WO2006037340A1

WO2006037340A1 - Method for increasing the bioavailability of calcium in milk

Info

Publication number: WO2006037340A1
Application number: PCT/DK2005/000646
Authority: WO
Inventors: Per Munk Nielsen; Ebbe L. Madsen
Original assignee: Chr Hansen AS; Novozymes AS
Current assignee: Chr Hansen AS; Novozymes AS
Priority date: 2004-10-08
Filing date: 2005-10-07
Publication date: 2006-04-13
Anticipated expiration: 2007-04-08

Abstract

The present invention relates to a method for increasing the bioavailability of calcium in milk by treating the milk with a protease

Description

METHOD FOR INCREASING THE BIOAVAILABILITY OF CALCIUM IN MILK

TECHNICAL FIELD

The present invention relates to a method for increasing the bioavailability of calcium in milk by treating milk with a protease to obtain controlled proteolysis of the milk without negative effects on milk appearance or organoleptic properties.

BACKGROUND OF THE INVENTION

Calcium is a mineral that is necessary for animals, including humans, in relation to many physiological functions, and is also an essential component of bone structure. Osteoporosis is a recognised health problem in many areas of the world and is often ascribed to an insuffi¬ cient intake of calcium. One of the major sources of calcium in the diet is milk and dairy prod- ucts, but only about 30% of the calcium in milk is absorbed when consuming milk. US 2002/0192333 (Christensen et al.) describes a method for increasing the calcium bioavailabil¬ ity of milk by controlled proteolysis. The method involves prolonged treatment of the milk. There is a need for a method for increasing the calcium bioavailability of milk that is economi¬ cal and can be easily implemented in a dairy proces system.

SUMMARY OF THE INVENTION

The inventors have found that the calcium bioavailability of milk can be improved when a pro¬ tease is added to cold milk, the milk is quickly heated to reaction temperature and held at this temperature for a short time and then pasteurised at a temperature where the protease is in¬ activated. Accordingly the present invention relates to a method for producing milk with in¬ creased calcium bioavailability comprising: i) adding a protease to a milk; ii) heating the milk from a temperature between 0 and 1O⁰C to a temperature between 45 and 70⁰C within less than 90 seconds; iii) holding the milk at the temperature achieved in step ii) for between 5 and 300 seconds; and iiii) inactivating the enzyme after step iii) by heating the milk to a tempera¬ ture sufficient to inactivate the enzyme; wherein step i) is conducted before, during, or imme¬ diately after step ii), before step iii).

DETAILED DESCRIPTION OF THE INVENTION

Milk processing

Milk according to the invention may be the lactal secretion of any mammal, e.g. cows , sheep, goats, or camels. Milk to be used for human consumption as such or in the form of a dairy product when produced industrially usually undergoes a number of processing steps. These may e.g. include centrifugation to control the fat content and/or the content of microorgan¬ isms, pasteurisation, and homogenisation. The milk used for the invention may be raw milk, or it may have been subjected to a heat treatment before use. The fat content and/or protein content may be the natural fat and/or protein content of the milk, or it may have been stan¬ dardised, e.g. by centrifugation and/or membrane filtration.

When the milk is stored at the dairy plant before processing it will usually be kept at a low temperature, such as e.g. between 0 and 10⁰C, to prevent excessive microbial growth.

If the fat content of the milk is standardised, this is often done by separation of the milk in a centrifuge into skim milk and cream and subsequent standardisation by adding cream back into the skim milk to obtain the desired fat content. Separation and standardisation is usually performed at elevated temperature, e.g. at between 45 and 60⁰C. If the milk is to be homoge¬ nised this is often performed in connection with separation and at the same temperature. Separation and homogenisation will usually be performed before pasteurisation, but homog¬ enisation may also be performed after pasteurisation, e.g. after UHT (Ultra High Tempera¬ ture) treatment of the milk.

Pasteurisation may be performed by quickly heating the milk to the pasteurisation tempera¬ ture, e.g. in a plate heat exchanger, and holding the milk at this temperature for the desired duration, e.g. at 70-95⁰C for 5-300 seconds. Pasteurisation may be performed as a UHT (Ul¬ tra High Temperature) treatment, e.g. at a temperature between 115 and 145°C for between 2 and 15 seconds.

Protease treatment

The present inventors have surprisingly found that a process for increasing the calcium bioavailability of milk by controlled proteolysis can be implemented in a usual milk processing system with short treatment time, and that the effect on the calcium bioavailability can be maximized by keeping the level of protease addition within certain ranges.

According to one embodiment of the invention the protease is added to cold milk, e.g. to milk directly from the storage tank of a dairy plant, the temperature of the milk may e.g. be be- tween 0 and 10°C, such as between 0 and 5°C, or between 2 and 4⁰C. At this temperature the protease will be inactive or the activity will be low. The milk is then heated to reaction temperature, e.g. between 45 and 60°C, such as between 50 and 65⁰C, or between 55 and 65°C, at which temperature the proteolysis of the milk will take place. Heating is performed quickly, e.g. within less than 90 seconds, such as less than 60 seconds, or less than 30 sec¬ onds, and may e.g. be performed in a plate heat exchanger. In one embodiment of the inven¬ tion the enzyme is added immediately after the milk has been heated to the reaction tempera- ture. The process may include a separation step to standardise the fat content of the milk. Standardisation may e.g. be performed by separating the milk into cream and skim milk in a centrifuge and adding cream back into the skim milk to obtain a desired fat content. If the milk is to be homogenised the process may additionally include a homogenisation step. The sepa¬ ration, standardisation, and/or homogenisation will often be performed at an elevated tem- perature, e.g. between 45 and 65°C, and in one embodiment of the invention separation, standardisation and/or homogenisation is performed after addition of the enzyme at the reac¬ tion temperature, so that the enzyme is active during separation, standardisation, and/or ho¬ mogenisation. The milk may be kept at the reaction temperature for e.g. between 5 and 300 seconds, such as between 10 and 120 seconds, between 10 and 60 seconds, between 10 and 55 seconds, or between 10 and 30 seconds. To stop the action of the protease the treated milk is heated to a temperature sufficient to inactivate the protease. The inactivation may e.g. be performed simultaneously with pasteurisation if pasteurisation is performed. The heat treatment may e.g. be performed at between 65 and 150°C for between 2 and 300 sec¬ onds, such as between 70 and 80°C for between 5 and 300 seconds, between 80 and 95°C for between 5 and 300 seconds, or between 115 and 145°C for between 2 and 15 seconds. In one embodiment of the invention the heating of the milk from reaction temperature to inactiva¬ tion temperature is effected within less than 90 seconds, such as less than 80 seconds, or less than 60 seconds, and may e.g. be performed in a plate heat exchanger, or by mixing the milk with steam, e.g. by steam injection. After heat treatment the milk may be cooled to stor- age temperature, or if the milk is to be used for the production of a dairy product, to a suitable production temperature. The milk may be packed in consumer packing immediately after pasteurisation.

Protease A protease to be used in the invention may be any protease capable of hydrolysing milk pro¬ tein, and may be of any source e.g. of microbial, vegetable or animal source. In one embodi¬ ment of the invention the protease is an alkaline protease. An alkaline protease according to the invention is a protease having pH optimum at a pH between 7.5 and 10. In one embodi¬ ment an alkaline protease is derived from a microorganism, e.g. from a fungus, such as an Aspergillus, e.g. A. oryzae; or a bacterium, such as a Bacillus, e.g. B. licheniformis. An exam¬ ple of an alkaline protease according to the invention is Subtilisin, e.g. from B. licheniformis. Subtilisin from S. licheniformis is available from Novozymes A/S, Bagsvaerd, Denmark, under the trade name Alcalase^®.

In one embodiment of the invention the protease is added in an amount of between 30 and 50 ppm, such as between 35 and 45 ppm. In another embodiment the protease is added in an amount corresponding to between 0.072 and 0.096 Anson Units per liter milk, such as be¬ tween 0.084 and 0.11 Anson Units per liter milk.

The amount of protease may depend on the time the milk is kept at the reaction temperature such that a higher amount of protease may be required for a short holding time compared to a longer holding time. Within the limits of the present invention the relationship between the holding time in step iii) above and the amount of protease may be substantially linear, so that a suitable amount of protease can be determined by dividing a predetermined numerical fac¬ tor with the holding time at the reaction temperature. The predetermined factor may be de¬ termined by multiplying a suitable amount of enzyme with the respective holding time. In one embodiment of the invention, the amount of protease added to the milk in ppm is between 1800 divided by the holding time of step iii) above in seconds and 3000 divided by the holding time of step iii) above in seconds, such as between 2100 and 2700 divided by the holding time of step iii) above in seconds. In another embodiment the amount of protease added to the milk in Anson Units per liter milk is between 4.3 divided by the holding time of step iii) above in seconds and 7.2 divided by the holding time of step iii) above in seconds, such as between 5.0 and 6.5 divided by the holding time of step iii) above in seconds.

Anson Units

One Anson Unit is defined as the amount of enzyme which under optimal conditions digests haemoglobin at an initial rate, so that there is liberated per minute an amount of TCA soluble product which gives the same colour with phenol reagent as one milliequivalent of tyrosine. Activity in Anson Units (AU) according to the present invention may be determined as the en¬ zyme activity when hydrolysing dimethylcasein at 5O⁰C, pH 8.3, relatively to an Alcalase stan¬ dard.

The conditions of the protease treatment should be selected so that the appearance and or¬ ganoleptic properties of the milk is not adversely affected, i.e. the milk should remain liquid without visible precipitation, and with taste and smell characteristic of milk. The treatment may increase the whiteness of the milk and/or give the milk a more cream-like taste, which is usu- ally increasing the attractiveness of the milk to the consumer. In one embodiment the inven¬ tion is directed at a method for increasing the calcium bioavailability of milk without adversely affecting the visible appearance and/or the organoleptic properties of the milk. Calcium bioavailability

Calcium absorption in mammals takes place via two routes: (a) Active, which is vitamin D de¬ pendent, saturable and varies with hormonal status and age. It depends finally on the synthe- sis of calcitriol. Active absorption takes place in the upper small intestine, (b) Passive. This is a non saturable mechanism thought to occur throughout the whole intestine via paracellular passage. This mechanism is independent of age, hormonal status and is stimulated by free calcium concentration in the lumen and the presence of proteolytic casein fragments called casein phosphopeptides (CPPs). It is the site of choice to help those in which the active mechanism is fading like postmenopausal women. High absorption is different from high bioavailability. The latter implies that the calcium entering the body is actually being useful for instance to build bone, and not lost immediately after absorption by excretion in the urine. To demonstrate bioavailability it is necessary to show high absorption and high deposition in bone. In other words, absorption by itself is insufficient to assess bioavailability. Calcium bioavailability may be determined by any suitable method known in the art.

Calcium bioavailability may e.g. be determined by determining the uptake of calcium by ani¬ mals or humans from milk treated according to the invention compared to milk not treated with a protease. E.g. mice may be fed milk where part of the calcium has been exchanged with ⁴⁵Ca, and the radioactivity of blood plasma and femurs after e.g. 2-5 hours may be used as an indication of the uptake of calcium from the samples.

Calcium absorption and bioavailability in milk may e.g. be determined by feeding mice 50 ng of calcitriol dissolved in 10 microliters of corn oil, and 2.5 hours later feeding the same mice milk samples radiolabeled with ⁴⁵Ca (180 microliters). 5 hours after the milk has been entirely ingested, the mice may be killed. Blood samples can be obtained by heart punction immedi¬ ately after death and collected in heparanized Eppendorf vials. After blood is drawn, the ani¬ mals may be dissected to obtain their femurs, which may be weighted and their radioactivity counted by liquid scintillation. To obtain the plasma samples the Eppendorf vials may be cen- trifuged and the red cells discarded. The effect of level of protease treatment of the milk on the level of radiolabeled calcium in plasma and femurs may be used as indication of absorp¬ tion and bioavailability of the calcium in the milk.

A method for determining the bioavailability of calcium is also given by: Heaney et al., Bioavailability of the calcium in fortified soy imitation milk, with some observations on method, Am J Clin Nutr 2000; 71 :1166-69. EXAMPLES

Example 1. Protease treatment of milk.

Protease: Alcalase^® AF 2.4L (Novozymes A/S, Bagsvasrd, Denmark), an alkaline protease derived from Bacillus licheniformis.

Milk treatment was performed in a pilot scale continuous milk processing system. Varying amounts of protease according to table 1 was added to 20 I batches of milk standard¬ ised to 0.5% fat, at 5⁰C. The milk with protease was heated in a plate heat exchanger within less than 25 seconds to a reaction temperature of 60°C and held at this temperature for 60 seconds during which period the milk was homogenized at 200 bar, and then immediately heated within less than 4O seconds in the plate heat exchanger to the pasteurization tempera- ture 90°C which was maintained for 5 minutes after which the milk was cooled in the plate heat exchanger to 5⁰C.

Tabel 1. Levels of protease addition.

Claims

1. A method for producing milk with increased calcium bioavailability comprising: i) adding a protease to a milk; ii) heating the milk from a temperature between 0 and 10⁰C to a temperature between

45 and 7O⁰C within less than 90 seconds; iii) holding the milk at the temperature achieved in step ii) for between 5 and 300 sec¬ onds; and iiii) inactivating the enzyme after step iii) by heating the milk to a temperature sufficient to inactivate the enzyme; wherein step i) is conducted before, during, or immediately after step ii), before step iii).

2. The method of claim 1 wherein the amount of protease added to the milk in Anson Units per litre milk is between 4.3 divided by the holding time of step iii) in seconds and 7.2 divided by the holding time of step iii) in seconds.

3. The method of claim 1 wherein the amount of protease added to the milk in ppm is be¬ tween 1800 divided by the holding time of step iii) in seconds and 3000 divided by the holding time of step iii) in seconds.

4. The method of any of the claims 1-3 wherein the protease is an alkaline protease.

5. The method of claim 4 wherein the protease is derived from a microorganism.

6. The method of claim 5 wherein the microorganism is belonging to the species Bacillus.

7. The method of claim 6 wherein the microorganism is Bacillus licheniformis.

8. The method of any of claims 1-3 wherein the protease is Trypsin.

9. The method of any of claims 1-8 wherein the temperature in step ii) is held for between 10 and 120 seconds.

10. The method of any of claims 1-9 wherein the milk is heated to a temperature between 50 and 65 °C in step ii).

11. The method of any of claims 1-9 wherein the heating of the milk in step iiii) is achieved within less than 90 seconds.