Food or edible material: processes – compositions – and products – Fermentation processes – Of milk or milk product
Reexamination Certificate
2000-08-17
2003-03-18
Hendricks, Keith (Department: 1761)
Food or edible material: processes, compositions, and products
Fermentation processes
Of milk or milk product
C426S047000, C426S056000
Reexamination Certificate
active
06534100
ABSTRACT:
FIELD OF THE INVENTION
This application relates to the treatment of foodstuffs using live cultures of protozoa, such as
Tetrahymena thermophila
. One aspect of the present invention is the provision of methods for the conversion of cholesterol present in foodstuffs into provitamin D3 and other sterols containing a double bond at position 7.
BACKGROUND OF THE INVENTION
Animal milk is a complex mixture of different compounds, including lipids, proteins, minerals, sugars and vitamins (Russof, L. L. (1970), J. Dairy Science 53:1296-1302). The calcium, phosphate and vitamin D content of milk make it an adequate source of nutrients for bone formation (Fox, P. F. and McSweeney, P. L. H. (1998a) Salts of milk. In “Dairy Chemistry and Biochemistry”, chapter 5, Blackie Academic & Professional, London). This may be a key aspect of its role in nature, allowing mammalian newborns to complete the formation of the skeleton after birth. Mineral and vitamin components of milk are also important to preserve bone structure in adulthood. Milk is also relatively economical, compared to other animal protein sources, and thus it makes a valuable contribution to the human diet (Russof, L. L. (1970), J. Dairy Science 53:1296-1302).
The lipid fraction of milk includes cholesterol, however, which has been implicated as a causative agent of coronary artery disease (Artaud-Wild, S. M., Connor, S. L., Sexton, G., Connor, W. E. (1993), Circulation 88:2771-2779). Other foodstuffs of animal origin such as eggs, which are commonly used in the preparation of a variety of food products, present the same problem. Because of the special organoleptic traits of milk and eggs, it is difficult to replace them by other products with less cholesterol content.
Patients with coronary heart disease (CHD) or hypercholesterolemia are commonly recommended to decrease their dietary cholesterol intake. Moreover, the general awareness of the risks associated with high blood cholesterol levels is an important factor limiting the consumption of eggs and dairy products by a health-conscious public. Cholesterol content is frequently indicated in the nutrition facts labels printed on food packages.
To address these problems, there is a need for methods to produce low-cholesterol versions of normally high-cholesterol foodstuffs, such as whole milk and eggs. Such methods should preferably not appreciably change the physical and organoleptic properties of the foodstuffs. The nutritional value of the treated foodstuffs should be preferably maintained, especially the levels of those components that are lipid-soluble and that are important for human nutrition (e.g., vitamins A and D, and essential fatty acids). Thus, the food treatment methods should yield products with lower cholesterol content but which are otherwise similar to the untreated foodstuffs. Additionally, the novel methods should preferably not require expensive equipment and materials or potentially toxic materials, such as organic solvents.
A number of methods have been described in patents in the US and other countries for reducing the cholesterol content of foodstuffs. For example, cholesterol can be removed from foodstuffs by the use of physicochemical methods. For instance, the use of supercritical fluids to produce liquid egg having reduced cholesterol content has been proposed (Ogasahara et al., U.S. Pat. No. 5,116,628). However, the high temperatures and pressures needed for the process can denature proteins present in the foodstuffs. Likewise, the production of low cholesterol butter oil by vapor sparging (Conte et al., U.S. Pat. No. 5,092,964) is another example of a method which, due to the extreme conditions used, is likely to denature proteins and alter organoleptic properties of the foodstuffs.
The use of organic solvents to extract cholesterol from foodstuffs has also been proposed. Thus, Fallis et al. (U.S. Pat. No. 4,104,286) have proposed the use of aqueous ethanol saponification and extraction with hydrocarbons and methanol to obtain free cholesterol, saponified fats and edible egg powder. This process uses extreme conditions and large quantities of organic solvents which may contaminate the processed foodstuffs. Extraction with liquid dimethylether (Yano et al., U.S. Pat. No. 4,234,619) is similarly inconvenient and does not appear to be selective for cholesterol as other neutral lipids are removed from the foodstuff. Johnson et al. (U.S. Pat. No. 4,997,668) applied solvent extraction to milk, but again the method does not appear to be selective for cholesterol and utilizes organic solvents which may contaminate foodstuffs.
A variation on the use of organic solvents is to employ oils to extract cholesterol from either aqueous or dry foodstuffs, like egg yolk and dairy products. (Bracco et al., U.S. Pat. No. 4,333,959; Keen, U.S. Pat. No. 5,039,541; Conte et al., U.S. Pat. No. 5,091,203; Merchant et al., U.S. Pat. No. 5,378,487; Jackeschky, U.S. Pat. No. 5,780,095). Again, these methods do not selectively extract cholesterol and oils contaminated with cholesterol are inevitably produced, which is undesirable.
Removal of cholesterol by formation of complexes with cyclodextrins has also been proposed for fatty substances of animal origin (Courregelongue et al., U.S. Pat. No. 4,880,573) and specifically in the case of dairy products (Chung Dae-Won, WO 9917620). The formation of complexes of cholesterol and saponin has also been described as a means to reduce cholesterol in milk (Richardson, U.S. Pat. No. 5,326,579). These methods are, however, too expensive for industrial applications.
A different approach is based on the use of enzymes that modify cholesterol. Thus, the use of cholesterol reductases, that modify cholesterol into poorly absorbed sterols, has been proposed (Beitz et al., U.S. Pat. No. 4,921,710; Ambrosius et al., U.S. Pat. No. 5,856,156). Another proposed enzymatic approach is the conversion of cholesterol into epicholesterol, which is then further modified by an epicholesterol dehydrogenase (Saito et al., U.S. Pat. No. 5,876,993). These methods are likely too expensive for industrial use, due to the cost of reasonably pure enzyme preparations, and they do not result in the conversion of cholesterol into useful compounds for human nutrition.
There is therefore a need for methods for treating foodstuffs to reduce the amount of cholesterol. Preferably, the cholesterol is converted to one or more substances that are useful for human nutrition, vitamin D, or a precursor of vitamin D such as the substances shown in FIG.
1
. For example, as shown in
FIG. 2
, desaturation of cholesterol at position 7 converts it into provitamin D3, which upon UV irradiation in the skin can be activated to vitamin D3.
Additionally, there is a need for methods to increase the level of essential unsaturated fatty acids in milk. For example, gamma-linolenic acid (18:3, n-6) is a precursor of arachidonic acid, a second messenger molecule, which in turn is the source of many other important physiological compounds, like prostaglandins. The polyunsaturated fatty acid content of milk is relatively small compared to other fatty acids. Increasing the amount of n-6 unsaturated fatty acids in milk is desirable, because such modified milk can be a sufficient source of this type of essential fatty acid. Differences in coronary mortality can be explained by differences in cholesterol and saturated fat intakes in 40 countries but not in France and Finland (See, Artaud-Wild, S. M. et al., Circulation 88:2771-2779). Moreover, an enhanced level of this type of unsaturated fatty acid promotes cardiovascular health.
Most species of protozoa of the genuses Tetrahymena and Colpidium have no sterol nutritional requirement, as shown by their growth in the absence of exogenous sterols. Under such conditions, the major unsaponifiable components are tetrahymanol, a pentacyclic triterpenoid alcohol, and diplopterol, an isomer of tetrahymanol. Other minor components found are squalene and ubiquinone (Holz, G. G. and Conner, R. L. (1973) The composition, metabolism and roles of lipids in Tetra
Florin-Christensen Jorge
Harris Joseph B.
Itzkovici Alejo
Valcarce German A.
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