EP0818957A2 - Kalzium zusätze und kalzium enthaldende getränke welche vitamin d beinhalten - Google Patents

Kalzium zusätze und kalzium enthaldende getränke welche vitamin d beinhalten

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Publication number
EP0818957A2
EP0818957A2 EP96911563A EP96911563A EP0818957A2 EP 0818957 A2 EP0818957 A2 EP 0818957A2 EP 96911563 A EP96911563 A EP 96911563A EP 96911563 A EP96911563 A EP 96911563A EP 0818957 A2 EP0818957 A2 EP 0818957A2
Authority
EP
European Patent Office
Prior art keywords
calcium
vitamin
beverage
concentrate
gum
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96911563A
Other languages
English (en)
French (fr)
Inventor
Terrence Bruce Mazer
Normanella Torres Dewille
Michael Allen Chandler
Robert John Ragan
Gregory Allan Snowden
Maureen Elizabeth Geraghty
Catherine Dubinin Johnson
Lonnie Richard Drayer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/418,393 external-priority patent/US5698222A/en
Priority claimed from US08/418,391 external-priority patent/US5597595A/en
Priority claimed from US08/418,729 external-priority patent/US5609897A/en
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of EP0818957A2 publication Critical patent/EP0818957A2/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A23L33/155—Vitamins A or D
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to calcium supplements and, in particular, to a solid supplement fortified with calcium glycerophosphate, vitamin D and vitamin C; to a beverage concentrate or additive (liquid or powder) containing calcium and vitamin D; and to a beverage made by reconstituting such beverage concentrates and additives to make a liquid nutritional product fortified with both calcium and vitamin D, and preferably having a low pH.
  • Calcium is an essential nutrient; it is a major component of mineralized tissues and is required for normal growth and development of the skeleton and teeth. Over the last decade calcium has enjoyed increased attention due to its potential role in the prevention of osteoporosis. Osteoporosis affects more than 25 million people in the United States and is the major underlying cause of bone fractures in postmenopausal women and the elderly. Optimal Calcium Intake", JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION, 272(24): 1942-1948 (1994).
  • osteoporosis refers to a reduction in the amount of bone mass.
  • Two important factors influencing the occurrence of osteoporosis are optimal peak bone mass attained in the first two to three decades of life and the rate at which bone mass is lost in later years.
  • Adequate calcium intake is critical to achieving optimal peak bone mass and modifies the rate of bone mass loss associated with aging. Wardlaw, "Putting osteoporosis in perspective", JOURNAL OF THE AMERICAN DIETETIC ASSOCIATION, 93(9): 1000-1006 (1993).
  • Dairy products are the major contributors of dietary calcium because of their high calcium content (e.g. approximately 250-300 mg/8 oz of cow's milk) and frequency of consumption.
  • milk is understood to refer to cow's milk
  • dairy products is understood to refer to food products derived from cow's milk.
  • Bioavailability (absorption) of calcium from food depends on the food's total calcium content and the presence of components which enhance or inhibit calcium absorption.
  • Bioavailability of minerals in food has been traditionally tested by the balance method, which estimates absorption from the difference between ingested intake and fecal output. This approach works well for many nutrients where the difference between intake and excretion is large, but is less well suited for an element such as calcium entering the digestive tract with its secretions. A decline in fractional absorption from 30% to 20% could have profound nutritional significance but would be difficult to detect using the balance method.
  • isotopic methods estimate absorption directly from the appearance of the ingested tracer in body fluids.
  • Future clinical evaluations of the bioavailability of calcium from the liquid nutritional product of the present invention will use a state-of-the-art isotope tracer method.
  • calcium carbonate calcium citrate, calcium glycerophosphate, calcium oxide, calcium pantothenate, calcium phosphate, calcium pyrophosphate, calcium chloride, calcium lactate, and calcium sulfate (56 FR at 60691).
  • Oxalic acid, fiber & phytates (only if Pregnancy & lactation achlorhydria present)
  • Vitamin D deficient individuals absorb less calcium than individuals whose vitamin D stores are adequate. Vitamin D metabolites enhance calcium abso ⁇ tion.
  • the major metabolite 1 ,25-dihydroxyvitamin D stimulates active transport of calcium in the small intestine and colon.
  • Deficiency of 1 ,25-dihydroxyvitamin D caused by inadequate dietary vitamin D, inadequate exposure to sunlight, impaired activation of vitamin D, or acquired resistance to vitamin D, results in reduced calcium abso ⁇ tion.
  • Vitamin D deficiency is associated with an increased risk of fractures.
  • Vitamin D deficiency because of insufficient vitamin D intake from their diet, impaired renal synthesis of 1,25-dihydroxyvitamin D, and inadequate sunlight exposure, which is normally the major stimulus for endogenous vitamin D synthesis. This is especially evident in homebound or institutionalized individuals. Supplementation of vitamin D intake to provide 600-800 lU/day has been shown to improve calcium balance and reduce fracture risk in these individuals. Sufficient vitamin D intake should be ensured for all individuals, especially the elderly who are at greater risk for development of a deficiency. Sources of vitamin D, besides supplements include sunlight, vitamin D-fortified liquid dairy products, cod liver oil, and fatty fish. Calcium and vitamin D need not be taken together to be effective.
  • Vitamin D may introduce risks such as hypercalciuria and hypercalcemia and should be avoided.
  • Anticonvulsant medications may alter both vitamin D and bone mineral metabolism particularly in certain disorders, in the institutionalized, and in the elderly. Although symptomatic skeletal disease is uncommon in noninstitutionalized settings, optimal calcium intake is advised for persons using anticonvulsants. "Optimal Calcium Intake”, JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION, 272(24): 1942-1948 (1994).
  • Simple sugars and organic acids also have an effect on bioavailability.
  • Fructose in orange juice and apple juice promoted positive calcium bioavailability from Calcium Citrate Malate (CCM) which is a combination of CaCO 3 , citric acid, malic acid: 5:1 :1 mol/mol/mol).
  • CCM Calcium Citrate Malate
  • the lactose in milk forms a soluble compound with calcium.
  • Organic acids such as citric acid, malic acid and ascorbic acid may also play a role in the favorable absorption of calcium from CCM.
  • Mehansho et al. "Calcium Bioavailability and Iron-Calcium Interaction in Orange Juice", JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION, 8(1 ):61 -68 (1989).
  • calcium supplements may be the preferred way to obtain optimal calcium intake.
  • calcium supplements are available in many salts, calcium carbonate is usually recommended because it contains more elemental calcium per gram than any of the other salts.
  • the disintegration and dissolution characteristics of commercial calcium carbonate preparations which vary widely, may produce important differences in calcium abso ⁇ tion.
  • Other problems with using large amounts of calcium carbonate is that it can lead to constipation and abdominal distention.
  • calcium lactate or calcium citrate are advised.
  • substitutions for calcium carbonate are also indicated for people with achlorhydria.
  • a popular commercially available calcium supplement is TUMS 500TM which is distributed by SmithKline Beecham, Pittsburgh, Pennsylvania, U.S.A. and is labeled as providing 500 mg of elemental calcium (from calcium carbonate per tablet).
  • the TUMS 500TM label does not indicate that this calcium supplement contains any vitamin D.
  • U.S. 4,786,510 and U.S. 4,992,282 disclose the use of calcium citrate malate in a beverage or dietary supplement fortified with iron, but do not disclose the addition of vitamin D to such a product.
  • WO 92/19251 and WO 92/21355 disclose the use of calcium citrate malate in a low pH beverage, and suggests that vitamin D be added to such a beverage along with oil flavors or weighing oil.
  • neither WO 92/19251 or WO 92/21355 disclose any other details about how to inco ⁇ orate vitamin D 3 into such a beverage.
  • EP 0486425 A2 discloses a liquid oral nutritional formulation which contains carbohydrates, protein, fat, fiber, calcium, and vitamin D, and has a pH of about 3.5 to 3.9.
  • this publication teaches that high amounts of micronutrients such as calcium or magnesium may impair the palatability of the product, and should contain the recommended daily allowance of these nutrients in about one liter or product.
  • this product contains only about 570 mg of calcium per liter and about 211 IU of vitamin D per liter.
  • a commercially available product in accordance with this patent publication is distributed by Sandoz Nutrition under the trade name CITRISOURCE® and is labeled as providing 570 mg of calcium and 210 IU of vitamin D per liter.
  • prototypes of a beverage according to the present invention contain about 1 ,408 mg of calcium per liter and about 338 IU of vitamin D 3 per liter.
  • U.S. 4,737,375 teaches beverage concentrates and beverages having a pH of 2.5 to 6.5, preferably 3.0 to 4.5, which contains calcium.
  • the use of vitamin D 3 in this beverage is not disclosed.
  • This patent does not teach the use of calcium glycerophosphate (which is used in preferred embodiments of the present invention, as a calcium source.
  • the acidulants used in this prior art beverage are chosen from mixtures of citric acid, malic acid and phosphoric acid, and the weight ratio of total acids to calcium is in the range of 4 to 7.
  • the calcium level is 0.06% to 0.15%, preferably 0.10% to 0.15% of the beverage, by weight.
  • prototypes of the beverage of the present invention have a weight ratio of total acids to calcium of about 5.1.
  • GB 2 196 253 A discloses a beverage containing calcium and vitamin D.
  • a water soluble non-toxic calcium salt is used in a quantity sufficient to provide in the final beverage a calcium ion content of from 1.0 x 10 2 to 40 x 10 '2 % w/w.
  • the beverage may contain up to 5 x 10" 6 w/w of vitamin D.
  • this published patent application does not teach the use of a gum, such as gum arabic or gum tragacanth, in such a beverage to improve vitamin D 3 stability.
  • the NIH Consensus Statement recommended that the private sector play an active role in promoting optimal calcium intake by developing and marketing a wide variety of calcium-rich foods to meet the needs and tastes of a multiethnic population.
  • Optimal Calcium Intake JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION. 272(24): 1942-1948 (1994).
  • a low pH beverage fortified with calcium and vitamin D 3 .
  • a liquid beverage concentrate fortified with calcium and vitamin D 3 .
  • a liquid beverage additive fortified with calcium and vitamin D 3 .
  • the invention comprises a beverage concentrate comprising: a) a source of calcium; b) vitamin D; c) a vegetable oil; and d) a gum.
  • This concentrate may be in dry powdered form or it may be in liquid form, in which case it further comprises a quantity of an aqueous solution, usually water or juice. Either concentrated form can be reconstituted/diluted with an aqueous solution to form the desired, final liquid beverage, which forms a second aspect of the invention.
  • Suitable solutions include water, fruit juices and vegetable juices, among others.
  • the source of calcium preferably is calcium glycerophosphate, but may also be calcium citrate malate or calcium carbonate or another food grade calcium salt.
  • the gum may preferably be selected from gum arabic, gum tragacanth and xanthan gum; whereas the vegetable oil may preferably be selected from corn oil and partially hydrogenated soybean oil.
  • compositions may further contain supplemental ingredients, such as vitamin C, lactic acid, an acidulant, a sweetener, a glucose polymer, potassium benzoate or a flavoring agent. Iff desired, the beverage may be carbonated.
  • the invention provides a calcium supplement in solid form comprising calcium glycerophosphate, vitamin D and vitamin C.
  • the calcium supplement in solid form comprises calcium glycerophosphate, vitamin D 3 , vegetable oil, vitamin C, and a gum selected from the group consisting of gum arabic, gum tragacanth and xanthan gum.
  • Figs. 1-7 are representative of the methodology used in determining vitamin D 3 levels.
  • Figs. 8-11 are representative of the methodology used in determining vitamin C levels.
  • the present invention provides high levels of calcium and vitamin D in a carbonated beverage, a noncarbonated beverage, a liquid beverage concentrate, a powdered beverage concentrate, a powdered beverage additive, beverages containing a powdered beverage concentrate or additive, or a calcium supplement.
  • liquid nutritional product and “beverage” are understood to be synonymous.
  • a "low pH beverage” is understood to refer to a beverage having a pH of less than 4.6.
  • Trial batches of low calorie lemon lime, orange, peach, and wild cherry flavored prototype carbonated beverages have been manufactured in accordance with the present invention. The prototype beverages were manufactured by preparing a beverage concentrate, then blending the beverage concentrate with treated water. The blends where then carbonated and filled into standard 12 ounce soda aluminum cans. (Soda aluminum cans are coated in accordance with accepted industry standards to substantially reduce migration of aluminum into the contents of the can.)
  • CaGP Calcium Glycerophosphate
  • FDA United States Food and Drug Administration
  • CaGP Calcium glycerophosphate
  • CaGP Another reason for selecting CaGP is its excellent calcium bioavailability.
  • Churella et al., "RELATIVE CALCIUM (CA) BIOAVAILABILITY OF CA SALTS USED IN INFANT FORMULAS", THE FASEB JOURNAL, 4(3):A788 (1990) reports a study which determined the calcium bioavailability of four calcium salts. Rats were fed various diets containing different calcium salts for three weeks. At the end of the study, the right femur was removed and tested for calcium. As compared to a control, the relative calcium bioavailability was as follows: tricalcium phosphate 110%, calcium citrate 110% and CaGP 106%.
  • CaGP calcium phosphate
  • monocalcium phosphate calcium chloride
  • tricaicium phosphate calcium citrate
  • calcium carbonate CaGP
  • D-gluconic acid hemicalcium salt
  • Aqueous solutions containing 500 mg of calcium per 237 mL (8 oz.) serving (2110 ppm) were prepared and the pH was adjusted to pH 3.5 and pH 5.0.
  • CaGP a beverage matrix containing this calcium salt requires the addition of less acid to achieve a pH below 4.0.
  • Acidity is desired in the liquid nutritional product of the present invention for several reasons such as: to maintain the calcium salt solubility, to complement flavor, to control microbial growth, and to enhance the role of preservatives, specifically potassium benzoate or sodium benzoate.
  • too much acidity can result in increased tartness and sourness that make the product undesirable from a sensory point of view.
  • the solution resists changes in pH and more acid is needed to bring down the pH than in commercially available sodas with no calcium fortification.
  • Aqueous solutions of various calcium salts were prepared to deliver 500 mg of elemental calcium per 12 oz. serving (1408 ppm) and the pH adjusted to pH 3.5 with citric acid.
  • Titratable acidity was determined by measuring the amount of 0.1 N NaOH needed to raise the pH to 8.3 in a 40g sample containing 1 ,409 mg/Kg of a calcium source.
  • the results presented in TABLE 4 indicate that, with the exception of calcium chloride, CaGP was the calcium salt that had the lowest titratable acidity. Titratable acidity is an indication of the total acidity of a beverage.
  • CaGP Calcium glycerophosphate
  • GaGP binds calcium with an approximate formation constant of 1.7.
  • CaGP when dissolved in water, dissociates readily to provide "free" calcium ions and protonated glycerophosphate species.
  • CaGP Yet another reason for selecting CaGP is the low aluminum content in commercially available CaGP. It has been theorized that chronic use of calcium supplements which have significant aluminum contents may constitute unnecessary metal exposure. Whiting, "Safety of Some Calcium Supplements Questioned", NUTRITION REVIEWS. 52(3):95-97 (1994). The aluminum content of some calcium sources is presented in TABLE 5. TABLE 5
  • Vitamin P As used herein and in the claims the terms “vitamin D” and “various forms of vitamin D” are understood to refer to vitamin D, cholecalciferol (D 3 ), ergocalciferol (D 2 ) and its biologically active metabolites and precursors such as, 1 ⁇ , 25- (OH) 2 vitamin D; 25 OH vitamin D, its biological precursor; and 1 ⁇ hydroxyvitamin D, and analogues of the dihydroxy compound. These materials promote intestinal abso ⁇ tion of calcium, contribute to plasma calcium regulation by acting on the remodeling processes of accretion and resorption and stimulate reabsorption of calcium by the kidney.
  • vitamin D 3 is cholecalciferol, it is understood that any of the various forms of vitamin D may be used in practicing the present invention, but vitamin D 3 is preferred in embodiments which are liquids. Dietary calcium and vitamin D are the natural mediators against bone loss. Vitamin D acts directly on bone cells (osteoblasts, osteoclasts) to alter bone mass. It also promotes gut uptake of calcium. Human skin activates pre-vitamin D molecules when exposed to ultra violet irradiation. In the summer, 15 minutes exposure to sunlight is sufficient to maintain adequate vitamin D levels. On the other hand, during winter, all day exposure to sunlight will produce negligible conversion of vitamin D. The thinner skin associated with aging is a less effective converter than the youthful skin.
  • the % recovery of vitamin D 3 of each batch was calculated by dividing the 0-time vitamin D 3 result by the theoretical fortification of each batch times 100%. (See Table 6). As used herein “theoretical fortification” refers to amount of vitamin D 3 added to the product. As used herein “0-time” refers to the time of initial vitamin D 3 analysis of the product. In Table 6, “% Recovery” is the percentage of theoretical fortification of vitamin D 3 remaining in the product after initial processing loss. Only batch 31 did not have the 0- time vitamin D 3 determined. Therefore, a projected result for this batch was extrapolated from the negative exponential regression curve generated from the stability data. T; ⁇ BLE ⁇
  • [D] Vitamin D 3 concentration (IU/KG) at time (t).
  • [D 0 ] Vitamin D 3 concentration (IU/KG) at 0-time.
  • e Exponential
  • k Rate constant (rate of loss of vitamin D 3 over time)
  • t Time (days)
  • the water dispersible vitamin D 3 used in this evaluation was a DRY VITAMIN D 3 Type 100-DS purchased from Roche Vitamins and Fine Chemicals, a division of Hoffman-LaRoche, Inc., Nutley, New Jersey, U.S.A., which contains vitamin D 3 (cholecalciferol USP-FCC), dicalcium phosphate, gum acacia, coconut oil, BHT, lactose, silicon dioxide, sodium benzoate and sorbic acid. It is a white powder and contains 100,000 lU/g of vitamin D 3 .
  • the loss of vitamin D 3 was primarily due to: (a) the fact that the vitamin D 3 was not homogenized into the product matrix; and (b) there was no emulsifier present that would assist in maintaining the vitamin D 3 in solution. Therefore, the vitamin D 3 was lost by the coating of the manufacturing equipment with vitamin D 3 .
  • the stability of Vitamin D 3 in these three batches was not acceptable over the shelf life of the product. As shown in Table 8, one half of the initial vitamin D 3 was lost in approximately 12.6 days.
  • Polysorbate 80 is a water soluble, non-ionic emulsifier used for various applications in the food industry. It is a polyoxyethylene derivative of sorbitan monooleate which interacts with the oil and aqueous phases in an emulsion to form a barrier at the interface that causes a reduction in Van der Waals forces and an improvement in emulsion stability. It was expected that the use of Polysorbate 80 to incorporate the vitamin D 3 would improve its recovery and stability by causing dispersion of the oil phase in the continuous aqueous phase.
  • Liquid beverage concentrates were prepared as described above, i.e., adding to water sodium benzoate (instead of potassium benzoate as a preservative), citric acid, potassium citrate, aspartame, calcium glycerophosphate, and vitamin D 3 in a premix containing Polysorbate 80 and propylene glycol.
  • the resultant liquid beverage concentrates were not homogenized and were diluted with five parts of water before carbonation.
  • the vitamin D 3 fortification level for each batch was 635 IU/KG of product. All batches contained vitamin C.
  • the variables in batches 4-6 are presented in Table 9. These variables were added in an attempt to improve vitamin C stability, since it has been found that cysteine, when added in a carefully controlled amount can overcome vitamin C deterioration in packaged beverages (U.S. Patent 3,958,017, May 18, 1976).
  • batch 6 also had slightly better initial vitamin D 3 Recovery than those batches in this series without protein. This suggested that a more rugged emulsion and some sort of matrix was needed as shown in Table 10.
  • WPC is not indicated if the product of the invention is desired to be low in calories or free of calories, but otherwise may be used in the practice of the invention. In an attempt to make a low calorie or calorie free product, the use of mechanical means such as homogenization was investigated.
  • Average Half Life (t,, 2 ) of Vitamin D, for Batches 4-6 is 626 Days.
  • the vitamin D 3 /Polysorbate premix was combined with the aqueous phase and the blend was emulsified by passing it through a two- stage Gaulin-L-100 homogenizer at a given pressure.
  • the pu ⁇ ose of this homogenization step is to break up, or evenly disperse, the oil phase into the aqueous phase so that the particle size of the emulsion is sufficiently small to retard coalescence of the oil phase and prevent separation.
  • a two-stage homogenization is needed since the fine particles formed during the first stage can clump.
  • the second stage, set at a lower pressure, is needed to break up the clumps, thereby making a more stable emulsion.
  • BVO Brominated vegetable oil
  • small quantities of gum arabic were added to the vitamin D 3 /Polysorbate premix prior to homogenization. This was done to increase the specific gravity of the oil phase and avoid phase separation, or oiling-off, of the emulsion.
  • BVO is used in the soda industry as a stabilizer for flavoring oils used in fruit flavored beverages.
  • BVO is a Food Additive (21, CFR 180.30) allowed in an amount not greater than 15 ppm of the finished beverage.
  • vitamin D 3 Two different sources of vitamin D 3 were used: (a) an oil soluble vitamin premix where the vitamin D 3 is dissolved in a small amount of corn oil; and (b) a vitamin D 3 premix where the vitamin D 3 is dissolved in Polysorbate 80 and propylene glycol (PG) (same as batches 4 through 6). One part of the complete concentrate was then dissolved with five parts of water before carbonation. The variables in batches 7-24 are presented in Table 11. TABLE 11
  • the gum arabic used in all batches was Nutriloid Gum Arabic from Tic Gums, Inc.
  • percent addition refers to percent of oil in the batch.
  • Batches contain either 3% or 6% extra Polysorbate 80 added.
  • the 20% and 40% refer to combinations of Polysorbate 80 and Propylene glycol where the Polysorbate 80 content is 3% and 6%.
  • Fructose was added to batch number 24 to see if it would extend the shelf-life of the product which is limited by the degradation of aspartame. In general, the fructose and the various levels of Polysorbate 80 did not affect the vitamin D 3 recovery as the homogenization step did.
  • VITAMIN D 3 (IU/KG OF PRODUCT) VERSUS DAYS
  • Average Half Life (t, ⁇ ) of Vitamin D 3 for batches 7-9 is 150 days.
  • VITAMIN D (IU/KG OF PRODUCT. VERSUS DAYS
  • Average Half Life (t 1/2 ) of Vitamin D 3 for batches 10-17 is 68.6 days.
  • Average Half Life (t 1/2 ) of Vitamin D 3 is 74.7 Days. (Batch 20 is a control and is not included in the average. 1 Days after initial vitamin D 3 testing. 0-time testing occurred 1 day after the product was manufactured.
  • gum arabic and gum tragacanth as emulsifying agents for flavor oils in soft drinks is well established in the soft drink industry. Melillo, "Physical Factors Governing the Stabilization of Cloudy Beverages", FOOD PRODUCTS DEVELOPMENT, June, 1977, pp. 108-110. While only gum arabic was used in the experiments, examples and prototypes disclosed herein, it is understood that one skilled in the art could substitute appropriate amounts of gum tragacanth, xanthan gum or any other appropriate gum into the products of the present invention, or that mixtures of gums may be used in the practice of the present invention.
  • Gum tragacanth is the dried, gummy exudation obtained from Astragalus gummifer or other Asiatic species of Astralag ⁇ s.
  • Tragacanth swells rapidly in either cold or hot water to a viscous colloidal sol or semi-gel.
  • the molecular weight of the gum is on the order of 840,000 and the molecules are elongated (4500A by 19A) which accounts for its high viscosity.
  • Tragacanth gum is compatible with other plant hydrocolloids as well as carbohydrates, most proteins, and fats. Viscosity is most stable at pH 4 to 8 with a very good stability down to pH 2.
  • Xanthan gum is an exocellular heteropolysaccharide produced by a distinct fermentation process.
  • the bacterium xanthomonas campestris generates the gum on specific organelles at the cell surface by a complex enzymatic process.
  • the molecular weight for xanthan gum is about two million.
  • Gum arabic also known as gum acacia, is the dried, gummy exudate from the stems or branches of Acacia Senegal or of related species of Acacia.
  • the most unusual property of gum arabic among the natural gums is its extreme and true solubility in cold or hot water.
  • Gum arabic is a complex calcium, magnesium, and potassium salt of arabic acid. It has a main backbone chain of (1 ⁇ 3) - linked D- galactopyranose units, some of which are substituted at the C-6 position with various side chains.
  • the side chains consist of D-galactopyranose, D-glucuronic acid and L- arabofuranose with additional side chains on the D-galactopyranose of L- rhamnopyranose.
  • the molecular weight is on the order of 250,000.
  • Gum Arabic is effective in stabilizing emulsions and inhibiting coalescence or phase separation by two mechanisms: (a) increasing the viscosity of the continuous (aqueous) phase; and, (b) forming strong films around the oil droplets. A small amount of protein is present in the gum arabic as a part of the structure.
  • the pH of the emulsions which contained gum arabic were lowered to pH 4.0 and sodium benzoate was added to preserve the emulsions for extended use.
  • the emulsions were then homogenized twice using a two-stage homogenizer at 1,500/600 PSI and 3,000/1,000 PSI, respectively.
  • batch 27 contained 50 grams of EMULGUM gum arabic hydrated in 950 grams of water, and upon cooling 77.2 milligrams of liquid vitamin D 3 in corn oil was blended into the gum solution in an amount giving a theoretical fortification of about 825 lU/Kg of finished beverage.
  • the emulsion was preserved by adding 0.3 g of sodium benzoate and the pH was lowered to 4.0 by adding 1.08 grams of citric acid.
  • vitamin D 3 emulsion manufactured by an outside contractor.
  • Tastemaker, Inc. of Cincinnati, Ohio, U.S.A., which is a provider of flavoring products, provided as a special order a vitamin D 3 emulsion containing water, gum arabic, partially hydrogenated soybean oil, citric acid, sodium benzoate and vitamin D 3 .
  • this commercially manufactured vitamin D 3 emulsion contains, per 10 Kg: (a) about 9.52 Kg of water; (b) about 0.35 Kg of gum arabic; (c) about 0.10 Kg of partially hydrogenated soybean oil; (d) about 0.02 Kg of citric acid; (e) about 0.01 Kg of sodium benzoate; and (f) and at least about 787,000 IU of vitamin D 3 .
  • Tastemaker considers the manufacturing procedure it used to be proprietary to it, and did not make that information available. While the commercially manufactured emulsion contained partially hydrogenated soybean oil, and the self-manufactured emulsion contained corn oil, (see description of batches 25-30) it is understood that the invention may be practiced using any suitable vegetable oil.
  • VITAMIN D 3 (IU/KG OF PRODUCT) VERSUS DAYS
  • Acids are commonly used in food and beverages to impart specific tart or sour tastes and to function as preservatives.
  • a combination of citric and lactic acids are used in the liquid nutritional product of the present invention.
  • Citric acid is the most widely used acid in fruit beverages in part because it blends well with these flavors. It is commercially manufactured by fermentation or by synthesis; either may be used in the practice of the present invention. When using fermented lactic acid, a purified form that is free of sugar residues is recommended due to its cleaner taste and clearer appearance.
  • Food grade lactic acid is available in aqueous and crystalline forms.
  • Sweetener The sweetener used in the prototype beverages described below is aspartame, but other artificial or natural sweeteners can be used in the practice of the present invention.
  • Artificial sweeteners that may be employed include saccharin, acesulfame-K and the like.
  • Natural sweeteners that may be employed include sucrose, fructose, high fructose corn syrup, glucose, sugar alcohols, dextrose, maltodextrins, maltose, lactose, and the like but other carbohydrates can be used if less sweetness is desired. Mixtures of natural sweeteners, or artificial sweeteners, or natural and artificial sweeteners can be used also.
  • the amount of the sweetener effective in a product according to any aspect of the present invention depends upon the particular sweetener used and the sweetness intensity desired. In determining the amount of sweetener, any sugar or other sweetener present in the flavor component or product matrix should also be taken into consideration.
  • Flavor includes both natural and artificial flavors.
  • the particular amount of the flavor component effective for imparting flavor characteristics to the beverage of the present invention can depend upon the flavor(s) selected, the flavor impression desired, and the form of the flavor component.
  • the amount of flavor employed in a product according to any aspect of the present invention is within the skill of one in the art and depends on the flavor intensity desired.
  • Preservatives Most microbial spoilage of low pH beverages is caused by aciduric and acidophilic organisms like certain varieties of yeasts and molds. For this reason, preservatives with anti-microbial activity such as benzoic and sorbic acids are added to soft drinks. Usage levels of these acids or their salts range from 0.025 to 0.050 percent, depending on the nutritive substances present and the pH of the finished beverage. The antimicrobial activity of these preservatives has been shown to be largely pH dependent. They are least effective under neutral conditions but their activity increases considerably with decreasing pH. For example, by reducing the pH value from 4.5 to 3.0, the preservative effect of benzoic acid is increased by nearly three times. Only beverages at low pH receive the full benefit from the addition of preservatives.
  • carbonation The amount of carbon dioxide in a beverage according to the present invention depends upon the particular flavor system used and the amount of carbonation desired. Usually, carbonated beverages of the present invention contain from 1.0 to 4.5 volumes of carbon dioxide. Preferred carbonated beverages contain from 2 to 3.5 volumes of carbon dioxide.
  • the beverages of the present invention can be prepared by standard beverage formulation techniques. To make a carbonated beverage carbon dioxide can be introduced either into the water mixed with the beverage syrup or into the drinkable diluted beverage to achieve carbonation. It should be understood, however, that carbonated beverage manufacturing techniques, when appropriately modified, are also applicable to noncarbonated beverages.
  • Tables 18-21 present bills of materials for manufacturing prototypes of low pH beverages fortified with calcium and vitamin D 3 in accordance with some aspects of the invention.
  • Treated Water 1 for beverage concentrate
  • Treated Water 1 for beverage concentrate
  • Vitamin D 3 Emulsion 2 10.000
  • treated water had had the chlorine, and alkalinity adjusted to levels commonly used in the soft drink industry. This emulsion is described above with regards to batch 31.
  • Treated Water 1 for beverage concentrate
  • Vitamin D 3 Emulsion 2 10.000
  • treated water has had the chlorine and alkalinity adjusted to levels commonly used in the soft drink industry. This emulsion is described above with regards to batch 31.
  • Treated Water 1 (for beverage concentrate) 138.02
  • Vitamin D 3 Emulsion 2 10.000
  • treated water has had the chlorine and alkalinity adjusted to levels commonly used in the soft drink industry. This emulsion is described above with regards to batch 31.
  • the concentrated mixture of ingredients that make up the beverage is referred to as the beverage concentrate.
  • the liquid beverage concentrate comprises at least water, a source of calcium, vitamin D 3 , gum arabic and vegetable oil.
  • the beverage concentrate also comprises vitamin C.
  • the beverage concentrate may also comprise: an acidulant, preservative(s), and/or flavoring agent(s), and/or acid stable coloring agent(s).
  • Prototypes of the beverage of the present invention have a weight ratio of total acids to calcium of about 5.1.
  • Prototype beverages of the present invention contained vitamin D 3 at levels of about 1.45 x 10 "6 to about 1.75 x 10 "® % w/w, and calcium at levels of about 1.46 x 10 1 to about 1.47 x 10 '1 w/w.
  • liquid beverage concentrate is prepared in a single vessel at ambient temperature by dissolving the ingredients in water using a blending tank equipped with vigorous agitation capability.
  • a specific order of addition, shown in Table 22, is followed to aid in dispersing the ingredients in an efficient manner. Each ingredient should be completely dissolved before the next ingredient is added.
  • Vitamin D 3 Emulsion (vitamin D 3 + gum arabic)
  • beverage concentrates In commercial beverage manufacturing, it is common for beverage concentrates to be prepared a day or more (often weeks or months) in advance of blending and filling containers with the final product. For this reason, the vitamin components may be added to the liquid beverage concentrate just prior to blending with water to complete the beverage in order to prevent unnecessary long term exposure to air.
  • PREPARATION OF LIQUID BEVERAGE CONCENTRATE Variations to the beverage concentrate manufacturing procedure described in EXAMPLE 1 can be made if available mixing vessel sizes are limited and no single mixing vessel is able to contain the required volume of beverage concentrate.
  • Beverages according to the present invention have been manufactured by preparing a plurality of beverage concentrate component slurries which were thereafter combined by pumping each beverage concentrate component slurry to a larger sized tank. The water was divided equally between five different beverage concentrate component slurries, all of which were constantly agitated. A first beverage concentrate component slurry was made by first adding potassium benzoate and then sodium citrate to the water.
  • a second beverage concentrate component slurry was made by adding to the water in the following order: (a) citric acid; (b) lactic acid: (c) aspartame; (d) calcium glycerophosphate.
  • a third beverage concentrate component slurry was made by adding the acid stable coloring agent(s) and then the flavoring agent(s) to the water.
  • a fourth beverage concentrate component slurry was made by adding the ascorbic acid to the water.
  • a fifth beverage concentrate component slurry was made by adding the vitamin D 3 emulsion to the water. The beverage concentrate component slurries are transferred to a single larger sized vessel in the order in which they have been described.
  • a liquid beverage concentrate in accordance with the invention should have a pH of 2.8-4.6, preferably 3.1-3.8.
  • the pH of the prototype beverage concentrates typically ranges from 3.1-3.8. If necessary, additional lactic acid is used to adjust the pH of the beverage concentrate to this range.
  • PREPARATION OF CARBONATED BEVERAGE Deareation and cooling increases the beverage's carbonation efficiency and stability because the solubility of carbon dioxide in water is directly proportional to carbon dioxide pressure and inversely proportional to temperature.
  • the extent of carbonation is expressed in terms of carbon dioxide gas volumes. The number of volumes can be determined by comparing sample readings with carbon dioxide temperature/pressure relationship charts. Since pressure gauges measure the sum of pressures from all gases, the presence of air in the carbonated mix can cause errors in CO 2 volume determination unless corrections are made.
  • a Zahm & Nagel air tester makes it possible to easily measure the pressure and air content of a sample.
  • the sample container is pierced, allowing head space gases to be released into a buret filled with 10-20% sodium or potassium hydroxide.
  • the carbon dioxide is absorbed by the basic solution, leaving only air inside the burette.
  • the total pressure reading is then corrected for the amount of air present in the burette, resulting in the corrected CO 2 pressure.
  • the gas volumes of the sample are then determined using the corrected pressure.
  • a beverage in accordance with the invention may be carbonated by either blending the beverage concentrate with carbonated water or blending the beverage concentrate with water followed by carbonation of the blend.
  • the prototype beverages were manufactured using a 5 to 1 ratio of beverage concentrate manufactured according to Example 2 to non-carbonated water. Carbonation levels in the finished beverage may range from about 1.0-4.5 volumes of CO 2 , depending on flavor or desired sensory attributes.
  • the product is then packaged and sealed in aluminum cans or tinted glass bottles.
  • separate in-stream lines of beverage concentrate and water were combined in the proper ratio by a continuous metering device known in the art as a volumetric proportioner and then deaerated.
  • the resulting mixture was transferred to a carbo- cooler where it was cooled and carbonated to approximately 2.5 volumes.
  • the pH of the finished beverage should be in the range of about 3.1-4, and the pH of the prototypes was about 3.7.
  • the finished product was then filled into standard 12 oz. aluminum soda cans.
  • Vitamin C 50% of RDI Calcium 50% of RDI Vitamin D 30% of RDI
  • An alternative embodiment of a liquid beverage concentrate may be prepared according to Example 1 or Example 2 excluding any ingredients other than the water, calcium source, vitamin D 3 and gum arabic (eg. the flavorant, and/or the colorant, and/or the sweetener may be omitted).
  • This liquid beverage concentrate may then be combined with another liquid beverage concentrate, such as a commercial soda pop concentrate, and the resultant blended beverage concentrate may thereafter be combined with carbonated water, or combined with non-carbonated water with the resultant beverage being carbonated in the manner described above in Example 3.
  • a liquid beverage concentrate may be prepared by blending a liquid beverage concentrate according to the present invention, such as described above in Examples 1 and 2, with non-carbonated water.
  • the resultant blend could then be placed into aluminum soda cans, or light reducing bottles, the head space flushed with nitrogen gas or carbon dioxide to eliminate oxygen which is harmful to vitamin and color stability, and sealing the cans in the usual manner.
  • An alternative embodiment of a liquid beverage concentrate may be prepared according to Example 1 or Example 2 excluding any ingredients other than the water, calcium source, vitamin D 3 and gum arabic (eg. the flavorant, and/or colorant, and or sweetener could be omitted), and thereafter blending the concentrate with fruit juice, vegetable juice, or any other suitable liquid matrix.
  • Example 7 POWDERED BEVERAGE CONCENTRATE
  • Vitamin D 3 Emulsion 1 350 g
  • Lactic Acid Powder (60% lactic acid) 181.3 g
  • a powdered beverage concentrate was prepared by placing the calcium glycerophosphate, sodium citrate, citric acid, lactic acid and ascorbic acid into the chamber of an Aeromatic Top Agglomerator. The powder was then blended for two minutes under medium fluidization. The temperature was brought to 70°C, the atomization was set at 1 Bar, the atomizing nozzle was placed at the highest level of three possible positions, and the fan capacity was set initially at 12 (nominal setting).
  • Aspartame was dissolved in approximately 800 ml of warm tap water and a small amount of citric acid was added to achieve a pH of approximately 4.
  • the vitamin D 3 emulsion and the flavor system were blended by hand with the aspartame solution to yield approximately 1200 ml of liquid.
  • the 1200 ml of liquid was placed on a stir plate and agitated under medium agitation while being sprayed onto the fluidized powder for approximately three hours.
  • a Kg of powdered beverage concentrate contained about 83.5 g of calcium, 12.9 g of vitamin C and 31 ,900 IU of vitamin D 3 .
  • the final powder particles were relatively large and brittle and were pulverized before reconstituting with water.
  • the powder was easily reconstituted (see Example 8) and flavor was typical of a powdered beverage concentrate product without the carbonation. Longer shelf life in this kind of beverage concentrate is anticipated because of the absence of water.
  • Example 7 Approximately 19.1 grams of the powdered beverage concentrate manufactured in Example 7 were dissolved in a sufficient amount of tap water to yield 1 Kg of beverage. A Kg of the resultant beverage is projected to contain about 1.4 g of calcium, about 0.25 g of vitamin C, and about 607 IU of vitamin D 3 .
  • the acid system can vary depending on the flavor selected.
  • a powdered beverage additive may be manufactured by the process described in Example 7, containing at least vitamin D 3 , a calcium source and vitamin C, but if desired omitting sweetener, acids, flavoring, etc.
  • the resultant powdered beverage additive could be added in appropriate quantities to a liquid matrix such as a fruit juice, blend of fruit juices, vegetable juices, coffee, tea or any suitable beverage.
  • the powdered beverage additive could be employed in bulk, (eg. at an orange juice processing facility), or on a serving by serving basis when provided in single serving size packets.
  • liquid or powdered beverage concentrate or beverage additive according to the invention is intended for use in a liquid matrix that may contain any dairy product, (for example, coffee or tea that may contain cream), a salt of ascorbic acid should be used in place of ascorbic acid to prevent curdling of the dairy product.
  • a calcium glycerophosphate/vitamin D 3 /vitamin C tablet supplement was prepared by placing about 291.6 g of calcium glycerophosphate and about 10.5 g of ascorbic acid (vitamin C) into the chamber of an Aeromatic laboratory batch agglomerator. The powder was then blended for three minutes under medium agitation. The temperature was brought to 55°C, the atomization was set to 1 bar, the atomizing nozzle was placed at the highest of three possible positions, and the fan capacity was set initially at 9 (nominal setting).
  • the peristaltic pump was set at 7 cc/minute and approximately 350 g of vitamin D 3 emulsion was sprayed onto the fluidized powder.
  • the commercially manufactured vitamin D 3 emulsion described above with respect to batch 31 was used in this calcium supplement.
  • any suitable dry blendable source of vitamin D preferably vitamin D 3 or D 2
  • the fan speed was incrementally increased to 12 over 55 minutes to maintain medium fluidization.
  • Temperature was also increased to 60°C after 16 minutes.
  • the heat was kept on and the powder was dried for three minutes.
  • a Kg of powder for tableting contained about 139.9 g of calcium, 26.4 g of vitamin C, and 39,600 IU of vitamin D 3 .
  • the final powder particle was a soft agglomerate. No excipients were added to the powder to facilitate the tableting process. Using a tablet die of approximately 1/2 inch diameter, 600 g of the final powder was compressed using a Carver model C laboratory press and an applied load of 200 pounds force. The tablet was easily removed from the die. This process was repeated using 1000 g and 1500 g of final powder to produce a total of three calcium supplement tablets, 600 g, 1000 g, and 1500 g, respectively.
  • a calcium supplement in solid form in accordance with the invention comprising calcium glycerophosphate, vitamin D, and vitamin C, is believed to be advantageous over prior art calcium supplements because it provides a source of calcium that has a low aluminum content as well as providing vitamin D.
  • the low pH beverage, the vitamin D 3 emulsion and the powder beverage are saponified with methanolic potassium hydroxide to destroy the fat and release the vitamin D3 for extraction.
  • the saponified samples are extracted with an ether/pentane mixture and the extracts are evaporated to dryness using nitrogen and reconstituted with iso-octane.
  • Sample extracts are eluted on a cleanup HPLC column (cyanopropyl bonded silica), and column switching is used to transfer a "slice" of the eluant containing vitamin D 3 onto an additional HPLC analytical column (aminopropyl bonded silica) for final quantitation.
  • the vitamin D 3 peak in the sample is quantitated using a linear regression external standard curve.
  • Teflon sleeves - sizes 24/40 (Cole-Parmer #N-06139-15).
  • Cooling tray large enough to accommodate centrifuge tube rack (#N 06737-40).
  • HPLC tubing - 0.040" stainless steel - 2 feet.
  • Refrigerator for storage of standards at 4( ⁇ 4) ⁇ C.
  • Detectors Cleanup system • fixed or variable wavelength capable of monitoring 254 nm or 264 nm ( Waters 440 or equivalent).
  • Analytical system - Variable wavelength detector capable of monitoring at 264 nm @ 0.0025 AUFS. Under normal operating conditions the short term noise should be less than 3% of the 5T vitamin D 3 standard peak height (Waters 486 or equivalent).
  • Switching Valve HPLC column switching valve with at least 6 ports. Has a working range up to 6000 psi (Micromeritics 732 or equivalent).
  • Recorder One 10 mV recording device for the cleanup HPLC output and either a recorder or an integrator for the analytical HPLC system. A data system capable of monitoring, acquiring, and reprocessing two channels of data is strongly recommended.
  • Pentane HPLC Grade recommend Burdick & Jackson #312.
  • 3T Pipet 3.0 ml of ISTD into a 100 ml volumetric flask and dilute to volume with iso-octane (approximately 0.8 lU/ml).
  • 15T Pipet 15.0 ml of ISTD into a 100 ml volumetric flask and dilute to volume with iso-octane (approximately 4.0 I U/ml).
  • 30T Pipet 30.0 ml of ISTD into a 100 ml volumetric flask and dilute to volume with iso-octane (approximately 8.0 I U/ml).
  • Detector 254 nm or 264 nm.
  • Run Time Approximately 35 minutes.
  • Recorder Recommend the use of an integrator or data system for reprocessing.
  • slice window i.e. transfer of vitamin D 3 from the cleanup column to the analytical column. This is done by setting the switching valve to switch the vitamin D 3 from the cleanup column to the analytical column at 0.10 minutes before the vitamin D 3 first elutes from the cleanup column until 0.10 minutes after the vitamin D 3 peak returns to baseline on the cleanup column. See Figures 4 and 5.
  • Slice window times should not exceed 1.0 minute - using a minimum among of time (generally 0.8 - 1.0 min.) necessary to collect all the vitamin D 3 while preventing the transfer of any other interfering components.
  • CALCULATIONS (Use only peak heights for reporting purposes)
  • Peak height is required for quantitation as small amounts of baseline noise can cause large area differences.
  • the peak heights of each respective level of the working standard are averaged.
  • a linear regression line is calculated by using the average peak heights (y-axis) and the concentration (x-axis) for the respective working standard.
  • the samples should be quantitated by bracketing the standards around the samples.
  • Vitamin D 3 (lU/kg) C) V) M000W
  • ICP-AES Inductively coupled plasma atomic emission spectrometry
  • the ICP-AES instrument consists of three components: sample introduction device, torchbox, and spectrometer. Most commonly, samples are introduced in the form of solutions which are nebulized (broken into tiny droplets), and passed into the torch with a stream of argon.
  • a plasma 1-2 kW of radio-frequency power is coupled from a copper coil (inductor) into a small region inside a quartz tube (torch), through which argon flows.
  • the power density in this region is high enough to heat the argon until it ionizes and, since the region is at atmospheric pressure, there are sufficient collisions with other argon atoms to instantly ignite a plasma with a temperature of about 10,000 K.
  • the micrometer-sized droplets from the nebulizer enter the bottom of the torch and pass through the cooler (6000 K), darker, central region of the plasma called the axial channel.
  • water is evaporated, and the remaining dry particles of analyte are vaporized and atomized (molecules broken down into atoms) by the heat of the plasma in just a few milliseconds.
  • Excitation and ionization of the outer electrons of the atoms occurs; the intensity of the emission that results from the deexicitation of these atoms and ions is proportional to the concentration of analyte in the original solution.
  • calibration consists of measuring the intensity of analyte emission for standards of known concentration.
  • Light emitted by the ICP is collected by a lens in the spectrometer and focused onto a diffraction grating which disperses the light into its component wavelengths.
  • the emitted radiation, wavelength resolved, from all the analyte elements is collected simultaneously by several detectors placed in front of the grating and converted into an electrical signal.
  • a data system relates these signals to the concentrations of the elements in the standards and calculates the analyte concentration in the samples.
  • the particular instrument used in this method features a movable entrance slit controlled by a high resolution stepper motor called SAMI (Scanning Accessory for Multielement Instrumentation). Moving the entrance slit slightly changes the angle of incidence upon the grating, and slightly changes the wavelengths incident upon the exit slits. This feature allows the user to perform background correction in the sample matrix by subtracting the emission background just off the peak center.
  • SAMI Sccanning Accessory for Multielement Instrumentation
  • This method employs a speedy dilution preparation of samples with a surfactant and dilute acid.
  • a special kind of nebulizer called a maximum dissolved solids nebulizer (MDSN), or high-solids nebulizer, is required to provide long term operation without clogging.
  • MDSN maximum dissolved solids nebulizer
  • an internal standard must be used to compensate for the poorer nebulization efficiency of the high solids samples.
  • Cobalt is added to each standard so that they are exactly 20.0 mg/L Co.
  • Calibration consists of measuring the analyte/Co ratio in the standards as a function of analyte concentration.
  • Instrument a. Inductively Coupled Argon Plasma Emission Spectrometer, ARL Model 3560 or Accuris b. Ryton V-groove nebulizer: ARL#173259-0000 or Precision Glass #510-50 only c. Spray chamber: ARL#173142-0003 or Precision Glass #110-34 or equivalent d. ICP torch: ARL#139009-0003 or Precision Glass #100-05 or equivalent
  • Plastic dispenser bottle fitted with a Teflon- constructed dispenser top with adjustable volume between 1- 10mL; dispenser may be fitted to concentrated HCl bottle directly j.
  • Magnetic stir plate and Teflon coated magnetic stir bars k.
  • Options 1 mL digital pipet with tips, Rainin EDP-Plus or 1 mL, glass volumetric pipet or equivalent
  • the following chemicals should be stored at room temperature. Their expiration date is one year after the date they are first opened. Upon expiration the chemicals must be either discarded or re-
  • the wavelengths that have been used are listed in the table below.
  • the instrument should be installed with identical channels if possible because the sensitivity of the line and the possibility of interferences can change if a different line is employed for analysis.
  • Typical ranges of operating conditions for the ARL 3560 are listed below. a. Incident power. 1200-1400 watts b. Reflected power ⁇ 5 watts c. Snout argon gas flow: on d. Coolant argon pressure: 30-40 psi e. Plasma argon pressure: 20-30 psi f. Nebulizer argon pressure: 30-46 psi
  • Peristaltic pump flow rate dial setting which corresponds to -2.5 mL/min. (depends on make and model of pump)
  • Peristaltic pump tubing 1.12 mm I.D. red/red P.V.C., Marprene, or equivalent
  • 2% HCl rinse Mix concentrated HCl with high purity water in the approximate ratio of 20 mL acid to 1000 mL total volume of solution. Use a plastic container of a size appropriate to the volume of solution prepared. For example, to prepare 20 L of 2% HCl, fill a 21 L carboy with high purity water to the 20 L mark and add 400 mL HCl to the water. Expiration: 6 months.
  • Triton X-100 solution (approximately 5%): Add about 700 mL high purity water to a 1 L plastic bottle containing a Teflon-coated stirring bar. Place the bottle on a magnetic stirrer and begin stirring at a moderate speed. Slowly add 50 mL Triton X-100 from a graduated cylinder. When the Triton is dissolved, fill the bottle approximately 1000 mL with high purity water. Transfer to 1L plastic bottle fitted with a Teflon-constructed dispenser with adjustable volume from 1-10 mL. Expiration: 6 months.
  • Standard blank solution Prepare 1 liter of a standard blank at the same time, and from the same reagent batches, as the above standards. Add 800 mL high purity water to a 1 L volumetric flask; 2.00 mL of 10,000 mg/L cobalt internal standard (Class A pipet), and 20 mL (repipetter) of hydrochloric acid to the flask. Add 50 mL of Triton X-100 solution from the dispenser to the flask and then fill the flask to volume with high purity water, slowly to avoid forming suds. Agitate well and transfer to a clean, dry 1 liter storage bottle. Dispense as needed into 125 mL storage bottles to use at the instrument. Expiration: 6 months.
  • ISRB Internal standard reference blank solution
  • the ISRB is analyzed before any standards or samples; the purpose is to subtract the intensity of analytes found in the reagents (Triton X-100 solution, HCl, and water) from the analyte intensities found in the standards and samples.
  • Standard Handling a. All bottles used for storage of standard solutions must first be soaked in 10% (v/v) HCl for a minimum of three hours, followed by multiple rinses with high purity water. Air dry or rinse several times with the standard. When reusing the bottles for a new batch of the same standard, no acid soak is necessary - simply rinse several times with high purity water and then several times with small portions of the fresh standard. b. As the working standards in the 125 mL bottles are used up, simply refill the bottles from the 1 L standards prepared in D. 3. c. Because there are many samples, the most efficient way to add the cobalt internal standard to the samples is with a 1 mL digital pipet.
  • Sample Preparation a. Refill the reagent containers before preparing samples so that the same batch of reagents can be used for all samples and the blank. b. Remove the caps and arrange the empty 50 mL tubes, with labels, in the rack beginning with the sample blank and two tubes for each sample. c. Transfer sample to a plastic storage container. Place these containers directly on a magnetic stirring plate and add a Teflon coated stirring bar. Set the stirrer at an intermediate speed. After a minimum of one minute of agitation begin to withdraw the sample for weighing with a disposable plastic transfer pipet. d. Carefully weigh and record to the nearest 0.0001 g, 5 g of sample into the plastic tubes. The sample blank tube is left empty at this point. Add the following reagents to each tube, including the blank, in this exact order
  • This procedure will insure that the SAMI motor is operating properly and that the calibration will always be performed near the exact center of each analyte peak. Perform this procedure before the first calibration is made, once during each 8-hour shift. Choose appropriate setup options and then run the profile. The measured peak centers for the element to be measured must be within ⁇ 6 SAMI units of the current SAMI profile position. If this result is not obtained, consult the supervisor: either a new default SAMI profile position needs to defined, or the instrument requires service. e. Select the appropriate task and the appropriate calibration sequence file name and begin calibration. Aspirate the standard solutions into the plasma starting with the ISRB solution prepared in D.5. The software prompts for each standard by name. Be alert to any error messages. If an error occurs, write down the message and consult the supervisor.
  • Samples should be as uniform and representative of the product as possible. Sampling should be performed immediately after a gentle mixing or stirring to prevent inaccurate sampling due to stratification. All sample weights must be recorded to at least three significant figures.
  • Sample sizes for low pH beverages are calculated from the following equation.
  • Sample Size is the theoretical sample size, in grams; E is the expected ascorbic acid concentration in milligrams per liter or kilogram, respectively, as is, and; 350 is the desired amount, in micrograms (meg), of ascorbic acid in the sample preparation.
  • the net conversion factor for micrograms to milligrams and kilograms to grams is unity.
  • the amount of L-ascorbic acid present in the sample is determined by coulometric titration.
  • a coulometric method of analysis measures the quantity of electricity required to carry out a chemical reaction. If the reaction is 100% efficient, the passage of one Faraday of electricity will cause the reaction of one equivalent weight.
  • iodine is coulometrically generated from iodide.
  • the iodine then oxidizes the L-ascorbic acid to dehydroascorbic acid.
  • an excess of iodine will occur.
  • This excess of iodine signals the equivalence point, and is detected by two constant potential electrodes.
  • the quantity of electricity used is given by the product of current times the time to reach the end point of the coulometric titration.
  • the amount of iodine used is equal to the number of equivalents of L-ascorbic acid, and the amount of L-ascorbic acid can be calculated.
  • Trichloroacetic acid is added to the sample to precipitate the protein and to maintain the acidic condition necessary for a quantitative reaction.
  • Double Platinum Wire Electrode Brinkmann cat. no. 20-92- 350-4, or equivalent
  • Isolation Tube outer diameter 20 mm, 125 mm long, Pore Size C, Ace Glass Company cat. no. 7209-16; OR outer diameter 12mm, 125 mm long, Pore Size E, Ace cat. no. 7209-10. Size of isolation tube depends on size of electrodes used.
  • Pipettor or dispenser 10 ml and 30 ml - Oxford, Wheateon, Lab Industries or equivalent
  • volumetric flasks 100, 500 ml, 1000 ml with stoppers D. REAGENTS
  • Degassed water should be used for the 0.1M potassium iodide solution to prevent air oxidation of I ' to l 2 .
  • Degas water by placing deionized water into a vacuum flask, and placing it under vacuum for 15 minutes with sonication.
  • Weight 142 ( ⁇ 0.5) g of sodium sulfate into a 1 liter volumetric flask. Add approximately 750 ml of deionized water and mix until dissolved. Dilute to volume with deionized water. This reagent may be stored for six months at room temperature.

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EP96911563A 1995-04-07 1996-04-04 Kalzium zusätze und kalzium enthaldende getränke welche vitamin d beinhalten Withdrawn EP0818957A2 (de)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US08/418,393 US5698222A (en) 1995-04-07 1995-04-07 Calcium supplement
US08/418,391 US5597595A (en) 1995-04-07 1995-04-07 Low pH beverage fortified with calcium and vitamin D
US418391 1995-04-07
US08/418,729 US5609897A (en) 1995-04-07 1995-04-07 Powdered beverage concentrate or additive fortified with calcium and vitamin D
US418393 1995-04-07
PCT/US1996/004601 WO1996031130A2 (en) 1995-04-07 1996-04-04 Calcium supplements and calcium containing beverages comprising vitamin d
US418729 1999-10-15

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NZ306211A (en) 1998-08-26
WO1996031130A3 (en) 1997-01-03
AU5441596A (en) 1996-10-23
NO974540D0 (no) 1997-10-01
WO1996031130A2 (en) 1996-10-10
NO974540L (no) 1997-12-08
MX9707584A (es) 1998-02-28
CA2217264A1 (en) 1996-10-10
JPH11503023A (ja) 1999-03-23
AU715227B2 (en) 2000-01-20

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