There are several forms of pasteurisation including batch or vat (treatment at 63°C for 30 minutes), high temperature short-time (treatment at noc for 1 5 seconds), ultra
3.2 MATERIALS AND METHODS
3.2.1 Whey proteins
Two (non-commercially available) whey protein preparations derived from pasteurised milk (treated at 72°C for 1 5 seconds; M. Pritchard, NZDRI, Palmerston North, New Zealand, pers. comm.) were used in this study. The p-Iactoglobulin (PT38 86, NZDB, Wellington, New Zealand) was supplied as a p-Iactoglobulin-enriched whey protein isolate, prepared by selective elution of a-lactalbumin from a standard whey protein isolate (M. Pritchard, NZDRI, pers. comm.). The a-lactalbumin (PT57 1 4, NZDB, Wellington, New Zealand) was supplied as an a-lactalbumin enriched isolate, prepared by loose ultra-filtration of a whey protein isolate (M. Pritchard, NZDRI, pers. comm.).
The total protein content and purity of the p-Iactoglobulin and a-lactalbumin sources used in this study are summarised in Table 3 . 1 . The typical microbiological estimates of the whey proteins evaluated in this study are given in Table 3.2. The following commercially available whey proteins prepared from pasteurised milk (treated at 1 40°C for a minimum of 8 hours and then shocked at 1 1 0°C for 1 5 seconds; Z. Dokonal, MP Biomedicals, Australia, pers. comm.) were also tested in this study; p-Iactoglobulin
(Catalogue number 1 5 1 536, ICN, Nutrition Biomedical, Cleveland, Ohio) isolated by chromatography (Z. Dokonal, MP Biomedicals, Australia, pers. comm.) and a lactalbumin (lactalbumin hydrolysate) (Catalogue number 1 02 1 29, ICN) isolated by ion exchange chromatography (Z. Dokonal, MP B iomedicals pers. comm.) and the minor whey protein, lactoferrin (Catalogue number 1 5 1 535, ICN). All whey proteins were diluted to required concentrations in sterile complete RPMI-1 640 medium ( Section 2. 1 ) and freshly prepared prior to use. All whey proteins were readily soluble in sterile complete RPMI- 1 640 and sterile filtered using a 0.22 Ilm filter (Millipore, MA, USA) prior to addition to cell cultures.
TABLE 3.1
Protein analysis of whey proteins evaluated in this study
Whey Protein Protein content % p-lac Isolates (TN*6.38) (0/0 of TN*6.38) J3-lactoglobulin 94% 8S% (NZDB) a-lactalbumin 92% 1 8% (NZDB) J3-lactoglobulin 99% (ICN) a-lactalbumin 95 - 98% (lCN) Lactoferrin (LF) 94.S% (ICN) Note:
1 . - indicates that the parameter was not analysed 2. TN = Total Nitrogen % a-lac (% of TN*6.38) 9% 7 1 % 80%
3. NZDB analyses conducted by reversed phase high pressure liquid chromatography (M. Pritchard, NZDRI)
4. ICN product details obtained from ICN B iomedicals, Sydney, Australia
TABLE 3.2
% LF
94%
Typical microbiological estimates for the whey proteins evaluated in this study
Whey Protein Standard plate Coli forms Yeasts & S. aureus Salmonella Isolates count (non-specific moulds
J3-1actoglobulin 730/g Neg/g < lO/g Neglg Neg/7S0 g
(NZDB)
a-lactalbumin 1000/g max Neg/g < l O/g Neglg Neg/7S0 g
(NZDB)
J3-lactoglobulin 1000/g max Neg/O . 1 g SO/g max Neg/0. 1 g Neg/S g
(lCN)
a-lactalbumin 1000/g rnax Neg/O. 1 g SO/g max Neg/O. l g Neg/S g (ICN)
Lactoferrin (ICN) 1000/g rnax Neg/O. l g SO/g rnax Neg/0. 1 g Neg/S g
Note:
1 . All counts calculated from number of colony forming units 2. Negative (Neg)
3. Staphylococcus aureus (s. aureus)
4. NZDB product details obtained from NZDB, Wellington, New Zealand S. ICN product details obtained from ICN B iomedicals, Sydney, Australia
3.2.2 Preparation of immune cell suspensions
Six to eight week old BALB/c mice were euthanased as described in Section 2.2. Spleens were removed aseptically from the mice, and single cell suspensions were prepared in complete RPMI- 1 640 medium as described in Section 2.3. For each individual whey protein the spleens of 1 2 BALB/c mice were pooled to provide sufficient cell numbers to assess splenocyte proliferative potential either in the presence or absence of mitogens.
3.2.3 Lymphocyte proliferation assay to assess the mitogenic activity of whey proteins
The in vitro mitogenic effect of whey proteins was determined by incubating spleen lymphocytes in the presence or absence of various concentrations of whey proteins. Cell
proliferation of splenic lymphocytes was measured using an enzyme-based
colourimetric cell proliferation kit (Boehringer Mannheim, Mannheim, Germany), as described in Section 2.4 with the following modification. After 50 )J.L of cells at a concentration of 4 x l 06cells/mL in complete RPMI- 1 640 medium were added to the wells of a 96-well flat-bottomed tissue culture plate (Nunc, Roskilde, Denmark) containing 1 00 )J.L of complete RPMI - 1 640, 50 )J.L of the required concentration of whey protein or complete RPMI-1 640 (control) was then added to the wells, resulting in a final volume of 200 )J.L/ well. No known mitogens (such as Con A, PHA and LPS) were added to the cell cultures.
Two fold serial dilutions of �-lactoglobulin and a-lactalbumin were added per well at concentrations ranging from 0.06 - 1 .8 8 )J.glmL or 3.75 - 1 20 )J.glmL. Two fold serial dilutions of lactoferrin were added per well at concentrations ranging from 0.003 - 0. 1 )J.glmL or 0.06 - 1 .88 )J.glmL. Control wells received 50 )J.L complete RPMI- 1 640 in place of whey protein (0 )J.glmL). The BrdU incorporation over the final 1 8 hours of a 96 hour culture period was measured as described in Section 2.4. The results were
expressed as mean (± SEM) absorbance of triplicate samples. Eight independent
3.2.4 Lymphocyte proliferation assay to assess the effect of whey proteins on the activity of various mitogens
The in vitro proliferative response of splenic lymphocytes to whey proteins and mitogens was determined by incubating mitogen-stimulated splenic lymphocytes with various concentrations of whey proteins. Lymphocyte proliferation was measured using an enzyme-based colourimetric cell proliferation kit (Boehringer Mannheim, Mannheim, Germany), as described in Section 2.4 with the following modifications; 1 00 J.lL of mitogen diluted in sterile RPMI- 1 640 was added to each well at the following final concentrations; 5 !Jg/mL LPS (B cell mitogen); 2.5 J.lg/mL Con A (T cell mitogen); or 1 3 .3 J.lg/mL PHA (T cell mitogen) resulting in a final volume of 200J.lLlwell. The BrdU incorporation over the final 1 8 hours of a 96 hour culture period was measured as
described in Section 2.4. The results were expressed as mean (± SEM) absorbance of
triplicate samples. Six independent experiments were performed.
3.2.5 Statistical analysis
The effects of each whey protein on mean splenic cell proliferation compared to the control group (no whey protein) were tested using analysis of variance (GraphPad Prism®, USA, 1 999). Individual protein concentrations were compared to controls (no whey protein) using a Dunnett's multiple comparison post-hoc test (GraphPad Prism®, USA, 1 999). Individual protein concentrations were compared to other protein concentrations using a Tukey's post-hoc test (GraphPad Prism®, USA, 1 999).
3 .3 RESULTS
3.3.1 The effect of p-Iactoglobulin (NZDB) on murine splenic lymphocyte proliferation
To investigate the mitogenic effect of p-Iactoglobulin (NZDB), the proliferation of murine spleen lymphocytes incubated with various concentrations of p-Iactoglobulin was measured. The p-Iactoglobulin used in this study exerted a dose-dependent stimulatory effect on the growth of splenic lymphocytes as shown in Figure 3 . 1 . Lymphocyte proliferation was 3 0 - 90% higher than the control (P < 0.05, 0.0 1 respectively) at concentrations ranging between 0.53 - 1 .8 8 ).lg/mL (Figure 3 . 1 A) and 250 - 3 00% greater than the control (P < 0.0 1 ) at higher concentrations ranging between 7.5 - 1 20 ).lg/mL (Figure 3 . 1 B). Maximum enhancement of splenic lymphocyte proliferation occurred when lymphocytes were incubated with 1 5 - 1 20 ).lg/mL of p lactoglobulin (Figure 3 . 1 B).
3.3.2 The effect of a-lactalbumin (NZDB) on murine splenic proliferation
To investigate the mitogenic effect of a-lactalbumin (NZDB), the proliferation of murine spleen lymphocytes incubated with various concentrations of a-lactalbumin was measured. The a-lactalbumin used in this study had no significant stimulatory effect on splenic lymphocytes at concentrations ranging between 0.06 - 1 .88 �g/mL (Figure 3 . 1 A). However, across the higher concentrations evaluated (3.75 - 1 20 ).lg/mL) the proliferation of splenic lymphocytes appeared to decline and at 1 20 ).lg/mL a lactalbumin significantly suppressed proliferation compared to the control (P < 0.0 1 ) (Figure 3 . 1 B).