Prebiotic composition and its methods of production

Abstract

The present invention relates to a prebiotic composition comprising a galacto oligosaccharide (GOS) produced from Lactobacillus plantarum, wherein the GOS acts as a selective growth medium for a chosen Lactobacillus plantarum probiotic bacterial strain, and wherein the GOS is substantially the same as the form produced by reverse β-galactosidase reaction in the chosen probiotic bacterial strain. The present invention also relates to methods of producing GOS and related composition incorporating the GOS.

Claims

1. A Lactobacillus synbiotic composition comprising Lactobacillus plantarum 2830 deposited at the European Collection of Cell Cultures (ECGC) under Accession Number 13110402 and galacto oligosaccharide (GOS) produced by Lactobacillus plantarum 2830 ECGC 13110402, wherein the GOS acts as a selective growth medium for Lactobacillus plantarum 2830 ECGC 13110402, and wherein the GOS is produced by reverse β-galactosidase reaction in Lactobacillus plantarum 2830 ECGC 13110402, and wherein at least one of: (a) the composition is encapsulated; (b) the composition further comprises an excipient or carrier compound; (c) the composition is in the form of a drinkable liquid or solid or liquid foodstuff; (d) the composition further comprises an additional active ingredient; and (e) the Lactobacillus plantarum 2830 ECGC 13110402 is freeze-dried.

2. The composition as claimed in claim 1 for use as a medicament.

3. The composition as claimed in claim 1 for use as a dietary supplement.

4. The composition as claimed in claim 1 for use in the management of cholesterol or in the treatment of high cholesterol.

5. The composition as claimed claim 1 for use in the management or treatment of a metabolic syndrome.

6. The composition as claimed in claim 1 for use in weight management.

7. The composition as claimed in claim 1 for use in the management or treatment of diabetes.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Embodiments of the present invention will now be described, by way of example only, in which:

(2) FIG. 1A is a graph of bacterial count over time using 0.1% lactose as a growth medium for L. plantarum;

(3) FIG. 1B is a graph of bacterial count over time using 0.1% lactose as a growth medium for L. casei;

(4) FIG. 1C is a graph of bacterial count over time using 0.1% lactose as a growth medium for L. salivarius;

(5) FIG. 1D is a graph of bacterial count over time using 0.1% lactose as a growth medium for L. fermentum;

(6) FIG. 1E is a graph of bacterial count over time using 0.1% lactose as a growth medium for L. rhanmosus;

(7) FIG. 1F is a graph of bacterial count over time using 0.1% lactose as a growth medium for L. delbrueckii;

(8) FIG. 2A is a graph of bacterial count over time using 5% lactose as a growth medium for L. plantarum;

(9) FIG. 2B is a graph of bacterial count over time using 5% lactose as a growth medium for L. casei;

(10) FIG. 2C is a graph of bacterial count over time using 5% lactose as a growth medium for L. salivarius;

(11) FIG. 2D is a graph of bacterial count over time using 5% lactose as a growth medium for L. delbrueckii;

(12) FIG. 2E is a graph of bacterial count over time using 5% lactose as a growth medium for L. rhanmosus;

(13) FIG. 2F is a graph of bacterial count over time using 5% lactose as a growth medium for L. acidophilus;

(14) FIG. 2G is a graph of bacterial count over time using 5% lactose as a growth medium for L. helveticus;

(15) FIG. 3 is a graph showing the results of different bacterial strains over 14 hours (OD.sub.600 measured every hour) in 0.4% oxgall and 100 mg/L cholesterol concentration in MRS media;

(16) FIG. 4 is a graph showing the results of different bacterial strains over 2 days prior to testing (OD.sub.600 measured every hour) in 0.4% oxgall and 100 mg/L cholesterol concentration in MRS media;

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

(18) 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;

(19) FIG. 7 is a graph showing the yield of GOS, lactose and monosaccharides by L. fermentum ATCC 11976 over 168 hours;

(20) FIG. 8 is a graph showing the yield of GOS, lactose and monosaccharides by L. fermentum NCIMB 30226 over 168 hours:

(21) FIGS. 9 & 10 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for L. fermentum ATCC 11976;

(22) FIGS. 11 & 12 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for L. fermentum NCIMB 30226;

(23) FIG. 13 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for 18U. L. fermentum ATCC 11976;

(24) FIG. 14 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for 18U. L. fermentum NCIMB 30226;

(25) FIG. 15 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for 30U. L. fermentum ATCC 11976;

(26) FIG. 16 shows graphs of the quantity of Sugars (GOS, Lactose and Monosaccharides) and GOS % over time for 30U. L. fermentum NCIMB 30226;

(27) 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;

(28) 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;

(29) FIG. 19 shows a graph of the OD.sub.420 measurement graph of Lactobacillus plantarum 2830 (ECGC 13110402) to assess B-gal activity;

(30) FIG. 20 shows the OD.sub.420 measurement graph of Lactobacillus fermentum NCIMB 30226 to assess B-gal activity;

(31) FIG. 21 shows the OD.sub.420 measurement graph of Lactobacillus fermentum ATCC 11976 to assess B-gal activity;

(32) FIG. 22 shows a chromatogram for Lactobacillus plantarum 2830 (ECGC 13110402) (GOS %=5.2 and Lactose %=89) used to assess initial GOS production;

(33) FIG. 23 shows a chromatogram for Lactobacillus fermentum NCIMB 30226 (GOS %=5.5 and Lactose %=82) used to assess initial GOS production:

(34) FIG. 24 shows a chromatogram for Lactobacillus fermentum ATCC 11976 (GOS %=6 and Lactose %=75) used to assess initial GOS production;

(35) FIG. 25 shows a chromatogram for a control strain of Lactobacillus delbruecki (GOS %=17 and Lactose %=32);

(36) FIG. 26 shows a chromatogram for Lactobacillus plantarum 2830 (ECGC 13110402) at 14 hours grown in 40% lactose at 50° C. with 6 units enzyme/ml;

(37) FIG. 27 shows a chromatogram for Lactobacillus plantarum 2830 (ECGC 13110402) at 24 hours grown in 40% lactose at 50° C. with 6 units enzyme/ml;

(38) FIG. 28 shows a graph of the yield of GOS, lactose and monosaccharides at 12 and 24 hours for Lactobacillus plantarum 2830 (ECGC 13110402)

(39) FIG. 29 is a graph showing the percentage of sugars for Lactobacillus fermentum NCIMB 30226 over 24 hr gown in 40% lactose at 50° C.

(40) FIG. 30 is a graph showing the percentage of sugars for Lactobacillus fermentum ATCC 11976 over 24 hr 40% lactose at 50° C.

(41) FIG. 31A and FIG. 31B show the comparatively different percentage of sugars (and GOS yield) between the Lactobacillus fermentum NCIMB 30226 (FIG. 31A) and Lactobacillus fermentum ATCC 11976 (FIG. 31B) strains when grown in 15% lactose at 40° C.; and

(42) FIG. 32A and FIG. 32B shows the comparatively different percentage of sugars (and GOS yield) between the Lactobacillus fermentum NCIMB 30226 (FIG. 32A) and Lactobacillus fermentum ATCC 11976 (FIG. 32B) strains when grown in 40% lactose at 40° C.

(43) 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.

(44) 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.

(45) 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 alteransucrase and may also occur with laevansucrase.

(46) 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.

(47) 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).

(48) 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 bifidobacteria), so a Lactobacilli GOS has the potentially enhance the growth, survivability, and health benefits of lactobacilli.

(49) 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.
The bacterial strains initially investigated during the first stage of the experiments are shown below in Table 1:

(50) TABLE-US-00001 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

(51) 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.

(52) 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.

(53) 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.

(54) Cholesterol was then included in the culture medium of the bacterial strains and each strain tested for quantity of cholesterol after incubation.

(55) The cholesterol assay used relies on the following formula:
% cholesterol×dry weight (g).sup.−1=(B−T/B×100)/W
Where B=cholesterol content in the uninoculated control mg/l.sup.−1, T=cholesterol in culture medium mg/l.sup.−1 and W=cells (dry weight g after 12 h of inc).

(56) The pellet weight of the culture was measured independently of the supernatant and the spent broth (evaporated residues) also measured. The cholesterol assay was run in triplicate in several runs.

(57) FIG. 3 shows the growth of different bacterial strains over 14 hours (OD.sub.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.sub.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.

(58) FIG. 4 is a graph showing the results of different bacterial strains over 2 days prior to testing (OD.sub.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.

(59) 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.

(60) TABLE-US-00002 TABLE 2 Bacteria 1.sup.st run 2.sup.nd run 3.sup.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

(61) It can be seen that L. casei, L. delbrueckii and L. acidophilus all had reliable BSH activity.

(62) 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.

(63) 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.

(64) 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 & heart disease; (c) Screening probiotic library using high throughput screening methodology; (d) Identifying strains with potential activity & 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 & probiotic to explore effect of combined pre & probiotic.
Evaluation of Anaerobic Utilisation of Novel L. reuteri GOS

(65) 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. Table 3 below shows the results of these experiments.

(66) TABLE-US-00003 TABLE 3 Melibiose Raffinose FOS GOS GOS + L. acidophilus GOS + 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)

(67) 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%.

(68) 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.

(69) Lactobacilli β-Galactosidase Screening Assay

(70) 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.

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

(72) 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.

(73) The yield of GOS, lactose and monosaccharides for L. fermentum ATCC 11976 is shown in the below in Table 4 and in FIG. 7:

(74) TABLE-US-00004 TABLE 4 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
The yield of GOS, lactose and monosaccharides for L. fermentum NCIMB 30226 is shown in the below in Table 5 and in FIG. 8:

(75) TABLE-US-00005 TABLE 5 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

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

(77) Table 6 below shows the sugars present at T0:

(78) TABLE-US-00006 TABLE 6 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
Table 7 below shows the sugars present at T24:

(79) TABLE-US-00007 TABLE 7 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

(80) 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.

(81) Protocol

(82) 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.sub.600; 7. Centrifuge cultures, 10 000 g×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

(83) As shown in FIGS. 9 to 12, there was a 30-45% lactose conversion and 10% GOS yield.

(84) Enzyme Activity

(85) 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.

(86) 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.sub.2 with same U of enzyme/reaction and activity analysed on an HPLC at 2 hour time points for 36 hours.

(87) The enzyme unit calculations were as follows in Table 8 below:

(88) TABLE-US-00008 TABLE 8 OD pre OD.sub.420 (enzyme) OD.sub.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

(89) As shown in FIGS. 13 to 16, there was a 40-50% lactose conversion and 15-20% GOS yield.

(90) Lactobacilli Specificity with GOS Purity

(91) 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.

(92) GOS Synthesis

(93) 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.

(94) Growth Curves 20% GOS Mixture

(95) 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.

(96) 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.

(97) GOS Synthesis Optimisation

(98) Optimisations studies were then conducted in respect Lactobacillus fermentum ATCC 11976, Lactobacillus fermentum NCIMB 30226 and Lactobacillus plantarum 2830 (ECGC 13110402).

(99) A modified MRS media having the following components was used as the growth medium: Bacteriological peptone 10 g/L; Meat extract 8 g/L; Yeast extract 4 g/L; Sodium phosphate monobasic 2 g/L; Sodium acetate 5 g/L; Triammonium citrate 2 g/L; MgSO.sub.4 0.2 g/L; MnSO.sub.4 0.05 g/L; Tween 80 1 ml/L; L cysteine HCL 0.5 g/L; rezasurin—4 ml/L; and supplemented with 2% lactose.

(100) Initial Steps

(101) Initial steps were taken to establish the optimum time for β-Galactosidase (B gal) expression during the growth phase. The method was as follows. Firstly, the cells were grown in the modified MRS (mMRS) (as detailed above) in 10 ml, anaerobically in hungate tubes at 37° C. Samples were then taken at 0, 2, 4, 6, 8, 12, 16 and 24 hour intervals and B gal activity assays conducted at each timepoint so as to establish the optimum times for enzyme expression.

(102) B Gal Activity Assay

(103) The following reagents were used: Sodium phosphate buffer (50 mM, pH 6.8); Magnesium chloride (50 mM); Sodium carbonate (1M); O—NPG (0.2M); and For the calibration curve-o-NP (10 mM). 250 μl o-NPG, 20 μl MgCl.sub.2, 200 μl buffer and 40 μl enzyme sample were incubated at 37° C. for 8 min at 120 rpm. 500 μl of sodium carbonate was then added and the sample measured at OD.sub.420 to assess the amount of o-nitrophenol and therefore establish the B gal activity by comparing the result against calibration curve. FIG. 19 shows the OD.sub.420 measurement of Lactobacillus plantarum 2830 (ECGC 13110402), FIG. 20 shows the OD.sub.420 measurement of Lactobacillus fermentum NCIMB 30226, whereas FIG. 21 shows the OD.sub.420 measurement of Lactobacillus fermentum ATCC 11976. B gal activity peaked at around 12 hours for Lactobacillus plantarum 2830 (ECGC 13110402), peaked at around 10 hours for Lactobacillus fermentum NCIMB 30226 and peaked at around 16 hours for Lactobacillus plantarum 2830 (ECGC 13110402).

(104) GOS Synthesis

(105) Based on the B gal activity studies, GOS was then synthesised in each species/strain by harvesting the cells at the peak activity time point. 50 ml pre-overnight cultures were prepared anaerobically at 37° C. in the modified MRS medium. Overnight cultures in 3 L batches were prepared anaerobic at 37° C. in the modified MRS medium. The cells were harvested at 12 hours for Lactobacillus plantarum 2830 (ECGC 13110402), 10 hours Lactobacillus fermentum NCIMB 30226 and 16 hours for Lactobacillus fermentum ATCC 11976.

(106) The harvested cells were then centrifuged at 10,000 g for 15 minutes. The cells then washed in sodium phosphate buffer (50 mM, pH 6.8) and centrifuged for a further 5 minutes at 10,000 g and then resuspend in 10 ml buffer. The cells were then lysed using liquid nitrogen freeze thaw for 3 cycles and a cell disrupter run at 45000 PSI for 2 passes. The lysate was then centrifuge at 15,000 g for 40 mins and stored at −80° C. prior to further analysis.

(107) Optimisation Analysis

(108) A range of potential optimisation conditions were assessed in each of the three organisms: 1, 2, and 4U of enzyme activity/ml; the growth medium containing 15, 20, 30, 40, 50% lactose; incubation temperatures of 40, 50, 55, 60, 65, 75° C.; and sampling taken at 0, 4, 8, 12, 24, 30 hour intervals. After incubation, the samples were heated at 95° C. for 5 min to denature enzymes and then filtered and stored at −20° C. prior to further analysis.

(109) The Standards used in this analysis were BiMuno® (a product marketed by Clasado Ltd containing galacto-oligosaccharide), lactose, glucose and galactose. The percentage of sugars were assessed using area under the curve analysis.

(110) No GOS production was established at 60, 65 and 75° C. (although the B gal activity assay has been carried out at these temperatures and did show activity). Generally low GOS yields, below 10% were found and this was likely due to the enzyme not being concentrated and higher levels of water reactions favours hydrolysis. There was evidence of GOS production from Lactobacillus plantarum 2830 (ECGC 13110402). FIGS. 22 to 25 shows chromatograms illustrating the evidence of GOS production in the selected organisms and Lactobacillus delbruecki as a control in 20% lactose, 2 units of enzyme, grown at 50° C. and sampled at 6 hours. FIG. 22 shows the results for Lactobacillus plantarum 2830 (ECGC 13110402) (GOS %=5.2 and Lactose %=89); FIG. 23 shows the results for Lactobacillus fermentum NCIMB 30226 (GOS %=5.5 and Lactose %=82); FIG. 24 shows the results Lactobacillus fermentum ATCC 11976 (GOS %=6 and Lactose %=75); and FIG. 25 shows the results for a control strain of Lactobacillus delbruecki (GOS %=17 and Lactose %=32) to establish that the reaction was working.

(111) Further synthesis reactions using B. bifidum as a control were undertaken. B. bifidum crude cell extracts were used to synthesize a GOS product similar to Bimuno® to assess whether the assay was working properly. Growth of the selected organisms was on 40% lactose at 50° C. and 6 units enzyme/ml. A plating out protocol was used to provide pure cultures after pre overnight and before harvesting the resultant 3 L cultures. FIG. 26 shows a chromatogram for Lactobacillus plantarum 2830 (ECGC 13110402) at 14 hours, whereas FIG. 27 shows a chromatogram for Lactobacillus plantarum 2830 (ECGC 13110402) at 24 hours. FIG. 28 shows a graph of the yield of GOS, lactose and monosaccharides at 12 and 24 hours for Lactobacillus plantarum 2830 (ECGC 13110402) and the results are also shows in Table 9 below:

(112) TABLE-US-00009 TABLE 9 Time GOS % Lactose % Monosaccharides % 0 0 100 0 14 30.49831 34.84451 34.65717 24 16.30435 29.34783 54.34783

(113) The results show that a transgalactosylation reaction is occurring and that lactose (substrate) is being depleted and monosaccharides (products) are increasing. GOS is synthesised by B gal but then after time is hydrolysed by B gal. From an optimisation point of view, it was established that 30.5% max GOS at 66% Lactose conversion at 14 hr was preferred.

(114) The optimised GOS production in Lactobacillus plantarum 2830 (ECGC 13110402) may be similar to two other strains, Lactobacillus plantarum 2691 (ECGC 13110401) and Lactobacillus plantarum 2828 (ECGC 13110403) and other strains of Lactobacillus plantarum which have been under investigation for the control of cholesterol, heart disease, diabetes or obesity:

(115) Similar experiments were conducted for both Synthesis reactions for both Lactobacillus fermentum NCIMB 30226 and Lactobacillus fermentum ATCC 11976. The data varies as enzyme units were not being controlled. FIG. 29 is a graph showing the percentage of sugars for Lactobacillus fermentum NCIMB 30226 over 24 hr gown in 40% lactose at 50° C. and also shown in greater detail in the Table 10 below:

(116) TABLE-US-00010 TABLE 10 Time % GOS % Lac % mono 0 0 100 0 2 5.940594 74.25743 19.80198 4 10.36036 58.10811 31.53153 9 14.0625 50 35.9375 11 31.64557 33.5443 34.81013 15 15.46943 37.42065 47.10992 17 15.88785 32.71028 51.40187 22 16.90821 31.40097 51.69082 26 18.44262 30.32787 51.22951

(117) The results show that a clear transgalactosylation reaction is occurring and an optimum 31% GOS yield at 66.5% lactose conversion at 12 hr was established.

(118) The results for the percentage of sugars for Lactobacillus fermentum ATCC 11976 over 24 hr 40% lactose at 50° C. is show in FIG. 30 and also in the Table 11 below:

(119) TABLE-US-00011 TABLE 11 Time % GOS % Lac % mono 0 0 100 0 2 8.252427 82.03883 9.708738 9 15.44715 56.09756 28.45528 11 16.90141 34.64789 48.4507 13 13.80952 50 36.19048 22 23.72881 36.27119 40

(120) The results show that GOS is being produced and an optimum 23% GOS yield at 64% lactose conversion at 22 hours was established.

(121) FIGS. 31A and 31B shows the comparatively different percentage of sugars (and GOS yield) between the Lactobacillus fermentum NCIMB 30226 and Lactobacillus fermentum ATCC 11976 strains when grown in 15% lactose at 40° C. and both show similar yields of GOS at similar time points. The results also demonstrate that little transgalactosylation is expected due to the low lactose concentration (15%). The reaction was carried out to ensure the enzyme was active which it was (hydrolysis) and also showed high monosaccharide production under these conditions.

(122) FIGS. 32A and 32B shows the comparatively different percentage of sugars (and GOS yield) between the Lactobacillus fermentum NCIMB 30226 and Lactobacillus fermentum ATCC 11976 strains when grown in 40% lactose at 40° C. Lactobacillus fermentum NCIMB 30226 showed a peak (51.08%) in GOS production at 18 hours which tailed off quickly thereafter. Lactobacillus fermentum ATCC 11976 showed a peak (40.47%) in GOS production at 2 hours and retained relatively high % of GOS (between 26 to 34%) for sometime thereafter.

(123) 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.

(124) Biological Deposits

(125) The application refers to and claims the following indication of deposited biological material: Name: European Collection of Cell Cultures Address: Public Health England Culture Collections Porton Down Salisbury SP4 0JG United Kingdom Date: 4 Nov. 2013 Accession Number: 13110401 —and— Name: European Collection of Cell Cultures Address: Public Health England Culture Collections Porton Down Salisbury SP4 0JG United Kingdom Date: 4 Nov. 2013 Accession Number: 13110402 —and— Name: European Collection of Cell Cultures Address: Public Health England Culture Collections Porton Down Salisbury SP4 0JG United Kingdom Date: 4 Nov. 2013 Accession Number: 13110403