Chemical extraction of chitin from American lobster (Homarus americanus) shells optimized through response surface methodology.

Chitin extraction from the shells of American lobsters (Homarus americanus) was optimized through the use of response surface methodology (RSM). The demineralization step was optimized to minimize the ash content of shell samples and the deproteination step was optimized to minimize the protein content of the chitin product. At a laboratory scale, one set of optimized conditions for the demineralization step was 7.35 % w/w acetic acid at a 40 mL/g of powdered lobster shell ratio for 15 min; this lowered the ash content from 39.62 % to 0.41 ± 0.08 %. A set of optimized conditions for the deproteination step at a similar scale was 4 % w/w sodium hydroxide at a 43 mL/g demineralized shell ratio heated to 95 °C for 83 min. These conditions were indicated to entirely remove protein from the resultant chitin. Average yields under optimized conditions were 23.43 ± 1.75 % for demineralization and 30.33 ± 0.02 % for deproteination, though a demineralization reaction with larger biomass input had a higher yield at 40.31 %.

Effect of glazing and rosemary (Rosmarinus officinalis) extract on preservation of mud shrimp (Solenocera melantho) during frozen storage.

In this study, glazing with water and rosemary (Rosmarinus officinalis) extract were applied on frozen mud shrimp (Solenocera melantho) and stored at -20 °C for 24 weeks. Quality loss and protein and lipid changes of shrimp were evaluated by total volatile basis nitrogen (TVB-N), drip loss, moisture distribution, sulfhydryl content (SH), disulfide bond, intrinsic fluorescence intensity, lipid content, free fatty acids (FFA), peroxide value (PV), fluorescent compounds and sensory characteristics. Results showed that unglazed mud shrimp exhibited significant quality decline after 16 weeks of frozen storage. Glazing treatment significantly reduced quality loss, protein degradation, and lipid oxidative damage of shrimp during the 24 weeks of frozen storage, compared to the unglazed control sample. Glazing with rosemary extract was more effective in controlling quality changes in frozen mud shrimp with lower TVB-N, drip loss, PV, FFA and higher lipid content and sensory scores.

MALDI-TOF/TOF Mass Spectrometric Determination and Antioxidative Activity of Purified Phosphatidylcholine Fractions from Shrimp Species.

Purification, characterization, and antioxidative activity in vitro of shrimp phosphatidylcholines (PCs) were investigated. The molecular structures of shrimp PCs were determined by MALDI-TOF/TOF MS. The MS2 fragments produced from protonated PC precursors and sodiated PC precursors were identified. The specific fragments including [M + Na - trimethylamine]+, [M + Na - 205]+, [M + Na - RCOOH - trimethylamine]+, and [M + H - RCOOH - trimethylamine]+ could distinguish the precursor type to confirm PC molecular structures. The antioxidative activities of purified shrimp PC fractions were evaluated by assay of DPPH free radical scavenging activity, and their effects on the oxidative stability of camellia oil were measured by monitoring changes in the peroxide value assay during oxidation. The PC fractions from Penaeus chinesis and Macrobranchium nipponense showed stronger antioxidative activities than those of other species. All of the shrimp PCs at 0.2% (w/w) improved the oxidative stability of camellia oil significantly (P < 0.05) compared to controls. The experimental findings suggest that shrimp PCs might be a valuable source of natural antioxidants for edible oils or other food dispersions.

New High-Performance Liquid Chromatography Coupled Mass Spectrometry Method for the Detection of Lobster and Shrimp Allergens in Food Samples via Multiple Reaction Monitoring and Multiple Reaction Monitoring Cubed.

Crustacean shellfish allergy ranks among the most frequent and severe food allergies for adults, demanding rugged and sensitive analytical routine methods. The objective of this study was therefore to develop a mass spectrometric approach for the detection of contamination with shrimp and lobster, two economically important types of crustaceans, in complex food matrices. Following a biomarker approach, we identified proteotypic peptides and developed a multiple reaction monitoring (MRM) method allowing for the identification and differentiation of shrimp and lobster in the food matrix at concentrations down to 0.1%. To further enhance sensitivity, we employed the MRM-cubed (MRM(3)) mode, which allowed us to detect crustaceans down to concentrations of 25 µg/g (crustacean/food, 0.0025%). We hereby present the first mass spectrometric method for the detection of shrimp and lobster in food matrices.

Large-field electron imaging and X-ray elemental mapping unveil the morphology, structure, and fractal features of a Cretaceous fossil at the centimeter scale.

We used here a scanning electron microscopy approach that detected backscattered electrons (BSEs) and X-rays (from ionization processes) along a large-field (LF) scan, applied on a Cretaceous fossil of a shrimp (area ∼280 mm(2)) from the Araripe Sedimentary Basin. High-definition LF images from BSEs and X-rays were essentially generated by assembling thousands of magnified images that covered the whole area of the fossil, thus unveiling morphological and compositional aspects at length scales from micrometers to centimeters. Morphological features of the shrimp such as pleopods, pereopods, and antennae located at near-surface layers (undetected by photography techniques) were unveiled in detail by LF BSE images and in calcium and phosphorus elemental maps (mineralized as hydroxyapatite). LF elemental maps for zinc and sulfur indicated a rare fossilization event observed for the first time in fossils from the Araripe Sedimentary Basin: the mineralization of zinc sulfide interfacing to hydroxyapatite in the fossil. Finally, a dimensional analysis of the phosphorus map led to an important finding: the existence of a fractal characteristic (D = 1.63) for the hydroxyapatite-matrix interface, a result of physical-geological events occurring with spatial scale invariance on the specimen, over millions of years.

Chitooligosaccharides as novel ingredients of fermented foods.

Chitooligosaccharides (COSs) have been clinically evaluated for their immunostimulating effects after oral intake. Similar to dietary supplements, prebiotics and biopreservatives, these water-soluble bioactives are easily incorporated into dairy products and beverages. Notwithstanding, the use of COS in fermented foods would be limited by its antimicrobial properties. In order to study the interaction with yoghurts as a model of fermented food, the effects of COS on chemical composition, viability, morphology and metabolism of lactic acid bacteria, fatty acid profiles and conjugated linoleic acid (CLA) were assessed over 28 days and after chemical digestion. There were no significant differences between the nutritional composition of controls and yoghurts supplemented with concentrations up to 0.1% w/w of COS. However, the acidification of milk decreased at 0.5% (p < 0.05) and the formation of yoghurt failed at 3.0%, without affecting viable counts. Lipid hydrolysis of yoghurts supplemented with 0.1% COS was not affected by chemical digestion. No significant differences were found between CLA percentages of controls and supplemented yoghurts after digestion. Although the nutritional composition, fatty acids and viable counts were not significantly modified after COS supplementation, the present study shows that COS diminishes bacterial acidification at concentrations higher than 0.1%, thus limiting the amounts that could be added to yoghurt.

Response surface methodology toward the optimization of high-energy carotenoid extraction from Aristeus antennatus shrimp.

High-energy assisted extraction techniques, like ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE), are widely applied over the last years for the recovery of bioactive compounds such as carotenoids, antioxidants and phenols from foods, animals and herbal natural sources. Especially for the case of xanthophylls, the main carotenoid group of crustaceans, they can be extracted in a rapid and quantitative way with the use of UAE and MAE. Response surface methodology (RSM) is used for the optimization of extraction methodologies, also being applied to optimize high energy techniques. Three independent variables, namely extraction time, ultrasound or microwave power and solvent/material ratio, were investigated for both methods by employing a 16-run three-level Box-Behnken design (BBD). Considering the extraction efficiency for carotenoids from Aristeus antennatus shrimp, the selected conditions for UAE were 5 min, 600 W and 10:1 mL g(-1). Acetone was the solvent of choice for the extraction procedure. For MAE, the best experimental values were 7 min, 30 W and 20:1 mL g(-1) using n-hexane:acetone:ethanol 2:1:1 (v/v/v) as extraction solvent. The determination of total carotenoid yield was carried out using the spectophotometric calibration curve (A=0.1646(±0.0061)C-0.005(±0.022), R(2)=0.996, n=3) of a standard mix solution of canthaxanthin, zeaxanthin and lutein at 452.1 nm. Under the selected conditions, the yield of total carotenoids for UAE was 23.4(±2.3) and 6.73(±0.56) mg of carotenoids per 100 g dry sample for the head and the body of shrimp, while for MAE was 23.92(±0.63) and 13.3(±1.1) mg of carotenoids per 100g dry sample, respectively. The recovery of both methods was calculated between 60 and 105%. The results indicate that high-energy extraction techniques are faster, less laborious, more repeatable and reproducible methods than the conventional approaches for the quantitative recovery of sensitive bioactive compounds. Moreover, the recovery of a high-added value group of bioactive molecules from natural sources, such as carotenoids, can constitute a profitable and valuable commercial alternative, as these compounds can be used as dietary supplements, food color enhancers and additives in animal feeds, functional foods, preservatives, pharmaceuticals and cosmetics.

Astaxanthin from shrimp by-products ameliorates nephropathy in diabetic rats.

AIM: This study investigated the hypoglycemic and antioxidant effects of shrimp astaxanthin on the kidney of alloxan-induced diabetic rats. METHODS: Animals were distributed into four groups of six rats each: a control group (C), a diabetic group (D), a diabetic group supplemented with Astaxanthin (D+As) dissolved in olive oil and a diabetic group supplemented with olive oil (D+OO). In vitro antidiabetic effect was tested in plasma and kidney tissue. RESULTS: The group D of rats showed significant (P < 0.05) increase of glycemia, creatinine, urea and uric acid levels compared to those of the control group (C). Moreover, plasma and kidney malondialdehyde (MDA) and protein carbonyl (PCO) levels for the rats of the group D were significantly increased compared to the control group. Contrariwise, antioxidant enzyme activities, such as catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and non-enzymatic levels of reduced glutathione, were significantly (P < 0.05) decreased in the plasma and kidney of diabetic rats compared to the control ones. The astaxanthin supplementation in rats diet improved the antioxidant enzyme activities and significantly decreased the MDA and PCO levels compared to diabetic rats. Indeed, no significant (P ≥ 0.05) improvement was observed for the fourth group (D+OO) compared to the control group (C). Histological analysis of kidney showed glomerular hypertrophy and tubular dilatation for the diabetic rats. For D+As rats, these histopathological changes were less prominent. CONCLUSIONS: Our results suggest that shrimp astaxanthin may play an important role in reduction of oxidative damage and could prevent pathological changes in diabetic rats suggesting promising application of shrimp astaxanthin in diabet treatment.

Effect of dietary astaxanthin on free radical scavenging capacity and nitrite stress tolerance of postlarvae shrimp, Pleoticus muelleri.

The aim of this study was to investigate the effect of astaxanthin feed supplementation and environmental nitrite stress in postlarvae of Pleoticus muelleri (15 ± 0.004 mg initial weight) under culture conditions. Diets containing three levels of astaxanthin, 0 mg kg(-1) of diet (C0), 100 mg kg(-1) of diet (C(100)), and 300 mg kg(-1) of diet (C(300)), were used. Postlarvae fed with each diet were exposed to different concentrations of nitrite (NO(2)Na) (0-200 mg L(-1)). The 96 h median lethal concentration (LC50) values of nitrite N were 76.3, 89.7, and 157 mg L(-1) for shrimps fed to C0, C(100), and C(300). The scavenging properties were evaluated against the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical by electron resonance spectroscopy (EPR). For all feed treatments, the extracts exhibited strong DPPH scavenging activity; however, shrimp fed with C(100) and C(300) showed the greatest activity to quench DPPH (62 and 59%, respectively) with respect to C0 (43%). It can be concluded that astaxanthin acts as a protector of nitrite stress in P. muelleri.

Chitin extraction from shrimp shell waste using Bacillus bacteria.

The ability of six protease-producing Bacillus species (Bacillus pumilus A1, Bacillus mojavencis A21, Bacillus licheniformis RP1, Bacillus cereus SV1, Bacillus amyloliquefaciens An6 and Bacillus subtilis A26) to ferment media containing only shrimp shell waste, for chitin extraction, was investigated. More than 80% deproteinization was attained by all the strains tested. However, demineralization rates not exceeding 67% were registered. Cultures conducted in media containing shrimp shell waste supplemented with 5% (w/v) glucose were found to remarkably promote demineralization efficiency, without affecting deproteinization rates. The antioxidant activities of hydrolysates, at different concentrations, produced during fermentation in medium supplemented with glucose, were determined using different tests: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method, reducing power assay and chelating activity. All hydrolysates showed varying degrees of antioxidant activity. Hydrolysate produced by B. pumilus A1 exhibited the highest DPPH radical scavenging activity, with an IC(50) value of 0.3 mg/ml. Highest reducing power (DO 700 nm=1.55 at 1.5 mg/ml) and metal chelating activity (98% at 5mg/ml) were obtained with B. pumilus A1 and B. licheniformis RP1 hydrolysates, respectively.

Bioactive marine peptides: nutraceutical value and novel approaches.

Marine organisms represent a valuable source of nutraceuticals and functional compounds. The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of novel active substances for the development of bioactive products. Recently, a great deal of interest has been expressed in marine-derived bioactive peptides because of their numerous beneficial health effects. Moreover, several studies have reported that marine bioactive peptides can be used as antihypertensive, antioxidative, anticoagulant, and antimicrobial components in functional foods or nutraceuticals and pharmaceuticals due to their therapeutic potential in the treatment or prevention of disease. In this chapter, we provide an overview of bioactive peptides derived from marine organisms as well as information about their biological properties and mechanisms of action with potential applications in different areas.

Immunocytochemical and molecular data guide peptide identification by mass spectrometry: orcokinin and orcomyotropin-related peptides in the stomatogastric nervous system of several crustacean species.

In order to identify new orcokinin and orcomyotropin-related peptides in crustaceans, molecular and immunocytochemical data were combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the crayfish Procambarus clarkii, four orcokinins and an orcomyotropin-related peptide are present on the precursor. Because these peptides are highly conserved, we assumed that other species have an identical number of peptides. To identify the peptides, immunocytochemistry was used to localize the regions of the stomatogastric nervous system in which orcokinins are predominantly present. One of the regions predominantly containing orcokinins was a previously undescribed olive-shaped neuropil region within the commissural ganglia of the lobsters Homarus americanus and Homarus gammarus. MALDI-TOF MS on these regions identified peptide masses that always occur together with the known orcokinins. Seven peptide ions occurred together in the peptide massspectra of the lobsters. Mass spectrometric fragmentation by MALDI-MS post-source decay (PSD) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI Q-TOF MS) collision-induced dissociation (CID) were used in the identification of six of these masses, either as orcokinins or as orcomyotropin-related peptides and revealed three hitherto unknown peptide variants, two of which are [His13]-orcokinin ([M+H]+ = 1540.8 Da) and an orcomyotropin-related peptide FDAFTTGFGHN ([M+H]+ = 1213.5 Da). The mass of the third previously unknown orcokinin variant corresponded to that of an identified orcokinin, but PSD fragmentation did not support the suggested amino acid sequence. CID analysis allowed partial de novo sequencing of this peptide. In the crab Cancer pagurus, five orcokinins and an orcomyotropin-related peptide were unambigously identified, including the previously unknown peptide variant [Ser9-Val13]-orcokinin ([M+H]+ = 1532.8 Da).

Characterization of a novel cellular retinoic acid/retinol binding protein from shrimp: expression of the recombinant protein for immunohistochemical detection and binding assay.

Members of the cellular retinoic acid (CRABP) and retinol binding (CRBP) proteins family are involved in the metabolic pathways of retinoic acid (RA) and retinal respectively. The objective of this study is to determine whether such proteins are present in crustaceans. We report here the cloning and isolation of a novel complementary DNA (cDNA) that showed characteristics of the CRABP/CRBP from the ovary and eyestalk of the shrimp. The cDNA is 0.9 Kb in size and the deduced shrimp protein is encoded for a protein of 14 kDa. Although it shows high amino acids sequence similarity to both the vertebrate and invertebrate CRABP, some conserved amino acids identified in other CRABPs were not found in MeCRABP. MeCRABP is expressed in the ovary, eyestalk, testis, epidermis and early larvae. The presence of MeCRABP in early larval stages suggests that the protein may be involved in the early larval development. Recombinant MeCRABP was produced and used to generate a polyclonal antibody. In the immunohistochemical detection study, anti-rCRABP antibody recognized the presence of CRABP in several cell types of the eyestalk as well as the smaller oocytes of the ovary. Although MeCRABP messenger RNA transcripts can be detected in the ovary throughout the ovarian maturation period, CRABP was detected only in the primary oocytes of the ovary. The results suggest that CRABP transcripts in the mature ovary are not translated and may be supplied to the oocyte as maternal messages. The binding property of the recombinant MeCRABP was also tested by a fluorometeric method. The result indicates that rMeCRABP binds to both RA and retinal with similar affinity. This study represents the first cloning and characterization of a cDNA that belongs to a member of retinoid/fatty acid binding protein family in crustaceans.

Effects of gamma irradiation on the flavor composition of food commodities.

Fifteen food products including potato, sweet potato, shallot, onion, garlic, ginger, papaya, mango, rice, tobacco, small red bean, mungbean, soybean, wheat, flour and spices have been approved for irradiation by the National Health Administration in Taiwan. Market tests (Wu et al., 1996) provided strong proof that Taiwanese consumers would accept irradiated foods. However, researchers in the food industry are concerned about the possibility of chemical changes, especially in volatile composition, during irradiation processing. This study considers several food commodities, including garlic, ginger, shiitake, onion, potato, day-lily, tilapia, silver carp and shrimp. Food samples were irradiated with optimum doses and then studied for possible occurrence of chemical changes and effects on compositional characteristics of foods.

Quantification of the major brown shrimp allergen Pen a 1 (tropomyosin) by a monoclonal antibody-based sandwich ELISA.

BACKGROUND: Among 13 allergens found in extracts of cooked brown shrimp (Penaeus aztecus) the 36 kd muscle protein tropomyosin has been identified as the only major shrimp allergen (Pen a 1). Cross-reacting molecules with similar molecular weights were detected in other crustacea species such as crab, lobster, and crawfish. Because Pen a 1 and Pen a 1-like allergens are important in crustacea allergy, the aim of this study was to develop a monoclonal antibody (mAb)-based sandwich ELISA to quantify Pen a 1 and to evaluate Pen a 1 levels in four commercial shrimp, crab, and lobster extracts. METHODS: Two Pen a 1-specific mAbs with different epitope specificities were selected. ELISA plates coated with captured mAb 3.2 were incubated with samples containing Pen a 1. Bound Pen a 1 was detected by a combination of biotinylated mAb 4.9.5 and alkaline phosphatase-labeled streptavidin. RESULTS: The optimized sandwich ELISA could detect Pen a 1 concentrations ranging from 4 to 125 ng/ml. Four commercial shrimp extracts demonstrated a 40-fold difference in Pen a 1 levels (24 to 920 microg/ml). Crab and lobster extracts contained detectable levels of Pen a 1-like proteins. No reactivity to cockroach, house dust mite, oyster, codfish, or peanut extracts was detected, which indicates that the developed assay is crustacea-specific. CONCLUSION: A sensitive sandwich assay was developed to quantify Pen a 1. This assay will be helpful to standardize shrimp extracts in regard to the content of the major allergen, Pen a 1, and to study cross-reactivities among and evaluate occupational exposure to different crustacea species.

[Simultaneous determination of tyrosine and tryptophan in amino acid injection, animal and plant extracts by polarization spectrofluorimetry].

A new method for the simultaneous determination of tyrosine and tryptophan by polarization spectrofluorimetry has been developed. The interference of phenylalanine and light scattering of solvent were eliminated by using vertically crossed polarizers. The overlapping of fluorescence spectra of the two amino acids was solved with filters. The linear ranges of determination were 0.02-12.0 mg.L-1(tyrosine) and 0.01-2.50 mg.L-1(tryptophan). The mean recoveries of tyrosine and tryptophan in samples were 95.4% and 101.7% (in amino acid injection). 99.2% and 100.0% (in wild jujube kernel), 95.3% and 97.2% (in shrimp shell) respectively. The proposed method has been used to determine tyrosine and tryptophan in amino acid injection, extracts of wild jujube kernel and shrimp shells with satisfactory results.

Practical applications of element-specific detection by inductively coupled plasma atomic emission spectroscopy and inductively coupled plasma mass spectrometry to ion chromatography of foods.

Three practical examples are presented to demonstrate the utility of element-selective detection for ion chromatography (IC). The determination of As species in a liquid health food supplement by IC with inductively coupled plasma atomic emission spectroscopy (IC-ICP-AES) is shown to confirm results obtained for total As. IC-ICP-AES is also used to investigate the identity of an unknown peak in a sample of shrimp commercially treated with tripolyphosphate. Finally, results are presented for the determination of residual bromate in baked goods by IC with inductively coupled plasma mass spectrometry detection.

Measurement of uranium content and its distribution in some crustacea of Mysidacea species by the fission track method.

Uranium distributions and contents in Mysidacea species were investigated in order to be subsequently correlated with some specific properties of these crustacea. The fission track micromapping technique was used in uranium distribution and content measurements. The investigated biological samples were: (1) Javanisomysis gutzui Bacescu, 1992 and (2) Mesopodopsis slabberi Van Beneden, 1861. The determined contents varied in the range of natural background values of uranium.