Prebiotic composition

Abstract

A composition comprising branched fructan molecules derived from a fructan extract of perennial high sugar grass (HSG) selected from the group consisting of AberMagic cultivar, AberDart cultivar, and S48 (Lolium spp), and obtained by enzymic endo-hydrolase hydrolysis, wherein said branched fructan molecules have a molecular weight of between 0.3 to 3 kDa, and wherein at least 50% of the branched fructan molecules present in the composition have a molecular weight of between 0.48 and 1.9 kDa is provided. Also provided are feed or food supplement comprising such a composition, a method of producing a composition comprising branched fructan molecules, and a method of making a feed or foodstuff.

Claims

1. A composition comprising branched fructan molecules derived from a fructan extract of perennial high sugar grass (HSG) selected from the group consisting of AberMagic cultivar, AberDart cultivar, and S48 (Lolium spp), and obtained by enzymic endo-hydrolase hydrolysis, wherein said branched fructan molecules have a molecular weight of between 0.3 to 3 kDa, and wherein at least 50% of the branched fructan molecules present in the composition have a molecular weight of between 0.48 and 1.9 kDa.

2. The composition according to claim 1, wherein the molecular weight of the branched fructan molecules of the composition is between 0.48 and 1.9 kDa.

3. A composition comprising the composition according to claim 1 and the probiotic microorganism Lactobacillus plantarum.

4. The composition according to claim 1, wherein at least 80% of the branched fructan molecules in the prebiotic composition have a molecular weight of between 0.48 and 1.9 kDa.

5. A feed or food supplement comprising the composition according to claim 1.

6. The food supplement according to claim 5 for human administration.

7. The feed supplement according to claim 5 for administration to at least one of sheep, poultry, swine, dog, cat, or fish.

8. The feed or food supplement according to claim 5 comprising at least 0.1% at least 0.5%; at least 1%; at least 2%; at least 3%; or at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 or 90% by weight of the composition.

9. A feedstuff or foodstuff comprising a feed or food material and the feed or food supplement according to claim 5.

10. The feedstuff or foodstuff according to claim 9, comprising at least 0.0001%, at least 0.0005%; at least 0.0010%; at least 0.0020%; at least 0.0025%; at least 0.0050%; at least 0.0100%; at least 0.020%; at least 0.100% at least 0.200%; at least 0.250%; at least 0.500%, at least 0.750%, at least 1.00%, at least 1.50%, at least 1.75%, at least 2.00%, at least 3.00%, at least 4.00%, at least 5.00%, at least 6.00%, at least 7.00%, at least 8.00%, at least 9.00%, or at least 10.00%, by weight of the feed or food supplement.

11. A method of producing a composition comprising branched fructan molecules, said method comprising the step of hydrolysing a fructan extract from one or more perennial high sugar grasses selected from the group consisting of AberMagic cultivar, AberDart cultivar, and S48 (Lolium spp), wherein said hydrolysis step comprises an enzymatic hydrolysis step performed by an endo-hydrolase enzyme on the fructan extract; wherein the branched fructans have a molecular weight of between 0.3 to 3 kDa, and wherein at least 50% of the branched fructan molecules present in the composition have a molecular weight of 0.48 to 1.9 kDa.

12. The method according to claim 11, further comprising subjecting the fructan extract to one or more purification steps prior to hydrolysis, optionally wherein said purification step comprises ethanol precipitation with 70% ethanol.

13. A method of making a feed or foodstuff comprising adding to a feed or food material a feed or food supplement according to claim 5 or the composition according to claim 1.

Description

(1) The invention will now be further described with reference to the following figures in which:

(2) FIG. 1 shows the prebiotic index of various grass extracts after 8 hours fermentation with 10% w/v faecal slurry.

(3) FIG. 2 shows the prebiotic index of various grass extracts after 24 hours fermentation with 10% w/v faecal slurry.

(4) FIG. 3 shows the prebiotic index of various grass extracts after 4 hours fermentation with 10% w/v faecal slurry.

(5) FIG. 4 shows the prebiotic index of various grass extracts after 24 hours fermentation with 10% w/v faecal slurry.

(6) FIG. 5 shows the effect of endo-hydrolase on the size of the branched fructan molecules obtained from grass juice.

(7) FIG. 6 shows the endo-hydrolase results from 6 species of grass. Similar profiles were obtained using the enzyme on Lolium, Festuca and Phleum (FIG. 6 panels A, B, C) while Barley, Wheat and Oat (FIG. 6 panels D, E, F) show a similar profile to each other. The difference in profile results from the fructans of Barley, Wheat and Oat being of lower molecular weight/short initial chain length.

(8) FIG. 7 shows the synbiotic effect of L. plantarum on two different fructan fractions.

(9) FIG. 8 shows the prebiotic index of various fraction of fructan molecules obtained grass after 8 and 24 hours.

(10) FIG. 9 shows the effect of various fructans fragments on the growth of two commercially used probiotic strains.

(11) FIGS. 10A and 10B show the effect of an added carbon source on the growth of two Lactobacillus strains.

(12) FIGS. 11A, B and C show the effect of an added carbon source on the growth of three Bacillus strains.

(13) FIG. 12 shows the effect of an added carbon source on the growth of a Bifidobacterium strain.

(14) FIGS. 13A and B show the effect of an added carbon source on the growth of two Lactococcus strains.

DETAILED DISCLOSURE OF THE INVENTION

(15) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range.

(16) Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to understand that this disclosure is not limited to particular embodiments described, as such may, of course, vary, it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

(17) It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context dearly dictates otherwise. Thus, for example, reference to “a fructan” includes a plurality of molecules and equivalents thereof known to those skilled in the art, and so forth.

(18) The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

(19) The fermentation reaction is an aerobic process in which the molecular oxygen needed is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel with a suitable oxygen partial pressure effective in assisting the microorganism species to grow in a thriving fashion. In effect, by using an oxygenated hydrocarbon substrate, the oxygen requirement for growth of the microorganism is reduced. Nevertheless, molecular oxygen must be supplied for growth, since the assimilation of the substrate and corresponding growth of the microorganisms, is, in part, a combustion process.

(20) In one aspect, preferably the probiotic fructan molecules for use in the present invention are in a purified form. The term “purified” means that the given component is present at a high level. The component is desirably the predominant component present in a composition. Preferably, it is present at a level of at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration. For some embodiments the amount is at least about 85% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration.

(21) In one aspect, preferably the probiotic fructan molecules for use in the present invention are used as a concentrate. The concentrate may be a concentrated form of the extract containing the fructans.

(22) Methods

(23) Procedure for the Isolation and Purification of Fructan from Grasses

(24) 1) Harvest Grass, chop and juice (9.15 kg S48, 9.32 Kg magic).

(25) 2) Juiced frozen until required.

(26) 3) Juice thawed in a warm water bath and then pasteurised (70-80° C.) 20 minutes.

(27) 4) Remove flocculated material by centrifugation.

(28) 5) Rotary evaporate to approx 0.25 of starting volume.

(29) 6) Precipitate Fructans with 90% EtOH and recovered by filtration.

(30) 7) Air dry.

(31) Crude magic fructan 370 g Crude S48 fructan 142 g

(32) 8) Dissolve 10 g of Fructan in 100 mL H.sub.2O (magic=60.55 mg mL; S48 28.5 mg mL).

(33) 9) Heat to 60° C. and add stoichiometric quantities of MgSO.sub.4.7H.sub.2O and Ca(OH).sub.2 and stirred for 10 minutes.

(34) 10) Clarify by centrifugation.

(35) 11) Repeat until solution is pale yellow. (S48 through a C18).

(36) 12) Precipitate by making up to 90% EtOH (−20° C.) to give clean slightly off white crystals.

(37) 13) Harvest by filtration and air dry.

(38) 14) Magic hygroscopic resuspended in 50 mL H.sub.2O. S48 remained as a powder.

(39) Magic=28.2 mg mL (14.1%) recovery

(40) S48=2 g fructan (33% recovery)

(41) Procedure for the Isolation of the Active Fructan Component from Grasses

(42) Purification of the 75% Fraction of Magic Fructan.

(43) Using the above method, low yields of purified fructan were obtained, and so we decided to exploit the properties of fructan solubility in ethanolic solutions. Based on results from ethanol ppt curves (data not shown) the 75% fraction was the most active.

(44) 1) Grind 10 g of dry Magic fructan (90% ethanol precipitate) to a fine powder.

(45) 2) Re-suspend powder in 50 v/w (500 mL) 95% ethanol, shake at 180 rpm, 30° C. for 24 h.

(46) 3) Remove supernatant by filtration (110 mm, Whatman 32 low ash) and discarded.

(47) 4) Re-suspend solid in 20 v/w of original weight (100 mL) 75% ethanol and shake at 180 rpm, 30° C. for 1 h.

(48) 5) Remove supernatant by filtration (55 mm, Whatman GF/A) and retain.

(49) 6) Re-suspend solid in 20 v/w of original weight (100 mL) 75% ethanol and shake at 180 rpm, 3° C. for 1 h.

(50) 7) Remove supernatant by filtration (55 mm, Whatman GF/A) and retain.

(51) 8) Re-suspend solid in 40 v/w of original weight (200 mL) 75% ethanol and shake at 180 rpm, 30° C. for 1 h.

(52) 9) Remove supernatant by filtration (55 mm, Whatman GF/A) retain and pool with the previous supernatants (400 mL). Add 100 mL of 75% ethanol to the solid and store at 4° C.

(53) 10) Wash 0.25 w/v (100 g) activated charcoal with 400 mL 75% ethanol and filter.

(54) 11) Add wet charcoal to the 400 mL of supernatant and re-suspend by stirring.

(55) 12) Filter the suspension and wash the charcoal with a further 400 mL 75% ethanol.

(56) 13) Pool the purified clear fractions, filter through a 0.45 μm membrane and remove the ethanol by rotary evaporation at 40° C.

(57) 14) Flash freeze the resulting concentrate, approx 78 mL, in a 500 mL glass beaker and freeze dry.

(58) Procedure for the Isolation of the Active Fructan Component from Grasses

(59) Modified for Use with Grass Pellets and the Use of the 70% Fraction which Incorporates the 75% Fraction with Some Additional Longer Chain Fructans

(60) 1) For 0.1 Kg of Aber Magic pellets, grind pellets to a fine powder.

(61) 2) Extract in 5 L of 70% ethanol overnight with stirring or shaking. (70% EtOH=700 mL EtOH+300 mL H.sub.2O).

(62) 3) Filter/centrifuge and retain supernatant.

(63) 4) Prepare 1 kg of activated charcoal by soaking in 5 L of 70% ethanol (or less if possible). If working in bulk, best to just let it settle out for a few hours/overnight and decant as much as you can. Keep the ethanol.

(64) 5) Add supernatant to the charcoal and stir for a few hours. Allow to settle and decant as much as possible and retain supernatant.

(65) 6) Wash the charcoal with an equal volume (v/v) of 70% ethanol, allow to settle, decant as much as possible and filter.

(66) 7) Combine supernatants and reduce volume (rotary evaporate).

(67) 8) Freeze dry to form your active fructan ready for P2 (P2—Bio-Rad gel filtration Column) clean-up (remove Glucose, Fructose and Sucrose (GFS)).

(68) 9) Column prep.

(69) Bed volume (cm.sup.3) of the column, flow rate and elution volume were determined from the parameters within the Bio-Rad Bio-Gel instruction booklet for a 20 mL sample.

(70) Prepare using 20 mM ammonium acetate.

(71) 10) Sample prep.

(72) Dissolve fructan (800 mgs) in 2.5 mls of ammonium acetate buffer (20 mM) and inject into column at a flow rate of 0.5 ml/min. using the ammonium acetate buffer as the mobile phase.

(73) 11) Collect fractions in 3 mls aliquots directly into 7 ml of 100% ethanol.

(74) 12) Combine fractions containing fructan but no GFS.

(75) 13) Dry and store desiccated.

(76) Procedure for the Isolation of the Active Fructan Component from Grasses

(77) Investigating the Decolourisation of 70% Ethanolic Extract Using Calcium Hydroxide and Activated Carbon

(78) Inulin derived from Jerusalem artichoke, is routinely de-coloured by a combination of calcium hydroxide and activated carbon processing steps. The purpose of using calcium hydroxide is to form calcium carbonate (CaCO.sub.3), in the presence of CO.sub.2, which, while being inert to water soluble carbohydrates, reacts with and precipitates many of the impurities that are associated to the pigmentation of the crude extract. This procedure has been modified for application to the high sugar grass variety Aber Magic.

(79) The over-all reaction gives:
CO.sub.2+Ca(OH).sub.2.fwdarw.CaCO.sub.3+H.sub.2O+heat (in the presence of water)

(80) Overall this reaction is slow and limited by the rate of solubility. Addition of sodium hydroxide, functions to catalyses the following reaction at high pH.

(81) 1) CO.sub.2+H.sub.2O.fwdarw.CO.sub.2 (aq)

(82) 2) CO.sub.2 (aq)+NaOH NaHCO.sub.3 (forming bicarbonate at high pH)

(83) 3) NaHCO.sub.3+Ca(OH).sub.2.fwdarw.CaCO.sub.3+H.sub.2O+NaOH

(84) 1) Grind 1 kg magic pellets and extract by shaking in 20 L of 70% ethanol overnight (16 h) at 30° C.

(85) 2) Remove the majority of particulates by filtering the ethanolic extract through a mashing sack and then through mirrar cloth and retain the supernatant.

(86) 3) De-water solids using a hydraulic press and filter through mirrar cloth, pool with the previously filtered supernatant.

(87) 4) In 4 L batches, raise the pH of the ethanolic extract to above pH 10 with approximately 8 mL of 5 M NaOH stirring constantly.

(88) 5) Add 50 mL aliquots of a 20% w/v suspension of calcium hydroxide (Ca(OH).sub.2) in 70% ethanol, and bubble carbon dioxide through the mix continuously.

(89) 6) Following the addition of 100 mL lots of Ca(OH).sub.2 remove 2 mL samples, centrifuged briefly to monitor colour.

(90) 7) When approximately 100 g of Ca(OH).sub.2 has been added monitor the colour until it changes from brown/green to pale green.

(91) 8) To the ethanolic extracted suspension add 200 g of powdered activated carbon in 500 mL 70% ethanol with continuous stirring for 5 minutes.

(92) 9) Monitor colour, if clear, filter (Whatman grade 1) under vacuum, and wash the carbon/calcium carbonate cake with 0.5 L 70% ethanol.

(93) 10) A white precipitate (possibly CaCO.sub.3) forms in the filtrate, which can be removed by subsequent filtration.

(94) 11) The ethanolic extract was concentrated using a Buchi Rotavapor R-153.

(95) 12) Sample sent to IFR

(96) Procedure for the Isolation of the Active Fructan Component from Grasses

(97) In this process, the large amounts of charcoal were replaced by using ascorbic acid to reduce formation of colour (reducing agent—preventing PPO activity etc.). This procedure also includes the use of HP-20 (hydrophobic resin) step included to remove polyphenolics.

(98) 1) Harvest grass, Chop and Juice.

(99) 2) Add Ascorbic acid (to 50 mM).

(100) 3) Centrifuge and reduce volume to 0.25 original volume (rot evap).

(101) 4) Crash fructan using 90% ethanol (−20° C. 2 hr).

(102) 5) Wash with 90% ethanol.

(103) 6) Partition fructan by re-solubilising the active fraction in 70% ethanol.

(104) 7) Centrifuge.

(105) 8) Add charcoal to 5% w/v.

(106) 9) Filter and wash with 70% ethanol.

(107) 10) Reduce volume by rotary evaporation.

(108) 11) Pass through a column containing HP-20 resin.

(109) 12) Wash column, combine washes and re-filter.

(110) 13) Dry.

(111) 14) Resuspend in min. volume of water and pass through P-2 (Biorad) column (removes GFS).

(112) 15) Dry and store desiccated.

(113) Procedure for the Isolation of the Active Fructan Component from Grasses

(114) An Alternative Method to Reduce Dependence on Charcoal and Ethanol—Cross-Flow Filtration

(115) 1) Harvest grass.

(116) 2) Chop and Juice.

(117) 3) Centrifuge juice using a continuous centrifuge.

(118) 4) Collect liquid and ferment for 24 hr with Yeast (ethanol red 0.1% inoc. at 30° C.)—removes GFS.

(119) 5) Centrifuge.

(120) 6) Pasteurise and precipitate protein by passing through 120° C. Bath (copper piping—200 ml/min).

(121) 7) Centrifuge and filter to give a clear liquid.

(122) 8) Pass liquid through a cross-filtration unit containing a 5 kDa membrane (with 6 washes).

(123) 9) Collect samples (a) retentate (>5 KDa) and (b) filtrate (<5 KDa)—containing active component.

(124) 10) Reduce volume using rotary evaporation.

(125) 11) Dry samples.

(126) 12) Re-suspend in min. amount of water and de-colour (anion exchange cartridge).

(127) Estimated time 2-3 days

(128) Production of Fructan at Pilot Scale

(129) 2.5 tonnes of fresh grass was processed through a hammer mill and fed through a juice press (capacity 1 T/h). A volume of 70 L was further processed. The juice was spun in a continuous centrifuge to remove particulate material including lipids etc, (CEPA at 25,000 rpm, at a rate of 100 L/h). The resulting supernatant was pasteurised at 90° C. by flow through a heated bed (20 L/h). Particulate (denatured protein etc.) was removed by continuous centrifugation (40,000 rpm—at a flow rate of 100 L/h). The supernatant was subjected to ultra-filtration (10 kDa, 4.5 m2 filter—recycling flow rate of 5000 L/h and a filtrate rate of 300 L/h at 34 psi) to retain large molecular weight fructan. Starting from an initial 70 L volume, the fructans were concentrated in 12 L. The filtrate subsequently underwent nano-filtration (approx. 1 kDa 2.4 m2 filter running at 1000 L/h recycling flow and 100 L/h filtrate flow rate) to separate smaller prebiotic fructans from small sugars/salts etc. resulting in a reduction in volume from the original 70 L to 5 L. The fructan samples were cleaned up by passing through an ion exchange resin (10 L volume). A clean product consisting of the selected size classes was produced.

(130) Increasing the ‘prebiotic’ fraction: Fructans from perennial ryegrass consist of long chains of fructose linked by β(2-1) or β(2-6) bonds. At any time, fructans in perennial ryegrass will consist of molecules ranging in length from 2 to over 150 fructose units. Initial studies demonstrated that fructans in the size range 10-50 chain length (gFOS75) showed the best prebiotic effect. However this component represents a small percent of the total fructan, with the majority having chain lengths in the 70-100 range. To increase the smaller fructan pool a number of fructan hydrolysing enzymes with either exo- and endo-activities were investigated. A Novozyme inulin endo-hydrolase enzyme preparation was found to completely convert large fructan to the size class found in gFOS75 (FIG. 5). This fraction will be referred to as (EHgFOS75).

(131) Consistency of product: To determine the reproducibility of the process, a series of grass samples were processed including: 1) same grass processed 3 times (batch to batch variation); 2) grass harvested at different times of the year; 3) grass harvested over 2 seasons. The latter two sets of samples will have variation in the distribution of size-classes of fructan molecules. Following digestion with the fructan endo-hydrolase and clean up, each sample showed the same fructan size and class profile (by Dionex).

(132) The results demonstrated that regardless of the initial fructan profile (variation due to environmental and seasonal effects) a consistent gFOS75 profile could be produced relatively easily following enzyme hydrolysis.

EXAMPLES

(133) Analysis of Crude Fractions for Prebiotic Activity

(134) Experimental Design

(135) A preliminary evaluation of the potential prebiotic effect of 6 different fractions was performed.

(136) The 6 fractions studied were: Inu 2 Inu 20 Magic 20% Ethanol Magic 50% Ethanol Magic 60% Ethanol Magic 75% Ethanol

(137) Batch Culture Fermentations

(138) To evaluate the potential prebiotic effect, static small scale 24 hour fermentations were performed using the above fractions as the sole source of carbohydrates. A vessel containing commercially available FOS prebiotic (Raftilose® P95) was included as a positive control. In the vessel used as a negative control the addition of carbohydrate was omitted. At time zero, basal medium was inoculated with 10% (w/v) faecal slurry. Faecal inoculum used was obtained from a healthy volunteer that had not taken any antibiotics pre- or probiotics in the previous 2 months. Eight fermentations were run in parallel at 37° C. and were maintained under oxygen-free atmosphere (10% H2; 10% CO2; 80% N2) using anaerobic cabinet (Don Whitley Scientific, Shipley, West Yorkshire, UK). Each vessel contained 1% (w/v) of the test fraction and samples were removed and processed at intervals over a 24 h period.

(139) Bacterial Enumerations

(140) Samples were taken at time 0, 4, 8 and 24 hours and were serially diluted with pre-reduced half strength peptone water, enriched with 0.5 g L-1 cysteine HCl (pH 7). 20 μL of each dilution were inoculated, in triplicate, onto agar plates designed to select for the total anaerobes as well as predominant groups of gut bacteria: enterobacteria, Bacteroides, bifidobacteria, lactobacilli and clostridia. The selective growth media used for the enumeration of major genera resident in the human large intestine were: Wilkins Challegren agar for total anaerobes; brucella bloodbased agar with addition of 75 mg L-1 kanamycin, 5 mg L-1 haemin, 75 mg L-1 vancomycin and 50 mL L-1 laked horse blood for bacteroides spp.; reinforced clostridia agar containing 8 mg L-1 novobiocin and 8 mg L-1 colistin for clostridium spp.; rogosa agar with 1.32 mL L-1 glacial acetic acid for Lactobacillus spp.; Columbia agar containing 5 g L-1 glucose, 0.5 g L-1 cysteine HCl and 0.5 mL L-1 propionic acid (adjusted to pH 5.0 using 4M NaOH) for Bifidobacterium spp.; Mac Conkey No 3 agar for Enterobacteria.

(141) Bacterial counts for the different samples tested were calculated (results not shown) From the bacterial counts the prebiotic index (PI) of each fraction was calculated.

(142) Calculation of Prebiotic index (PI).
Prebiotic Index=[(Bifidobacterium/Total)+(Lactobacillus/total)]−[(Bacteroides/total)+(Clos/Total)]

(143) PI was calculated for each fraction after 8 hours of culture and after 24 hours of culture as shown in Table 1.

(144) TABLE-US-00001 TABLE 1 PI values for the different fractions tested at 8 and 24 hours fermentations. 8 Hours culture 24 hours culture Inu 2 0.95 3.65 Inu 20 1.076 3.19 Magic 20 0.265 2.942 Magic 50 0.831 4.333 Magic 60 1.082 3.228 Magic 75 1.524 17.1 Positive control 0.806 4.382 Negative control 0.587 1.607

(145) The PI values for the different fractions tested at 8 and 24 hours fermentations was calculated and is shown in FIGS. 1 and 2 respectively.

(146) The results of PI at 8 and 24 hours are considered separately because the experiments were performed under static batch conditions where pH is not controlled. In this type of batch fermentation the bacterial numbers observed at 24 h may be affected by a drop in pH.

(147) FIG. 1 shows that the PI values of fractions Inu2, Inu20 and 75% at T8 are considerably higher than the positive control (commercially available FOS prebiotic Raftilose® P95).

(148) FIG. 2 shows the PI values at T24, these results clearly show a significantly greater PI value for the fraction extracted by 75% ethanol.

(149) To determine more exact PI values for the most interesting fraction (75% Ethanol extracted) further studies with large scale pH-controlled faecal bacterial batch culture fermentation were performed using the full 3 stage continuous colon model.

(150) Further Analysis of 75% Ethanol extracted fraction in the full 3 stage continuous colon model

(151) Experimental Design

(152) In this study two fractions of the original 75% Ethanol extracted carbohydrates were examined. These sub-fractions were:

(153) a) Juice Fructans DT01381/53/1

(154) Fructans extracted using the processes detailed herein using wet grass as the raw material

(155) b) Pellet Fructan

(156) Fructans extracted using the processes detailed herein using dried pelleted grass as the raw material

(157) Batch Culture Fermentations

(158) Previously static small scale 24 h fermentations were performed due to quickly determine those fractions with the greatest prebiotic activity. For this study a significantly larger sample was used to perform full 24 h batch fermentations using the above sub-fractions as a source of carbohydrates.

(159) At time zero water-jacketed fermenters were filled with basal medium and each vessel inoculated with 1% (w/v) of the test fraction. A vessel containing the commercially available prebiotic FOS (Raftilose® P95) was included as a positive control. In the vessel used as a negative control the addition of carbohydrate was omitted. Each vessel was also inoculated with 10% (w/v) faecal slurry. Faecal inoculum used was obtained from a healthy volunteer that had not taken any antibiotics pre- or probiotics in the previous 2 months. Four fermentations were run in parallel at 37° C. maintained by a circulating water bath. Culture pH was controlled automatically and maintained at pH 6.8. Anaerobic conditions were maintained by sparging the vessels with oxygen-free nitrogen gas. Samples were removed and processed at intervals over a 24 h period.

(160) Bacterial Enumerations

(161) Samples were taken at time 0, 4, 8 and 24 hours and were serially diluted with pre-reduced half strength peptone water, enriched with 0.5 g L-1 cysteine HCl (pH 7). 20 μL of each dilution were inoculated, in triplicate, onto agar plates designed to select for the total anaerobes as well as predominant groups of gut bacteria: enterobacteria, Bacteroides, bifidobacteria, lactobacilli and clostridia. The selective growth media used for the enumeration of major genera resident in the human large intestine were: Wilkins Challegren agar for total anaerobes; brucella blood-based agar with addition of 75 mg L-1 kanamycin, 5 mg L-1 haemin, 75 mg L-1 vancomycin and 50 mL L-1 laked horse blood for bacteroides spp.; reinforced clostridia agar containing 8 mg L-1 novobiocin and 8 mg L-1 colistin for clostridium spp.; rogosa agar with 1.32 mL L-1 glacial acetic acid for Lactobacillus spp.; Columbia agar containing 5 g L-1 glucose, 0.5 g L-1 cysteine HCl and 0.5 mL L-1 propionic acid (adjusted to pH 5.0 using 4M NaOH) for Bifidobacterium spp.; Nutrient agar for total aerobes, Mac Conkey No 3 agar for Enterobacteria.

(162) Methods Relating to Bacterial Growth Results Shown in FIGS. 10 to 13 Inclusive

(163) Bacterial Strains and Culture Media

(164) The following bacterial cultures obtained from an in-house culture collection and were used for pure culture growth experiments: Lactobacillus rhamnous and Lactobacillus plantarum were grown in MRS media and incubated at 37° C. without shaking. Lacococcus lactis subsp. lactis and Lactococcus lactis subsp. cremorium were grown in GM17 media and incubated at 30° C. without shaking. Bifidobacterium longum was grown in BHI media in anaerobic cabinet at 37° C. without shaking. Bacillus subtilis was grown in L-broth, Bacillus amyloliquifaciens was grown in BHI media and Bacillus niacini was cultured in TSB (tryptone soya broth). All Bacillus strains were incubated at 30° C. in a shaking incubator (200 RPM).

(165) Measurement of Bacterial Growth Demonstrated in FIGS. 10 to 13 Inclusive

(166) Bacteria were grown overnight (18 h) in the corresponding liquid media and were used to inoculate (1% v/v) into fresh media with a final volume 300 ul in Bioscreen honeycomb 100-well plates in triplicates (Thermo Life Sciences, Basingstoke, UK). The carbon source added was either glucose or fructan fraction at a final concentration of 1% (w/v). The cultures were incubated at the appropriate temperature for 20-24 h in the Labsystems Biosceen C (Thermo Life Sciences) and growth was measured at OD.sub.600 by taking readings at 15 minute intervals. The plates were shaken for 5 s prior to measurement of the optical density. For growth of Bacillus the plates were continuously shaken. For growth of Bifidobacteria the Biscreen C equipment was kept at 37° C. in an anaerobic cabinet (Don Whitley anaerobic workstation).

(167) Results

(168) Bacterial counts for the different samples tested were calculated (results not shown) From the bacterial counts the prebiotic index (PI) of each fraction was calculated as shown in Table 2.

(169) TABLE-US-00002 TABLE 2 Values of PI for the different fractions tested at 4 and 24 hours fermentations. 4 Hours culture 24 hours culture Juice fructans 0.864 1.284 Pellet fructans 0.156 0.909 Positive control 1.338 0.999 Negative control 0.385 0.109

(170) The calculated PI values at 4 and 24 hours are shown in FIGS. 3 and 4.

(171) By conclusion of the fermentation at T24 both sub-fractions had a P.I. that was at least equivalent to the positive control (commercially available FOS prebiotic Raftilose® P95) with the juice fructans showing a higher P.I. than the positive control. Thereby demonstrating that prebiotic materials can be extracted from both wet and dried pelleted grasses by the protocols detailed herein and which have equivalent prebiotic potential as the commercially available FOS prebiotic Raftilose® P95.

(172) The results shown in FIGS. 1 and 2 show that of the total fructan molecules extracted from the grass, only a specific size range gave a significant prebiotic effect i.e that extracted using 70-75% ethanol.

(173) Use of Endo-Hydrolase to Increase Prebiotic Fraction

(174) Using an endo-fructan hydrolysing enzyme, which cleaves fructan molecules at specific points in the chain, the remaining non-prebiotic fraction can be converted to fructan with shorter chain lengths as shown in FIG. 5. Using this method a fructan molecule size profile similar to that of the 70-75% prebiotic fraction can be obtained.

(175) The digests were shown to be consistent and independent of starting material as demonstrated with grass samples from different harvests over the season and between different, data not shown.

(176) FIG. 6 shows the endo-hydrolase results from 6 species of grass. As can be seen from panels A, B and C, similar profiles were obtained using the enzyme on Lolium, Festuca and Phleum (FIG. 6 panels A, B, C) while Barley, Wheat and Oat (FIG. 6 panels D, E, F) show a similar profile to each other. The difference in profile results from the fructans of Barley, Wheat and Oat being of lower molecular weight/short initial chain length.

(177) The ability of a number of commercial probiotic strains, as well specific members of the human gut bacteria, to utilise the isolated oligosaccharide fractions as a source of carbon and energy was investigated.

(178) Synbiotic Effect of the Prebiotic Fructan Fragment.

(179) A number of experiments were set up to determine the prebiotic effect of grass fructans and a possible synergistic effect by including the probiotic L. plantarum.

(180) To test whether the inclusion of the bacterium L. plantarum as a probiotic enhances the biological activity of the prebiotic fructans initial batch culture experiments were undertaken. As shown in FIG. 7, the PI of gFOS75 (labelled as fraction 2) or large fructan size-classes (labelled as fraction 3), in the presence or absence of L. plantarum, in a culture containing a complex mixed faecal ecosystem after 8 and 24 hours fermentation was calculated. L. plantarum is known to produce an extracellular fructan exo-hydrolase enzyme. The results show that the growth of L. plantarum was stimulated to a higher degree by gFOS75 (fraction 2). The results demonstrate that L. plantarum plays a symbiotic role in both fractions, however, it acts as a better probiotic with fraction 2 than with fraction 3 and that fraction 2 (gFOS75) is a better prebiotic than fraction 3.

(181) Prebiotic Activity of EHgFOS75

(182) To test whether the fraction (EHgFOS75) produced following hydrolysis of large fructan to a size class equivalent to gFOS75 has prebiotic activity further fermentation experiments were undertaken. Four fructan fractions were used. Fraction 2—gFOS75 component; Fraction 3—large chain fructan; Fraction 4—large chain fructans but treated with boiled enzyme, and Fraction 5—EHgFOS75. Raftilose, a commercial prebiotic was used as a positive control and culture media without glucose as a negative control.

(183) Following fermentation in static cultures, EHgFOS75 (Fraction 5) showed a higher prebiotic index compared to gFOS75, see FIG. 8, indicating that the enzyme hydrolysis method could be used to increase the quantity of prebiotic fructan from grasses.

(184) It appears that Fraction 5 (EHgFOS75) shows a higher prebiotic index than both the gFOS75 fraction (Fraction 2) and the positive control.

(185) Effect of Prebiotic Fructans on Probiotic Strains

(186) To test the effect of fructans on a selection of probiotic strains a number of strains were selected to carry out pure culture fermentations. Strains selected were either commercial probiotic strains or in-house strains with potential probiotic properties. Each strain was grown in the presence of either fraction 3 (large fructans) or fraction 5 (EHgFOS75). Raftilose was used as a positive control and MRS media without glucose as a negative control. FIG. 9 shows that better growth was observed for L. casei immunitas and L. casei shirota in the presence of fraction 5 (EHgFOS75) than fraction 3. Fraction 5 also performed better than the commercial product.

(187) Referring to the methods and measurements relating to bacterial growth shown in FIGS. 10 to 13 inclusive, positive results were obtained with L. Rhamnous and L. plantarum as both growth curves in FIGS. 10A and 10B show that culture OD is higher with inclusion of fructans. Positive controls of addition of glucose show high growth as expected but increased growth with fructans indicates that the fructans are exerting a prebiotic effect insofar that both Rhamnous and L. plantarum can utilise the fructans as a carbon source.

(188) Bacillus media has to contain glucose for the bugs to grow—the line in FIGS. 11A to 11C relating to “with glucose” refers to additional glucose being added to the media to supplement growth. Positive results were seen with B. amyloliquifaciens and B. subtilis as addition of fructan is actually stimulating growth over and above addition of extra glucose to the media—conclusively demonstrating that the fructans are a very suitable carbon source for two out of the three Bacillus species. There is also a slight improvement with B. niacini.

(189) Referring to FIG. 12, the results are similar to Bacillus, the media contained glucose before extra supplementation. Positive results are demonstrated with B. longum. Referring to FIGS. 13A and 13B, no glucose in the unsupplemented media was required with these strains of Lactococcus. Very positive results seen with L. cremorium and slight positive with L. lactis. Ultimately whilst the I. cactis reading is low and a fraction of that with the glucose supplementation, the final OD for the samples is nearly twice that of the “media alone” reading (0.38 vs 0.23 or 165% that of the control).

(190) The inventors have provided at least one example of prebiotic stimulation of growth for each of the bacterial species tests. With the data presented as final OD of “plus fructan” vs “media alone” as in Table 3 below the results suggest that at least some of the fructan is being utilised.

(191) TABLE-US-00003 TABLE 3 Maximum OD Percentage Strain Media alone Plus fructan increase Lactobacillus rhamnosus 0.8 0.8 0** Lactobacillus plantarum 0.35 0.5 43 B. amyloliquifaciens 0.8 2.25 280 B. subtilis 0.31 0.375 21 B. niacini 0.65 0.67 3 Bifidobacterium longum 0.7 0.85 21 L. lactis cremorium 0.55 0.9 64 Lactococcus lactis 7000716 0.25 0.35 40 TABLE 3 Results from FIGS. 10 to 13 inclusive presented as % increase in final OD. **Initial rapid increase in OD seen with/without frutans but frutans appeared to maintain culture OD at a much higher level over time.

(192) It has been demonstrated that the total fructan from grass can be converted to the prebiotic form using a fructan endo-hydrolysing enzyme, and that there are synergistic enhancements of the prebiotic activity by the inclusion of, for example, a Lactobacillus strain that has the capability of hydrolysing fructan through the action of an extracellular fructan exo-hydrolase.

(193) Enhancements of the prebiotic activity have also been shown by the inclusion of Bacillus, Bifidobacterium and Lactococcus strains.

(194) All publications mentioned in the above specification are herein incorporated by reference in their entirety. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.