CHEMISTRY OF MILK

Lesson 20. CHEMICAL REACTION OF LACTOSE-PART I9

Module 5. Carbohydrates of milk

Lesson 20
CHEMICAL REACTION OF LACTOSE-PART I

20.1 Introduction

It is a well known fact that under rigorous conditions of processing carbohydrates undergoes extensive complex chemical changes. Similarly lactose also undergoes changes even in mild conditions. The chemical reaction of lactose mainly involves four sites.

• The hemiacetal linkages between carbon 1 and 5 of the glucose moiety.
• β-1,4 glycosidic linkages between the two sugars.
• The hydroxyl groups of both the glucose and galactose units
• The carbon to carbon bonds

20.2 Oxidation

The extent to which lactose may be oxidized will vary depending up on the particular reagent, its concentration, and other reaction conditions. Thus by selection of conditions it is possible to derive oxidation products from lactose which range from relatively simple alterations of the reducing carbon in the glucose portion of the molecule to a carboxylic acid group to complete degradation with the end products being CO₂ and water.

20.2.1 Mild oxidation

Mild oxidation of lactose with such reagents as alkaline copper, iodine, or picrate forms lactobionic acid (4-0-β-D-galactopyranosyl-D-gluconic acid), which has a carboxyl group at C-1 of the glucose unit. Such oxidation re­actions often are used to determine reducing sugars quantitatively. Lactose also can be oxidized to lactobionic acid by mild chemical dehydrogenation and by lactose dehydrogenase produced by cer­tain species of bacteria of the genus Pseudomonas. Lactobionic acid readily forms lactones by esterification of the carboxyl with hydroxyls at C-4 or C-5.

In methods for the quantitative measurement of lactose in which its reducing property serves as the basis of measurement where lactobionic acid is the resultant product. Such compounds as lactobionic acid have a profound tendency to form lactones through inter esterification with hydroxyl groups of the number 4 or 5 carbon.

20.2.2 Vigorous oxidation

Somewhat more vigorous oxidation of lactose with dilute nitric acid ruptures the glycosidic linkage and produces dicarboxylic acid deriv­atives of the two sugars. The dicarboxyl derivative of galactose, galactaric or mucic acid, was formerly much used as a crystalline derivative for iden­tification of galactose.

20.2.3 Biological oxidation

Biological oxidation of lactose to CO₂ and water can be brought about by mixed cultures of bacteria and protozoa obtained from sewage sludge. Such processes are useful in decomposing lactose-containing wastes from dairy factories.

20.3 Lactulose(4-O-β-D-Galactopyranosyl-D-Fructose)

Lactulose is a compound found in heated milk product in which the fructose moiety occurs predominantly in the pyranose and partly in the furanose form.

Its concentration in commercial evaporated milk will be up to 1%. It is an isomer of lactose that is formed by molecular rearrangement, usually under alkaline conditions where by the terminal aldose residue of lactose is converted into a ketose. Preparation of lactulose with calcium hydroxide has long been known but preparation of ketoses by this method is time-consuming, yields are less than 20%, and the keto sugar must be isolated from un-reacted starting materials, alkaline degradation products, and metal salts. A method used to prepare lactulose in nearly 90% yield is by treatment of lactose with boric acid in an aqueous solution made basic by tertiary amines. Ultra filtration can also be used to obtain lactulose from sweet whey which can be made into a non hygroscopic lactulose by some treatments. Lactulose is extremely soluble in water and polar solvents such as methanol. It is difficult to crystallize, especially when traces of other sugars are present.. Lactulose is unstable in alkaline solution, degrading by alkaline peeling and β -elimination reactions to yield galactose, isosaccharinic acids, and other acid products.Amines can bring about dehydration and degradation reactions. Lactulose is similar to sucrose in humectant’s properties. Lactulose has several important uses in the food and drug industries. There is much information on lactulose utilization in infant nutrition. The presence of lactulose in infant feeding encourages the development of Bifidobacterium bifidum in the intestinal flora, imitating flora in the guts of breast-fed infants. There has been some concern about the possible laxative effects of lactulose, especially in infants; a low colonic pH might be a contributing factor to this effect. It is currently believed that lactulose cannot be digested by human alimentary enzymes, so even lactose tolerated individuals cannot digest lactulose some of the research workers suggested that lactulose could partially replace sucrose and corn sweeteners in intermediate-moisture foods. Only limited amounts could be tolerated in foods because of its laxative properties.

Lactose may be hydrolyzed to D-glucose and D-galactose with mineral acids, with cation exchange resins in the acid form, and with β-galactosidases (often called lactases). The β-1,4linkage between the two sugar residues is much more resistant to hydrolysis than is the 1,2 linkage between glucose and fructose in sucrose. Treatments such as an hour at 90°C with 1.5 M HCI, or at 150°C with 0.1 M HCI, are required to hydrolyze lactose completely.

20.4 Acid Hydrolysis

Lactose is resistant to acid hydrolysis compared to other disaccharides such as sucrose. In fact,organic acids, such as citric acid, that easily hydrolyze sucrose are unable to hydrolyze lactose under similar conditions. This is useful in analyzing a mixture of these two sugars, because the quantity of sucrose can be measured by the extent of these changes in the optical rotation of reducing power as a result of mild acid hydrolysis.

The speed of hydrolysis of lactose varies with time, temperature, and concentration of the reactant; some of the research workers have shown that 5 to 40% lactose solutions (w/w)can readily be hydrolyzed with 1 to 3 N hydrochloric acid or sulfuric acid. Ninety percent of the lactose could be hydrolyzed to the constituent monosaccharides at relatively low temperatures (60°C) and long reaction times (up to 36 hr). Due to degradative side reactions producing high levels of off flavor and color this process could not be used for whey concentrate. Lactose hydrolysis can also be brought about with 0.1 N hydrochloric acid in short reaction times at 121°C. Sulfonic acid-type ion exchange resins have been used to catalyze lactose hydrolysis. The resin was equally effective on lactose solutions and acid whey permeates.

The hydrolysis is carried out at temperatures ranging from 90°C to 98°C. The advantages of this method are continuous operation, short reaction times, and no mineral acid to be removed from the hydrolyzed product. High temperature and low pH eliminate problems with microbial contamination. Best reaction rates were achieved with strong acid granular-type cation exchange resins with low degrees of cross linking. The formation of oligosaccharides during acid hydrolysis seem to be much less than during enzymatic hydrolysis

20.5 Enzymatic Hydrolysis

The hydrolysis of lactose using β-galactosidase (lactase) enzymes is most common method available. There has been significant progress in this field, and several processes are almost commercially feasible. There are three major approaches to enzymatic hydrolysis

• “Single use’’ or “throwaway” lactase systems;
• Lactase recovery systems based on membranes to retain the lactase for reuse; and
• Immobilized systems in which the enzyme is physically or chemically bound to a solid matrix.

Several lactases are available which are suitable for industrial processing of whey or lactose. The enzyme prepared from the yeast Kluveromyceslactis has a pH optimum between 6 and 7 and a temperature optimum of about 35°C. The lactase from K. fragilis has a pH optimum of 4.8 and a temperature optimum of about 50°C. A batch processing operation is the simplest method of achieving enzymatic lactose hydrolysis but suffers from the disadvantage that a large amount of recoverable enzyme is needed. For small users or manufacture on an irregular basis, the single-use enzyme procedure is probably the method of choice. Membrane reactor systems in which the enzyme is recovered by ultra filtration of the reaction mixture after hydrolysis is complete have been developed.

Lactose hydrolysis with immobilized systems is the method of choice when regular production of hydrolyzed syrups on a large scale is required. The best known of these is the Corning immobilized system, which uses lactase from Aspergillus niger covalently bound to a controlled-poresilica carrier. The particle size is 0.4 to 0.8 mm, the wet bulk density is0.6, the activity is near 500 U/g at 50°C, and the optimal pH of operation is between 3.2 and 4.3. The rate of hydrolysis is dependent on the mineral, lactose, and galactose concentrations, as well as on the temperature and pH. Inhibition of hydrolysis can be caused by galactose or sodium and calcium ions, so demineralization is often necessary. Because immobilized systems are designed for long-term use, adequate techniques must be developed to ensure sanitary operations. Common techniques use back flushing with water,acetic acid, milk alkali, and detergents with bactericidal activity. At least 10 di and oligosaccharides have been detected during β-D-galactosidasehydrolysis of lactose Three of the disaccharides have been identified as 3-O-β-galactopyranosyl-D-glucose, 6-O-β-D galactopyranosyl-D-glucose, and 6-β-D-galactopyranosyl- D glucose. Galactose is primarily involved in the formation of the oligosaccharides, which accounts for the lower concentration of free galactose than of free glucose during hydrolysis. The use of hydrolyzed lactose syrups has been proposed as an alternative sweetener to corn syrup solids.

20.6 Fermentation of Lactose

Lactose is metabolized by various microorganisms to compounds of lower molecular weight. In dairy field the most important is lactic acid fermentation. Lactic acid bacteria are present everywhere. Their activity in milk intended for liquid consumption is not desirable while it is important in the preparation of cultured products, butter, and cheese. Some microorganism can produce as much as 1.5% lactic acid in milk. These bacterial are classified as homofermentative if they produce only lactic acid and heterofermentative if they produce acetic acid, alcohol,and carbon dioxide along with lactic acid.

Homofermentative bacteria have phosphotransferase system in the cell membrane that phosphorylates lactoseat C-6 of the galactose moiety as it enters the cell. A β-D- phosphogalactosidase then hydrolyses the lactose-P to glucose and galactose-6-P. The glucose is phosphorylated at C-6 and metabolized to pyruvate via Embden-Meyerh of pathway of glycolysis. The galactose-Pconverted via a fructose isomer, tagatose-P to the triose-P stage of glycolysis and then to pyruvate.

Other homofermentative bacteria such as S. thermophilus and Lactobacillus bulgaricus do not phosphorylate entering lactose but rather hydrolyse it withan intracellular β-galactosidase. Heterofermentative organisms lack aldolase enzyme to cleave the six carbon atom to two three carbon units in the Embden-Meyerhof pathway. They use a pathway known as hexose-monophophateshunt pathway in which 6-phosphogluconic acid is formed from glucose and thenit is decarboxylated and the resulting five carbon unit, ribose-5-phosphate is split to acetyl phosphate and glyceraldehyde-3- phosphate. The former yields acetic acid and / or ethanol and the latter yields pyruvic acid and then lacticacid.