Date of Award:

5-1978

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences

Committee Chair(s)

D. K. Salunkhe

Committee

D. K. Salunkhe

Committee

T. M. Farley

Committee

A. W. Mahoney

Committee

E. McCullough

Committee

V. T. Mendenhall

Abstract

The objectives of this investigation have been to characterize black gram (Phaseolus mungo L.) and rice (Oryza sativa L.) proteins and to study changes in their nutritional value due to fermentation. Black gram, the legume chosen for this work, is one of the most important legume crops throughout a large part of the tropics.

The protein content of 60 mesh, dehydrated, defatted black gram meal was 28.5 percent. Sodium carbonate (0.5-1.0 percent), tetra-sodium pyrophosphate (0.5 percent), and sodium dodecyl sulfate (SDS) (0.5-5 percent) proved to be the potential protein solubilizers as they extracted more than 76 grams of Lowry's protein per 100 grams Kjeldahl protein. On the considerations of contaminating residue in the final product and disruption of native structure of the proteins, these chemical agents were unsuitable. Sodium sulfate at the 10 percent level was judged to be the best protein solubilizer. Proteins separated on polyacrylamide gel using a phenol-acetic acid-mercaptoethanol-urea (PAMU) system were run on the flat bed gel containing SDS. The proteins were separated in 13 constituents and the molecular weights of the major ones were 140,000 and 55,000.

Solubilized proteins contained 81 percent globulins, 13 percent albumins, 4 percent prolamins, and 2 percent glutelins. Sulfur containing amino acids and threonine were deficient in total proteins of the seeds with 27.6 and 78.8 as their respective chemical scores. Chemical scores of the albumin, globulin, prolamin, and glutelin fractions were 64, 0, 56, and 70.7, respectively. The predicted biological values in human nutrition varied from 0 for globulins to 110 for glutelins, and was 14.9 for total proteins in the seeds. The constituents of the protein fractions were isoelectrically focused in the acidic pH range with the exception of two globulins for which the isoelectric points were 8.42 and 8.65.

The trypsin inhibitor from black gram was isolated using affinity chromatography gel with 19.5 fold purification. The inhibitor had 75 amino acid residues and contained one disulfide bridge. Chemical studies assigned an important role for the hydrogen bonds and demonstrated vital importance of the disulfide bridge in retaining the inhibiting activity. The inhibitor was stable and retained 35 percent of the activity when heated at 100°C for 60 minutes at pH 11. Chemical modification of amino acid residues suggested the involvement of lysine and arginine residues at the active site of the inhibitor. Lysine and arginine moieties at the active site have been proposed to be present as alanyllysine and histidylarginine.

Inhibition of bovine pancreatic trypsin by the inhibitor was kinetically studied. The kinetic constants Km and Vmax were 2.7 x 10-5M and 6 x 10-3M/min, respectively. The dissociation constant for the enzyme-inhibitor complex (Ki) was 4 x 10-7M, whereas that for the enzyme-inhibitor-substrate complex (Ki ' ) was 1.89 x 10-6M. The inhibition was a mixture of partial competitive and pure-noncompetitive systems.

Rice and black gram form the integral parts of a fermented snack food of the Indian subcontinent—idli. Amino acid composition of black gram and rice were complementary. Leucine, lysine, and sulfur containing amino acids were the most limiting amino acids in rice with 65.1, 66.3, and 67.9 as their respective scores. The estimates of biological values of rice proteins in human nutrition qualified albumins as superior and prolamins as inferior proteins.

The PAMU system in polyacrylamide gel electrophoresis was more efficient in resolving protein subunits than the SDS gel system. The PAMU system was not sensitive to the ionic strength of the sample. Mobilities of rice and black gram proteins in SDS and PAMU systems were based on the related parameters. In the PAMU system, the mobilities of most proteins seemed to depend on their molecular size. The PAMU system on gel electrophoresis was judged superior to the SDS system.

Fermentation of the black gram-rice blend was kinetically studied for changes in physicochemical characteristics and nutritional functionality. Trypsin inhibiting activity was unaffected, but chymotrypsin inhibiting activity was reduced to 3 percent after 20 hours fermentation. Significant increases were noted in the contents of sulfur containing amino acids during fermentation. These amino acids seemed to be bioavailable. In vitro digestion with pepsin and pancreatin indicated improvement in digestibility of proteins after fermentation. The digestibility was further enhanced by steaming.

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