Biochemical characterization of esterase from soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.)

Esterases are enzymes that present good potential in industrial application, and soybean seed can represent an alternative source for this enzyme. The extraction and esterase activity of Brazilian soybean seeds (Glycine max L.) were evaluated. Esterase activity was found in both the germinated and non-germinated seeds at 2.90 and 1.70 U/mg, respectively, with a concentration in the powdered extract (freeze dried) of 100 mg/mL. The enzyme showed a preference for the hydrolysis of short chain fatty acids (120.02 U/mL) and optimum pH for activity was pH 8.0 with optimum temperatures of 40 and 80°C. The enzyme showed stability at 70°C showing 60% of residual activity and activity increased with the addition of the following salts: NaNO₃, K₂SO₄, and Na₂SO₄ in the reaction medium.     

The antioxidative activity of summer savory (Satureja hortemis L.) and rosemary (Rosmarinus officinalis L.) in dressing stored exposed to light or in darkness

For an oil-in-water emulsion dressing, addition of 0.15% of dried leaves of summer savory (Satureja hortensis L.) or more significantly of rosemary (Rosmarinus officinalis L.), resulted in a significantly better antioxidative protection than addition of 80 ppm propyl gallate (standard concentration for this type of product) during dark storage at 19 °C for up to 24 weeks, as determined by development of conjugated dienes, peroxide value, head space hexanal and thiobarbituric acid-reactive substances. Addition of freeze-dried methanol extract of the two spices, in an equivalent concentration, had less antioxidative effect, but was comparable to the effect of propyl gallate. Exposure to fluorescent light (850 lux) during storage had a clear pro-oxidative effect for dressing with or without spice or spice extract added, when compared to dark storage. For dressing with extract of savory added, the antioxidative effect found for dark storage was during light exposure, changed to a pro-oxidative effect, and a hexanal level as high as 600 mg kg⁻¹ oil was detected after 8 weeks of storage, while the net antioxidative effect of rosemary was maintained for storage exposed to light.     

Mate (<Emphasis Type="Italic">Ilex paraguariensis</Emphasis>) as a source of water extractable antioxidant for use in chicken meat

Aqueous extract of mate, made from dried leaves of Ilex paraguariensis, St. Hilaire, was shown to be effective during chilled storage for up to 10 days in protecting lipids and vitamin E against oxidation in pre-cooked meat balls made from chicken breast added 0.5% salt and packed in atmospheric air. Extracts made with water, methanol, ethanol or 70% aqueous acetone were evaluated by comparing (1) total phenolic content, (2) radical scavenging capacity, (3) effect on lipid oxidation in a food emulsion model, and in liposomes. Based on the three-step evaluation, aqueous mate extract was preferred for food use. Dried leaves were further compared to dried rosemary leaves in chicken meat balls, and mate (0.05 and 0.10%) found to yield equal or better protection than rosemary at the same concentration against formation of secondary lipid oxidation products.     

Extracts of plant cell cultures of <Emphasis Type="Italic">Lavandula vera</Emphasis> and <Emphasis Type="Italic">Rosa damascena</Emphasis> as sources of phenolic antioxidants for use in foods

Ethanolic extracts of plant cell cultures of lavender (Lavandula vera) and rose (Rosa damascena) have been examined as potential food antioxidants. The L. vera cell extract quenched the radicals Fremy’s salt, DPPH (2,2-diphenyl-1-picrylhydrazyl radical), and ABTS^·+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic) radical) more efficiently than the R. damascena extract. Also the L. vera extract inhibited lipid oxidation in a methyl linoleate emulsion more efficiently than the R. damascena extract. However, the L. vera extract had a prooxidative effect on the iron-based Fenton reaction in an aqueous model system. A similar effect was observed for pure rosmarinic acid, but not for the R. damascena extract. The addition of L. vera extract to minced chicken meat reduced lipid oxidation (measured as thiobarbituric acid reactive species) and the loss of α-tocopherol during cold storage after the meat was cooked. This suggests the antioxidative properties of L. vera extracts dominate in a real food system.     

Inhibition of butter oxidation by <Emphasis Type="Italic">N</Emphasis>-acetyl-cysteine and glutathione

The ability of N-acetyl-cysteine and glutathione to inhibit the oxidation of butter was evaluated. Peroxide value and thiobarbituric acid reactive substances were monitored during storage of butter at 50 °C for up to 84 days and at 110 °C for up to 12 h. N-Acetyl-cysteine and glutathione inhibited butter oxidation at 50 °C and at 110 °C. They were active at 10 mg/L and in a higher degree at 20 mg/L. N-Acetyl-cysteine appeared to be more active than glutathione. At 50 °C, N-acetyl-cysteine at 20 mg/L exhibited antioxidant activity comparable to that of butylated hydroxyanisole (BHA) at 200 mg/L. Volatile aldehydes were also determined during storage of butter at 50 °C for 10 days. N-Acetyl-cysteine and glutathione, each at 20 mg/L, inhibited the formation of several volatile aldehydes such as hexanal to an extent equal to BHA at 200 mg/L. Present results indicate that N-acetyl-cysteine and glutathione, especially the former, may be taken into account as antioxidants in butter during storage and cooking.     

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.     

<Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> fermented yam (<Emphasis Type="Italic">Dioscorea opposita</Emphasis> Thunb.) and its preventive effects on gastric lesion

This study was carried out to find the possibility of fermenting yam (Dioscorea opposita Thunb.) with Lactobacillus acidophilus and the protective role on stomach lesion of rats. The solutions of raw and extracted yam used were 2 and 6%, and they were fermented by L. acidophilus at 37°C for 0, 12, 24, and 36 h. At intervals (0, 12, 24, and 36 h), viscosity, titratable acidity (TA), allantoin, and diosgenin were examined. In the results, it was observed the most desirable data at 12 h with 6% fermented raw yam (FRY) without any change in the content of allantoin and diosgenin. In the animal study by rats (Sprague-Dawley), the inhibition ratios on gastric lesion were 53.41% in FRY. Based on these data, it confirmed that raw yam powder fermented by L. acidophilus would be a functional food with the supplement of viable cells and nutraceutical component of allantoin and diosgenin.     

Modeling the Inactivation of <Emphasis Type="Italic">Listeria innocua</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> in Fresh-Cut Tomato Treated with Pulsed Light

The effectiveness of pulsed light (PL) treatments to inhibit microorganisms on fresh-cut tomatoes (Lycopersicon esculentum Mill., cv. Daniela) was investigated. Tomato slices inoculated with Escherichia coli or Listeria innocua were exposed to PL treatments (4, 6, or 8 J cm^?2 fluence) and kept cold at 4 °C for 20 days. L. innocua and E. coli counts, gases in the headspace of the containers (O₂ and CO₂), pH, titratable acidity, and soluble solid content were monitored throughout the cold storage. The PL treatments reduced significantly (p < 0.05) initial loads of both microbes. The effect of the PL fluence on the survival number of microoganisms was described by a log-linear model (R ² = 0.849–0.999). At any fixed time within the cold storing, the microbial counts for untreated samples were always higher than those cut tomatoes that had been previously PL-treated. The behavior of L. innocua and E. coli during the storage were well adjusted (R ² > 0.930) by Gompertzian models; the studied microorganisms exhibited different patterns during the storage period. On the other hand, O₂ and CO₂ partial pressures in containers with fresh-cut tomatoes were also significantly affected by PL treatments (p < 0.05). The highest PL fluence caused the greatest changes of O₂ and CO₂ contents. In addition, the application of PL triggered an acceleration of the O₂ consumption during the cold stage. PL treatments might be used to effectively extend the safety of fresh-cut tomatoes over 12 days of storage against E. coli and L. innocua growth.     

Tyramine reduction by tyrosine decarboxylase inhibitor in <Emphasis Type="Italic">Enterococcus faecium</Emphasis> for tyramine controlled <Emphasis Type="Italic">cheonggukjang</Emphasis>

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.     

Control of <Emphasis Type="Italic">Penicillium Expansum</Emphasis> and <Emphasis Type="Italic">Botrytis Cinerea</Emphasis> on Apple Fruit by Mixtures of Bacteria and Yeast

Antagonistic activity of the mixtures Rahnella aquatilis–Rhodotorula glutinis and R. aquatilis–Cryptococcus laurentii was assessed against Penicillium expansum (cause of blue rot) and Botrytis cinerea (cause of grey rot) on apple fruit at 4 °C and 95% relative humidity (RH). Under these cold-storage conditions, the mixture R. aquatilis–R. glutinis inhibited the development of B. cinerea and P. expansum in apples stored for 40 days and reduced the incidence of disease produced by these moulds to nearly zero. The other mixture, R. aquatilis–C. laurentii, was less effective; the incidences of the grey rot and the blue rot were about 25% and 15%, respectively. Population dynamics of the mixtures showed that the growth of R. aquatilis was strongly stimulated by the presence of the yeast R. glutinis, but in the case of the mixture R. aquatilis–C. laurentii, the same effect could not be observed. In this study, it was demonstrated that the combination of two microorganisms with different requirements and antagonistic abilities resulted in a successful mixture against the two pathogen molds, B. cinerea and P. expansum. In addition, it was also proved that it is possible to improve the biocontrol of these pathogens without increasing the inoculum size of the antagonist (alone or in mixtures), which was always 10⁶cell/ml, despite the high concentration of the pathogen (10⁶ conidia/ml) utilized.     

Influence of blanching and freezing broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">italica</Emphasis>) prior to storage and cooking on glucosinolate concentrations and myrosinase activity

Glucosinolates contribute to the chemoprotective effects of Brassica vegetables. The influence of blanching and freezing broccoli, followed by storage or cooking, on its glucosinolate concentration and myrosinase activity was investigated. Myrosinase activity was reduced by 93%, while glucosinolate concentration was unaltered after blanch-freezing broccoli. Blanch-frozen Brassica retained the glucosinolate content of its fresh counterpart after storage at T = −20 °C for up to 90 days. Fresh or blanch-frozen broccoli was stir-fried, boiled, boiled and kept hot for 2 h, or left uncooked. Stir-frying retained the highest glucosinolate concentration and myrosinase activity, regardless of pre-cooking. Boiling and keeping blanch-frozen broccoli hot completely denatured myrosinase and decreased glucosinolate concentration by 42%. Aromatic and indole glucosinolates were reduced to a larger extent than aliphatic glucosinolates, especially when boiled broccoli was kept hot. The final concentration of glucosinolates in Brassica is influenced by cooking, while the myrosinase activity is modified by pre-treatment and subsequent cooking.     

Antitumor activities of liposome-incorporated aqueous extracts of <Emphasis Type="Italic">Anodonta </Emphasis><Emphasis Type="Italic">woodiana</Emphasis> (Lea, 1834)

To acquire the effectiveness of oral treatments, aqueous extracts of Anodonta woodiana (HB) were incorporated into liposome and its antitumor activities evaluated in vivo. The HB-loaded liposomes (HBL) were prepared at a mean size of 14.85 μm by reverse-phase evaporation method. Referring to the maximum tolerated doses test, mice with orally administrated HBL, at a 3 g/kg body weight dosage, showed no obvious acute toxic sign. Furthermore, the tumoricidal activities of HBL against C26 murine colon carcinoma, Lewis lung tumor, human QGY hepatic carcinoma and MKN-45 gastric tumor were examined, respectively. In contrast to free HB, HBL possessed remarkable antitumor activity. Simultaneously, the effect of HBL was observed in a dose-dependent manner. For C26 murine colon carcinoma and Lewis lung tumor, the inhibitory ratios of HBL were 54.36 and 51.97% at a dose of 400 mg/kg, respectively. The suppression of tumor growth and the reduction in body weight were more pronounced in human QGY hepatic carcinoma and MKN-45 gastric tumors inoculated mice by treatment of HBL during 30 days. All these promising results implied that liposome-incorporated aqueous extracts of Anodonta woodiana had a more potential application as a natural antitumor and immunomodulator formulation.     

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.     

Analysis of the survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in food-grade lubricants

Listeria monocytogenes is a ubiquitously occurring Gram-positive bacterium, which is associated with raw ready-to-eat foods, in particular with smoked or graved salmon. Due to its tenacity and psychrotrophic growth properties, it may persist on the equipment and machinery used in food production. In this study, we analysed the survival of three L. monocytogenes strains in eight H1 lubricants, seven greases and one oil, applicable for food-processing machinery. None of the native lubricants contained Listeria spp. above the detection limit of 10³ cfu/g. In artificially contaminated lubricants, the viable counts of different L. monocytogenes strains decreased by more than 99.9% within 7 days, and the reduction rates were found to dependent on the composition of the respective lubricant as well as on the L. monocytogenes strain. Chemical binding of either benzoate or acetate to native grease 3 caused a similar anti-listerial activity, but a decreased effect was observed when stearic acid was used. When benzoate, acetate, salicylate or parabene was added at a concentration of 0.5% to native grease 1, similar anti-listerial effects were observed. The water content (0, 1 and 5%) and the age of the lubricant did not influence the results. Used lubricants originating from dismantled industrial machinery did not contain L. monocytogenes, though this microorganism was re-isolated from the lubricants following artificial contamination. Thus, there is no evidence that L. monocytogenes replicate or persist in the H1 lubricants used in this study.     

Development of a nucleic acid lateral flow immunoassay for simultaneous detection of <Emphasis Type="Italic">Listeria</Emphasis> spp. and <Emphasis Type="Italic">Listeria</Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> in food

We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5 h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15 min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food samples.     

Non-structural carbohydrates in processed soft fried onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.)

The effects of curing, removal of top and root, peeling, storage of raw and peeled onion on the content of non-structural carbohydrates and the ability of onion to develop a brown colour were studied. Glucose, fructose, sucrose, 1-kestose, neokestose, nystose, 1^F--d-fructofuranosylnystose and four unknown oligofructosides were quantified using high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Field drying affected the concentration of fructose only. Glucose, fructose and an unknown fructan decreased in concentration while the concentration of, e.g. 1-kestose, neokestose and nystose increased by removal of root and top of the onions when stored at 15 °C for one week. Peeling had minor influence on changes in non-structural carbohydrates. Storage of non-peeled (raw) onions at 5 °C and storage of mechanically damaged and peeled onion at 20 °C promoted fructan hydrolysis and browning of soft fried onion. Similar changes were produced by long-term storage of cultivars of common onion. Dry matter and fructose increased during storage of peeled onions at both 5 and 20 °C. The increase in fructose content during storage was due to a significantly decrease in the content of 1-kestose, neokestose, 1^F--d-fructofuranosylnystose, an unknown fructan and total fructans. It was concluded that the missing ability of industrially processed onions to develop a brown colour may be overcome by storage of the peeled onions for a few hours or even overnight at 5 °C.     

Effect of glasswort (<Emphasis Type="Italic">Salicornia herbacea</Emphasis> L.) and rice (<Emphasis Type="Italic">oryza sativa</Emphasis> L.) on quality of <Emphasis Type="Italic">Ganjang</Emphasis> (Korean Soy Sauce)

Four types of meju were made from 100%(w/w) defatted soybean (DFS), a mixture of 80%(w/w) defatted soybean, and 20%(w/w) glasswort (DFS-G), a mixture of 80%(w/w) defatted soybean and 20%(w/w) rice (DFS-R), and a mixture of 60%(w/w) defatted soybean, 20%(w/w) glasswort, and 20%(w/w) rice (DFS-GR). Four types of Korean traditional soy sauce were prepared from the 4 types of meju. Mineral and antioxidant contents in the soy sauce made of DFS-G and DFS-GR were significantly higher than others. Citric, malic, succinic, lactic, and pyroglutamic acid contents in soy sauce made of DFS-R and DFS-GR were 1.3–1.5 times higher than others. Total nitrogen and free amino acid contents in soy sauce were correlated with DFS concentration in the meju. The bacterial community in the non-fermented meju-making ingredients was replaced largely by Bacillus sp. in the fermented meju. The use of glasswort and rice in the meju-making process did not alter the bacterial community responsible for the fermentation of meju.     

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.     

Lactic acid fermentation of<Emphasis Type="Italic"> Brassica rapa</Emphasis>: chemical and microbial evaluation of a typical Italian product (<Emphasis Type="Italic">brovada</Emphasis>)

Brovada is an ancient traditional product from the northeast Italian region, included in any list of typical Italian products, which is obtained by natural fermentation of turnips (Brassica rapa). Turnips are cleaned and put in vats, alternating them with a layer of grape skins. Before covering the vat a mix of water and salt or water only is added. The vegetable fermentation is a spontaneous process caused by the microorganisms present on the various components (turnip and grape skins). A total 225 lactic acid bacteria (LAB) and 63 yeasts were isolated during this natural fermentation process. The main heterofermentative population was identified as Lactobacillus spp. while the major homofermentative LAB were Pediococcus spp., Candida spp. was the main yeast present. During the first 24 h of fermentation, the pH decreased rapidly to 3.7. At different steps of fermentation the headspace aroma compounds of brovada were analyzed by solid-phase-micro-extraction/gas chromatography/flame ionization detection to define the characteristic aroma profile of this particular product.