FIELD OF THE INVENTION

The invention disclosed herein relates to the use of dairy lipid to lower the GlycA level in a subject; to a composition comprising a lipid fraction for use in lowering the GlycA level in a subject. The invention further relates to a method for lowering the GlycA level in a subject comprising a step of administering a composition comprising a lipid fraction to the subject. Optionally the use, composition for use and the method additionally relate to reducing the risk of inflammation in a subject.

BACKGROUND

Breast feeding is the best way to ensure healthy growth and development of infants during the first months of life. It is recommended by the WHO to exclusively provide breast feeding during the first six months of life and the introduction of safe and appropriate complementary feeding thereafter to supplement continued breast feeding up to two years of age or beyond. However, when mothers cannot or choose not to breastfeed for whatever reason a safe alternative to breast feeding is required, there is a legitimate role for breast milk substitutes, produced according to strict international compositional and safety standards.

Glycoprotein acetyls (GlycA) is a unique inflammatory biomarker with analytic and clinical attributes that may complement or provide advantages over existing clinical markers of systemic inflammation. It is composed of a complex of heterogeneous NMR signal containing N-acetyl sugar groups originating from multiple acute-phase circulating glycoproteins. A simplified description how the GlycA ‘peak’ on NMR relates to systemic inflammation is given hereafter: In the setting of inflammation, irrespective of the trigger, macrophages are recruited to the site of inflammation where they secrete a variety of cytokines. These cytokines act locally to induce an inflammatory response aimed at removing the insulting trigger and promoting subsequent tissue recovery. However, some of these cytokines also enter the systemic circulation and reach the liver, where they induce an increased production and secretion of several so-called acute phase reactants, as well as various glycosylation-mediating enzymes, known as glycosyltransferases, which alter the glycosylation patterns of the latter acute phase reactants. The acute phase reactants themselves, and their glycosyltransferase-modified derivatives contribute to the GlycA peak seen on the NMR spectrum (R.A. Bailout and A.T. Remaley J Lab Precis Med 2020;5:17 I http://dx.doi.org/10.21037/jlpm.2020.03.03). Accordingly, there is a need to keep the level of GlycA low in order to reduce the risk for inflammation.

It was found that eating may result in an increase of the GlycA level. It is desired to have the GlycA levels return to their basal level as fast as possible in order to lower the risk of inflammation or any other condition related to increased levels of GlycA. It was surprisingly found that this so-called postprandial response is dependent on the diet being consumed. More surprisingly, it was found that GlycA returns faster to its basal level after consumption of milk fat as compared to consumption of vegetable fat.

It is an objective of the present invention to provide a further use of milk fat and or milk lipids, to provide a further composition for use and to provide a further method comprising a step of administering milk fat or milk lipids. It is an objective to better address at least one of the aforementioned desires and/or needs.

SUMMARY OF THE INVENTION

One or more objectives of this invention are achieved by the use, composition for use and method as defined in the claims. According to an aspect of the invention this is achieved by the features of claim 1. There is provided a use of dairy lipid to reduce the risk of inflammation in a subject and/or to lower the GlycA level in a subject; preferably, to lower the postprandial GlycA level in a subject. In addition and/or alternatively, according to a further aspect of the invention there is provided a composition comprising a lipid fraction for use in the reduction of the risk of inflammation in a subject and/or to lower the GlycA level in a subject; wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10 wt%.

According to a further aspect of the invention there is provided a method for reducing the risk of inflammation in a subject and/or to lower the GlycA level in a subject comprising a step of administering a composition comprising a lipid fraction to the subject, wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10 wt%.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the invention relates to the use of a lipid fraction to lower the Glycoprotein acetyls (GlycA) level in a subject wherein the amount of C 15:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of 017:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; wherein the lowering of GlycA is a reduction of the postprandial time needed for the GlycA level to return to its basal level in the subject after consumption of a composition comprising a lipid fraction and optionally wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%.

In one embodiment the invention relates to the use of a lipid fraction to reduce the risk of inflammation in a subject and/or to lower the GlycA level in a subject wherein the amount of 015:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of C17:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; and optionally wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%. In one embodiment the invention relates to the use of a lipid fraction to reduce the risk of inflammation in a subject and/or to lower the GlycA level in a subject wherein the amount of 015:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of C17:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; and wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%.

As referred to in different embodiments and aspects of the invention, the amount of C15:0 in the lipid fraction expressed as percent by weight of total fatty acids preferably is at least 0.20% and the amount of C17:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.12%; more preferably the amount of C15:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.30% and the amount of C17:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.18%. As used herein, the amount of C15:0 in the lipid fraction is referring to amount of pentadecanoic acid that is esterified to the glycerol backbone, likewise, the amount of Cl 7:0 is referring to the amount of heptadecanoic acid that is esterified to the glycerol backbone.

The term "treatment", in relation to a given disease or disorder, includes, but is not limited to, inhibiting the disease or disorder, for example, arresting the development of the disease or disorder; relieving the disease or disorder, for example, causing regression of the disease or disorder; or relieving a condition caused by or resulting from the disease or disorder, for example, relieving, preventing or treating symptoms of the disease or disorder.

The term "prevention" in relation to a given disease or disorder means preventing the onset of disease development if none had occurred, preventing the disease or disorder from occurring in a subject that may be predisposed to the disorder or disease but has not yet been diagnosed as having the disorder or disease, and/or preventing further disease/disorder development if already present.

A reduction of the risk of a disease or disorder means a lower likelihood that a given disease or disorder develops if none had occurred, and/or a lower likelihood that a given disease or disorder further develops if already present, and/or a lower likelihood that a given disease or disorder will be diagnosed.

As used herein, “dairy lipid” consists of dairy fat (i.e. milk fat) and dairy phospholipids (i.e. milk phospholipids). As such the total lipid fraction consists of dairy fat, vegetable fat, dairy phospholipid and vegetable phospholipid. The “dairy lipid” as used in this document may be obtained from milk of ruminants using methods known in the art, preferably from bovine milk fat, more preferably cow’s milk fat. The milk fat source can in principle be any available ruminant milk fat source, such as whole milk, cream, anhydrous milk fat (AMF) or milk fat fractions resulting from dry fractionation, critical CO2 extraction or other fractionation methods known in the art. The dairy lipid source can also be a source of milk fat globular membrane (MFGM). Likewise, the dairy lipid source can be a combination of different sources. It was, however, found particularly suitable to use whole milk, AMF, cream and/or MFGM as the dairy lipid source. Preferably the ruminant milk lipids are bovine milk lipids and the lipids are selected from the group consisting of anhydrous milk fat (AMF), cream, MFGM, and whole milk. More preferably, the lipids are selected from AMF, cream, or mixtures thereof. Even more preferably, the bovine milk lipids are cow milk lipids. In one embodiment the dairy lipid is bovine whole milk or cream. In one embodiment, the dairy lipid is bovine whole milk, in another embodiment the dairy lipid is bovine cream, in still another embodiment the dairy lipid is bovine AMF. Preferably, the dairy lipid source is bovine AMF and/or bovine MFGM. As used herein, MFGM is referring to an MFGM enriched milk fraction. AMF and MFGM enriched milk fractions are readily commercially available from milk processing companies like FrieslandCampina, Aria, or Fonterra e.g. VMF100 (FrieslandCampina), Vivinal® MFGM (FrieslandCampina), Lacprodan® MFGM- 10 (Aria), SureStart™ MFGM Lipids (NZMP). Conventional techniques can be applied to produce different milk lipid sources like whole milk, cream, anhydrous milk fat (AMF) or milk fat fractions resulting from dry fractionation, critical CO2 extraction or other fractionation methods. Such milk lipid sources are also readily commercially available. If a thermization or pasteurization step is needed e.g. to meet legal requirements, this is possible using methods known in the art, for example at temperatures of about 63 °C (145 °F) maintained for 30 minutes or, heating to a higher temperature, e.g. 72 °C (162 °F), and holding for 15 seconds or, alternatively, e.g. an ultra-high temperature (UHT) treatment at not less than 135 °C in combination with a suitable holding time. Alternatively, the ruminant milk lipids may be obtained from fresh milk. The term “cream” as used herein is referring to the fat portion from whole milk, preferably bovine milk. This fat portion may be separated from the whole milk using methods known in the art. Cream is comprising triglycerides and milk fat globular membrane (MFGM) fragments (such fragments may also be referred to as MFGM components). The MFGM fragments comprise milk phospholipids and a large variety of (glyco)proteins. The milk fat globules comprise triglycerides surrounded with a membrane comprised of MFGM fragments.

As used herein “postprandial lipemia” refers to a rise in circulating triglyceride- containing lipoproteins following consumption of a meal, postprandial lipemia has been recognized as a risk factor for the development of cardiovascular disease and other chronic diseases.

The term "subject" as used herein preferably refers to a human. The term "subject" refers to both the male and female sex unless one sex is specifically indicated. The human subject can be an infant (< 2 years old), a juvenile, an adolescent, an adult or an elderly subject. In one preferred embodiment the human subject is aged 0-36 months. In a particularly preferred embodiment of the invention the human subject is aged 18 years and above, e.g. at least 25 years, at least 30 years, at least 35 years, at least 40 years, or at least 45 years. More preferably, the human subject is at least 50 years old, at least 55 years old, at least 60 years old or at least 65 years of age. There is no particular upper limit although in practice, human subjects treated in accordance with the invention will typically be at most 110 years of age, e.g. at most 100 or at most 90 years of age.

As is shown in the examples, human consumption of fat results in an increase of the GlycA level. The increase being determined relative to the basal level. The basal level being the GlycA level in the subject in a fasted state of at least 10 hours. During the fasting state, the subject is not allowed to eat or drink, except water. As such, the lowering of the GlycA level as referred to in the different aspects and/or embodiments of the invention is a reduction of the postprandial time needed for the GlycA level to start decreasing, preferably a reduction of the postprandial time needed for the GlycA level to return to its basal level in the subject after consumption of a composition comprising a lipid fraction. As used herein, “postprandial time” is defined as the time after consumption of a consumption comprising a lipid fraction. Preferably, the lipid fraction in the composition comprising a lipid fraction as referred to in the different aspects and/or embodiments of the invention, comprises at least 10 wt% dairy lipid as determined to the total weight of lipid in the lipid fraction.

Alternatively, in other embodiments of the different aspects of the invention, the lowering of the GlycA level is one or more selected from the group consisting of i.

- v: i. a reduction in the postprandial time needed for the GlycA level to return to its basal level in the subject after consumption of a composition comprising a lipid fraction as compared to the postprandial time needed for the GlycA level to return to its basal level in the subject after consumption of the same amount of vegetable oils is reduced with at least 5%, preferably at least 10%, more preferably at least 15% most preferably at least 20%; and/or ii. a decrease in the GlycA level in the subject between 5 and 6 hours post prandial, preferably between 4 and 6 hours post prandial, most preferably between 3 and 6 hours post prandial; and/or iii. the GlycA level being back at the basal level at 6 hours post prandial; and/or iv the GlycA level at 4.5 hours post prandial being at 60% or less of the GlycA level at 3 hours post prandial, preferably 50% or less; and /or v. the Area Under the Curve (AUG) of the delta postprandial response GlycA being reduced by at least 10%, preferably at least 20%.

As used herein, the Area Under the Curve (AUG) of the delta GlycA postprandial response is referring to a curve like Figure 2, wherein the postprandial GlycA level subtracted with the basal GlycA level is plotted as a function of time between t=0 (basal level) and t=6 hours.

The amount of dairy lipid as determined relative to the total weight of the lipid fraction in the composition comprising a lipid fraction as referred to in different embodiments and/or aspects of the current invention can be at least 20 wt%, preferably at least 30 wt%, more preferably at least 50 wt%, particularly preferably at least 75 wt%, most preferably at least 90wt%, such as at least 95 wt% or 99 wt%.

The reduced risk of inflammation and/or the lowering of the GlycA level is preferably determined relative to the subject consuming a composition comprising a lipid fraction wherein the lipid fraction consist of vegetable oils instead of dairy lipid. More preferably, the vegetable oils consist of a blend of palm olein oil, rapeseed oil low in erucic acid, palm kernel oil and sunflower oil.

The composition comprising a lipid fraction as referred to in different embodiments of the invention may be further comprising a carbohydrate fraction and a protein fraction; preferably wherein the composition comprising the lipid fraction is selected from the group consisting of infant nutrition product, adult nutrition product, a sports nutrition product, drink, candy, and a food supplement. Such products are well-known in the art and the amount of dairy lipid may be increased in such products by replacing other sources of lipid, using methods known in the art.

In another aspect, the invention relates to a composition comprising a lipid fraction for use in lowering the GlycA level in a subject; wherein the amount of 015:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of 017:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; wherein the lowering of GlycA is a reduction of the postprandial time needed for the GlycA level to return to its basal level in the subject after consumption of a composition comprising a lipid fraction and optionally wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%.

In one embodiment the invention relates to a composition comprising a lipid fraction for use in the reduction of the risk of inflammation in a subject and/or to lower the GlycA level in a subject; wherein the amount of 015:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of 017:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; and optionally wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%. In one embodiment, the invention relates to a composition comprising a lipid fraction for use in the reduction of the risk of inflammation in a subject and/or to lower the GlycA level in a subject; wherein the amount of C15:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of Cl 7:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; and wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%. Preferably, wherein the lowering of GlycA is a reduction of the postprandial time needed for the GlycA level to return to its basal level in the subject after consumption of a composition comprising a lipid fraction.

In some jurisdictions the invention may also be worded as the use of a composition comprising a lipid fraction in the manufacture of a medicament for use in lowering the GlycA level in a subject; wherein the amount of C15:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of Cl 7:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; wherein the lowering of GlycA is a reduction of the postprandial time needed for the GlycA level to return to its basal level in the subject after consumption of a composition comprising a lipid fraction and optionally wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%.

In one embodiment the invention relates to the use of such a composition comprising a lipid fraction in the manufacture of a medicament for use in the reduction of the risk of inflammation in a subject and/or to lower the GlycA level in a subject; wherein the amount of C15:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of C17:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; and optionally wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%. In one embodiment, the invention relates to a use of a composition comprising a lipid fraction for use in the manufacture of a medicament for the reduction of the risk of inflammation in a subject and/or to lower the GlycA level in a subject; wherein the amount of C15:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of 017:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; and wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%. Preferably, wherein the lowering of GlycA is a reduction of the postprandial time needed for the GlycA level to return to its basal level in the subject after consumption of a composition comprising a lipid fraction.

In yet another aspect, the invention relates to a method for lowering the GlycA level in a subject comprising a step of administering a composition comprising a lipid fraction to the subject, wherein the amount of 015:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of C17:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; and wherein the lowering of the GLycA level is a reduction of the postprandial time needed for the GlycA level to return to its basal level in the subject after consumption of a composition comprising a lipid fraction and optionally wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%.

In one embodiment the invention relates to a method for reducing the risk of inflammation in a subject and/or to lower the GlycA level in a subject comprising a step of administering a composition comprising a lipid fraction to the subject, wherein the amount of 015:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of C17:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; and optionally wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%. In one embodiment, the invention relates to a method for reducing the risk of inflammation in a subject and/or to lower the GlycA level in a subject comprising a step of administering a composition comprising a lipid fraction to the subject, wherein the amount of 015:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.10% and the amount of C17:0 in the lipid fraction expressed as percent by weight of total fatty acids is at least 0.06%; and wherein the amount of dairy lipid as determined relative to the total weight of the lipid fraction is at least 10% wt%. Preferably, wherein the lowering of the GlycA level is a reduction of the postprandial time needed for the GlycA level to return to its basal level in the subject after consumption of a composition comprising a lipid fraction.

In one embodiment, the use, composition for use, and method of the invention as defined in the different aspects and embodiments of the invention relate to a reduction in the risk of inflammation in a subject and a lowering of the GlycA level in a subject.

In one embodiment the invention relates to the use, the composition for use, or the method of the invention wherein the GlycA level is lowered and wherein the risk of inflammation is reduced; preferably wherein the reduced risk of inflammation is determined relative to the subject consuming a composition comprising a lipid fraction wherein the lipid fraction consist of vegetable oils instead of dairy lipid.

It is also to be understood that this invention is not limited to the specific embodiments and methods described herein, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way.

It must also be noted that, as used in the specification and the appended claims, the singular form "a", "an," and "the" comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components.

It will be understood that within this disclosure, any reference to a weight, weight ratio, and the like pertains to the dry matter, in particular the dry matter of the composition.

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "comprising", which is synonymous with "including" or "containing", is open-ended, and does not exclude additional, unrecited element(s), ingredient(s) or method step(s), whereas the term "consisting of' is a closed term, which excludes any additional element, step, or ingredient which is not exphcitly recited.

As used herein, the term “essentially consisting of’ is a partially open term, which does not exclude additional, unrecited element(s), step(s), or in gre dient (s), as long as these additional element(s), step(s) or ingredient(s) do not materially affect the basic and novel properties of the invention.

As used herein, the term “comprising” (or “comprise(s)”) hence includes the term “consisting of’ (“consist(s) of’), as well as the term “essentially consisting of’ (“essentially consist(s) of’). Accordingly, the term “comprising” (or “comprise(s)”) is, in the present application, meant as more particularly encompassing the term “consisting of’ (“consist(s) of’), and the term “essentially consisting of’ (“essentially consist(s) of’).

Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and/or use are to be understood as modified by the word "about" in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary: percent, "parts of," and ratio values are by weight; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies, mutatis mutandis, to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.

The invention is hereinafter illustrated with reference to the following, nonlimiting, examples.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 Differential effects of interventions on postprandial metabolites. Flowchart of A) the study design and B) the number of metabolites significantly changed in the interventions. Shake*time effect was tested using linear mixed models for repeated measures. Significant findings were corrected for multiple testing using FDR-correction (FDR p-value<0.05). A LSD posthoc test was performed on metabolites with a significant FDR p-value for shake*time to identify differences between the shakes and shake*time effects at specific time points.

Figure 2 Delta postprandial response of GlycA. Delta postprandial response of glycoprotein acetyls (GlycA). All values represent mean ± SD. Shake*time effect in linear mixed models analysis (FDR p-value = 0.019). Significant difference between VEGE and AMF (p<0.001). and VEGE and CREAM (p=0.003). a and b indicate significant differences between shakes for shake*time effect at specific timepoints. All values represent mean ± SD.

EXAMPLES

Example 1

Fatty acid determination

[0068]The content of the different fatty acids in the lipid of the composition of the invention can be determined by standard method ISO 15884/IDF 182:2002 (Milk fat - Preparation of fatty acid methyl esters) and ISO 15885/IDF 184 (Milk fat - Determination of the fatty acid composition by gas-liquid chromatography). These ISO methods allow for determination of molar concentration of a fatty acid relative to total moles of this fatty acid in TAG ([FA- TAG]). The distribution of fatty acids over the glycerol backbone can be determined according to the method disclosed in JOCS/AOCS Official Method Ch 3a- 19 Determination of the composition of fatty acids at the 2-position of oils and fats - Enzymatic Transesterification method using Candida antarctica lipase (Approved 2019) In essence, this method involves hydrolysis of triacylglycerols (TAG) by a sn-1,3 specific pancreatic lipase (porcine). The required 2 -monoacylglycerols formed are isolated by thin layer chromatography and these are subsequently methylated for gas chromatographic analysis and quantified in molar concentrations relative to the total moles of fatty acids at the sn-2 position ([FA(sn-2)]).

Determination of the GlycA level in a subject

GlycA levels were determined using the Nightingale Health platform (https://research.nightingalehealth.com/ under Blood biomarker analysis) as described by Wurtz et al ( P. Wurtz et al Quantitative Serum Nuclear magnetic Resonance metabolomics in Large-Scale Epidemiology: A Primer on -Omics Technologies, Am. J. Epidemiol. 2017 vol 186(9) pp 1084-1096). Briefly, using a A high-throughput proton (1H) nuclear magnetic resonance (NMR) metabonomics approach as described by Soininen et al (Soininen et al High-throughput serum NMR metabonomics for cost-effective holistic studies on systemic metabolism; Analyst, 2009, vol 134, pp 1781-1785).

Clinical study

A study was performed aimed to explore the postprandial response of plasma metabolites to high fat shake consumptions, including vegetable fats and bovine milk fats with and without milk fat globular membranes.

Study population

The study population consisted of 40 healthy men and women aged 40-70, with a BMI of 22-27 kg/m ² . Participants were excluded if they were suffering from any chronic metabolic, gastrointestinal, inflammatory, or another chronic disease, if they had (a history) of gastro-intestinal complaints or surgery, renal or hepatic malfunctioning (determined by ALAT, ASAT, and creatinine measurements), or if they were using any medication that may influence the study results by affecting intestinal motility. Additionally, participants were excluded if they were (wishing to become) pregnant, using soft/hard drugs, drank >14 alcohol consumptions/week, smoked, or had an unstable body weight. Lastly, participants were also excluded from the study if they were following a vegan diet or had food allergies to any of the products used in the study. All participants provided their written consent before participation in the study.

Study Design

The study was a double-blind, randomized acute intervention study (Figure la). Each participant visited the research unit three times, with a wash-out period of at least one week. During each study visit, the participants underwent a dietary lipid challenge test in the form of a high-fat shake. Participants were randomly assigned to a sequence of shakes.

The evening before each study visit, participants consumed a standardized meal (ad libitum) and were not allowed to eat or drink anything except water until the next day. The following day, participants appeared at the research unit in a fasted state for a minimum of 10 hours.

The study was conducted at Wageningen University, the Netherlands, from 07- 01-2020 until 10-03-2020. The experimental protocol and procedures were approved by the Medical Ethical Committee of Wageningen University and were in accordance with the Helsinki declaration of 1975 as revised in 1983. The study was registered at clinicaltrials.gov as NCT04178681 ‘Postprandial Effects of Milk Fats (POEMI)’.

Dietary lipid challenge test

During each study visit, participants underwent a dietary lipid challenge test. In this test, participants consumed a liquid shake containing skimmed milk (0.5 L) and 95 grams of fat from different sources (Table 1). Table 1. Nutritional information on the high-fat shakes

Amounts per 618 gram of shake (consisting of water, skimmed milk, and fat shake powder.

Constituent VEGE AMF Cream

Fat

Vegetable fat blend 95.0 0 0 (g)

Anhydrous Milk Fat (AMF) 0 95.0 0 (g)

Cream 0 0 95.0 (g)

Fat (total) * 95.5 95.5 95.5 (g)

Protein 20.9 20.9 21.2 (g)

Carbohydrate 42.3 42.3 42.5 (g)

0.5 g of fat via skimmed milk

The shakes were prepared by dissolving the fat blend in 500 ml of skimmed milk.

Three types of fat were consumed during this study, namely: 100% vegetable fat blend (VEGE), 100% anhydrous milk fat (AMF, bovine milk fat), and 100% cream (CREAM) (AMF + milk fat globular membranes). The shakes were isocaloric and differed solely in their fat source, as the remaining constituents were identical. The fats were provided by FrieslandCampina in powder form (Table 2). The shakes were prepared by dissolving the fat powders in skimmed milk.

Table 2. The ingredient list of the three different fat powders

Fat powder Ingredients

Vegetable blend Vegetable fat blend (blend of palm olein, rapeseed oil (VEGE) low in erucic acid, palm kernel oil and sunflower oil), glucose syrup, milk protein (Na-caseinate), stabilizer (E451i: Pentasodium triphosphate), free-flowing agent (E551: silicon dioxide) Fat powder Ingredients

Anhydrous milk fat Anhydrous milk fat, glucose syrup, milk protein (Na- (AMF) caseinate), stabilizer (E451i: Pentasodium triphosphate), free-flowing agent (E551: silicon dioxide) CREAM Cream, milk protein (Na-caseinate), glucose syrup,

(AMF+MFGM) stabilizer (E451i: Pentasodium triphosphate), free- flowing agent (E551: silicon dioxide)

Fatty acid composition of the lipid blend

The fatty acid composition of the different fat blends as given in Table 3, was determined using the method described elsewhere in this example. The amounts listed are expressed as percent by weight of the total amount of fatty acids (w/w%) in the lipid fraction.

Table 3 Fatty acid composition of the different fat shakes

Fatty acid (w/w%)* VEGE AMF Cream

C15:0 <0.1 1.1 1.1

C17:0 <0.1 0.5 0.5

* amounts listed are expressed as percent by weight of the total amount of fatty acids (w/w%) in the lipid fraction.

The shakes were provided to the participants in a tinted cup with an opaque straw to hide their content. Participants had to consume the shake within ten minutes and were not allowed to eat or drink anything except for water (ad libitum) unless the last blood sample was drawn. During that time frame, participants were also not allowed to engage in physical exercise.

Blood collection

During each study visit, a catheter cannula was inserted in the participant’s antecubital vein. Thirty minutes after the cannula insertion, blood was drawn from the catheter cannula for the baseline measurement (t=0). After this baseline measurement, participants consumed a high-fat shake. Subsequently, blood was drawn at t=l, 2, 3, 4, 5, 6, 7, and 8 hours after consumption. All blood samples were stored at -80°C until further analysis.

Metabolomics

Blood samples collected at 0 hours (baseline), 3 hours, and 6 hours were processed by Nightingale Health. This platform performs high-throughput proton Nuclear Magnetic Resonance (NMR). Details on the analysis have been described previously (Soininen, P., Kangas, A. J., Wurtz, P., Suna, T., and Ala-Korpela, M. (2015). Quantitative serum nuclear magnetic resonance metabolomics in cardiovascular epidemiology and genetics. Circ. Cardiovasc. Genet. 8, 192-206; Wurtz, P., Kangas, A. J., Soininen, P., Lawlor, D.A., Davey Smith, G., and Ala- Korpela, M. (2017). Quantitative Serum Nuclear Magnetic Resonance Metabolomics in Large-Scale Epidemiology: A Primer on -Omic Technologies.

Am. J. Epidemiol. 186, 1084-1096); Soininen et al High-throughput serum NMR metabonomics for cost-effective holistic studies on systemic metabolism; Analyst, 2009, vol 134, pp 1781-1785). The analysis allows for simultaneous detection and quantification of 249 metabolites and their corresponding ratios within one experimental set-up. These include lipoprotein subclasses and constituents, their relative ratios, lipids, fatty acids, amino acids, ketone bodies, glycolysis-related metabolites, and various other low-molecular-weight metabolites.

Statistical analysis

Statistical analyses of the metabolites were performed on log2-transformed data. Differences in the postprandial responses between the three different shakes from the dietary lipid challenge test were analyzed using linear mixed models for repeated measures. Participants were specified as subjects. Shake and time were specified as repeated measures. The delta values (t=3h-t=0h and t=6h-t=0h) of the 249 metabolites were selected as the dependent variable in the model. Shake, time, and the interaction between shake*time were included as fixed effects. A first-order autoregressive covariance structure was selected for the model. An additional LSD posthoc test was performed on metabolites with a significant shake*time effect to identify differences between the shakes and specify shake*time effects at specific time points. The linear mixed model’s findings were corrected for multiple testing using Benjamini-Hochberg’s FDR correction (FDR p-value 0.05) ([ Benjamini, Y. and Hochberg, Y. (1995) Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. R. Stat. Soc. Ser. B 57, 289-300). All statistical analyses were performed using IBM® SPSS® Statistics (version 28.0.1.0). Figures were created in GraphPad Prism (Version 9.3.1) and Cytoscape (version 3.9.1).

Results Participant characteristics

Of the 40 participants that entered this study, 37 completed the study. One participant dropped out after the first study visit, and two other participants dropped out after the second study visit (Figure 1A). Data from the dropouts were still included in the analysis. The participant characteristics of the 40 participants can be found in Table 4.

Table 4. Participant characteristics of the 40 included participants

Data are presented as mean ± standard deviation.

Characteristic

Gender (m/f) 9/31

Age (years) 57.3 ± 8.0

Weight (kg) 71.5 ± 8.5

Height (cm) 172.2 ± 7.7

BMI (kg/m ² ) 24.1 ± 1.7

Waist circumference (cm)

Men 92.2 ± 5.9

Women 81.8 ± 6.2

Hip circumference (cm)

Men 102.6 ± 4.6

Women 102.2 ± 6.0

WH R

Men 0.90 ± 0.05 Characteristic

Women 0.80 ± 0.05

Hb (mmol/L) 8.4 ± 0.6

ALAT (uL) 24.7 ± 11.8

ASAT (uL) 21.5 ± 7.3

Creatinine (umol/L) 71.6 ± 11.1

Abbreviations: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), body mass index (BMI), hemoglobin (Hb), waist-hip ratio (WHR)

AMF, CREAM and VEGE impact postprandial metabolites differently

The effect of the 3 fat sources on postprandial plasma metabolite levels (between shake comparison) was investigated. Of the 249 metabolites measured in total, 102 metabolites were differently changed postprandially by the three shakes. Posthoc tests were performed to identify the differences between shakes at a specific time point. At 3 hours postprandially, 37 metabolites were significantly different between VEGE and AMF, 39 metabolites were significantly different between VEGE and CREAM, and no metabolites were significantly different between AMF and CREAM. At 6 hours postprandially, 41, 29, and 8 metabolites were identified as significantly different for the above comparisons, respectively (Figure lb).

Significant changes of metabolites in between-shake comparisons and within- shake comparisons

Differences between the three shakes were tested using linear mixed models for repeated measures. A significant shake*time effect was found for 102 of the 239 metabolites. Significant findings were corrected for multiple testing using FDR- correction (FDR p-value<0.05). Additional LSD posthoc tests were performed to identify the differences between shakes and shake*time effects at a specific timepoint (Figure lb).

AMF and CREAM consumption led to a more rapid decline in the postprandial increase of the inflammatory marker GlycA GlycA is a biomarker of systematic inflammation and has been identified with many metabolic disorders and diseases. Surprisingly, the postprandial response of GlycA differed significantly between the three shakes. Specifically, CREAM and AMF consumption resulted in a lower GlycA level 6 hours post prandially compared to VEGE. CREAM and AMF consumption resulted in a rapid return of the postprandial increase of GlycA to its basal level, which did not occur after VEGE consumption (Figure 2a). As used herein, the “GlycA level” is referring to the delta values of the measured GlycA level at a specific postprandial time point and the GlycA level at baseline (t=3h-t=0h and t=6h-t=0h). CREAM and AMF have their highest GlycA level 3 hours postprandially, while VEGE increases up to 6 hours postprandially. It is further noted that at t=6, the GlycA level has returned to baseline level for both CREAM and AMF.

It is observed that the postprandial increase of low-grade chronic inflammation marker glycoprotein acetyls (GlycA) was lower and almost back to baseline, 6 hours postprandial after CREAM and AMF consumption while VEGE still seems to rise.

Example 2

An example of a nutritional composition (e.g. an infant formula) suitable to be used in different embodiments or aspects of the invention is given in the below table 5.

Table 5 ingredients of formulas according to the invention

* Milk serum protein concentrates (SPC) are proteins found in cheese whey that are removed directly from milk. Because SPC are not exposed to the cheesemaking process, enzymatic or chemical reactions that can lead to off-flavors are reduced.

Example 3

An example of a nutritional composition (e.g. an adult nutrition product) suitable to be used in different embodiments or aspects of the invention is given below: Ingredients: Water, milk protein (SPC), glucose syrup, vegetable oils, (sunflower oil, rapeseed oil), milk fat, sucrose, fiber (fructooligosaccharides, inulin, galactooligosaccharides), minerals, fish oil, emulsifier, vitamins, carnitine, stabilizer, flavor, taurine.

The composition comprises per serving size of 100.0ml:

Energy: 550 - 800 kJ

Protein: 5 - 15 g (from SPC)

Dietary Fiber: 1 - 5 g

Fat: 5 - 10 g

Carbohydrate: 10 - 15 g

Alpha Linolenic Acid: 150 - 200 mg,

Calcium: 200 - 350 mg

Poly Un Saturated Fat: 3 - 5 g.