Abstract:
The present invention relates to a prebiotic composition comprising a microbially produced oligosaccharide, wherein the oligosaccharide is characterised by being selective for a pre-determined probiotic bacterial strain and also capable of being produced by the pre-determined probiotic bacteria by reverse enzyme action. The present invention also relates to methods of screening a composition suitable for use as a prebiotic and methods for screening of prebiotics for incorporation into synbiotic formulations.

Description:
TECHNICAL FIELD OF THE INVENTION 
       [0001]    The invention relates to a prebiotic composition which is specific for the growth of a desired probiotic bacterial strain. 
       BACKGROUND TO THE INVENTION 
       [0002]    Probiotics are bacteria which confer health benefits to a host. Typically, cultures of probiotic bacterial strains are consumed or administered to individuals in order to supplement the naturally occurring bacteria population of the gut. A number of health benefits have been associated with probiotics, including reducing the incidence of cancer, diarrhoea and irritable bowel syndrome to name a few. Preliminary studies also indicate that probiotics can be useful in reducing serum levels of cholesterol and blood pressure and help modulate diabetes. 
         [0003]      Lactobacilli  are common probiotics in diary products and make up approximately 75% of probiotics currently sold. However, it has been estimated that only 2% of  Lactobacilli  dose survives be effective in the gut. 
         [0004]    Prebiotics are dietary ingredients which can selectively enhance beneficial indigenous gut microbiota, such as  lactobacilli  or bifidobacteria, and are finding much increased application into the food sector. Prebiotics are non digestible food ingredients that are selectively metabolised by colonic bacteria which contribute to improved health. As such, their use can promote beneficial changes within the indigenous gut microbial milieu and they can therefore help survivability of probiotics. They are distinct from most dietary fibres like pectin, celluloses, xylan, which are not selectively metabolised in the gut. Criteria for classification as a prebiotic is that it must resist gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption, it is fermented by intestinal microflora and selectively stimulates the growth and/or activity of intestinal bacteria associated with health and well-being. 
         [0005]    Fructo-oligosaccharides (FOS, inulin and oligofructose) and galactooligosaccharides (GOS) have been demonstrated to fulfil the criteria for prebiotic classification repeatedly in human intervention studies. Currently, no prebiotic for  Lactobacilli  exists. 
         [0006]    It is an object of the present invention to provide a prebiotic composition which allows for the specific growth of a given probiotic bacteria. It would also be desirable if the prebiotic targeted a beneficial strain of prebiotic such as a  Lactobacilli.  A yet further object of the present invention is to provide a screening method to identify and produce prebiotic compositions which are selective for certain probiotic bacterial strains. 
       SUMMARY OF THE INVENTION 
       [0007]    In accordance with a first aspect of the present invention, there is provided a prebiotic composition comprising a microbially produced oligosaccharide, wherein the oligosaccharide is characterised by being selective for a pre-determined probiotic bacterial strain and also capable of being produced by the pre-determined probiotic bacterial strain by reverse enzyme reaction. 
         [0008]    The enzyme may comprise a saccharolytic enzyme. Such an enzyme may be one selected from one of following: β-galactosidases, α-galactosidases, α- and β-glucosidases, α-mannosidases, or β-xylosidases. 
         [0009]    The prebiotic composition may comprise galacto oligosaccharide (GOS). 
         [0010]    The pre-determined bacterial strain preferably comprises a  Lactobacilli  and may comprise a strain selected from:  Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillusdelbrueckii  ssp.  bulgaricus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus salivarius  ssp.  salivarius, Lactobacillus fermentum  or  Lactobacillus helveticus.  It is preferred that the GOS form is substantially the same as the form produced by reverse β-galactosidase reaction of the bacterial strain. 
         [0011]    The prebiotic composition will preferably be present in the composition in an effective amount so as to elicit a change in the proportions of the desirable indigenous gut microbiota and in particular the preferred probiotic bacterial strain. Higher amounts may be utilised if change in the microbiota is required quickly or if the composition is being used to help seed the gut with a new bacterial strain not currently present. 
         [0012]    The prebiotic composition may be encapsulated. Many encapsulation techniques will be apparent to the skilled addressee and the one employed will be tailored to the required stability of the prebiotic growth medium during digestive transit. 
         [0013]    The prebiotic composition may further comprise an excipient or carrier compound to enable it to pass through the gastrointestinal environment of the body and be efficiently delivered to, and released in the lower gut. The prebiotic may be concentrated and/or freeze dried. The composition may be in a number of formats, such as a drinkable liquid and/or powder which can be mixed with a solid or liquid food stuff. 
         [0014]    The prebiotic composition may be combined with one or more active ingredients, such as vitamins, minerals, phytochemicals, antioxidants, and combinations thereof. 
         [0015]    Vitamins may include fat soluble vitamins such as vitamin A, vitamin D, vitamin E, and vitamin and combinations thereof. In some embodiments, vitamins can include water soluble vitamins such as vitamin C (ascorbic acid), the B vitamins (thiamine or B 1, riboflavoin or B25 niacin or B3, pyridoxine or B6, folic acid or B9, cyanocobalimin or B12, pantothenic acid, biotin), and combinations thereof. 
         [0016]    Minerals may include but are not limited to sodium, magnesium, chromium, iodine, iron, manganese, calcium, copper, fluoride, potassium, phosphorous, molybdenum, selenium, zinc, and combinations thereof. 
         [0017]    Antioxidants may include but are not limited to ascorbic acid, citric acid, rosemary oil, vitamin A, vitamin E, vitamin E phosphate, tocopherols, di-alpha-tocopheryl phosphate, tocotrienols, alpha lipoic acid, dihydrolipoic acid, xanthophylls, beta cryptoxanthin, lycopene, lutein, zeaxanthin, astaxanthin, beta-carotene, carotenes, mixed carotenoids, polyphenols, fiavonoids, and combinations thereof. 
         [0018]    Phytochemicals may include but are not limited to cartotenoids, chlorophyll, chlorophyllin, fiber, flavanoids, anthocyamns, cyaniding, delphinidin, malvidin, pelargonidin, peonidin, petunidin, flavanols, catechin, epicatechin, epigallocatechin, epigailocatechingallate, theaflavins, thearubigins, proanthocyanins, flavonols, quercetin, kaempferol, myricetin, isorhamnetin, flavononeshesperetin, naringenin, eriodictyol, tangeretin, flavones, apigenin, luteolin, lignans, phytoestrogens, resveratrol, isoflavones, daidzein, genistein, glycitein, soy isoflavones, and combinations thereof. 
         [0019]    In accordance with a further aspect of the present invention, there is provided a prebiotic composition for use in the management of cholesterol or the treatment of high cholesterol. Alternatively or additionally, the composition may be for use in the management or treatment of a metabolic syndrome, weight management or obesity or diabetes. The composition comprising a microbially produced oligosaccharide, wherein the oligosaccharide is characterised by being selective for a pre-determined probiotic bacterial strain and also capable of being produced by the pre-determined probiotic bacterial strain by reverse enzyme reaction a prebiotic composition as herein above described for use as a medicament or pharmaceutical and/or a dietary supplement. 
         [0020]    In accordance with a further aspect of the present invention, there is provided a prebiotic composition for the treatment of high cholesterol, a metabolic syndrome, obesity or diabetes, the composition comprising a microbially produced oligosaccharide, wherein the oligosaccharide is characterised by being selective for a pre-determined probiotic bacterial strain and also capable of being produced by the pre-determined probiotic bacterial strain by reverse enzyme reaction. 
         [0021]    In a yet further aspect of the present invention, there is provided a use of a prebiotic composition, in the manufacture of a medicament for the treatment of high cholesterol, a metabolic syndrome, obesity or diabetes, the composition comprising a microbially produced oligosaccharide, wherein the oligosaccharide is characterised by being selective for a pre-determined probiotic bacterial strain and also capable of being produced by the pre-determined probiotic bacterial strain by reverse enzyme reaction. 
         [0022]    It will be apparent to the skilled addressee that the features of the prebiotic as described in the first aspect of the invention will also be applicable and interchangeable for the composition for the management of cholesterol. 
         [0023]    Alternative (or additionally) to a pharmaceutical or medicament, the composition may be used as a dietary supplement, a nutraceutical or a functional food. A yet further aspect of the present invention may be a prebiotic composition for a dietary supplement, a nutraceutical or a functional food, the composition comprising a microbially produced oligosaccharide, wherein the oligosaccharide is characterised by being selective for a pre-determined probiotic bacterial strain and also capable of being produced by the pre-determined probiotic bacterial strain by reverse enzyme reaction. 
         [0024]    It will again be apparent to the skilled addressee that the features of the prebiotic in connection with the first aspect of the invention will also be applicable and interchangeable for the composition for a dietary supplement, a nutraceutical or a functional food. 
         [0025]    Furthermore, the composition could be incorporated into an existing food, such as yoghurt or as a powder which can be easily blended with foodstuffs or made into a liquid drink. 
         [0026]    In accordance with another aspect of the present invention, there is provided a method of screening a composition which is suitable for use as a prebiotic comprising the steps:
       (a) assembling a panel of probiotic bacterial strains;   (b) selecting a strain found to have oligosaccharide activity;   (c) inducing the selected probiotic strain to produce a oligosaccharide prebiotic composition by reverse enzyme reaction; and   (d) isolating the oligosaccharide prebiotic composition.       
 
         [0031]    The method may further comprise:
       (e) assessing growth and/or survivability of the selected probiotic bacterial strain using the isolated oligosaccharide prebiotic composition.       
 
         [0033]    By utilising reverse enzyme reaction in the probiotic bacterial strain to produce a prebiotic which is specific to the probiotic, the subsequent use of the prebiotic allows for greater specificity of growth promotion for the desired probiotic strain at the expense of other bacterial strains. 
         [0034]    It is preferred that the oligosaccharide comprises GOS. 
         [0035]    The method may be used in the identification and production of a prebiotic composition as herein above described. 
         [0036]    In accordance with yet a further aspect of the present invention, there is provided a method of screening for a synbiotic formulation comprising the steps:
       (a) assembling a panel of probiotic bacterial strains;   (b) selecting strains found to have oligosaccharide activity;   (c) inducing the selected probiotic strains to produce a oligosaccharide prebiotic composition by reverse enzyme reaction;   (d) isolating the oligosaccharide prebiotic compositions for each selected strain;   (e) combining each selected strain and corresponding isolated oligosaccharide prebiotic composition in a formulation; and   (f) assessing the improved growth and/or survivability of the selected probiotic bacterial strain for each formulation in a gut model and identifying the formulation showing improved growth and/or survivability.       
 
         [0043]    The gut model may comprise an in vivo method of investigating the gut microbial flora of an individual before and after administration of a given formulation. In the alternative, the gut model may comprise an in vitro method which substantially mimics the conditions of the gut. 
         [0044]    It will be apparent to the skilled addressee that the features of the prebiotic in connection with the first aspect of the invention will also be the desired attributes of the composition forming part of the screened formulation. 
     
    
     
       DETAILED DESCRIPTION OF THE INVENTION 
         [0045]    Embodiments of the present invention will now be described, by way of example only, in which: 
           [0046]      FIG. 1A  is a graph of bacterial count over time using 0.1% lactose as a growth medium for  L. plantarum;    
           [0047]      FIG. 1B  is a graph of bacterial count over time using 0.1% lactose as a growth medium for  L. casei;    
           [0048]      FIG. 1C  is a graph of bacterial count over time using 0.1% lactose as a growth medium for  L. salivarius;    
           [0049]      FIG. 1D  is a graph of bacterial count over time using 0.1% lactose as a growth medium for  L. fermentum;    
           [0050]      FIG. 1E  is a graph of bacterial count over time using 0.1% lactose as a growth medium for  L. rhanmosus;    
           [0051]      FIG. 1F  is a graph of bacterial count over time using 0.1% lactose as a growth medium for  L. delbrueckii;    
           [0052]      FIG. 2A  is a graph of bacterial count over time using 5% lactose as a growth medium for  L. plantarum;    
           [0053]      FIG. 2B  is a graph of bacterial count over time using 5% lactose as a growth medium for  L. casei;    
           [0054]      FIG. 2C  is a graph of bacterial count over time using 5% lactose as a growth medium for  L. salivarius;    
           [0055]      FIG. 2D  is a graph of bacterial count over time using 5% lactose as a growth medium for  L. delbrueckii;    
           [0056]      FIG. 2E  is a graph of bacterial count over time using 5% lactose as a growth medium for  L. rhanmosus;    
           [0057]      FIG. 2F  is a graph of bacterial count over time using 5% lactose as a growth medium for  L. acidophilus;    
           [0058]      FIG. 2G  is a graph of bacterial count over time using 5% lactose as a growth medium for  L. helveticus;    
           [0059]      FIG. 3  is a graph showing the results of different bacterial strains over 14 hours (OD 600  measured every hour) in 0.4% oxgall and 100 mg/L cholesterol concentration in MRS media; 
           [0060]      FIG. 4  is a graph showing the results of different bacterial strains over 2 days prior to testing (OD 600  measured every hour) in 0.4% oxgall and 100 mg/L cholesterol concentration in MRS media; 
           [0061]      FIG. 5A-5C  are graphs show the results of a range of lactobacilli species which were screened for β-galactosidase activity measured at OD 420  in A MRS broth, B 1% lactose basal media and C 5% lactose basal media; 
           [0062]      FIG. 6A-6C  are graphs show the results of a range of lactobacilli species which were screened for β-galactosidase activity measured at uM of o-NP in A MRS broth, B 1% lactose basal media and C 5% lactose basal media; 
           [0063]      FIG. 7  is a graph showing the yield of GOS, lactose and monosaccharides by  L. fermentum  ATCC 11976 over 168 hours; 
           [0064]      FIG. 8  is a graph showing the yield of GOS, lactose and monosaccharides by  L. fermentum  NCIMB 30226 over 168 hours; 
           [0065]      FIGS. 9 &amp; 10  shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS% over time for  L. fermentum  ATCC 11976; 
           [0066]      FIGS. 11 &amp; 12  shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS% over time for  L. fermentum  NCIMB 30226; 
           [0067]      FIG. 13  shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS% over time for 18U.  L. fermentum  ATCC 11976; 
           [0068]      FIG. 14  shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS% over time for 18U.  L. fermentum  NCIMB 30226; 
           [0069]      FIG. 15  shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS% over time for 30U.  L. fermentum  ATCC 11976; 
           [0070]      FIG. 16  shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS% over time for 30U.  L. fermentum  NCIMB 30226; 
           [0071]      FIG. 17  is a graph illustrating the relative growth profiles of a range of bacteria grown on a GOS mixture produced from  L. fermentum  ATCC 11976; and 
           [0072]      FIG. 18  is a second graph illustrating the relative growth profiles of a smaller range of bacteria grown on a GOS mixture produced from  L. fermentum  ATCC 11976. 
       
    
    
       [0073]    Mechanistically glycosidases are all transferases that use water as their preferred acceptor molecule. Under appropriate circumstance, however, such as high concentrations of substrate carbohydrate, these enzymes will transfer monosaccharide moieties from the substrate (acting as glycosyl donor) to other substrate or non-substrate carbohydrates (acting as glycosyl acceptor). Typically, the products of these reactions are complex mixtures containing all possible glycosidic linkages but in differing amounts. As the reactions are kinetically controlled, the linkage profile synthesised should map onto the rate constants for hydrolysis of those linkages by the producing enzyme. Consequently the oligosaccharides may be more readily metabolised by the producing organisms than by others in the gastrointestinal ecosystem. This approach has shown promise in laboratory testing. 
         [0074]    It is possible, however in many enzyme synthesis reactions to include other carbohydrates which will act as acceptors in addition to the lactose. In this way, novel mixtures containing novel structures could be built up. 
         [0075]    Probiotic species such as lactobacilli and bifidobacteria are highly saccharolytic and they frequently produce a range of glycosidase enzymes. These enzymes may have transfer activity and be able to synthesise oligosaccharides. This activity is widely reported for β-galactosidases but has not been as intensively studied for other enzymes such as α-galactosidases, α- and β-glucosidases, α-mannosidases, or β-xylosidases. It is also possible to synthesise oligosaccharides using sucrose dependant glycosyltransferases. These transfer either the fructose or glucose moiety from sucrose to sucrose acceptors and build up long polysaccharide chains. In the presence of suitable acceptors, however, they frequently synthesise hetero-oligosaccharides. This has been shown to occur with dextransucrase and alternansucrase and may also occur with laevansucrase. 
         [0076]    The experiments sought to explore a strategy to use the products of one synthesis reaction as acceptors in a subsequent reaction. If a probiotic produces a β-galactosidase and a laevan sucrase, for instance, an enzyme extract could be used to synthesise galactooligosaccharides. This product mixture could then be used with the same extract and sucrose as glycosyl donor to bring about the synthesis of fructans—many of which would be built up on the galacto-oligosaccharides which would act as acceptors. In this way novel complex mixtures could be produced that should have a highly tailored fermentation by the producing organism. 
         [0077]    The basis of the present experiments was to reversibly use β-galactosidases in microorganisms so as to produce a novel GOS. Ordinarily, β-galactosidases would digest lactose. However, by changing the reaction conditions, in terms of substrate and temperature, the enzyme acts reversibly and generates an oligosaccharide version of the lactose (GOS). 
         [0078]      Lactobacilli  are more frequently used as probiotics than are bifidobacteria, yet no prebiotic selective to lactobacilli exists. As these probiotics also harbour β-galactosidase activity, the experiments induced the production of GOS which was specific to these probiotics. The metabolism of prebiotics like GOS are species specific (as evidenced by Bi-Immuno and Bifido bacteria), so a  Lactobacilli  GOS has the potentially enhance the growth, survivability, and health benefits of  lactobacilli.    
         [0079]    The experiments undertaken were as follows:
       1. Assemble and test a range of probiotic  lactobacilli  for their capacity to generate GOS and measuring β-galactosidase activities;   2. Generate a prebiotic GOS using the reverse enzyme procedure;   3. Scale up of the novel molecule to allow in vitro testing;   4. Compare survival and growth of lactobacilli in the absence and presence of the prebiotic in a series of ‘gut model’ experiments that test the probiotics and synbiotics;   5. Assess the possibility for using GOS as encapsulation material for the lactobacilli; and   6. Test delivery properties of the encapsulation material.       
 
         [0086]    The bacterial strains initially investigated during the first stage of the experiments are shown below in Table 1: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Strain 
                 Number 
                 Origin 
               
               
                   
                   
               
             
             
               
                   
                 
                   Lactobacillus acidophilus 
                 
                 NCIMB 30184 
                 Human 
               
               
                   
                 
                   Lactobacillus rhamnosus 
                 
                 NCIMB 30188 
                 Human 
               
               
                   
                 
                   Lactobacillus plantarum 
                 
                 NCIMB 30187 
                 Pickled cabbage 
               
               
                   
                 
                   Lactobacillus delbrueckii 
                 
                 NCIMB 30186 
                 Yogurt 
               
               
                   
                 ssp.  bulgaricus   
               
               
                   
                 
                   Lactobacillus casei 
                 
                 NCIMB 30185 
                 Cheese 
               
               
                   
                 
                   Lactobacillus salivarius 
                 
                 NCIMB 30225 
                 Human 
               
               
                   
                 ssp.  salivarius   
               
               
                   
                 
                   Lactobacillus fermentum 
                 
                 NCIMB 30226 
                 Dairy 
               
               
                   
                 
                   Lacobacillus helveticus 
                 
                 NCIMB30224 
                 Dairy 
               
               
                   
                 
                   Lactobacillus fermentum 
                 
                 ATCC11976 
                 Human 
               
               
                   
                 
                   Lactobacillus salivarius 
                 
                 ATCC 11741 
                 Human 
               
               
                   
                   
               
             
          
         
       
     
         [0087]    Bacterial growth curve determination was undertaken by sampling cultures at 0 h, 3 h, 5 h, 8 and 24 h intervals using a 100 μL of dilution series of culture in 900 μL PBS. 20 μL of each series was spread onto a jar and with a negative control and growth assessed. 
         [0088]    Bacterial count of several of the strains was assessed by using 0.1% lactose as the growth medium.  FIGS. 1A-1F  show that bacterial count over time using 0.1% lactose as a growth medium for  L. plantarum, L. casei, L. salivarius, L. fermentum, L. rhanmosus,  and  L. delbrueckii  all resulted in a steady growth curve from approximately 6.5 log 10 CFU/ml to just over 9.5 log 10 CFU/ml at around 13 hours and growth tailed off as it did not increase by 25 hours. 
         [0089]    Bacterial count of several of the strains was assessed by using 5% lactose as the growth medium.  FIGS. 2A-2G  show the bacterial count over time using 5% lactose as a growth medium for  L. plantarum, L. casei, L. salivarius, L. delbrueckii, L. rhanmosus, L. acidophilus  and  L. helveticus.  Again, all resulted in a steady growth curve from approximately 6.5 log 10 CFU/ml to just over 9.5 log 10 CFU/ml at around 13 hours and growth was then flat as it did not increase by 25 hours. 
         [0090]    Cholesterol was then included in the culture medium of the bacterial strains and each strain tested for quantity of cholesterol after incubation. 
         [0091]    The cholesterol assay used relies on the following formula: 
         [0000]      % cholesterol×dry weight (g) −1 =( B−T/B× 100)/ W  
 
         [0000]    Where B=cholesterol content in the uninoculated control mg/I −1 , T=cholesterol in culture medium mg/l −1  and W=cells (dry weight g after 12 h of inc). 
         [0092]    The pellet weight of the culture was measured independently of the supernanent and the spent broth (evaporated residues) also measured. The cholesterol assay was run in triplicate in several runs. 
         [0093]      FIG. 3  shows the growth of different bacterial strains over 14 hours (OD 600  measured every hour) in 0.4% oxgall and 100 mg/L cholesterol concentration in MRS media and shows that some bacterial strains were much more effective at growing in this media.  L. Planatarum  showed the best growth profile, followed by  L. delbrueckii, L. casei  and  L. fermentum.    FIG. 3  shows the growth of different bacterial strains over 12 hours (OD 600  measured every hour) in 0.4% oxgall and 100 mg/L cholesterol concentration in MRS media and shows that some bacterial strains were much more effective at growing in this media.  L. planatarum  showed the best growth profile, followed by  L. delbrueckii, L. casei  and  L. fermentum.    
         [0094]      FIG. 4  is a graph showing the results of different bacterial strains over 2 days prior to testing (OD 600  measured every hour) in 0.4% oxgall and 100 mg/L cholesterol concentration in MRS media.  L. fermentum  showed the best growth profile, followed by  L. rhanmosus, L. halveticus, L. halveticus  and  L. salivarius.    
         [0095]    Direct plate assay tests were then conducted on the strains to further measure cholesterol activity. Resting cell Bile Salt Hydrolase (BSH) activity was measured to assess the release of amino acids from hydrolysis of conjugated bile acids. Bile salt deconjugation (based upon the release of free cholic acid) was measured and finally co-precipitation of cholesterol with deconjugated bile assessed. Table 2 below shows the results of the direct plate assay. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Bacteria 
                 1 st  run 
                 2 nd  run 
                 3 rd  run 
               
               
                   
                   
               
             
             
               
                   
                 
                   L. casei 
                 
                 Y 
                 Y 
                 Y 
               
               
                   
                 
                   L. delbrueckii 
                 
                 Y 
                 Y 
                 Y 
               
               
                   
                 
                   L. acidophilus 
                 
                 Y 
                 Y 
                 Y 
               
               
                   
                 
                   L. fermentum 
                 
                 X 
                 Y 
                 Y 
               
               
                   
                 
                   L. salivarius 
                 
                 X 
                 X 
                 X 
               
               
                   
                 
                   L. halveticus 
                 
                 Y 
                 X 
                 X 
               
               
                   
                 
                   L. rhamnosus 
                 
                 X 
                 X 
                 X 
               
               
                   
                 
                   L. plantarum 
                 
                 X 
                 Y 
                 Y 
               
               
                   
                   L. salivarius * 
                 X 
                 X 
                 X 
               
               
                   
                   L. fermentum * 
                 X 
                 X 
                 Y 
               
               
                   
                   
               
             
          
         
       
     
         [0096]    It can be seen that  L. casei, L. delbrueckii  and  L. acidophilus  all had reliable BSH activity. 
         [0097]    By comparing the results of the strains being able to grow in media containing cholesterol and those strains having BSH activity  L. casei  and  L. delbrueckii  appear to be suitable candidates for producing and identifying a specific prebiotic GOS. 
         [0098]    The GOS prebiotic generated by a specific strain has optimised metabolism not just to produce the GOS, but also to metabolise it (as its generated from a reverse enzyme procedure). The GOS can therefore be incorporated with the probiotic into a synbiotic that would create a highly selective environment for the probiotic. As a probiotic can have a specific health benefits then a synbiotic formula which is tailored to a specific health benefit can be generated. 
         [0099]    A screening method for identifying and formulating a synbiotic composition in accordance with an aspect of the invention follows the steps of:
   (a) Identifying health need;   (b) Identifying key interjection points for probiotic action e.g BSH activity, cholesterol assimilation &amp; heart disease;   (c) Screening probiotic library using high throughput screening methodology;   (d) Identifying strains with potential activity &amp; health benefits;   (e) Optimising expression of activity using fermentation processes;   (f) Screening strains for beta galactosidase activity;
       (g) Generating a novel GOS;   (h) Scaling up to allow in vitro testing;   (i) Comparing survival and growth of the probiotic in the absence and presence of the prebiotic using in vitro plate assays and gut model. If strain characterised then use molecular methodologies to study population changes over time. This will see if affect due to increasing number or increasing activity; and   (j) Combining pre &amp; probiotic to explore effect of combined pre &amp; probiotic.
 
Evaluation of Anaerobic Utilisation of Novel  L. reuteri  GOS
   
       
 
         [0110]    In these experiments, anaerobic cultures were tested to evaluate the in vitro utilisation of a novel  Lactobacillus reuteri  galactooligosaccharide by monitoring the populations of gut bacterial groups at 24 hours using fluorescent in situ hybridisation, and short-chain fatty acid (SOFA). Fructooligosaccharides (FOS), melibiose and raffinose were used as reference carbohydrates. The table below shows the results of these experiments. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                   
                   
                 GOS +  
                   
               
               
                   
                   
                   
                   
                   
                   
                 
                   L.  
                 
                 GOS +  
               
               
                   
                   
                 Melibiose 
                 Raffinose 
                 FOS 
                 GOS 
                 
                   acidophilus 
                 
                 
                   L. reuterri 
                 
               
             
          
           
               
                   
                   
                   
                 24 hr 
                   
                 24 hr 
                   
                 24 hr 
                   
                 24 hr 
                   
                 24 hr 
                   
                 24 hr 
               
               
                   
                   
                   
                 % 
                   
                 % 
                   
                 % 
                   
                 % 
                   
                 % 
                   
                 % 
               
               
                 Group 
                 Inoculum 
                 24 
                 change 
                 24 
                 change 
                 24 
                 change 
                 24 
                 change 
                 24 
                 change 
                 24 
                 change 
               
               
                   
               
             
          
           
               
                 Total count 
                 8.84 
                 
                   9.14 
                 
                 
                   103% 
                 
                 
                   9.19 
                 
                 
                   104% 
                 
                 
                   9.2  
                 
                 
                   104% 
                 
                 9.12 
                 103% 
                 
                   9.55 
                 
                 
                   108% 
                 
                 
                   9.34 
                 
                 
                   106% 
                 
               
               
                 
                   Bifidobacteria 
                 
                 6.85 
                 7.33 
                 107% 
                 
                   7.69 
                 
                 112% 
                 
                   7.47 
                 
                 
                   109% 
                 
                 
                   7.69 
                 
                 
                   112% 
                 
                 
                   7.83 
                 
                 
                   114% 
                 
                 
                   8.19 
                 
                 
                   120% 
                 
               
               
                 
                   Bacteroides 
                 
                 7.98 
                 7.9  
                  99% 
                 8.08 
                 
                   101% 
                 
                 8.08 
                 101% 
                 7.95 
                 100% 
                 8.01 
                 100% 
                 7.89 
                  99% 
               
               
                 
                   Lactobacilli 
                 
                 7.15 
                 
                   7.43 
                 
                 
                   104% 
                 
                 
                   7.45 
                 
                 
                   104% 
                 
                 7.32 
                 102% 
                 
                   7.69 
                 
                 
                   108% 
                 
                 
                   7.67 
                 
                 
                   107% 
                 
                 
                   7.73 
                 
                 
                   108% 
                 
               
               
                 
                   Clostridia 
                 
                 7.55 
                 7.65 
                 101% 
                 7.81 
                 103% 
                 8   
                 106% 
                 7.23 
                  96% 
                 7.48 
                  99% 
                 7.2  
                  95% 
               
               
                 
                   E. coli 
                 
                 8.14 
                 
                   7.66 
                 
                 
                    94% 
                 
                 8.03 
                  99% 
                 
                   7.85 
                 
                 
                    96% 
                 
                 8.04 
                  99% 
                 8.24 
                 101% 
                 7.96 
                  98% 
               
               
                 
                   Eubacteria 
                 
                 8.06 
                 7.84 
                  97% 
                 
                   8.69 
                 
                 
                   108% 
                 
                 8.27 
                 103% 
                 
                   7.75 
                 
                 
                    96% 
                 
                 8.16 
                 101% 
                 
                   8.28 
                 
                 
                   103% 
                 
               
               
                   
               
               
                 (Key: 
               
               
                 BOLD = Significant Increase; 
               
               
                 Italics = Significant Decrease) 
               
             
          
         
       
     
         [0111]    The results show the  Lactobacillus reuterri  GOS showed a significant increase in bifidobacteria and  lactobacilli  population numbers exhibiting a prebiotic affect. In addition, the GOS increased the growth rate of  lactobacilli  by 108%, more than any other sugar suggesting a genus specificity. Addition of a strain of  Lactobacillus reuterri  increased the prebiotic affect, increasing the bifidobacterium population by 120%. 
         [0112]    This suggests that the addition of a GOS producing organism to the GOS produced by that organism had a greater effect on the gut microflora population than the GOS alone. 
         [0113]      Lactobacilli  β-Galactosidase Screening Assay 
         [0114]    In these experiments, 10  lactobacilli  species were screened for β-galactosidase activity in triplicate using standard enzyme assay with o-NPG as substrate. The experiments were carried out in 3 different media; MRS, 1% and 5% lactose in basal media, as lactose is the primary substrate for β-galactosidase it was expected to exhibit highest activity. Activity was measured at time points between time 0-24 hrs, highest activity was shown after 24 hrs. As shown in  FIGS. 5-6 , in general, 5% lactose exhibits highest enzyme activity and tends to be higher than in MRS broth (contains only glucose as carbon source). High enzyme activity is essential for generating GOS, the 3 organisms which show overall high activity include both  L. fermentum  strains and  L. casei.    
         [0000]    GOS Produced from  L. fermentum  ATCC 11976 and  L. fermentum  NCIMB 30226 in a Long Time Period 
         [0115]    In these experiments,  L. fermentum  ATCC 11976 and  L. fermentum  NCIMB 30226 were assessed for their production (and consumption) of GOS, lactose and monosaccharides over 168 hours. 
         [0116]    The yield of GOS, lactose and monosaccharides for  L. fermentum  ATCC 11976 is shown in the below and in  FIG. 7 : 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                 Time 
                   
                   
                   
                   
                   
               
               
                 point 
                 GOS 
                 lactose 
                 Monosaccharides 
                 Total 
                 GOS %= 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 0 
                 0.601 
                 85 
                 1.464 
                 87.065 
                 0.690289 
               
               
                 16 
                 15.65 
                 30.077 
                 18.92 
                 64.647 
                 24.20839 
               
               
                 22 
                 183 
                 130 
                 75 
                 388 
                 47.16495 
               
               
                 36 
                 14.4 
                 25.6 
                 11.45 
                 51.45 
                 27.98834 
               
               
                 48 
                 14 
                 33 
                 10 
                 57 
                 24.5614 
               
               
                 168 
                 27.4 
                 32.971 
                 0.5 
                 60.871 
                 45.01322 
               
               
                   
               
             
          
         
       
     
         [0117]    The yield of GOS, lactose and monosaccharides for  L. fermentum  NCIMB 30226 is shown in the below and in  FIG. 8 : 
         [0000]                                                                          Time                           point   GOS   lactose   Monosaccharides   Total   GOS %=                                0   2.206   53.309   2.538   58.053   3.799976       16   20.789   74.275   24.481   119.545   17.3901       22   15.066   53.918   15.713   84.697   17.78812       36   9.699   30.672   6.977   47.348   20.4845       48   13.971   47.341   7.944   69.256   20.17298       168   9.3   28.125   0.521   37.946   24.50851                    
GOS Produced from  L. fermentum  ATCC 11976 in a 20% Lactose Medium Over 24 Hours
 
         [0118]    In this experiment, GOS synthesis from  L. fermentum  ATCC 11976 β-galactosidase was investigated. After lysis, the crude extract was incubated in 20% lactose over 24 hr and samples taken at time 0 and 24. 
         [0119]    The table below shows the sugars present at T0: 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Ret.  
                   
                   
                   
                   
                   
                   
               
               
                   
                 Time 
                 Height 
                 Width 
                   
                 Asym. 
                 Plates 
                   
               
               
                 No. 
                 min 
                 v 
                 min 
                 Type 
                 (EP) 
                 (EP) 
                   
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                  0.226 
                 0.397 
                 n.a. 
                 BM 
                 n.a. 
                 n.a. 
                   
               
               
                 2 
                  0.689 
                 0.283 
                 n.a. 
                 MB 
                 n.a. 
                 n.a. 
                   
               
               
                 3 
                  6.912 
                 1.743 
                 n.a. 
                 Ru 
                 n.a. 
                 n.a. 
                   
               
               
                 4 
                  8.436 
                 1.465 
                 n.a. 
                 Ru 
                 n.a. 
                 n.a. 
                   
               
               
                 5 
                  9.072 
                 1.234 
                 n.a. 
                 Ru 
                 n.a. 
                 n.a. 
                   
               
               
                 6 
                 10.716 
                 13.758  
                  1.419  
                 BMb 
                 0.87 
                 851 
                   
               
               
                 7 
                 14.403 
                 0.605 
                 n.a. 
                 Ru 
                 n.a. 
                 n.a. 
                   
               
               
                 8 
                 18.457 
                 16.603  
                 n.a. 
                 bM 
                 n.a. 
                 n.a. 
                   
               
               
                 9 
                 18.694 
                 17.001  
                 n.a. 
                 M 
                 n.a. 
                 n.a. 
                   
               
               
                 10 
                 22.318 
                 0.373 
                 n.a. 
                 Ru 
                 n.a. 
                 n.a. 
                   
               
               
                 11 
                 24.168 
                 29.345  
                 29.609  
                 M 
                 n.a. 
                 n.a. 
                   
               
               
                 
                   12 
                 
                 
                   28.157 
                 
                 
                   150.287 
                 
                 
                    1.544 
                 
                 
                   MB 
                 
                 
                   n.a. 
                 
                 
                   5436 
                 
                 
                   Lactose 
                 
               
               
                 n.a. 
                 n.a. 
                 n.a. 
                 n.a. 
                 n.a. 
                 n.a. 
                 n.a. 
                   
               
               
                 Av- 
                   
                 19.424  
                 10.857 
                   
                 0.87 
                 3144 
                   
               
               
                 erage: 
               
               
                   
               
             
          
         
       
     
         [0120]    The table below shows the sugars present at T24: 
         [0000]                                                                                          Ret.                                    Time   Height   Width       Resol.   Asym.   Plates           min   v   min   Type   (EP)   (EP)   (EP)                                 2.506   0.010   n.a.   BMB   n.a.   1.52   128            6.903   0.097   n.a.   BM   n.a.   n.a.   n.a.           10.624   10.367    1.121   M   1.75   n.a.   1425           15.062   3.082   3.812   MB   2.17   n.a.   232           20.868   1.220   1.268   BMB   2.66   0.65   3522             24.177       10.614        1.097       BMb       3.50       1.57       7869       GOS           28.167       73.205        1.207       bM       n.a.       1.45       8860       Lactose         29.600   5.009   2.231   M   n.a.   n.a.   n.a.             32.806       10.232        1.873       M       1.05       n.a.       5038       Glucose           34.822       8.609       2.038       M       n.a.       n.a.       4812       Ga-                                       lactose         41.161   0.867   n.a.   M   n.a.   n.a.   n.a.           43.560   0.590   n.a.   M   n.a.   n.a.   n.a.           46.616   0.386   n.a.   M   n.a.   n.a.   n.a.           49.693   0.107   n.a.   MB   n.a.   n.a.   n.a.           51.010   0.006   n.a.   bMB   n.a.   n.a.    n.a.           54.025   0.006   n.a.   BMB   1.18   1.41   774387           54.751   0.008   n.a.   BMB   n.a.   1.27    48500           n.a.   n.a.   n.a.   n.a.   n.a.   n.a.    n.a.               7.319   1.831       2.05   1.31   85477                        
GOS Produced from  L. fermentum  ATCC 11976 and  L. fermentum  NCIMB 30226 in a Short Time Period
 
         [0121]    In this experiment, GOS was produced from  L. fermentum  ATCC 11976 and  L. fermentum  NCIMB 30226 and the enzyme activity of the sugars vs the % GOS assessed over 50 hours as this was when most activity took place during the previous experiments. 
       Protocol 
       [0122]    GOS was produced using the following protocol:
       1. Set up 50 ml overnight cultures in modified MRS broth supplemented with 2% lactose for  L. fermentum  ATCC 11976 and  L. fermentum  NCIMB 30226;   2. Suspend 50 ml of overnight culture in 1 L of mMRS broth with 2% lactose;   3. Incubate in anaerobic cabinet at 37° C.;   4.  L. fermentum  ATCC 11976 for 14 hours;   5.  L. fermentum  NCIMB 30226 for 8 hours;   6. Measure OD 660 ;   7. Centrifuge cultures, 10 000g×10 mins;   8. Make up 40% lactose in sodium phosphate buffer. 400 g/L;   9. Pour off supernatant;   10. Resuspend pellets in sodium phosphate buffer (50 mM, pH 6.8);   11. Pool pellets in 50 ml falcons;   12. Freeze thaw in Liquid Nitrogen ×3;   13. French Press, 30,000 PSI, 1 pass, 5 drops/min;   14. Spin down lysate—15,000 g×45 min;   15. Pour supernatant into fresh falcon;   16. Carry out β gal activity assay to work enzyme concentrations;   17. Incubate the free cell extract with 40% lactose/sodium phosphate buffer;   18. Sample 200 μl every 2 hours over 50 hours;   19. Freeze samples;   20. Filter sterilise all samples through 0.2 μm filter;   21. Analyse on HPLC.       
 
       Results—GOS Production 
       [0144]    As shown in  FIGS. 9 to 12 , there was a 30-45% lactose conversion and 10% GOS yield. 
       Enzyme Activity 
       [0145]    A further experiment was conducted in order to ascertain the enzyme activity (and therefore efficiency) of the GOS produced from  L. fermentum  ATCC 11976 and  L. fermentum  NCIMB 30226. 
         [0146]    Cultures were grown for 8 hrs F, 14 hr for F* in 1 L and harvested at 12,000 g×10 min. The cells were lysed and cell extract spun down 15,000 g×45 min. This was then incubated at 40° C. in 40% lactose sodium phosphate buffer +MgCl 2  with same U of enzyme/reaction and activity analysed on an HPLC at 2 hour time points for 36 hours. 
         [0147]    The enzyme unit calculations were as follows: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                 OD pre 
                 OD 420  (enzyme) 
                 OD 420  (enzyme) 
                 Enzyme 
               
               
                 Organism 
                 harvest 
                 after french press 
                 after final spin 
                 U/15 ml 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 F*1 
                 0.83 
                 2.4605 
                 2.3315 
                 18.23977 
               
               
                 F*2 
                 0.86 
                 1.83 
                 3.1955 
                 30.17002 
               
               
                 F1 
                 0.94 
                 1.833 
                 3.812 
                 30.0665 
               
               
                 F2 
                 1.13 
                 1.5739 
                 6.0115 
                 47.63684 
               
               
                   
               
               
                 Where F*1, F2 18 U/reaction, F*2, F1 30 U/reaction. 
               
             
          
         
       
     
       Results 
       [0148]    As shown in  FIGS. 13 to 16 , there was a 40-50% lactose conversion and 15-20% GOS yield. 
         [0000]      Lactobacilli  Specificity with GOS Purity 
         [0149]    In this experiment, GOS produced from  L. fermentum  ATCC 11976 used as part of the growth media for a range of bacteria to see if this species specific GOS provided any growth specificity. 
       GOS Synthesis 
       [0150]      L. fermentum  ATCC 11976 was grown in modified MRS supplemented with 2% lactose in 1 L cultures for 14 hours. The culture was spun down and re-suspend in a sodium phosphate buffer. The cells were lysed using liquid Nitrogen and a French Press and the lysate spun to obtain free cell extract. The free cell extract was incubated with 40% Lactose and a sample taken every 2 hours over 50 hours. Samples were loaded on HPLC after every time point for analysis. 
       Growth Curves 20% GOS Mixture 
       [0151]    1% of the impure GOS produced earlier was added to 9 ml mMRS hungates. The growth of a range of organisms were on this mixture were analysed:  Clostridium difficile, Bifidobacterium bifidum, Bifidobacterium longum, Lactobacillus fermentum  ATCC 11976,  Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus casei  &amp;  Lactobacillus delbrueccki.  Experiments were conducted in 3 repeats in triplicate with enumeration at 0, 3, 6, 8, 16 and 24 hours. 
       Results 
       [0152]    As shown in  FIGS. 17 and 18 , little growth was found in  C.difficile,  whereas the best growth was found in  L.rhamnosus.  The 20% GOS mixture as generally more selective towards  lactobacilli.    
         [0153]    The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.