WO2004080200A1

WO2004080200A1 - Probiotic micro-organisms and uses thereof

Info

Publication number: WO2004080200A1
Application number: PCT/CA2004/000368
Authority: WO
Inventors: Ben Saïda Ali HALIDI; Blaise Ouattara
Original assignee: Inatech International Inc.
Priority date: 2003-03-11
Filing date: 2004-03-11
Publication date: 2004-09-23
Also published as: CA2559183A1

Abstract

The present invention relates to a probiotic food compositions. Particularly, the probiotic composition of the invention comprises microbial spores. The spores can be additionally mixed with digestives enzymes or other biological or biochemical active agents useful for modulating the digestion in a human or an animal.

Description

PROBIOTIC MICRO-ORGANISMS AND USES THEEEOF

TECHNICAL FiΕ JD)

This invention relates to a new natural food additive for animal such as swine, poultry and the like. More particularly, the invention relates to a probiotic composition including a carrier fraction and microbial spores, a method for modulating digestion in a human or an animal and the use of microbial spores in the manufacture of the probiotic composition.

BACKGROUND ART Probiotic micro-organisms are micro-organisms which beneficially affect a host by improving its intestinal microbial balance (Fuller, 1989; Drago et al., 1997). Many probiotic strains are able to significantly reduce disorders caused by infectious agents including Escherichia coli, Listeria monocytogenes, Salmonella, Shigella, Yersinia, and Campylobacter (Con et al, 2001; Drago et al., 1997). Extensive literature is also available on the beneficial effect of probiotic micro-organisms intestinal metabolic activities (hypocholesterolemic effect, improvement of lactose digestion), inhibition of carcinogenesis, and stimulation of immune response (Klaver and ven der Meer, 1993). Reported beneficial effects on cattle, pigs, and chickens include improved general health, more efficient feed utilization, faster growth rate, and increased milk and egg production (Fuller, 1992). Probiotics used for human and animal nutrition include Lactobacillus, bifidobacterium, Bacillus, Treptococcus, Pediococcus, Enterococcus and yeast such as Saccharomyces cerevisiae and S. boulardii .

The mechanism by which probiotic bacteria achieves antipathogenic activity involves active colonisation of attachment sites on the mucosa of the gut wall and a resulting out competition of potential pathogens. Probiotics also produce i) organic acids (lactic and acetic acids) which show antimicrobial activity towards many microorganisms, decrease the pH and the redox potential in the gut, creating suboptimal conditions for pathogens, ii) metabolites such as volatile fatty acids and hydrogen peroxide which is antagonistic to some pathogens through inhibition of their ability to take up nutrients and ions , and iii) bacteriocins and bacteriocin-like products which exert their effects by disruption of energy production systems, macromoleculβ synthesis and membrane permeability.

Therefore there is considerable interest in including probiotic micro-organisms into foodstuffs. For example, many fermented milk products which contain probiotic micro-organisms are commercially available. Usually these products are in the form of yogurts and an example is the LC1.RTM. yogurt (Societe des Produits Nestle SA). Several infant and follow-up formulas which contain probiotic micro-organisms are also commercially available. Similarly, for animals, there has been interest in including probiotic micro-organisms into animal feeds.

Canadian patents 2,275,507, 2,222,758 and US patents 5,688,502 and 6,241,983 Bl disclose processes and food additives formulations based on probiotic bacteria and various other ingredients including prebiotics and enzymes.

However, to achieve probiotic status, microorganisms incorporated into foodstuffs must fulfill a number of criteria including ability to survive in the gastrointestinal tract and during prolonged periods of storage. Therefore, the stability of commercial probiotic strains is important to assure that stated levels of viable cells are delivered in probiotic products. The survival of ingested probiotics at different levels of the gastrointestinal tract differs between strains. Although some strains such as Bifidobacteria or Lactobacillus can pass through the entire gut at very high concentrations, it is well known that vegetative forms of probiotics bacteria are highly sensitive to environmental conditions.

During the last years, extensive research activities have been conducted to improve survival of bacteria, including, i) strain selection techniques, ii) addition of growth promoting factors of prebiotics such as starch and oligo-saccharides, iii) or buffering liquid and semi-solid foods. However, these have had only a limited success. Recent, developments of encapsulation techniques using various materials and methods led to more efficient effects by segregating bacterial cells from their adverse environment, thus potentially reducing cell loss. Unfortunately, the stability of encapsulated and microencapsulated probiotics are widely influenced by the type of materials used. From the report of Siuta-Cruce and Goulet (Food Technology, 2001, 55), only 58 % survival rate was found after 50 days in microencapsulated L. acidophilus stored at 40°C and 75% relative humidity.

Until now no disclosure is published on the development of food ingredients containing spores-forming products, for example, lactic acid bacteria combined with selected natural compounds for synergistic effects.

It would be highly desirable to be provided with new food or oral formulations for modulating the digestion in a human or animal. Preferably, the formulation should be a food composition.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide a probiotic composition comprising a carrier fraction and microbial spores.

Another aspect of the present invention is to provide a method for modulating digestion in a human or an animal comprising orally administrating to said human or animal a portion of a probiotic food composition as defined above in a quantity sufficient to obtain a desired level of modulation.

The modulation may improve intake of nutrients, or reduce or improve the growth of microorganisms. In accordance with the present invention there is provided the use of microbial spore in the manufacture of a composition to be orally administered to a human or an animal.

Alternatively, ,the probiotic composition of the present invention comprises at least one digestive enzyme. The carrier fraction can be a food component or composition.

It will be recognized from the present description that the spores can originate from bacteria or from fungi. The spores can also be spores of lactic acid bacteria and can be thermoresistant. It will be recognized to someone skilled in the art that the spores can be at least one of endospores or conidia, and can be from microorganisms selected from the group consisting of Lactobacillus, Bifidobacterium, Lactobacillus, Saccharomyses, and Bacillus.

Preferably, the composition of the invention contains the spores at a concentration between 3 to 8 log CFU/g.

In another aspect of the invention, the spores and digestive enzymes can be encapsulated or coated into a biodegradable or bioresorbable polymer or a biopolymer in order to protect them against digestive enzymes and conditions into the digestive tube.

The enzyme contained in the probiotic composition of the present invention can be selected from but are not limited to, the group of amylase, phytase, xylanase, glucanase, and galactosidase.

The probiotic food composition of the present invention may comprise in addition, for example, a biological or biochemical active agent, such as a food additive or a supplement, a vitamin or cofactors, an antibiotic, an antifungal compound, an antibody, an antimicrobial product, a pH neutralizing agent. For the purpose of the present invention the following terms are defined below.

The term "probiotic" as used herein is intended to mean live microbial feed supplements which beneficially affect the host animal by improving its intestinal microbial balance. It will be recognized by someone skilled in the art that the probiotic microorganisms of the present invention are found in the form of spores, or accompanied by microbial spores. A spore is generally defined in the art as being a type of reproductive cell produced by some plants, fungi and protozoa. Bacterial spores are thick-walled, dormant forms which are capable of surviving unfavorable conditions.

The term "endospore" as used herein is intended to mean a spore intracellularly formed. It will be understood that spore includes endospores and exospores. DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter with reference to preferred embodiments of the invention. This invention, may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The use of spore-forming lactic acid producing bacteria is an interesting alternative in the development of resistant probiotic food formulations. A number of species allocated to the genus Bacillus are known to produce lactic acid. Most of them have been isolated from soil, spoiled food or milk, and from intestine of crayfish. Due to their ability of forming endospores resistant to air drying and other stresses, and which enable them to survive long terms under adverse conditions, spore-forming lactic acid bacteria are good candidates for the development of heat and pressure resistant probiotic foods. Strains described so far belong to the genus Bacillus (B. coagulans, B. licheniformis, B. subtilis, B. stearotherrnophilus, B. smithii) and Sporolactobacillus (Spl. inulinus).

One embodiment of the present invention is to provide a food, probiotic composition comprising thermoresistant probiotic bacteria that can survive during the extrusion process and be used as supplements for extrudable food (swine, chicken, turkey).

Examples of suitable probiotic-organisms are spore-forming lactic acid bacteria including Bacillus subtilis, B. licheniformis, Bacillus cereus var toyoi, Lactobacillus sporogenes or a mixture of them. The probiotic micro-organisms are preferably in powdered dried form. It is also an object of the invention to encapsulate or microencapsulate vegetative forms of probiotic micro-organisms to increase their probability of survival in gastro-intestinal conditions (low pH, bile) and under adverse processing and storage conditions (high temperature and pressure, relative humidity). For example, the micro-organisms may be incorporated in a sugar matrix, a fat matrix or a polysaccharide matrix. Suitable vegetative probiotic micro-organisms for encapsulation or microencapsulation processes are Lactobacillus acidophilus, Lb rhamnosus, Lb salivarius, Lb reuteri, Bifidobacterium bifidum, B. animalis, B. longum, B. infantis, Enterococcus faecium, E. faecalis, Pediococcus acidilactis, Sacchaomyces cerevisiae, S. boulardii.

It is also an object of the present invention to provide a composition of enzymatic formulation that, when incorporated into animal food, results in an enhancement of the digestion of carbohydrate and/or protein fraction. In particular the enzymatic activity is directed toward soluble and insoluble arabinoxylan and β-glucan, starch, and antinutritional factors such as phytic acid. Suitable enzymes for that application include xylanase, β-glucanase, amylase, and phytase from different sources including bacterial and fungal. The enzymes may be protected against thermal inactivation by ii) incorporation into a crosslmked polymer matrix or ii) by co-incorporation of thermoprotectant compounds together with the selected enzymes. Examples of thermoprotectants are glycine, betaine, choline, sorbitol, mannosylglycerate, or a mixture of these compounds.

Another embodiment of the invention is to provide a new formulation of animal food supplement based on a synergistic and/or additional beneficial effect between probiotic, prebiotic, and other ingredients such as enzymes, vitamins, and minerals. The different constituents of the supplement may be used directly or may be treated following the thermoprotection procedure described previously.

There are disclosed herein nutritional supplements in powdered form comprising prebiotic, enzymes and a probiotic system comprising Lactobacillus acidophilus, Pediococcus acidilactici, Enterococcus faecium, Bacillus licheniforniis, Bacillus subtilis, and Bacillus toyoi. The powdered nutritional supplements contain at least 10⁸ viable organism grams. The bacteria in the supplement are stable and can withstand room temperature storage and extreme environmental conditions such as heat, moisture and pH. The probiotic bacteria in the supplement are also stable during the preparation of premixes and complete pelleted feed.

There is further provided according to the present invention a method of improving the intestinal digestibility, the growth rate, and the feed conversion of various animal species by incorporating different concentrations of the nutritional supplement. Animal species include but are not limited to pigs, chickens, turkeys. The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I Inhibition of pathogenic bacteria

Selected probiotic bacteria were evaluated for their inhibitory effects against pathogenic bacteria. Probiotic bacteria under study included one or several strains of the following species: Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus salivarius, Pediococcus acidilactici, Enterococcus faecium, Bifidobacterium bifidum, and Bifidobacterium longum. Target pathogenic bacteria included Escherichia coli O157:H7, Salmonella typhimurium, Salmonella enteritidis, Yersinia enterocolitica, Listeria monocytogenes, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Shigella sonei. Agar spot test was used to determine the inhibitory efficacy of probiotic bacteria. Antagonistic effects between probiotic strains were also evaluated. The results indicated that most of probiotic bacteria had consistent inhibitory effects against the target pathogenic bacteria. The most efficient of the strains appeared to be Lb acidophilus, Lb rhamnosus, P. acidilactici, and. Ec. faecium with more than 10 mm inhibition zone for all the pathogenic bacteria tested. Lb Rhamnosus INRH-12 did not have any antibacterial effect (only 2 mm inhibition zone). B. longum and B. bifidum were not efficient against Listeria monocytogenes.

Table 1 Inhibition zones in mm of pathogenic bacteria by selected probiotic bacteria

ST: Salmonella typhimurium, SE: Salmonella enteritidis, EC: Escherichia coli O157:H7,

YE: Yersinia enerocolitica, LM: Listeria monocytogenes.

(*) The experiment has been carried out in two independent replications.

EXAMPLE II

Tolerance to high temperature and pressure

A feed mixture is supplemented with powdered probiotic preparations, moistened and submitted to different temperatures (70 to 100°C) and times of exposure (0 to 1 min). Viable counts of probiotic bacteria were determined in control samples and samples were submitted to temperature and pressure treatment in order to evaluate the residual percentage of viable cells.

The test for high temperature tolerance was performed at Comptoir Agricole de St-Hyacintlie (Quebec, Canada). The probiotic formulation containing Bacillus subtilis and Bacillus licheniformis was incorporated in poultry food and pelleted at a temperature of 78°C for 45s. Samples before and after heat treatment were collected for viable probiotic numeration. The microbiological analysis were done by Bodycote Essais de Materiaux Canada Inc. (Pointe-Claire, Quebec, Canada). The results obtained are presented in Table 2. No significant reduction of viability was observed between samples submitted to heat treatment and control samples. Probiotic counts before heat treatment was 5.59 ± 0.16 lg CFU/g compared to 5.02 ± 0.19 lg CFU/g after heat treatment.

Table 2

Heat tolerance of probiotic formulation containing Bacillus subtilis and Bacillus licheniformis

(*) The experiment was performed in three independent replications. EXAMPLE HI

Stability during preparation and storage of probiotic supplement and complete feed

Probiotic supplement and complete animal feed containing the probiotic supplement are evaluated for stability during storage. The test preparations consisted of both pelleted and non pelleted feed. Samples are stored at 2 different temperatures (25°C and 45°C) and relative humidity (56 % and 100 %). Residual viable counts were performed monthly or every two months during a total storage period of one year.

Two probiotic supplements were submitted to a stability test during storage at ambient temperature (25 ± 2°C) during 6 mois: i) formulation 1 contained a mixture of vegetative probiotic bacteria Lactobacillus acidophilus, Enterococcus faecium and Pediococcus acidilactici; ii) formulation 2 contained sporulated probiotic bacteria Bacillus subtilis and Bacillus licheniformis. The initial bacterial concentration was determined and other microbial tests were performed after 2 weeks 4 weeks and 6 months. The results of the stability tests are presented in Table 3.

Table 3 Stability test during storage of two probiotic formulations.

Results are expressed in log CFU/g of viable probiotic bacteria in the formulations

EXAMPLE IV

Growth performance improvement

Trials are performed using 1 day old chickens and young piglets until weaning.

The animals were separated in 3 groups. One group is fed with complete chicken or piglets feed incorporated with probiotic supplements at the concentration of 1 kg/ton of animal feed. The second group is fed with animal feed supplemented with an antibiotic growth promoter. The third group is fed with the animal feed without any additive. The animals were weighted periodically and the amount of feed consumed was recorded is order to calculate the growth performance and the food conversion rate in each treatment group.

Experiment 1

Experimental design

The experiment was carried out with Hywhite Ross male chickens (hatchery Scott Jonction, Quebec, Canada) fed with a maize-based diet. Day old chickens were randomly divided into four (4) dietary treatments groups. Group 1 was fed with diet containing antibiotic supplement (virginiamycine, 11 ppm), group 2, group 3 and group 4 were fed with Nutraflore-F™ at 1, 5, or 10 kg/ton, respectively. Each treatment group comprised 25 chickens and the overall experiment was done in 3 replications. For all the birds, the diets were given ad libitum (day 1-21 starter diet; day 22-35 grower diet, and day 36-42 finisher diet). At day 1, 7, 18, 28 and 42, five (5) the chickens were randomly selected in each group and weighted.

Statistical analysis

Analysis of variance (ANOVA) was done using the GLM procedure of the SAS statistical package (SAS Institute, Gary, NC) and the Duncan test was used to differentiate treatments. Differences between averages were considered significant when p < 0.05.

Results

The results of experiment 1 are summarized in table 3. Treatment 3 significantly improved the body weight compared to control group at day 7, 18, and 28. Table 3

Treatment l=Control, 11 ppm virginiamycine; Treament 2=Nutraflore-F, 1 kg/ton; Treament 3=Nutraflore-F, 5 kg/ton, treatment 4=Nutraflore-F, 10 kg/ton. Averages in the same row followed by different letters (a, b, ab) are significantly different p < 0.05.

Experiment 2

Experimental design

Experiment 2 was performed only with the most interesting concentration of Nutraflore-F™ obtained from experiment 1 i.e 5 kg / ton. Two treatment groups comprising 25 chicken each were tested: treatment 1 (control, 11 ppm virginiamycin) and treatment 2 (Nutraflore-F™, 5 kg/ton). The experiment was performed in four separate replications. For all the broilers, the diets were given ad libitum (day 1-21 starter diet; day 22-35 grower diet, and day 36-42 finisher diet). At day 1, 7, 18, 28 and 42, five (5) chickens were randomly selected in each group and weighted. For experiment 2, the global weights of all the chicken was also taken and divided by the number of chickens to obtain the average body weight in each experimental group.

Statistical analysis

Analysis of variance (ANOVA) was done using the GLM procedure of the SAS statistical package (SAS Institute, Gary, NC) and the student t-test was used to differenciate treatments. Differences between averages were considered significant when p < 0.05. Results

For both types of measurement ((Five broilers randomly selected or average of the group body weight), treatment 3 (5 kg of Nutraflore-F^TM/ton) resulted in significant improvement of growth performance at day 18 Table 2). After day 18, no significant difference was observed between control and treatment 3.

Table 4

Effect of treatment 3 (Nutraflore-F™, 5 kg/ton) on the weight gain of Hywhite Ross male broilers.

Treatment l=Control, 11 ppm virginiamycine; Treament 3=Nutraflore-F, 5 kg/ton. For each type of measurement (Five broilers randomly selected or Average of the group body weight), averages in the same row followed by different letters (a, b) are significantly differentρ < 0.05.

Experiment 3 The experiment was carried out with Hywhite Ross male chickens (Hatchery

Scott Jonction, Quebec, Canada) fed with a maize-based diet. Day old chickens were randomly divided into two dietary treatments, each comprised of 75 birds. Each treatment was replicated with 3 subgroups. One group received a diet supplemented with the viable micro-organism supplement (Nutraflore-R, 1 kg per tonne) containing sporulated probiotic bacteria Bacillus subtilis and Bacillus licheniformis. The second group (control group) was fed with an antibiotic supplement (virginiamycine, 11 ppm). The diets were given ad libidum (day 1-21 starter diet; day 22-35 grower diet, and day 36 - 42 finisher diet). Weight gain of 5 birds randomly selected and global weight gains of all the birds were recorded. Results

The results, as shown in Table 5, reveal that the composition of the present invention significantly allows to improve the growth of birds.

Table 5

Assessment on chicken

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

CLAIMS:

1. A probiotic composition comprising a carrier fraction and microbial spores.

2. The probiotic composition of claim 1 comprising at least one digestive enzyme.

3. The probiotic composition of claim 1, wherein said carrier fraction is a food component or composition.

4. The probiotic composition of claim 1, wherein said spores are selected from bacteria or fungi.

5. The probiotic composition of claim 1, wherein said spores are spores of lactic acid bacteria.

6. The probiotic composition of claim 1, wherein said spores are thermoresistant.

7. The probiotic composition of claim 1, wherein said spores are at least one of endospores or conidia.

8. The probiotic food composition of claim 1, wherein said spores are from microorganisms selected from the group consisting of lactobacillus, bifidobacterium, lactobacillus, saccharomyses, and bacillus.

9. The probiotic food composition of claim 1, wherein said spores are in concentration between 0.5 to 80% (w/w).

10. The probiotic food composition of claim 1, wherein said spores are coated with a polymer or a biopolymer.

11. The probiotic food composition of claim 2, wherein said digestive enzyme is encapsulated into a biodegradable or bioresorbable polymer or a biopolymer.

12. The probiotic food composition of claim 2, wherein said enzyme is selected from the group consisting of amylase, phytase, xylanase, glucanase, and galactosidase.

13. The probiotic food composition of claim 1 comprising a biological or biochemical active agent.

14. A method for modulating digestion in a human or an animal comprising orally administrating to said human or animal a portion of a probiotic composition as defined in claim 1 in a quantity sufficient to obtain a desired level of modulation.

15. The method of claim 14, wherein said modulation consists of improving intake of nutrients, or reducing or improving the growth of microorganisms.

16. Use of microbial spores in the manufacture of a composition for oral administration to a human or an animal.