COMPOSITIONS AND METHODS WITH A PROBIOTIC AND A N-3 FATTY ACID FOR THE PREVENTION OR TREATMENT OF MASTITIS

Abstract

The invention provide a mix or a nutritional compostion comprising a probiotic Lactobacillus Fermentum CECT-5716 and a n-3 fatty acid selected from the group consisting of docosahexaenoic acid (DHA) and 18:3 n-3 octadecatrienoic acid (alpha-linolenic acid); and optional a further nutrient or mineral selected from the group consisting of iron, manganese and magnesium, or combination of two of more thereof; for use in treating or preventing mastitis in a subject, preferably a pregnant or lactating woman.

Claims

1. A method for use in treating or preventing mastitis in a subject comprising administering to the subject a mix comprising n-3 fatty acid and a probiotic Lactobacillus Fermentum CECT-5716, wherein the fatty acid is selected from the group consisting of docosahexaenoic acid (DHA) and 18:3 n-3 octadecatrienoic acid (alpha-linolenic acid).

2. The method of claim 1 wherein the mix further comprises a protein selected from the group consisting of alpha-lactalbumin, lactoferrin and albumin for use in treating or preventing mastitis in a subject.

3. The method of claim 1 wherein the mix further comprises a mineral selected from the group consisting of iron, manganese, magnesium, and a combination of two of more thereof.

4. The method of claim 1 wherein the mix comprises administering vitamin E.

5. The method of claim 1 wherein the mix further comprises phosphatidylcholine and/or lecithin.

6. The method of claim 1, wherein mix is in the form of a nutritional composition.

7. The method of claim 1, wherein the mastitis is sub-clinical mastitis or clinical mastitis.

8. The method of claim 1, wherein the subject is at risk of suffering from sub-clinical mastitis or clinical mastitis.

9. The method of claim 1, wherein the treatment or prevention increases the probability of initiating and/or continuing breastfeeding by the subject; and/or increases the probability of the subject exclusively breast-feeding her infant and/or increases the duration of breastfeeding of the subject.

10. The method of claim 1, wherein the subject is able to breast-feed for at least 4 months.

11. The method of claim 1, wherein the treatment or prevention increases the quality and/or quantity of the subject's breast milk.

12. (canceled)

Description

DESCRIPTION OF THE DRAWINGS

[0095] FIG. 1

[0096] Comparison of iron concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0097] FIG. 2

[0098] Comparison of manganese concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0099] FIG. 3

[0100] Comparison of magnesium concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0101] FIG. 4

[0102] Comparison of copper concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0103] FIG. 5

[0104] Comparison of zinc concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0105] FIG. 6

[0106] Comparison of selenium concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0107] FIG. 7

[0108] Comparison of calcium concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0109] FIG. 8

[0110] Comparison of phosphorous concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0111] FIG. 9

[0112] Comparison of docosahexaenoic acid (DHA) concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0113] FIG. 10

[0114] Comparison of 18:3 n-3 octadecatrienoic acid (alpha-linolenic acid) concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0115] FIG. 11

[0116] Comparison of alpha-lactalbumin concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0117] FIG. 12

[0118] Comparison of lactoferrin concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

[0119] FIG. 13

[0120] Comparison of albumin concentrations between the milk of mothers with sub-clinical mastitis and the milk of normal mothers at 6 time-points post-partum (V1=0-3 d; V2=17±3 d; V3=30±3 d; V4=60±5 d; V5=90±5 d; and V6=120±5 d).

DETAILED DESCRIPTION OF THE INVENTION

[0121] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including” or “includes”; or “containing” or “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms “comprising”, “comprises” and “comprised of” also include the term “consisting of”.

Mastitis

[0122] Mastitis is an inflammation of the mammary gland tissue, which can be classified as sub-clinical or clinical depending on the degree of inflammation.

[0123] Clinical mastitis is a form of mastitis associated with reduced milk secretion, visible signs of inflammation of the breast and, changes in the appearance of milk, which may be accompanied by systemic signs. Sub-clinical mastitis is a form of mastitis characterised by reduced milk secretion and a high milk bacterial count in the absence of evident inflammatory changes, including pain (Fernandez, L. et al. (2014) Beneficial Microbes 5: 169-183).

[0124] Concentrations of sodium and potassium in milk are commonly used in the diagnosis of sub-clinical mastitis. For example, a number of studies have found that Na:K ratios in the milk of healthy women at 1 month post-partum generally average 0.6 or less. This corresponds to average human milk sodium and potassium concentrations ranging between 5-6 mmol/L and 13-14 mmol/L, respectively. In contrast, the mean sodium concentration in mastitis milk is greater than 16 mmol/L. Accordingly, a Na:K ratio of less than or equal to 0.6 is considered to be normal; a Na:K ratio of greater than 0.6 but less than or equal to 1.0 is considered to be moderately raised; and a Na:K ratio of greater than 1.0 is considered to be greatly raised.

[0125] Mastitis may occur at any time during lactation and is experienced by up to about 33% of lactating women. Occurrence is particularly prevalent during the second and third week post-partum.

[0126] Sub-clinical mastitis (SCM) is an inflammatory condition of the lactating breast that is understood to be caused by milk stasis and/or infection, and has been associated with elevated risk of lactation failure and poor infant weight gain.

[0127] Staphylococcus infections, in particular S. aureus and S. epidermidis infections, are understood to be a primary cause of mastitis.

[0128] Mastitis can result in curtailment or even lack of initiation of breast-feeding of an infant. Furthermore, the composition of breast milk may change during mastitis, for example increasing in content of sodium and inflammatory mediators, which may adversely affect the nutrition provided to the infant.

Mineral

[0129] In one aspect, the invention provides a mineral selected from the group consisting of iron, manganese, magnesium, and a combination of two of more thereof, for use in treating or preventing mastitis in a subject.

[0130] In one embodiment, the mineral is in combination with one or more further minerals selected from the group consisting of copper, zinc, selenium, calcium and phosphorus.

[0131] The minerals may be used in any form suitable for ingestion by animals, preferably humans (e.g. are non-toxic). The minerals may be used, for example in compositions such as nutritional compositions, in any appropriate amount. The skilled person will be able to determine appropriate amounts depending on the desired dosage of the mineral. Dosages may depend on factors such as the age, size and health status of the woman to whom they are administered, on her lifestyle, as well as on her genetic heritage. Dosages may be in line with the recommended daily intakes (RDA) developed by organisations such as the Food and Nutrition Board of the National Academy of Sciences.

[0132] The skilled person can readily determine an appropriate dose of one of the agents of the invention to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage that will be most suitable for an individual subject and it will depend on a variety of factors including the activity of the specific agent employed, the metabolic stability and length of action of that agent, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition and the individual undergoing therapy. There can of course be individual instances where higher or lower dosage ranges are merited.

[0133] In one embodiment, the dosage of iron is about 2.7-45, 5-25 or 9-10 mg/day. A dosage of about 9-10 mg/day may be preferred for breast-feeding women.

[0134] In another embodiment, the dosage of iron is about 30-60 mg/day. A dosage of about 30-60 mg/day may be preferred for pregnant women.

[0135] In one embodiment, the dosage of iron is at least 9.1 mg/day. In a further embodiment, the dosage of iron is at least 9.5 mg/day. In a still further embodiment, the dosage of iron is ranging from 9.5 to 60 mg/day, for example from 9.5 to 50 mg/day, for example 9.5 to 40 mg/day.

[0136] In one embodiment, the dosage of iron for a lactating woman is at least 9.1 mg/day. In a further embodiment, the dosage of iron is at least 9.5 mg/day. In a still further embodiment, the dosage of iron is ranging from 9.5 to 60 mg/day, for example from 9.5 to 30 mg/day, for example 9.5 to 20 mg/day.

[0137] In one embodiment, the dosage of iron is at least 11.6 mg/day. In a further embodiment, the dosage of iron is at least 12 mg/day. In a still further embodiment, the dosage of iron is ranging from 12 to 60 mg/day, for example from 12 to 50 mg/day, for example 12 to 40 mg/day.

[0138] In one embodiment, the dosage of iron for a lactating woman is at least 11.6 mg/day. In a further embodiment, the dosage of iron is at least 12 mg/day. In a still further embodiment, the dosage of iron is ranging from 12 to 60 mg/day, for example from 12 to 30 mg/day, for example 12 to 20 mg/day.

[0139] The iron may be comprised in any form suitable for ingestion by a woman such as a pregnant woman, a woman trying to conceive or a lactating woman. For example, iron may be comprised in the form of iron sulfate, iron citrate, iron choline citrate, iron ammonium citrate, iron chloride, iron fumarate, iron gluconate, iron pyrophosphate or a mixture thereof.

[0140] In one embodiment, the dosage of manganese is about 1.8-11, 2-3 or 2.5-2.7 mg/day. A dosage of about 2.5-2.7 mg/day may be preferred for breast-feeding women. A dosage of about 1.9-2.1 mg/day may be preferred for pregnant women.

[0141] In one embodiment, the dosage of manganese is at least 2.1 mg/day. In a further embodiment, the dosage of manganese is at least 2.3 mg/day. In a still further embodiment, the dosage of managese is ranging from 2.1 to 4 mg/day, for example from 2.3 to 3.5 mg/day.

[0142] In one embodiment, the dosage of manganese for a lactating woman is at least 2.1 mg/day. In a further embodiment, the dosage of manganese is at least 2.3 mg/day. In a still further embodiment, the dosage of managese is ranging from 2.1 to 4 mg/day, for example from 2.3 to 3.5 mg/day.

[0143] In one embodiment, the dosage of manganese is at least 2.6 mg/day. In a further embodiment, the dosage of manganese is at least 3.0 mg/day. In a still further embodiment, the dosage of managese is ranging from 2.6 to 4 mg/day, for example from 3.0 to 3.5 mg/day.

[0144] In one embodiment, the dosage of manganese for a lactating woman is at least 2.6 mg/day. In a further embodiment, the dosage of manganese is at least 3.0 mg/day. In a still further embodiment, the dosage of managese is ranging from 2.6 to 4 mg/day, for example from 3.0 to 3.5 mg/day.

[0145] The manganese may be comprised in any form suitable for ingestion by a woman such as a pregnant woman, a woman trying to conceive or a lactating woman. For example, manganese may be comprised in the form of manganese gluconate, manganese sulfate, manganese ascorbate, manganese amino acid chelates, manganese aspartate, manganese picolinate, manganese fumarate, manganese malate, manganese succinate, manganese citrate or a mixture thereof.

[0146] In one embodiment, the dosage of magnesium is about 35-350, 200-350 or 300-350 mg/day. A dosage of about 300-350 mg/day may be preferred for breast-feeding women.

[0147] In one embodiment, the dosage of magnesium is at least 270 mg/day. In a further embodiment, the dosage of magnesium is at least 300 mg/day. In a still further embodiment, the dosage of magnesium is ranging from 270 to 350 mg/day, for example from 300 to 350 mg/day.

[0148] In one embodiment, the dosage of magnesium for a lactating woman is at least 270 mg/day. In a further embodiment, the dosage of magnesium is at least 300 mg/day. In a still further embodiment, the dosage of magnesium is ranging from 270 to 350 mg/day, for example from 300 to 350 mg/day.

[0149] In one embodiment, the dosage of magnesium is at least 302 mg/day. In a further embodiment, the dosage of magnesium is at least 305 mg/day. In a still further embodiment, the dosage of magnesium is ranging from 302 to 350 mg/day, for example from 305 to 350 mg/day.

[0150] In one embodiment, the dosage of magnesium for a lactating woman is at least 302 mg/day. In a further embodiment, the dosage of magnesium is at least 305 mg/day. In a still further embodiment, the dosage of magnesium is ranging from 302 to 350 mg/day, for example from 305 to 350 mg/day.

[0151] The magnesium may be comprised in any form suitable for ingestion by a woman such as a pregnant woman, a woman trying to conceive or a lactating woman. For example, magnesium may be comprised in the form of magnesium chloride, magnesium citrate, magnesium sulfate, magnesium oxide, magnesium hydroxide, magnesium amino acid chelates (e.g. chelates of glycinate, lysinate, orotate, taurate, aspartate, threonate and/or malate) or a mixture thereof.

[0152] In one embodiment, the dosage of copper is about 0.1-10, 0.1-2 or 0.5-1.5 mg/day.

[0153] In one embodiment, the dosage of copper is at least 1.250 mg/day. In a further embodiment, the dosage of copper is at least 1.30 mg/day. In a still further embodiment, the dosage of copper is ranging from 1.250 to 10 mg/day, for example from 1.30 to 2 mg/day, for example from 1.30 to 1.50 mg/day.

[0154] In one embodiment, the dosage of copper for a lactating woman is at least 1.250 mg/day. In a further embodiment, the dosage of copper is at least 1.30 mg/day. In a still further embodiment, the dosage of copper is ranging from 1.250 to 10 mg/day, for example from 1.30 to 2 mg/day, for example from 1.30 to 1.50 mg/day.

[0155] In one embodiment, the dosage of copper is at least 1.46 mg/day. In a further embodiment, the dosage of copper is at least 1.48 mg/day. In a still further embodiment, the dosage of copper is ranging from 1.46 to 10 mg/day, for example from 1.46 to 2 mg/day, for example from 1.48 to 1.50 mg/day.

[0156] In one embodiment, the dosage of copper for a lactating woman is at least 1.46 mg/day. In a further embodiment, the dosage of copper is at least 1.48 mg/day. In a still further embodiment, the dosage of copper is ranging from 1.46 to 10 mg/day, for example from 1.46 to 2 mg/day, for example from 1.48 to 1.50 mg/day.

[0157] The copper may be comprised in any form suitable for ingestion by a woman such as a pregnant woman, a woman trying to conceive or a lactating woman. For example, copper may be comprised in the form of copper oxide, copper chloride, copper gluconate, copper sulfate, copper amino acid chelates or a mixture thereof.

[0158] In one embodiment, the dosage of zinc may be about 5-40, 7-13 or 9.5-12 mg/day.

[0159] In one embodiment, the dosage of zinc is at least 9.5 mg/day. In a further embodiment, the dosage of zinc is at least 10 mg/day. In a still further embodiment, the dosage of zinc is ranging from 9.5 to 12 mg/day, for example from 9.5 to 11.5 mg/day, for example from 10 to 11 mg/day.

[0160] In one embodiment, the dosage of zinc for a lactating woman is at least 9.5 mg/day. In a further embodiment, the dosage of zinc is at least 10 mg/day. In a still further embodiment, the dosage of zinc is ranging from 9.5 to 12 mg/day, for example from 9.5 to 11.5 mg/day, for example from 10 to 11 mg/day.

[0161] The zinc may be comprised in any form suitable for ingestion by a woman such as a pregnant woman, a woman trying to conceive or a lactating woman. For example, zinc may be comprised in the form of zinc acetate, zinc chloride, zinc citrate, zinc gluconate, zinc lactate, zinc oxide, zinc sulfate, zinc carbonate or a mixture thereof.

[0162] In one embodiment, the dosage of selenium may be about 20-400, 25-250, 26-85 or 60-70 μg/day.

[0163] In one embodiment, the dosage of selenium is at least 131 μg/day. In a further embodiment, the dosage of selenium is at least 135 μg/day. In a still further embodiment, the dosage of selenium is ranging from 131 to 400 μg/day, for example from 140 to 250 μg/day, for example from 150 to 200 μg/day.

[0164] In one embodiment, the dosage of selenium for a lactating woman is at least 131 μg/day. In a further embodiment, the dosage of selenium is at least 135 μg/day. In a still further embodiment, the dosage of selenium is ranging from 131 to 400 μg/day, for example from 140 to 250 μg/day, for example from 150 to 200 μg/day.

[0165] The selenium may be comprised in any form suitable for ingestion by a woman such as a pregnant woman, a woman trying to conceive or a lactating woman. For example, selenium may be comprised in the form of sodium selenite, sodium hydrogen selenite or a mixture thereof.

[0166] In one embodiment, the dosage of calcium is about 100-2500, 500-2000 or 1000-1500 mg/day.

[0167] In one embodiment, the dosage of calcium is at least 750 mg/day. In a further embodiment, the dosage of calcium is at least 850 mg/day. In a still further embodiment, the dosage of calcium is ranging from 750 to 2500 mg/day, for example from 850 to 2000 mg/day, for example from 900 to 1500 mg/day.

[0168] In one embodiment, the dosage of calcium for a lactating woman is at least 750 mg/day. In a further embodiment, the dosage of calcium is at least 850 mg/day. In a still further embodiment, the dosage of calcium is ranging from 750 to 2500 mg/day, for example from 850 to 2000 mg/day, for example from 900 to 1500 mg/day.

[0169] In one embodiment, the dosage of calcium is at least 860 mg/day. In a further embodiment, the dosage of calcium is at least 900 mg/day. In a still further embodiment, the dosage of calcium is ranging from 860 to 2500 mg/day, for example from 900 to 2000 mg/day, for example from 900 to 1500 mg/day.

[0170] In one embodiment, the dosage of calcium for a lactating woman is at least 860 mg/day. In a further embodiment, the dosage of calcium is at least 900 mg/day. In a still further embodiment, the dosage of calcium is ranging from 860 to 2500 mg/day, for example from 900 to 2000 mg/day, for example from 900 to 1500 mg/day.

[0171] The calcium may be comprised in any form suitable for ingestion by a woman such as a pregnant woman, a woman trying to conceive or a lactating woman. For example, calcium may be comprised in the form of calcium citrate, calcium carbonate or a mixture thereof.

[0172] In one embodiment, the dosage of phosphorous is about 70-4000, 100-1500 or 250-1250 mg/day.

[0173] In one embodiment, the dosage of phosphorus is at least 1275 mg/day. In a further embodiment, the dosage of phosphorus is at least 1300 mg/day. In a still further embodiment, the dosage of phosphorus is ranging from 1300 to 4000 mg/day, for example from 1300 to 2000 mg/day, for example from 1300 to 1500 mg/day.

[0174] In one embodiment, the dosage of phosphorus for a lactating woman is at least 1275 mg/day. In a further embodiment, the dosage of phosphorus is at least 1300 mg/day. In a still further embodiment, the dosage of phosphorus is ranging from 1300 to 4000 mg/day, for example from 1300 to 2000 mg/day, for example from 1300 to 1500 mg/day.

[0175] In one embodiment, the dosage of phosphorus is at least 1250 mg/day. In a further embodiment, the dosage of phosphorus is at least 1275 mg/day. In a still further embodiment, the dosage of phosphorus is ranging from 1250 to 4000 mg/day, for example from 1275 to 2000 mg/day, for example from 1300 to 1500 mg/day.

[0176] In one embodiment, the dosage of phosphorus for a lactating woman is at least 1250 mg/day. In a further embodiment, the dosage of phosphorus is at least 1275 mg/day. In a still further embodiment, the dosage of phosphorus is ranging from 1250 to 4000 mg/day, for example from 1275 to 2000 mg/day, for example from 1300 to 1500 mg/day.

[0177] The phosphorous may be comprised in any form suitable for ingestion by a woman such as a pregnant woman, a woman trying to conceive or a lactating woman. For example, phosphorous may be comprised in the form of sodium phosphate.

[0178] In a further embodiment of the present invention, the dosage of iron is ranging from 9.5 to 60 mg/day, for example from 9.5 to 30 mg/day, for example 9.5 to 20 mg/day; the dosage of managese is ranging from 2.1 to 4 mg/day, for example from 2.3 to 3.5 mg/day; the dosage of magnesium is ranging from 270 to 350 mg/day, for example from 300 to 350 mg/day; the dosage of copper is ranging from 1.250 to 10 mg/day, for example from 1.30 to 2 mg/day, for example from 1.30 to 1.50 mg/day; the dosage of calcium is ranging from 750 to 2500 mg/day, for example from 850 to 2000 mg/day, for example from 900 to 1500 mg/day; and the dosage of phosphorus is ranging from 1300 to 4000 mg/day, for example from 1300 to 2000 mg/day, for example from 1300 to 1500 mg/day. In such embodiment, the subject receiving the mineral combination or composition comprising it is for example a lactating woman.

Vitamins, Fatty Acids and Proteins

[0179] The minerals disclosed herein may be used in combination with further agents, in particular vitamin E, n-3 fatty acids (preferably selected from the group consisting of docosahexaenoic acid (DHA) and 18:3 n-3 octadecatrienoic acid (alpha-linolenic acid)) and/or a protein selected from the group consisting of alpha-lactalbumin, lactoferrin and albumin.

[0180] In another aspect, the invention provides an n-3 fatty acid for use in treating or preventing mastitis in a subject, preferably wherein the fatty acid is selected from the group consisting of docosahexaenoic acid (DHA) and 18:3 n-3 octadecatrienoic acid (alpha-linolenic acid). In one embodiment, the n-3 fatty acid is DHA.

[0181] In one embodiment the n-3 fatty acid (eg. DHA) of the is present in an amount able to deliver an amount of 20 to 1000 mg n-3 fatty acid (eg. DHA) per day, preferably 50 to 500, more preferably 100 to 200 mg/n-3 fatty acid (eg. DHA) per day. In one embodiment the n-3 fatty acid (eg. DHA) amount in the composition of the invention is from 1 to 800 mg/g of composition, alternatively 5 to 400 mg, or 10 to 100 mg. In one embodiment where the compositions of the invention is a complete nutritional composition, the n-3 fatty acid (eg. DHA) amount may be from 5 to 200 mg/g of composition, alternatively 10 to 100 mg. In one embodiment the amount of n-3 fatty acid (eg. DHA) is from 1 to 20 mg/100 kcal of composition, alternatively from 2 to 10 mg or 2 to 6 mg.

[0182] In another aspect, the invention provides a protein selected from the group consisting of alpha-lactalbumin, lactoferrin and albumin for use in treating or preventing mastitis in a subject.

[0183] Such agents (vitamin E, n-3 fatty acids, alpha-lactalbumin, lactoferrin and albumin) may be used in any form suitable for ingestion by animals, preferably humans (e.g. are non-toxic). The agents may be used, for example in compositions such as nutritional compositions, in any appropriate amount. The skilled person will be able to determine appropriate amounts depending on the desired dosage of the agent. Dosages may depend on factors such as the age, size and health status of the woman to whom they are administered, on her lifestyle, as well as on her genetic heritage. Dosages may be in line with the recommended daily intakes (RDA) developed by organisations such as the Food and Nutrition Board of the National Academy of Sciences.

[0184] In one embodiment, the dosage of vitamin E is about 11-1000, 7.5-300 or 11-19 mg/day.

[0185] In one embodiment, the dosage of vitamin E is at least 8.1 mg/day. In a further embodiment, the dosage of phosphorus is at least 8.5 mg/day. In a still further embodiment, the dosage of phosphorus is ranging from 8.1 to 300 mg/day, for example from 8.5 to 19 mg/day, for example from 9.5 to 19 mg/day.

[0186] In one embodiment, the dosage of vitamin E for a lactating woman is at least 8.1 mg/day. In a further embodiment, the dosage of phosphorus is at least 8.5 mg/day. In a still further embodiment, the dosage of phosphorus is ranging from 8.1 to 300 mg/day, for example from 8.5 to 19 mg/day, for example from 9.5 to 19 mg/day.

[0187] The vitamin E may be, for example, in the form of a tocopherol or a mixture of different tocopherols. For example, the vitamin E may be alpha-tocopherol, gamma-tocopherol or a mixture of alpha-tocopherol and gamma-tocopherol.

[0188] The vitamin E may be comprised in any form suitable for ingestion by a woman such as a pregnant woman, a woman trying to conceive or a lactating woman, for example, alpha-tocopherol and/or gamma-tocopherol, and/or may be comprised in the form of tocopherol concentrate mix, L-vitamin E, D,L-vitamin E, tocopherols mixed pure, D,L-alpha-tocopherol, D,L-alpha tocopheryl acetate, tocopherol rich extract or a mixture thereof.

[0189] In one embodiment, the vitamin E is alpha-tocopherol.

[0190] In one embodiment, the dosage of docosahexaenoic acid (DHA) is less than or equal to 1000 mg/day, preferably about 500-1000 mg/day.

[0191] In one embodiment, the dosage of alpha-linolenic acid is less than or equal to 2000 mg/day, preferably about 500-1000 mg/day.

[0192] In one embodiment, the dosage of phosphatidylcholine is about 1500-1750 mg/day.

[0193] In one embodiment, the dosage of lecithin is about 1500-1750 mg/day.

[0194] In one embodiment, the dosage of lactoferrin is about 5-500 mg/day, preferably about 100-500 mg/day.

[0195] With respect to dosages defined herein as amounts per daily dose the amount of nutrient in a composition administered to the subject may vary depending upon whether it is intended to be consumed once a day, or more or less frequently.

Methods of Treatment

[0196] The term “combination”, or terms “in combination”, “used in combination with” or “combined preparation” as used herein may refer to the combined administration of two or more agents simultaneously, sequentially or separately.

[0197] The term “simultaneous” as used herein means that the agents are administered concurrently, i.e. at the same time.

[0198] The term “sequential” as used herein means that the agents are administered one after the other.

[0199] The term “separate” as used herein means that the agents are administered independently of each other but within a time interval that allows the agents to show a combined, preferably synergistic, effect. Thus, administration “separately” may permit one agent to be administered, for example, within 1 minute, 5 minutes or 10 minutes after the other.

[0200] It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment. The treatment of mammals, particularly humans, is preferred. Both human and veterinary treatments are within the scope of the invention.

[0201] The minerals, fatty acids, proteins, combinations and compositions disclosed herein may be administered to a woman desiring to get pregnant, to a pregnant woman and/or to a lactating woman.

[0202] If administration is to a woman desiring to get pregnant, administration may be for example during at least 1, 2, 3 or 4 months preceding the pregnancy or desired pregnancy.

[0203] If administration is to a pregnant woman, administration may be for example for at least 4, at least 8, at least 12, at least 16, at least 20, at least 24, at least 28 or at least 36 weeks during pregnancy. As the nutritional requirements increase in the second and third trimester of pregnancy, it may be particularly beneficial if administration is throughout the second and/or third trimester of pregnancy.

[0204] Administration pre-pregnancy and/or during pregnancy may enable a woman to build up a store of one or more of the minerals, fatty acids and/or proteins before lactation.

[0205] If administration is to a lactating woman, administration may be for example for any part of the lactation period for example up to 2 years, up to 1 year, up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 months post birth.

[0206] In one embodiment, administration is to a woman desiring to get pregnant, to a pregnant woman and/or to a lactating woman.

Composition

[0207] The term “maternal nutritional composition” as used herein refers to any composition that has been specifically manufactured for consumption by a pregnant woman, a woman trying to conceive or a lactating woman, or a composition that is specifically marketed at pregnant women, women trying to conceive or lactating (e.g. breast-feeding) women.

[0208] The maternal nutritional composition may be, for example, a food product, a functional food product, a drink (beverage), a dairy product or dairy substitute product, a pharmaceutical formulation or a supplement.

[0209] The “mix” as used in the present document refers to the concomitant presence of relevant ingredients or molecules. The mix is not limited to the actual mixing of the relevant ingredients or molecules. For example, DHA and the probiotic used in the invention can be present in a nutritional composition of the invention (i.e. the “mix” is present) without having been actually mixed together separately at any stage of the manufacturing of the composition.

[0210] The term “dairy product” as used herein refers to food products produced from animals such as cows, goats, sheep, yaks, horses, camels and other mammals. Examples of dairy products are low-fat milk (e.g. 0.1%, 0.5% or 1.5% fat milk), fat-free milk, milk powder, whole milk, whole milk products, butter, buttermilk, buttermilk products, skim milk, lactose-free products, high milk-fat products, condensed milk, créme fraiche, cheese, ice cream and confectionery products, probiotic drinks or probiotic yoghurt-type drinks. A dairy substitute product may be a soya, almond or vegetable-based dairy substitute, e.g. a milk or yoghurt substitute.

[0211] The term “pharmaceutical formulation” as used herein refers to a composition comprising at least one pharmaceutically-active agent, chemical substance or drug. The pharmaceutical formulation may be in solid or liquid form and can comprise at least one additional active agent, carrier, vehicle, excipient or auxiliary agent identifiable by the skilled person. The pharmaceutical formulation may be in the form of a tablet, capsule, granules, powder, liquid or syrup.

[0212] The term “beverage product” as used herein refers to a nutritional product in liquid or semi-liquid form that may be safely consumed by an individual. The beverage product may be a water-based product, such as a product in which the agents of the invention are dissolved or suspended in water.

[0213] The term “food product” as used herein refers to any kind of product that may be safely consumed by a woman, in particular a pregnant woman, a woman trying to conceive or a lactating (e.g. breast-feeding) woman. Said food product may be in solid, semi-solid or liquid form and may comprise one or more nutrients, foods or nutritional supplements. For example, the food product may further comprise one or more of the following nutrients and micronutrients: a source of protein, a source of lipid, a source of carbohydrate, vitamins and minerals. The composition may also contain anti-oxidants, stabilisers (when provided in solid form) or emulsifiers (when provided in liquid form).

[0214] The term “functional food product” as used herein refers to a food product providing an additional health-promoting or disease-preventing function to the individual. Food products and functional food products include, for example, cereal-based products, yoghurts or other milk-derived products and bars.

[0215] The term “supplement” as used herein refers to a nutritional product that provides nutrients (e.g. vitamins and/or minerals) to an individual that may otherwise not be consumed in sufficient quantities by said individual. Supplements may be, for example, provided in the form of a pill, a tablet, a lozenge, a chewy capsule or tablet, a capsule, or a powder supplement that can be, for example, dissolved in water or milk, or sprinkled on food. Supplements typically provide selected nutrients without providing a significant portion of the overall nutritional needs of a subject. Typically supplements do not represent more than 0.1%, 1%, 5%, 10% or 20% of the daily energy need of a subject. In the context of the present invention the subject may be, for example, a woman trying to get pregnant, a pregnant woman and/or a lactating woman.

[0216] The term “pregnancy supplement” as used herein refers to a supplement that is specifically formulated for administration to a woman who is trying to conceive and/or to a woman who is pregnant, or marketed towards a woman who is trying to conceive and/or a woman who is pregnant.

[0217] The term “lactation supplement” as used herein refers to a supplement that is specifically formulated for administration to a woman who is lactating, or marketed toward a woman who is lactating. Consumption of lactation supplements may be advised to commence during pregnancy.

[0218] The compositions of the invention may also comprise ingredients commonly used in maternal nutritional compositions. Non-limiting examples of such ingredients include: other probiotics, lipids, carbohydrates, pharmaceutically-active agents and conventional additives, such as anti-oxidants, stabilisers, emulsifiers, acidulants, thickeners, buffers or agents for pH adjustment, chelating agents, colourants, excipients, flavour agents, osmotic agents, pharmaceutically-acceptable carriers, preservatives, sugars, sweeteners, texturisers, emulsifiers and water.

[0219] It may also be beneficial if the compositions of the invention comprise probiotics. Probiotics may help nutrients pass through the gut.

[0220] The term “probiotic” as used herein refers to live probiotic bacteria, non-replicating probiotic bacteria, dead probiotic bacteria, non-viable probiotic bacteria, fragments of probiotic bacteria, such as DNA, metabolites of probiotic bacteria, cytoplasmic compounds of probiotic bacteria, cell wall materials of probiotic bacteria, culture supernatants of probiotic bacteria, and combinations of any of the foregoing.

[0221] The probiotic may be live probiotic bacteria, non-replicating probiotic bacteria, dead probiotic bacteria, non-viable probiotic bacteria and any combination thereof.

[0222] The mix and/or composition of the invention comprises a particular Lactobacillus Fermentum strain, namely Lactobacillus Fermentum CECT-5716. The strain Lactobacillus Fermentum CECT5716 is known and its genome has been sequenced (see: “Complete Genome Sequence of Lactobacillus Fermentum CECT 5716, a Probiotic Strain Isolated from Human Milk”—JOURNAL OF BACTERIOLOGY, September 2010, p. 4800, vol. 192, N° 18, by Esther Jiménez, Susana Langa, Virginia Martin, Rebeca Arroyo, Rocio Martin, Leónides Fernández, and Juan M. Rodriguez).

[0223] The strain has been deposited under the reference CECT-5716 at the Coleccion Espanola de Cultivos Tipo, under the Budapest treaty (address: c/Catedrático Agustin Escardino, 9. 46980 Paterna (Valencia), Spain). The strain is the subject of the granted patent EP1565547B1 which comprises claims to the strain L. fermentum CECT-5716 itself (applicant: Biosearch S.A., Camino de Purchil 66, 18004 Granada/ES). L. fermentum CECT-5716 is commercialized by Biosearch S.A., in particular under the tradename/trademark “Hereditum LC-40®”. The strain L. fermentum CECT-5716 has also been fully described in a request for a US GRAS approval by the US Food & Drug Administration (approval for ingredients “Generally Recognized as Safe”) under reference number GRN-531, publically available under the following link:

[0224] https://www.accessdata.fda.gov/scripts/fdcc/?set=GRASNotices&id=531&sort=GRN No&or der=DESC&startrow=1&type=basic&search=531.

[0225] The strain L. fermentum CECT-5716 has been well studied, and has in particular been linked to a reduction in the pathogenic load and to a positive effect on mastitis. See in particular the following scientific articles: [0226] Study protocol: evaluation of the probiotic Lactobacillus Fermentum CECT5716 for the prevention of mastitis in breastfeeding women: a randomised controlled trial; Bond et al.; BMC Pregnancy and Childbirth (2017) 17:148 DOI 10.1186/s12884-017-1330-8 [0227] Treatment of Infectious Mastitis during Lactation: Antibiotics versus Oral Administration of Lactobacilli Isolated from Breast Milk, by Rebeca Arroyo, Virginia Martin, Antonio Maldonado, Esther Jiménez, Leónides Fernández, Juan Miguel Rodriquez; Clinical Infectious Diseases, Volume 50, Issue 12, 15 Jun. 2010, Pages 1551-1558, https://doi.org/10.1086/652763.

[0228] The effect on mastitis has however been sometimes challenged (see in particular the scientific opinion by an EFSA panel dated Jun. 27, 2007—“Lactobacillus Fermentum CECT-5716 and a reduction of the Staphylococcus load in breast milk which reduces the risk of infectious mastitis: evaluation of a health claim pursuant to Article 14 of Regulation (EC) No 1924/2006”, EJ EFSA Journal, doi: 10.2903/j.efsa.2017.4917). The inventors may hypothesize that the evidences in some of the studies may have been partly or entirely shaded by the non controlled intake of n-3 fatty acids, namely docosahexaenoic acid (DHA) and/or 18:3 n-3 octadecatrienoic acid (alpha-linolenic acid). On contrary the present invention builds on the synergy between the specific strain in a context of a relatively significant amount of n-3 fatty acid (eg DHA), and especially in a population of women at risk of mastitis or sub-clinical mastitis (i.e. partly deficient in DHA). Without being bound by the theory it is believed that the effect of the CECT-5716 probiotic on balancing out potential pathogenic bacteria, (including influencing their growth), and the beneficial effect of n-3 fatty acids (e.g DHA), for example on the immune system and/or on the inflammatory status, can synergize to reveal and/or enhance a better handling against mastitis—both at the prevention level avoiding the occurrence of sub-clinical mastitis and at the direct reduction of the occurrence or severity (treatment level).

[0229] In one embodiment, the probiotic strain L. fermentum CECT-5716 may be present in the mix or in the composition of the invention in an amount of 10.sup.2 cfu to 10.sup.12 cfu per g of composition (or mix). Alternatively 10.sup.3 to 10.sup.10 cfu/g or 10.sup.5 to 10.sup.8 cfu/g or 10.sup.6 to 10.sup.7 cfu/g.

[0230] In one embodiment, one or more other probiotics are also present in the mix or composition of the invention (“other probiotics” meaning “other than Fermentum CECT-5716”, which may still be present as per the present invention).

[0231] Generally, the probiotics may help establishing a healthy gut microbiota and strengthen natural immune defenses. The probiotics may also stimulate a development of the immune system at introduction of weaning food and prevent diarrhea. By stimulating the immune system the probiotics can synergize with the other essential components of the invention.

[0232] Examples of suitable probiotic micro-organisms include bacteria such as the genera Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus.

[0233] Specific examples of suitable probiotic micro-organisms are: Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei subsp. casei, Lactobacillus casei Shirota, Lactobacillus curvatus, Lactobacillus delbruckii subsp. lactis, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus rhamnosus (Lactobacillus GG), Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus halophilus, Streptococcus faecalis, Streptococcus thermophilus, Staphylococcus carnosus, and Staphylococcus xylosus.

[0234] Probiotic bacterial strains useful in the context of the present invention may include Lactobacillus rhamnosus ATCC 53103 obtainable from Valio Oy of Finland under the trade mark LGG, Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCM 1-2116, Bifidobacterium lactis CNCM 1-3446 sold inter alia by the Christian Hansen company of Denmark under the trade mark Bb 12 and Bifidobacterium longum ATCC BAA-999 sold by Morinaga Milk Industry Co. Ltd. of Japan under the trade mark BB536, Bifidobacterium adolescentis IVS-1 (Danwell Technology, Garden Grove, Calif.), Bifidobacterium adolescentis MG10502, (obtainable from Belgian Coordinated Collection of Microorganisms (BCCM/LMG), Bifidobacterium infantis Rosen-33, Bifidobacterium infantis (in particular strain LMG 8811, species name: Bifidobacterium longum ATCC 1569,

[0235] In one embodiment of the invention the strain mentioned in the above paragraph are used in replacement of the strain L. fermentum CECT-5716, in combination of the n-3 fatty acid of the invention.

Subject

[0236] The term “subject” as used herein refers to either a human or non-human animal. The non-human animal may be, for example, a livestock animal or a companion animal.

[0237] A “companion animal” is any domesticated animal, and includes, without limitation, cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cows, goats, sheep, donkeys, pigs and the like.

[0238] In one embodiment, the subject is a human subject. In another embodiment, the subject is a companion animal. Preferably, the subject is a human.

[0239] In one embodiment, the subject is at risk of mastitis and/or subclinical mastitis. In another embodiment, the subject is a lactating animal.

[0240] In one embodiment, the human subject is a woman.

[0241] In a further embodiment, the human subject is a lactating woman. In another embodiment, the human subject is a pregnant woman. It is believed that the compositions and mix of the invention can have a long term effect which pre-establishes adequate body conditions during pregnancy in order to prevent or treat mastitis during later lactation.

[0242] In a still further embodiment, the human subject is a woman at risk of mastitis and/or of subclinical mastitis.

[0243] In another embodiment, the human subject is a lactating woman at risk of mastitis and/or of subclinical mastitis.

[0244] In an additional embodiment, the subject is a European lactating woman. In an additional embodiment, the subject is a Caucasian lactating woman. In an additional embodiment, the subject is a European Caucasian lactating woman.

Treating and Preventing

[0245] The term “prevent” as used herein includes prevention and reducing the risk of a condition.

[0246] The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0247] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0248] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature.

EXAMPLES

Example 1

Methods

[0249] Within the framework of a multicentre European observational study to characterise the human milk (HM) composition in the first four months of lactation (Atlas of Human Milk Nutrients study), we set out to understand whether there are differences in the HM composition between lactating women with subclinical mastitis (SCM) versus those without based on the sodium/potassium (Na/K) ratios in the HM.

Study Protocol

[0250] The ATLAS study was conducted in seven countries across Europe (France, Italy, Norway, Portugal, Romania, Spain and Sweden) as a longitudinal, observational, cohort in which HM as well as multiple maternal and infant parameters were collected at six time points post-partum (0-3 d, 17±3 d, 30±3 d, 60±5 d, 90±5 d and 120±5 d). Institutional and local Ethical boards of each centre approved the study. The participants provided a written informed consent form to participate in the study after receiving explanations and having read and understood the purpose and the objective of the study in their respective local languages. Pregnant women were recruited before delivery, generally during the last trimester of pregnancy. Inclusion criterial for this study were: (a) pregnant women between ages of 18 and 40 years; (b) BMI between 19 and 29, inclusive; (c) intention to breastfeed at least until 4 months post-partum; and (d) agreement to the study protocol and signed informed consent form. Exclusion criteria for this study were: (a) currently participating in another trial; (b) presenting conditions that contraindicate breastfeeding; (c) medical conditions or on medications for conditions such as metabolic and cardiovascular abnormalities; (d) dietary probes such as anorexia or bulimia; and (e) subjects not able to comply to the study procedures. Dedicated, trained and certified research nurses and assistants collected all data for this study. Maternal data included: demography, anthropometry, medical history, history of dietary supplements and three-day food diaries. Infant data included: demography, anthropometry, history of medication use, body composition (one centre in France and one in Sweden) and infant intake diary (three centres in France only).

Standardised Human Milk Sampling

[0251] HM sampling was standardised for all subjects. Milk was collected at 11h00±2h00 using an electric breast pump (Medela Symphony). For each mother, milk was collected from the same breast for the entire study and mothers were requested to empty the breast in the previous feed. This collected single full breast milk samples were mixed and an aliquot of 10-40 mL HM for each time point was collected. For colostrum, or the first time point 5-10 mL was collected. The remainder of the HM was returned to the mother for feeding to the infant at a later time point, if so required. Each collected HM sample was transferred to freezing tubes, labelled with subject number and collection information, stored at −18° C. in the home freezer, transferred to the hospital for storage at −80° C. and then shipped on dry ice to the Nestle Research Centre (Lausanne, Switzerland) where it was stored at −80° C. until analysis. The frozen HM samples were thawed once for aliquoting into 15 individual small volume fractions (0.2 mL to 2 mL) in separate polypropylene tubes dedicated to the different analyses.

Assessment of SCM Status

[0252] Lactating women were categorised in to two groups: those having any SCM (defined as Na/K ratio>0.6) and those normal (defined as Na/K ratio 0.6) based on the Na/K ratios in HM in early lactation (days 2, 17 and 30). Lactating women having at least 1 instance of SCM during any of these three time points were classified as having any SCM, while those in the normal category did not have any instance of SCM in any of these time points.

Fatty Acid Quantification in HM

[0253] Fatty acid profiles were determined by preparing the methyl esters of fatty acids (FAMEs). A direct transesterification of HM was performed with methanolic chloridric acid solution as described by Cruz-Hernandez et al. (Cruz-Hernandez, C. et al. (2017) J Sep Sci 40: 3289-3300). Briefly, into a 10 mL screw cap glass test tube, milk (250 μL) was added and mixed with 300 μL of internal standard FAME 11:0 solution (3 mg/mL) and 300 μL of internal standard TAG 13:0 solution (3 mg/mL). After addition of 2 mL of methanol, 2 mL of methanolic chloridric acid (3 N) and 1 mL of hexane, the tubes were heated at 100° C. for 90 min. To stop the reaction 2 mL of water was added and after centrifugation (1200 g×5 min) the upper phase (hexane) was transferred into gas chromatography vials. The analysis of FAMEs was performed by GC using a CP-Sil 88 capillary column (100 m, 0.25 mm id. 0.25 μm film thickness) and their identification by comparison of retention time with authentic standards (GC standard Nestle 36 from NuCheck-Prep, Elysan Minn. USA).

Protein Quantification in HM

[0254] Total protein content in HM was measured using the colorimetric bicinchoninic acid (BCA) method according to the protocol provided with the BCA assay kit (ThermoFisher Scientific).

[0255] The four major HM proteins alpha-lactalbumin, lactoferrin, serum albumin and caseins were quantified using a LabChip system as described previously (Affolter et al. (2016) Nutrients 8: 504).

Mineral Quantification in HM

[0256] Quantification of minerals was realised using Inductively Coupled Plasma Mass Spectrometry (ICP-MS).

[0257] For Sodium (Na), Magnesium (Mg), Phosphorous (P), Potassium (K), Calcium (Ca), Manganese (Mn), Iron (Fe), Copper (Cu), Zinc (Zn) and Selenium (Se), 0.7 mL of human breast milk was transferred into PFA vessels and mineralised in a CEM® Microwave digestion system using HNO.sub.3/H.sub.2O.sub.2. Mineralised samples were transferred to PE tubes, diluted with MQ water and Germanium (Ge) and Tellurium (Te) were added as internal standards. Quantification was realised by ICP-MS using He as collision gas.

[0258] Certified Reference Materials (CRM) were added to all analytical series to control the quality of the quantification.

Results

[0259] The concentrations of iron, manganese, magnesium, copper, zinc, selenium, calcium, phosphorous, DHA, 18:3 n-3 octadecatrienoic acid, alpha-lactalbumin, lactoferrin and albumin in both the milk of mothers with sub-clinical mastitis and the milk of normal mothers at the 6 time-points post-partum are shown in FIGS. 1-13 and Tables 1-3.

[0260] Women with sub-clinical mastitis have higher concentrations of iron, manganese, magnesium, copper, zinc and selenium; and lower concentrations of calcium and phosphorous in their milk in comparison to normal women.

[0261] The n-3 fatty acids docosahexaenoic acid (DHA) and 18:3 n-3 octadecatrienoic acid (alpha-linolenic acid) are present at lower concentrations in the milk of women with sub-clinical mastitis in comparison to normal women.

[0262] In addition, alpha-lactalbumin, lactoferrin and albumin are present at higher concentrations in the milk of women with sub-clinical mastitis in comparison to normal women.

[0263] Based on those unexpected findings, the inventors have developed and refined the inventions: by re-establishing an adequate level of n-3 fatty acids—for example docosahexaenoic acid (DHA) and/or 18:3 n-3 octadecatrienoic acid (alpha-linolenic acid), and by providing a known effector probiotic able to modulate the occurrence of mastitis, the inventors synergistically potentialize the body response in order to treat and prevent mastitis.

TABLE-US-00001 TABLE 1 Mineral and trace element concentrations of human milk across lactation by presence or absence of sub-clinical mastitis (SCM). Overall Day 2 Day 17 Day 30 Day 60 Day 90 Day 120 P value Iron Any SCM 601 ± 392 461 ± 310 346 ± 162 292 ± 141 264 ± 140 216 ± 103 <0.0001 (μg/L) (n = 91) (n = 97) (n = 84) (n = 78) (n = 74) (n = 71) Normal 415 ± 193 389 ± 186 326 ± 137 278 ± 132 245 ± 127 262 ± 502 (n = 111) (n = 192) (n = 174) (n = 166) (n = 156) (n = 148) Manganese Any SCM 6.6 ± 3.9 4.4 ± 1.6 3.7 ± 1.1 4.5 ± 3.4 3.4 ± 0.8 3.7 ± 0.9 <0.0001 (μg/L) (n = 79) (n = 41) (n = 26) (n = 22) (n = 19) (n = 13) Normal 5.8 ± 3.1 3.9 ± 1.3 3.5 ± 1.0 3.4 ± 0.9 3.3 ± 0.7 4.0 ± 2.2 (n = 92) (n = 103) (n = 75) (n = 45) (n = 36) (n = 37) Magnesium (mg/L) Any SCM 38.5 ± 8.5 35.0 ± 6.2 34.2 ± 5.5 37.4 ± 5.8 38.8 ± 5.4 38.4 ± 5.4 0.076 (n = 87) (n = 94) (n = 80) (n = 76) (n = 72) (n = 69) Normal 38.0 ± 6.5 34.3 ± 5.7 33.9 ± 5.2 36.5 ± 5.6 38.6 ± 5.9 39.3 ± 6.2 (n = 106) (n = 171) (n = 149) (n = 162) (n = 154) (n = 145) Copper Any SCM 533 ± 267 529 ± 135 423 ± 86 325 ± 74 268 ± 72 221 ± 69 <0.001 (μg/L) (n = 91) (n = 97) (n = 84) (n = 78) (n = 74) (n = 71) Normal 472 ± 175 522 ± 111 422 ± 89 304 ± 76 256 ± 77 228 ± 89 (n = 111) (n = 192) (n = 174) (n = 166) (n = 156) (n = 148) Zinc Any SCM 7975 ± 3093 3234 ± 1274 2422 ± 906 1616 ± 1208 1113 ± 521 996 ± 520 <0.0001 (μg/L) (n = 91) (n = 97) (n = 84) (n = 78) (n = 74) (n = 71) Normal 7178 ± 2734 3590 ± 1051 2686 ± 872 1599 ± 692 1219 ± 571 1031 ± 563 (n = 111) (n = 192) (n = 174) (n = 166) (n = 153) (n = 148) Selenium Any SCM 37 ± 19 19 ± 6 15 ± 3 12 ± 3 10 ± 2 9 ± 2 <0.0001 (μg/L) (n = 91) (n = 97) (n = 84) (n = 78) (n = 74) (n = 71) Normal 26 ± 12 18 ± 3 15 ± 3 12 ± 2 10 ± 2 10 ± 6 (n = 111) (n = 192) (n = 174) (n = 166) (n = 156) (n = 148) Calcium Any SCM 256 ± 73 281 ± 54 296 ± 47 299 ± 41 288 ± 45 271 ± 36 0.003 (mg/L) (n = 91) (n = 97) (n = 84) (n = 78) (n = 74) (n = 71) Normal 295 ± 72 293 ± 55 296 ± 51 301 ± 45 297 ± 48 284 ± 42 (n = 111) (n = 192) (n = 174) (n = 166) (n = 156) (n = 148) Phosphorus Any SCM 109 ± 41 150 ± 31 149 ± 29 138 ± 21 128 ± 18 126 ± 20 <0.0001 (mg/L) (n = 91) (n = 97) (n = 84) (n = 78) (n = 74) (n = 71) Normal 144 ± 32 169 ± 28 155 ± 24 139 ± 21 133 ± 20 131 ± 22 (n = 111) (n = 192) (n = 174) (n = 166) (n = 156) (n = 148)

TABLE-US-00002 TABLE 2 Fatty acid concentrations of human milk across lactation by presence or absence of sub-clinical mastitis (SCM). Overall Day 2 Day 17 Day 30 Day 60 Day 90 Day 120 P value DHA Any SCM 11.5 ± 7.8 12.6 ± 9.0 10.5 ± 5.8 10.3 ± 6.1 9.5 ± 10.0 8.4 ± 5.9 0.0001 (mg/100 mL) (n = 103) (n = 103) (n = 89) (n = 78) (n = 77) (n = 74) Normal 13.7 ± 8.8 15.1 ± 10.2 13.6 ± 11.0 11.3 ± 8.4 10.8 ± 6.9 11.7 ± 15.8 (n = 158) (n = 187) (n = 168) (n = 155) (n = 144) (n = 137) 18:3 n-3 Any SCM 13.9 ± 11.2 21.8 ± 19.4 24.5 ± 18.7 27.8 ± 24.7 27.3 ± 27.6 23.6 ± 18.8 <0.0001 octadecatrienoic (n = 100) (n = 104) (n = 90) (n = 80) (n = 77) (n = 76) acid Normal 17.0 ± 11.3 29.1 ± 21.9 29.3 ± 23.7 32.8 ± 33.8 27.9 ± 24.7 32.3 ± 30.4 (mg/100 mL) (n = 159) (n = 187) (n = 171) (n = 161) (n = 154) (n = 144)

TABLE-US-00003 TABLE 3 Protein concentrations of human milk across lactation by presence or absence of sub-clinical mastitis (SCM). Overall Day 2 Day 17 Day 30 Day 60 Day 90 Day 120 P value Alpha-lactalbumin Any SCM 3232.8 ± 3131.2 ± 2893.1 ± 2469.0 ± 2249.5 ± 2089.4 ± <0.0001 (ng/μL) 685.0 548.4 506.6 466.5 422.3 415.8 (n = 102) (n = 104) (n = 91) (n = 81) (n = 77) (n = 75) Normal 3043.6 ± 3036.6 ± 2740.7 ± 2278.1 ± 2126.7 ± 2015.8 ± 597.1 544.4 499.1 372.4 374.1 378.0 (n = 128) (n = 187) (n = 170) (n = 162) (n = 156) (n = 147) Lactoferrin Any SCM 7053.9 ± 3315.6 ± 2430.8 ± 1757.1 ± 1543.1 ± 1510.4 ± <0.0001 (ng/μL) 3832.6 2486.1 2176.9 955.4 699.2 1502.3 (n = 102) (n = 104) (n = 91) (n = 81) (n = 77) (n = 75) Normal 5340.2 ± 2379.4 ± 1765.9 ± 1339.0 ± 1298.6 ± 1468.2 ± 2372.7 913.4 680.7 485.6 810.1 3269.1 (n = 128) (n = 187) (n = 170) (n = 162) (n = 156) (n = 147) Albumin (ng/μL) Any SCM 1534.6 ± 729.2 ± 659.1 ± 536.5 ± 474.7 ± 483.8 ± <0.0001 1821.9 671.1 655.3 190.5 130.2 388.8 (n = 102) (n = 104) (n = 91) (n = 81) (n = 77) (n = 75) Normal 813.3 ± 551.8 ± 529.7 ± 469.2 ± 455.8 ± 516.8 ± 1342.5 160.6 301.8 123.3 126.8 894.1 (n = 128) (n = 187) (n = 170) (n = 162) (n = 156) (n = 147)

Example 2

Methods

Study Population

[0264] This study used data from ‘ATLAS’, a longitudinal, observational study across seven European countries between December 2012 and January 2016. The study was approved by the institutional and local ethical boards for each center and was registered at ClincalTrials.gov with identifier NCT01894893. Maternal and infant demographics, anthropometry, and medical history were collected by trained and certified research nurses and assistants.

[0265] Human milk was collected from 305 women in 7 European countries. Of those, 185 provided information on dietary intake. 8 women were further excluded due to missing information, resulting in a final sample size of 177 women. Written informed consent was obtained from all women in their respective local languages.

SCM Analysis

[0266] Milk samples were obtained using an electric breast pump (Medela Symphony, Switzerland) from the same breast throughout the study period, at 11:00 h±2:00 h to avoid circadian influence. Samples were first frozen at −18° C. until delivery to the Nestle Research Centre (Lausanne, Switzerland) and then at −80° C. for further analysis.

[0267] SCM was assessed in early lactation, at visits 1 (0-3 days postpartum), 2 (17 days postpartum±3 days), and 3 (30 days postpartum±3 days). SCM was defined as having a sodium potassium ratio (Na/K) in breastmilk higher than 0.6 at any of the three visits. Moderate SCM was defined as Na/K ratio between >0.6 and 1 while severe SCM as Na/K ratio>1.

Dietary Intake Data

[0268] Dietary intake was assessed with 3-day food diaries at visits 2 (V2) and 3 (V3). The dietary information was then translated to nutrient and food group intakes by Nutrilog using the French food group classification and nutrient composition database (CIQUAL).

[0269] Diets which contained less than 1074.8 kcal or more than 4776.9 kcal of energy were considered outliers and were removed. After removing the outlier diets from the dataset, we considered each visit in V2-V3 in turn and the subset of subjects who attended that visit. For each visit, we removed a subject and all associated dietary information from a given visit if fewer than two non-outlier diets were reported for that subject across the three-day survey period for that visit. For example, if a subject S1 attended visit V2 and reported one outlier diet and one non-outlier diet for that visit, then we removed S1 and all her reported diets for V2 from the dataset. 177 subjects who attended at least one visit were retained for analysis.

[0270] Once the mean daily consumption was calculated, it was then normalized by the mean daily energy intake for that visit in kcal/day. This adjusted consumption was then averaged over all visits that each individual subject attended within the subset. As a final step, we then performed normalization to zero mean and standard deviation of 1. An example pipeline for a subject who attended all visits in S2 (visits V2-V3)

[0271] Dietary reference values for nutrients intake for lactating women were extracted from European Food Safety Authority (EFSA)'s summary report on Dietary Reference Values for nutrients.

Statistical Analysis

[0272] 1) We used multivariable regression to examine the association between nutrient intake in relation to SCM. Wilcox-test was used to calculate the p-values.

[0273] Analyses were run with R.

[0274] Results are reported in Table 4.

[0275] 2) We also used multivariable regression to examine the association between nutrient intake in relation to SCM. The statistical model has SCM status, Country and Mode of Delivery as covariates and contrast estimates were calculated to show the differences between SCM group and the No SCM group. A logarithmic transformation was applied as the nutrient intake data generally has a skewed distribution.

[0276] Analyses were run with the statistical software R ver 3.2.1 and packages mass and contrast were used for modelling and estimation.

Results are reported in Table 5.

Results

[0277] Table 4 reports the median intake of certain nutrients for women with SCM (i.e. those with human milk sodium potassium (Na/K) ratio>0.6 during any of the following visits: days 2, 17, and 30), and for women with no SCM (i.e. those with no SCM are defined as having a Na/K ratio ≤1.6 during any of the following visits: days 2, 17, and 30).

[0278] These data show that in the group of women with subclinical mastitis certain minerals (namely iron, manganese, magnesium, copper, calcium, phosphorous) were present at lower amounts in the diet as compared to the diet of women not having subclinical mastitis.

[0279] Similarly, data show that in the group of women with subclinical mastitis certain vitamin E was present at lower amounts in the diet as compared to the diet of women not having subclinical mastitis.

[0280] Table 5 reports the median intake of zinc and selenium for women with SCM (i.e. those with human milk sodium potassium (Na/K) ratio>0.6 during any of the following visits: days 2, 17, and 30), and for women with no SCM (i.e. those with no SCM are defined as having a Na/K ratio ≤1.6 during any of the following visits: days 2, 17, and 30).

[0281] These data show that in the group of women with subclinical mastitis certain minerals in addition to those previously mentioned (namely zinc and selenium) were present at lower amounts in the diet as compared to the diet of women not having subclinical mastitis.

[0282] These findings thus provide additional evidence that supplementation of one or more of such nutrients in the diet can prevent and/or treat subclinical mastitis.

TABLE-US-00004 TABLE 4 Median intakes of energy and nutrients Women Women Difference with with in median SCM* at SCM at intakes any visit no visit wilcox- between Average diet n = 52 n = 125 test SCM and of V2-V3 Median Median p value no SCM Magnesium, mg 265 313 0.01 −48.003 Phosphorus, mg 1254 1340 0.01 −85.926 Calcium, mg 744 974 0.00 −229.719 Manganese, mg 2 3 0.02 −0.498 Iron, total, mg 9 11 0.06 −1.632 Copper, mg 1.2 1.4 0.01 −0.213 Vitamin E, mg 8 10 0.02 −1.467

TABLE-US-00005 TABLE 5 Median intakes of energy and nutrients Women Women with with SCM* at SCM at Difference any visit no visit in geometric n = 52 n = 125 mean intakes Estimated Estimated between Average diet Geometric Geometric Contrast SCM and of V2-V3 Mean Mean p value no SCM Zinc, mg 9.46 10.84 0.0325 −1.39 Selenium, μg 130.96 156.64 0.0064 −25.68 Further example: The mix of the invention or the composition of the invention can, for example, be a commercially available, conventional, nutritional composition for pregnant women, to which the selected probiotic L. Fermentum CECT-5716 and the n-3 fatty acid are incorporated.

[0283] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the disclosed agents, compositions, uses and methods of the invention will be apparent to the skilled person without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention, which are obvious to the skilled person are intended to be within the scope of the following claims.