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.     

Fortification of dairy products with vitamin D3

The purpose of this study was to analyse emulsions for vitamin D₃ delivery in yoghurt and sour cream. Oil‐in‐water emulsions stabilised by whey proteins alone and by whey proteins plus carboxymethylcellulose were used. No change in vitamin D₃ added to the yoghurt and sour cream in the form of both emulsions was observed after storage at 7 days in light and 14 days in dark at 4 °C. The results of bioavailability tests, using rats, for vitamin D₃ from the fortified emulsions and yoghurt indicated that it is feasible to use stabilised emulsions as delivery systems of vitamin D₃ in fortified products.     

The determination of vitamin D3 and 25-hydroxyvitamin D3 in early lactation and seasonal bovine milk

There is a need to account for the content of 25-hydroxyvitamin D₃ (25OH-D₃) in foods to more accurately estimate dietary vitamin D intake, given its higher biological activity. A high-performance liquid chromatography–tandem mass spectrometry method was applied to the determination of vitamin D₃ and 25OH-D₃ in bovine milk obtained during early lactation and over the course of a full milking season. In this seasonal study of bovine milk, vitamin D₃ levels ranged from 167 ng L⁻¹ in winter to 615 ng L⁻¹ in summer, whereas the content of 25OH-D₃ in bovine milk was <50 ng L⁻¹ and showed little variation. This study will provide manufacturers with data concerning endogenous vitamin D content that will enhance formulation capability related to the production of bovine-milk-based paediatric products.     

Short communication: Technological and seasonal variations of vitamin D and other nutritional components in donkey milk

Vitamin D is an essential nutrient that plays a crucial role in calcium homeostasis and bone metabolism and also acts as a hormone. Although several studies on the content of vitamin D in bovine milk have been conducted, little information is available regarding donkey milk. In the context of the nutritional assessment of donkey milk, the aim of this study was to assess the vitamin D content in donkey milk and its chemical profile, with particular reference to seasonal and technological modifications after pasteurization. The study was conducted on a dairy farm that produces donkey milk for human consumption located in central Italy. At sampling time, an aliquot of total bulk milk production was sampled before and after pasteurization (63°C for 30 min without homogenization) with a total of 20 raw and 20 pasteurized milk samples. The samples were collected for 10 mo, every 15 d, from May to February 2017. All the samples were analyzed for the chemical composition and vitamin D₂ and D₃ content by HPLC after saponification. The donkey milk analyzed showed a higher average vitamin D content (raw milk: vitamin D₂ = 1.68, vitamin D₃ = 0.60 μg/100 mL; pasteurized milk: vitamin D₂ = 1.38, vitamin D₃ = 0.30 μg/100 mL) than reported for bovine and human milk. The results of the effect of pasteurization on milk did not highlight significant differences in the total content of vitamin D. However, vitamin D₃ has a poor thermal stability, which led to a significant reduction in content in pasteurized milk compared with raw milk. The total vitamin D content of donkey milk did not show significant variations between seasons; however, a higher concentration of vitamin D₃ was found in spring and summer. In conclusion, raw and pasteurized donkey milk showed a high content of vitamin D, which could be useful in meeting the deficiencies of this vitamin in humans. Further investigations are needed to improve the vitamin D content in donkey milk by increasing its endogenous synthesis or its transfer in milk and to clarify other variability factors.     

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.     

Production of novel vitamin D3 loaded lipid nanocapsules for milk fortification

Lipid nanoparticles are carriers to improve stability, solubility, and efficacy of bioactive compounds. In this paper, novel vitamin D₃ loaded lipid nanocapsules (LNC) were produced by phase inversion method. The produced nanocapsules were characterised by particle size, polydispersity index, zeta potential, encapsulation efficiency, and encapsulation load. LNC showed sizes in the range of 31.43 to 36.66 nm. Optimum LNC formulation was selected for further analysis (such as morphological study, analysis of chemical structure, release study, and sensory evaluation). Transmission electron microscopy revealed that particles had approximately spherical shape. The Fourier transform infrared spectra indicated that no adverse reactions occurred between vitamin D₃ and lipid nanocapsules. About 9.6% of vitamin released in gastric simulated solution (pH: 1.2), which indicated that LNC can protect vitamin against acidic conditions. Sensory evaluation revealed the potential application of produced vitamin D₃ loaded LNC for development of fortified milk.     

Intramammary 25-hydroxyvitamin D3 treatment modulates innate immune responses to endotoxin-induced mastitis

Vitamin D signaling in response to pathogen-associated molecules contributes to activation of innate immune responses of bovine monocytes. We hypothesized that lipopolysaccharide (LPS) of bacteria associated with mastitis in dairy cows activates the vitamin D pathway in innate immune cells of the udder and that increasing availability of 25-hydroxyvitamin D₃ [25(OH)D₃] would augment expression of vitamin D-associated genes. The objective of this experiment was to determine the effects of intramammary LPS and 25(OH)D₃ treatments on activation of the vitamin D pathway and innate immune responses of mammary immune cells. Individual mammary quarters of 5 lactating cows were treated with placebo control, 100 μg of 25(OH)D₃, 5 μg of LPS, or a combination of 100 μg of 25(OH)D₃ and 5 μg of LPS. Somatic cells from milk were evaluated for percentage of neutrophil and macrophage populations and expression of genes associated with vitamin D metabolism and innate immunity. Data from samples collected from 4 to 12 h after challenge were analyzed for main effects of LPS and 25(OH)D₃ treatments, treatment interactions, and simple effects of 25(OH)D₃ treatment. Data from samples collected at the time of challenge were used as covariates. The percentages of neutrophils in milk at 8 h postchallenge were 58 ± 10, 82 ± 11, 89 ± 10, and 63 ± 10% of total cells in milk from control, 25(OH)D₃, LPS, and LPS plus 25(OH)D₃ glands, respectively, such that the interaction of LPS and 25(OH)D₃ was significant. Expression of the vitamin D 1α-hydroxylase (CYP27B1) and vitamin D receptor genes was upregulated by LPS treatment in total cells, macrophages, and neutrophils in milk. In addition, expression of the vitamin D 24-hydroxylase (CYP24A1) gene in milk somatic cells was upregulated by 25(OH)D₃ and LPS treatments. The inducible nitric oxide synthase (iNOS), chemokine (C-C-motif) ligand 5 (CCL5), β-defensin 3 (DEFB3), DEFB7, and DEFB10 genes were upregulated by LPS treatment in total cells and neutrophils from milk. Expression of iNOS in milk somatic cells tended to be affected by the interaction between LPS and 25(OH)D₃, such that 25(OH)D₃ tended to increase iNOS in the absence of LPS but not in the presence of LPS. Furthermore, expression of CCL5 in macrophages was downregulated by 25(OH)D₃. In conclusion, intramammary endotoxin challenge activates the vitamin D pathway in mammary macrophages and neutrophils, and intramammary 25(OH)D₃ treatment alters the percentage of neutrophils and expression of immune genes in milk somatic cells.     

Increased dietary vitamin D3 and calcium partially alleviate heat stress symptoms and inflammation in lactating Holstein cows independent of dietary concentrations of vitamin E and selenium

Twelve multiparous Holstein cows (42.2 ± 5.6 kg of milk/d; 83 ± 27 d in milk) were used in a split-plot design testing the effects of mineral and vitamin supplementation on the time course of animal performance, metabolism, and inflammation markers during heat stress. The main plot was the average concentrations of dietary vitamin E and Se (adequate: 11.1 IU/kg of vitamin E and 0.55 mg/kg of Se, and high: 223 IU/kg of vitamin E and 1.8 mg/kg of Se, respectively). Within each plot, cows were randomly assigned to (1) heat stress (HS) with adequate concentrations of vitamin D₃ and Ca (1,012 IU/kg and 0.73%, respectively), (2) HS with high concentrations of vitamin D₃ and Ca (HS+D₃/Ca; 3,764 IU/kg and 0.97%, respectively), or (3) pair-feeding (PF) in thermoneutrality with adequate concentrations of vitamin D₃ and Ca (1,012 IU/kg and 0.73% Ca) in a Latin square design with 14-d periods and 7-d washouts. The highest rectal temperature was recorded at 1700 h for HS (39.4°C; mean of d 1 to 14), being 1.2 and 0.8°C greater than for PF and HS+D₃/Ca, respectively. Respiratory rate and water intake were higher in HS (73 breaths/min and 115 L/d, respectively) relative to PF (28 breaths/min and 76 L/d). Heat stress decreased dry matter intake progressively, reaching a nadir on d 5 to 7 (33% reduction) and was not different between treatments. Milk yield decreased progressively in all treatments, but remained greater in PF relative to HS from d 3 to 14 (10%), whereas HS and HS+D₃/Ca were not different. Milk fat, protein, and lactose concentrations and yields were lower in HS relative to PF from d 3 to 14, but not different between HS and HS+D₃/Ca. Relative to PF, preprandial insulin concentrations were increased in HS, whereas plasma nonesterified fatty acids were decreased on d 7 and 14. Plasma lipopolysaccharide-binding protein concentrations increased in HS cows on d 7 and 14, respectively, relative to PF, whereas they were reduced in HS + D₃/Ca on d 14. Plasma C-reactive protein, tumor necrosis factor-α, and fecal calprotectin were increased in HS relative to both PF and HS+D₃/Ca on d 7 and 14. Rectal temperature was positively associated with plasma lipopolysaccharide-binding protein (r = 0.72), tumor necrosis factor-α (r = 0.74), C-reactive protein (r = 0.87), and with milk somatic cells (r = 0.75). Plasma 8-hydroxy-2-deoxyguanosine concentrations presented a 3-way interaction, where 8-hydroxy-2-deoxyguanosine was lower in HS than in PF on d 7 and 14, and lower in HS+D₃/Ca relative to HS on d 14 in the adequate vitamin E and Se treatment, but no effects were observed in the high vitamin E and Se group. Plasma superoxide dismutase concentrations increased over time, and were higher in HS relative to PF on d 14, whereas HS+D₃/Ca was similar to HS. Heat stress markedly reduced milk production and milk components while increasing markers of leaky gut and inflammation. In contrast, vitamin D₃ and Ca supplementation reduced hyperthermia (d 7–14), markers of leaky gut, and inflammation independent of dietary concentrations of vitamin E and Se.     

The effect of vitamin D(3) supplementation on texture and oxidative stability of beef loins from steers treated with zilpaterol hydrochloride

In this study, 10 young steers received no beta agonist (C), 50 animals all received zilpaterol hydrochloride (Z), with 1 group receiving Z while the other 4 groups received Z and vitamin D₃ at the following levels (IU/animal /day) and durations before slaughter: 7 million for 3 days (3D7M) or 6 days (6D7M), 7 million for 6 days with 7 days no supplementation (6D7M7N) and 1 million for 9 days (9D1M). LD samples were vacuum-aged 14 days post mortem, and repacked in high-oxygen modified atmosphere (70% O₂/30% CO₂) for a further 7 days. Parameters included Warner Bratzler shear force (WBSF), myofibril fragment length, thiobarbituric acid reactive substances, free thiol levels, muscle fat, and instrumental colour parameters. When allowing for a conversion period of vitamin D₃ to its active metabolites, supplementing the zilpaterol treated steers with a high dosage of vitamin D₃ (6D7M7N) resulted in improved colour stability, higher stability towards protein oxidation and lower stability towards lipid oxidation.     

The determination of vitamin D3 in bovine milk by liquid chromatography mass spectrometry

A renewed international interest in vitamin D status has revealed significant deficiencies in several populations, including Australia. Vitamin D exists in two forms, cholcalciferol (D₃) and ergocalciferol (D₂). The main source of vitamin D₃ is from exposure of 7-dehydrocholesterol present in the skin to UV irradiation. However, there is an absolute requirement for vitamin D through proper dietary intake if humans live in the absence of sunlight or exclusively indoors. Bovine milk is considered to be a good dietary source of vitamin D₃, even though the levels are quite low. This paper describes robust methods using liquid chromatography–linear ion trap mass spectrometry (LC–MSⁿ) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to measure the levels of vitamin D₃ in fresh bovine milk (0.05 μg/100 ml), commercial (natural and fortified) milk samples (0.01–2 μg/100 ml) and a dairy based infant formula (8 μg/100 g), without the need for extensive clean-up procedures. The limits of quantification (LOQ) are 0.01 μg/100 ml and 0.02 μg/100 ml for LC–MSⁿ and LC–MS/MS, respectively. Recoveries of vitamin D₃ added to the samples prior to saponification were satisfactory (range 60–90%). 25-Hydroxyvitamin D₃ was not present in any of the samples analysed (LOQ = 0.01 μg/100 ml, recovery range 30–40%).     

Nanoliposomes coencapsulating curcumin and vitamin D3 produced by hydration of proliposomes: Effects of the phospholipid composition in the physicochemical characteristics of vesicles and after incorporation in yoghurts

Nanoliposomes coencapsulating curcumin and vitamin D₃ (VD3) using different compositions of purified and unpurified lecithins were produced by hydration of proliposomes and characterised over 15 days of storage. The dispersions were incorporated to pineapple yoghurts produced in laboratory‐scale, which were also characterised. Results showed that curcumin and vitamin D₃ were retained in the nanovesicles up to 86.1% and 94.1%, respectively. Most of the parameters assessed for the yoghurt samples were within the limits required by the Brazilian legislation and the Codex Alimentarius. Finally, the enriched product was well accepted by the panellists with the purchase intention ranging from 53 to 75%.     

Influence of ice-cream mix components on the thermal stability of bovine milk γ-glutamyl transpeptidase and Listeria innocua

The influence of various components of ice cream mixes (milk fat, milk-solids nonfat, sucrose, stabilizer) on the thermal stability of bovine milk γ-glutamyl transpeptidase (TP) and Listeria innocua (LN) was determined. A factorial Screening Design experiment revealed that D values for TP at 71, 74, and 77 °C were significantly (P ≤ 0.05) increased in ice-cream mixes over the values found in whole milk, with an average 8.9-fold increase for D₇₁. In contrast, D values for LN at 60, 63, and 66 °C were not significantly influenced by mix components, although an average 2.4-fold increase was found for D₆₀ indicating that mix components exert some protective effect toward LN. Likewise, the TP z-value was not affected by the components of the mix. The Screening Design was augmented into a Central Composite Design and a response surface model for TP was derived for log₁₀D₇₁. A total of 90% of the variation in the data was explained by the model, which incorporated linear, quadratic and interactive effects of the four mix components.     

Effects of storage conditions,microencapsulation and inclusion in chocolate particles on the stability of probiotic bacteria in ice cream

Three technological approaches to enhance viable counts of probiotic bacteria in ice cream were examined: post-freezing inoculation, use of a microencapsulated culture (spray-coating technology) and inclusion of cultures in chocolate or tablet particles. When a free-cell powder (FCP) of Bifidobacterium longum R0175 was added to the soft ice cream before hardening, a drop of almost 3 log cfu g⁻¹ occurred during production and storage, while it was of only 0.43 log cfu g⁻¹ for Lactobacillus rhamnosus R0011. However, inoculation with a powder of microencapsulated cells (MEP) improved stability of B. longum. The viability of probiotics was further improved when the MEP was incorporated into chocolate particles, which were subsequently blended into the ice cream. Viability losses of the FCP culture during storage at −16 °C in a household freezer having periodic defrost cycles were 10 times higher than when constantly maintained at −20 °C.     

Producing ice cream using a substantial amount of juice from kiwifruit with green,gold or red flesh

Ice cream prepared using a substantial amount of juice from kiwifruit with green, gold or red flesh may have consumer appeal, through the combination of kiwifruit's unique color, natural flavor and health-promoting constituents. The aqueous fractions from purees of kiwifruit with green, gold and red flesh (AFKWs) were added at 49% v/v to a basic low-fat ice cream mix that contained no commercial flavoring and coloring agents. The resultant ice creams were subjected to comparative product evaluation (e.g. overrun, melting behavior and rheological properties) and chemical analyses of bioactives (e.g. total extractable polyphenol content (TEPC), vitamin C, antioxidant capacity, polyphenol (PP) and carotenoid composition). Results revealed that both the pH pre-adjustment and pre-heating of the AFKW played critical roles in ice cream making. The ice creams retained the PP and vitamin C contents as well as natural color flavor of the kiwifruit used. The rheological properties of all ice creams showed non-Newtonian flow behavior, and the storage modulus G′ decreased in the same pattern following the order of green > gold > red. The melting rate, overrun and vitamin C content of the ice cream with green AFKW were the fastest, lowest and least, respectively. The TEPC and antioxidant capacity in the ice cream with red AFKW were the highest. The amounts of PPs and vitamin C were encouragingly high. Health beneficial compounds, dimethyl-caffeic acid hexoside, caffeic acid derivatives, protocatechuic acid, syringic acid, salicylic acid/ο-coumaric acid, lutein and beta-carotene, were detected in the final products. Thus, there are commercial possibilities for using AFKW which should be further evaluated.     

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.     

The influence of latitude on the concentration of vitamin D3 and 25-hydroxy-vitamin D3 in Australian red meat

There is little information on the vitamin D content of Australian red meat or on the possible influence of latitude on this content. To determine the content of vitamin D₃ and 25-hydroxy-vitamin D₃ (25OHD₃), lamb and beef were analysed from 34° S with LC–IT-MS. To investigate the possible influence of latitude on vitamin D in meat, the lean meat and fat from five cuts of beef were analysed from 17° S and 41° S. Lamb contained 0.10 μg vitamin D₃/100 g and 0.20 μg 25OHD₃/100 g lean meat, while beef contained 0.12 μg vitamin D₃ and 0.27 μg 25OHD₃/100 g (lean meat). Latitude had no effect on the vitamin D₃ (P = 0.21) or 25OHD₃ (P = 0.29) content of lean beef, but fat from cattle in the 17° S latitude group contained significantly higher (P < 0.01) concentrations of vitamin D₃ than fat from the 41° S group of cattle.     

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].     

Performance of whey protein hydrolysate–maltodextrin conjugates as emulsifiers in model infant formula emulsions

Model infant formula emulsions containing 15.5, 35.0 and 70.0 g L⁻¹ protein, soybean oil and maltodextrin (MD), respectively, were prepared. Emulsions were stabilised by whey protein hydrolysate (WPH) + CITREM (9 g L⁻¹), WPH + lecithin (9 g L⁻¹) or WPH conjugated with MD (WPH–MD). All emulsions had mono-modal oil droplet size distributions post-homogenisation with mean oil droplet diameters (D_4,3) of <1.0 μm. No changes in the D_4,3 were observed after heat treatment (95 °C, 15 min) of the emulsions. Accelerated storage (40 °C, 10 d) of unheated emulsions resulted in an increase in D_4,3 for CITREM (2.86 μm) and lecithin (5.36 μm) containing emulsions. Heated emulsions displayed better stability to accelerated storage with no increase in D_4,3 for CITREM and an increase in D_4,3 for lecithin (2.71 μm) containing emulsions. No increase in D_4,3 over storage was observed for unheated or heated WPH–MD emulsion, indicating its superior stability.     

Effects of dietary supplementation of vitamins D(3) and E on quality characteristics of pigs and longissimus muscle antioxidative capacity

The effects of addition of vitamin D₃ and vitamin E to pig diets on blood plasma calcium concentration, meat quality (longissimus muscle) and antioxidative capacity were investigated. Two treatments consisted of supplementation with vitamin D₃ (500,000 IU/d) for 5 days separately (group D) and a combination of vitamin E (500 mg α-tocopheryl acetate/kg diet) for 30 days and vitamin D₃ (500,000 IU/d) for 5 days (group D + E) to growing-finishing pigs before slaughter. Pigs fed with vitamin D₃ had higher (P < 0.01) plasma calcium concentration compared with control pigs. Dietary supplementation of vitamin E significantly (P < 0.05) increased the concentration of α-tocopherol in meat (longissimus muscle). Vitamin D₃ supplementation resulted in higher (P = 0.07) a^∗ values of loin chops at 5 days of storage. Vitamin D₃ and vitamin E supplementation did not affect other meat quality characteristics or tenderness (quantified by Warner–Bratzler shear force). Antioxidative capacity (measured as MDA production after incubation of longissimus muscle homogenates with Fe²⁺/ascorbate) was improved by vitamin E and partly by vitamin D₃ supplementation.