Abstract

Nutrition in early infancy and childhood can significantly impact growth and development as well as immediate and later health.  The World Health  Organization recommends exclusive, breastfeeding for the first six months of life, followed by continued breastfeeding with appropriate, complementary foods for up to two years or beyond. Breastfeeding and/or nutritional intervention during early life can help prevent both infectious and non-communicable disease risks from childhood till being adult.

Infant formulas aim to mimic the composition and functionality of human milk by providing ingredients reflecting those of the latest human milk insights, such as oligosaccharides, bacteria, and bacterial metabolites. The objective of this mini-review article is to discuss the most recent developments in infant formula with a  special focus on human milk oligosaccharides and postbiotics.

Keywords:

Human milk  oligosaccharides;  probiotics;  prebiotics;  synbiotics;  postbiotics;  2′-fucosyllactose (2′-FL); lacto-N-neotetraose (LNnT); 3′-galactosyllactose (3′-GL); breastfeeding; infant formula.

Introduction:

Breastfeeding is natural and the optimal basis of infant nutrition and development, with many benefits for maternal health. Human milk is a dynamic fluid fulfilling an infant’s specific nutritional requirements and guiding the growth, developmental, and physiological processes of the infant. Human milk is considered unique in composition, and it is influenced by several factors, such as maternal diet and health, body composition, and geographic region. Human milk stands as a model for infant formula providing nutritional solutions for infants not able to receive enough mother’s milk. Infant formulas aim to mimic the composition and functionality of human milk by providing ingredients reflecting those of the latest human milk insights, such as oligosaccharides, bacteria, and bacterial metabolites. The objective of this narrative review is to discuss the most recent developments in infant formula with a special focus on human milk oligosaccharides and postbiotics.

Postbiotics are “bioactive compounds produced by food-grade microorganisms during a fermentation process.” The ISAPP has stated their official definition is a “preparation of inanimate microorganisms and/or their components that confers a health benefit on the host”. Exactly what does all that mean in layman’s terms? “Postbiotics is an umbrella term for the variety of metabolites and by-products that form from the action of probiotic bacteria in the gut,” explains Jennifer Martin-Biggers,

Many of us are familiar with probiotics, such as certain yogurts and fermented foods, full of “good” bacteria that can keep the gut healthy.

You might even have heard of prebiotics, foods rich in complex carbohydrates (dietary fiber) that help foster good bacteria in the large intestine. Popular prebiotic foods include oats, nuts and legumes.

But what about postbiotics? What are they and how do they affect our gut health?

1. Bacteria in Human Milk

Human milk is also an important source of beneficial bacteria (naturally occurring ‘probiotics’) that help colonize the infant’s gut and contribute to the composition of favorable gut microbiota, including Bifidobacterium spp. and  Lactobacillus spp. , with genus Bifidobacterium dominating the gut microbiota of a vaginally delivered infant. Bacteria in human milk are anticipated to be bioactive components regulating the development of an infant’s immune system and attenuating inflammation processes. In general, the microbiome of human milk is a recent field of research, as explored elsewhere [The total number of bacteria in human milk significantly differs according to detection methods. It has been estimated that human milk contains median values between 10 3 and  106 bacteria per milliliter]. The difference in number caused by different detection methods could be due to the fact that in molecular-based methods,

2. Microbial Metabolites in Human Milk

Besides bacteria, their  metabolites (e.g.,  butyrate  and  other  short-chain fatty  acids (SCFAs),

peptides,  oligosaccharides)  may also naturally pass into human milk,  and this can be detected through metabolomics research using nuclear magnetic resonance spectroscopy Most recently these metabolites have gained more research attention that is focused on their possible role in shaping the growth and development of the infant. A widely accepted definition still needs to be agreed on,  but in general,  they may also be referred to as ‘natural postbiotics’ (comprising both inactivated bacterial cells and metabolites)  and are anticipated to stimulate both healthy gut microbiota composition and function as well as immune functioning and development.

3. Postbiotics

A provisional definition has been formulated that stipulates that postbiotics  are compounds produced by microorganisms and released from  food components or microbial constituents,  including non-viable cells that, when administered in adequate amounts, promote health and well-being

Examples of postbiotics have been summarised as follows

• Compounds deriving from bacterial metabolisms, such as exopolysaccharides, vitamins, lactic

acid, bacteriocins, enzymes, surfactants, antioxidants, and SCFAs.

• Complex molecules released  from  food  compounds  (enzymatically  produced  during  food

fermentation), such as peptides and galactooligosaccharides, e.g., 3’-GL and 6‘-GL.

• Components released from lysed  cells including  DNA, RNA,  cell  walls and,  perhaps, other

cytoplasmic components, and surface layer proteins.

A consensus definition of postbiotics is currently being formulated by ISAPP

4. Postbiotics through fermentation

For many thousands of years, fermentation of food has been applied as a natural process to generate foods with particular properties, palatability,  taste, and health benefits. Most

benefits of fermented functional foods are accomplished by either the live microorganisms ingested or by postbiotics deriving from these microorganisms.

For cow’s milk-based infant formulas, fermentation processes typically use lactic acid-producing

bacteria as a starter culture. In addition to lactose consumption during fermentation, microbial enzymatic transgalactosylation can transform lactose into other lactose-based biomolecules, with 3’-GL as the major trisaccharide produced by Streptococcus thermophilus (e.g., Streptococcus thermophilus 065) from lactose fermentation. The fermentation process  is  usually  followed  by  physical treatment, which may include homogenization, pasteurization, sterilization,  and/or  spray-drying

Recently, a  specific fermentation process  has  been developed for  infant  nutrition, using two

specific types of food-grade lactic acid-producing microorganisms (Bifidobacteriumbreve C50  and Streptococcus thermophilus 065) and naturally delivering postbiotics.

5. Benefits of postbiotics in infant formula

In a mouse model, infant formula containing postbiotics deriving from a specific fermentation process combined with prebiotic scGOS/lcFOS (9:1) stimulated morphological (i.e., crypt-villus length in the ileum) and functional (i.e., ilealsucrase activity, gut permeability) gut maturation more similar to the mother-fed situation than infant formula without pre-  or postbiotics.

Gut permeability measured by fluorescein isothiocyanate-dextran was similar in mice receiving post- and prebiotics and mother-fed mice, while it was significantly lower in mice receiving the control infant formula without post- or prebiotics. An early decrease in permeability, as observed in the control group, could have a lasting detrimental effect on health by changing immune maturation.

6. Synbiotics

Beneficial synergistic effects may be expected from a combination of probiotics and prebiotics,

which are called synbiotics, using prebiotics to selectively increase the abundance of both endogenous beneficial bacteria and beneficial microbes in the infant’s gut.

Given the evidence for aberrant gut microbiota in infants with allergies] and the key

role of the gut microbiota on immune system maturation there is a  strong rationale for developing a suitable pre- and probiotic (synbiotic) blend for use in infant formula for infants at high risk of allergy and infants with already developed allergy.

A synbiotic mixture of prebiotics (scGOS/lcFOS or scFOS/lcFOS in a ratio of 9:1) and the probiotic strain Bifidobacterium breve M-16V already demonstrated promising results.  This synbiotic combination compensated for delayed bifidobacteria colonization in infants delivered by cesarean section (compared to vaginally delivered infants)  in an exploratory, randomized,  double-blind,  controlled study with  153  infants with detection of total fecal bifidobacteria as the primary study outcome.  Secondary outcomes demonstrated a lower proportion of potential pathogens (e.g., clostridia-related species) [118], lower fecal pH, and significant changes in SCFA pattern (e.g., higher acetate and lower butyric, isobutyric, and isovaleric acids). Furthermore, this synbiotic blend led to a significant decrease in SCORAD (Scoring Atopic Dermatitis) score in infants with atopic dermatitis (AD) and greater improvement of SCORAD score in infants with IgE (immunoglobulin E)-associated AD. At the 1-year follow-up of this study, children with AD in the synbiotics group showed less asthma-like symptoms (frequent wheezing and wheezing and/or noisy breathing apart from colds) and asthma medication use.

However, even if some synbiotic combinations already display promising clinical results.

For infants, more randomized trials with longer follow-ups are needed for the determination of their physiological and metabolic impact on the host.

7. HMOs 2′-FL and LNnT

2’-FL and  LNnT  have been anticipated  as  candidate prebiotics   Although  manufactured

HMOs are structurally identical to their counterparts in human milk, and regulatory approval is required for novel foods by the European Union (Commission Implemented Regulation (EU)

The EFSA Scientific Panel on Nutrition recently assessed 2’-FL, difucosyllactose  (DFL), LNnT, and LNT as safe for use in infant formula [94–96]. Furthermore, 2’-FL, DFL, LNnT, and LNT have been added to the GRAS-list derived from the FDA [97].

Preclinical research has shown that synthetic 2’-FL (with an identical structure as the 2’-FL found in human milk and often prepared from lactose) have prebiotic effects  and may deliver functional benefits in infants. In preclinical trials, 2’-FL promoted the growth of specific bifidobacteria [98,99],  blocked the growth of pathogens [100–102], and supported gut maturation and the stimulation of the gut-intestinal barrier [103]. Furthermore, 2’-FL impacted neuronal-dependent gut migrating motor  complexes, suggesting beneficial effects on the central nervous system.

8. HMO 3’-GL

HMO 3’-GL is naturally present in human milk and was already isolated from human milk in

1988. Recently, a single chromatography run using an improved sample pre-treatment method and ultrahigh-pressure liquid chromatography with fluorescence detection quantified 15 sialylated and neutral HMOs with high sensitivity, identifying 3′-GL and 6′-GL in colostrum, transitional, and mature human milk.

Human milk from mothers with preterm delivery revealed  3’-GL concentrations of 4–28.82 µg/mL (median 10.44) at 1 w postpartum, whereas concentrations ranged from 4–32.97 µg/mL (median 12.34) in colostrum and 4–20.73 µg/mL (median 4) at 8 w postpartum from mothers with term delivery.

Further human milk analyses of mothers with term delivery revealed 47–79 µg/mL of 3’-GL in colostrum [109], 5.08±0.45 µg/mL (mean ± SD) in colostrum, and 4.84±0.48 µg/mL (mean ± SD) at 100 d of lactation [39], and 0.5–39 µg/mL (median 4.6) in milk pooled until 21 d of lactation.

In preclinical studies, 3’-GL, 4’-GL, and 6’-GL prepared from colostrum individually accounted

for specific immunomodulation of polyinosinic: polycytidylic acid-induced interleukin-8 levels in an immature human intestine tested at a concentration of 200 µg/mL each. Another study reported that a solution of  5  mg galactooligosaccharides/mL,  which was synthesized from lactose and comprised of 14 % 3’-GL, 8 % 4’-GL, and 12 % 6’-GL, demonstrated ex vivo anti-inflammatory effects by attenuating the nuclear transcription factor κB inflammatory signaling in human intestinal epithelial cells [38]. In a model for intestinal barrier function using human intestinal epithelial Caco-2 cell monolayers grown in a transwell system, 3’-GL chemically synthesized from lactose was able to protect the intestinal barrier against breakdown of intestinal integrity, whereas alpha-3’-GL (with an α1-3 glycosidic linkage), 4’-GL, and 6’-GL (all chemically synthesized from lactose) demonstrated no significant results.

Previously,  a  commercial mixture of galactooligosaccharides also demonstrated a barrier-stabilizing effect and resulted in improved integrity of the intestinal barrier function and reduced inflammatory response by using the same in vitro method.

Although these first pre-clinical data may indicate that certain galactosyllactoses, such as 3’-GL,

have protective and immunomodulatory effects in the gut, more research is needed to further explore their role as HMOs.

Conclusion: 

Breastfeeding is the optimal way of feeding an infant. Current evidence still focuses on infant formula aiming to more closely resemble the composition and functionality of human milk, with some already comprising probiotics,  prebiotics,  synbiotics,  and postbiotics.

Type of feeding is a key issue in human gut development, the diversity of the microbiome, and intestinal function at any age in life. Whereas breastfeeding is the reference, the increasing efforts industry is making in the production of IFs that may qualitatively resemble and act as close as possible to HM have led to the supplementation of different bioactive ingredients, including probiotics, prebiotics, synbiotics, postbiotics, and HMOs. Concomitantly, scientific data on the benefits of MIF is continuously growing, with much evidence indicating overall positive effects on microbiome composition and metabolic activity.

Human milk naturally provides these components by delivering  HMOs  (‘natural prebiotics’)  and beneficial bacteria (‘natural probiotics’) and their metabolites (‘natural postbiotics’).  In infant formula, these nutritional concepts are provided by different  pre- and probiotic mixtures and a mixture of both (synbiotics) and, additionally, by using partly fermented infant formula with beneficial compounds produced by microorganisms that are released from food components or microbial constituents, including non-viable cells (postbiotics). One of the most current examples of such compounds is 3′-galactosyl lactose (3′-GL),  which is present in human milk and is a  natural derivative of milk fermentation. Although such developments may pave the way for future infant formula alternatives for those infants who are not able to be (fully) breastfed, human milk feeding will always remain the unmatched goal for infant nutrition and development as well as provide many benefits for maternal health

Abbreviations:

2′-FL

2′-fucosyllactose

3‘-GL  3′-galactosyl lactose

4’-GL  4’-galactosyl lactose

AD  atopic dermatitis

AE adverse event

DFL  difucosyllactose

DP  degree of polymerization

CFU  colony forming units

FDA  U.S. Food & Drug Administration

FL fucosyllactoses

FOS  fructooligosaccharides

FUT2  fucosyltransferase 2

GOS  galactooligosaccharides

GL galactosyl lactose

GRAS is generally recognized as safe

HM  human milk

HMO  human milk oligosaccharide

IF infant formula

IgE  immunoglobulin E

ISAPP  International Scientific Association for Probiotics and Prebiotics

kDakilodalton

LA Lactobacillus acidophilus

LB  lysogeny broth

LNnT  lacto-N-neotetraose

LNT  lacto-N-tetraose

NCDO  National Collection of Dairy Organism

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Biography:

Dr. Said is a consultant pediatrician whose experience in the field spans 20 years, backed by a higher education degree from the royal college of pediatrics’ child health in the UK, in addition to a master’s degree from Ain Shams University in Egypt one of the oldest and top ranking universities in the MENA region. He is pioneering an open and contextual evaluation model based on constructive responses, which has led to the creation of new methods to improve pediatric healthcare, neonatology, and pediatric nutrition.