionary strategies. This study aimed to boost galactose utilization in B. subtilis using adaptive laboratory evolution (ALE) and to elucidate the genetic and metabolic changes underlying the observed enhancements. The strains of B. subtilis underwent multiple rounds of adaptive laboratory evolution (approximately 5000 generations) in an environment that favored the use of galactose. This process resulted in an enhanced specific growth rate of 0.319 ± 0.005 h-1, a significant increase from the 0.03 ± 0.008 h-1 observed in the wild-type strains. Upon selecting the evolved strain BSGA14, a comprehensive whole-genome sequencing revealed the presence of 63 single nucleotide polymorphisms (SNPs). Two of them, located in the coding sequences of the genes araR and glcR, were found to be the advantageous mutations after reverse engineering. The strain with these two accumulated mutations, BSGALE4, exhibited similar specific growth rate on galactose to the evolved strain BSGA14 (0.296 ± 0.01 h-1). Furthermore, evolved strain showed higher productivity of protease and ß-galactosidase in mock soybean biomass medium. ALE proved to be a potent tool for enhancing galactose metabolism in B. subtilis. The findings offer valuable insights into the potential of evolutionary strategies in microbial engineering and pave the way for industrial applications harnessing enhanced galactose conversion.

Consumer Preference of Traditional Korean Soy Sauce (Ganjang) and Its Relationship with Sensory Attributes and Physicochemical Properties.

This study aimed to investigate the physicochemical characteristics, sensory attributes, and consumer acceptance of the Certification of Quality of Traditional Food (CQT) ganjang samples produced in different provinces of Korea. Wide variations in physicochemical properties were found among the samples, especially in lipids, total nitrogen, acidity, and reducing sugar. Traditional fermented foods are known to be closely tied to regional features, but the composition and characteristics of CQT ganjangs might be influenced much more by individual ganjang producers than by region. Preference mapping was performed to understand consumer behavior towards ganjang, and most consumers tended to have similar preferences, implying shared a common sensory ideal. The results of the partial least squares regression revealed drivers of liking for ganjang among sensory attributes, free amino acids, and organic acids. Overall, sensory attributes such as sweetness and umami were positively associated with acceptability, while the terms related to fermentation were negatively associated. In addition, amino acids, such as threonine, serine, proline, glutamate, aspartate, and lysine, and organic acids, such as lactate and malate, were positively associated with consumer acceptance. The important implications of the findings of this study for the food industry can be utilized to develop and optimize traditional foods.

The Effect of Vacuum Films on Physicochemical and Microbiological Characteristics of Hanwoo (Korean Native Cattle).

The objective of this study was to investigate the physicochemical and microbiological characteristics of round of Hanwoo by vacuum packaging film materials, polyvinylidene chloride (PVDC) and ethylene vinyl alcohol (EVOH). The packaged beef samples were stored in refrigerated conditions (2±1°C) for 12 weeks. Physicochemical analysis with pH, surface color, thiobarbituric acid reactive substances (TBARS) values, and volatile basic nitrogen (VBN) values and microbiological analysis with aerobic plate count (APC) and metagenomic analysis of packaged beef samples were performed. The pH and surface color did not change substantially during the 12 weeks and EVOH-packaged beef tended to be lower than those of PVDC-packaged beef. PVDC- and EVOH-packaged samples showed low TBARS and VBN values below standard limits. APC did not exceed 7 Log CFU/g for both samples during storage. In metagenomic analysis, Firmicutes and Lactobacillaceae were dominant phylum and family of the PVDC- and EVOH-packaged beef. In both packaged samples, Dellaglioa algida was the dominant species during storage, with the notable difference being the presence of Lactococcus piscium. Therefore, this study provided the information on the quality of vacuum-packaged beef according to different vacuum films for long-term refrigerated storage.

Untargeted Metabolomics reveals Doenjang metabolites affected by manufacturing process and microorganisms.

Doenjang is a traditional Korean fermented soybean-based food that is now produced industrially as traditional Doenjang and modernized Doenjang, depending on the conditions of the manufacturing process. In this study, the effect of the production process on Doenjang metabolites was analyzed using untargeted capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS). Of the 247 metabolites detected in 19 traditional and three modernized Doenjang samples, the level of 55 metabolites were significantly different between the two production groups. The S-plot of the orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed that nine compounds (tryptamine, 2-phenylethylamine, citrulline, gamma-aminobutyric acid, putrescine, tyramine, 2-aminoisobutyric acid, stachydrine, and N5-ethylglutamine) were highly distributed in the traditional group, and six compounds (arginine, citric acid, choline, cytidine, hypoxanthine, and glucaric acid) were considered distinguishable metabolites of the modernized group. Microbial community analysis indicated that the levels of these metabolites were significantly altered by the presence of Bacillus spp., Enterococcus faecium, Tetragenococcus halophilus, Aspergillus oryzae, Penicillium spp., and Saccharomyces cerevisiae. These findings will give better understanding of the influence of the manufacturing process on Doenjang production in terms of both microbial activity and metabolite profiles.

Characteristics of Bacterial Strains with Desirable Flavor Compounds from Korean Traditional Fermented Soybean Paste (Doenjang).

To identify and analyze the characteristics of the microorganisms involved in the formation of the desirable flavor of Doenjang, a total of 179 strains were isolated from ninety-four Doenjang collected from six regions in South Korea, and fourteen strains were selected through a sensory evaluation of the aroma of each culture. The enzyme activities of amylase, protease and lipase was shown in the various strains. Bacillus sp.-K3, Bacillus sp.-K4 and Bacillus amyloliquefaciens-J2 showed relatively high protease activity, at 317.1 U, 317.3 U and 319.5 U, respectively. The Bacillus sp.-K1 showed the highest lipase activity at 2453.6 U. In the case of amylase, Bacillus subtilis-H6 showed the highest activity at 4105.5 U. The results of the PCA showed that Bacillus subtilis-H2, Bacillus subtilis-H3, and Bacillus sp.-K2 were closely related to the production of 3-hydroxy-2-butanone (23.51%~43.37%), and that Bacillus subtilis-H5 and Bacillus amyloliquefaciens-J2 were significantly associated with the production of phenethyl alcohol (0.39% and 0.37%). The production of peptides was observed to vary among the Bacillus cultures such as Val-Val-Pro-Pro-Phe-Leu and Pro-Ala-Glu-Val-Leu-Asp-Ile. These peptides are precursors of related volatile flavor compounds created in Doenjang via the enzymatic or non-enzymatic route; it is expected that these strains could be used to enhance the flavor of Doenjang.

Physiological Characteristics and Anti-Diabetic Effect of Pediococcus pentosaceus KI62.

The purpose of this study is to examine the physiological characteristics and anti-diabetic effects of Pediococcus pentosaceus KI62. The α-amylase and α-glucosidase inhibitory activity of P. pentosaceus KI62 was 94.86±3.30% and 98.59±0.52%, respectively. In MRS broth containing 3% maltodextrin inoculated by P. pentosaceus KI62, the amounts of short chain fatty acids (SCFA) were propionic acid 18.05±1.85 mg/kg, acetic acid 1.12±0.07 g/100 mL, and butyric acid 2.19±0.061 g/kg, and those of medium chain fatty acids (MCFA) were C8 0.262±0.031 mg/kg, C10 0.279±0.021 mg/kg, and C12 0.203±0.009 mg/kg. Compared to sixteen antibiotics, P. pentosaceus KI62 had the highest sensitivity to penicillin-G and rifampicin, as well as the highest resistance to vancomycin and ampicillin. The strain also showed higher leucine arylamidase and valine arylamidase activities than other enzyme activities, but it did not produce ß-glucuronidase which is carcinogenic enzymes. The survival rate of P. pentosaceus KI62 in 0.3% bile was 91.67%. Moreover, the strain showed a 98.63% survival rate in pH 2.0. P. pentosaceus KI62 exhibits resistance to Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus at rates of 29.41%, 38.10%, 51.72%, and 50.47%, respectively. P. pentosaceus (23.31%) showed a similar adhesion ability to L. rhamnosus GG, the positive control (24.49%). These results show that P. pentosaceus KI62 has possibility as a probiotic with anti-diabetic effects.

Complete genome sequence of Paenibacillus xylanexedens PAMC 22703, a xylan-degrading bacterium.

Paenibacillus is widely distributed in various environments and has the potential for use as a biotechnological agent in industrial processes. Here, we report the complete genome sequence of the marine bacterium, Paenibacillus xylanexedens PAMC 22703, which utilizes xylan. The P. xylanexedens PAMC 22703 strain was isolated from marine sediments. P. xylanexedens PAMC 22703 utilizes xylan as a carbon source to grow. The genome sequence clarified that this strain possesses genes for utilizing xylan. The complete genome sequence contained one chromosome (7,053,622 bp with 46.0% GC content) and one plasmid (44,617 bp with 44.1% C + G content). The genome harbored genes that fully deploy the xylan assimilation pathway. The complete genome sequence of P. xylanexedens PAMC 22703 would prove useful in acquiring information for its application with xylan in various industries.

In Vitro Antidiabetic and Antiobesity Activities of Traditional Kochujang and Doenjang and Their Components.

In this study we investigated the antidiabetic and antiobesity effects of aqueous ethanol extracts of traditional kochujang and doenjang. The average α-glucosidase inhibitory activity and adipogenesis inhibitory activity for the kochujang samples were 29.6% and 20.8%, respectively, while those of the doenjang samples were 46.3% and 11.6%, respectively. Therefore, antidiabetic activity is high in doenjang and antiobesity activity is high in kochujang. Kochujang and doenjang components responsible for suppressing the functional effects were investigated by metabolomic analysis. For kochujang, p-coumaric acid, N6,N6,N6-trimethyllysine, threonine, and methionine positively correlated with inhibition of adipogenesis activity, whereas for doenjang, betaine and betaine aldehyde were thought to be responsible for the antidiabetic effects. As p-coumaric acid and betaine were the most probable candidates with functional effects, these two compounds were selected for further analysis. Inhibition of adipogenesis was shown to be 14.0±1.85% for betaine chloride and 38.3±3.27% for p-coumaric acid, suggesting that p-coumaric acid is more effective than betaine against obesity. However, betaine exhibited higher α-glucosidase inhibitory activity than p-coumaric acid. Our results suggest that both kochujang and doenjang can be used against diabetes and obesity. However, clinical trials are necessary to support these results.

Short communication: Physicochemical features and microbial community of milk kefir using a potential probiotic Saccharomyces cerevisiae KU200284.

The aim of this study was to analyze the ß-glucan contents, physicochemical features, and microbial communities in milk kefir prepared using Saccharomyces cerevisiae KU200284 isolated from cucumber jangajji, a fermented vegetable commonly eaten in Korean. Three types of milk kefir were manufactured, with (1) activated kefir grain, (2) activated kefir grain with commercial S. cerevisiae BOF, and (3) activated kefir grain with S. cerevisiae KU200284. ß-Glucan contents of milk kefir using kefir grain and kefir grain with S. cerevisiae strains BOF and KU200284 were 8.29, 8.59, and 8.57%, respectively. The pH, titratable acidity, viscosity, Brix level, and alcohol contents of milk kefir using kefir grain with S. cerevisiae strains were acceptable compared with milk kefir using only kefir grain. In milk kefir produced using kefir grains and S. cerevisiae strains, 16S rRNA reads showed representative strains of Lactobacillus kefiranofaciens (>72% relative abundance) and Acetobacter fabarum (>16% relative abundance). In particular, milk kefir using kefir grain with S. cerevisiae KU200284 had the highest relative abundance of L. kefiranofaciens. In addition, the internal transcribed sequence (ITS) rRNA reads in tested milk kefir showed representative strains of Kluyveromyces marxianus (>52% relative abundance) and Saccharomyces cerevisiae (>16% relative abundance). In contrast, milk kefir using S. cerevisiae strains had higher relative abundance of S. cerevisiae (>37%). The ß-glucan production, physicochemical properties, and microbial community profiling indicate that S. cerevisiae KU200284 could be used in functional dairy products as a starter culture.

Protein Extraction from Porcine Myocardium Using Ultrasonication.

Porcine myocardium is regarded as a byproduct in slaughterhouses and is rarely used as a food source due to its unsuitability for processing and consumption. In this study, we sought to develop an efficient ultrasonication method to extract protein from porcine myocardium. Comparisons of protein yield using various ultrasonication conditions with porcine myocardium revealed that treatment with 0.2 M NaCl, with pH 8.0, at an extraction temperature of less than 40 °C and an amplitude of 60% to 80% was optimal, yielding an extraction rate of 90%. In addition, SDS-PAGE analysis showed that increasing the time interval for ultrasonication increased the presence of myosin heavy chain and actin protein content. Functional analysis of the physiological properties of the isolated proteins using an ATPase assay showed that Ca and Mg ATPase activity was virtually undetectable in the early stages of ultrasonic treatment and that the proteins denatured rapidly. An analysis of protein digestion also showed that the digestive capacity of proteins treated by ultrasonication methods was greater. These results demonstrate that the ultrasonication method is effective for high-yield protein extraction from cardiac myofibrils of porcine myocardium with low salt concentrations, low Ca and Mg ATPase activities, and high digestive capacities.

Complete genome sequence of Leuconostoc garlicum KCCM 43211 producing exopolysaccharide.

Leuconostoc garlicum KCCM 43211 isolated from traditional Korean fermented food is an intensive producer of exopolysaccharide (EPS). Here we report the first complete genome sequence of L. garlicum KCCM 43211. The genome sequence displayed that this strain contains genes involved in production of EPS possibly composed of glucose monomers. An uncharacterized EPS from the L. garlicum KCCM 43211 strains was also produced during fermentation in the sucrose medium. The MALDI-TOF results displayed the typical mass spectrometry pattern of dextran. This uncharacterized EPS may have use in commercial prebiotics, food additives, and medical purposes. The complete genome sequence of L. garlicum KCCM 43211 will provide valuable information for strain engineering based on the genetic information.

Comparative Study on the Characteristics of Weissella cibaria CMU and Probiotic Strains for Oral Care.

Probiotics have been demonstrated as a new paradigm to substitute antibiotic treatment for dental caries, gingivitis, and chronic periodontitis. The present work was conducted to compare the characteristics of oral care probiotics: Weissella cibaria CMU (Chonnam Medical University) and four commercial probiotic strains. Survival rates under poor oral conditions, acid production, hydrogen peroxide production, as well as inhibition of biofilm formation, coaggregation, antibacterial activity, and inhibition of volatile sulfur compounds were evaluated. The viability of W. cibaria CMU was not affected by treatment of 100 mg/L lysozyme for 90 min and 1 mM hydrogen peroxide for 6 h. Interestingly, W. cibaria produced less acid and more hydrogen peroxide than the other four probiotics. W. cibaria inhibited biofilm formation by Streptococcus mutans at lower concentrations (S. mutans/CMU = 8) and efficiently coaggregated with Fusobacterium nucleatum. W. cibaria CMU and two commercial probiotics, including Lactobacillus salivarius and Lactobacillus reuteri, showed high antibacterial activities (>97%) against cariogens (S. mutans and Streptococcus sobrinus), and against periodontopathogens (F. nucleatum and Porphyromonas gingivalis). All of the lactic acid bacterial strains in this study significantly reduced levels of hydrogen sulfide and methyl mercaptan produced by F. nucleatum and P. gingivalis (p < 0.05). These results suggest that W. cibaria CMU is applicable as an oral care probiotic.

Effect of Fermentation Temperature on the Volatile Composition of Kimchi.

This study was conducted to evaluate the effect of fermentation temperature on the volatile composition in Kimchi. Kimchi was fermented at 2 temperature conditions (4 and 20 °C). Volatile compounds of Kimchi samples were analyzed during the fermentation periods using the dynamic headspace gas chromatography-mass spectrometry method. The optimum ripening time for the Kimchi fermented at 4 °C was 35 d, and that of 20 °C was 2 d. The pH at the optimum ripening time was 4.97 and 4.41, and the titratable acidity was 0.59% and 0.76% for the Kimchi fermented at 4 and 20 °C, respectively. Forty different types of volatile compounds, including alcohol, aldehyde, ester, and sulfur compounds, were identified. The Kimchi fermented at 20 °C produced greater amounts of volatile compounds than that at 4 °C. The amounts of most volatiles increased as the fermentation time increased, but those of aldehydes decreased rapidly during both 4 and 20 °C fermentation. Organic acids, ester, and nitriles were detected only in Kimchi fermented at 20 °C. The amounts of dimethyl disulfide, methyl-2-propenyl disulfide, and di-2-propenyl disulfide produced from the Kimchi fermented at 20 °C were more than 2-times of those at 4 °C. Therefore, it is concluded that the strong pungent odor of Kimchi fermented at 20° C is probably due to the high amount of organic acids (low pH) and sulfur compounds (dimethyl disulfide, methyl-2-propenyl disulfide, and di-2-propenyl disulfide) between the 2 Kimchi.

Optimization of ultrasonic extraction of phenolic antioxidants from green tea using response surface methodology.

Response surface methodology (RSM) has been used to optimize the extraction conditions of antioxidants with relatively low caffeine content from green tea by using ultrasonic extraction. The predicted optimal conditions for the highest antioxidant activity and minimum caffeine level were found at 19.7% ethanol, 26.4 min extraction time, and 24.0 ° C extraction temperature. In the predicted optimal conditions, the experimental values were very close to the predicted values. Moreover, the ratio of (EGCg + ECg)/EGC was identified a major factor contributing to the antioxidant activity of green tea extracts. In this study, ultrasonic extraction showed that the ethanol concentration and extraction time used for antioxidant extraction could be remarkably reduced without a decrease in antioxidant activity compared to the conventional extraction conditions.

Inactivation of pathogens inoculated into prepared seafood products for manufacturing kimbab, steamed rice rolled in dried seaweed, by gamma irradiation.

Three prepared seafood products for manufacturing a laver (dried seaweed) roll, a traditional and rapidly growing ready-to-eat meal in Korea, were selected and the effects of irradiation treatment for eliminating pathogens of public health significance were investigated. The pathogens tested were Salmonella Typhimurium, Escherichia coli, Staphylococcus aureus, and Listeria ivanovii. The radiation sensitivity (D10-values or the dose required to inactivate 90% of a population) of these organisms ranged from 0.23 to 0.62 kGy in imitation crab leg, 0.31 to 0.44 kGy in surimi gel, and 0.27 to 0.44 kGy in dried seaweed. The growth of all four test organisms inoculated (10(8) CFU/g) into these foods was inhibited by irradiation during 24 h of postirradiation storage regardless of the temperature (10, 20, and 30 degrees C). L. ivanovii was not detected after a 3-kGy treatment, but the other pathogens were not detected following irradiation at 2 kGy. These studies indicated that low-dose irradiation (2 kGy or less) of prepared seafood materials can keep them microbiologically safe before manufacturing a ready-to-eat prepared meal, a laver roll.