Tyramine production of technological important strains of <Emphasis Type="Italic">Lactobacillus</Emphasis>, <Emphasis Type="Italic">Lactococcus</Emphasis> and <Emphasis Type="Italic">Streptococcus</Emphasis>

The aim of this paper was to study the biogenic amines (histamine, tyramine, putrescine, cadaverine, agmatine, spermine and spermidine) production of selected technological important lactic acid bacteria (strains of the genera Lactococcus, Lactobacillus and Streptococcus). Three methods (ion-exchange chromatography (IEC), PCR and cultivation method with pH indicator) were used. Within the 39 strains of lactic acid bacteria tested, the production of tyramine (formed by tyrosine decarboxylase) was detected in eight strains (3 strains of Lactococcus lactis subsp. lactis, three strains of Lactococcus lactis subsp. cremoris, 1 strain of Streptococcus thermophilus and 1 strain of Lactobacillus delbrueckii subsp. bulgaricus). The other tested biogenic amines were not detected. Cultivation in decarboxylation broth seems to be the least accurate method for the detection of biogenic amines due to enhanced risk of false-positive reactions. Therefore, in order to detect bacteria producing biogenic amines, the combination of PCR and chromatographic methods (e.g. IEC) can be recommended.     

Estrogenic effects of various extracts from <Emphasis Type="Italic">Chamdanggui</Emphasis> (<Emphasis Type="Italic">Angelica gigas</Emphasis> Nakai) and <Emphasis Type="Italic">sogdan</Emphasis> (<Emphasis Type="Italic">Phlomis umbrosa</Emphasis> Turcz)

The various extracts from chamdanggui (Angelica gigas Nakai) and sogdan (Phlomis umbrosa Turcz) were evaluated for estrogenic activity and characterized according to HPLC profile. Chamdanggui and sogdan were individually extracted with 4 solvents (hot water, 70% ethanol, n-butanol, and dichloromethane) of differing polarities. Estrogenic activity was determined by E-screen using an estrogen-dependent MCF-7 BUS cell. Although almost all extracts showed estrogenic effects in a concentrationdependent manner, the hot water extract from chamdanggui (250 μg/mL) had the higher effect (138%). Among 90 fractions using HPLC separation of the hot water extract from chamdanggui, fraction 21 and 28 produced the highest estrogenic effects of 178 and 163% at 10 μg/mL, respectively. The results imply that the hot water extract from chamdanggui could be useful as an alternative hormone replacement therapy.     

Molecular diversity among natural populations of <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>/<Emphasis Type="Italic">paraplantarum</Emphasis> strains isolated from autochthonous dairy products

The diversity of 87 Lactobacillus paracasei and Lactobacillus plantarum/paraplantarum strains, previously identified from different autochthonous dairy products, was investigated by phenotypic and genotypic approaches. The increased resolution obtained using phenotypic and genotypic characterization allowed the level of strain heterogeneity detection to be widened. Phenotypic diversity was evaluated by studying biochemical characteristics of technological interest, including antimicrobial and proteinase activities, resistance to nisin, aggregation ability, production of exopolysaccharides, acetoin and diacetyl, citrate utilization, and antibiotic susceptibility. Genotypic diversity was generally evaluated by PCR amplification of repetitive bacterial DNA element fingerprinting using the (GTG)₅ primer [(GTG)₅-PCR]. Moreover, in cases where strains were not discriminated by (GTG)₅-PCR combined with phenotypic analysis, pulsed-field gel electrophoresis (PFGE) analysis was performed. The results indicate that L. plantarum/paraplantarum and L. paracasei natural isolates from artisanal dairy products are a gold mine in terms of diversity of strains and could be potentially interesting to dairy companies for the formulation of functional starter cultures in the production of innovative foods.     

Characterization of brewing microorganisms isolated from Korean traditional <Emphasis Type="Italic">nuruk</Emphasis> for <Emphasis Type="Italic">Cheongju</Emphasis> production

The objective of this study was to select and identify yeasts and molds isolated from traditional nuruk and to investigate their brewing characteristics for Cheongju production. The yeast strains Y190 (Accession ID-KACC 93251P), Y263 (Accession ID-KACC 93252P), and Y270 (Accession ID-KACC 93253P) showing high alcohol and flavor productivity were isolated and identified by phylogenetic inference based on an internal transcribed spacer 2 region sequence analysis. In addition, Aspergillus oryzae 83-10 (Accession ID-KACC 93254P) showing the highest enzyme activity was isolated. This study provides basic data for the production of Korean Cheongju and assesses the applicability of these three yeast strains and A. oryzae 83-10 isolated from traditional nuruk.     

Characterization of mannanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp., a novel <Emphasis Type="Italic">Codium fragile</Emphasis> cell wall-degrading bacterium

Seaweeds are considered as a health food partly due to the polysaccharide composition of the cell wall. Because conventional extraction methods have low yields and lead to environmental pollution, enzymatic methods have been proposed. In this study, a new strain of Bacillus sp. was isolated from cattle feces that produced a mannanase, a polysaccharide-degrading enzyme active against the green seaweed Codium fragile. The purified 39-kDa mannanase exhibited maximum activity at 55 °C and pH 6.0, and maintained its catalytic activity stably at temperatures up to 60 °C and at a broad pH range (5.0–11.0). Enzymatic activity was slightly enhanced by Cu²⁺ and Na⁺ but strongly inhibited by Fe²⁺, Ag⁺, and EDTA. The mannanase showed the highest specificity to the inexpensive substrates such as konjac powder and locust bean gum, and efficiently released various manno-oligosaccharides. This novel mannanase can thus be applicable in the food, feed, and pulp industries.     

Prevalence,genetic diversity,and antibiotic susceptibility of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. (<Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>) isolated from <Emphasis Type="Italic">Sunshik</Emphasis>, its ingredients and soils

The prevalence, genetic diversity, and antibiotic susceptibility of Cronobacter species (Enterobacter sakazakii) isolated from sunshik products, its ingredients, and root vegetable farm’s soils were investigated to analyze main reservoirs and contaminated sources of Cronobacter spp. Cronobacter spp. was isolated from 9 of 15 sunshik products, 26 of 72 its ingredients, and 2 of 39 soils. The root vegetables such as sweet potato and carrot showed higher contamination rate (70%) than the other sunshik ingredients. All isolates showed 929 bp band amplified from 16S rRNA and resistant to ampicillin, amoxicillin/clavulanic acid, and cefazolin. All isolates showed diverse random-amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) band patterns. However, 3 cases of RAPD banding patterns between clinical strains and isolates from sunshik products and root vegetables (yam, carrot) were related with similarities level of 80%. These studies indicated that root vegetables can be an important contamination source of Cronobacter spp. in sunshik products. Thus, the preparation of root vegetables for manufacturing sunshik products used as a weaning diet was handled with more care than the other sunshik ingredients.     

In vivo study of antiallergenicity of ethanol extracts from <Emphasis Type="Italic">Sargassum tenerrimum</Emphasis>, <Emphasis Type="Italic">Sargassum cervicorne</Emphasis> and <Emphasis Type="Italic">Sargassum graminifolium turn</Emphasis>

Food allergy has becoming the serious threat in the world for which the search of an effective anti-allergic drug is the demand of time. Keeping in view of the potentiality of seaweeds, the ethanol extracts from Sargassum tenerrimum (ST), Sargassum cervicorne (SC), and Sargassum graminifolium turn (SG) have been studied in vivo for its antiallergenicity through passive cutaneous anaphylaxis (PCA) and active cutaneous anaphylaxis (ACA) in female BALB/c mice. Intraperitoneal administration of these ethanol extracts inhibit mouse PCA and ACA in a dose-dependent manner using ovalbumin (OVA) and shrimp allergen as triggering agents to induce allergenicity over mice. The extract of ST containing phlorotannin has been found most active over the suppression of PCA triggered by OVA and shrimp with IC₅₀ values of 25.64 and 40.98 mg/kg, respectively and an efficacy comparable to that of an anti-allergic drug disodiumcromoglycate. Similarly, ST inhibits ACA triggered by ova and shrimp allergen in the mouse, with 50% suppression at 25.5 and 43.53 mg/kg, respectively. The results presented here show that these extracts are active on the studied models among which ethanol extract of ST was the most potent, leading toward the promising development of a new class of anti-allergic drugs.     

Pressurized acidified water extraction of black carrot [<Emphasis Type="Italic">Daucus carota</Emphasis> ssp. <Emphasis Type="Italic">sativus</Emphasis> var. <Emphasis Type="Italic">atrorubens</Emphasis> Alef.] anthocyanins

The anthocyanins present in black carrot were extracted with pressurized water acidified with sulfuric, citric and lactic acids. Anthocyanin degradation became significant above 100 °C and there was no improvement when extraction pressure was increased to 100 bar. Therefore, the extraction from black carrot was carried out at temperatures 50, 75 and 100 °C under 50 bar pressure. The extraction efficiencies in terms of acylated and non-acylated anthocyanins were comparable for all three acids used to acidify water at 50 °C, while similar results were observed at 75 °C for both citric and lactic acids. Water acidified with lactic acid showed significantly higher extraction efficiency at 100 °C compared to water acidified with sulfuric and citric acids. Highest degree of polymerization together with increasing degree of browning was observed within extracts when sulfuric acid was used. On the other hand, when organic acids were used to acidify water, a higher extraction efficiency of anthocyanins, accompanied with a relatively low polymerization and browning was observed, with lactic acid giving the best results.     

Effects of different <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Enterococcus</Emphasis> strains and chemical acidification regarding degradation of gluten proteins during sourdough fermentation

Dough quality and baking performance of wheat dough are significantly affected by the qualitative and quantitative composition of the gluten. Therefore, the degradation was studied of specific fractions of gluten proteins in sourdough as affected by starter cultures. Doughs were fermented for 0, 5, and 24 h at 30 °C after addition of Lactobacillus sakei, L. plantarum, L. sanfranciscensis or Enterococcus faecalis. Chemically acidified doughs were used as controls. All doughs were analyzed quantitatively for their content of albumins, globulins, gliadins, glutenins, and glutenin macropolymer by means of a combined extraction/HPLC procedure. Protein degradation during sourdough fermentation was primarily due to acidic proteases present in flour. While L. sakei, L. plantarum and L. sanfranciscensis were mostly non-proteolytic, E. faecalis clearly contributed to gluten proteolysis. Single gluten protein types were clearly different in their resistance to proteolytic activities of the dough system and E. faecalis, and, in contrast to total glutenins, the amounts of gluten macropolymer were significantly reduced already after 5 h of incubation. When longer fermentation times were applied, gluten was substantially degraded. The strongest decrease was found for the glutenin fraction leading to an increase of alcohol soluble oligomeric proteins in the gliadin fraction. The extent of the decrease of monomeric gliadins was strongest for the γ-type followed by the α- and the ω-types. This indicates that dough properties residing in specific types of gluten fractions can be influenced by the duration of fermentation and the application of proteolytic strains.     

Thermal inactivation kinetics of quality-related enzymes in cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">botrytis</Emphasis><Emphasis Type="Bold">)</Emphasis>

Thermal inactivation of quality-related enzymes in both cauliflower crude enzyme extracts and fresh tissue samples was studied in temperature range 50–100 °C. For crude enzyme extracts, several parameters, reaction rate constants (k) and activation energy (E _a) as well as decimal reduction time (D) and (z) values, were used to characterize the thermal stability. The rates of inactivation were found to follow first-order inactivation kinetics. Activation energies varied between 101.18 and 208.42 kJ mol⁻¹ with z values of 10.59–24.09 °C. The examined kinetics indicated that lipoxygenase was the most heat resistant followed by peroxidase, polyphenol oxidase, pectin methyl esterase and ascorbic acid oxidase. Furthermore, the obtained results from the blanched fresh tissues indicated that inactivation of lipoxygenase secured disappearing of any other enzyme activities. Therefore, this study recommends using lipoxygenase as an indicator enzyme to optimize the thermal treatments of cauliflower products.     

A New Multiplexed Real-Time PCR Assay to Detect <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, <Emphasis Type="Italic">C. coli</Emphasis>, <Emphasis Type="Italic">C. lari</Emphasis>, and <Emphasis Type="Italic">C. upsaliensis</Emphasis>

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 10² genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.     

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.     

Xanthan gum production under different operational conditions by <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv <Emphasis Type="Italic">vesicatoria</Emphasis> isolated from pepper plant

The influence of operational conditions (pH, stirrer speed, and temperature) used in the process of xanthan production by Xanthomonas axonopodis pv vesicatoria (XCVA3-1) isolated from pepper plant were evaluated through yield of xanthan and compared with control strain Xanthomonas campestris NRRL-B 1459. Different conditions used during the fermentation affected the xanthan production. In this study the best combination of yield was obtained, reaching 1.325 g/100 mL with the use of pH 7.0, 30°C, and 250 rpm during fermentation. Increased yield of xanthan production can be obtained at high agitation values, with the maximum at 400 rpm. Higher yields of gum production can be obtained at 30°C and the optimum pH was found 7.0. This results were similar for the X. campestris NRRL-B 1459.     

Antioxidant and emulsion properties of freshwater carps (<Emphasis Type="Italic">Catla catla</Emphasis>, <Emphasis Type="Italic">Labeo rohita</Emphasis>, <Emphasis Type="Italic">Cirrhinus mrigala</Emphasis>) protein hydrolysates prepared using flavorzyme

Fish protein hydrolysates (FPHs) were prepared from freshwater carps (Catla catla, Labeo rohita, and Cirrhinus mrigala) using flavorzyme at different degrees of hydrolysis (DHs) ranging from 5 to 20%. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical-scavenging activity of the FPHs prepared from the three species were in the range of 50–82%; the ferric reducing power of the FPHs prepared from catla was the highest. The linoleic acid peroxidation inhibition activity of the prepared FPHs varied from 71 to 91%. The emulsion activity index of the FPHs prepared from catla and rohu decreased significantly with an increase in the DH (p < 0.05). The emulsion stability index of the FPHs prepared from the three species was the highest at 20% DH. FPHs prepared from freshwater carps possess good antioxidant and surface-active properties and are therefore suitable to be used as natural antioxidants in health-food formulation and as water-soluble antioxidants in the food-processing industry.     

The ability of <Emphasis Type="Italic">Lactobacillus</Emphasis> adhesin EF-Tu to interfere with pathogen adhesion

Probiotic bacteria play an important role in preventing widespread colonization of enteropathogens in human gastrointestinal tract. Lactobacillus plantarum CS24.2, isolated from child fecal sample, and L. rhamnosus GG, a widely accepted commercial probiotic strain, were examined in vitro for their ability to inhibit the colonization of enteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhi (S. Typhi) on human intestinal epithelial cell line (Caco-2). Different adhesion assays such as competitive inhibition, adhesion inhibition, and displacement were carried out for the assessment of antagonistic activity of probiotic bacteria toward adhesion of enteropathogens to Caco-2 cells. Both the probiotic bacteria were able to inhibit pathogen colonization between 30–90% under different assay conditions. The gene coding for the known Lactobacillus adhesion factor—elongation factor Tu (EF-Tu)—was amplified from L. plantarum CS24.2 and cloned into E. coli expression vector, pET30(a). The partially purified recombinant EF-Tu was able to inhibit 50% adhesion of L. plantarum CS24.2 to Caco-2 cells but there was no effect on L. rhamnosus GG adhesion under competitive inhibition assay. To investigate the functional role of lactobacilli EF-Tu in pathogen inhibition to Caco-2 cells, competitive adhesion assay was carried out and there was significant reduction in the adhesion of E. coli (35.3%) and S. Typhi (47.7%) to Caco-2 cells.     

Nutritional properties of mussels <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis>

The aim of this study was to determine the quality indices and assess the influence of diets supplemented with mussels from contaminated (MCont) and non-contaminated areas (MNCont) on some indices of protein and lipid metabolism in rats fed with cholesterol. A wide range of in vitro tests demonstrated that mussels from contaminated area have a higher content of proteins, lipids and higher antioxidant capability. In the in vivo experiment, 28 male Wistar rats were divided into 4 diet groups each of 7 and named Control, Cholesterol (Chol), Chol/MCont and Chol/MNCont. During 30 days of the experiment rats of all four groups were fed basal diet (BD), supplemented with 1% of Chol, 1% of Chol and 5.6% of mussel’s dry matter (DM) from contaminated and 1% Chol and 5.6% of mussel DM from non-contaminated areas for Chol/MCont and Chol/MNCont groups, respectively. At the end of the experiment, a high digestibility coefficient of DM and crude protein in the Chol/MCont and Chol/MNCont groups of rats was found: 93.56 and 93.87% and 91.64 and 91.36%, respectively, and the differences were not significant (P > 0.05). However, the protein efficiency ratio was significantly higher in the Chol/MCont and Chol/MNCont than in the Control and Chol diet groups. Also the level of nitrogen retention as shown by feces and urine examination was higher in the Chol/MCont and Chol/MNCont groups. Diet, supplemented with mussel dry matter from both contaminated and non-contaminated areas significantly hindered the rise in plasma lipids and also hindered the decrease in plasma antioxidant activity. Minor changes were fixed in the protein profile of rat’s plasma after both mussels’ diet. In conclusion, supplementation of diets, containing cholesterol with mussels’ DM from both contaminated and non-contaminated areas improves animals’ protein metabolism and positively affects plasma lipid profile and plasma antioxidant activity. Antioxidant tests can be used as an additional index for the quality of mussels.     

Exposure assessment of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in <Emphasis Type="Italic">Kimbab</Emphasis>, a ready-to-eat Korean food

The exposure assessment was carried out for Staphylococcus aureus in kimbab by predicting growth of S. aureus and the production of enterotoxin using Food MicroModel® program. Environmental parameters selected were pH 5.5, Aw 0.999, and storage temperatures in the range of 10 to 30°C. It was predicted that 6.3 hr could be a critical time for enterotoxin production while kimbab was stored at 30°C. Mild case scenario analysis showed that enterotoxin could not be produced if kimbab was kept at 10°C during preparation and distribution and then left at 25°C for 4 hr before consumption. In the worst case scenario, the keeping time at 25°C was assumed to be 7.0 hr. The level of S. aureus in the worst case was predicted to be 6.8×10⁶ CFU/g which is lower than the critical level (7.8×10⁶ CFU/g) for toxin production.     

Ethanolic fermentation from red ginseng extract using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Saccharomyces carlsbergensis</Emphasis>

The pH of red ginseng extracts fermented with Saccharomyces cerevisiae and Saccharomyces carlsbergensis decreased rapidly during 3 days of fermentation, with no further significant change thereafter. After 20 days of fermentation, a relatively small difference remained in the acidity of extracts fermented with S. cerevisiae (0.54%) and S. carlsbergensis (0.58%). Reducing sugar in the S. cerevisiae and S. carlsbergensis extracts decreased from 258.6 to 45.4 and 43.2 mg/mL glucose equivalents, respectively; and ethanol contents increased from 1.5% at day 0 to 16.0 and 15.0%, respectively, at day 20. Ginsenosides Rb₁, Rb₂, Rc, Re, Rf, and Rg₁ decreased during the fermentation with S. cerevisiae, but Rd and Rg₃ increased by day 12. Ginsenosides Rb₁, Rb₂, Rc, Re, and Rg₁ decreased gradually in the extract with S. carlsbergensis, but Rd and Rg₃ were increased at day 6 and 9, respectively.     

Functionality of the S-layer proteins from <Emphasis Type="Italic">Lactobacillus</Emphasis> in the competitive against enteropathogens infection

The interest of probiotics as remedy for a broad number of infectious diseases has gained wide interest over the last few years, but little is known about their underlying mechanism of action. In this study, five selected Lactobacillus isolated from human intestinal and ferment milk were preliminarily identified by 16S rDNA gene sequencing and assessed the ability to inhibit the adhesion of enteropathogens using HT-29 cells model through a process which may be related to specific components of the bacterial surface. Surface layer proteins are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. Removal of S-layer proteins from the Lactobacillus (treated with 5 M LiCl) reduced inhibition activity as revealed in exclusion, competition and displacement assays, which suggested that S-layer proteins had involved in the adhesion of probiotics. SDS-PAGE analysis confirmed the presence of S-layer proteins with dominant band which was approximately 60 kDa. Further analysis of S-layer proteins revealed that the hydrophobic amino acids accounted for 43.2 % of the total amino acid for the Lactobacillus paracasei M7. So, these probiotics could be used in the health-promoting food products, which could prevent the diarrhea caused by pathogens.