K.M. Shahani

The Effect of Processing and Storage on Key Enzymes, B Vitamins, and Lipids of Mature Human Milk I. Evaluation of Fresh Samples and Effects of Freezing and Frozen Storage

Summary: A study was initiated to evaluate the effects of several methods of processing and storage on key enzymes, B vitamins, and lipid components of mature human milk. In order to establish standard values for the nutrient components with which to compare processed samples, a total of 30 individual raw samples of mature human milk were analyzed. There was considerable sample to sample variation as indicated by the large range of values for each component Freezing and frozen storage had little effect on the enzymes of pooled samples of milk. Lactoperoxidase activity decreased from 36 in raw pooled samples to 17 in pooled samples slow frozen and stored for 3 months at −25°C (P < 0.05). Similarly, quick freezing and storage for 3 months significantly decreased the lactoperoxidase activity of pooled samples from 93 to 14 (P < 0.05). Quick freezing and frozen storage tended to increase lipase activity although the changes were not significant. Freezing and frozen storage did not significantly affect the levels of biotin, niacin, and folic acid. Similarly, the total lipid fatty acid level and relative % of each fatty acid were not significantly different in the frozen samples as compared to the raw samples.Speculation: Our data suggest that freezing and frozen storage for up to 3 months can be used to preserve mature human milk with minimum loss of its biologic activity. Slow freezing is more preferable because this method requires less effort and equipment but affords the same storage stability as quick freezing.

Human milk ribonuclease.

Two components having ribonuclease (EC 3.1.27.5) activity were isolated from human milk. Each component of human milk ribonuclease (RNAase) moved at a slightly different rate when electrophoresed on polyacrylamide gel but at the same rate when ultracentrifuged. The major component had a molecular weight of approx. 14 000, an isoelectric point of pH 7.9, and exhibited a broad absorbance maximum between 277 and 281 nm. Human milk RNAase hydrolyzed yeast RNA, poly(cytidylic acid) and poly(uridylic acid) but not DNA, poly(adenylic acid) or poly(guanylic acid). Maximum activity occurred at pH 7.7 and 60 degrees C. Amino acid analysis of the major component revealed a large number of alanine, valine, glycine and aspartic acids but no tryptophan or free sulfhydryl groups. Lysine was the N-terminal amino acid. Tryptic hydrolysis yielded 18 peptides, some of which are similar to those from bovine pancreatic RNAase. Human milk RNAase activity was increased in the presence of NaCl, KCl and sodium citrate and decreased by CaCl(2), MgCl(2), FeSO(4), ZnSO(4) and CuSO(4).

Reduction and reactivation of human and bovine milk lysozymes.

Abstract Reduction of the disulfide bonds in human milk lysozyme (HML) and bovine milk lysozyme (BML) with 2-mercaptoethanol destroyed their lytic activity. Titration with p -mercuribenzoate (PMB) revealed six free SH groups in the reduced lysozymes. Enzymic activity was rapidly regenerated during reoxidation at 37 °C in Tris-chloride buffer (pH 8.5). Regeneration of HML yielded a maximum of 84% of its native activity while regeneration of BML yielded 328% activity. Addition of 2-mercaptoethanol to the reoxidation medium decreased both the rate and extent of BML reactivation. Reoxidized HML and BML possess no free SH groups. The data indicate that reoxidized HML has the same structure and specific activity of the native enzyme. BML, when compared to the native enzyme, was found to have same electrophoretic mobility but differed markedly in its spectral properties and specific activity. Possible explanations for the altered properties of BML are discussed.