Human milk is an optimum source of nutrients acting in a unique innate medicinal role for infants. The bactericidal activity of human milk is the combined action of various biocomponents, some of which have already been identified and the others remain to be determined (6). Major antimicrobial factors of human milk affecting specific and assorted interactions with pathogenic microorganisms include viable cells, secretory immunoglobulin (Ig)A, IgG, IgM, lactoferrin, lysozyme, complements, probiotic properties (eg, Bifidobacterium spp, Lactobacillus spp), gangliosides, cytokines, and lipid products (6).
The combined action of all of the bioactive components exerting bactericidal activity is quantified in the present study. We used 2 different species of bacteria. The strain of E coli, which is a common enteric pathogen in infants, was selected because it is commonly used (10,13). Also, we added a strain of P aeruginosa, which is a serious Gram-negative pathogen in infants, especially during the neonatal period. It is also noteworthy that P aeruginosa is an important nosocomial sepsis agent, especially for premature infants (14).
The E coli and P aeruginosa strains were inhibited in all of the fresh milk samples because growth of the bacteria was of a lower magnitude than in the control bacteria mixture. These findings are consistent with previous reports (10,13). Studies using P aeruginosa to analyze bactericidal activity of human milk are extremely rare in the literature (2,12). Dolan et al (2) showed inhibition of bacterial pathogens in human milk compared with infant formula in a broad range of bacteria, including P aeruginosa.
Numerous studies have been made to disclose the effect of storage condition on antimicrobial components of human milk showing different degrees of alteration in certain durations (11,15). These various antimicrobial components may be altered or irreversibly damaged by the processes such as refrigeration, freezing, thawing, and heating.
The influences of refrigeration on the bactericidal activity of human milk are well documented. It has been shown that the stability of bactericidal activity during refrigeration is protected for at least 48 hours (13,16). Although decreasing beyond 48 hours, bactericidal activity was detected up to 7 days with reduced levels, especially in colostrum milk (10). Therefore, the Human Milk Banking Association of North America recommends up to 8 days of refrigerator storage, considering the alterations of bioactive components, bacterial growth, and bactericidal effect of human milk (7,17). The advice of a certain storage time for human milk by refrigeration is controversial. Slutzah et al (18) recommended 4 days of storage by refrigeration at 4°C in NICU conditions.
Freezing is recommended if prolonged storage of human milk is required (approximately >1 week). Freezing has been carried out at −20°C, which is within the range of a home freezer, and at −70°C, which is within the range of a laboratory freezer (9,19,20). Breast milk can be stored at −20°C for up to 12 months (9,19). This recommendation is based on the knowledge that nutrient constituents remain stable at −20°C for up to 12 months; however, the information about bactericidal activity of human milk after long-term freezing is limited.
It has been shown that there are some effects of freezing on breast milk properties: it reduces the numbers and functions of bioactive cells (21) and increases lipolysis (11,22). In a study by Reynolds et al (23), 19% of cells in human milk remained viable, whereas the levels of IgA, IgM, IgG, lactoferrin, lysozyme, C3 and C4 complement components, and concentrations of amino acids and fatty acids were largely preserved or slightly changed after freezing at −20°C for 1 month. Akinbi et al (12) reported reduced concentrations of lysozyme, lactoferrin, lactoperoxidase, and secretory IgA at −20°C. Determination of bactericidal activity as a sum of these certain antimicrobial components in fresh milk and alterations by freezing at different temperatures and durations was the goal of the present study.
Bactericidal activity was stable for E coli at −20°C following freezing for 1 month; however, it declined after 3 months of storage in the present study. Ogundele (10) found decreasing bactericidal activity against E coli at −20°C at 28 days of storage. Hernandez et al (24) reported a significant loss of antimicrobial action of breast milk after 21 days of freezing, but the temperature was comparably high (−4°C). Silvestre et al (16) showed unaltered bactericidal activity of human milk frozen at −20°C for 7 days.
Milk can be stored safely for an unlimited time or >12 months at −70°C (6,9). Unchanged bactericidal activity of human milk at −70°C has been shown by Hernandez et al (24); however, human milk was freshly frozen at −70°C for 2 minutes. In the present study, bactericidal activity of human milk against E coli remained stable during storage at −80°C for 1 and 3 months. We suggest that the bioactivity of antimicrobial components is better protected by storage at −80°C. These data serve evidence for the consideration of storage safety at −80°C for a long period.
Freezing at −20°C and −80°C for 1 and 3 months did not cause any significant change in bactericidal activity against P aeruginosa compared with fresh samples in the present study. We do not have any explanation for why bactericidal activity for E coli was altered while bactericidal activity for P aeruginosa was not affected by freezing. We can say only that different antibacterial components probably act as bactericides for E coli and P aeruginosa and these components imply different sensitivities to freezing.
The effect of nursing stage on the bactericidal activity of milk was also examined and no significant differences were observed between fresh colostrum and transitional milk. The alterations in bactericidal activity of colostrum and transitional milk were similar during freezing. Ogundele (10) found that bactericidal activity of colostrum was more stable compared with transitional milk after refrigeration.
It is expected that the bactericidal properties of colostrum must be higher than those of transitional milk; however, in the present study we did not find any difference. It is probable that limited numbers of colostrum samples affected our results. Further studies are needed to clarify this issue.
In conclusion, human milk possesses a bactericidal function that remains stable during the first month of freezing storage; however, bactericidal activity is significantly reduced after 3 months of storage at −20°C. Bactericidal activity does not change significantly by freezing at −80°C for 3 months. Storage by freezing at −80°C is more appropriate to keep the bactericidal capacity of stored human milk >1 month if affordable and available, especially in NICU settings.
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Keywords:© 2012 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,
bactericidal activity; freezing of human milk; human milk; storage of human milk