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The LP system can be used to prevent bacterial deterioration of milk when refrigeration is not available.  It can also be used to prolong the safe storage life of refrigerated milk.  Arguably the LP-system, immunoglobulins and lactoferrin have potential to be of value in neonate nutrition.  The remaining section largely concerns the exploitation of the LP-system in the protection of neonates.

Manufacture of milk powders containing a functional LP system

That milk provides neonates with nutrients and  various protective antimicrobial factors has been discussed previously.  Because many of these factors are denatured by the heat treatments used in milk replacer manufacture commercial products, unless specially produced, generally do not contain antimicrobial proteins in active form.

The author and colleagues at the Scottish Agricultural College (SAC) at Auchincruive developed milk replacers containing a functional LP system, in powder form, stable at ambient temperature for at least five months and suitable for use on the farm.  These were the result of a collaborative project between SAC, the former Scottish Milk Marketing Board (SMMB) and the former Swedish animal nutrition company Astra-Ewos.

Milk replacers containing active antimicrobial proteins can be produced by two main methods.  Method 1 involves careful control of time-temperature treatments employed during replacer manufacture to avoid denaturation of the antimicrobial agent.  In the second, the agent is extracted from milk or whey e.g. lactoferrin, and is dry-mixed with other ingredients to give a final milk replacer.

Calf milk replacers

Conventional calf milk replacers do not contain a functional LP system. LP containing replacers can be produced by dry mixing a low heat treated skim milk powder of high LP content, with fat filled powder, and mineral and vitamin premixes. Extenders e.g. whey powder or other protein sources along with 'denaturing' agents such as starch may also be added.  A hydrogen peroxide donor, or a hydrogen peroxide generating system, and thiocyanate must also be added.

Production of skim milk powder containing high levels of LP activity

Raw milk contains high but variable levels of LP activity (e.g. Korhonen et al., 1977). LP activity equivalent to enzyme protein concentrations  between 20-40 mg/L is found frequently. These are markedly in excess of the levels (0.5 μg/ml in absence of catalase) required for efficient LP system activation (Björck, 1978).  Powders of high LP activity can be produced by carefully controlling the temperatures at key stages during the drying process.  The temperatures used for pasteurisation, and preheating prior to evaporation are the major critical control points as far as conservation of LP activity is concerned.  The author has found that if temperatures at these points should exceed 75°C that this will result in significant loss of LP activity.  Another activity critical control point is the temperature of the concentrate in the 'temperature controlled" jacketed pipe going to the atomiser.

Low heat skim milk powder can easily be produced commercially, using modern plant, containing approximately 50% of the LP activity of raw milk.  Powder of high LP content has a non-casein nitrogen/total nitrogen ratio of >0.2 and a heat or casein number of <80.

Selection of hydrogen peroxide donor or hydrogen peroxide generating system

The LP system in milk replacers has been activated by addition of hydrogen peroxide in liquid form, generation of hydrogen peroxide in situ by use of glucose oxidase and by the hydrolysis of magnesium peroxide.  The author and colleagues have also investigated other methods for example the use of hydrogen peroxide producing lactic acid bacteria, for LP system activation.

The characteristics of an ideal hydrogen peroxide donor are shown in table 1. Magnesium peroxide meets most of these criteria.  This material is available commercially as a mixture containing Mg (OH) 2 (75% w/w) and Mg02 (25% w/w).  Hydrolysis of this compound in milk is pH dependant; hydrogen peroxide production requires the pH to be lowered to 6 or less. Consequently replacers containing magnesium peroxide produce hydrogen peroxide in the abomasium of the calf.  The use of magnesium peroxide for LP system activation is described in British Patent No. 1546747.  Optimal LP system activation requires magnesium peroxide to be added to milk replacers to as to give a concentration of 0.03% (w/v) in the reconstituted replacer.  

Table 1: Characteristics of a hydrogen peroxide donor for use in milk replacer formulation

1. It should be available in powder form.

2. It should be un-reactive with replacer components during storage.

3. It should release hydrogen peroxide at a controlled rate in the reconstituted replacer.

4. It should be relatively inexpensive.

5. It should be non-toxic and have no detrimental effect on the palatability of the replacer

Sodium thiocyanate addition

Optimal activity of the LP system requires equi-molar concentrations of hydrogen peroxide and thiocyanate (Björck, 1978).  Thiocyanate levels in milk replacers are generally less than <0.1 mM and while high enough to give partial activation of the LP system, supplementation with additional thiocyanate is required for maximum anti-microbial activity against E. coli and other pathogens.

Thiocyanate must be added to mineral and vitamin pre-mixes and carefully mixed with the other ingredients to ensure homogenous distribution of the SCN.

Fat source

The quality of the fat source is very important for the nutrition of the calf.  However, it is outside the scope of this communication to discuss this area.  There is a range of fat filled powders available commercially and whatever powder is chosen it should be pre-tested prior to incorporation in the milk replacer to ensure that it does not contain LP system inhibitors and that it meets the quality requirements for neonate feeding.

Extenders

These can cause major problems. A range of extenders can be used but those used must be chosen carefully so not to inhibit the antimicrobial activity of the LP system e.g. low heat-treated whey powder can be used.

The composition of a typical LP milk replacer is shown in table 2.

Table2. Typical composition (1) of a calf milk replacer containing an
active lactoperoxidase system

Ingredient

% w/w

Low heat skim milk powder

31

Fat filled milk powder (36-38% fat)

53

Low heat whey powder

13

Mineral and vitamin premix (2)

0.65

Starch (3)

2

1. Figures have been 'rounded' and do not add up to 100%.
2. Pre-mixes contained 1.5 g/kg 'magnesium peroxide' (25% Mg02, 75% Mg (OH) 2) and 300 mg/kg of sodium thiocyanate.
3. Starch was added as a 'denaturing' agent for EU subsidy purposes.


Anti-microbial activity of LP-containing milk replacers

Reconstituted milk replacer, adjusted to pH 5.8 (to hydrolyse the magnesium peroxide and release hydrogen peroxide) has been shown to be bactericidal to a range of pathogens (Mullan et al., 1982b).  Typical data showing the effectiveness of the system against presumptive pathogens isolated from calves at post mortem by staff at the veterinary laboratory at the SAC at Auchincruive are shown in table 3.

Table 3.  Effect of the LP system in calf milk replacers on the viability of strains of E. coli isolated from post-mortem examination of calves

Strain

T0 Inoculum (CFU/ml)

CFU/ml after 2 hours at 37 °C

 

No cysteine added

+ 1 mM cysteine

% Kill

1

1.3 x 107

3.3 x 106

6.84 x 107

74.6

8

3.9 x 106

2.39 x 105

5.84 x 107

93.9

9

1.49 x 107

4.2 x 106

5.58 x 107

71.8

 

1.72 x 107

1.1 x 106

6.72 x 107

93.6

NCDO 2328

9.5 x 106

3.2 x 102

2.68 x 107

99.99

NCDO 904

3.1 x 107

7.1 x 106

5.84 x 107

76.87

2

7.0 x 106

3.5 x 105

7.2 x 106

50

3

1.0 x 107

2.6 x 106

1.03 x 109

74

4

6.0 x 107

3.0 x 107

1.45 x 109

50

5

7.6 x 106

1.3 x 106

5.1 x 106

83

6

6.0 x 107

7.0 x 105

7.5 x 108

98.8

7

3.0 x 107

2.0 x 105

5.6 x 108

99.3

Notes: Milk replacer was reconstituted to give 10% TS and was adjusted to pH 5.8. Reconstituted replacer contained 0.03% (w/w) Mg02, LP activity equivalent to 7 μg LP/ml and 0.35 mM SCN. E. coli strains were isolated from lesions in calves at post-mortem and were presumed to be calf pathogens. Mullan and Waterhouse, unpublished data.

 

Shelf life of lactoperoxidase-containing calf-milk replacers

There can be marked differences in the decline of enzyme activity in milk powders stored at ambient temperatures. LP concentrations in powders after 5 months storage generally ranged from 0.05 — 0.5 ug/ml of the reconstituted replacer.  While 0.05 ug/ml of LP is too low to give optimal bactericidal activity it is high enough to reduce a population of 1 x 107 CFU/ml of a sensitive E. coli by up to 3 log cycles (Mullan and Waterhouse, unpublished data).

 Quality assurance of LP containing milk replacers

Some of the main concepts have been reported by the author and colleagues. Because of the low heat treatment required to produce milk powder containing high levels of LP activity it is important to use high quality raw milk.   This milk should have a low concentration of spore formers and thermoduric organisms at the point of manufacture.

In vitro anti-microbial tests using a suitable strain of E. coli (Extrand et al., 1985) are required to ensure that the LP system is functioning in the milk replacer.

Since antibiotics can also inhibit the growth of E. coli  it is important that all materials are screened for the presence of antibiotics and that antibiotic-contaminated materials are rejected for use in milk replacer manufacture.

LP activity in milk powders and milk replacers must be monitored. Enzyme assays have been discussed previously.

Effects of feeding milk replacers containing an active LP system on neonate growth and mortality

There have been 14 trials of LP containing milk replacers at the SAC at Auchincruive. In 12, significant effects on growth rate and concentrate intake were found. Diarrhoea (scouring) was also reduced in most trials for calves in experimental groups (receiving LP replacers) compared with controls.

In 1982/83 the SAC, funded by the Scottish Milk Marketing Board, undertook a major evaluation of LP-milk replacers (Waterhouse et al., 1983) on 8 commercial farms in S.W. Scotland. This study involved 396 calves and, amongst many findings, revealed that feeding LP-milk replacers led to an increase in growth rate over the period 0-4 weeks compared with controls.

More recently, a trial in which the LP-system and lactoferrin have been used to successfully treat induced enterotoxic colibacillocis in calves has been reported (Still et al., 1990).

Some research on the use of immunoglobulin concentrates in calf feeding has been published. The limited data suggest that inclusion of non-specific antibodies in milk replacers does not improve calf health or performance (De Gregorio and Anexstad, 1991) but  suggest that concentrates containing antibodies specific for pathogens may have positive effects (Annexstad et al., 1991).

Improved human milk replacers

The in vitro antibacterial and antiviral properties of human milk have been well documented (Reiter, 1989). The lower incidence of enteric infections in naturally fed infants has been attributed to the activities of these and other antimicrobial substances (Reiter, 1978).

There is interest in the addition of the antimicrobial proteins of bovine milk to infant formulae. However, published data on the effectiveness of these additions are not available and the benefits remain speculative.

Milk powders containing an active LP system may have advantages as far as protecting the health of the newborn when formula feeds are reconstituted with contaminated water under unsanitary conditions (Banks and Board, 1985).

Human milk contains 10-100 times more lactoferrin than bovine milk (Masson, 1970). Because unsaturated lactoferrin has been shown to have a bacteriostatic effect on the growth of some enteric pathogens (see the references of Reiter, 1978) there has been speculation on the potential for the prophylactic use of lactoferrin in infant nutrition.

Bovine immunoglobulins possess both antibacterial and antiviral activity, e.g. antirotavirus activity (Goldman et al., 1985). There is some research interest in the isolation of immunoglobulins from milk e.g. cheese whey and the addition of immunoglobulin concentrates to human milk replacers. If these antimicrobial proteins are to be effective then a local immune response within the gastrointestinal tract of the infant must be considered as the mechanism responsible; this remains to be demonstrated.

Acknowledgements

The LP project at the WSAC at Auchincruive (now SRUC) was a collaborative project involving the Dairy Technology, Animal Husbandry and Veterinary departments, the Scottish Milk Marketing Board and Astra-Ewos. Please see the acknowledgement's section.


How to cite this article

Mullan, W.M.A. (2003) . [On-line]. Available from: https://www.dairyscience.info/index.php/exploitation-of-anti-microbial-proteins/168-lactoperoxidase-system.html . Accessed: 6 December, 2016. Revised August 2009.

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