WO2023208925A1

WO2023208925A1 - Nutritional composition for use in preventing programmed obesity in female infants

Info

Publication number: WO2023208925A1
Application number: PCT/EP2023/060796
Authority: WO
Inventors: Anniek Lidewij SCHIPPER; Gertjan VAN DIJK
Original assignee: N.V. Nutricia; Rijksuniversiteit Groningen
Priority date: 2022-04-25
Filing date: 2023-04-25
Publication date: 2023-11-02

Abstract

The invention relates to a nutritional composition selected from infant milk formulation, follow-on milk formulation and growing-up milk formulation for use in preventing or reducing programmed obesity after early childhood in female subjects that are overfed during infancy, wherein the nutritional composition comprises phospholipids, sphingolipids and cholesterol.

Description

NUTRITIONAL COMPOSITION FOR USE IN PREVENTING PROGRAMMED OBESITY IN FEMALE INFANTS

TECHNICAL FIELD OF THE INVENTION

The invention relates to a nutritional composition selected from infant milk formulation, follow- on milk formulation and growing-up milk formulation for use in preventing or reducing programmed obesity after early childhood in female subjects that are overfed during infancy.

BACKGROUND OF THE INVENTION

Human milk is the uncontested gold standard concerning infant nutrition. However, in some cases breastfeeding is inadequate or unsuccessful for medical reasons or because of a choice not to breastfeed. For such situations infant or follow on formulas have been developed. Commercial infant formulas are commonly used today to provide supplemental or sole source of nutrition early in life. These formulas comprise a range of nutrients to meet the nutritional needs of the growing infant, and typically include fat, carbohydrate, protein, vitamins, minerals, and other nutrients helpful for optimal infant growth and development. Commercial infant formulas are designed to mimic, as closely as possible, the composition and function of human milk.

Since long it has been appreciated that breastfed infants have a different weight gain pattern or trajectory compared to formula-fed infants. After the first week of life, in which breastfed infants initially tend to lose more weight than formula-fed infants and take slightly longer to regain their birth weight, the weight gain patterns are similar between breastfed and formula fed infants for the first 4 months of life. Breastfed infants tend to have slightly higher weight at 3 months age (Andres et al, 2013, J Pediatrics 163: 49-54). After about 4 months of age, the rate of weight gain diverges markedly between breastfed and formula-fed infants. The difference in average weight at 12 months approximates up to 500-650 g (Dewey et al., 1993, Am J Clin Nutr 57: 140-145). Numerous studies performed in various regions from all over the world have reported that breastfed infants have a slower weight gain between 4 and 12 months of life in Western developed countries as well as in non-Western developing countries. Length gain tends to differ less between breastfed and formula-fed infants and as a result breastfed infants are leaner at 12 months of age (Dewey et al., 1993). Thus, in the art it has been indicated that the growth curve of infants fed with commercial infant formula differs from the growth curve of breastfed infants. Typically the infant formula has a growth accelerating effect in the first year of life, in particular from 4 months of age onwards, resulting in a higher weight at 12 months of age (Dewey et al., 1993; Dewey et al, 1992 Pediatrics 89:1035).

This difference in growth curve continues after infancy. Breast fed infants have a decreased chance of becoming obese later in life, compared to standard formula fed infants, but little is known about the effects of ingredients in the infant formula on obesity later in life. The term “programmed obesity” is used to refer to later-in-life obesity that is induced by the diet during infancy.

Obesity is a major health problem in the Western world. It is a medical condition in which excess fat has accumulated to the extent that it may have an adverse effect on health, leading to reduced life expectancy and it is associated with many diseases, particularly heart disease and type 2 diabetes. Obesity is a leading preventable cause of death worldwide, with increasing prevalence in adults and children, and authorities view it as one of the most serious public health problems of the 21st century. It has been established that a higher visceral to subcutaneous fat ratio is a marker strongly associated with higher risk of cardiometabolic disease (Kaess et al. The ratio of visceral to subcutaneous fat, a metric of body fat distribution, is a unique correlate of cardiometabolic risk. Diabetologia. 2012 Oct;55(10):2622-2630. doi: 10.1007/S00125-012-2639-5. Epub 2012 Aug 17. PMID: 22898763; PMCID: PMC3636065.). Thus, there is a need to improve upon infant formulation to prevent obesity later in life.

A problem with feeding infants with infant formula is that parents are inclined to overfeed their child. When bottle fed, the parents often dictate the amount of milk to be received, while breastfeeding heavily relies on the feedback of the child. Furthermore, the required caloric daily intake for infants is dependent on the age of the infant, while the same infant formula is used during the first 6 months. In contrast, studies have shown that breast milk composition changes from the first to second and third month of lactation for example. Consequently, parents have difficulty in administering the proper amount of calories using one-fits-all infant formulae available in the market.

Furthermore, infant formulae neglect the sex of the infant. Powe et al. have shown that there is a difference in energy content in breast milk based on the gender of the new born infant. Milk secreted from the mothers of male infant was reported to be 25 % greater in energy content when compared with the milk of mothers of infants. Boys and girls have different physiological needs based upon the World Health Organization (WHO) standard growth curves for boys and girls. Consequently, the tendency to overfeed infants is even more prevalent in girls, because the composition of infant formula administered t bottle fed infants is the same regardless of the gender.

The following data were extracted “Human energy requirements, Report of a Joint FAO/WHO/UNU Expert Consultation”, Rome 17-24 October 2001 (Table 3.2, Energy requirements of infants during the first year of life). This data shows that the caloric requirements of girls are significantly lower than those of boys and that the caloric requirements continuously increase with age.

WO 2007/073194 relates to a nutritional composition comprising a lipid, protein and digestible carbohydrate component and :

(a) 0.5 to 20 wt.% phospholipids based on total lipid;

(b) 0.1 to 20 wt.% sphingolipids based on total lipid; and (c) 0.005 to 10 wt.% cholesterol based on total lipid; which nutritional composition is to be administered to a non-obese infant with the age below 36 months for the prevention of obesity and/or diabetes type 2. WO 2015/86170 relates to a synthetic nutritional composition for a male infant or child comprising: a. 60-85 kcal/100ml energy, b. 3.9-6 g/100ml lipid, and c. 600-810 mg/100ml linoleic acid.

It also relates to a synthetic nutritional composition for a female infant or child comprising: a. 57-70 kcal/100ml energy, b. 3.5-4.5 g/100ml lipid, and c. 410-580 mg/100ml linoleic acid.

Baars et al. (Milk fat globule membrane coating of large lipid droplets in the diet of young mice prevents body fat accumulation in adulthood. British Journal of Nutrition 2016 Vol 115 Is 11 P1930-1937) report that in their mouse model, feeding a diet containing large lipid droplets coated with phospholipids (Nuturis®) in early life protected against excessive body fat later in life.

Metabolic programming is defined as a biological phenomenon that determines the association between physical and chemical stimuli in early life and future functional status. Overnutrition during early postnatal life represents a risk factor for persistent obesity and associated metabolic and cardiovascular disturbances. It has been shown that animals raised in small litters (SL) have an accelerated body weight gain before weaning, which is associated with permanent modulation of adiposity and hypothalamic circuits that control food intake and energy balance in adulthood (Rodrigues, et al. (2009), Postnatal early overnutrition changes the leptin signalling pathway in the hypothalamic-pituitary-thyroid axis of young and adult rats. The Journal of Physiology, 587: 2647-2661.)

Ye et al. (Neonatal Milk Fat Globule Membrane Supplementation During Breastfeeding Ameliorates the Deleterious Effects of Maternal High-Fat Diet on Metabolism and Modulates Gut Microbiota in Adult Mice Offspring in a Sex-Specific Way. Frontiers in Cellular and Infectious Microbiology 2021 Vol. 11 Art 621857) report the effects of milk fat globule membrane (MFGM) supplementation to the offspring of mothers consuming an high fat diet during pregnancy and lactation in a mouse model. Sex of the offspring was found to be a relevant parameter.

Oosting et al. (Size and phospholipid coating of lipid droplets in the diet of young mice modify body fat accumulation in adulthood, PEDIATR RES, 2012 vol. 72, no. 4, 1) describe the programming effect of infant formula comprising large, phospholipid-coated lipid droplets on the metabolic phenotype later in life.

US 2017/151203 describes a method of developing cognitive or behavioural performances, including fine motor skills and visual acuity, in a human subject, the method comprising administration of a nutritional composition to the huma subject wherein the composition comprises lipid globules with a core comprising vegetable lipids and a coating comprising phospholipids and polar lipids.

SUMMARY OF THE INVENTION

The inventors have unexpectedly observed that, despite metabolic challenges early in life, programmed obesity in females can be minimised effectively by feeding these females during infancy a nutritional composition that comprises a lipid fraction, wherein the lipid fraction comprises:

(a) 0.5 to 20 wt.% phospholipids based on total lipid;

(b) 0.1 to 20 wt.% sphingolipids based on total lipid; and

(c) 0.005 to 10 wt.% cholesterol based on total lipid.

Since it is extremely difficult to prevent overfeeding of infants, especially during early infancy, the present invention provides an important option for minimising programmed obesity in human females.

Drawings

Fig. 1 : Cumulative (PN0-PN14) food intake per pup of dams with a small or normal litter. Data are presented as means with SEMs. ***= p < 0.001.

Figure 2. Average weight development of the nests per pup (PN2-PN21). Error bars represent the standard error of the mean (SEM). *=p<0.05, **=p<0.01 .

Figure 3: Body weight of female mice between 22 and 126 days of age. Data represent mean +/- SEM. Unless specified otherwise, the reported effect on the graph are significant.

Figure 4: Body fat % of the mice determined by echo MRI at (A) PN98 and (B) PN112. Data are expressed as mean+/-SEM. a significant difference compared to healthy reference group; #a trending compared to healthy reference group; b. significant difference compared to SL CTR group. Figure 5: Total fat mass of adult males and females on the WSD diet (PN126). Data are presented as means with standard error of the mean (SEM; *=p<0.05).

Figure 6: Fat to lean mass of adult males and females on the WSD diet (PN126). Data are presented as means with standard error of the mean (SEM; *=p<0.05).

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the invention relates to a nutritional composition selected from infant milk formulation, follow-on milk formulation and growing-up milk formulation for use in preventing or reducing programmed obesity after 8 years of age in female subjects that are overfed during infancy, said use comprising orally administering the nutritional composition to female subjects during infancy; the composition comprising a lipid fraction, wherein the lipid fraction comprises:

(a) 0.5 to 20 wt.% phospholipids based on total lipid;

(b) 0.1 to 20 wt.% sphingolipids based on total lipid; and

(c) 0.005 to 10 wt.% cholesterol based on total lipid.

In other words, the present invention concerns a method preventing or reducing programmed obesity after 8 years of age in female subjects that are overfed during infancy, said method comprising orally administering the nutritional composition to female subjects during infancy, said nutritional composition being selected from infant milk formulation, follow-on milk formulation and growing-up milk formulation; wherein the lipid fraction comprises:

(a) 0.5 to 20 wt.% phospholipids based on total lipid, preferably 1 to 10 wt.% phospholipids based on total lipid, even more preferably 4 to 8 wt.% phospholipids based on total lipid;

(b) 0.1 to 20 wt.% sphingolipids based on total lipid, more preferably 1 to 10 wt.% sphingolipids based on total lipid, even more preferably 2 to 8 wt.% sphingolipids based on total lipid;

(c) 0.005 to 10 wt.% cholesterol based on total lipid, more preferably 0.01 to 5 wt.% cholesterol based on total lipid, even more preferably 0.05 to 1 wt.% cholesterol based on total lipid.

Worded differently, the present invention concerns the use of lipids in the manufacture of a nutritional composition selected from infant milk formulation, follow-on milk formulation and growing-up milk formulation for preventing or reducing programmed obesity after 8 years of age in female subjects that are overfed during infancy, wherein the lipid fraction comprises: (a) 0.5 to 20 wt.% phospholipids based on total lipid, preferably 1 to 10 wt.% phospholipids based on total lipid, even more preferably 4 to 8 wt.% phospholipids based on total lipid; (b) 0.1 to 20 wt.% sphingolipids based on total lipid, more preferably 1 to 10 wt.% sphingolipids based on total lipid, even more preferably 2 to 8 wt.% sphingolipids based on total lipid;

The present invention may also be defined as the use or method for non-therapeutic, or a non- therapeutic use or method for preventing or reducing programmed obesity after 8 years of age in female subjects that are overfed during infancy, said method comprising orally administering the nutritional composition to female subjects during infancy, said nutritional composition being selected from infant milk formulation, follow-on milk formulation and growing-up milk formulation; wherein the lipid fraction comprises:

In preferred embodiments, the composition comprises (d) 78 to 99.3 wt.% fat based on total lipid.

The term “programmed obesity” as used herein refers to obesity that occurs after early childhood and that is induced during infancy. “Programmed obesity” induced during infancy can be achieved by different ways: increased caloric density (while the total daily volume intake remains the same); increased total daily volume intake (while the composition’s caloric density remains the same); or a combination thereof.

Preferably, ‘programmed obesity’ induced during infancy is achieved by the increased total daily volume intake. Herein, the increase is in comparison with the intake by a breastfed infant of the same gender and age.

Preferably, ‘programmed obesity’ induced during infancy is achieved by overfeeding the infant, i.e. feeding the infant a volume of infant formula, follow-on formula, or growing up milk that is at least 5%, preferably at least 10%, more preferably at least 20% higher than the volume of infant formula, follow-on formula, or growing up milk required by an infant of the same gender and age. Preferably, the infant is overfed for a period of at least 1 month, more preferably at least 2 months, most preferably at least 4 months.

Preferably, ‘programmed obesity’ induced during infancy is achieved by overfeeding the infant, i.e. feeding the infant with a caloric amount that is at least 5%, preferably at least 10%, more preferably at least 20% higher than the caloric amount required by an infant of the same gender and age. Preferably, the infant is overfed for a period of at least 1 month, more preferably at least 2 months, most preferably at least 4 months.

‘Reducing programmed obesity’ as used herein refers to having a lower chance of developing obesity after early childhood in an infant who is overfed in infancy compared with a breastfed infant of the same gender and age, preferably by reducing weight gain, fat tissue deposition, or combinations thereof after early childhood.

‘Overfed during infancy’ as used herein refers to bottle fed infants, preferably bottle fed female infants. Preferably, the bottle fed infant's diet comprises infant formula, follow-on formula, or growing up milk. In one embodiment, the infant’s diet comprises both breast milk and infant formula. In another embodiment, the infant’s diet consists of infant formula. Preferably at least 10%, more preferably at least 20%, even more preferably at least 40% of the infant’s diet is infant formula, follow-on formula, or growing up milk.

The terminology “after early childhood” means after the age of 8 years, preferably after the age of 5 years, more preferably after the age of 3 years, even more preferably after the age of 2 years, most preferably after the age of 1 year.

The term “infancy” as used herein refers to the period of life between birth and the age of 36 months, preferably between birth and the age of 24 months, more preferably between birth and the age of 18 months, even more preferably between birth and the age of 12 months.

The term “fat” as used herein refers to glycerides selected from the group consisting of triglycerides, diglycerides, monoglycerides and combinations thereof. Preferably, the fat is vegetable fat.

The term “lipid” as used herein refers to the sum of fat as defined here before and other lipids such as phospholipids, sphingolipids and cholesterol.

The term “phospholipids” as used herein refers to derivatives of glycerol phosphate comprising one or two fatty acid residues. The term “phospholipids” as used herein does not encompass sphingolipids. Examples of phospholipids include phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol.

Phospholipids used in the present invention may be derived from animal or vegetable sources. Preferably, the composition of the invention comprises egg phospholipids, soy bean phospholipids or mammalian milk phospholipids. More preferably the nutritional composition according to the invention comprises phospholipids derived from mammalian milk, most preferably derived from non-human mammalian milk. Phospholipids derived from non-human mammalian milk include phospholipids isolated from milk lipid, cream lipid, cream serum lipid, butter serum lipid beta serum lipid, whey lipid, cheese lipid and/or buttermilk lipid. The buttermilk lipid is typically obtained during the manufacture of buttermilk. The butter serum lipid or beta serum lipid is typically obtained during the manufacture of anhydrous milk fat from cream or butter. Preferably the phospholipids are obtained from milk cream. The phospholipids are preferably derived from milk of cows, mares, sheep, goats, buffalos, horses and camels, most preferably from cow’s milk. It is most preferred to use a lipid extract isolated from cow’s milk. A suitable source of phospholipids derived from nonhuman mammalian milk is the fraction that can be isolated from milk called milk fat globule membrane (MFGM). Hence in one embodiment, the phospholipids to be used in the nutritional composition in the method or use according to the present invention are derived from or form part of the milk fat globule membrane (MFGM), or are provided as MFGM, preferably cow’s milk MFGM.

The term “milk fat globule membrane” or “MFGM” as used herein refers to the combination of substances that surrounds milk fat globules secreted from the milk producing cells of humans and other mammals. MFGM comprises multiple bioactive compounds, including phospholipids, glycosphingolipids and cholesterol.

The term “sphingolipid” as used herein refers to a class of lipids derived from the aliphatic amino alcohol sphingosine. Examples of sphingolipids include sphingomyelins, ceramides, and glycosphingolipids.

According to the present invention the risk of being overfed of the above defined ‘at risk’ infants is compared to the feeding of the same infants (same gender, age and for the same period of time) who did not receive increased volume and/or caloric density and/or did not consume the nutritional composition according to the invention but instead consumed a standard or control nutritional composition. In some embodiments of the present invention, the nutritional composition comprises lipid globules. Preferably, the lipid globules comprise components (a), (b) and (c), more preferably (a), (b), (c) and (d). When in liquid form these lipid globules are emulsified in the aqueous phase. Alternatively the lipid globules are present in a powder and the powder is suitable for reconstitution with water or another food grade aqueous phase, preferably to provide a ready to drink formula.

The lipid globules as used herein comprise a core and a surface. Preferably, at least 90 wt.% of the lipid is contained in lipid globules comprising a core of fat surrounded by a coating comprising phospholipids. The core preferably comprises vegetable fat and preferably comprises at least 90 wt.% triglycerides and more preferably essentially consists of triglycerides.

Not all vegetable lipids that are present in the composition need necessarily be comprised in the core of lipid globules, but preferably a major part is, preferably more than 50% wt.%, more preferably more than 70 wt.%, even more preferably more than 85 wt.%, even more preferably more than 95 wt.%, most preferably more than 98 wt.% of the vegetable lipids that are present in the composition are comprised in the core of lipid globules. In one embodiment the core of the lipid globules comprises at least 40 wt.% triglycerides of vegetable origin, more preferably at least 50 wt.%, even more preferably at least 70 wt.% triglycerides of vegetable origin, more preferably the core of the lipid globules comprises at least 85 wt.%, more preferably at least 95 wt.% triglycerides of vegetable origin.

The nutritional composition of the present invention preferably is selected from an infant milk formulation and a follow-on milk formulation. Most preferably, the nutritional composition is an infant milk formulation.

The nutritional composition of the present invention may be provided, for instance, in the form of a powder or a liquid. Preferably, the nutritional composition of the present invention is a powder. Oral administration of the nutritional composition in powder form requires that the composition is combined with aqueous liquid before it is administered. Preferably, the nutritional composition comprises 3 to 7 g lipid/100 kcal, 1.25 to 5 g protein/100 kcal and 6 to 18 g digestible carbohydrate/100 kcal.

Preferably, the nutritional composition of the present invention preferably comprises 4 to 6 g lipid/100 kcal, most preferably of 4.5 to 5.5 g lipid/100 kcal. The protein content of the nutritional composition is preferably in the range of 1.35 to 4 g protein/100 kcal, more preferably 1.5 to 3 g protein/100 kcal, more preferably 1.25 to 2.5 g protein/100 kcal, more preferably 1.25 to 2.25 g/100 kcal, even more preferably 1.25 to 2.1 g protein/100 kcal.

Digestible carbohydrate is preferably contained in the nutritional composition in a concentration of 8 to 16 g digestible/100 kcal, more preferably 10 to 15 g digestible carbohydrate/100 kcal.

The nutritional composition to be administered in the method or use according to the present invention comprises carbohydrates, protein and lipids wherein preferably the lipids provide 30 to 60 % of the total calories, the protein provides 5 to 20 % of the total calories and the carbohydrates provide 25 to 75 % of the total calories. Preferably the nutritional composition comprises 10 to 50 wt.% lipids based on dry weight of the total composition.

Preferably, all the fat present in the composition is in the form of lipid globules. The lipid globules are preferably contained in the nutritional composition in a concentration 10-40 g/100g composition (dry powder), more preferably in a concentration of 15-35 g lipid globules/100 g and most preferably 18-35 g lipid globules/100 kcal.

In a particularly preferred embodiment, the invention relates to a nutritional composition that contains 3 to 7 g lipid/100 kcal, 1.25 to 5 g protein/100 kcal and 6 to 18 g digestible carbohydrate/100 kcal; wherein the lipid comprises:

(a) 0.5 to 20 wt.% phospholipids based on total lipid;

(b) 0.1 to 20 wt.% sphingolipids based on total lipid;

(c) 0.005 to 10 wt.% cholesterol based on total lipid;

(d) 78 to 99.3 wt.% fat based on total lipid; and wherein the composition comprises lipid globules containing components (a), (b), (c) and (d).

Preferably, the composition comprises 80 to 98.5 wt.% fat based on total lipid, more preferably 85 to 98 wt.% fat based on total lipid.

Together, the components (a), (b) and (c) preferably represent 0.6 to 25 wt.%, more preferably 1 to 15 wt.%, most preferably 3.0 to 10 wt.% of the total amount of lipid in the nutritional composition. According to a particularly preferred embodiment, at least 50 wt.%, more preferably at least 75 wt.% and most preferably at least 90 wt.% of the components (a), (b), (c) and (d) is contained in the lipid globules.

The lipid globules in the nutritional composition in the method or use of the present invention have a volume-weighted mode diameter above 1.0 pm, preferably above 3.0 pm, more preferably 4.0 pm or above, preferably between 1.0 and 10 pm, more preferably between 2.0 and 8.0 pm, even more preferably between 3.0 and 8.0 pm, most preferably between 4.0 pm and 8.0 pm. Preferably in addition the size distribution is in such a way that at least 45 volume %, preferably at least 55 volume %, even more preferably at least 65 volume %, even more preferably at least 75 volume % has a diameter between 2 and 12 pm. More preferably at least 45 volume %, preferably at least 55 volume %, even more preferably at least 65 volume %, even more preferably at least 75 volume % has a diameter between 2 and 10 pm. Even more preferably at least 45 volume %, preferably at least 55 volume %, even more preferably at least 65 volume %, even more preferably at least 75 volume % has a diameter between 4 and 10 pm. Preferably less than 5 volume % has a diameter above 12 pm. The percentage of lipid globules is based on volume of total lipid. The mode diameter relates to the diameter which is the most present based on volume of total lipid, or the peak value in a graphic representation, having on the X-as the diameter and on the Y-as the volume (%).

The volume of the lipid globule and its size distribution can suitably be determined using a particle size analyzer such as a Mastersizer (Malvern Instruments, Malvern, UK), for example by the method described in Michalski et al, 2001 , Lait 81 : 787-796.

The lipid globules that are contained in the nutritional composition of the present invention preferably comprise a core of fat surrounded by a coating comprising phospholipids, and preferably sphingolipids and/or cholesterol.

Accordingly, the lipid globules that are present in the nutritional composition for use according to the present invention are preferably at least partly coated on the surface with phospholipids. By ‘coating’ it is meant that the outer surface layer of the lipid globule comprises phospholipids, whereas these phospholipids are virtually absent in the core of the lipid globule. The presence of phospholipids as a coating or outer layer of the lipid globule in the diet administered was found to advantageously fully prevent programmed obesity in the tested animal model. Not all phospholipids that are present in the composition need necessarily be comprised in the coating, but preferably a major part is. Preferably more than 30 wt.%, more preferably more than 50 wt.%, more preferably more than 70 wt.%, even more preferably more than 85 wt.%, most preferably more than 95 wt.% of the phospholipids that are present in the composition are comprised in the coating of lipid globules.

Methods for obtaining lipid globules with an increased size and coating with phospholipids are disclosed e.g. in WO 2010/0027258, WO 2010/0027259 and WO 2013/135738.

Preferably at least 50 wt.% of the lipid in the nutritional composition is contained in the lipid globules, more preferably at least 75 wt.% of the lipid is contained in the lipid globules, even more preferably 90-100 wt.% of the lipid is contained in lipid globules.

The nutritional composition preferably contains, calculated by weight of fat, at least 75 wt.%, more preferably at least 80 wt.% and most preferably at least 85 wt.% triglycerides

The nutritional composition preferably comprises, calculated by weight of fat, 10-100 wt.% of mammalian milk fat, more preferably 20-80 wt.% of mammalian milk fat, most preferably 30- 70 wt.% of mammalian milk fat, even more preferably 35-65 wt.% of mammalian milk fat.

The mammalian milk fat is preferably obtained from mammalian milk selected from cow’s milk, ewe’s milk, mare milk, goat milk, camel milk and combinations thereof. Most preferably the mammalian milk fat is obtained from cow’s milk (e.g. milk lipid, cream lipid, cream serum lipid, butter serum lipid beta serum lipid, whey lipid, cheese lipid and/or buttermilk lipid).

The nutritional composition preferably comprises, calculated by weight of fat, 0-90 wt.% of vegetable fat, more preferably 20-80 wt.% of vegetable fat, most preferably 30-70 wt.% of vegetable fat, even more preferably 35-65 wt.% of vegetable fat.

Preferably the vegetable fat is selected from linseed oil, rapeseed oil, sunflower oil (including high oleic sunflower oil), safflower oil, soybean oil, coconut oil, palm oil, palm kernel oil and combinations thereof.

Preferably the lipid in the nutritional composition comprises a fat source comprising long chain poly-unsaturated fatty acids (LC-PUFA), selected from the group consisting of fish oil, marine oil, algal oil, microbial oil and single cell oil, more preferably in an amount of 0.5 to 10 wt.%, calculated by weight of the total amount of fat.

Preferably, the nutritional composition preferably comprises 3-35 wt.% polyunsaturated fatty acids (PLIFA) based on total fatty acids, more preferably the composition comprises 5-30 wt.% PLIFA based on total fatty acids, even more preferably the composition comprises 10-25 wt.% PLIFA based on total fatty acids, most preferably the composition comprises 15-20 wt.% PLIFA based on total fatty acids.

The nutritional composition preferably comprises linoleic acid (LA) and alpha-linolenic acid (ALA), preferably the composition comprises LA to ALA in a weight ratio of 5 to 16, most preferably the composition comprises LA and ALA in a weight ratio of 7-14, even more preferably 8 to 12.

The nutritional composition preferably comprises palmitic acid, preferably the composition comprises at least 10 wt.% palmitic acid based on total fatty acids, more preferably the composition comprises 12-30 wt.% palmitic acid based on total fatty acids, most preferably the composition comprises 15-25 wt.% palmitic acid based on total fatty acids. Preferably, palmitic acid content in the nutritional composition is 2-10g/100g dry composition (powder), more preferably 3-6g/100 g composition.

In a preferred embodiment, at least 15 wt.% of the palmitic acid is esterified to the sn-2 position of a triglyceride based on total palmitic acid, even more preferably at least 30 wt.% of the palmitic acid. Accordingly to one embodiment, at most 40 wt.% of the palmitic acid is esterified to the sn-2 position of a triglyceride based on total palmitic acid, even more preferably at most 35 wt.% of the palmitic acid is esterified to the sn-2 position of a triglyceride based on total palmitic acid. Most preferably 16-24 wt.% of the palmitic acid is esterified to the sn-2 position of a triglyceride based on total palmitic acid.

Preferably the combination of phosphatidylethanolamine and phosphatidylcholine represents at least 30 wt.% of total phospholipids, more preferably 35-70 wt.% of the phospholipids, even more preferably 40-65 wt.% of the phospholipids.

Preferably the combination of sphingomyelin and glycosphingolipids represents at least 10 wt.% of the total phospholipids, more preferably 15-35 wt.% of the phospholipids, even more preferably 18-30 wt.% of the phospholipids. According to a preferred embodiment, all sphingolipids were sphingomyelin. Preferably, sphingomyelin is present between 0.05- 3g/100g dry nutritional composition (powder), more preferably 0.08-2g/100g composition.

According to one embodiment, the nutritional composition does not comprise glycosphingolipids. Glycosphingolipids are ceramides with one or more sugar residues joined in a p-glycosidic linkage at the 1 -hydroxyl position. Glycosphingolipids may be further subdivided into cerebrosides, globosides and gangliosides.

Preferably the present composition contains gangliosides, more preferably at least one ganglioside selected from the group consisting of GM3 and GD3.

The nutritional composition according to the present invention preferably comprises MFGM. More preferably the composition comprises 0.1-5 wt.% MFGM based on total lipid, more preferably the composition comprises 0.5-3 wt.% MFGM based on total lipid, most preferably 1-2.5 wt.% MFGM based on total lipid. Preferably, the nutritional composition comprises 1-8g phospholipids per 100 g of dry composition (powder), more preferably 2-6g/100 g of dry composition (powder).

The MFGM is preferably obtained from milk that was obtained from a mammal selected from cows, sheep, mares, goats, camels and combinations thereof. Most preferably the MFGM is obtained from cow’s milk.

According to a particularly preferred embodiment, the use of the present nutritional composition comprises oral administration of the nutritional composition to a female subject in the age range of 0 to 12 months, more preferably in the age range of 0 to 6 months, most preferably in the age range of 0 to 4 months.

The use of the nutritional composition preferably comprises at least once daily administration, more preferably at least twice daily administration during a period of at least one month, more preferably during a period of at least 3 months.

The use of the present nutritional composition to prevent or reduce programmed obesity preferably comprises oral administration of the nutritional composition to a female subject suffering from overweight. More preferably, the use comprises oral administration of the nutritional composition to a female subject suffering from obesity.

The World Health Organization has published the following BMI-for-age data for girls of 0-24 months.

Infants are considered overweight if they have a BMI greater than 2 standard deviations above the WHO Child Growth Standards median; and obese if they have a BMI greater than 3 standard deviations above the WHO Child Growth Standards median.

Preferably, the use of the nutritional composition to prevent or reduce programmed obesity comprises oral administration of the nutritional composition to a female subject having; an average BMI at the age of 0-1 month of at least 16.8, preferably at least 18.4; and/or an average BMI at the age of 1-2 months of at least 18.3, preferably at least 19.9; and/or an average BMI at the age of 2-3 months of at least 19.4 preferably at least 21.1 ; and/or an average BMI at the age of 3-4 months of at least 19.9, preferably at least 21 .8; and/or an average BMI at the age of 4-5 months of at least 20.1 , preferably at least 22.1 ; and/or an average BMI at the age of 5-6 months of at least 20.3, preferably at least 22.3; and/or an average BMI at the age of 6-7 months of at least 20.3, preferably at least 22.3; and/or an average BMI at the age of 7-8 months of at least 20.3, preferably at least 22.3; and/or an average BMI at the age of 8-9 months of at least 20.2 preferably at least 22.2; and/or an average BMI at the age of 9-10 months of at least 20.0, preferably at least 22.0; and/or an average BMI at the age of 10-11 months of at least 19.9, preferably at least 21.9; and/or an average BMI at the age of 11-12 months of at least 19.7, preferably at least 21.8; wherein the BMI is calculated by dividing the weight (kg) of the subject by the square of the length (m) of the subject.

The use of the nutritional composition preferably comprises administration to a female subject having an energy intake of: • on average at least 487 kcal/day, more preferably on average at least 499 kcal/day, most preferably on average 510-696 kcal/day at the age of 0-1 month; and/or

• on average at least 543 kcal/day, more preferably on average at least 556 kcal/day, most preferably on average 569-776 kcal/day at the age of 1-2 months; and/or

• on average at least 578 kcal/day, more preferably on average at least 591 kcal/day, most preferably on average 605-825 kcal/day at the age of 2-3 months; and/or

• on average at least 564 kcal/day, more preferably on average at least 577 kcal/day, most preferably on average 591-806 kcal/day at the age of 3-4 months; and/or

• on average at least 600 kcal/day, more preferably on average at least 613 kcal/day, most preferably on average 628-857 kcal/day at the age of 4-5 months; and/or

• on average at least 629 kcal/day, more preferably on average at least 643 kcal/day, most preferably on average 659-899 kcal/day at the age of 5-6 months; and/or

• on average at least 633 kcal/day, more preferably on average at least 649 kcal/day, most preferably on average 664-906 kcal/day at the age of 6-7 months; and/or

• on average at least 660 kcal/day, more preferably on average at least 676 kcal/day, most preferably on average 692-944 kcal/day at the age of 7-8 months; and/or

• on average at least 685 kcal/day, more preferably on average at least 701 kcal/day, most preferably on average 717-978 kcal/day at the age of 8-9 months; and/or

• on average at least 710 kcal/day, more preferably on average at least 727 kcal/day, most preferably on average 744-1004 kcal/day at the age of 9-10 months; and/or

• on average at least 729 kcal/day, more preferably on average at least 746 kcal/day, most preferably on average 763-1041 kcal/day at the age of 10-11 months; and/or

• on average at least 748 kcal/day, more preferably on average at least 765 kcal/day, most preferably on average 783-1068 kcal/day at the age of 11-12 months.

The use of the nutritional composition preferably comprises administration to a female subject having an energy intake of:

• on average at least 112 kcal/kg/day, more preferably on average at least 115 kcal/kg/day, most preferably on average 118-171 kcal/kg/day at the age of 0-1 month; and/or

• on average at least 106 kcal/kg/day, more preferably on average at least 109 kcal/kg/day, most preferably on average 111-152 kcal/kg/day at the age of 1-2 months; and/or

• on average at least 99 kcal/kg/day, more preferably on average at least 102 kcal/kg/day, most preferably on average 103-141 kcal/kg/day at the age of 2-3 months; and/or • on average at least 88 kcal/kg/day, more preferably on average at least 90 kcal/kg/day, most preferably on average 92-126 kcal/kg/day at the age of 3-4 months; and/or

• on average at least 87 kcal/kg/day, more preferably on average at least 89 kcal/kg/day, most preferably on average 91-125 kcal/kg/day at the age of 4-5 months; and/or

• on average at least 86 kcal/kg/day, more preferably on average at least 88 kcal/kg/day most preferably on average 90-123 kcal/kg/day at the age of 5-6 months; and/or

• on average at least 82 kcal/kg/day, more preferably on average at least 84 kcal/kg/day, most preferably on average 86-117 kcal/kg/day at the age of 6-7 months; and/or

• on average at least 82 kcal/kg/day, more preferably on average at least 84 kcal/kg/day, most preferably on average 86-117 kcal/kg/day at the age of 7-8 months; and/or

• on average at least 82 kcal/kg/day, more preferably on average at least 84 kcal/kg/day, most preferably on average 86-117 kcal/kg/day at the age of 8-9 months; and/or

• on average at least 83 kcal/kg/day, more preferably on average at least 85 kcal/kg/day, most preferably on average 87-119 kcal/kg/day at the age of 9-10 months; and/or

• on average at least 83 kcal/kg/day, more preferably on average at least 85 kcal/kg/day, most preferably on average 87-119 kcal/kg/day at the age of 10-11 months; and/or

• on average at least 83 kcal/kg/day, more preferably on average at least 85 kcal/kg/day, most preferably on average 87-119 kcal/kg/day at the age of 11-12 months.

Expressed differently, the use of the nutritional composition preferably comprises administration to a female subject receiving a daily energy intake of at least 5% in excess of the recommended daily energy intake for her age, according to “Human energy requirements, Report of a Joint FAO/WHO/UNU Expert Consultation”, Rome 17-24 October 2001 (Table 3.2, Energy requirements of infants during the first year of life), more preferably of at least 7% in excess of the recommended daily energy intake for her age, even more preferably 10-50% in excess of the recommended daily energy intake for her age.

In a preferred embodiment, the female subject is exposed to a Western style diet later in life. In yet a further preferred embodiment according to the invention, preventing or reducing programmed obesity after 8 years of age, in female subjects that are overfed during infancy, upon exposure to a Western style diet after early childhood. A Western-style diet is known in the art to be a general unhealthy diet, characterized by a large content of (unhealthy) lipids and a large content of rapidly digestible carbohydrates, in particular sucrose. A Western-style diet may also be referred to as a “high-fat diet”, as a “cafeteria diet” or as “unhealthy diet”. Typically, a Western-style diet is high in fat and high in saturated fat. In the context of the present invention, the Western-style diet is defined as containing more than 45 % lipid, based on total calories of the daily food intake, wherein the lipid contains at least 50 % by weight of saturated fats, such as present in for example cream, cheese, butter, ghee, suet, tallow, lard, and fatty meats. Further, the Western-style diet is defined as containing more than 50 % carbohydrate, based on total calories of the daily food intake, wherein the carbohydrate contains at least 75 % by weight of rapidly digestible carbohydrates selected from the group consisting of glucose, fructose, sucrose, lactose and starch.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1 : Effects on prevention of programmed obesity

The effects of the compositions according to the invention on the prevention of programmed obesity was investigated using an animal model of metabolic challenge early in life (i.e. postnatal overfeeding exposure by small litter rearing [SL]) to increase the vulnerability to adult diet-induced obesity, followed by adult Western Style Diet (WSD). The small litter causes excessive supply of calories to the suckling pups. The excessive supply is caused by relative increased supply of milk to the pups (quantity). Small litters may also affect the composition of milk, i.e. higher fat content (quality), as well as changes in hormones and other compounds in the milk.

Animals & care

Experimental animals (C57BL/6J) were bred in-house. Primiparous breeder dams and males were obtained from Charles River Laboratories (Sulzfeld, Germany). After arrival, all animals were habituated for 2 weeks in the facilities and housed in Makrolon type III cages, containing Aspen wood shavings and a shelter (red house: Techniplast, Va, Italy) in a controlled environment (21 °C, -55% humidity, 12/12h light-dark cycle) with ad libitum access to (weekly weighed) standard chow (Altromin 1414, Altromin GmbH, Germany) and water, unless specified otherwise. Breeding was performed by placing a female mouse in the cage of a male mouse for 3 consecutive days after being exposed to male scent by exposure to soiled bedding from a male cage for 3 days. Mice were never tail-handled, but always tube (tunnel) handled to reduce stress and anxiety levels. At the end of gestation, pregnant dams were daily inspected without disturbing them and day of delivery was noted as postnatal (PN) day 0.

Diets

IMF diets were supplied by Sniff (Soest, Germany) and contained a micro and macronutrient composition similar to AIN-93-G (Reeves, et al “AIN-93 Purified Diets for Laboratory Rodents: Final Report of the American Institute of Nutrition Ad Hoc Writing Committee on the Reformulation of the AIN-76A Rodent Diet,” J. Nutr., vol. 123, no. 11 , pp. 1939-1951 , Nov. 1993, doi: 10.1093/jn/123.11 .1939) with the lipid fraction entirely derived from lipids in IMF powder (Nutricia Research, Utrecht, the Netherlands). The 3 IMF diets were isocaloric and consisted of 28.3% w/w IMF powder, complemented with additional protein, carbohydrates and micronutrients to mimic AIN-93G standard rodent diet composition. The total amount of lipid in the diets was 7 wt.% based on the dry weight.

The 3 experimental IMFs comprised: (i) control IMF, (ii) IMF-MFGM added with milk fat globule membrane (MFGM) component and (iii) IMF-Test (Nuturis®). The IMFs were isocaloric with similar nutritional composition, but differed in physical properties of lipid globules and phospholipid content due to the addition of MFGM-phospholipids and adjusted processing.

IMF-Test (iii) was prepared in a similar way as diet 6 described in example 1 of WO 2011/115491. Diet (ii) (IMF-MFGM) was prepared in a similar way as diet 3 described in in example 1 of WO 2011/115491. Diet (i) (control) is a commercially available IMF with a low phospholipid content. The lipid globule characteristics of the 3 diets are specified in table B.

Table A - Lipid globule characteristics of the IMFs

Experimental design

At postnatal day 2 (PN2), pups were randomly redistributed among the available dams and culled to either a small litter (SL) (n=48) or normal litter (NL) (n=14) size. A SL consisted of 3 pups (1 male - 2 females, or 2 males - 1 female) and a NL consisted of 6 pups (mixed sex: 2 males - 4 females, or 4 males - 2 females).

From PN16 onwards all nests received the experimental rodent diets containing IMFs varying in supramolecular lipid structure. These diets were provided daily in the form of a dough ball and placed directly in the cage (standard chow was removed at this point from the food hopper). This resulted in three different early life groups with n=24 per group (per sex):

NL with control IMF diets (NL), small litter with control IMF diet (SL - Control) and small litter with IMF-Test diet; (SL - Test).

At weaning (PN21) pups were weighed individually, ear-clipped and pair housed (2 littermates of same sex per cage) at random with cage enrichment (red house, red tube; Techniplast, Va, Italy). Animals remained on the IMF diet until day 42, after which all animals were fed the same semi-synthetic diet based on AIN-93M diet (AIN-93M; control diet: 10% kCal from fat) and a moderate modern Western style diet (WSD) with an adjusted lipid fraction (WSD; 40% kcal from fat, containing 20 wt.% lipid of which 17 wt.% lard, 3 wt.% soy oil and 0.1 wt.% cholesterol, based on total lipid). Half of the animals (random) were subjected to the control diet or WSD, obtaining an n=12 per group in each sex, with always maintaining the pairwise housing of littermates. The WSD was provided during adulthood until the end of the study.

At the end of the adult experiment (PN126), animals were sacrificed under isoflurane anesthesia by cardiac-puncture and decapitation. Subsequently, organs and fat-pads (visceral and subcutaneous) were extracted from the carcass and weighed.

Mice that were raised in normal sized litters (6 pups; offspring sex ratio M:F per litter = 3:3) and exposed to the control IMF diet and not subjected to WSD during adulthood served as a healthy reference group. Groups are summarized in Table B below.

Table B - Study groups

Statistical analysis

Statistical analyses were performed using SPSS 26 (IBM Software, Armonk, USA) and graphical design using GraphPad Prism (GraphPad Software, Inc). All data were analyzed per sex by one-way ANOVA with multiple comparisons and considered significantly different when p<0.05. Statistical trends (P value between 0.05 and 0.1) were also reported. All data are presented as means ± SEMs.

Results

Cumulative food intake calculated per pup on PN0-PN14 shows that dams in a small litter size (SL) had a higher food intake (p<0.001) compared to dams with a normal litter size (NL) (Fig. 1). Between PN2 and PN21 , average pup weight (derived from total nest weight) was increased in SL compared to NL from PN14 onwards (Fig. 2; PN14; p<0.01 , PN21 ; p<0.05). At PN22 all mice raised in small litters showed higher body weight than animals in the reference group, confirming overfeeding of the SL-raised mice.

Exposure to I MF -MF GM and IMF-Test diets early in life reduced adult body weight (gain) in female animals: body weight of SL female animals was higher than that of the healthy female reference group from PN56 onwards for SL-animals raised on CTR diet (Fig. 3). In particular, during adulthood (PN98 and PN112), the body fat % of female animals was increased in SL- CTR compared to the healthy reference (figures 4A and 4B), confirming the programmed obesity model. Surprisingly, both IMF-MFGM and IMF-Test diets reduced adult fat accumulation compared to control IMF in the model for programmed obesity: IMF-Test exposure prevents programmed obesity in female mice in this model at PN98 and PN112, bringing body fat % back to values observed for animals raised in normal litters and not exposed to adult WSD (healthy reference) (Figure 4A and B). At PN126, female animals fed with IMF-Test presented a significantly lower total fat mass as compared to the SL-control group, and actually close to the NL-healthy group (Fig. 5). The fat to lean ratio was also significantly lower in SL-Test female animals as compared to SL-control, and substantially the same as in the healthy reference group (Fig. 6).

At dissection at PN126, all 3 SL groups showed increased deposition of visceral and subcutaneous fat compared to the healthy reference group. In addition, SL resulted in a higher visceral to subcutaneous fat ratio, a marker strongly associated with higher risk of cardiometabolic disease (Table C). Surprisingly, early life dietary exposure to IMF-MFGM as well as IMF-Test reduced the visceral and subcutaneous adipose tissue deposition compared to the SL-CTR group, the strongest effect was observed by IMF-Test. Moreover, both IMF- MFGM and IMF-Test diets exposure resulted in SL animals in a visceral to subcutaneous fat ratio that was close to that of the healthy reference. The visceral to subcutaneous fat ratio of IMF-Test animals was significantly lower than that of SL-CTR animals, which is indicative of a reduced risk for cardiometabolic disease. Table C. Fat tissue deposition PN126

Data are expressed as mean+/-SEM. A. significant difference compared to healthy reference group; b. significant difference compared to SL CTR group; #b. trending effect compared to SL CTR group.

Example 2

An exemplary infant formula (such as IMF Test) comprising large lipid globules comprises per 100 g powder 477 kcal, 24.6 g lipid, 54.1 g digestible carbohydrates, 5.8 g non-digestible oligosaccharides (galacto-oligosaccharides and long chain fructo-oligosaccharides in a weight ratio of 9/1), 9.7 g protein, and vitamins, minerals, trace elements as known in the art. The lipid blend comprises vegetable oil and tuna fish oil. Also milk derived phospholipids is used. The lipid composition is such that 0.40 wt.% of the powder is composed of phospholipids (1.64 wt.% of powder is composed of phospholipids based on total lipids) and 0.1 wt.% of the powder is composed of glycosphingolipids (0.41 wt.% of powder is composed of phospholipids based on total lipids). The composition comprises about 0.03 wt.% cholesterol based on total lipids. About 96-98 % of the lipid is vegetable lipid, the rest being milk fat, fish oil and microbial oil. The ARA content is 0.32 wt.%, DHA content is 0.21 wt.% and EPA 0.045 wt.% based on total fatty acids. The LA/ALA ratio is 5.4.

IMF-Test is prepared in a process similar to the one described in WO2013135738 (A1). The volumetric mode diameter is above 1 pm. The volume % of lipid globules with a size between 2 and 12 pm is above 45 vol.% based on total lipid volume. The lipid globules were covered by a layer of polar lipids.

Example 3

An exemplary infant formula comprising MFGM (such as IMF MFGM) has the same composition as Infant Formula of Example 2. However, the production process of this MFGM- IMF formula is different: The IMF-Test is prepared in a process similar to the example 1 IMF 3 in US 10548869 B2. The volumetric mode diameter is below 1 pm. The volume % of lipid globules with a size between 2 and 12 pm is below 5 vol.% based on total lipid volume. Polar lipids were present as free components, not attached to the lipid globules.

Claims

1. A nutritional composition selected from infant milk formulation, follow-on milk formulation and growing-up milk formulation for use in preventing or reducing programmed obesity after the age of 8 years in female subjects that are overfed during infancy, said use comprising orally administering the nutritional composition to female subjects during infancy; the composition comprising a lipid fraction, wherein the lipid fraction comprises:

(a) 0.5 to 20 wt.% phospholipids based on total lipid;

(b) 0.1 to 20 wt.% sphingolipids based on total lipid; and

(c) 0.005 to 10 wt.% cholesterol based on total lipid.

2. The composition for use according to claim 1 , wherein the composition comprises (d) 78 to 99.3 wt.% fat based on total lipid.

3. The composition for use according to claims 1 or 2, wherein the composition comprises lipid globules.

4. The composition for use according to claim 3, wherein the lipid globules comprise components (a), (b) and (c), preferably (a), (b), (c) and (d).

5. The composition for use according to any one of the preceding claims, wherein the composition comprises milk fat globule membrane (MFGM).

6. The composition for use according to claim 5, wherein the composition comprises 0.1-5 wt.% MFGM based on total lipid.

7. The composition for use according to any one of claims 3-6, wherein the lipid globules have a volume-weighted mode diameter of at least 1 .0 pm.

8. The composition for use according to any one of claims 3-7, wherein at least 40 vol.% of the lipid globules have a diameter from 2 to 12 pm.

9. The composition for use according to any one of claims 3-8, wherein at least 30 wt.% of the phospholipids that are present in the composition are comprised in the coating of lipid globules. The composition for use according to any one of the preceding claims, wherein the lipid comprises 5 to 35 wt.% poly-unsaturated fatty acids (PLIFA), based on total fatty acids. The composition for use according to any one of the preceding claims, wherein the lipid comprises at least 10 wt.% palmitic acid based on total fatty acids. The composition for use according to claim 11, wherein at least 10 wt.% of the palmitic acid is esterified to the sn-2 position of a triglyceride based on total palmitic acid. The composition for use according to any one of the preceding claims, wherein the composition is administered to a female subject in the age range of 0 to 12 months. The composition for use according to any one of the preceding claims, wherein the composition is administered to a female subject suffering from overweight. The composition for use according to any one the preceding claims, the nutritional composition is administered to a female subject having;

• an average BMI at the age of 0-1 month of at least 16.8; and/or

• an average BMI at the age of 1-2 months of at least 18.3; and/or

• an average BMI at the age of 2-3 months of at least 19.4; and/or

• an average BMI at the age of 3-4 months of at least 19.9; and/or

• an average BMI at the age of 4-5 months of at least 20.1 ; and/or

• an average BMI at the age of 5-6 months of at least 20.3; and/or

• an average BMI at the age of 6-7 months of at least 20.3; and/or

• an average BMI at the age of 7-8 months of at least 20.3; and/or

• an average BMI at the age of 8-9 months of at least 20.2; and/or

• an average BMI at the age of 9-10 months of at least 20.0; and/or

• an average BMI at the age of 10-11 months of at least 19.9; and/or

• an average BMI at the age of 11-12 months of at least 19.7; wherein the BMI is calculated by dividing the weight (kg) of the subject by the square of the length (m) of the subject.