Simultaneous Identification of Different Domains of Calpain from Blood and Turkey Meat Samples Using Casein Zymography

The aim of this study was to develop a simple method for simultaneous identification of two different domains of calpains (μ- and m-calpains) in blood and turkey meat samples. The method is based on Tris-based extraction techniques followed by casein zymography detection. The extracts were purified by dialysis, and target compounds were separated on Diethylaminoethyl (DEAE)-Sephacel anion-exchange chromatography. Results indicated that a buffer pH of 6.7 produced optimum extraction efficiency. The separation of calpastatin, μ- and m-calpains on column chromatography was carried out using stepwise elution profiles of 100, 200, and 400 mM NaCl solutions, respectively. Casein zymography study clearly demonstrated the presence of μ- and m-calpains in the crude extracts of breast and blood samples; however, μ-calpain was absent in the thigh sample. Similarly, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis identified both the calpains from muscle and blood samples, but presence of calpastatin was observed in both the breast and thigh muscle samples only. The activity analysis of purified fractions indicated that both the μ- and m-calpain concentrations were differed significantly (P?<?0.05) amongst breast, thigh and blood samples. Collectively, it was concluded that Tris buffer (pH 6.7) and DEAE-Sephacel column for extraction and separation of μ- and m-calpains and casein zymography and SDS-PAGE detection methods were most suitable for accurate identification of calpains and calpastatin from blood and turkey meat samples.     

Purification and characterization of <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Oenococcus oeni</Emphasis> 31MBR

This study was designed to characterize a β-glucosidase from Oenococcus oeni 31MBR, a strain widely used in Chinese winemaking. An intracellular β-glucosidase (EC 3.2.1.21) was partially purified using a combination of ammonium sulfate precipitation and chromatographic methods. A single band was obtained in SDS-PAGE electrophoresis, indicating that the enzyme was highly purified and had an estimated molecular mass of 38.9 kDa. The enzyme exhibited highest activity at pH 4.5–5.0. The optimum temperature was 45 °C. Ethanol promoted the activity of this enzyme up to three times. Among the several metal ions assayed, only Mn²⁺ exhibited a partial promotion effect. The K _m and V _max values for p-nitrophenyl-β-d-glucopyranoside were 1.05 mmol/L and 0.957 nmol/min, respectively. Up to now, this study contains the first characterization of a native β-glucosidase purified from crude extracts of O. oeni 31MBR.     

Probiotic characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> P223 isolated from kimchi

Probiotic characteristics of Bacillus subtilis P223 isolated from kimchi were investigated in this study. Spore cells of B. subtilis P223 showed high tolerance to artificial gastric juice (pH 2.5, 0.3% pepsin, 3 h) and bile salts (0.3% oxgall, 24 h). Spore cells of B. subtilis P223 showed more adherence to intestinal cells (HT-29 cells) than vegetative cells. In addition, B. subtilis P223 showed high autoaggregation ability, similar to a commercial strain (Bacillus clausii ATCC 700160). Moreover, its coaggregation abilities with pathogens were strong. The adherence of three pathogens (Salmonella enteritidis ATCC 13076, Listeria monocytogenes ATCC 15313, and Escherichia coli ATCC 25922) to HT-29 cells was inhibited by B. subtilis P223. It was found that B. subtilis P223 could not produce β-glucuronidase, a carcinogenic enzyme. However, it had amylase and protease activities. Antibiotic susceptibility was measured using disk diffusion assay. It was revealed that B. subtilis P223 was only resistant to streptomycin among eight kinds of antibiotics. In addition, B. subtilis P223 showed no hemolysis activity. It did not have enterotoxin genes. Results of this study suggest that B. subtilis P223 isolated from kimchi has potential as a probiotic strain.     

Development and characterization of reconstituted hydrogel from <Emphasis Type="Italic">Aloe vera</Emphasis> (<Emphasis Type="Italic">Aloe barbadensis</Emphasis> Miller) powder

Structural and rheological characterization of reconstituted hydrogels developed from A. vera non-fibrous alcohol insoluble residue (NFAIR) powder using different methods [viz., shaking (S), heating-shaking (HS), and heating (H)] and concentrations (viz., 0.2–1.6 %, w/v) was carried out. Functional group distribution by FTIR spectroscopy and Congo red (CR) method revealed the presence of acetylated acemannan in A. vera powder. Dynamic oscillation studies of A. vera (NFAIR) fluids at all concentrations of 0.2–1.6 %, w/v, showed gel strength in the order of H > HS > S method. However, in H method, increase in concentration from 0.2 to 1.6 %, w/v showed the conformational transition from semi-diluted solution to weak gel nature. Rheological models described the effect of heating temperatures (HT); 30–90 °C, and times (Ht); 15–60 min on viscoelastic behavior in reconstituted A. vera fluids. The reconstituted A. vera hydrogel prepared with a concentration of 1.6 %, w/v using 50 °C (HT) and 30 min (Ht) condition showed a good agreement with the Power law (storage modulus, G′) and Weak gel model (complex modulus, G*) fitted data (R² > 0.94) resulting higher viscoelastic moduli intercepts; G′₀ (71.5 Pa s ^n′), G″₀ (33.5 Pa s ^n″), lower slopes; n′ (0.22), n″ (0.06), higher network strength (A _F , 121.3 Pa s^1/z ) and number of network (z, 5.3) values. The obtained results suggested that heating at 50 °C/30 min can develop aqueous weak gel networks of A. vera with enhanced gel strength which may be utilized as a novel gelling agent for wide variety of targeted applications in food and pharmaceutical sectors.     

Effect of heat-treated <Emphasis Type="Italic">Nuruk</Emphasis> on the quality characteristics of aged <Emphasis Type="Italic">Yakju</Emphasis>

Long-term aging of Yakju, a traditional Korean liquor made of rice and Nuruk (a fermentation agent), causes browning and odor and flavor development. This study investigated the effects of heat-treated Nuruk (50–80 °C, 30 min) on Yakju quality. The saccharogenic powers and glucoamylase, α-amylase, and carboxypeptidase activities were similar in non-heat-treated Nuruk and that treated at 50 °C. However, acidic protease and alcohol dehydrogenase decreased above 50 °C. The content of nitrogen-containing compounds was inversely proportional to the heat-treatment temperature. Compounds that cause off-flavors decreased at 50–60 °C, but increased at 70–80 °C, whereas compounds that provide fragrance increased at 50–60 °C. Sensory evaluation indicated that bad taste attributes were higher in Yakju produced using non-heat-treated Nuruk. Therefore, heat treatment of Nuruk at 50 °C can be adopted as a method for improving Yakju quality, as enzymatic activities that affect color, aroma, and taste are regulated.     

Physicochemical properties of enzyme-treated waxy rice flour and expansion properties of <Emphasis Type="Italic">Gangjung</Emphasis> (a traditional Korean oil-puffed rice snack)

Long steeping process of waxy rice involved in Gangjung production is essential for obtaining good quality Gangjung, but it causes many related problems. The objective was to examine physiochemical properties of waxy rice flour and expansion properties of Gangjung manufactured using waxy rice flour treated with protease (P-flour), α-amylase (A-flour), or in combination of the two (PA-flour) over different durations. While crude protein and reducing sugar contents of P-flour and Aflour differed depending on enzyme treatment time, it was not observed for PA-flour. Gangjung made with P-flour or A-flour could show similar expansion rate to that of Gangjung made with waxy rice flour steeped under the optimal condition, however, Gangjung made with PA-flour showed lower expansion rate than of the optimal one. These findings indicate that treatment of waxy rice flour with either protease or α-amylase, but not both, could reduce the steeping time involved in Gangjung production.     

Glycerides isolated from the aerial parts of <Emphasis Type="Italic">Malva verticillata</Emphasis> cause immunomodulation effects via splenocyte function and NK anti-tumor activity

A preliminary study revealed that a 10 µg/mL n-BuOH fraction of Malva verticillata aerial parts significantly enhanced splenocyte proliferation and induced significant enhancement of natural-killer (NK) cell activity against tumor cells (YAC-1). This study was initiated to identify the principal components that exhibited these activities, and four glycerides were isolated through repeated SiO₂ and ODS column chromatography. Structures of compounds 1–4 were determined to be (2S)-1-O-palmitoyl glyceride, (2S)-1-O-stearoyl glyceride, (2S)-1-O-linolenoyl glyceride, and (2S)-1,2-di-O-linoleoyl glyceride, respectively. Compounds 1–3 showed potential immune-enhancing activity in murine splenocyte and natural-killer (NK) cells at 10 µM. In contrast, compound 4 showed weak activity, indicating the monoacyl glycerides (1–3) are more effective than diacyl glyceride (4). Also, the longer the carbon number of the fatty acid in monoacyl glyceride, the better the activity, and the monoacyl glyceride including an unsaturated fatty acid (3) is more effective than the glycerides including the saturated fatty acids (1–2).     

RETRACTED ARTICLE: Antimicrobial and antifungal effects of green tea extracts against microorganisms causing vaginitis

Green tea extracts (GTE) including catechins and caffeine possess strong antimicrobial activity against a variety of microorganisms. In this study, we examined their possible antimicrobial and antifungal effects on bacteria (Proteus mirabilis and Streptococcus pyogenes) and yeast species (Candida albicans) that cause vaginitis. The GTE and ethyl acetate fractions exhibited strong growth inhibitory activity against P. mirabilis and S. pyogenes, whereas the aqueous fraction possessed poor antimicrobial activity. Among the catechins, epigallocatechin gallate showed strong antibacterial activity. C. albicans was potently inhibited by the methylene chloride fraction and caffeine, while antifungal activity of GTE was contributed by caffeine. Furthermore, we evaluated whether in vitro activity of GTE included purified solvent fractions that were stable to heat and pH. Antimicrobial activity was potent in alkaline environment for P. mirabilis and acid environment for S. pyogenes and C. albicans. Herein GTE can be a valuable therapeutic agent for the treatment of vaginal-related pathogens.     

Purification,characterization and action mechanism of plantaricin JY22, a novel bacteriocin against <Emphasis Type="Italic">Bacillus cereus</Emphasis> produced by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> JY22 from golden carp intestine

A novel bacteriocin-producing strain, Lactobacillus plantarum JY22 isolated from golden carp intestine, was screened and identified by its physiobiochemical characteristics and 16S rRNA gene sequence analysis. This bacteriocin, named plantaricin JY22, was purified using ethyl acetate extraction and gel filtration. Its molecular weight was approximately 4.1 kDa by SDS-PAGE analysis. The partial amino acid sequence of plantaricin JY22 was DFGFDIPDEV. It was highly heat-stable and remained active at pH range from 2.5 to 5.5, but was sensitive to protease. Plantaricin JY22 had a bactericidal mode. Scanning electron microscope analysis indicated that plantaricin JY22 damaged the morphology of cells and spores for Bacillus cereus. Moreover, the plantaricin JY22 destroyed cell membrane integrity as confirmed by the leakage of electrolytes, the losses of Na⁺K⁺-ATP, AKP, nucleic acids (OD_260nm) and proteins. SDS-PAGE of B. cereus proteins further demonstrated that plantaricin JY22 had a remarkable effect on bacterial proteins.     

Properties of a fibrinolytic enzyme secreted by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> JS2 isolated from saeu (small shrimp) <Emphasis Type="Italic">jeotgal</Emphasis>

Bacillus species were screened to be used as starters for jeotgals, salted and fermented Korean sea foods. A strain, JS2, showing strong fibrinolytic activity was isolated from saeu (small shrimp) jeotgal, and identified as Bacillus subtilis. Bacillus subtilis JS2 grew well at 20% (w/v) NaCl concentration. SDS-PAGE of culture supernatant from JS2 showed 3 major bands of 27, 29, and 60 kDa in size. Fibrin zymography showed that the 27 kDa band was the major fibrinolytic protein. The gene, aprEJS2, was cloned and introduced into B. subtilis WB600 using pHY300PLK. A B. subtilis transformant harboring pHYJS2 showed higher fibrinolytic activity than B. subtilis JS2. aprEJS2 was overexpressed in Escherichia coli BL21 (DE3). The optimum pH and temperature for AprEJS2 were pH 8.0 and 40 °C, respectively. Km and V_max values were determined. AprEJS2 has strong α-fibrinogenase activity and moderate β-fibrinogenase activity.     

Gene Expression Analysis as a Method to Predict OTA Accumulation in Dry-Cured Meat Products

Dry-cured meat products are frequently colonised by toxigenic Penicillium nordicum and Penicillium verrucosum throughout their ripening that can produce ochratoxin A (OTA). The predictive nature of the molecular analyses can be used to determine the risk of toxigenic species. For this reason, the objective of the present work was to analyse the temporal changes in the expression of the otapks and otanps genes by P. nordicum and P. verrucosum in relation to OTA production on slices of dry-fermented sausage salchichón and dry-cured ham. Gene expression was higher in P. nordicum than in P. verrucosum in both meat matrices. The otapks gene was overexpressed by both Penicillium species, especially in salchichón. OTA was only detected in inoculated salchichón regardless of the ochratoxigenic species used. The high significant correlation found between the early relative expression of the otapks gene and OTA production in salchichón leads to propose the relative gene expression of the otapks gene as a good indicator to predict OTA accumulation throughout the ripening of this product.     

Influence of keeping system on the fatty acid composition in the <Emphasis Type="Italic">longissimus</Emphasis> muscle of bulls and odorants formed after pressure-cooking

German Simmental bulls were kept either in a stable (group 1) or on a pasture (group 2). For both groups the intramuscular fat content of the longissimus muscle and the fatty acid composition in phospholipids and triacylglycerols were investigated. Significant influences of feeding were shown in most fatty acids measured in both lipid classes. In concentrate-fed bulls a higher content of linoleic acid (C18:2n-6) was detected, corresponding to the large amount of linoleic acid found in concentrate used as feed. In contrast, bulls fed grass proved to have a higher linolenic acid (C18:3n-3) content, also correlating well with the larger amount of linolenic acid found in grass. Pressure-cooking of the beef followed by an analysis of eight important meat odorants revealed that, in line with the higher concentrations of the precursor fatty acids C18:3n-3 and C18:1 in muscle of group 2, (E,Z)-2,6-nonadienal (from C18:3n-3) as well as nonanal and octanal (from C18:1) were much higher in the processed meat of animals fed grass. On the other hand, the much higher amounts of C18:2n-6 in bulls fed concentrate were well-reflected by higher concentrations of five aroma compounds known to be formed by a thermal degradation of this acid, e.g. (E,E)-2,4-decadienal.     

Identification of bitter components from <Emphasis Type="Italic">Artemisia princeps</Emphasis> Pamp.

Identification of bitter components from the aerial parts of Artemisia princeps Pamp. was performed to search for a method to eliminate the bitter taste from A. princeps products. The aerial parts of A. princeps were extracted in an aqueous EtOH solution, and the obtained extracts were partitioned into essential-oil, flavonoid-rich, n-BuOH, and aqueous fractions. Two purified bitter sesquiterpenoids were identified through repeated column chromatography of the bitterest fraction, the flavonoid-rich fraction, through an activity-guided fractionation method. The compounds were identified to be 1α,6α,8α-trihydroxy-5α,7βH-guaia-3,9,11(13)-trien-12-oic acid and artecalin, respectively, based on the interpretation of NMR, MS, and IR spectroscopic data. Both compounds were 50 times bitterer than caffeine and had similar bitterness to quinine HCl. Neither eupatilin nor jaceosidin, the major active components of A. princeps, showed any bitterness.     

Effect of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> Bauer and <Emphasis Type="Italic">Bifidobacterium animalis</Emphasis> ssp. <Emphasis Type="Italic">lactis</Emphasis> BB12 on proteolytic changes in dry-cured loins

The effect of the potentially probiotic bacteria strain of Lactobacillus acidophilus Bauer and probiotic bacteria Bifidobacterium animalis ssp. lactis BB12 on proteolytic changes of proteins in dry-cured loins during fermentation and cold storage was studied. Results of the conducted tests demonstrated that the use of probiotic bacteria for the production of dry-cured meats impacts the generation of products of protein proteolysis with high antioxidant activity. The highest antioxidant activity of peptides after fermentation and cold storage was observed in the loin with the strain B. animalis ssp. lactis BB12 and the loin with the mixture of strains L. acidophilus Bauer and B. animalis ssp. lactis BB12. Qualitative analysis of peptides demonstrated that peptides with weight below 3.5 kDa are characterized by the highest capacity of quenching the ABTS cation radical, including the peptides in loins with the strain B. animalis ssp. lactis BB12.     

The Comparison of Cinnamomi Cortex and <Emphasis Type="Italic">Cinnamomum burmannii</Emphasis> Blume Using <Superscript>1</Superscript>H NMR and GC-MS Combined with Multivariate Data Analysis

Cinnamomi Cortex (CC, rougui) is the stripped trunk bark of Cinnamomum cassia Presl (CCP) and commonly is used to treat dyspepsia, gastritis, blood circulation disturbances, and inflammatory diseases. However, Cinnamomum burmannii Blume (CBB), an edible ingredient in food from a different Cinnamomum, sometimes replaces CC due to their similar functions and appearance. To identify and distinguish CC from CBB, we investigated the metabolite differences in polar and non-polar extracts of these species by high-resolution ¹H nuclear magnetic resonance (¹H NMR) spectroscopy and gas chromatography–mass spectrometry (GC-MS) combined with multivariate statistical analyses. The results showed a significantly higher separation in GC-MS analysis (non-polar extracts) than ¹H NMR analysis (polar extracts). One-way ANOVA revealed that polar extracts (acetate, α-glucose, sucrose, glycerol, fructose) and non-polar extracts (trans-cinnamaldehyde, α-copaene, δ-cadinene, 2-methoxycinnamaldehyde, (?)-calamenene, α-muurolene, γ-muurolene, α-calacorene, cubenol and α-muurolol) contributed greatly to the comparison of CC and CBB. These results indicate that the ¹H NMR- and GC-MS-based metabolomic approach can effectively differentiate the phytochemical compositions among different species in plants, which in turn could identify important metabolites with known pharmacological activity.     

Molecular analysis of bacterial community dynamics during the fermentation of <Emphasis Type="Italic">soy-daddawa</Emphasis> condiment

Bacterial community dynamics during soy-daddawa fermentation was investigated using culture-dependent and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) molecular methods. The total titratable acidity (TTA), pH, and bacterial counts (BCs) were monitored daily during a 72-h fermentation period. Bacteria were characterized based on 16S rRNA gene sequencing. TTA ranged from 0.08 to 0.26 mg lactic acid/g, whereas pH ranged from 7.01 to 8.19. BCs increased from 3.9 to 10.61 log CFU/g. Fifty-eight isolates were obtained by culture method and clustered into seven operational taxonomic units (OTUs) at 97% sequence similarity, whereas four OTUs were obtained from the PCR-DGGE method. Taxonomic identification revealed that bacteria belonged to the genera Bacillus, Enterobacter, Enterococcus, and Staphylococcus with B. subtilis being present throughout fermentation. Medically significant isolates, including B. anthracis, Enterococcus casseliflavus, and Enterobacter hormaechei were detected. These results emphasize the need for starter culture utilization and offer a platform for starter culture screening and selection.     

Integrated Analytical Methods to Characterize Lipids from <Emphasis Type="Italic">Prosopis</Emphasis> spp. and <Emphasis Type="Italic">Ceratonia siliqua</Emphasis> Seed Germ Flour

Flour from seed germ of European carob (Ceratonia siliqua) and South American algarrobo (Prosopis spp.) is a potential ingredient for health-promoting baked products. Herein, lipids from germ of three Argentinean Prosopis (P. alba, P. nigra, and P. ruscifolia) and one European carob species were characterized in detail, exploiting an array of up-to-date analytical techniques. Total lipids ranged from 7.1 to 8.1% (w/w). Linoleic acid (C18:2, ω-6) predominated the GC flame ionization detector profiles of fatty acid in all samples (43.3–50.6%). Prosopis spp. contained 6–7% of C20–C24 fatty acids and, consistently, C56–C60 triacylglycerols, as detected by MALDI-TOF mass spectrometry (MS), which were practically missing in C. siliqua germ. Phospholipids were isolated by hydroxyapatite chromatography, characterized by MALDI-TOF MS and grossly quantified by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Because of a relatively high content (9.5–11.8%, v/v), phospholipids might increase the antioxidant potential and improve the baking performances of flour fortified with carob and algarrobo seed germ.     

A Comparative Biochemical Characterization of Microbial Transglutaminases: Commercial vs. a Newly Isolated Enzyme from <Emphasis Type="Italic">Streptomyces</Emphasis> Sp.

A new microbial transglutaminase (MTGase or MTG, EC 2.3.2.13) from a Streptomyces sp. strain isolated from Brazilian soil samples was characterized in crude and purified forms. The aim of this work is to provide relevant information about a new transglutaminase and to compare its characteristics with the well-known commercial transglutaminase from Ajinomoto Co. Inc. (Activa® TG-BP). The enzyme from Streptomyces sp., in both crude and pure forms, exhibited optimal activity in the 6.0–6.5 pH range and at 35–40°C. The results for the commercial enzyme were the same. A second maximum of activity was observed at pH 10.0 with both the crude Streptomyces sp. enzyme and the commercial enzyme. This interesting fact has not been reported in the literature previously. The fact that this second maximum of activity does not appear on the purified form of the enzyme may suggest the presence of an isoenzyme on the crude extract. All of the enzymes tested were stable over the pH range from 4.5 to 8.0 and up to 45°C. The decline in activity of the commercial transglutaminase above 45°C and pH 8.0 was more gradual. The activities of all the MTG samples were independent of Ca⁺² concentration, but they were elevated in the presence of K⁺, Ba²⁺, and Co²⁺ and inhibited by Cu²⁺ and Hg²⁺, which suggests the presence of a thiol group in the MTG’s active site. The purified enzyme presented a K _m of 6.37 mM and a V _max of 1.7 U/mL, while the crude enzyme demonstrated a K _m of 6.52 mM and a V _max of 1.35 U/mL.     

Effects of gardenia seed,green tea,and cactus pear in rice batter on the chemical quality of lotus root <Emphasis Type="Italic">bugak</Emphasis> and frying oil

This study investigated the effects of the addition of gardenia seed, green tea, or cactus pear (Opuntia ficus-indica) to rice batter at 10% on the lipid oxidation, pigments, antioxidants, and antioxidant activity of lotus root bugak and frying oil. Lipid oxidation was evaluated based on the conjugated dienoic acid and p-anisidine values. Lipid oxidation and tocopherol degradation were significantly reduced in the gardenia seed-added bugak and frying oil, whereas the cactus pear-added bugak and frying oil showed an increase. The addition of green tea had no significant effects on the lipid oxidation of bugak and frying oil. The in vitro antioxidant activity of lotus root bugak significantly increased with the addition of gardenia seed, green tea, or cactus pear. The results suggested that green tea and gardenia seed could improve the health and food functionality of antioxidation for lotus root bugak, respectively.