Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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Storage of human breast-milk by freezing or refrigeration of milk with and without heating have been recommended (43). Storage of HBM, at least for a limited period can hardly be avoided because of the peculiar social circumstances of most mothers who may be working or schooling, as well as unavoidable peculiar needs some pre-term babies to be fed with expressed breast-milk. The question of keeping them safe for the babies´ consumption continues to gain such an importance as never before, not only in the industrialized nations, but also increasingly so in the urbanized developing communities. Significant changes occurring in the breast-milk during storage at temperatures at or above -20°C include lipolysis and release of free fatty acids (FFA). This leads to a drop of the pH and changes in the divalent ions solubility (28). This would explain the observed changes in pH during milk storage.

Bacteriological examination of refrigerated milk have proven their safety for human consumption even up to 72 hours (17-20,23,24). Interestingly, most of the studies demonstrated decrease in the bacterial count in the breast-milk during in vitro storage both at 4°C and -20°C. This suggests that some anti-microbial processes are activated during this period. In addition to the well documented lipolysis-induce cytolytic effects (31), the possibility of other complement-dependent mechanisms have been detected in the present studies. One possible explanation for this phenomenon would be the activation of the alternative pathway of complement, which is said to preferentially take place at 4°C (32).

Since similar bactericidal FFA are also released after the digestion of cow´s milk-based formula feed, the recognised differences between the occurrences of gastrointestinal infections between the breastfed and artificial fed infants tend to throw doubt on the possible physiological significance of the anti-microbial effect of these lipids (33). On the other hand, a deficient production of local complement components has been associated with an increased risk of developing mastitis in lactating mothers (34). Skin sepsis in the suckling infant, as well as inflammation of the lactating breast (mastitis), are associated with an increased level of complement secretion in the breast-milk (5). Though these are no proofs of cause-effect relationships, they point to the possible physiological role of the complement system on the mucosa surfaces.

The complement content of the HBM has been previously shown to induce a respiratory burst (as evidenced by increased chemo-illuminiscence) in PMN leucocytes phagocytizing adherent bacteria, but not primarily responsible for its opsonizing properties (7). It has also been shown to be bacteriocidal for Salmonella (2). The bacteriocidal effect of bovine milk colostrum and specific antibodies have been suggested to account for the favourable influence of breastfeeding on gut colonization (27) It is also possible that the physiological significance of breast-milk complement system, might contribute to the generally observed trend of reduced total faecal bacterial load in the breastfed infant, particularly within the first week of life (35).

Immunochemical and haemolytic levels of C system in the HBM have been fairly well studied by several authors. Using radial immunodiffusion assays, levels of C3 and C4 comparable to those in the normal serum have been found in the early colostrum (11,12). These levels fall rapidly after a few days of lactation, and then more slowly in transitional milk to a stable level in mature breast-milk, C3 averaging 6-12% that of the normal human serum in most reported studies. By using high degree of concentration, C1q was later detected in the human colostrum (36). The level of factor B that has been measured immuno-chemically and other evidence for a complete APC have been documented (12,13). The classical studies by Nakajima and other showed the presence of haemolytic activity of all the nine components of the classical complement components in the colostrum, ranging from 0.03 to 7% that of the normal human serum (13).

The lipid content of HBM consists mainly of triglycerides enveloped in complete tri-laminar units of biological cell membranes, the milk fat globule membrane (MFGM) ranging from 4 to 20µm in diameter. This membrane is derived from the apical region of the mammary gland epithelial cells and are budded off around the milk lipids as they are being secreted by the cells (37). Electron microscopic studies of the outer layer of the human MFGM has shown the presence of numerous thin filaments radially oriented around the membrane into the aqueous phase of milk, many of which appear branched. They are said to be absent from the MFGM of other mammals (38). However, their functions and physiological significance have not been clearly identified. It has been suggested that the filaments may protect the MFGM against degradation by milk proteases and in the digestive tract, and act to prevent the fusion of the globules to each other. Other proposed functions of the MFGM include transportation of milk lipid along the upper intestinal tract towards the site of enzymatic digestion in the stomach and small intestine, to prevent adhesion to the upper intestinal wall. They appear to be involved in activating the breast-milk neutrophils, leading to increased surface expression of CD11b, by mechanisms partly involving their phagocytosis (39). The bacteria-adhesive role of high molecular weight mucus components of MFGM have been implicated in preventing the colonization and infection of epithelial mucosa by certain meningitis-causing bacterial strains in neonates (40). The present studies provide evidence that this property of adhesion is even enhanced during in-vitro storage, by mechanisms involving activated complement factors. The potential ability of rapid freeze-thawing to disrupt MFGM (30), would suggest that refrigeration of milk would be preferable for short-term storage. For a storage over longer periods even up to one month, freezing could still be recommended, since up to two-third of bactericidal activities are still preserved up to this period (Fig 3). A number of protective breast-milk components may be altered or irreversibly damaged by such processes as freeze-thawing or heating of the milk. The possibility of accumulation of undesirable bacterial products such as enterotoxins, bacterial enzymes and reactive amines has been stated to be very unlikely at refrigerator temperatures, since most bacteria are metabolically inactivated at this temperature and the other antibacterial systems in the milk act to prevent any significant bacterial growth and activity (19). The expressed fears arising from increased titrable acidity of such stored milk samples, which were suspected to be due to formation of lactic acid by bacterial fermentation of milk sugars, is no more tenable, since it has been shown to be mainly attributable to levels of FFA (22).

It was interesting to observe that the loss in mainly complement-mediated bactericidal activities in refrigerated milk is more than well compensated for by its enhanced bacteria sequestration ability. This would suggest that while inflammatory processes are diminishing, non-inflammatory protective mechanisms are gaining greater importance in stored HBM. This agrees with the proposed anti-inflammatory properties of HBM (41).

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