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Effects of Refrigeration on the Bactericidal Activity of Human Milk: A Preliminary Study
Abstract
This study analyzed the bactericidal activity of human milk and how it is influenced by refrigerated storage. Nine samples of mature human milk were collected and divided into 3 aliquots. One was analyzed immediately, and the other 2 were refrigerated at 4 degrees C to 6 degrees C for 48 and 72 hours, respectively. All of the fresh samples exhibited bactericidal activity with an average value of 83.47% +/- 18.37%. Refrigeration for 48 hours did not cause significant modifications, whereas storage beyond 72 hours significantly lowered the degree of bacteriolysis versus fresh milk. In conclusion, human milk possesses bactericidal activity that remains stable during the first 48 hours of refrigerated storage, but it is significantly reduced beyond 72 hours.
... 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. ...
... 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). ...
... 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). ...
... 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. ...
... 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). ...
... 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). ...
Storage of human milk by freezing has been recommended for long-term storage. The present study analyzed the bactericidal activity of human milk on Escherichia coli and Pseudomonas aeruginosa and determined the changes in bactericidal activity following freezing at -20°C and -80°C for 1 month and 3 months.
Forty-eight milk samples were collected from 48 lactating mothers. Each sample was divided into 10 aliquots. Two of the samples were processed immediately and the others were stored at both -20°C and -80°C until analysis after 1 month and 3 months of freezing.
All of the fresh milk samples showed bactericidal activity against E coli and P aeruginosa. Freezing at -20°C for 1 month did not cause statistically significant alteration in bactericidal activity (P > 0.017), whereas storage for 3 months lowered the degree of bactericidal activity significantly (P < 0.017) against E coli. Bactericidal activity was protected when the samples were stored at -80°C. There was no statistically significant difference in the bactericidal activity of human milk against E coli between freezing at -20°C and -80°C for 1 month (P > 0.017); however, when milk was stored for 3 months, -80°C was significantly more protective (P < 0.017). Freezing at -20°C and -80°C for 1 month and 3 months did not cause any significant change in bactericidal activity against P aeruginosa (P > 0.05).
Storage by freezing at -80°C is more appropriate to keep bactericidal capacity of stored human milk >1 month if affordable and available, especially in intensive care settings.
... Others stored by immersing the container of breast milk in cold water (37.8%) or at room temperature (30 o C) (10.2%). Studies [13,14] have shown that the best method of storing expressed breast milk is in a refrigerator (at 4 o C) especially in hot climates. However, in a developing country like Nigeria, refrigerators are not usual household equipment in many homes. ...
The use of expressed breast milk has been advocated as an effective way of encouraging and maintaining lactation when the mother is separated from the baby for a while. However, prospects of storage of expressed breast milk for any considerable period of time is hindered by the possibility of bacterial contamination and growth of infectious pathogens in the stored milk. Previous research worldwide has focused on optimal conditions for storing expressed breast milk. This study investigated bacterial growth in expressed breast milk stored at two different conditions and time periods. A total of 240 randomly selected lactating mothers participated in a survey used to elicit information on their practices concerning expressed and stored breast milk. Out of this, 20 lactating mothers were willing to donate their breast milk , which was used for microbial studies. Using antiseptic procedures, breast milk samples were collected from these 20 lactating mothers by manual expression and put into sterile containers. One half was stored at room temperature (30 o C), while the other half was immersed in a container of water (26 o C) for 0, 3, 6 and 9hrs. The samples at different time periods in both storage conditions were plated on three different culture media (Nutrient agar , MacConkey agar and Chocolate agar) and incubated for 24hrs. Bacterial load was enumerated and isolates were characterized and identified. Data was analyzed using one way ANOVA and presented as frequencies, percentages means and standard deviations. Results showed that in all the culture media, bacteria l load was higher in breast milk samples stored at room temperature for up to 9hrs compared to that immersed in water (p<0 .05). However, the average colony counts were within acceptable limits (<10 4 CFU/ml). Enterococcus faecalis , Escherichia coli and Staphylococcus aureus were the predominant bacteria isolated in the breast milk samples under both storage conditions. The findings from this study revealed that storage of breast milk either at room temperature or immersed in a container of water appeared to be safe for up to 9 hours of storage in a tropical environment . Generally, the number of microbes was within levels considered acceptable in expressed breast milk .
... The findings achieve in the study showed that after 2 Study has shown that the best method of storing expressed breast milk is in refrigerator at 4 o C temperature, especially in hot climate (Marin et al 2009, Martinez-Costa et al 2007. Other studies (Tully 2000, Zinn 2000) have been carried out in optimum conditions of storage noted that EBM could last for up to 3-8 days in the refrigerator. ...
Storing EBM at room temperature in several hours before consuming, frequently found in Indonesia. Based on Academy of Breastfeeding Medicine guidelines EBM can last for 6 to 8 hours in room temperature (25oC or 77oF). However, currently there hasn’t been study in tropical country especially Indonesia for the guidelines. This study aimed to assess microbiological quality of EBM on room temperature, including bacterial growth and major bacterial found on EBM for health care and society recommendations. An observational study of 30 expressed breast milk samples provided by 30 healthy women with term baby below 6 month old. The samples were kept sterile and laid at plates for 0 hours, 2 hours, 4 hours and 6 hours in room temperature (26°-32° C) and used drop plate technique on several culture media. Data was analyzed by Chi-square and paired sample T-test. Thirty of unheated fresh EBM from 30 lactating mothers were stored at room temperature, examined for the degree of bacterial contamination at 0 hour, 2 hours, 4 hours, and 6 hours. All the EBM samples were contaminated at 2 hour. Bacterial species identified was Coagulase-negative Staphylococcus (CNS), Escherichia coli, Klebsiella pneumoniae and Streptococcus faecalis, range of growth 109 cfu/ml-63 x 109 cfu/mm3 after 6 hour of storage. The EBM exposed at room temperature (30-36 0C) for more than two hour reduce the quality and do not recommended to be given to the infants.
... Study has shown that the best method of storing expressed breast milk is in refrigerator at 4 0C temperature, especially in hot climate (Martınez-Costa et al 2007, Marın et al 2009. Other studies (Zinn 2000, Tully 2000) have been carried out in optimum conditions of storage noted that EBM could last for up to 3-8 days in the refrigerator. ...
Expressed breast milk (EBM) has been advocated as an effective way for encouraging and maintaining lactation when the mother is separated from the baby for a while. Storing EBM at room temperature in several hours before consuming, frequently found in Indonesia. Based on WHO guidelines EBM can last for 6 to 8 hours in room temperature. But currently there hasn’t been study in tropical country especially Indonesia for the guidelines. Assesing microbiological quality of EBM on room temperature, including bacterial growth and major bacterial found on EBM for health care and society recommendations. An observational study of 30 expressed breast milk samples provided by 30 healthy women with term baby below 6 month old. EBM were collected by electric breast pump swing model. The samples were kept sterile and laid at plates for 2 hours, 4 hours and 6 hours in room temperature (26o- 32oC) and used drop plate technique on several culture media. Data was analyzed by Chi-square and paired sample T-test. Thirty of unheated fresh EBM from 30 lactating mothers were stored at room temperature, examined for the degree of bacterial contamination at 0 hour, 2 hours, 4 hours, and 6 hours. All the EBM samples were contaminated at 2 hour. There were strong correlation between 2 hours storing with 4 hours storing (p=0.004) and total colony (p=0.000). There were also significant difference between bacterial colony and the duration itself (p=0.026). Bacterial species identified was Coagulase-negative Staphylococcus (CNS), Escherichia coli, Klebsiella pneumoniae and Streptococcus faecalis, range of growth 109 cfu/ml - 63 x 109 cfu/mm3 after 6 hour of storage. Evaluation result that the EBM exposed at room temperature (30-36 0C) for more than two hour reduce the quality and do not recommended to be given to the infants because bacterial counts > 104 cfu/ml and the present of pathogens E. coli, Streptococcus faecalis and Klebsiella pneumoniae.
... In such cases, stricter requirements were used in comparison to milk for home use. In addition, almost all of these studies were focused on the microbiology Knoop, Schutt-Gerowitt, & Matheis, 1985;Sosa & Barness, 1987), bactericidal activity (Martinez-Costa et al., 2007;Ogundele, 2002;Silvestre, López, March, Plaza, & Martínez-Costa, 2006), macronutrients (Bertino et al., 2013;Kaylegian, Lynch, Fleming, & Barbano, 2007), and the high abundance proteins. (Bertino et al., 2013;Kaylegian et al., 2007). ...
... The findings achieve in the study showed that after 2 Study has shown that the best method of storing expressed breast milk is in refrigerator at 4 o C temperature, especially in hot climate (Marin et al 2009, Martinez-Costa et al 2007. Other studies (Tully 2000, Zinn 2000) have been carried out in optimum conditions of storage noted that EBM could last for up to 3-8 days in the refrigerator. ...
Storing EBM at room temperature in several hours before consuming, frequently found in Indonesia. Based on Academy of Breastfeeding Medicine guidelines EBM can last for 6 to 8 hours in room temperature (25oC or 77oF). However, currently there hasn’t been study in tropical country especially Indonesia for the guidelines. This study aimed to assess microbiological quality of EBM on room temperature, including bacterial growth and major bacterial found on EBM for health care and society recommendations. An observational study of 30 expressed breast milk samples provided by 30 healthy women with term baby below 6 month old. The samples were kept sterile and laid at plates for 0 hours, 2 hours, 4 hours and 6 hours in room temperature (26°-32° C) and used drop plate technique on several culture media. Data was analyzed by Chi-square and paired sample T-test. Thirty of unheated fresh EBM from 30 lactating mothers were stored at room temperature, examined for the degree of bacterial contamination at 0 hour, 2 hours, 4 hours, and 6 hours. All the EBM samples were contaminated at 2 hour. Bacterial species identified was Coagulase-negative Staphylococcus (CNS), Escherichia coli, Klebsiella pneumoniae and Streptococcus faecalis, range of growth 109 cfu/ml-63 x 109 cfu/mm3 after 6 hour of storage. The EBM exposed at room temperature (30-36oC) for more than two hour reduce the quality and do not recommended to be given to the infants.
... The evaluation of antibacterial effect of breast milk has been investigated by past workers [10][11][12] which clearly shows its useful potential against bacterial infections. However, not much work has been focussed into studying the effect of mother's lactation age and storage of expressed milk on the antibacterial effect against the common neonatal pathogens. ...
The present study evaluates the effect of mothers lactation age and storage on the
antibacterial potency of breast milk against common pathogens (E.coli, S.aureus and coagulase
negative staphylococci) affecting newborns and toddlers. Breast milk expressed at four months
and twelve months post-partum was subjected to antibacterial assay by agar well diffusion
against the above bacterial isolates in comparison to cow milk. Also, breast milk stored at 4°C
was evaluated for its antibacterial effect to study the effect of storage. Results indicated that
human breast milk was far more superior in its anti-bacterial effect seen (in terms of zone
diameter) than cow milk against all the five strains tested. Also, early post partum milk (four
months post-partum) was significantly superior (in terms of zone size) than twelve months post
partum milk. Storage studies showed that breast milk retained its activity upto 24 hours of
storage at 4°C against the five test strains.The present study indicates that even late lactation
milk has enough anti-bacterial properties to protect babies beyond one year and toddlers as well
thus promoting the concept of continued breast feeding for one year and beyond. Further, study
highlighted that lactating mother can pump the milk and refrigerate it for later consumption
without compromising on the antibacterial potency of their milk for upto 24 hours.
... For instance, the constituents of pumped breast milk are different from directly acquired breast milk [30,48]. Decreases in lutein, vitamins C, B6, folic acid, lipase, immune cells, and fat have been reported [29,30,[49][50][51][52], as well as the growth of foreign pathogens [53,54]. In addition, freezing and re-heating of pumped milk may result in a breakdown of milk constituents [29]. ...
Background:
Modes of infant feeding such as direct and indirect breastfeeding, and formula feeding, and their combinations may play a role in child health.
Objective:
The aim was to investigate which feeding patterns in the first 6 months pose risks of eczema/skin allergy in children up to 6 years compared to direct breastfeeding for at least 3 months.
Methods:
The Infant Feeding Practices Study II in the United States and its 6-year follow-up provided data on feeding modes in infancy and doctor's diagnosed eczema/skin allergy in the first 6 years of life (1387 infants), based on parental reports. Different feeding patterns were identified. Log-linear models were used to estimate prevalence ratios (PRs) of feeding patterns for doctor's diagnosed eczema/skin allergy in the first 6 years of life, adjusting for confounders.
Results:
Compared to "direct breastfeeding for at least 3 months" (DBF3m), the combination of "direct feeding at the breast (DBF), pumping and feeding breast milk (BM), and formula (FF) in the first months" (DBF/BM/FF) showed a statistically significant higher risk of eczema/skin allergy in the first 6 years of life (PR = 1.46), adjusting for confounders. DBF combined with BM for the first 3 months followed by mixed feeding also had an increased risk (PR = 1.26), although not statistically significant. Formula feeding introduced since birth had no effect on eczema. Among the confounders, paternal eczema and race/ethnicity (Hispanic vs White) were associated with a higher risk of eczema/skin allergy.
Conclusions & clinical relevance:
Mixed infant feeding may carry a higher risk of eczema/skin allergy compared to direct feeding at the breast. The recent epidemic of pumping and feeding in the United States and the use of mixed infant feeding modes requires additional studies to provide appropriate and renewed assessments of the risks of feeding modes for the future development of allergies.
... No estudo realizado por Martínez-Costa et al., 13 foi demonstrada atividade bactericida média de 83,47% em amostras de leite materno, média muito similar à obtida neste estudo. Eles também não observaram alterações significativas em razão da refrigeração das amostras. ...
O leite materno é o melhor alimento para os bebês, provendo nutrição, proteção imunológica e aumentando o laço afetivo entre a mãe e o bebê. O trabalho propôs avaliar a capacidade bactericida do leite materno para Escherichia coli. Poucos estudos avaliaram aspectos relativos ao armazenamento do leite em refrigerador e congelador, considerando a possível interferência na capacidade microbicida, por isso este estudo tornou-se relevante. O leite materno foi coletado de doadoras com idade entre 18 e 35, foram coletadas amostras de cerca de 12 mL de lactantes em amamentação exclusiva. As amostras foram centrifugadas por 10 minutos para separação da camada lipídica. Constatou-se que as médias de morte de bactérias foram de 80,3% para leite refrigerado sem gordura, 70% para leite congelado sem gordura e 67,3% tanto para leite refrigerado e congelado com gordura. Os resultados indicaram que, mesmo armazenado na geladeira, o leite é capaz de matar bactérias, reforçando a importância da prática do aleitamento materno como meio de redução de infecções em lactentes.
... In fact, global acceptance of human milk by neonates is directly correlated with its aroma, a parameter influenced by odorous volatile compounds present in this biological matrix. In this respect, several studies have shown that newborns are very sensitive to olfactory changes, being able to recognise specific odours with high reliability (Martínez-Costa et al., 2007;Terry, 2004). Table 1 shows the volatile compounds (represented by arbitrary area units AAU Â 10 3 ), grouped by chemical families, detected in the headspace of human milk samples subjected to different preservation treatments (HoP and HPP). ...
... The loss of antibacterial effect of human mature milk after cold storage has no bearing on the actual bacterial growth in the stored milk. However, the current results are contradictory to the findings of Martinez-Costa et al. [37], that refrigeration at 4-6°C for 48 h had no significant effect on the bactericidal activities of nine mature milk samples on another strain of E. coli (O111). Hence, the results suggest that future studies should measure the bacterial loading in milk specimens, whereas the bacterial killing activity against individual milk components should be tested with a wider range of pathogenic bacteria for infants. ...
The nutritional and immunological qualities of human milk from Hong Kong mothers were profiled.
A total of 25 colostrum (≤ 3 days postpartum) and 11 mature (30-45 days postpartum) milk specimens were collected from healthy Chinese women in Hong Kong. Parameters including total protein, whey, casein, triglycerides, lactose, lysozyme, secretory immunoglobulin A, lactoferrin and antibacterial activity of human milk were quantified.
Breast milk of Hong Kong Chinese mothers is nutritionally comparable to that of western mothers for colostrum and mature milk, with protein (25.0±11.3 g/l vs 10.1±1.4 g/l) and lactose (44.2±7.5 g/l vs 66.6±5.5 g/l) for growth and energy supply in infants, respectively. The milk of Hong Kong mothers is however characterized by its exceptionally high levels of sIgA (806.3±792.6 mg/dl and 1545.9±334.6 mg/dl) and low levels of triglycerides (10.8±9.3 mmol/l and 11.1±7.7 mmol/l). The human milk in Hong Kong was also shown to be superior to bovine formula milk, particularly in terms of lower total protein level, the high whey to casein protein ratio, rich immunological active contents, and significant antibacterial activity against 2 common Escherichia coli strains.
Preliminary data also indicated that mature milk could be refrigerated for at least 3 days in order to maintain the major nutritional constituents and antibacterial activity.
... Human milk loses its bactericidal capacity when refrigerated for .72 hours. [34][35][36] Temperature and time can be carefully monitored using the refrigerator logger with a probe placed in a bottle of water to indicate the core temperature. The environmental temperature in the inside of the refrigerator or shown on the display is indicative only of the core temperature. ...
With the increasing use of human milk and growing evidence of the benefits of mother's milk for preterm and ill newborns, guidelines to ensure its quality and safety are an important part of daily practice in neonatal intensive care units. Operating procedures based on hazard analysis and critical control points can standardize the handling of mother's expressed milk, thereby improving nutrition and minimizing the risk of breast milk-induced infection in susceptible newborns. Because breast milk is not sterile, microorganisms can multiply when the milk is not handled properly. Additional exogenous contamination should be prevented. Strict hygiene and careful temperature and time control are important during the expression, collection, transport, storage, and feeding of maternal milk. In contrast to formula milk, no legal standards exist for the use of expressed maternal milk. The need for additional measures, such as bacteriological screening or heat treatment, remains unresolved.
... In fact, different factors concur to determine such an uncertainty, among them the shortage of studies and different biochemical 5,6,8,17 and microbiological 2,17 criteria used to fix a cut off point beyond which stored human milk is no longer good/safe. Arbitrariness mainly derives from different attitudes of authorities and health workers in defining an expiration date that, at the current state of the art, continues to be the result of a compromise; we do not want babies consume human milk judged as biologically inadequate, while still promoting breastfeeding and therefore mother-infant health. ...
... This result is not strange because skim milk has been widely used as a bacterial cryopreservative. Some studies have shown that refrigeration up to 48 hours and freezing and cold storage of human milk do not eliminate its antimicrobial properties (19,25,26), whereas, in contrast, the antimicrobial activity exhibited by fresh human milk is lost after the standard heat treatment (27). The fact that vegetative bacterial cells remain viable after cold storage but are destroyed after pasteurization may explain, at least partly, such differential effect on the antimicrobial properties of breast milk. ...
In the last few years, it has been proved that human milk contains bacteria that constitute an important factor in the initiation and development of the neonatal gut microbiota. In this context, the objective of this study was to evaluate the effect of cold storage on the natural bacterial composition of breast milk.
Breast milk samples provided by 34 healthy women and collected either by manual expression (n = 27) or breast pump (n = 7), were plated onto several culture media immediately after arrival at the laboratory (day 0) and after storage at -20 degrees C for 6 weeks. A high number of isolates from 8 of the women were identified at the species level.
No statistically significant differences were observed between the counts obtained at both sampling times in those media in which growth was detected. In all of the culture media, bacterial counts in pump-collected samples were higher than in those obtained by manual expression. Staphylococci and streptococci were the predominant bacteria in both fresh and frozen samples, Staphylococcus epidermidis being the most abundant species at both sampling times. Lactic acid bacteria and bifidobacteria were also present in fresh and frozen breast milk samples, but among them, only 1 species (Lactobacillus gasseri) could be isolated at both sampling times.
The results of this study suggest that cold storage of milk at -20 degrees C for 6 weeks does not significantly affect either the quantitative or the qualitative bacterial composition of breast milk.
... It is reported that expressed human milk can be stored up to 4 – 8 h at 258C or 2 – 5 days at 48C [3 – 5]. The bactericidal activity of human milk has been reported to decrease only beyond 72 h of refrigerated storage [6] and bacterial growth was not found significantly different after 4 days at 48C [7], presumably due to the high lysozyme, lactoferrin, and other bacteriostatic agents in human milk [8]. Some authors report that for short-term storage, breast milk may also be stored safely at 158C for 24 h [9 – 11]. ...
Information about lipid oxidation in fresh and stored human milk compared with infant formulas is scarce. We aimed to assess n-6 and n-3 PUFA oxidation in these milks by measuring the 4-hydroxynonenal (4-HNE) and 4-hydroxyhexenal (4-HHE) content. Human milk samples (n = 4), obtained from volunteer mothers, were analyzed fresh and after 1 wk at 4 degrees C or 24 h at 18 degrees C. Vitamin E and malondialdehyde (MDA) were measured by HPLC and fatty acid profile by GC. The 4-HHE and 4-HNE contents were measured by GC-MS. Infant formulas (n = 10) were tested; their fat droplet size was measured by laser light scattering and observed by confocal laser scanning microscopy. Human milk samples contained 31.0 +/- 6.3 g/L of lipids and 1.14 +/- 0.26 mg/L of vitamin E. Fat droplets were smaller in infant formulas than reported in human milk. The (4-HHE/n-3 PUFA) ratio was 0.19 +/- 0.01 microg/g in fresh human milk (unchanged after storage) versus 3.6 +/- 3.1 microg/g in dissolved powder formulas and 4.3 +/- 3.8 microg/g in liquid formula. (4-HNE/n-6 PUFA) was 0.004 +/- 0.000 microg/g in fresh milk (0.03 +/- 0.01 microg/g after storage) versus 1.1 +/- 1.0 microg/g in dissolved powder formulas and 0.2 +/- 0.3 microg/g in liquid formula. Infant formulas also contained more MDA than human milk. n-3 PUFA were more prone to oxidation than n-6 PUFA. Whether threshold levels of 4-HHE and 4-HNE would be of health concern should be elucidated.
Background
Neonatal infections with Cronobacter sakazakii have recently been associated with the consumption of expressed human milk.
Study Aims
(1) To evaluate whether human milk has antimicrobial capacity against C. sakazakii and (2) to determine the stability of its capacity when it is subjected to various treatments.
Methods
The antimicrobial capacity of human milk against C. sakazakii was evaluated using an observational, cross-sectional, comparative design. Mature human milk samples ( N = 29) were subjected to different treatments. After incubation at 37°C for 72 hr, samples were compared with fresh milk on the stability of their antimicrobial capacity. Two-way analysis of variance (ANOVA) was performed.
Results
In fresh milk, counts of C. sakazakii were reduced by 47.26% ( SD = 6.74) compared to controls. In treated milk, reductions were: refrigeration at 4°C for 72 hr ( M = 33.84, SD = 13.84), freezing at –20°C for 1, 2, and 3 months ( M = 40.31, SD = 9.10; M = 35.96, SD = 9.39; M = 26.20, SD = 13.55, respectively), Holder pasteurization ( M = 23.56, SD = 15.61), and human milk bank treatment with ( M = 14.37, SD = 18.02) and without bovine fortifier ( M = 3.70, SD = 23.83). There were significant differences ( p < .05) between fresh and treated milk.
Conclusions
Human milk has antimicrobial capacity against C. sakazakii. However, its capacity is negatively influenced by common preservation and hygienization methods. Milk should be stored refrigerated for a maximum of 72 hr or frozen for a short period of time.
This review evaluated the effect of various storage and handling conditions on the fat composition of expressed breast milk (EBM). Three databases PubMed, Embase and Scopus were searched in April 2019 with words from the three key components: human milk, handling process (i.e. storage and/or pasteurization), and fatty acid composition. The comparisons were EBM subjected to handling processes versus fresh EBM or versus EBM subjected to another handling processes. Both intervention and observational studies were included, and the outcomes measured included total fat and lipid classes of the EBM. We included 42 studies (43 reports), 41 of which were assessed to be of good quality. Relative changes to the fat composition of EBM subjected to handling processes were calculated based on the data provided in the included studies, and the results were synthesized narratively. The total fat content and total fatty acid composition of EBM was not generally influenced by storage and handling process, with most changes less than 10%, which is likely a result of methodological variation. A reduction in EBM triglyceride concentration and concomitant increase in free fatty acid concentration were seen after exposing to various conditions, probably due to endogenous lipase.
Mother's milk is the recommended food for premature infants cared for in the NICU. In the cases presented in this article, mothers pumped their milk into food-grade aseptic plastic containers. Milk was refrigerated before use. In Case 1, an infant developed Pseudomonas aeruginosa sepsis. In Case 2, an infant developed methicillin-resistant Staphylococcus aureus. Both cases were attributed to contaminated mother's milk. Proper cleaning and sterilization of pump parts is essential to prevent milk contamination.
Numerous advantages for infants, their mothers, families, and society from breastfeeding and the use of expressed human milk have been documented by extensive research. These benefits extend beyond the infant’s health and development by contributing to the mother’s wellness and that of families and the general community by raising healthier individuals [1]. Consumption of human milk is advantageous to the environment, as manufacturing and transport of infant formulas demands energy and produces waste. There are appreciable cost savings from a reduction in formula purchasing [2–4].
Lyophilization appears to be a viable method for storing human milk, assuring no microbiological contamination and preserving its health benefits and antibacterial properties.
To evaluate and compare the effects of different storage methods (lyophilization and freezing at -20°C and -80°C) and maternal factors (gestational length or time postpartum) upon the microbiological contents and bactericidal activity of human milk. The possible relationship between bactericidal activity and the content of certain nutrients and functional components is also investigated.
Microbiological content, bactericidal activity, sialic acid and ganglioside contents, as well as protein, fat and lactose concentrations were assessed in 125 human milk samples from 65 healthy donors in the Human Milk Bank of La Fe (Valencia, Spain).
Lyophilization and storage at -80°C significantly reduced the content of mesophilic aerobic microorganisms and S. epidermidis when compared to storage at -20°C. Bactericidal activity was not significantly modified by lyophilization when compared to freezing at either -20°C or -80°C. Bactericidal activity was not correlated with fat, protein or lactose content, but was significantly correlated to ganglioside content. The bactericidal activity was significantly greater (p < 0.05) in mature milk and in milk from women with term delivery than in milk from early lactation (days1-7 postpartum) and milk from women with pre-term delivery, respectively.
Lyophilization and storage at -80°C of human milk yields similar results and are superior to storage at -20C with regard to microbial and bactericidal capacities, being a feasible alternative for human milk banks.
Objective:
There is an increasing evidence of the immunological role of breast milk (BM) microbiota on infant health. This study aims to analyze several determining factors of milk microbiota.
Study design:
A total of 96 milk samples from 32 healthy mothers (19 preterm vs 13 at term gestations; and 15 vaginal deliveries vs 17 Cesarean sections) were longitudinally collected. Microbiota composition was studied by quantitative PCR and the influence of lactation stage, gestational age and delivery mode was evaluated.
Result:
Globally, Lactobacillus, Streptococcus and Enterococcus spp. were the predominant bacterial groups. Total bacteria, Bifidobacterium and Enterococcus spp. counts increased throughout the lactation period. At all lactation stages, Bifidobacterium spp. concentration was significantly higher in milk samples from at term gestations than in preterm gestations. Higher bacterial concentrations in colostrum and transitional milk were found in Cesarean sections. Nevertheless, Bifidobacterium was detected more frequently in vaginal than in Cesarean deliveries.
Conclusion:
Lactation stage, gestational age and delivery mode all influence the composition of several bacteria inhabiting BM: Bifidobacterium, Lactobacillus, Staphylococcus, Streptococcus and Enterococcus spp., and, consequently, may affect the infant's early intestinal colonization.
Human milk is rich in polyunsaturated fatty acids (PUFA) that are prone to oxidation. We have measured a global (MDA) and specific end-markers of n–6 and n–3 PUFA oxidation, respectively: 4-HNE and 4-HHE, from 7 breast milk samples. Analyses were performed on milk fresh or stored 1 day at 18°C or 7 days at 4°C. Ten infant formulas, liquids or in powder, some supplemented in long chain PUFA, were also studied. The ratio [4-HHE/n–3 PUFA] was 20-fold higher in liquid infant formula than in human milk (0.19 ±0.01μg.g−1). The ratio [4-HNE/n–6 PUFA] was 300-fold higher in liquid infant formula than in human milk (0.004 ±0.000μg.g−1). Storage of human milk did not increase significantly lipid peroxidation, on the contrary to infant formula where MDA increased. The native structure of fat globules in breast milk can take part in its greater protection against oxidation compared to the artificial fat droplets in infant formulas. The health consequences of chronic infant exposure to low dietary doses of lipid peroxidation products should be investigated and infant formulas should be stored in appropriate conditions.
Background:
Refrigeration of human milk has been recommended for its short-term storage. It has been shown that some nutritional, immunological, and bioactive properties and bactericidal activity of human milk can be altered during refrigeration. Pyrex bottles and polyethylene bags are 2 commonly used containers for human milk storage.
Objective:
The aim of this study was to compare the association between storage container type on the bactericidal activity of human milk for different durations of refrigeration (fresh, and at 24 and 48 hours).
Methods:
Forty-four samples of human milk were collected from 22 lactating mothers. Two samples of breast milk (approximately 10 mL each) were obtained by manual expression from each mother. One was collected directly into sterile Pyrex bottles and the other into polyethylene bags. One mL of human milk from each container was processed immediately after arrival to the laboratory. The remaining human milk was kept in the Pyrex and polyethylene containers at 4°C until analysis at 24 and 48 hours. The bactericidal activity of each sample was studied. A strain of Escherichia coli ATCC 25922 was used to determine the bactericidal effect of human milk.
Results:
Bactericidal activity was significantly reduced in milk samples stored in polyethylene bags compared to those stored in Pyrex bottles when milk samples were stored at 4°C for 24 and 48 hours (P < .05).
Conclusion:
Short-term storage of human milk in Pyrex bottles is more appropriate than polyethylene bags for preserving its bactericidal activity against E coli.
Medication administration is the final step in the medication delivery cycle before a prescribed drug reaches a patient. In
ambulatory settings, the patient, or in the case of children, the parent or guardian is responsible for accepting prescribed
medications from the pharmacist and following directions regarding direct administration of drug doses. In inpatient settings,
it is principally the bedside nurse who administers prescribed medications. A study of inpatient settings suggests that interventions
by pediatric clinical pharmacists, while effective for intercepting prescribing errors, may be ineffective in intercepting
harmful administration errors.1
Breastfeeding is believed to impart major beneficial effects on babies, due to various human-specific components. Compatibility of family and profession, however, often necessitates the pumping of breast milk and its storage under diverse conditions, either in a refrigerator, freezer or for brief periods at room temperature. The present study investigates the formation of a characteristic fish-like and metallic off-odour during the conventional storage of human milk that does not develop during cow milk storage, and elucidates the molecular factors for these specific aroma changes. Odorants in fresh and stored human milk containing the specific fishy-metallic off-odour were characterized by means of gas chromatography–olfactometry and identified based on their mass spectrometric data and specific chromatographic and sensory characteristics.
A central goal of The Academy of Breastfeeding Medicine is the development of clinical protocols for managing common medical problems that may impact breastfeeding success. These protocols serve only as guidelines for the care of breastfeeding mothers and infants and do not delineate an exclusive course of treatment or serve as standards of medical care. Variations in treatment may be appropriate according to the needs of an individual patient.
Human neonates are born with an immature and naive acquired immune system, and many of the innate components of mucosal immunity are not fully developed. Thus, the innate immune system of human milk is an important complement to the mucosal barrier of the developing gut. The nursing mother provides her infant many protective agents through milk, a growing number of which have been identified as isolates of milk in laboratory models of infection. The number, the potency, and the importance of these protective agents are probably greater than previously thought. For example, many potent protective agents are not found in milk until digestion releases antimicrobial agents such as fatty acids and peptides. An alternate conformer of alpha-lactalbumin forms from milk in the stomach and inhibits cancer cells. Many of the protective constituents of human milk inhibit different aspects of a pathogenic process, creating a synergy, where much lower concentrations of each component become protective. Some components have a temporal and a spatial specificity that would cause their protective role to go unrecognized by most laboratory models of infection. Some protective components had remained underappreciated because of technical challenges in their isolation and testing. Recent reports suggest that human milk contains a highly potent mixture of protective agents that constitute an innate immune system, whereby the mother protects her infant from enteric and other diseases. These human-milk components may represent a rich source of novel classes of therapeutic agents against human pathogens.
Breastfed infants have lower morbidity and mortality due to diarrhea than those fed artificially. This had been attributed primarily to the secretory antibodies and prebiotic factors in human milk. Oligosaccharides are the third largest component of human milk. They were initially considered to be functionless by-products of glycoprotein and glycolipid synthesis during milk production. However, in the past few decades it has become apparent that the human milk oligosaccharides are composed of thousands of components, at least some of which protect against pathogens. Oligosaccharide protection against infectious agents may result in part from their prebiotic characteristics, but is thought to be primarily due to their inhibition of pathogen binding to host cell ligands. Most human milk oligosaccharides are fucosylated, and their production depends on enzymes encoded by the genes associated with expression of the Lewis blood group system. The expression of specific fucosylated oligosaccharides in milk thus varies in relation to maternal Lewis blood group type, and is significantly associated with the risk of infectious disease in breastfed infants. Specific fucosylated moieties of oligosaccharides and related glycoconjugates (glycans) are able to inhibit binding and disease by specific pathogens. This review presents the argument that specific glycans, especially the oligosaccharides, are the major constituent of an innate immune system of human milk whereby the mother protects her infant from enteric and other pathogens through breastfeeding. The large input of energy expended by the mother in the synthesis of milk oligosaccharides is consistent with the human reproductive strategy of large parental input into rearing relatively few offspring through a prolonged period of maturation. These protective glycans may prove useful as a basis for the development of novel prophylactic and therapeutic agents that inhibit diseases caused by mucosal pathogens.
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Human breast milk samples were collected from lactating mothers, and aliquots were maintained at room temperature, frozen, and pasteurized. Samples were inoculated with 10 to 50 colony-forming units per milliliter (cfu/ml) of Escherichia coli or group B streptococcus, and incubated at 37 C. Quantitative growth was measured at eight and 24 hours. No inhibitory activity was demonstrated by control broth, commercial formula, and pasteurized breast milk, which had a rapid logarithmic growth to a maximum of 10(8) to 10(9) cfu/ml at 24 hours. Compared with these controls, fresh breast milk, fresh frozen breast milk, and breast milk frozen for 21 days demonstrated a significant inhibition of bacteria growth. A trend toward gradual loss of inhibiting activity was noted with prolonged freezing of breast milk. Although freezing may quantitativly decrease the amount of some breast milk host-defense factors, it cannot be assumed that comparable functional reductions will necessarily result.
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 degrees C (P less than 0.05). Similarly, quick freezing and storage for 3 months significantly decreased the lactoperoxidase activity of pooled samples from 93 to 14 (P less than 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.
This paper reviews the effects of storage and bacterial content contaminating human milk on some milk constituents. Moreover, it reviews the inhibitory effect of refrigeration and freezing on bacterial growth. Our results suggest that the type and length of storage have an effect on some milk constituents, that this effect is modulated by the bacterial contamination of the milk and that refrigeration has a significant inhibitory effect on bacterial growth which is not observed after freezing. This stresses the importance of collecting noncontaminated milk and justifies the choice of refrigeration at 0-4 degrees C for storage up to 8 days.
Preformed lipid peroxidation products present in the feed may contribute to the total reactive oxygen radical load infants have to deal with and may play a role in the pathogenesis of necrotizing enterocolitis and bronchopulmonary dysplasia. In this study, the occurrence of lipid peroxidation in human milk and feeding formulas for preterm babies was evaluated in vitro. Free linoleic acid (18:2) and its hydroperoxide (18:2OOH) were measured by gas chromatography-mass spectrometry and the concentration of 18:2OOH and the 18:2OOH/18:2 ratio were used as indices of peroxidation. In all feeds peroxidation products were present, but the proportion of peroxidized 18:2 was greater in infant formula. Storage of human milk (+4 degrees C for four days) increased lipid peroxidation. Exposure to light during tube feeding increased peroxidation in infant formula but not in human milk. Different procedures for preparation, storage and feeding may decrease the concentration of these potentially toxic peroxidized lipids in human milk and infant formula.
Effects of steel and glass storage containers, duration of storage up to seven hours, and storage temperature on the concentration of humoral and cellular fac tors in 357 samples of human breastmilk were examined. Leukocytic functions were estimated by E-rosetting of sheep RBCs by T-lymphocytes and phagocytosis of S. cere visieae by phagocytes. Immunoglobulin (Ig) levels were studied by single radial immu nodiffusion technique (SRID).
Viability and cell counts reduced during the storage period, but were greater than 60 percent of baseline values. IgG, IgA, and IgM levels in milk did not show significant decline after storage (p>0.01). Milk stored in glass yielded a greater number of func tional cells after storage at 4°C. JHL12:31-35, 1996.
Human milk provides the infant with protection against infectious diseases. This protection is conferred through several mechanisms: specific antibody targeted protection against pathogens in the infant's environment (through milk IgA, IgG, and IgM) and broad-spectrum, nonspecific protection provided through several distinct mechanisms. These are: bactericidal effects (lactoferrin), bacteriostatic action (lactoferrin, lysozyme), lysis of microorganisms (lysozyme), antiviral effects (lactoferrin, products of milk fat digestion), antiprotozoan activity (free fatty acids produced during gastric and intestinal digestion of milk fat), and ligand action (inhibition of Helicobacter pylori adhesion to gastric mucosa by kappa-casein). In addition to these protective functions of the proteins and lipids of human milk, several enzymes present in human milk might provide protection by generating components that are bactericidal (bile salt dependent lipase, peroxidase), prevent inflammatory reactions (platelet-activating factor acetylhydrolase), or protect the integrity of milk proteins (antiproteases).
Human milk provided by healthy and well-nourished mothers is believed to cover the infant's nutrient requirements during the first half year of life. It is composed of a mixture of nutritive components as well as other bioactive factors with relevant physiologic effects in the neonate infant. Human milk composition has a dynamic nature and varies with time postpartum, during a nursing, and with the mother's diet and certain diseases. The changes of human milk composition with time of lactation seem to match the changing needs of the growing infant over time. Human milk proteins are a source of peptides, amino acids, and nitrogen for the infant, but also in the protein fraction reside other properties of human milk that may benefit the breastfeeding infant. Specific whey proteins are involved in the development of the immune response (immunoglobulins), whereas others participate in the nonimmunologic defense (lactoferrin). In addition, human milk contains a complex mixture of oligosaccharides that are present only in minute amounts in other mammal's milk. They may act as inhibitors of bacterial adhesion to epithelial surfaces, and thus play an important role in preventing infectious diseases in the newborn infant. Oligosaccharides may also promote the development of a so-called bifidus flora. In the next years, future research will lead to improved characterization of human milk components and elucidation of their individual mechanisms of action, which will increase our knowledge about the properties of human milk and the benefits of breastfeeding for the infant.
The storage of human milk for use later by the mother's own infant or an unrelated recipient has an impact on its constituents. These effects involve the storage container, heating, cooling and freezing the milk. Overall, glass is the least destructive container. Milk can be safely refrigerated for 72 h with little change. Freezing destroys cellular activity and reduces vitamins B6 and C. Boiling, in addition, destroys lipase and reduces the effect of immunoglobulin A and secretory immunoglobulin A. The nutrient value of human milk is essentially unchanged, but the immunological properties are reduced by various storage techniques.
Storage of human breast milk by freezing or refrigeration of milk with and without heating have been recommended. This can hardly be avoided because of the social circumstances of most mothers who are regularly separated from their infants because of work or schooling as well as the particular needs of some pre-term or sick babies to be fed with expressed breast milk. The greatest fear that has hindered the prospects of in-vitro storage of breast milk for any considerable period of time is the possibility of bacterial contamination and growth of infectious pathogens in the stored milk, thereby rendering them unsafe for human consumption. Bacteriological examination of refrigerated milks has proven their safety for human consumption for even up to 72 h. For a storage over longer periods up to 1 month, freezing at −20 °C could be recommended, but the most preferred method, especially for longer storage would be fresh freezing at −70 °C, if affordable or available. The expressed fears arising from increased titratable acidity of such stored milk samples have been unfounded, since it has been shown to be mainly attributable to levels of free fatty acids, rather than lactic acid, which might have been produced by bacterial fermentation of milk sugars.
Conclusion Evidence shows that temporary storage of human milk under appropriate conditions is not dangerous for babies and infants. This would further encourage the practice of prolonged exclusive breastfeeding and allow the families to reap its multi-fold benefits.
The immunoprotective constituents of human milk are stable when stored at room temperature for 8 hours, when stored at 0 degree-4 degrees C for three days, or when frozen at -20 degrees C for 12 months. They are also stable during pasteurization at 56 degrees C for 30 minutes. Sonification may reduce levels of sIgA and lysozyme and the ability of milk to inhibit growth of E coli. The number of cells in human milk is reduced by storage, freezing, pasteurizing, microwaving and sonification, and the functional capacity of surviving cells is also reduced.
Storage of human milk for limited periods of time is unavoidable in neonatal units and also in the home where increasing numbers of mothers go back to work soon after delivery. Many mothers, convinced of the importance of prolonged exclusive or complementary breast-feeding, often express and store human milk for use during the period of separation. This study examines the effects of different storage methods on the pH and some antibacterial activities of human milk. Portions of milk and colostrum samples from healthy lactating mothers were stored at 4 degrees C and -20 degrees C for periods ranging from one day to 12 weeks. The stored milk samples were analysed for pH, bactericidal and bacteria sequestration activities against a serum-sensitive Escherichia coli, and compared with freshly collected samples, with and without EDTA. Milk became progressively more acidic during storage. While the bactericidal activities of refrigerated samples diminished rapidly, up to two-thirds of the original activity level was maintained by freezing for up to three months. The ability of milk fat globule membrane to adhere to suspended bacteria was gradually lost in frozen milk samples, while it was greatly enhanced during the first few days in refrigerated samples, before declining sharply. This study shows that loss of bactericidal activity in refrigerated milk is well compensated for by enhanced bacteria sequestration activity, and allays any fears that might arise concerning the suitability of stored human milk for infant consumption.
Mammary fluids, colostrum and milk, deliver nature's first host defense systems upon birth, and these essential liquids are critical for survival of the neonate. The identification and characterization of anti-infectious proteins were among the early scientific discoveries and this group of proteins has long been recognized for promoting health benefits in both newborns and adults. Among the more widely studied are the immunoglobulins, lactoperoxidase, lysozyme, and lactoferrin. Recently, it was shown that alpha--lactalbumin may also function in a protective capacity dependent upon its folding state. Some of these, especially lactoferrin, also display an immunomodulatory role in which case a totally separate cascade of host defense responses is initiated. It was noted that the mechanism of action for this cluster of sentry proteins does vary; thus, this protective strategy provides for a broad range of responsive reactions to infection. Presently, there is a major focus on the discovery of novel peptides that can be generated from existing milk proteins via proteolytic reactions. To date, this substrate list includes alpha--lactalbumin, beta-lactoglobulin, all casein fractions, and lactoferrin. Again, the immunoregulatory effects prompted as a result of the appearance of these peptides are currently being defined. Herein, we review the principal biological properties associated with each of these contributing milk components with a special emphasis on the role of biodefensive milk peptides. We envision future contributions emerging from this research field as an opportunity to develop effective new therapies to be used in treating infectious diseases and promoting health benefits in vivo.
The survival of a nonimmune infant faced with a new pathogen depends in part on an array of specific and nonspecific human milk factors. Human milk protects via multiple redundant strategies. It targets shared virulence mechanisms to provide cross protection. By interfering with fundamental processes such as attachment to glycoconjugates, human milk anticipates new mutations and new pathogens that utilize carbohydrates as receptors. Furthermore, human milk IgA present appears to reflect long-term maternal immunologic memory. These overlapping protective strategies endow human milk with the unique ability to protect infants from organisms that mutate rapidly and are present in vast numbers.
Human milk contains a wide array of proteins that provide biologic activities ranging from antimicrobial effects to immunostimulatory functions. Proteins like lactoferrin, secretory IgA, kappa-casein, lactoperoxidase, haptocorrin, lactadherin and peptides formed from human milk proteins during digestion can inhibit the growth of pathogenic bacteria and viruses and therefore protect against infection. At the same time, proteins like lactoferrin, bile-salt stimulated lipase, haptocorrin, kappa-casein, and folate-binding protein can facilitate the absorption of nutrients in the neonatal gut. However, the proteins in human milk themselves also provide adequate amounts of essential amino acids to the growing infant. This suggests a highly adapted digestive system, which allows the survival of some proteins and peptides in the upper gastrointestinal tract, while still allowing amino acid utilization from these proteins further down in the gut. It is now possible to produce recombinant human milk proteins in transgenic plants and animals, which makes it possible to further study the bioactivity of these proteins. Provided these proteins can be produced in large scale at low cost, that they show biologic activity and pose no safety concerns, it may be possible to add some human milk proteins to infant diets, such as formula and complementary foods. Human milk proteins produced in rice or potatoes, for example, could be added without much purification, because these staples commonly are used in weaning foods. Thus, some qualities provided by human milk may be included into other diets, although it is highly unlikely that all unique components of human milk can be copied this way.
Breastfeeding and human milk are widely accepted as optimal for human infants' nutrition. Nowadays lifestyle often makes it difficult to maintain or even initiate human lactation. This situation is mostly related to the workload of women away from home. New approaches are needed to enable maternal lactation under these circumstances. Human breastmilk storage for differed use is one possibility. The aim of this study was to assess changes in glutathione peroxidase (GPx) activity and in the concentration of the lipid peroxidation marker, malondialdehyde (MDA), when human milk was kept refrigerated or frozen. Thirty-two human milk samples were assayed for GPx activity and MDA concentration. Samples were divided in three aliquot portions, the first to be immediately analysed, the second to be refrigerated at 4 degrees C and analysed 24 h thereafter, and the third to be frozen at -20 degrees C and assayed after 10 days. GPx activity was significantly decreased in refrigerated and in frozen milk, when compared to their control samples. MDA was increased only in refrigerated milk but not in frozen samples. Thus, freezing seems better than refrigeration in order to prevent lipid peroxidation in stored human milk samples.
Breast-feeding can reduce the incidence and the severity of gastrointestinal and respiratory infections in the suckling neonate by providing additional protective factors to the infant's mucosal surfaces. Human milk provides protection against a broad array of infectious agents through redundancy. Protective factors in milk can target multiple early steps in pathogen replication and target each step with more than one antimicrobial compound. The antimicrobial activity in human milk results from protective factors working not only individually but also additively and synergistically. Lipid-dependent antimicrobial activity in milk results from the additive activity of all antimicrobial lipids and not necessarily the concentration of one particular lipid. Antimicrobial milk lipids and peptides can work synergistically to decrease both the concentrations of individual compounds required for protection and, as importantly, greatly reduce the time needed for pathogen inactivation. The more rapidly pathogens are inactivated the less likely they are to establish an infection. The total antimicrobial protection provided by human milk appears to be far more than can be elucidated by examining protective factors individually.
Nutritional and biochemical properties of human milk: I. General aspects, proteins and carbohydrates
- Kunz
Lipid peroxidation in human milk and infant formula: effect of storage, tube feeding and exposure to phototerapy
- Van Zoeren-Grobben