John Karkalas

SOME FACTORS DETERMINING THE THERMAL PROPERTIES OF AMYLOSE INCLUSION COMPLEXES WITH FATTY ACIDS

Abstract The thermal properties of water-insoluble amylose-stearic acid (18:0) complexes prepared under various conditions were studied by differential scanning calorimetry (DSC). Complexes were studied normally at a concentration of 5% in water at pH ∼7. Type I complexes formed at ≤ 60°C had dissociation temperatures (Tm) in the range 96–104°C. Type IIa polymorphs formed at ≥ 90°C had Tm = 114–121°C. Various ratios of types I and IIa were formed at 80°C depending on the duration of heating, but no intermediate form was detected. Annealing of the type IIa complex at 105°C and at 115°C gave rise to increasing proportions of type IIb polymorphs with Tm = 121–125°C and dissociation enthalpies of 32–34 J/g of amylose, depending on the temperature and time of annealing. Conversion into the higher polymorphs was retarded at a higher concentration (10%) of the complex under identical conditions, and was delayed at pH ∼ 4.7. The dissociation temperatures of amylose complexes with the cis-unsaturated fatty acids oleic (18:1), linoleic (18:2), and linolenic (18:3) also depended on the temperature of formation, and three distinct types were obtained (I, IIa, and IIb). Significant decreases in the Tm of the three polymorphs were observed for each double bond in the fatty acid guest molecule. When type I and type II complexes were made using various proportions of 18:0 and 18:2, mixed acid complexes were obtained with Tm values intermediate between those of the monoacid complexes. The origin of the endothermic transitions on heating the three types of complexes is discussed.

Thermal dissociation of amylose-fatty acid complexes

Abstract Complexes saturated with fatty acids of increasing chain length (10:0 to 20:0), prepared by neutralisation of an alkaline solution of amylose, were shown by differential scanning calorimetry not to contain uncomplexed fatty acids. Insoluble complexes, prepared by dilution with water of a solution of amylose in methyl sulphoxide, contained uncomplexed fatty acids. The dissociation temperature of the complexes increased with increasing chain length of the fatty acids, but the dissociation enthalpy was practically independent of the chain length. Dissociation of the complexes (originating from alkaline solution) at 135°, followed by annealing for 16 h at 90° and rapid cooling, increased the dissociation temperature. The results are discussed in the light of a stoichiometric relationship between fatty acids and amylose.

Quantitative aspects of amylose-lipid interactions

Abstract The formation of complexes between amylose and the potassium salt of fatty acids in 0.01 M KOH (pH 12) was studied by capillary viscometry at 20°. The viscosity number of the clear solutions decreased with increasing concentration of fatty acids (12:0 to 22:0 with an even number of carbon atoms) and reached a constant value presumably because of saturation of the amylose helix. At saturation, the molar ratio of fatty acid/amylose was dependent on the chain length of the fatty acid; the longer the chain length the lower the saturation molar ratio. Insoluble amylose-fatty acid complexes were obtained after neutralisation of the alkaline solutions and adjustment of the pH to 4.6. Essentially the same saturation molar ratios were found by determination of the precipitated complexed amylose. Fatty acids were not readily extractable with diethyl ether from the precipitated complexes and their extractability decreased with increasing molecular weight. Unsaturated fatty acids, with 14 double-bonds (all cis ), were slightly less effective in forming complexes than their saturated counterparts, apparently because of steric constraints. Cyprylic (8:0) and capric acids (10:0) did not form complexes at pH 12, although they did form insoluble complexes at pH 4.6. Monoglycerides of fatty acids, as liposomes, readily formed insoluble complexes at pH 4.6. Experimental techniques for the determination of the complexing ability of fatty acids and their monoglycerides in model systems are presented.

Resistance to acid hydrolysis of lipid-complexed amylose and lipid-free amylose in lintnerised waxy and non-waxy barley starches

Abstract Waxy barley starches (0.8–4.0% lipid-complexed amylose = L·AM, 0.9–3.4% lipid-free amylose = F·AM) and non-waxy barley starches (6.1–7.2% L·AM, 23.1–25.9% F·AM) were lintnerised by steeping in 2 M HCl at 35°C for 140 h. Material solubilised from the waxy starches was estimated to be 70.7% of their amylopectin (AP) plus 3.7% of their L·AM and F·AM, and material solubilised from the non-waxy starches was estimated to be 70.7% of their AP plus 28.9% of their L·AM and F·AM. The polysaccharide components of the insoluble residue were characterised by HPLC, GPC, and λ max of the polyiodide complex. I was concluded that short chain-length (CL 16) material was from external chains of AP, intermediate material (modal CL 46) was from retrogated F·AM, and longer chain residues (CL 77, 120–130_ were from lipid-complexed segments of L·AM. The starch lysophospholipids were completely hydrolysed to free fatty acids which remained complexed with L·AM residues. This was shown by the 13 C CP/MAS-NMR spectrum which had a clear resonance at 31 ppm from mid-chin methylene carbons of fatty acids in complexes. The C-1 signal of the L·AM residues also included a feature at 104 ppm indicative of single V 6 AM helices. The wide-angle X-ray diffraction patterns of the residues of non-waxy starches were Cc-type ( = mixed A + B types), whereas the spectra of the original starches were A-type. It is suggested that, during the early stages of lintnerisation, amorphous (F·AM was partially hydrolysed into material (CL 13 C CP/MAS-NMR spectra, which were consistent with a mixture of double helices and V-type glycosidic conformations, with only a small proportion of non-ordered regions. Broad DSC endotherms were found for both waxy (50–110°C) and non-waxy (50–110°C) lintner residues, which were assigned to disordering of double helices from short chains (modal CL 16) for waxy residues, together with disordering of longer chains (modal CL 46) in double-helix residues of F·AM and also V-helix residues of L·AM for non-waxy starch residues.

Solid state13C NMR investigation of lipid ligands in V-amylose inclusion complexes

The characteristics of the ligands in inclusion complexes formed from stearic, palmitic, oleic, linoleic, linolenic and docosahexaenoic acids, glycerol monooleate (GMO), glycerol monopalmitate (GMP) and lysophosphatidylcholine (LPC) have been studied by13C NMR in dry and hydrated forms of the complexes, with13C labels being used for the car☐yl and C-1(3) glycerol carbons in stearic acid and GMO, respectively.13C NMR provides definitive proof that V-amylose inclusion complexes have been formed with the mono-car☐ylic fatty acids of varying degrees of unsaturation, GMO, GMP and LPC. The chemical shift of the mid-chain methylenes in stearic acid moves about 1.5 ppm upfield upon complexation with the1H rotating frame relaxation times becoming identical for the lipid and amylose. With the exception of docosahexaenoic acid, the mid-chain methylenes inside the V-helical segments have essentially the same chemical shift for all the other unsaturated fatty acids and lipids investigated. The cross-polarisation dynamics for the car☐yl and glycerol groups in stearic acid and GMO, respectively, have indicated that these bulky polar groups occupy highly mobile conformations in the hydrated complexes which must lie outside the V-helical segments adjacent to the amorphous domains.

Properties of damaged starch granules. I. Comparison of a micromethod for the enzymic determination of damaged starch with the standard AACC and Farrand methods

An enzymic colorimetric micromethod for the determination of damaged starch in samples weighing 50 mg was used. Oligosaccharides in filtered fungal α-amylase digests of damaged granules were hydrolysed with amyloglucosidase to yield glucose which was measured with a glucose oxidase—peroxidase—chromogen reagent. Sixteen manual determinations were performed in 3 h, or 32 determinations in 3 h if automated flow injection analysis of the digests was used. A comparative study of the American Association of Cereal Chemists (AACC) and the Farrand methods, both of which determine damaged starch as reducing sugars by non-specific methods, provided evidence that the AACC method overestimates damaged and gelatinized starch by about 30 % (α-glucan basis), while the Farrand method gives corresponding values which are about three times greater. These methods quantify, as ‘maltose’, only 64–70 % of the starch digested by α-amylase, and substantial correction factors are then used to calculate the total damaged starch content. Since amylose contplexed with endogenous lipids of cereal starches is not digested by fungal α-amylase, a small correction factor should be used for damaged or gelatinized cereal starches; no correction is required for the lipid-free waxy cereal starches and non-cereal starches. The micromethod can also be used to quantify gelatinized starch in processed cereal foods.

The occurrence of non-volatile amines in chilled-stored rainbow trout (Salmo irideus)

Abstract The effect of holding freshly caught whole rainbow trout (Salmo irideus) at 0, 10, 20 and 30°C for a period of 6 h prior to chilled storage was studied. Whole and eviscerated samples were analysed for putrescine, cadaverine, histamine, spermidine and spermine by high-performance liquid chromatography (HPLC) of their benzoyl derivatives after 2, 4, 7, 10 and 14 days of storage at 0°C. The concentrations of putrescine, cadaverine and histamine increased during storage, while those of spermidine and spermine decreased after an initial rise during the first 4 days. Tyramine was not detected in any of the samples. In general, holding of the samples at temperatures above 0°C for 6 h prior to chilling gave rise to increased concentrations of non-volatile amines. Eviscerated fish contained lower concentrations of amines than whole samples. Evidence is provided that the concentration of putrescine, and possibly that of cadaverine and histamine, in the flesh of chilled-stored rainbow trout, can be used as a criterion for the assessment of the freshness of the fish.

Continuous enzymic determination of α-glucans in eluates from gel-chromatographic columns

A method was developed for the continuous measurement of α-glucans in weak alkaline eluates from the gel permeation chromatography of the components of entire or debranched starch. A modified flow injection analysis system was used comprising the continuous neutralization of the alkali with dilute acid, the hydrolysis of α-glucans with amyloglucosidase and the in-flow spectrophotometric determination of the resulting glucose with an improved glucose oxidase-peroxidase-chromogen reagent. The system is sensitive, reliable and safe compared with hitherto used colorimetric methods relying on hot concentrated sulphuric acid.

Automated enzymic determination of starch by flow injection analysis

A method is described for the continuous enzymic determination of starch by unsegmented flow injection analysis (FIA). Pre-weighed starch-containing samples were treated with thermostable bacterial alpha -amylase for 10 min at 100 °C, followed by filtration and loading of the filtrates onto an autosampler. The filtrates were then injected sequentially into the calibrated FIA apparatus, and hydrolysed continuously with fungal amyloglucosidase at pH 4·5 and 60 °C. The resulting glucose was measured colorimetrically at 505 nm by the continuous addition of a reagent stream containing glucose oxidase, peroxidase and a chromogen that gives a pink colour. The systems throughput is 32 samples per hour and the consumption of reagents was low in comparison with manual methods.