Mednarodni znanstveni simpozij Vloga humanega mleka v razvoju èrevesne mikrobiote dojenèka

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1 Moje-MLEKO Moje-MLEKO My-MILK Mednarodni znanstveni simpozij Vloga humanega mleka v razvoju èrevesne mikrobiote dojenèka International scientific symposium on The role of human milk in development of breast fed child's intestinal microbiota Ljubljana, 18. oktober 2011 Zbornik prispevkov / Symposium proceedings

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3 Moje-MLEKO: Zbornik prispevkov / Mednarodni znanstveni simpozij "Vloga humanega mleka v razvoju črevesne mikrobiote dojenčka", Ljubljana, Slovenija, My MILK: Symposium proceedings / International scientific symposium on "The role of human milk in development of breast fed child's intestinal microbiota", Ljubljana, Slovenia, Izdala in založila: Univerza v Ljubljani Biotehniška fakulteta Oddelek za zootehniko Inštitut za mlekarstvo in probiotike Groblje 3, 1230 Domžale Uredniški odbor: Irena Rogelj Nataša Fidler Mis Bojana Bogovič Matijašić Tehnični urednik/oblikovanje: Gorazd Tompa Tisk: SKALA IN OSTALI, grafične dejavnosti in komerciala d.n.o. Naklada: 250 izvodov CIP - Kataloţni zapis o publikaciji Narodna in univerzitetna knjiţnica, Ljubljana (082) (082) MEDNARODNI znanstveni simpozij Vloga humanega mleka v razvoju črevesne mikrobiote dojenčka (2011 ; Ljubljana) Moje-mleko : zbornik prispevkov = My-milk : symposium proceedings / Mednarodni znanstveni simpozij Vloga humanega mleka v razvoju črevesne mikrobiote dojenčka, Ljubljana, Slovenija, = International Scientific Symposium of Breast Fed and Child's Intestinal Microbiota, Ljubljana, Slovenia, ; [uredniški odbor Irena Rogelj, Nataša Fidler Mis, Bojana Bogovič Matijašić]. - Ljubljana : Biotehniška fakulteta, Oddelek za zootehniko, Inštitut za mlekarstvo in probiotike, 2011 ISBN Gl. stv. nasl. 2. Vzp. stv. nasl. 3. Vloga humanega mleka v razvoju črevesne mikrobiote dojenčka 4. International Scientific Symposium of Breast Fed and Child's Intestinal Microbiota 5. Rogelj, Irena, Moje MLEKO Zbornik prispevkov

4 Kazalo / Contents Zgodnja prehrana dolgoročni vpliv na zdravje in dobro počutje 4 Early nutrition long-term impact on health and well-being Berthold Koletzko, Brigitte Brands, Hans Demmelmair, Veit Grote, Wolfgang Peissner, Peter Rzehak, Martina Weber Mikrobiota mlečne žleze: od kod prihaja? 15 Mammary microbiota: where it comes from? Juan Miguel Rodriguez Prehrana v maternici in debelost potomca ter presnovni izidi 27 In Utero Nutrition and Offspring Obesity and Metabolic Outcomes Eric L. Ding Kratka predstavitev raziskovalnega projekta "Vloga humanega mleka 30 v razvoju črevesne mikrobiote dojenčka" A short presentation of the research project "The role of human milk in development of breastfed child's intestinal microbiota" Borut Bratanič, Nataša Fidler Mis, Evgen Benedik, Irena Rogelj Metodološki pristopi k študiju mikrobiote 35 Methodological approaches to study microbiota Irena Rogelj, Primož Treven, Anja Mavrič, Tanja Obermajer, Gorazd Tompa, Bojana Bogovič Matijašić Prehrana matere in maščobno-kislinska sestava humanega mleka 40 Maternal diet and the fatty acid composition of human milk Nataša Fidler Mis, Evgen Benedik Spletna aplikacija za oceno prehranskega stanja OPKP 45 Web-based dietary evaluation tool OPKP Barbara Koroušić Seljak Izzivi antropometrije nosečnic 49 Anthropometry challenges in pregnant women Petra Golja Vsebnosti škodljivih in koristnih elementov v materinem mleku 52 pri slovenski populaciji mater Levels of toxic and essential elements in breast milk of mothers from Slovenia Janja Snoj Tratnik, Darja Mazej, Milena Horvat Zahvala 56 Sponzorji simpozija Moje-MLEKO / My-MILK 57 Symposium proceedings My-MILK

5 Zgodnja prehrana - dolgoročni vpliv na zdravje in dobro počutje Early nutrition long term impact on health and well-being Berthold Koletzko, Brigitte Brands, Hans Demmelmair, Veit Grote, Wolfgang Peissner, Peter Rzehak, Martina Weber Dr. von Hauner Children s Hospital, University of Munich Medical Centre, Lindwurmstr. 4, München, Germany Correspondence: Professor Berthold Koletzko, MD PhD, Dr. ; Introduction The developmental origins of health and disease hypothesis has gained a high level of interest among researchers of various disciplines after more than three decades of research on programming effects of early nutrition on later health outcomes. A wide range of metabolic factors acting pre- and postnatal during sensitive time periods were found associated with lasting adverse health outcomes and risk of later disease of the newborn, which is referred to as metabolic programming [1, 2]. Evidence from epidemiological studies and animal models indicates that maternal nutritional status during gestation and lactation can programme central and peripheral systems that regulate energy balance in the developing offspring. Smoking of the mother [3], maternal obesity or diabetes and maternal weight gain during pregnancy are some of the prenatal environmental risk factors for the development of obesity in the offspring [4, 5]. Moreover, nutritional deficiencies during pregnancy as well as placental insufficiency can lead to intrauterine growth retardation of the foetus which has been shown to increase the probability of adverse health outcomes. The interest of research in early programming of long-term health coincides with the worldwide increasing incidence of obesity and related diseases such as type 2 diabetes, cardiovascular disease and the metabolic syndrome [6-8]. Since childhood obesity has shown an alarming increase of both prevalence and severity in developed countries as well as in less-privileged populations during the last decades, it is more and more becoming an economic burden and considered a global public health concern [9-12]. One of the possible strategies to contribute to the reduction of obesity risk in later life could be the improvement of maternal nutrition during pregnancy and lactation, and of infant nutrition [2, 13]. Early risk factors for the development of obesity The success of treating obesity in children is less than satisfactory [14], therefore the development and implementation of effective prevention strategies is urgently needed [15, 16]. The aetiology of obesity is multifactorial and involves genetic and environmental factors, and their interactions. Besides the links of obesity to socioeconomic status and current lifestyle components such as low physical activity and 4 Moje MLEKO Zbornik prispevkov

6 high energy intake, variables during early life were shown to be associated with the later likelihood to develop obesity [17]. Maternal obesity prior to and during pregnancy, as well as maternal smoking during pregnancy were identified as potential risk factors for the development of obesity in childhood in epidemiological studies [3, 18-20]. In addition to maternal overweight before and during pregnancy, maternal diabetes during gestation is also associated with offspring obesity, possibly partly due to genetic factors but most likely also due to effects on the uterine metabolic environment, such as increased glucose and insulin levels that affect fetal growth and body composition. A recent review by Morisett et al. which examined studies published between 1975 and 2009 concluded that maternal obesity, as assessed by pre-pregnancy BMI and gestational weight gain, are both associated with an increased risk of gestational diabetes mellitus in the mother a physiological disorder which may be related to a whole set of adverse health outcomes in the newborn infant, including obesity [21]. For example, in a prospective birth cohort study in Denmark (n= 4234) a greater gestational weight gain was found associated with a higher body mass index (BMI) from childhood trough adulthood, with an increased risk for obesity at adult age [22]. These findings suggest that enhancing strategies to avoid excessive weight gain during pregnancy might be of benefit. However, controversial results have been obtained on the effectiveness of limiting gestational weight gain with respect to reducing obesity risk in the offspring [23, 24]. Women with gestational diabetes mellitus were found to have an almost four times higher risk of delivering a large for gestational age baby in weight, but not large in length [5]. In females, a high birth weight is associated with an increased risk of being obese at childbearing age, which implies that maternal overweight, rapid weight gain in pregnancy and gestational diabetes all may induce a vicious trans-generational circle accelerating the obesity epidemic in subsequent generations [25]. The importance of a suitable prenatal nutrient supply for long term health is also supported by the observation, that both excessive and suboptimal nutrient supply are associated with an increased risk for obesity and metabolic diseases. Several systematic reviews and recent observational studies confirmed that rapid weight gain in infancy and the first 2 years of life is a further significant risk factor for later adiposity [26-30]. A recent study of Goodell et al. examining the relationship between birth weight, rapid weight gain (RWG), and early childhood obesity in a low-income minority population in the US found that children who experienced RWG during the first year of life were 9.24 times (CI: ) as likely to become obese compared to those who did not experience RWG. In contrast, low birth weight did not increase the odds of becoming obese but predicted underweight at the age of months instead [30]. Although these findings highlight the importance of early growth patterns for later obesity risk, an identification of underlying mechanisms, e.g. genetics, environmental influences or early programming, was not possible in these studies. A high weight gain during the first 2 years of life has been shown to be the best overall predictor of overweight at school entry compared to other anthropometric markers and time intervals a conclusion drawn from an observational cohort study in Bavaria, which related growth measures at the age of 5-6 years of 4,235 children to corresponding anthropometric data obtained at birth, 6, 12 and 24 months [31]. As excessive weight gain in infancy seems directly related to the risk for later adiposity, identification of modifying factors could help to reduce obesity risk [32, 33]. Symposium proceedings My-MILK 5

7 Is there a role of infant feeding in obesity prevention? The first years of life have been identified as a critical period for the prevention of later obesity. Research in this field aims to disentangle in detail the relations of infant diet (breast milk, formula) and the mode of feeding (breast, bottle) to the risk of later obesity. A report by The World Health Organisation (WHO) in 2007 on Evidence of the Long-Term Effects of Breastfeeding concluded that breastfeeding may have a small protective effect on the prevalence of later obesity [34]. Three meta-analyses of observational studies reveal that breast feeding, both exclusivity and duration, reduces the odds ratio for obesity at school age by about 20%, relative to formula feeding, even after adjustment for biological and socio-demographic variables [35-37]. In a recent cohort study Baird et al showed that infants who were exclusively breastfed from birth to 6 months gained less weight, length and adipose tissue than formula-fed infants, independent of maternal factors (e.g. education attainment, smoking) and the age at introduction of solid foods [38]. Observational studies applying sophisticated statistical methods enable a detailed description of the association between breastfeeding and later obesity, but for the establishment of causal relationships and the identification of mechanisms interventional studies are needed. Potential mechanisms underlying the protective effect of breastfeeding on obesity A recent review by Bartok et al. evaluated some of the potential mechanisms that may mediate the association of breastfeeding and decreased obesity risk [44]. One possible explanation could be that confounding factors such as maternal weight status, education, socioeconomic status or age may be the true cause of this protective effect influencing infantile length and weight gain indirectly. Thus, not only the infant feeding experiences themselves but also lifestyle factors might be influencing variables. A general approach in epidemiological studies is to adjust for such confounding factors. This necessitates that potential confounding factors are known and that information can be collected with sufficient accuracy, but some relevant lifestyle factors such as physical activity cannot be well assessed in cohort studies. A possible strategy to overcome this limitation of confounding has been pursued by the PROBIT study in Belarus, a study that cluster randomized hospitals to a program of breastfeeding promotion or no active intervention [43, 45]. Results showed a significantly longer duration of breastfeeding when it was actively promoted. However, when the children were revisited at the age of 6.5 years, measures of obesity in the intervention and control group were not different. While this approach clearly benefits from removing some of the bias or confounding, this study did not compare breastfeeding with formula feeding since almost all children had been breastfed. A further limitation of the PROBIT study is that the prevalence of obesity was rather low in the population and that in Belarus different milk substitutes are used than common in other parts in the world. Moreover, Rückinger et al. showed that the study lacked the statistical power to detect effects of breastfeeding on obesity risk [40]. Therefore, while this cluster randomized trial is large and impressive, it does not allow drawing final conclusions on the effects of infant feeding on later obesity. 6 Moje MLEKO Zbornik prispevkov

8 An further possible explanation for the association of breastfeeding and reduced obesity risk considered by Bartok et al. is the influence of behavioural factors [44]. Encouraging the infant s capabilities of self-regulation of intake or reducing problematic feeding behaviours on the caregivers side, that interfere with the infant s self-regulation of intake might be possible causal factors. Finally, the compositional aspects of breast-milk, such as the lower protein content as compared to infant formula, or the different content of lipids, LC-PUFA or other bioactive compounds, might affect the regulation of energy intake and expenditure and the growth pattern of the infant. It has also been proposed that endocrine agents such as insulin-like -growth factor 1 (IGF-1), leptin, ghrelin and adiponectin, which are present in human milk, are related to an infant s growth [46-49]. However, undisputable proof for a causal protective effect of breast milk components remains difficult to obtain. Understanding those causalities may strengthen the conclusions on the protective effects of breastfeeding, and, moreover, this knowledge might help to improve the practice of feeding formula and complementary foods for infants who are not breastfed for long periods. Reduction of dietary protein intake in early life modifies infant growth patterns Epidemiological studies have positively related high protein intake in early childhood, but not high intakes of energy from fat or carbohydrates, to adipose tissue development and a high BMI in childhood, after correction for parental BMI [50, 51]. Most infant formulas have a slightly higher energy density (kcal/100ml) than average values for mature human milk, and energy intakes per kilogram of body weight in formula-fed infants aged 3-12 months were reported to be about % higher than those of breastfed babies [52]. However, the protein supply in formula-fed babies is even much larger than in breastfed infants. In animal models, high protein supply in the postnatal period of rodents has been shown to induce increased body fat and weight in adult animals [53, 54]. The Early Protein Hypothesis postulates that a high protein intake with infant formula feeding above metabolic requirements might induce increased circulating concentrations of insulin-releasing amino acids, which in turn may stimulate the secretion of IGF-1, thereby inducing an increased weight gain during the first 2 years of life as well as increased adipogenic activity [55]. We examined in a randomized double-blind clinical trial whether higher or lower protein intakes during the first year of life influence growth until the age of 2 years and obesity risk at later ages. This multicenter clinical trial is being conducted in 5 European countries that differ substantially in the practice of infant feeding and the prevalence of adult obesity. A breastfed reference group is included in each country to allow for a comparison of the effects of breastfeeding and formula feeding. Growth from birth to the age of 2 years was chosen as the primary outcome variable on the basis of our previous finding that this measure is a predictive early biomarker of later obesity risk [31]. Results show that length was not different between randomized groups at any time, whereas the weight-for-length z scores of infants in the lower protein formula group increased less from the first half year of life onwards and were 0.20 (0.06, 0.34) lower at 24 months of age than that of the higher protein group, and similar to the breastfed reference group [56]. These results indicate that limiting the protein content of infant and follow-on Symposium proceedings My-MILK 7

9 formula could be an important approach to normalizing early growth and to reducing the risk of childhood overweight and obesity. Conclusions and perspectives There is convincing evidence that genetics and environmental factors are complemented by programming effects during the perinatal period in influencing later body weight development as well as adult health and disease. Observational and animal studies indicate the importance of nutrient supply during this period for the manifestation of programming effects, although other factors can not be excluded. Among the dietary components currently only for early infantile protein intake lasting effects on anthropometric development and on early indicators of obesity risk have been demonstrated in a randomized intervention study. This could be the basis for the development of a component in a multifactorial approach for early obesity prevention, if confirmed in further studies. Obesity and associated disorders are also the focus of the new European Commission funded collaborative research project EarlyNutrition, running from with a total budget of 10,7 Mio EUR. This project brings together 36 partners from academia, industry and the SME sector from 12 European countries, the USA and Australia. EarlyNutrition investigates study populations of more than 450,000 individuals to explore three principal hypotheses: The fuel mediated in utero hypothesis The accelerated postnatal weight gain hypothesis The mismatch hypothesis. Using existing cohort studies, ongoing and novel intervention studies and a basic science programme, EarlyNutrition will provide the scientific foundations for evidence based recommendations for optimal early nutrition that incorporate long-term health outcomes, focusing on 4 Target Groups: women before pregnancy; pregnant women; infants (incl. breastfeeding) and young children. Scientific and technical expertise in placental biology, epigenetics and metabolomics will provide understanding at the cellular and molecular level, and refined strategies for intervention in pregnancy and early post natal life to prevent obesity. The project s impact comprises definitive evidence on early nutrition effects on health, enhanced EU and global policies, major economic benefits through obesity prevention and value-added nutritional products, and practical recommendations on optimal nutrition in Target Groups. Acknowledgements The work of the authors is financially supported in part by the Commission of the European Communities, Specific RTD Programme Quality of Life and Management of Living Resources, within the 6th Framework Programme (Food-CT ) and the 7th Framework Programme (contract ) and FP7 (FP and FP7-KBBE ). This manuscript does not necessarily reflect the views of the Commission and in no way anticipates future policy in this area. Berthold Koletzko is the recipient of a Freedom to Discover Award of the Bristol-Myers-Squibb Foundation, New York, NY. The authors declare no conflicts of interest. 8 Moje MLEKO Zbornik prispevkov

10 Slovenski povzetek znanstvenega prispevka Hipoteza o razvojnem izvoru zdravja in bolezni je deleţna velikega zanimanja med raziskovalci različnih disciplin po več kot 30-ih letih raziskav vpliva zgodnjega programiranja prehrane na kasnejše zdravje. Odkrita je bila vrsta presnovnih dejavnikov, ki delujejo v občutljivem časovnem obdobju pred in po porodu in so povezani z dolgotrajnimi neugodnimi vplivi in tveganjem za različna obolenja kasneje v ţivljenju, kar imenujemo presnovno programiranje (angl. metabolic programming). Rezultati epidemioloških študij in študij na ţivalskih modelih kaţejo, da prehrana matere v nosečnosti in med dojenjem lahko programira centralni in periferni sistem, ki uravnavata energijsko ravnovesje razvijajočega se potomca. Kajenje, debelost ali diabetes matere in pridobivanje telesne teţe med nosečnostjo, so nekateri izmed prenatalnih okoljskih dejavnikov tveganja za razvoj debelosti pri potomcu. Poleg tega lahko prehranske pomanjkljivosti, med nosečnostjo kot tudi insuficienca (nezadostna zmogljivost ) placente povzroči intrauterini zastoj rasti ploda, kar je bilo dokazano, da poveča tveganje za neugodne zdravstvene posledice. Poleg tega lahko prehranske pomanjkljivosti, med nosečnostjo kot tudi insuficienca placente povzroči intrauterini zastoj rast ploda, kar dokazano poveča verjetnost za neugoden učinek na rezultat. Zanimanje za raziskave o zgodnjem programiranju dolgoročnega zdravja sovpada z naraščajočo incidenco debelosti, in z njo povezanimi boleznimi kot so diabetes tip 2, bolezni srca in oţilja ter presnovni sindrom, v svetu. Ker je debelost pri otrocih alarmantno v porastu tako glede prevalence kot resnosti v razvitih drţavah kot tudi v manj privilegiranih populacijah v zadnjih desetletjih, vse bolj in bolj postaja gospodarsko breme in je postala globalna skrb javnega zdravja. Ena od moţnih strategij, ki bi prispevala k zmanjšanju tveganja debelosti kasneje v ţivljenju bi lahko bilo izboljšanje prehrane mater med nosečnostjo in dojenjem ter prehrane dojenčkov. Zgodnji dejavniki tveganja za razvoj debelosti Uspeh za zdravljenje debelosti pri otrocih je manj kot zadovoljiv, zato je nujno potreben razvoj in izvajanje učinkovitih preventivnih strategij. Etiologija debelosti je večstranska in vključuje genetske in okoljske dejavnike ter tudi medsebojne vplive obojih. Poleg povezave debelosti s socialno-ekonomskim statusom ter načinom ţivljenja, kot je nezadostna telesna aktivnost in visok energijski vnos, so spremenljivke v zgodnjem ţivljenju pokazale povezavo s kasnejšim nagnjenjem za razvoj debelosti. Debelost matere pred in med nosečnostjo, kakor tudi kajenje med nosečnostjo sta se v epidemioloških študijah pokazala kot moţna dejavnike tveganja za razvoj debelosti v otroštvu. Poleg prekomerne telesno teţe matere pred in med nosečnostjo, je nosečniški diabetes prav tako povezan z debelostjo potomca, delno morda kot posledica genetskih dejavnikov, najverjetneje pa zaradi vplivov na presnovno okolje v maternici, kot je povečana raven glukoze in inzulina v krvi, ki vplivajo na rast ploda in na sestave telesa. Symposium proceedings My-MILK 9

11 V nedavnem preglednem članku Morisetta s sod., ki so preučevali študije, objavljene med leti 1975 in 2009 so zaključili, da sta tako debelost matere, ocenjena na osnovi ITM pred nosečnostjo, kot tudi povečanje telesne mase med nosečnostjo, povezani z večjim tveganjem za gestacijski diabetes mellitus matere - fiziološko motnjo, ki je lahko povezana s celo vrsto neugodnih zdravstvenih izidov novorojenčka, vključno z debelostjo. V prospektivni študiji kohorte rojstev na Danskem (n = 4.234) je bil večji porast telesne mase med nosečnostjo povezan z višjim indeksom telesne mase (ITM) od otroštva v odraslo dobo ter s povečanim tveganjem za debelost v odrasli dobi. Ta odkritja sugerirajo, da bi lahko bile koristne strategije, ki bi omejile prekomeren porast telesne teţe v času nosečnosti. Pri potomcih ţenskega spola je visoka porodna teţa povezana s povečanim tveganjem za debelost v rodni dobi, kar pomeni, da debelost matere, hiter porast teţe med nosečnostjo ter gestacijski diabetes vodijo v začarani krog epidemije debelosti v naslednjih generacijah. Pomen ustrezne prenatalne oskrbe s hranili za dolgoročno zdravje podpira tudi ugotovitev, da sta tako prekomerna kot pomanjkljiva oskrba s hranili povezani s povečanim tveganjem za debelost in presnovne bolezni. Ali ima prehrana dojenčka vlogo pri preprečevanju debelosti? Prva leta ţivljenja veljajo za kritično obdobje za preprečevanje debelosti. Raziskave na tem področju so usmerjene k proučevanju povezav med prehrano dojenčka (dojenje, mlečne formule) in načinom hranjenja (dojka, flaška) na tveganje za kasnejšo debelost. Tri meta-analize opazovalnih študije kaţejo, da dojenje, tako ekskluzivnost kot trajanje, zmanjša tveganje za debelost v šolski starosti za okoli 20%, glede na hranjenje z mlečno formulo, tudi po prilagoditvi bioloških in socio-demografskih spremenljivk. V nedavni kohortni študiji so Baird s sod. pokazali, da so dojenčki, ki so bili izključno dojeni od rojstva do 6 mesecev starosti pridobili manj teţe, dolţine in maščobnega tkiva kot dojenčki, hranjeni z mlečno formulo, neodvisno od dejavnikov matere (npr. izobrazbe, kajenja) in starost ob uvajanju mešane prehrane. Medtem ko opazovalne študije omogočajo podroben opis povezave med dojenjem in kasnejšo debelostjo, pa so za ugotavljanje vzročnih odnosov in identifikacijo mehanizmov potrebne interventne študije. Potencialni mehanizmi zaščitnega učinka dojenja proti debelosti Ena moţnih razlag za povezavo dojenja z manjšim tveganjem za debelost je, da razen samega načina hranjenja pomembno vpliva tudi način ţivljenja, pogojen s socialnim statusom, starostjo matere, teţo matere in izobrazbo. Vplivajo lahko tudi vedenjski dejavniki, kot na primer spodbujanje oziroma oviranje zmoţnosti dojenčka, da sam regulira vnos hrane. Pomembna pa je tudi razlika v sestavi mlečnih formul v primerjavi z materinim mlekom. Zmanjšanje vnosa beljakovin v zgodnjem življenju spreminja vzorec rasti dojenčka Epidemiološke raziskave so pokazale pozitivno povezavo med visokim vnosom beljakovin, ne pa višjim vnosom energije iz maščob ali ogljikovih hidratov, v zgodnjem otroštvu in razvojem maščobnega tkiva ter visokim ITM v otroštvu, po korekciji za 10 Moje MLEKO Zbornik prispevkov

12 ITM staršev. Večina mlečnih formul ima nekoliko večjo energijsko gostoto (kcal/100ml) od povprečne vrednosti zrelega humanega mleka. Vnos energije na kilogram telesne teţe pri zalivančkih starostih 3-12 mesecev, je pribliţno od 10 do 18% višji kot pri dojenih dojenčkih. Še večji pa je razkorak v oskrbi z beljakovinami, ki jih otroci, hranjenih s formulami, prejmejo več. Predpostavljajo, da lahko previsok vnos beljakovin v mlečnih formulah povzroči povečano koncentracije insulin-sproščajočih aminokislin, kar lahko spodbudi izločanje IGF-1 in posledično večjo pridobivanje telesne mase in kopičenje maščob v prvih dveh letih ţivljenja. Rezultati nedavne randominizirane dvojno-slepe multicentrične klinične študije, ki so jo izvedli v 5 evropskih drţavah, so pokazali, da zmanjšanje vsebnosti beljakovin v začetnih in nadaljevalnih formulah lahko ugodno vpliva na zmanjšanje tveganja za prekomerno telesno teţo in debelost v otroštvu. Sklepi in perspektive Obstajajo prepričljivi dokazi, da genetske in okoljske dejavnike za razvoj telesne teţe ter zdravja in bolezni v odrasli dobi, dopolnjujejo učinki zgodnje prehrane med perinatalnim obdobjem, oziroma tako imenovano presnovno programiranje. Opazovalne študije in raziskave na ţivalskih modelih kaţejo, kako pomembno je hranjenje v tem obdobju za izraţanje učinkov programiranja, čeprav tudi drugih dejavnikov ni mogoče izključiti. Debelost in s tem povezane motnje so tudi osrednja tema novega projekta EarlyNutrition, ki ga financira Evropska Komisija. Ta projekt zdruţuje 36 partnerjev iz akademskih krogov, industrije in sektorja malih in srednjih podjetij iz 12 evropskih drţav, ZDA in Avstralije. Potekal bo od , zanj pa so namenili kar 10,7 milijonov EUR. Z raziskavami, v katere bo vključenih več kot posameznikov, ţelijo postaviti priporočila za optimalno zgodnjo prehrano, ki vključuje dolgoročne ugodne učinke na zdravje, ki bodo temeljila na znanstvenih dokazih. Posvetili se bodo štirim ciljnim skupinam: ţenskam pred nosečnostjo, nosečnicam, dojenčkom (ter doječim materam) in majhnim otrokom. S pomočjo znanja na področju placentalne biologije, epigenetike in metabolomike bodo proučevali mehanizme na celični in molekularni ravni. Med pričakovanimi učinki pa so pridobitev dokazov o vplivih zgodnjega prehranjevanja na zdravje, izboljšano EU in svetovno politiko, gospodarske koristi zaradi preprečevanja debelosti in zaradi dodane vrednosti prehranskih izdelkov in praktična priporočila za optimalno prehrano omenjenih ciljnih skupin. References / Literatura 1. Sullivan, E.L. and K.L. Grove, Metabolic Imprinting in Obesity, in Frontiers in Eating and Weight Regulation2010, Karger: Basel. p Koletzko, B., Early nutrition and its later consequences: new opportunities. Adv Exp Med Biol, : p Ino, T., Maternal smoking during pregnancy and offspring obesity: Meta-analysis. Pediatrics International, (1): p de Novaes, J.F., et al., Environmental factors associated with childhood overweight. Revista De Nutricao-Brazilian Journal of Nutrition, (5): p Ahlsson, F., et al., Gestational diabetes and offspring body disproportion. Acta Paediatrica, (1): p Wells, J.C.K., Historical cohort studies and the early origins of disease hypothesis: making sense of the evidence. Proc Nutr Soc, (2): p Symposium proceedings My-MILK 11

13 7. Langley-Evans, S.C. and S. McMullen, Developmental Origins of Adult Disease. Medical Principles and Practice. 19(2): p Symonds, M.E., et al., Nutritional programming of the metabolic syndrome. Nature Reviews Endocrinology, (11): p Wang, Y., et al., Is obesity becoming a public health problem in India? Examine the shift from under- to overnutrition problems over time. Obesity Reviews, (4): p Fall, C., Maternal nutrition: Effects on health in the next generation. Indian Journal of Medical Research, (5): p Karnieli, E., Preface. Endocrinology & Metabolism Clinics of North America, (3): p. xvii -xviii. 12. Withrow, D. and D.A. Alter, The economic burden of obesity worldwide: a systematic review of the direct costs of obesity. Obes Rev, Koletzko, B., et al., Infant feeding and later obesity risk. Adv Exp Med Biol, : p Oude Luttikhuis, H., et al. Interventions for treating obesity in children. Cochrane Database of Systematic Reviews, DOI: / CD pub Koletzko, B., Childhood obesity: time for treatment or prevention? European Journal of Lipid Science and Technology, (5): p Hesketh, K.D. and K.J. Campbell, Interventions to Prevent Obesity in 0-5 Year Olds: An Updated Systematic Review of the Literature. Obesity, : p. S27-S Parsons, T.J., et al., Childhood predictors of adult obesity: a systematic review. Int J Obes Relat Metab Disord, Suppl 8: p. S Butte, N.F., Impact of Infant Feeding Practices on Childhood Obesity. Journal of Nutrition, (2): p. 412S-416S. 19. Toschke, A.M., et al., Paternal smoking is associated with a decreased prevalence of type 1 diabetes mellitus among offspring in two national British birth cohort studies (NCDS and BCS70). J Perinat Med, (1): p von Kries, R., et al., Maternal smoking during pregnancy and childhood obesity. Am J Epidemiol, (10): p Morisset, A.S., et al., Prevention of gestational diabetes mellitus: a review of studies on weight management. Diabetes-Metabolism Research and Reviews, (1): p Schack-Nielsen, L., et al., Gestational weight gain in relation to offspring body mass index and obesity from infancy through adulthood. International Journal of Obesity, (1): p Bodnar, L.M., et al., Severe obesity, gestational weight gain, and adverse birth outcomes. Am J Clin Nutr. 24. Dodd, J.M., et al., Antenatal interventions for overweight or obese pregnant women: a systematic review of randomised trials. BJOG. 25. Freeman, D.J., Effects of maternal obesity on fetal growth and body composition: implications for programming and future health. Seminars in Fetal and Neonatal Medicine, (2): p Lamb, M.M., et al., Early-Life Predictors of Higher Body Mass Index in Healthy Children. Annals of Nutrition and Metabolism, (1): p Monteiro, P.O.A. and C.G. Victora, Rapid growth in infancy and childhood and obesity in later life - a systematic review. Obesity Reviews, (2): p Ong, K.K. and R.J.F. Loos, Rapid infancy weight gain and subsequent obesity: Systematic reviews and hopeful suggestions. Acta Paediatrica, (8): p Baird, J., et al., Being big or growing fast: systematic review of size and growth in infancy and later obesity. British Medical Journal, (7522): p Moje MLEKO Zbornik prispevkov

14 30. Goodell, L.S., D.B. Wakefield, and A.M. Ferris, Rapid Weight Gain During the First Year of Life Predicts Obesity in 2-3 Year Olds from a Low-income, Minority Population. Journal of Community Health, (5): p Toschke, A.M., et al., Identifying children at high risk for overweight at school entry by weight gain during the first 2 years. Archives of Pediatrics & Adolescent Medicine, (5): p Koletzko, B., et al., Protein intake in the first year of life: A risk factor for later obesity? The EU Childhood Obesity project, in Advances in Experimental Medicine and Biology:PERINATAL PROGRAMMING OF ADULT HEALTH - EC SUPPORTED RESEARCH, B. Koletzko, et al., Editors. 2005, Springer-Verlag Berlin. p Koletzko, B., Long-term consequences of early feeding on later obesity risk, in Protein and Energy Requirements in Infancy and Childhood, J. Rigo and E.E. Ziegler, Editors. 2006, Karger: Basel. p Horta B, B.R., Martines J, Victora C, Evidence of the Long-Term Effects of Breastfeeding: Systematic Reviews and Meta-Analysis.. World Health Organization Publication: Geneva, Switzerland, 2007, Arenz, S., et al., Breast-feeding and childhood obesity--a systematic review. Int J Obes Relat Metab Disord, (10): p Harder, T., et al., Duration of breastfeeding and risk of overweight: a meta-analysis. Am J Epidemiol, (5): p Owen, C.G., et al., Effect of infant feeding on the risk of obesity across the life course: a quantitative review of published evidence. Pediatrics, (5): p Baird, J., et al., Milk feeding and dietary patterns predict weight and fat gains in infancy. Paediatric and Perinatal Epidemiology, (6): p Kwok, M.K., et al., Does breastfeeding protect against childhood overweight? Hong Kong's 'Children of 1997' birth cohort. Int J Epidemiol, (1): p Ruckinger, S. and R. von Kries, Breastfeeding and reduced risk of childhood obesity: will randomized trials on breastfeeding promotion give the definite answer? American Journal of Clinical Nutrition, (2): p Beyerlein, A., A.M. Toschke, and R. von Kries, Breastfeeding and Childhood Obesity: Shift of the Entire BMI Distribution or Only the Upper Parts? Obesity, (12): p Owen, C.G., et al., The effect of breastfeeding on mean body mass index throughout life: a quantitative review of published and unpublished observational evidence. Am J Clin Nutr, (6): p Kramer, M.S., et al., Effects of prolonged and exclusive breastfeeding on child height, weight, adiposity, and blood pressure at age 6.5 y: evidence from a large randomized trial. American Journal of Clinical Nutrition, (6): p Bartok, C.J. and A.K. Ventura, Mechanisms underlying the association between breastfeeding and obesity. Int J Pediatr Obes, (4): p Kramer, M.S., et al., A Randomized Breast-feeding Promotion Intervention Did Not Reduce Child Obesity in Belarus. Journal of Nutrition, (2): p. 417S-421S. 46. Savino, F., et al., Ghrelin, leptin and IGF-I levels in breast-fed and formula-fed infants in the first years of life. Acta Paediatr, (5): p Elmlinger, M.W., et al., Insulin-like growth factors and binding proteins in early milk from mothers of preterm and term infants. Horm Res, (3): p Martin, L.J., et al., Adiponectin is present in human milk and is associated with maternal factors. Am J Clin Nutr, (5): p Bouret, S.G., Early life origins of obesity: role of hypothalamic programming. J Pediatr Gastroenterol Nutr, Suppl 1: p. S31-8. Symposium proceedings My-MILK 13

15 50. Scaglioni, S., et al., Early macronutrient intake and overweight at five years of age. International Journal of Obesity, (6): p Hoppe, C., et al., Protein intake at 9 mo of age is associated with body size but not with body fat in 10-y-old Danish children. Am J Clin Nutr, (3): p Heinig, M.J., et al., Energy and protein intakes of breast-fed and formula-fed infants during the first year of life and their association with growth velocity: the DARLING Study. Am J Clin Nutr, (2): p Kim, S.H., et al., Selection of carbohydrate to protein ratio and correlations with weight gain and body fat in rats allowed three dietary choices. Int J Vitam Nutr Res, (2): p Metges, C.C., Longterm effects of pre- and postnatal exposure to low and high dietary protein levels - Evidence from epidemiological studies and controlled animal experiments, in Early Nutrition and Its Later Consequences: New Opportunities - Perinatal Programming of Adult Health - Ec Supported Research, B. Koletzko, et al., Editors. 2005, Springer-Verlag Berlin: Berlin. p Koletzko, B., et al., Can infant feeding choices modulate later obesity risk? Am J Clin Nutr, (5): p. 1502S-1508S. 56. Koletzko, B., et al., Lower protein in infant formula is associated with lower weight up to age 2 y: a randomized clinical trial. Am J Clin Nutr, (6): p Moje MLEKO Zbornik prispevkov

16 Mikrobiota mlečne žleze: od kod prihaja? Mammary microbiota: where it comes from? Juan M. Rodríguez Dpt. Nutrition, Food Science and Food Technology, Complutense University of Madrid, Madrid, Spain Introduction Human milk constitutes the best feeding during the first months of life since it usually satisfies all the nutritional requirements of the rapidly-growing infant; additionally, it educates the infant immune system and confers a certain degree of protection against pathogens. Therefore, it is not strange that breastfeeding of preterm newborns is associated to a significantly lower incidence of necrotizing enterocolitis and septicaemia, a faster tolerance to enteral nutrition and a lower dependence on parenteral nutrition. These effects result from the synergistic action of many bioactive molecules, present in colostrum and milk, including immunoglobulins, immunocompetent cells, fatty acids, polyamines, oligosaccharides, lysozyme, lactoferrin, and other glycoproteins, and antimicrobial peptides, which inactivate pathogens individually, additively, and synergistically. More recently, several studies have revealed that colostrum and breast milk are continuous sources of commensal and potentially probiotic bacteria to the infant gut; therefore, such biological fluids are also key elements of the defence system that this biological fluid offers to the infant. This is a relevant finding since human milk was traditionally considered to be sterile despite the lack of scientific evidences supporting such assumption. In fact, human milk constitutes one the main sources of bacteria to the infant gut since it has been estimated that a baby consuming approximately 800 ml/day of milk will ingest between 1 x 10 5 and 1 x 10 7 bacteria daily. This explains why the bacterial composition of the infant gut microbiota reflects that of breast milk. Bacterial colonization of the human gut is a complex process that seems to starts, at a small scale, during the foetal period. Contact with microorganisms belonging to the vaginal, intestinal and mammary microbiota of the mother, and to the surrounding environment of the neonate, leads to a notable intensification of this process after birth. As a consequence, factors such as composition of the maternal microbiota, place and way of birth and/or feeding pattern play key roles in a process that exerts a strong influence on host functions so important as nutrient absorption, formation of host barriers against pathogens, or maturation of the immune system. Symposium proceedings My-MILK 15

17 Bacterial diversity of human milk During decades, microbiological studies focused on human milk were restricted to the identification of potential pathogenic bacteria in stored milk and in clinical cases of maternal or infant infection. The first descriptions of the bacterial diversity of breast milk in healthy women were based on the use of culture media and showed that predominance of staphylococci, streptococci, lactic acid bacteria and closely related Gram -positive bacteria. More recently, human milk has been shown to be a source of live bifidobacteria to the infant gut. The fact that bacteria belonging to such genera are easily isolated from fresh milk of healthy women from distant countries suggests that their presence in this substrate is a common event. Therefore, they should be considered as components of the natural microbiota of human mammary gland, instead of mere contaminant bacteria. This mammary microbiota has the peculiarity that has a transient nature: its development starts during the last third of pregnancy and sharply declines at weaning. Subsequently, the application of culture-independent molecular techniques, and particularly those based on 16S rrna genes, allowed a complementary assessment of the biodiversity of the human milk microbiota. Typically, this approach involves extraction of the DNA from the biological samples, PCR amplification of 16S rrna gene fragments with universal or group-specific bacterial primers, analysis of PCR products by fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) and/or, the construction of clone libraries to assess the variety of 16S rrna gene sequences present. Culture-independent techniques have confirmed the dominance of staphylococci, streptococci, lactic acid bacteria and bifidobacteria, and have also revealed the presence of DNA belonging to other bacterial groups, such as some Gram-negative bacteria. These Gram-negative bacteria may have their origin in the mammary gland but also can be the result of the use of contaminated milk pumps when these devices are employed for sample collection. On the other hand, it has shown the strong influence of this biological fluid on the bacterial colonization of the neonatal gut. In a study, samples of fresh breast milk, vaginal swabs and infant feces collected from five mother-infant pairs whose neonates were born by vaginal delivery and from other five that had their babies by programmed caesarean section were submitted to PCR amplification using Lactobacillus group-specific primers. Subsequently, the amplicons were analyzed by DGGE and, parallel, clone libraries were constructed to describe the Lactobacillus-group microbial diversity. Interestingly, none of the species detected among the vaginal samples were found in breast milk-derived libraries and only a few were detected in 16S rrna gene libraries from infant feces. In contrast, the profiles of Lactobacillus sequences retrieved from infant feces were more similar to those retrieved from breast milk of the respective mothers. Such results are also in agreement with recent molecular studies which have shown that bacterial colonization is not significantly related to the delivery method and that vaginal delivery has a minor role in the development of the infant gut microbiota. Similarly, a molecular epidemiological study on the transmission of vaginal Lactobacillus species from mother to the newborn infant showed, that only less than one-fourth of the infants acquired maternal vaginal lactobacilli at birth, and that 1 month later, they had 16 Moje MLEKO Zbornik prispevkov

18 been replaced by lactobacilli associated with human milk. In fact, some studies have revealed the presence of the same bacterial strains (as determined by genetic profiling) in both breast milk and infant feces of different mother-infant pairs. The bacterial strains shared by the mother-infant pairs included a variety of species belonging to the genera Staphylococcus, Enterococcus, Lactobacillus, and Bifidobacterium. Therefore, colostrum and milk may play key roles in the initiation, development and composition of the infant gut microbiota. Up to the present, our research group has isolated more than 200 different bacterial species, belonging to approximately 50 different genera, from human milk. In addition, human milk has been the source of new bacterial species, such as Streptococcus lactarius. However, the number of bacterial species found simultaneously in the milk of a healthy woman seems to be low and ranges from 2 to 18 different species. This fact can explain why the gut microbiota of healthy breast-fed infants usually comprises of a narrow species spectrum and, also, why the development of a more diverse microbiota coincides with the weaning period. Infant gut microbiota is greatly influenced by diet and the introduction of solid food, together with the progressive elimination of breast milk, leads to major changes in its composition. Probably, this fact is responsible for the differences observed between the gut microbiota of breast-fed and formula-fed infants. Although infant nutrition has experimented notable advances in the last decades, there are still big qualitative and quantitative differences between human milk and any infant formula, particularly regarding the microbiological composition. It has to be had in account that human milk bacteria may play several functions in the infant gut. More recently, microbial identification techniques based on pyrosequencing of the 16S ribosomal RNA gene were used to obtain a first description of the human milk microbiome. Results indicated that milk bacterial communities are more complex than expected, with several genera represented greater than 5% of the relative community abundance. Staphylococci (particularly Staphylococcus epidermidis) and streptococci were the dominant genera. Bacterial populations were usually, although not always, stable over time within the same individual. The application of the omics concept (metagenomics, transcriptomics, proteomics, metabolomics) to the study of the human mammary microbiota is already in progress and there is no doubt that the results provided by such techniques will open new perspectives in the understanding of the initiation and development of the infant gut microbiota. Staphylococci and streptococci Staphylococcus epidermidis seems to be the most prevalent species both in human milk and in feces of breast-fed infants while it is practically absent in those of formula fedinfants. Therefore, S. epidermidis can be considered as a differential trait of the fecal microbiota of breast-fed infants. Studies carried 20 years ago already described that staphylococci were common in feces of breast-fed infants. More recently, it has been shown that coagulase-negative staphylococci colonized 100% of breast-fed Western infants from day 3 onwards. Such staphylococci colonized vaginally and cesarean sectiondelivered infants equally early. Some authors suggest that, in fact, staphylococcal colonization of the infant gut has increased from the 70s to the present. It has been speculated that this may be an effect of a highly hygienic lifestyle which leads to a delayed acquisition of traditional fecal bacteria, such as enterobacteria. In their Symposium proceedings My-MILK 17

19 absence, staphylococci become the first gut colonizers and some works suggest that breast milk could be the main source. Globally, most of the S. epidermidis strains isolated from human milk harbour several adhesion factors but not antibiotic resistance or virulence determinants. Since staphylococcal strains provided first by colostrum and, later, by breast milk may successfully compete with potentially pathogenic strains found in the hospital environment, their application as probiotics in neonatal units could be considered in the future if works in progress (including complete genome sequencing and analysis) confirm the safety of selected strains. In fact, exclusion of pathogenic staphylococci that use to be highly prevalent in hospital environments by commensal staphylococci found in human milk may be one of the reasons explaining the success of the kangaroo mother method in the management of preterm neonates (unpublished results). Commensal streptococci can be also useful to reduce the acquisition of undesired pathogens by infants exposed to hospital environments. For example, it has been shown that viridans streptococci inhibit oral colonization by methicillin-resistant S. aureus in high-risk newborns exposed to hospital environments. In contrast to the atopic infant gut, it is interesting to note that the presence of viridans streptococci seems to be a feature of the healthy infant gut. In addition, the oral streptococcal species S. sanguinis is recognized for its putative antagonistic role in dental caries and periodontal diseases and its artificial implantation in the oral cavity has been already proposed as a probiotic approach to prevent caries, including measures that foster a mother-to-child transmission. Lactic acid bacteria Some species belonging to the Lactobacillus group (which includes the genera Lactobacillus, Pediococcus, Leuconostoc and Weissella) are commonly found in milk; however, their prevalence may be somehow underrated due to the fact that their isolation of is in some cases difficult with the culture media and/or conditions currently available. A recent molecular survey on the Lactobacillus species present in the gut microbiota of Swedish infants showed that they were significantly more often isolated from the feces of infants receiving breast milk, than from weaned infants. Similarly, it has been observed that at 6 months, Lactobacillus counts are significantly higher in breastfed than formula-fed infants. Lactococci and enterococci are also frequently isolated from breast milk. In fact, nisinproducing Lactococcus lactis isolates were found in 30% of the breast milk samples provided by healthy Finnish women, and such strains showed potential in using them as bio-therapeutic agents preventing neonatal and maternal breast infections caused by Staphylococcus aureus. A study showed that all the Enterococcus faecium strains isolated from breast milk of seven healthy women were devoid of virulence factors, both at the genotypic and phenotypic levels. Bifidobacteria Bifidobacteria were first isolated a century ago from infant feces and were quickly associated with a healthy infant gut because of their predominance in breast-fed infants 18 Moje MLEKO Zbornik prispevkov

20 in comparison to formula-fed ones. Since then, it was widely accepted that bifidobacteria represent one of the most important bacterial groups in the infant gut. Some studies have suggested that infants with delayed bifidobacterial colonization and/or decreased bifidobacterial numbers may be more susceptible to a variety of gastrointestinal or allergic conditions. Different studies revealed the presence of bifidobacterial DNA in breast milk. More recently, the first report describing the physical isolation of bifidobacteria (Bifidobacterium breve, B. adolescentis, and B. bifidum) from this biological fluid was published. Bifidobacteria could be isolated only from 8 of the 23 milk samples. This may reflect the fastidious growth requirements of other bifidobacterial species or strains that could be also present in the samples and/or their relative low concentration in this biological fluid. In this context, the fact that bifidobacteria are more easily isolated from a infant feces may be the consequence of their rather different concentration (>10 7 cfu/g in feces versus <10 3 cfu/ml in breast milk). Gram-negative bacteria E. coli, Klebsiella spp. and other enterobacteria can be also isolated from breast milk of healthy women. Molecular studies have shown that Escherichia coli was found to be one of the first colonizer of the infant gut, where it can coexist with Gram-positive bacteria. Previously, evidence for mother-to-infant transmission of Enterobacteriaceae from was obtained in four out of five cases. The species E. coli comprises non-pathogenic as well as pathogenic bacteria and commensal strains generally represent normal and ecologically important inhabitants of the human mucosal surfaces. In fact, E. coli strain Nissle 1917 (O6:K5:H1) forms the basis of an infant probiotic preparation and different studies have shown that its oral application to full-term and premature infants reduces the number and incidence of infections, significantly stimulates specific humoral, and cellular responses and simultaneously induces non-specific natural immunity. Connotations for Milk Banks Human milk is generally considered the Gold Standard for infant nutrition and for a correct colonization of the neonatal gastrointestinal tract. Although criteria to define a bacterial contamination vary considerably between Milk Banks (from 10 3 to 10 5 cfu/ml), such counts are usually met in fresh milk obtained from healthy women. Despite the results of a variety of culture-based studies that indicate that Gram-negatives, enterococci, staphylococci or streptococci are normal inhabitants of human milk, the mere presence of these species would be enough to consider the milk unfit for consumption. Fortunately, cultures are thought to be unnecessary when extracted milk is used to feed the mother s own baby. Therefore, it is not strange that a recent bacteriological screening of expressed breast milk in Chinese women showed that between 63 and 86% of the samples would be considered contaminated following the criteria of different Milk Banking Associations. However, and to the surprise of the medical staff, this contaminated milk was the one that provided the highest benefits to neonates and infants in a Chinese orphanage, including the lowest rates of necrotizing enterocolitis. Symposium proceedings My-MILK 19

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