Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

PHYSICOCHEMICAL CHANGES IN BREASTMILK DURING IN VITRO STORAGE AT VARIOUS TEMPERATURES

The normal pH of the colostrum averaged 7.60. The transitional and mature milk samples were slightly more acidic, averaging 7.44 and 7.29 respectively. Partial or total de-fattening of fresh milk led to a slight increase in the pH. PBS caused no significant change in the pH of the breast-milk samples (Table 1). The pH of the breast-milk progressively fell during storage both at -20°C and +4°C (Table 2).

Table 2  pH changes during storage of different human breast-milk samples at 4°C. Values are mean ± SD.  

No. of storage 
 days at 4°C               0             3             7            14            21            28
Colostrum             7.60 ± 0.44   7.35 ± 0.40   7.05 ± 0.35   6.80 ± 0.52   6.55 ± 0.60   6.40 ± 0.50
Transitional milk     7.44 ± 0.50   7.10 ± 0.32   6.95 ± 0.25   6.75 ± 0.42   6.50 ± 0.35   6.35 ± 0.52
Mature milk           7.29 ± 0.40   7.25 ± 0.35   6.95 ± 0.24   6.61 ± 0.20   6.28 ± 0.42   6.37 ± 0.32

EFFECT OF EDTA ON PH CHANGES IN HUMAN BREAST-MILK

EDTA was used to prevent ongoing activation of the complement system reaction cascade in the HBM samples. Addition of EDTA to HBM samples led to an immediate significant fall in the pH, compared to when a comparable volume of PBS buffer is added This drop in the pH was partly reversed by de-fattening the breast-milk through centrifugation (Table 1). It is proposed that EDTA, being a detergent, destroyed the milk fat globule membranes and led to a release of free fat into the aqueous phase of the milk.

Table 1:  Effect of added PBS and EDTA on the pH of different human breast-milk samples. 
Values are mean ± SD.
                 Whole-milk alone   Whole-milk + PBS    After centrifugation    Whole-milk + EDTA   After centrifugation
Colostrum          7.60 ± 0.44        7.63 ± 0.54           7.70 ± 0.65            6.80 ± 0.35          7.00 ± 0.74
Transitional milk  7.44 ± 0.50        7.46 ± 0.45           7.66 ± 0.45            6.93 ± 0.23          7.12 ± 0.61
Mature milk        7.29 ± 0.40        7.30 ± 0.53           7.40 ± 0.49            6.97 ± 0.36          7.05 ± 0.42

INCREASED SEQUESTRATION OF SUSPENDED BACTERIA BY REFRIGERATED HUMAN BREAST-MILK

The ability of whole-milk samples stored by refrigeration at 4°C in the absence of anticoagulants, to adhere to suspended bacteria under in-vitro conditions which favoured C activation, was compared to those frozen at -20°C or -70°C. It was observed that the refrigerated samples sequestered significantly more viable bacteria within the first two weeks of storage than fresh milk samples; followed by a progressive fall, gradually approaching the level cultured from the normal saline control .

Figure 1 Effects of storage temperature on the sequestration of bacteria by human colostrum samples.

This enhancement of bacteria sequestration could be attributed to the presence of accumulation of activated C fragments in the stored milk, since they were not obtained in similarly stored milk samples containing EDTA. The other possible explanation for this observation might be the effect of free fatty acids (FFA) released by ongoing lipolysis (28). This however seems unlikely since their levels which are expectedly higher in milk samples frozen at -20°C than at -70°C, did not result in a corresponding enhancement of sequestration at this temperature.

Previous studies have confirmed the accumulation of C split products, resulting from ongoing C activation, in biological samples stored at temperatures below -70°C (29). The accumulation of these split products in HBM stored by either refrigeration or freezing in the absence of EDTA have also been recently documented. The levels are generally higher in refrigerated than in frozen milk (Unpublished data). The rapidly declining degree of bacteriolyis during the period of refrigeration, within the first 48 hours in mature milk and within one week in colostrum, further attest to ongoing loss of native C components through non-specific activation .

Figure 2 Comparison of the effects of refrigeration on the bactericidal and sequestration activities of human colostrum samples.

The subsequent fall in sequestrating abilities of refrigerated milk samples observation might reflect the ultimate loss of intact MFGM due to ongoing in-vitro lipolysis (29), as well as the ultimate degradation of the C-activation products. This suggests the necessity of an intact MFGM, independent of the presence of activated C fragments, to effect bacterial sequestration by milk samples.

EFFECT OF FREEZING AND THAWING ON THE SEQUESTRATION OF SUSPENDED BACTERIA BY MILK FAT

Freeze-thawing of whole milk by repeated freezing at -20°C and subsequent thawing are known to be destructive to biological membranes (30). Frozen whole-milk at -20°C showed a fairly constant level of bacterial sequestrating capacities, slightly lower than those of fresh milk and those stored at -70°C, despite the presence of accumulating C split products .

Figure 1 Effects of storage temperature on the sequestration of bacteria by human colostrum samples.

This contrasts with the enhanced sequestration of suspended bacteria by refrigerated milk samples. This failure of enhanced sequestration at -20°C might suggest that freezing either alters the surface of the MFGM, so that they are unable to bind to the limited level of C activation products in the milk samples, or the freeze-thawing procedures rapidly lead to their destruction.

EFFECT OF IN-VITRO STORAGE ON THE BACTERICIDAL ACTIVITY OF HUMAN BREAST-MILK

The bactericidal activity of the colostrum against E. coli decreased during in-vitro storage at both 4°C and freezing at -20°C. The loss of bactericidal activity was however more rapid in the refrigerated samples compared to the frozen samples .

Figure 3 Effect of storage at different temperatures on the bactericidal activity of human colostrum samples.

While the bactericidal activity in most refrigerated mature or transitional milk was lost rapidly within 48 hours of storage (results not shown), the activity in colostrum fell more slowly, up to half of which was still measurable until after 7 days. The observed bactericidal activities of various human breast-milk (HBM) samples tested were found to be mainly mediated by their complement system components. Most of the activities were abolished by heating at 56°C for 30 minutes, or by addition of EDTA. Centrifugation of the milk samples also led to some loss of bactericidal activities, suggesting the involvement of the milk fat globule membrane (MFGM) in the activation of the milk C .

Figure 4 Comparison of the effects of centrifugation and complement-inactivation by heating or EDTA on the bactericidal activities of different human breast-milk samples against E coli 0111.

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