FR2488137A1 - Bifido:bacterium multiplication-promoting agent - contg. oligosaccharide prepd. by treating lactose with beta-glucosidase produced by Aspergillus oryzae - Google Patents

Abstract

Objective multiplication-promoting composition is characterised by containing the oligosaccharide represented by general formula GaLn-Glc (I) (where Gal is galactose, GIC is glucose and (=2-5) as the effective ingredient and can be prepared by treating lactose or lactose-containing substance with beta-glucosidase produced by Aspergillus oryzae. The treatment is practiced at 20-50 degrees C at pH 3-6.5 adapting substrate concentration 10-50% and enzyme concentration 1-100 units/ml and the enzymic activity can be ceased by heating the reaction mixture at above 90 degrees C for 5-10 minutes. During the enzymic treatment at first oligosaccharide content in the reaction mixture increases linearly and after a prescribed time it decreases gradually. The oligosaccharide (I) shows the multiplication-promoting affect not only in vitro but alos in vivo and can promote the multiplication of in-intestine fixable Biphidobacterium regardless of its variety such as B. brieve, B. biphidum. B. infantis, B. andrescens, etc.

Description

 The present invention essentially relates to a composition promoting the growth of bifidobacteria, and a process for the manufacture thereof.

Many substances capable of promoting the growth of bifidobacteria (hereinafter referred to as Bifidus growth factors) have been proposed, as well as numerous and varied processes for the manufacture of these substances, corresponding to powdered milk containing these substances, as has been has been described in the patents
Japanese N-32908/66, 6510/70, 6865/70, 21606/70, 40956/74 and 40957 / 74.However, the effectiveness of most of the growth factors of Bifidus already proposed has only been tested in cultures. in a non-living environment (conducted outside the living body), the activity of these factors outside the living body is not satisfactorily elucidated.

 Bifidobacteria are useful bacteria that live in the human intestines and have a very well-known biological significance. Great efforts have already been made to increase the proportion of these bacteria in the number of bacteria living in the intestines of infants fed artificially. In addition to the use of Bifidus growth factors for growth of bifidobacteria in a non-living environment, these growth factors of Bifidus voluntarily contained in food or a culture medium must also show positive and significant effects in living bodies.

The growth factors of Bifidus proposed by the invention make it possible to satisfy this demand and have effects, in living medium, greatly improved.

 An object of the present invention is to provide Bifidus growth factors which exhibit high activity in living medium and therefore meet the above demand.

 This object can be achieved by a composition of the present invention for promoting the growth of bifidobacteria containing as an effective component an oligosaccharide of the general formula Gal- (Gal) n -Glc, wherein Gal represents a galactose residue, Glc a glucose residue , and n an integer of 1 9 4.

 The present invention also provides a process for the manufacture of this composition for accelerating the growth of bifidobactdriescharacterisd in that lactose or a lactose-containing product is treated with β-galactosidase produced by Aspergillus oryzae.

The invention will be better understood and other objects, features, details and advantages thereof will appear more clearly in the following explanatory description made with reference to the graphs and exesples annexis given only as an example illustrating a mode. embodiments of the invention and in which
FIG. 1 is a graph showing the relationship between the saccharide yield and the reaction time, in the case where the lactose is treated with galactosidase
FIG. 2 is a graph showing the results of the artel filtration experiment by oligosaccharides;
FIG. 3 is a graph showing the results of the example 2 experiment
FIG. 4 is a graph illustrating the results of the experiment of example 3
FIG. 5 is a graph illustrating the results of the experiment of example 4; and
FIG. 6 is a graph showing the results of the experiment of example 5.

Most of the oligosaccharides mentioned above, and which are the effective constituents of the composition of the invention for promoting the growth of bifilobacteria or explicitly the growth agents of Bifidus, have been discovered by the inventors of the present invention during the course of the invention. their research on transfer reactions occurring with a hydrolysis reaction when lactose is subjected to the action of -galactosidase. Many reports have been made on these transfer reactions, for example by
Wallenfels in 1951.The transferred oligosaccharides, whose insulating properties have already been studied, are disaccharides and mainly Gal- (-1,2) -Glc, Gal- (ss-1,3) -Glc, Gal- ( B-1,6) -Glc, Gal- (-1,3) -Gal and Gal- (-1,6) -Gal and trisaccharides, mainly
Gal- (ss-1,6) -Gal- (ss-1,4) -Glc and Gal- (- ss-1,6) -Gal- (-1,6) -
Glc, in which the type of binding between galactosides is indicated in the parentheses. The presence of tetrasacharrides has been confirmed by Hubert et al., But the structure of these has not been clearly established. The production of polysaccharides comprising pentasaccharides has not been revealed. The contents of these reports are limited to the explanation of the transfer mechanism and to the study of oligosaccharide structures, so no communication has been carried out on the relationship. transferred oligosaccharides and growth of bifidobacteria.

 The oligosaccharide, a growth factor of Bifidus, of the general formula mentioned above (hereinafter, this specific oligosaccharide will be designated by the term oligosaccharidei may be produced by treatment of lactose with ss-galactosidase produced by Aspergillus oryzae After this treatment, the reaction medium comprises lactose which has not reacted and also galactose and glucose produced by hydrolysis.Thus, to obtain a product having a high activity as a growth factor of Bifidus, it is necessary to raising the oligosaccharide concentration in a suitable refining step after enzymatic treatment, or by a choice of enzymatic treatment conditions to produce as high a proportion as possible of oligosaccharides or a combination of these two steps.

 The method of the present invention for the manufacture of a composition for promoting growth of bifidobacteria is explained in detail below.

 Commercially available lactose can be used without special purification. It is also possible to use lactose-containing starting material of whole milk or creamed milk.

 For enzymatic treatment, the substrate concentration is from 10 to 50%, the pH from about 3 to about 8, the enzyme concentration from about 1 to about 100 units, and the preferred temperature from about 20 to about 500C.

The reaction time has a significant influence on the oligosaccharide yield. FIG. 1 illustrates the relationship between the reaction time T in hours and the saccharide concentration obtained in a specific enzymatic treatment. Curve G represents the glucose concentration variation as a function of the reaction time, the H curve the galactose curve, the O curve corresponds to the variation of the oligosaccharide concentration during the reaction and the curve
L corresponds to the lactose concentration during this reaction.

 The concentrations of glucose, galactose and oligosaccharide increase rapidly and linearly at the beginning of the reaction and then its variations describe fairly complex curves, it should be noted that the concentration of oligosaccharide decreases after a certain reaction time. The reaction time for which the oligosaccharide concentration is maximum also depends on other factors, but the maximum reaction time factor is preferably selected for the following experiments.

 The oligosaccharides contained in the reaction medium are separated from the other constituents by thin layer chromatography and determined by the Anthrone method.

 The enzymatic reaction can be completed by heating the reaction medium for about 5 to 10 minutes at a temperature above 90 ° C.

 The reaction medium produced by the enzymatic treatment can be used as a growth factor for Bifidus or in solution in dairy products, for example containing, with a convoluted concentration or dried in powder form. The reaction medium can be purified to increase the concentration of the effective component, mainly the oligosaccharides.

This purification can be carried out in many ways, such as, for example, by preliminary purification of the reaction medium with an ion exchange resin after passage through an activated carbon column for the adsorption of the oligosaccharides, and solution with an aqueous solution of 'alcohol. It is also possible to inoculate fermentation microorganisms of mono and disaccharides in the reaction medium for the culture and consumption of mono- and disaccharides to facilitate the isolation of oligosaccharides.

 An exemplary example of a composition of the invention containing oligosaccharides is described below with reference mainly to Example 1, described below and carried out under standard conditions.

 a) Molecular weight distribution.

Figure 2 shows the result of gel filtration with a Bio-Gel P-2. This curve represents the absorbance at 660 mm of the various fractions collected. The peaks A, B and C correspond respectively to tri-, grouse, pentasaccharides. The proportion of each of these compounds is calculated by measuring the area of each peak and is about 5596 for the trisaccharide, about 32% for the tetrasaccharide, and about 13% for the pentasaccharide and polysaccharides:
b) saccharide component.

 The lactose, the oligosaccharides (not separated in their constituents) and the tri- and tetra-acharides of the oligosaccharide are hydrolysed at 100 ° C. for 4 hours in a 0.5 N HCl solution. The same product is also hydrolysed at 500 ° C. 4 hours with 6-galactosi dase. The molar proportions of the saccharides obtained are summarized in Table 1 below for the different cases. This table shows that the glucose to galactose molar ratio for the tri- and tetra- and pentasaccharides of the oligosaccharide is 1: 2, 1: 3 and 1: 4, respectively.

Table 1.

<Tb>

<SEP> galactose / glucose <SEP>
<tb><SEP> Hydrolysis <SEP> Decomposition
<tb><SEP> acid <SEP> enzymatic
<tb> Lactose <SEP> 0.98 <SEP> 0.98
<tb> Oligosaccharide <SEP> 2.44 <SEP> 2.42
<tb> Trisacoharide <SEP> 1.94 <SEP> 1.92
<tb> Tetrasaccharide <SEP> 2.88 <SEP> 2.84
<tb> Pentasaccharide <SEP> 3.82 <SEP> 3.80
<Tb>
c) Mode of binding of the different saccharides.

The presence of lactose and Gal - (- 1,6) - has been confirmed
Gal in addition with glucose and galactose in a product obtained by partial acid hydrolysis of the main compounds of the trisaccharide (separated by activated carbon column chromatography). From this result, the glucose to galactose ratio in the fully hydrolyzed product being 1: 2, the estimated structure of the trisaccharide e is GalAss1,6) -Gal- ((ss-1,4) -Glc.

 Following the result of our similar analyzes of many oligosaccharide constituents, we have discovered that the oligosaccharide has the general formula mentioned above and that the galactosegalactose bonds are ss-1,3; ss-1,4 bonds with an r8 bond -1.6 predominant, and the galactose glucose bonds are ss -1,3 tus1,4 or p -1,6 bonds with a predominant p -1,4 bond.

 As discussed above, and to the extent that the oligosaccharides of the composition of the invention are isolated, it can be stated that the elemental oligosaccharides each act as growth factors of Bifidus, and of course a mixture of various oligosaccharides may act excellent way to facilitate the growth of doebifidobacteria.

 The particular advantage of the oligosaccharide contained in the composition of the invention and it operates with great efficiency not only in an environment outside the living body but also inside said living body.

 The particular growth-promoting operations of the bifidobacteria specific to the composition of the present invention have substantially no relation to the species of bifidobacteria and may be effective for the bifidobacterium breve, bifidobacterium bifidum, bifidobacterium infantis, Bifidobacterium adolescentis, etc. the human intestines.

 The composition of the invention may be reaction products obtained by treating highly purified oligosaccharides or other mixtures containing oligosaccharides such as lactose, or lactose-containing product with β-galactosidase. Fermented or powdered milk or similar foods or drugs comprising a secondary product such as a reaction product or other desired foods or drugs are added to the purified oligosaccharides.

 The present invention is better described with reference to the various examples in which the bifidobacterium is referred to as "B".

 These examples should in no way be considered as limiting the scope of the present invention, because many variations and modifications are possible.

 Example 1

 3.6 kg of lactose are dissolved in approximately 6 liters of hot water, and 50 ml of a 1M acetic acid buffer solution (pH 4.6), 100,000 units of galactosidase and a quantity of water for a solution of 10 liters. This solution reacts for 5 hours at 370C. This reaction solution is heated to denature the enzymes; the denatured proteins being extracted by filtration. The remaining reaction solution is poured into an anion exchange resin column and a cation exchange resin column. The filtrate is left in contact overnight in a 30x30 cm column filled with activated charcoal. The activated carbon is washed with 60 liters of deionized water to elute the monosaccharides. The activated charcoal is washed successively with 60 liters of water containing 5% of ethanol and 60 liters of water containing 50% of ethanol.

The eluate obtained from the elution solution containing 50% ethanol is concentrated to about 7 liters and axenically filtered on a filter membrane.
(0.2 r pore size), and passed back into an ion exchange column to obtain a clear saccharide solution. This solution is concentrated under vacuum until a viscous solution of about 2.5 liters is obtained which is filtered again on a filter membrane; the filtrate solution is prepared by freezing to give a white oligosaccharide powder (hereinafter referred to as TOS).

 Example 2

Six Fischer-specific axenic adult rats were administered different bacteria (including B. breve) extracted from human excreta. These bacteria settle in the intestines. We then administer
a) an aqueous solution of 3% lacturose,
b) an aqueous solution of 3% of TOS obtained in Example 1 above and,
(c) an aqueous solution of 3% lactose, daily for a period of one week following the order at c. These solutions are administered in amounts of 0.6 g per day as a solid product, and intermediate periods of one week are observed between each change of solutions administered. During this interval, the excrements of the rats are taken to determine the number of B. breve living bacteria contained therein. The result is shown in Figure 3. This figure represents the decimal logarithm of many bifidobacteria in 1g of exer- spleens according to the number of days from the first administration of a solution according to the invention. This curve is divided into different sectors that correspond to the water administration or solutions a, d and o described above.

 As shown in this figure, the number of B. breve is approximately multiplied by 10 during the period of administration of the TOS solution (b). A minor increase in the number of bacteria may be noted during the period of administration of the lactose solution (c), while this number does not vary during the period of administration of the lacturose solution (a).

 ExesDle 3.

 The following experiment was conducted with a series of six adult common fishser rats. The bifidobacteria used are B. breve or B. infantis which are obtained in such a way that the bacteria resulting from a culture on VL-G medium of 48 hours, and after centrifugal separation are suspended in a dairy culture medium and administered. under this form. The amount administered is 2 × 10 8 per day, expressed as the number of bacteria, or 20 ml per day of a 5% aqueous solution of TOS.

 a) the rats only drink the bacteria solution for a single day.

 b) each day of the second week only, each rat is given the solution of bacteria.

 c) During the third week, the bacterial solution and the TOS solution are administered simultaneously.

 d) During the fourth week only the TOS solution is administered daily.

 Figure 4 illustrates the results and the measured number of live bacteria of B bacteria (of the species administered) contained in rat excrement.

 This figure represents the decimal logarithm of the number of bacteria contained in 1 g of rat excrement as a function of the number of days elapsed since the first administration of a solution of bacteria. The curve D represents the number of B. breve and the curve E the number of B. infants. Moreover, this figure is divided into sectors each corresponding to the administration of the solution of bacteria (e) or solution TOS (d).

As illustrated in Figure 4, the number of
B. bacteria in the intestines increases remarkably when administering the solution of bacteria (e) and the solution TOS (d) and it is also remarkable that the number of bacteria can be maintained at a high value beyond interrupting the administration of the bacteria solution (e).

 Example 4

 The following experience is conducted with 5 healthy adult men. The bacterial solutions administered are the B. breve solutions obtained in a manner analogous to those described in Example 3.

The course of the experiment is
- first week: administration of the solution of bacteria (f) alone,
- second week: administration of the solution of bacteria (f) and solution TOS (d) (dose: 3 g per day),
- third week: administration of the solution of bacteria (f) and the solution TOS (d) (dose: 10g per day).

 Number of bacteria administered: 109 per day.

 Method of administration of TOS solution (d) extended in lukewarm water and drunk after lunch.

 Measurement: On the third, fifth and seventh day of each week, the number of B. breve bacteria is measured in the excrements of each man subjected to autest. An average value is calculated for each respective week.

 The results of these experiments are illustrated in Figure 5. This graph represents the decimal logarithm of the number of B. breve contained in lg of exctes as a function of the number of days elapsed after the first administration. This graph is divided into three sectors corresponding to the three weeks of experience and the different administrations of solutions.

According to this figure it is concluded that the number of
B bacteria is remarkably increased by administering the TOS solution (d).

 Example 5

 The following experiment is conducted with 5 healthy adult men.

Conduct of the experiment
- first week: no administration,
- second week: administration of 3 grams per day of a TOSÇd solution),
- third week: administration of 10 grams per day of a solution TOS (d),
The mode of administration of the solution TOS is identical to the mode described in example 4.

 Measurement: the total number of debifidobacteria contained in the excrements is measured as in Example 4.

 The results of this experiment are summarized in FIG. 6. Like FIGS. 3 to 5, this figure represents the logarithm of the number of bifidobacteria contained in excrement as a function of the number of days elapsed after the first administration, and each sector of this graph corresponds the different weeks of experimentation and the different products administered.

This figure shows that the number of live bifidobacteria in the intestineshimains can be increased by the administration of a TOS solution.

 Example 6

 1000 milliliters of skim milk reduced to 10 million units of Aspergillus oryzae s-galactosidase. The reaction mixture is kept at 40 ° C. for 2 hours and heated to deactivate the enzymes and sterilize the solution. The resulting product is transferred to a culture tank. After having inoculated a proliferation initiator of bifidobacteria, the culture is maintained at 37 ° C. for 24 hours, and sweeteners, etc. are then added, and the solution is then homogenized to form a fermented milk containing oligosaccharides and bifidobacteria.

 Of course, the invention is not limited to the embodiments described and shown which have been given by way of example. In particular, it includes all the means constituting technical equivalents of the means described, and their combinations, if they are executed according to its spirit and implemented in the context of the protection as claimed.

Claims (3)

 1. Composition for promoting the growth of bifidobacteria, characterized in that it contains as effective component an oligosaccharide of general formula Galal) nGlc, in which Gal represents a galactose residue, Glc a glucose residue and n an integer of 1 at 4.  A process for the manufacture of the composition for promoting the growth of bifidobacteria according to claim 1, characterized in that the lactose or a product containing lactose is treated with the β-galactosidase produced by Aspergillus oryzae.  3. Pharmaceutical composition especially used to increase the number of bifidobacteria in the human body, characterized in that it comprises a therapeutically effective dose of a composition according to claim 1, in a pharmaceutical vehicle acceptable for the body and compatible with him.