WO1990014773A1

WO1990014773A1 - Nutritional composition containing an enzyme

Info

Publication number: WO1990014773A1
Application number: PCT/FR1990/000374
Authority: WO
Inventors: Jean-Luc Barry
Original assignee: Institut National De La Recherche Agronomique (Inra)
Priority date: 1989-05-30
Filing date: 1990-05-29
Publication date: 1990-12-13
Also published as: FR2647638A1

Abstract

A composition containing a food product for human or animal consumption, said product containing a constituant which does not decompose when digested by the organism, and an enzyme capable of hydrolyzing said constituant. The invention further relates to a method for valorizing such a product for human or animal consumption.

Description

Food composition containing an enzyme

The present invention relates to the field of human or animal nutrition. It particularly relates to an original food composition and a process for enhancing the value of certain products already used in food.

It is well known that certain substances are resistant to digestion. Higher organisms have enzymatic complexes in their digestive tract that make it possible to degrade most of the organic compounds in food. By higher organisms is understood, within the meaning of this description, the man or the animals of production, company or laboratory.

Proteins, fats and most carbohydrates are quickly hydrolyzed into small molecules which are then absorbed and used by the body. However, certain substrates, mainly of a carbohydrate nature, escape digestion due to a deficiency of enzymes necessary for their hydrolysis.

It is mainly alpha-galactosides, parietal carbohydrates which are the major constituents of fibers, such as cellulose or hemi-celluloses, pectins, lignin. Other substances are also interested in this type of problem, even if these are found in lesser quantities or anecdotally, these include phytates, polyphenols, tannins, cutins, or even inulin found for example in large quantities in Jerusalem artichoke or in artichokes. Other substances forming part of the food bolus but having been introduced for a non-nutritional purpose, for example for the purpose of preserving or coloring the food, are also affected by this problem. Chemically these additives can be classified among gums, mucilages, βlginates and car raghénates.

Alpha-galactosides are defined as oligosaccharides of the general formula: (galactose) _n- sucrose. These small, polymers due to a deficiency of higher organisms in alpha-galactosidase, are not hydrolyzed in the small intestine. They therefore arrive intact in the colon where they are fermented under the action of numerous microorganisms with gas production. This results in two major drawbacks, on the one hand an overall reduction in the energy value of the food, and on the other hand this fermentation is a source of discomfort for humans or animals, and it can cause digestive disorders. A depolymerization of alpha-galactosides to galactose and sucrose easily metabolizable by the body would solve these problems. However, the destruction of these alpha-galactosides requires the use of enzymes. Indeed, these compounds are very resistant and cannot be altered by the usual chemical and physical processes.

In the Applicant's state of knowledge, the processing of animal feed with a view to the degradation of substances which cannot be digested under normal physiological conditions in the digestive tract has always taken place before the animal's ingestion of its food. By way of illustration, a process for the degradation of alpha-galactosides has been the subject of a European patent application No. 0.081.262. This relates to a strain of Saccharomyces cerevisiae capable of producing an alpha-galactosidasic activity as well as a method of preparation of this microorganism. The subject of this application is also a method of degrading the oligosaccharides and monosaccharides contained in soy milk, said method being characterized in that fermentation extracts of the strain claimed are brought into contact with soy. This prior document describes a reduction in soy milk of the rate of stachyosis, passing from 5.5% to 0.1%, and of raffinose, passing from 1.5% to 1%, after treatment.

All the degradation processes by enzymatic treatment of non-degradable substances, known to date, are treatments carried out before ingestion of the food by humans or animals. The fact of having such a degradation step inserted before ingestion represents a major drawback for the food industry, since in particular it requires large quantities of water; moreover, the monomers thus released can then be degraded before ingestion by the animal.

The present invention relates to a composition comprising at least one product for human or animal consumption and at least one enzyme, said enzyme allowing the in situ degradation of undigested substances under normal physiological conditions, as well as a process for the enhancement of 'A product for human or animal food, said product containing at least one component escaping digestion under normal physiological conditions.

The object of the invention is therefore a composition containing a product for human or animal food and an enzyme whose activity allows degradation in situ, that is to say in the digestive tract of man or of the animal substances which escape non-microbial digestion under normal physiological conditions. This enzyme at least partially resists degradation by proteases of the digestive system and has a significant activity of degradation of the substances of which it is specific. Said enzyme functions optimally under physiological conditions of the digestive tract and moreover does not present toxicity either for the man or for the animal.

The object of the invention is therefore a composition comprising at least one product for human or animal food, said product containing at least one constituent escaping degradation during non-microbial digestion by the body, said composition being characterized in that it further comprises at least one enzyme capable of hydrolyzing said constituent during digestion tion, said enzyme not being naturally present in the digestive system and being substantially without effect on said constituent before ingestion of the composition.

Within the meaning of the present description, the expression "enzyme not naturally present in the digestive tract" means that this enzyme is not synthesized, or is synthesized in insignificant quantity, by the organism under normal conditions of digestion. This expression therefore excludes enzymes originating from host microorganisms in the digestive tract.

It was by no means foreseeable that the enzyme found in dry form, within the food composition, could exert its activity effectively during digestion. It is known in this connection that, in many cases, the enzymes are degraded in the anterior part of the digestive system to such an extent that they lose their specific activity. Another difficulty is that the enzyme, even after having resisted the first stages of digestion, can exercise its activity under the pH and temperature conditions specific to it.

In a preferred embodiment of the invention, the composition is presented in a dry form and in particular in the form of flour or in agglomerated form, such as granules, or other forms of presentation usual in human or animal food. . This type of presentation is of notable interest for animal nutrition.

Products suitable for food can be in particular grains, seeds, oil cakes and other industrial by-products.

According to another embodiment of the invention, the enzyme is specific to the constituent (s) to be hydrolyzed in said product for human or animal food, and said enzyme is present in an amount effective for at least partially hydrolyzing said consti killing during digestion. According to another embodiment of the invention, this enzyme is chosen from specific enzymes whose activity is optimal under the conditions, in particular of pH and temperature, of digestion in the organism. This enzyme can be in a purified form, or can be mixed with other enzymes having other activities.

The enzyme can also be added in the form of a preparation of microorganisms capable of producing said enzyme.

For the purposes of the invention, it is possible to use an enzyme of any origin, or mixtures of such enzymes, the only condition being that the enzymatic preparation has the desired enzymatic activity. It is thus possible to use enzymes originating from bacteria, yeasts or fungi or even synthetic enzymes.

According to another embodiment of the invention, said enzyme is heat-resistant, in particular with a view to withstanding the temperatures involved in the preparation of the composition.

Said composition is prepared by methods known in themselves by those skilled in the art, by intimate mixing of the specific enzyme and of the product for human or animal consumption, optionally followed by agglomeration treatment.

The subject of the invention is also a process for the enhancement of a product for human or animal consumption, containing at least one constituent which escapes digestion in the body, said process being characterized in that one mixes intimately to said product an effective amount of at least one enzyme capable of hydrolyzing said constituent during digestion, said enzyme not being present naturally in the digestive tract and being substantially without effect on said constituent before ingestion of the composition. The description which follows gives by way of illustration and without limitation an example of application of the invention to alpha-galactosides,

EXAMPLE

Comparison of the efficacy of four diets in rats

a) Composition of diets

The following four regimes were used:

- the basic diet (B), the composition of which appears in Table 1, was designed so as to contain a minimum of fermentable substances and to cover the needs of growing rats. This regime served as the basis for the constitution of the other experimental regimes.

- the BE diet was obtained by adding 5% of the enzyme to the B diet.

the BR diet was carried out from the B diet by adding 5% raffinose.

- the BRE diet was formed by adding 5% raffinose and 5% enzyme to diet B.

Cellulose is a wood type cellulose

COLMALCEL. The composition of the mineral supplement (CM) is given in table 2 and that of the vitamin supplement (CV) is given in table 3.

In the case of the BR and BRE regimes, the raffinose was supplied by the SIGMA company (R0250) and the enzymatic preparation is a cellulase preparation of the Dutch company GI5T-BROCADES, containing a secondary alpha-galactosidasic activity.

The galactosidasic activity was determined by the method of BERGMEYER (1974 - Methods of enzymatic analysis - Académie Press, New York). The activity is assayed by release of phenol after action of the enzyme on para-nitro-phenyl-gaiactose (PNPGal). 0.4 ml of a 4 mM solution of PNPGal in 0.1 mM phosphate buffer at pH 6.5 and 0.4 ml of a 0.01 M enzyme solution in 0.1 phosphate buffer mM at pH 6.5 are incubated at 40 ° C for 0, 10, 20 and 30 minutes. Then 0.1 ml of the reaction medium is added to 0.6 ml of a 1M sodium carbonate solution. The optical density is read at 400 nm. The slope of the line obtained as a function of the duration of the incubation makes it possible to estimate the alpha-galactosidasic activity.

The enzyme used has an activity corresponding to the degradation of 52 mg of raffinose per mg of enzyme in 2 hours at 40 ° C and pH 6.5.

The raffinose contents were determined on BR and BRE foods. In order to take into account the changes likely to occur at 25 ° C during the measurement period, the food was kept for 3 days at this temperature before analysis.

The raffinose was extracted with an ethanol / water mixture (80/20) at boiling point and at reflux; the use of ethanol makes it possible to rapidly inactivate any alpha-galactosidasic activity. After extraction, the raffinose was determined by chroma togreph ie high pressure liquid according to the QUEMENER method (1988 - Improvement in the high pressure liquid chromatographic determination of amino-sugars and alpha-gaiactosides in Faba bean, lupine and pea. J. Agric. Food. Chem -36: 754 - 759).

The raffinose content was measured in BR and BRE foods in order to determine the rate of hydrolysis under the possible action of alpha-galactosidase. The raffinose content of the two foods is identical and equal to 5.15% of dry matter (DM): the enzyme does not hydrolyze raffinose in the food and the animals received identical amounts of raffinose.

During the course of the experiment, the food was kept cold at 4 ° C.

b) - Rat feeding cycles

The experiment was carried out on 4 male rats of the WISTAR breed receiving successively, in a random order, according to a Latin square device (Table 4), four regimes (PI, PII, PIII, PIV) differing in their alpha- galactosides and alpha-galactosidase. The animals were 8 weeks old at the start of the experiment and 15 weeks old at the end of the experiment.

For each animal, the experiment therefore includes 4 periods corresponding to each of the 4 diets. Each period includes an adaptation period of 9 days, carried out in individual growth cages and a measurement period of 3 days.

c) - Food consumption of animals

During the adaptation periods, the animals were fed ad libitum; food was distributed daily. Refusals were collected daily and grouped over the duration of the period. The DM contents of the food and the rejects were determined by drying at constant weight at 100 ° C.

During the measurement periods, the food was distributed at once at the rate of 110% of the 3-day consumption measured during the adaptation period. Refusals were collected at the end of the measurement period. The DM content of the food and the refusals have been determined.

The general behavior of the animals was very satisfactory. Food consumption, shown in Table 5, is close to all experimental diets and close to 20 g DM / d / animal. The diets were well accepted, with no detrimental effect from the presence of raffinose or the enzyme preparation. In particular, no case of diarrhea was noted. The growth of the animals (Figure 1) was measured for information. Not statistically different depending on the diet, it is on average 4.8 g / d / animal.

Figure 1 is a graph illustrating the influence of the nature of the diets on animal growth. The figures corresponding to the growth of the rats were plotted on the ordinate, expressed in grams calculated over three days.

The consumption index averages 4.4. This high value essentially reflects a low growth of the animals; the conditions of restraint imposed by the presence in the respiratory chamber may explain this result.

d) - Determination of intestinal gas production

The measurement of intestinal gas production following the experimental regimes was carried out by means of a respiratory chamber, the diagram of which is shown in Figure 1. The experimental animal is placed in an enclosure (1) where it has at its water at will and the food tested. Air renewal and homogenization are ensured by a diaphragm pump (2). Carbon dioxide (CO ₂ ) released is captured by bar kicking in an aqueous solution of potash (3). The water from respiration is fixed by passage over ACTIGEL silica gel (4). An oxygen electrode (5), placed at the outlet of the cage, measures the concentration of the oxygen circuit and makes it possible to keep it constant by action on a regulator (6) connected to a solenoid valve (7), itself connected to a cylinder of oxygen (8). The direction of gas flow in the device is indicated by the arrow (F).

During the measurement periods, the temperature was maintained at 25 ° C; the humidity, continuously monitored, was between 50 and 60%.

During the measurement periods, two daily air samples were taken from the breathing chamber for analysis of hydrogen and methane.

The hydrogen and methane concentrations were determined under the following conditions:

* gas chromatography (QUINTRON)

* column filled (2m, room temperature) * phase: molecular sieve

* carrier gas: medical air.

The slope of the regression of the gas concentration as a function of time was used to determine the theoretical concentration of each of the gases from intestinal fermentations after 3 days.

Since the production of hydrogen is the only one recognized as linked to the fermentation of indigestible sugars, the results relating to the production of methane have not been taken into account.

The hydrogen concentrations for the different regimes are shown in Table 6. The BR regime gives very high values and significantly different from the other regimes (p <0.05). The presence of alpha-galactosidase leads to an almost total disappearance of the fermentations, lowered to the level of the basic regime. The B and BE diets give very similar results: the enzyme preparation added to the diet has no effect on the parietal fraction of the food. Its only detectable action therefore consists in a degradation of the raffinose contained in the experimental regimes.

The major result of this experiment is the demonstration of a reduction in the fermentations linked to the ingestion of raffinose following the incorporation of an alpha-galactosidasic activity in the food. The absence of fermentation translates to a total hydrolysis of raffinose which no longer reaches the colon. Raffinose is not hydrolyzed in the food where it was found intact: the action of the enzyme is located in the digestive tract. It is also shown in this experiment that the level of incorporation of the enzyme and its conditions of action, close to those existing in the small intestine, led to a total hydrolysis of the added raffinose. The conditions of action of the enzyme, close to those existing in the small intestine and the level of incorporation of the enzyme into the diet are favorable parameters. This experiment demonstrates the activity in the digestive tract of an enzyme incorporated into the food.

The experience also shows that the alpha-galac tos idase studied resists degradation by endogenous enzymes and expresses its potential in the digestive tract, thus allowing, added to a diet to completely destroy the alpha-galactosides.

Claims

1. Composition comprising at least one product for human or animal food, said product containing at least one constituent escaping degradation during non-microbial digestion by the organism, said composition being characterized in that it also comprises at least one enzyme capable of hydrolyzing said constituent during digestion, said enzyme not being present naturally in the digestive system and being substantially without effect on said constituent before ingestion of the composition.

2. Composition according to claim 1, characterized in that it is presented in dry form.

3. Composition according to claim 2, characterized in that it is presented in the form of flour or in agglomerated form, such as granules, or other forms of usual presentation existing for human or animal food.

4. Composition according to any one of claims 1 to 3, characterized in that the product suitable for food is chosen from grains, seeds, oil cakes, and other industrial by-products.

5. Composition according to any one of claims 1 to 4, characterized in that the enzyme is specific to the constituent (s) to be hydrolyzed in said product for human or animal food.

6. Composition according to any one of claims 1 to 5, characterized in that said enzyme is present in a mixture of enzymes having various activities.

7. Composition according to any one of claims 1 to 6, characterized in that said enzyme is present in an amount effective to at least partially hydrolyze said constituent during digestion.

8. Composition according to any one of the claims cations 1 to 7, characterized in that said enzyme is chosen from specific enzymes under the conditions in particular of pH and temperature of digestion in the organism.

9. Composition according to any one of claims 1 to 8, characterized in that the component resistant to degradation is an alpha-galactoside and in that said enzyme is an alpha-galactosidase.

10. Composition according to any one of claims 1 to 9, characterized in that said enzyme is heat-resistant in particular in order to resist the temperatures involved in the preparation of the composition.

11. Process for the va lori sa ti on of a pro du it for human or animal food, containing at least one constituent escaping non-microbial digestion in the organism, said process being characterized in that it is intimately mixed said product produces an effective amount of at least one enzyme capable of hydrolyzing said constituent during digestion before the end of the small intestine, said enzyme not being present naturally in the digestive system and being substantially without effect on said constituent before ingestion of the composition.