The histochemical basis of quantitative histology*

The interaction between histochemistry and microscopy for quantifying the morphology and function of cells in sections of tissue is reviewed. In principle, the morphological parameters of cells can be measured in suitably-stained sections using image analysers. In practice, however, the individual cells often cannot be distinguished from each other in such systems. This problem might be overcome, at least in muscle, by visualizing types III and IV collagen in the connective tissue and basement membranes surrounding the cells. The functional capacities of cells within tissues can be inferred by measuring the activities of their key enzymes with quantitative histochemical techniques. At present, the wrong enzymes are frequently chosen for this purpose. Flux-generating and physiologically significant enzymes are preferable, but this requires new histochemical techniques to be devised and validated quantitatively for use with the computer-assisted analytical microscopes that are now becoming available commercially.     

微波处理对哈密瓜汁中酶活性的影响研究

初步研究了微波处理对哈密瓜汁中POD、PPO、LOX酶活性的抑制作用。结果表明,控制微波处理温度不超过48℃时,最优处理组合为微波处理时间40s、微波处理9次、pH值3.5。     

Chemically and enzymatically catalyzed synthesis of C6–C10 alkyl benzoates

The esterification of benzoic acid with n‐hexanol, n‐octanol, 2‐ethyl hexanol and n‐decanol was investigated in detail. An analysis of the reaction kinetics of esterification in the presence of different commercially available chemical catalysts was carried out. The effects of catalyst type and loading on the reaction rate were studied. Although the considered reaction is bimolecular, it showed a first‐order behavior, and a linear dependence with respect to the catalyst concentration was observed. Hence, a new approach is presented to describe the reaction kinetics accurately over a wide range. The application of biotechnological synthesis applying different enzymes as catalysts offers an interesting alternative besides chemical synthesis. Especially an esterase from Bacillus subtilis immobilized on Sepabeads EC‐EP showed high stability and was applied for 2 days in the synthesis of hexyl benzoate. Nevertheless, the chemical reaction route remains superior with respect to the catalyst activities under the applied conditions, which were 25 kU/g for the chemical reaction and 0.7 kU/g for the best enzymatic conversion.     

Thermally Responsive Reversed Micelles for Immobilization of Enzymes

In this study, thermally responsive alkyl end‐capped poly(N‐isopropylacrylamide‐co‐acrylic acid) amphiphilic copolymers were synthesized, and successfully utilized to fabricate reversed micelles and immobilize enzymes. Transmission electron microscopy (TEM) study showed that the reversed micelles were spherical in nature. The activity of Candida rugosa lipase immobilized in the reversed micelles towards catalyzing the esterification of lauric acid and 1‐propanol was analyzed in comparison with naked enzyme. Immobilized lipase provided much greater catalytic activity. In addition, the effects of pH, water content, polymer and enzyme concentration on the catalytic activity were investigated. The optimized fabrication conditions of lipase‐loaded reversed micelles, under which lipase gave the highest activity, were as follows: polymer concentration, 12 mg mL^–1; enzyme concentration, 25 mg mL^–1 phosphate buffered saline (PBS); pH, 7.4; W₀ ([water]/[surfactant]), 83.3. Lipase immobilized in these micelles was much more stable than that in conventional sodium bis(2‐ethylhexyl) sulfosuccinate micelles. More importantly, the size of lipase‐immobilized micelles decreased, and the enzyme solution precipitated from the reaction mixture when the temperature increased to a value slightly higher than the lower critical solution temperature (LCST) of the polymer. The recovery rate of enzyme was about 75 %. The α‐helix structure of the recovered lipase remained intact. In addition, the enzymatic reaction was terminated after raising the environmental temperature slightly above the LCST. These thermally responsive micelles may make a promising system for enzyme immobilization.     

The effect of different enzymes on the quality of high-fibre enriched brewer’s spent grain breads

The brewing industry produces large quantities of waste co-products. There is increasing pressure to ensure total utilisation of such products to address economic and environmental concerns. Brewer’s spent grain (BSG) the main by-product of the brewing industry is rich in dietary fibre and has a strong potential to be recycled. The overall objective of this study was to incorporate BSG into wheat flour breads together with a range of different enzymes (Maxlife 85, Lipopan Extra, Pentopan Mono BG and Celluclast) and evaluate the bread quality. A number of nutritional and textural properties of the finished product were studied. The incorporation of BSG significantly (P < 0.0001) improved the dietary fibre but the major difficulty encounted was to achieve a good structure and high loaf volume. Increasing the level of dietary fibre significantly (P < 0.001) increased dough development time, dough stability and crumb firmness but decreased the degree of softening and loaf volume. It was found that addition of Lipopan Extra (LE), Pentopan Mono (PE) and a mixture of Pentopan Mono and Celluclast (PCE) enzymes improved the texture, loaf volume and shelf life while Maxlife 85 enzyme (ME) was not significantly different from control samples (wheat flour breads containing 0%, 10%, 20% and 30% BSG). Image analysis of the bread structure obtained from the C-cell analyzer showed that the most significantly (P < 0.001) open network was obtained using LE, followed by PE and PCE.     

Effect of mild pressure treatments and thermal blanching on yellow bell peppers (Capsicum annuum L.)

The effect of thermal blanching, in conditions carried out at industrial level, and pressure treatments of 100 and 200 MPa on quality of yellow bell peppers was compared. While the soluble protein content was reduced from 13 to 34% by the blanching treatments, the pressure treated peppers showed equal (100 MPa) to higher levels (up to 33%) for 200 MPa. The ascorbic acid (AA) content of the peppers was not affected by thermal blanching, with the exception of the more severe condition (98 °C/150 s), whereas all pressurized samples showed an increase of AA content in the range of 11-48%. Polyphenol oxidase activity was found mainly in the soluble fraction (around 80%) and decreased progressively as the temperature and time of blanching increased, till reaching about 50% the value of unprocessed peppers, while the pressure treatments showed no effect. Peroxidase activity was practically absent in the ionically-bound fraction (only 1% of total activity) and was reduced by the blanching treatments by 80%-100%, while the pressure treatments only reduced peroxidase activity from 5 to 10%. Activity of pectin methylesterase was undetectable in fresh and both thermally and pressure processed peppers. Firmness of peppers measured from the skin side was about 2.5 fold higher than from the flesh side. After the thermal blanching and the pressure treatments, firmness of peppers was better retained when measured from the flesh side. Upon freezing the pressure treated and the thermally blanched peppers showed similar relative decreases in firmness.     

Delaminated Zeolites: An Efficient Support for Enzymes

Delaminated zeolites (see, e.g., Figure) have been proposed as excellent supports for enzymes. This type of material presents suitable physicochemical properties such as a very high, hydroxylated, and ordered external surface, and good thermal stability. Here we describe the method of preparation of delaminated materials and surface immobilization of enzymes, as well as the remarkable activity and stability of the final products.     

Effect of high intensity pulsed electric fields on enzymes and vitamins in bovine raw milk

Pulsed electric fields (PEF) were applied to fresh bovine raw milk using a laboratory-scale continuous PEF system to study the impact on selected native enzyme activities and on vitamin degradation. The activities of protease, lipase, alkaline phosphatase and lactoperoxidase following PEF treatment at electric field strengths ranging from 15 to 35 kV/cm for treatment times of 12.5 µs to 75 µs were examined. Reductions in enzyme activity, even under the most severe conditions employed, were quite modest, amounting to 14%, 37% and 29% for lipase, protease and alkaline phosphatase, respectively, while the activity of lactoperoxidase remained unchanged. The PEF treatment had no effect on the levels of thiamin, riboflavin, retinol and α-tocopherol in the milk .     

Effect of prefreezing hams on endogenous enzyme activity during the processing of Iberian dry-cured hams

The use of frozen/thawed raw material in the processing of Iberian dry-cured ham has been studied to determine its effect on the sensory quality of the final product. The proteolysis and lipolysis processes were measured by the proteolytic and lipolytic enzyme activities and free amino acids and free fatty acids. The thawed Iberian hams had lower salt contents throughout the process. The use of thawing raw material did not affect the proteolytic enzymes, cathepsins, aminopeptidases and dipeptidylpeptidases, only the activity of dipeptidylpeptidase III was reduced due to thawing. Moreover, there were no differences in the content of free amino acids between fresh and thawed hams during the whole process. However, the use of thawing hams affected the lipolytic activity. The activity of phospholipase and neutral lipase were significantly higher in the thawed hams and also the content of free fatty acids, at all the stages analyzed. Consumer sensory analysis showed thawed Iberian hams had the lowest hardness, probably due to an intense proteolysis. The acceptability of the Iberian hams was similar between fresh and thawed hams.