Fortification of cheese with vitamin D(3) using dairy protein emulsions as delivery systems

Vitamin D is an essential vitamin that is synthesized when the body is exposed to sunlight or after the consumption of fortified foods and supplements. The purpose of this research was to increase the retention of vitamin D(3) in Cheddar cheese by incorporating it as part of an oil-in-water emulsion using a milk protein emulsifier to obtain a fortification level of 280 IU/serving. Four oil-in-water vitamin D emulsions were made using sodium caseinate, calcium caseinate, nonfat dry milk (NDM), or whey protein. These emulsions were used to fortify milk, and the retention of vitamin D(3) in cheese curd in a model cheesemaking system was calculated. A nonemulsified vitamin D(3) oil was used as a control to fortify milk. Significantly more vitamin D(3) was retained in the curd when using the emulsified vitamin D(3) than the nonemulsified vitamin D(3) oil (control). No significant differences were observed in the retention of vitamin D(3) when emulsions were formulated with different emulsifiers. Mean vitamin D(3) retention in the model system cheese curd was 96% when the emulsions were added to either whole or skim milk compared with using the nonemulsified oil, which gave mean retentions of only 71% and 64% when added to whole and skim milk, respectively. A similar improvement in retention was achieved when cheese was made from whole and reduced-fat milk using standard manufacturing procedures on a small scale. When sufficient vitamin D(3) was added to produce cheese containing a target level of approximately 280 IU per 28-g serving, retention was greater when the vitamin D(3) was emulsified with NDM than when using nonemulsified vitamin D(3) oil. Only 58±3% of the nonemulsified vitamin D(3) oil was retained in full-fat Cheddar cheese, whereas 78±8% and 74±1% were retained when using the vitamin D(3) emulsion in full-fat and reduced-fat Cheddar cheese, respectively.     

Comparison of different methods for fortifying Cheddar cheese with vitamin D

Fortification of Cheddar cheese with vitamin D was tested using three different addition methods to cheesemilk at a final concentration of 400 IU L⁻¹: addition of a commercial water-soluble emulsion of vitamin D (Vitex D); homogenization of crystalline liposoluble vitamin D in a portion of cream used for cheesemilk standardization; and addition of water-soluble vitamin D entrapped in multilamellar liposomes (Prolipo-Duo^TM). The recovery of vitamin D in cheese curd, losses in whey and stability of vitamin D during cheese making and ripening over a 7 months period were measured. The method of vitamin D addition did not affect significantly the composition of experimental cheeses (protein, fat, moisture and salt), which was not different from that of control cheeses made without vitamin D. The recovery of vitamin D in cheese was significantly higher when vitamin D was entrapped in liposomes (61.5±5.4%) than for vitamin D homogenized in cream (40.5±2.2%) and for Vitex D (42.7±1.7%). Vitamin D concentration in experimental cheeses was stable for 3–5 months of ripening depending on the addition method, but decreased thereafter, particularly with liposome-encapsulated vitamin D. Vitamin D concentration after 7 months of ripening was very similar for all experimental cheeses, and corresponded to approximately 60, 89 and 84% of that measured after production in cheese fortified by vitamin D in liposomes, cream, and Vitex D, respectively.     

Evaluation of increased vitamin D fortification in high-temperature, short-time-processed 2% milk, UHT-processed 2% fat chocolate milk, and low-fat strawberry yogurt

The objective of this study was to determine the effect of increased vitamin D fortification (250 IU/serving) of high-temperature, short-time (HTST)-processed 2% fat milk, UHT-processed 2% fat chocolate milk, and low-fat strawberry yogurt on the sensory characteristics and stability of vitamin D during processing and storage. Three replicates of HTST pasteurized 2% fat milk, UHT pasteurized 2% fat chocolate milk, and low-fat strawberry yogurt were manufactured. Each of the 3 replicates for all products contained a control (no vitamin D fortification), a treatment group with 100 IU vitamin D/serving (current level of vitamin D fortification), and a treatment group with 250 IU vitamin D/serving. A cold-water dispersible vitamin D₃ concentrate was used for all fortifications. The HTST-processed 2% fat milk was stored for 21 d, with vitamin D analysis done before processing and on d 0, 14, and 21. Sensory analysis was conducted on d 14. The UHT-processed 2% fat chocolate milk was stored for 60 d, with vitamin D analysis done before processing and on d 0, 40, and 60. Sensory analysis was conducted on d 40. Low-fat strawberry yogurt was stored for 42 d, with vitamin D analysis done before processing, and on d 0, 28, and 42. Sensory analysis was conducted on d 28. Vitamin D levels in the fortified products were found to be similar to the target levels of fortification (100 and 250 IU vitamin D per serving) for all products, indicating no loss of vitamin D during processing. Vitamin D was also found to be stable over the shelf life of each product. Increasing the fortification of vitamin D from 100 to 250 IU/serving did not result in a change in the sensory characteristics of HTST-processed 2% fat milk, UHT-processed 2% fat chocolate milk, or low-fat strawberry yogurt. These results indicate that it is feasible to increase vitamin D fortification from 100 to 250 IU per serving in these products.     

Short communication: Production of cottage cheese fortified with vitamin D

The availability of alternative food products fortified with vitamin D could help decrease the percentage of the population with vitamin D deficiency. The objective of this study was to fortify cheese with vitamin D. Cottage cheese was selected because its manufacture allows for the addition of vitamin D after the draining step without any loss of the vitamin in whey. Cream containing vitamin D (145 IU/g of cream) was mixed with the fresh cheese curds, resulting in a final concentration of 51 IU/g of cheese. Unfortified cottage cheese was used as a control. As expected, the cottage cheese was fortified without any loss of vitamin D in the cheese whey. The vitamin D added to cream was not affected by homogenization or pasteurization treatments. In cottage cheese, the vitamin D concentration remained stable during 3 weeks of storage at 4°C. Compared with the control cheese, the cheese fortified with vitamin D showed no effects of fortification on cheese characteristics or sensory properties. Cottage cheese could be a new source of vitamin D or an alternative to fortified drinking milk.     

Bioavailability of vitamin D from fortified process cheese and effects on vitamin D status in the elderly

We conducted 2 studies to determine the effect of vitamin D-fortified cheese on vitamin D status and the bioavailability of vitamin D in cheese. The first study was designed to determine the effect of 2 mo of daily consumption of vitamin D₃-fortified (600 IU/d) process cheese on serum 25-hydroxyvitamin D (25-OHD), parathyroid hormone (PTH), and osteocalcin (OC) concentrations among 100 older (≥60 yr) men and women. Participants were randomized to receive vitamin D-fortified cheese, nonfortified cheese, or no cheese. Serum levels of 25-OHD, PTH, and OC were measured at the beginning and end of the study. There were no differences in 25-OHD, PTH, or OC after 2 mo of fortified cheese intake. The vitamin D-fortified cheese group had a greater decrease in 25-OHD than other groups, due to higher baseline 25-OHD. A second study was conducted to determine whether the bioavailability of vitamin D₂ in cheese (delivering 5880 IU of vitamin D₂/56.7-g serving) and water (delivering 32,750 IU/250 mL) is similar and whether absorption differs between younger and older adults. The second study was a crossover trial involving 2 groups of 4 participants each (younger and older group) that received single acute feedings of either vitamin D₂-fortified cheese or water. Serial blood measurements were taken over 24 h following the acute feeding. Peak serum vitamin D and area under the curve were similar between younger (23 to 50 yr) and older (72 to 84 yr) adults, and vitamin D₂ was absorbed more efficiently from cheese than from water. These studies demonstrated that vitamin D in fortified process cheese is bioavailable, and that young and older adults have similar absorption. Among older individuals, consuming 600 IU of vitamin D₃ daily from cheese for 2 mo was insufficient to increase serum 25-OHD during limited sunlight exposure.     

Vitamin D content and variability in fluid milks from a US Department of Agriculture nationwide sampling to update values in the National Nutrient Database for Standard Reference

This study determined the vitamin D₃ content and variability of retail milk in the United States having a declared fortification level of 400 IU (10 μg) per quart (qt; 1 qt = 946.4 mL), which is 25% daily value per 8 fluid ounce (236.6 mL) serving. In 2007, vitamin D₃ fortified milk (skim, 1%, 2%, whole, and 1% fat chocolate milk) was collected from 24 statistically selected supermarkets in the United States. Additionally, 2% milk samples from an earlier 2001 USDA nationwide collection were reanalyzed. Vitamin D₃ was determined using a specifically validated method involving HPLC with UV spectroscopic detection and vitamin D₂ as an internal standard. Quality control materials were analyzed with the samples. Of the 120 milk samples procured in 2007, 49% had vitamin D₃ within 100 to 125% of 400 IU (10 μg)/qt (label value), 28% had 501 to 600 IU (12.5-15 μg)/qt, 16% had a level below the label amount, and 7% had greater than 600 IU (15 μg)/qt (>150% of label). Even though the mean vitamin D₃ content did not differ statistically between milk types, a wide range in values was found among individual samples, from nondetectable [<20 IU (0.5 μg)/qt] for one sample to almost 800 IU (20 μg)/qt, with a trend toward more samples of whole milk having greater than 150% of the labeled content. On average, vitamin D₃ in 2% milk was higher in 2007 compared with in 2001 [473 vs. 426 IU (11.8 vs. 10.6 μg)/qt].     

Estimation and fortification of vitamin D3 in pasteurized process cheese

The objective of this study was to develop methods for the estimation and fortification of vitamin D3 in pasteurized Process cheese. Vitamin D3 was estimated using alkaline saponification at 70 degrees C for 30 min, followed by extraction with petroleum ether:diethyl ether (90:10 vol/vol) and HPLC. The retention time for vitamin D3 was approximately 9 min. A standard curve with a correlation coefficient of 0.972 was prepared for quantification of vitamin D3 in unknown samples. In the second phase of the study, pasteurized Process cheeses fortified with commercial water- or fat-dispersible forms of vitamin D3 at a level of 100 IU per serving (28 g) were manufactured. There was no loss of vitamin D3 during Process cheese manufacture, and the vitamin was uniformly distributed. No losses of the vitamin occurred during storage of the fortified cheeses over a 9-mo period at 21 to 29 degrees C and 4 to 6 degrees C. There was an approximately 25 to 30% loss of the vitamin when cheeses were heated for 5 min in an oven maintained at 232 degrees C. Added vitamin D3 did not impart any off flavors to the Process cheeses as determined by sensory analysis. There were no differences between the water- and fat-dispersible forms of the vitamin in the parameters measured in fortified cheeses.     

Increasing retention of vitamin D3 in vitamin D3 fortified ice cream with milk protein emulsifier

This study aimed to develop vitamin D₃ fortified ice cream by incorporating vitamin D₃ in an emulsified form using milk protein as emulsifier. Physicochemical stability of vitamin D₃ emulsions using different milk protein emulsifiers including nonfat dry milk, sodium caseinate (Na-Cas), and whey protein isolate was investigated. Emulsion using Na-Cas had the smallest oil droplet size and the lowest creaming index throughout the storage time (P < 0.05) and was selected to fortify in full-fat, reduced-fat, and low-fat ice creams at 250 IU per serving. Vitamin D₃ retention in each ice cream was determined after 0, 7, 14, 28 and 56 d of storage at −20 °C. The results indicated that the emulsified form of vitamin D₃ remarkably improved vitamin D₃ stability in all ice cream formulations.     

Fish oil fortification of soft goat cheese

Soft goat cheese was fortified with four levels of purified fish oil (0, 60, 80, and 100 g fish oil per 3600 g goat milk) prior to curd formation to deliver high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) per serving. The cheese was evaluated for proximate composition, EPA+DHA content, oxidative stability, color, pH, and consumer acceptability. The cheese was partially vacuum packed and stored at 2 °C for four weeks. The fat content was significantly (p < 0.05) higher in the fortified treatments compared to the control, but was not significantly different among fortified treatments. Likewise, EPA+DHA contents were not significantly different among fortified samples, averaging 127 mg EPA+DHA per 28 g serving. No significant lipid oxidation was detected by thiobarbituric acid reactive substances (TBARS) or hexanal and propanal headspace analyses over the four week refrigerated shelf-life study for any treatments. The fortified cheeses were all liked 'moderately' by consumers (n = 105) for overall acceptability, although the 60 g fortification level did rate significantly higher. The control cheese and the 60 g fortification level had no significant differences in consumer purchase intent. These results demonstrate that fortification levels of up to 127 mg EPA+DHA per serving may be added to soft cheese without negatively affecting shelf-life or consumer purchase intent. PRACTICAL APPLICATION: Omega-3 fatty acids have been shown to have strong associations with health and well-being, and fish oil is a rich source of these fatty acids. In this study, goat cheese was successfully fortified to deliver 127 mg omega-3 fatty acids per 28 g serving without affecting shelf life or consumer purchase intent.     

Vitamin D3 fortification and quantification in processed dairy products

The purpose of this research was to examine the fortification and retention of vitamin D₃ added to lab-scale Cheddar cheese-like matrix, yogurt and ice cream, using two means of incorporation: pre-dissolved crystalline vitamin D₃ and emulsified vitamin D₃. An improved extraction method was developed, which yielded near total recovery from all samples, and in particular, from difficult-to-extract cheese samples. With the cheese, the loss of vitamin D₃ into whey was 7–9% (w/w). Storage stability of the vitamin in the cheeses differed: over three months, the emulsified form was more stable than the crystalline form. Conversely, both forms of vitamin D₃ were stable in experimental yogurts and ice creams during the expected shelf life of these products. These processed dairy products appear to be suitable for vitamin D₃ fortification.     

Yield and functionality of Cheddar cheese as influenced by homogenization of cream

Cheese milk was standardized (casein-to-fat ratio of 0.7) by blending 0.64% fat milk and 35% fat cream. Cream was homogenized at 0/0 MPa (CO), 3.5/3.5 MPa (H05), 6.9/3.5 MPa (H10) or 10.4/3.5 MPa (H15). Cream homogenization did not influence rennet-clotting time, but it increased rate of curd firming and increased curd firmness of cheese milk. Moisture and salt in moisture phase of cheese increased with homogenization. Moisture (37%) and salt (1.5%) adjusted yield increased 1.42, 3.44 and 3.85% in H05, H10 and H15, respectively, over CO. Homogenized treatment cheeses melted faster with age. Free oil in 1 week old cheeses was lowest in H10 and highest in H05 and increased in all treatments with age. Cheese hardness was not influenced by homogenization but decreased with age. Cheeses with homogenized cream had improved body and texture and flavor. Cream homogenized at 6.9/3.5 MPa was optimal for enhancing Cheddar cheese yield and functionality.     

Evaluation of processed cheese fortified with fish oil emulsion

Processed cheese fortified with fish oil is an excellent food for the delivery of omega-3 long-chain polyunsaturated fatty acids (omega-3 LC PUFA). However, oxidation and the “fishy” flavour of fish oil limit the level of fortification. The physical properties, lipid oxidation, and sensory perception of model processed cheese slices fortified with a fish oil emulsion (encapsulated fish oil) were examined and were compared with those of samples fortified with straight fish oil. Peroxide values, the results of thiobarbituric acid reactive substances (TBARS) tests, and propanal values showed that cheese samples fortified with fish oil emulsion had lower levels of oxidation than cheese samples fortified with non-encapsulated fish oil. A sensory panel detected a “fishy” flavour at a higher level of fish oil addition in the samples fortified with fish oil emulsion. This suggests that a fish oil emulsion made with a milk protein complex is a useful carrier for elevating the fortification level of omega-3 LC PUFA in processed cheese products.     

Vitamin Fortification of Fluid Milk

Vitamin concentrates with vitamins A and D are used for fortification of fluid milk. Although many of the degradation components of vitamins A and D have an important role in flavor/fragrance applications, they may also be source(s) of off‐flavor(s) in vitamin fortified milk due to their heat, oxygen, and the light sensitivity. It is very important for the dairy industry to understand how vitamin concentrates can impact flavor and flavor stability of fluid milk. Currently, little research on vitamin degradation products can be found with respect to flavor contributions. In this review, the history, regulations, processing, and storage stability of vitamins in fluid milk are addressed along with some hypotheses for the role of vitamin A and D fortification on flavor and stability of fluid milk.     

The effect of vitamin concentrates on the flavor of pasteurized fluid milk

Fluid milk consumption in the United States continues to decline. As a result, the level of dietary vitamin D provided by fluid milk in the United States diet has also declined. Undesirable flavor(s)/off flavor(s) in fluid milk can negatively affect milk consumption and consumer product acceptability. The objectives of this study were to identify aroma-active compounds in vitamin concentrates used to fortify fluid milk, and to determine the influence of vitamin A and D fortification on the flavor of milk. The aroma profiles of 14 commercial vitamin concentrates (vitamins A and D), in both oil-soluble and water-dispersible forms, were evaluated by sensory and instrumental volatile compound analyses. Orthonasal thresholds were determined for 8 key aroma-active compounds in skim and whole milk. Six representative vitamin concentrates were selected to fortify skim and 2% fat pasteurized milks (vitamin A at 1,500–3,000 IU/qt, vitamin D at 200–1,200 IU/qt, vitamin A and D at 1,000/200–6,000/1,200 IU/qt). Pasteurized milks were evaluated by sensory and instrumental volatile compound analyses and by consumers. Fat content, vitamin content, and fat globule particle size were also determined. The entire experiment was done in duplicate. Water-dispersible vitamin concentrates had overall higher aroma intensities and more detected aroma-active compounds than oil-soluble vitamin concentrates. Trained panelists and consumers were able to detect flavor differences between skim milks fortified with water-dispersible vitamin A or vitamin A and D, and unfortified skim milks. Consumers were unable to detect flavor differences in oil-soluble fortified milks, but trained panelists documented a faint carrot flavor in oil-soluble fortified skim milks at higher vitamin A concentrations (3,000–6,000 IU). No differences were detected in skim milks fortified with vitamin D, and no differences were detected in any 2% milk. These results demonstrate that vitamin concentrates may contribute to off flavor(s) in fluid milk, especially in skim milk fortified with water-dispersible vitamin concentrates.     

Vitamin D Stability in Milk

A method was developed to determine vitamin D₃ in milk. It includes saponification, solid phase extraction and HPLC. Recovery of added vitamin D₃ was 93%. Vitamin D₃ concentrations in commercial milks were variable. Stability studies showed that on exposure to light, there was a slight loss of vitamin D₃ from fortified milk. Air exposure did not affect stability in milk. Upon standing there was some stratification of the vitamin in milk containers with slightly more vitamin D₃ in the top layer of milk than at the bottom.     

Effect of fat content on sensory and physico‐chemical properties of laboratory‐pasteurised calcium‐ and vitamin D‐fortified mixture of cow and buffalo milk

The influence of milk fat on physico‐chemical properties of calcium and vitamin D‐fortified milk was investigated. Sensory scores, curd tension, viscosity, rennet coagulation time and TBA value increased with the increase in fat content. Calcium and vitamin D fortification had no effect on sensory scores, whereas a significant increase was observed in curd tension and viscosity. The TBA value of fortified milk was significantly lower than that of the unfortified milk. The rennet coagulation time of milk increased significantly with addition of calcium phosphate, whereas calcium citrate fortification had no significant effect. All milk samples were stable to alcohol.     

Gas production by Paucilactobacillus wasatchensis WDCO4 is increased in Cheddar cheese containing sodium gluconate

Paucilactobacillus wasatchensis can use gluconate (GLCN) as well as galactose as an energy source and because sodium GLCN can be added during salting of Cheddar cheese to reduce calcium lactate crystal formation, our primary objective was to determine if the presence of GLCN in cheese is another risk factor for unwanted gas production leading to slits in cheese. A secondary objective was to calculate the amount of CO₂ produced during storage and to relate this to the amount of gas-forming substrate that was utilized. Ribose was added to promote growth of Pa. wasatchensis WDC04 (P.waWDC04) to high numbers during storage. Cheddar cheese was made with lactococcal starter culture with addition of P.waWDC04 on 3 separate occasions. After milling, the curd was divided into six 10-kg portions. To the curd was added (A) salt, or salt plus (B) 0.5% galactose + 0.5% ribose (similar to previous studies), (C) 1% sodium GLCN, (D) 1% sodium GLCN + 0.5% ribose, (E) 2% sodium GLCN, (F) 2% sodium GLCN + 0.5% ribose. A vat of cheese without added P.waWDC04 was made using the same milk and a block of cheese used as an additional control. Cheeses were cut into 900-g pieces, vacuum packaged and stored at 12°C for 16 wk. Each month the bags were examined for gas production and cheese sampled and tested for lactose, galactose and GLCN content, and microbial numbers. In the control cheese, P.waWDC04 remained undetected (i.e., <10⁴ cfu/g), whereas in cheeses A, C, and E it increased to 10⁷ cfu/g, and when ribose was included with salting (cheeses B, D, and F) increased to 10⁸ cfu/g. The amount of gas (measured as headspace height or calculated as mmoles of CO₂) during 16 wk storage was increased by adding P.waWDC04 into the milk, and by adding galactose or GLCN to the curd. Galactose levels in cheese B were depleted by 12 wk while no other cheeses had residual galactose. Except for cheese D, the other cheeses with GLCN added (C, E and F) showed little decline in GLCN levels until wk 12, even though gas was being produced starting at wk 4. Based on calculations of CO₂ in headspace plus CO₂ dissolved in cheese, galactose and GLCN added to cheese curd only accounted for about half of total gas production. It is proposed that CO₂ was also produced by decarboxylation of amino acids. Although P.waWDC04 does not have all the genes for complete conversion and decarboxylation of the amino acids in cheese, this can be achieved in conjunction with starter culture lactococcal. Adding GLCN to curd can now be considered another confirmed risk factor for unwanted gas production during storage of Cheddar cheese that can lead to slits and cracks in cheese. Putative risk factors now include having a community of bacteria in cheese leading to decarboxylation of amino acids and release of CO₂ as well autolysis of the starter culture that would provide a supply of ribose that can promote growth of Pa. wasatchensis.     

Thermal inactivation of chymosin during cheese manufacture

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100 +/- 21 min and a z-value of 5.9 +/- 0.3 degrees C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.     

Vitamin D2 stability in milk during processing,packaging and storage

Stability of vitamin D2 in milk was determined in vitamin D2 fortified milk. Reverse phase high pressure liquid chromatography was used to determine the vitamin D₂ loss during processing, packaging and under light. The percentage losses during pasteurization, boiling and sterilization were demonstrated to be statistically insignificant. Milk was stored for seven days in both glass and plastic bottles under refrigerated temperature, non significant loss of vitamin D₂ was observed, whereas, when stored in polyethylene pouches significant loss was observed as vitamin D₂ decreased from 596.66 to 548.04 IU. This clearly indicated that vitamin D₂ was sorbed up by polyethylene material during storage resulting in its loss. Milk samples were stored for 32 h under three different light intensities (14,852,970 and 4455 lux). Non significant loss of vitamin D₂ was observed in glass packaging, whereas significant loss was observed in polyethylene pouches. In milk fortified with both calcium and vitamin D₂, non significant effect of calcium was observed on the loss of vitamin D₂.