Raymond R. Mahoney

Galactosyl-oligosaccharide formation during lactose hydrolysis: A review

Enzymatic hydrolysis of lactose is accompanied by galactosyl transfer to other sugars, thereby producing oligosaccharides. These are hydrolyzed slowly, both in vitro and in vivo. They can be thought of as low molecular weight, non-viscous, water-soluble, dietary fibre. They are considered to be physiologically functional foods which promote the growth of bifidobacteria in the colon and a wide variety of health benefits has been claimed in connection with this effect. This article reviews the mechanism of oligosaccharide formation, and then discusses the amount and nature of the products as well as the factors which influence them. The appearance and disappearance of oligosaccharides is explored through consideration of the kinetics of tranferase activity. The consequences of oligosaccharide formation for dairy processing, food analysis, nutrition and health are then briefly discussed.

Formation of oligosaccharides by β-galactosidase from Streptococcus thermophilus

Abstract The β-galactosidase of Streptococcus thermophilus exhibited substantial glycosyl transferase activity during the hydrolysis of lactose in milk. The new sugars formed accounted for 25% of the total sugars when the lactose had been 94% hydrolysed. Sixty per cent of the new sugars were identified as allolactose and 30% as 6-O-β- d -galactopyranosyl- d -galactose . No other disaccharides, trisaccharides or higher oligosaccharides were detected. The ratio of free glucose to free galactose during hydrolysis was as high as two to one.

Iron chelation by digests of insoluble chicken muscle protein: the role of histidine residues

The role of histidine residues in the chelation of iron at neutral pH by peptides from chicken muscle was investigated to see if it could contribute to the effect of muscle tissue on iron absorption. When ferric iron was chelated by L-histidine at pH 6, the ratio of iron chelated to loss of reactive histidine was 1:1. Chelation of iron by soluble peptides in a digest of insoluble chicken muscle protein was accompanied by a small loss of reactive histidine (4–7%) in the peptides. When peptides were modified with diethylpyrocarbonate, increasing loss of histidine led to a progressive decrease in iron chelation. However, even 89% loss of histidine only reduced iron chelation by 30%. It was concluded that histidyl residues do contribute to iron chelation and therefore could by involved in the promotion of iron absorption by muscle tissue. However, other amino acid residues are likely to be involved. © 2000 Society of Chemical Industry

PURIFICATION AND THERMOSTABILITY OF BETA-GALACTOSIDASE (LACTASE) FROM AN AUTOLYTIC STRAIN OF STREPTOCOCCUS SALIVARIUS SUBSP. THERMOPHILUS

beta-Galactosidase from an autolytic strain of Streptococcus salivarius subsp. thermophilus was purified 109-fold to near homogeneity. The yield of purified enzyme was 41% and the specific activity was 592 o-nitrophenyl beta-D-galactopyranoside U/mg at 37 degrees C. Two isozymes were present, but only one subunit was detected, having a mol. wt of 116,000. Enzyme stability was 37-83 times greater in milk than in buffer in the range 60-65 degrees C. At 60 degrees C the half-life in milk was 146 min. Denaturation in buffer was first-order, but in milk the overall reaction order with respect to enzyme concentration was approximately 0.5. The activation energy for denaturation was 453 kJ/mol in milk and 372 kJ/mol in buffer. In milk the activation energy for lactose hydrolysis was 35.1 kJ/mol.

Studies on the thermostability of lactase ( Streptococcus thermophilus ) in milk and sweet whey

The stability of lactase from Streptococcus thermophilus at 55 °C increased 7-fold, 2-fold and 1·5-fold in the presence of lactose, galactose and glucose respectively; maltose had no effect. Total stability over an 8 h period was more than 10-fold better in milk and sweet whey than in lactose solution, owing to the stabilizing influence of the milk proteins and the milk salts. Ovalbumin and reduced glutathione provided some extra stability but were not as effective as the milk components. In the absence of lactose the enzyme was less stable in milk and was not protected at all by sweet whey constituents. None of the milk protein fractions was as effective in the absence of lactose as when it was present. Enhanced thermostability of the enzyme in milk and sweet whey is due to contributions by all major milk components, but binding of lactose to the enzyme is the major factor controlling the extent of stabilization by other components.

Kinetic properties of α-D-galactosidase from Lactobacillus fermenti

The kinetics of α- d -galactosidase (α- d -galactoside galactohydrolase, EC 3.2.1.22) from Lactobacillus fermenti have been investigated to determine the optimum conditions for the hydrolysis of raffinose sugars in soymilk. PNPG activities of the enzyme were highest in late growth on glucose; maximum productivity was 17.8 units l−1 h−1 after 24 h growth. Maximum and specific activities of the enzyme were 1.1 units ml−1 and 4.3 units mg−1 protein, respectively, at 45°C, pH 5.0. Activity and specific activity of the enzyme in 24 h glucose cultures at 37°C, pH 5.8, were 0.6 units ml−1 and 2.2 units mg−1 protein, respectively. The enzyme was inhibited competitively by galactose, non-competitively by sucrose and fructose and retained 64% of its initial activity following 30 days storage at 4°C. After 5 h at 37°C, 94% of the raffinose and 68% of the stachyose were hydrolysed by 0.114 units of enzyme. These sugars were completely hydrolysed in a commercial soymilk of pH 5.2 or 6.5 at 37°C in 7 h by 0.454 units of enzyme.

Iron solubilisation by chicken muscle protein digests

The objective of this work was to compare the solubilisation of iron by in vitro digests of soluble and insoluble protein fractions from chicken muscle. Chicken breast muscle was extracted to provide dilute salt-soluble protein (DSSP) and dilute salt-insoluble protein (DSIP) fractions. These fractions together with casein and ovalbumin were subjected to in vitro digestion in the presence of ferric iron. After proteolytic digestion, soluble iron increased fourfold for DSSP, 20-fold for DSIP, twofold for casein and 0.5-fold for ovalbumin. 64% of the soluble iron in the DSSP digest and 30% of the soluble iron in the DSIP digest were ferrous; in the casein and ovalbumin digests, less than 6% was ferrous. Dialysable iron was less than 5% of the soluble iron for all proteins and was mostly ferric iron. DSIP solubilised twice as much iron as DSSP but much less than casein or ovalbumin digests. It was concluded that muscle proteins solubilise iron by reduction and chelation to mostly large (non-dialysable) peptides resulting from digestion.

The activity of lactase (Streptococcus thermophilus) in milk and sweet whey

Abstract Activity of lactase (Streptococcus thermophilus) in milk, sweet whey and milk salts was 33, 27 and 18%, respectively, of the activity in phosphate buffer containing a similar level of magnesium ions. The decrease is due primarily to the unfavorable ionic environment in milk products. Milk proteins activated the enzyme only slightly in buffer (6–14%), but seemed to ameliorate the inhibiting effect of the ionic environment in milk and whey. Activity in raw milk was 6–8% less than in the same milk heated to 63 or 85°C for 30 min.

Binding of iron by chicken muscle protein digests: the size of the iron‐binding peptides

The dilute salt-insoluble proteins (DSIPs) were extracted from chicken muscle and digested in the presence of ferric iron. The size of the peptides was determined by ultrafiltration using membranes with various molecular weight cut-offs (MWCOs). Over 90% of the peptides in both the pepsin and pepsin–pancreatin digestions were smaller than 10 kDa and more than 45% were smaller than 3.5 kDa. Pancreatin digestion increased the amount of small peptides (<3.5 kDa) as compared to pepsin digestion alone. Most of the iron was in a form too large to pass the 10 kDa MWCO membrane and was presumably bound to a few large peptides. About 10% of the iron was smaller than 3.5 kDa, but there was very little in the intermediate range (3.5–10 kDa). Pancreatin digestion produced more soluble iron than pepsin alone, but the size distribution was similar for both digestions. The smaller peptides (<10 kD) bound very little iron during digestion but could bind iron if separated from the large peptides before iron addition. The large iron-binding peptides were separated by ultrafiltration, and a 22-fold increase in specific activity (iron mg−1 peptide) was achieved using a 10 kDa MWCO membrane. © 2000 Society of Chemical Industry

Lyophilization decreases the formation of dialyzable iron by extraction and digestion of chicken breast muscle.

We studied the effect of lyophilization of chicken breast muscle on the formation of dialyzable iron from ferric iron. Chicken breast muscle was used chilled, frozen or lyophilized and was analyzed for sulfhydryl and histidine content. It was then homogenized and mixed with ferric iron. The mixture was extracted with acid or digested with pepsin and pancreatin. The extracts and digests were analyzed for dialyzable ferrous and dialyzable total iron and also for protein. In the chilled muscle, similar amounts of dialyzable iron were formed after acid extraction and after proteolytic digestion; however, digestion led to more dialyzable ferrous iron. Freezing had no effect but lyophilization of the homogenized muscle caused large decreases in dialyzable iron and dialyzable ferrous iron for both extraction and digestion processes. Lyophilization also resulted in decreased extraction of peptides, decreased digestion of muscle proteins and reduced levels of sulfhydryl and histidine residues. Our results demonstrate that dialyzable iron is produced both by acid-soluble low molecular weight muscle component(s) and also by peptides resulting from digestion of muscle proteins: both of which reduce and chelate iron. Reduced formation of dialyzable iron by both mechanisms following lyophilization could be explained by sulfhydryl oxidation and impaired digestion due to protein crosslinking.