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Preserving Milk Proteins while Inactivating Pathogens

    human donor milk, babies, pre-term infants, pathogens, pasteurization

    Written by: Sandeep Ravindran, Ph.D. | Issue # 117 | 2023

    • Milk banks use Holder pasteurization to inactivate pathogens in human milk.
    • Holder pasteurization also degrades many bioactive milk proteins important for infant health.
    • High-pressure processing preserves human milk proteins while inactivating pathogens.

    Human milk provides important health and growth benefits for infants, particularly preterm infants. But parents of preterm infants are not always able to provide them with sufficient milk, in which case preterm infants are often fed donor human milk from nonprofit milk banks [1-3]. 

    To reduce the risk of transmitting pathogens to infants through donor milk, milk banks apply a heat treatment known as Holder pasteurization (HoP) to donor human milk. HoP involves heating the milk to 62.5 ºC for 30 minutes, and results in a more than 5-log—or 100,000-fold—reduction in the major bacterial and viral pathogens in milk, inactivating 99.999% of them [4]. 

    HoP is thus excellent at making donor milk safe from most pathogens. But at the same time, it damages some bioactive milk proteins, including enzymes such as bile salt-stimulated lipase (BSSL), lactoferrin, and immune proteins such as immunoglobulin (Ig) A and IgG [5-9]. Unfortunately, this degradation of human milk proteins by HoP means that preterm infants may not be getting all the health benefits of consuming human milk.

    “I’ve been really interested in all the bioactives of human milk for a long time, particularly the proteins, and I’m interested in what all those things can do but also how to best preserve them so that all those proteins stay in the intact forms and have the best opportunity to improve the health of infants,” says David Dallas, Associate Professor in the nutrition program of the College of Health at Oregon State University. “Holder pasteurization is actually quite a good method, but there are some bioactive proteins that are still lost by Holder pasteurization, so the question was, can we improve upon that?”

    Preserving some of the bioactive compounds in human milk could help improve the growth of preterm infants. “One of the key proteins that we were interested in is called bile salt-stimulated lipase, and it contributes to a large degree to fat digestion in preterm infants,” says Dallas. BSSL degradation by HoP could affect infant fat absorption and growth. “There have been some studies that show that if you compare very premature infants fed their own parents’ milk versus donor pasteurized milk, the ones fed parents’ own milk grow much better and they also have shown they have a 30–40% higher lipid absorption compared with Holder-pasteurized milk,” he says. “I thought that was really cool because growth is a hugely important thing for preterm infants and is still somewhat of an unsolved problem in preterm infant nutrition, and lipids are the highest calorie source of milk, so it’s really important that lipids are fully absorbed,” says Dallas.

    Preserving milk proteins could also benefit infant immunity. Holder pasteurization partially degrades immune proteins called immunoglobulins that are very important for protecting infants from pathogens, says Dallas. “So there’s a lot of key proteins and the idea is let’s preserve them as well as we can,” he says. “As I learned about how heat processing can denature and cause other changes to proteins, I’ve been interested in trying to find better ways to process milk, and we heard about high-pressure processing as another method that has some promise for human milk and decided to examine that,” he says.

    High-pressure processing (HPP) uses water to transmit pressure without applying heat [10]. It is currently used in the food industry to inactivate microorganisms in products such as juice and some meats and seafoods. HPP is thought to inactivate microbes by disrupting their cell membranes and some microbial proteins [11].

    In a new study, Dallas investigated the minimum HPP conditions required to inactivate pathogenic bacteria and examined how this treatment affected bioactive milk proteins [12]. He and his colleagues spiked donor milk with bacteria and bacterial spores and examined what HPP conditions would deliver a 5-log reduction in pathogenic bacteria. “I think this is the first paper that did the actual spiking experiments with human milk as opposed to just using the natural abundances of bacteria that might be present in the milk, and we did that to ensure that we could achieve the same kind of standards as Holder pasteurization,” says Dallas. The researchers pooled raw human donor milk and inoculated it with several relevant pathogens such as Enterococcus faecium, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii, as well as spores of Bacillus subtilis and Paenibacillus spp.

    Dallas discovered HPP conditions that inactivated these microbes just as well as HoP. “We used 500 megapascals of pressure for nine minutes and we kept the temperature as close to zero as possible, and that was sufficient to create a greater than five-log reduction of the bacteria that we tested,” he says.

    Dallas then examined how HPP affected the bioactive proteins in human milk, in collaboration with Oregon State food microbiologist Joy Waite-Cusic, a co-author on the study. “We found better preservation of certain proteins that we tested including better preservation of IgA, IgG, IgM, lactoferrin, elastase, polymeric immunoglobin receptor, and BSSL activity,” he says. “So we were pretty pleased with those results,” he says. In contrast to HPP, HoP eliminates lactoferrin, elastase, and BSSL in human milk and causes a significant decrease in IgA, IgG, and IgM antibodies and polymeric immunoglobin receptor.

    These findings suggest that high-pressure processing of human donor milk could preserve more bioactive proteins than HoP and thus improve infant health outcomes. Improved immunoglobulin and lactoferrin levels could provide better protection against pathogenic bacteria and viruses, better elastase retention could improve protein digestion, and improved BSSL activity could improve lipid digestion and infant growth [13-15].

    Dallas says more studies need to be done before milk banks start using HPP on milk. “I think there’s still more work to do to verify that it’s the right technique, that it will be beneficial,” he says. “I think that for milk banks, probably the  number one concern is safety, so you have to have a really good safety profile,” says Dallas. Among other things, he plans to look at how HPP affects viruses. “In order to be as good as Holder pasteurization, it doesn’t necessarily have to be able to get rid of all viruses, but it should probably at least be able to do the equivalent of what Holder pasteurization does to convince milk banks, so that’s key,” says Dallas. 

    Milk banks also have practical and logistical considerations to consider, given that HPP equipment is large and expensive. “What we do is we pick up milk from a milk bank and then we take it to a high-pressure processing company, they charge for processing, and you tell them what settings you want to do and they’ll do that for you,” says Dallas. “That’s what we’re doing for our feeding studies, and I think that could be a feasible solution for milk banks,” he says.

    In follow-up experiments, Dallas plans to test the effects of HPP on other nutrients and milk components, including milk fat globules and exosomes. “It’s possible that by preserving those things better, we could have even better health outcomes,” he says. “Then we need to have testing in infants to show some sort of improvement, whether that’s growth or gut health outcomes,” says Dallas. 

    Dallas is currently doing a study where preterm infants in the neonatal intensive care unit are fed HPP and HoP milk. “We’re feeding the same baby one day with high-pressure processed milk, and the next day with Holder-pasteurized milk, and then we are looking at their stool samples and we are going to be comparing which one has better lipid absorption, so which one has the least amount of fat still in their stool,” he says. “The hope is that because we preserve the bile salt-stimulated lipase better, that we should have better lipid absorption in high-pressure processed milk-fed infants, so that’s what we’re working on now,” says Dallas. If he’s able to show better lipid absorption, he plans to then move on to a longer-term feeding study to look for changes in growth patterns between babies fed HoP milk and HPP milk.

    In the future, Dallas plans to continue refining milk treatment methods to preserve even more milk proteins and components while inactivating more pathogens. For example, neither HoP nor HPP can inactivate spore-forming bacteria, and the search is on for methods that can do that without degrading milk proteins. “If you’re doing some sort of mechanical process that degrades bacteria, chances are it can also degrade other things in milk, so it’s a really hard challenge,” says Dallas. “Ideally we could create a process that gets rid of bacteria and spores and also better preserves proteins,” he says.

    But HPP already improves upon HoP by providing an equivalent inactivation of pathogens while doing a better job of retaining key bioactive proteins. “It’s definitely  moving towards something that’s a better preservation technique,” says Dallas. “It shows that there is more flexibility in the human milk space to try some innovative techniques, so we need creativity in this space and it would be great to have more engineers and more food scientists entering this space,” he says.

    References

    1. Hill PD, Aldag JC, Chatterton RT, Zinaman M. Comparison of milk output between mothers of preterm and term infants: the first 6 weeks after birth. J Hum Lact. 2005 Feb;21(1):22-30.
    2. Unger S, Gibbins S, Zupancic J, O’Connor DL. DoMINO: Donor milk for improved neurodevelopmental outcomes. BMC Pediatr. 2014 May 13;14:123.
    3. Wight NE. Donor human milk for preterm infants. J Perinatol. 2001 Jun;21(4):249-54.
    4. Kontopodi E, Hettinga K, Stahl B, van Goudoever JB, M van Elburg R. Testing the effects of processing on donor human Milk: Analytical methods. Food Chem. 2022 Mar 30;373(Pt A):131413.
    5. Koh J, Victor AF, Howell ML, Yeo JG, Qu Y, Selover B, Waite-Cusic J, Dallas DC. Bile salt-stimulated lipase activity in donor breast milk influenced by pasteurization techniques. Front Nutr. 2020 Nov 12;7:552362.
    6. Silvestre D, Miranda M, Muriach M, Almansa I, Jareño E, Romero FJ. Antioxidant capacity of human milk: effect of thermal conditions for the pasteurization. Acta Paediatr. 2008 Aug;97(8):1070-4.
    7. Paulaviciene IJ, Liubsys A, Eidukaite A, Molyte A, Tamuliene L, Usonis V. The effect of prolonged freezing and Holder pasteurization on the macronutrient and bioactive protein compositions of human milk. Breastfeed Med. 2020 Sep;15(9):583-8.
    8. Demazeau G, Plumecocq A, Lehours P, Martin P, Couëdelo L, Billeaud C. A new high hydrostatic pressure process to assure the microbial safety of human milk while preserving the biological activity of its main components. Front Public Health. 2018 Nov 6;6:306.
    9. Espinosa-Martos I, Montilla A, de Segura AG, Escuder D, Bustos G, Pallás C, Rodríguez JM, Corzo N, Fernández L. Bacteriological, biochemical, and immunological modifications in human colostrum after Holder pasteurisation. J Pediatr Gastroenterol Nutr. 2013 May;56(5):560-8.
    10. Viazis S, Farkas BE, Jaykus LA. Inactivation of bacterial pathogens in human milk by high-pressure processing. J Food Prot. 2008 Jan;71(1):109-18.
    11. Peila C, Emmerik NE, Giribaldi M, Stahl B, Ruitenberg JE, van Elburg RM, Moro GE, Bertino E, Coscia A, Cavallarin L. Human milk processing: A systematic review of innovative techniques to ensure the safety and quality of donor milk. J Pediatr Gastroenterol Nutr. 2017 Mar;64(3):353-61.
    12. Liang N, Mohamed HM, Kim BJ, Burroughs S, Lowder A, Waite-Cusic J, Dallas DC. High-pressure processing of human milk: A balance between microbial inactivation and bioactive protein preservation. J Nutr. 2023 Sep;153(9):2598-611.
    13. Lönnerdal B. Bioactive proteins in human milk: health, nutrition, and implications for infant formulas. J Pediatr. 2016 Jun;173 Suppl:S4-9.
    14. Kell DB, Heyden EL, Pretorius E. The biology of lactoferrin, an iron-binding protein that can help defend against viruses and bacteria. Front Immunol. 2020 May 28;11:1221.
    15. Koh J, Victor AF, Howell ML, Yeo JG, Qu Y, Selover B, Waite-Cusic J, Dallas DC. Bile salt-stimulated lipase activity in donor breast milk influenced by pasteurization techniques. Front Nutr. 2020 Nov 12;7:552362.

    Contributed by

    Dr. Sandeep Ravindran

    Freelance Science Writer

    Sandeepr.com