Determination of <Emphasis Type="SmallCaps">d</Emphasis>,<Emphasis Type="SmallCaps">l</Emphasis>-Amino Acids in Collagen from Pig and Cod Skins by UPLC Using Pre-column Fluorescent Derivatization

A simple and sensitive analytical method for the quantification of d,l-amino acids by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection is described. The reaction of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS] with d,l-hydroxyproline (Hyp), glycine (Gly), d,l-aspartic acid (Asp), and d,l-proline (Pro) effectively proceeds at 55 °C for 20 min in the presence of 3% TEA to produce the corresponding fluorescent diastereomers (excitation at 460 nm, emission at 550 nm). The mixture was simultaneously separated within 20 min on an ACQUITY UPLCTM BEH C18 (1.7 μm, 100 mm?×?2.1 mm i.d.) by gradient elutions using water–acetonitrile containing 0.2% formic acid as the mobile phase. Peak resolution was in the range of 1.62 (d,l-Asp), 2.99 (d,l-Pro), and 6.74 (d,l-Hyp). A good linearity was achieved from the calibration curves (r²?>?0.9984) in the range of 1.0~100 pmol; the limit of detection (S/N?=?3) was 42.0–250 fmol, the inter-day and intra-day assay precisions were all less than 6.23%, and the mean recoveries (%) of the d,l-amino acids spiked in the collagen from pig and dried cod skins were 87.58–107.41%. The derivatives of the free d,l-amino acids in the collagen were successfully identified by the proposed procedure. To the best of our knowledge, for the first time, these three kinds of d-amino acids, which were d-Asp, d-Pro, and d-Hyp, were detected in the collagen samples.     

Improvement of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> by genome shuffling for the efficient production of arabitol from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

Arabitol is used in the food industry as a low-calorie sweetener. It is produced by yeasts during the biotransformation process of l-arabinose. Genome shuffling was performed in Candida parapsilosis DSM 70125, an efficient producer of arabitol, to obtain fusants with improved arabitol production ability. Four mutants from the parental library were used for the first round of genome shuffling. The best fusants, GSI-1 and GSI-10A, were subjected to a second round of genome shuffling. Finally, two fusants, GSII-3 and GSII-16, produced concentrations of arabitol that were 50% higher than that of the wild-type strain during selection culture. Under the optimal conditions established for C. parapsilosis, the two fusants produced 11.83 and 11.75 g/L of arabitol and were approximately 15–16% more efficient than the wild-type strain. Flow cytometry analysis showed that the ploidy of the new strains did not change.     

Simultaneous Determination of Melatonin, <Emphasis Type="SmallCaps">l</Emphasis>-Tryptophan,and two <Emphasis Type="SmallCaps">l</Emphasis>-Tryptophan-Derived Esters in Food by HPLC with Graphene Oxide/SiO<Subscript>2</Subscript> Nanocomposite as the Adsorbent

In this research, a dispersive solid-phase extraction (dSPE) with graphene oxide@SiO₂ (GO@SiO₂) nanocomposites as the adsorbent followed by high-performance liquid chromatography analysis was developed for simultaneous determination of melatonin, l-tryptophan, and two l-tryptophan-derived esters in food (black sesame seed (Sesamum indicum L.) was selected in this case). The GO@SiO₂ nanocomposite was prepared by one-pot aggregation in aqueous phase with sol-gel technique. The structure and morphology of the GO@SiO₂ were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform-infrared spectroscopy, and X-ray diffraction. The extraction conditions of dSPE including the ratio of material to liquid, adsorption and desorption time, desorption temperature, and desorption solvents were investigated, respectively. The detection limits of the developed method for the analysis of melatonin, l-tryptophan, l-tryptophan methyl ester, and l-tryptophan ethyl ester were achieved below 0.1 μg mL^?1. The established method was successfully applied to the analysis of the target analytes in black sesame seed, which provided a simple, low-cost, and sensitive approach for the determination of trace compounds in complex samples.     

Effects of <Emphasis Type="SmallCaps">l</Emphasis>-lysine on thermal gelation properties of chicken breast actomyosin

The effects of l-lysine (l-Lys) on the water holding capacity (WHC) and texture of actomyosin (AM) gel and the possible mechanisms were investigated. l-Lys increased the WHC and hardness of the AM gel. These effects may be related to the even and continuous microstructure of the gel according to the scanning electron microscopy analysis. Furthermore, l-Lys increased the surface hydrophobic residues and the reactive sulfhydryl groups. l-Lys decreased the storage modulus at the first transition temperature but increased it at the second transition temperature and the third transition enthalpy. These results suggested that l-Lys varied the thermal behaviors and the microstructure of the AM gel by increasing the surface hydrophobicity and reactive sulfhydryl groups, ultimately contributing to the increased WHC and hardness. The changes in pH did not fully explain the results from the present study. The results were useful for understanding previous findings and may serve as a reference for the preparation of reduced-sodium and phosphate-free meat products.     

Antioxidant capacity and antioxidative compounds in barley (<Emphasis Type="Italic">Hordeum</Emphasis><Emphasis Type="Italic">vulgare</Emphasis> L.) grain optimized using response surface methodology in hot air roasting

Response surface methodology was used to predict optimum conditions for hot air roasting of barley grains (temperature, time, and amount). Antioxidant capacity in the grains was highest under optimum conditions of 250 °C, 63.5 min and 42 g (one and a half layers). A correlation of R ² = 0.74 (p < 0.05) was found between 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and total phenolic contents. Ethanol and aqueous extracts were prepared from grains roasted under optimum conditions and assessed for antioxidant capacity. Antioxidative compounds in the extracts were then identified using GC–MS. The IC₅₀ value of ethanol extract was significantly lower (11.45 μg mL^–1) than that of aqueous extract (33.54 μg mL^–1) and α-tocopherol (12.6 μg mL^–1) but higher than BHT (9.59 μg mL^–1). The same trend was observed in linoleic acid assay. In reducing power, the ethanol extract and α-tocopherol were not significantly different. Phenolic acids p-hydroxybenzaldehyde, vallinic and gallic acids were identified as the major compounds in the extracts. The results obtained from this study show that it is possible to optimize antioxidant capacity in barley grains during roasting.     

Optimization of culture medium for yellow pigments production with <Emphasis Type="Italic">Monascus anka</Emphasis> mutant using response surface methodology

In our laboratory, one Monascus anka mutant able to produce high yield of yellow pigments screened by physical and chemical combination mutagenesis was obtained. This study evaluated peptone, NH₄NO₃ and KH₂PO₄ as the most significant variables for Monascus yellow pigment production by this M. anka mutant MYM in submerged fermentation. Response surface methodology (RSM) optimized these nutrient parameters for maximum yellow pigment production (87.24 OD units), which resulted at 10.3 g/L peptone, 11.9 g/L NH₄NO₃ and 4.7 g/L KH₂PO₄ in the medium. According to the fitting equation, through five replication experiments under the optimized conditions, the average yellow pigment production obtained was 88.14 OD units for flask cultivation and 92.45 OD units for 5 L fermenter cultivation. Meanwhile, the citrinin could not be detected by HPLC method.     

Contamination of commercially available <Emphasis Type="SmallCaps"> L-</Emphasis>tryptophan by related substances

In 1989 a new autoimmune disease, the eosinophilia myalgia syndrome (EMS), was traced back to the intake of L-tryptophan (Trp) of a single manufacturer, Showa Denko (SD). In an epidemiological study, six minor contaminants (10-1,000 mg/kg Trp) were related to the occurrence of EMS. In this study the pattern of contaminants in pharmaceutical- and feed-grade Trp was investigated in a market survey of 21 lots of Trp raw materials of six different manufacturers, as well as one bioconversion broth, and four tablets. For total Trp-related impurities all UV_{220 nm} detectable substance peaks separated from Trp by RP-HPLC were calculated as N -acetyl-tryptophan. In all samples 4-,5-, 6-, and 7-OH-Trp, 1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (THCC), 1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (MTHCC) and 1-(3-indolylmethyl)-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (IMTHCC) as well as 2-[2,3-dihydroxy-1-(3-indolyl)-propyl]- L- tryptophan (dhPIT) were determined, since the last four are known to be major contaminants in biotechnologically manufactured Trp. Additionally, four EMS-related substances were been analysed: 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrroloindole-2-carboxylic acid (PIC), 2-(3-indolylmethyl)- L- tryptophan (IMT), 1,1'-ethylidenebis-( L- tryptophan) (EBT) and 3-(phenylamino)alanine (PAA). EBT was only detectable in a single SD-Trp sample known to be EMS associated. Low amounts of PAA (20 mg/kg) were detected in feed-grade Trp of one manufacturer as well as in SD-Trp. IMT was found in small amounts in recent pharmaceutical-grade Trp (10-25 mg/kg Trp), but in higher amounts in feed-grade Trp samples (120-1,400 mg/kg) as well as in pharmaceutical-grade Trp and Trp tablets manufactured prior to 1989 (70-660 mg/kg). PIC is a primary oxidation product of Trp; thus we detected 4-50 mg/kg of the earlier-eluting PIC diastereomer in recent pharmaceutical-grade Trp and up to 2,500 mg/kg in feed-grade Trp. Non-EMS correlated THCC, MTHCC, IMTHCC, and dhPIT proved to be the main contaminants in amounts of <10-13,500 mg/kg.     

A Novel Technological Process of Extracting <Emphasis Type="SmallCaps">l</Emphasis>-Tyrosine with Low Fluorine Content from Defatted Antarctic Krill (<Emphasis Type="Italic">Euphausia superba</Emphasis>) By-product by Enzymatic Hydrolysis

Antarctic krill (Euphausia superba) offers a high potential for development of innovative food products due to its large reserves and satisfied nutritional values. Defatted krill powder by-product generates from the process of krill oil, which is rich in various types of amino acids. This study aimed at recovering tyrosine from defatted krill by-product through enzymatic hydrolysis. Defatted krill by-product was hydrolyzed with three types of protease under optimum conditions with different incubation time. Free tyrosine dissociated from protein through hydrolysis by trypsin and was detected in the protein hydrolysates. Content of tyrosine increased with the increasing of incubation time till 5 h. Tyrosine extract contained 6.43 mg/kg fluorine and over 95 % of tyrosine, which demonstrated its high purity and safety. Tyrosine extract showed rod-shaped microstructure through scanning electron microscope analysis. Crystalline structure and thermal properties of tyrosine extract agreed well with those of l-tyrosine standard. During this process, both tyrosine of high purity and bioactive peptide products were obtained.     

Content and distribution of vicine,convicine and <Emphasis Type="SmallCaps">l</Emphasis>-DOPA during germination and seedling growth of two <Emphasis Type="Italic">Vicia faba</Emphasis> L. varieties

This work examined the content and distribution of the pyrimidine glucosides vicine and convicine, and the nonproteic amino acid l-DOPA in cotyledons and embryo axes along the germination and seedling growth of Vicia faba L. vars. Alameda and Brocal. The behaviour of these compounds was similar in both varieties. Vicine and convicine, implicated in favism disease, slowly declined in cotyledons. In embryo axis, vicine levels were sharply reduced and convicine amount was slightly increased as assay progressed. Total pyrimidine glucosides remained unchanged in the whole plant during the study. l-DOPA only appeared in the embryo axis of V. faba seeds and the highest content was observed in Brocal variety at 6 DAI (days after imbibition). The information provided in this study could be valuable for a possible role of embryo axis, rich in l-DOPA, for the treatment of Parkinson’s disease and also as a choice ingredient for functional foods or as nutraceutical.     

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.     

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.     

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.     

<Emphasis Type="SmallCaps">l</Emphasis>-Cysteine Functionalized Silica Gel as an Efficient Adsorbent for the Determination of Heavy Metals in Foods by ICP-MS

A novel l-cysteine functionalized silica gel adsorbent (SG-Cys) was successfully synthesized and characterized by Fourier transform infrared (FTIR) spectra and elemental analysis. An efficient method for simultaneous determination of heavy metals [V (V), Cr (VI), Cu (II), As (V), Cd (II), and Pb (II)] was developed by inductively coupled plasma-mass spectrometry (ICP-MS) coupled with pre-concentration with the prepared SG-Cys adsorbent. The experimental parameters, including the solution pH, sample flow rate, eluent volume, eluent type, and concentration, have been systematically optimized to obtain higher enrichment factors of target ions. Under the optimum conditions, the enrichment factors of V (V), Cr (VI), Cu (II), As (V), Cd (II), and Pb (II) reached 123, 100, 86, 106, 97, and 94, respectively, and the detection limits were as low as 1.8–3.7 ng L^?1. The proposed method has been applied to the determination of trace heavy metals in water, rice, wheat, corn, and tea samples, which exhibited a satisfactory recovery in the range of 90.6–106.0 %.     

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.     

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.     

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.     

Lipase-catalyzed acylation of <Emphasis Type="SmallCaps">l</Emphasis>-carnitine with conjugated linoleic acid in [Bmim]PF<Subscript>6</Subscript> ionic liquid

Improving significantly the acylation reaction of l-carnitine with conjugated linoleic acid catalyzed by lipase needs to select an efficient and suitable reaction medium that is not only polar but also hydrophobic. [Bmim]PF₆ which is satisfied with the above two requirements was applied as the reaction medium. The optimal reaction conditions were: Novozyme 435 as the catalyst, 5:1 of molar ratio of fatty acid to l-carnitine, 40 g L⁻¹ lipase, 0.22 a_W of initial water activity, 125 g L⁻¹ molecular sieves (they were added within 0–3 h of reaction time), 60 °C of reaction temperature, 200 rpm of rotation speed, 32 h of reaction time. The overall conversion could reach 98.27%. The conversion in [Bmim]PF₆ was 2.13 and 1.56 times higher than that in acetonitrile and in solvent-free system, respectively. The lipase in the present work could be used eight times. [Bmim]PF₆ as the reaction medium was better than acetonitrile and solvent-free system.