WO2005067962A2 - Composition derived from milk goat comprising growth factors and oligosaccharides - Google Patents

Composition derived from milk goat comprising growth factors and oligosaccharides Download PDF

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Publication number
WO2005067962A2
WO2005067962A2 PCT/EP2005/000429 EP2005000429W WO2005067962A2 WO 2005067962 A2 WO2005067962 A2 WO 2005067962A2 EP 2005000429 W EP2005000429 W EP 2005000429W WO 2005067962 A2 WO2005067962 A2 WO 2005067962A2
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Prior art keywords
lactose
oligosaccharides
composition
milk
sialyl
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PCT/EP2005/000429
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English (en)
French (fr)
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WO2005067962A3 (en
Inventor
Luis BARÓ RODRÍGUEZ
Julio Boza Puerta
Juristo FONOLLÁ JOYÁ
Emilia Guadix Escobar
Jesús Jiménez López
Eduardo LÓPEZ-HUERTAS LEÓN
Antonio MARTÍNEZ-FÉREZ
Jordi Xaus Pei
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Puleva Biotech, S.A.
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Publication of WO2005067962A2 publication Critical patent/WO2005067962A2/en
Publication of WO2005067962A3 publication Critical patent/WO2005067962A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C21/00—Whey; Whey preparations
    • A23C21/06—Mixtures of whey with milk products or milk components
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/14—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment
    • A23C9/142—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration
    • A23C9/1422—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration by ultrafiltration, microfiltration or diafiltration of milk, e.g. for separating protein and lactose; Treatment of the UF permeate
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/20—Dietetic milk products not covered by groups A23C9/12 - A23C9/18
    • A23C9/203—Dietetic milk products not covered by groups A23C9/12 - A23C9/18 containing bifidus-active substances, e.g. lactulose; containing oligosaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C2210/00—Physical treatment of dairy products
    • A23C2210/20—Treatment using membranes, including sterile filtration
    • A23C2210/202—Treatment of milk with a membrane before or after fermentation of the milk, e.g. UF of diafiltration
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a composition of growth factors and oligosaccharides from goat milk, to nutritional products containing these oligosaccharides, to a process to obtain that composition, and also to the use of this composition in the preparation of nutritional products and products to be used in the prevention of infections and intestinal disorders.
  • Milk is nature's designer food to fulfil the nutritional needs for the growth and development of the neonate.
  • Milk oligosaccharides are thought to be beneficial for the breastfed infant with regard to their prebiotic and anti-infective properties. The interest showed by researchers for oligosaccharides started recently after observing that these could promote bifidogenic flora in children fed with breast milk.
  • oligosaccharides are not digested in the gastrointestinal tract (they constitute the "soluble fibre” of breast milk), they provide nutrients for all colonic bacteria in children fed with breast milk, therefore contributing to a beneficial increase in the PH and bacterial flora differences in breastfed children compared to those fed with infant formulae (McVeagh P, Miller JB, 1997, J Paediatr Child Health 33: 281-286).
  • Oligosaccharides may resemble cellular receptors for pathogenic micro organisms, producing specific interactions between oligosaccharides and pathogens which indicates oligosaccharides play a role as intestinal mucosa cell protectors against pathogens. They may constitute an additional defence mechanism for newborn children, whose gastric pH is less acidic than in adults, and whose immunity system is not yet mature. In fact, among breastfed children there is a lower incidence of diarrhoea, otitis and respiratory diseases compared to non-breastfed children (Kunz C et al, 2000, Ann Rev Nutr 20: 699-702).
  • sialyl oligosaccharides (especially 3- and 6- sialyl-lactose) have been reported to have special importance and play a major role in prevention of infections (Hirmo S et al., 1998, Anal Biochem; 257: 63-66).
  • galactose is a major component of some very important brain lipids including myelin and it has been suggested that galactose derived oligosaccharides may play a role in neonatal brain development (Kunz C et al. 1999, Br J Nutr ; 82: 391-399).
  • the US6045854 patent is the first appeared patent that relates the use of human milk oligosaccharides in nutritional products.
  • This patent describes the use of 3 fucosyl-lactose, lacto-N-fucopentaose III, lacto-N-fucopentaose II, difucosyl- lactose, lacto-N-fucopentaose I, lacto-N-neotetraose, lacto-N-fucopentaose V and lacto-N-tetraose, all of them structures that are present in human milk.
  • the authors since isolation of these oligosaccharides from human milk is so difficult, the authors used preferably chemically synthesised oligosaccharides, instead of natural sources.
  • the authors describe oligosaccharide mixtures that produce health benefits that are close to those produced by human milk oligosaccharides, although the patent does not describe any specific proof or data of this activity for the described mixtures. It is a fact that colostrums and breast milk contain multiple hormones, bioactive peptides and growth factors. These hormones have direct influence on a wide range of biological systems.
  • hypothalamic-hypophyseal system since milk contains prolactin, somatostatin, oxytocin, etc.
  • thyroid gland since milk contains the thyroid stimulant hormone, thyroxine and calcitonin
  • sexual glands since milk contains estrogens and progesterone
  • pancreatic and adrenal glands Koldovsky O and Strbak V, 1995, In: Handbook of Milk Composition; Jensen, RG, Ed Academic Press, New York.
  • EGF Epidermal Growth Factor
  • TGF- ⁇ The Transforming Growth Factor ⁇
  • TGF- ⁇ The Transforming Growth Factor ⁇
  • TGF- ⁇ implicated in numerous processes, such as the development and differentiation of the intestinal epithelium; the growth, carcinogenesis and regulation of the immune response system. With respect to the immune system, it has an essential effect in two important parts of the intestinal mucus immune system: e.g., a production and induction of oral tolerance (Kalliomaki M et al, 2000, J Allergy Clin Immunol; 104: 1251-1257).
  • IGF-I Insulin Growth Factor I and II
  • IGF-II Insulin Growth Factor I and II
  • GH Growth Hormone
  • IgA vasoactive peptide
  • T cells stimulating T cells
  • macrophages activating macrophages and producing IL-12 by P substance
  • P substance Goldman AS et al, 2000, J Nutr, 130: 426S- 431 S.
  • - CD14 an anchor glycoprotein of 53-55 kDa present on the surface of monocytes and polymorphonuclear leukocytes that is able to be linked to the bacterial LPS initiating the cellular response to infections through mediators and cytokines. They have not been detected in goat milk, but in human milk (Jarvinen KM, et al, 1999, PediatrRes; 45: 76-81 ).
  • the isolation or production processes of some of these growth factors present in human milk have been patented, not only for dietetic products, but also for pharmaceutical preparations.
  • the NZ518217 patent describes the extraction process of TGFU2 and IGF-1 of a dairy product by chromatographic techniques.
  • the patent application WO01/24813 describes the use of TGF ⁇ 2 e IGF- 1 , isolated from mammal milk in products to treat and/or prevent intestinal mucosa damage as a result of chemotherapy, radiotherapy or gastrointestinal diseases.
  • TGFU2 coming from mammal milks or colostrums, in products for enteral nutrition or pharmaceutical preparations for the treatment of inflammatory bowel diseases, diarrhoeas and allergies have been claimed in the E0527283.
  • the first aspect of the invention provides a composition comprising growth factors coming from goat milk.
  • the invention provides a process to obtain the compositions of the invention.
  • a third aspect of the inventions relates to a food product, dietetic supplement or nutritional supplement comprising a composition of the invention, as well as the use of that composition for the preparation of these products.
  • the invention also provides the use of this food product, dietetic supplement or nutritional supplement in the prevention or treatment of diseases, as well as its cosmetic use.
  • Figure 1 It illustrates the strategy used to isolate the goat milk oligosaccharides and growth factors described in the invention.
  • Figure 2. It shows a Western blot of bovine and goat milk samples, samples of the concentrate of the invention and positive controls using polyclonal antibodies to TGF ⁇ 2, EGF, IGF-1 and CD-14 (GM: goat milk; GC: goat milk concentrate).
  • Figure 3. It illustrates the effect (inhibition) of growing concentrations of the composition of the invention on the bacterial adhesion of intestinal epithelial Caco 2 cells.
  • Figure 4 It shows the results of the RT-PCR related to the gene expression of MUC2, MUC3 and rRNA (control) of caco 2 cells incubated in the absence (-) or presence (+) of the composition of the invention.
  • the first aspect of the invention relates to a composition obtained from goat milk comprising growth factors.
  • This composition is characterised because these growth factor are in concentrations to be physiologically active and because these factors are selected from a list that comprises: Epidermal growth factor (EGF), transforming growth factor ⁇ 2(TGF ⁇ 2), insulin like growth factor type 1 (IGF-1 ) and anchored glycoprotein CD-14.
  • EGF Epidermal growth factor
  • TGF ⁇ 2 transforming growth factor ⁇ 2(TGF ⁇ 2)
  • IGF-1 insulin like growth factor type 1
  • anchored glycoprotein CD-14 anchored glycoprotein CD-14.
  • the patent WO01/42263 describes oligosaccharide mixtures coming from one or more ruminant milks, claiming an effect which is close to that of human milk oligosaccharides, in particular, anti-infective properties.
  • the present invention describes a method to obtain and use a mixture of compounds coming from goat milk with biological activity, including not only the above mentioned oligosaccharides, but also growth factors, also present in human milk. The ratios found among the growth factors described in the invention are similar to those found in human milk
  • TGF ⁇ 2, IGF-1 or CD14 are claimed in several patents or patent applications (NZ518217, WO01/24813, E0524283 or WO022945). These documents claim the extraction process from different milks and/or the use in the prevention or treatment of gastrointestinal diseases.
  • the present invention describes a method to obtain a mixture that include these four growth factors TGF ⁇ 2, IGF-1 , EGF and CD14, apart from the oligosaccharides, as it is described later.
  • the growth factors are in the composition of the invention in the following ratios (by weight): IGF-1/TGF ⁇ 2: 10-30000/1 , preferably, 10-2000/1 , more preferably, 1-20/1 ; CD14/TGF ⁇ 2 ratio: 2-5000/1 , preferably, 10-50/1 ; and EGF/TGF ⁇ 2: 0.1-2000/1 , preferably 1-20/1.
  • the composition of the invention contains in addition oligosaccharides , in weight % of at least 0.5% and these oligosaccharides are selected from a list that comprises: 3-sialyl-lactose, 6- sialyl-lactose, 3- galactosyl-lactose, 6-galactosyl-lactose, lacto-N-tetraose (LNT), lacto-N-hexaose (LNH), sialyl-LNH, N-acetyl-glucosaminyl (NAcG)-LNH, disialyl-lactose, di-NAcG lactose, sialyl-NAcG lactose, sialyl-hexosyl-lactose, NAcG-hexosyl-lactose, sialyl- NAcG-hexosyl lactose, disialyl-hexosyl lac
  • the chemical composition and structure of oligosaccharides isolated from goat milk is given in Table 2. About 70% of the oligosaccharides described in the goat milk of the invention have been found for the first time in this milk, including 18 new oligosaccharide structures and, furthermore, about 80% of the oligosaccharides described in the invention were found to be present in human milk, which is especially rich in them.
  • the oligosaccharide level found in goat milk was about 250- 300 mg per litre of milk which represents 4 to 10 times more the amount of oligosaccharides reported for other commercial milks (sheep or cow).
  • the present invention uses a natural source of oligosaccharides which is a significant difference to the above mentioned patent (US6045854) which claims the use of oligosaccharide structures that are present in human milk, but produced, preferably, by chemical synthesis.
  • the most abundant oligosaccharide structures present in goat milk we found were 1 ) N-acetyl-glucosyl lactose (70 mg/l), 2) Galactosyl-lactose (20 mg/l) and 3) N- acetyl-neuraminyl lactose (65 mg/L). These three represent about 77% of the total goat milk oligosaccharide mixture.
  • composition of the invention contains lactose.
  • fraction of oligosaccharides and growth factors accounts for an amount in weight that is higher than 0.5% up to 90% of the total of the composition.
  • Another aspect of the invention relates to the amount of sialyl oligosaccharides found in goat milk which is especially rich.
  • they are preferred compositions those in which the amount of sialyl oligosaccharides are between 5 and 85% in weight of the total of the composition, especially those in which this amount are between 40 and 45% in weight of the total of the composition.
  • Goat milk oligosaccharides are specially enriched in galactose which as mentioned above may have very important implications in neonatal brain development.
  • a preferred realisation of the invention consists of sialyl-lactose, galactosyl-lactose and N-acetyl-glucosaminyl- lactose ratios (in weight) of 2-7/1/1-5, preferably 2.9/1/2.6.
  • the invention provides a process to obtain the compositions above mentioned, based on the application of membrane ultra-filtration technology to goat milk. The process is made in such conditions that it is assured the presence of growth factors and oligosaccharides in their biologically active form and in adequate concentrations for maximum functionality.
  • this comprises the following steps: a. to make an initial fractionation of skimmed goat milk at slightly acid pH using a 15 to 50 kDa cut off membrane, preferably a 50 kDa cut off membrane. b. using the permeate of the step a), a new ultrafiltration step using a 1 to 5 kDa cut off membrane, preferably a 1 to 3 kDa cut off membrane, and ideally a 1 kDa cut off membrane. c.
  • a final purification step can be made with the retentate obtained in step b) to remove lactose and salts including one or more of the following processes: active charcoal chromatography, ion-exchange chromatography and electrodialysis.
  • the mentioned process can use goat milk whey as raw material.
  • a third aspect of the invention relates to a nutritional product to which the composition described in the invention above has been added.
  • the nutritional products of the invention preferably comprise from 0.1 mg to 10g of the added goat milk oligosaccharide mixture per 100 ml or 100 g of nutritional product, more preferably, they comprise from 1 mg to 500 mg of the composition of the invention.
  • Nutritional products containing this amount of the composition can be milk, dairy products, yoghurts, fermented milks, fruit and vegetable juices, biscuits, cakes, infant food and dehydrated food.
  • the addition of the composition of the invention to the nutritional products is effected by mixture and homogenisation according to the technological procedure of each product. Other components such as vitamins can be added to the nutritional products.
  • vitamins A, B 6 , B 12 , C, D, E or folic acid or a mixture of one or more thereof can be added to nutritional products according to the present invention.
  • the addition of these compounds can be effected either previously or after the heat treatment according to the specific technological procedure of each nutritional product.
  • An additional aspect of the invention relates to the use of a composition of the invention for the preparation of food products, dietetic supplements or nutritional supplements.
  • a final aspect of the invention is related to the use of these food products, dietetic supplements or nutritional supplements in the prevention of bacterial or viral infections, in gastrointestinal infections treatment and neonatal brain development.
  • composition of the invention can be also used for the preparation of cosmetic products.
  • the process for preparing the goat milk oligosaccharides mixture described above comprises three stages: 1. Initial fractionation of skimmed goat milk which is performed at a slightly acidic pH (6-7) using a multichannel ceramic tubular membrane Ceram Inside ® (TAMI Industries, France) made by zirconium and titanium with a molecular weight cut-off between 50-15 kDa, preferably 50 kDa. Temperature is kept below 50 °C to avoid thermal aggregation of ⁇ -lactalbumin. Under these conditions caseins, whey proteins and large peptides are retained by the membrane due to both steric and electrostatic interactions. The smaller components including oligosaccharides, growth factors, salts and lactose pass through the membrane and are collected in the filtrate.
  • a process of concentration and subsequent discontinuous diafiltration with a concentration factor of 2 and a minimum number of 5 cycles is carried out to obtain an oligosaccharide fraction with a yield of about 95%.
  • a channel circulation velocity of 4 m/s is used.
  • the filtrate product from the previous stage is fractionated again using a similar membrane with molecular weight cut-off of 5 to 1- kDa, preferably 3- and 1- kDa, most preferably 1-kDa.
  • An optimal separation of salts and lactose is achieved using a working pH of 6 to 8, preferably 7 to 7.5, and a temperature below 50 °C.
  • a process of concentration and subsequent discontinuous diafiltration with a concentration factor between 1 and 3, preferably 2 or 3, most preferably 3, and a number of cycles from 2 to 7, preferably 4 is carried out to obtain a retentate containing almost all goat milk oligosaccharides and growth factors.
  • CA CA, USA
  • CarboPac PA-100 column 250 x 4.6 mm i.d.
  • a guard column 250 x 4.6 mm i.d.
  • Model ED50A Electrochemical detector NaOH solution (19 mol/l; low in carbonate) was purchased from Baker (Philadelphia, PA, USA).
  • Sodium acetate of analytical grade was from Merck (Darmstadt, Germany).
  • Sephadex G25 was obtained from Pharmacia (Uppsala, Sweden).
  • Thin-layer plates (Silica-gel 60, 100x100 mm) were purchased from Merck. All other reagents were of analytical grade.
  • Carbohydrate-containing fraction was applied to a Sephadex G-25 column (900 x 25 mm i.d.) connected to a FPLC system and eluted with water to reduce levels of lactose and salts. Then, salt-free but carbohydrate-positive fractions were lyophilized and resuspended in 500 ⁇ l of water before further analysis. These fractions sometimes contained small amounts of lactose which is difficult to separate completely from the oligosaccharide fraction as lactose is present in milk in very large amounts and its molecular mass is very similar to the rest of the other small oligosaccharides occurring in milk.
  • the first step for yoghurt production is the milk standardisation, the milk intended must be of the highest bacteriological quality and must not contain antibiotics, bacteriophages, residues of CIP solution or sterilising agents.
  • the fat and dry solids contents of the milk are normally standardised according to the formulation. A sample is described below:
  • the milk continues to homogenisation to prevent creaming during incubation period and to assure uniform distribution of the milk fat. Homogenisation also improves the stability and consistency of cultured milks, even those with low fat contents. As a general recommendation, the milk should be homogenised at 20-25 Mpa and 65-70 °C to obtain optimum physical properties in the product.
  • the homogenised milk flows now to the heat treatment before being inoculated with the starter in order to improve the properties of the milk as a substrate for the bacteria culture, ensure that the coagulum of the finished yoghurt will be firm and reduce the risk of whey separation in the end product.
  • Optimum results are achieved by heat treatment at 90-95 °C and a holding time of about 5 minutes. That temperature/time combination denatures about 70-80 % of whey proteins which interacts with the casein, helping to give the yoghurt a stable "body”.
  • the starter (0,1 g/Kg) is metered into the stream of milk as it is pumped from an intermediate storage tank to the filling machine. Following packaging in the filling machine, the packages after crating and palletising, are trucked into the system for incubation and cooling.
  • the incubation room is able to accommodate a large number of filled pallets and maintain a temperature of about 40-45 °C during the fermentation process, about 5-6 hours.
  • the pallets are trucked to a conveyor passing through the cooling sections enclosed in a tunnel.
  • the normal target temperature is 18-20 °C; it is important to stop further growth quickly, which means that a temperature of about 35°C should be reached within 30 minutes, and 18-20 °C after another 30-40 minutes.
  • composition of the invention was tested using an in vitro fermentation approach based on culture and further growth quantification of different species of Bifidobacterium and Lactobacillus.
  • Bacteria was cultured in microtiter plates at 10 6 cfu/ml dilution for 24 h in the presence or absence of different concentrations of purified goat milk oligosaccharides obtained as described in the invention as an unique source of carbohydrates.
  • the culture media contains a pH indicator that allows for the quantification of the pH fall due to fermentation process, and an aliquot of the culture was plated to count the increase in bacterial cfu (colony forming units).
  • Table 3 Effect of the composition of the invention on fermentation and proliferation of several species of Lactobacillus and Bifidobacterium.
  • the Caco-2 cells cultured as above were used to study the influence of the composition of the invention on in vitro adhesion of different dilutions of pathogenic bacteria (Escherichia coli, Salmonella typhi or S. typhimurium).
  • pathogenic bacteria Esscherichia coli, Salmonella typhi or S. typhimurium
  • goat oligosaccharide fractions reduce binding capabilities of these pathogens up to 60% (Figure 3). Since bacterial adhesion to the intestinal epithelium is a required step for most pathogenic infections, our results clearly indicate that the composition of the invention have anti-infectious effect that may resemble the one observed for human milk in breast-fed infants.
  • the very high amount of sialyl- and fucosyl-oligosaccharides in goat milk may also explains the inhibitory effect that the composition of the invention have on the adhesion capabilities of pathogenic bacteria to human intestinal epithelial cells.
  • composition of the invention also blocks the effect of the heat-stable enterotoxin of E. coli which is responsible for the diarrheic effect observed upon infection.
  • To test the anti-diarrheic properties of the composition of the invention we treated Balb/c mice with 10 ⁇ g/ml of purified heat-stable enterotoxin from E. coli with or without the composition of the invention. Preliminary results showed that 75% of mice treated with enterotoxin in absence of oligosaccharides develop a diarrheic episode before 12 hours whereas only a 32 % due it when treated with the composition of the invention.
  • Caco-2 cells were grown in culture media in the presence or absence of the composition of the invention, obtained as previously described in the invention (10 mg/dish). At day 21 , culture medium was aspirated a cell homogenate was prepared and centrifuged at 100 000 x g for 30 min at 4° C to obtain a membrane pellet.
  • Goat milk oligosaccharides and growth factors can also bind to carbohydrate receptors on intestinal epithelial cells and modulate some of their physiological functions.
  • the results obtained show that human intestinal epithelial cells treated with purified goat milk oligosaccharides for 24 and 48 hours induce the expression of MUC 2 and MUC 3 genes ( Figure 4). It has been described that production of intestinal mucins is one of the first lines of protection against bacterial invasion in the intestine and therefore the MUC2 MUC3 expression induction observed here could represent another infection protection mechanism of the composition of the invention.
  • the modulating effect of the gut inflammatory response of the composition of the invention was studied in intestinal epithelial cells (Caco 2 and HT-29 cells).
  • Intestinal epithelial cells (Caco-2 and HT-29) were cultured as described above and incubated with or without the composition of the invention.
  • the expression of inflammatory cytokines Tumor Necrosis Factor-alpha and interleukins 1 and 8 were studied and quantified upon incubation with two pathogenic bacteria E.coli and Salmonella sp. We found a significant reduction of these cytokines at the protein (ELISA) and expression level by RT PCR which indicates that the composition of the invention may modulate gut inflammatory response induced by pathogens.
PCT/EP2005/000429 2004-01-13 2005-01-12 Composition derived from milk goat comprising growth factors and oligosaccharides WO2005067962A2 (en)

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US9872869B2 (en) 2006-03-10 2018-01-23 N. V. Nutricia Use of non-digestible saccharides for giving an infant the best start after birth
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