Studies on the foaming properties of proteins : the role of soluble leaf proteins and other surfactants in the persistence of bloat foams : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University

Abstract

Methods were developed for the isolation of the soluble leaf proteins in as pure a form as possible and free of any phenoloxidase products. This protein material was separated into two fractions (Fraction 1 and Fraction 2 proteins). A detailed study of the foaming properties of these soluble protein fractions was made so that the conditions necessary for the production of stable foams from these solutions could be evaluated. The nature of the foams derived from bovine salivary secretions and the soluble proteins of the holotrich protozoa were also examined. The foams derived from the leaf and protozoal proteins were rigid and of high stability only when the foams were of high compressive strength. In contrast the salivary secretions produced foams of low compressive strength but high persistence. For protozoal proteins and Fraction 1 protein of white clover and red clover the optimum pH for foam production was close to pH 5.8 to 5.9 and for the plant Fraction 2 proteins in the range 5.1 to 5.4. The foams derived from bovine salivary mucoprotein was unaffected by changes in pH over the range 3.5 to 7.5. The foams generated in vitro from rumen liquor were of low compressive strength but extremely high persistence, and their properties were very different from those of the foams generated from either the plant or protozoal proteins except that they showed maximum foam persistence in a similar pH range. The concentration of Fraction 1 protein in the rumen liquor was below the minimum concentration required to produce stable Fraction 1 protein foams. Of this low concentration only 24% was surface denatured in production of these very stable rumen foams. The significance of this result is discussed. Apart from the low level of Fraction 1 protein, other low molecular weight proteins together with a major component containing carbohydrate as well as protein, were observed on analysing the rumen liquor by acrylamide gel electrophoresis and cellulose acetate electrophoresis. This major component resembled salivary mucoprotein in its schlieren profile in an analytical ultracentrifuge. This material was isolated by preparative ultracentrifugation and some of its properties examined. It was not precipitated by trichloracetic acid, unlike the protozoal and plant proteins, but was precipitated by 60% ammonium sulphate, 80% ethanol, and an equal volume of 1% cetavlon. The antibody to this material gave a positive precipitin reaction with the salivary mucoprotein, the sensitivity of which could be increased by incubating the salivary mucoprotein with neuraminidase, an enzyme which removes the sialic acid from the mucoprotein. molecule. The significance of these findings in relation to other work is discussed. The action of various surfactants that have been implicated in the bloat syndrome on the foaming properties of Fraction 1 protein foams was examined. Thus calcium was found to increase the rigidity of Fraction 1 protein foams, slightly increase the rigidity of Fraction 2 protein foams at high calcium concentrations only, but was without effect on salivary mucoprotein foams. Sodium polygalacturonate increased the persistence of Fraction 1 protein foams at concentration greater than 0.04% w/v. Two salivary secretions were examined for their effect on Fraction 1 protein foams. The first of these was bovine salivary mucoprotein, which whilst increasing the persistence of the foam, decreased its rigidity. Foams of maximum persistence were produced from solutions containing Fraction 1 protein/mucoprotein in the ratio 2/1 , w/w. The second salivary secretion examined was the oesophageal mucin. This material did not produce stable foams by itself, but was an extremely effective stabilizing agent of Fraction 1 protein foams. The most effective antifoaming agent of the polar lipids of red clover examined in this thesis, was phosphatydyl choline which at a concentration of 50 µg ml-1 completely inhibited the production of Fraction 1 protein foams. Addition of mucoprotein to lipid/Fraction 1 protein mixtures which would not support stable foams, resulted in production of extremely persistent foams. Both Fraction 1 protein and mucoprotein were essential for the formation of these foams which resembled the properties of the foams generated in vitro from rumen liquor. From this study it appeared that neither the plant nor the protozoal proteins by themselves could account for the properties of the rumen foams. The properties of the rumen foams could be reproduced by generating foams from mixtures of Fraction 1 protein/plant lipid and salivary mucoprotein. The soluble proteins and the foaming properties of extracts of bloat and non-bloat provoking legume pastures were examined. It was found that the temperate non-bloating legumes contained condensed tannins which precipitated the soluble leaf proteins and thus by removing the plant foaming agents from solution inhibited foam production from these extracts. These tannins were isolated from Lotus pedunculatus Cav., and were shown to form insoluble complexes, not only with the soluble leaf proteins, but with protozoal proteins, salivary mucoprotein, and the protein present in rumen liquor. Tannins were common in the Lotus species, but of the Trifolium species examined, they were found only in Trifolium arvense L.. The significance of incorporation of tannins into bloating pastures in an attempt to eliminate bloat is discussed. The non-bloating tropical legumes, apart from the Desmodium species, did not contain tannins but were lower in soluble leaf protein, and the bloat potential could be correlated with the compressive strength of the foams derived from extracts of these plants.