AU661089B2

AU661089B2 - Ganglioside-GM3-fortified nutrient composition

Info

Publication number: AU661089B2
Application number: AU32004/93A
Authority: AU
Inventors: Eiki Deya; Goro Hanagata; Tadashi Idota; Yuji Murakami; Taku Nakano; Norihumi Sato; Kiyoshi Tatumi
Original assignee: Snow Brand Milk Products Co Ltd
Current assignee: Megmilk Snow Brand Co Ltd
Priority date: 1992-03-31
Filing date: 1993-01-25
Publication date: 1995-07-13
Anticipated expiration: 2013-01-25
Also published as: AU3200493A; JPH05276894A; JP3002850B2

Description

66 108,94

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): SNOW BRAND MILK PRODUCTS CO., LTD.

invention Title: GANGLIOSIDE-GM3 -FORTIFIED NUTRIENT COMPOS IT ION The following statement is a full description of this invention, including the best method of performing it known to me/us:

SPECIFICATION

TITLE OF THE INVENTION Ganglioside-GM3-Fortified Nutrient Composition BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nutrient composition which is fortified with ganglioside GM3.

2. Description of the Prior Art Gangliosides, the general term for glycosphingolipids containing sialic acid, are amphiphilic molecules consisting of a hydrophilic saccharide chain moiety and a hydrophobic ceramide moiety, and vary in terms of molecular structures and properties due to variation in their saccharide moiety and ceramide moiety.

Gangliosides are present on the surfaces of cell membrane where the ceramide moiety projects into the lipid bilayer. Gangliosides exist in large quantities on tissue cells of the nervous system, but various kinds of gangliosides from other tissue cells have been isolated and identified. Lately, biological functions of gangliosides are being elucidated, and gangliosides have been spotlighted as factors 2 responsible in the message receiving mechanism. For example, gangliosides are deeply 'nvolved in such biological phenomena as the prolifiration, differentiation and canceration of cells; furthermore, gangliosides on the surfaces of cells have a fundamental role as receptors for many physiological ligands. The function of receptors is expressed in a great variety of ways, which reflects the variation of ganglioside molecules.

Ganglioside GM3 (hereinafter referred to as GM3), according to the present invention, is a ganglioside in which neuraminic acid is in the N-acetyl (Neu- Ac) form and is presented in a abbreviated form: Neu Ac alpha 2 3 Gal beta 1 4 Glc beta 1 1 Cer (wherein Neu is a neuraminic acid residue, Gal is a galactose residue, Glc is a glucose residue, and Cer is a ceramide residue).

As mentioned above, the functions of gangliosides as receptors are varied and the mode of expression of a function is considered to be different for different gangliosides, namely GM3, GM2, GM1, GD3, GD1b, GD1a, GT1b and the like.

Functions known to date about GM3 are as follows: Cell membrane GM3 functions as a tumorspecific antigen and acts as a functional receptor for 3 influenza virus involving invasion into cells via membrane infusion (KAGAKU TO SEIBUTSU, 25(8), 496-504, 1984).

Cell membrne GM3 binds to a receptor of fibroblast growth factor (FGF) itself or to its neighboring membrane protein to affect the function of the FGF receptor (TAMPAKUSHITU, KAKUSAN, KOSO, 29(12), 1146-1159, 1984).

Cell membrane GM3 acts on EGF (epithelial growth factor) receptors of human epithelial carcinoma cell strain and human ovarian epithelial carcinoma cell strain directly or indirectly via other cells, suppresses activation of EGF receptor protein kinase by EGF, or changes the specificity of EGF receptor self phosphorylation site as a substrate so as to inhibit 5. the action of protein kinase. GM3 is reported to be involved in regulation of cell growth through this mechanism (SAIBO KOGAKU, 616- 627, 1986).

Various kinds of gangliosides are present in cow's milk and goat milk, but the major ganglioside is GD3. On the other hand, in human milk, GD3 and GM3 are the major ganglioside components, but the amounts of GD3 and GM3 characteristically change during the lactation period; in colostrum, GD3 is the major single ganglioside component, but from day 7 after delivery, GD3 gradually starts to decrease and GM3 complementari- 4 ly starts to increase, and then 60 days after delivery GM3 becomes the single major ganglioside component. It was discovered that the difference in ganglioside components in different animal species is mainly in the minor ganglioside components J. Dairy and Food Sci., 37(6), 1988).

Furthermore, as to GM3 and GD3 content in human milk, it was reported that 810 micrograms/100 ml of GM3 and no GD3 was found in combined samples of human milk secreted 2 to 10 months after delivery (Pediatric Research, 20(5), 416, 1986).

The above-mentioned facts are summarized as follows: GM3 exists within cell membranes and acts by e 0 itself as a receptor, or regulates functions of other receptors so as to act as a target for viral attack, or as a target for killer T-cells to destroy abnormal cells. In addition, GM3 acts on EGF receptor and regulates the cellular growth function. However, these CeC actions and effects are related to GM3 fixed within the cell membrane and found in vitro; actions and effects ~of free GM3 (not fixed within cell membranes) in vivo, such as in oral administration, remain unknown.

Furthermore, although it was revealed that GM3 was characteristically found in large quantities in human milk from 2 to 10 months after delivery, the GM3 content changes depending on the period of lactation 5 and the nutritional significance of GM3 ingested orally remains unknown.

That is, no attempt has been made to integrate GM3 into a nutrient composition as an additive, and there is no information on GM3 as an additive as to physiological functions, an effective amount to be added or potential synergistic effects associated with other components.

Furthermore, there are serious problems in utilizing GM3 as an additive. That is, GM3, like other gangliosides in general, exists only in extremely small quantities within cell membranes and cannot be obtained in a sufficient quantity for industrial use by means of extraction and recovery from tissues, and the technology of chemical synthesis of GM3 has not yet reached the production stage on an industrial scale, which makes GM3 scarcely available.

Furthermore, it is difficult to calculate an appropriate amount of GM3 to be added because the amount of GM3 contained in nutritional compositions, especially in dairy products infant formulas) is not known; that is, GM3 is unevenly distributed or GM3 is destroyed during processing of milk into dairy products so that the amounts of GM3 remaining in the final products are not known.

6 SUMMARY OF THE INVENTION In consideration of the above-mentioned problems in the prior art, the present inventors first studied the in vivo physiological action of GM3, which is present in large quantfties in human milk during a specific lactation period, in order to determine the physiological significance of GM3 ingested orally, which is expected to be different from that of GM3 within cell membranes as a functional receptor, and found that GM3 ingested orally prevents adhesion of viruses and bacteria to mucosal cells of the digestive tracts. This could mean that GM3 has an important effect as a protective factor against infection, particularly in infants who are susceptible to infections caused by viruses and bacteria from outside the body because of their insufficient immune functions, although it is not known why GM3 is scarcely found in cow's milk or goat milk, or human milk secreted in the first 2 months after delivery.

The effect of GM3 to prevent aChesion of bacteria to mucosal cells of the digestive tracts becomes conspicuous when GM3 is present in a specified concentration at the time of oral ingestion.

Furthermore, considering that it is desirable to continue ingestion of GM3 in a specified concentration in order to maintain this effect, addition of GM3 to 7 nutrient compositions is extremely useful, and particularly in infants formula, synergistic effects associated with nutrient compositions such as other gangliosides derived from cow's milk can be expected.

Further, amounts of GM3 and GD3 present in infant formulas are reported for the first time in the present invention.

The effect of GM3 with respect to the protective function against infection described above is a direct effect of GM3 ingested orally. However, other effects of GM3 can also be expected; that GM3 ingested orally is metabolized wherein gangliosides such as GM3 within the cell membrane increase, thereby regulating ~cell growth function, a significant physiological function.

Further, the problem that GM3 is scarce, and not available in quantities sufficient for use as an additive can be solved by enzyme treatment or acid treatment of GD3 derived from cow's milk to produce GM3.

Accordingly, the present invention is characterized in that a nutrient composite for oral ingestion containing proteins, carbohydrates and/or fats as major 4* components is fortified with ganglioside GM3, and, preferably, the admixing ratio of GM3 is m =r f .Or prfnr-hly 0.4-50 mg per 100 g solid weight 8 of the fortified nutrient composition. In the case where the nutrient composite is an infant formula, this range is based on new findings by the present inventors that the concentration of GM3 in infant formula is less than the minimum detectable amount (about 20 micrograms/i g solid and that nutrient composites in general contain no GM3, and that viral and bacterial infections through alimentary tracts can be prevented by orally administering GM3 along with a nutrient composite in advance. Here, GM3 is preferably the one which is prepared by treating GD3 derived from milk with enzyme or acid.

DETAILED DESCRIPTION AND THE PREFERRED EMBODIMENTS As mentioned above, not only GM3, but gangliosides in general attract attention because of their role in the message receiving mechanism within the cell membrane, and their biological significance within the cell membrane is becoming understood.

S e However, since GM3 and other gangliosides are localized in quantity in limited cells such as nerve cells and carcinoma cells, the physiological significance of oral ingestion of GM3 and other gangliosides is still being studied. Since the effects of oral ingestion cannot be revealed by in vitro study with cultured cells and, since very little GM3 is available, much remains un- 9 known with respect to the effect of oral ingestion of GM3.

As mentioned above, it was reported in KAGAKU TO SEIBUTSU, 25(8), 496-504, 1984, that GM3 within the membrane is recognized by influenza virus and is involved invasion of the virus into the cells. The virus is attracted to the GM3, which is fixed in the cell membrane lipid bilayer, and attaches to the surface of the cells. Said GM3 fixed in the cell membrane also has a role in membranous fusion. However, it is difficult to predict the actions and effects of GM3 which is ingested from external sources and is not fixed within the cell membrane, on the basis of the S findings mentioned above. In considering that GM3 in the membrane is recognized by influenza virus, it is expected that GM3 ingested from external sources would be adhesive to the influenza virus. However, for example, it was totally unknown whether GM3 could inhibit the binding between receptors for viruses, which are considered to exist on cells of the digestive system, and the viruses. Furthermore, even if GM3 could inhibit the binding, information with respect to GM3 concentration or the like which is necessary for the inhibition was not available.

The present inventors have solved the abovementioned unsolved problems and have clarified the 10 physiological effect of GM3 ingested orally, which is the significance of the present invention. The present invention clearly shows that, by allowing GM3 to be present in the digestive system or the like by oral administration, adhesion of viruses, bacteria or the like to cells of the digestive system can be inhibited, thereby infection being prevented; however the mechanism of the physiological action of GM3 still remains unknown.

GM3 is a ganglioside whose structure is presented in the general formula: Neu 5 Ac alpha 2 3 Gal beta 1 4 Glc beta 1 1 Cer. Any GM3 of this formula can be used and it is not necessary for the ceramide to have a specific fatty acid composition.

In considering the difficulty in obtaining GM3, it is known that a GD3 composition can be prepared e in large quantities from milk (see Japanese Patent Laid-Open 269992/1988). A GD3 composition is prepared by this method, and the GD3 composition is desialized with the hydrolysis of only one sialic acid molecule, e* thereby GM3 being produced; or a dairy product containing GD3 is treated with an acid such as hydrochloric acid, lactic acid and citric acid at 37'C .I for 1-5 hours, at 80'C for 5-20 minutes or at 100'C for minutes to desialize with the hydrolysis of only one sialic acid molecule, thereby GM3 being produced.

11 GM3 thus prepared in large quantities can be added to a nutrient composite, particularly to an infant formula, to simulate human milk in its composition.

In addition, GD3 prepared from whey protein concentrate (WPC), buttermilk or the like can be used.

According to an analysis by the present inventors, it is revealed that GD3 exists in large quantities in cow's milk (880 micrograms/100 ml), and in particular GD3 content is relatively high in dairy products such as buttermilk (294 micrograms/1 g solid) and WPC (406 micrograms/1 g solid).

Furthermore, instead of using a GD3 composition as the starting material, it is possible to treat milk or dairy products such as WPC and buttermilk *04. directly with sialidase or acid in order to obtain milk or dairy products which contain a GM3 composition in *s accordance with the invention.

In the method using sialidase, the GD3 S composition to be desialized and the sialidase for desialization are suspended or dissolved in a buffer Go*: solution to carry out sialic acid enzymatic decomposition, thereby GM3 being obtained, or the GD3 composition to be desialized is suspended or dissolved in a buffer solution having an appropriate pH and then treated with sialidase to hydrolyze sialic acid, thereby GM3 being obtained.

12 Any sialidase preparations, which are commercially available today, derived from Vibrio cholerae, Clostridium, Arthrobacter or the like can be used for GM3 production.

Furthermore, as to the buffer solution, any buffer solution which has buffering action in the pH range in which the enzyme is active, which include acetate buffer, phosphate buffer, citrate buffer and maleate buffer solutions, can be used. The concentration of the buffer solution is preferably 200 mM.

Furthermore, the concentration of the GD3 composition as a substrate is preferably about 0.5-5 mg /I ml.

As to the amount of enzyme, if the amount is excessive, degradation proceeds further to make lactosylceramide; if the amount is too small, the reaction stops. Accordingly, the amount of enzyme is preferably 2-20 m unit per 1 mg substrate.

In such a method using sialidase, three se conditions, namely, appropriate buffer concentration, substrate concentration and amount of enzyme, are important.

On the other hand, in the hydrolysis method with the use of acid, the relationship among pH of the reaction solution, reaction temperature and reaction 13 time is extremely important. By selecting a low pH and a high reaction temperature, the rate of GM3 production increases but at the same time, the rate of GM3 degradation also increases. An appropriate reaction time as a function of pH, ranging from 2 to 5, and reaction temperature, ranging from 37 to 100 0 C, must be selected.

According to the method of the present invention, a GM3 composition can be easily prepared in large quantities from a GD3 composition which is available in large quantities, and at relatively low cost. Furthermore, GM3 according to the present invention is Lonsidered to have a higher activity as a *receptor for a virus or the like than those obtained by other methods (see Reference Examples in Example S thereinafter).

Further, for the GM3 so obtained, the structure of the saccharide chain can be identified by proton nuclear magnetic resonance spectrometry, sialic acid is identified by methylation analysis, the binding site of the saccharide chain is identified by methylation analysis, and fatty acid composition is identified by GLC.

**In GM3 obtained by the methods described above, the major components of ceramide in general are long-chain bases which include sphingosine (dl8:1) 14 hexadecasphingenine (d16:1) sphinganine and hexadecasphinganine (d16:0) and fatty acids which include docosanoic acid (C22:0), tricosanoic acid (C23:0), tetracosanoic acid (C24:0) and tetracosanoic acid (C24:1), the fatty acid ratio being 20- 40%, 10-30% and 5-15% for C22:0, C23:0, C24:0 and C24:1, respectively. The GM3 contains a large number of long chain fatty acids having 22-24 C atoms.

However, GM3 to be used in the present invention is not limited to those obtained by the methods described above.

Furthermore, a nutrient composite to which GM3 is added contains proteins, carbohydrates and/or fats as major components. It generally contains all the nutrients, namely proteins, carbohydrates, lipids, vitamins and minerals; however, the combination of admixture can be altered depending on the purpose and use. For example, if the nutrient composite has a U. dietetic purpose, a defatted material may be used; if a primary purpose is to supply vitamins and minerals, these contents may be increased. Furthermore, if the e 9991 nutrient composite is an infant formula, various trace elements may be added to simulate the composition of human milk.

Nutrient composites to be ingested orally can be in any form. A solid in the forms of a powder, 15 granules, blocks and bars, gels, semi-liquid, liquid or the like can be used as desired.

Nutrient composites to which GM3 is added are preferably powdered milk for infants whose immune system is not mature, particularly from the protection against infection, infant powder milk and powdered formula for premature infants, as well as follow- up milk, special powdered milk for medicinal purposes and, naturally, ordinary powdered milk. Furthermore, GM3 can be used also as an additive to dairy products such as cheese and to ordinary food, and in pharmaceuticals and functional food. Further, in an infant formula, approximately 13-14% solid content is preferable; however, in a nutritionally fortified food, the content rer can be as high as approximately In order to introduce physiological effects of GM3 in nutrient compositions and other food products, the amount of GM3 to be added to the nutrient composites is preferably 9t--e mere, mere t0.4-50 mg, and most preferably 0.75-15 mg, Gs:'"o per 100 g of solid product. If less than 0.1 mg is some used, effects of the additive are too small and thus 0.4 mg or more is normally preferred. Amounts over mg do not produce any better results and are not preferable in terms of an economical viewpoint.

-The admixture ratio of GM3 is given herein by 16 solid weight of product based on the findings that the effect in obstructing viral and bacterial adhesion to tissue cells depends on the GM3 concentration. That is, in the case where the same amount of GM3 is ingested, GM3 ingested as an admixture with a nutrient composite is more effective than GM3 singly ingested.

Furthermore, in order that GM3 be present consistently in digestive tracts, it is quite effective to ingest GM3 in the form of an admixture with a nutrient composite.

Results of a test for infection prevention is shown in an Example following, which indicates that oooo •eg infection can be prevented in mice by oral administraoooo S. tion of a composition containing GM3, 15 minutes before exposure to viruses. This is considered to be a direct action of GM3; that is, viral and bacterial adhesion to tissue cells can be prevented by providing GM3 on internal walls of tissue of the digestive system or the like where viral or bacterial infection is expected.

Thus, the GM3 concentration in oral ingestion is important. This is because, unlike the concentration in blood, the concentration in the digestive tract or the like is related to the concentraLion ingested and, furthermore, it is possible to extend the time for GM3 to remain in the digestive tract or the like by simultaneous ingestion with other nutrient compositions.

17 In order to attain the most effective prevention of infection in the digestive system, it is desirable that GM3 is orally ingested 5-60 minutes before exposure to infection; furthermore, considering that infection occurs unexpectedly, it is mostly desirable to ingest GM3 daily with a nutrient composite.

Further, it is probable that a certain amount of GM3 is already contained in a nutrient composite.

If so, the amount of GM3 to be admixed may be adjusted to meet the range per 100 g solid, as discussed above.

Next, admixing of GM3 to an infant formula, which is a preferable embodiment, will be explained.

Conventionally, the GM3 content in infant formula is not known. This is mainly because research focused on GM3 has not been started and physiological significance of GM3 present in human milk 2-10 months after delive-y was unknown. The significance of the presence of GM3 has not been completely clarified; however, the present inventors have found that GM3 does s** inhibit the viral and bacterial adhesion to cells.

S0% Sir-.e almost no GM3 is found even in human milk secreted in the first 7 days after delivery, including colostrum, and in cow's milk, there may be specific physiological significance other than the above-mentioned effects. However, the inventors paid special attention to the above-mentioned effect and thus completed the 18 present invention. In this regard, the present invention is not a simple simulation of infant formulas to human milk. The GM3 content in human milk (2-10 months after delivery) is approximately 6.2 mg/100 g solid (assuming that milk solid content is In terms of effect in obstructing viral and bacterial adhesion to cells, 0.4-50 mg/100 g solid is required as mentioned above.

According to an analysis by the inventors, the GM3 content in infant powdered milk is less than the minimum detectable amount (approximately 20 micrograms/i g solid), but the GD3 content in solution was 260-635 micrograms/100 ml (2-4.9 mg/100 g solid). As to cow's milk, GM3 and GD3 contents are micrograms/100 ml (0.5 mg/100 g solid), and 880 micrograms/100 ml (6.8 mg/100 g solid), respectively.

The reason why GM3 exists in cow's milk o S(although in a minute amount) but not in infant formula is probably that raw materials for infant formula, that is skimmed milk, butter milk, WPC or the like, are all separated fractions from milk and accordingly GM3 is distributed unevenly, or destroyed during processing since GM3 is susceptible to degradation under acid conditions.

From these results, it is revealed that almost no GM3 but GD3 in large quantities is contained 19 in infant milk powders and cow's milk. That is, the GM3 to GD3 ratio found in cow's milk is greatly different from that-found in human milk. Accordingly, it is also revealed that addition of GM3 is necessary for simulation of infant powdered milk to human milk.

By doing so, the nutrient composition can be expected to regulate cell proliferation and, in particular, to suppress the growth of carcinoma cells, because of GM3 being contained therein.

The ratio of GM3 to GD3 preferably is GM3/GD3 0.05-2 (by weight).

Example The present invention will bi, explained in detail by Examples and Reference Examples.

Results of measurement of GM3 and GD3 contents in infant powder milks and dairy products by the present inventors, and results of the infection preventing test for GM3-added powder milk against Campylobacter are shown in the Reference Examples.

Reference Example 1: GM3 and GD3 contents of infant powder milks and dairy products Methods Cow's milk and dairy products used as samples were treated to extract lipid components with 20 chloroform:methanol:water=4:8:3 and the resultant extract was subjected to ion exchange chromatography on DEAE-Sephadex A-25 (a product of Pharmacia).

Gangliosides adsorbed on the ion exchange resin were eluted with a 0.5 N sodium acetate solution and the pH of the eluate was adjusted to 9.0 with 0.1N sodium hydroxide to open the lactone ring. Subsequently, the resultant solution was neutralized with 0.5 N acetic acid and dialyzed to desalt; the desalted solution was subjected to silica-gel column chromatography using latro beads 6RS 8060 (a product of Yatron) and a fraction eluted with chloroform:methane=2:8 was obtained Sand concentrated. After concentration, the concentrated sample was subjected to thin layer chromatography on Sa silica-gel plate No. 13749 (a product of Merck), the solvent used for development was chloroform:methanol:0.2% calcium chloride solution 55:45:10, the color development was carried out with orcinol sulfuric acid and resorcinol hydrochloric acid, Sand the measurements of GM3 and GD3 were made by a densitometer (minimum detectibility: approximately micrograms/ g solid).

Results Results of the measurements are shown in Table 1, in which figures for infant powder milks and cow's milk are given in micrograms per 100 ml liquid 21 and for other dairy products in micrograms per ig solid.

Table 1 Sample Infant powder milk Example 1 Example 2 Example 3 Cow's milk Skimmed milk Butter milk

WPC

GM3 content (micrograms) Not detected Not detected Not detected 70 Not detected Not detected Not detected GD3 content (micrograms) 635 260 400 880 36 294 406

D

r r o These results indicated that in all the samples, almost no GM3 was contained and the GM3 content was much lower as compared with human milk as described above. On the other hand, it was also shown that in dairy products as well as infant powdered milks, the GD3 content was higier than the GM3 content, which indicates that cow's milk and diary products are a good source of GD3.

0 0 0 Reference Example 2: Preparation of GM3 22 One gram of ganglioside GD3 (derived from cow's milk) (prepared by the method described in Japanese Patent Laid-Open 269992/1988) and 5 units of sialidase (derived from Arthrobacter) were suspended in 500 ml of a 0.1 M acetate buffer solution (pH Without delay the enzyme reaction was started and continued at 40'C for 24 hours. During the reaction, the amount of ganglioside GM3 produced was measured at intervals by thin-layer chromatography. When the reaction completed, the enzyme was inactivated by placing the reaction mixture in a boiling water bath for 1 minute. Subsequently, a white powder, which was obtained from the reaction mixture by drying under reduced pressure, was subjected to ion exchange chromatography on DEAE-Sephadex A-25 (a product of Pharmacia) and further to silica-gel chromatography o, latro beads 6RS 8060 (a product of Yatron) to isolate a reaction product. The reaction product thus obtained was lyophilized and then 415 mg of white powder was obtained.

*5t55* S* Analysis by thin-layer chromatography showed that this powder has the same Rf value as a standard GM3 (derived from the bovine brain). Furthermore, NMR and IR analyses showed a close analogy of the white powder with the standard GM3.

From these results, the white powder was confirmed to be GM3.

23 The fatty acid composition of GM3 thus obtained was determined using GC-MS after treatment with HCl-methanol to produce free fatty acids. Table 2 is the resultant fatty acid composition.

Table 2 Fatty Acid Composition Fatty Acid composition C14:0 0.2 C16:0 1.9 Trace C18:0 C18:1 Trace C20:0 0.8 C21:0 0.7 C22:0 29.2 C22:1 4.9 C23:0 24.3 C23:1 C24:0 21.5 C24.1 0*S* C25:0 C25:1 1.2

S*

C26:0 0.8 C26:1 Not detected Total 100.0 Reference Example 3: Preparation of GM3 24 HCl was added to a 10% whey protein concentrate solution (WPC solution) containing 40 mg of GD3 per 1 liter to adjust the pH to 2.5 and the reaction was carried out at 37'C for 8 hours. After completion of the reaction, the reaction solution was neutralized with sodium hydroxide. A fatty acid fraction was extracted from the resulting solution and subjected to analysis by thin-layer chromatography. By the analysis, it was confirmed that 10 mg of GM3 component Tvas contained in 1 liter of the solution.

Reference Example 4: Preparation of GM3 S. Citric acid was added to a 10% butter milk solution containing 28 mg of GD3 per 1 liter to adjust the pH to 4 and the reaction was carried out at 100'C for 15 minutes. After completion of the reaction, the reaction solution was neutralized with sodium hydroxide. The analysis by thin-layer chromatography as in Example 3 showed that 46 mg of GM3 component was S" contained in 1 liter of the solution.

Reference Example 5: Recognition of influenza virus receptor by GM3 (in vitro) Specificity of recognition of influenza virus receptor was determined by using the GM3 component obtained in Reference Example 2 and a commercially 25 available GM3 (GM3-Ganglioside (trade name), a product of Bio Curve). The method of Suzuki et al Biol.

Chem., 260, 1362-1365, 1985) was used for determination. Results are shown in Table 3.

From the results, it was confirmed that GM3 obtained in Reference Example 2 had higher recognition specificity for influenza virus than the conventional GM3 preparation, but both GM3s showed higher specificity than GD3.

Table 3 Recognition Specificity of Gangliosides Tested by Influenza Virus Gangliosides Recognition Specificity GM3 (Reference Example) 21 4 GM3 (Commercial product) 13 3 GD3 (Cow's milk) 0 (Measurement unit is nmol/mg protein/min.) e e 9* Example 1 S" To a mixture of 6.8 kg of casein, 70.6 kg of whey powder and 1 kg of a vitamin and mineral component was added a solution of 1500 mg of a GM3 composition (containing 30% GM3), which had been prepared by treating GD3 derived from cow's milk with sialidase to hydrolyze only one molecule of sialic acid in the same manner as described in Reference Example 2, in 700 kg 26 water, and the resultant solution was mixed with 23.9 kg of vegetable oil and the mixture was homogenized.

The solution thus obtained was pasteurized, concentrated and dried in an ordinary manner, thereby 100 kg of powder milk being obtained. The GM3 content of the powder milk was 450 micrograms per 100 g. (Further, the GD3 content was 2 mg per 100 g.) Example 2 To a mixture of 52.7 kg of whey powder and 1 kg of a vitamin and mineral component (same as described in Example 1) was added a solution of 45g of a GM3 composition (containing 60% GM3), which had been prepared by treating GD3 derived from butter milk with *o hydrochloric acid to hydrolyze only one molecule of sialic acid in the same manner as described in Reference Example 4, and 23.9 kg of skimmed milk powder in 700 kg water, and the resultant solution was mixed with 23.9 kg of vegetable oil, and the mixture was homogenized. The solution thus obtained was pasteurized, concentrated and dried in an ordinary manner, thereby 100 kg of powder milk being obtained. The GM3 content e.

of the powder milk was 45 mg per 100 g. (Further, the GD3 content was 40 mg per 100 g.) Example 3 27 To a mixture of 527 g of whey powder and 10 g of a vitamin and mineral component (same as described in Example 1) was added a solution of 100 mg of GM3 composition (containing 28.5% GM3) which had been prepared by treating GD3 derived from WPC with citric acid to hydrolyze only one molecule of sialic acid, and 239 g of skimmed milk powder in 5 kg water, and the resultant solution was mixed with 239 g of vegetable oil and the mixture was homogenized. The solution thus obtained was pasteurized, concentrated and dried in an ordinary manner, thereby 1 kg of powder milk being obtained. The GM3 content of the powder milk was 10 mg per 100 g. (Further, the GD3 content was 5 rng per 100 g.) Test Example 1 Test for prevention of Campylobacter infection by GM3-added powder milk in the rat GM3 prepared from GD3 derived from cow's milk in the same manner as described in Reference Example 2 was added to a commercial powdered milk (containing 13% protein, 27.8% fats, 54.2o carbohydrates, 2.2% ash and S. 2.8% water) in various concentrations to prepare samples for the test. In an infection experiment, the samples thus prepared and the compositions obtained in Examples 1, 2 and 3 were orally administered to rats (4-week old SD rats) and then the rats were infected 28 with Campylobacter bacteria, which were isolated from human intestinal mucosal tissue, by oral inoculation of 0.1 ml of 101 0 cells/ml bacterial culture. More precisely, GM3 was added to milk preparations containing 13% infant powdered milk to give the final concentrations of 0-50 mg/100 ml (0-400 mg/100 mg solid) and the samples thus prepared were orally administered to rats forcefully so as to make the amount of ingestion 3 ml/100 g body weight, and then in minutes, a specified amount of Campylobacter culture was orally given to each animal. Incidence of diarrhea was examined. (Milk preparations containing 13% infant milk powder used in this experiment contained 0.6 mg/100 ml GD3).

Test results Results are given in Table 4.

Table 4 GM3 added Incidence, mg/100 ml (mg/100 g solid) 0 \oo 0.01 (0.08) S" 0.05 0.1 29 (40) 10.0 (80) 50.0(400) Example 1 (0.45) Example 2 (45) Example 3 (10) As distinctly shown in Table 3, when GM3 prepared from GD3 derived from cow's milk was added, incidence of diarrhea caused by Campylobacter infection was low as compared to the case where non GM3 was 600* added, in particular, when the amount of added GM3 was 0.05 mg/100 ml (0.4 mg/100 g) or more, the incidence was markedly suppressed. However, addition of mg/100 ml or more showed no better result. Further, when no GM3 was added, incidence of diarrheas due to Campylobacter could not be prevented despite the presence of GD3 at a concentration of 0.6 mg/100 ml.

Claims

1. A nutrient composition for oral ingestion having a function to protect against infections in the digestive system, characterized in that a nutrient composite comprising proteins, carbohydrates and/or fats as major components is fortified with ganglioside GM3.

2. A nutrient composition as set forth in Claim 1, wherein the concentration of ganglioside GM3 in the admixture ranges from 0.4 to 50 mg per 100 g solid of nutrient composition.

3. A method of preparing a ganglioside GM3 preparation comprising changing ganglioside GD3 which is derived from cow's milk so as to hydrolyze and desialate only one sialic acid molecule at the non-reducing terminal 15 by contacting with sialidase or an acid.

4. A nutrient composition of any one of claims 1 or 2 characterized in that the ganglioside GM3 is prepared by Sa method of claim 3. The nutrient composition as set forth in any one of claims 1, 2 or 4, wherein said nutrient composite is a powdered infant milk. DATED THIS 5TH DAY OF MAY 1995. SNOW BRAND MILK PRODUCTS CO., LTD By its Patent Attorneys: GRIFFITH HACK CO Fellows Institute of Patent Attorneys of Australia staWahleerVkeep/32004,93.speci.sw 14 4 1 1 ABSTRACT OF DISCLOSURE f A nutrient composition is prepared by forti- fying with ganglioside GM3 at a concentration of pref- erably a o 0.4-50 mg/100 g solid, with a nutrient composite com- prising proteins, carbohydrates and/or fats as major components. The nutrient composition has improved physiological functions such as protection against viral and bacterial infection, particularly the preven- tion of diarrhea caused by Campylobacter infection, and other physiological activities.