Controlling hemoglobin-mediated lipid oxidation in herring (Clupea harengus) co-products via incubation or dipping in a recyclable antioxidant solution

Applying value-adding techniques to fish co-products is rendered difficult due to their high susceptibility to hemoglobin (Hb)-mediated lipid oxidation. In this study, we investigated a dipping technology with a solution containing Duralox MANC 213- a mixture of rosemary extract, ascorbic acid, tocopherols and citric acid – to control lipid oxidation during storage at 0 \ub0C and 20 \ub0C. The possibilities to re-use the antioxidant solution was also analyzed, along with studies on the link between Duralox MANC and Hb-form. Dipping in Duralox MANC largely increased the oxidation lag phase; from <0.5 to >3.5 d at 20 \ub0C, and from <1 d to >11 d at 0 \ub0C. Even after re-use of the solution up to 10 times, lipid oxidation was completely inhibited at 0 \ub0C. Duralox MANC could prevent auto-oxidation and hemin loss of herring Hb; which are suggested as the main mechanisms behind the observed stabilization of herring co-products against lipid oxidation

Effect of antioxidants on lipid oxidation in herring (Clupea harengus) co-product silage during its production, heat-treatment and storage

Provided high product quality, ensilaging can be used to valorize fish filleting co-products into a silage suitable for food applications. However, a documented challenge for products from hemoglobin-rich fish raw materials is the high susceptibility to lipid oxidation, calling for stabilization by antioxidants. In a comparison among different rosemary-containing antioxidants and isoascorbic acid, we here found that the commercial mixture Duralox MANC-213 (MANC) provided the best protection against peroxide value and 2-thiobarbituric acid reactive substances (TBARS) development during ensilaging of herring filleting co-products (0–7\ua0days, 22\ua0\ub0C), and also during subsequent heat-treatment (30\ua0min, 85\ua0\ub0C). Increasing MANC concentration from 0.25 and 0.75 to 1.25% lowered TBARS values from 43.53 and 25.12 to 18.04 \ub5mole TBARS/Kg silage, respectively, after 7\ua0days of ensilaging. During storage at 4\ua0\ub0C/22\ua0\ub0C in presence of MANC, 1.25% provided the highest protection with 87–90% and 66–73% lower TBARS, at 4\ua0\ub0C and 22\ua0\ub0C, respectively, at 6\ua0months compared to the controls. At this time point, heat-treated silages had lower protein degree of hydrolysis and free amino acids values than the non-heat-treated one. Regardless of antioxidant addition, total volatile basic nitrogen (TVB-N) formation still increased during the storage, but, overall, TVB-N values in silages were below the acceptable limit of 30\ua0mg TVB-N/100\ua0g fish for human consumption. Together with lipid oxidation data, this suggests that herring silage produced in presence of antioxidants can be used both for high-quality feed and food applications

Effect of antioxidants on lipid oxidation in herring (Clupea harengus) co-product silage during its production, heat-treatment and storage

Provided high product quality, ensilaging can be used to valorize fish filleting co-products into a silage suitable for food applications. However, a documented challenge for products from hemoglobin-rich fish raw materials is the high susceptibility to lipid oxidation, calling for stabilization by antioxidants. In a comparison among different rosemary-containing antioxidants and isoascorbic acid, we here found that the commercial mixture Duralox MANC-213 (MANC) provided the best protection against peroxide value and 2-thiobarbituric acid reactive substances (TBARS) development during ensilaging of herring filleting co-products (0-7 days, 22 degrees C), and also during subsequent heat-treatment (30 min, 85 degrees C). Increasing MANC concentration from 0.25 and 0.75 to 1.25% lowered TBARS values from 43.53 and 25.12 to 18.04 mu mole TBARS/Kg silage, respectively, after 7 days of ensilaging. During storage at 4 degrees C/22 degrees C in presence of MANC, 1.25% provided the highest protection with 87-90% and 66-73% lower TBARS, at 4 degrees C and 22 degrees C, respectively, at 6 months compared to the controls. At this time point, heat-treated silages had lower protein degree of hydrolysis and free amino acids values than the non-heat-treated one. Regardless of antioxidant addition, total volatile basic nitrogen (TVB-N) formation still increased during the storage, but, overall, TVB-N values in silages were below the acceptable limit of 30 mg TVB-N/100 g fish for human consumption. Together with lipid oxidation data, this suggest that herring silage produced in presence of antioxidants can be used both for high quality feed and food applications

Understanding the effect of temperature and time on protein degree of hydrolysis and lipid oxidation during ensilaging of herring (Clupea harengus) filleting co-products

The aims of this study were to investigate the effect of temperature, time and stirring on changes in protein degree of hydrolysis (DH), free amino acids (FAA), lipid oxidation and total volatile basic nitrogen (TVB-N) during ensilaging of herring (Clupea harengus) filleting co-products. Results showed that temperature and time, and in some cases the interaction effect between these two factors, significantly influenced all the studied responses. Increasing ensilaging temperature and time from 17 to 37 \ub0C and 3 to 7 days, respectively, increased DH, FAA, and TVB-N content from 44.41 to 77.28%, 25.31 to 51.04 mg/g, and 4.73 to 26.25 mg/100 g, respectively. The lipid oxidation marker 2-thiobarbituric acid reactive substances (TBARS) did not increase with time at temperatures above 22 \ub0C, while 2-pentylfuran increased up to 37 \ub0C. Based on the process parameters and responses investigated in this study, and considering energy requirements, it was suggested to perform ensilaging at ambient temperatures (i.e. around 20 \ub0C) with continuous stirring at 10 rpm for 1-3 days; the exact length being determined by the desired DH

Hemoglobin-mediated lipid oxidation of herring filleting co-products during ensilaging and its inhibition by pre-incubation in antioxidant solutions

The aims of this study were to investigate the role of hemoglobin (Hb) in lipid oxidation development during ensilaging of herring filleting co-products, and, to inhibit this reaction by pre-incubating the co-products in water or physiological salt, with/without different antioxidants. Results showed that both peroxide value (PV) and 2-thiobarbituric acid reactive substances (TBARS) gradually increased during 7\ua0days of ensilaging at 22\ua0\ub0C in absence of antioxidants. The increase in TBARS was proportional to the Hb levels present, while PV was less affected. A Hb-fortified Tris-buffer model system adjusted to pH 3.50 confirmed that Hb changed immediately from its native oxyHb to the metHb state, which facilitated heme group release and thus probably explains the increased PV and TBARS during ensilaging. Pre-incubating the co-products for 30\ua0s in a solution containing 0.5% rosemary extract was the most promising strategy to inhibit lipid oxidation both in the co-products during pre-processing storage and during the actual ensilaging. The solution could be re-used up to ten times without losing its activity, illustrating that this methodology can be a scalable and cost-effective strategy to extend the oxidative stability of herring co-products allowing for further value adding e.g., into a high-quality silage

A recyclable dipping strategy to stabilize herring (Clupea harengus) co-products during ice storage

Applying value-adding techniques to fish filleting co-products is rendered difficult due to their high susceptibility to lipid oxidation, microbial spoilage, and amine formation. In this study, a recyclable dipping strategy was developed and investigated for its ability to stabilize herring (Clupea harengus) co-products (head, backbone, caudal fin, intestines, belly flap, skin, and in some cases roe) against oxidation and microbial spoilage. From initial screening of seven antioxidative components/formulas in minced herring co-products during ice storage, an oil-soluble rosemary extract (RE-B) and isoascorbic acid (IAA) were identified as most promising candidates. These compounds were then formulated to a recyclable solution to be used for dipping of the herring co-products. The commercial Duralox MANC antioxidant mixture was used as a positive control. Dipping in 0.2% RE-B solution \ub1 0.5% IAA or in 2% Duralox MANC solutions remarkably increased the oxidation lag phase from 12\ua0days during subsequent storage on ice (0–1\ua0\ub0C) of minced or intact co-products, respectively, even when the antioxidant solutions were re-used up to 10 times. The dipping also reduced microbiological growth and total volatile basic nitrogen, but the effect became weaker with an increased number of re-using cycles. The presented dipping strategies could hereby facilitate more diversified end use of herring co-products from current fish meal to high-quality minces, protein isolates, or oils for the food industry

Pilot-Scale Ensilaging of Herring Filleting Co-Products and Subsequent Separation of Fish Oil and Protein Hydrolysates

In this study, ensilaging of herring (Clupea harengus) flleting co-products was taken from lab-scale to pilot scale (1500 L) while monitoring the protein degree of hydrolysis (DH) and lipid oxidation. Subsequently, the possibility of recovering fish oil and protein hydrolysates using batch centrifugation at diferent g-forces/times was investigated. Around 38% DH was recorded after 2-day pilot-scale ensilaging of herring co-products at ambient temperature (i.e., ~22 \ub0C), which was similar to the DH found in lab-scale (40% after 2 days; 22 \ub0C). The lipid oxidation marker 2-thiobarbituric acid reactive substances (TBARS) reached 20 \ub5mole TBARS/kg silage after 2-day ensilaging. Centrifugation of the silage at 3000–8500 7g for 2–20 min revealed successful separation into fsh oil and protein hydrolysates. Heat-treating the silage (85 \ub0C; 30 min) prior to centrifugation resulted in signifcantly higher oil and hydrolysates recoveries; the same being true for increased g-force.At 8500 7g, the recovery of oil and hydrolysates were 9.7 and 53.0% w/w, respectively, from heat-treated silage, while recoveries were 4.1 and 48.1% w/w, respectively, from non-heat treated silage. At 4500 7g, being a more scalable approach, corresponding numbers were 8.2 and 47.1% (w/w) as well as 2.0 and 40.2% (w/w). The recovered fsh oil contained 8% EPA and 11% DHA of total fatty acids. Free fatty acids (FFA), peroxide value (PV), p-anisidine value (p-AV), and total oxidation (TOTOX) values of oils were in the range of 4–7% (FFA), 3.6–3.7 meq/kg oil (PV), 2.5–4.0 (p-AV), and 9.9–11.1 (TOTOX), respectively, which were within the acceptable limits for human consumption specifed by the GOED voluntary monograph.The recovered protein hydrolysates contained peptides in the molecular weight range 0.3–6 kDa (~ 37%) and 11–34 kDa (~ 63%). Also, the remaining solids contained 15–17% (w/w) protein, having 44–45% essential amino acids. Overall, the results suggest that herring co-product silage is a valuable source of fsh oil and protein hydrolysates, paving the way for ensilaging based-biorefning of herring co-products into multiple products

Combined effects of isolation temperature and pH on functionality and beany flavor of pea protein isolates for meat analogue applications

The combined effects of isolation temperature (20, 30 and 40\ua0\ub0C) and pH (2.0–12.0) on yield, techno-functional properties, and beany flavor of pea protein isolates were investigated. Increasing pH from 2.0 to 9.5 and 11.0 increased yields from 37\ua0% to 75\ua0% and 79\ua0%, respectively, at 20\ua0\ub0C. At a constant pH, increasing temperature from 20 to 40\ua0\ub0C did not increase protein recovery; rather, negatively affected the techno-functional properties such as protein solubility, foaming and gelation. Protein isolated at pH 11.0 (20\ua0\ub0C) provided a higher fat absorption, gelation capacity, gel hardness, cohesiveness, chewiness, and gumminess than at pH 9.5, due to higher protein denaturation as supported by their higher surface hydrophobicity. Volatile beany flavor marker hexanal was predominant in all isolates than the starting material, irrespective of isolation temperature, probably due to lipid oxidation. The results provide a basis for tuning the isolation process for producing pea protein isolates with desired techno-functional properties for meat analogue applications

Valorization of herring filleting co-products to silage - Control of protein hydrolysis and lipid oxidation during ensilaging and possibilities for separating herring silage into multiple products

Industrial processing of herring (Clupea harengus) into convenience products such as fillets generates around 60% co-products being rich in both protein and n-3 polyunsaturated fatty acids (PUFAs). A promising cost-efficient strategy to valorize these raw materials into food and/or feed ingredients would be to apply ensilaging, i.e., proteolysis mediated by endogenous proteases under acidic conditions. Although an ancient technique, very little is still known about factors affecting the protein degree of hydrolysis (DH) and lipid oxidation during ensilaging. In this work, the effect of e.g. temperature and time on DH and lipid oxidation during ensilaging of herring co-products was investigated with the aim to maximize the DH while keeping the formation of unwanted free amino acids (FAA) and lipid oxidation to a minimum. Also, the role of hemoglobin (Hb) in ensilaging-induced lipid oxidation was studied, along with different ways of introducing antioxidants to the silage. Finally, possibilities to separate pilot scale-produced herring silage into oil and hydrolysates were investigated.Ensilaging for 1-7 days between 7-47\ub0C revealed that the highest DH was noticed at 32\ub0C, but the DH increased over time at all studied temperatures, which also applied to FAA. At ambient temperature (i.e. 22\ub0C), being the main focus in this thesis, DH and FAA reached 60% and 14%, respectively, at day 7. Heat-treating the silage for 30 min at 85\ub0C prior to storage (0-6 months; 4\ub0C or 22\ub0C) stopped hydrolysis, and is thus a route to minimize FAA formation. Lipid oxidation proceeded during ensilaging at all temperatures, however, ≥ 22\ub0C, the secondary oxidation product marker malondialdehyde (MDA) underwent hydrolytic cleavage or interacted with proteins/peptides/amino acids, preventing its accumulation. Instead, non-enzymatic browning reaction products e.g. 2-ethylfuran and 2-pentylfuran developed, along with saturated aldehydes as pentanal and hexanal. Upon adjusting the pH from physiological to 3.5 (i.e. ensilaging pH), it was documented that trout oxyHb changed to metHb, facilitating heme group release, suggesting heme-mediated peroxide cleavage was a dominating mechanism behind the ensilaging-induced lipid oxidation. To minimize lipid oxidation, two different strategies were evaluated; (i) pre-incubating the co-products in antioxidant solutions, or (ii), direct addition of 0.25-1.25% antioxidants at the start of ensilaging. Both strategies were effective, and among all the antioxidants studied, the commercial rosemary extract-based antioxidant Duralox MANC-213 provided best protection against lipid oxidation during ensilaging, heat-treatment and storage of silage. The total volatile basic nitrogen (TVB-N) level in silages remained below the acceptable limit of 30 mg TVB-N/100 g fish for human consumption, suggesting that silage can be used in both food and feed. Scaling up the ensilaging to a 1500-liter batch size, ~38% DH was recorded after 2 days at ~22\ub0C, which was similar to the DH found in lab-scale (40% after 2 days; 22\ub0C), suggesting lab-scale data well simulated what happens in pilot scale. Subjecting the silage to centrifugation (3000-8500 x g; 2-20 min) showed that it can be successfully separated into fish oil and protein hydrolysates.To summarize, these studies provide valuable information on optimal process parameters to use in on site valorization of herring co-products into a high-quality peptide-rich silage, and/or fish oil and hydrolysates, paving the way for silage based-biorefining of herring side streams into multiple products

Towards valorization of herring filleting by-products to silage 2.0: Effect of temperature and time on lipid oxidation and non-enzymatic browning reactions

The objective of this study was to investigate the effect of temperature and time on lipid oxidation and non-enzymatic browning reactions during ensilaging of herring by-products. Ensilaging for 0–7 days between 7 and 47\ua0\ub0C revealed an increasing development rate for peroxide value (PV) and 2-thiobarbituric acid reactive substances (TBARS) from 7\ua0\ub0C to 22\ua0\ub0C followed by a decreasing trend until 47\ua0\ub0C. Similarly, specific oxidation markers, e.g. malondialdehyde (MDA), 4-hydroxy-hexenal (HHE), and 2,4-heptadienal had equal development rate at 7 and 22\ua0\ub0C; but, substantially lower development rate at 47\ua0\ub0C. At 47\ua0\ub0C, there was on the other hand a more pronounced formation of saturated aldehydes and non-enzymatic browning products. This study thus showed that the degree of oxidation in silages was higher at elevated temperatures, although not visible in the classic oxidation analyses PV and TBARS. Therefore, addition of e.g. 2-pentylfuran as an oxidation marker is suggested when targeting the production of high-quality silage “2.0”