COMPOSITION & METHODS OF SCREENING

Abstract

The present invention relates to a symbiotic composition comprising a probiotic bacterial strain and a prebiotic growth medium which is specific to the growth of the probiotic bacterial strain, wherein the bacterial strain is capable of producing the same growth medium by reverse enzyme reaction. The present invention also relates to methods of producing and screening for such compositions.

Claims

1-22. (canceled)

23. A symbiotic composition comprising: a Lactobacilli probiotic strain comprising a -galactosidase enzyme producing a 1-4 linked galacto-oligosaccharide (GOS) through a reverse -galactosidase enzyme reaction; and a prebiotic growth medium comprising 1-4 linked GOS produced by the Lactobacilli probiotic strain through the reverse -galactosidase enzyme reaction.

24. The composition of claim 23, wherein the concentration of the prebiotic growth medium is determined by the probiotic strain.

25. The composition of claim 23, wherein the Lactobacilli probiotic strain is in an amount in the range of 10.sup.5 cfu/g to 10.sup.12 cfu/g.

26. The composition of claim 23, wherein the Lactobacilli probiotic strain and/or the growth medium is encapsulated.

27. The composition of claim 26, wherein the growth medium encapsulates the Lactobacilli probiotic strain.

28. The composition of claim 23, wherein the composition further comprises an excipient or carrier compound that enables the Lactobacilli probiotic strain and/or growth medium to pass through the gastrointestinal environment of the body.

29. The composition of claim 23, wherein the Lactobacilli probiotic strain is concentrated and/or freeze dried.

30. The composition of claim 23, wherein the composition is in the form of a drinkable liquid and/or can be mixed with a solid or liquid food stuff.

31. The composition of claim 23, wherein the growth medium comprises 20% or more of the 1-4 linked GOS produced by the Lactobacilli probiotic strain.

32. A medicament comprising the composition of claim 23.

33. A dietary supplement comprising the composition of claim 23.

34. A method of producing a synbiotic composition comprising: selecting a Lactobacilli probiotic strain comprising a -galactosidase enzyme producing a 1-4 linked galacto-oligosaccharide (GOS) through a reverse -galactosidase enzyme reaction, the Lactobacilli probiotic strain producing a prebiotic growth medium comprising the 1-4 linked GOS; establishing a concentration of the prebiotic growth medium to provide growth selectivity of the desired quantity of the probiotic bacterial strain; and combining the bacterial strain with the established concentration of growth medium to form the symbiotic composition.

35. The method of claim 34, further comprising producing the prebiotic growth medium with the Lactobacilli probiotic strain.

36. The method of claim 35, wherein the growth medium comprises 20% or more of the 1-4 linked GOS produced by the Lactobacilli probiotic strain.

37. The method of claim 34, further comprising encapsulating the symbiotic composition.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0046] Embodiments of the present invention will now be described, by way of example only and with reference to the following Figures:

[0047] FIG. 1A-C are graphs show the results of a range of lactobacilli species which were screened for -galactosidase activity measured at OD.sub.420 in A MRS broth, B 1% lactose basal media and C 5% lactose basal media;

[0048] FIG. 2A 2C are graphs show the results of a range of lactobacilli species which were screened for -galactosidase activity measured at uM of o-NP in A MRS broth, B 1% lactose basal media and C 5% lactose basal media;

[0049] FIG. 3 is a graph showing the yield of GOS, lactose and monosaccharides by L. fermentum ATCC 11976 over 168 hours;

[0050] FIG. 4 is a graph showing the yield of GOS, lactose and monosaccharides by L. fermentum NCIMB 30226 over 168 hours;

[0051] FIGS. 5 & 6 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for L. fermentum ATCC 11976;

[0052] FIGS. 7 & 8 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for L. fermentum NCIMB 30226;

[0053] FIG. 9 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for 18U. L. fermentum ATCC 11976;

[0054] FIG. 10 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for 18U. L. fermentum NCIMB 30226;

[0055] FIG. 11 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for 30U. L. fermentum ATCC 11976;

[0056] FIG. 12 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for 30U. L. fermentum NCIMB 30226;

[0057] FIG. 13 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

[0058] FIG. 14 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.

[0059] 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.

[0060] 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.

[0061] 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 -galactosidases but has not been as intensively studied for other enzymes such as -galactosidases, - and -glucosidases, -mannosidases, or -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.

[0062] 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 -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 galactooligosaccharides 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.

[0063] One particular experiment was conducted to reversibly use -galactosidases in microorganisms. Ordinarily, these 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).

[0064] Lactobacilli are more frequently used as probiotics than are bifidobacteria, yet no prebiotic selective to lactobacilli exists. As these probiotics also harbour -galactosidase activity, GOS which was specific to these probiotics was produced. 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. Ultimately, by combining the prebiotic and probiotic an efficacious synbiotic was generated (which would improve gut survival of the former).

[0065] The experiments undertaken were as follows: [0066] 1. Assemble and test a range of probiotic lactobacilli for their capacity to generate GOS. This would involve measuring -galactosidase activities; [0067] 2. Generate a prebiotic GOS using the reverse enzyme procedure; [0068] 3. Scale up of the novel molecule to allow in vitro testing; [0069] 4. Compare survival and growth of lactobacilli in the absence and presence of the prebiotic. This would involve a series of gut model experiments that test the probiotics and synbiotics; [0070] 5. Research the possibility for using GOS as encapsulation material for the lactobacilli; and [0071] 6. Test delivery properties of the encapsulation material.

[0072] 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.

[0073] A screening method for identifying and formulating a synbiotic composition in accordance with an aspect of the invention follows the steps of: [0074] (a) Identifying health need; [0075] (b) Identifying key interjection points for probiotic action e.g BSH activity, cholesterol assimilation & heart disease; [0076] (c) Screening probiotic library using high throughput screening methodology; [0077] (d) Identifying strains with potential activity & health benefits; [0078] (e) Optimising expression of activity using fermentation processes; [0079] (f) Screening strains for glycosidase (e.g beta galactosidase) activity; [0080] (g) Generating a novel oligosaccharide (e.g GOS) [0081] (h) Scaling up to allow in vitro testing; [0082] (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 [0083] (j) Combining pre & probiotic to explore Optibiotic affect of combined pre & probiotic.
Evaluation of Anaerobic Utilisation of Novel L. reuteri GOS

[0084] 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 (SCFA). Fructooligosaccharides (FOS), melibiose and raffinose were used as reference carbohydrates. The table below shows the results of these experiments.

TABLE-US-00001 GOS + L. GOS + L. Melibiose Raffinose FOS GOS acidophilus 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)

[0085] 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%.

[0086] 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.

Lactobacilli -Galactosidase Screening Assay

[0087] In these experiments, 10 lactobacilli species were screened for -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 -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. 1-2, 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.

GOS Produced from L. fermentum ATCC 11976 and L. fermentum NCIMB 30226 in a Long Time Period

[0088] 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.

[0089] The yield of GOS, lactose and monosaccharides for L. fermentum ATCC 11976 is shown in the below and in FIG. 3:

TABLE-US-00002 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

[0090] The yield of GOS, lactose and monosaccharides for L. fermentum NCIMB 30226 is shown in the below and in FIG. 4:

TABLE-US-00003 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

[0091] In this experiment, GOS synthesis from L. fermentum ATCC 11976 -galactosidase was investigated. After lysis, the crude extract was incubated in 20% lactose over 24 hr and samples taken at time 0 and 24.

[0092] The table below shows the sugars present at TO:

TABLE-US-00004 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. Average: 19.424 10.857 0.87 3144

[0093] The table below shows the sugars present at T24:

TABLE-US-00005 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 Galactose 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

[0094] 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

[0095] GOS was produced using the following protocol: [0096] 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; [0097] 2. Suspend 50 ml of overnight culture in 1 L of mMRS broth with 2% lactose; [0098] 3. Incubate in anaerobic cabinet at 37 C.; [0099] 4. L. fermentum ATCC 11976 for 14 hours; [0100] 5. L. fermentum NCIMB 30226 for 8 hours; [0101] 6. Measure OD.sub.660; [0102] 7. Centrifuge cultures, 10 000 g10 mins; [0103] 8. Make up 40% lactose in sodium phosphate buffer. 400 g/L; [0104] 9. Pour off supernatant; [0105] 10. Resuspend pellets in sodium phosphate buffer (50 mM, pH 6.8); [0106] 11. Pool pellets in 50 ml falcons; [0107] 12. Freeze thaw in Liquid Nitrogen 3; [0108] 13. French Press, 30,000 PSI, 1 pass, 5 drops/min; [0109] 14. Spin down lysate15,000 g45 min; [0110] 15. Pour supernatant into fresh falcon; [0111] 16. Carry out gal activity assay to work enzyme concentrations; [0112] 17. Incubate the free cell extract with 40% lactose/sodium phosphate buffer; [0113] 18. Sample 200 l every 2 hours over 50 hours; [0114] 19. Freeze samples; [0115] 20. Filter sterilise all samples through 0.2 m filter; [0116] 21. Analyse on HPLC.

ResultsGOS Production

[0117] As shown in FIGS. 5 to 8, there was a 30-45% lactose conversion and 10% GOS yield.

Enzyme Activity

[0118] 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.

[0119] Cultures were grown for 8 hrs F, 14 hr for F* in 1 L and harvested at 12,000 g10 min. The cells were lysed and cell extract spun down 15,000 g45 min. This was then incubated at 40 C. in 40% lactose sodium phosphate buffer +MgCl.sub.2 with same U of enzyme/reaction and activity analysed on an HPLC at 2 hour time points for 36 hours.

[0120] The enzyme unit calculations were as follows:

TABLE-US-00006 OD OD.sub.420 (enzyme) OD.sub.420 (enzyme) Enzyme Organism pre 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 18U/reaction, F*2, F1 30U/reaction.

Results

[0121] As shown in FIGS. 9 to 12, there was a 40-50% lactose conversion and 15-20% GOS yield.

Lactobacilli Specificity with GOS Purity

[0122] 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

[0123] 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 resuspend 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

[0124] 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 & Lactobacillus delbrueccki. Experiments were conducted in 3 repeats in triplicate with enumeration at 0, 3, 6, 8, 16 and 24 hours.

Results

[0125] As shown in FIGS. 13 and 14, little growth was found in C. difficile, whereas the best growth was found in L. rhamnosus. The 20% GOS mixture was generally more selective towards lactobacilli.

[0126] 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.