The process of storage and sampling was conducted according to the method of Wu, Ghirmai, et al.(2020).2.4. Total heme pigment measurementThe herring co-product minces were processed according to themethod of Wu, Ghirmai, et al. (2020) using liquid nitrogen. Total hemecontent was then measured using the acetone-based method of Wu et al.(2020). Bovine Hb was used to construct a standard curve, and resultswere expressed as μmol Hb/kg mince.2.5. Analyses of lipid oxidationTotal lipids were extracted from 1-g samples of co-product minceusing chloroform:methanol (2:1) (Cavonius & Undeland, 2017). Thelower phase (chloroform) was collected for peroxide value (PV) analysisas described by (Larsson, Almgren, & Undeland, 2007). The upper phase(water–methanol) was used to determine TBA-reactive substances(TBARS) according to the method of Wu, Xiao, Yin, Zhang, and Richards(2021). Results are expressed as μmol peroxides or μmol TBARS/kgmince.2.6. Preparation of hemolysateHerring blood was obtained as described by Ghirmai, Eriksson, Wu,Axelsson, and Undeland (2020), and hemolysate was prepared from theblood mixed with 1 vol of anticoagulant (sodium heparin) according toFyhn et al. (1979) by washing the red blood cells four times in 1 mM Tris(pH 8) containing 0.9% NaCl and then lysing them in 1 mM Tris (pH 8).The method of quantify the Hb levels in hemolysate was described byWu, Yin, Zhang, and Richards (2017).2.7. Determination of auto-oxidation and hemin loss of herring HbHb (5 μM) was incubated with or without Duralox MANC (0.5 g/L) ina sodium phosphate buffer (50 mM, pH 6.3) and stored at 4 ◦C. The pHwas selected based on fsh post-mortem pH (6.3–6.9). The percentage ofmethemoglobin was calculated according to the equations of Benesch,Benesch, and Yung (1973). The metHb was prepared as described previously (Wu et al., 2017). The hemin loss from metHb was measuredaccording to the method described by Maestre, Pazos, and Medina(2009).2.8. StatisticsIn each experiment, minced samples from all treatments were storedin duplicate Erlenmeyer fask (n = 2). PV, TBARS and total Hb are reported as mean ± maximum-minimum value/2 from these duplicatesamples (Larsson, Harrysson, Havenaar, Alminger, & Undeland, 2016).An unpaired t-test or ANOVA with Tukey’s HSD test was used todetermine signifcant differences between the different samples at aspecifc storage point, or, between different time points for a specifcsample. Differences are regarded as signifcant when p < 0.05.To investigate how different raw material batches infuenced some ofour main conclusions, key samples like the un-treated controls and theco-products incubated or dipped in 0.9% NaCl with or without 2% or 5%Duralox MANC, were included in 8 and 4 separate experiments,respectively. Data are shown in Supplementary information, Fig. 1, andillustrate that conclusions were the same, regardless of raw materialbatch.
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