Matrix metalloproteinases (MMPs) inhibitory effects of an octameric oligopeptide isolated from abalone Haliotis discus hannai

Abalone (Haliotis discus hannai) is a marine gastropod, and an important fishery and food industrial resource that is massively maricultured in Asia, Africa, Australia and America. However, its health benefits have rarely been studied for nutraceutical and pharmaceutical application. In this study, the purified abalone oligopeptide (AOP) with anti-matrix metalloproteinases (anti-MMPs) effects was isolated from the digests of abalone intestine using recycle HPLC with a JAI W253 column and an OHpak SB-803 HQ column. The AOP was identified as Ala-Glu-Leu-Pro-Ser-Leu-Pro-Gly (MW = 782.4 Da) with a de novo peptide sequencing technique using a tandem mass spectrometer. The AOP exhibited a specific inhibitory effect against MMP-2/-9 activity and attenuated protein expression of p50 and p65 in the human fibrosarcoma (HT1080) cells, dose-dependently. The results presented illustrate that the AOP could inhibit MMP-2/-9 expression in HT1080 cells via the nuclear factor-kappaB (NF-κB)-mediated pathway. This data suggest that the AOP from H. discus hannai intestine may possess therapeutic and preventive potential for the treatment of MMPs-related disorders such as angiogenesis and cardiovascular diseases.     

Impact of heat processing on the detection of the major shellfish allergen tropomyosin in crustaceans and molluscs using specific monoclonal antibodies

The major heat-stable shellfish allergen, tropomyosin, demonstrates immunological cross-reactivity, making specific differentiation of crustaceans and molluscs for food labelling very difficult. The aim of this study was to evaluate the application of allergen-specific monoclonal antibodies in differential detection of shellfish-derived tropomyosin in 11 crustacean and 7 mollusc species, and to study the impact of heating on its detection. Cross-reactive tropomyosin was detected in all crustacean species, with partial detection in molluscs: mussels, scallops and snails but none in oyster, octopus and squid. Furthermore, we have demonstrated that heating of shellfish has a profound effect on tropomyosin detection. This was evident by the enhanced recognition of multiple tropomyosin variants in the analysed shellfish species. Specific monoclonal antibodies, targetting the N-terminal region of tropomyosin, must therefore be developed to differentiate tropomyosins in crustaceans and molluscs. This can help in correct food labelling practices and thus protection of consumers.     

Antioxidant activity of hydrolysates obtained from scallop (Patinopecten yessoensis) and abalone (Haliotis discus hannai Ino) muscle

Scallop (Patinopecten yessoensis) and abalone (Haliotis discus hannai Ino) muscle were hydrolysed with commercially available food-grade proteases. The resulting hydrolysates showed DPPH and hydroxyl radicals scavenging abilities, reducing power, and ferrous ion chelating capacity. The antioxidant activities of hydrolysate of abalone foot muscle (HAFM) increased with increasing incubation time during the whole hydrolysis process in 180 min. Whereas, the antioxidant activities of hydrolysate of scallop adductor muscle (HSAM) increased at initial stage and peaked after 25-30 min of hydrolysis, and then gradually decreased thereafter. Compared with HAFM, HSAM with comparable hydrolysis time contained more free amino acids (FAA) and small-sized peptides (below 500 Da), which may account for the differences in antioxidant activities versus hydrolysis time curves of the two hydrolysates. The above results indicate that limited hydrolysis of proteins can increase their antioxidant activity, whereas extensive hydrolysis can decrease it.     

Identification and characterisation of the major allergen of Chinese mitten crab (Eriocheir sinensis)

Tropomyosin (TM) from Chinese mitten crab (Eriocheir sinensis) was purified to homogeneity and TM genes were amplified from three species of crab (Chinese mitten crab, mud crab and swimming crab), respectively. Sequence analysis showed that all three cloned DNA fragments had open reading frames of 855 bp, predicted to encode proteins with 284 amino acid residues. Sequence alignment revealed that the three tropomyosins share high homology to tropomyosins from other crustaceans. Chinese mitten crab TM gene was further recombined with the vector of pGEX-4T-3 and expressed in Escherichia coli JM109. The expressed protein revealed a band of about 62 kDa on SDS–PAGE, suggesting the successful expression of glutathione S-transferase–tropomyosin (GST–TM) fusion protein. Immunoblotting analysis using sera from subjects with crustacean allergy confirmed that the expressed fusion protein reacted positively with these sera, indicating tropomyosin is a major allergen of Chinese mitten crab.     

即食皱纹盘鲍腌制配方及食盐渗透规律

利用皱纹盘鲍生产即食食品,运用感官模糊综合评判法确定最佳腌制配方,并对腌制过程不同温度条件下食盐渗透规律进行了研究。正交实验结果表明,最佳腌制配方为食盐添加量5%,白糖添加量9%,味精添加量1%,食醋0.15%,此腌制配方所得即食皱纹盘鲍风味、质地、色泽均较好。按照最佳腌制配方,通过对不同温度下（30、40、50℃）腌制过程中食盐渗透量的数据进行拟合,得到相关方程,温度越高渗透速度常数越高;30、40、50℃渗透速度常数分别为0.6224、0.7431、0.8777;要得到食盐含量3.8%的鲍鱼,在最佳腌制配方的腌制溶液中的腌制条件为:30℃-3.78h、40℃-2.95h、50℃-1.99h。本研究为合理控制即食鲍鱼的加工时间及其扩大化工业生产提供一定的基础。      

Structural analysis and CCK-releasing activity of a sulphated polysaccharide from abalone (Haliotis Discus Hannai Ino) viscera

A fraction of water-soluble sulphated polysaccharide conjugate, termed AHP-2, was obtained from abalone (Haliotis Discus Hannai Ino) viscera by protease-assisted aqueous extraction followed by precipitation with ethanol and purification with gel filtration chromatography. Analysis by high performance liquid chromatography (HPLC) coupled to a TSK-gel G4000PW_XL column and gel filtration chromatography on Sepharose CL-6B indicated AHP-2 is homogenous with an average molecular weight (MW) of about 11.0 kDa. The structure of AHP-2 was revealed by chemical methods, Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. Results indicated AHP-2 is a heteroglycan consisting of glucose, fucose, xylose, rhamnose and galactose with molar ratio of 1.0:2.0:3.9:6.7:7.4. The backbone of AHP-2 consists of 1,3-linked rhamnose and 1,3,6-linked galactose, with glucose, fucose, xylose and galactose of different linkage types distributing in branched chains. Meanwhile, AHP-2 was found to increase cholecystokinin (CCK) release in CCK-secreting STC-1 cells.     

Chemical composition and free radical scavenging activities of a sulphated polysaccharide extracted from abalone gonad (Haliotis Discus Hannai Ino)

Crude polysaccharides (CPs) were prepared from Haliotis Discus Hannai Ino gonad by protease-hydrolysis combining boiling-methanol extraction. They were purified to homogeneity by gel-filtration chromatography on Sephacryl-S 100 and Sephacryl-S 200, and semi-preparation high performance liquid chromatography (semi-RP-HPLC) equipped with a SOURCE 30Q anion exchange column. The sulphated polysaccharide obtained, named as PAGP, has an average molecular weight (MW) of 12.5 kDa determined by HPLC coupled to a TSK-gel G4000PW_XL column. Chemical composition analysis indicated it was composed of carbohydrate (50.7%), sulphated group (31.1%), aminohexose (7.1%), aronic acid (6.2%) and protein (2.0%). The carbohydrate part of PAGP was composed of rhamnose (Rha), fucose (Fuc), and galactose (Gal) in molar ratio of 1.0:1.6:2.3. The protein part of PAGP contained 13 kinds of amino acids. Antioxidant assays demonstrated that PAGP possessed stronger 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and hydroxyl radical scavenging activities than positive controls vitamin C (VC) and vitamin E (VE).     

Expanded bed adsorption as a fast technique for the large-scale purification of the complete isoform pool of Ber e 1, the major allergen from Brazil nuts

A new, fast, large-scale purification method for Ber e 1, the major allergen from Brazil nuts, using expanded bed adsorption (EBA) chromatography, is presented. Using EBA, crude extracts can be applied to a fluidized column, which allows the unhindered passage of particulate impurities, thereby avoiding time-consuming centrifugation or filtration steps. With this new purification method, 2.8 g of Ber e 1 was obtained from 85 g defatted Brazil nut meal, essentially within 1 day. Various structural as well as immunochemical characteristics of the purified protein were determined, and compared to those of Ber e 1 purified using conventional chromatographic techniques. The complete pool of Ber e 1 isoforms was collected using EBA. The most abundant isoforms were observed to have pI around 8 and heterogeneity was observed in both the large and the small subunit of the heterodimeric protein. Ber e 1 has a highly ordered secondary structure. No apparent differences in immune reactivity were observed between EBA purified Ber e 1 and conventionally purified Ber e 1, using IgE-binding experiments. Thus, using EBA, Ber e 1 can be purified fast and on gram-scale, while having purity equal to that of conventionally purified Ber e 1.     

Identification of antioxidants in Fructus aurantii and its quality evaluation using a new on-line combination of analytical techniques

A new on-line method for simultaneous identification and monitoring of antioxidants in Fructus aurantii was established by coupling high performance liquid chromatography-diode array detector-electrospray ionisation-ion trap-time of flight-mass spectrometry with post-column derivatisation and luminol–potassium ferricyanide chemiluminescence (HPLC-DAD-ESI-IT-TOF-MS-PCD-LPFCL). While the HPLC fingerprint, structural identification and radical scavenging profile were rapidly obtained by an on-line assay using ultraviolet (UV) absorption, MS and LPFCL, details of the precise substitution patterns of various structures were achieved through UV absorption using PCD addition of shift reagents. Twenty-five flavonoids were identified by either their PCD and MS data or comparison with reference substances. Data collected both from chromatograms and activity profiles of 12 samples revealed significant differences among samples from different habitats. The results showed that this method was rapid and precise, and therefore would be an effective and sensitive method for biocompounds analysis and quality evaluation for complex food and medicinal samples.     

Identification of Lactobacillus curvatus TMW 1.624 dextransucrase and comparative characterization with Lactobacillus reuteri TMW 1.106 and Lactobacillus animalis TMW 1.971 dextransucrases

Recently, it was affirmed that the exopolysaccharides (EPSs) of Lactobacillus curvatus TMW 1.624, Lactobacillus reuteri TMW 1.106 and Lactobacillus animalis TMW 1.971 improve the quality of gluten-free breads and that they can be produced in situ to levels enabling baking applications. In this study we provide insight into the molecular and biochemical background of EPS production of these three strains. EPS formation strongly correlated with growth and took place during the exponential phase. Gtf genes were heterologously expressed, purified and their enzymatic properties as well as the structures of the EPSs formed were compared. Structural comparison of EPS formed by heterologously expressed glucosyltransferases (Gtfs) and of those formed by the wildtype lactobacilli confirmed that the respective genes/enzymes were identified and examined. The glucan formed by L. animalis Gtf was identified as a linear low molecular weight dextran. Optimal enzymatic conditions were pH 4.4 and 45 °C for the L. reuteri Gtf and pH 4.4 and 31 °C for L. curvatus Gtf. The Gtf from L. animalis had an optimal pH of 5.8 and displayed more than 50% of activity over a broad temperature profile (22–59 °C). The three Gtfs were stimulated by various mono- and divalent metal ions, dextran, as well as levan to different extents.     

Identification and analysis of isothiocyanates and new acylated anthocyanins in the juice of Raphanus sativus cv. Sango sprouts

The freeze-dried sprouts’ juice of Raphanus sativus (L.) cv. Sango was prepared and analysed for the first time. HPLC analysis of total isothiocyanates, after protein displacement, resulted in 77.8 ± 3.0 μmol/g of dry juice while GC–MS analysis of hexane and acetone extracts showed E- and Z-raphasatin (8.9 and 0.11 μmol/g, respectively) and sulforaphene (11.7 μmol/g), summing up to 20.7 ± 1.7 μmol/g of free isothiocyanates. Sprouts’ juice contained an unprecedented wealth of anthocyanins and a new fractionation methodology allowed us to isolate 34 mg/g of acylated anthocyanins (28.3 ± 1.9 μmol/g), belonging selectively to the cyanidin family. Analysis was performed by HPLC–PDA–ESI–MSⁿ and extended to deacylated anthocyanins and aglycones, obtained, respectively, by alkaline and acid hydrolysis. This study identified 70 anthocyanins, 19 of which have never been described before and 32 of which are reported here in R. sativus for the first time. Sango radish sprouts are exceptional dietary sources of heath-promoting micronutrients.     

Tyrophagus putrescentiae (Schrank) (Astigmata: Acaridae) as a new predator of Lasioderma serricorne (F.) (Coleoptera: Anobiidae) in tobacco stores in Greece

While studying Lasioderma serricorne in tobacco stores, during the years 2002 and 2003, the presence of the cosmopolitan mite species Tyrophagus putrescentiae¹ preying on live L. serricorne larvae was noted. At intervals during this 2-year period, samples of dried tobacco leaves infested with L. serricorne were taken and the rate of parasitism by the mite was measured. The percentage of L. serricorne predated by T. putrescentiae, depending on time and tobacco store, was about 20%. Tyrophagus putrescentiae is reported for the first time as a predator of L. serricorne in tobacco stores in Greece.     

Use of a new milk-clotting protease from Thermomucor indicae-seudaticae N31 as coagulant and changes during ripening of Prato cheese

Prato cheeses were manufactured using coagulant from Thermomucor indicae-seudaticae N31 or a commercial coagulant. Cheeses were characterised using the following analysis: yield; fat; acidity; moisture; ash; salt; pH; total nitrogen; total protein; NS-pH 4.6/NT*100; NS-TCA 12%/NT*100; casein electrophoresis; and RP-HPLC. The results were statistically analysed and revealed that the proteolytic indices were not significantly different throughout the 60 days of ripening of cheeses made with either coagulant. Even though there were some quantitative differences in the peptide profile of cheeses, the enzyme from T. indicae-seudaticae N31 was used in the production of good quality Prato cheese without having to change the established technological parameters of the process.     

Pulsed UV light as an intervention strategy against Listeria monocytogenes and Escherichia coli O157:H7 on the surface of a meat slicing knife

The main objective of this work was to explore the applicability of the Intense Light Pulses (ILP) for decontamination of a stainless steel meat contact surface, exemplified by a slicing knife, as a function of time between contamination and decontamination, number of light pulses applied, and the prior contact with different meat matrices. Listeria monocytogenes and Escherichia coli O157:H7 were chosen as the challenge microorganisms. The ILP system was a laboratory-scale four-lamp batch system generating 3 J/cm² with an input voltage of 3000 V. The results obtained demonstrate successful application of ILP treatment for reduction of L. monocytogenes and E. coli O157:H7 on a surface of stainless steel slicing knife. The inactivation effectiveness depended on the type of meat product that was in the contact with the treated surface and on the time between the contamination and the ILP treatment. Statistical analysis showed the significant interaction between the time and type of meat product on the effectiveness of ILP treatment. The highest effectiveness of the ILP (the complete inactivation of 6.5 log CFU/side of knife) was obtained when the knife surface was in contact with the products containing lower fat and protein content and when it was treated with pulsed light as fast as possible after the contamination (within 60 s). The decontamination efficacy of ILP treatment could not be improved by multiple light pulses if lost due to the extended time between the moment of contamination and ILP treatment. Results showed that the suggested approach can be very effective as an intervention strategy along meat processing lines preventing cross-contamination between the equipment and the final product.     

Phenolic components of Olea europea: Isolation of new tyrosol and hydroxytyrosol derivatives

Two new phenolic compounds were isolated from fruits of Olea europaea, Hojiblanca cultivar. The first compound is the methyl acetal of the aglycone of ligstroside, while the second derivative, not yet reported in the literature, is the β-hydroxytyrosyl ester of methyl malate. These microcomponents may be responsible for hedonistic-sensorial characteristics of olive products.