by HR Schlotterer · Cited by 1 — fortifier has a more significant impact on bioactive components in fortified HPDHM than does storage time. Our findings of lack of negative impact of

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The effects of fortification and refrigerated storage on bioactive proteins in Holder -pasteurized donor human milk By: Hannah R. Schlotterer, Boriana Parvez, and Maryanne T. Perrin This is a non -final version of an article published in final form in Schlotterer H, Parvez B, Perrin MT. The effects of fortification and refrigerated storage on bioactive proteins in Holder -pasteurized donor human milk. Journal of Pediatr ic Gastroenterology and Nutrition September 2019 – Volume 69 – Issue 3 – p 370 -374 doi: 10.1097/MPG.0000000000002386 Made available courtesy of Lippincott, Williams & Wilkins: http://dx.doi.or g/10.1097/MPG.0000000000002386 ***© 2019 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition . Reprinted with permission. No further reproduction is authorized without written permission from Lippincott, Williams & Wilkins . This version of the document is not the version of record. Figures and/or pictures m ay be missing from this format of the document. *** Abstract: Objective: The aim of the study was to assess the total protein, lysozyme, and immunoglobulin A (IgA) content of unfortified and fortified Holder -pasteurized donor human milk (HPDHM) during 96 hours of refrigerated storage. Study Design: HPDHM was prepared in a hospital feeding room and subjected to treatment with 3 dif ferent fortifiers: an acidic, bovine -based (F -ACID), a neutral, bovine -based, and a human milk Œderived (F -HUM) fortifier. Unfortified HPDHM served as the control (CONTROL). Samples were stored at 4°C, and every 24 hours, a 1-mL aliquot was removed for anal ysis. Results: At baseline, there was a significant difference in protein (mean, standard deviation) concentration (g/dL) between control (1.3, 0.1) and all other treatments (F -ACID = 2.0, 0.2; neutral, bovine -derived fortifier = 2.2, 0.1; F -HUM = 2.5, 0.1; P < 0.001). Lysozyme and IgA were significantly lower in the F -ACID group ( P < 0.001). Lysozyme and IgA were significantly higher in the F -HUM group ( P < 0.001). There was no significant effect of storage time ( P > 0.9) for all dependent variables. Concl usion: The type of fortifier has a more significant impact on bioactive components in fortified HPDHM than does storage time. Our findings of lack of negative impact of refrigeration storage time on the protein and bioactive components of donor milk streng then the recent recommendations to extend storage time to 48 hours. Keywords: bioactives | donor human milk | donor milk | fortified | storage Article:

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What Is Known Unfortified Holder -pasteurized donor human milk retains lysozyme and immunoglobulin A activity for up to 7 days of refrigerated storage. Neonatal intensive care units in the United States routinely fortify human milk for very -low -birth -weight infants. What Is New Acidic fortifiers significantly lower the lysozyme and immunoglobulin A activity in Holder -pasteurized donor human milk. Human milk Œderived fortifiers significantly increase the lysozyme and immunoglobulin A activity of Holder -pasteurized donor human milk. Refrigerated storage for up to 96 hours had no impact on the total protein, lysozyme activity, and immunoglobulin A activity in fortified Holder -pasteurized donor human milk. The use of pasteurized donor human milk is supported by the World Health Organization, the American Academy of Pediatrics, and the Un ited States Surgeon General as an important strategy for improving health outcomes in premature infants when their mother’s milk is not available (1Œ3) . In the United States, donor milk use is on the rise, with more than 65% of neonatal intensive care unit s (NICUs) reporting use in 2015 (4Œ6) . To ensure the safety of donor milk, the Human Milk Banking Association of North America (HMBANA) and other international milk bank networks have issued guidelines, including appropriate storage temperatures and durati ons. Current best practices for the refrigerated storage of Holder -pasteurized donor human milk (HPDHM) state that thawed milk should be stored in the refrigerator and used within 48 hours (7) . Evidence is emerging that unfortified HPDHM remains free of mi crobial growth during refrigerated storage over 4 days, suggesting an opportunity to extend the expiration date and reduce unnecessary waste of a costly and valuable resource (8Œ12) . Affordability was the most frequently cited barrier of nonuse in a 2013 s urvey of 183 level 3 NICUs (5) . Research to evaluate the feasibility of extending defrosted HPDHM expiration dates has the potential to reduce cost barriers associated with a short shelf life and product waste. Although bacteria levels of HPDHM are one ma rker of product quality and safety, additional research is needed regarding the retention of nutrients and immune factors in HPDHM during extended refrigerated storage to inform evidence -based guidelines on appropriate clinical use. In addition, it is now common practice in the NICU to fortify mother’s milk and HPDHM with human milk fortifiers (HMF) to increase protein and energy content (4) . The effects of refrigerated storage time on the retention of macronutrients and other bioactive components in fortif ied HPDHM remain largely unstudied (13) . The objective of this study is to assess total protein, lysozyme, and immunoglobulin A (IgA) in unfortified and fortified HPDHM during 96 hours of refrigerated storage. Lysozyme and IgA were selected for this study because of their antimicrobial properties (14) and their partial retention after pasteurization (15) . A 96 -hour window was selected as the maximum amount of time a hospital might take to use a standard 4 oz bottle of HPDHM when starting preterm infants at a low feeding volume (15 Œ20 mL · kg 1 · day 1) and advancing at 10 to 20 mL · kg 1 · day 1(16) . METHODS

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HPDHM was acquired through an HMBANA milk bank (The New York Milk Bank, Valhalla, NY). Twelve unique batches were subject to 4 treatments: unfortified HPDHM (CONTROL) served as the control; fortification to 24 kcal/oz with a liquid, acidic, bovine milk Œbased whey hydrolysate fortifier (F -ACID) (Enfamil Human Milk Fortifier Acidified Liquid, Mead Johnson, Chicago, IL); fortification to 24 kcal/oz with a liquid, neutral, bovine milk Œbased fortifier (F -NEUT) (Similac Human Milk Fortifier Concentrated Liquid, Abbot, Chicago, IL); and fortification to 28 kcal/oz with a liquid, neutral, human milk Œbased fortifier (F -HUM) (Prolact+8, Prolacta Bioscience, City o f Industry, CA). Sample Preparation Fortification and handling of the milk samples occurred in the hospital milk preparation room at Westchester Medical Center (Valhalla, NY) between September 2016 and December 2016 by a trained Milk Technician. Each uni que batch of HPDHM was mixed according to the protocol outlined here: F -ACID was fortified with 1 vial (5 mL) of Enfamil HMF added to 25 mL HPDHM to reach 24 kcal/oz; F -NEUT with 1 envelope (5 mL) of Similac HMF fortifier plus 25 mL HPDHM to reach 24 kcal/ oz; and F -HUM with Prolact+8 HMF (40 mL) plus 60 mL HPDHM, to reach 28 kcal/oz. Once mixed, all samples were stored in clean containers labeled with the HPDHM batch number and a unique letter code corresponding to fortification type. With the exception of the milk technician, all researchers were blinded to treatment type until after all samples were analyzed and all data were collected. A 5 mL aliquot from each control and fortification group was stored in the refrigerator in a clean, glass container. Time 0 for the fortified samples began once the samples had been fortified and placed in the refrigerator. Time 0 for the CONTROL began once the unfortified HPHDM was aliquoted in the glass container and refrigerated. Samples were stored in the refrigerator throughout the study and a daily log of refrigerator temperatures was kept. Every 24 hours, each 5 mL aliquot was opened, and 1 mL of milk was removed using a sterilized pipette and transferred to a clean, glass bottle. The samples were reseal ed and returned to the refrigerator. The 1 mL aliquot was labeled with the storage time (0, 24, 48, 72, 96 hours) and immediately identified the treatment group . When the 96 -hour sample was added to the specimen bag, the bag was labeled with the date on which the last sample was collected. When all 12 batches had been processed, they were packaged on dry ice and shipped from Westchester Medical Center to the Nutr ition Department at the University of North Carolina Greensboro (Greensboro, NC) for analysis. One batch was missing the F -NEUT treatment; therefore, the study generated 235 total samples for analysis, with a total of 60 samples across 5 time points for th e unfortified, F -ACID, and F -HUM treatments, and 55 samples across 5 time points for the F -NEUT treatment. In the laboratory, samples were thawed at room temperature and with the help of body heat, being held in the hands and pockets of the lab technician , mixed using a vortexer for until analysis. Samples were stored frozen for between 4 and 15 months, and similar thawing techniques were employed before sample an alysis. Sample Analysis

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For each sample collected, IgA activity, lysozyme activity, and total protein content were assessed. IgA activity was measured by enzyme -linked immunosorbent assay, which has previously been described in detail (17,18) . Escherichi a coli acquired from the STEC Center (Michigan State University, East Lansing, MI) were used to prepare an antigen for coating the wells of a microplate. IgA antibodies from human milk bind to the E coli antigens and also bind with anti -human -IgA antibody labeled with horseradish peroxidase (part number A0295, Sigma -azino -bis -3-ethylbenzothiazoline -6-sulfonic acid (part number A1888, Sigma Aldrich) was used as a colorimetric substrate. Absorbance was meas ured at 405 nm via spectrophotometry (Synergy HT, BioTek Instruments, Winooski, VT) and IgA activity was computed based on a known human colostrum IgA standard (part number I2636, Sigma Aldrich). Lysozyme activity was analyzed by changes in turbidity to a microbial suspension of Micrococcus lysodeikticus (part number NC9310237, Fisher Scientific, Hampton, NH) at 450 nm over the course of 7 minutes, measured by spectrophotometry (Synergy HT, Bio -Tek Instruments) (19,20) . Total protein was measured by the b icinchoninic acid (BCA) method (part number PI23225, Fisher Scientific) (21) . This assay measures the reduction of Cu+ 2 by the acidic side chains of human milk proteins and the resulting color change, induced by BCA, which exhibits a strong absorbance at 5 62 nm. Samples were diluted 1:20 with deionized water and analyzed via spectrophotometry (Synergy HT, Bio -Tek Instruments) alongside known bovine serum albumin standards. For each control sample, all corresponding fortified samples and all time points (4 treatments × 5 time points = 20 samples total) were measured on the same 96 -well plate to eliminate interassay variability. Statistical Analysis Statistical analysis was performed using SAS software 9.4 Enterprise Edition (SAS Institute, Inc, Cary, NC). Descriptive statistics were computed for the main effects of treatment and time. Repeated measures were assessed using the mixed procedure to determine the effects of treatment, time, and an interaction between treatment and time. Main effects that were statistically different were evaluated using an analysis of variance with a Tukey adjustment for multiple comparisons. P values were set at 0.05. This study was classified as exempt by the institutional review board at the University of North Carolina Greens boro (protocol 16 -0411). RESULTS Each analytical test was performed in triplicate and the resulting average coefficients of variation for assays were as follows: total protein, 2.9%; lysozyme activity, 9.4%; and IgA activity, 2.3%. For IgA activity, all R2 values were >0.996, and for lysozyme, all R2 values were >0.979. The F -NEUT treatment group was missing in sample 1; therefore, analysis was performed with the incomplete dataset of 12 samples, and with a complete dataset of 11 samples. None of the

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sign ificant conclusions changed; therefore, all data presented represent analysis using the 11 samples in which all treatments and time points were available. Effect of Fortification at Baseline (Time 0) Using the repeated measures analysis, there was a high ly significant impact of treatment ( P < 0.001) for all dependent variables. Descriptive statistics by treatment at baseline (time 0) are summarized in Table 1. Table 1. Descriptive statistics by treatment at baseline (time = 0) Control F-ACID F-NEUT F-HUM Samples 11 11 11 11 Protein, g/dL 1.2 (0.1)* 2.0 (0.2) ƒ 2.2 (0.1) ƒ 2.5 (0.1) ⁄ Lysozyme, units/mL 5270 (890)* 3340 (1660) ƒ 4530 (1150) ⁄ 6230 (500) § IgA, mg/dL 80.3 (22.4)* 70.0 (12.3) ƒ 89.5 (18.1)* 144.4 (16.1) ⁄ Data represent means and standard deviations. Differences between groups were assessed by analysis of variance (ANOVA) analysis with a Tukey test for multiple comparisons. Entries in the same row with different superscripts are statistically different (P<0.05). F-ACID = acidic bovine Œderived fortifier; F -HUM = human milk Œderived fortifier; F -NEUT = neutral bovine Œderived fortifier; IgA = immunoglobulin A; UNFORT = unfortified. All 3 fortifiers significantly increased the total protein concentration of HPDHM (F -ACID 2.0 g/dL, F -NEUT 2.2 g/dL, F -HUM 2.5 g/dL, P < 0.05) compared to control (1.2 g/dL). Total protein was not significantly different between F -ACID and F -NEUT treatments ( P = 0.15). As expected F -HUM treatment group had a significantly higher protein concentrati on compared with all other treatments ( P < 0.0001). Lysozyme activity was significantly different in all treatment groups ( P < 0.001). Fortification with F -ACID resulted in a significant decrease in lysozyme activity compared to all other treatments ( P < 0.001), with a 37% reduction compared to control. Lysozyme was undetectable at all time points in the F -ACID treatment group of sample 5. Mean lysozyme activity was significantly higher in F -HUM group compared with all other treatments ( P < 0.001), with an 18% increase compared to control. IgA activity was not significantly different between F -NEUT and control samples ( P = 0.23). There was significant decrease in mean IgA activity with F -ACID treatment ( P < 0.001) compared to all other treatments, with a 1 3% reduction compared to control. There was a significantly increase in mean IgA activity in F -HUM treatment group ( P < 0.001) compared to the other treatments, with an 80% increase compared to control. Box and whisker plots of the baseline data (time 0) a re presented in Figure 1. PAGE - 6 ============ Figure 1. Holder -pasteurized donor human milk (n = 11 per treatment) at baseline (time 0) based on fortification status. Distributions with the same letter are not statistically different ( P > 0.05) using an analysis of variance (ANOVA) analysis with a Tukey test for multiple comparisons. In the box and whisker plots, the box spans the interquartile range, the line represents the median, the diamond represents the mean, and the top and bottom lines extend to t he highest and lowest observations.

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due to the fact that these samples were mixed to 28 kcal/oz due to clinical need at the time of sample collection, compared to the other treatments, which were mixed to 24 kcal/oz. At baseline, HPDHM fortified with F -ACID exhibited si gnificant declines in both lysozyme and IgA activity compared to all other treatments. Anytime acidity is increased, the risk for deactivating and denaturing proteins increases. It is quite likely that this is the case here. Although the pH of the HPDHM in this study was not measured due to small sample volume, Donovan et al (23) found that fortification with an acidic HMF resulted in a pH of 4.96 in preterm HPDHM and a pH of 5.10 in term HPDHM. Quan et al (26) reported a 19% reduction in the lysozyme activ ity, and no change in IgA, in raw human milk fortified with a human milk fortifier that was available in the early 1990s, although no additional information on the fortifier characteristics were provided. Reduction of the bioactive factors in HPDHM through the use of acidic fortifiers may have immune consequences for the vulnerable preterm neonate. Emerging evidence also suggests acidic fortifiers lead to higher rates of feeding intolerance and metabolic acidosis in the preterm infant (27) . Study Limitations In order to aliquot and complete analyses in the laboratory, the HPDHM samples underwent 4 freeze -thaw cycles, which is 2 more than traditionally encountered in normal use (12,28) . Each freeze/thaw cycle brings with it the risk of destabilized casein micelles and the altered quaternary structure of whey proteins, which can result in the formation of precipitates (29) . In our study, the F-ACID treatment resulted in precipitates in the samples which required additional handling steps so as not to interfere with spectrophotometry readings. These samples were thawed in a shaking water bath at 35°C and 80 rpm for 60 minutes to improve homogenization. It is possible that the multiple freeze -thaw cycles contributed to this phenomenon. That being said, all samples were subject to the same number of freeze -thaw cycles, which allows for comparison across the study. In the hospital feeding room, the F -HUM was mixed to 28 kcal/oz, although both the F -ACID and F -NEUT were mixed to 24 kcal/oz. The human milk Œbased fortifier used in this study was a higher calorie and protein fortification than originally planned due to clinical needs in the hospital during the study period, and it would be expected to provide more protein than the bovine milk Œbased fortifiers. This explains the average higher protein concentrations measured in this treatment group compared to the others. However, it is also likely that, had the human milkŒbased HMF been mixed to 24 kcal/oz like the F -ACID and F -NEUT HMF, the levels of lysozyme a nd IgA activity would still have been significantly higher than all other treatments, due to the fact that only F -HUM would likely contain meaningful quantities of these antimicrobial proteins. The BCA assay over -reports actual protein content; however, it has been validated as reliable for measuring human milk ( R2 > 0.99) and is therefore appropriate for assessing differences between groups (21) . We did not evaluate bacterial growth in our study, which is an important consideration in the NICU setting. Gaps in Literature and Future Research Implications Very few studies have specifically evaluated the impact of long -term refrigerated or frozen storage of HPDHM. Many address long -term storage in combination with other pasteurization methods, treatments, an d outcomes, which can make the results specific to storage difficult to

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assess. In addition, small sample size and the use of milk samples from a single donor rather than samples of pooled HPDHM are common limitations. In order to more accurately represent HPDHM found in the NICU, future studies should use pooled milk samples in clinically relevant volumes and be designed to emulate clinical practices, with the opening and closing of refrigerators and containers. More research is also needed into how fortif iers impact essential micronutrients in HPDHM. CONCLUSIONS As expected, fortification with liquid bovine and human milk Œbased fortifies significantly increases the protein concentration in donor human milk. Human milk Œbased fortifier provides additional IgA and lysozyme which may be beneficial. Our findings of lack of negative impact of refrigeration storage time on the protein and bioactive components of donor milk strengthen the recent HMBANA recommendation to extend storage time to 48 hours. The observ ed decline in IgA and lysozyme with acidic fortification is concerning and warrants further studies. REFERENCES 1. AAP SECTION ON BREASTFEEDING. Breastfeeding and the use of human milk. Pediatrics 2012; 129:e827 Œe841. 2. Lowe NK. The surgeon general’s call to action to support breastfeeding. J Obstet Gynecol Neonatal Nurs 2011; 40:387 Œ389. 3. WHO. Infant and Young Child Nutrition: Biennial Progress Report. World Health Assembly 61.20. Geneva, Switzerland: World Health Organization; 2008. 4. Perrin MT. Donor human mil k and fortifier use in United States level 2, 3, and 4 neonatal care hospitals. J Pediatr Gastroenterol Nutr 2018; 66:664 Œ669. 5. Parker MGK, Barrero -Castillero A, Corwin BK, et al. Pasteurized human donor milk use among US level 3 neonatal intensive care uni ts. J Hum Lact 2013; 29:381 Œ389. 6. Perrine CG, Scanlon KS. Prevalence of use of human milk in US advanced care neonatal units. Pediatrics 2013; 131:1066 Œ1071. 7. The Human Milk Banking Association of North America. Best Practice for Expressing, Storing and Hand ling Human Milk in Hospitals, Homes, and Child Care Settings. 4th edFort Worth, TX: Human Milk Banking Association of North America; 2019. 8. Cohen RS, Huang C -FR, Xiong SC, et al. Cultures of Holder -pasteurized donor human milk after use in a neonatal intens ive care unit. Breastfeed Med 2012; 7:282 Œ284. 9. Vickers AM, Starks -Solis S, Hill DR, et al. Pasteurized donor human milk maintains microbiological purity for 4 days at 4°C. J Hum Lact 2015; 31:401 Œ405. 10. Meng T, Perrin MT, Allen JC, et al. Storage of unfed an d leftover pasteurized human milk. Breastfeed Med 2016; 11:538 Œ543. 11. Taylor E, Labbok M. Donor human milk access and use in the United States: Findings and recommendations. Carolina Global Breastfeeding Institute.

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28. Section VI. Milk bank procedures. In: Guidelines for the Establishment and Operation of a Donor Human Milk Bank . FortWorth, TX: The Human Milk Banking Association of North America; 2018:34 Œ45. 29. García -Lara NR, Escu der -Vieco D, García -Algar O, et al. Effect of freezing time on macronutrients and energy content of breastmilk. Breastfeed Med 2012; 7:295 Œ301.

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