Evaluation of Fetal Intestinal Cell Growth and Antimicrobial Biofunctionalities of Donor Human Milk After Preparative Processes

Abstract

Background:

Donor human milk is considered the next best nutrition following mother's own milk to prevent neonatal infection and necrotizing enterocolitis in preterm infants who are admitted at neonatal intensive care unit. However, donor milk biofunctionalities after preparative processes have rarely been documented.

Objective:

To evaluate biofunctionalities preserved in donor milk after preparative processes by cell-based assays.

Materials and Methods:

Ten pools of donor milk were produced from 40 independent specimens. After preparative processes, including bacterial elimination methods (holder pasteurization and cold-sterilization microfiltration) and storage conditions (−20°C freezing storage and lyophilization) with varied duration of storage (0, 3, and 6, months), donor milk biofunctionalities were examined by fetal intestinal cell growth and antimicrobial assays.

Results:

At baseline, raw donor milk exhibited 193.1% ± 12.3% of fetal intestinal cell growth and 42.4% ± 11.8% of antimicrobial activities against Escherichia coli. After bacteria eliminating processes, growth promoting activity was better preserved in pasteurized donor milk than microfiltrated donor milk (169.5% ± 14.3% versus 146.0% ± 11.8%, respectively; p < 0.005), whereas antimicrobial activity showed no difference between groups (38.3% ± 14.1% versus 53.7% ± 17.3%, respectively; p = 0.499). The pasteurized donor milk was further examined for the effects of storage conditions at 3 and 6 months. Freezing storage, but not lyophilization, could preserve higher growth-promoting activity during 6 months of storage (163.0% ± 9.4% versus 72.8% ± 6.2%, respectively; p < 0.005). Nonetheless, antimicrobial activity was lost at 6 months, regardless of the storage methods.

Conclusions:

This study revealed that fetal intestinal cell growth and antimicrobial assays could be applied to measure donor milk biofunctionalities and support the utilization of donor milk within 3 months after preparative processes.

Introduction

All preterm infants should receive human milk to prevent deleterious outcomes.^1,2 Human milk provides protective effects against several illnesses, that is, late-onset sepsis (LOS) and necrotizing enterocolitis (NEC),^3–5 which are of particular importance for premature infants admitted to the neonatal intensive care unit (NICU). When mother's own milk is unavailable, the next best option is donor human milk prepared and distributed by Human Milk Bank (HMB).^6,7

For the best possible outcome, donor milk needs to be collected, prepared, and stored by the established HMBs to ensure safety regarding microbiological issues and to preserve nutrients and bioactive factors.⁸ Criteria for selection and screening of milk donors are similar to blood donation. After collection, holder pasteurization (HoP, process at 62.5°C for 30 minutes) is usually performed to inactivate viral and bacterial contaminants, while freezing storage at −20°C with a maximal storage duration up to 6 months is generally applied by most HMBs. However, previous studies showed that HoP may cause degradation of several bioactive proteins.^9–14 It has remained unclear what length of storage duration is most appropriate for the processed donor milk. Although a number of studies evaluated the effects of HoP and freezing storage on donor milk nutrients and bioactive components,^13,15–18 the evidence regarding biofunctionalities in donor milk after preparative processes is rarely documented. In addition, it was unknown whether or not methods such as cold-sterilization microfiltration (MF) and lyophilization, which are commonly used in pharmaceutical industries and research laboratories to sterilize and keep bioactive compounds, would improve the quality of donor milk in terms of biofunctionality preservation.

Fetal intestinal cell growth measurement and antimicrobial assay against Escherichia coli may be applicable to evaluate donor milk biofunctionalities with clinical relevance. NEC is an excessive inflammatory process of gastrointestinal tract developed from multifactorial insults on top of the intestinal immaturity, accompanied by abnormal bacterial colonization in the intestine. A previous study using an animal model showed that breast milk could protect against NEC development by the activation of epidermal growth factor (EGF) receptor, which attenuated Toll-like receptor 4 signaling-induced mucosal injury.¹⁹ Therefore, fetal intestinal cell growth promotion is an important human milk biofunctionality related to NEC prevention. Gram-negative bacteria, including E. coli, are commonly colonized along the intestinal tract. Abnormal colonization of E. coli possesses the risk of both NEC and LOS development in preterm infants. The measurement of antimicrobial potency against E. coli is a reasonable method to evaluate the donor milk biofunctionality.

This study therefore adopted fetal intestinal cell growth and antimicrobial assays to evaluate donor milk biofunctionalities after different preparative processes, including bacterial elimination methods (HoP and MF), storage conditions (freezing storage and lyophilization), and duration of storage (0, 3, and 6 months).

Materials and Methods

Study design

After the informed consent, donor milk sample was collected by using a breast pump at Ramathibodi HMB and was immediately kept in a −20°C freezer until pooling. All specimens were obtained within the 1-month period. Mothers of full-term infants, during 2-week to 9-month lactation period, were eligible to enroll into this study. Exclusion criteria included the presence of any underlying diseases, abnormal results of anti-HIV, HBsAg, or VDRL, or receiving antibiotics within the 2-week period before enrollment. This study was conducted following the principles of the Declaration of Helsinki and was approved by the Ethical Clearance Committee on Human Rights Related to Research Involving Human Subjects, Faculty of Medicine Ramathibodi Hospital, Mahidol University (protocol ID 10-57-27).

Donor milk preparation

Donor milk samples (60 mL each) were pooled to produce 10 distinct batches (4 individual samples per batch). The pooled donor milk specimens were then divided into 5-mL aliquots after bacterial eliminating processes and used throughout this study. HoP was performed by a milk sterilizer at 62.5°C for 30 minutes. The pasteurized donor milk was then rapidly cooled down and kept at 4°C until further processing. MF is a cold sterilization method that can separate microorganisms and any particulates from the filtrated liquid based on the pore size of the filter membrane. In this study, MF was performed by using a 0.1 μm pore size, Minisart high flow microfilter (Sartorius AG, Goettingen, Germany) to separate all bacteria (0.5–5.0 μm in size) from the microfiltrated donor milk. The microfiltrated donor milk was then kept in a sterile container at 4°C until further processing.

Donor milk storage conditions

For freezing storage, the pasteurized donor milk was maintained at the rear of the upright −20°C freezer until the indicated time. For lyophilization (or freeze drying), the pasteurized donor milk was kept in a compatible container at −70°C for 2 hours and subsequently placed in a freeze-dried chamber of the lyophilizer. Lyophilization was performed for ∼24 hours or until the frozen milk was completely dried. The lyophilized donor milk powder was harvested and kept at room temperature (RT), 4°C, or −20°C until used. At the indicated time, the donor milk powder was reconstituted with an equal volume of prelyophilized milk using the sterile deionized water at 37°C with gentle agitation.

Fetal intestinal cell growth assay

Fetal intestinal cell growth-promoting activity was measured for a biofunctionality related to donor milk protective effect against NEC. Fetal intestinal cells (FHs74Int) were purchased from ATCC (ATCC^®CCL241). FHs74Int cells were propagated and maintained in the 25-cm² (T-25) culture flask containing Dulbecco's modified Eagle's medium (DMEM; Gibco, Paisley, Scotland) supplemented with 10% fetal calf serum at 37°C, 5% CO₂. Since the FHs74Int cell line was slow growing, additional supplements, including EGF (a final concentration of 30 ng/mL) and human recombinant insulin (a final concentration of 10 μg/mL), were used to enhance cell proliferation rate during propagation. The medium was changed every 2–3 days. When cells reached 80–90% confluency (usually within 7–9 days), cells were detached by 0.1% trypsin/0.5 mM ethylene diamine tetraacetic acid (EDTA) solution at 37°C for 5 minutes. After neutralizing the trypsin/EDTA activity by adding complete media, cells were aspirated by gentle pipetting, centrifuged to remove the supernatant and resuspended the pellet in complete media, and then subcultured into T-25 culture flasks with the split ratio of 1:2–1:3. Cells between the passage 4 and 10 were used for fetal intestinal cell growth assay. For long-term storage of seed stocks, FHs74Int cells (7–9 × 10⁵ cells/cryovial) were kept in liquid nitrogen using 10% dimethylsulfoxide in fetal bovine serum as a preservative.

Fetal intestinal cell growth assay was performed as described previously.²⁰ FHs74Int cells were placed in DMEM (Gibco) containing 10% fetal calf serum at a concentration of 5 × 10³ cells/well in a 96-well plate for 24 hours at 37°C, 5% CO₂. Thereafter, the cells were washed with serum-free DMEM to eliminate serum protein contamination. The growth-promoting activity was examined at a final concentration of 10% donor milk in DMEM (Gibco) and the cells were further cultured for 24 hours. Cell proliferation was measured by the MTT assay at an absorbance of 690 nm (AU₆₉₀) as per the manufacturer's instructions (Promega, Madison, WI). All experiments were performed in triplicate. The growth-promoting activity was calculated by the following equation: growth promoting activity (%) = 100 × (AU₆₉₀ of milk supplement/AU₆₉₀ of serum-free media).

Antimicrobial assay

The antimicrobial activity was measured for a biofunctionality related to donor milk protective effect against LOS. The antimicrobial assay was performed as described previously.¹⁶ Fifty microliters of E. coli strain ATCC 25922 at 0.5 McFarland turbidity standard (∼8 × 10⁶ colony-forming unit [CFU]) was aliquoted into 200 μL of milk specimens or phosphate-buffered saline (blank control) and incubated at 37°C for 24 hours. Thereafter, the number of bacterial colonies was measured by plate colony counting. All experiments were performed in triplicate. The antimicrobial activity was measured by the reduction in total number of viable bacteria in milk specimen compared to that of blank control as follows: antimicrobial activity (%) = 100 × [1 − (bacteria in milk (CFUs/mL)/bacteria in blank control (CFUs/mL))].

Statistical analysis

SPSS version 18 (SPSS, Inc., Chicago, IL) was used for statistical analysis. Continuous data were presented as percentage or mean ± standard deviation as appropriate. The difference between groups was assessed by unpaired Student's t-test, one-way ANOVA, or nonparametric test as appropriate. p-Value <0.05 was considered statistical significance.

Results

Demographic data of the milk donors are shown in Table 1. A total of 40 specimens, donated from eight milk donors, were collected at Ramathibodi HMB. Four milk samples were pooled to produce a distinct batch, and a total of 10 different batches was used throughout the entire study.

Table 1.

Demographic Data of Eight Milk Donors

Characteristics	n = 8 donors
Maternal age (year), mean ± SD	33.6 ± 2.8
Body weight (kg), mean ± SD	54.6 ± 6.5
Parity, n (%)
G1	5 (62.5)
G2	2 (25.0)
G3	1 (12.5)
Gestational age (week), mean ± SD	38.0 ± 0.5
Birth weight (g), mean ± SD	3,057 ± 410
Day of lactation (day), mean ± SD	168 ± 93

SD, standard deviation.

Each batch of pooled donor milk was submitted to bacterial eliminating processes, either HoP or MF (n = 10 pooled donor milk per group). Thereafter, the growth-promoting activity on FHs74Int fetal intestinal cells and antimicrobial effect against E. coli were measured (Table 2). No significant difference in the antimicrobial activity was observed among raw donor milk, pasteurized donor milk, and microfiltrated donor milk (Table 2). Both HoP and MF caused a significant decrease in the growth-promoting activity of donor milk compared to that of raw donor milk. Nonetheless, this growth-promoting activity was significantly higher in the pasteurized donor milk than the microfiltrated donor milk (Table 2). Therefore, only pasteurized donor milk was further evaluated for the effects of storage conditions.

Table 2.

Effects of Bacterial Elimination Processes on Fetal Intestinal Cell Growth and Antimicrobial Activities of Donor Milk (n = 10 Pooled Milk Samples)

Condition	Growth promoting activity (%)	Antimicrobial activity (%)
Raw donor milk	193.1 ± 12.3	42.4 ± 11.8
Pasteurized donor milk	169.5 ± 14.3^*	38.8 ± 14.1
Microfiltrated donor milk	146.0 ± 11.8^*^,†	53.7 ± 17.3

p < 0.005 compared to raw donor milk.

†

p < 0.005 compared to pasteurized donor milk.

The pasteurized donor milk was allocated to −20°C freezing storage or lyophilization. Lyophilization also varied in the storage temperature, that is, RT, 4°C, and −20°C. Thereafter, donor milk biofunctionalities were measured at time zero (prestorage condition) as baseline values and then evaluated at 3- and 6-month storage duration to determine the effects of different storage methods.

The result showed that lyophilization caused a gradual loss of growth-promoting activity of the pasteurized donor milk over time (up to 89% reduction at 6 months), whereas freezing storage well preserved the growth-promoting activity up to 6 months of storage (Table 3). Surprisingly, the antimicrobial activity of the pasteurized donor milk was only maintained for a 3-month duration, regardless of the storage conditions. The results showed that the pasteurized donor milk with freezing storage or lyophilization entirely lost the antimicrobial activity during 6 months of storage (Table 4). These negative values of antimicrobial activity reflected the increase in bacterial growth.

Table 3.

Effects of the Storage Conditions and the Storage Time of Fetal Intestinal Cell Growth Promoting Activity of the Pasteurized Donor Milk (n = 10 Pooled Milk Samples)

	Growth promoting activity (%)
Condition	Time zero	3 month	6 month
Pre-storage	162.5 ± 30.4	—	—
−20°C freezing storage	—	158.6 ± 6.5	163.0 ± 9.4
Lyophilization
RT	—	117.1 ± 10.3^*^,†	65.2 ± 4.3^*^,§,‡
4°C	—	120.1 ± 12.4^*^,†	70.6 ± 8.0^*^,§,‡
−20°C	—	121.1 ± 9.7^*^,†	72.8 ± 6.2^*^,§,‡

p < 0.005 compared to prestorage condition.

†

p < 0.005 compared to 3-month freezing storage.

p < 0.005 compared to 6-month freezing storage.

‡

p < 0.005 compared to 3-month storage (at −20°C) after lyophilization.

RT, room temperature.

Table 4.

Effects of the Storage Conditions and the Storage Time of Antimicrobial Activity Against Escherichia coli of the Pasteurized Donor Milk (n = 10 Pooled Milk Samples)

	Antimicrobial activity (%)
Condition	Time zero	3 month	6 month
Prestorage	35.9 ± 14.2	—	—
−20°C freezing storage	—	34.0 ± 13.5	−76.1 ± 23.5^*^,§
Lyophilization
RT	—	11.4 ± 17.1^†	−62.8 ± 16.8^*^,‡
4°C	—	24.4 ± 14.8	−72.2 ± 18.7^*^,‡
−20°C	—	39.6 ± 10.7	−79.7 ± 28.3^*^,‡

p < 0.005 compared to prestorage condition.

†

p < 0.05 compared to prestorage condition.

p < 0.005 compared to 3-month freezing storage.

‡

p < 0.005 compared to 3-month storage (at −20°C) after lyophilization.

RT, room temperature.

Discussion

Knowledge of donor milk biofunctionalities after different preparative processes is necessary to improve the quality of donor milk prepared and distributed by HMBs. Since one of the major reasons of donor milk utilization in preterm infants admitted in NICU is disease prevention against NEC and LOS, fetal intestinal cell growth and antimicrobial assays were then adopted to evaluate donor milk biofunctionalities. Using this approach, we demonstrated that HoP and −20°C freezing storage, which is routinely used in most HMBs, were superior to alternative procedures, that is, cold-sterilization MF and lyophilization, regarding the preserved biofunctionalities in donor milk. In addition, our biofunctional data provided a supportive evidence to the practice of HMB utilization of donor milk within the 3 months of freezing storage.

Previous studies showed that heating effect of HoP reduced the concentrations of insulin-like growth factor 1 (IGF-1), IGF-2, IGF binding protein-2 (IGFBP-2), IGFBP-3, and transforming growth factor-β2 in donor milk as measured by immunological-based assay.^10,14 Our study confirmed those findings by using the cellular functional study. Approximately 23% of growth-promoting activity of donor milk was reduced after HoP (Table 2). The remaining growth-promoting activity in the pasteurized donor milk may be contributed by EGF, which is a heat-stable protein, corresponding to the previous study that HoP did not affect EGF concentration in donor milk.¹⁰ Nevertheless, cold-sterilization MF caused a more unfavorable effect to donor milk with ∼46% reduction in the growth-promoting activity (Table 2), even though there is no heating effect. It is postulated that some growth factors contained in human milk fat globules, particularly milk fat globule epidermal growth factor-8 (MFGE-8),²¹ may be lost during the MF process.²² Milk fat globules have average diameters of 3–5 μm, while the pore size of MF used in this study was 0.1 μm for the sterilization purpose.

The effects of freezing storage and lyophilization on antimicrobial activity were demonstrated by Salcedo et al.,²³ in which lyophilization and storage at −80°C were superior to the standard −20°C freezing storage with regard to the antimicrobial activity in nonpasteurized human milk, which was stored up to 6 weeks. In our practice, donor milk is usually stored after pasteurization. This study, therefore, examined the antimicrobial activity, together with the growth-promoting effect on fetal intestinal cells, in pasteurized donor milk, which was stored up to 6 months by either freezing storage or lyophilization. In contrast to a previous study,²³ our findings demonstrated that lyophilization was not compatible with long-term storage of pasteurized donor milk due to the reduction of measured biofunctionalities at 3- and 6-month duration (Tables 3 and 4).

Comparison of antimicrobial activity preserved in pasteurized donor milk after storage at the different time points provided supportive information to improve the HMB protocol for donor milk storage and distribution. In the current practice of HMB,²⁴ pasteurized donor milk can be stored up to 6 months in the freezer at −18°C or lower. However, our results from the antimicrobial activity assay (Table 4) suggested that the pasteurized donor milk with −20°C freezing storage should be kept no longer than 3 months, which is consistent with previous studies.^16,17 The decrease in lactoferrin and ganglioside levels after a 3-month freezing storage may be responsible for this phenomenon.^17,23

Recently, Ahrabi et al., reported that human milk with freezing storage at −20°C for 9 months was associated with decreased bacterial count and thus supported the practice of the freezing storage of human milk up to 9 months.¹⁸ However, the outcome measure in that study was the numbers of colony formation of the “contaminated” bacteria in raw, nonsterilized human milk, but not the measurement of antimicrobial activity against the “added” bacteria into pasteurized donor milk. It is unknown whether antimicrobial activity in raw, nonsterilized human milk can be preserved longer than 3 months in storage. Since donor milk provided by HMBs requires a bacterial elimination process for safety reasons and its indication of use in preterm infants relates to the protective effects, we, therefore, support the practice that HBM should utilize the pasteurized donor milk within 3 months of freezing storage. This practice is already adopted and employed by our HMB.

This study had limitations. First, we measured the biofunctionalities in the pooled donor milk, but not the independent milk specimens, which caused loss of information such as the data outlier and variation to some extent. However, the pooling of donor milk is a general practice of milk processing in HMBs, as per the guidelines of the Human Milk Banking Association of North America (HMBANA). For a smooth translation of laboratory findings into the HMB practice, the obtained data from the pooled donor milk are a reasonable trade-off. Second, our study did not measure the changed amounts of the particular bioactive molecules in donor milk after processes, which may be a drawback in a pharmaceutical viewpoint. Instead, we utilized the so-called “functional-guided strategy” to evaluate the functional-related outcomes of donor milk after preparation and storage. As aforementioned, donor milk feeding in preterm infant is indicated by the clinical benefits. This functional-guided strategy may be developed as functional biomarkers of the qualified donor milk for utilizations in either clinical research or practice. Third, this study only examined the antimicrobial activity of donor milk against E. coli. Nonetheless, other bacteria such as Clostridial and Klebsiella species have also been reported in NEC cases. These pathogens therefore serve as valuable targets for future evaluation of antimicrobial effect of donor milk. Fourth, this study did not evaluate novel approaches to milk preparation, for example, high-temperature short-time pasteurization,²⁵ high-pressure processing,²⁶ ultraviolet light technology,²⁷ and the deep freezing storage at −80°C.¹⁶ It would be interesting to apply the functional-guided strategy, in addition to the measurement of nutrients and bioactive molecules, to assess the capability of those novel methods on donor milk preparation. Information on functional aspects, together with nutrients and biomolecular concentrations, would shed light into improvement of donor milk preparation for HMB practice in the future.

In conclusion, different preparative processes of donor milk had unequally preserved biofunctionalities as evaluated by fetal intestinal cell growth and antimicrobial assays. Conventional methods, that is, HoP and freezing storage, provided better outcomes than the comparators, that is, MF and lyophilization. This study supported the utilization of donor milk within 3 months after pasteurization and freezing storage.

Footnotes

Acknowledgments

We thank all mothers who donated their breast milk to Ramathibodi HMB. We also thank Kanuengnit Emrat, Sirimon Kongthaworn, Achara Tangnoo, and Numtip Tongsawan for their invaluable help on milk donating process. Finally, we thank Tassanee Lohnoo and Paisan Jitthrontham for their technical assistance. P.K. was a pediatric resident during initiation and completion of the study. This study was supported by the International Health Policy Program Foundation (a grant number RF58047 to S.C.). S.C. was also supported by International Health Policy Program Foundation, Ministry of Public Health, Thailand (grant number IHPF59102559 to S.C.).

Authors' Contributions

S.S. initiated the ideation. N.A., C.M., S.S., P.N., and S.C. developed the design. P.K. gave informed consent to participants and collected specimens. P.K., N.P., N.A., and C.M. performed experiments. P.K., N.P., N.A., C.M., P.N., and S.C. analyzed the data. P.K. and N.P. prepared the tables and wrote the article. N.A., C.M., S.S., P.N., and S.C. revised the article. S.S. contributed to overall research strategy. S.C. finalized the article. All authors read and approved the final version of the article.

Disclosure Statement

No competing financial interests exist.

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