WO1987005469A1

WO1987005469A1 - Method of deactivating heat-stable enzymes

Info

Publication number: WO1987005469A1
Application number: PCT/GB1987/000181
Authority: WO
Inventors: Anthony Russell Bucky; Patrick Robert Hayes; David Stuart Robinson
Original assignee: Minister of Agriculture Fisheries and Food UK
Current assignee: Minister of Agriculture Fisheries and Food UK
Priority date: 1986-03-17
Filing date: 1987-03-16
Publication date: 1987-09-24
Anticipated expiration: 1988-09-17
Also published as: GB2209919A; GB8821555D0; GB8606524D0; DK601187A; DK601187D0; AU7125087A; GB2209919B

Abstract

A method of treating dairy and other food products by enhancing the deactivation of underisable heat-stable enzymes, in a nutrient, comprising the steps of sterilising the nutrient by a UHT process, cooling the nutrient and subsequently maintaining the temperature of the nutrient within the temperature range 45 - 95°C, resulting in an enzyme deactivated product with an increased consumer lifetime which may be milk, cream, soup, fruit juice or any other heat-stable enzyme containing nutrient.

Description

Method of Deactivating Heat-Stable Enzymes

This invention relates to a method of deactivating heat-stable enzymes present in liquid foodstuffs and beverages, ana in particular relates to a method of deactivating heat-stable upases present in milk.

One of the greatest causes of economic loss of refrigerated food are the psychrotrophs and their enzymes. Psychrotrophs are microorganisms including bacteria, yeasts, and molds, which fluorish at the temperatures above 0ºC typically used for food refrigeration. Heat treatments such as pasteurisation or sterilisation can destroy most or all of these unwanted microorganisms and thus enhance the shelf-life of the food, but the enzymes of these microorganisms produced prior to heat treatment can provide a more intractable problem because many are markedly heat stable and can cause subsequent spoilage even in a sterile food product. This problem is of particular concern to the dairy industry.

Freshly drawn milk is likely to contain psychrotrophic bacteria which are mainly derived from milk handling equipment such as storage tanks and pipework. These bacteria have the ability to multiply relatively quickly at the low storage temperatures (4-7ºC) employed by the dairy industry for raw milk. Such milk may well be stored for up to 3 days at these temperatures before processing at the dairy. During this storage period the psychrotrophs (mainly Pseudomonas spp.) can reach fairly high levels, typically up to 10 million bacteria per ml of milk. This growth is accompanied by the production of extra cellular enzymes of which lipases are of especial concern, because lipases attack milk fat (lipids) with the production of fatty acids which have a rancid or soapy flavour.

At the dairy raw milk is conventionally pasteurised by the High Temperature Short Time (HTST) process, which typically consists of maintaining the milk at a temperature of 71.6°C for 15 seconds. This effectively kills all pathogenic bacteria as well as the psychrotrophs which are typically very heat sensitive. However, extracellular lipases produceα by the psychrotropic flora (chiefly pseudomonads) are markedly heat stable and are largely unaffected by the process, although with pasteurised milk they are not significantly involved in spoilage of flavour. Shelf life is limited in this case by certain heat resistant bacteria which survive the HTST process, grow in the milk and consequently cause quality defects over a few days' storage.

Milk of a much longer shelf life, extending over some 3-4 months, can be obtained using the Ultra High Temperature (UHT) process which involves (in accordance with present UK legal requirements) heating the milk under pressure to a temperature of not less than 132.2°C for at least 1 second. Other countries have similar legal requirements for this process. In practice, a temperature of approximately 140°C for 2-5 seconds is generally used. In the UK, such milk (usually referred to as UHT milk) must satisfy a prescribed colony count test. Unopened packets of UHT milk are incubateα at 30-32°C for 24 hours, and a standard loopful (0.01 ml) is then removed and incubated at 30-37°C for 48 hours on ϊeastrel Milk Agar. To satisfy the test, no more than 10 colonies should grow. In practice the product must be sterile to prevent subsequent bacterial growth during storage, hence the general use of 140ºC for 2-5 seconds. The UHT milk is then cooled rapidly to about 20ºC and filled into special cartons under aseptic conditions. Milk so produced is sterile and should remain acceptable for consumption for several months. A further advantage is that the flavour of UHT milk is close to that of pasteurised milk and does not suffer from the 'cooked' flavour associated with sterilized milk (produced by heat treatment at 100ºC for an extended period) which it has largely superseded. This is largely because the UHT treatment is normally of very short duration which is insufficient to generate such unnatural flavours.

However, with UHT milk lipases produced previously by the pseudomonads are of particular concern, since typically 80-90% of the original lipase activity may remain in the milk after this process. If sufficient lipase is produced by psychrotrophs in the raw milk before heat treatment some deterioration in the quality of UHT milk can manifest itself within the stipulated storage period, giving rise to rancid or soapy flavours in the product. Obviously much will depend on the numbers of lipase-producing bacteria in the raw milk before processing, and these numbers, in turn, mainly reflect the cleanliness of the milk handling equipment, and the temperature and storage time of the raw milk.

Thus the problem of spoilage due to the presence of lipases applies equally to UHT milk, to products prepared from UHT milk such as ready-to-eat desserts, flavoured milk, ice cream, cream and other dairy products, and to other consumable fat-containing liquids which are suitable for treatment by a UHT process to lengthen their shelf life. The problem also applies to other UHT-sterilised liquid foodstuffs and beverages, where spoilage is additionally or alternatively caused by other heat-stable enzymes (such as proteinases) produced by psychrotrophs. However, lipases are of especial concern because they can cause spoilage when present at only very low concentrations.

It is an object of the present invention to provide, in part at least, a solution to the above problem. According to the present invention there is provided a method of enhancing the deactivation of heat-stable enzymes present in a liquid nutrient under aseptic conditions, which comprises the steps of sterilising the nutrient by a UHT process, cooling the nutrient and subsequently maintaining the temperature of the nutrient within the temperature range 45 to 95°C, preferably 50°C to 90°C, for at least 30 seconds and preferably for not more than 10 minutes. Most preferably the temperature range is 57 to 60°C for at least 2 minutes and not more than 10 minutes.

The term "liquid nutrient" used in this specification means any liquid foodstuff or beverage which is suitable for sterilisation by a UHT process and which contains a substrate, such as fats (lipids), proteins or carbohydrates, capable of conversion to flavour-spoiling products by enzymic action. Examples of liquid nutrients are soups, broths, fruit juices and milk or milk products eg cream. The nutrient is preferably one containing fats (lipids) dissolved or dispersed therein, because the present method is particularly effective at deactivating heat-stable lipases. Any such fatcontaining nutrient will be susceptible to spoilage due to the presence of fatty acids produced by the action of the lipases on the lipids. The nutrient is more preferably a dairy product, and is most preferably milk, especially bovine milk. The term "UHT process" used in this specification encompasses any heat treatment process which involves heating the nutrient to above 100°C for 0.5 seconds to 2 minutes, preferably 115-160°C for 0.5 seconds to 2 minutes, more preferably 125-165°C for 1-10 seconds, and preferably at a pressure above atmospheric pressure sufficient to suppress vapourisation (especially boiling) of the nutrient. Where the nutrient consists (in whole or part) of milk, the process will involve heating the nutrient for at least 1 second, preferably 2 to 10 seconds, most preferably 2 to 5 seconds, at a temperature of at least 130°C, preferably 132.2°C to 160°C, most preferably

135°C to 145°C. In practice, a temperature of at least 135°C for at least 2 seconds is normally required to ensure that the milk is sterilised rather than merely pasteurised.

The nutrient is preferably subsequently maintained for at least 1 minute, in the temperature range 45 to 95°C. The nutrient is more preferably maintained within this range for 2 to 6 minutes, most preferably for 3 to 5 minutes. Typically, the inventors have found that at a subsequent temperature of 55-65°C lipase activity declines rapidly for about 4 minutes and thereafter remains fairly constant. Long heat treatments are undesirable because they may require the use of large nutrient holding apparatus (eg storage tanks) and may detrimentally alter the nutrient's quality and flavour. For this reason, the nutrient is preferably maintained in the 50-95°C temperature range for not more than 6 minutes. The temperature at which the nutrient is subsequently maintained is preferably from 50 to 90°C, more preferably from 55 to 85°C, and is most preferably from 57 to 60°C. For a given length of subsequent heat treatment, the level of lipase deactivation has been found by the present inventors to be remarkably constant over the range 60°C to 80°C at least, which is especially surprising since at temperature in excess of 100°C lipase deactivation increases with increasing temperature. It is especially desirable to keep the temperature of the nutrient at or below 75°C during the subsequent heat treatment step, since it is well known in conventional pasteurisation that nutrients such as milk can be kept at or below this temperature for several minutes without suffering an undue impairment of flavour. The nutrient is preferably maintained at a substantially constant temperature which being held within the 45°C to 95°C temperature range. This provides for better control of the degree of enzyme deactivation brought about by the present method. The nutrient is preferably cooled directly from the UHT temperature to the subsequent heat treatment temperature without being held during an intervening period below 45°C.

Once the liquid nutrient is .heat treated within the temperature range 45-95°C for the specified period of time, the nutrient is preferably cooled under aseptic conditions to below 35°C within 5 minutes, preferably within 2 minutes, most preferably within 1 minute. Short cooling times will normally be desirable to keep holding times to a minimum and to preserve the flavour of the nutrient. If desired, the nutrient may at this point be packaged by asceptically filling the nutrient into sterile containers (such as cartons or bottles) and sealing the containers against the ingress of contaminants. It is important to cool the nutrient below 35°C, preferably below 25°C, before filling collapsible containers (eg plastic or cardboard cartons) or containers having collapsible seals (eg plastic or glass bottles with foil tops), because after these containers are sealed, subsequent cooling of the nutrient to room temperature or below will create vacuum conditions inside the containers and may cause them to distort, leak, or rupture. In order to facilitate the performance of the present invention on a commercial scale within, for example, a dairy, the present method may be carried out as a continuous or semi-continuous aseptic process.

This will preferably involve cooling a flow of UHT-treated liquid nutrient to within the 45-95°C temperature range employing a heat exchanger, conveniently a plate-type heat exchanges or a vacuum or flash cooler. The flow of nutrient will preferably be held within the 45-95°C temperature range in a holding tank or an array of pipes, and will thereafter preferably be cooled to below 45°C using a further, preferably plate-type, heat exchanger. Alternatively a heat echanger may be used to cool a flow of UHT-treated liquid nutrient to below 45°C. The nutrient may then be held within a storage tank at below 45°C for subsequent re-heating to 45-95°C and then recooling using two further heat exchangers.

The present method is especially useful in that it provides a simple and cost-effective way of increasing the potential shelf life of liquid nutrients such as UHT milk. The findings of the present inventors are surprising, since significant lipase deactivation does not occur when milk is subject to a conventional UHT process, to a pasterurisation process at 55-80°C, or to a process consisting of the latter followed by the former. Although the present invention is not limited in any way by this explanation, it is believed that the UHT process sensitises the heat-stable enzymes to thermal deactivation at a lower temperature.

According to other aspects of the present invention there are provided

(a) a container, especially a carton, containing a liquid nutrient, especially milk, treated in accordance with the method of the first aspect,

(b) a dairy product, such as ice cream, yogurt, butter, cheese, dairy dessert, cream, or flavoured milk, prepared from milk treated in accordance with the method of the first aspect, and

(c) a milk constituent isolated from UHT milk treated, in accordance with the method of the first aspect. Since the enhanced deactivation of lipases leads to a significant increase in the potential acceptable shelf life of UHT milk, it is evident that the potential shelf life of cartoned milk, milk constituents and milk products prepared from UHT milk treated by the method of the present invention will also be significantly enhanced.

The present invention will now be described by way of Example only with reference to tables 1 to 8 which show times and temperatures for thermal deactivation of lipase and protease and figures 1 to 5 which show the deactivation of lipase and protease. General Procedure

In order to assess the effectiveness of the invention, high lipase activity was artifically induced in samples of milk by growing isolated strains or pseudomonads in sterile milk originally free of lipase activity. Thus the lipase activity induced in the samples was wholly attributable to the bacterial strain grown in the milk. The general procedures used to prepare the milk samples, assay the lipase, and treat the samples in accordance with the invention, are all given below.

1. Culture selection identification and maintenance

The bacteria used were isolated from samples of a range of regrigerated raw bovine milks taken from a wide range of sources. The milks were diluted, after storage for 24 to 48 hours at 10ºC, and plated out onto a solid growth medium containing butterfat and an indicator dye. Plates of this meαium were incubated at 7ºC for 7 days, when active lipase producers were selected by the removal of single bacterial colonies which gave the strongest reaction. Isolates were then checked for purity and tested for their ability to produce heat stable lipase in UHT milk cultures.

The selected isolates (pseudomonad3 46, 53 and 55) were classified as non-fluorescent pseudomonads on the basis of physical and biochemical tests. Cultures were stored under liquid nitrogen, a fresh one being used for each culture run. 2. Culturing conditions

300ml batches of UHT milk were dispersed into 1 litre conical flasks which were then autoclaved at 115ºC for 10 minutes. No lipase activity was measurable by the assay method given below in the resulting autoclaved milk media. The milk batches, were then inoculated with 1 x 10⁴ bacteria ml^-1 from a culture of one of the selected pseudomonads which had been grown for 24 hours ait 10ºC in 10 ml of UKT milk. The milk batches so inoculated were then incubated under shaken conditions at 10*C maintained using a refrigated orbital incubator revolving at 100 revolutions per minute. Samples (5 ml) were removed at regular intervals for measurement of both viable bacterial numbers by the spread plate technique and lipase activity. 3. Lipasr assay

Lipase was assayed using uncentrifuged culture samples with triolein as substrate. The assay mixture contained 2 ml of triolein emulsion (10% w/v triolein emulsified in 10% w/v gum arabic using an Ultra-Turrax homogeniser at maximum speed for 10 minutes) 0.5 ml of culture medium and 2 ml of 0.2M Tris-HC1 buffer pH 8.5.

A 1 ml portion of the reaction mixture was transferred to 5 ml of Doles reagent (propan-2-ol, n-heptane and 2M H₂SO₄,40:10: 1 by volume) before and after incubation at 40ºC for 1 hours. The amount of oleic acid released was determined by the method of Dole and Meinertz (Journal of Biological Chemisty (1960),235(9),2595-2599) using oleic acid as standard. One unit of lipase activity is defined as the amount of enzyme releasing 1 x 10^-6 mol of oleic acid per minute. 4. Heat treatments

Lipase heat stability was assessed using samples removed from milk cultures towards the end of logarithmic growth when maximum lipase activity was approached. The culture samples (1.5 ml) were injected into coiled stainless steel tubing (2.38mm inside diameter, 0.15mm wall thickness) and the apparatus pressurised using a nitrogen cylinder to 4.2 kg cm (g) to prevent evaporation during heat treatment. For heat treatments above 100°C, the tubing was totally immersed in a heated polyethylene glycol bath. For heat treatments below 100°C, the tubing was totally immersed in a heated water bath. After reaching the appropriate temperature(s) and holding for the prescribed time period(s) given in the Examples below, the whole apparatus was plunged into a water bath at 10ºC to facilitate rapid cooling. Lipase activity was measured immediately afterwards. Examples 1-15

15 samples of milk each containing one of the three Pseudomonas strains 46, 53 and 55 were subjected to a double heat treatment consisting of, firstly, 130ºC for 5 seconds followed by, secondly, 60ºC to 80ºC for 3 minutes. Each sample was quenched in cold water to 10ºC between the first and second heat treatments. Lipase activity was assayed immediately before and after the double heat treatment. Residual lipase activity after heat treatment is given in Table 1 below. Table 1

Example Organism 2nd Heat % residual lipase Treatment Temperature activity (mean of triplicate determinations)

1 46 60ºC 60

2 46 65ºC 61

3 46 70ºC 60

4 46 75ºC 64

5 46 80ºC 62

6 53 60ºC 63

7 53 65ºC 65

8 53 70ºC 63

9 53 75ºC 62

10 53 80ºC 63

11 55 60ºC 66

12 55 65ºC 68

13 55 70ºC 63

14 55 75ºC 65

15 55 80ºC 64

Examples 16-30

The double heat treatment method described under Examples 1-15 was repeated on 15 further samples, except that the first heat treatment consisted of 140ºC for 5 seconds rather than 130ºC for

5 seconds. The results of this method in terms of residual lipase activity is given in Table 2 below.

Table 2

Example Organism 2nd Heat % residual lipase

Treatment Temperature activity (mean of triplicate determinations)

16 46 60ºC 46

17 46 65ºC 45

18 46 70ºC 47

19 46 75ºC 49 20 46 80ºC 50

21 53 60ºC 50

22 53 65ºC 52

23 53 70ºC 48

24 53 75ºC 53

25 53 80ºC 51

26 55 60ºC 56

27 55 65ºC 53

28 55 70ºC 54

29 55 75ºC 52

30 55 80ºC 56

Examples 31-45

15 samples of milk each containing one of the three Pseudomonas strains 46, 53 and 55 were subjected to a double heat treatment consisting of, firstly, 140ºC for 5 seconds followed by, secondly, 60ºC for 10 second to 10 minutes. Each sample was cooled directly between heat treatments from the upper temperature of 140ºC to the lower temperature of 60ºC by quenching the hot tubing in the water bath at 60ºC. Cooling to 60ºC in this manner took about 10 seconds. Lipase activity was assayed immediately before and after the double heat treatment. Residual lipase activity after heat treatment is given in Table 3.. Table 3

Example Organism 2nd Heat % residual lipase Treatment Time activity (mean of triplicate determinations)

31 46 10 sees 75

32 46 30 sees 62

33 46 3 mins 42

34 46 5 mihs 38

35 46 10 mins 35

36 53 10 sees 74

37 53 30 βecs 67

38 53 3 mins 53

39 53 5 mins 46

40 53 10 mins 44

41 55 10 sees 77

42 55 30 sees 65

43 55 3 mins 47

44 55 5 mins 40

45 55 10 mins 38

Examples 31 to 45 illustrate that lipase deactivation is further enhanced if the milk is cooled directly from the UHT temperature to the second heat treatment temperature. They further illustrate that the optimum time for lipase deactivation during the second heat treatment is aboutv 3-5 minutes, since the greatest rate of deactivation was generally found during the first 3 minutes of treatment. Examples 46-63 (comparative)

18 samples of milk each containing one of the Pseudomonas strains 46, 53 and 55 were held at a temperature of 60 to 80ºC for either 3 or 10 minutes. Lipase activity was assayed immediately before and after heat treatment. Residual lipase activity after heat treatment is given in Table 4 below. Table 4 % residual lipase activity (means of triplicate determinations)

Heat Treatment Temperature 60ºC 70ºC 80ºC

Heat Treatment Time 3 min 10 min 3 min 10 min 3 min 10 min

Organism 46 98 95 93 90 85 82

Organism 53 96 95 95 93 90 88 Organism 55 96 95 96 94 90 89

The above Table 4 shows that conventional pasteurisation has little effect on lipase activity.

Examples 64-66 (comparative)

Three samples of milk each containing one of the Pseudomonas strains 46, 53 and 55 were held at a temperature of 140ºC for 5 seconds. Lipase activity was assayed immediately before and after heat treatment. Residual lipase activity after heat treatment is given in Table 5 below, which shows the lipase enzyme to be extremely heat stable.

Table 5

Example Organism % residual lipase activity

(means of triplicate determinations)

64 46 90

65 53 81

66 55 85

Examples 67-69 (comparative)

Three samples of milk each containing one of the three Pseudomonas strains 46, 53 and 55 were subjected to a double heat treatment of 60ºC for 3 minutes followed by 140ºC for 5 seconds. Lipase activity was assayed immediately before and after the double heat treatment. Residual lipase activity after heat treatment is given in Table 6. Table 6

Example Organism % residual lipase activity (mean of triplicate determinations)

67 46 88

68 53 79

69 55 83

The above Table 6 shows that a double heat treatment described above does not enhance lipase deactivation significantly over the single conventional UHT process used in Examples 64-66.

Legends to Figures

Figure 1 Thermal deactivation-time curves for Pseudomonas strains P.46(a), P.53(b) and P.55(c) lipases in whole milk cultures at 130°C ( · ) and 140°C ( + ).

Figure 2 Thermal deactivation-time curves for Pseudomonas strains P.46 ( · ), P.53 ( + ) and P.55 ( X ) lipases in whole milk cultures at 60°C following 140°C for 5 sec

A further set of experiments was performed to investigate the deactivation of proteinase enzymes as well as lipase enzymes in milk using the method of the invention, and also to monitor any changes in the treated milk which occurred on long term storage. For lipases, residual lipase activity was determined by the rate of rancid short chain fatty acid production during milk storage rather than by assaying activity using triolein as substrate. The former method more closely simulates actual conditions and was therefore chosen as the preferred technique. In addition, milk storage was necessary to evaluate the heat stabilities of proteinases in whole milk since the bacteria of commercial concern often produce insufficient enzyme in this medium to make accurate rapid assay methods possible.

Initially three strains of pseudomonads, isolated from raw milk and shown to be active lipase and proteinase producers, were cultured in resterilised UHT whole milk at 5°C under shaken conditions (110 rpm). When the bacterial count reached approximately 1 x 10⁸ cells ml^-1 samples were heat treated and placed in bottles containing Thimerosal, a biocide added to prevent subsequent bacterial contamination, and shown to have no effect on the activity of a range of bacterial lipases and proteinases. Residual lipolytic and proteolytic activities were measured during storage for up to 8 weeks at 20°C. As a control, uninoculated resterlised milk was heat treated and stored for an equivalent period. Lipase activity was determined by measurement of rancid short chain fatty acids (C4:0, C6:0, C10:0, C12:0) using gas liquid chromatography. Proteolytic activity was measured using a colorimetric method for detection of free amino groups in acid soluble short peptides produced by the action of proteinase on milk proteine.

A range of double heat treatments was used. The minimal treatment (140°C for 5 sec followed by colling directly to 60°C within 10 sec and holding for 5 min) which resulted in greatest enhancement of lipase and proteinase deactivation for each of the three bacterial cultures was selected for further study using raw milk cultures. The effectiveness of this dual heat treatment was compared to a standard UHT treatment using two different raw milk samples obtained from bulk tanks of a local dairy. These milks were cultured in sterilised conical flasks at 5°C under shaken conditions (75 rpm) until the bacterial numbers reached either approximately

3.5 x 10⁶, 6 x 10⁶, 5 x 10⁷ or 1 x 10⁸ cells ml^-1. The milks were then pasteurised at 72°C for 15 sec to deactivate the 'natural' milk lipase, homogenised at 15-20°C, and finally heat treated (either 140°C for 5 sec or 140°C for 5 sec followed by 60°C for 5 min). The treated milks were stored at 20°C in the presence of Thimerosal, with samples removed either weekly for the two milks which originally contained the higher bacterial numbers, or every two months for those which originally contained the lower numbers of bacteria. Control samples of milk removed on arrival at the laboratory which contained approximately 3 x 10⁴ bacteria ml^-1 were also treated as described above and sampled every two months, for six months.

Results for the effect of various heat treatments on lipase and proteinase activities for the milks inoculated with the three pseudomonads are shown in Table 7. The values given represent the means of relative activities measured over the total storage period. Whilst not illustrated, it should be noted that fatty acids and peptides were produced at a near linear rate for each sample. These pure culture studies indicate that the enzymes may be deactivated effectively by a dual heat treatment comprising 140°C for 5 sec followed by cooling to

60°C and holding at this temperature for 5 min. Extending the low temperature treatment beyond this time or increasing the temperature to 65°C apparently has no advantage, whilst reducing the heating time or temperature to 2 min or 57°C respectively, may result in signifi- cantly less deactivation. The proteinases were in general more resistant than the lipases to the low temperature deactivation, although substantial enhanced activity loss was noted in all cases. Any apparent discrepancy between results in Table 7 are not significant and are due to experimental variation. Results for the thermal deactivation of lipases and proteinases in raw milks which contained a natural mixed bacterial population are given in Table 8. The production of fatty acids and peptides in heat treated samples of raw milk 'B' which had contained approximately 1 x 10⁸ bacteria ml^-1 are shown graphically in Figs 3, 4 and 5. These graphs show the typical near linear rates of lipolysis and proteolysis which are common to all samples which demonstrate such activities. The raw milk study confirms the value of the dual heat treatment process in extending the shelf life of UHT milk, and again illustrates the greater sensitivity of lipases, in general, to the process. Rancid defects may be detected in whole milk when short chain free fatty acids reach a level of approximately 0.25 umol ml^-1

(Scanlan et al. 1965). The raw milk culture 'B' which contained approximately 6 x 10⁶ bacteria ml^-1 would be expected to reach this level after some 9 weeks at 20°C following conventional UHT treatment. However, the same milk after dual heat treatment was only expected to become rancid after approximately 20 months.

It is more difficult to correlate accurately proteolytic activity with flavour defects in whole milk. However, McKellar (1981), using the same method of analysis as used here found that the mean level of proteolysis required to cause off-flavours in milk was approximately 0.4 umol free amino groups ml^-1. The levels of proteinase measured in the two raw milks A and B containing either 3.5 x 10⁶ or 6 x 10⁶ bacteria ml^-1 before UHT heat treatment would be expected to cause such a defect after storage at 20°C for 9 weeks and 3 weeks, respectively. Based on the proteinase activities in Table 8 the dual heat treatment may be expected to increase storage times to 10 months and 2 months, respectively, confirming the potential value of the process.

Thus overall a minimum 3-fold increase in shelf-life of UHT milk and derived products may be anticipated after the dual heat treatment process described herein. References

McKellar, R.C. (1981). Development of off-flavours in ultra high temperature and pasteurized milk as a function of proteolysis. Journal of Dairy Science, 64, 2138-2145.

Scanlan, R.A., Sather, L.A. and Day, E.A. (1965). Contribution of free fatty acids to the flavour of rancid milk. Journal of Dairy Science, 48,

1582-1584.

Legends to Figures

Figure 3 Production of free fatty acids C4:0, C6:0 (X),

C8:0 (+), C10:0, C12:0 (0) in Raw Milk B which had contained approximately 1 x 10⁸ bacteria ml^-1 before heat treatment at 140°C for 5 sec.

Figure 4 Production of free fatty acids C4:0, C6:0 (X),

C8:0 (+), C10:0, C12:0 (0) in Raw Milk B which had contained approximately 1 x 10⁸ bacteria ml^-1 before heat treatment at 140°C for 5 sec followed by

60°C for 5 min.

Figure 5 Production of acid soluble peptides measured as free amino groups in Raw Milk B which had contained approximately 1 x 10⁸ bacteria ml^-1 before heat treatment at 140°C for 5 sec or 140°C for 5 sec followed by 60°C for 5 sec min (X).

Table 7. Relative residual lipase and proteinase activities following dual heat treatments, compared to a standard UHT treatment (140°C for 5 s) as control, for three pseudomonads cultured in whole milk.

Heat treatment Organism 1 Organism 2 Residual activity (%) Residual activity (%) Lipase Proteinase Lipase Proteinase

140°C 5 sec 100 100 100 100

140°C 5 sec +

57°C 5 min 12 33 11 11

60°C 2 min 12 36 10 9

60°C 5 min 7 17 5 5

60°C 10 min 7 20 5 5

65°C 5 min 5 18 5 4

Heat treatment Organism 3

Residual activity (%)

Lipase Proteinase

114400°°CC 55 sseecc 110000 110000

140°C 5 sec +

57°C 5 min 9 30

60°C 2 min 10 32

60°C 5 min 4 18

60°C 10 min 4 20

65°C 5 min 4 20

Claims

1. A method of enhancing the deactivation of heat-stable enzymes present in a liquid nutrient under asceptic conditions characterised in that it comprises the steps of sterilising the nutrient by a UHT process, cooling the nutrient and subsequently maintaining the temperature of the nutrient within the temperature range 45°C to 95°C for at least 30 seconds and not more than 10 minutes.

2. A method according to Claim 1 wherein the preferred liquid nutrient is a dairy product.

3. A method according to Claim 1 characterised in that the UHT process is performed above 100°C for between 0.5 seconds to 2 minutes.

4. A method according to Claim 1 characterised in that the UHT process is performed between 115°C and 160°C for 0.5 seconds to 2 minutes.

5. A method according to Claim 1 characterised in that the UHT process is performed between 125°C and 165°C for 1 to 10 seconds.

6. A method according to Claim 1 characterised in that the UHT process is performed at, at least 132.2°C and not more than 160°C for a minimum of 1 seconds and not more than 10 seconds.

7. A method according to Claim 1 characterised in that the temperature of the heat treatment subsequent to the UHT process is in the range 50°C - 90°C.

8. A method according to Claim 7 characterised in that the temperature of the heat treatment subsequent to the UHT process is in the range 55°C to 85°C.

9. A method according to Claim 7 characterised in that the temperature of the heat treatment subsequent to the UHT process is in the range 55°C to 75°C.

10. A method according to Claim 7 characterised in that the temperature of heat treatment subsequent to the UHT process is in the range 57°C to 60°C.

11. A method according to Claim 1 characterised in that the duration of subsequent heat treatment is for at least 1 minute and not more than

10 minutes.

12. A method according to Claim 1 characterised in that the duration of subsequent heat treatment is in the range 2 to 6 minutes.

13. A method according to Claim 1 characterised in that the duration of subsequent heat treatment is in the range 3 to 5 minutes.

14. A method according to Claim 1 characterised in that the nutrient is cooled directly to the subsequent heat treatment temperature range or temperature.

15. A method according to Claim 1 characterised in that the nutrient is cooled below the required subsequent heat treatment temperature range or temperature before reheating the nutrient to the subsequent heat treatment temperature range or temperature.

16. A method according to Claim 1 characterised in that during the subsequent heat treatment the temperature is maintained substantially constant.

17. A method according to any one of the preceeding claims characterised in that after the subsequent heat treatment the nutrient is cooled to below 35°C within 5 minutes.

18. A method according to Claim 1 characterised in that it is performed as a continuous or semi-continuous process.

19. A liquid nutrient or a product prepared from a liquid nutrient characterised in that the liquid nutrient has been sterilised by a UHT process and then subsequently subjected to a temperature in the range 45°C to 95°C for at least 30 seconds and not more than 10 minutes wherein residual lipase and/or protease activity in the liquid nutrient is less than 80% of the lipase and/or protease activity before the UHT sterilisation and subsequent heat treatment.

20. A liquid nutrient or product according to Claim 19 characterised in that the residual activity of lipase and/or protease is less than 35% of the lipase and/or protease activity before the UHT sterilisation and subsequent heat treatment.

21. A liquid nutrient according to Claim 14 or 15 characterised in that the liquid nutrient is a dairy product.