Moisture in proteins

Lin and Hsu [1.85] described the determination of residual moisture in protein pharmaceuticals in sealed glass vials by near-infrared (NIR) spectroscopy. Five proteins were studied recombinant humanized monoclonal antibody (rhuMAb) E25, rhuM-Ab HER2, rhuMAb CDlla, TNKase and rt-PA. Higher moisture contents (RM) were obtained by adding appropriate amounts of MilliQ water to the wall of the vial in a... 

Broadhead J, Rouan SKE, Hau 1, Rhodes CT. The effect of process and formulation variables on the properties of spray-dried gaiactosidas. J Pharm Pharmacol 1994 64 458-67. Hagcman MJ. The role of moisture in protein stability. Drug Dev Ind Pharm 1988 14 2047-70. 

Ml Hageman. The role of moisture in protein stabihty. Drug Dev Ind Pharm 14 2047-2070, 1988. 

Infrared spectra of fats and oils are similar regardless of their composition. The principal absorption seen is the carbonyl stretching peak which is virtually identical for all triglyceride oils. The most common appHcation of infrared spectroscopy is the determination of trans fatty acids occurring in a partially hydrogenated fat (58,59). Absorption at 965 - 975 cm is unique to the trans functionaHty. Near infrared spectroscopy has been utilized for simultaneous quantitation of fat, protein, and moisture in grain samples (60). The technique has also been reported to be useful for instmmental determination of iodine value (61). 

Defatted peanuts are high in protein, low in moisture, contain only 20% of the naturally occurring fat, and have better stability than whole peanuts. Monosodium glutamate (MSG) has been used as a flavor enhancer for defatted nuts, but the result has not been entirely satisfactory as the addition of MSG produces a meaty rather than nutty flavor. This meaty flavor is more compatible with salted butter and nuts than with candy. 

In addition to the additives used in a formulation to help stabilize the protein to freezing, the residual moisture content of the lyophilized powder needs to be considered. Not only is moisture capable of affecting the physicochemical stability of the protein itself, equally important is the ability of moisture to affect the Tg of the formulation. Water acts as a plasticizer and depresses the Tg of amorphous solids [124,137,138]. During primary drying, as water is gradually removed from the product, the Tg increases accordingly. The duration and temperature of the secondary drying step of the lyophilization process determines how much moisture remains bound to the powder. Usually lower residual moisture in the finished biopharmaceutical product leads to enhanced stability. Typically, moisture content in lyophilized formulations should not exceed 2% [139]. The optimal moisture level for maximum stability of a particular product must be demonstrated on a case-by-case basis. 

In agriculture and food, NIR has been a powerful tool for decades. All shipments of grain leaving US ports are analyzed for moisture, fat, protein, and starch via NIR. Processed foods are also a prime venue for NIR percent of fat in cheese spread, hardness of wheat, and freshness of meats are just some of the applications in food. 

The first NIR instruments were, in reality, developed for the UV and Vis regions of the spectrum. They were made by seven companies Beckman, Cary (now owned by Varian), Coleman, Perkin-Elmer, Shimadzu, Unicam, and Zeiss. Based on the work of Karl Norris and coworkers in the USDA, the Illinois Department of Agriculture solicited bids from companies to produce a pure NIR instrument, capable of measuring protein, oil, and moisture in soybeans. 

The same functions used in agriculture can be applied to processed foods. In baked goods, wheat gluten, various additives, starch damage, and water absorption are just some of the parameters measured [21-24]. Dairy products are also important and often analyzed by NIR. Moisture, fat, protein, lactose, lactic acid, and ash are common analytes in the dairy industry [25-28]. 

As seed moisture in field peas decreases, there is a decrease in starch fraction yield, protein content of starch fraction, protein content of protein fraction, and percent starch separation efficiency, and a concurrent increase in protein fraction yield, percent starch in starch fraction, percent starch in protein fraction, percent protein separation efficiency, and percent neutral detergent fiber in the protein fraction. Lower moisture content of field peas improves milling efficiency and results in more complete separation of protein and starch fractions, which could explain the increase in protein fraction yield and percent starch in starch fraction, improved protein separation efficiency and less protein in the starch fraction. The decrease in starch separation efficiency was probably due to the increased starch content of protein fraction and increased protein fraction yield with lower seed moisture. 

Food-control laboratories seeking to be accredited for the purposes of the Directive should include, as a minimum, the following techniques in generic protocols HPLC, GC, atomic absorption and/or ICP (and microscopy). A further protocol on sample preparation procedures (including digestion and solvent dissolution procedures) should also be developed. Other protocols for generic methods which are acceptable to UKAS may also be developed. Proximate analyses should be addressed as a series of specific methods including moisture, fat, protein and ash determinations. 

E. Bouveresse, D.L. Massart and P. Dardenne, Calibration transfer across near-infrared spectroscopic instruments using Shenk s algorithm effects of different standardisation samples. Anal. Chim. Acta, 297, 405 16 (1994). B.G. Osborne and T. Feam, Collaborative evaluation of universal cahbrations for the measurement of protein and moisture in flour by near-infrared reflectance, /. Food TechnoL, 18, 453 60 (1983). 

Another important factor proved to be the influence of trace quantities of moisture in the compacts, from both the environment and also within the protein itself. Dry proteins adsorb water with avidity because of the exposed hydrophilic regions within the protein structure, changing the properties of the hydrogen bonding within the structure and, therefore, the flexibility of the overall structure. Excessive drying, on the other hand, can result in the collapse of an otherwise water-soluble protein to the point where it is effectively denatured and will no longer dissolve. 

Cheeses made from milks with high fat contents tend to have higher moisture levels in relation to protein content. This ratio, also called moisture in the nonfat substance (MNFS), is an important compositional factor influencing the quality of cheese (Pearce and Gilles 1979). It is the relative wetness of the casein in the cheese, rather than the percentage of moisture in the cheese as a whole, that influences the course of the ripening process (Lawrence and Gilles 1980). 

The ability of MIR to monitor fundamental vibrations of several functional groups provides a new tool for researchers to look at minor compounds in cheese. Some of its early applications were focused on the analysis of macromolecules in cheese such as fat, moisture, and protein (Chen et ah, 1998 McQueen et ah, 1995). More recently, the chemical parameters of cheese (Martfn-del-Campo et ah, 2007), composition (Rodriguez-Saona et ah, 2006), protein structure and interactions during ripening (Mazerolles et ah, 2001), and ripening of Swiss cheese (Martin-del-Campo et ah, 2009) were analyzed with improved techniques. Almost all attempts have been directed toward the determination of macromolecules in cheese. This is mainly because of difficulties in sampling procedures and the heterogeneous nature of cheese (McQueen et ah, 1995) that make analysis of minor compounds difficult. 

Osborne, B.G. and Fearn, T., Collaborative Evaluation of Universal Calibrations for the Measurement of Protein and Moisture in Flour by Near-Infrared Reflectance /. Food Technol. 1983, 18, 453-460. 

Iin, T.P., Hsu, Ch.C Determination of residual moisture in lyophilized protein pharmaceuticals using a rapid and non-invasive method near-infrared spectroscopy. PDA J. Pharm. Sci. Technol. 56, pp. 196-205, 2002... 

Dambergs, N. (1963). Extractives of fish muscle. 3. Amounts, sectional distribution and variations of fat, water-solubles, protein and moisture in cod fillets. Journal of the Fisheries Research Board of Canada 20,909-918. 

Suppes, C., Tiemeier, O.W. and Deyoe, C.W. (1967). Seasonal variations of fat, protein and moisture in channel catfish. Transactions cfthe Kansas Academy of Science 70, 349-358. 

As can be seen, as protein concentration increased, blandness decreased. Overall it was concluded that DSF and G were the blandest protein sources evaluated while SC was the least bland. Perhaps the differences in protein contents within the same series (DSF and SPC WPC and SC) produced these observed differences. It was also apparent that the two high temperature sets of samples were not as bland as the low temperature series. Within each set, high moisture produced blander tasting products than low moisture. More flavorful compounds were produced with high temperature, apparently due to thermal decomposition reactions. Perhaps, high moisture produced starch/protein structures do not permit the rapid and/or complete release of flavor compounds in the mouth. 

Uses of partial-waxy and waxy wheat in Asian noodles, bread and tortillas have been reviewed.269,270,298 French bread made from double-null partial waxy wheat flour retained 1-2% more moisture in its crumb in 24 hours compared to bread from a wild type flour. French bread made from the mutant wheat had a softer crumb immediately and up to 48 hours after baking.299 Wheat noodles are made from flour and low levels of salts, so starch plays a major role in noodle quality. Flours from partial waxy wheats with —10% protein and 21-24% apparent amylose (starch basis) are favored for white salted (sodium chloride) noodles because partial waxy starch swells during cooking somewhat more than non-waxy wheat starch. For good appearing noodles, the flour should be from a white wheat low in polyphenol oxidase, and... 

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