Fresh Anchovies Research Articles

Effect of the electrostatic field on fish deterioration: the case of the European anchovy (Engraulis encrasicolus)

SummaryThe European anchovy (Engraulis encrasicolus) being a species very rich in long‐chain fatty acids, it is subject to rapid spoilage with consequent sensory losses. Therefore, this study aimed to verify the effect of a low‐voltage electrostatic field (DENBA+) on the quality of fresh anchovies in a refrigerated environment. During 9 days of storage, anchovy samples were analysed from a chemical (TVB‐N and TBARS), physical (texture analysis), microbiological (microbial count) and sensory (QIM) point of view and compared to control samples (without the electrostatic field). DENBA+ technology was able to reduce of 20% the QI score increment rate with respect to the control sample justified by a better quality of the samples after 7 days of storage. In addition, loads of spoiling microbes were significantly lower in DENBA+ after 9 days of storage. This non‐thermal technology has shown positive effects for long periods of storage.

Microbiome dynamics, antibiotic resistance gene patterns and spoilage-associated genomic potential in fresh anchovies stored in different conditions

Fresh fish is a highly perishable product and is easily spoiled by microbiological activity and chemical oxidation of lipids. However, microbial spoilage is the main factor linked with the rapid fish sensorial degradation due to the action of specific spoilage organisms (SSOs) that have the ability to dominate over other microorganisms and produce metabolites responsible for off-flavours.We explored the microbial dynamics in fresh anchovies stored in different packaging (air, modified atmosphere, under vacuum) and temperatures (0, 4 and 10 °C) using shotgun metagenomics, highlighting the selection of different microbial species according to the packaging type. Indeed, Pseudoalteromonas nigrifaciens, Psychrobacter cryohalolentis and Ps. immobilis, Pseudomonas deceptionensis and Vibrio splendidus have been identified as the main SSOs in aerobically stored anchovies, while Shewanella baltica, Photobacterium iliopiscarium, Ps. cryohalolentis and Ps. immobilis prevailed in VP and MAP. In addition, we identified the presence of spoilage-associated genes, leading to the potential production of biogenic amines and different off-flavors (H2S, TMA). In particular, the abundance of microbial genes leading to BA biosynthesis increased at higher storage temperature, while those related to H2S and TMA production were enriched in aerobically and VP packed anchovies, suggesting that MAP could be an effective strategy in delaying the production of these compounds. Finally, we provided evidence of the presence of a wide range of antibiotic resistance genes conferring resistance to different classes of antibiotic (β-lactams, tetracyclines, polymyxins, trimethoprims and phenicols) and highlighted that storage at higher temperature (4 and 10 °C) boosted the abundance of ARG-carrying taxa, especially in aerobically and MAP packed fish.

The Effect of Freezing Raw Material on the Quality Changes and Safety of Salted Anchovies (Engraulis encrasicolus, Linnaeus, 1758) at Cold Storage Conditions

This study demonstrates the effects of the freezing and frozen storage of anchovies prior to brining and dry-salting on quality changes and food safety during refrigerated storage (4 ± 1 °C). Fresh anchovies were divided into two groups, one of which was used as a control representing fresh raw material; the other was the experimental group and consisted of frozen and thawed anchovies stored at −18 °C for a year. Five different salt concentrations were used for brining (10, 15, 20, 25 and 30%) in addition to the dry-salting method. Microbiological, chemical, physical and sensory analyses were carried out during storage. Salt concentration and salting method had significant effects on the shelf-life of salted anchovy products, with the highest shelf-life corresponding to dry-salted anchovies (p < 0.05). The effect of using frozen and thawed raw materials for salting on the shelf-life depended on the processing method since the experimental dry-salted group had a shelf-life one month longer than that of the control group, while the opposite situation occurred for the brined samples, with one exception. Strong correlations were usually found between sensory values and chemical quality parameters (R2: 0.83–0.99 for the control group and 0.63–0.99 for the experimental group). The results demonstrated that the experimental group, with some exceptions, had better values for most quality and food safety parameters in comparison to the control group, indicating the advantage of using frozen and thawed raw materials before salting to prevent spoilage and enhance food safety. Considering that the experimental group was produced from one-year-stored raw material compared to the control group, the advantage of the freezing and frozen storage of anchovies can be accepted as much higher versus when freezing is not implemented. The positive effect is due to the fact that frozen anchovies absorb salt faster after thawing, especially in groups with high salt concentrations. Therefore, it can be concluded that frozen salted anchovies can be utilized for longer as they have a longer shelf-life, particularly when using either the 30% brining or dry-salting method.

Price volatility transmission in the value chain of fresh anchovies in Spain

This study examines the price volatility transmission of fresh anchovies (Engraulis encrasicolus) among different markets along the value chain in Spain. For this purpose, the prices in the first-hand sale, wholesale, and retail markets are considered. A vector autoregressive (VAR) model and an asymmetric multivariate generalised autoregressive conditional heteroskedasticity (MGARCH) model are used to analyse the relationship of price volatility among the markets in the value chain. The results indicate that the retail market has the lowest volatility. Therefore, volatility in the first-hand sale and wholesale markets is only minimally transmitted to consumers. Finally, asymmetric effects are observed in the price volatility transmission along the fresh anchovy value chain.

PROFIL ASAM AMINO DAN KANDUNGAN MINERAL IKAN TERI (Stolephorus commersonii) SEGAR DAN KERING DARI DESA SIAHONI KABUPATEN BURU

Anchovy (Stolephorus commersonii) is a fish that is mostly produced in Siahoni Village, both in the form of fresh and dried anchovies which are traditionally dried by direct sun drying. This traditional drying method is unhygienic and easily contaminated, so people have to develop appropriate technology, namely closed sun drying method. The purpose of this study is to examine the amino acid profile and mineral content (Ca, P, Fe, and I) of fresh and dried anchovy (Stolephorus commersonii) from Siahoni Village, Buru Regency. The method used in this study is experimental method and laboratory analysis which includes proximate analysis, amino acid analysis, and mineral analysis (Ca, P, Fe, and I). The Predicted Protein Efficiency Ratio (P-PER) was used for protein quality analysis. The results of this study indicate that based on the proximate value, there is an increase in the protein content of dried anchovy, where the protein content of dried anchovy which is dried in closed sun drying is higher than the protein content of dried anchovy which is traditionally dried. The highest essential amino acid content in fresh anchovy and traditional dried anchovy is lysine, and in sun dried anchovy is arginine. While the highest non-essential amino acid content in fresh anchovies, traditional dried anchovies and solar drying is glutamic acid. The estimated value of the protein efficiency ratio (P-PER) indicates that the protein quality of fresh anchovy is relatively poor, while traditional dried anchovy and solar drying have good protein quality. Fresh anchovies and dried anchovies also contain high and good minerals calcium (Ca), phosphorus (P), iron (Fe), and iodine (I).

Effect of organic acids on the formation of biogenic amines in fermented anchovy sauce comprising raw anchovy materials with different levels of freshness.

We investigated the effect of different levels of organic acids on the formation of biogenic amines in anchovy fish sauce. Fish sauce samples were prepared with fresh anchovies used immediately after being caught (F), and anchovies left at ambient temperature for 4h (4h), 18h (18h), or 24h (24h). Anchovies from each of the four groups were mixed with salt at a 4:1 ratio and then fermented at ambient temperature for varying periods of time. The F and 4h anchovies contained higher levels of acetic acid, succinic acid, and lactic acid, and the levels increased during the fermentation process. The histamine content of the fish sauce samples prepared using F anchovies ranged from 44.0 to 9.2mg/kg at 1 and 24months of fermentation. The histamine content of fish sauce samples prepared using 4h, 18h, and 24h anchovies was 111.5-37.0mg/kg (4h), 780.1-560.3mg/kg (18h), and 880.6-675.7mg/kg (24h). Our findings indicated that the histamine and other biogenic amine levels were closely associated with the degree of freshness of the raw anchovy material. These findings indicate that lower pH levels resulting from organic acids generated by the fresh raw material can effectively inhibit histamine formation.

A 5-Years (2015–2019) Control Activity of an EU Laboratory: Contamination of Histamine in Fish Products and Exposure Assessment

Histamine contamination was evaluated on 474 batches (3130 determinations) of fish products collected in Puglia and Basilicata (southern part of Italy) during the years 2015–2019, using a high-throughput two-tier approach involving a screening (ELISA test) and confirmatory method (HPLC/FLD with o-phthalaldehyde derivatization). Histamine concentration >2.5 mg kg−1 was detected in 51% of total batches with the 2.5% of non-compliance. Except for two samples of fresh anchovies, all non-compliant samples were frozen, defrosted and canned tuna. Among 111 fresh tuna batches, 9 had a content of histamine between 393 and 5542 mg kg−1, and scombroid poisoning cases were observed after their consumption. Good quality canned tuna and ripened anchovies sold in Italy was observed. Furthermore, the analysis of the processing technology and storage practice critical points were reported in this study, with useful considerations to minimize the histamine risk for consumers. Finally, based on these results, several considerations about risk exposure were reported.

Monitoring the characteristics of cultivable halophilic microbial community during salted-ripened anchovy (Engraulis anchoita) production

The halophilic microbial community of the salted-ripened anchovy process was studied. Samples from raw materials (salt and fresh anchovies) and from the stages of brining and ripening were collected and analyzed for their bacterial counts at 15 and 20% NaCl. No halophilic colonies were found in fresh anchovy and counts of about 103 CFU/g were determined in salt samples. A fluctuation of bacterial counts during the process was found. At the end of brining, ~104 CFU/g were determined in anchovy samples and this value was reduced to not detectable counts at the beginning of the ripening stage. After one month, counts increased to ~104 CFU/g and remained stable until the end of the process.From each sample, colonies having different morphotypes were isolated and submitted to a macro and microscopic characterization, a study of salt requirement for growth, and biochemical and phenotypic tests. The results were submitted to Univariate, Bivariate and Multiple Correspondence Factorial Analysis (MCFA). A total of 79 colonies were isolated during the salting-ripening anchovy process. Among the isolates, about 40–50% was positive for indole production and lipolytic activity and a 25% showed ability to produce H2S and proteolytic capacity. Proteolytic and lipolytic activities were well balanced along the process and resulted independent from the isolation stage, which is a desirable condition due to the contribution of microbial proteolysis and lipolysis to the development of texture and final aroma, respectively. H2S and indole producers practically were not detected during ripening. This fact is important because indole and H2S are associated with the development of off-flavors and spoilage in salted fish products. MFCA and Cluster Analyses complemented the Bivariate Analyses. The factor map showed proximity between the isolates from salt samples and from ripening. Isolates were statistically clustered in two groups. Cluster 1 grouped non-desirable activities (H2S and indole production) with cultures proceeding from brining whereas Cluster 2 related isolates mainly from salt samples and during ripening with some desirable microbial capacities (Cytochrome oxidase activity and non-H2S and non-indole production). These results would indicate that during the ripening process of salted anchovies, a natural selection of beneficial microorganisms for the development of the typical product sensory attributes occurred.

Anisakis spp. larvae in different kinds of ready to eat products made of anchovies (Engraulis encrasicolus) sold in Italian supermarkets

In this study the occurrence of visible anisakid larvae in semi-preserved anchovy products sold on the Italian market was investigated. Totally, 107 ready to eat products (33 salted-ripened, 49 in oil and 25 marinated) were sampled. Each sample was digested, then the digested material was observed under natural and UV light. Parasites were counted, collected and microscopically identified to genus level. A representative subset was molecularly identified using the cox2 gene. At least one visible Anisakis sp. larva was found in 54.2% of the total 107 products analysed and totally 1283 dead larvae were collected. Anisakis sp. larvae were found in all the 33 salted products and 1139 (88.8%) larvae were collected, with a range of 1–105 parasites per product. Larval density per gram was 0.13. Anisakis sp. larvae were found in 49.0% of the products in oil and 143 (11.1%) larvae were isolated, with a range of 0–28 and a density of 0.03. Only 1 larva was found in the 25 marinated products (4.0%, density 0.00). A highly significant difference between all the product categories in respect of number of larvae per product, frequency of products contaminated by at least one larva and larval density per gram was found. Within the subset of larvae molecularly analysed (n=122), 92 (75.4%) were identified as A. pegreffii and 30 (24.6%) as A. simplex. This study showed that semi-preserved anchovy products heavily contaminated with Anisakis spp. larvae reach the market. Beyond the negligible risk for anisakidosis, the presence of dead visible parasites may cause immediate rejection in consumers. In addition, the potential risk related to allergic reactions in sensitized individuals needs to be further assessed. In order to avoid commercialization of obviously contaminated products, fresh anchovies' batches intended for the production of such products should be accurately selected by the processing industry applying inspection methods.

The Effectiveness of Fresh Anchovies Diet and Calcitriol Supplementation to Prevent Osteoporosis in Ovariectomized Rats

This study was conducted to assess the effectiveness of a diet contained fresh anchovies and calcitriol supplementation for 6 weeks to prevent osteoporosis in ovariectomized rats. Fifteen Wistar rats aged 8 weeks were divided randomly into 3 groups (normal/K, ovariectomized/Ov, andovariectomized + calcitriol/OVD), 5 mice each. Group K and Ov rats were fed with fresh anchovies, while the OVD group was fed with freshanchovies + calcitriol. At the age of 15 weeks, all mice were done for euthanasia, then left femur was collected for immunohistochemistryexamination of tartrate resistant acid phosphatase5b (TRAP5b). The detection of (TRAP5b) was conducted using a monoclonal ant ibody antiTRAP5b, and detected using a streptavidin-biotin. The results showed that the metaphysis part of distal femur bone of mice group K, Ov, andOVD were positive TRAP5b stained with brown color on trabecular bone in bone marrow cavity and trabecular spiculum surface, but rats ingroup K and Ov had extensive bone marrow cavity and normal trabecular spiculum, whereas OVD group showed bone marrow cavity dilation,accumulation of adipocytes in the bone marrow cavity, and shorter the spiculum of trabeculae. It can be concluded that fresh anchovies diet andcalcitriol supplementation for 6 weeks are not effective in preventing osteoporosis in ovariectomized rats.Key words: calcitriol, femur, ovariohisterectomy

Assessment of a Sampling Plan Based on Visual Inspection for the Detection of Anisakid Larvae in Fresh Anchovies (Engraulis encrasicolus). A First Step Towards Official Validation?

The presence of anisakid larvae in fish is a public health issue, and effective risk management procedures are needed to avoid that heavily infected products reach the market. Currently, an official sampling plan for fresh fish defining sample size, inspection methods, and criteria to accept or reject the merchandise is lacking at the European and Italian level. In this study, we compared the visual inspection proposed by the sampling plan of the Lombardy Region (Italy) to the UV press method and to an optimized digestion procedure with the aim to assess its ability in detecting visible parasites. Thirty-one batches of Engraulis encrasicolus, each composed of ∼30 specimens, were collected and subsequently analyzed with the three techniques. The mean abundance (MA) was calculated after each procedure and compared on the basis of a threshold value. The results showed that the visual inspection performed similarly to the digestion method, with a sensitivity of 93 %, a specificity of 100 %, and an accuracy of 97 %. Overall, the comparison showed that, in the proposed sampling plan, the visual inspection is effective in rejecting unmarketable anchovies and in preventing the commercialization of unsafe products. This method is simple, less demanding than digestion in terms of time and equipment, and thus suitable as a standardized procedure to be routinely applied by food business operators. The hazard characterization, performed by sequencing the mtDNA cox2 gene, has identified the visible larvae as Anisakis pegreffii in 98 % of the cases, highlighting the zoonotic potential of the parasites found and the need for preventive measures.

ELECTRONIC NOSE FOR ANCHOVY FRESHNESS MONITORING BASED ON SENSOR ARRAY AND PATTERN RECOGNITION METHODS: PRINCIPAL COMPONENTS ANALYSIS, LINEAR DISCRIMINANT ANALYSIS AND SUPPORT VECTOR MACHINE

An electronic nose based system, which employs an array of six inexpensive commercial gas sensors based on tin dioxide (Figaro Engineering Inc., Japan), has been used to analyse the freshness states of anchovies. Fresh anchovies were stored in a refrigerator at 4 ± 1°C over a period of 15 days. Electronic nose measurements need no sample preparation and the results indicated that the spoilage process of anchovies could be followed by using this technique. Conductance responses of volatile compounds produced during storage of anchovy were monitored and the result were analysed by multivariate analysis methods. In this paper principal component analysis (PCA) and linear discriminant analysis (LDA) were used to investigate whether the electronic nose was able to distinguishing among different freshness states (fresh, moderated and non-fresh samples). The loadings analysis was used to identify the sensors responsible for discrimination in the current pattern file. Therefore, the support vector machines (SVM) method was applied to the new subset, with only the selected sensors, to confirm that a subset of a few sensors can be chosen to explain all the variance. The results obtained prove that the electronic nose can discriminate successfully different freshness state using LDA analysis. Some sensors have the highest influence in the current pattern file for electronic nose. Support vector machine (SVM) model, applied to the new subset of sensors show the good performance.

Shelf Life of Anchovy Products (Engraulis Encrasicolus): Evaluation of Sensory, Microbiological and Chemical Properties.

Fishery products have always been an important food in Italy. In the past, increased consumption was mainly due to the good quality of the products, easiness of use and their beneficial effects on health. Recently, owing to the national financial crisis, there has been a decline in the consumption of fish. In fact, in 2013, according to data from ISMEA, the consumption of fresh fish suffered a sharp contraction (-5%). This decline also concerns anchovy (Engraulis encrasicolus). This species, partly because of its low price, is a mainstay of traditional Italian food. The aim of this study was to evaluate sensorial, chemical and microbiological properties of anchovy-based (Engraulis encrasicolus) products during storage at 4 and -20°C. Fresh anchovies, obtained from the wholesale fish market of Pozzuoli (Southern Italy) were cut into small pieces and hand-prepared using bread, eggs, cheese and lemon juice. Samples were analysed after 0, 2, 4, 6 and 8 days of storage at 4°C. An aliquot was quickly frozen and analysed after 34 days at -20°C. Sensory assessment, microbiological (specific spoilage organisms, Listeria spp. and Salmonella spp.) and chemical (histamine, total volatile basic nitrogen, trimethylamine, thiobarbituric acid, pH and aw) analyses were performed. Results showed that the shelf life of anchovy products was less than 5 days for the samples stored at 4°C. At -20°C, all anchovies preparations showed good sensory, microbiological and chemical properties for 34 days.

Identification, technological and safety characterization of Lactobacillus sakei and Lactobacillus curvatus isolated from Argentinean anchovies (Engraulis anchoita)

In this study, the identification and characterization of Lactobacillus previously isolated from fresh anchovies (Engraulis anchoita) are investigated. 16S rDNA partial sequencing assigned all the isolates to belong to the Lactobacillus sakei/curvatus group. Fourteen out of 15 isolates were identified as L. sakei by phenotypic traits: they exhibited catalase activity and fermented melibiose, although only 10 of them hydrolyzed arginine. These results were confirmed by multiplex PCR-based restriction enzyme analysis with HindIII and by restriction fragment length polymorphic (RFLP) analysis of the 16S-23S rDNA intergenic spacer region with TaqI. Among identified isolates, four L. sakei strains and the sole L. curvatus strain showing sensitivity to chloramphenicol, erythromycin and tetracycline and exhibiting high tolerance to NaCl (10-18%) were unable to produce neither dextran nor biogenic amines. Based on technological and safety features, L. sakei SACB704 and L. curvatus SACB03a naturally present in fresh anchovies may be promising strains for the development of a starter culture to accelerate and control the fermentation of salt fermented anchovy-based products.

Characterization of halophilic Archaea isolated from different hypersaline ecosystems

Halobacterial representatives were isolated from salted fish, naturally occurring salt pans as well as artificial saline poolsin the Mediterranean area. The isolation techniques experimented proved successful, allowing halophilic archaea to be retrieved iralmost 72% of analysed samples. About 65% of strains could be presumptively ascribed to the speciellaloarcula marismortui by RFLP (Restriction Fragment Length Polymorphism) grouping and 16S rRNA gene sequence comparison. Nevertheless, cluster analysis of RAPD-PCR patterns revealed a wide heterogeneity among isolated strains. Biochemical features of technological interest, such as proteclic, lipolytic and decarboxylase activity, were investigated in order to elucidate the role of archaea during ripening of salted anchovies. Proteolytic activity was only evidenced on the sarcoplasmic fraction extracted from fresh anchovies and for only 4 strains out of 2 No strain revealed either lipolytic capability on cod liver oil or proteolytic activity on fresh anchovies myofibrillar extract. Finally, only one strain exhibited decarboxylase activity, minimizing the potential responsibility of cultivable archaea microflora in the spoilage of salted anchovies.

Sensory analysis to assess the freshness of Mediterranean anchovies ( Engraulis encrasicholus) stored in ice

A quality index method and a sensory scheme were developed to assess raw and cooked Mediterranean anchovies ( Engraulis encrasicholus), respectively. Fresh anchovies were stored in ice for 7 days in three trials. The changes in sensory punctuations showed satisfactory correlations with time of storage. Enterobacteria were the first microorganisms that exceeded the maximum allowable limit for marketable fish according to the Spanish regulations, reaching more than 10 3 colony former units/g after 5 days of storage. Receiver operating characteristic curves of the sensory scores of raw and cooked anchovy against Enterobacteria counts were used to corroborate the suitability of the schemes. Areas under the curves were satisfactory and the estimated rejection values ranged from 0.43 to 0.51 and from 6.6 to 7.1 for raw and cooked anchovies, respectively.

A New Procedure for Marinating Fresh Anchovies and Ensuring the Rapid Destruction of Anisakis Larvae

The consumption of marinated anchovies is the main route of transmission of anisakiasis in Spain. Because this country is one of the world's major tourist destinations, this traditional food also poses a potential health risk to millions of foreign visitors. Anisakis larvae are not destroyed by the traditional marinating procedure, and alternative methods, such as long-term storage in brine, freezing, or hydrostatic pressure treatment, all present major difficulties. In this study, we used high food-grade acetic acid concentrations (10, 20, 30, and 40% [vol/vol] in line with the quantum satis rule) to destroy these larvae rapidly, and we report data on the survival of Anisakis larvae exposed directly to different marinades and when the larvae are placed under the fish musculature. The percentage of salt and acetic acid in the fish tissue water phase was also determined. A marinating procedure is proposed that ensures the rapid death of Anisakis through the use of strong acetic acid concentrations. Posttreatment washes with water reduce these to levels acceptable to consumers. The sensory characteristics of the product were shown to be satisfactory. The actual selection of an acetic acid concentration for marinating depends on costs and the processing time available. The physiological stress of the larvae exposed to the different marinades was determined by measuring the levels of their stress proteins. The latter are good indicators of injury and might reflect the infectivity of larvae. In addition, we also used a rat model to determine the infectivity of larvae considered microscopically dead.

Effect of delayed gutting on biogenic amine contents during ripening of European anchovies

The effect of delayed gutting on biogenic amine formation during ripening of European anchovies (Engraulis encrasicholus) was studied. The main difference between the two processes was the use of gutted or ungutted fish in the first step of ripening. Biogenic amines and microbial counts were studied in seven batches of samples taken during manufacturing from fresh anchovies and from anchovies throughout ripening. The formation of amines commonly related to fish spoilage (histamine, tyramine, cadaverine, putrescine and agmatine) was significantly higher in the first step of ripening of batches in ungutted fish. In contrast, no differences were observed between the content of the natural polyamines spermine and spermidine.

Liquid Chromatographic Method for Determination of Biogenic Amines in Fish and Fish Products

A liquid chromatographic method with postcolumn derivatization is described for determination of biogenic amines in fish and fish products. Histamine, tyramine, serotonin, beta-phenylethylamine, tryptamine, putrescine, cadaverine, agmatine, spermine, and spermidine can be determined in less than 60 min. Routine sample preinjection treatment implies only 2 extractions with 0.6N perchloric acid and filtration through a 0.45 micron filter. Lack of interferences from volatile amines, amino acids, and dipeptides was verified. Results of reliability study were satisfactory. The proposed method was linear for each amine between 0.25 and 8.00 mg/L. Average recoveries ranged from 92 to 103%. Precisions (coefficients of variation) ranged from 0.70 to 9.75%. Determination limits were < or = 1 mg/kg. A modification in LC conditions was necessary to apply the method to ripened fish products to avoid interferences from food matrixes. In addition, stability of biogenic amines in fresh anchovies and hake during short-term frozen storage was studied. Results showed that, except for agmatine, frozen storage is suitable for keeping samples before analysis.