IL37866A - Enzymatic scission of milk lactose and the products obtained thereby - Google Patents

Description

Enzymatic scission of milk lactose and the products obtai ed thereby NAM PROGBTfI S.p.A.

This invention relates to a process for the enzymaiiic scission of lactose contained in milk or a derivative of milk, and to the product, namely the milk having a lower lactose content.

It is well known that it is advantageous to obtain milk in a form wherein the lactose is partially hydrolyzed into glucose and galactose. There are the dietetic reasons of a better tolerance, which can broaden the consumer field. Also, the introduction of the hydrolyzed milk into the diets of animals produces remarkable advantages in the growth and feeding of the animals.

Furthermore there are very important technological reasons for reducing the lactose content; for instance, when milk is concentrated to a solid content greater than >0/¾, it is very difficult to preserve such a milk owing to the crystallization of part of the lactose, thereby causing e protein coagulation. The same difficulty is found in the ice-cream industry, where it is not possible to use milk having a high solids content owing to the crystallization of lactose which is difficult to redissolv^.

Some methods have been proposed in order to overcome these drawbacks, for example the hydrolysis of lactose at high temperatures, the treating of milk with ion exchange resins, and the adding of riboflavin^.

However, the first process causes protein coagulation, the second alters the natural balance of salts in the milk, and the third, although avoiding lactose precipitation, employs a very expensive additive and does not *s*esolve the dietetic problems.

Therefore the enzymatic hydrolysis method appears favourable. Although the employment of enzymes in solution "has found many applications, the addition of the more readily available enzymes gives the milk a disagreeable taste. Moreover, at the end of the operation, the enzyme has to be made inactive and it remains in the milk as denatured enzyme. Furthermore, the process requires a great amount of enzymes (2-4% v/ith respect to the weight of lactose, according to its activity), wnich influences the cost of the process.

Another factor limiting the application of this process consists of the microbiological complications caused by the hydrolysis.

It is an object of the present invention to provide a reasonably priced process for the scission of the lactose contained in the milk and its derivatives, in whic process -the aforesaid drawbacks are reduced or obviated. Another object of the present invention is to provide a milk or derivative thereof, wherein the lactose content is reduced owing to a partial or total scission into glucose and galactose, without the addition of external agents.

According to one aspect of the present invention, there is provided a process for the enzymatic scission of - - ^ lactose in milk or a derivative of milk, whic comprises contacting the milk or derivative of milk with a f lamentous structure in which is present the enzyme β-giLactosidase, the β-galactosidase being present in the filamentous structure i a manner such that it can cause the at least partial hydrolysis of lactose and it is retained substantially in the filament.

It is possible economicall and simply to obtain filamentous structures englobing. the enzyme in, a÷ steady and,, lasting manner, for example as described in Italian Patent Ho. 836,462.

The f lamentous structure may be in the orm of a bundle of filaments or an article formed from a plurality of filaments, for example a staple fibre, a yarn, a skein or a woven article.

When the filamentous structure is arranged in a column, the reaction may be carried out in a continuous manner, which can easily be made automatic. Such a continuous operation remarkably simplifies the baoteriological problems because it is possible to employ precautions which are difficult or impossible with other methods.

The filamentous structure can occasionall be washed with a suitable detergent. The washing liquid may be admixed with a bactericide to disinfect the filamefc, the ducts and the vessels employed.

Among possible bactericides, the quaternary ammonium salts give rise to very good results, because many of them have no harmful action on the incorporated enzyme.

Purthennore, some of them have both a bactericidal activity and a detergent activity* and this is very important because the bactericidal actio of the quaternary ammonium salts can be strongly hindered by organic substance residues* A great advantage of the process of the present inventio is that the milk does not retain any additive, as in the ease of hydrolysis carried out with soluble enzymes; therefore, it is not necessary to carry out a heating process to denature the enzyme and there is no disagreeable odour in the treated milk* A more important advantage is the possibility of using the incorporated enzyme for a long time, and therefore of treating a large amount of material without any substantial decay of its hydrolytic activity* It was found that the activity of a sample of enzyme-incorporatin filaments, after bein used in successive cycles of hydrolysis of lactose i milk, remained substantially unchanged after 60 days. Similarly, a similar sample of enzyme-incorporatin filament, when arranged in a glass column, continuously hydrolysed the lactose in milk for more than one month without any decay in activity.

The enzyme-englobing method allows filaments to be obtained having a variable activity, by whieh it is possible to shorten the contact time between the material to be treated and the enzyme carrier.

The process of the present invention may be carried out on. milk as such (i.e. whole milk), on partiall skimmed milk or on concentrated milk. The process may be also applied to whey.

- The filamentous structure may be constituted by *cel-fulosic substances such as cellulose acetate, or by a synthetic polymer or copolymer such as polyamide, poly-^ acrylonitrile , polyacrylates , polymethacrylates , polyvinyl-chloride, polyvinylesters and . Very good results were obtained by employing istee. cellulose triacetate.

The present invention is now illustrated by the following Examples.

Example 1 Preparation of the incorporated enzyme: 24.6 g of cellulose triacetate (Fluka) were dissolved with stirring in 260 g of methylene chloride (C. rba), in a reactor.

Separately, an enzyme solution was prepared as follov/s: 1.4-5 6 °f β-galactosidase (BDH) were dissolved in a glycerol-water mixture (20:80 v/v) so as to bring the final volume to 50 ml. The solution was centrifug'sefced in a refrigerated centrifuge (-10°C). From the clear solution $2 ml were withdrawn, and these were added to the piymer solution.

The emulsion of the two phases was performed by means of vigorous stirring at 0°C for JO minutes. The emulsion was poured into a spinning apparatus maintained at 6°C and was spun under ^ nitrogen pressure. The filament was coagulated in toluene at room temperature and recovered on a winder. It was dried under vacuum for one night in order to remove toluene.

Example 2 In this Example, use was made of a column having an internal diameter of 25 mm and a length of 4-00 mm. The column was filled with 50 g of an enzyme-incorporating filament obtained according to Exaaiple 1; the filament was arranged in a random way and compressed so as to fill the whole volume of the column. The column was provided with a thermostatic jacket, through which water was passed at 25°C.

By means of a metering pump, milk having a lactose content of 4,9% and a lipids content of 1,1% was drawn from a t vessel kept at +4-°C. The milk passed through a heat exchanger, wherein its temperature was brought to 25°C, entered the bottom of the column, contacted the enzyme filament and left from the top of the column, passing through a heat exchanger which, cooled it to +4-°C. The flow pump was kept constant at 150 ml/hour. When the column was running 53% Pf "bbe lactose of the milk was hydrolyzed.

Example 3 By working according to Example 2, the column was fed with milk, but the flow rate (Q) was varied. The degree of hydrolysis of the lactose of the effluent milk was determined, and is shown belo :-Flow (Q) ml/h Hydrolyzed lactose % 150 53.00 300 28.00 600 18.20 1200 ^ 9.20 3200 2.50 .

Example This test was performed by employing the same 50 g of enzyme-incorporating filament arranged in the same column of Example 1. 380 ml of the same milk (lactose originally = 4-.95 - > lipids originally = 1.1 t) were continually recirculated and passed over the filament. The hydrolysis degree of lactose was determined at various intervals as a function of the flow (¾). The working temperature was kept at 25°C. The obtained data were: Hydrolyzed lactose % Time (minutes) Q = 800 ml/hour Q = 3200 ml/hour 11.80 10.80 21. 50 19.10 60 25.70 23.60 - Hydrolyzed lactose % Time (minutes) Q = 800 ml/hour Q = 3200 ml/hour 90 35.70 35.00 120 4-1.00 44. 50 150 47.00 50.20 . 180 52.50 55.00 210 59.00 63.00 240 63. 50 67.00 Example " >■ By using the same 0 g of enzyme-incorporating filament as described in Example 1, arranged in the same column as described in Example 2 , the influence of the temperature on the process was studied, 380 ml of the same milk were continuously recirculated over the enzyme-incorporating filament, and the degree of hydrolysis was determined after one hour. The flow pump was kept constant at 3200 ml/hour, whilst the working temperature was changed.

Temperature Hydrolyzed lactose % after 1 hour °C 17.4- 25°C . . 22.80 °c . 29.5^ Exaaple 6 Use was made of the same 50 g of filament as described in Example 1 arranged in the same. glass column as 3* described in Example 2. 380 ml of a partially skimmed milk (lipids = l.l/s) were continuously passed through the column. The degree of hydrolysis was determined as a function of time. The pump flow and the working temperature were kept constant. The data show variations in the degree of hydrolysis of the lactose during time. (hours) Hydrolyzed lactose 1 20.60 36.30 3 7. 0 4 56.20 62.40 6 73Λ 7 79.40 8 88.00 Example 7 By usin the method described in Example 1 , 50 g of an enzyme-incorporating filament were prepared containing 5 nig of β-galactosidase (BDH ) per gram. The filament was arranged in a glass column having an internal diameter of 25 mm and a length of 400 mm, in a random way.

A partially skimmed milk (lipids content = 1.1ό; lactose content = 4.8%) was fed at a flow rate of 1 ml/ hour; the temperature was kept at 25°C. When the column was running 81.4% of the lactose of the milk was hydrolyze Exam le 8 The procedure of Example 7 was again carried ou , but this time with feeding the column with whole milk (3.5% of lipids and 4-, 85% of lactose). 6% of the lactose was hydrolyzed.

Example 9 The procedure of Example 7 was repeated except that the column was fed with entirely skimmed milk (lipids = 0.5% lactose = 5.10%). 8% of the lactose of the' milk was hydrolyzed.

Example 10 The procedure of Example 7 was repeated except that the column was fed with whey (lactose = 4-, 9%) at a pH of 6.7. The whey lactose was 72 hydndLyzed.

Example 11 0 g of enzyme-incorporating filament were prepared according to the procedure of Example 1, containing 75 nig of β-giactosidase (BDK).

When preparing the emulsion use was made of an aqueous phase volume (inl)/dry polymer weight (g) ratio than in the preceding Examples equal to 1.6. The filament, which was more porous/, was arranged in a column having an internal diameter = 25 mm, and height = 250 mm. The column was fed with milk (lipids content = 1.1%·) at a flow rate Q = 300 ml/hour and maintained at 25°C.

V/hen the column was running, the lactose in the effluent milk had "been 66% hydrolyzed.

Example 12 2g of an enzyme-incorporating filament were contacted with 50 ml of a partially skimn:ed milk in a 150 ml flask.

The flask was kept on a shaking bath, maintained at 25°C.

After 4- hours the milk was separated from the filament; the bacteriological content of the milk and the per cent of hydrolyzed lactose were determined.

The well pressed enzyme filament was well washed by a suitable solution of detergent and bactericide. Pressed and dried between two filter paper sheets, the filament was again used under the same conditions. Subsequent working cycles were again performed, again measuring the lactose hydrolysis degree and the bacteriological content of the treated milk at the end of each cycle. Several quaternary ammonium salts were tested according to the aforesaid method, to test their effect on the enzyme and the microorganisms, among them the following were included: Fardi 7 (Farmitalia) , Straminol (Bracco), benzalkonium chloride and Anfocid (Gianni), Tego 51 (Italian Tego), Steramine H (Formenti).

Also tested were some other antibacterial agents such as phenol, hydrogen dioxide, hypochlorite, iodoform and antibiotics. - The better results were obtained using the quaternary ammonium salts, particularly Steramine H and Fardi 7.

Using the aforesaid batch working, the enzyme acti-v was determined at different times. The same sample of filament carried out 80 hydrolysis cycles whilst keeping activity constant.

Example 13 Use was made of a column having an internal diameter of 2 mm and a height of 00 mm, and of 50 E of enzyme-incorporating filament obtained as in Example 1, which was used for several days. After each working the filament was washed with an aqueous solution containing 0.25% of Steramine H, at room temperature. After the washing liquid had been discharged, 500 ml of milk were passed in a closed circuit. The pump flow was 00 ml/hour and the working temperature 25°C. At intervals, the degree of hydrolysis and the bacteriological content of the treated milk were determined. The data were as follows:-' Time (minutes) Hydrolyzcd lactose % Microorganisms per ml 60 18.8 120.000 95 28.4- I70.OOO 120 33.6 300.000

Claims (1)

A process for the enzymatic scission of lactose in a derivative of which contacting the milk derivative of milk with a filamentous structure in which present the enzyme the being present in the ilamentous strueture in a manner suc tha it cause the at least partial hydrolysis of lactose and it retained substantially in A process according to Claim wherein the contacting i carried out in a continuous A process according to Claim or wherein the enzymatic reaction is intercalated by a washing operation wit process accordin to Claim wherein in addition to t detergent there present a A process to Claim wherein the bactericide a quaternary ammonium A process according to any preceding wherein enzyme is incorporated in a filamentous structure formed from cellulose derivative or a synthetic polymer or A process according to Claim wherein the filamentous structure incorporating the enzyme is formed of cellulose A process according to Claim wherein the filamentous structure incorporating the enzyme is formed of a a a a a polyvin a or a A process according to
1. Claim 1 substantially as described in any of the foregoing 1 to Compositions obtained by a process according to any one of Claims 1 to milk or a derivative a reduced concentration of and an amount of glucose galactose corresponding to the reduction in and having no significant enzymatic Compositions according to Claim wherein the milk is partially Compositions according to Claim wherein the milk is concentrated according to Claim the is Compositions according to C substantially described in any of the Examples 2 to the Applicants insufficientOCRQuality