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* Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan 84322-4815
Department of Nutrition and Food Sciences, Utah State University, Logan 84322-8700
Bioproducts, Inc., Fairlawn, OH 44333
2 Corresponding author: trdhiman{at}cc.usu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: milk • fatty acid • fish oil • conjugated linoleic acid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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9-desaturase. Based on a 30-yr epidemiological study, Kromhout (1989) reported that low levels of eicosapentaenoic acid (EPA; C20:5 n-3) and docosahexaenoic acid (DHA; C22:6 n-3) result in a significant decrease in the incidence of coronary heart disease. The n-3 FA have also been shown in animal and human studies to be necessary for growth, development, immunity, and insulin activity (Vessby, 2000; Calder, 2001; Uauy et al., 2001).
Increasing the concentrations of beneficial dietary FA (CLA, VA, and n-3 FA) and maintaining a favorable ratio between n-3 and n-6 FA in milk will enhance the nutritive and therapeutic value of dairy products. Previous research has shown that feeding fish oil at 2% of dietary DM to dairy cows increased the concentrations of CLA and VA in milk FA and decreased daily feed intake and fat content of milk (Chouinard et al., 1999; Donovan et al., 2000; Baer et al., 2001; Ramaswamy et al., 2001). A feed source of fish oil and soybean oil that increases the proportions of healthful FA on milk but does not negatively affect feed intake and milk fat content and is easy to handle will be more acceptable at the farm level.
Our hypothesis is that feeding feedstuffs rich in linoleic acid and partially ruminally inert source of fish oil will supply substrate in the rumen for CLA synthesis and source of n-3 FA postruminally for direct absorption in the small intestine without negatively affecting feed intake and milk fat content. Our objective was to enhance CLA and n-3 FA while maintaining feed intake, milk fat content, and comparable flavor characteristics of milk and cheese that are acceptable to consumers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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. Extruded full-fat soybeans and oil supplements were added by replacing corn and soybean meal in the basal diet. Diets were formulated to meet the nutrient requirements of cows producing 50 kg of 3.5% FCM/d according to NRC (2001) recommendations and fed as a TMR. The soybean oil was stored in a cold room at 4° C and was added fresh to the diet each morning to avoid oxidation. The Ca-PFO was stored in 22.7-kg airtight bags, in a covered, cool, and dry feed barn. Cows were housed in a tie-stall barn and fed individually once daily ad libitum (0700 h); amounts fed and refused were recorded daily. Orts were restricted to 5 to 10% of daily intake on an as-fed basis. Animal care and procedures were approved and conducted under established standards of the Utah State University Institutional Animal Care and Use Committee.
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Dried feed samples were ground through a Wiley mill (1-mm screen; Arthur H. Thomas, Philadelphia, PA) and analyzed for chemical composition. The CP content of feed samples was determined using the macro Kjeldahl nitrogen test using AOAC method 954.01 (AOAC, 2000) with a Kjeltec digester 20 and Kjeltec System 1026 distilling unit (Tecator AB, Hoganas, Sweden). The NDF and ADF contents were determined with the ANKOM200 Fiber Analyzer (Ankom Technology Corporation, Fairport, NY), using the basic procedure of Van Soest et al. (1991). Sodium sulfite was not used in the procedure for NDF determination, but pre-treatment with heat-stable amylase (Type XI-A from Bacillus subtilis, Sigma-Aldrich Corporation, St. Louis, MO) was included. Weekly samples of dietary ingredients were analyzed for total FA content and FA profile (Sukhija and Palmquist, 1988). During analysis, the samples were further dried at 105° C for 8 h to determine the absolute DM, and chemical analyses were expressed on the basis of this final absolute DM.
The chemical composition of the TMR was calculated from the chemical composition of individual ingredients of the diet. Daily DMI for individual cows was calculated by subtracting the weekly mean of orts from the weekly mean of feed offered. The NEL content of the diet was calculated by using the NEL table values (NRC, 2001) for the individual dietary ingredients (Table 1
). Weekly mean NEL intakes were calculated by multiplying the NEL values of the diet by the mean DMI of the individual cows for that week. The CP, NDF, and FA intakes were calculated by subtracting CP, NDF, and FA amounts in orts from feed offered. The amount of CP, NDF, and FA in orts was calculated by multiplying weekly mean orts for individual cows by treatment average CP, NDF, and FA content in orts during that week.
Diet Composition
Chemical composition of the treatment diets is presented in Table 1. Mean DM content of the treatment diets ranged between 62.0 and 62.5 g/100 g throughout the experiment. The NEL content of the FO, FOESM, and FOSO treatment diets was higher than the control diet because of the high-energy value of the oil. Average protein, NDF, and ADF contents of the treatment diets were within recommended ranges for the production level of lactating dairy cows used in the present study (NRC, 2001). Total FA content for the FO, FOESM, and FOSO diets was 42.2% higher than for the control diet (4.61 vs. 6.56 g/100 g). Dietary FA levels of 6 to 7% on a DM basis are acceptable in lactating dairy cow diets (NRC, 2001). Fatty acid composition of the treatment diets and Ca-PFO is presented in Table 2. The Ca-PFO had higher proportions of C16:0 and C18:1 cis-9, and soybean products were rich in C18:2 FA. As expected, the addition of Ca-PFO increased the proportions of C16:0 and C18:1 cis-9 in diets compared with the control treatment.
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Cheddar Cheese Manufacturing
Cheddar cheese was prepared using the small-scale manufacturing procedure used at the Dairy Products Laboratory, Utah State University, in stainless-steel, steam-jacketed rectangular cheese vats. Each cheese vat was filled with approximately 230 kg of pasteurized milk adjusted to 31° C for ideal culture growth. Lyophilized cultures of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris blend plus selected adjunct cultures as required for specific flavor profiles were added to the vat and were allowed to ripen for 30 min. Calcium chloride was added to aid coagulation after the inoculant, during the ripening process, and diluted with cold water to a ratio of 10:1. Color was added using single strength annatto (DSM Food Specialties, Heerlen, NL) at a level of 20 mL/100 kg of milk and diluted with cold water to 20:1. Double-strength Maxiren coagulant (Maxiren double strength, DSM Food Specialties, Menomonee Falls, WI) was added and diluted to 20:1 with cold chlorine-free water and stirred in the vat. The contents of the vat sat undisturbed for 30 min and then were cut into 3-dimensional cube curd. The curd was heated slowly over 10 min to 33° C and then to 39° C over the next 20 min. The curd was cooked for 45 min, and the whey was drained. The curd mat was then cut and rotated to keep it warm. The cheese mat was cut into pieces using a cheese mill and salted over 3 applications at a rate of 6.27 kg of salt/1,000 kg of milk. Salted cheese curd (11.4 kg) was placed in each of 2 sanitized stainless-steel, 10-kg cheese hoops with disposable plastic cheesecloth. Hoops were labeled with the treatment identification and pressed overnight. Two vats of cheese (one from each treatment) were made on the same day (during wk 5 and 6) with a total of eight 10-kg blocks of cheese being produced. Cheddar cheese was vacuum-packaged in air-tight plastic bags and stored at 4° C until evaluated for sensory parameters at 21, 30, 90, and 180 d of aging.
Compositional Analysis of Milk and Cheese
Individual milk samples were analyzed for fat, true protein, lactose, and urea by the Rocky Mountain DHIA Laboratory (Logan, UT) with mid-infrared wavebands (2 to 15 µm) procedures using a Bentley 2000 (Bentley Instruments, Chaska, MN). The infrared instrument was calibrated weekly using raw milk standards based on chemistry analysis (Eastern Laboratory Services Limited, Fairlawn, OH). The fat measurement channel used was a combination of Fat A and Fat B. Final milk composition for each week was expressed on weighted milk yield of a.m. and p.m. samples. Average fat and protein yields were calculated by multiplying milk yield from the respective week by fat and protein content of the milk on an individual cow basis. Energy-corrected milk was calculated on an individual cow basis using milk yield, fat, and protein content (Tyrrell and Reid, 1965). Gross feed efficiency was calculated by dividing daily ECM by DMI on an individual cow basis.
Weekly weighted composite milk samples from individual cows were analyzed for FA composition, including CLA, VA, and n-3 FA. Milk fat was extracted by boiling the milk in a detergent solution (Hurley et al., 1987) using the procedures and gas chromatography conditions described by Dhiman et al. (1999b, 2002). Samples containing methyl esters in hexane (1 to 3 µL) were injected through the split-less injection port onto a Supelcowax 10, fused silica capillary column (100 m x 0.32 mm., 0.25-µm film thickness; Supelco Inc., Bellefonte, PA). Fatty acids were identified by comparing the retention times with methylated FA standards including CLA (Nu-Chek Prep, Elysian, MN; Matreya, Pleasant Gap, PA; Supelco Inc.). Hepta-decanoic acid was used as a qualitative internal standard. The CLA reported is CLA-1 and CLA-2. Percentage of each FA was calculated by dividing the area under the FA peak (minus the area under the peak for heptadecanoic acid) by the sum of the areas under the total reported FA peaks. Fatty acids were reported as grams/100 g of FA methyl esters. Different C18:1 trans and cis FA were not identified because of the limitations of column used in the gas chromatography. In the present study, an acidic catalyst was used for FA methyl ester preparation. Studies have shown that recovery of CLA is lower when using an acid catalyst as compared with a base catalyst (Kramer et al., 1997; Murrieta et al., 2003). Though the values reported in this study are lower than would be determined using a basic catalyst for methylation, relative comparisons among treatments should still be valid. Treatment differences in CLA levels in muscle were not affected by use of acidic methylation catalysts (Murrieta et al., 2003).
To compare the influence of methylation procedures on FA profiles, cheese samples were methylated using both acidic (Chin et al., 1992) and alkaline (Chouinard et al., 1999) procedures. Extracted fat was derivatized to methyl esters using an alkaline methylation procedure by mixing 40 mg of fat with a sodium methoxide methylation reagent (NaOCH3/MeOH) as described by Chouinard et al. (1999) with minor modifications. After FA methyl esters were formed, anhydrous calcium chloride pellets were added and allowed to stand for 1 h to remove water in the sample. Samples were then centrifuged at 2,600 x g at 5° C for 5 min. Separation of FA was achieved by gas chromatography (model 6800 Series II, Hewlett Packard Co., Avondale, PA) fitted with a flame-ionization detector. The FA methyl esters in hexane were injected through a 40:1 split injection port with He as a carrier gas. Oven temperature was set for 70° C and held for 4 min then was ramped to 120° C at 8° C/min. The temperature was then ramped to 200° C at 7° C/min and held for 10 min and then ramped to 225° C at 4° C/min and held for 18 min. Finally, the temperature was ramped to 235° C at 8° C/min. Injector and detector were set at 250° C. Heptadecadenoic acid was used as qualitative internal standard. Each peak was identified using FA and FA methyl esters (Nu-Chek Prep; Matreya; and Supelco 37 Component FAME mix, Supelco Inc.). Individual cheese samples were tested for FA profiles every 4 wk for 24 wk to determine the influence of aging of cheese on CLA content. The CLA yield was calculated by multiplying CLA content with total fat yield corrected for glycerol content (Chouinard et al., 2001) on an individual cow basis.
The VA is converted to CLA in the mammary gland (Corl et al., 2001) via the 9-desaturase enzyme. The 9-desaturase index was calculated for selected milk FA using product-to-substrate ratios of FA. The FA ratios used to determine the 9-desaturase index were C14:1:C14:0, C16:1:C16:0, C18:1 cis-9:C18:0, and C18:1 trans- 11:CLA-1.
Fatty acids can promote or prevent atherosclerosis and coronary thrombosis based on their effects on serum cholesterol and low density lipoprotein-cholesterol concentrations (Ulbright and Southgate, 1991). The equations proposed by Ulbright and Southgate (1991) for the atherogenic (AI) and thrombogenic indices (TI) indicated that the C12:0, C14:0, and C16:0 FA are atherogenic and that C14:0, C16:0, and C18:0 are thrombogenic. The n-3, n-6, and monounsaturated FA are anti-atherogenic and antithrombogenic. The ratio between the 2 is used to calculate the AI and TI.
The AI and TI were calculated in the present study using the equations described by Ulbright and Southgate (1991). In equations, the C14:0 FA is considered to be 4 times more atherogenic than the other FA; thus, the coefficient "4" has been assigned to it. The C18:1 n-6 and monounsaturated FA have been assigned coefficients of 0.5 because they are less antiatherogenic than the n-3 FA, which have been assigned a coefficient of 3.
Sensory Evaluation of Milk and Cheese
A trained panel of judges evaluated pasteurized and homogenized fluid milk samples of wk 5 and 6 from each treatment on 3 and 10 d of storage, for acid, barny, cooked, feed, fermented, foreign, stale, oily, oxidized, rancid, overall quality, and specific flavors on a scorecard patterned after the American Dairy Science Association scoring guide (Hammond et al., 1986). Seven judges were selected from a group of people who had been regularly exposed to training and judging a variety of dairy products, including fluid milk. Judges were not specifically trained for the current study, but were familiar with the scorecard for fluid milk flavor. The sensory testing was conducted by using a continuous 9-point scale for flavor characteristics, where 9 = highly pronounced flavor, 7 = moderate, 5 = slight, 3 = barely perceptible, and 1 = none. Overall quality of milk was rated on a continuous scale of 1 to 10, where 10 = excellent in overall quality, 5 = average, and 1 = undesirable and unfit for sale. Whole milk fortified with vitamin D in a clear plastic container was purchased from the store with at least a 14-d shelf-life and was used as a positive control. Reference samples for specific attributes were provided for all flavor characteristics during sampling.
Refrigerated (22.0 ± 1.0° C) fluid milk (20 mL) was served in plastic cups to a trained panel. Random code numbers were assigned for identification of each sample, but the order of sample presentation was not randomized among panelists. The evaluation of milk from wk 5 and 6 was conducted in individual booths under fluorescent white light. Water and spittoons were provided to panelists to cleanse the palate between samples. This was repeated after a 20-min break. Sample position was changed each time samples were offered to judges.
Nine experienced panelists evaluated Cheddar cheese samples from each treatment on 21, 30, 90 and 180 d of storage for acid, bitter, color, feed, fermented, flat, oily, oxidized, rancid, sulfide, unclean, and overall quality using a modified official American Dairy Science Association scorecard (Hammond et al., 1986) for cheese flavors. The panelists were selected from among people who had been regularly exposed to training on dairy products, including Cheddar cheese. The panelists were previously trained for quantitative descriptive sensory analysis, including training on the Cheddar cheese lexicon and the American Dairy Science Association scorecard. The scale used for specific flavors, uniformity of color, and overall quality of cheese by the trained panel was the same as was used for milk.
For evaluation, cheese blocks were cut into cubes (1 cm3) and served to a panel of 9 judges. Cheese was served in plastic cups at room temperature (22° C) with random code numbers assigned to each sample. All samples of cheese were tested the same day with breaks allowed for judges between trays of samples. All judges tested all samples. Flavor compounds were provided as reference for specific flavor attributes to the panelists while performing the test. Testing conditions were the same as described previously for milk. Judges were not provided information regarding the treatment of each sample in any of the previous evaluations.
Estimation of Ruminal Protection of Fat in Ca Salts of Fish Oil
The Ca-PFO were analyzed for ruminal protection of FA using a simple in vitro procedure. Rumen digesta was collected from ruminally cannulated cows fed a TMR containing 46% forage and 54% grain mixture in a thermos flask flushed with CO2. Digesta was strained through cheesecloth and mixed with McDougall’s buffer in a ratio of 50:50. Duplicate samples of Ca-PFO were incubated (1.20 g/100 mL of incubation solution) in flasks in a 39.5° C water bath for 0 and 24 h. Flasks were manually shaken every 2 to 3 h. At the end of each time, flask contents were freeze-dried and analyzed for FA profile (Sukhija and Palmquist, 1988) as described earlier. To determine the level of fat protection, sum of polyunsaturated FA (C18:2 cis-9,12; C18:3 cis-9,12,15; C18:3 cis-6,9,12, C20:3 cis-8,11,14, C20:3 cis- 11,14,17; C20:5 cis-5,8,11,14,17; C22:4 cis-7,10,13,16; C22:5 cis-7,10,13,16,19) as a proportion of total FA at 0 h were compared with the 24-h time point. At 0 h, the total polyunsaturated FA level was 13.19% of the total FA. At the 24-h time point, the total polyunsaturated FA level decreased to 11.65%. This suggests that polyunsaturated FA in the Ca-PFO were approximately 88.3% inert in the rumen degradation when incubated in the rumen fluid for 24 h. In a review by Demeyer and Doreau (1999), it is stated that when diets are high in unsaturated FA and rumen pH is low, these factors will increase Ca salt dissociation in the rumen. Therefore, these data need careful interpretation because ruminal protection level for cows fed high-grain diets may actually be < 88.3%. Calcium salts of unsaturated FA are more dissociated than Ca salts of saturated FA at any given ruminal pH; this disassociation can also weaken the physical barrier surrounding the FA in a real-time ruminal situation (Lundy et al., 2004) and result in lower ruminal protection than observed in vitro.
Statistical Analyses
All statistical analyses were performed using the Mixed statistical procedure of SAS (1999–2000). Analyses on diet composition data were performed with treatment and week as the fixed effects. The significance level was declared at P < 0.05 unless otherwise noted. Trends for significance were declared at P = 0.05 to 0.10. Analysis of intake data, milk yield, milk composition, FA composition, 9-desaturase enzyme index, AI, and TI were done using Proc Mixed in a repeated measures design. Treatment, block, week, and treatment x week were included in the model as fixed effects with week as the repeated measure on cows. Covariance structure was autoregressive (1). In the case of cheese FA profile, treatment, age, and treatment x age were included in a factorial model; week was the replicate. For the sensory panel on milk and cheese, treatment, judge, age, treatment x judge, treatment x age, judge x age, and treatment x judge x age were included in the model; batch was used as the replicate. For the methylation procedure, the model was built in a stepwise manner deleting the factors with 
> 0.3. Final model included treatment, method, and treatment x method.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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. The DMI was not different among treatments despite the high level of fat in the FO, FOESM, and FOSO compared with the control diet. The addition of oil to the diet can depress DMI because of the increased energy content or its ability to coat fiber in the rumen, decreasing the activity of fibrolytic bacteria (Jenkins, 1993). No significant treatment differences in DMI resulted in similar intakes of NEL, CP, and NDF among treatments. Fat intake was higher for FO, FOESM, and FOSO than for the control because of the higher FA content in the diets of those treatments (Table 1).
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). Other researchers (Chouinard et al., 1999; Baer et al., 2001; Whitlock et al., 2002) have reported a significant decrease in milk fat with the addition of fish oil to the diet at 2% of the dietary DM. In the present study, there was a slight tendency for lower milk fat content in FOSO treatment, probably because of unsaturated FA from soybean oil (Dhiman et al., 1999b). Dairy farmers are paid premium price for higher milk fat. Therefore, even a small decrease in milk fat could be of economic value to dairy producers. Ramaswamy et al. (2001) reported that fish oil in combination with soybeans did not depress milk fat. Donovan et al. (2000) reported no decrease in milk protein yield if fish oil was supplemented at < 3% of the diet. In contrast, Whitlock et al. (2002) reported that in lactating cow diets, the addition of fish oil at 2% of the diet decreased the milk protein yield. Milk urea nitrogen was higher in FOESM, probably because of the numerically higher CP content of the diet for this treatment. No significant negative effects were observed in the present study on nutrient intake, milk production, and milk composition, which is probably due to the fact that partially ruminally inert fat supplement was used in the present study instead of free fish oil.
FA Composition of Milk and Cheese
The treatment by week interaction was nonsignificant for FA in milk and cheese; therefore average values of 4, 5, and 6 wk of the experiment for milk FA composition are presented in Table 4. Data for FA composition of cheese are not presented because the trends among treatments for most FA were very similar to milk FA profile. The C4:0 FA was not reported because of low recovery during the gas chromatography analysis. The proportion of short-chain FA (C6:0 to C12:0) decreased in milk and cheese for the FO, FOESM, and FOSO compared with the control. This supports the findings of Baer et al. (2001) who reported a decrease in short-chain FA as fish oil or soybean oil (Dhiman et al., 1999b) was added to the diet of lactating dairy cows. A decrease in several medium-chain FA (C14:0, C15:0, and C17:1) was also observed in milk and cheese in FO, FOESM, and FOSO compared with the control. The FO, FOESM, and FOSO also showed an increase in long-chain FA (VA, C18:1 cis-9, C22:4, C22:5, and DHA) compared with the control for both milk and cheese, which is consistent with Baer et al. (2001), who reported the increase in long-chain FA with the addition of fish oil to the diet. The proportion of the C18:0 FA in milk tended to decrease with the addition of Ca-PFO alone or in combination with soy-bean products. The proportion of the FA C20:2, C20:3 cis-11,14,17, and EPA in milk showed a tendency toward increasing with the addition of Ca-PFO alone or in combination with soybean products compared with the control.
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9-desaturase enzyme (Kay et al., 2004).
The CLA-1 in milk FA increased 2-, 2.4-, and 3-fold in the FO, FOESM, and FOSO treatments, respectively, compared with the control (Table 4). The proportion of CLA-1 in cheese was 0.58, 1.07, 1.34, and 1.63 (P < 0.001) g/100 g of FA methyl esters in the control, FO, FOESM, and FOSO, respectively. Some of the differences seen in the FA composition of the milk and cheese can be attributed to the decreased variability of the cheese, as milk from all cows was pooled for each treatment and samples were taken from each batch of cheese for analysis in contrast with milk analysis, which was conducted on milk samples from individual cows (n = 18 per treatment vs. 2; SEM = 0.03 vs. 0.13). Previous research has shown that feeding unsaturated oils increases CLA in milk (Dhiman et al., 1999b). The higher levels of CLA found in the FOESM and FOSO in milk and cheese compared with FO is due to available linoleic acid in the oil for ruminal synthesis of CLA. The proportion of CLA-1 was highest in milk FA when Ca-PFO was fed along with free soybean oil (FOSO) compared soybean oil through full-fat extruded soybeans (FOESM) or when Ca-PFO was fed alone. This was probably because there was more VA available for the mammary gland to convert to CLA in this treatment. Total CLA (CLA-1 and CLA-2) was higher in milk and cheese for the FO, FOESM, and FOSO treatments compared with the control. As mentioned earlier, previous studies have shown that addition of fish oil in free form alone or in combination with soybeans increases CLA, usually accompanied by the depression of DMI, milk yield, or milk fat and protein content (Chouinard et al., 1999; Baer et al., 2001; Whitlock et al., 2002). In the present study, addition of Ca-PFO alone or in combination with extruded full-fat soybeans or soybean oil increased the CLA content of milk and cheese while maintaining similar DMI, milk yields, and protein contents as the control, except there was a tendency for decreased fat content in the FOSO treatment. As discussed earlier, even a small decrease in milk fat could be of significant economic value to dairy producers. The Ca-PFO is in granular form and is easy to handle on the farm. Thus, from a practical point of view, adding Ca-PFO is more convenient and did not cause any negative effects in the present study. However, its use will depend on the economic returns, which needs further investigation.
The VA is a source for CLA synthesis in the mammary gland. Higher CLA levels are correlated with higher levels of VA in Table 4. It is important to estimate the index of the 9-desaturase enzyme, which is responsible for the conversion of VA to CLA. Corl et al. (2001) concluded, in research focusing on the enzyme 9-desaturase, that the major site for synthesis of this enzyme is the mammary gland in lactating cows. An increase in the CLA content of milk can be partially attributed to the increased index of the 9-desaturase. There was no treatment by week interaction for the 9-desaturase enzyme index, so average values for 4, 5, and 6 wk are given in Table 5. The 9- desaturase index for C14:1:C14:0 and C16:1:C16:0 was not different among treatments. However, there was a increase in 9-desaturase index in the FO, FOESM, and FOSO over the control for C18:1 cis-9:C18:0 and VA:CLA- 1. The increase in CLA-1 content of milk in FO, FOESM, and FOSO may be partially attributed to the increase in 9-desaturase enzymatic activity in the mammary gland of cows in these treatments. It is important to point out here that higher levels of CLA-1 in FOSO without further increase in 9-desaturase index compared with FO suggest that source of CLA-1 in this treatment was through the rumen rather than through the mammary gland.
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The ratio between n-3:n-6 FA tended to increase (Table 4) in the FO, FOESM, and FOSO treatments in milk and was higher in cheese (average = 0.13; P < 0.01) compared with the control (0.12). It is not known with certainty what the optimum or desirable dietary ratio of n-3:n6 FA is for humans. However, current evidence suggests that the ratio between n-3:n-6 FA of 0.20 or lower is probably reasonable, whereas a value of 0.02 is undoubtedly excessive (Givens et al., 2000). Feeding Ca-PFO alone or in combination with extruded full-fat soybeans or soybean oil decreased the proportion of saturated FA and increased the unsaturated FA in milk and cheese compared with the control. This decrease in saturated FA and increase in unsaturated FA was greater in FOESM and FOSO compared with the FO treatment, reflecting the dietary supply. The average unsaturated:saturated FA ratio in the present study was 0.50, 0.66, 0.73, and 0.71 in the control, FO, FOESM, and FOSO treatments, respectively (Table 4). The relationship between saturated FA intake and low density lipoprotein in humans is direct and progressive, increasing the risk of cardiovascular disease (Clarke et al., 1997). The increase in the proportions of CLA, VA, n-3, and unsaturated FA in milk by feeding Ca-PFO alone or in combination with soybean products resulted in milk and cheese with higher nutritive and therapeutic value.
Dietary fats and oils have been associated with increased incidence of cardiovascular disease, namely coronary heart disease that is caused primarily by atherosclerosis or coronary thrombosis. The link between dairy fat consumption and risk of coronary heart disease is weak (Ness et al., 2001). None the less, dairy products with lower AI and TI indicate that they are less likely to cause atherosclerosis or coronary thrombosis, thus being potentially healthier for humans. The AI and TI of milk and cheese FA in the FO, FOESM, and FOSO were lower than in the control (Table 6), indicating that milk and cheese from treatment cows fed Ca-PFO alone or in combination with soybean products are less likely to lead to atherosclerosis and coronary thrombosis than milk from cows fed the control treatment.
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The recommended daily intakes of EPA + DHA for humans were presented in the workshop on the essentiality of these FA and were agreed upon by the workshop participants (Simopoulos et al., 1999). According to the workshop, the recommended daily intake for EPA + DHA is 650 mg/d, and one 500-mL serving of milk from cows on the control treatment would meet 1.2% of this requirement. Milk from cows fed the FO, FOESM, and FOSO treatments would meet 2.9, 2.3, and 2.0% of the recommended daily intake. By manipulating cow’s diet in the present study, we were not able to enrich milk with EPA and DHA to a level that would provide a significant portion of recommended daily intake, probably because of inherently low EPA and DHA content of milk and low transfer efficiency from diet to milk fat.
Although the acid methylation procedure was used for the esterification of milk FA, it is recognized as being less efficient for CLA analysis than the alkaline methylation. This decreased precision is due to the isomerization of CLA and the possible production of methoxy artifacts that can occur in the acid methylation procedure (Park et al., 1999). Cheese samples for each treatment were also analyzed using an alkaline methylation procedure and compared with the proportion of CLA and n-3 FA recovered using acid methylation. There was no treatment by method interaction, so results for VA, CLA, and n-FA are presented as an average across treatments (Table 7). When comparing the two methylation procedures, there was an increase in the recovery of CLA-1, CLA-2, total n-3 FA, and n-3:n-6 ratio by 10.7, 33.3, 20.7, and 18.8% with the use of an alkaline methylation procedure, respectively. A similar increase in the proportion of CLA and n-3 FA compared with acid methylation could also be expected in milk FA when using an alkaline methylation procedure (Table 4).
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). Lower scores by the taste panelists indicate a less pronounced physical or flavor characteristic. Trained taste panelists did not observe any off flavor in milk from all treatments including the control. The overall quality of milk was the same across treatments. However, store milk had barny and feed flavor characteristics compared with the treatment milk. There was a tendency toward overall lower quality of the store milk compared with the treatment milk. This may partially be because milk purchased at the store was in a non-light shielding plastic container. We recommend that store milk in a colored plastic jug or paperboard container should be used in future experiments to assure the quality of milk.
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The taste panelists found no flavor difference among treatments in cheese except for the acid flavor (below slightly perceptible level), which was lower in the FO treatment than in the FOESM treatment, but not different than the control or FOSO treatments (Table 8). Although statistically there was no difference in overall quality scores for the treatments, there was a tendency for decrease in the FO, FOESM, and FOSO treatments compared with the control. Some of the characteristics detected by the panelists, but not included on the scorecard were soft, bland, salty, citric, and metallic, which were placed in the "other" category. Results from the present study suggest that sensory scores and consumer acceptability characteristics of milk and cheese were comparable for FO, FOESM, and FOSO with those from the control treatment.
As cheese ages, residual coagulant and enzymes from starter culture and nonstarter lactic acid bacteria hydrolyze the milk proteins converting them into peptides, amino acids, and other flavor compounds. Some breakdown of triglycerides also occurs, releasing free FA, although lipolytic activity is generally less than proteolytic activity in a cheese such as Cheddar cheese. The longer the cheese is stored, the more distinctive the cheese flavor becomes. Along with the generation of flavor compounds that impart the characteristic flavor of Cheddar cheese, there can also be other compounds produced that impart off-flavors such as bitter, oxidized, and rancid flavors.
There was a significant treatment by age interaction observed for flavor characteristics of the cheeses as they were aged from mild (21 to 30 d of storage) to sharp (180 d of storage). There were no pronounced changes in off-flavors detected by the sensory panel for the control treatment cheese during the 180 d of aging, and only slight (but nonsignificant) increase in the flat and oxidized flavors were observed in the FO and FOESM treatment cheeses (data not shown). The FOSO treatment cheese exhibited the most change in off-flavor. For the FOSO treatment cheese, there were significant changes in feed, oily, oxidized, rancid, and sulfide flavor characteristics, although these were still only at the level of 3 (barely perceptible) or less (Table 9). The largest change was for oxidized flavor, which was most pronounced in the medium cheese and then diminished back to its original level when further aged to sharp. Changes in the other flavor characteristics followed a similar pattern but did not reach the same level of pronouncement as oxidized flavor. This increase in oxidized flavor may reflect the increased level of unsaturated FA in the milk from cows fed supplemental fat, especially those supplemented with soybean oil in addition to Ca-PFO. The decrease in sensory scores for these flavor characteristics as the cheese was aged from 90 to 180 d probably came about because as the overall cheese flavor increases to become a "sharp" rather than "medium" cheese, the off-flavors would be less apparent.
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. Results from the present study suggest that there were no major changes in the flavor characteristics and CLA or n-3 FA of CLA enriched cheese when it was aged to sharp Cheddar.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Partially ruminally inert Ca salts of palm and fish oil can be used at a level of 2.7% of dietary DM in combination with extruded full-fat soybeans or soybean oil for feeding to dairy cows to enhance the CLA, VA, unsaturated, and n-3 FA in milk fat without negatively impacting the animal performance and overall quality and consumer acceptability of milk and cheese.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Received for publication March 20, 2005. Accepted for publication August 21, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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); the basal diet + 2.7% Ca salts of palm and fish oil (Ca-PFO; 
); the basal diet + 2.7% Ca-PFO + 5% full-fat extruded soybeans (
); or the basal diet + 2.7% Ca-PFO + 0.75% of dietary DM as soybean oil (
).