Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-08-07
2004-06-15
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C426S656000
Reexamination Certificate
active
06750035
ABSTRACT:
BACKGROUND
1. Field of the Invention
The present invention generally relates to assays to determine the digestibility of protein-containing compositions. More specifically, the present invention relates to in-vitro digestibility assays using proteases.
2. Description of Related Art
The digestibility of protein-containing human food or non-human animal feed can vary greatly. This is particularly true for food or feed ingredients utilized in food or feed processing. Additionally, variations in digestibility can be magnified during processing of these ingredients.
Processing is often performed during production of protein-containing human food or non-human animal feed to make the food or feed more digestible, more nutritious, more preservable, or more palatable. Common processing steps include heating, storing, cooling, drying, wetting, subjecting to pressure, fermenting, particle-size reducing (e.g. by grinding), constituent separating, preservative adding, and mixing of ingredients.
Processing of protein-containing food or feed can affect the digestibility of the food or feed. For example, improper heat or pressure treatment can adversely affect digestibility of vegetable- or animal-based feed and food ingredients (Dudley-Cash, 1999, .Feedstuffs 71:10; Wang et al., 1998, Poultry Sci. 77:834). Also, digestibility of food or feed can be affected by storage conditions (Culver et al. 1998, J Dairy Sci. 72:2916) and the quality of the new raw ingredients (Wang et al., Id).
Digestibility may be measured using in vivo or in vitro assays. See generally Swaisgood et al., 1981, Adv. Food Nutr. Res. 35:185. Several in vivo digestibility assays have been developed, including the rat true digestibility assay (Chang et al., 1990, J. Agric. Food Chem. 38:1016), ileal nitrogen digestibility assays using pigs (Jaguelin et al., 1994, Proc. 6
th
Int. Symp. Digestive Physiol. in Pigs, Bad Dobran, Germany, 114) or dogs (Johnson et al., 1998, J. An. Sci. 76:1112), and precision-fed cockerel assays using conventional or cecectomized animals (Han et al., 1990, Poult. Sci. 69:1544; Wang et al., 1998, Poult. Sci. 77:834). Because these in vivo assays are time consuming, expensive, and difficult to perform, several in vitro digestibility assays have been developed.
In vitro digestibility assays utilize proteolytic enzymes to correlate with in vivo protein digestion. Most of these assays utilize mammalian gastric and/or pancreatic and intestinal enzymes to more closely mimic mammalian digestion. Some assays use one enzyme, usually pepsin, to digest the test protein in solution although papain has also been used (Buchanan, 1969, Br. J. Nutr. 23:533; Buchanan and Byers, J. Sci. Food Agric., 20:364). The digestion is followed by an assay for amino acids. More commonly, the in vitro assays utilize more than one enzyme. Results from these multiple enzyme assays usually correlate more closely to in vivo results than single enzyme assays. Examples of these assays are described in Jaguelin et al., 1994, Proc. 6
th
Int. Symp. Digestive Physiol. in Pigs, Bad Dobran, Germany, 114 and W, McDonough et al., 1990, J. Assoc. Off. Anal. Chem. 73:622.
A variant of these in vitro enzyme digestibility assays, IDEA
I
mmobilized
D
igestive
E
nzyme
A
ssay), utilizes enzymes which are covalently immobilized on large-pore diameter (200 nm) glass beads via an amide linkage (Porter et al., 1984, J. Agr. Food Chem. 32:334; Chang et al., 1990, J. Agric. Food Chem. 38:1016). By employing immobilized enzymes, this assay has the following advantages over soluble enzyme assays: autolysis (digestion of the enzymes by the enzymes themselves) is prevented, the stability of the enzymes can be increased, the digest is not contaminated with the enzymes or their autolysis products, the digest is easily separated from the enzyme, and the immobilized enzymes can be used in multiple assays. The IDEA method uses two bioreactors. The first contains immobilized pepsin and the second contains immobilized trypsin, chymotrypsin, and intestinal mucosal peptidases. The bioreactors employ either a recirculating design (Chang et al., 1992, J. Food Biochem. 16:133) or a fluidized bed design (Culver et al., 1989, J. Dairy Sci. 72:2916). Digestion of the test sample proceeds in the pepsin bioreactor at low pH (~2) for 18-20 hr at 37° C. The pH of the pepsin digest is then adjusted to 7.5 with Na
2
HPO
4
then treated with the second biodigester for 24 hr at 37° C. The free a amino groups are then determined using o-phthalaldehyde (OPA) in the presence of mercaptoethanol, which yields adducts which absorb strongly at 340 nm (Church et al., 1985, Anal. Biochem. 146:343). This value is compared the total peptide bonds present in the test sample, determined by complete acid hydrolysis of the sample followed by OPA determination. Digestibility is defined as the fraction of the total peptide bonds present which were hydrolyzed by the digesters.
The IDEA assay has proved useful for digestibility determinations of various complex food compositions (Chang et al., 1990, J. Agric. Food Chem., 38:1016; Thresher et al., 1989, Plant Foods for Hum. Nutr. 39:59; Chang et al., 1992, J. Food Biochem. 16:133). However, the assay takes about two days and requires an elaborate heated recirculation system. Thus, while the IDEA assay is simpler and less costly than the in vivo digestibility assays and has several advantages over the in vitro assays employing soluble enzymes, it is still rather difficult to use for routine determination of digestibility of, e.g., raw materials coming into a processing plant, food or feed at various stages of processing, or finished food or feed. The IDEA system would also be prohibitively time consuming and complex for use in conjunction with animal feed processing; it has not been suggested for that use. Therefore, there is a need for digestibility assays suitable for routine use by, e.g., food and feed processors.
SUMMARY
Among the several aspects of the present invention may be noted the provision of methods for determining the digestibility of food and non-human-animal feed. A more specific aspect of the invention is the provision of such methods in the form of assays which are more rapid and/or simpler to execute than previously known assays. It is also an aspect of the invention to provide digestibility assays wherein the digestibility of a protein-containing composition is determined by visually comparing the assay results with a standard result. A further aspect of the invention is the provision of methods for determining the acceptability of non-human-animal feed ingredients by rapid and simple digestibility assays.
Briefly, therefore, the present invention is directed to a method of measuring digestibility of a protein-containing composition. The method comprises (a) mixing the protein-containing composition with an aqueous liquid to form an aqueous composition; (b) incubating the aqueous composition with a first immobilized protease in a reaction vessel and causing movement of the aqueous composition relative to the immobilized protease in such a manner that none of the aqueous by liquid is removed from the reaction vessel to metabolize a first portion of the protein-containing composition into it, hydrolyzed peptide bonds; and (c) estimating the hydrolyzed peptide bonds.
Another embodiment is directed to a method of measuring digestibility of a protein-containing composition comprising (a) mixing the protein-containing composition with an aqueous liquid to form an aqueous composition; (b) incubating the aqueous composition with a first immobilized protease in a reaction vessel and causing movement of the aqueous composition relative to the immobilized protease in such a manner that none of the aqueous liquid is removed from the reaction vessel to metabolize a first portion of the protein-containing composition into hydrolyzed peptide bonds;(c) separating the composition from the first immobilized protease; (d) incubating the aqueous composition with at least one additional immobilized protease in a reaction vessel and moving the aqu
Schasteen Charles S.
Wu Jennifer
Gitomer Ralph
Novus International, Inc.
Senniger Powers Leavitt & Roedel
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