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    Issue Date: September 2022

    Baby Backwash Can Trigger Immune Response in Milk

    • When a human infant suckles, some of their saliva gets pulled back into the nipple and can travel into the milk ducts. Researchers believe infant backwash could inform the mammary gland about the infant’s health.
    • A new study experimentally infected mice pups, who also backwash while suckling, with an enteric virus and discovered that the pups transmitted this virus while nursing to their mother’s mammary gland, which resulted in an increase in milk antibody concentration.
    • The increase in immune factors in milk following mammary infection could be an evolved response to protect the mammary gland, to protect the baby, or protect both the mammary gland and the baby.

    Mothers have many ways to tell if their infant might be sick—an increase in body temperature, a decrease in appetite, or even small changes in sleep habits could all hint that an illness is brewing. But milk researchers have long suspected that nursing infants have another, more direct means of communicating their health status to their mothers: backwash.

    When a human infant suckles, their tongue movements create a vacuum that draws milk from the ducts to the mouth. Suckling also creates negative pressure that pulls fluids in the reverse direction, taking milk and saliva from the infant’s mouth through the nipple and into the milk ducts [1-3]. Researchers believe that if this milky backwash—called retrograde duct flow—contains information that signals an illness, it could trigger a local response in the mammary gland to increase the production of immune factors [4].

    Can a sick infant’s backwash actually change the composition of their mother’s milk? A handful of studies [5–8] on human milk have attempted to test this intriguing hypothesis. Immune factors in milk from healthy mothers were found to increase during times of infant illness, supporting the idea that infants can communicate their health status to mammary gland receptors. However, these studies stopped short of singling out baby backwash as the source of communication (after all, infants can share a lot of bodily fluids with their mothers, especially during times of illness).

    A new study [9] on suckling mice pups—who also backwash during nursing [10]—adds in the important piece missing from human research by tracking a single signal from infant saliva to the mammary gland, to milk. The investigators were interested in determining whether enteric viruses, such as norovirus, replicate in salivary glands. To demonstrate that saliva transmits these types of viruses, the study team performed a very clever set of experiments involving suckling pups, mammary glands, and milk composition [9].

    In the first part of the study, mice pups were inoculated with either murine norovirus (MNV-1) or rotavirus (EDIM), but their mothers (dams) were not. After three days, the team identified a spike in the levels of secretory immunoglobulin A (sIgA) in the intestines of the pups and in the dam’s milk. Moreover, they found a 105-fold increase in viral RNA in dam mammary glands (specifically in the cells lining the milk ducts) [9]. To rule out that the dams were infected by the infants through contact with fecal material, they also orally inoculated a group of dams that were pup-free. Although these dams’ intestines had an increase in viral RNA (signaling infection), there was no detectable viral RNA in their mammary glands nor was there a surge of sIgA in their milk [9].

    To further demonstrate an oral route of transmission, the study team also conducted a cross-over experiment. A group of mouse pups (pups A) were orally inoculated with EDIM and allowed to stay with their mother (dam A) for one day. Then, the researchers switched dams; dam A was placed with uninoculated (healthy) pups (pups B), while dam B (also healthy) was placed in the cage with pups A. Two days later, the researchers identified a 104-fold increase in viral genome levels in mammary glands from both dam A and dam B and a 106-fold increase in the viral genome levels of both pups A and B [9]. Dam A showed no evidence of infection and is presumed to have passed on the virus to pups B via infected mammary glands (courtesy of nursing pups A for one day).

    Both experiments demonstrate that saliva is a route of transmission for enteric viruses, but they are also the most clear-cut evidence to date that pathogens backflow from infants’ mouths to their mother’s mammary gland during nursing, and that the resulting localized infection in the mammary gland triggers an immune response in milk [9]. Perhaps even more incredible, this immune response was quicker than when the dam was infected, and milk antibodies were maternally derived via the entero-mammary pathway.

    The benefits of passing on pathogens through retrograde duct flow to the infant are clear; the surge in immune factors (many that are specific to the pathogen causing the infection) likely contributes to helping them clear that infection. However, in a previous SPLASH! piece, lactation biologist Foteini Kakulas (né Hassiotou) made the important observation that the increase in immune cells in human milk not only functions to protect the infant, they also protect the lactating breast. Kakulas believes the surge in immune factors in milk following mammary infection could be an evolved response to maternal infection, infant infection, or maternal and infant infection. But regardless of the why, the response of the mammary gland provides yet another example of how milk protects infants, be they mice or (hu)man.



    1. Geddes DT, Sakalidis VS. Breastfeeding: how do they do it? Infant sucking, swallowing, and breathing. Infant. 2015; 11(5): 146-50
    2. Gardner H, Kent JC, Hartmann PE, Geddes DT. Asynchronous milk ejection in human lactating breast: case series. Journal of Human Lactation. 2015 May;31(2):254-9.
    3. Geddes DT, Kent JC, Mitoulas LR, Hartmann PE. Tongue movement and intra-oral vacuum in breastfeeding infants. Early Human Development. 2008 Jul 1;84(7):471-7.
    4. Bode L, McGuire M, Rodriguez JM, Geddes DT, Hassiotou F, Hartmann PE, McGuire MK. It’s alive: microbes and cells in human milk and their potential benefits to mother and infant. Advances in Nutrition. 2014 Sep;5(5):571-3.
    5. Breakey AA, Hinde K, Valeggia CR, Sinofsky A, Ellison PT. Illness in breastfeeding infants relates to concentration of lactoferrin and secretory Immunoglobulin A in mother’s milk. Evolution, Medicine, and Public Health. 2015 Jan 1;2015 (1): 21-31.
    6. Bryan DL, Hart PH, Forsyth KD, Gibson RA. Immunomodulatory constituents of human milk change in response to infant bronchiolitis. Pediatric Allergy and Immunology. 2007 Sep;18(6):495-502.
    7. Hassiotou F, Hepworth AR, Metzger P, Lai CT, Trengove N, Hartmann PE, Filgueira L. Maternal and infant infections stimulate a rapid leukocyte response in breastmilk. Clinical & Translational Immunology. 2013 Apr;2(4), e3.
    8. Riskin A, Almog M, Peri R, Halasz K, Srugo I, Kessel A. Changes in immunomodulatory constituents of human milk in response to active infection in the nursing infant. Pediatric Research. 2011 Feb;71(2): 220-5.
    9. Ghosh S, Kumar M, Santiana M, Mishra A, Zhang M, Labayo H, Chibly AM, Nakamura H, Tanaka T, Henderson W, Lewis E. Enteric viruses replicate in salivary glands and infect through saliva. Nature. 2022 Jul;607(7918):345-50.
    10. Ramsay DT, Kent JC, Owens RA, Hartmann PE. Ultrasound imaging of milk ejection in the breast of lactating women. Pediatrics. 2004 Feb;113(2):361-7.






    Bring Back the Fat in Milk: Kids’ Edition

    • Current dietary guidelines recommend children two to 18 years old consume low- or non-fat milk to limit the risk for overweight and obesity.
    • A large prospective cohort study found consumption of higher fat milk during early childhood was associated with a lower chance of being overweight or obese during early adolescence.
    • Dairy fats can have positive effects on heart health and the risk for metabolic diseases.
    • Scientific research on adults and children does not support dietary advice to limit or avoid saturated fats from milk.

    An apple a day keeps the doctor away. Feed a cold, starve a fever. Eating carrots will improve your eyesight. Everyone knows these are old wives’ tales and not actual dietary advice. But what happens when actual dietary advice becomes an old wives’ tale?

    For decades, the Dietary Guidelines for Americans (DGA) [1] has recommended that individuals over two years old consume low-fat or non-fat dairy products to limit their intake of saturated fats, despite mounting scientific evidence that dairy fat has positive effects on adult heart health and the risk for developing other chronic diseases, including diabetes.

    New research [2-4] on children and adolescents is now calling into question the dairy fat guidance for these age groups as well. Currently, children aged two to 18 are encouraged to include low-fat or non-fat dairy foods, particularly milk, in their diet, to reap the nutritional benefits they offer for growth and development and to avoid the higher calorie count and saturated fat intake provided by whole milk [1]. The concern for weight gain in this age group is justified; 41% of children and adolescents in the U.S. are overweight or obese [1]. However, the association of milk fat content with weight gain in children is still not well understood [2].

    Going against the conventional wisdom that low-fat milk has a protective effect toward obesity, a team of pediatricians and nutritionists [3] hypothesized that higher-fat cow’s milk consumption (whole or 2%) during early childhood would be associated with lower measures of adiposity and cardiometabolic risk during early adolescence than lower-fat (1% or non-fat) cow’s milk consumption. This is a tricky hypothesis to test because so many factors contribute to body composition, including diet, genetics, physical activity, and socio-economic status. To determine the influence of just one factor (milk fat content) required the collection of a large amount of additional data and advanced statistical modeling to account for potential confounding factors. Of particular concern was a type of bias called confounding by indication. Parents of young children that have a higher body mass index (BMI) may be more likely to select low-fat or non-fat milk to reduce their child’s body weight, whereas parents of children with a lower BMI may be more likely to continue providing whole milk. BMI during childhood (the indication) could bias the milk choice and imply an association when in fact there isn’t one [3].

    The 796 study participants were all part of a prebirth, Boston-area prospective cohort called Project Viva [3]. Participants were first evaluated at an early childhood visit that took place close to the child’s third birthday. Using a food-frequency questionnaire about the previous month’s diet, the team determined cow’s milk fat intake and cow’s milk intake frequency for each child. Nearly a decade later (mean age = 13.1 years), participants were measured for body composition (including lean mass, total fat mass, and truck fat mass) and cardiometabolic health (including blood pressure, HDL cholesterol, and blood triglycerides) [3].

    The study did not find any protective effect of lower-fat cow’s milk on adiposity or cardiometabolic risk during adolescence [3]. Instead, after controlling for multiple confounding variables and adjusting for baseline anthropometrics to reduce confounding by indication, the consumption of whole or 2% milk during early childhood was associated with lower odds of overweight or obesity in early adolescence. The investigators also found no association between the frequency of milk consumption at age three and any trends in body mass or measurements of cardiometabolic health in adolescence [3]. These results were consistent with a 2021 prospective cohort study that found children 9 months to 8 years old that drank whole compared with reduced fat milk had lower odds of overweight two years later [4].

    A simple explanation for these somewhat counterintuitive findings is greater satiety (or a feeling of fullness) from whole or 2% milk compared with lower-fat milks [3]. Children drinking full-fat milk with a meal may eat less than those drinking non-fat milk and therefore consume fewer calories overall. But the study authors also point to the fact that milk fats are biologically different from saturated fats found in other foods [3]. Compared with red meat, milk has a higher proportion of short- and medium-chain fatty acids, which could positively influence cholesterol levels, and more odd-chain saturated fatty acids, which have been shown to have positive effects on heart health and the risk for developing type 2 diabetes. Moreover, these beneficial types of fatty acids have unique packaging in milk: membrane-bound bubbles called globules. The membrane that surrounds the milk fat globule has been found to have immune-boosting properties, including interacting with probiotic gut bacteria [5]. It is possible that these probiotic effects have a positive influence on metabolism like that of probiotics provided by fermented foods.

    If saturated fats from milk are not associated with weight gain and can have positive effects on health, why are nutritional guidelines still asking adults and children to avoid them? Old habits die hard; saturated fats have been the bad guys of the nutritional world for decades and large-scale policy changes to encourage their consumption only in particular foods may not be successful. The Project Vida study team believe that the best policy change would be to remove any guidance about milk or yogurt fat content after age two [3]. This change could go a long way in taking the stigma off milk fat and would move nutritional advice in the direction of being food-based rather than based on the particular type of fat.


    1. US Department of Agriculture and US Department of Health and Human Services. Dietary Guidelines for Americans, 2020-2025. 9th Washington, DC: US Government Publishing Office; 2020.
    2. O’Sullivan TA, Schmidt KA, Kratz M. Whole-fat or reduced-fat dairy product intake, adiposity, and cardiometabolic health in children: a systematic review. Advances in Nutrition. 2020 Jul 1;11(4):928-50.
    3. McGovern C, Rifas-Shiman SL, Switkowski KM, Woo Baidal JA, Lightdale JR, Hivert MF, Oken E, Aris IM. Association of cow’s milk intake in early childhood with adiposity and cardiometabolic risk in early adolescence. The American Journal of Clinical Nutrition. 2022 Aug;116(2):561-71.
    4. Vanderhout SM, Keown-Stoneman CD, Birken CS, O’Connor DL, Thorpe KE, Maguire JL. Cow’s milk fat and child adiposity: A prospective cohort study. International Journal of Obesity. 2021 Dec;45(12):2623-8.
    5. Kosmerl E, Rocha-Mendoza D, Ortega-Anaya J, Jiménez-Flores R, García-Cano I. Improving human health with milk fat globule membrane, lactic acid bacteria, and bifidobacteria. Microorganisms. 2021 Feb 9;9(2):341.

    Yogurt Enhanced with Prebiotics and Probiotics Improves Calcium Uptake in Young Women

    • Previous studies have suggested that prebiotics and probiotics can have a positive impact on calcium absorption in the gut, but few studies have examined how the combination of the two (synbiotics) influences calcium absorption.
    • A crossover study gave 30 young women either a treatment of yogurt enhanced with synbiotics or a control for three weeks before switching treatments.
    • Two-thirds of participants had 24% improved calcium uptake on average.

    Yogurt can be a great source of calcium, with one 170-g tub containing over 200 mg of the critical nutrient [1]. Calcium is vital in building healthy bones and teeth, as well as muscle, heart, and nerve function, but most calcium from food can’t be retained by the gut. Dietary calcium is not always in a form that is easily soluble, and only around 30% is absorbed into the body [2]. Luckily, a new study conducted on young women shows this absorption rate can be improved 24% on average by consuming yogurt supplemented with synbiotics—a combination of pre- and probiotics [3].

    Previous animal studies have shown that prebiotics—fibers that are beneficial for gut bacterial growth—aided in calcium retention in the gut [4], but results have been murkier in human trials [5, 6]. Studies showing the effects of probiotics—beneficial gut bacteria and yeast—on calcium retention in humans are also few and far between [7, 8].

    “There were no studies in the literature testing this particular combination of prebiotic and probiotic functional dairy foods on calcium absorption,” says Carmen Donangelo, a researcher at the Universidad de la Republica in Montevideo, Uruguay, and a lead author of the new paper. Donangelo and her colleagues investigated whether consuming yogurt supplemented with synbiotics affected calcium absorption in young adult women.

    For the study, published in The Journal of Nutrition, Donangelo and her colleagues recruited 30 women between the ages of 21 and 33. Half of the study participants were given yogurt supplemented with inulin, a common plant prebiotic, as well as the probiotic Lactobacillus rhamnosus. The other half of study participants were given the control—yogurt without the synbiotic supplement. After consuming the yogurt for three weeks, study participants were given an isotopic calcium tracer dissolved in orange juice, and calcium retention was studied by measuring how much excess calcium was excreted in the urine over a 36-hour period. The first treatment was then followed by a three-week “washout” period so that participants could return to their baseline calcium absorption levels, and afterwards, the participants that had the fortified yogurt were given control yogurt and vice versa for three weeks before measuring calcium retention again. The results between treatment sessions were compared for each participant [3].

    Donangelo says that a 24% increase in calcium absorption was predictable, given what was known from previous literature. She explains that the majority of calcium is absorbed in the small intestine, whereas only about 5% of dietary calcium is absorbed in the colon.

    The synbiotics likely work, she explains, by creating conditions that are more conducive to healthy gut bacterial growth in the large intestine. These beneficial bacteria could work by reducing pH in the gut, which helps with absorption, improving the health of the mucosal lining of the large intestine, or by creating short-chain fatty acids that form molecular complexes with calcium and aid in passive absorption.

    The study also found that participants who did have improved calcium retention generally had a higher intake of vegetables, potassium, calcium, and protein compared with those who did not have a response. This was likely because a diet higher in vegetables makes the microbiotic environment more conducive to calcium uptake [3].

    Donangelo says one of the study’s main limitations was that they did not measure the absolute amount of calcium in the bloodstream, a method that would require an intravenous injection of calcium. Rather, calcium was measured less invasively through urine samples and compared between treatments in the same participant to give a relative measure.

    In the future, Donangelo says it would be ideal to study the long-term effects of synbiotic yogurt consumption, especially the effects on bone health. However, recruiting participants for such a lengthy study would be difficult. In the meantime, shorter-term studies could examine how synbiotics affect uptake of other vital nutrients, or how other co-consumed nutrients such as vitamin D might influence calcium absorption.



    1. Yoghurt, whole milk, plain nutrition facts and analysis [Internet]. Available from:
    2. The Nutrition Source. Calcium | The Nutrition Source| Harvard T.H. Chan School of Public Health [Internet]. Available from:,heart%20rhythms%20and%20nerve%20functions.
    3. Cornes R, Sintes C, Pena A, Ablin S, O’Brien K, Abrams S, Donangelo CM. Daily intake of a functional synbiotic yogurt increases calcium absorption in young adult women. J. Nutr 2022;152(7):1647-54.
    4. Scholz-Ahrens KE, Ade P, Marten B, Weber P, Timm W, Asil Y, Glüer CC, Schrezenmeir J. Prebiotics, probiotics, and synbiotics affect mineral absorption, bone mineral content, and bone structure. J Nutr 2007;137(3):838S–46S.
    5. Van den Heuvel EG, Schaafsma G, Muys T, van Dokkum W. Nondigestible oligosaccharides do not interfere with calcium and nonheme-iron absorption in young, healthy men. Am J Clin Nutr 1998;67(3):445–51.
    6. Tahiri M, Tressol JC, Arnaud J, Bornet FR, Bouteloup-Demange C, Feillet-Coudray C, Brandolini M, Ducros V, Pépin D, Brouns F, Roussel AM. Effect of short-chain fructooligosaccharides on intestinal calcium absorption and calcium status in postmenopausal women: a stable-isotope study. Am J Clin Nutr 2003;77(2):449–57.
    7. Narva N, Nevela R, Poussa T, Korpela R. The effect of Lactobacillus helveticus fermented milk on acute changes in calcium metabolism in postmenopausal women. Eur J Nutr 2004;43(2):61–8.
    8. Asemi Z, Esmaillzadeh A. Effect of daily consumption of probiotic yogurt on serum levels of calcium, iron, and liver enzymes in pregnant women. Int J Prev Med 2013;4:949–55.

    Remarkable Diversity of Milk-digesting Bacteria in Hunter-gatherer Infants

    • People from non-industrialized lifestyles have a more diverse gut microbiome, which is associated with fewer autoimmune diseases.
    • Researchers took stool samples from infants and mothers from the Hadza hunter-gatherers of Tanzania and performed a metagenomic analysis of gut microbiomes.
    • Samples taken from infants from industrialized lifestyles had less diversity in milk-digesting bacteria overall, a finding that influences the trajectory of gut bacterial growth into adulthood.
    • Much of the diversity in the non-industrialized microbiome is also transmitted vertically from family members and the local environment.

    The Hadza people in northern Tanzania are regarded as one of the last true hunter-gatherer societies left in the world [1]. They typically live in social groups of between five and 30 people, and they spend much of their day hunting wildlife or foraging for honey, tubers, or fruit [2]. Reflecting a unique and rarefied lifestyle is also their diverse and complex gut microbiome.

    The gut microbiota of people living in non-industrialized societies—societies characterized by a lack of processed food, modern antibiotics and medicine, and without excessive sanitization—typically are much more diverse and healthier than those of people from industrialized societies [3]. A more diverse microbiome is associated with all sorts of health outcomes, ranging from maintaining a healthy weight, to brain health, to preventing autoimmune disorders [4], and the development of the microbiome from birth to adulthood is well characterized in industrialized populations [5]. However, the same information is lacking for non-industrialized people [6]. In a new paper published in Science, researchers performed a metagenomic analysis of Hadza fecal samples to determine the characteristics of gut microbiomes starting from birth [2].

    Matthew Olm, a postdoctoral researcher at Stanford University and lead author of the paper [2], says one of the main questions the study was trying to answer was how the complex microbiome of Hadza hunter-gatherers came to be. “We know those steps in industrialized populations like Americans or Europeans, but the adults of industrialized and non-industrialized populations are very different,” he explains. “So, the goal is to look at the microbiome of infants in these non-industrialized contexts to see the building the blocks that would establish adult microbiomes.”

    For the study, 39 Hadza infant fecal samples were analyzed, as well as samples from 23 infant and mother pairs. “The unique thing about this study was the amount of information we generated based on these samples,” says Olm. A large metagenomic analysis was performed using the samples, which was able to give a detailed and simultaneous view of the genetics in the gut microbiota. “That let us see the more rare species,” says Olm. “You get a more in-depth scope of what’s going on.”

    Overall, the study found that gut microbiota across fecal samples from people living in industrialized and non-industrialized countries were most similar at birth, but microbiomes quickly diverged within the first few months of life. Hadza infants were found to have a more diverse microbiome with 745 species of bacteria detected, 175 of which were new to microbiome science [2]. Hadza infants also had more of a beneficial gut bacterium, a probiotic called Bifidobacterium longum subsp. infantis, which has been known to play an important role in immunological development [7]. In contrast, infants from industrialized societies tended to have more Bifidobacterium breve, which is thought to competitively exclude the growth of B. infantis [2].

    “It’s interesting that there’s a strain that we’re using as a probiotic in industrialized countries, but it’s just naturally there in Hadza microbiomes,” says Olm. “It just points to how potentially industrialization has impacted our microbiome for the worse.”

    The analysis also revealed that samples from people living non-industrialized lifestyles had a higher diversity and incidence of genes associated with digesting human milk sugars, even when controlling for breastfeeding duration. The genes, or human milk oligosaccharide degradation clusters, are associated with improved immunological health and fewer autoimmune disorders [8]. Olm says that this suggests that even among the B. infantis bacteria, the Hadza may have strains that are more beneficial.

    “The human microbiome is important for a bajillion reasons but it seems to be especially important in early life—specifically for this training of the immune system,” says Olm, adding that one of the key takeaways from the study is that the microbiome is determined very early on in life, which has cascading effects into adulthood. Furthermore, analyzing mother-infant fecal samples and comparing samples between group members show that many of the microbiota obtained in early life are transmitted vertically from the parents and from the local community. “With the Hadza there’s a lot of pre-mastication of food, so you easily imagine strains being shared that way,” explains Olm, adding that children are also typically raised communally. “It’s interesting thinking about how different ways of raising kids is going to impact their microbiome.”



    1. National Geographic Society. Hadza| National Geographic Society [Internet]. Available from:
    2. Olm MR, Dahan D, Carter MM, Merrill BD, Yu FB, Jain S, Meng X, Tripathi S, Wastyk H, Neff N, Holmes S, Sonnenburg ED, Jha AR, Sonnenburg JL. Robust variation in infant gut microbiome assembly across a spectrum of lifestyles. Science. 2022; 376: 1220-1223.
    3. Stewart CJ, Ajami, NJ, O’Brien JL.Temporal development of the gut microbiome in early childhood from the TEDDY study. Nature. 2018;562583–588.
    4. com. Why the Gut Microbiome is Crucial for Your Health [Internet]. Available from:
    5. Fragiadakis GK, Smits SA, Sonnenburg ED, Van Treuren W, Reid G, Knight R, Manjurano A, Changalucha J, Dominguez-Bello MG, Leach J, Sonnenburg JL. Links between environment, diet, and the hunter-gatherer microbiome. Gut Microbes. 2019; 10(2): 216-227.
    6. de Goffau, MC, Jallow AT, Sanyang C, Prentice AM, Meagher N, Price DJ, Revill PA, Parkhill J, Pereira A, Wagner J. Gut microbiomes from Gambian infants reveal the development of a non-industrialized Prevotella-based trophic network. Nat Microbiol. 2022; 7: 132–144.
    7. Henrick BM, Rodriguez L, Lakshmikanth T, Pou C, Henckel E, Arzoomand A, Olin A, Wang J, Mikes J, Tan Z, Chen Y, Ehrlich AM, Bernhardsson AK, Mugabo CH, Ambrosiani Y, Gustafsson A, Chew S, Brown HK, Prambs J, Bohlin K, Mitchell RD, Underwood MA, Smilowitz JT, German JB, Frese SA, Brodin P. Bifidobacteria-mediated immune system imprinting early in life. Cell. 2021; 184(15): 3884-3898.e11.
    8. Underwood MA, German JB, Lebrilla CB, Mills DA. Bifidobacterium longum subspecies infantis: champion colonizer of the infant gut. Pediatr Res. 2015; 77(1-2): 229-235.

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