ALBERT

Notes: A simpler and less expensive process than the process of Helbig et al. (1980) for preparation of a nonbitter, desalted milk hydrolysate is presented. Skim milk was hydrolyzed with Alcalase (subtilisin Carlsberg) at 50°C in 3 hr. The resin in OH form was added to milk batchwise to simultaneously raise the pH to 8.5 for optimum proteolysis and exchange the anions in milk which contributed to saltiness. These modifications resulted in a shorter hydrolysis time and a more economical resin regeneration than those of the Helbig process. The dried milk hydrolysate was added to soft drinks and fruit juices at solids concentrations up to 10% (3% protein) without adverse effects on their taste, color, viscosity and clarity.

Notes: Tetramethylurea (TMU) was used to delipidate very low density lipoprotein from egg yolk granules [VLDL(MF)] and dissociate the apoproteins into subunits prior to gel electrophoresis. Electrophoretic analysis indicated the presence of three apoproteins, one of which was a complex of the other two. SDS disc gel electrophoresis of the proteins extracted from the TMU gels demonstrated that each band was heterogeneous, containing many polypeptide bands. SDS polyacrylamide gel electrophoresis dissociated apoVLDL(MF) into 22 polypeptides (molecular weights ranging from 9,600-136,000) in the presence of urea, dodecyl sulphate and 2-mercaptoethanol. Fourteen of these bands were glycoproteins reacting positively with the Schiff reagent. This heterogeneity of polypeptides is due in part to slight differences in the carbohydrate bound to the apoprotein. Electrophoresis devoid of 2-mercaptoethanol resulted in resolution into only 18 bands, most of which contained higher molecular weight aggregates not present in the reduced samples. The apo-VLDL(MF) was fractionated on 6% agarose columns containing 8M urea or 2 mM dodecyl sulphate. TWO apoprotein fractions were eluted from the SDS column, and three from the urea column. The protein eluted at the void volume of the urea column was a complex aggregate of the other two proteins; urea was not as good a dissociating agent as dodecyl sulphate. A similarity was found between the fractions eluted from the SDS column and the proteins extracted from the TMU gels; the polypeptides identified by SDS gel electrophoresis had identical electrophoretic patterns. The molecular weights of the two apoproteins from the SDS column were analyzed by sedimentation equilibrium techniques. The molecular weight range for the high molecular weight component was 33,300-165,800 and 5,800-28,100 for the low molecular weight component. It appears that the apoprotein isolated from egg yolk granules has similar protein moiety to that from egg yolk plasma and hen's plasma.

Notes: The interaction between αs1, -casein and K-carrageenan was investigated by sedimentation velocity and sedimentation equilibrium ultra-centrifugation, frontal chromatography, fluorescence polarization, viscosity, and turbidity experiments. Schlieren patterns of αs1-casein-K-carrageenan mixtures during sedimentation velocity ultracentrifugation experiments (pH 6.6, μ= 0.08) revealed a large αs1 -casein containing peak followed by a slower sedimenting peak which was thought to be residual, uncomplexed k-carrageenan. The S2 0, w of the interaction peak was greater than the S2 0, w of αs1 -casein alone under identical conditions. The effects of pH, ionic strength, temperature and 6.OM urea suggested that the interaction observed during sedimentation velocity ultracentrifugation and viscometry of αs1 -casein and K-carrageenan was mediated by hydrogen bonding. Fluorescence polarization, frontal chromatography, and molecular weight distributions calculated from sedimentation equilibrium data, however, showed that αs1 -casein and K-carrageenan did not interact in calcium-free systems (pH 6.6, μ= 0.08). DNS-αs1 -casein and DNS-K-carrageenan were employed as the labelled components during the fluorescence polarization experiments. αs1 -Casein-K-car-rageenan mixtures eluted from a controlled pore glass column (170 Å pore diameter) as the individual components with elution volumes identical to those obtained when αs1 -casein and K-carrageenan were chromatographed separately. The molecular weight distributions of αs1 -casein-K-carrageenan mixtures (pH 6.6, μ= 0.08), subjected to sedimentation equilibrium ultracentrifugation, contained a major peak in the molecular weight range corresponding to unreacted αs1, -casein. Thus the “interaction” revealed by sedimentation velocity and viscosity data was not a chemical interaction but, rather a physical entrapment of αs1 -casein by K-carrageenan. Fluid flow through the capillary during viscosity measurements and the intense gravitational fields generated during sedimentation velocity ultracentrifugation probably induced physical entanglement of the K-carrageenan. Under these conditions, αs1 -casein-K-carrageenan mixtures flowed as a “porous-plug” where αs1 -casein, larger than the pores of the K-carrageenan network, was trapped giving rise to the observed “interaction” peak. Physical entanglement of αs1 -casein within the K-carrageenan system which causes a pseudo-interaction was not detected during frontal chromatography, sedimentation equilibrium, and fluorescence polarization measurements. Thus, αs1 -casein and K-carrageenan do not chemically interact in calcium-free systems.

Notes: Graphic illustration of response surface is difficult to carry out for cases with more than two factors as the graphs are of higher orders than three dimensions. An approximation was made by dividing the level values of each factor into four groups of equal intervals, and then the response values plotted were linked to each other when all other factor levels were “in the same groups” of experimental conditions, instead of “under the same conditions” in the case of two or three dimensional graphics. When applied to mathematical models, this new mapping simplex optimization significantly (p 〈 0.01) reduced the number of experiments required for reaching the optimum as compared to the Morgan-Deming simplex optimization. Most experiments on food analysis and processing have been optimized within 25 – 35 iterative experiments depending on the number of factors. Computer-directed optimization in conjunction with the mapping technique followed by a simultaneous shift of factor levels greatly facilitated research supervision.

Notes: The super-simplex optimization was modified by incorporating a quadratic regression subroutine. The new algorithm was found to be more efficient in converging at the optimum than the following algorithms: fractional factorial designs, sequential single-factor search, pattern-search method, Morgan-Deming simplex and super-simplex. The efficiency of the modified super-simplex optimization was dependent on the‘fitness of the regression equation to the model equation. Response surface analysis was accurate only when the model equation was quadratic factorial and the boundary covered the optimum. When a constraint was imposed to restrict the search within the boundary, unlike other simplex algorithms including the weighted centroid method, the modified super-simplex algorithm circumvented a problem of the search stalling at the boundary. Since it is not unusual for optimization to be restricted within boundaries, the modified super-simplex algorithm could be useful in optimizing food processing and analysis. When the modified super-simplex technique is applied to -food research, even higher optimization efficiency could be expected by incorporating backward stepwise multiple regression analysis instead of the quadratic regression subroutine with regression equations of set formulae. To obtain a regression equation fit better to the true response surface, all information, including graphics, should be utilized.

Notes: The very low density lipoproteins VLDL(MF) were isolated from the yolk granules of hen's egg by a combination of preparative ultracentrifugation and agarose gel filtration chromatography. Ultra-centrifugal, electrophoretic and chromatographic analyses were performed to characterize and assess the purity of the VLDL(MF) and delipidated VLDL. Flotation velocity. analysis of VLDLCMF) indicated a heterogeneous preparation with two major floating boundaries with S°f, rates of 75s and 207S Alkylation of VLDL(MF) with iodoacetamide prevented aggregation of VLDL(MF) due to oxidation of sulfhydryl groups and decreased S°f rates to 41s and 108s. Concanavalin A affinity chromatography of VLDL(MF) produced a retained and an unretained fraction. The retained fraction contained twice as much total carbohydrates as the unfractionated VLDL(MF), while the unretained VLDL(MF) contained half as much carbohydrate. All fractions were glycoproteins containing hexose, hexosamine and sialic acid residues. VLDL(MF) was partially delipidated with n-heptane which preferentially extracted the neutral lipids and preserved the solubility of the phospholipid-protein residues. Two fractions of the partially delipidated VLDL(MF) were isolated by gel filtration and characterized by analytical ultracentrifugation and by phospholipid/protein ratios. VLDL(MF) was totally delipidated with sodium deoxycholate (NaDOC) and ethanol-ether procedures. Removal of lipids by both methods produced aggregated apoproteins; however, the NaDOC apoprotein remained soluble, whereas that obtained from ethanol-ether precipitated, but could be solubilized in sodium dodecylsulfate or urea.

Notes: Adsorption methods for debittering skim milk hydrolysates were compared in an attempt to develop acid-soluble milk solids for fortification of beverages which appear and taste like the original beverages after fortification of the milk solids up to the level of skim milk. Preliminary studies using casein hydrolysates indicated that the bitter peptides were mostly hydrophobic as they were almost completely eliminated by hydrophobic chromatography on hexylepoxy Sepharose, Activated carbon, talc, and flcyclodextrin were more effective in adsorbing bitter peptides than Sephadex G-10. With skim milk hydrolysate, it was observed that activated carbon, glass powder or fiber, Sephadex LH-20, and phenoxyacetyl cellulose were effective in this order in reducing the bitter flavor of skim milk hydrolysate. 100% carbon vs protein completely eliminated the bitterness of Pronase- or ficin-hydrolyzed skim milk; the carbon requirement was reduced by 70% when Pronase hydrolysates were pretreated with glass fiber. About 60% of the riboflavin and less than 5% of the protein were lost during treatment; no substantial decrease in lactose, calcium or ash content was observed. The essential amino acid pattern compared well with the FAO/WHO (1973) provisional reference pattern except for the total aromatic amino acid content which was 81% that of the reference pattern. Sensory analysis of the product indicated that orange crush, orange juice, and grapefruit soda were not significantly preferred over beverages containing 10% hydrolysate solids, while apple juice was preferred to the fortified juice due to a faint whey-like salty flavor of the fortified juice. No difference in the appearance was detected between the fortified and the original beverages except a slight turbidity developed when clear apple juices were fortified. The two bitter peptides were isolated from the butanol extract of the activated carbon used in debittering Pronase-hydrolyzed skim milk. It is likely that these peptides were derived from casein especially αS1-casein.

Notes: Surface hydrophobicity (So) of crude salt extracts of meat was determined fluorometrically using cis-parinaric acid as a probe. Upon heating the extracts to 70°C, So increased two- to threefold, whereas protein solubility (s) decreased to 20%. Further heating (to 85°C) resulted in slight decreases in both So and s. Backwards stepwise multiple regression equations for prediction of emulsifying activity index (EAI) and emulsifying capacity (EC) were: EAI = 2.658 + 0.0001308 sSo, and EC =−35.30 + 6.459 s + 0.001317 sSo− 0.05463 s2. So was most important for predicting emulsifying properties of samples with high (〉50%) solubility, whereas solubility parameters were more influential for samples with low (〈50%) solubility.

Notes: Nɛ-Protected lysine derivatives and threonine were covalently attached to wheat gluten using 1-ethyl-3-(3-dimethylaminopro-pyl)carbodiimide. Influential reaction factors were studied using fractional factorial experiments. Lysine content increased 4.0-fold and 6.5-fold after reaction of pepsin-hydrolyzed gluten with NE-benzylidene lysine and NE-acetyl lysine respectively, but product yields were low (40–65%). Using unhydrolyzed gluten as the starting material, 1.6-fold and 2.5-fold lysine increases resulted using NC-benzylidene and NE-acetyl lysine, respectively, with 95% yield. At least 4-fold increase in threonine could be obtained from reaction of threonine with pepsin-hydrolyzed gluten, but with only 30–50% product yield. Yields increased to 96% by reacting threonine with unhydrolyzed gluten, with 1.8-fold increase in threonine.

Notes: Four methods (absorbance at 280 nm; the Lowry method; the fluor-escamine method, and the trinitrobenzenesulfonic acid method) for determining hydrolysis of milk proteins were compared. Each method was applied to the trichloracetic acid soluble fraction of milk protein, which had been digested with trypsin for various periods of time. Detectability was measured as the ratio between standard error of estimate and slope calculated from the linear regression analysis of Deming for cases when both variables were subject to error. Although it was nondimensional, the detectability thus calculated was simple and reliable for comparing assay methods which were based on different analytical principles. Detectability as well as the detection limit measured according to Schwerdtfeger showed that, of the methods compared, the fluorescamine method was most reliable and sensitive.