Enzymatic degradation of oligosaccharides in peas and pea flours
Karoutis, Apostolos Ioannou
A high resolution gas chromatographic (HRGC) method was developed for the analysis of oligosaccharides in pea flours which circumvented some of the shortcomings of existing packed-column GC and HPLC methods. Aqueous methanol (80%) extracts of pea flour were dried and derivatized with either trimethylsilylimidazole (TMSI) or N-methyl-bis (trifluoroacetamide) (MBTFA). Separation of the oligosaccharide derivatives was achieved on a 10-metre DB5-60W capillary column. The effects of carrier gas (He) flow rate and split ratio on analysis time, resolution and reproducibility were studied. TMS derivatives of sucrose, raffinose, stachyose, and verbascose gave satisfactory results over a wide range of carrier flow rates, with the fastest analysis and the least peak broadening at 6.7 ml/min. Reproducibility was best at a flow rate of 3.7 ml/min and a split ratio of 1:50. Even shorter analysis times were achieved with the more volatile MBTFA derivatives, but discrimination in the split injector of the gas chromatograph caused serious reproducibility problems. HRGC analysis proved to be a rapid, sensitive method for quantitation of oligosaccharides in pea flours. In the course of establishing conditions for the assay of Î±-galactosidase activity in pea flours, it was determined that the use of extraction temperatures in the range of 0-5Â°C yielded extracts substantially higher in Î±-galactosidase activity than those obtained at ambient temperature. Enzyme activity remained stable for 10 minutes in cold-extracted preparations held on ice prior to assay. A 20 minute incubation of crude Î±-galactosidase extracts with p-nitrophenyl Î±-D-galactopyranoside as substrate was shown to be satisfactory for determination of a-galactosidase activity in pea flour, air-classified pea protein and flour from germinated peas. Steeping of peas in deionized water for 12 hours at 25Â°C, with the intent of increasing the rate and uniformity of germination, resulted in the disappearance of a portion of their sucrose and oligosaccharide content, only part of which was recovered in the steepwater. Raffinose showed the largest percentage decrease (29.4%) whereas, in absolute terms, stachyose showed the largest decline (from 1.73% of seed dry weight to 1.35%). Following steeping (12 hr, 25Â°C), a substantial reduction in the oligosaccharide concentrations of peas occurred during germination for 96 hours, which was accompanied by a large increase in the sucrose concentration. Alpha-galactosidase activity showed two activity maxima (at 12 and 48 hours, respectively) during the 96 hour germination period. This was attributed to the formation/activation of two enzyme types. Osmotic priming (restricted hydration) of peas in either polyethylene glycol (PEG 8000) or potassium nitrate (KNO3) solutions was less effective than germination in inducing Î±-galactosidase activity and in reducing oligosaccharide concentrations. Autolysis of whole peas (5:1 water to seed ratio) for 12 hours at 45 or 65Â°C, and of 10 and 20% (w/v) air-classified pea protein slurries for 6 and 12 hours, respectively, at 25,45 or 65Â°C, resulted in substantial reductions in oligosaccharide concentrations. Microbial loads in the autolysed samples were monitored since the extent of microbial growth, if significant, would influence the practicality of a particular treatment. Standard plate counts increased exponentially in samples autolyzed at 25 or 45Â°C, whereas at 65Â°C microbial loads decreased for the first 6 hours of autolysis followed by slow increases between 6 and 24 hours. Addition of a microbial α-galactosidase preparation to 10 and 20% (w/v) airclassified pea protein slurries resulted in essentially complete hydrolysis of oligosaccharides after 60 and 90 minutes, respectively, at 25, 45 or 65Â°C. Little effect of enzyme concentration on the rate or degree of hydrolysis was seen at the levels of α-galactosidase added (3.3-13.9 units/g of slurry).