Physicochemical and sensory properties of milk supplemented with lactase microcapsules coated with enteric coating materials.

In this paper, we report the physicochemical and sensory properties of milk supplemented with a powder of microencapsulated lactase. The core material was lactase (ß-galactosidase), the primary coating material was medium-chain triglyceride (MCT), and the secondary (enteric) coating material was either hydroxypropyl methylcellulose phthalate (HPMCP) or shellac, comparing both against market milk as a control. The physicochemical properties of both types of microcapsules were analyzed, including the particle size, zeta potential, and in vitro release behavior. To survey the stability of the microcapsules in milk during storage, we studied the residual lactose content and pH. Furthermore, to determine the properties of milk supplemented with the microcapsules, changes in color and sensory properties were evaluated during storage. The particle sizes (volume-weighted mean; D[4,3]) of the microcapsules coated with HPMCP or shellac were 2,836 and 7,834 nm, respectively, and the zeta potential of the capsules coated with shellac was higher than the zeta potential of those coated with HPMCP. The pH levels of milk supplemented with the lactase microcapsules were similar to those of the control (unsupplemented market milk); however, for milk supplemented with HPMCP-coated microcapsules, the pH was slightly lower. The core material, lactase, was released from the microcapsules during 12-d storage, and 18.82 and 35.09% of lactose was hydrolyzed in the samples for HPMCP- and shellac-coated microcapsules, respectively. The sensory characteristics of milk containing microcapsules coated with HPMCP did not show significant differences from the control, in terms of sweetness or off-taste, until 8 d of storage. However, shellac-coated microcapsules showed significant difference in sweetness and off-taste at d 8 and 6 of storage, respectively. The color of milk containing HPMCP-coated microcapsules did not show a significant difference during storage. However, that containing shellac-coated microcapsules was somewhat higher in color values than others. In particular, it showed significance from 0 to 4 d storage in L* and C* values. In conclusion, a powder of lactase microcapsules coated with HPMCP can be suitable as a supplement for milk.

The preventive effects of nanopowdered red ginseng on collagen-induced arthritic mice.

This study was carried out to investigate the efficiency of red ginseng nanopowder in preventing collagen-induced arthritis (CIA) in mice. The mice were divided into five groups: normal group (no immunisation), control (CIA), powdered red ginseng (PRG), nanopowdered red ginseng (NRG) and methotrexate (MTX). Administering MTX, PRG and NRG to arthritic mice significantly decreased spleen indexes, clinical and histological scores compared to control group. Serum analysis of NRG and MTX groups showed a reduction in the cytokines such as the levels of tumour necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1ß (IL-1ß) in comparison to PRG group. The levels of immunoglobulin M (IgM) and immunoglobulin G1 (IgG1) in the NRG group were significantly lower than those of the PRG group. In summary, the present study indicated that NRG can be effective in preventing type II collagen-induced rheumatoid arthritis in mice.

Design of microemulsion system suitable for the oral delivery of poorly aqueous soluble beta-carotene.

Beta-carotene is a potent antioxidant for maintaining human health. However, its oral absorption is low due to poor aqueous solubility of less than 1 µg/ml. A microemulsion delivery system was designed to solubilize beta-carotene toward enhancing its oral bioavailability. From seven pseudoternary diagrams constructed, three systems were selected with large microemulsion areas suitable for oral administration and dilution in the predominately aqueous gastrointestinal fluids. Conductivity and rheology characterization were conducted along four dilution lines within the selected systems. Three pseudoternary-phase diagrams were selected with large microemulsion regions, >60% of the total phase diagram area, which provide microemulsions with higher drug-loading capacity. A phenomenon was observed by which both propylene glycol and Capmul MCM EP stabilize the microstructure of the microemulsions has been proposed based on the characterization studies. An optimal bicontinuous microemulsion formulation was selected comprising 12% orange oil, 24% Capmul MCM, 18% Tween 20, 6% Labrasol, 20% propylene glycol and 20% water, with a high beta-carotene loading capacity of 140.8 µg/ml and droplet size of 117.4 nm. In conclusion, the developed novel microemulsion formulation allows solubilizing beta-carotene and is a promising basis for further development as a functional beverage.

Highly bioavailable nanocalcium from oyster shell for preventing osteoporosis in rats.

Oyster shell is one of the foremost natural sources of calcium and is used as an alternative treatment for osteoporosis. In this study, we demonstrated that zinc-activated nanopowdered oyster shell (Zn-NPOS) effectively reduced bone loss compared with powdered oyster shell (POS) in an ovariectomized rat (OVX) model. As a result of nanosizing, the solubility and bioavailability of the oyster shell were greatly improved, and its effectiveness was further enhanced by zinc activation. Bone analysis indicated greater recovery from ovariectomy-induced bone loss following Zn-NPOS treatment. Moreover, Zn-NPOS treatment resulted in higher bone strength and superior trabecular architecture compared with NPOS and POS treatments. Furthermore, Zn-NPOS showed greater efficiency in increasing bone formation and reducing bone resorption markers. Therefore, nanosizing with zinc activation could be a viable strategy for improving the efficiency of oyster shells used for osteoporosis prevention.

Physicochemical and Sensory Properties of Red Ginseng Extracts or Red Ginseng Hydrolyzates-added Asiago Cheese during Ripening.

This study was carried out to investigate physicochemical properties of different concentrations (0.1%, 0.3%, and 0.5%) of red ginseng hydrolyzates (RGH)- or red ginseng extract (RGE)-added Asiago cheeses (AC) during ripening at 14°C for 4 months. The moisture content significantly increased with increasing concentrations of both RGH- and RGE- added AC (p<0.05). While RGHAC and RGEAC were more yellow and darker with increasing concentrations than that of control (p<0.05), the color was not influenced from the hydrolysis. In texture analysis, hardness, cohesiveness, and chewiness of RGHAC and RGEAC significantly decreased compared to the control during the ripening (p<0.05). In sensory analysis, bitterness and ginseng flavor and taste scores increased significantly with increasing the concentrations of RGH and RGE during ripening (p<0.05). In conclusion, the addition of RGH and RGE into cheese slightly influenced the properties of Asiago cheese, and similarities were observed between RGHAC and RGEAC. Thus, the lower concentrations (0.1% to 0.3%) of RGH and RGE added to AC were preferred for color, texture, and sensory during the ripening, therefore, these cheeses would be worth developing commercially.

Physicochemical, microbial, and sensory properties of nanopowdered eggshell-supplemented yogurt during storage.

This study was carried out to investigate the possibility of adding nanopowdered eggshell (NPES) into yogurt to improve the functionality of yogurt and the effects of adding NPES on the physicochemical, microbial, and sensory properties of the products during storage. The pH and mean lactic acid bacteria counts of NPES-added (0.15-0.45%, wt/vol) yogurt ranged from 4.31 to 4.66 and from 6.56 × 10(8) to 8.56 × 10(8)cfu/mL, respectively, whereas these values ranged from 4.13 to 4.44 and 8.46 × 10(8) to 1.39 × 10(9), respectively, for the control samples during storage at 5 °C for 16d, which indicates a prolonged shelf-life with NPES-supplemented yogurt. Color analysis showed that the lightness (L*) and position between red and green (a*) values were not significantly influenced by the addition of NPES. However, the position between yellow and blue (b*) value significantly increased with the addition of the concentration (0.45%, wt/vol) of NPES at d 16 of storage. Sensory evaluation revealed that NPES-added yogurts showed a notably less sourness score and a higher astringency score than the control. An earthy flavor was higher in 0.45% NPES-supplemented yogurt compared with the control. Based on the results obtained from the current study, the concentration (0.15 to 0.30%, wt/vol) of NPES can be used to formulate NPES-supplemented yogurt without any significant adverse effects on the physicochemical, microbial, and sensory properties.

Physicochemical and biofunctional properties of crab chitosan nanoparticles.

The physicochemical and biofunctional properties of crab chitosan nanoparticles of two different sizes (Nano A and B) manufactured by dry milling method were evaluated for commercialization. The deacetylation degrees (DD) of Nano A, B and the control chitosan were 90.9, 93.0, and 92.7% respectively whereas their molecular weights (M(w)) were 43.9, 44.7 and 208.8 kDa. The average sizes of the dispersed Nano A, B and the control chitosan in cetyltrimethylammonium chloride were 735.9, 849.4 and 2,382.4 nm, respectively, which were lower than 1441.7, 2935.6 and 6832.9 nm of the intact chitosans. Chitosan nanoparticles had mild tyrosinase, antioxidant and angiotensin I converting enzyme (ACE), but weak collagenase, elastase and beta-glucuronidase inhibitory activity. However, Nano A had strong alpha-glucosidase inhibitory activity, which was comparable to that of acarbose, a commercial alpha-glucosidase inhibitor. In addition, the minimum inhibitory concentrations (MICs) of chitosan and its nanoparticles ranged from 30 to > 200 microg/mL against each four gram-positive and gram-negative bacteria. Therefore, crab chitosan nanoparticles could be used as a nutraceutical, cosmeceutical or pharmaceutical product.

Optimization of water-in-oil-in-water microencapsulated ß-galactosidase by response surface methodology.

This study was carried out to determine the optimum conditions for water-in-oil-in-water (W/O/W) microencapsulated lactase (ß-galactosidase) in order to prevent the intolerance of lactose in milk. The core material was lactase and the coating materials were medium-chain triglyceride for W/O phase, and whey protein isolate (WPI), maltodextrin, gum arabic, and its mixtures for W/O/W phase. Polyglycerol polyricinoleate (PGPR) was used as a primary emulsifier, and polyoxyethylene sorbitan monolaurate (PSML) was selected as a secondary emulsifier based on emulsion stability index. To determine the most efficient conditions for the W/O/W-lactase microencapsulation, the ratio of core to coating materials and amounts of emulsifiers were investigated by response surface methodology. The optimum ratio of core to coating materials in W/O, amount of PGPR, ratio of core to coating material in W/O/W, and amount of PSML were found to be 0.5-9.5, 0.75% (w/v), 1.7-8.3, and 0.25% (w/v), respectively. The average size of the microcapsules was about 10 µm under optimum conditions. Microcapsules of 30% (w/v) WPI as a secondary coating material could evenly distribute the pocket of lactase. Based on the data obtained from this study, lactase microcapsules could effectively be produced by the method of W/O/W double emulsion.

Comparison of Physicochemical and Sensory Properties between Cholesterol-removed Gouda Cheese and Gouda Cheese during Ripening.

This study was performed to compare physicochemical and sensory properties of cholesterol-removed Gouda cheese (CRGC) and Gouda cheese made in the laboratory during ripening. Composition, short-chain free fatty acids (SCFFA), texture, color, and sensory properties were measured. In chemical composition analyses, moistures were significantly different between control cheeses (42.86%) and sample cheese (48.32%) (p<0.05). But fat and protein in the control and the sample were 32.77, 22.45 and 31.35, 20.39%, respectively, and were not significantly different (p>0.05). The amount of cholesterol in control was 82.52 mg/100 g and the percentage of cholesterol removal was 90.7%. SCFFA increased gradually during ripening and its level of CRGC increased and significantly different from that of control (p<0.05). The texture, hardness, gumminess, and chewiness were significantly increased, but cohesiveness and springiness were not increased in both cheeses during ripening periods (p>0.05). In comparison of the control and sample cheeses, hardness, and springiness were not significantly different, but cohesiveness, gumminess, and chewiness were different (p<0.05). In color measurement, all color values were not different between CRGC and control (p>0.05). However, L* value decreased, while a* and b* values tended to increase significantly (p<0.05). In sensory properties, appearance, aroma, flavor and taste, and texture were significantly increased except buttery and nutty in aroma and sweetness in taste in both cheeses, and were not significantly different between the control and sample cheeses during ripening (p>0.05). Therefore, this study suggests that the quality of cholesterol-removed Gouda cheese is not different from the control cheese.

Single- and 14-day repeat-dose toxicity of cross-linked ß-cyclodextrin in rats.

The present study was carried out to investigate the oral single and repeat toxicity of cross-linked ß-cyclodextrin (ß-CD) made with adipic acid. Ten each of female and male rats were orally administered a 5000 mg/kg single dose. At this dose, mortality or macroscopic changes of internal organs were not observed. Dose levels 500, 1000, or 2000 mg/kg of ß-CD were given daily to 5 each of female and male rats by oral gavage for 14 days. Body and organ weights were not significantly different during the study (P < .05). Hematology and clinical chemistry did not show any toxicity from the cross-linked ß-CD. Macroscopic or microscopic changes were not observed between the controls and the treated rats. Based on these results, the cross-linked ß-CD did not produce any signs of toxicity or other adverse effects at dose levels up to 2000 mg/kg per d for 14 days.

Physicochemical, Microbial, and Sensory Properties of Queso Blanco Cheese Supplemented with Powdered Microcapsules of Tomato Extracts.

The present study examined the physical, chemical, microbial, and sensory characteristics of Queso Blanco cheese supplemented with powdered microcapsules containing tomato extracts (0.5-2.0%) during storage at 7°C for 60 d. The lactic acid bacterial count and lycopene concentrations in Queso Blanco cheese supplemented with powdered microcapsules were significantly higher than those of the control. In a texture analysis, the gumminess, chewiness, and hardness values for Queso Blanco cheese were significantly higher with increasing concentrations of the powdered microcapsules containing tomato extracts. Total short-chain fatty acids in Queso Blanco cheese supplemented with powdered microcapsules containing tomato extracts were not significantly altered compared to the control. Sensory evaluation scores for the yellowness, tomato taste, and firmness of Queso Blanco cheese were significantly higher after supplementation with powdered microcapsules containing tomato extracts.

Physicochemical and Sensory Properties of Appenzeller Cheese Supplemented with Powdered Microcapsule of Tomato Extract during Ripening.

The objective of this study was to determine the physicochemical and sensory properties of Appenzeller cheese supplemented with different concentrations (0, 1, 2, 3, and 4%, w/w) of powdered microcapsules of tomato extracts (PMT) during ripening at 14℃ for 6 mon. The particle sizes of PMT ranged from 1 to 10 m diameter with an average particle size of approximately 2 m. Butyric acid (C4) concentrations of PMT-added Appenzeller cheese were significantly higher than that of the control. Lactic acid bacteria counts in the cheese were not significantly influenced by ripening time from 0 to 6 mon or the concentrations (0-4%, w/w) of PMT. In terms of texture, the hardness of PMT-added Appenzeller cheese was significantly increased compared to the control. The gumminess and chewiness of PMT-added Appenzeller cheese were similar to those of the control. However, both cohesiveness and springiness of PMT-added Appenzeller cheese were slightly decreased. In sensory analysis, bitterness and sourness of Appenzeller cheese were not significantly changed after supplementation of PMT, but sweetness of the cheese was significantly increased after increasing the ripening time from 0 to 6 mon and increasing the concentration from 1 to 4% (w/w). Based on these results, the addition of the concentrations (1-4%, w/w) of PMT to Appenzeller cheese can be used to develop functional Appenzeller cheese.

Physicochemical properties of whey protein, lactoferrin and Tween 20 stabilised nanoemulsions: Effect of temperature, pH and salt.

Oil-in-water nanoemulsions were prepared by emulsification and solvent evaporation using whey protein isolate (WPI), lactoferrin and Tween 20 as emulsifiers. Protein-stabilised nanoemulsions showed a decrease in particle size with increasing protein concentration from 0.25% to 1% (w/w) level with Z-average diameter between 70 and 90 nm. However, larger droplets were produced by Tween 20 (120-450 nm) especially at concentration above 0.75% (w/w). The stability of nanoemulsions to temperature (30-90°C), pH (2-10) and ionic strength (0-500 mM NaCl or 0-90 mM CaCl2) was also tested. Tween 20 nanoemulsions were unstable to heat treatment at 90°C for 15 min. WPI-stabilised nanoemulsions exhibited droplet aggregation near the isoelectric point at pH 4.5 and 5 and they were also unstable at salt concentration above 30 mM CaCl2. These results indicated that stable nanoemulsions can be prepared by careful selection of emulsifiers.

Application of microencapsulated isoflavone into milk.

This study was designed to develop a microencapsulated, water-soluble isoflavone for application into milk and to examine the hypocholesterolemic effect of such a milk product in a rat diet. The coating material was medium-chain triglyceride (MCT) and the core material was water-soluble isoflavone. The microencapsulation efficiency was 70.2% when the ratio (w/w) of coating material to core material was 15:1. The isoflavone release from the microcapsules was 8% after 3-day storage at 40 degrees C. In in vitro study, 4.0-9.3% of water-soluble isoflavone in simulated gastric fluid was released in the pH range of 2 to 5 after 60 min incubation; however, in simulated intestinal fluid at pH 8, 87.6% of isoflavone was released from the capsules after 40 min incubation time. In sensory analysis, the scores of bitterness, astringency, and off-taste in the encapsulated isoflavone-added milk were slightly, but not significantly, different from those in uncapsulated, isoflavone-added milk. In blood analysis, total cholesterol was significantly decreased in the isoflavone-added group compared with that in the control after 6-week feeding. Therefore, this study confirmed the acceptability of MCT as a coating material in the microencapsulation of water-soluble isoflavone for application into milk, although a slight adverse effect was found in terms of sensory attributes. In addition, blood total cholesterol was lowered in rats which had been fed a cholesterol-reduced and microencapsulated, isoflavone-added milk for 6 weeks.

Antioxidant activity and bioaccessibility of size-different nanoemulsions for lycopene-enriched tomato extract.

Lycopene nanoemulsions were prepared to protect the antioxidant activity and improve the bioaccessibility of lycopene-enriched tomato extract (containing 6% of lycopene) by an emulsification-evaporation method. Lycopene nanoemulsions, with droplet sizes between 100 and 200 nm, exhibited higher anti-radical efficiency and antioxidant activity, than did those smaller than 100 nm. Strong protectability of lycopene in droplets smaller than 100 nm was associated with relatively slower rates of DPPH and ABTS reactions. In vitro bioaccessibility values of lycopene-enriched tomato extract, lycopene nanoemulsions with droplets larger than 100 nm (approximately 150 nm on average), and lycopene nanoemulsions with droplets smaller than 100 nm (69 nm on average) were 0.01, 0.53, and 0.77, respectively. Interestingly, nanoemulsions with droplets smaller than 100 nm showed the highest in vitro bioaccessibility, which could be interpreted as evidence of nanoemulsification enhancing the in vitro bioaccessibility of lycopene.

The microencapsulated ascorbic acid release in vitro and its effect on iron bioavailability.

The present study was carried out to examine the stability of microencapsulated ascorbic acid in simulated-gastric and intestinal situation in vitro and the effect of microencapsulated ascorbic acid on iron bioavailability. Coating materials used were polyglycerol monostearate (PGMS) and medium-chain triacylglycerol (MCT), and core materials were L-ascorbic acid and ferric ammonium sulfate. When ascorbic acid was microencapsulated by MCT, the release of ascorbic acid was 6.3% at pH 5 and 1.32% at pH 2 in simulated-gastric fluids during 60 min. When ascorbic acid was microencapsulated by PGMS, the more ascorbic acid was released in the range of 9.5 to 16.0%. Comparatively, ascorbic acid release increased significantly as 94.7% and 83.8% coated by MCT and PGMS, respectively, for 60 min incubation in simulated-intestinal fluid. In the subsequent study, we tested whether ascorbic acid enhanced the iron bioavailability or not. In results, serum iron content and transferring saturation increased dramatically when subjects consumed milks containing both encapsulated iron and encapsulated ascorbic acid, compared with those when consumed uncapsulated iron or encapsulated iron without ascorbic acid. Therefore, the present data indicated that microencapsulated ascorbic acid with both PGMS and MCT were effective means for fortifying ascorbic acid into milk and for enhancing the iron bioavailability.

Microencapsulation of water-soluble isoflavone and physico-chemical property in milk.

This study was carried out to investigate the addition of water-soluble isoflavone into milk by means of microencapsulation technique. The yield of microencapsulation, sensory attributes, and capsule stability of water-soluble isoflavone microcapsules in milk were measured. Coating materials used was polyglycerol monostearate (PGMS), and core material was water-soluble isoflavone. The encapsulation yield of water-soluble isoflavone with PGMS was 67.2% when the ratio of coating material to core material was 15:1. The rate of water-soluble isoflavone release from capsules was 18, 19, and 25% when stored at 4, 20, and 30 degrees C for 12 days in milk, respectively. In sensory evaluation, beany flavor and color of microencapsuled water-soluble isoflavone added milk were significantly different from uncapsuled water-soluble isoflavone added milk, however, bitterness was not significantly different. In vitro study, micro-capsules of water-soluble isoflavone in simulated gastric fluid with the range of 3 to 6 pHs were released 3.0-15.0%, however, the capsules in simulated intestinal fluid with pH 7 were released 95.7% for 40 min incubation time. In conclusion, this study provided that PGMS as coating materials was suitable for the microencapsulation of water-soluble isoflavone, and the capsule containing milk was almost not affected with sensory attribute.

The Preventive Effects of Nanopowdered Peanut Sprout-added Caciocavallo Cheese on Collagen-induced Arthritic Mice.

The present study was carried out to investigate the effects of nanopowdered peanut sprout-added Caciocavallo cheese (NPCC) on the prevention and treatment of rheumatoid arthritis in DBA/IJ mice immunized with type II collagen. After the induction of arthritis, the mice were being divided into five groups: (1) normal, no immunization; (2) CIA, collagen-induced arthritis; (3) MTX, collagen-induced arthritis treated with methotrexate (0.3 mg/kg body weight); (4) CC, collagen-induced arthritis treated with Caciocavallo cheese (0.6 g/d); (5) NPCC, collagen-induced arthritis treated with nanopowdered peanut sprout-added Caciocavallo cheese (0.6 g/d). Nanopowdered peanut sprout was ranged from 300 to 350 nm, while regular powdered peanut sprouts were ranged from 50 to 150 µm. The NPCC group had considerable reductions of clinical scores and paw thicknesses at the end of experiment as compared to the CIA group. In the serum analysis, the TNF-α, IL-1ß, IL- 6 and IgG1 levels in the NPCC group have decreased by 69.4, 75.9, 66.6, and 61.9%, respectively, when compared to the CIA group. The histological score and spleen index of the NPCC group were significantly lower than the CIA group. In conclusion, the feeding NPCC method could delay and/or prevent the rheumatoid arthritis in the collagen-induced arthritis mouse model. Based on this study, nanopowdered peanut sprouts could be applied to various functional cheeses.

Physicochemical and Sensory Properties of Nanopowdered Chitosan-Added Maribo Cheese during Ripening.

Nanopowdered chitosan (NPC) has high biological activities, such as blood cholesterol lowering effect and antidiabetic activity. This study is carried out to determine the effects of nano-powdered chitosan-added Maribo cheese (NCMC) for the physicochemical properties and sensory analysis during its ripening at 14℃ for 6 mon. From the results, the moisture and fat levels are not significantly influenced from the addition of chitosan (p>0.05), but ash contents increased with increasing chitosan concentrations and the protein contents decreased with increasing chitosan concentrations. In the short-chain fatty acids analysis during the ripening, the total production is initially 13.79 ppm in 0.2% NCMC and 13.81 ppm in control, and their levels have steadily increased to 59.94 and 53.11 ppm, respectively. For the color levels, the L* values decreased, while the a* and b* values significantly increased during ripening for all samples (p<0.05). In texture analysis, the hardness and gumminess of NCMC significantly decreased as compared to the control during ripening (p<0.05), while the cohesiveness, springiness and chewiness were not significantly different among the treatments (p>0.05). In sensory analysis, the butyric off-flavor and bitterness increased slightly with increasing concentrations of NCMC during ripening. The overall acceptability of 0.2% NCMC held the highest score amongst the samples during the ripening. From the results obtained, the 0.2% NCMC was preferred during the ripening and observed the possibility of functional cheese.

Effect of isoflavone-enriched milk on bone mass in ovariectomized rats.

This study examined the effect of isoflavone-enriched milk on bone loss in ovariectomized (OVX) rats. Thirty 6-week-old Sprague-Dawley female rats were divided into two groups: sham-operated and OVX. The OVX group was subdivided into three dietary groups (OVX, non-isoflavone-enriched milk; OVX+Iso, isoflavone-enriched milk; and OVX+Iso+Vit+Ca, isoflavone-, vitamins D and K-, and Ca-enriched mik). After 19 weeks of feeding, the food efficiency ratio and body weight gain in the sham-operated group were significantly lower compared with those in the other groups. The bone alkaline phosphatase and total alkaline phosphatase activities were significantly higher in isoflavone-enriched groups (OVX+Iso and OVX+Iso+Vit+Ca) when compared with the sham-operated group. Urinary excretions of deoxypyridinoline and hydroxyproline were significantly higher with ovariectomy, but mostly normalized in the OVX+Iso and OVX+Iso+Vit+Ca groups. The rats in the OVX+Iso and OVX+Iso+Vit+Ca groups showed higher femur and tibia weights. A significant increase was found in bone density of femur and trabecular bone area in the OVX+Iso+Vit+Ca group, which almost reached that of the sham-operated group, whereas no difference was found among the OVX and OVX+Iso groups. The histological areas of the proximal tibia sections showed highly filled trabecular bone in both isoflavone-enriched groups (OVX+Iso and OVX+Iso+Vit+Ca). The present study indicated that isoflavone-enriched milk may have a partial preventive effect on ovariectomy-induced bone loss; however, vitamins D and K and Ca enrichment with isoflavone may enhance effectiveness for increasing bone mass in OVX rats.