Trout Fillets Research Articles

Combined effect of an oxygen absorber and oregano essential oil on shelf life extension of rainbow trout fillets stored at 4 °C

In the present study the combined effect of an O 2 absorber and oregano essential oil (0.4% v/w) on shelf life extension of rainbow trout fillets ( Onchorynchus mykiss) stored under refrigeration (4 °C) was investigated. The study was based on microbiological [TVC, Pseudomonas spp., Lactic Acid Bacteria, H 2S-producing bacteria including Shewanella putrefaciens, Enterobacteriaceae and Clostridium spp.), physicochemical (pH, PV, TBA, TVBN and Drip loss) and sensory (odor, taste) changes occurring in the product as a function of treatment and storage time. Aerobically-packaged rainbow trout fillets stored at 4 °C were taken as control samples. Results showed that TVC exceeded 7 log cfu/g on day 4 of storage for control samples, day 7–8 for samples containing oregano oil, day 9 for samples containing the O 2 absorber and day 12–13 for samples containing the O 2 absorber and oregano oil. Pseudomonas spp., Enterobacteriaceae and LAB were only partially inhibited by the O 2 absorber and/or the oregano oil. In all cases the inhibition effect was more pronounced when the combination of O 2 absorber with oregano essential oil was used. pH decreased from an initial value of 6.65–6.09 and subsequently increased to 6.86 due to formation of protein decomposition products. % Drip loss ranged between 7% and 11–12% at the end of the product shelf life. PV values ranged between 11.4 and 27.0 meq O 2/kg oil while malondialdehyde (MDA) ranged between 9.6 and 24.5 mg/kg. TVBN ranged between 10.6 and 54.6 mg/kg at the time of sensory rejection. Sensory shelf life was 4 days for the control samples, 7–8 days for samples containing oregano oil, 13–14 days for samples containing the O 2 absorber and 17 days for samples containing the O 2 absorber plus oregano oil.

Effects of Phosphate Dip Treatments on Chemical, Microbiological, Color, Textural, and Sensory Changes of Rainbow Trout (Onchorhyncus mykiss) Fillets During Refrigerated Storage

The influence of dipping rainbow trout fillets for 10 min in 5% solutions containing sodium monophosphate (MSP), sodium diphosphate (DSP), and sodium triphosphate (TSP) on microbiological, chemical, color, textural, and sensory analyses of fish fillets were assessed. Treatment of rainbow trout fillets with DSP and TSP were effective against the proliferation of total mesophilic, psychrotrophic bacteria, and coliform bacteria counts. Phosphate treatments did not affect lipid oxidation (TBA), TVB-N values, and textural qualities. According to the results of microbiological analyses, DSP and TSP extended the shelf-life of trout fillets an additional three days over the control and MSP during refrigerated storage. DSP and TSP treatments were determined more effective than MSP for inhibition of microbial growth. DSP and TSP treatments can be an alternative way to improve the shelf-life of fish and fish products.

Microbiological, Chemical, Sensory, Color, and Textural Changes of Rainbow Trout Fillets Treated with Sodium Acetate, Sodium Lactate, Sodium Citrate, and Stored at 4°C

The influence of dipping rainbow trout fillets for 10 min in 2.5% solutions containing sodium acetate (NaA), sodium lactate (NaL), and sodium citrate (NaC) on microbiological, chemical, color, textural, and sensory analyses of fish fillets were assessed. According to the results of microbiological analyses, total mesophilic counts for rainbow trout fillets exceeded 7 log cfu/g, which is considered the maximum level of acceptability for freshwater and marine fish after 6 days for the control, and 9 days for NaA, NaL, and NaC treatment. The shelf life of the treated trout fillets was extended by 3 days. NaA, NaL, and NaC were efficient in preventing the proliferation of total mesophilic bacteria (TMC), psychrotrophic (PC), and coliform bacteria (CB) counts. The general order of antibacterial activity of different organic salts used was: NaA > NaL > NaC. Organic salts treatment did not affect lipid oxidation and textural qualities. NaA, NaL, and NaC treatment can be an alternative way to improve the shelf life of fish and fish products.

Effect of fish freezing on selected parameters of smoked product quality

In the circumstances of small processing facilities for smoked fish production, and a particularly good fishing season, there occurs the necessity for freezing the catch in order to process it and preserve it in this way, at times when the season is not so good. To this end, the goal of our research was to examine the effect of freezing fish on the selected parameters of cold smoked, vacuum-packed trout fillets. To conduct the experiment, two groups of trout were formed: the control one, out of fresh fish, and the experimental one, out of previously frozen fish. In the course of a three week period of smoked fish storage at the temperature of 4 o C the variation of the total number of bacteria, and the total number of lactobacilli were monitored. Also, at the end of processing, the content value of water and salt were determined, as well as the content value of salt in the water phase, and aw value (water activity). By testing the bacteriological status of two groups of products during the storage period, it was determined that the total number of both bacteria and lactobacilli was smaller in the samples produced from frozen fish. The average content value of water and NaCl, as well as the salt content value in the water phase, in the vacuum-packed, smoked fillets produced from frozen fish was statistically, considerably larger than the content value of water, salt, and salt content value in the water phase in the samples manufactured from fresh fish. Our research findings show that using the raw material that was previously frozen for producing smoked fish fillets is not only appropriate, but also recommended.

Effect of edible coatings on the quality of frozen fish fillets

The objective of this study was to determine the changes in the quality of coated trout fillets after coating with edible materials. Fillets were coated and stored at −18 °C for a period lasting up to 7 months. Coating materials were applied in three different stages (first, second, and last coatings). The coated fillets were fried and analyzed for oil absorption and moisture content throughout the storage period. Sensorial attributes and the physical–biochemical changes were also measured before the frying process in each month. It was observed that it is more advantageous to use gluten as the first coating, xanthan gum as the second coating, and wheat (W) and corn (C) flours in the ratio of 1:1 or 2:1 as the last coating. In terms of the fillet quality, the following results were obtained in the analyses conducted before frying. The lowest pH found was 6.25 in zein-containing samples and 6.30 in guar-containing samples. The effects of the last coatings on pH were unimportant (P > 0.05). The lowest thiobarbituric acid levels found were 2.07 mg kg in the fillets coated with casein mixture, 2.44 mg kg in the fillets coated with xanthan gum, and 2.25 mg kg in the fillets coated with W:C flour mixture in the ratio of 2:1. The lowest total volatile basic nitrogen levels found were18.06 mg 100 g in the fillets coated with casein mixture, 18.62 mg 100 g in the fillets coated with xanthan gum, and 18.47 mg 100 g in the fillets coated with W:C flour mixture at 1:1 ratio. In the sensorial analysis, the coated samples were much more preferred than those not coated. As a result of the effects of all the materials, the coating layers on the meat surface provided more resistance against mass transfer during storage.

Stable isotope ratio analysis as a tool to discriminate between rainbow trout (O. mykiss) fed diets based on plant or fish-meal proteins.

The use of stable isotope ratio analysis (SIRA) as a rapid analytical tool to characterize and discriminate farmed fish on the basis of the feedstuffs included in the diet formulation is discussed. Two isoproteic (44.8%) and isolipidic (19.6%) extruded diets were formulated: a fish-meal-based diet (FM diet), containing fish meal as the sole protein source; a plant-protein-based diet (PP diet), where pea protein concentrate and wheat gluten meal replaced 80% of fish meal protein. The diets were fed to eight groups of rainbow trout (initial body weight: 106.6g) for 103 days in two daily meals under controlled rearing conditions. Growth performance (final body weight: 318.5 g; specific growth rate: 1.06%) and feed-to-gain ratio (0.79) were not affected by the dietary treatment. The differences in isotopic values of the two diets were clearly reflected in the different carbon and nitrogen isotopic values in rainbow trout fillets. The delta(13)C and delta(15)N values of muscle of farmed rainbow trout showed differences between farmed fish fed a fish-protein-based diet (-20.47 +/- 0.34 and 12.38 +/- 0.57 for delta(13)C and delta(15)N, respectively) and those fed a plant-protein-based diet (-23.96 +/- 0.38 and 7.15 +/- 0.51 for delta(13)C and delta(15)N, respectively). The results suggest that SIRA provides a robust and verifiable analytical tool to discriminate between fish fed on a plant or a fish protein diet.

Flesh characteristics of pan-size triploid and diploid rainbow trout (<i>Oncorhynchus mykiss</i>) reared in a commercial fish farm

Abstract. Sexual maturation of rainbow trout during production has a negative effect on the slaughter yields and flesh quality. Induction of triploidy is a method to prevent the development of fertile trout. In many investigations sexually mature triploid and diploid rainbow trout had been compared. In the present study the slaughter characteristics and flesh quality of female immature diploid and triploid rainbow trout reared up to 66 and 75 weeks (pan-size-format) were compared. Considering the age the old fish had significantly higher body and fillet weights accompanied with larger muscle fibres. The fillets of the old trout were brighter and redder, had a reduced water-holding capacity (WHC) and contained more fat and protein. With regard to the ploidy status the triploid trout had higher body and fillet weights and larger muscle fibres. The fillets of the triploid fish were brighter, had a reduced WHC and lower fat contents. With regard to the age-ploidy status interaction the young and old triploid trout had better growth characteristics but the WHC and the fillet fat concentration was reduced in these animals. The colour values were inconsistent. The pH and electrical conductivity values indicated no higher pre- or peri-mortal stress and/or different stress susceptibility of the investigated young as well as old triploid and diploid trout. In conclusion, it could be suggested that induction of triploidy positively influences body and fillet development mainly by muscle fibre hypertrophy, but the triploid fillets showed slightly worse flesh characteristics (Brightness, WHC).

Contribution of cathepsins B, L and D to muscle protein profiles correlated with texture in rainbow trout ( Oncorhynchus mykiss)

Post-mortem softening of fish tissue often results in low yield and decreased product quality. In this study, proteolytic profiles of trout stored 5 days on ice were obtained by SDS–PAGE. The link between protein band intensities and firmness of trout fillets was examined through a correlation study. In parallel, trout extracts were incubated with cathepsin B, cathepsin L and cathepsin D, alone or in combination, in order to evaluate the effect of each cathepsin on the texture-related proteins. Proteins from both myofibrillar (α-actinin, actin, MLC1, MLC2, and N-terminal 70 kDa MHC fragment) and sarcoplasmic (glycogen phosphorylase, creatine kinase, and TPI) fractions correlated closely with firmness. Cathepsins D, B and L affected, respectively, 10, 9 and 4 out of the 17 protein bands correlating with firmness, and most changes induced by cathepsin D were unfavourable to firmness. This implies that cathepsin D is likely to be involved in textural change of trout, due to breakdown of the muscle structure.

Microbial quality of raw aquacultured fish fillets procured from Internet and local retail markets.

The microbial quality of raw fillets of aquacultured catfish, salmon, tilapia, and trout was evaluated. A total of 272 fillets from nine local and nine Internet retail markets were tested. Mean values were 5.7 log CFU/g for total aerobic mesophiles, 6.3 log CFU/g for psychrotrophs, and 1.9 log most probable number (MPN) per gram for coliforms. Differences in these microbial levels between the two kinds of markets and among the four types of fish were not significant (P > 0.05), except that Internet trout fillets had about 0.8-log higher aerobic mesophiles than did trout fillets purchased locally. Although Escherichia coli was detected in 1.4, 1.5, and 5.9% of trout, salmon, and tilapia, respectively, no sample had > or = 1.0 log MPN/g. However, E. coli was found in 13.2% of catfish, with an average of 1.7 log MPN/g. About 27% of all fillets had Listeria spp., and a positive correlation between the prevalence of Listeria spp. and Listeria monocytogenes was observed. Internet fillets had a higher prevalence of both Listeria spp. and L. monocytogenes than did those fillets purchased locally. L. monocytogenes was present in 23.5% of catfish but in only 5.7, 10.3, and 10.6% of trout, tilapia, and salmon, respectively. Salmonella and E. coli O157 were not found in any sample. A follow-up investigation using catfish operation as a model revealed that gut waste exposed during evisceration is a potential source of coliforms and Listeria spp.

Acidified Sodium Chlorite Solution as an Antimicrobial Treatment for Rainbow Trout (Oncorhynchus mykiss) Fillets

Minimizing microbial growth and maintaining overall quality are priorities for intervention strategies that extend the shelf life of fresh, aquatic foods. Four treatments included a control (fresh fillets), water, 50 ppm of acidified sodium chlorite (ASC), and 1,000 ppm of ASC. Fillets were stored at 1 to 2°C for 0, 8, and 15 days. A significant (P < 0.05) interaction between treatment and storage time was observed for psychrotrophic counts. The increase in psychrotrophic counts with storage time was less for fillets treated with ASC, regardless of ASC concentration. Aerobic plate counts were not affected (P > 0.05) by intervention; however, a significant increase in counts was observed during storage (P < 0.05). Fillet pH, moisture, fat, thiobarbituric acid–reactive substances, fatty acid composition, color, cook yield, and shear force were not affected (P > 0.05) by intervention. Thiobarbituric acid–reactive substances decreased (P < 0.05) during storage. Percentages of individual fatty acids were constant, with the exception of C15 and C20:2; they decreased with storage to 15 days. Percent fat, L* (lightness) and b* (yellowness) values, and cook yield increased (P < 0.05) during storage. Fillet pH, moisture, a* (redness) value, and shear force did not change (P > 0.05) with storage to 15 days. Based on these data, 50 ppm of ASC performed equally as well as 1,000 ppm of ASC. The value of ASC is as a decontaminant; however, fillets in this study had low psychrotrophic counts pretreatment (2.3 log CFU/cm2) and posttreatment (2.03 log CFU/cm2), which did not demonstrate ASC's effectiveness as a decontaminant.

Argon Modified Atmosphere Packaging for Fillets of Rainbow Trout (Oncorhynchus mykiss) Fed Astaxanthin or Canthaxanthin

ABSTRACT Fillets from rainbow trout fed astaxanthin or canthaxanthin were packaged under air and modified atmosphere, 60% N2/40% CO2 and 60% Ar/40% CO2, and stored at +2°C for 26 days. Fish samples were analyzed at day 0, 5, 9, 13, 16, 19, 22, and 26 after processing. Drip loss, internal pH, surface color, carotenoid content, total lipid content, thiobarbituric acid reactive substances, and microbial contents of fish fillets were determined. A significant positive effect was found between pigment fed, gas used, and time of storage for the analyzed parameters. The use of argon led to a positive effect on the preservation of trout fillet quality and to an extension of trout fillet storage life.

Contaminant Levels in Rainbow Trout, Oncorhynchus mykiss, and their Diets from Missouri Coldwater Hatcheries

Organochlorine and metal contaminants often occur in commercial fish diets and can accumulate in fish to levels of concern for human consumption. Contaminant levels were investigated in diet and rainbow trout fillets from Missouri coldwater hatcheries used in "put and take" fisheries. The average fillet:diet ratio was <0.1 for lead and cadmium, 0.4-0.6 for organochlorine compounds, and about 0.8 for mercury. Trout fillet concentrations for all contaminants were low (<50 ng/g) and below Missouri's fish consumption advisory trigger levels.

Improvement in the Production of Smoked Trout Fillets (Salmo Gairdnerii) Steamed with Liquid Smoke

A fish smoking process was applied using a combination of liquid smoke and steaming at pressures up to 1 bar above atmospheric pressure. Drying and brining prior to smoking have not shown any significant effect on the quality of the trout fillets. The nondried and dried for 4 h trout fillets prior to processing were assessed as slightly more acceptable products, processing yield, sensory analysis, instrumental color, pH, available lysine, polycyclic aromatic hydrocarbons (PAHs) and preservation time were estimated. The losses due to processing of the nondried samples were 23.7%, while 28.8% for those dried for 4h. The fillets processed at 1.5 and 2 bar steam pressure were assessed as highly acceptable. Lightness (L*) showed a decreasing trend due to pressure while redness (a*), and yellowness (b*) an increasing one. The destruction of available lysine was low (13.20% ± 1.01) and it was slightly dependent upon the process. No PAHs were detected. The preservation time, studied via total viable count, lactic acid bacteria, existence of Listeria monocytogenes, and sensory analysis, extended to more than 98 days at 4 ± 1 °C. The drying prior to processing seems to affect the preservation time. This processing technique is much simpler, faster, environmental effective, and leads to high quality smoked products.

Effect of water activity on the inactivation kinetics of Escherichia coli O157:H7 by electron beam in ground beef, chicken breast meat, and trout fillets

SummaryWater activity (aw) of ground beef, chicken breast meat, and trout fillets was modified to intermediate (aw 0.98–0.99) and lowest (aw 0.94–0.96) levels. The meat samples with modified and unaltered (native, aw 1.00) aw were inoculated with Escherichia coli O157:H7 and subjected to electron beam (e‐beam). Survivor curves were plotted and the D10‐values were calculated. The D10‐values ranged from 0.22 kGy for trout at native aw to 0.33 kGy for beef at intermediate, and chicken and trout at lowest aw. Regardless of the species, aw reduction increased E. coli resistance to e‐beam, suggesting that even small depletion of unbound water from food increases survival. The difference of the D10‐values between the samples at intermediate and lowest aw was insignificant. E‐beam could be used before aw‐reducing techniques are applied to food products. However, this would require stringent microbial control following e‐beam processing. The ‘tailing’ of survivors was observed for some samples with reduced aw.

Characterisation of the lipid fractions obtained by proteolytic and chemical extractions from rainbow trout ( Oncorhynchus mykiss) roe

Lipids from rainbow trout ( Oncorhynchus mykiss) roe were extracted by chemical and enzymatic methods. Enzymatic hydrolysis by Alcalase ®, Neutrase ® and Protamex ® yielded three fractions after centrifugation: a light phase (oil), an intermediate fraction (water-soluble material) and a residual heavy phase. Best results were obtained with Alcalase ® where the roe proteins hydrolysis degree was 7.8% compared with 19% for trout fillets. The difference was explained by the particular composition of the proteins of the fish egg chorion. The yield of oil extracted after proteolysis was 3.7% vs. 9.6% by chemical technique. The polyunsaturated fatty acids (PUFAs) amount in the oil phase was high (41.8%) with 34.7% n−3 PUFA accounting for total lipids. High-concentration of PUFA was found in the polar lipid fraction of the heavy phase where docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) accounted for 25.6 and 10.9% of total lipids, respectively.

Peptides reproducibly released byin vivodigestion of beef meat and trout flesh in pigs

Characterisation and identification of peptides (800 to 5000 Da) generated by intestinal digestion of fish or meat were performed using MS analyses (matrix-assisted laser desorption ionisation time of flight and nano-liquid chromatography electrospray-ionisation ion trap MS/MS). Four pigs fitted with cannulas at the duodenum and jejunum received a meal exclusively made of cooked Pectoralis profundus beef meat or cooked trout fillets. A protein-free meal, made of free amino acids, starch and fat, was used to identify peptides of endogenous origin. Peptides reproducibly detected in digesta (i.e. from at least three pigs) were evidenced predominantly in the first 3 h after the meal. In the duodenum, most of the fish- and meat-derived peptides were characteristic of a peptic digestion. In the jejunum, the majority of peptides appeared to result from digestion by chymotrypsin and trypsin. Despite slight differences in gastric emptying kinetics and overall peptide production, possibly in relation to food structure and texture, six and four similar peptides were released after ingestion of fish or meat in the duodenum and jejunum. A total of twenty-six different peptides were identified in digesta. All were fragments of major structural (actin, myosin) or sarcoplasmic (creatine kinase, glyceraldehyde-3-phosphate dehydrogenase and myoglobin) muscle proteins. Peptides were short ( < 2000 Da) and particularly rich in proline residues. Nineteen of them contained bioactive sequences corresponding mainly to an antihypertensive activity. The present work showed that after fish or meat ingestion, among the wide variety of peptides produced by enzymic digestion, some of them can be reproducibly observed in intestinal digesta.

Parvalbumin mRNA in raw and processed fillet of rainbow trout Oncorhynchus mykiss: determination of copy number and stability against heat and acid

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) has been developed for determination of short sequences (∼120 nucleotides) of parvalbumin mRNA. The assay consisted of two steps. At first mRNA was transcribed to cDNA using primers specific for parvalbumin RNA. In the second step cDNA was quantified by real-time PCR with the LightCycler FastStart DNA Master SYBR Green I (Roche Applied Science, Mannheim, Germany). High copy numbers of mRNA, up to 109/g wet weight, have been measured in fillets of rainbow trout. Degradation of mRNA was not observed in cases of cooked, hot-smoked or acidified fish muscle, but storage of minced and washed raw muscle led to some reduction of copy numbers.

Protein Recovery from Rainbow Trout (Oncorhynchus mykiss) Processing Byproducts via Isoelectric Solubilization/Precipitation and Its Gelation Properties As Affected by Functional Additives

Solubility of rainbow trout proteins was determined between pH 1.5 and 13.0 and various ionic strengths (IS). Minimum solubility occurred at pH 5.5; however, when IS = 0.2, the minimum solubility shifted toward more acidic pH. Isoelectric solubilization/precipitation was applied to trout processing byproducts (fish meat left over on bones, head, skin, etc.), resulting in protein recovery yields (Kjeldahl, dry basis) between 77.7% and 89.0%, depending of the pH used for solubilization and precipitation. The recovered protein contained 1.4-2.1% ash (dry basis), while the trout processing byproducts (i.e., starting material) 13.9%. Typical boneless and skinless trout fillets contain 5.5% ash, and therefore, the isoelectric solubilization/precipitation effectively removed impurities such as bones, scales, skin, etc., from the trout processing byproducts. The recovered proteins retained gel-forming ability as assessed with dynamic rheology, torsion test, and texture profile analysis (TPA). However, the recovered proteins failed to gel unless beef plasma protein (BPP) was added. Even with BPP, the recovered protein showed some proteolysis between 40 and 55 degrees C. Addition of potato starch, transglutaminase, and phosphate to the recovered proteins resulted in good texture of trout gels as confirmed by torsion test and TPA. Higher ( P < 0.05) shear stress and strain were measured for gels developed from basic pH treatments than the acidic counterparts. However, proteins recovered from acidic treatments had higher ( P < 0.05) lipid content than the basic treatments. This is probably why the gels from acidic treatments were whiter ( L* - 3 b*) ( P < 0.05) than those from the basic ones. Our study demonstrates that functional proteins can be efficiently recovered from low-value fish processing byproducts using isoelectric solubilization/precipitation and subsequently be used in value-added human foods.

Protein and Lipid Oxidation during Frozen Storage of Rainbow Trout (Oncorhynchus mykiss)

This study aimed at investigating protein and lipid oxidation during frozen storage of rainbow trout. Rainbow trout fillets were stored for 13 months at -20, -30, or -80 degrees C, and samples were analyzed at regular intervals for lipid and protein oxidation markers. Lipid oxidation was followed by measuring lipid hydroperoxides (PV), as well as secondary oxidation products (volatiles) using dynamic headspace GC-MS. Free fatty acids (FFA) were measured as an estimation of lipolysis. Protein oxidation was followed using the spectrophotometric determination of protein carbonyls and immunoblotting. Significant oxidation was observed in samples stored at -20 degrees C, and at this temperature lipid and protein oxidation seemed to develop simultaneously. FFA, PV, and carbonyls increased significantly for the fish stored at -20 degrees C, whereas the fish stored at -30 and -80 degrees C did not show any increase in oxidation during the entire storage period when these methods were used. In contrast, the more sensitive GC-MS method used for measurement of the volatiles showed that the fish stored at -30 degrees C oxidized more quickly than those stored at -80 degrees C. Detection of protein oxidation using immunoblotting revealed that high molecular weight proteins were oxidized already at t = 0 and that no new protein oxidized during storage irrespective of the storage time and temperature. The results emphasize the need for the development of more sensitive and reliable methods to study protein oxidation in order to gain more explicit knowledge about the significance of protein oxidation for food quality and, especially, to correlate protein oxidation with physical and functional properties of foods.

Microbiological Changes Occurring in Trout Fillets (Oncorhynchus mykiss W. 1792) Salted and Treated with Potassium Sorbate During Production and Storage

In this study, microbiological changes during processing and storage of salted-cured trout fillets treated with potassium sorbate were investigated. For this purpose, 10 and 15% (w/w) NaCl and 1, 5 and 10% (w/v) potassium sorbate were applied to the fillets. The processed fillets were vacuum-packed and storage at 4 degrees C. The samples were analyzed in some periods of production and in the storage days of 7, 14, 28, 42, 56, 70 and 84 for numbers of total mesophilic aerob, psycrophylic, yeast and mould. In conclusion, the microbiological quality of all samples treated with 15% NaCl and potassium sorbate were found better. Consequently, it can be concluded that the usage of potassium sorbate may be useful and a synergistic effect between salt and potassium sorbate determined.