Use of a composition comprising a phospholipid-enriched milk fraction and SPC in the prevention of an infection with influenza virus

Abstract

Synthetic nutritional composition comprising a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) for use in the prevention of an influenza virus and / or the prevention of further development of a viral infection with influenza virus.

Description

[0001] USE OF A COMPOSITION COMPRISING A PHOSPHOLIPID-ENRICHED MILK FRACTION AND SPC IN THE PREVENTION OF AN INFECTION WITH INFLUENZA VIRUS

[0002] This invention relates to a synthetic nutritional composition comprising a milk fraction enriched in phospholipids for use in the prevention of an infection of the respiratory tract with influenza virus.

[0003] Background

[0004] Influenza virus infections are a major disease burden for humans, both in infants and adults. Symptoms of an influenza infection range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. These symptoms begin one to four (typically two) days after exposure to the virus and last for about two to eight days. Diarrhea and vomiting can occur, particularly in children. Influenza may progress to pneumonia from the virus or a subsequent bacterial infection. Other complications include acute respiratory distress syndrome, meningitis, encephalitis, and worsening of pre-existing health problems such as asthma and cardiovascular disease.

[0005] There are four types of influenza virus: types A, B, C, and D. Aquatic birds are the primary source of influenza A virus (IAV), which is also widespread in various mammals, including humans and pigs. Influenza B virus (IBV) and influenza C virus (ICV) primarily infect humans, and influenza D virus (IDV) is found in cattle and pigs. Influenza A virus and influenza B virus circulate in humans and cause seasonal epidemics, and influenza C virus causes a mild infection, primarily in children. Influenza D virus can infect humans but is not known to cause illness.

[0006] Human influenza A viruses impose a constant health threat in babies, infants and adults. The viruses mutate rapidly and cause seasonal epidemics; around a billion cases annually, including 3-5 million cases of severe illness, according to WHO and occasionally pandemics. Influenza can cause acute respiratory infections and spreads easily. It is common in all parts of the world, and vaccinations are available. However in 2018, among children under 5 years globally the influenza virus accounted for 7% of the acute lower respiratory infections (ALRI) cases, 5% of ALRI hospital admissions and 4% of ALRI deaths. Human influenza A viruses are classified into subtypes based on the structures of the HA and NA proteins (e.g. H1 N1 , H3N2) which are both present on the virus envelope. Influenza strain subtype H1N1 caused 2 global influenza pandemics (‘Spanish flu’ in 1918 and ‘swine flu’ in 2009) and many influenza epidemics.

[0007] Young infants e.g. up to an age of six months during which they are largely dependent on maternal transferred immunity, and patients with pre-existing health problems are most susceptible to severe influenza symptoms after an infection .

[0008] It has been considered for a long time by nutritionists that the best food or nutrition supplied to an infant is its own mother's milk; i.e. fresh human milk. It is recognized, however, that many situations arise wherein the infant cannot be fed mother's milk and as a result cows’ milk based formulas have been prepared and used to nourish an infant. These formulas contain a mixture of casein and whey proteins to provide an amino acid profile as close as possible to that of mother's milk. Much effort has been made to improve infant milk formulas to more closely mimic mother's milk. This resemblance of mother’s milk may reside in the composition of the infant formula. Additionally and / or alternatively it may reside in the growth and development of the infant being more similar to infants exclusively being fed with mother’s milk.

[0009] Sustained high levels of actively produced anti-influenza IgA in breast milk and the decreased infant episodes of respiratory illness with fever suggest that breastfeeding may provide local mucosal protection for the infant for at least 6 months (Schlaudecker EP, et al. (2013) PLoS ONE 8(8): e70867. doi:10.1371 / journal.pone.0070867). Nevertheless, many children fully rely on bovine milk based infant formulas that do not seem to offer a similar level of protection.

[0010] It is therefore an object of the present invention to provide an ingredient or a mix of ingredients for such infant formulas that offers protection or improved protection against influenza virus infection. It is another object of the present invention to provide a composition that offers protection or improved protection against influenza virus infection.

[0011] W02008016108A1 discloses an agent for preventing infection of influenza virus or a food and drink for preventing infection of influenza virus. The agent for preventing infection of influenza virus and food and drink for preventing infection of influenza virus is containing a fat globule membrane component as an active ingredient, in particular a phospholipid with sphingosine and / or a derivative thereof, particularly sphingomyelin as an active ingredient.

[0012] Phospholipids are the main part of the polar lipids fraction in milk, having a polar (charged) head group and a-polar tail. Glycerophospholipids contain two fatty acids on a glycerol backbone and a phosphate head group. Phosphosphingolipids have a sphingosine backbone of which the amide group is N-linked to a fatty acid and a phosphate head group. The five types of phospholipids present in milk are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin (SPM). Phospholipids are strongly amphiphilic and are practically insoluble, but dispersible, in water and oil. They are highly surface active.

[0013] Phospholipids are present in the fat globule membrane in milk. Milk fat globules are composed of a triglyceride-rich core surrounded by a tri-layer membrane. This membrane is known as milk fat globule membrane (MFGM). MFGM is a complex mixture of said phospholipids, cholesterol, and bio-active proteins enclosed in a unique th-layered structure.

[0014] MFGM has been associated with various health benefits - e.g. improved brain function and development, maturation of the gut, immune modulation, and antiviral action against, e.g., rotavirus. The ability of MFGM or phospholipid-enriched milk fractions to inhibit influenza has, however, not been described before.

[0015] Details of the invention

[0016] It has now been found that a synthetic composition comprising a phospholipid-enriched milk fraction and further comprising a milk serum protein concentrate (SPC) is better able to prevent infections with influenza virus, in other words, lower levels of protein of this mixture are needed to obtain a predefined level of virus inhibition. Accordingly, in a first aspect the invention relates to a synthetic nutritional composition comprising a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) for use in the prevention of an influenza virus infection and / or the prevention of further development of a viral infection with influenza virus. In a preferred aspect the invention relates to a synthetic nutritional composition comprising a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) for use in the prevention of an influenza virus infection.

[0017] Preferably, the influenza virus is influenza A virus (IAV).

[0018] A “better prevention” as used herein refers to an inhibition of influenza virus obtained with a composition of the invention which is better or higher as compared to an identical composition not comprising SPC, while the amount of phospholipid-enriched milk fraction in the composition not comprising SPC is equal to or lower as compared to the composition of the invention.

[0019] The present invention therefore relates to synthetic nutritional compositions comprising a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) for use in the prevention of a viral infection with influenza virus and / or prevention of the further development of a viral infection with influenza virus.

[0020] The nutritional composition of the invention is a synthetic nutritional composition, i.e. it is produced by humans. The nutritional composition of the invention is not milk from a mammal, such as human milk.

[0021] The term “phospholipid-enriched milk fraction” refers to dairy products that have been obtained by subjecting milk to separation techniques and thereby contain a higher concentration of phospholipids, based on dry matter, than the milk it has been derived from.

[0022] The total phospholipid content of raw bovine milk, based on dry matter, is generally around 0.25-0.30 wt%. The phospholipid-enriched milk fraction used in the present invention preferably has a total phospholipid content, based on dry matter, of at least 1.0 wt%, preferably at least 1.5 wt%, more preferably at least 2.5 wt%, even more preferably at least 4 wt%, particularly at least 6 wt%, most preferably at least 7 wt%. In one embodiment the total phospholipid content is in the range of 1-71 wt%, preferably between 5-50wt%, more preferably between 7 and 20 wt%, and most preferably in the range 7-15 wt%.

[0023] This total phospholipid content can be determined by lipid extraction using a Rose-Gottlieb method and the subsequent determination of phosphorus in the lipid extract using ICP (Induced Coupled Plasma). In one embodiment, the invention relates to a synthetic nutritional composition comprising a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) for use in the prevention of an influenza virus infection and / or the prevention of further development of a viral infection with influenza virus wherein the nutritional composition is having a phospholipid content, based on total lipids, between 0.5wt% and 2.5wt%, and a casein macropeptide (CMP) level per 100 g of composition (total solids) being between 100 mg and 500 mg.

[0024] In a preferred embodiment, the total phospholipid content refers to the content of dairy phospholipids, i.e. phospholipid originating from milk. The phospholipid content and sphingomyelin content of a milk or milk like composition (e.g. an infant formula) can be determined by quantifying the polar lipid content of a composition using31P-NMR. Quantification of polar lipids by31P-NMR is highly selective and allows to distinguish the total level of phospholipids, and levels of individual phospholipids like sphingomyelin (Giuffrida et al, Quantification of phospholipids classes in human milk; Lipids, Volume 48, Issue 10, Pages 1051 - 1058 October 2013). The sphingomyelin content, based on total phospholipids, is preferably at least 19 wt%, preferably between 19-35 wt%, particularly preferably between 21-31 wt%, more preferably between 25.0 and 28.0 wt%, most preferably between 26.0 wt% and 27.0 wt%.

[0025] In one embodiment, the phospholipid content, based on total lipids, of the synthetic nutritional composition of the invention preferably is between 0.1 and 25 %, more preferably between 0.2 and 20%, even more preferably between 0.25 and 15%, most preferably between 0.25 and 10%. In another embodiment it is between 0.26 wt% and 0.8 wt% based on total lipids, and hence similar to the phospholipid content in human breast milk.

[0026] Milk Serum Protein Concentrate (SPC) and Whey Protein Concentrate (WPC) are both the result of separating skimmed milk into a casein-rich and a whey protein-rich fraction; either by renneting (i.e. cheese making), acidification, or microfiltration.

[0027] Whey protein concentrate (WPC) is a product obtained by ultrafiltration and / or reverse osmosis and optionally demineralization of acid or cheese whey. By ultrafiltration, a large part of the water, lactose and ash are removed from the product, thereby concentrating the whey proteins. Reverse osmosis can be used to remove water and to further concentrate the WPC. WPC is readily available from several milk processing companies such as FrieslandCampina, Aria, Valio, and Fonterra.

[0028] Serum protein concentrate (SPC) is also a concentrated protein product and differs from WPC in the origin of the whey fraction. Instead of acid or cheese whey, the proteins in SPC result from microfiltration of skimmed milk. Said microfiltration results in a concentrated casein retentate fraction and a serum fraction containing most of the whey proteins as the permeate fraction. Conventionally, this permeate fraction is then subjected to ultrafiltration and / or reverse osmosis in order to remove lactose, ash, and water. SPC is readily available from several milk processing companies such as FrieslandCampina (Hiprotal® Milkserum 60 Liquid or H iprotal® Milkserum 38 Liquid), Valio, Aria, and Fonterra. Likewise, MFGM enriched ingredients are readily commercially available.

[0029] In one embodiment, the composition comprises a therapeutically effective amount of the phospholipid-enriched milk fraction and SPC.

[0030] The milk from which the phospholipid-enriched milk fractions can be obtained is preferably a ruminant milk. The term “ruminant” includes true ruminants, like cattle, sheep, cows and goats, and pseudo-ruminants, like camels. The milk to be used for the production of phospholipid-enriched milk fractions is preferably obtained from cattle or goats, meaning that bovine milk and goat milk are the preferred sources of the phospholipid-enriched milk fraction, in particular cow’s milk and goat milk. Bovine milk, more in particular cow’s milk, is the most preferred source.

[0031] The milk from which the SPC can be obtained is preferably a ruminant milk. The term “ruminant” includes true ruminants, like cattle, sheep, cows and goats, and pseudoruminants, like camels. The milk to be used for the production of SPC is preferably obtained from cattle or goats, meaning that bovine milk and goat milk are the preferred sources of SPC. Bovine milk, more in particular cow’s milk, is the most preferred source.

[0032] The phospholipid content, based on total lipids of the synthetic nutritional composition for use according to the present invention is in one embodiment between 0.5wt% and 2.5wt%, preferably between 0.8wt% and 2.2wt%, more preferably between 1.0wt% and 1.8wt%, most preferably between 1.2wt% and 1.6wt%. The phospholipid content, based on total protein of the synthetic nutritional composition for use according to the present invention in another embodiment is between 1.0wt% and 15.0wt%, preferably between 3.0wt% and 12.0wt%, more preferably between 6.0wt% and 11.0wt%, most preferably between 5.0wt% and 10.0wt%.

[0033] Sphingomyelin is considered an important type of phospholipid. Hence instead of expressing the total level of phospholipid, in yet another embodiment, the sphingomyelin content, based on total lipids, of the synthetic nutritional composition for use according to the present invention is at least 0.15 wt%, preferably between 0.20 wt% and 0.60 wt%, more preferably between 0.25 wt% and 0.55 wt%, most preferably between 0.30 wt% and 0.50 wt%.

[0034] In still another embodiment, the sphingomyelin content, based on total protein of the synthetic nutritional composition for use according to the present invention is at least 0.45 wt%, preferably between 0.50 wt% and 1.05 wt%, more preferably between 0.55 wt% and 1.00 wt%, most preferably between 0.60 wt% and 0.95 wt%.

[0035] In still another embodiment, the sphingomyelin content based on total phospholipids in the synthetic nutritional composition for use according to the present invention is at least 14 wt%, such as at least 19 wt%, preferably between 19-35 wt%, particularly preferably between 21-31 wt%, more preferably between 25.0 and 28.0 wt%, most preferably between 26.0 wt% and 27.0 wt%.

[0036] When milk is separated in protein-rich, fat-rich (cream), and lactose-rich fractions, the phospholipids mainly end up in the fat-fraction of whey or the whey-fraction of cream. Examples of such phospholipid-enriched fractions which can be used according to the present invention are cream, sweet buttermilk, alpha-serum, beta-serum, cream serum, and whey protein phospholipid concentrate.

[0037] The synthetic nutritional composition for use according to the invention can be in liquid or dry form. A dry form of the composition may be reconstituted with water to obtain a liquid form. The composition for use of the present invention may be obtained by mixing suitable amounts of the phospholipid-enriched milk fraction and milk serum protein concentrate (SPC). These ingredients may be mixed in a wet stage or as a powder. Mixing in the wet stage being preferred as it allows for the easy addition of other ingredients. Afterwards, the composition may be heat treated e.g. pasteurised, and optionally dried e.g. spray dried.

[0038] Sweet buttermilk is obtained by skimming of milk to obtain skim milk and cream (about 40% fat), followed by churning of the cream fraction into butter and sweet buttermilk. When said cream is subjected to centrifugation, it can be separated into a cream with a higher fat content (at least about 65%) and a serum fraction. This serum fraction is called alpha-serum. When the obtained cream is subsequently subjected to phase inversion with a homogeniser, it can be separated into butter oil or anhydrous milk fat (AMF) and another serum fraction: beta-serum.

[0039] Cream serum is the mixture of alpha-serum and beta-serum.

[0040] The serum fractions can be further concentrated by, e.g., ultrafiltration and optionally dried to obtain a powder.

[0041] Whey protein phospholipid concentrate can be obtained by separating the fat fraction of sweet whey protein concentrate or acid whey protein concentrate using one or more microfiltration steps, optionally followed by drying to obtain a powder. Such a process is disclosed in WO 2017 / 194068 and WO 2022 / 112552.

[0042] In a preferred embodiment, the phospholipid-enriched milk fraction is selected from beta-serum and whey protein phospholipid concentrate, as these milk fractions have the highest phospholipid content.

[0043] Most preferably, the phospholipid-enriched milk fraction is a whey protein phospholipid concentrate, as that gives the best protection against influenza virus infection.

[0044] The phospholipid-containing compositions can be used in the form of a powder or a liquid concentrate.

[0045] Examples of commercially available phospholipid-enriched milk fractions are Lacprodan® MFGM-10, Vivinal® MFGM, HilmarTM7500 MFGM, SureStart™ MFGM, SureStart™ Lipid 70, SureStart™ Lipid 100, Lacprodan® PL-20, Tatua’s PLC1 and BSP2, Fonterra’s BPC-50, and Corman’s SM2.

[0046] In a preferred embodiment, the composition of the invention added to or is part of an nutritional composition in order to reach subjects that are most vulnerable to influenza infections: people with pre-existing health problems such as asthma and / or cardiovascular disease. Such a composition may be in the form of a bar, cookie, snack, or drink. Preferably in the form of a milk-based drink.

[0047] In another preferred embodiment, the composition of the invention added to or is part of an infant formula in order to reach subjects that are most vulnerable to influenza infections: infants and young children, especially infants up to 6 months of age. In other words, in another embodiment the synthetic nutritional composition for use of the present invention is an infant formula. The amount of the phospholipid-enriched milk fraction present in such infant formula is preferably in concentrations of 0.1-10 wt%, preferably 0.5-8 wt%, even more preferably 1-7 wt%, and most preferably 2-5 wt%, based on dry weight.

[0048] The term "infant formula" as used herein refers to a nutritional composition intended for infants and as defined in Codex Alimentarius, (Codex STAN 72-1981) and Infant Specialties (incl. Food for Special Medical Purpose) as defined in Codex Alimentarius, (Codex STAN 72-1981). It also refers to a foodstuff intended for particular nutritional use by infants during the first months of life and satisfying by itself the nutritional requirements of this category of person (Article 2(c) of the European Commission Directive 2006 / 141 / EC of 22 December 2006 on infant formulae and follow-on formulae). The infant formulas can encompass the starter infant formulas, the followup or follow-on formulas, and young child formulas. Generally, a starter formula is for infants from birth as breast-milk substitute, and a follow-up or follow-on formula from the 6th month onwards. A young child formula - also called ‘growing-up milk’ - is tailor-made to support the nutritional needs of children from 1 year of age onwards, preferably 1-6 years. It differs from both starter formula (for infants from 0-6 months) and follow-on formula (for infants from 6-12 months). So, an infant formula may be dedicated for infants of 0 to 6 months, 6 to 12 months, 12 months and older.

[0049] In addition to the phospholipid-enriched milk fraction, the infant formula will contain its regular ingredients, including a carbohydrate fraction, a protein fraction, and a lipid fraction.

[0050] The protein fraction in the composition for use of the current invention may comprise plant proteins or milk proteins such as whey proteins and / or casein. The carbohydrate fraction may comprise digestible and non-digestible saccharides. Digestible saccharides include glucose, galactose, sucrose, maltose, dextrin, and lactose. Non-digestible oligosaccharides can be selected from the group consisting of fructo-oligosaccharides, galacto-oligosaccharides, arabino-oligosaccharides, arabinogalacto-oligosaccharides, gluco-oligosaccharides, chito-oligosaccharides, glucomanno-oligosaccharides, galactomanno-oligosaccharides, mannanoligosaccharides, fucosylated oligosaccharides, sialylated oligosaccharides and N-acetylglucosamine-functional oligosaccharides; more preferably selected from the group consisting of fructo-oligosaccharides and galacto-oligosaccharides. As used herein, fructo-oligosaccharides include inulin. All such non-digestible oligosaccharides are readily commercially available.

[0051] The carbohydrate fraction may also comprise one or more human milk oligosaccharides (HMOs), as these are known for their various health effects. Preferably the one or more HMOs are selected from the group consisting of 2’-fucosy I lactose (2’-FL), 3-fucosyl lactose (3-FL), di-fucosy I lactose (DFL), 3’-galactosyllactose (3’-GL), 6’-galactosyllactose (6’-GL), 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT), and combinations thereof. Preferably, the total amount of HMOs in the infant formula is at least 0.1 wt%, more preferably at least 0.5 wt%. The total amount is preferably less than 10 wt%, more preferably less than 5 wt%.

[0052] The lipid fraction may comprise a mixture of different fats and oils, such as a mixture of vegetable oils, or a mixture of vegetable oils and milk fat. Preferably, long chain poly unsaturated fatty acids (LC-PUFA) such as DHA, ARA, and / or EPA are present in the infant formula. In one embodiment, the lipid fraction has a fatty acid composition wherein at least 0.6 wt% of the fatty acid acyl groups consists of 4 carbon atoms, preferably at least 1.2 wt%, more preferably at least 1.5 wt%; and / or wherein at least 10% of the fatty acid acyl groups present in the lipid fraction is a palmitoyl group (CH3(CH2)i4C(O)) and at least 30% of the palmitoyl groups is esterified to the sn-2 position of a triglyceride based on total palmitoyl groups.

[0053] Vegetable oils for human consumption are well known in the art. Suitable milk fat for the composition of the invention is ruminant milkfat, preferably from cow’s milk, sheep milk, or goat milk, most preferably from cow’s milk. For example whole milk, cream, butter, anhydrous milkfat and the like. In one embodiment the amount of milk fat in the lipid fraction of the composition of the invention is at least 20 wt% of the lipid fraction, preferably at least 30 wt%, more preferably at least 40 wt%, even more preferably at least 60 wt%.

[0054] In one embodiment at least 5wt% of the total protein content in the composition of the invention originates from SPC. Preferably it is more than 8 wt%, more preferably more than 15 wt%, even more preferably more than 25 wt%, most preferably more than 30 wt%.

[0055] In a particular embodiment the phospholipid content in the composition of the invention is, based on dry matter, at least 1.0 wt% and the amount of SPC in the synthetic composition for use of the present invention is in one embodiment more than 5wt% as determined relative to the dry weight of the total protein content in the composition. In one embodiment, the phospholipid content in the composition of the invention is, based on dry matter, in the range 7-15 wt% and amount of SPC in the synthetic composition for use of the present invention is in one embodiment more than 5wt% as determined relative to the dry weight of the total protein content in the composition, preferably the SPC content is more than 10 wt%, more preferably more than 20 wt%, even more preferably more than 30 wt%, most preferably more than 40 wt%.

[0056] In still another embodiment, the phospholipid content in the composition for use of the invention is, based on dry matter, in the range 7-15 wt%; and the sphingomyelin content, based on total phospholipids is in the range 20-35wt. In one embodiment the amount of SPC in in the synthetic composition for use of the present invention is more than 5wt% as determined relative to the dry weight of the total protein content in the composition; preferably the SPC content is more than 10 wt%, more preferably more than 20 wt%, even more preferably more than 30 wt%, most preferably more than 40 wt% as determined relative to the dry weight of the total protein content in the composition.

[0057] In a particularly preferred embodiment, the phospholipid enriched milk fraction is a whey protein fraction enriched in milk fat globular membrane (MFGM) components, even more preferably it is MFGM.

[0058] Due to the combined use of SPC and the phospholipid-enriched milk fraction the composition of the invention may, in still another embodiment, further have a relative low amount of casein macropeptide (CMP), which may also be referred to as glycomacropeptide (GMP). The low level of CMP per 100 g of composition (total solids) being between 100 mg and 500 mg, preferably between 100 mg and 300 mg, more preferably between 105 mg and 250 mg, most preferably between 105 mg and 225 mg. Lower levels of CMP are beneficial in the prevention of hyperthreonina in formula fed infants.

[0059] In another aspect, the invention relates to the use of a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) in the prevention of an influenza virus infection and / or the prevention of further development of a viral infection with influenza virus, optionally wherein the phospholipid enriched milk fraction and the SPC are comprised in a synthetic nutritional composition wherein the nutritional composition is as defined elsewhere herein.

[0060] The composition for use of the present invention as herein described may in some jurisdictions also be referred to as a composition comprising a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) for use in the manufacture of a medicament for use in the prevention of a viral infection with influenza virus and / or the prevention of further development of a viral infection with influenza virus. They may also be referred to as the use of such compositions for use in the manufacture of a medicament for use in the prevention of a viral infection with influenza virus and / or the prevention of further development of a viral infection with influenza virus.

[0061] In a preferred embodiment, the invention relates to a synthetic nutritional composition comprising a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) for use in the prevention of an influenza virus infection and / or the prevention of further development of a viral infection with influenza virus wherein the phospholipid enriched milk fraction is a whey protein fraction enriched in milk fat globular membrane (MFGM) components and wherein the ratio between proteins from the MFGM fraction and SPC fraction is between 0.5 and 1.0, preferably between 0.5 and 0.9, more preferably between 0.6 and 0.8, most preferably between 0.63 and 0.70.

[0062] In another preferred embodiment, the invention relates to a synthetic nutritional composition comprising a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) for use in the prevention of an influenza virus infection and / or the prevention of further development of a viral infection with influenza virus wherein the phospholipid enriched milk fraction is milkfat globular membrane (MFGM) and wherein the ratio between proteins from the MFGM fraction and SPC fraction is between 0.5 and 1.0, preferably between 0.5 and 0.9, more preferably between 0.6 and 0.8, most preferably between 0.63 and 0.70.

[0063] FIGURES

[0064] Figure 1 : The total amount of protein of SPC, MFGM or a mixture of SPC and MFGM needed (in mg / ml) in order to get 30, 50, or 70 percentage inhibition of Influenza infection.

[0065] EXAMPLES

[0066] Example 1 Inhibition of influenza by MFGM, SPC, or MFGM + SPC

[0067] In vitro experiments were performed to investigate the capacity of various commercial milk fractions to inhibit influenza A virus strain Influenza A H1N1 subtype A / WSN / 33 infection in an A549 (ATCC CRM-CCL-185) human alveolar basal epithelial cell line. Different commercial milk protein concentrates were used: Vivinal® MFGM FrieslandCampina (phospholipid content on dry matter: 7.4 wt%) and Hiprotal® Milkserum 60 Liquid (i.e. SPC60 (63.5 g protein / 100 g solids).

[0068] A Whey Protein Isolate (WPI, Nutri Whey Isolate, typically 88% m / m) protein, FrieslandCampina, The Netherlands), the whey protein isolate counterpart of Vivinal MFGM, was used as a negative control (no influenza A inhibition).

[0069] A solution (mixture I mix) was made of Vivinal® MFGM (71.9 g protein / 100 g powder) and Hiprotal® Milkserum 60 Liquid (i.e. SPC60 (63.5 g protein / 100 g solids) - both from FrieslandCampina -, with the ratio between proteins from MFGM and SPC being 35.6 : 52.8, respectively.

[0070] The milk fractions were solubilized in a complete DMEM medium (DMEM+ 0.3% BSA, 0.1 %FCS, penicillin / streptomycin, and 20 mM HEPES). The samples were solubilized by incubating overnight at 4°C followed by 2h at 40°C with frequent vortexing.

[0071] At Virology Research Services (UK), A549 cells (human alveolar basal epithelial) were pre-incubated 24 hours with the ingredients in different concentrations (10 mg protein / ml as highest concentration followed by 7x two-fold serial dilutions thereof), followed by 24 hours infection with Influenza A H1N1 subtype A / WSN / 33.. After infection, cells were fixed and processed for immunostaining. In parallel, incubations were performed to determine cytotoxicity using the MTT assay. For each tested milk fraction the percentage influenza inhibition was plotted as a function of the tested concentration. Using a nonlinear fit (Graphpad software) an IC50 was calculated. The IC50 value indicates the protein concentration (mg / ml or ug / ml) required to inhibit the infection by 50% of the total infectious capacity. The lower the IC50, the more effective the stream / component is in inhibiting the virus. The IC30 and IC70 were determined similarly.

[0072] The total amount of protein needed (in mg / ml) in order to get 30, 50, or 70 percentage inhibition of Influenza infection is shown in Figure 1. The figure clearly shows that lower protein concentrations are needed to obtain a certain inhibition level when the mixture of MFGM and SPC is used compared to the inhibition of MFGM or SPC in isolation. This has a several advantages. Firstly, a lower dosage of the mixture is required for a certain level of inhibition. Secondly, considering that SPC may be cheaper than MFGM, the price per g of composition is lower than the price of gram of MFGM, in other words it is cheaper to get a certain level of inhibition. Moreover, since the product for use of the present invention requires less phospholipid enriched milk fraction (like MFGM), it provides a wider formulation space, in other words it makes it easier to prepare a recipe providing the desired nutrient levels of the composition e.g. infant formula. A wider formulation space may be considered helpful in defining more sustainable compositions.

[0073] In the same experiment, other milk components were tested to unravel which milk component was responsible for the influenza inhibition. Immunoglobulins, lactoferrin and osteopontin were tested at similar concentrations as present in the MFGM, SPC or mix. No inhibition influenza was observed for these components.

[0074] Example 2 Exemplary formulations

[0075] An exemplary infant formula comprising both MFGM and SPC for use according to the present invention is given below.

[0076] The composition of a cow’s milk based formula according to the invention comprising a native whey protein concentrate (H iprotal® Milkserum 60 Liquid, FrieslandCampina) and a whey protein concentrate rich in phospholipids (Vivinal® MFGM, FrieslandCampina) is exemplified in the table below.

[0077] The product was designed to meet the nutritional requirements of infants (0 to 6 months). The nutritional composition of the formulae is presented in Table 1 A and 1 B.

[0078] Table 1 A. Nutrient composition of an infant formula according to the invention per 100 ml of ready to drink product

[0079] Composition per 100 ml Infant formula 0-6 months

[0080] Energy (kcal) 66

[0081] Protein1(g) 1.4

[0082] - Casein1(g) 0.5

[0083] - Whey protein1(g) 0.9

[0084] Fat (g) 3.5

[0085] Carbohydrates (g) 7.0

[0086] Galacto-oligosaccharides (g) 0.4

[0087] bLac (g) 0.4

[0088] Urea (mg) 6

[0089] Sphingomyelin (mg) 13

[0090] Phospholipids (mg) 49

[0091] Sialic acid (mg) 14

[0092] Casein MacroPeptide (CMP) (mg) 29

[0093] Sphingomyelin per 100g of fat (mg) 368

[0094]

[0095] 1: N*6.25 Table 1 B. Nutrient composition of an infant formula according to the invention per 100 gram powder.

[0096] Composition per 100 g powder Infant formula 0-6 months

[0097] Energy (kcal) 499

[0098] Protein1(g) 10.1

[0099] - Casein1(g) 3.7

[0100] - Whey protein1(g) 6.4

[0101] Fat (g) 26.4

[0102] Carbohydrates (g) 53

[0103] Galacto-oligosaccharides (g) 3

[0104] bLac (g) 3.1

[0105] Urea (mg) 47

[0106] Sphingomyelin (mg) 97

[0107] Phospholipids (mg) 369

[0108] Sialic acid (mg) 107

[0109] Casein MacroPeptide (CMP) (mg) 217

[0110]

[0111] 1: N*6.25

[0112] Other milk-based compositions meeting the requirements of the present invention may readily be prepared by those skilled in art by replacing some of the protein-providing ingredients with a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC).

Claims

CLAIMS1. Synthetic nutritional composition comprising a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) for use in the prevention of an influenza virus infection and / or the prevention of further development of a viral infection with influenza virus;preferably wherein the nutritional composition is having a phospholipid content, based on total lipids, between 0.5wt% and 2.5wt%, and a casein macropeptide (CMP) level per 100 g of composition (total solids) being between 100 mg and 500 mg.

2. Synthetic nutritional composition for use according to claim 1 wherein the influenza virus is influenza A virus.

3. Synthetic nutritional composition for use according to any of the preceding claims, wherein the phospholipid-enriched milk fraction is obtained from ruminant milk, preferably bovine milk or goat milk, more preferably bovine milk.

4. Synthetic nutritional composition for use according to any of the preceding claims, wherein the SPC is obtained from ruminant milk, preferably bovine milk or goat milk, more preferably bovine milk.

5. Synthetic nutritional composition for use according to any of the preceding claims having a phospholipid content, based on total lipids, between 0.5wt% and 2.5wt%, preferably between 0.8wt% and 2.2wt%, more preferably between 1.0wt% and 1.8wt%, most preferably between 1.2wt% and 1.6wt%.

6. Synthetic nutritional composition for use according to any of the preceding claims having a phospholipid content, based on total protein, between 1.0wt% and 15.0wt%, preferably between 3.0wt% and 12.0wt%, more preferably between 6.0wt% and 11.0wt%, most preferably between 5.0wt% and 10.0wt%.

7. Synthetic nutritional composition for use according to any of the preceding claims having a sphingomyelin content, based on total lipids, of at least 0.15 wt%,preferably between 0.20 wt% and 0.60 wt%, more preferably between 0.25 wt% and 0.55 wt%, most preferably between 0.30 wt% and 0.50 wt%.

8. Synthetic nutritional composition for use according to any of the preceding claims having a sphingomyelin content, based on total protein of at least 0.45 wt%, preferably between 0.50 wt% and 1.05 wt%, more preferably between 0.55 wt% and 1.00 wt%, most preferably between 0.60 wt% and 0.95 wt%.

9. Synthetic nutritional composition for use according to any one of the preceding claims wherein the sphingomyelin content, based on total phospholipids, is at least 14 wt%, preferably between 19-35 wt%, particularly preferably between 21- 31 wt%, more preferably between 25.0 and 28.0 wt%, most preferably between 26.0 wt% and 27.0 wt%.

10. Synthetic nutritional composition for use according to any one of the preceding claims wherein the phospholipid-enriched milk fraction is selected from the group consisting of whey protein phospholipid concentrate, beta-serum, alpha-serum, cream serum, cream, and sweet buttermilk, preferably is selected from the group consisting of whey protein phospholipid concentrate and beta-serum, and most preferably is a whey protein phospholipid concentrate.

11. Synthetic nutritional composition for use according to any one of the preceding claims wherein said composition is one or more of the following:a) in liquid or dry form;b) used as part of an infant formula.

12. Synthetic nutritional composition for use according to any of the preceding claims wherein the phospholipid-enriched milk fraction is present in said infant formula in a concentration of 0.1-10 wt%, preferably 0.5-8 wt%, even more preferably 1- 7 wt%, and most preferably 2-5 wt%, based on dry weight.

13. Synthetic nutritional composition for use according to any of the preceding claims wherein at least 5wt% of the total protein content in the composition of the invention originates from SPC.

14. Synthetic nutritional composition for use according to any of the preceding claims wherein the amount of casein macropeptide (CMP) per 100 g of composition being between 100 mg and 500 mg based on total solids.

15. The use of a phospholipid-enriched milk fraction and a milk serum protein concentrate (SPC) in the prevention of an influenza virus infection and / or the prevention of further development of a viral infection with influenza virus, optionally wherein the phospholipid enriched milk fraction and the SPC are comprised in a synthetic nutritional composition wherein the nutritional composition is as defined in anyone of claims 1 - 14.

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