CA2275507A1 - Prebiotics and probiotics - Google Patents
Prebiotics and probiotics Download PDFInfo
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- CA2275507A1 CA2275507A1 CA002275507A CA2275507A CA2275507A1 CA 2275507 A1 CA2275507 A1 CA 2275507A1 CA 002275507 A CA002275507 A CA 002275507A CA 2275507 A CA2275507 A CA 2275507A CA 2275507 A1 CA2275507 A1 CA 2275507A1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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Abstract
A method of enhancing a resident population of lactic acid-producing microorganisms, preferably lactobaccillus, in the gastrointestinal tract of an animal, the method comprising providing to the animal a beta-glucan, compounds derived therefrom, or mixtures thereof, either alone or in combination with other prebiotics and/or with one or more probiotic microorganisms such that upon ingestion the beta-glucan passes through the gastrointestinal tract substantially unutilised until it reaches a site in the gastrointestinal tract where it is utilised by a resident population of microorganism and/or the introduced probiotic microorganisms, if provided, thereby causing an increase in number and/or activity of the resident population of lactic acid-producing microorganisms and the probiotic microorganisms, if provided.
Description
Prebiotics and probiotics Technical Field This invention relates generally to the use of prebiotic and probiotic preparations to improve intestinal health and conditions related to microbial populations in the gastrointestinal tract of host humans and animals, and thereby improve the health and well being of the host.
Background Art The concept of probiotics was in use in the early 1900s, however, the term was only coined in 1965 and has subsequently evolved and numerous definitions have been proposed. Initially it was used to refer to the stimulation of the growth of one microbe by another i.e. the opposite of antibiotic. Today it is generally agreed that a probiotic is a preparation "of live microorganisms which, applied to human or animal, beneficially affects the host by improving the properties of the indigenous microbiota". Lactic acid bacteria and particularly lactobacilli are frequently used as probiotics.
Bifidobacteria are also extensively used as probiotics. Furthermore, microbes of gastrointestinal origin or strains related to such microbes may also be used as probiotics.
The gastrointestinal (indigenous) microbiota contributes significantly to the health and well being of the host. It is a complex and diverse population which can reach up to 101' bacteria in an individual and may have both beneficial and harmful effect; on the host. Some of the beneficial and harmful effects of the gastrointestinal microbiota are summarised in Table 1. It is envisaged that these parameters may be influenced by probiodc administration.
The gut microflora of an healthy subject protects the host from pathogen invasion, however, in the young, the elderly, and the compromised patient, this protective barrier is less effective. An individual can be compromised to various degrees, varying from minor stress related events to extreme cases such as in immunocompromised patients and patients undergoing therapy.
The term prebiotic has been coined to refer to carbohydrate preparations that may be preferentially used to provide a carbohydrate source for desirable microbes.
Background Art The concept of probiotics was in use in the early 1900s, however, the term was only coined in 1965 and has subsequently evolved and numerous definitions have been proposed. Initially it was used to refer to the stimulation of the growth of one microbe by another i.e. the opposite of antibiotic. Today it is generally agreed that a probiotic is a preparation "of live microorganisms which, applied to human or animal, beneficially affects the host by improving the properties of the indigenous microbiota". Lactic acid bacteria and particularly lactobacilli are frequently used as probiotics.
Bifidobacteria are also extensively used as probiotics. Furthermore, microbes of gastrointestinal origin or strains related to such microbes may also be used as probiotics.
The gastrointestinal (indigenous) microbiota contributes significantly to the health and well being of the host. It is a complex and diverse population which can reach up to 101' bacteria in an individual and may have both beneficial and harmful effect; on the host. Some of the beneficial and harmful effects of the gastrointestinal microbiota are summarised in Table 1. It is envisaged that these parameters may be influenced by probiodc administration.
The gut microflora of an healthy subject protects the host from pathogen invasion, however, in the young, the elderly, and the compromised patient, this protective barrier is less effective. An individual can be compromised to various degrees, varying from minor stress related events to extreme cases such as in immunocompromised patients and patients undergoing therapy.
The term prebiotic has been coined to refer to carbohydrate preparations that may be preferentially used to provide a carbohydrate source for desirable microbes.
Influences of the human intestinal microbiota on the host Beneficial effects Harmful effects Inhibition of pathogens Constipation Stimulation of immune system Diarrhoea Synthesis of vitamins Infections Aid in digestion Liver damage Produce metabolic fuel for enterocytesCancer Maintain stability of ecosystem Flatulence Metabolise drugs Irritable bowel Ulcerative colitis Crohn's disease Glucans are cell wall polysaccharides with glucose as the main component sugar having a beta-bonding with mostly beta 1,3 links and also beta 1,6 links. While originally isolated from black fungi, glucans have since been found in large quantities in yeast cell walls. Glucans have been shown to propagate indigenous bifidus bacteria in intestines and have been proposed to be useful as a pharmaceutical agent for conditioning the intestine. It has been reported that glucans can function as an immunostimulator (e.g. Brattgjerd et al 1994) when intraperitoneally administered.
Probiotic bacteria have been described to exert antimicrobial effects which refers to the actions of the probiotic preparation on another microbe or group of microbes. Some probiotic bacteria are directly applicable for enhancing resistance against intestinal pathogens, prevention of diarrhoea and constipation (reviewed by Fernandes et al. ; 1987; Katelaris, 1996;
Sanders, 1994). The types of interactions include competitive colonisation as well as adhesion and growth inhibition.
Competitive colonisation refers to the fact that the probiotic strain can successfully compete with the pathogen for either nutrients or the site of colonisation. As many gastrointestinal pathogens attach to the intestinal mucosa as the first step in infection, it would be beneficial to the host if this adhesion could be inhibited . There are reports that lactobacilli produce Received 31 March 1999 components which inhibit attachment of enterotoxigenic Escherichia coli to intestinal mucosa (Blomberg et al., 1993), however, there is no evidence as yet that this occurs in the digestive traca. In addition, various compounds produced during growth of the probiotiic have beef shown to inhibit pathogen growth (reviewed by Fernand.es et al., 1987; Klaenhammer, 1988;
Mishra and Lambert, 1996). These include organic acids such as lactic and acetic acid, reuterin and bacteriocins ('ragg et al., 1976). The organic acids lower the pH and thereby can indirectly affect growth of the pathogen. In addition, the lactic and acetic acids produced by these organisms can be toxic to microbes. Reuterin which inhibits the growth of a very broad range of cells (Lindgren & Dobrogosz, 1990), i~;s produced by Lactobacillus reuteri when grown in the presence of glycerol. Numerous bacteriocins have been reported to be produced by lactobacilli e.g. Acidophilin, Acidolin, Lactocidin, Bacteriocin, Bulgarican, Lactolin, Lactobacillin and Lactobrevin (reviewed by z5 Fernandes et al., 1987; Klaenhalnmer) 1988). Bacteriocins can either have a very broad range of activity or alternatively specifically inhibit the growth of a very limited range of closely related microbes. For example, Lactobacillus sp exhibited specific antagonistic effects towards Clostridium ramosum (McCormick & Savage, 1983).
The present inventors have surprisingly found that beta-glucans can be used to influence pathogen adhesion, alter microbial populations in the gastrointestinal tract, improve the effecaiveness of probiotic compositions, and promote growth of a probiotic microorganism. A range of crude and pure preparations of beta-glucans have been shown to be effective.
Disclosure of the Invention In a first aspect, the present invention consists in a method of enhancing a resident population of lacl:ic acid-producing microorganisms in the gastrointestinal tract of an animal, the method comprising providing to the animal a beta-glucan derived from cell sacs in combination with other prebiotics and/or with one or more probiotic microorganisms, such that upon ingestion, the beta-glucan passes through the gastrointestinal tract substantially unutilised until the beta-l;lucan and the probiotic microorganisms reach a site in the gastrointestinal tract where the beta-glucan is utilised by the resident population of lactic acid-producing nucroorganisms and the probiotic microorganisms, thereby causing an increase in number and/or activity of the resident population of lactic acid-AMENDED SHE
. _.....___ .._~._.~E~A~ ' ~ _ . .
.t Received 31 March 1999 producing microorganisms and the probiotic microorganisms, wherein the combination of the beta-glucan and thf: one or more probiotic microorganisms provides a synergistic effect upon the increase in number and/or activity of the resident population of lactic acid-producing microorganisms.
In a second aspect, the present invention consists in a method of suppressing an undesired population of microorganisms in a total microorganism population in the gastrointestinal tract of an animal, the method comprising providing to the animal a beta-glucan derived from cell sacs in combination with other prebiotics and/or with one or more probiotic microorganisms, such that upon ingestion, the beta-glucan passes through the gastrointestinal tract substantially unutilised until the beta-glucan and the probiotic n>scroorganisms reach a site in the gastrointestinal tract where the beta-glucan is utilised by a resident population of lactic acid-producing microorganisms and the probiotic microorganisms thereby causing an increase in number and/or activity of the resident population of lactic acid-producing microorganisms and the probiotic microorganisms and suppressing the growth and/or activity of the undesired population of microorganisms, wherein the combination of the beta-glucan and the one or more probiotic microorganisms provides a synergistic effect upon the increase in number and/or activity of tlhe resident population of lactic acid-producing microorganisms.
In a preferred form, the undesired population of microorganisms is enteric pathogens. As will be appreciated, there are a large number of pathogens including Graln negative bacteria like Salmonella, Escherichia coli, Campylobacter, Helicobacter, Vibrio, and Pseudomonas; Gram positive bacteria like Clostridium; viruses like Norwalk virus, Norwalk-like viruses and Rotavirus; and protozoa like Crytosporidium, Entamoeba, Giardia, and Dientamoeba. The present invention vvould be suitable for the suppression of 3o many of these pathogens in the gastrointestinal tract of animals.
Suitable beta-glucans include those originating from (a) plants including cereals such as oats and barley, (b) fungi, (c) yeast, and (d) bacteria. In addition, nucrobial cell will preparations and whole cells rich in beta-glucans are also suitable sources for beta-glucan preparations useful for the present invention. Monomer residues in glucans can have 1-3 and 1-4, or 1-3 and 1-6 linkages (that is the mononner units are joined through 1.3, 1,4 or . ._..__.E:~ ~-'~ ....._ . . . _..
IPEAIAU
Received 31 March 1999 1,6 bonds) and the percent of each type can vary. Preferably, beta-glucans derived from yeast, particularly from 5'accharomyces, preferably Saccharomyces cerevisae, are used for the present invention. It will be appreciated, however, that other beta-~;lucans would also be suitable. A
5 concentration of up to about 10% when combined with other prebiotics and/or probiotic nucroorganisms can be used. One percent has been found to be particularly suitable.
The resident lactic acid-producing microorganism is preferably a lactobacillus, and more preferably Laci:obacillus fermentum.
1o In a preferred form, the present :invention further includes one or more oligosaccharides, polysaccharides or other prebiotics. A concentration of oligosaccharides, polysaccharides or other prebiotics of about 0.001 g to 10 g per kg body weight per day is preferred.
Preferably, the probiotic microorganism is selected from the group consisting of lactic acid-producing microorganisms, bifidobacterium, yeast, and mixtures thereof. Preferably, the lactic acid-producing microorganism is a lactobacillus, more preferably Lactobacillus fermentum. Preferably, the yeast is a SaccharomycE~s sp. Typical concentration range of probiotic microorganisms is 103 to 1013 cells per day.
Although oligosaccharides have been shown to promote bifidobacteria numbers in animals, prior to the present invention, it was thought that lactobacillus numbers cannot be changed in a similar manner. Gibson et al 1995 found that oligofructose and inul:in, when fed to humans, selectively stimulated the growth of bifidobacteria without influencing the numbers of lactobacillus. From these results and observations by others in the field, it would not be expected that beta-glucans would positively effect the growth and/or activity of lactic acid-producing; microorganisms, particularly lactobacillus, in vivo as found by the present inventors.
It should be further emphasised that the present invention relates to 3o the synergist relationship between the beta-glucans and probiotic preparations both in terms of proliferation of the probiotics themselves and inhibition of pathogens. The synergy between beta-glucans and probiotics preparations results in selective increase in the numbers of specific beneficial microorganism including probiotic strains in the presence of glucans. Furthermore, a reduction in numbers of pathogens in the presence of beta-glucans and additional probiotics is surprisingly significantly greater . _ _._ ___ _AMEN17SD.SMEE~- . . . _..
IF~EA/AU
Received 31 March 1999 ~6 additive effects of beta glucans or probiotics individually, in reducing pathogen numbers. This enhancement is not explainable by an increase in the numbers of some of the probiotics since the synergistic effect is demonstrable with probiotics which are not enhanced by the inclusion of glucan".
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples and drawings.
Brief Description of Drawings Figure 1 shows the results of Iac;tobacillus in the gastrointestinal tract of animals fed beta-glucan or control diet; and Figure 2 shows faecal beta-glucanolytic microorganisms of animals fed beta-glucan or control diet. Analysis o~f beta-glucanolytic activity on agar plates containing beta-glucan fTOIIl yeast cell sacs.
Modes for Carrying Out the Invention This has been studied by enumerating coliforms, bifidobacterium and lactobacilli in vitro in the presence of beta-glucan. The beta-glucan used in this example was prepared from yeast cell sacs. More specifically, aliquots (1 n ll) of human faecal homogenates (10 ~; per 100 ml diluent) were added to diluted WC broth (diluted 50:50 with (1.05M phosphate buffer) to which were added the beta-glucan and a lactobacillus or bifidobacterium strain and various combinations. For each of the combinations, parallel tubes were prepared with one set being inoculated with Bifidobacterium spp or Lactobacillus spp. All mixtures were tlhen incubated for up to 24 hours and bacterial numbers enumerated. Results (Table 2) are expressed as the numbers of coliforms, total lactobacilli., total bifidobacterium, or total anaerobes when enumerated as colony forming units. It can be seen that coliform numbers are unchanged or reduced, while numbers of bifidobacteria and lactobacilli can be increased up to 10-fold, and selected groups within a genus can be enhanced.
AMENDED SHEET
. . _ EB'ect of 1% beta-glucan on Lactobacillus and Bifidobacterium Bacterial numbers (CFU/ml Lactobacillus Culture mix Coliforms Total Large Bifidobacterium Colon Faecal homogenate 3.2 x 10 3.4 x 10 1.2 x < 2 (FH) 10' FH + 1% glucan 5.2 x 106 5.3 x 105 4.8 x < 2 FH + Bifidobacterium (SUB) NA 1VA NA 1.0 x 106 FH + SUB + 1% glucan NA IVA NA 1.2 x 10' FH + L. fermentum NA 2.8 x 205 2.8 x NA
(Lf) 105 FH + Lf + 1% glucan NA 1.2 x 106 1.2 x NA
NA - not analysed This example demonstrates that dietam supplementation with beta-glucan stimulates gastrointestinal populations of Lactobacillus. In this study.
animals (8 per group) were provided either bei~a-glucan diet or control diet (Table 3). The beta-glucan used in this examF~le was prepared from yeast cell sacs. After 6 weeks feeding. animals were euthanised and luminal content collected from stomach, ileum, caecum and colon. Collected material was homogenised and diluted in Wilkens-Charlgren broth (Oxoid). Dilutions were spread on to Rogosa gear (Oxoid) and incubated anaerobically for 48 h.
Beta-glucan-rich diet resulted in significant increase (P<0.0005) of Lactobacill us, particularly in the gastric region where an 100 fold increase was detected (Fig. 1). The increase of lactobacilli numbers in the subsequent intestinal tract corresponded to about 1.5 log units.
Composition of beta-glucan and sucrose diet Ingredient Amount Amount (Glucan diet) Control diet Beta-glucan 450 0 Sucrose 150 600 Casein 200 200 Sunflower 25 25 Canola oil 25 25 Gelatin 20 20 Wheat bran 100 100 Choline chloride 2 2 Methionine 3 3 Standard mineral 35 35 mix Standard vitamin 10 10 mix In addition to studying effects on microbial growth, effects on adhesion have been investigated. Desirable bacteria such as lactobacillus, as well as undesirable pathogenic E. coli and Salmonella sp have been grown either in a standard laboratory broth or in this broth supplemented with beta-glucans. The beta-glucan used in this example was prepared from yeast cell sacs. . Bacterial suspensions were incubated with mucosal pieces from various regions of the gastrointestinal tract. These mucosal pieces were either used directly or after pre-treatment with the beta-glucan. Results are shown in Table 4. While adhesion of the pathogen was adversely affected, the adhesion of the lactobacillus was increased by the presence of the beta-glucan.
Effect of beta-glucans on adhesion Bacteria adhering (per mg tissue) Bacteria Control tissueTissue + 1% lucanReduction (%) S. typhimurium 1.69 x 108 5.53 x 10' -- 65 E. coli K88 6.07 x 10' 3.43 x 10' --- 50 L. ermentum 5.37 x 108 6.2 x 108 0 This example demonstrates that dietary supplementation with beta-glucan increases resistance to gastrointestinal infection by Clostridium difficile and S, typllimurium and that this effect is enhanced when glucan-treated mice were also orally dosed with. Saccharomyces sp cell suspension.
The beta-glucan used in this example w;is acid and alkali washed cell sacs of yeast. The Saccharomyces sp alone reduced numbers of C. difficile to some extent and markedly reduced S. typhim urium numbers, Groups of animals (4 groups, 8 animals per group) were provided either beta-glucan diet or control diet (Table 3). After feeding diets far four weeks, four groups were supplied drinking water containing cefoxitin (0.0;32 g/L) and D-cycloserine (1.0 g/L) and subsequently challenged with C. dif~icile (10-8 CFU). Another four groups were suppled drinking water containing streptomycin (5 g/L) and neomycin (5 g/L). These animals were subsequently challenged with S. typhimurium (10-' CFU). After pathogen challenge, animals were orogastrically dosed with either Saccharnmyces sp (strain FII 542900)(108 CFU in YPG broth), or with fresh YPG broth. Development of infections were monitored daily by analysis of faecal S. typhimurium or C. difficile. Faecal material was homogenised and diluted in Wilkins-Charlgen broth (Oxoid).
Dilutions were spread on to either YSA (Oxoid) or C. difficile selective agar (Oxoid) for analysis of faecal S. typhimuuum and or C. difficile, respectively.
Animals fed a beta-glucan rich diet were significantly more protected against colonisation by C. difficile (Table 5), The faecal levels of C.
difficile were, in animals fed beta-glucan, about 2 log units less than what was detected in animals fed the control diet, one day post challenge (P<0.05). A
non-significant reduction of S. typhimurium numbers was noted in beta-:LO
Received 31 March 1999 glucan treated mice, while a log 3 reduction in mice dosed with Saccharomyces sp alone (P<0.05). The combination of beta-glucan diet and Saccharomyces sp oral administration resulted in a significant reduction (P<0.05) in faecal numbers of both pathogens when compared to both the control diet and the Saccharomyces sp dosed mice (Table 5). This reduction corresponded to 3 and >4 log units for C. difficile and S. typhimurium, respectively. This corresponded to a marked improvement in weight loss and other signs of severe infection in animals treated with the combination of beta-glucan and Saccharomyces sp. In summary, when the beta-glucan is s0 provided together with probiotic preparations e.g. Saccharomyces, a surprising synergistic enhancement in the protective effect against pathogens is noted where greater than log 4 reduction is noted for the combination of beta-glucan plus probiotics, while the beta-glucan alone or the probiotic alone reduce pathogen levels by log 1-2. It can be concluded that a beneficial s5 synergistic effect can be achieved by beta-glucan alone and enhanced by the combinations of beta-glucan and Sacci'laromyces sp.
TAttLE 5 Reduction of pathogens by oral administration of Saccaromyces sp to beta-20 glucan fed mice that were chal:(enged with either C. difficile or S. typh~imurium Reduction to CFLT
er faeces Diet C. di 'cile S. ty himurium beta-glucan 1.5 * <1 beta-glucan and Saccharomyces sp 3 * * > 4 Saccharom ces s 0.8 3 * Significantly different from beta-glucan free diet (P<0.05) 25 * * Significantly different from beta-glucan-free diet and Saccharomyces sp alone.
~MEi'~DED SHEET
. _ ~._.___~ ,._~.._._. ~,p~AU.. .~._ . .. __ This example demonstrates that:
Received 31 March 1999 (i) beta-glucanolytic microorganisms a:re present in the gastrointestinal tract;
(ii) dietary supplementation with beta-glucan stimulates gastrointestinal populations of beta-glucan degrading microorganisms (includes lactic acid-producing microorganisms).
In this study, animals (8 per gro,ap) were provided either beta-glucan or control diet (Table 3). After 4 weeks fE;eding, faecal material was collected, homogenised and diluted in Wilkins-Charlgen broth (Oxoid). Diluted material was spread on to beta-glucan agar (Table 6) and incubated anaerobically for 75 h. Clear zones de~~eloped around colonies formed by beta-glucanolytic microorganisms.
Relative to animals fed the control diet, density of beta-glucanolytic population was up to 100 fold greater i.n animals fed beta-glucan diet (Fig.
2).
TAH~LE 6 Comuosidon of beta-~lucan a>;ar In redient Amount L
Yeast extract 2.5 Trytone 5.0 Peptone 7.5 beta-glucan 10 Cysteine 0.5 NaCI 2.0 KZHPO,~ 2.0 KHZPO,~ 1.0 NaHC03 2.0 MgCl2 0.2 CaClz 0.2 CoCl2 0.02 MnClz 0.02 FeSO~ 0.005 Tween 80 2 Hemin 0.005 Vitamin B1z 0.001 Vitamin K 0.0005 A ar 13.0 . _ ~._ _.__ .._._AME~O;p~p.~H~ . . _..
IPEA/AU
This example demonstrates that a range of beta-glucans induce the observed effects. Beta-glucan prepared from a number of Saccharomyces cerevisae strains and of bacterial origin were used. Both crude forms and more purified forms were tested, The crude forms were whole cell sacs that were washed 2-3 times in water and spray dried. The more purified forms were prepared from the water washed preparations that were subsequently alkali washed, then acid washed and then water washed prior to spray drying. When these various beta-glucan forms were incubated with faecal homogenates diluted in laboratory medium, all forms were degraded (Table 7) and were selectively utilised by lactobacilli when tested using lactobacillus pure cultures on glucan agar (Table 6).
Fermentation of beta-glucan preparations by mixed fecal slurries Beta-lucan sam le Degradation (%) 50 >60 >60 >70 >50 Cell sac per field:
0 hour 156 NA NA clumped NA
72 hours 9.2 NA NA 75 NA
Dry residues ( ) 0.3048 0.2630 0.3097 0.3950 NA
Total anaerobes 2.9 x 3.1 x 10' 1.4 x 1.9 x 10' NA
10~ 10' Beta-glucan 1 - water-washed Saccharomyces strain A cell sacs Beta-glucan 2 - alkali- and acid-washed Saccharomyces strain B cell sacs (preparation 1) Beta-glucan 3 - alkali- and acid-washed Saccharomyces str ain B cell sacs (preparation 2) Beta-glucan 4 - water-washed Saccharomyces strain B cell sacs Beta-glucan 5 - bacterial-origin cell sacs NA - not analysed This example demonstrates that yeast sac beta-glucan is poorly degraded in the upper regions of the mouse digestive tract and that most of the degradation occurs in the caecum a.nd colon of the animal where the microbial population is greatest. Two groups of three SPF Balb/c mice were fed for 60 hours with a diet containing 40% w/w yeast cell sac beta-glucan.
After the mice were sacrificed, the stomach, duodenum, ileum, jejunum, caecum and faeces were sampled. Gas1_rointestinal contents were squeezed from the gut sections and faecal samples were individually homogenised with 50 mM phosphate buffer and a drop of the homogenate was placed on microscope slides. The degradation of the beta-glucan from mouse intestinal tract samples was examined using phase-contrast microscopy. The beta-glucan granules were degraded by less i_han 20% in the stomach and small intestine while greater than 70% degradation was noted in the caecum and faecal homogenates (Table 8).
Degradation of beta-glucan in mouse intestinal tract Sam le source Stomach Duodenum Ilium Jejunum Caecum Faeces Degra-dation < 10% NO < 20% < 20% > 70% > 80%
NO - Not Observed: Few yeast cell sacs were observed in that area, probably because of the rapid transit time.
USES
The present invention can be applied to all conditions in which microbes are identified or proposed as causative agents of disease in both human and animals and which can be advantageously effected by altering the numbers and/or activities of beneficial microflora of the gastrointestinal tr act.
As infective diarrhoea has been shown to be improved by probiotic dosage, the present invention can be used to enhance the effect of the probiotic by itself and to reduce the microbial-induced diarrhoea when orally dosed alone . In addition, the present invention may be used effectively to improve non-infective diarrhoea which has not been shown to be influenced by probiotics alone. It could be effective in reducing the effects of dietary related diarrhoea problems.
Infective diarrhoea refers to all cases of diarrhoea, both acute and chronic, in which the causative agents can be shown to be microbial, including bacterial, viral and protozoan. Such infective diarrhoea can manifest itself in a number of ways e.g. (a) infantile and geriatric diarrhoea;
(b) antibiotic associated diarrhoea; (c) traveller's diarrhoea; (d) stressed-induced diarrhoea.
Both prophylactic and therapeutic uses are described in the present specification. The former can relate to prevention when the individual can be exposed to potential problems e.g. (i) investigative gastrointestinal examination when the bowel is decontaminated and can then be recolonised by an undesirable microbial population; (ii) travellers exposed to an altered pathogen load or an alteration of the gut ecosystem because of various factors including diet change, which can predispose the individual to a lower infective dose of a pathogen. Therapeutic uses relate to the treatment of established conditions related to an undesirable balance of the gut microflora or an established pathogen infection.
In summary, glucan preparations singly or together with probiotics and/or prebiotics such as oligosaccharides can be included in foods or other preparations for therapeutic or prophylactic use in the follows situations:
i. As a general gut microflora stabiliser ii. In clinical conditions directly or indirectly related to gastrointestinal microflora e.g. irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), Crohn's disease, diarrhoea and constipation, colon cancer, sleep disorders, rheumatoid arthritis, chronic fatigue syndrome, hyperactivity, and ulcerative colitis iii. improved intestinal health iv. directly influence the metabolism of the microbes such that the 5 beneficial activities are enhanced e.g. elevated levels of antimicrobials or butyrate, and the harmful activities are reduced. e.g. reduced toxin production v. directly influence numbers of desirable microbes either directly or indirectly in the complex mix of gastrointestinal microbes 10 vi. directly influence numbers of undesirable microbes either directly or indirectly in the gastrointestinal systerr~.
The present inventors have found that beta-glucans and oligosaccharides are carbohydrates which can be selectively used by beneficial microbes in the gut which in turn can suppress the numbers of the 15 undesirable microbes.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiment=s without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
References Brattgjerd, S, Evensen, O, Lauve, A. (1994). Effect of injected yeast glucan on the activity of macrophages in Atlantic salmon, Salmo salar L., as evaluated by in vitro hydrogen peroxide production and phagocytic capacity.
Immunology 83:288-94.
Blomberg, L., Henriksson, A. & Conway, P. L. (1993). Inhibition of Escherichia coli K88 to piglet ilea) mucus by Lactobacillus spp. Applied and Environmental Microbiology. 59, 34-39.
Fernandes, C. F., Shahani, K. M. & Amer, M. A. (1987). Therapeutic role of dietary lactobacilli fermented dairy products. FEMS Microbiology Reviews. 46. 343-356.
Gibson, G. R., Beatty, E.R., Wang, X. & Cummings, J.H. (1995).
Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology 108, 975-982.
Katelaris, P. H. (1996). Probiotic control of diarrhoea) disease. Asia Pacific Journal of Clinical Nutrition. 5, 39-43.
Klaenhammer, T. R. (1988). Bacteriocins of lactic acid bacteria.
Biochemie. 70, 337-349.
Lindgren. S. E. & Dobrogosz, W. J. (1990). Antagonistic activities of lactic acid bacteria in food and feed fermentations. FEMS microbiology reviews. 87. 149-164.
McCormick, E. L. & Savage, D. C. (1983). Characterisation of Lactobacill us sp. strain 100-3 7 from the murine gastrointestinal tract:
ecology.
plasmid content. and antagonistic activity toward Clostridum ramosum H1.
Appl. Environ. Microbiol. 46. 1103-.
Mishra, C. & Lambert, J. (1996). Production of antimicrobial substances by probiotics. Asia Pacific journal of Clinical Nutrition. 5, 20-24.
Sanders, M. E. (1994). Lactic acid bacteria as promoters of human health. In Functional foods, pp. 294-322. Edited by I. Goldberg. New York:
Chapman and Hall.
Tagg, J. R., Dajani, A. S. & Wannamaker, L. W. (1976). Bacteriocins of gram positive bacteria. Bacteriol. Rev. 40, 722-756.
Probiotic bacteria have been described to exert antimicrobial effects which refers to the actions of the probiotic preparation on another microbe or group of microbes. Some probiotic bacteria are directly applicable for enhancing resistance against intestinal pathogens, prevention of diarrhoea and constipation (reviewed by Fernandes et al. ; 1987; Katelaris, 1996;
Sanders, 1994). The types of interactions include competitive colonisation as well as adhesion and growth inhibition.
Competitive colonisation refers to the fact that the probiotic strain can successfully compete with the pathogen for either nutrients or the site of colonisation. As many gastrointestinal pathogens attach to the intestinal mucosa as the first step in infection, it would be beneficial to the host if this adhesion could be inhibited . There are reports that lactobacilli produce Received 31 March 1999 components which inhibit attachment of enterotoxigenic Escherichia coli to intestinal mucosa (Blomberg et al., 1993), however, there is no evidence as yet that this occurs in the digestive traca. In addition, various compounds produced during growth of the probiotiic have beef shown to inhibit pathogen growth (reviewed by Fernand.es et al., 1987; Klaenhammer, 1988;
Mishra and Lambert, 1996). These include organic acids such as lactic and acetic acid, reuterin and bacteriocins ('ragg et al., 1976). The organic acids lower the pH and thereby can indirectly affect growth of the pathogen. In addition, the lactic and acetic acids produced by these organisms can be toxic to microbes. Reuterin which inhibits the growth of a very broad range of cells (Lindgren & Dobrogosz, 1990), i~;s produced by Lactobacillus reuteri when grown in the presence of glycerol. Numerous bacteriocins have been reported to be produced by lactobacilli e.g. Acidophilin, Acidolin, Lactocidin, Bacteriocin, Bulgarican, Lactolin, Lactobacillin and Lactobrevin (reviewed by z5 Fernandes et al., 1987; Klaenhalnmer) 1988). Bacteriocins can either have a very broad range of activity or alternatively specifically inhibit the growth of a very limited range of closely related microbes. For example, Lactobacillus sp exhibited specific antagonistic effects towards Clostridium ramosum (McCormick & Savage, 1983).
The present inventors have surprisingly found that beta-glucans can be used to influence pathogen adhesion, alter microbial populations in the gastrointestinal tract, improve the effecaiveness of probiotic compositions, and promote growth of a probiotic microorganism. A range of crude and pure preparations of beta-glucans have been shown to be effective.
Disclosure of the Invention In a first aspect, the present invention consists in a method of enhancing a resident population of lacl:ic acid-producing microorganisms in the gastrointestinal tract of an animal, the method comprising providing to the animal a beta-glucan derived from cell sacs in combination with other prebiotics and/or with one or more probiotic microorganisms, such that upon ingestion, the beta-glucan passes through the gastrointestinal tract substantially unutilised until the beta-l;lucan and the probiotic microorganisms reach a site in the gastrointestinal tract where the beta-glucan is utilised by the resident population of lactic acid-producing nucroorganisms and the probiotic microorganisms, thereby causing an increase in number and/or activity of the resident population of lactic acid-AMENDED SHE
. _.....___ .._~._.~E~A~ ' ~ _ . .
.t Received 31 March 1999 producing microorganisms and the probiotic microorganisms, wherein the combination of the beta-glucan and thf: one or more probiotic microorganisms provides a synergistic effect upon the increase in number and/or activity of the resident population of lactic acid-producing microorganisms.
In a second aspect, the present invention consists in a method of suppressing an undesired population of microorganisms in a total microorganism population in the gastrointestinal tract of an animal, the method comprising providing to the animal a beta-glucan derived from cell sacs in combination with other prebiotics and/or with one or more probiotic microorganisms, such that upon ingestion, the beta-glucan passes through the gastrointestinal tract substantially unutilised until the beta-glucan and the probiotic n>scroorganisms reach a site in the gastrointestinal tract where the beta-glucan is utilised by a resident population of lactic acid-producing microorganisms and the probiotic microorganisms thereby causing an increase in number and/or activity of the resident population of lactic acid-producing microorganisms and the probiotic microorganisms and suppressing the growth and/or activity of the undesired population of microorganisms, wherein the combination of the beta-glucan and the one or more probiotic microorganisms provides a synergistic effect upon the increase in number and/or activity of tlhe resident population of lactic acid-producing microorganisms.
In a preferred form, the undesired population of microorganisms is enteric pathogens. As will be appreciated, there are a large number of pathogens including Graln negative bacteria like Salmonella, Escherichia coli, Campylobacter, Helicobacter, Vibrio, and Pseudomonas; Gram positive bacteria like Clostridium; viruses like Norwalk virus, Norwalk-like viruses and Rotavirus; and protozoa like Crytosporidium, Entamoeba, Giardia, and Dientamoeba. The present invention vvould be suitable for the suppression of 3o many of these pathogens in the gastrointestinal tract of animals.
Suitable beta-glucans include those originating from (a) plants including cereals such as oats and barley, (b) fungi, (c) yeast, and (d) bacteria. In addition, nucrobial cell will preparations and whole cells rich in beta-glucans are also suitable sources for beta-glucan preparations useful for the present invention. Monomer residues in glucans can have 1-3 and 1-4, or 1-3 and 1-6 linkages (that is the mononner units are joined through 1.3, 1,4 or . ._..__.E:~ ~-'~ ....._ . . . _..
IPEAIAU
Received 31 March 1999 1,6 bonds) and the percent of each type can vary. Preferably, beta-glucans derived from yeast, particularly from 5'accharomyces, preferably Saccharomyces cerevisae, are used for the present invention. It will be appreciated, however, that other beta-~;lucans would also be suitable. A
5 concentration of up to about 10% when combined with other prebiotics and/or probiotic nucroorganisms can be used. One percent has been found to be particularly suitable.
The resident lactic acid-producing microorganism is preferably a lactobacillus, and more preferably Laci:obacillus fermentum.
1o In a preferred form, the present :invention further includes one or more oligosaccharides, polysaccharides or other prebiotics. A concentration of oligosaccharides, polysaccharides or other prebiotics of about 0.001 g to 10 g per kg body weight per day is preferred.
Preferably, the probiotic microorganism is selected from the group consisting of lactic acid-producing microorganisms, bifidobacterium, yeast, and mixtures thereof. Preferably, the lactic acid-producing microorganism is a lactobacillus, more preferably Lactobacillus fermentum. Preferably, the yeast is a SaccharomycE~s sp. Typical concentration range of probiotic microorganisms is 103 to 1013 cells per day.
Although oligosaccharides have been shown to promote bifidobacteria numbers in animals, prior to the present invention, it was thought that lactobacillus numbers cannot be changed in a similar manner. Gibson et al 1995 found that oligofructose and inul:in, when fed to humans, selectively stimulated the growth of bifidobacteria without influencing the numbers of lactobacillus. From these results and observations by others in the field, it would not be expected that beta-glucans would positively effect the growth and/or activity of lactic acid-producing; microorganisms, particularly lactobacillus, in vivo as found by the present inventors.
It should be further emphasised that the present invention relates to 3o the synergist relationship between the beta-glucans and probiotic preparations both in terms of proliferation of the probiotics themselves and inhibition of pathogens. The synergy between beta-glucans and probiotics preparations results in selective increase in the numbers of specific beneficial microorganism including probiotic strains in the presence of glucans. Furthermore, a reduction in numbers of pathogens in the presence of beta-glucans and additional probiotics is surprisingly significantly greater . _ _._ ___ _AMEN17SD.SMEE~- . . . _..
IF~EA/AU
Received 31 March 1999 ~6 additive effects of beta glucans or probiotics individually, in reducing pathogen numbers. This enhancement is not explainable by an increase in the numbers of some of the probiotics since the synergistic effect is demonstrable with probiotics which are not enhanced by the inclusion of glucan".
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples and drawings.
Brief Description of Drawings Figure 1 shows the results of Iac;tobacillus in the gastrointestinal tract of animals fed beta-glucan or control diet; and Figure 2 shows faecal beta-glucanolytic microorganisms of animals fed beta-glucan or control diet. Analysis o~f beta-glucanolytic activity on agar plates containing beta-glucan fTOIIl yeast cell sacs.
Modes for Carrying Out the Invention This has been studied by enumerating coliforms, bifidobacterium and lactobacilli in vitro in the presence of beta-glucan. The beta-glucan used in this example was prepared from yeast cell sacs. More specifically, aliquots (1 n ll) of human faecal homogenates (10 ~; per 100 ml diluent) were added to diluted WC broth (diluted 50:50 with (1.05M phosphate buffer) to which were added the beta-glucan and a lactobacillus or bifidobacterium strain and various combinations. For each of the combinations, parallel tubes were prepared with one set being inoculated with Bifidobacterium spp or Lactobacillus spp. All mixtures were tlhen incubated for up to 24 hours and bacterial numbers enumerated. Results (Table 2) are expressed as the numbers of coliforms, total lactobacilli., total bifidobacterium, or total anaerobes when enumerated as colony forming units. It can be seen that coliform numbers are unchanged or reduced, while numbers of bifidobacteria and lactobacilli can be increased up to 10-fold, and selected groups within a genus can be enhanced.
AMENDED SHEET
. . _ EB'ect of 1% beta-glucan on Lactobacillus and Bifidobacterium Bacterial numbers (CFU/ml Lactobacillus Culture mix Coliforms Total Large Bifidobacterium Colon Faecal homogenate 3.2 x 10 3.4 x 10 1.2 x < 2 (FH) 10' FH + 1% glucan 5.2 x 106 5.3 x 105 4.8 x < 2 FH + Bifidobacterium (SUB) NA 1VA NA 1.0 x 106 FH + SUB + 1% glucan NA IVA NA 1.2 x 10' FH + L. fermentum NA 2.8 x 205 2.8 x NA
(Lf) 105 FH + Lf + 1% glucan NA 1.2 x 106 1.2 x NA
NA - not analysed This example demonstrates that dietam supplementation with beta-glucan stimulates gastrointestinal populations of Lactobacillus. In this study.
animals (8 per group) were provided either bei~a-glucan diet or control diet (Table 3). The beta-glucan used in this examF~le was prepared from yeast cell sacs. After 6 weeks feeding. animals were euthanised and luminal content collected from stomach, ileum, caecum and colon. Collected material was homogenised and diluted in Wilkens-Charlgren broth (Oxoid). Dilutions were spread on to Rogosa gear (Oxoid) and incubated anaerobically for 48 h.
Beta-glucan-rich diet resulted in significant increase (P<0.0005) of Lactobacill us, particularly in the gastric region where an 100 fold increase was detected (Fig. 1). The increase of lactobacilli numbers in the subsequent intestinal tract corresponded to about 1.5 log units.
Composition of beta-glucan and sucrose diet Ingredient Amount Amount (Glucan diet) Control diet Beta-glucan 450 0 Sucrose 150 600 Casein 200 200 Sunflower 25 25 Canola oil 25 25 Gelatin 20 20 Wheat bran 100 100 Choline chloride 2 2 Methionine 3 3 Standard mineral 35 35 mix Standard vitamin 10 10 mix In addition to studying effects on microbial growth, effects on adhesion have been investigated. Desirable bacteria such as lactobacillus, as well as undesirable pathogenic E. coli and Salmonella sp have been grown either in a standard laboratory broth or in this broth supplemented with beta-glucans. The beta-glucan used in this example was prepared from yeast cell sacs. . Bacterial suspensions were incubated with mucosal pieces from various regions of the gastrointestinal tract. These mucosal pieces were either used directly or after pre-treatment with the beta-glucan. Results are shown in Table 4. While adhesion of the pathogen was adversely affected, the adhesion of the lactobacillus was increased by the presence of the beta-glucan.
Effect of beta-glucans on adhesion Bacteria adhering (per mg tissue) Bacteria Control tissueTissue + 1% lucanReduction (%) S. typhimurium 1.69 x 108 5.53 x 10' -- 65 E. coli K88 6.07 x 10' 3.43 x 10' --- 50 L. ermentum 5.37 x 108 6.2 x 108 0 This example demonstrates that dietary supplementation with beta-glucan increases resistance to gastrointestinal infection by Clostridium difficile and S, typllimurium and that this effect is enhanced when glucan-treated mice were also orally dosed with. Saccharomyces sp cell suspension.
The beta-glucan used in this example w;is acid and alkali washed cell sacs of yeast. The Saccharomyces sp alone reduced numbers of C. difficile to some extent and markedly reduced S. typhim urium numbers, Groups of animals (4 groups, 8 animals per group) were provided either beta-glucan diet or control diet (Table 3). After feeding diets far four weeks, four groups were supplied drinking water containing cefoxitin (0.0;32 g/L) and D-cycloserine (1.0 g/L) and subsequently challenged with C. dif~icile (10-8 CFU). Another four groups were suppled drinking water containing streptomycin (5 g/L) and neomycin (5 g/L). These animals were subsequently challenged with S. typhimurium (10-' CFU). After pathogen challenge, animals were orogastrically dosed with either Saccharnmyces sp (strain FII 542900)(108 CFU in YPG broth), or with fresh YPG broth. Development of infections were monitored daily by analysis of faecal S. typhimurium or C. difficile. Faecal material was homogenised and diluted in Wilkins-Charlgen broth (Oxoid).
Dilutions were spread on to either YSA (Oxoid) or C. difficile selective agar (Oxoid) for analysis of faecal S. typhimuuum and or C. difficile, respectively.
Animals fed a beta-glucan rich diet were significantly more protected against colonisation by C. difficile (Table 5), The faecal levels of C.
difficile were, in animals fed beta-glucan, about 2 log units less than what was detected in animals fed the control diet, one day post challenge (P<0.05). A
non-significant reduction of S. typhimurium numbers was noted in beta-:LO
Received 31 March 1999 glucan treated mice, while a log 3 reduction in mice dosed with Saccharomyces sp alone (P<0.05). The combination of beta-glucan diet and Saccharomyces sp oral administration resulted in a significant reduction (P<0.05) in faecal numbers of both pathogens when compared to both the control diet and the Saccharomyces sp dosed mice (Table 5). This reduction corresponded to 3 and >4 log units for C. difficile and S. typhimurium, respectively. This corresponded to a marked improvement in weight loss and other signs of severe infection in animals treated with the combination of beta-glucan and Saccharomyces sp. In summary, when the beta-glucan is s0 provided together with probiotic preparations e.g. Saccharomyces, a surprising synergistic enhancement in the protective effect against pathogens is noted where greater than log 4 reduction is noted for the combination of beta-glucan plus probiotics, while the beta-glucan alone or the probiotic alone reduce pathogen levels by log 1-2. It can be concluded that a beneficial s5 synergistic effect can be achieved by beta-glucan alone and enhanced by the combinations of beta-glucan and Sacci'laromyces sp.
TAttLE 5 Reduction of pathogens by oral administration of Saccaromyces sp to beta-20 glucan fed mice that were chal:(enged with either C. difficile or S. typh~imurium Reduction to CFLT
er faeces Diet C. di 'cile S. ty himurium beta-glucan 1.5 * <1 beta-glucan and Saccharomyces sp 3 * * > 4 Saccharom ces s 0.8 3 * Significantly different from beta-glucan free diet (P<0.05) 25 * * Significantly different from beta-glucan-free diet and Saccharomyces sp alone.
~MEi'~DED SHEET
. _ ~._.___~ ,._~.._._. ~,p~AU.. .~._ . .. __ This example demonstrates that:
Received 31 March 1999 (i) beta-glucanolytic microorganisms a:re present in the gastrointestinal tract;
(ii) dietary supplementation with beta-glucan stimulates gastrointestinal populations of beta-glucan degrading microorganisms (includes lactic acid-producing microorganisms).
In this study, animals (8 per gro,ap) were provided either beta-glucan or control diet (Table 3). After 4 weeks fE;eding, faecal material was collected, homogenised and diluted in Wilkins-Charlgen broth (Oxoid). Diluted material was spread on to beta-glucan agar (Table 6) and incubated anaerobically for 75 h. Clear zones de~~eloped around colonies formed by beta-glucanolytic microorganisms.
Relative to animals fed the control diet, density of beta-glucanolytic population was up to 100 fold greater i.n animals fed beta-glucan diet (Fig.
2).
TAH~LE 6 Comuosidon of beta-~lucan a>;ar In redient Amount L
Yeast extract 2.5 Trytone 5.0 Peptone 7.5 beta-glucan 10 Cysteine 0.5 NaCI 2.0 KZHPO,~ 2.0 KHZPO,~ 1.0 NaHC03 2.0 MgCl2 0.2 CaClz 0.2 CoCl2 0.02 MnClz 0.02 FeSO~ 0.005 Tween 80 2 Hemin 0.005 Vitamin B1z 0.001 Vitamin K 0.0005 A ar 13.0 . _ ~._ _.__ .._._AME~O;p~p.~H~ . . _..
IPEA/AU
This example demonstrates that a range of beta-glucans induce the observed effects. Beta-glucan prepared from a number of Saccharomyces cerevisae strains and of bacterial origin were used. Both crude forms and more purified forms were tested, The crude forms were whole cell sacs that were washed 2-3 times in water and spray dried. The more purified forms were prepared from the water washed preparations that were subsequently alkali washed, then acid washed and then water washed prior to spray drying. When these various beta-glucan forms were incubated with faecal homogenates diluted in laboratory medium, all forms were degraded (Table 7) and were selectively utilised by lactobacilli when tested using lactobacillus pure cultures on glucan agar (Table 6).
Fermentation of beta-glucan preparations by mixed fecal slurries Beta-lucan sam le Degradation (%) 50 >60 >60 >70 >50 Cell sac per field:
0 hour 156 NA NA clumped NA
72 hours 9.2 NA NA 75 NA
Dry residues ( ) 0.3048 0.2630 0.3097 0.3950 NA
Total anaerobes 2.9 x 3.1 x 10' 1.4 x 1.9 x 10' NA
10~ 10' Beta-glucan 1 - water-washed Saccharomyces strain A cell sacs Beta-glucan 2 - alkali- and acid-washed Saccharomyces strain B cell sacs (preparation 1) Beta-glucan 3 - alkali- and acid-washed Saccharomyces str ain B cell sacs (preparation 2) Beta-glucan 4 - water-washed Saccharomyces strain B cell sacs Beta-glucan 5 - bacterial-origin cell sacs NA - not analysed This example demonstrates that yeast sac beta-glucan is poorly degraded in the upper regions of the mouse digestive tract and that most of the degradation occurs in the caecum a.nd colon of the animal where the microbial population is greatest. Two groups of three SPF Balb/c mice were fed for 60 hours with a diet containing 40% w/w yeast cell sac beta-glucan.
After the mice were sacrificed, the stomach, duodenum, ileum, jejunum, caecum and faeces were sampled. Gas1_rointestinal contents were squeezed from the gut sections and faecal samples were individually homogenised with 50 mM phosphate buffer and a drop of the homogenate was placed on microscope slides. The degradation of the beta-glucan from mouse intestinal tract samples was examined using phase-contrast microscopy. The beta-glucan granules were degraded by less i_han 20% in the stomach and small intestine while greater than 70% degradation was noted in the caecum and faecal homogenates (Table 8).
Degradation of beta-glucan in mouse intestinal tract Sam le source Stomach Duodenum Ilium Jejunum Caecum Faeces Degra-dation < 10% NO < 20% < 20% > 70% > 80%
NO - Not Observed: Few yeast cell sacs were observed in that area, probably because of the rapid transit time.
USES
The present invention can be applied to all conditions in which microbes are identified or proposed as causative agents of disease in both human and animals and which can be advantageously effected by altering the numbers and/or activities of beneficial microflora of the gastrointestinal tr act.
As infective diarrhoea has been shown to be improved by probiotic dosage, the present invention can be used to enhance the effect of the probiotic by itself and to reduce the microbial-induced diarrhoea when orally dosed alone . In addition, the present invention may be used effectively to improve non-infective diarrhoea which has not been shown to be influenced by probiotics alone. It could be effective in reducing the effects of dietary related diarrhoea problems.
Infective diarrhoea refers to all cases of diarrhoea, both acute and chronic, in which the causative agents can be shown to be microbial, including bacterial, viral and protozoan. Such infective diarrhoea can manifest itself in a number of ways e.g. (a) infantile and geriatric diarrhoea;
(b) antibiotic associated diarrhoea; (c) traveller's diarrhoea; (d) stressed-induced diarrhoea.
Both prophylactic and therapeutic uses are described in the present specification. The former can relate to prevention when the individual can be exposed to potential problems e.g. (i) investigative gastrointestinal examination when the bowel is decontaminated and can then be recolonised by an undesirable microbial population; (ii) travellers exposed to an altered pathogen load or an alteration of the gut ecosystem because of various factors including diet change, which can predispose the individual to a lower infective dose of a pathogen. Therapeutic uses relate to the treatment of established conditions related to an undesirable balance of the gut microflora or an established pathogen infection.
In summary, glucan preparations singly or together with probiotics and/or prebiotics such as oligosaccharides can be included in foods or other preparations for therapeutic or prophylactic use in the follows situations:
i. As a general gut microflora stabiliser ii. In clinical conditions directly or indirectly related to gastrointestinal microflora e.g. irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), Crohn's disease, diarrhoea and constipation, colon cancer, sleep disorders, rheumatoid arthritis, chronic fatigue syndrome, hyperactivity, and ulcerative colitis iii. improved intestinal health iv. directly influence the metabolism of the microbes such that the 5 beneficial activities are enhanced e.g. elevated levels of antimicrobials or butyrate, and the harmful activities are reduced. e.g. reduced toxin production v. directly influence numbers of desirable microbes either directly or indirectly in the complex mix of gastrointestinal microbes 10 vi. directly influence numbers of undesirable microbes either directly or indirectly in the gastrointestinal systerr~.
The present inventors have found that beta-glucans and oligosaccharides are carbohydrates which can be selectively used by beneficial microbes in the gut which in turn can suppress the numbers of the 15 undesirable microbes.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiment=s without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
References Brattgjerd, S, Evensen, O, Lauve, A. (1994). Effect of injected yeast glucan on the activity of macrophages in Atlantic salmon, Salmo salar L., as evaluated by in vitro hydrogen peroxide production and phagocytic capacity.
Immunology 83:288-94.
Blomberg, L., Henriksson, A. & Conway, P. L. (1993). Inhibition of Escherichia coli K88 to piglet ilea) mucus by Lactobacillus spp. Applied and Environmental Microbiology. 59, 34-39.
Fernandes, C. F., Shahani, K. M. & Amer, M. A. (1987). Therapeutic role of dietary lactobacilli fermented dairy products. FEMS Microbiology Reviews. 46. 343-356.
Gibson, G. R., Beatty, E.R., Wang, X. & Cummings, J.H. (1995).
Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology 108, 975-982.
Katelaris, P. H. (1996). Probiotic control of diarrhoea) disease. Asia Pacific Journal of Clinical Nutrition. 5, 39-43.
Klaenhammer, T. R. (1988). Bacteriocins of lactic acid bacteria.
Biochemie. 70, 337-349.
Lindgren. S. E. & Dobrogosz, W. J. (1990). Antagonistic activities of lactic acid bacteria in food and feed fermentations. FEMS microbiology reviews. 87. 149-164.
McCormick, E. L. & Savage, D. C. (1983). Characterisation of Lactobacill us sp. strain 100-3 7 from the murine gastrointestinal tract:
ecology.
plasmid content. and antagonistic activity toward Clostridum ramosum H1.
Appl. Environ. Microbiol. 46. 1103-.
Mishra, C. & Lambert, J. (1996). Production of antimicrobial substances by probiotics. Asia Pacific journal of Clinical Nutrition. 5, 20-24.
Sanders, M. E. (1994). Lactic acid bacteria as promoters of human health. In Functional foods, pp. 294-322. Edited by I. Goldberg. New York:
Chapman and Hall.
Tagg, J. R., Dajani, A. S. & Wannamaker, L. W. (1976). Bacteriocins of gram positive bacteria. Bacteriol. Rev. 40, 722-756.
Claims (20)
1. A method of enhancing a resident population of lactic acid-producing microorganisms in the gastrointestinal tract of an animal, the method comprising providing to the animal a beta-glucan derived from cell sacs in combination with other prebiotics and/or with one or more probiotic microorganisms, such that upon ingestion, the beta-glucan passes through the gastrointestinal tract substantially unutilised until the beta-glucan and the probiotic microorganisms reach a site in the gastrointestinal tract where the beta-glucan is utilised by the resident population of lactic acid-producing microorganisms and the probiotic microorganisms, thereby causing an increase in number and/or activity of the resident population of lactic acid-producing microorganisms and the probiotic microorganisms, wherein the combination of the beta-glucan and the one or more probiotic microorganisms provides a synergistic effect upon the increase in number and/or activity of the resident population of lactic acid-producing microorganisms.
2. A method of suppressing an undesired population of microorganisms in a total microorganism population in the gastrointestinal tract of an animal, the method comprising providing to the animal a beta-glucan derived from cell sacs in combination with other prebiotics and/or with one or more probiotic microorganisms, such that upon ingestion, the beta-glucan passes through the gastrointestinal tract substantially unutilised until the beta-glucan and the probiotic microorganisms reach a site in the gastrointestinal tract where the beta-glucan is utilised by a resident population of lactic acid-producing microorganisms and the probiotic microorganisms thereby causing an increase in number and/or activity of the resident population of lactic acid-producing microorganisms and the probiotic microorganisms and suppressing the growth and/or activity of the undesired population of microorganisms, wherein the combination of the beta-glucan and the one or more probiotic microorganisms providers a synergistic effect upon the increase in number and/or activity of the resident population of lactic acid-producing microorganisms.
3. The method according to claim 2 wherein the undesired population of microorganisms is an enteric pathogen.
4. The method according to any one of claims 1, to 3 wherein the beta-glucan is a preparation derived from yeast or bacteria.
5. The method according to claim 4 wherein the beta-glucan is derived from yeast cell sacs rich in beta-glucans.
6. The method according to claim 5 wherein the yeast is a Saccharomyces sp.
7, The method according to claim 6 wherein the Saccharomyces sp is Saccharomyces cerevisae,
8. The method according to any one of claims 1 to 7 wherein the beta-glucan is provided at a concentration of up to 10% (w/w).
9. The method according to claim 8 wherein the beta-glucan is provided at a concentration of 1% (w/w).
10. The method according to any one of claims 1 to 9 wherein the resident population of lactic acid-producing microorganisms is lactobacillus.
11. The method according to claim 10 wherein the lactobacillus is Lactobacillus fermentum.
12. The method according to any one of claims 1 to 11 wherein the probiotic microorganisms are selected from the group consisting of lactic acid-producing microorganisms, bifidobacterium, yeast, and mixtures thereof,
13. The method according to claim 12 wherein the lactic acid-producing microorganisms are lactobacillus.
14. The method according to claim 13 wherein the lactobacillus is Lactobacillus fermentum.
15. The method according to claim 12 wherein the yeast is a Saccharomyces sp.
16. The method according to any one of claims 1 to 15 wherein the probiotic microorganism is administered at a concentration of 10 3 to 10 13 cells per day.
17. Use of a synergistic combination of a beta-glucan derived from cell sacs with one or more probiotic microorganisms and optionally one or more other prebiotics for enhancing a resident population of lactic acid-producing microorganisms in the gastrointestinal tract of an animal.
18. The use according to claim 17 wherein upon ingestion, the beta-glucan passes through the gastrointestinal tract substantially unutilised until the beta-glucan and the probiotic microorganisms reach a site in the gastrointestinal tract where the beta-glucan is utilised by the resident population of lactic acid-producing microorganisms and the probiotic microorganisms, thereby causing an increase in number and/or activity of the resident population of lactic acid-producing microorganisms and the probiotic microorganisms, wherein the combination of the beta-glucan and the one or more probiotic microorganisms provides a synergistic effect upon the increase in number and/or activity of the resident population of lactic acid-producing microorganisms.
19. Use of a synergistic combination of a beta-glucan derived from cell sacs with one or more probiotic microorganisms and optionally one or more other prebiotics for suppressing an undesired population of microorganisms in a total microorganism population in the gastrointestinal tract of an animal.
20. The use according to claim 19 wherein upon ingestion, the beta-glucan passes through the gastrointestinal tract substantially unutilised until the beta-glucan and the probiotic microorganisms reach a site in the gastrointestinal tract where the beta-glucan is utilised by a resident population of lactic acid-producing microorganisms and the probiotic microorganisms thereby causing an increase in number and/or activity of the resident population of lactic acid-producing microorganisms and the probiotic microorganisms and suppressing the growth and/or activity of the undesired population of microorganisms, wherein the combination of the beta-glucan and the one or more probiotic microorganisms provides a synergistic effect upon the increase in number and/or activity of the resident population of lactic acid-producing microorganisms.
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| PCT/AU1997/000859 WO1998026787A1 (en) | 1996-12-19 | 1997-12-18 | Prebiotics and probiotics |
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| WO (1) | WO1998026787A1 (en) |
Families Citing this family (33)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19751907B4 (en) * | 1997-11-22 | 2004-07-08 | Pharma-Zentrale Gmbh | Use of Escherichia coli strain DSM 6601 for the treatment of diarrhea in the veterinary sector |
| DE19836339B4 (en) | 1998-08-11 | 2011-12-22 | N.V. Nutricia | carbohydrate mix |
| DE19861497B3 (en) | 1998-08-11 | 2017-03-30 | N.V. Nutricia | Baby food and its use to promote the human colon flora |
| NL1010770C2 (en) * | 1998-12-09 | 2000-06-13 | Nutricia Nv | Preparation containing oligosaccharides and probiotics. |
| DE19962427A1 (en) | 1999-12-22 | 2001-07-12 | Nutrinova Gmbh | Encapsulated multifunctional, biologically active food component, process for their production and their application |
| JP4499979B2 (en) * | 2001-05-21 | 2010-07-14 | コンビ株式会社 | Composition for controlling pathogen infection |
| US20050272694A1 (en) * | 2003-03-07 | 2005-12-08 | Aureo Co., Ltd | Composition containing beta-glucan and constipation-relieving drug, immunopotentiatior, and skin moistening agent using the composition |
| JP4054697B2 (en) * | 2003-03-07 | 2008-02-27 | 株式会社アウレオ | Constipation improving agent |
| JP4000078B2 (en) * | 2003-03-07 | 2007-10-31 | 株式会社アウレオ | Skin moisturizer |
| US6936598B2 (en) * | 2003-11-21 | 2005-08-30 | Hill's Pet Nutrition, Inc. | Composition and method |
| EP1597978A1 (en) | 2004-05-17 | 2005-11-23 | Nutricia N.V. | Synergism of GOS and polyfructose |
| US8252769B2 (en) | 2004-06-22 | 2012-08-28 | N. V. Nutricia | Intestinal barrier integrity |
| US8992999B2 (en) | 2004-06-25 | 2015-03-31 | Alltech, Inc. | Methods and compositions for controlling parasitic infections of animals |
| ATE473636T1 (en) * | 2005-02-15 | 2010-07-15 | Barry R Goldin | FOODS CONTAINING PROBIOTIC AND ISOLATED BETA-GLUCAN AND METHOD OF USE THEREOF |
| US20090263416A1 (en) * | 2006-06-16 | 2009-10-22 | Dawson Karl A | Reduction of antibiotic resistance in bacteria |
| US20150147298A1 (en) * | 2006-07-24 | 2015-05-28 | CortControl, Inc. | Sleep enhancement with cortisol reduction medical food |
| CA2705642A1 (en) * | 2007-11-13 | 2009-05-22 | Biotec Pharmacon Asa | Methods of treating or preventing inflammatory diseases of the intestinal tract |
| WO2010008272A1 (en) * | 2008-07-15 | 2010-01-21 | N.V. Nutricia | Treatment of gut motility disorders |
| JP5320965B2 (en) * | 2008-10-09 | 2013-10-23 | ダイソー株式会社 | Gastrointestinal mucosa protective agent or diarrhea inhibitor using β-1,3-1,6-D-glucan |
| RU2580040C2 (en) | 2009-12-22 | 2016-04-10 | Проби Аб | Unfermented compositions containing cereal fraction and probiotic, and using them |
| US8906668B2 (en) | 2012-11-23 | 2014-12-09 | Seres Health, Inc. | Synergistic bacterial compositions and methods of production and use thereof |
| CA3212772A1 (en) | 2012-11-23 | 2014-05-30 | Seres Therapeutics, Inc. | Synergistic bacterial compositions and methods of production and use thereof |
| EP2951283A4 (en) | 2013-02-04 | 2017-01-25 | Seres Therapeutics, Inc. | Compositions and methods |
| EP3584308A3 (en) | 2013-02-04 | 2020-03-04 | Seres Therapeutics, Inc. | Compositions and methods |
| WO2014136982A1 (en) * | 2013-03-08 | 2014-09-12 | 学校法人東京理科大学 | Lactobacillus proliferation promoter, regulatory t cell proliferation agent, lactobacillus proliferation promoting method, method for proliferation of regulatory t cells, method for evaluating effectiveness of regulatory t cell proliferation, and method for evaluating effectiveness of lactobacillus proliferation promotion |
| WO2014153194A2 (en) * | 2013-03-14 | 2014-09-25 | Seres Health, Inc. | Methods for pathogen detection and enrichment from materials and compositions |
| WO2014145958A2 (en) | 2013-03-15 | 2014-09-18 | Seres Health, Inc. | Network-based microbial compositions and methods |
| RU2722482C2 (en) | 2013-11-25 | 2020-06-01 | Серес Терапеутикс, Инк. | Synergistic bacterial compositions and methods for preparing and using them |
| WO2015095241A2 (en) | 2013-12-16 | 2015-06-25 | Seres Health, Inc. | Bacterial compositions and methods of use thereof for treatment of immune system disorders |
| JP2020530840A (en) | 2017-08-14 | 2020-10-29 | セレス セラピューティクス インコーポレイテッド | Compositions and Methods for Treating Cholestasis Diseases |
| CN110025639A (en) * | 2019-03-07 | 2019-07-19 | 浙江工业大学 | Application of the yeast dextran composition in prevent constipation |
| CN113841901A (en) * | 2021-09-15 | 2021-12-28 | 广西大学 | Preparation method of high-activity synbiotics preparation freeze-dried powder |
| WO2023241390A1 (en) * | 2022-06-16 | 2023-12-21 | Nutricia Early Life Nutrition (Shanghai) Co., Ltd. | Compositions and uses thereof |
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|---|---|---|---|---|
| US4962094A (en) * | 1988-10-28 | 1990-10-09 | Alpha Beta Technology, Inc. | Glucan dietary additives |
| IT1274654B (en) * | 1995-02-28 | 1997-07-18 | Newpharma Srl | ASSOCIATIONS OF LACTIC FERMENTS AND SACCHAROMETIC LYSATES THEIR THERAPEUTIC USE AND COMPOSITIONS CONTAINING THEM |
| US5576015A (en) * | 1995-03-02 | 1996-11-19 | Donzis; Byron A. | Substantially purified beta (1,3) finely ground yeast cell wall glucan composition with dermatological and nutritional uses |
| AUPN398295A0 (en) * | 1995-07-05 | 1995-07-27 | Carlton And United Breweries Limited | Chemical compounds and processes for their production |
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1996
- 1996-12-19 AU AUPO4304A patent/AUPO430496A0/en not_active Abandoned
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1997
- 1997-12-18 JP JP52712098A patent/JP2001506129A/en active Pending
- 1997-12-18 CA CA002275507A patent/CA2275507A1/en not_active Abandoned
- 1997-12-18 WO PCT/AU1997/000859 patent/WO1998026787A1/en active Application Filing
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| Publication number | Publication date |
|---|---|
| JP2001506129A (en) | 2001-05-15 |
| WO1998026787A1 (en) | 1998-06-25 |
| AUPO430496A0 (en) | 1997-01-23 |
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