Use of phospholipid-rich milk fraction in prevention of RSV infection

Abstract

A phospholipid-rich milk fraction for use in the prevention of viral infection of respiratory syncytial virus (RSV) and / or in the further development of the prevention of viral infection of respiratory syncytial virus (RSV) is disclosed.

Description

[0001] The present invention relates to a milk fraction enriched in phospholipids for use in preventing respiratory syncytial virus (RSV) infection of the respiratory tract.

[0002] RSV infections are a major disease burden in infants. They are the second most common cause of death in children – particularly in low- and middle-income countries – and a major seasonal burden on healthcare systems.

[0003] Infants under six months of age are most susceptible to RSV infection, during which time they rely heavily on maternally transferred immunity.

[0004] S. Geoghegan et al. (AJ Respir. Crit. Care Md. (2017) 195:96-103) have shown that breastfeeding reduces the severity and incidence of RSV infection in children. However, many children rely exclusively on cow's milk-based infant formulas, which do not appear to provide similar levels of protection.

[0005] It was therefore an object of the present invention to provide an ingredient for such an infant formula which provides protection against RSV.

[0006] It has now been found that milk fractions enriched in phospholipids are able to prevent RSV infection.

[0007] Phospholipids make up the majority of the polar lipid fraction in milk and have a polar (charged) head group and a nonpolar tail. Glycerophospholipids contain two fatty acids attached to a glycerol backbone and a phosphate head group. Sphingomyelins have a sphingosine backbone with an amide group N-linked to the fatty acid and phosphate head groups. The five types of phospholipids found in milk are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SPM). Phospholipids are strongly amphiphilic and practically insoluble in water and oil, but dispersible in both. They are highly surface-active.

[0008] Phospholipids are found in the membranes of milk fat globules. Milk fat globules consist of a triglyceride-rich core surrounded by three membrane layers. This membrane is called the milk fat globule membrane (MFGM). MFGM is a complex mixture of these phospholipids, cholesterol, and bioactive proteins encapsulated in a unique three-layer structure.

[0009] MFGM has been associated with various health benefits – such as improved brain function and development, intestinal maturation, immune modulation, and antiviral effects against, for example, rotavirus. However, the ability of MFGM or phospholipid-rich milk fractions to inhibit RSV has not been previously described.

[0010] The present invention therefore relates to a milk fraction enriched in phospholipids for use in the prevention of viral infections by respiratory syncytial virus (RSV) and / or in the further development of the prevention of viral infections by respiratory syncytial virus (RSV).

[0011] The term "phospholipid-enriched milk fraction" refers to milk products obtained by subjecting milk to separation techniques, these milk products having a phospholipid concentration (on a dry matter basis) that is higher than the phospholipid concentration of the milk from which they are derived.

[0012] The total phospholipid content of raw bovine milk is generally about 0.25-0.30 wt % based on dry matter. The total phospholipid content of the phospholipid-enriched milk fraction for use according to the invention is preferably at least 1.0 wt %, preferably at least 1.5 wt %, more preferably 2.5 wt %, even more preferably at least 4 wt %, more preferably at least 6 wt %, even more preferably at least 7 wt %, even more preferably in the range of 7-20 wt %, and most preferably in the range of 7-15 wt %, based on dry matter.

[0013] The total phospholipid content can be determined by performing lipid extraction using the Rose-Gotlieb method and subsequently determining phosphorus in the lipid extract using ICP (Inductively Coupled Plasma).

[0014] In a preferred embodiment, the total phospholipids refer to the content of milk-derived phospholipids (i.e., phospholipids derived from milk). 13 The sphingomyelin content (phospholipids not present in plant phospholipid sources) is determined by P-NMR analysis. The sphingomyelin content is preferably at least 20 wt%, more preferably in the range of 20-35 wt%, most preferably in the range of 20-30 wt%, based on total phospholipids.

[0015] The milk from which the phospholipid-rich milk fraction can be obtained is preferably ruminant milk. The term "ruminant" includes true ruminants, such as cattle, sheep, and goats, as well as pseudo-ruminants, such as camels. The milk used to produce the phospholipid-rich milk fraction is preferably obtained from cattle or goats, meaning that bovine and goat milk are preferred sources of the phospholipid-rich milk fraction. Bovine milk, more particularly dairy cow's milk, is the most preferred source.

[0016] When milk is separated into protein-rich, fat-rich (cream), and lactose-rich fractions, phospholipids are primarily found in the fat fraction of the whey or the whey fraction of the cream.

[0017] Examples of such phospholipid-rich fractions that can be used according to the invention are cream, sweet buttermilk, alpha-serum, beta-serum, cream serum, and whey protein phospholipid concentrate.

[0018] Sweet buttermilk is obtained by skimming milk to obtain skim milk and cream (about 40% fat), and then churning the cream fraction into butter and sweet buttermilk.

[0019] When the cream is centrifuged, it can be separated into a cream fraction with a higher fat content (at least about 65%) and a serum fraction. This serum fraction is called α-serum. When the resulting cream is subsequently phase-inverted using a homogenizer, it can be separated into butter or anhydrous milk fat (AMF) and another serum fraction: β-serum.

[0020] Cream serum is a mixture of alpha-serum and beta-serum.

[0021] The serum fraction can be further concentrated, for example, by ultrafiltration, and optionally dried to obtain a powder.

[0022] 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 methods are disclosed in WO 2017 / 194068 and WO 2022 / 112552.

[0023] In a preferred embodiment, the phospholipid-rich milk fractions are selected from beta-serum and whey protein phospholipid concentrates, since these milk fractions have the highest phospholipid content.

[0024] Most preferably, the phospholipid-rich milk fraction is whey protein phospholipid concentrate, as this provides the best protection against RSV infection.

[0025] The phospholipid-containing composition may be used in the form of a powder or a liquid concentrate.

[0026] Commercially available phospholipid-enriched milk fractions are Lacprodan® MFGM-10, Vivinal® MFGM, Hilmar TM 7500 MFGM, SureStart TM MFGM, SureStart TM Lipid 70, SureStart TM Lipid 100, Lacprodan® PL-20, Tatua’s PLC1 and BSP2, Fonterra’s BPC-50, and Corman’s SM2.

[0027] In a preferred embodiment, the phospholipid-rich milk fraction is added to or is part of an infant formula so that it can be targeted to the subjects most susceptible to RSV infection: infants and young children, especially infants under six months of age.

[0028] The phospholipid-rich milk fraction is preferably present in such 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% on a dry weight basis.

[0029] As used herein, the term "infant formula" refers to nutritional compositions intended for use by infants as defined in the Codex Alimentarius (Codex STAN 72-1981), and infant specialties (including foods for special medical purposes) as defined in the Codex STAN 72-1981. It also refers to foods intended for special nutritional use by infants in the first few months of life and which meet the nutritional needs of such persons (Article 2(c) of the European Commission Directive 2006 / 141 / EC of 22 December 2006 on infant formulae and follow-on formulae). Infant formula may encompass a first-stage infant formula, an older infant formula or follow-on formula, and a toddler formula. Generally speaking, first-stage formula is used as a breast milk substitute for infants from birth, and follow-on formula or follow-on formula is used from the 6th month onwards. Therefore, infant formula can be specifically for infants 0 to 6 months, 6 to 12 months, or 12 months and above.

[0030] In addition to the phospholipid-rich milk fraction, infant formula will also contain its conventional ingredients, including a carbohydrate fraction, a protein fraction and a lipid fraction.

[0031] The protein fraction in the composition for use according to the invention may comprise vegetable proteins or milk proteins, such as whey proteins and / or caseins.

[0032] The carbohydrate fraction can contain both readily digestible and non-digestible sugars. Easily digestible sugars include glucose, galactose, sucrose, maltose, dextrin, and lactose. Non-digestible oligosaccharides can be selected from the group consisting of fructooligosaccharides, galacto-oligosaccharides, arabino-oligosaccharides, arabino-galacto-oligosaccharides, glucose oligosaccharides, chito-oligosaccharides, glucomannan oligosaccharides, galacto-oligomannan oligosaccharides, manno-oligosaccharides, fucosylated oligosaccharides, sialylated oligosaccharides, and N-acetylglucosamine functional oligosaccharides; more preferably, selected from the group consisting of fructooligosaccharides and galacto-oligosaccharides. As used herein, fructooligosaccharides include inulin. All such non-digestible oligosaccharides are readily commercially available.

[0033] The carbohydrate fraction may include one or more human milk oligosaccharides (HMOs), as these oligosaccharides are known to have various health effects. Preferably, the one or more HMOs are selected from the group consisting of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose (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%.

[0034] The lipid fraction may comprise a mixture of different fats and oils, such as a mixture of vegetable oils or a mixture of vegetable oil and milk fat. Preferably, long chain polyunsaturated fatty acids (LC-PUFAs) 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 consist 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 are palmitoyl groups (CH3(CH2) 14 C(O)), and at least 30% of these palmitoyl groups, based on the total palmitoyl groups, are esterified at the sn-2 position of the triglyceride.

[0035] Vegetable oils for human consumption are well known in the art. Suitable milk fats for the compositions of the present invention are ruminant milk fats, preferably from cow's milk, sheep's milk or goat's milk, most preferably from cow's milk. For example, whole milk, cream, butter, anhydrous milk fat, etc.

[0036] In one embodiment the amount of milk fat in the lipid fraction of the composition of the invention is at least 20 wt%, preferably at least 30 wt%, more preferably at least 40 wt%, even more preferably at least 60 wt% of the lipid fraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Shown are the percentage inhibition of RSV infection in vitro as a function of the concentration of different phospholipid-enriched milk fractions.

[0038] Figure 2 Shown are the percentage inhibition of RSV infection in vitro as a function of phospholipid concentration in two phospholipid-rich milk fractions (whey protein phospholipid concentrate and beta-serum).

[0039] Figure 3 Shown are the percentage inhibition of RSV infection in vitro as a function of protein concentration for two whey protein compositions: phospholipid-enriched whey protein concentrate and conventional whey protein isolate.

[0040] Examples

[0041] In vitro experiments were performed to investigate the ability of various commercial phospholipid-rich milk fractions to inhibit infection by the RS virus strain RSV-A2 in the A549 (ATCC CRM-CCL-185) cell line.

[0042] Different whey protein phospholipid concentrates were used: FrieslandCampina’s Vivinal® MFGM (phospholipid content based on dry matter: 7.4 wt%), and three commercially available competitor products: A (phospholipid content: 7.5 wt%), B (phospholipid content: 5.7 wt%), and C (phospholipid content: 6.4 wt%).

[0043] In addition, β-serum was used as a milk fraction rich in phospholipids.

[0044] Commercial whey protein isolate (WPI) was used as a comparison.

[0045] Phospholipid-rich milk fractions and comparative WPI were dissolved in complete F12 medium (F12 Nut mix + glutamax + 0.3% BSA, 0.1% FCS, penicillin / streptomycin, and 20 mM HEPES). After sample dissolution (30 min at 50°C with frequent vortexing), Figure 1 The samples shown in Figure 3 were centrifuged at 3220 g for 30 minutes and filter sterilized through a 0.2 uM PES filter. Figure 2 and Figure 3 The samples shown were not centrifuged and filter sterilized after lysis.

[0046] Serial dilutions of the samples were preincubated with RSV-A2 for 1 hour. A549 cells were serially incubated with the component / virus premix (2 hours) and infection medium without the component / virus (22 hours), all at 37°C, after which infectivity was determined by immunostaining.

[0047] Figure 1 The percentage inhibition of RSV infection is shown as a function of product concentration compared to the control (set as 100%). This figure clearly demonstrates that various commercial whey protein phospholipid concentrates are able to inhibit RSV infection.

[0048] Figure 2 Shown are the percentage inhibition of RSV infection as a function of the phospholipid content of whey protein phospholipid concentrate ("whey-derived MFGM") and beta-serum ("beta-serum MFGM") compared to the control (set as 100%).

[0049] Figure 3 The percentage inhibition of RSV infection is shown as a function of product concentration compared to the control (set as 100%). This figure clearly shows that conventional WPI is not able to inhibit RSV infection compared to whey protein phospholipid concentrate.

Claims

1. A phospholipid-rich milk fraction for use in the prevention of viral infection by respiratory syncytial virus (RSV) and / or in the prevention of the further development of viral infection by respiratory syncytial virus (RSV).

2. The phospholipid-rich milk fraction for use according to claim 1, wherein the phospholipid-rich milk fraction is obtained from ruminant milk, preferably cow's milk or goat's milk, more preferably cow's milk.

3. The phospholipid-rich milk fraction for use according to claim 1 or 2, having a phospholipid content of at least 1.0 wt%, preferably at least 1.5 wt%, more preferably 2.5 wt%, even more preferably at least 4 wt%, more preferably at least 6 wt%, even more preferably at least 7 wt%, even more preferably in the range of 7-20 wt%, and most preferably in the range of 7-15 wt%, based on dry matter.

4. A phospholipid-enriched milk fraction for use according to any one of the preceding claims, wherein the sphingomyelin content is at least 20 wt%, preferably in the range of 20-35 wt%, most preferably in the range of 20-30 wt%, based on total phospholipids.

5. The phospholipid-rich milk fraction for use according to any of the preceding claims, wherein the phospholipid-rich milk fraction is selected from the group consisting of whey protein phospholipid concentrate, beta-serum, alpha-serum, cream serum, cream and sweet buttermilk, preferably from the group consisting of whey protein phospholipid concentrate and beta-serum, and most preferably is whey protein phospholipid concentrate.

6. The phospholipids-enriched milk fraction for use according to any one of the preceding claims, wherein the phospholipids-enriched milk fraction is in liquid or dry form.

7. The phospholipids-rich milk fraction for use according to any one of the preceding claims, wherein the phospholipids-rich milk fraction is used as part of an infant formula.

8. The phospholipids-rich milk fraction for use according to claim 7, wherein the phospholipids-rich milk fraction is present in the 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% on a dry weight basis.

9. The phospholipids-rich milk fraction for use according to any one of the preceding claims, wherein the phospholipids-rich milk fraction is administered to a young child or infant, preferably to an infant, most preferably to an infant under six months of age.

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