Use of phospholipid-enriched milk fractions in the prevention of infection with RSV

Abstract

A phospholipid-enriched milk fraction for use in preventing viral infection with respiratory syncytial virus (RSV) and / or preventing the further progression of viral infection with respiratory syncytial virus (RSV).

Description

[Technical Field]

[0001] The present invention relates to a phospholipid-enriched milk fraction for use in the prevention of infection of the respiratory tract by respiratory syncytial virus (RSV). [Background technology]

[0002] RSV infection is a major disease burden among infants, is the second leading cause of death among children, especially in low- and middle-income countries, and represents a major seasonal burden on health systems.

[0003] Infants are most vulnerable to RSV infection up to the age of 6 months, during which time they are highly dependent on maternal 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. Nevertheless, many children rely entirely on cow's milk-based infant formulas, which do not appear to offer the same level of protection. Summary of the Invention [Problem to be solved by the invention]

[0005] It is therefore an object of the present invention to provide ingredients for such infant formulas that confer protection against RSV.

[0006] It has now been found that phospholipid-enriched milk fractions can prevent infection with RSV.

[0007] Phospholipids are the major component of the polar lipid fraction in milk, possessing a polar (charged) head group and a non-polar tail. Glycerophospholipids contain two fatty acids on a glycerol backbone and a phosphate head group. Phosphosphingolipids have a sphingosine backbone with an amide group 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 have strong amphiphilic properties and are virtually insoluble but dispersible in water and oil. Phospholipids are highly surface active.

[0008] Phospholipids are present in the milk fat globule membrane. Milk fat globules consist of a triglyceride-rich core surrounded by a three-layer membrane. This membrane is known as 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 a variety of health benefits, including improved brain function and development, intestinal maturation, immunomodulation, and antiviral effects, such as against rotavirus. However, the ability of MFGM or phospholipid-enriched milk fractions to inhibit RSV has not previously been described. [Means for solving the problem]

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

[0011] The term "phospholipid-enriched milk fraction" refers to a dairy product obtained by subjecting milk to separation techniques and containing a higher concentration of phospholipids on a dry matter basis than the milk from which it is derived.

[0012] The total phospholipid content of untreated cow's milk, based on dry matter, is generally about 0.25-0.30% by weight. The phospholipid-enriched milk fraction for use according to the present invention preferably has a total phospholipid content, based on dry matter, of at least 1.0%, preferably at least 1.5%, more preferably 2.5%, even more preferably at least 4%, more preferably at least 6%, even more preferably at least 7%, even more preferably in the range of 7-20% by weight, and most preferably in the range of 7-15% by weight.

[0013] This total phospholipid content can be determined by lipid extraction using the Rose-Gotlieb method and subsequent determination of phosphorus in the lipid extract using ICP (inductively coupled plasma).

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

[0015] The milk from which the phospholipid-enriched milk fraction can be obtained is preferably ruminant milk.The term "ruminant" includes true ruminants such as cows, sheep and goats, and pseudoruminants such as camels.The milk used to produce the phospholipid-enriched milk fraction is preferably obtained from cows or goats, which means that cow's milk and goat's milk are the preferred source of the phospholipid-enriched milk fraction.Cow's milk, more specifically 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 contained in the whey fat fraction or the cream whey fraction. Examples of such phospholipid-enriched fractions that can be used in accordance with the present invention are cream, sweet buttermilk, alpha-serum, beta-serum, cream serum and whey protein phospholipid concentrate.

[0017] Sweet buttermilk is obtained by removing the cream from milk to obtain skim milk and cream (about 40% fat), followed by churning the cream fraction to form butter and sweet buttermilk. Centrifuging the cream separates it into a higher fat cream (at least about 65%) and a serum fraction, called alpha-serum. The resulting cream can then be phase-inverted using a homogenizer to separate it into butter oil or anhydrous milk fat (AMF) and another serum fraction: beta-serum.

[0018] Cream serum is a mixture of alpha- and beta-serums.

[0019] The serum fraction can be further concentrated, for example, by ultrafiltration, and optionally dried to obtain a powder.

[0020] The whey protein phospholipid concentrate can be obtained by separating the fat fraction of a sweet whey protein concentrate or an acid whey protein concentrate using one or more microfiltration steps, optionally followed by drying to obtain a powder. Such processes are disclosed in WO 2017 / 194068 and WO 2022 / 112552.

[0021] In a preferred embodiment, the phospholipid-enriched milk fraction is selected from beta-serum and whey protein phospholipid concentrate, these milk fractions having the highest phospholipid content.

[0022] Most preferably, the phospholipid-enriched milk fraction is a whey protein phospholipid concentrate, which provides the best protection against RSV infection.

[0023] The phospholipid-containing composition can be used in the form of a powder or a liquid concentrate.

[0024] Commercially available phospholipid-enriched milk fractions are Lacprodan® MFGM-10, Vivinal® MFGM, Hilmar™ 7500 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.

[0025] In a preferred embodiment, the phospholipid-enriched milk fraction is added to or is part of infant formula to reach those most vulnerable to RSV infection: infants and young children, particularly those up to 6 months of age.

[0026] The phospholipid-enriched milk fraction is preferably present in such infant formulas at a concentration of 0.1 to 10% by weight, preferably 0.5 to 8% by weight, even more preferably 1 to 7% by weight, and most preferably 2 to 5% by weight, based on dry weight.

[0027] The term "infant formula" as used herein refers to nutritional compositions 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 foodstuffs intended for specific nutritional use by infants from birth to the first few months of life and which by themselves meet the nutritional needs of this category of persons (Article 2(c) of European Commission Directive 2006 / 141 / EC of December 22, 2006 on infant formulas and follow-on formulas). Infant formulas can include starter infant formulas, follow-up or follow-on formulas, and toddler formulas. Generally, starter formulas are formulas used with infants from birth as a substitute for breast milk, and follow-up or follow-on formulas are formulas from six months of age onwards. Thus, infant formulas may be designated specifically for infants aged 0-6 months, 6-12 months, or 12 months and older.

[0028] In addition to the phospholipid-enriched milk fraction, infant formula contains its usual components, including a carbohydrate fraction, a protein fraction, and a lipid fraction.

[0029] 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 casein.

[0030] The carbohydrate fraction may contain digestible and non-digestible sugars. Digestible sugars include glucose, galactose, sucrose, maltose, dextrin, and lactose. The non-digestible oligosaccharides may be selected from the group consisting of fructooligosaccharides, galactooligosaccharides, arabinooligosaccharides, arabinogalactooligosaccharides, glucooligosaccharides, chitooligosaccharides, glucomanooligosaccharides, galactomannooligosaccharides, mannanoligosaccharides, fucosylated oligosaccharides, sialylated oligosaccharides, and N-acetylglucosamine-functional oligosaccharides; more preferably, they may be selected from the group consisting of fructooligosaccharides and galactooligosaccharides. As used herein, fructooligosaccharides include inulin. All of these non-digestible oligosaccharides are readily available commercially.

[0031] The carbohydrate fraction may also contain one or more human milk oligosaccharides (HMOs), which are known for their various health benefits. 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% by weight, more preferably at least 0.5% by weight. The total amount is preferably less than 10% by weight, more preferably less than 5% by weight.

[0032] The lipid fraction may comprise a mixture of various fats and oils, for example, a mixture of vegetable oils or a mixture of vegetable oils and dairy fats. 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 comprises at least 0.6% by weight, preferably at least 1.2% by weight, more preferably at least 1.5% by weight of the fatty acyl groups consisting of 4 carbon atoms; and / or at least 10% of the fatty acyl groups present in the lipid fraction comprise a palmitoyl group (CH3(CH2) 14 C(O)) and at least 30% of the palmitoyl groups are esterified at the sn-2 position of the triglyceride, based on total palmitoyl groups.

[0033] Vegetable oils suitable for human consumption are well known in the art. The milk fat suitable for the composition of the present invention is preferably ruminant milk fat derived 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.

[0034] In one embodiment, the amount of milk fat in the lipid fraction of the compositions of the present invention is at least 20% by weight of the lipid fraction, preferably at least 30% by weight, more preferably at least 40% by weight, and even more preferably at least 60% by weight. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 shows the percentage of inhibition of in vitro RSV infection as a function of concentration of various phospholipid-enriched milk fractions. [Figure 2] FIG. 2 shows the percentage of in vitro RSV infection inhibition as a function of phospholipid concentration for two phospholipid-enriched milk fractions: whey protein phospholipid concentrate and beta-serum. [Figure 3] FIG. 3 shows the percentage of in vitro RSV infection inhibition as a function of protein concentration for two whey protein compositions: a phospholipid-enriched whey protein concentrate and a regular whey protein isolate. DETAILED DESCRIPTION OF THE INVENTION

[0036] Example In vitro experiments were performed to investigate the ability of various commercially available phospholipid-enriched milk fractions to inhibit respiratory syncytial virus strain RSV-A2 infection in the A549 (ATCC CRM-CCL-185) cell line.

[0037] Various whey protein phospholipid concentrates were used: Vivinal® MFGM ex-FrieslandCampina (phospholipid content on dry matter basis: 7.4 wt.%) and three commercially available competitive products: A (phospholipid content: 7.5 wt.%), B (phospholipid content: 5.7 wt.%) and C (phospholipid content: 6.4 wt.%).

[0038] Additionally, beta-serum was used as a phospholipid-enriched milk fraction.

[0039] A commercially available whey protein isolate (WPI) was used as a comparison.

[0040] The phospholipid-enriched milk fraction and comparative WPI were solubilized in complete F12 medium (F12 Nut mix + glutamax + 0.3% BSA, 0.1% FCS, penicillin / streptomycin, and 20 mM HEPES). After solubilizing the samples at 50°C for 30 minutes with frequent vortexing, the samples shown in Figure 1 were centrifuged at 3220g for 30 minutes and filter-sterilized through a 0.2uM PES filter. The samples shown in Figures 2 and 3 were not centrifuged or filter-sterilized after solubilization.

[0041] Serial dilutions of samples were preincubated with RSV-A2 for 1 h. A549 cells were subsequently incubated with component / virus premix (2 h) and component / virus-free infection medium (22 h), all incubation steps were performed at 37°C, and then infectivity was determined by immunostaining.

[0042] Figure 1 shows the percentage of RSV infection inhibition compared to the control (set at 100%) as a function of product concentration. This figure clearly demonstrates that various commercially available whey protein phospholipid concentrates can inhibit RSV infection.

[0043] FIG. 2 shows the percentage of RSV infection inhibition compared to the control (set at 100%) as a function of phospholipid content of whey protein phospholipid concentrate ("Whey-derived MFGM") and beta-serum ("Beta-serum MFGM").

[0044] Figure 3 shows the percentage of RSV infection inhibition compared to the control (set at 100%) as a function of product concentration. This figure clearly shows that regular WPI, in contrast to whey protein phospholipid concentrate, is unable to inhibit RSV infection.

Claims

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

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

3. 3. A phospholipid-enriched 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 to 20 wt.-%, most preferably in the range of 7 to 15 wt.-%, based on dry matter.

4. 4. A phospholipid-enriched milk fraction for use according to any one of claims 1 to 3, wherein the sphingomyelin content is at least 20% by weight, preferably in the range of 20 to 35% by weight, most preferably in the range of 20 to 30% by weight, based on total phospholipids.

5. 5. A phospholipid-enriched milk fraction for use according to any one of claims 1 to 4, 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 selected from the group consisting of whey protein phospholipid concentrate and beta-serum, most preferably whey protein phospholipid concentrate.

6. A phospholipid-enriched milk fraction for use according to any one of claims 1 to 5, wherein the phospholipid-enriched milk fraction is in liquid or dry form.

7. A phospholipid-enriched milk fraction for use according to any one of claims 1 to 6, wherein the phospholipid-enriched milk fraction is used as part of an infant formula.

8. 8. The phospholipid-enriched milk fraction for use according to claim 7, wherein the phospholipid-enriched milk fraction is present in the infant formula at a concentration of 0.1 to 10% by weight, preferably 0.5 to 8% by weight, even more preferably 1 to 7% by weight, most preferably 2 to 5% by weight, based on dry weight.

9. 9. A phospholipid-enriched milk fraction for use according to any one of claims 1 to 8, wherein the phospholipid-enriched milk fraction is administered to a toddler or infant, preferably an infant, most preferably an infant up to 6 months of age.

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