Fortification of pork loins with docosahexaenoic acid (DHA) and its effect on flavour
© Meadus et al.; licensee BioMed Central Ltd. 2013
Received: 10 June 2013
Accepted: 19 November 2013
Published: 20 November 2013
Pork is traditionally low in docosahexanoic acid (DHA, C22:6n-3) and deficient in omega-3 fats for a balanced human diet. DHA as triglycerides was commercially prepared from the microalgae Schizochytrium and injected into fresh pork loins. Treatments of a mixed brine control (CON), 3.1% sunflower oil in mixed brine (SF) and a 3.1% DHA oil in mixed brine (DHA) were injected into pork loins at 10 mL/100 g and grilled at 205°C. After cooking, the CON and SF pork loins contained 0.03 to 0.05 mg DHA/g of pork and the DHA injected loins contained approximately 1.46 mg DHA/g. This also changed the fatty acid profile of omega-6: omega-3 from, 5 to 1 in the CON pork, to a ratio of 1.7 to 1 in DHA pork. The appearance, odor, oxidation rates and sensory taste, as judged by a trained panel, determined the DHA injected meat to be, 'slightly desirable’ and gave lower 'off flavour’ scores, relative to the CON and SF injected pork. Pork can be fortified with DHA oil to 146 mg/100 g serving, which would meet half the recommended daily omega 3 fatty acid requirements for adult humans and would be desirable in taste.
Pork is viewed as a lean healthy food, providing good nutrition; however, there are concerns about the quantity and types of fat it possesses. According to the USDA, a typical pork chop contains 11.3 g of fat/100 g of meat, of which 1.3 g is polyunsaturated fat and essentially no omega-3 fats . Humans require the essential fatty acids omega-6 linoleic acid (C18:2n-6) and omega-3 α-linolenic acid (C18:3n-3) in their diet. Human adults are recommended to consume at least 1 g/d of omega-3 fat for proper cardiovascular health [2, 3]. The long chain omega-3 fatty acid, docosahexaenoic acid (C22:6n-3), is particularly important, since it comprises ~14% of the cerebral cortex [4, 5]. To improve the omega-3 nutritional content of pork, researchers have fed plants such as, flax , soybeans and canola  which are high in α-linolenic acid; however, α-linolenic acid is only weakly converted to DHA . Pork can be selectively enriched with DHA by feeding fish oils such as tuna  or by feeding microalgae biomass Schizochytricium. However, there are problems with 'off’ flavours and trimethylamine odors caused by fish sources [8, 10, 11] or with achieving adequate concentrations of expensive pure sources of dietary grade DHA. The option of directly injecting the DHA into the meat as a brine marinade, may overcome some of these issues.
Injecting water for moisture into pork has been in practice since 1960 . The addition of a polyphosphate to a brine mixture further improves the juiciness, tenderness and flavour after cooking ; however, some discoloration has been noted. In addition to brine, injection of fats and oils  may improve the eating experience of pork. In North America, lean pork loins are averaging less than 2% intramuscular fat (IMF), the minimal IMF for consumer acceptance is > 3% . The IMF adds flavour and juiciness and has a minor improvement on tenderness . Beef injections with conjugated linoleic acid has recently been done to improve the nutrition but also to improve the eating quality experience of beef . This study was done to improve the nutritional profile of pork by injecting lean pork loins with DHA oil and to assess consumer perceptions of eating quality and to examine if any off flavours would be generated by the DHA oil.
Docosahexaenoic acid oil was supplied by Martek Bioscience Corporation (Boulder, CO, USA). Sunflower oil 100% was purchased from Compliments Company (Mississauga, ON, Canada). Sodium tripolyphoshate and salt was supplied by the Food Supplies Company (Winnipeg, Manitoba, Canada). The soy lecithin was from Solae, St. Louis, MO USA. Alpha tocopherol acetate was from Aquas Chem. Intl. (Torrance, CA, USA). Thiobarbituric acid, propyl gallate, ethylenediaminetetraacetic acid (EDTA), malonaldehyde, tetraethoxypropane, 1-hexanal, butanoic acid were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada).
Animals used in this study were cared for and slaughtered, according to Canadian Council for Animal Care guidelines . Barrows were selected from the Lacombe Research Centre f1 pig herd produced from Large White X Duroc mating. Pigs were given water ad libitum and fed a standard finisher diet comprised of 35% corn, 25% peas, 19% barley, 17% canola and 4% vitamin premix including 100 IU/kg of α-tocopherol (vitamin E) and 0.5 mg/kg selenium . The animals (n = 20) were slaughtered at the Lacombe Research Centre abattoir at 120 kg after a 24 h feed withdraw but with full access to water. Carcasses were split and cooled for 24 h at 4°C, then 24 carcass halves were selected and cut into primals, according to Canadian Meat Council guideline . The ~10 kg boneless loins were removed from both sides of the carcass, weighed, and distributed for treatment. Loins were evaluated and judge equal, based on visual colour and marbling scores .
Injection treatment of pork loins
Three treatments were allocated to the 24 h boneless loins (n = 8 loins/treatment). The treatments were an injection of 10 mL/100 g loin (longissimus dorsi muscle) of mixed brine solution (CON) containing phosphate, sodium chloride, a 3.1% sunflower oil in the mixed brine solution (SF) or a 3.1% DHA oil in mixed brine solution (DHA). The mixed brine consisted of, 4.8% sodium tripolyphosphate Na5P3O10 (BCCHEM, PQ, Canada), 4.8% sodium chloride, 0.01% α-tocopherol, and 0.15% precept 8140 powdered soy lecithin in distilled water. The SF oil consisted of the control brine mixed with 3.1% of mid oleic grade sunflower oil (Compliments, ON, Canada). The DHA oil consisted of the control brine mixed with 3.1% of DHA-S oil (Martek Bioscience Corp, Boulder, CO, USA). DHA-S oil was comprised of 35% docosahexaenoic acid extracted from microalgae mixed with 65% high oleic sunflower oil, 0.02% α- tocopherol and 0.01% soy lecithin.
The injection of pork loins with 3.1% DHA or 3.1% sunflower oil in a tripolyphosphate brine solution would add approximately 0.31 mL of oil/100 g of pork. The brine mixtures were injected using 4 mm needles spaced 2.8 cm in an Inject Star BI-72 unit (J Redmond & Sons, Northampton, UK), set at 2 bar and 56 strokes/min. After injection, the loins were allowed to equilibrate for 18 h at 2°C and then cut into 1 inch chops from the center, yielding 8–10 chops/loin, and 8 loins/treatment. The fluid loss was not measured at cutting. The chops from the three treatment groups, were packaged individually in polystyrene trays on dri-loc pads (UZ Soaker Ultra Zap Pads, Paper Pak Industries Washington, GA, USA), overwrapped with oxygen permeable film (8,000 mL/m2/24 h vitafilm choice wrap (Goodyear Canada Inc, Toronto, ON, Canada) and stored for an additional 24 h at +4°C. Day 1 raw chops (approximately 66 h post mortem) were selected (2/loin/treatment) for evaluation by trained panellists for visual colour, striping caused by the injections and odours and measured for color and thiobarbituric acid (TBARS) and again after 3 d, under refrigeration at 2°C, the maximum reasonable limit for retail display [21, 22]. A portion of the chops from each of the three treatments were sealed (n = 12/treatment) immediately after cutting, in vacuum packages (Multivac AGW; Multivac Inc., Kansas City, MO, USA) and stored in a -20°C freezer for the FAME analysis.
Fatty acid analysis (FAME) of oil and raw pork loins
Fatty acid methyl ester profile of the DHA oil and Sunflower oil preparations
DHA oil, (mg/g)
Sunflower oil, (mg/g)
Total FAME mg/g
Fatty acid methyl esters profile of the raw and cooked injected pork loins between Control brine (CON), Sunflower (SF) and DHA treatments
mg/g wet tissue
The colour of each loin treatment section was measured using a Minolta CM2002 color meter (Minolta Canada Inc., ON, Canada). Chops were cut from the injected treated loin and allowed to oxygenate at 4°C for 20 min before taking the colour measurements directly from the meat surface. The CIE L*, a*, b* colour coordinates were recorded along with Chroma and hue values and illuminated using a Minolta CR-300 color meter on the raw injected chops at days 0, 1 and 3 according to the manufacturers specification (Konica Minolta, Ramsay, NJ, USA).
Thiobarbituric Acid Reactive Substances (TBARS)
The free meat juice purge (1 mL) was collected from the drip trays (n =8/treatment) of the raw 1d and 3d, injected loin chops and then the chops were diced into 1 g cubes and blended with an Ultra Turax in 10 mL of extraction solution: trichloroacetic acid (75 g of TCA/L in water), propyl gallate (1 g/L) and EDTA (1 g/L). The extraction solution was filtered through a Whatman no. 42 filter then 2.5 mL of the filtered extract was mixed with 2.5 mL of thiobarbituric acid (TBA) (2.88 g/L) and heated to 94°C for 40 min. in closed glass vials. The samples were immediately cooled, and the absorbance was measured at 531 nm. TBARS values were determined relative to a standard curve of malonaldehyde generated with 1 g/L of tetraethoxypropane and 20 mmol/L to 90 mM TBA solution .
Sensory and odours evaluation of raw loin chops
Panellist (n = 8) were informed, selected and trained, according to the American Meat Science Associations guidelines . The panellist were asked evaluate the visual display of the 0 d and 3 d raw loin chops and give rating based on a 8-point hedonic scale for: overall retail appearance (8 = extremely desirable to 1 = extremely undesirable) and descriptive scales for lean muscle color (1 = pale pink/grey and white to 6 = dark purplish red), colour of striping (1 = none to 7 = yellow/brown),% striping (1 = none to 7 = 100%), spoilage colour (1 = none to 7 = brown),% surface spoilage (1 = none to 7 = 100%), and visual marbling score (1 = devoid to 6 = abundant).
Odour rating was completed using a 4-point descriptive scale for Off odor intensity (4 = prevalent to 1 = no off odours) a 5-point hedonic scale for odour acceptability (5 = unacceptable to 1 = acceptable), and a 9-point descriptive classification for Off odours (9 = other, 8 = unidentified, 7 = fishy, 6 = rancid/painty, 5 = stale/cardboard, 4 = piggy/barn like, 3 = metallic, 2 = off/sour, 1 = none). The panellist were also asked to rate the 3 brine mixtures.
Sensory and odours evaluation of cooked loin chops
Assessment of cooked chops was performed on 1 d loins, 24 h after brine injection and approximately 66 h post mortem. Each treated loin was weighed after removal from the vacuum pack and the percentage cooking loss was calculated based on the weight, before and after cooking. The injected loin chops, 8/treatment, were sliced into 1 inch chops and then cooked on a preheated Garland electric grill ED-30B at 205°C. The chops internal temperature was monitored every 5 s with a type T thermo-coupled temperature probes until the internal temperature reached 71°C. The cooked chops were allowed to cool for 3 min then trimmed of all outside edges and fat. Chops were cut into 1.3 cm cubes avoiding connective tissue and placed into 250 mL glass jars pre-warmed at 68°C. The samples were served to the panellist under 180-lux light in well ventilated partitioned booths. Panellist cleaned their palates between each sample with unsalted crackers and filtered water.
The panellist were asked to rate the samples on 9-point descriptive scale for initial and overall tenderness (9 = extremely tender to 1 = extremely tough), initial and sustained juiciness (9 = extremely juicy to 1 = extremely dry), and salt intensity (1 = no salt to 10 = extremely salty). Flavour desirability and overall palatability were rated on a 9-point hedonic scale (1 = not desirable to 9 = extremely desirable). Off flavour intensity was rated on a 9-point scale (9 = extremely intense to 1 = bland) and if off flavours were present, the panellist were asked to identify the most predominant descriptive classification for 'off odours’ (9 = other, 8 = unidentified, 7 = fishy, 6 = rancid/painty, 5 = stale/cardboard, 4 = piggy/barn like, 3 = metallic, 2 = off/sour, 1 = none).
For all meat treatment group variables, least square means were generated and were tested for significance (P < 0.05) within GLM and ANOVA. The lipid profiles were analyzed using the MIXED procedure and significance was determined using the DIFF option and Duncan’s test to identify differences between the groups means, CON, SF, and DHA and by raw and cooked treatment effect . The statistical model included the treatment effect at 1d or 3d interaction. An ordinate scale was used for the panellist evaluations of the sensory measures using Friedman test and the nominal scale was used for the biochemical measurement values, using Tukey’s HSD test.
Results and discussion
Pork fatty acid content
The injected loin treatments were primarily performed to determine if the DHA oil could be added at a concentration of 1 mg/g of fresh pork, without adversely affecting aroma or taste. Regular pork loins from pigs fed a standard finisher diet of, corn, barley, peas, and canola, would have ~0.5 mg of omega-3 FAME/g of meat and only ~0.02 mg of DHA FAME/g of meat . Injection of the 3.1% DHA brine mixture at 10 mL/100 g into the boneless meat, increased the DHA (C22:6n-3) content 50-fold, to an estimated concentration of 1.05 mg/g of pork. This changed the fatty acid profile of omega-6: omega-3 from, 5 to 1 in the CON pork, to a ratio of 1.7 to 1 in DHA pork. The actual concentration in the loin chops was 1.16 mg/ g of raw pork (Table 2). In a previous study, 1.22 mg DHA/g of raw bacon was achieved after feeding pigs, a diet containing 0.11% DHA for 25 d –the equivalent of approximately 825 g DHA . This trial achieved the 1.16 mg DHA/g level in a 10 kg loin, by injecting approximately 3.1% DHA, equivalent to approximately 32 g DHA/10 kg loin. The retention of DHA was higher after cooking at 1.46 mg/g of cooked pork. This increase was probably due to water loss evaporation by grill cooking (Table 2). The average cooking loss for all three treatments was 21.5 ± 3.04%. Conservatively, this would adjust the estimated level of DHA to approximately 0.82 mg/g of pork, if the oil was retained evenly but usually, free fatty acid content is increased by cooking . The amount of 18:1cis9 and 18:2n-6 was also significantly increased in the SF and DHA treatments (Table 1) but the final concentration of 18:1cis9 and 18:2n-6 was increased less than 2-fold in the actual raw and cooked pork (Table 2).
Panellist assessment of raw loin chops for retail display, visual marbling, color, striping, and odours between injection treatments
Measurement of oxidation
The effect of injection treatments on raw loin chops ( n = 24) for TBARS estimated oxidation and Colour meter measurements L*, a*, b*, Chroma
Oxidation in purge(TBARS)1
Oxidation in meat (TBARS)1
Panellist assessment on cooked loin chops for, tenderness, juiciness, saltiness, and overall palatability between injection treatments
Cook time (min.)
Sensory taste panel
The odour and sensory evaluations were made with a eight member trained taste panel. Before the trial, the panellist were asked to evaluate the chemicals: 1-hexanal, butanoic acid, docosahexanoic acid (DHA) oil, sunflower oil (SF) and DHA or SF oil plus soy lecithin. The chemicals 1- hexanal and butanoic acid were chosen as probable breakdown products of oxidation of DHA, caused by air exposure which leads to oxygen cleavage at double bonds [34, 35]. The 1-hexanal was described as 'stale’ and 'grassy’ and the butanoic acid was describes as 'rancid butter’. The DHA and DHA plus lecithin was initially odourless and nutty but was later described as fishy after exposure to air for >1 h. The SF and SF + lecithin was odourless then described as 'oily’ or 'stale’ after being exposed to air for more than 1 h. The raw chops were allowed to reach room temperature after 1 h before the chops were evaluated for odours. On day 1 and 3, the vitafilm wrapped, raw loin chops were assessed for odours. The raw chops were rated as generally acceptable for overall odours on day 1 and day 3 (Table 3). There was a noticeable drop in the 'unacceptable odours’ score by day 3 but this was still within the partially acceptable to neutral range and consistent between all treatments. There was no difference between the treatments and the scores were very low and unchanged for 'off’ odours in the 1 d and 3 d chops.
Chops were cooked, 24 h after injection, and were offered to the panellists which evaluated them for palatability and sensory flavours. The amount of cooking loss (%) was not significantly different between the CON (22.2 ±2.8%), the SF (19.9 ±3.6%) or the DHA (22.4 ±2.2%) treatments. There was very little difference between the treatments, according to the taste panellist as well (Table 5). The injected cooked chops rated highly for scores of, initial and sustained juiciness, initial and overall tenderness, and salt intensity. Initial juiciness was scored the highest in the DHA injected chops (Table 5). Salt intensity score was reduced by the addition of DHA and should be investigated further. The addition of tripolyphosphate and water to the chops has been used in the pork industry for over a decade and in the poultry industry since 1954 . It has been proposed that polyphosphate has two effects of depolymerisation of myosin filaments and weakening the binding of myosin with actin, thus promoting muscle fibre relaxing . This also would permit polyphosphate treated meat to retain more water. The panellists did not score any differences in the flavour of the 1d cooked chops between the treatment groups. The flavours were scored as bland, regardless of the treatments, and the overall palatability and pork flavour scored as 'slightly desirable’ to 'neutral’ in the trial. This is in agreement with previous sensory studies  which noted that the brine injected meat has only a minimal increase on saltiness scores and a less intense, pork flavour. It has been speculated that the flavour of brine injection, dilutes the carbohydrates, proteins and lipids and washes away the Maillard reactions complexes, which give meat its’ roasted flavour . If a panellist did mark the injected chops for 'off flavours’, they scored the sample as very low and gave a description as 'stale’ or 'piggy’ and surprisingly, the off flavours score were higher in the untreated CON and SF injected cooked pork than the DHA injected cooked pork (Table 5). It has been noted that DHA triacylglycerol can impart umami and flavour and supress bitterness in certain taste panels .
The injection of pork loins with 3.1% DHA in a tripolyphosphate brine mixture was rated to be 'desirable’, by trained taste panellist. The trained taste panel also scored the cooked DHA injected pork better at surviving against 'off’ flavours, than CON and SF injected pork. Increasing the lipid content ~ 0.3% by weight in the loins may have improved the juiciness of the cooked loin, especially since the IMF of pork was >2%. DHA content was improved approximately 50-fold to 1.16 mg DHA/g of raw pork, which converts to 116 mg of DHA in a typical 100 g serving of pork. The DHA content was further improved by cooking on a grill to 146 mg of DHA/100 g of pork. This would meet over half the adult human recommended daily requirements for DHA omega-3 fatty acid . The injection of DHA oil added to the nutritional value of the pork and will help in reducing plasma triglycerides of consumers .
Financial support was provided by Agriculture and Agri-Food Canada. The assistance with the raising and maintenance of pigs used in the study was provided by the staff at the Lacombe Center piggery, Sheri Nelson, Michelle Hambly and Rob Hambly. Processing of the pig carcasses was provided by Chuck Pimm, Darcy Schatschneider, and Jeremy Sealock at the Lacombe abattoir. We would also like to thank, Sophie Zawadski and Glynnis Croken, who were instrumental in the preparation of the injected pork and the sensory taste panel work.
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