Technological field of invention

Food technology - dietetic products Technological problem

Gout is a disease accompanied by severe pain and complications due to accumulation and sedimentation of uric acid or its salts-urates. Therapy consists of drugs which inhibit the production of uric acid and of 'apurine diet' which does not contain purine-rich food, primarily meat, processed meats and offal. Milk is recommended as the only source of essential proteins. However, a very important fact is often neglected: milk is significantly rich in nucleic acids, as well as in mononucleotide and nucleoside derivatives, in other words, it is rich jn free purine bases (hypoxantine, xantine), and particularly in uric acid itself! The uric acid which was formed in digestive tract or was obtained from food does not metabolize in the human organism and it is resorbed into the bloodsteam, directly contributing to blood uric acid increase!

State of the Art

According to literature, the amount of uric acid in cow milk samples is around 200 mol/L and the amount of other purine bases is around 20 μιτιοΙ/L. Milk is rich in purine ribonucleotides, substrates of nuclease enzyme which are present in milk, and particularly in the digestive tract. According to literature, the amount of present ribonucleotides in the form of polynucleotides and oligonucleotides is around Θ8+/-55 μ/ηοί/L, in the form of mononucleotides it is around 84 +/- 25 μιηοΙ/L and 10 +/- 2 pmol/L in the form of nucleosides. There exists a certain amount of free purine bases, such as hypoxanthine and xanthine up to the amount of 20 pmol/L. The mentioned derivatives inevitably succumb to hydrolytic breakdown in the digestive tract, which means that 200 pmol/L is the minimum amount of uric acid that can be approximately produced and/or consumed from 1 L of milk.

The content of uric acid varies in milk samples (Table 1 , 2 and 3), but it is never below 100 pmol/L, although our results confirm that manipulation which activates xanthine oxidase (homogenisation) or an analysis of full fat, non-skimmed milk returned high levels of uric acid which amounts even up to 300 pmol/L. Our results vary depending on the specificity of the methodology as well as the type of milk. Since we obtained around 330 pmol/L in milk samples using spectrophotometric technique, we applied a more precise chromatographic technique, HPLC method. The results showed that the values of uric acid were around 100-200 pmol/L, whereas the concentration of purine bases (adenine, hypoxanthine, xanthine and guanine) and bases within ribonucleic acids amounted to 50 pmol/L in cow milk, but more in goat milk. The results show the exact values of uric acid and purine compounds measured by the HPLC method using an appropriate standard.

Milk is rich in xanthine oxidase, a key enzyme in the production of uric acid from which it was first originally isolated and analyzed. Commercial preparations of purified xanthine oxidase are still produced from milk. As a significant source of free radicals, it appears to be bactericidal, but its detrimental effect is mirrored in high production of uric acid from available exogenous substrates. Having in mind the fact that milk is rich in purine ribonucleotides, as well as nucleases, it is obvious that the degree of sample manipulation, such as the length and temperature of homogenisation, significantly influence the increase in the amount of nucleosides, free bases and uric acid in milk.

Procedures recommended for isolation of purine nucleotides from milk mostly aimed at more precise determination of the type of present purine and pyrimidine bases, which means that they were mentioned solely for exploration purposes but not for the purpose of elimination from food, i.e. for dietetic purposes. The literature describes the isolation of purine bases on specific ion exchange columns. The column usually contains cation resin, most often DOWEX, which binds anions such as uric acid. In order to isolate the mentioned bases, it is first of all necessary to perform the deproteinisation of the sample. Otherwise, the cation resin would bind the proteins from milk which also have anion properties, whereby the degree of column sample purity would be decreased but in the wider sense, milk would also be poor in proteins. Therefore, this method is predominantly analytihical. In the process of analysis of the type and characteristics of the purine bases and derivatives in milk, we used HPLC chromatography so that the milk sample had to be previously deproteinized.

Other ways of uric acid isolation imply the use of dialysis techniques. In this sense, a majority of small, diffusible molecules such as lactose, urea, uric acid, creatinine, amino acids and ions can be isolated from milk. If performed in the laboratory, the technique is very demanding because it is necessary to use specific dialysers for small molecules. The technique should not be neglected in medicine because of the results obtained from patients with gouty renal insufficiency in whom uric acid increases above 700 pmol/L accompanied by renal insufficiency. After the dialysis, these patients show a significant decrease in the concentration of uric acid in plasma, as well as urea. If milk was to undergo dialysis during the production process, this would mean that a dairy would have to possess a dialyser as well as a prepared dialyzing liquid causing an increase in production costs. If milk dialysis is performed under appropriate conditions, the use of dialysis membrane made of cellophane along with dialyzing liquid which possesses no other anions except for hydroxyl group (because of the Donovan's law), the dialysis provides weak results because there is neither pressure nor constant flow of the dialyzing liquid. Besides, milk is exposed to external factors in the open system so the mentioned method is not entirely appropriate for the technological procedure of uric acid isolation in industry and it is very expensive.

References:

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2. Roubenoff R, Klag MJ, Mead LA, Liang KY, Seidler AJ, Hochberg MC.

Incidence and risk factors for gout in white men. JAMA 1991 ;266:3004-3007. Kramer HM, Curhan G. The association between gout and nephrolithiasis: the National Health and Nutrition Examination Survey III, 1988-1994. Am J Kidney Dis 2002;40:37-42.

Emmerson BT. The management of gout. N Engl J Med 1996;334:445-451. Fam AG. Gout, diet, and the insulin resistance syndrome. J Rheumatol 2002;29:1350-1355.

Gibson T, Rodgers AV, Simmonds HA, Court-Brown F, Todd E, Meilton V. A controlled study of diet in patients with gout. Ann Rheum Dis 1983;42:123- 127.

Matzkies F, Berg G, Madl H. The uricosuric action of protein in man. Adv Exp Med Biol 1980;122:227-231.

Loenen HM, Eshuis H, Lowik MR, et al. Serum uric acid correlates in elderly men and women with special reference to body composition and dietary intake (Dutch Nutrition Surveillance System). J Clin Epidemiol 1990;43:1297- 1303.

Garrel DR, Verdy M, PetitClerc C, Martin C, Brule D, Hamet P. Milk- and soy- protein ingestion: acute effect on serum uric acid concentration. Am J Clin Nutr 1991 ;53:665-669.

Ghadirian P, Shatenstein B, Verdy M, Hamet P. The influence of dairy products on plasma uric acid in women. Eur J Epidemiol 1995;11 :275-281. Clifford AJ, Riumallo JA, Young VR, Scrimshaw NS. Effect of oral purines on serum and urinary uric acid of normal, hyperuricemic and gouty humans. J Nutr 1976;106:428-434.

Zollner N, Griebsch A. Diet and gout. Adv Exp Med Biol 1974;41 :435-442. Griebsch A, Zollner N. Effect of ribomononucleotides given orally on uric acid production in man. Adv Exp Med Biol 1974;41 :443-449.

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Campion EW, Glynn RJ, DeLabry LO. Asymptomatic hyperuricemia: risks and consequences in the Normative Aging Study. Am J Med 1987;82:421-426. Nugent CA. Renal urate excretion in gout studied by feeding ribonucleic acid. Arthritis Rheum 1965;8:671-685. 16. Gibson T, Highton J, Potter C, Simmonds HA. Renal impairment and gout. • Ann Rheum Dis 1980;39:417-423.

17. Glynn RJ, Campion EW, Silbert JE. Trends in serum uric acid levels 1961- 1980. Arthritis Rheum 1983;26:87-93.

18. Raiziss GW, Dubin H, Ringer AI. Studies in endogenous uric acid metabolism.

J Biol Chem 1914;19:473-485.

19. Dessein PH, Shipton EA, Stanwix AE, Joffe Bl, Ramokgadi J. Beneficial

effects of weight loss associated with moderate calorie/carbohydrate restriction, and increased proportional intake of protein and unsaturated fat on serum urate and lipoprotein levels in gout: a pilot study. Ann Rheum Dis 2000;59:539-543

20. Shadick NA, Kim R, Weiss S, Liang MH, Sparrow D, Hu H. Effect of low level lead exposure on hyperuricemia and gout among middle aged and elderly men: the Normative Aging Study. J Rheumatol 2000;27:1708-1712.

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disease: the Framingham Study. J Clin Epidemiol 1988;41 :237-242.

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behaviour in patients with acute gout. Can Med Assoc J 1984;131 :563-567.

24. Kearns J. Charcoal Filtration Basics 2007; 1-7.

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Description of New Invention

The above referenced technical problem is solved by composite dietetic milk draught and method for production of composite dietetic milk draught with reduced level of uric acid and purine compounds. The draught according to this invention comprises the following properties:

• to eliminate uric acid, nuclic acids, nuclotides and free purine bases from milk

• to contain substances which would enable a better elimination of uric acid in the kidneys - to achieve uricosuric effect

• to eliminate heavy metals which have a toxic and destructive effect on the tubular system of the kidneys and present an additional cause of hyperuricemia because they inhibit active secretion of uric acid

• to eliminate xanthine oxidase which is present in milk in high levels so that the remaining amount, for the most part, be irretrievably inhibited and that the presence of natural xanthine oxidase inhibitors lowers the activity of this enzyme in the digestive tract

• to contain antioxidants which would reduce oxidative stress and naturally inhibit xanthine oxidase

Technological procedure according to this invention is designed for achieving the above referenced properties with goal to in non-toxic way eliminates uric acid as much as possible, as well as purine ribonucleotides, desoxyribonucleotides, nucleosides and free purine bases (adenine and guanine) which are present in milk. Everybody can consume this type of dietetic milk draught but it is primarily produced for people with gout and increased content of uric acid in blood.

The technique of purine bases and nucleotide isolation by charcoal proved useless under experimental conditions because adsorption of uric acid and purine bases is so stable that they cannot be eluted from the adsorbents even by the more aggressive treatments (such as NaOH), in other words they cannot be detected and quantified. This is when our method of separating the uric acid from milk is useful because such high affinity inhibits the release of purine bases and uric acid once bound to charcoal. Charcoal acts as a strong adsorbent and binds a number of elements, especially purine and pyrimidine derivatives. In this way, uric acid is eliminated irreversibly, as well as other purine and pyrimidine derivatives because charcoal has a high power of adsorption of these elements which depends on charcoal concentration, minimum exposure time, but also physical conditions such as temperature. Charcoal is considered non-toxic to human organism because it is not resorbed in the digestive tract so it had a therapeutic use in different kinds of poisoning, especially heavy metal poisoning. Charcoal is a very powerful adsorbent because it is familiar that 1 g has an active surface of around 1000m ² . The effectiveness of charcoal is also proved by the fact that drinking water is purified by charcoal water filters, which means that it is one of the least toxic materials.

Charcoal drugs are still used in the treatment of various poisonings, but also in the treatment of hypelipidemia because they effectively bind lipids, especially cholesterol. Earlier, when there were no drugs for the treatment of gout, charcoal was mentioned in the literature as a way of treatment, but during that time large doses were used because it was necessary to provide constant presence of charcoal in the digestive tract. Large amounts were used, up to 35 g a day, but in this way only temporary elimination of uric acid from the digestive tract was provided. Daily consumption of such great amounts of charcoal was difficult and pointless because it is quickly eliminated via gut peristaltic. Apart from the elimination of purine and pyrimidine nucleotides, charcoal also helps remove the unpleasant smell coming from hydrogen sulfide. The charcoal is not used routinely in the treatment of hypelipidemia, although it has been shown that it reduces the values of cholesterol and aterogenous lipoproteids in blood up to 30%.

Having in mind all the findings, we have arrived at the idea to use charcoal for binding uric acid and purine derivatives in milk. An additional gain is the binding of heavy metals as well as lipids which is a positive effect of this dietetic product considering that toxic metals damage tubules whereby secretion of uric acid at the level of kidney tubule is aggravated. The lipids are one of the causes of secondary hyperuricemia in obesity and diabetes.

In case of additive substitution, the suggested formula refers to the composite dietetic milk or its derivative products. The formula is shown in Table 1 and can be further modified based on the form of the draught (milk or fermented milk draught - yogurt). Milk may be in the form of liquid or condensed composite milk. In the technological procedure, the separation of uric acid, as well as other purine and pyrimidine derivatives is performed by the method of adsorption by charcoal.

Dietetic justification of the discovery: The potential significance of the new milk product is best described by the fact that the FDA (Food and Drug Administration program) announced the expectations that 'in the future, milk will become functional food chosen by the consumers according to their needs'. Besides, the 21st century is the century of functional food and genomics. As part of genomics, nutrigenomics has its place in functional food because the development of modern scientific trends opens up the possibility for a modern dietetic approach. Through daily diets, this approach will significantly model individual predispositions to diseases thus preventing the predictive risk of diseases.

Gout is a relatively common disease caused by sedimentation of uric acid and its salts in joints and other parts of the body, predominantly kidneys. Prevalence of gout is larger in developed countries. During 1999, the incidence of gout in America was 41 , out of 1000 patients, whereas in England it was 14. It is assumed that only in America there are around 5 million people with gout. American data show that around 10% of people has hyperuricemia but it cannot be predicted with certainty when and who will develop gout. The number of patients with gout is rising. People over 65 years of age belong to predisposed population. At younger age, gout usually appears as a consequence of congenital defects in allosteric regulation of purine synthesis. The consequence of enormous synthesis is an enormous degradation and subsequent uric acid production. The Lesch-Nyan syndrome caused by a deficiency of the HGPRT enzyme is also characterized by enormous uric acid production and gout as a common aggravating condition. Compared to women, gout is more common in men with the ratio of 9:1 but due to secondary hyperuricemia, caused by obesity or diabetes, later in life, the ratio changes to 3.6:1. It is often a family disease. These families have members suffering from diabetes, obesity or kidney stone. Sedimentation of uric acid and its salts causes damage and permanent deterioration of joint cartilage which, in some cases, affects joint mobility, causes deformities and limited working and physical ability. Although gout is a chronic disease, acute gout attack appears in crises. Patients suffering from gout also show a number of subsequent co-morbidities such as coronary disease, hypertension, diabetes and kidney diseases. It is believed that hyperuricemia is of secondary character.

Data published in the New England Journal of Medicine concerning the effect nutritive factors have on the development of gout, show that the incidence of gout is lower in people who consume skimmed, low-fat milk. This is logical if we consider the fact that xanthine oxidase is located in fat drops. Our findings confirm a lower level of uric acid in this kind of milk. Many web sites and instructions on healthy diets of patients with gout or secondary hyperuricemia allow the consumption of milk and put it on the list of the so-called 'apurine diet'. But is that true? Certainly not, for many reasons. Having in mind the frequency of the disease, potential risks, as well as the significance of milk as the only source of essential proteins (caseine and lactalbumin), there is a need for the production of the whole line of different dietetic milk products for this population.

The significance of the mentioned additives has been proved in other researches which show that people who drink at least 1.500 milligrams of vitamin C a day lower the risk of gout by 45%, as opposed to those whose daily intake of the vitamin is less than 250 milligrams. Coenzyme Q10 (ubichinon, ubidecarenone, coenzyme Q) is an important coenzyme of the respiratory chain of the mitochondria but also a strong antioxidant. It has been shown that the coenzyme Q10 treatment is especially useful in patients with coronary disease, hypertension and cardiac insufficiency.

Considering the detrimental effect that free radical have in adults, the question arises on how to evade the activity of xanthine oxidase in cow milk. The enzyme in cow milk is very stable and it is destroyed by sterilisation at 80°C for at least 10 seconds, i.e. UHT sterilisation. If the enzyme is still present, the oxidation activity will change the taste and organoleptic properties of milk. Sometimes, the measurement of xanthine oxidase activity in milk can give false negative results because the enzyme is localized in specific milk fat globules where it is often protected and inactive, thus' unavailable to activity measurement. Homogenisation processes cause the destruction of milk fat globules and the enzyme is released and becomes an active producer of free radicals. On average, fresh milk has an activity of around 50- 60 IJ/L whereas the fat part has an activity of around 200 IJ/L. In connection to this, during the late 1980's of the 20th century, there appeared a theory on the direct aterogenous effect and increased cardiovascular risk, caused by the consumption of homogenized milk, because the available xanthine oxidase can be resorbed and have negative toxic effects on the endothelium of blood vessels. Particularly sensitive are cell structures which contain complex lipids-plazmalogens.

Detailed description of formula preparation Detailed description of formula preparation is presented in phases through which milk passes in the dairy, staring from the collection sites.

Phase I Primary sample: For the technological procedure of milk processing, dairies take the milk obtained from healthy, properly fed and regularly milked cows or goats, at least 15 days before and 5 days after calving, without adding or subtracting anything from the milk. Average chemical content of domestic cow milk is: water (87.3%), dry matter (12.7%). Dry matter contains: proteins, mostly casein (3.55%); fat (3.8%); lactose (4.7%); minerals (0.65%). Acidity should not be above 7.60 SH, milk should be kept at optimally low temperature and should be microbiologically safe with proper smell and taste. Milk is obtained from collection dairy.

Phase II Elimination of uric acid, nucleic acids and nucleotides: This phase is marked by the separation of uric acid and purine and it starts by taking 1 L of milk from the collected milk. 50-100 g of granulated charcoal (from coconut) is added to 1 L of milk sample, i.e. from -10% sample. The sample is mixed and placed at 25- 30°C for 2-4 hours.

Phase III Homogenisation. Milk samples undergoe homogenisation with the aim to increase the stability of milk emulsion by reducing the average diameter of fat globules. This is a routine procedure in milk processing which contributes to better and longer adsorption. Further, we shall address homogenisation as a negative factor in the sense of release of xanthine oxidase from inactive form which is at this point released from fat globules and becomes active in producing uric acid. Since charcoal is already present, all newly-formed uric acid will be bound. Table 2 and sheme 1 show the influence of various physical factors on the final residual concentration of the uric acid in the sample.

Phase IV Fat standardisation to 0.5%. Depending on the race and individual properties of the herd, cow milk contains between 2.5 and 6.0% of milk fat, so milk fat is a necessary ingredient of milk. Suggested product should contain 0.5% milk fat. This suggestion is the result of exploration of uric acid content in samples of pasteurized or UHT milk available in our market. The analyzed milk samples had from 3.25 to 0.5% milk fat and it was established that the lowest level of uric acid was in the samples of milk with the lowest level of fat. This is logical if we have in mind that xanthine oxidase is located in fat drops. Centrifugal separator is used for separation of milk fat. The separation is based on the difference between the specific mass of milk fat and other milk components (0.93g/cm ³ to 1.032g/cm ³ ). The suggested milk draught contains 0.5% milk fat. The process is called 'in line' fat standardisation and it is composed of the following elements: density transmitter, flow transmitter, control valve, control board and turn off valve. It is a standard apparatus and every dairy possesses one. The specific apparatus is described based on the one owned by the Dairies.

Phase V Charcoal elimination: Bactofuge is an apparatus used only for removing bacteria, dirt and spores which is its standard purpose in the industrial milk processing. Milk sample is processed in a bactofuge when charcoal is eliminated by centrifuging because larger particles are sedimented. In addition to bactofuge milk goes through KOFIL S5/100 (mm/g/m ² ) filter and then through Whatman Paper and the smallest particles of charcoal are detained on the paper.

Phase VI Addition of additives: The suggested additives are added to the milk sample with reduced content of uric acid, nucleic acids and purine. Additives are hydro-soluble matters which dissolve well in water and also in milk. On the basis of their physicochemicai properties it has been established that there is no interaction with milk components. The suggested additives are:

• Vitamin C-L-Ascorbic acid 1000mg/L

• Coenzyme Q10 (natural) 200mg/L

• Arginine 500 mg/L

An appropriate amount of L-Ascorbic acid, Arginine and Coenzyme Q10 is added to 1 L milk sample and mixed well. If a larger amount of milk is taken initially, the concentration of additives is calculated according to the formula:

recommended dose x liters of milk sample

Since lactose is also to a small degree adsorptive of charcoal, its values are somewhat lower than in the standard milk products, which is favorable for patients suffering from obesity and diabetes.

Phase VII Deodorisation. The use of partial vacuum for removing unpleasant smells from milk represents a standard procedure. Addition of charcoal is favorable in this phase because charcoal is used with the aim of removing unwanted odor. Phase VIII UHT sterilisation. Sterilisation implies that milk had been homogenized, cooled to the temperature of 4 to 1°C, purified and heated to the temperature of 135 to 150°C at least 24 hours after milking, after which it is cooled and primed under aseptic conditions. During UHT sterilisation, there is no change in physicochemical properties of the mentioned vitamins but an irreversible inhibition of xanthine oxidase takes place. This is a standard procedure performed by every dairy so that this step need not be described in detail because it is neither new nor modified.

During the analysis of charcoal adsorption we have paid particular attention to the effects of the following factors: ^' charcoal concentration in the sample, milk exposure duration and temperature. In each of the variables, mechanical stirring contributes to a better adsorption which is also the basis of the physical procedure of adsorption. Table shows various physical factors which influence the final-residual amount of uric acid in the sample. It can be confirmed that milk exposure duration and temperature have more influence than the amount of milk fat present. This should be expected because adsorptive surface of charcoal is large and the present fats have no significant effect.

Phase IX Packaging. Nonreturnable plastic or cardboard containers are recommended. Tetra pak or Tetrabrik represent the most suitable cardboard packaging. Wrapping and original package are shown in Picture 4. The package would provide the originality of the product. When the product is put on the market in the original package, the declaration would contain the following data:

) name of product

2) name and place of manufacturer

3) production date and expiration date

4) net weight (volume) of package

5) list of ingredients and additives

6) storing conditions

Table 1. Percentage of uric acid in cow milk according to different height above sea level.

Table 2a, b and c. Percent of uric acid and purine derivatives according to the conditions of charcoal treatment a)

Legend:

H milk sample was maintained on cold conditions (H2 for 2 hours, H4 for 4 hours)

T milk sample was maintained on thermal conditions of 25-30°C (T2 for 2 hours,

T4 for 4 hours)

Numbers in front represent the percent of active charcoal:

OH and 0T means without charcoal;

Numbers 1 , 2, 3, 4, 5 in front of H or T mean , 1%,2%,3%,4%,5% of prepared sample according to the percent of active charcoal

6T means 10% sample. Types of milk investigated

Goat milk -(z bregov) Vindija, importer ERFA Ljubljana 3,0% milk fat;

2) Alpen milk- Polno alpsko mleko, 3,5% milk fat (Ljubljanske mlekarne);

3) Milk- Mleko Spar 3,5% milk fat