Protein stability in In Vitro Digestibility Assays

Proteins widely differ in their stability to digestion in the gastrointestinal tract. Normal proteolytic digestion of consumed food proteins starts with pepsin-mediated hydrolysis in the acidic environment of the stomach, and continues with neutral pH enzymatic digestion in the small intestine. Some proteins quickly degrade to amino acids, providing great nutritional value and representing no safety concern associated with their consumption, whereas other proteins are relatively stable or they yield stable fragments (e.g., histone proteins).34 Many food allergens are stable to digestion with pepsin in a low-pH environment of the stomach,35 hence increasing a possibility that undigested allergens or their fragments would be presented to the intestinal immune system, leading to a variety of gastrointestinal and systemic manifestations of immune-mediated allergy36 (for allergenicity assessment, see Chapter 8).

Adverse reactions to food that are not mediated by the immune system are usually caused by toxic and pharmacologically active proteins contained in the consumed food. These proteins have an ability to survive the acidic environment of the stomach and proteolytic degradation with pepsin and pancreatin in biologically active forms,37-39 thereby causing a severe adverse reaction in the gut or an adverse systemic response as a result of entry into the systemic circulatory system by absorption across the intestinal epi-thelium.36,38 Consequently, evaluation of a protein's intrinsic sensitivity to proteolytic digestion with the enzymes of the gastrointestinal tract is a part of the protein safety assessment. In vitro tests have been developed to examine digestion of proteins with pepsin in simulated gastric fluid (SGF). The method was recently reevaluated during an interlaboratory study, resulting in the generation of the standardized method.40

Proteins exposed to SGF can also be exposed to simulated intestinal fluid (SIF) containing a mixture of proteases (known as pancreatin) to enhance an understanding of the protein fate during digestion in vivo. The SIF is usually prepared according to the method described in The United States Pharmacopoeia.41 Prior to the addition to SIF, the low pH and pepsin activity of the SGF assay must be neutralized. After digestion in SIF, proteins are separated using SDS-PAGE and can be either visualized by direct staining or transferred onto a PVDF or cellulose membrane and incubated with protein-specific antibodies to detect immunoreactive fragments. If a protein is digested rapidly during an exposure to SGF alone, or during short exposure to SIF following digestion in SGF, the probability of being absorbed by epithelial cells of the small intestine in a biologically active form would be extremely low. Although an in vitro digestibility assay can provide useful information regarding the intrinsic stability of introduced protein, results of these tests should be interpreted with caution, since there are oversimplified assessments of true human digestion, and only in conjunction with other components of the safety evaluation.

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