Milk Fat Globule Membrane: A Unique and Valuable Dairy Component for Young Animal Feed - PARTNERS' R

Dr. Evi Croes, Nukamel BV, The Netherlands.

The Science Behind Young Animal Nutrition

✔ Nukamel is the first animal nutrition company ever to consider MFGM, as a potent bioactive milk component to support the growth of new-born piglets, calves, and even broilers.

TABLE OF CONTENT

• Introduction

• There's more to milk fat than just energy

• MFGM: Potential within its complexity

• A minor ingredient in milk that can have major effects on development in young mammals

• MFGM prevents infection and improves gut barrier integrity in challenging conditions

• MFGM promotes beneficial bacteria colonization and immune development

• MFGM improves brain development

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• MFGM improves fat digestibility and metabolism Page 18

• Applications of MFGM in enhancing formulae for new-borns

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• About the author Page 23

MILK FAT GLOBULE MEMBRANE: A UNIQUE AND VALUABLE DAIRY COMPONENT FOR YOUNG ANIMAL FEED

Nature’s creations are amazing! A deep look into the structure of a natural product, such as milk and its constituents shows this clearly. Whole milk is not just a simple assembly of fat, proteins, carbohydrates and minerals. No, nature has built it up in such a complex, but ingenious way, that it provides unique properties, from a physical, nutritional and physiological point of view.

The Science Behind Young Animal Nutrition

THERE'S MORE TO MILK FAT THAN JUST ENERGY.

For decades, scientists have studied human breast milk to enhance understanding of its complexities and enable closer mirroring of its composition and functionalities in improving infant formulae. Initially, research focused predominantly on the role of dairy proteins, microbial safety, and later, on the unique bioactivity of human milk oligosaccharides.

Previously, the main focus with regard to fats in infant nutrition was the amount of fat that can be tolerated and digested by infants, while the significance of the composition of dietary fat received little attention (Forsyth, 2007). However, there is more to fat than its role as a key fuel in energy metabolism. Lipids are essential for tissue growth, cardiovascular health, enteric nervous system (brain-gut)- and immune system development throughout life. Consequently, in later years, recommendations were made on the fatty acid composition, especially for lauric-, α-linolenic-, docosahexaenoic- and arachidonic acids (Uauy and Castillo, 2003; Tai et al., 2013; and Crawford et al., 2015).

Thus, vegetable oils, such as corn-, soy- or sunflower oil, are added to replace milk fat in infant formula because of their availability, nutritional properties and relative costs. One underestimated milk component by doing so, is the milk fat globule membrane (MFGM),

which is naturally inherent in all mammalian milks. Clinical evidence from recent years has indicated the beneficial effects of MFGM on infant health and development. Adding this fraction of milk back into infant formula further closes the gap that is lost in the switch to vegetable oils.

With extensive and convincing literature on the role of MFGM in the development and health of new-born infants, Nukamel investigated the usage of this valuable dairy component in milk replacers and other nutritional solutions for young animals. The first weeks, months or even years of an animal’s life are crucial in establishing the foundations for its future health. In addition, it is also the period when they are most vulnerable and more susceptible to infections. At this time, bioactive dairy molecules contribute to a healthy start that extends later into life.

Nukamel is the first animal nutrition company ever to consider MFGM, as a potent bioactive milk component to support the growth of new-born piglets, calves, and even broilers.

Before elaborating on Nukamel’s applications, we would like to introduce you to this extraordinary creation of nature and its array of benefits in early life nutrition.

The Science Behind Young Animal Nutrition

MFGM: POTENTIAL WITHIN ITS COMPLEXITY

MFGM surrounds the triacylglycerol (TAG) lipid core of milk fat globules (Figure 1), thereby protecting it from coalescence and degradation. It is a complex, three-layered membrane of about 10-50 nm thick that consists of polar lipids (phospholipids and glycolipids), apolar lipids (cholesterol and cerebrosides), proteins (mainly glycoproteins) and carbohydrates (sialylated oligosaccharides and glycoconjugates) (Dewettinck et al., 2008; Wang et al., 2022). MFGM-enriched dairy ingredients can be produced commercially from either whey or cream concentrates. The specific compositions of MFGM fractions depend upon their origin.

Figure 1: Schematic structure of milk fat globules, containing a TAG lipid core (yellow) and surrounded by MFGM (adapted from Gallier et al., 2014).

✔ Proteins

Although MFGM proteins account for only 1–4% of total milk proteins and 1% of the total globule mass, more than five hundred proteins with diverse functions have been identified by proteomics analysis (Cavaletto et al., 2008).

The major proteins in bovine MFGM have been identified as mucin 1 (MUC1), mucin 15 (MUC15), xanthine oxidoreductase (XDH/XO), cluster of differentiation 36 (CD36), butyrophilin (BTN), adipogenic differentiation-associated protein (ADPH), lactadherin (LADH) and fatty acid binding protein (FABP) (Dewettinck et al., 2008; Wang et al., 2022).

Proteins originating from the mammary epithelial cell plasma membrane are generally glycosylated (Brink and Lönnerdal, 2020). Glycosylated MFGM proteins are more resistant to digestion, which may explain their immune benefits and bacterial adhesion.

✔ Lipids

Polar lipids within the MFGM structure include phospholipids and sphingolipids, both of which have an amphiphilic nature. Phospholipids consist of a glycerol backbone, to which two fatty acids are esterified, and a phosphate group, attached to the third hydroxyl group. When coupled to a e.g. serine, choline or ethanolamine, the molecules are defined as phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), among others.

The ratio of the different phospholipid classes within the MFGM structure slightly varies among different mammalian species, but overall PC, PE, and sphingomyelin (SM) are the most abundant (Wang et al., 2022). This strongly contrasts well-known vegetable sources of phospholipids, such as soy and sunflower (Figure 2). SM is uniquely present in animal sources (Chai et al., 2022). Data from studies have demonstrated its importance in intestinal health and in maturation of the nervous system (Cas et al., 2020). Gangliosides, which are sphingolipids with sialic acid containing oligosaccharides attached, and cholesterol, a non-polar lipid, are also present in MFGM (Brink and Lönnerdal, 2020).

✔ Carbohydrates

The sugars in MFGM are mainly combined with proteins or lipids and exist in the form of glycoprotein or glycolipid. Within the structure, carbohydrate moieties from glycolipids and glycoproteins are orientated outwards and appear to be uniformly distributed over the external MFGM surface (Dewettinck et al., 2008; Wang et al., 2022).

Figure 2: Relative proportion of PL species (%) from different sources. Mature human and bovine milk, egg, soy, sunflower. SM, sphingomyelin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PA, phosphatidic acid (in the case of sunflower); PL, phospholipids (Chai et al., 2022).

Figure 3: The different components making up the MFGM (adapted from Gallier et al., 2014).

The Science Behind Young Animal Nutrition

A MINOR INGREDIENT IN MILK THAT CAN HAVE MAJOR EFFECTS ON DEVELOPMENT IN YOUNG MAMMALS

MFGM is a very good example of a unique structure that is present in mammalian milk in minor amounts, and yet can have significant health effects on the new-born. Extensive research has been performed on the role of its various components on different aspects of health and development (Figure 4). Growing interest in supplementing infant formula with MFGM is not only derived from its individual components, which have proven bioactivities, but also from the benefits associated to the combination of these components and their structural organisation.

The lipid-lipid and lipid-protein interactions within the MFGM structure afford these molecules a degree of persistence in the gastro-intestinal lumen. Through this, protective bioactive molecules can be conveyed to the large intestine, where they perform specific roles in early colonization and development of immune education. An example of this is how SM resists upper gastrointestinal digestion.

SM is partially cleaved to ceramide and sphingosine, two bioactive lipids, by intestinal alkaline sphingomyelinase in the small intestine. This favours the formation of sphingomyelin–cholesterol complexes in the proximal part of the intestine and exposes the lower small intestine and colon to SM and its bioactive metabolites (Bourlieu and Michalski, 2015). As evidence of just how much remains undegraded, it has been established that 25% of the SM found in faeces of rats was discovered in an undegraded form (10%), a ceramide form (30–90%), and a free sphingosine form (3–6%).

A more recent human ileostomy content study has shown that 19% to 25% of dietary SM was unabsorbed after ingestion (Norris et al., 2019).

Figure 4: Bioactivities of the components in MFGM (Wang et al., 2022).

The Science Behind Young Animal Nutrition

MFGM PREVENTS INFECTION AND IMPROVES GUT BARRIER INTEGRITY IN CHALLENGING CONDITIONS

MFGM-enriched baby milk reduced the number of days with fever in young children (Veereman-Wauters et al., 2012) and showed a preventative effect against infections in babies of 2- to 6-months of age (Timby et al., 2014). MFGM-embedded glycoproteins (such as butyrophilin, lactadherin, mucins, lactoferrin) and lipids (gangliosides) may participate in the defence against infections by preventing pathogen adhesion to epithelium. Secondly, part of the mechanism is probably due to a direct antimicrobial activity (Brink and Lönnerdal, 2020). A study of MFGM fractions from buttermilk and cream showed inhibition of rotavirus in vitro (Fuller et al., 2013). Gangliosides have also been shown to inhibit adhesion of both enterotoxic and enteropathogenic E. coli strains in an in vitro Caco-2 cell mode.

In a rat study, MFGM-supplemented diets fed from Postnatal Day 5 resulted in a better restoration of the intestinal villus and crypt architecture and improved expression of tight junction proteins. In mice, challenged with lipopolysaccharide, the membrane-rich milk fat exerted a protective role against gastrointestinal leakiness. Protection may be provided by the presence of phospholipids (such as lyso-phosphatidyl choline) and gangliosides. In addition, the inflammatory response in the animals fed the MFGM-rich diet was significantly lower than in those fed the control diet. Serum levels of different cytokines were decreased and signify a reduced stress on the gut barrier (Snow et al., 2011).

Also using neonatal piglets, MFGM showed evidence of increasing mucosal growth and protection against harmful bacteria (Le Huërou-Luron et al., 2018a). Even more specifically, the administration of MFGM in the first week of neonatal piglets showed enhanced protection against the invasion of pathogens through upregulated expressions of tight junctions (e.g. occludin, claudin-4) and mucins (Wu et al., 2021). MFGM supplementation in formula milk for rat pups showed many beneficial effects on the intestinal architecture at small intestinal and colonic sites. The villus heights and crypt depths were similar to mother’s milk fed pups, while the control diet fed pups yielded significant deficits in intestinal development (Figure 4, Bhinder et al., 2017). In challenging conditions against pathogenic bacteria, such as Clostridium difficile or Listeria monocytogenes, MFGM has

been attributed to conferring protection, probably by stimulating mucin secretion and preventing adherence of pathogens to the intestinal mucosa in rats (Bhinder et al., 2017).

Figure 4: (A) Body weights (g) of rat pups in diet groups fed either mother’s milk (MM), MFGM at 1.2 g/L and 6 g/L or control type (CTL) formula were similar after ten days of supplementation. n ≥ 10 (B) Villus lengths, measured in μm, in the jejunum (left) and ileum (right). Dose dependent increases in length in the MFGM supplemented group were observed. n = 8–10. Crypt depth in the jejunum (left) and ileum (right) (C) and colon (D) with CTL formula fed pups showing significantly decreased depths compared to all other groups. n = 8–10. The graphed data presented are the mean ± SEM, analyzed by One-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. (Bhinder et al., 2017).

The Science Behind Young Animal Nutrition

MFGM PROMOTES BENEFICIAL BACTERIA COLONIZATION AND IMMUNE DEVELOPMENT

The infant immune system is inexperienced. Complex lipids help prime and balance the immune system so that it doesn’t overreact or underreact to challenges. Immunoregulation consists of both enhancement and suppression of the immune system. The final immune response is a result of the balance between enhancing (helper) and suppressive influences (Krakauer and Clough, 1980). Nutrients might modulate immune maturation and responses and provide factors that influence the intestinal microbiome.

Cytokines play an important role as mediators in the immunological response. A study using spleen cell cultures showed a clear anti-inflammatory effect of MFGM, mainly due to the production of cytokines. In pathologic situations, the inflammatory response can become chronic and lead to tissue injury. Downregulation may be beneficial to survival, and hence, specific inhibitors of inflammation-related products. So, MFGM could be a potential antagonist preventing inflammatory reaction (Zanabria et al., 2014).

Intestinal colonization of the gut with beneficial microbiota influences vital functions and has profound effects on the adult health status of the host. Microflora plays a role in gut barrier, metabolism and the maturation of the host’s immune responses. Wu and co-workers (2021) studied the effect of early life administration of MFGM to piglets and found a positive relationship with plasma IgG levels and active colonization of the gut with short-chain fatty acid-producing bacteria.

Particular glycosylated proteins from MFGM could be a substrate for microbial fermentation, and the metabolites formed may exert further alterations in the microbiota. In rat pups, MFGM-enriched formulae impacted the microbiome as well, similar to the pups fed mother’s milk rather than that seen in their littermates that received a control formulae type (Bhinder et al., 2017). Le Huërou-Luron and co-workers (2018b) concluded that both milk fat and MFGM fragments changed the faecal microbiota composition of piglets. An increase in Proteobacteria and Bacteroidetes and a decrease in Firmicutes phyla were found.

The addition of dairy lipids also accelerated the maturation of the intestinal immune system that was closer to the one observed in mother-fed piglets. Long-term effects were shown in adult pigs, receiving a dairy lipid-supplemented diet in infancy. Compared to the control diet animals that were fed vegetable oils, they showed decreased proinflammatory cytokine secretions in response to a lipopolysaccharide challenge, confirming the beneficial impact of dairy lipids on the intestinal immune system (Le Huërou-Luron et al., 2018a).

MFGM IMPROVES BRAIN DEVELOPMENT

Phospholipids and gangliosides are presumed to be important components in neuronal and cognitive development. Dietary choline, which is found attached to the phosphate group of PC in MFGM, has been studied in animal trials for its role in gestation on neurodevelopmental outcomes in the offspring (Zeisel, 2006). Choline is a methyl donor nutrient that has been shown to affect DNA methylation and long-term potentiation in the developing hippocampus of foetal rats (Niculescu et al., 2006).

In the brain, SM is an essential component of myelin that surrounds neuronal axes. Considering that myelination is closely associated with the maturation of brain networks, the coordination of information processing, and in the cognitive performance, nutritional provision of these particular molecules, e.g. through the supplementation in formula milk, is hypothesized to be highly relevant in infants and children (Cas et al., 2020). Tanaka et al. (2013) demonstrated improvements in cognitive development in very low birth weight pre-term infants fed with SM-fortified milk. Infants at 18-months old, performed better in mental-, motor- and behavioural tests.

Gangliosides are concentrated within the brain’s grey matter and constitute approximately 6% to 10% of the total human brain lipid mass. Additionally, gangliosides are enriched at the synaptic membrane of neurons, and are functionally involved in neurotransmission and synapse formation (Yu et al., 2009). Liu et al. (2014) studied brain development and spatial learning and memory in neonatal piglets. Piglets that were fed with a formula containing milk phospholipids and gangliosides to mimic levels in human milk made choices more rapidly and with fewer errors in a cognitive test compared to controls, implying improved spatial learning.

Similarly, Vickers et al. (2009) demonstrated that rats showed significant improvements in learning and memory tasks when fed MFGM. Also term infants, receiving formula milk, supplemented with MFGM, rich in gangliosides, exhibited higher cognitive scores than the control group, and not significantly different from the breastfed reference group (Gurnida et al., 2012; Timby et al., 2014).

MFGM fragments were evaluated as a nutritional intervention to modulate stress-induced dysfunction, e.g. visceral hypersensitivity, and brain development. Intervention with MFGM, in combination with a prebiotic blend significantly impacted the composition of the microbiota, as well as ameliorating some of the long-term effects of early-life stress (O’Mahony et al., 2020).

MFGM IMPROVES FAT DIGESTIBILITY AND METABOLISM

MFGM acts as a natural emulsifying agent, preventing the flocculation and coalescence of fat globules in milk and protecting against enzymatic action (Contarini and Povolo, 2013). So, due to its amphiphilic nature, MFGM is considered to be an excellent emulsifier. Also, MFGM can reside on the interface of liquid-air, thereby, acting as a foam stabilizer.

Among other bioactive proteins, bovine MFGM also possesses fatty acid-binding proteins (Fong et al., 2007) that may promote fatty acid transfer across the intestinal tract. Emulsification of fat droplets with milk phospholipids had a positive effect on lipid digestion and postprandial lipid metabolism in rats (Lecomte, et al., 2015). Bezelgues et al. (2009) observed, by an in vitro digestion study with tocopherol and lycopene, that MFGM-purified fractions had a higher ability of incorporation of fat-soluble molecules into bile salts micelles than other conventional emulsifying milk proteins.

Fat absorption in young animals and infants is generally less efficient relative to their adult counterparts. This is even more pronounced in pre-term piglets or pre-term infants, who have an absorption rate of 70–80% or even lower, depending upon the fat composition of their diet. The fact that lipid digestion in formula milk is reduced compared to mother’s milk is (partly) due to the presence of MFGM. Currently, soy lecithin is commonly used as an emulsifier and stabilizer in infant formulae. Soy lecithin mainly consists of phosphatidylcholine and does not contain SM or cholesterol.

Using a pre-term neonatal piglet model, Knudsen et al. (2021) demonstrated that piglets fed milk supplemented with MFGM ‘emulsifiers’ showed higher plasma triglyceride levels relative to the soy lecithin supplemented group. Lipid droplets, emulsified with bovine dairy emulsifiers increase the fat digestion in milk, compared to droplets with an interfacial layer, composed mainly of phosphatidylcholine (i.e., the soy lecithin emulsions).

The lipolysis with MFGM is faster and coincides with the findings from earlier studies suggesting that the pancreatic lipase has a higher affinity for a lipid surface covered with casein or whey proteins.

Obesity has become a major health problem around the world. It is a risk factor for metabolic disorders, such as type 2 diabetes, insulin resistance, hyperglycaemia and cardiovascular diseases. Some studies have shown that MFGM and its components have beneficial effects against obesity and related metabolic disorders. Diets of young mice supplemented with MFGM prevented body fat accumulation (by preventing adipocyte hypertrophia) in adulthood (Oosting et al., 2014).

Moreover, MFGM counteracts the negative impact of high-fat diets rich in saturated fat in obese adults. The protective effects of SM and PC against obesity and metabolic dysfunction have been studied (Li et al., 2020). SM affects different aspects of cholesterol transport and metabolism, suggesting that it may influence atherosclerosis (Contarini and Povolo, 2013). In addition, in rodents, piglets, and baboons, as well as infants, it appears that the dietary provision of MFGM, as a source of cholesterol, suppress de novo synthesis, and show long-term benefits in altering the cholesterol metabolism later in life.

Fil and co-workers (2019) also found higher circulating high-density lipoprotein (HDL) concentrations in neonatal pigs fed MFGM. Current evidence from pre-clinical studies indicates that dietary milk SM has protective properties against dysfunctional lipid metabolism, gut dysbiosis, and inflammation. A reduction of serum and hepatic lipid concentrations by the supplementation of milk SM is observed in animal studies. Human studies show similar effects, although more research is needed to fully understand the role of these molecules in humans (Norris et al., 2019).

APPLICATIONS OF MFGM IN ENHANCING FORMULAE FOR NEW-BORNS

✔ Human nutrition

For decades, there has been a focus on mimicking the composition of human breast milk in infant formulae (Gallier, et al., 2015). Several research groups and infant formula manufacturers have investigated the addition of ingredients rich in MFGM or complex lipids into infant formulae to improve several growth and development outcomes in infants (Billeaud et al., 2014; Guan et al., 2015; Hernell et al., 2016; Oosting et al., 2014; Reis et al., 2016; Timby et al., 2017).

Mead Johnson Nutrition Inc. (Enspire™), Fonterra Co-operative Group Ltd (Anmum™), Arla Food Ingredients (Lacprodan® MFGM-10), Hilmar Ingredients (Hilmar TM 7500) and Semper AB (Baby Semp) are currently selling, in different markets, infant formulae containing MFGM or complex milk lipids alone or in combination with other bioactive nutrients. One of the main drivers for this is ample research evidence on the benefits for brain development and cognition (Guan et al., 2015; Mudd, et al., 2016; Reis, et al., 2016; Timby et al., 2014;2017).

Alongside this, the addition of MFGM is promoted due to its decreasing risk of infections, acting as a decoy for pathogens and viruses (Hamosh et al., 1999) and immunomodulatory effects (Le Huërou-Luron et al., 2018a, 2018b). Moreover, MFGM supplementation in infancy is hypothesized to have programming effects that may influence circulating lipid levels later in life.

Older adults want to stay active and energetic and maintain mental agility. New research indicates that MFGM may play a supportive role in maintaining brain health and physical performance. MFGM also improves intestinal health and skin condition, combats hypercholesterolaemia, obesity and inflammation. A recent study in mice reported that MFGM in combination with exercise was effective in suppressing age-associated muscle mass and strength deterioration. MFGM supplementation, in combination with exercise, has been shown to improve the physical performance of healthy middle-aged adult humans (Sugita et al., 2021; Ota et al., 2015; Abd El-Salam and El-Shibiny, 2020).

Patients with chronic inflammatory diseases of the gastrointestinal tract may benefit from dietary products that can counteract the autoimmune host cellular responses and hence limit localized inflammation (Cross and Gill, 1999).

Sphingolipids in MFGM can not only treat and improve cardiovascular diseases, but also participate in the inflammatory process of atherosclerosis and insulin resistance. Therefore, it can be inferred that dietary sphingolipids have great potential for the treatment of various aspects of metabolic syndromes, such as dyslipidemia, insulin resistance, and cardiovascular diseases (Wang et al., 2022).

✔ Nukamel: Harnessing the full potential of MFGM in young animal nutrition

Studies on rodent and piglet models have proven the impact of MFGM on infection, inflammation, brain composition, and gut barrier integrity, as well as intestinal development. The overall findings indicate MFGM’s potential as a functional ingredient for modulating microbial populations and improving intestinal health and functions in mammalian neonates. Nevertheless, all nutritional and health studies on MFGM performed on animals had a single goal of providing a model for human-, and more specifically, infant nutrition.

It is, however, very likely that MFGM is also important for milk-fed calves, piglets and lambs. Young animals must thrive every day to achieve optimal growth. The early stage of life sets the pace for later. Less antibiotic treatments, and more efficient growth result from good nutrition.

With a view to providing additional support for young animals, Nukamel has invested in diverse studies focused on exploring the benefit of supplementing milk replacers, or pre-starter diets with MFGM-enriched ingredients. As a young animal nutrition and dairy ingredient specialist, Nukamel wants to leverage the full benefit of this unique milk constituent. In infant nutrition, vegetable oils are currently used to replace milk fat in the diets for new-born animals due to availability, practicality and economics. However, the formulation of the diets can be improved further through the supplementation of this natural, and highly valuable functional milk ingredient. Clever formulation of young animal

The Science Behind Young Animal Nutrition

nutritional solutions that makes full use of the intrinsic strength and bioactivity of milk, is something that Nukamel strives for every day.

It must be said: “This is one of the biggest revolutions in infant formula over the last few years”. And yet, despite MFGM being a normal addition in infant formulae nowadays, it still novel in young animal nutrition. Future work is likely to be addressed towards a deeper comprehension of MFGM usage in early life nutrition across different species. Nukamel is the first to pioneer this.

✔ NUKAMEL, AGAIN THE FIRST, STILL THE BEST!!

Further details on the nutritional developments Nukamel explored in relation to the usage of MFGM in calves, piglets, or even broilers can be found on our website: www.nukamel. com. The following concepts are explained in more detail:

• for calves: ProtéGO;

• for piglets, Piglait,

• and for all young animals, including broilers: Volamel Compass.

ABOUT THE AUTHOR

After her Phd in Bio Engineering at the KU Leuven, Dr. Evi Croes became a Research Scientist at the Proefcentrum voor Fruitteelt. Since 2010 she has been employed at Nukamel NV, first as Product Manager and since 2015 as R&D Manager.

Dr. Evi Croes is a regular key note speaker at international conferences and seminars. She is considered an expert on young animal nutrition and regularly publishes articles and whitepapers.

She adressed this specific topic during her key note speach at the European Calf Conference in 2020.

DR. EVI CROES

R&D Manager at Nukamel NV

Emai: Evi.croes@nukamel.com