Extraction of Carotenoprotein from Shrimp Process Wastes with the Aid of Trypsin from Atlantic Cod

Atlantic cod trypsin or bovine trypsin were used to aid the extraction of carotenoprotein from shrimp wastes at 4°C. When 25 mg% cod trypsin was added to extraction medium containing 0.5N ethylene diaminetetraacetic acid (EDTA) 64% of the astaxanthin and 81% of the protein of shrimp waste was recovered as carotenoprotein in 24 hr. With 25 mg% bovine trypsin, under otherwise identical conditions, the carotenoprotein recovered represented 49% of the astaxanthin and 65% of the protein of the waste. Semi-purified cod trypsin was not as effective as pure trypsin in facilitating recovery of carotenoprotein from shrimp waste. The recovery of carotenoprotein from shrimp waste, during extraction at 4°C with or without trypsin, was facilitated by EDTA.     

Characterisation of trypsin purified from the viscera of Tunisian barbel (Barbus callensis) and its application for recovery of carotenoproteins from shrimp wastes

Trypsin was purified from the viscera of barbel by precipitation using ammonium sulphate (0-80%), Sephadex G-100, and Mono Q-Sepharose ion exchange chromatography. The trypsin was purified 27-fold, with 79 U/mg specific activity and 31% recovery. The enzyme had a molecular weight of 24 kDa; purified trypsin appeared as a single band on native-PAGE. The optimum pH and temperature for enzyme activity were pH 10.0 and 55 °C with BAPNA used as a substrate. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECTPYSQ. The Michaelis-Menten constant (K_m) and catalytic constant (k_cat) values of the enzyme were 0.018 mM and 1.21 s⁻¹, respectively. The study also investigated the effects of purified trypsin on the recovery of carotenoproteins from shrimp (Parapenaeus longirostris) shells through hydrolysis using 1.0 U barbel trypsin/g shrimp shells for 1 h at 30 °C. The freeze-dried carotenoproteins recovered contained 71.09% protein, 16.47% lipid, 7.78% ash, and 1.79% chitin.     

Albacore tuna (Thunnus alalunga) spleen trypsin partitioning in an aqueous two-phase system and its hydrolytic pattern on Pacific white shrimp (Litopenaeus vannamei) shells

Partitioning behaviors of trypsin from the spleen of albacore tuna (Thunnus alalunga) in an aqueous two-phase system (ATPS) were investigated. Partitioning behaviors of proteins were influenced by polyethylene glycol (PEG) molecular mass and concentration, types and concentration of salts, NaCl concentration, and temperature. Trypsin was preferentially partitioned into the PEG-rich top phase. The best ATPS conditions for trypsin partitioning from albacore tuna spleen were 15% PEG 4000–15% NaH₂PO₄ at 40°C without the addition of NaCl, which increased the purity by 5.54-fold with the recovered activity of 71.92%. Based on SDS-PAGE, the enzyme after ATPS separation was near homogeneity and the result of SDS-substrate gel electrophoresis revealed that the band intensity of enzyme in ATPS fraction increased, indicating the enhanced specific activity of splenic extract. The study further investigated the effect of fractionated trypsin on the hydrolysis of Pacific white shrimp (Litopenaeus vannamei) shells. Electrophoretic study revealed that trypsin after ATPS separation was a potential enzyme for extraction of carotenoprotein from Pacific white shrimp shell waste. Therefore, ATPS was an effective method for purification and recovery of trypsin from the spleen of albacore tuna and it could be used as an alternative cheap proteinase for the extraction of carotenoprotein from shrimp shells.     

Amino acid profile and enhancement of the enzymatic hydrolysis of fermented shrimp carotenoproteins

Amino acid profiles of carotenoproteins extracted from fermented and non-fermented shrimp waste were analysed. Fermented carotenoproteins were hydrolysed with a protease and a combination of a protease and a lipase. Essential amino acids in fermented and non-fermented carotenoproteins were 49% and 47%, respectively, with respect to total amino acids. The highest carotenoprotein hydrolysis (900 and 66 mg/g soluble protein and total carotenoids, respectively) was obtained by a combination of 15 proteolytic units together with 10 lipolytic units. The most efficient treatment using only protease was obtained with 15 proteolytic units (852 and 48 mg/g of soluble protein and total carotenoids, respectively). A relatively protein-free form of astaxanthin derived from shrimp waste carotenoproteins may be of interest for applications in salmon culture, and in natural health products and cosmetics. Furthermore, fermented carotenoproteins could be used in human and animal diets due to their high essential amino acids concentration.     

CAROTENOPROTEIN FROM TROPICAL BROWN SHRIMP SHELL WASTE BY ENZYMATIC PROCESS

While extraction of carotenoprotein from brown shrimp (Metapenaeus monoceros) shell waste, trypsin showed maximum recovery (55%) of carotenoid pigment in 4 hours at (28±2°C); but pepsin and papain showed about 50% recovery during the same period. The yield of protein paste by trypsin was maximum. The average protein content in the protein paste was about 450 g kg^-1. The percent of recovery of protein by papain and pepsin was close to that of trypsin. During storage at ambient temperature (28±5°C) loss of carotenoids from cake prepared by trypsin was minimum. The cost of trypsin is twenty times that of papain. Thus papain, easily available and the cheapest enzyme, can be used suitably for moderate recovery of carotenoids and good recovery of protein from shrimp shell waste at tropical ambient temperature. The dried colorless solid residue after extraction of carotenoprotein and protein, can be used as raw materials for chitin/chitosan.     

A simplified method for the determination of several fish drugs in edible fish and shrimp by high-performance liquid chromatography

A simplified HPLC method was used to recover antibiotic and antibacterial agents from 11 cultured fish and one shrimp. The average recoveries of oxytetracycline, oxolinic acid, miloxacin, and sulfamonomethoxine were 77, 92, 89 and 80%, respectively. The detection limit was 0.02–0.04 μg/g. We also examined the recovery of oxytetracycline and oxolinic acid used for cultured black tiger shrimp, which is a popular imported shrimp in Japan. To demonstrate the usefulness of this technique for detecting drug residues in cultured fish, the method was used to examine the muscle tissues of 12 commercial fish and shrimp obtained from several fish shops. No agents were found in any of the fish tested.     

Importance of conformation for the IgE reactivity of sarcoplasmic calcium-binding protein from the black tiger shrimp Penaeus monodon

Sarcoplasmic calcium-binding protein (SCP) is an EF-hand Ca²⁺-binding protein recently identified as a new crustacean allergen. Some EF-hand Ca²⁺-binding allergens, such as parvalbumin (fish allergen) and Bet v 4 (birch pollen allergen), have been shown to contain conformational-type IgE epitopes associated with the Ca²⁺-chelating. This study was performed to clarify the relationships between IgE reactivity and structure of the black tiger shrimp SCP. When analyzed by ELISA in the absence and presence of EGTA, the IgE reactivity of the black tiger shrimp SCP was found to be considerably reduced by Ca²⁺-depletion. Furthermore, circular dichroism spectral data showed a conformational difference between Ca²⁺-bound and Ca²⁺-depleted forms of the black tiger shrimp SCP. On the other hand, the synthetic 18 peptides spanning the entire amino acid sequence of the black tiger shrimp SCP were all assessed to have little IgE-binding ability by ELISA. Our results demonstrate that the IgE reactivity of the black tiger shrimp SCP is attributable mostly to conformational-type IgE epitopes, at least part of which is maintained by Ca²⁺-chelating.     

Comparative studies on chemical composition and thermal properties of black tiger shrimp (Penaeus monodon) and white shrimp (Penaeus vannamei) meats

Chemical composition and thermal properties of meat from two species of shrimps, black tiger shrimp (Penaeus monodon) and white shrimp (Penaeus vannamei), were comparatively studied. White shrimp meat had higher protein and ash contents than had black tiger shrimp meat (p < 0.05). Fractionation of nitrogenous constituents revealed that myofibrillar protein was the major component in the muscles; myosin heavy chain (MHC) and actin were the predominant proteins. White shrimp meat comprised higher stromal protein with greater pepsin-soluble collagen and insoluble collagen contents than did black tiger shrimp meat. Muscle proteins from black tiger shrimp, especially MHC, had higher thermal stability than those from white shrimp as indicated by the higher transition temperature (T_max) as well as the lower inactivation rate constant (K_D). Phospholipid was the predominant lipid (72–74%) in both shrimps, followed by triglyceride. Polyunsaturated fatty acids were found as the major fatty acids with the range of 42.2–44.4%. DHA (22:6)/EPA (20:5) ratio in black tiger shrimp (2.15) was higher than that in white shrimp (1.05). Mg was the dominant mineral in both shrimps. Ca and Fe were also found at high concentrations. Arginine was the most abundant amino acid, while leucine, isoleucine and proline were predominant in both shrimps. Glutamic acid and glycine contents were greater in black tiger shrimp meat; however, white shrimp meat had higher hydroxyproline content. Different compositions might govern the different characteristics as well as thermal properties of both species.     

EFFECTS OF FEED DIETS ON DIGESTIVE PROTEASES FROM THE HEPATOPANCREAS OF WHITE SHRIMP (Penaeus vannamei)

Protease activities in the hepatopancreas extract (HP) from white shrimp (Penaeus vannamei) fed one of seven test diets for 30 days were evaluated by several methods. The test diet contained 85% of a reference ration for shrimp and 15% of either anchovy meal, tuna waste meal, deboned white fish meal, langostilla meal, soybean meal, and two menhaden meals as a protein replacer. One of the menhaden fish meals tested (B) had the lowest quality as a shrimp feed based on amino acid analysis. SDS-PAGE zymograms of HP from each of the seven diet groups showed similar proteins activity patterns with casein as substrate. The degree of hydrolysis of casein, measured by pH-stat, was also the same for HP from the seven diet groups (P > 0.05). However, total protease activity measured by azocasein hydrolysis (units/g HP) was higher for the diet group fed the test ration containing tuna waste as a replacer (P > 0.05). Trypsin and chymotrypsin activities measured with synthetic substrates (units/mg protein; units/g HP) from animals reared on the diet with menhaden meal B replacer were greater than in the other diet groups (P < 0.05). This study shows that a relatively small amount (15%) of a specific protein replacer in white shrimp rations can influence the protease activity of shrimp HP. Given that digestive proteases such as trypsin can leach into the muscle of postharvest shrimp and thereby cause softening of the meat, the impact of the rearing diet on postharvest shelf-life should be considered along with the standard measures of feed quality that are used by the fish farmer, i.e. animal growth and heatlh.     

Salmonella prevalence in seafood imported into Japan

A total of 353 samples of 29 types of seafood were tested for Salmonella prevalence and total microbial population. Salmonella enterica serotype Weltevreden was isolated from 2 of 47 black tiger prawn samples. The contamination levels of Salmonella were in a range of <30 to 40 most probable number per 100 g. In addition, one sample of black tiger prawns and two samples of white shrimp were positive for Salmonella invA gene on PCR assay. Although the mean aerobic bacterial count was greater than 4 log CFU/g in most of the sample types, those in the two Salmonella-isolated samples of black tiger prawn were 7.48 and 5.18 log CFU/g, respectively. These results indicate the possibility that shrimp and prawns contribute to foodborne infections. The improvement of seafood quality is an important issue, and the information on contamination by pathogens should be provided as feedback to the originating country, with the aim of increasing safety.     

Antioxidant activity of extracts produced by solvent extraction of almond shells acid hydrolysates

The ethyl acetate-soluble fraction generated during acid hydrolysis of almond shells was evaluated for radical-scavenging capacity and protection against fish oil oxidation. The influence of the operational conditions during acid hydrolysis on: (i) the total phenolics produced; (ii) the recovery yield of ethyl acetate solubles; (iii) the phenolic content in the ethyl acetate extracts; (iv) the antioxidant activity of extracts was assessed. A one-at-a-time variation study of the hydrolysis time and sulfuric acid concentration was carried out. For a given temperature and hydrolysis time, the influence of the acid concentration was noticeable; whereas the maximal phenolics production, measured in the hydrolyzate (2.2 g gallic acid equivalents/100 g shells) was achieved with 2% sulfuric acid, the maximal recovery in the organic phase required at least 5% acid. The crude extracts showed DPPH radical-scavenging activities (EC₅₀ < 0.5 g/l) comparable to those of synthetic antioxidants, and protected labile lipid systems, such as fish oils and fish oil-in water emulsions, from oxidation as efficiently as did propyl gallate.     

Recovery of Components from Shrimp (Xiphopenaeus kroyeri) Processing Waste by Enzymatic Hydrolysis

ABSTRACT:  Industrial shrimp waste is a good source of protein, chitin, and carotenoids. In general, this waste is discarded with no attempt to use it, thus contributing to environmental pollution. This study was aimed at recovering the 3 main components of industrial shrimp waste, protein, chitin, and astaxanthin, using enzymatic treatment with Alcalase and pancreatin. An increase in the degree of hydrolysis (DH) from 6% to 12% resulted in 26% to 28% protein recovery. Alcalase was more efficient than pancreatin, increasing the recovery of protein from 57.5% to 64.6% and of astaxanthin from 4.7 to 5.7 mg astaxanthin/100 g of dry waste, at a DH of 12%. The enzymatic hydrolysis of the industrial waste from Xiphopenaeus kroyeri shrimp using Alcalase allowed for 65% protein recovery in the form of hydrolysates, in addition to providing suitable conditions for the recovery of astaxanthin and chitin.     

Comparative studies on the effect of the freeze–thawing process on the physicochemical properties and microstructures of black tiger shrimp (Penaeus monodon) and white shrimp (Penaeus vannamei) muscle

The effects of different freeze–thaw cycles (0, 1, 3 and 5) on the physicochemical properties and microstructures of black tiger shrimp (Penaeus monodon) and white shrimp (Penaeus vannamei) muscle were investigated. White shrimp had greater exudate loss and higher α-glucosidase (AG), as well as β-N-acetyl-glucosaminidase (NAG) activities, than did black tiger shrimp, especially when the number of freeze–thaw cycles increased (P < 0.05). The decreases in Ca²⁺-ATPase activity, sulfhydryl group content and protein solubility with concomitant increases in disulfide bond formation and surface hydrophobicity were more pronounced in white shrimp muscle, than in black tiger shrimp muscle, particularly after five cycles of freeze–thawing (P < 0.05). The shear force of both shrimps was decreased after five freeze–thaw cycles (P < 0.05). The microstructure study revealed that the muscle fibers were less attached, with the loss of Z-disks, after subjection to five freeze–thaw cycles. Therefore, the freeze–thawing process caused denaturation of proteins, cell disruption, as well as structural damage of muscle in both shrimps. White shrimp generally underwent physicochemical changes induced by the freeze–thawing process to a greater extent than did black tiger shrimp.     

RECOVERY OF FISH BONE FROM HOKI (JOHNIUS BELENGERI) FRAME USING A PROTEOLYTIC ENZYME ISOLATED FROM MACKEREL INTESTINE

A new enzymatic treatment procedure was devised to recover and further utilize fish bones from processing which are normally discarded. This procedure includes the use of an enzyme preparation isolated from raw mackerel intestine by acetone precipitation. The caseinolytic activity of mackerel intestine crude enzyme (MICE) was approximately 0.58 U/mg protein, which was comparable to those of trypsin and α‐chymotrypsin, or even higher than those of papain and Aspergillus saitoi protease. The optimum pH, temperature and enzyme/substrate ratio of MICE for hydrolysis of protein with hoki (Johnius belengeri) frames were determined to be pH 9.0, 40C and 1:100 (w/w). When the hoki frame was treated by MICE for 6 h under the optimum reaction conditions, the yield of bone recovery was approximately 90%. Compared to Alcalase, trypsin, α‐chymotrypsin and Neutrase, the yield of bone recovery using MICE was higher. These results suggest that MICE may be utilized for bone recovery from fish frame.     

Effect of heating on physical properties and microstructure of black tiger shrimp (Penaeus monodon) and white shrimp (Penaeus vannamei) meats

Effects of heating on cooking loss, texture, colour and microstructure of black tiger shrimp (Penaeus monodon) and white shrimp (Penaeus vannamei) meats from different parts were evaluated. Cooking loss increased sharply when the samples were heated for longer time, particularly more than 1 min (P < 0.05). Among all parts, tail part of both shrimps had the highest cooking loss. The shear force of all samples increased markedly when the samples were heated for longer time, especially more than 0.5 min (P < 0.05). Black tiger shrimp and white shrimp meats had the slight differences in shear force either before or after heating. L*, a* and b*‐values increased when heating time increased up to 1 min (P < 0.05). Similar microstructures between raw meats of black tiger shrimp and white shrimp were found. Cooked meats of both species had more compact fibre arrangement with the shrinkage of sarcomere, compared with raw samples. Generally, tail portion showed the denser structure than other parts.     

Carotenoproteins from lobster waste as a potential feed supplement for cultured salmonids

Lobster waste (including the head and hard carapace, viscera, mandibles and gills) contains approximately 54 μg/g total astaxanthin, 29% protein, 23% chitin, 34% ash and 2.2% crude fat on a dry weight basis. Trypsin from bovine pancreas was applied to facilitate the recovery of carotenoid pigments and protein as carotenoprotein complex, which was subsequently air‐dried to a stable powder form at 45°C and 15% relative humidity. The product obtained was found to contain 60% protein, 15% crude fat, 6% ash, 8% chitin and 295 μg/g total astaxanthin. Thus, the process achieved a substantial reduction in the levels of anti‐nutrients associated with lobster waste (i.e., ash and chitin) while elevating the levels of carotenoid pigments and essential nutrients such as protein and fat in the recovered product These characteristics of the final product suggest that it could be used as an inexpensive source of pigment and protein in diets of cultured salmonid species.     

Effect of storage temperature and packaging on quality and shelf‐life of high pressure processed black tiger shrimp (Penaeus monodon)

The effect of high pressure processing (361 MPa/12 min/46 °C) on the quality and shelf‐life of black tiger shrimp was investigated. Changes in physical, biochemical, microbiological, and sensory attributes of samples packed in low density polyethylene (LDPE), ethylene vinyl alcohol (EVOH), and multilayer metalized polyester (MMP) pouches were examined during storage at 4, 15, and 25 °C for 30 days. The estimated shelf‐life of pressure treated samples based on sensory, chemical, and microbiological data was found to be 30 days for EVOH and MMP samples and 18 days for LDPE samples at 4 °C. The control and high pressure samples at 15 °C reach the unacceptable limit by 3rd and 9th day of storage, respectively. However, the samples at 25 °C showed shelf‐life of less than 3 days. Among the packages employed in the study, EVOH film was adjudged to be best in maintaining the quality of shrimp.

Practical applications

In global seafood market, shrimp is a commercially traded important commodity. The conducted study detailed about the quality changes occurred due to microbial activity and biochemical reactions in high pressure processed black tiger shrimp under different storage and packaging conditions and their impact on the shelf‐life. The provided data will be useful in the selection of appropriate conditions to preserve the valuable catch, establish high quality and safety requirements to enhance the marketing potential of shrimp.     

Ensilage Treatment of Crawfish Waste for Improvement of Astaxanthin Pigment Extraction

Acid ensilage treatment preserves crawfish heat-processed waste under ambient temperature conditions with stabilization of the astaxanthin pigment present. Implementation of acid ensiling prior to pigment extraction increased concentration of the astaxanthin oil extract by 40–50%, and oil recovery by 10%. A twofold increase in free amino nitrogen, and a 70% reduction in exoskeleton calcium carbonate, were observed in crawfish silage (pH 4.2) from acid/enzymatic hydrolysis, compared with controls. A correlation was seen between solubilization of calcium carbonate and pigment release in relation to silage pH. The role of acid-resistant proteolytic microorganisms in breakdown of carotenoprotein complex is discussed. Application of ensilage process for ultimate commercial production of astaxanthin-enriched vegetable or fish oils from pigment rich crustacean wastes is postulated.     

EXTENDED VALIDATION OF A SIMPLIFIED EXTRACTION AND GRAVIMETRIC DETERMINATION OF TOTAL FAT TO SELECTED FOODS

A simplified method for chloroform/methanol extraction and gravimetric determination of total fat previously published for diet composites was tested on additional foods, including milk, cheese, fried snack foods, nuts/seeds, salad dressings, baked goods, meat, fish/shellfish and fruits/vegetables. Homogenized food composites and certified reference materials (CRMs) were analyzed using the existing method involving, briefly, orbital shaking of a subsample with chloroform/methanol/buffer in specific proportions, then recovery of total lipid in the chloroform extract. For meat, fish, shrimp, cheese and fried plantains, total fat recovery was low and/or variability was high with the unmodified assay versus results from other standard total fat methods (e.g., ether extraction, acid hydrolysis) or to CRM assigned values. Reducing the sample size or adding a homogenization step during extraction resulted in relative standard deviations of < 3% for all matrices and also values consistent with those generated by other standard total fat methods and within assigned ranges for total fat in most CRMs.

PRACTICAL APPLICATIONS

The method allows for routine batch analysis of multiple samples of a range of food types with minimized hands-on time, using commonly available laboratory equipment. The resulting total lipid extract can be subdivided for analysis of various specific lipid analytes as well as for gravimetric measurement of total fat.     

Enumeration of coliforms and Escherichia coli in frozen black tiger shrimp Penaeus monodon by conventional and rapid methods

Conventional (most probable number, MPN) and rapid methods-including Chromocult coliform agar (CCA), Fluorocult(R) LMX broth (LMX), and Petrifilm Escherichia coli count plates (PEC) for enumeration of coliforms and E. coli in frozen black tiger shrimp from Thailand were compared in order to assess the possibility of using one of the rapid methods for routine analysis. Enumeration of coliforms and E. coli from 18 samples of regular frozen black tiger shrimp and 156 samples of frozen black tiger shrimp experimentally contaminated with coliforms or E. coli at concentrations of approximately 10, approximately 10(2), and approximately 10(3) CFU g(-1) revealed that at the level of approximately 10 CFU g(-1), coliform numbers ranked as LMX>CCA>MPN=PEC and E. coli as MPN=LMX=PEC=CCA. At the level of approximately 10(2) CFU g(-1), coliform numbers ranked as LMX>MPN=PEC=CCA and E. coli as MPN=LMX>PEC=CCA. At the level of 10(3) CFU g(-1), coliforms ranked as LMX>MPN=CCA>PEC and E. coli as MPN>LMX>CCA>PEC. Agreements with the conventional MPN method for coliforms were LMX 108%, PEC 87.2%, and CCA 91.2% and agreements for E. coli were LMX 101%, PEC 95.7%, and CCA 96.3%. Sensitivities (%) ranked LMX>MPN>CCA=PEC for coliforms and E. coli, whereas equal specificities (100%) of all methods for coliforms and E. coli were demonstrated. Rankings for the other parameters compared were: convenience, PEC>CCA=LMX>MPN; time to detection, MPN>LMX=PEC=CCA; expense, MPN=PEC>CCA>LMX; labor, MPN>LMX=CCA>PEC; accuracy for coliforms, PEC>CCA>MPN>LMX; and accuracy for E. coli, PEC=CCA>LMX>MPN.