Fish processing industry residues: a review of valuable products extraction and characterization methods

Fish processing industry has experienced significant growth, playing an important role in the world economy. The increased exploration of marine resources contributes to the generation of considerable amounts of biowaste, which ends up as discards. In the face of the resultant disposal and environmental problems, many efforts have been made to deal with the fishery waste in more efficient ways. Nowadays, these by-products are regarded as important sources of high added value compounds, such as hydroxyapatite, collagen, gelatin, lipids, enzymes, hydrolysates and bioactive peptides, with great potential for human health applications. The present paper aims to review the current methods of extraction and characterization of added value products from fish by-products, as well as their actual and potential applications.This work was supported by FCT-Fundação para a Ciência e a Tecnologia (projectPEst-OE/QUI/UI0674/2019, CQM, Portuguese Government funds), and through Madeira 14–20 Program, project PROEQUIPRAM—Reforço do Investimento em Equipamentos e Infraestruturas Científicas na RAM (M1420-01-0145-FEDER-000008) and by ARDITI-Agência Regional para o Desenvolvimento da Investigação Tecnologia e Inovação, through the project M1420-01-0145- FEDER-000005—Centro de Química da Madeira—CQM+ (Madeira 14-20). The work was also performed in the frame of project MarineBlueRefine PROCiência2020 (Portaria nº 371/2015, de 16/12), M1420- 01-0247-FEDER-000006; Pedro Ideia is the recipient of a PhD Grant under the project M1420-09-5369-FSE-000001.info:eu-repo/semantics/publishedVersio

ANTIOXIDANT ACTIVITY OF PROTEIN HYDROLYSATE PRODUCED FROM TUNA EYE (Thunnus sp.) BY ENZYMATIC HYDROLYSIS

Tuna (Thunnus sp.) by-products from frozen loin and canning industry especially the eye is rich in proteins and in lipids consisting of polyunsaturated fatty acids (PUFA). That requires protective agent (antioxidant) to inhibit the oxidation naturally present and predicted to be protein peptides. Enzymatic hydrolysis of protein is an appropriate method to produce bioactive peptide with such nutraceutical/pharmaceutical function such as an antioxidant peptide. This study aimed to produce protein hydrolysate having a function as anwith an antioxidant activity from eye of tuna through enzymatic hydrolysis and determining the antioxidant activity by DPPH methods. Protein soluble content of tuna’s eye protein hydrolysate (TEPH) ranged from 59.98±0.130 to 94.90±0.002%. The degree of hydrolysis (DH) of TEPH was about 9.10±0.28 to 16.14±0.09%. The highest inhibition of DPPH radical scavenging activity was 93.57±0.05% (at 5 mg/mL) was obtained with a DH of 11.35±0.002% at the concentration 0.1% of papain for 6 hours hydrolysis. The IC50 value of was 1.08±0.008 mg/m

Functional and Bioactive Components from Mackerel (Scomber scombrus) and Blue Whiting (Micromesistius poutassou) Processing Waste

The use of fish processing waste from an oily fish model (mackerel, Scomberscombrus) and a white fish model (blue whiting, Micromesistius poutassou) as a source of functional and bioactive compounds was investigated.Gelatines were extracted from fish heads and skins using pre-treatment withdifferent organic acids (acetic, citric, lactic, tartaric and malic acids). Gelatines from fish bones were extracted after pre-treating the bones either chemically or enzymatically. The type of pre-treatment affected the yield, colour, turbidity, amino acid profiles as well as functional properties of fish gelatines. Mackerel gelatines formed stronger and more stable gels than blue whiting. Acetic acid pre-treatment of mackerel and blue whiting heads and skins produced stronger gelatine gels. The enzymatic pre-treatment of fish bones resulted in gelatines with poor rheological properties.Hydrolysates obtained from the hydrolysis of mackerel head and skingelatines with pepsin exhibited the highest antioxidant activity (DPPH ~ 80 %). These hydrolysates had high anti-inflammatory activity (inhibition of SSAO activity by 50 %) and antihypertensive activity (inhibition of ACE activity by 75 %). Skin gelatine hydrolysates, were also able to inhibit platelet aggregation.The hydrolysis of mackerel viscera with Flavourzyme produced hydrolysateswith high antioxidant activity (73 %) and inhibited the SSAO activity by 45.8 % and ACE by 60 %.Different procedures (chemical, physical or enzymatic) were developed to extract oil rich in ω-3 fatty acids from mackerel heads and skins. The yield varied significantly (p \u3c 0.05) depending on the extraction method. The physicochemical properties of the majority of oils extracted were acceptable according to the Codex Alimentarius criteria. Polar oil fractions extracted from mackerel heads after enzymatic hydrolysis with Alcalase had high DPPH radical scavenging activity which was due to the presence of carotenoids

Enzymatic fish protein hydrolysates in finfish aquaculture: A review

In intensive farming systems, fish are held at high densities, which may increase stress, leading to susceptibility to diseases that result in economic losses. Therefore, effective feeding practices incorporating health‐promoting compounds such as proteins, hydrolysates and bioactive peptides that can stimulate the defence mechanisms of fish and achieve better growth are some of the priorities for sustainable aquaculture development. Globally, the fish processing industries generate and discard a large volume of waste every year, estimated at up to 60% of the harvested biomass. This waste can be converted to value‐added products such as fish protein hydrolysate (FPH) with the addition of various proteolytic enzymes. FPH from fish processing waste including skin, heads, muscle, viscera, liver and bones is a good source of protein, amino acids, peptides and antioxidants and has been found to possess desirable functional and bioactive peptides. A moderate inclusion of FPH in aquafeeds has the potential to improve growth, feed utilization, immune functions and disease resistance of fish. Production of FPH, targeted to more precise molecular weight ranges, has superior functionalities that are in high demand. With interest in FPH as an aquafeed supplement, this review aimed to summarize the source, production processes and functional properties of FPH and the reported impact of FPH in aquafeed supplement on fish growth, survival, feed utilization, immune response and disease resistance. Possible limitations of using FPH and future research potential as an opportunity for the use of processing fish waste are also discussed

Combined Neutrase-Alcalase Protein Hydrolysates from Hazelnut Meal, a Potential Functional Food Ingredient

Consumers\u27 interest in functional foods has significantly increased in the past few years. Hazelnut meal, the main valuable byproduct of the hazelnut oil industry, is a rich source of proteins and bioactive peptides and thus has great potential to become a valuable functional ingredient. In this study, hazelnut protein hydrolysates obtained by a single or combined hydrolysis by Alcalase and Neutrase were mainly characterized for their physicochemical properties (SDS-PAGE, particle size distribution, Fourier-transform infrared (FTIR) spectroscopy, molecular weight distribution, etc.) and potential antiobesity effect (Free fatty acid (FFA) release inhibition), antioxidant activity (DPPH and ABTS methods), and emulsifying properties. The impact of a microfluidization pretreatment was also investigated. The combination of Alcalase with Neutrase permitted the highest degree of hydrolysis (DH; 15.57 \ub1 0.0%) of hazelnut protein isolate, which resulted in hydrolysates with the highest amount of low-molecular-weight peptides, as indicated by size exclusion chromatography (SEC) and SDS-PAGE. There was a positive correlation between the DH and the inhibition of FFA release by pancreatic lipase (PL), with a significant positive effect of microfluidization when followed by Alcalase hydrolysis. Microfluidization enhanced the emulsifying activity index (EAI) of protein isolates and hydrolysates. Low hydrolysis by Neutrase had the best effect on the EAI (84.32 \ub1 1.43 (NH) and 88.04 \ub1 2.22 m2/g (MFNH)), while a negative correlation between the emulsifying stability index (ESI) and the DH was observed. Again, the combined Alcalase-Neutrase hydrolysates displayed the highest radical scavenging activities (96.63 \ub1 1.06% DPPH and 98.31 \ub1 0.46% ABTS). FTIR results showed that the application of microfluidization caused the unfolding of the protein structure. The individual or combined application of the Alcalase and Neutrase enzymes caused a switch from the β-sheet organization of the proteins to α-helix structures. In conclusion, hazelnut meal may be a good source of bioactive and functional peptides. The control of its enzymatic hydrolysis, together with an appropriate pretreatment such as microfluidization, may be crucial to achieve the best suitable activity

AKTIVITAS ANTIOKSIDAN HIDROLISAT PROTEIN MIOFIBRIL BELUT (Synbranchus bengalensis) YANG DIHIDROLISIS DENGAN ENZIM PAPAIN

Protein hydrolysates contain bioactive peptides which have several function in order to maintain health and reduce the risk of disease. One of the functions of bioactive peptides is as an antioxidant. This study aims to characterize eel myofibril (Synbranchus bengalensis), determine the effect of papain enzyme ratio and hydrolysis time on eel myofibril protein hydrolysates, determine the antioxidant activity of eel myofibril hydrolysates by using DPPH and reducing power assay, and characterize the hydrolysate of myofibril proteins which have the best antioxidant activity. The results showed that eel myofibril protein contained moisture, fat, protein and ash of  85.91, 0.15, 0.14 and 12.78% respectively and contained the highest glutamic acid and lysine. Hydrolysates of eel myofibril proteins treated using various enzyme ratio and hydrolysis time had hydrolysates yields ranging from 3.53-9.68%, the value of hydrolysis degrees 5.20 - 16.193%, IC50 14.24 - 30.26 mg/mL for DPPH assay and absorbance 0.125 - 0.190 at a concentration of 5 mg/mL for reducing power assay. Hydrolysis eel myofibril protein using ratio papain 0.20:100 for 2 hours produced the highest antioxidant activities measuring by DPPH scavenging and reducing power iron. The hydrolysate had molecular weight approximately 19.51 kDa. Glutamic acid and lysine became the dominant amino acids of the hydrolysate. Keywords: antioxidant, hydrolysates, myofibril, Synbranchus bengalensis, papai

Enzymatic protein hydrolysis of residual raw material from Atlantic cod: Selectivity of proteases, outcome and bioactivities

Global trends show that interest in fish products and fish consumption are increasing, while marine fishery resources are decreasing. Fish processing industry produces a high amount of residual raw materials that have nutritious proteins and other valuable compounds. An optimization of residual raw materials’ utilization can help meet the growing demand for fish products and help reduce environmental problems. A promising valorization method is enzymatic protein hydrolysis. In this project, enzymatic protein hydrolysis was performed to produce protein hydrolysates from complex material of Atlantic cod heads. Three types of material from cod heads (muscle, skin and bone) were hydrolyzed by 23 different proteases. The produced hydrolysates were analyzed and evaluated based on yield, molecular weight (determined by SEC and SDS-PAGE), selectivity of proteases towards collagen and myofibrillar proteins (selectivity ratio), and bioactivity properties (anti-proliferative, antioxidant and anti-inflammatory). It was determined that the highest yield from muscle was produced by Tail 191, from skin by Tail 194, and from bone by Tail 190. Different proteases produced hydrolysates with different average MW. Notably, Tail 189 produced hydrolysates with the lowest average MW from all three types of raw material. The SDS-PAGE patterns of the hydrolysates from skin indicated that Tail 193 and Endocut 01 might have selectivity towards peptide bonds they cleave. The selectivity ratio identified that Endocut 01 had the highest selectivity towards myofibrillar proteins and Flavourzyme was the only enzyme selective to collagen. The results of bioactivity assays showed no antiproliferative or anti-inflammatory activity of the hydrolysates, however, all hydrolysates demonstrated antioxidant activity. The hydrolysates made from muscle showed higher antioxidant activity than the hydrolysates prepared from skin and bone. Based on the results, conditions for a scale-up experiment (from 5 g of raw material to 250 g) were suggested, which included recommendation of several enzymes per material, adjustment of temperature to optimal for each enzyme, monitoring of hydrolysis process and determination of ash content in the product

Innovative non-thermal technologies for recovery and valorization of value-added products from crustacean processing by-products—an opportunity for a circular economy approach

The crustacean processing industry has experienced significant growth over recent decades resulting in the production of a great number of by-products. Crustacean by-products contain several valuable components such as proteins, lipids, and carotenoids, especially astaxanthin and chitin. When isolated, these valuable compounds are characterized by bioactivities such as antimicrobial, antioxidant, and anti-cancer ones, and that could be used as nutraceutical ingredients or additives in the food, pharmaceutical, and cosmetic industries. Different innovative non-thermal technologies have appeared as promising, safe, and efficient tools to recover these valuable compounds. This review aims at providing a summary of the main compounds that can be extracted from crustacean by-products, and of the results obtained by applying the main innovative nonthermal processes for recovering such high-value products. Moreover, from the perspective of the circular economy approach, specific case studies on some current applications of the recovered compounds in the seafood industry are presented. The extraction of valuable components from crustacean by-products, combined with the development of novel technological strategies aimed at their recovery and purification, will allow for important results related to the long-term sustainability of the seafood industry to be obtained. Furthermore, the reuse of extracted components in seafood products is an interesting strategy to increase the value of the seafood sector overall. However, to date, there are limited industrial applications for this promising approach

Protease de Bacillus licheniformis : produção, purificação, caracterização e aplicação da enzima na hidrólise das proteínas de arroz e ervilha

Orientador: Hélia Harumi SatoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: Os efeitos da ampliação de escala e de diferentes variáveis de processo (temperatura e agitação) sobre a produção de proteases por Bacillus licheniformis LBA 46 foram avaliados utilizando meio de cultura composto por melaço de cana¿de¿açúcar (32 g/L), água de maceração de milho (6 g/L), extrato de levedura (2 g/L) e soro de leite (20 g/L), pH 7. Os resultados obtidos mostraram que o aumento da escala de fermentação de frascos Erlenmeyer contendo 100 mL de meio de cultura para reator de bancada contendo 3 L de meio resultou em aumento de 2,3 vezes na atividade enzimática, sendo obtido 2.448 U/mL de protease após 48 h de fermentação e que as condições mais adequadas de temperatura e agitação foram 30 ºC e 300 rpm, respectivamente. O extrato bruto de proteases foi submetido à precipitação com sulfato de amônio (80% de saturação), diálise e liofilização para posterior caracterização bioquímica. A preparação enzimática semi¿purificada apresentou atividade ótima na faixa de 60 e 65 °C em pH 7 e reteve 78%, 39% e 9% da atividade inicial após 120 min de tratamento a 50, 60 e 70 ºC, respectivamente. A protease foi purificada 3,33 vezes por precipitação com 80% de sulfato de amônio e cromatografia em coluna de DEAE¿Sepharose, apresentando uma massa molecular de 40 kDa estimada por eletroforese por SDS¿PAGE. A protease purificada apresentou atividade ótima na faixa de 50 e 60 °C. A enzima purificada apresentou alta atividade (> 80%) na faixa 6,5 a 9, tendo atividade ótima em pH 8,5; bem como mostrou¿se estável na faixa de pH 5 a 10 após 24 h de incubação a 4 ºC, retendo mais de 86% da atividade inicial. A enzima purificada também se mostrou estável após 1 h de incubação a 40 ºC e reteve 85% da atividade inicial após 1 h a 50 ºC. O tratamento da protease semi¿purificada (obtida por precipitação com 80% de sulfato de amônio) em pH 4, 7 e 9, com homogeneização a alta pressão (50 ¿ 200 MPa) não aumentou sua atividade e estabilidade em 40, 60 e 90 °C. Os hidrolisados de proteína de arroz preparados com a protease de B. licheniformis LBA 46 e protease comercial Alcalase 2.4L apresentaram atividade antioxidante superior para as respostas avaliadas pelos métodos de ORAC e FRAP em comparação com proteína de arroz não hidrolisada. Os hidrolisados de proteína de ervilha preparados com a protease de B. licheniformis LBA 46 e protease comercial Alcalase 2.4L apresentaram atividade antioxidante para todas as respostas avaliadas pelos métodos DPPH, ORAC e FRAP. A atividade antioxidante dos hidrolisados de proteína de ervilha medida em termos de DPPH e FRAP foi menor em comparação com a atividade antioxidante da amostra de proteína não hidrolisada. No entanto, a atividade antioxidante medida em termos do ORAC teve um aumento significativo de 7,33 e 9,27 vezes para os hidrolisados da protease LBA e para os hidrolisados da Alcalase 2.4L, respectivamente, comparada com a proteína não hidrolisada. As condições de hidrólise foram validadas e, na condição selecionada como mais adequada (pH 10 e 100 U/mL de protease), foi possível confirmar que os modelos definidos foram capazes de predizer os resultados alcançadosAbstract: The effects of scale amplification and different process variables (temperature and agitation) on the production of proteases by Bacillus licheniformis LBA 46 were evaluated using culture medium composed of sugar cane molasses (32 g/L), corn steep liquor (6 g/L), yeast extract (2 g/L) and dried whey (20 g/L), pH 7. The results obtained showed that the increase in the fermentation scale of Erlenmeyer flasks containing 100 mL of broth culture medium to a bench reactor containing 3 L of medium resulted in a 2.3¿fold increase in enzyme activity, obtaining 2,448 U/mL protease after 48 h of fermentation and the most suitable conditions of temperature and agitation were 30 ºC and 300 rpm, respectively. The crude protease extract was submitted to ammonium sulphate precipitation (80% of saturation), dialysis and lyophilization for subsequent biochemical characterization. The semi¿purified enzymatic preparation presented optimum activity in the 60 and 65 ºC range at pH 7 and retained 78%, 39% and 9% of the initial activity after 120 min of treatment at 50, 60 and 70 ºC, respectively. The protease was purified 3.33 times by precipitation with 80% ammonium sulfate and DEAE¿Sepharose column chromatography, presenting a molecular mass of 40 kDa estimated by SDS¿PAGE electrophoresis. The purified protease presented optimum activity in the range of 50 and 60 ºC. The purified enzyme presented high activity (> 80%) in the range of 6.5 to 9, and had optimal activity at pH 8.5; as well as showed to be stable in the pH range of 5 to 10 after 24 h of incubation at 4 ºC, retaining more than 86% of the initial activity. The purified enzyme also showed to be stable after 1 h of incubation at 40 ºC and retained 85% of the initial activity after 1 h at 50 ºC. Treatment of semi¿purified protease (obtained by precipitation with 80% ammonium sulphate) at pH 4, 7 and 9, with high pressure homogenization (50 ¿ 200 MPa) did not increase its activity and stability at 40, 60 and 90 ºC. Rice protein hydrolysates prepared with the B. licheniformis protease LBA 46 and commercial protease Alcalase 2.4L showed superior antioxidant activity for the responses evaluated by the ORAC and FRAP methods compared to unhydrolysed rice protein. Pea protein hydrolysates prepared with B. licheniformis protease LBA 46 and commercial protease Alcalase 2.4L showed antioxidant activity for all responses evaluated by the DPPH, ORAC and FRAP methods. The antioxidant activity of pea protein hydrolysates measured in terms of DPPH and FRAP was lower in comparison with the antioxidant activity of the non-hydrolyzed protein sample. However, the antioxidant activity measured in terms of the ORAC had a significant increase of 7.33 and 9.27 times for the LBA protease hydrolysates and for the Alcalase 2.4L hydrolysates, respectively, compared to the non-hydrolyzed protein.The hydrolysis conditions were validated and, in the condition selected as the most appropriate (pH 10 and 100 U/mL protease), it was possible to confirm that the defined models were predictiveDoutoradoCiência de AlimentosDoutora em Ciência de Alimento