Bacterial growth enhancer

Abstract

We describe the production and use of an extract obtained from Musa spp., preferably from bananas, in the promotion of growth of Gram-positive bacteria such as lactic acid bacteria. The extract is also useful for growth enhancement of environmentally-stressed Gram negative bacteria. Fermented foods containing such extracts are also described.

Claims

1. A method of enhancing or promoting growth or viability of bacteria selected from the group consisting of: Gram positive bacteria, and lactic acid bacteria, the method comprising: preparing an extract obtained from Musa spp by: blending at least a portion of a Musa fruit in a suitable diluent to form a liquidized intermediary; centrifuging the liquidized intermediary to form a juice and a debris pellet; decanting the juice from the debris pellet to form the extract; and autoclaving the extract; introducing an enhanced bacterial growth medium consisting of a serum-free bacterial growth medium supplemented with the extract to said bacteria, wherein the extract is present in said medium at a concentration of between 0.01 and 10%; and culturing an isolated bacterial sample of said bacteria on said growth medium to thereby enhance or promote growth or viability of said bacteria over that which would be obtained with a medium typically used to grow said bacteria in the absence of said extract; wherein said bacterial growth medium is capable of enhancing growth of said bacteria from an OD600 value of 0.1 to at least 0.145 over an incubation period of at least 1480 minutes, or wherein said bacterial growth medium is capable of enhancing viability of said bacteria from 10.sup.5 colony forming units (CFU)/ml to at least 10.sup.7 CFU/ml over 7 days.

2. The method of claim 1 wherein the extract is obtained from banana skin and/or banana pulp.

3. The method of claim 1 wherein the bacteria are lactic acid bacteria.

4. The method of claim 1 wherein the bacteria are environmentally stressed prior to growth with the extract.

5. The method of claim 1, wherein the culturing of the bacteria is in a non-anaerobic environment.

6. The method of claim 1, wherein the sample is taken from a food product, or a brewing or winemaking product.

7. The method of claim 1, wherein preparing the extract comprises any or all of the following additional steps: filtering the extract; drying or lyophilizing the extract; and freezing the extract.

8. A method, comprising: introducing a bacterial growth medium selected from the group consisting of deMann Rogan Sharpe (MRS) and Luria, the bacterial growth medium supplemented with an extract obtained from a plant selected from the group consisting of: Musa spp, and a species of apple, orange, plum, carrot, tea, or coffee to bacteria, wherein the extract is present in said medium at a concentration of 0.01 to 10%; and culturing an isolated bacterial sample of said bacteria on said growth medium to thereby enhance adherence to a substrate, or to promote viability, or viscosity, said bacteria being selected from the group consisting of: Gram positive bacteria and lactic acid bacteria, wherein said bacterial growth medium is capable of enhancing viability of said bacteria from 10.sup.5 colony forming units (CFU)/ml to at least 10.sup.7 CFU/ml over 7 days.

9. The method of claim 8, wherein the extract is obtainable or obtained from Musa spp.

10. The method of claim 8, wherein the bacteria are lactic acid bacteria.

11. The method of claim 8, wherein the extract is present in said medium at a concentration of between about 0.1 to 5%.

12. The method of claim 11, wherein the extract is present in said medium at a concentration of between about 1 to 2%.

13. The method of claim 8, wherein the medium does not comprise serum, and wherein the supplement comprises no additional nutrient components.

14. The method of claim 8, wherein the extract does not substantially comprise catecholamines and/or fructo-oligosaccharides.

15. The method of claim 8, further comprising preparing the extract by: blending at least a portion of a Musa fruit in a suitable diluent to form a liquidized intermediary; centrifuging the liquidized intermediary to form a juice and a debris pellet; decanting the juice from the debris pellet to form the extract; and autoclaving the extract.

16. The method of claim 1, wherein the extract is present in said medium at a concentration of between about 1 to 2%.

17. The enhanced bacterial growth medium of claim 1, wherein the bacterial growth medium is selected from the group consisting of MRS and Luria.

18. The method of claim 1, wherein the extract is autoclaved at 121° C. and 103 kPa for 15 minutes.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) These and other aspects of the present invention will now be described by way of example only with reference to the accompanying figures, in which:

(2) FIG. 1 shows the heat stability of banana extract after autoclaving

(3) FIG. 2 shows the growth response of L. casei to dietary catecholates compared with banana extract. Tannic acid was used at 50 μg/ml, the other compounds were all used at 100 μM, final concentration; BF was used at 2% v/v. Cultures were grown statically for 24 hrs 37° C. in a humidified incubator; the values shown are representative data from several similar analyses and are means of triplicate plate counts.

(4) FIG. 3 shows growth of L. casei on agar plates with and without banana extract

(5) FIG. 4 shows bacterial growth after 16 hours of 50 μl of L. casei (10.sup.8 CFU/ml suspension) streaked out onto Luria agar with (right hand picture) and without (left hand picture) banana extract at 2% v/v.

(6) FIG. 5 shows bacterial growth under anaerobic conditions with banana extract

(7) FIG. 6 shows growth of environmentally stressed E. coli with banana extract

(8) FIG. 7 shows a time course of lactic acid bacterial growth under aerobic conditions with and without banana extract

(9) FIG. 8 shows the growth response of L. casei to various fruit, vegetable and beverage extracts compared with banana extract

(10) FIG. 9 shows the effect of banana extract on the viability of probiotic lactic acid bacteria

(11) FIG. 10 shows the effect of banana extract on the adherence of lactic acid bacteria to a surface

DETAILED DESCRIPTION OF THE FIGURES

(12) Production of Banana Extract

(13) We refer herein to the banana extract as ‘banana factor’ or BF. The extract may be produced from banana pulp, or from banana skin.

(14) Banana Pulp

(15) 1. Peeled banana pulp is liquidised with water in a food blender to a smooth paste, at a ratio of 100 ml of distilled water per 250 g of fruit pulp. The liquidised pulp is then centrifuged for 20 minutes at 8000 rpm to remove pips and other debris. The resulting viscous liquid, a crude banana juice, is then decanted. This juice can then be further processed in one of 4 ways:

(16) 2. Added directly with no further processing to culture media, which must then be sterilised (see 4. below). At the recommended supplementation of 1-2% v/v, this produces culture media which has a few speckles of fruit tissue, but which does not affect visualisation of colonies. It is also possible to lyophilise to dryness this crude pulp juice, and reduce the dried material (which is a greyish honeycomb like substance) to powder using a coffee grinder. This dried banana powder can also be directly incorporated into dry culture media.

(17) 3. With no further processing, the crude pulp can be stored at −20° C. (to prevent microbial contamination and minimise oxidative modifications from endogenous oxidases), until required for use (such as detailed in 2. above).

(18) 4. Sterilisation of the crude banana pulp can be achieved by passage of the extract through a 0.2 or 0.45 micrometer filter, though since phenolate oxidases will still be active in such preparations, it is recommended that either this extract be added directly to media, which must then autoclaved, or it is stored at −20° C.

(19) 5. Autoclaving can be used to preserve the banana extract, as this will inactivate most degradative enzyme activities, as well as sterilising the pulp extract. The active growth inducing agents in BF are fully stable to autoclaving. However, it will be clear to one skilled in the art that consecutive pasteurisation and filtration steps could also provide a satisfactory, less energy consuming method of combined stabilisation and sterilisation.

(20) 6. For preservation by autoclaving, the extract is decanted into heatproof glass bottles and heated for 15 minutes, at 121° C., 15 lbs in.sup.−2 (103 kPa). After autoclaving, the resulting preparation is aseptically decanted into sterile centrifuge tubes, and centrifuged at 8000 rpm to remove any precipitated material. This extract, now termed BF, is aseptically decanted into fresh, sterile tubes or bottles, which are stable to storage at 4° C. or −20° C.

(21) 7. A second batch of BF activity can also be obtained by re-extracting the pip and cellular debris pellet described in 1. above with water, at a ratio of half the volume of banana pulp centrifuged e.g. 500 ml of water to extract banana debris from an original pulp volume of 1000 ml. The extraction procedure involves re-suspending the debris pellet in the water, followed by vigorous mixing for around 5 minutes. This second BF extract is re-centrifuged for 20 minutes at 8000 rpm. Further extractions are possible, though these contain considerably less activity and may therefore not be cost-effective to pursue.

(22) 8. Activity recoveries from a typical extraction are shown in Table 1. BF activity levels are measured using a biological growth assay, defined as follows: A “Unit of Activity” of BF activity is defined as follows: A unit of activity (U) is the quantity of BF preparation required to stimulate the growth of test strain E. coli E2346/89 from an initial inoculum of approximately 10.sup.2 CFU/ml to 10.sup.7 CFU/ml, under the following culture conditions: 18 hours static growth at 37° C. in a 5% CO.sub.2/air humidified incubator in serum-SAPI medium. Serum-SAPI medium is 6.25 mM NH.sub.4NO.sub.3, 1.84 mM KH.sub.2PO.sub.4, 3.35 mM KCl, 1.01 nM MgSO.sub.4 and 2.77 mM glucose (pH 7.5) supplemented with 30% (v/v) adult bovine serum.

(23) 9. BF can be used directly, or lyophilised to dryness, and re-constituted with water at volumes of up to 20% of the original volume of extract (i.e. concentrated by a factor of at least 5-fold).

(24) Preparation of Banana Skin Extracts

(25) 1. Pulp-free banana skins are chopped into approximately 1 cm pieces, and then blended with water at a ratio of 100 ml of water per 100 g of skin until a smooth but fibrous extract is obtained. This extract is then centrifuged for 20 minutes at 8000 rpm to remove cellulose and other debris. The resulting liquid is then decanted.

(26) 2. Although this extract is less viscous than the pulp extract, and can therefore be readily filter sterilised, because of high endogenous oxidase activity in the skin, it is recommended that the extract be incorporated into culture media and autoclaved immediately, or that the skin extract be autoclaved before use.

(27) 3. Although banana skin possesses less growth inducing activity than the pulp, a media supplementation of 1-2% v/v of the skin extract is sufficient to induce high level growth stimulation. It is possible to concentrate lyophilised skin extract by a factor of at least 10-fold.

(28) TABLE-US-00001 TABLE 1 Typical purification data Activity Total Recovery Stage Total volume U/ml Activity U % Crude pulp juice Extraction 1 800 1330 1 064 000 100 Extraction 2 500 1000   500 000 100 After autoclaving Extraction 1 800 1400 1 120 000 100 Extraction 2 500 950   475 000 95 After lyophilization Based on re- Ext. 1 1250 1 000 000 94 and final constitution Ext. 2 900   450000 90 preparation (diluted of 10 ml to original vol) test volume
Stability Analyses
Heat Stability

(29) The banana juice extract is highly stable to heat treatment and can be autoclaved (121° C. for 20 minutes) without significant loss of activity. This is demonstrated in FIG. 1, which shows the growth response of test strain E. coli E2346/89 in serum-SAPI medium to a dilution series of control and autoclaved BF. Growth conditions were as described in section 8 above (18 hrs incubation at 37° C.); the values shown are means of duplicate plate counts.

(30) Preliminary Analysis of the Growth-Enabling Components within BF

(31) 1. Comparison of BF with Dietary Phenolates

(32) BF is prepared from the pulp of the banana fruit, though banana skin also possesses some growth enhancement activity. Banana pulp extracts contain a variety of compounds that have been shown by various researchers of being able to modulate growth of LAB, including sugars, minerals and various dietary phenolates. Shown in FIG. 2 is the growth response of L. casei to certain of these phenolates, compared with growth response to the BF preparation. All the phenolate compounds shown are suitable for human consumption, though highly expensive when used in purified form and individually are less effective than the much more economical BF. Note that the growth analyses shown below were in vitro assays performed in serum-based SAPI, a minimal salts microbiological culture medium which provides an intentionally stressful culture environment containing immunological and nutrient challenges reflective of those which will be experienced in vivo. In this medium the LAB grow normally poorly when inoculated with low cell numbers. However substantial log-fold improvements in growth were obtained when cultures were supplemented with BF, as well as the individual phenolates present in BF, suggesting that BF is likely to be functional in LAB growth enhancement in vivo if co-consumed with probiotic LAB species. However, while BF is able to enhance growth in non-serum-based media, the dietary phenolates shown in FIG. 2 are not (Freestone, data not shown). This suggests that while the individual dietary phenolates may increase growth in serum-supplemented medium, their contribution to the enhancement of LAB or other species growth in laboratory culture media such as MRS or Luria broth is functionally less important.

(33) 2. Analyses of the Mechanism by which BF and Dietary Phenolates Induce Lab Growth

(34) A paper (Lyte, 1997) was published in 1997 detailing the ability of banana pulp extracts (created by blending and filter-sterilisation only) to induce growth of pathogenic enteric bacteria. The author, Mark Lyte found that banana skin and pulp extracts had no effects on Gram-positive bacteria (LAB are classified as being Gram-positive). The conclusion from the results of several experiments was that the active components in banana pulp and skin were the catecholamines noradrenaline and dopamine. All of these analyses performed in this publication were done in serum-based media. While LAB do show around 2 log-fold increases in response to noradrenaline and dopamine, this growth enhancement only occurs in serum-based media (our unpublished data). Previous work has shown that the catecholamines noradrenaline and dopamine induce growth in iron-restricted media such as serum via provision of Fe from host iron-sequestering proteins such as transferrin or lactoferrin (Freestone et al 2000, Journal of Bacteriology 182: 6091-6098; Freestone et al 2002 Shock 18:465-470; and Freestone et al 2003 FEMS Microbiol. Lett. 222: 39-43) (note that the main bacteriostatic factor in serum is transferrin, and in mucosal secretions lactoferrin). While the catecholamines and other phenolate compound present within banana extracts can deliver iron from host iron binding proteins to Gram-negative species such as E. coli (Table 2) it is clear that the same compounds are not acting in a similar manner, that is deliverance of Fe, with respect to the Gram-positive L. casei. Further evidence for a different method of growth induction by banana-derived compounds comes from our demonstration of the utility of autoclaved BF to enhance bacterial growth in standard non-serum-containing LAB-specific and other microbial culture media (see FIGS. 3-7). It is clear therefore, that the mechanism of growth stimulation by BF, specifically the dietary phenolates and catecholamines contained within BF, is different in Gram-positive LAB species from that demonstrated in Gram-negative bacteria such as E. coli.

(35) TABLE-US-00002 TABLE 2 Ability of BF and dietary catecholamines and catecholates to mediate delivery of lactoferrin-complexed Fe to L. casei and E. coli .sup.55Fe incorporation/ml .sup.55Fe incorporation/ml of culture of culture Addition L. casei E. coli None 66 376 Noradrenaline 29 8542 Dopamine 48 6815 Caffeic acid 29 11385 Catechin 57 3931 Chlorogenic acid 28 7374 Tannic acid 28 13952 Banana (BF) 409 14220

(36) L. casei and E. coli were added at around 5×10.sup.6 CFU/ml to triplicate 1 ml serum-SAPI medium containing the additions shown in Table 2, and 2×10.sup.5 cpm/ml of .sup.55Fe-complexed human lactoferrin. Tannic acid was used at 50 μg/ml, the other compounds were all used at 100 μM, final concentration; BF was used at 2% v/v. Cultures were incubated as described in the legend to FIG. 2, and growth enumerated on MRS agar (L. casei) or Luria agar (E. coli). .sup.55Fe incorporation was determined by scintillation counting of triplicate washed bacteria cultures.

(37) All of the cultures were similar in terms of final cell numbers, although when centrifuged and washed the banana (BF)-supplemented LAB culture giving the higher .sup.55Fe incorporation count was found to have produced large amounts of exopolysaccharide and were very viscous, possibly explaining the higher incorporation of .sup.55Fe (due to trapping of .sup.55Fe-lactoferrin within the sticky exopolysaccharide produced by the LAB under this particular set of growth conditions).

(38) Agar Plate Assays

(39) FIGS. 3 to 6 show photographs of agar plates with and without banana extract used to grow test strains of Lactobacillus casei under various conditions and at various dilutions. The photographs show the growth promoting properties of banana extract.

(40) FIG. 3 shows agar plates made with MRS medium (left photograph) with the addition of BF (right photograph). MRS medium (a very widely used LAB-specific culture medium) was solidified with 1.5% bacteriological agar (hereafter referred to as MRSAgar). The LAB used were taken directly from a Danacol brand L. casei probiotic supplement suspension and serially diluted in MRS liquid medium, in steps of 1:100 [−2 dilution], 1:1000 [−3], 1:10,000 [−4] and 1:100,000 [−5]. 50 μl of each dilution was then pipetted onto the MRSAgar plates, which were then incubated for 36 hours at 37° C. in a humidified CO.sub.2 incubator.

(41) Even though MRS medium is the preferred media for culture of the nutritionally fastidious LAB, it is typical for healthy non-environmentally stressed LAB to take 3 days or more to shown visible growth of individual colonies. In contrast, supplementing MRSAgar with BF enables growth to visible levels of all dilutions at this time point.

(42) The ability of LAB to subsist within acidic, anaerobic environments hostile to most microbial species, plus their fermentation of available sugars to lactic acid, makes them undesirable souring agents in the brewing and wine making industries (LAB also cause sliminess in certain vinegar generators). Currently, quality control (QC) analysis of beer and wine involves plating test samples on MRSAgar, for between 3 and 7 days (control LAB strains can take up to 3 days to grow on MRSAgar; environmental isolates can take up to 7 days or even longer). The economic advantages in reducing the time to detection to around 1 day are obvious.

(43) FIG. 4 shows plates with Luria agar only (left image) and Luria agar plus BF (right image). Luria agar (LA) is a general laboratory medium for less nutritionally fastidious bacteria such as E. coli and Salmonella. Though LA is considerably cheaper than MRSAgar it is not normally used for culture of LAB because the LAB will grow very slowly on it, taking at least 5 days to grow to visible colony size. However, when the LA was supplemented with 2% v/v BF extract, strong, visible growth of LAB was seen after a single overnight incubation at 37° C. The significance of this experiment is that LAB, such as L. casei, can now be grown on less complex (and substantially cheaper) media with BF extract supplementation. Furthermore, this result begs the question of just how simple the media composition for growing LAB could become when supplemented with BF extracts.

(44) FIG. 5 demonstrates the growth of bacteria on MRSAgar without BF (left image) and with BF (right image) under anaerobic conditions. In this experiment (MRSAgar only [−ve on the image] or MRSAgar with 2% BF [B on the image]) there can be seen the growth levels of serially-diluted L. casei (from a probiotic beverage brand name Danacol, manufacturer Danone) after 36 hours under anaerobic conditions at 30° C. While it is clear that there is growth on the MRSAgar only plates, there is also substantially more on the BF-supplemented medium. This result combined with the data from FIGS. 3, 4, 6 and 7 indicates that BF is likely to be functional in most aerobic and anaerobic LAB culture protocols.

(45) FIG. 6 demonstrates the enhancement of growth of environmentally stressed bacteria in the presence of BF. A stationary phase Luria Broth culture of enteropathogenic E. coli (EPEC) strain E2348-69 was stored continuously at 0-4° C. for 3 months. This culture was then serially diluted in sterile phosphate buffered saline and plated onto MRSAgar+/−2% BF as shown (MRSAgar only, left image; with BF, right image). Plates were incubated at 37° C. for 16 hours. The results show that BF helps to resuscitate the “viable but non culturable” population within this aged and cold damaged/nutrient starved culture.

(46) Time Course of LAB Growth

(47) The time course of growth of LAB grown aerobically in the presence of BF is shown in FIG. 7. LAB taken directly from a commercial yoghurt drink preparation (Danacol brand, Danone manufacturer) were serially diluted into MRS medium+/−2% BF. Duplicate cultures were incubated aerobically with shaking and growth monitored continuously for 24 hours. The viable count per ml of original yoghurt was 1.06e8 CFU/ml, which means that the initial cell numbers at the beginning of each time course of growth are as follows: −3 Diln (1:1000 dilution) is equal to approximately 1.06e5 CFU/ml; −4 Diln (1:10000 dilution) approximately 1.06e4 CFU/ml; −5 Diln (1:100 000 dilution) approximately 1060 CFU/ml and the −6 Diln (1:1000 0000 dilution) is equivalent to around 100 CFU/ml. Viable counts of the end points of each of the time courses allows interpretation of the OD600 value of 0.25 to be equivalent to bacterial cell count value of around 5-6 e7 CFU/ml, indicating that in the presence of BF growth of a starting inoculum of around 100 LAB was enhanced more than 100,000-fold.

(48) Note that there is no significant growth in the absence of BF in MRS media under these fully aerobic conditions, even after 24 hours incubation. Data for LAB from other probiotic supplements and yoghurt preparations shows similar results.

(49) Applications of BF in LAB and Other Species Growth Enhancement Processes

(50) Our data suggests the utility of banana extract, both pulp and peel, to enhance the growth of a variety of lactic acid bacteria strains commonly used in probiotic supplements, such as the Lactobacilli and Bifidobacteria. We have characterised the stability of banana extracts to conditions involved in typical media preparation, such as autoclaving (heating for up to 30 minutes at 121° C., 15 lbs in.sup.−2) and freeze-drying. We have also shown that incorporation of banana extract can convert cheaper, non-specialised culture media, both liquid and solid agar based, into media on which the lactic acid bacteria can grow as comfortably as expensive bespoke LAB media. We also have data showing the utility of BF in speeding up growth of food poisoning agents such as Listeria monocytogenes and pathogenic E. coli. Thus, we have ample proof of principle data for the creation of a new and much improved media for the growth of the economically important beneficial lactic acid bacteria, which may also have applications for more rapid detection of those microbial species that can pose danger to humans.

(51) Growth Promotion by Other Edible Plant-Derived Extracts of LAB and Other Microbial Species

(52) Although extracts prepared from Musa fruit contain highly potent growth enhancement compounds as indicated by the data shown in FIGS. 3-7, we also have discovered that extracts prepared from other fruit, vegetables and plant tissues used in beverage preparation also possess activities which may be beneficial to growth and resuscitation of LAB and other bacterial species. FIG. 8 shows the effects of juice (2% v/v) from apple, orange, plum, carrot, and infusions (2% v/v) from tea (prepared from 1 teabag infused for 60 seconds in 200 ml of boiling water) and coffee (prepared from 3 g of instant coffee dissolved in 200 ml boiling water) on the growth of L. casei in serum-SAPI medium; responses of the same culture dilutions to a banana (BF) extract are shown for comparison. Cultures were incubated as described in the legend to FIG. 2, and growth enumerated on MRS agar. Given that the extracts shown generally induce comparable levels of growth to banana, their potential for use in applications similar to those suggested for Musa-derived extracts is clear.

(53) Yoghurt Viability and Efficacy Improvement Through Use of BF

(54) In order to demonstrate the potential benefits of BF improving the quality and durability of probiotic yoghurts a series of tests was set up using a range of market leading probiotic yoghurts and drinks.

(55) 1. We analysed effects on leading once-a-day probiotic yoghurts to investigate whether the addition of BF could have a beneficial effect on the overall numbers of viable bacteria persisting within a refrigerated once-a-day dose of probiotic bacteria.

(56) The experimental set up involved aseptically decanting commercially available suspensions of probiotic yoghurt bacteria, aliquotting them into triplicate sterile 25 ml plastic tubes supplemented with and with 1% (15 units/ml) of BF. The covers of these tubes were loosely closed, to maintain exposure to atmospheric oxygen without compromising microbiological sterility, and stored upright in a refrigerator set at 4° C. At the times indicated, culture samples were aseptically withdrawn and viability counts determined using culture on MRS agar; all viable counts were performed in triplicate, and showed standard errors of less than 2%. All experiments were also performed on at least 2 separate occasions, and are fully reproducible.

(57) It should be noted that for the yoghurt examined, the manufacturer's website estimation of the initial viable count (colony forming units, CFU/ml) of the preparation was around 10.sup.8 CFU/ml. However, initial measurements at time 0, shown in FIG. 9, indicate a viable count of nearer 10.sup.5 CFU/ml. Over the 7 days of the time course shown in FIG. 9 there was no significant reduction in viability within the control cultures. However, data from the yoghurt samples with added BF indicates that there is a significant difference (P<0.001) in the level of viable cells occurring in the presence of BF. After about 3 days pre-incubation with BF there was a significant increase in the overall viability of the bacteria within the yoghurt suspension, peaking at about 7 days (the experiment was continued for a further 4 days, with no more significant changes in viable count, data not shown). In total, BF increased the level of viable lactic acid bacteria (as determined by plate culture) by over 140-fold. It is unlikely that the increase in viable cell count observed in FIG. 9 represents an increase in numbers as a consequence of cell division as lactic acid bacteria, particularly probiotic strains, are mesophilic in terms of optimal growth temperature. Also, the optical density readings of the cultures did not increase during the course of the experiment, which would be expected if a >100-fold increase in cellular biomass had occurred. The data in FIG. 9 therefore indicates that a significant proportion of the bacteria in the yoghurt preparation investigated were dormant and that BF is bringing back the culture to a level of viability present when the yoghurt was initially manufactured.

(58) 2. Additional tests of the culture in the yoghurt preparation shown in FIG. 9 also indicates that BF appears to be augmenting the viscosity and ability of the probiotic lactic acid bacteria examined to attach to surfaces; with the effect being seen within 3 days of incubation at 4° C. (FIG. 10). The pictures show adherence of probiotic lactic acid yoghurt suspensions 1 and 5 minutes after inversion. In the presence of BF, it can clearly be seen that attachment of bacteria to the rear of the plastic tube is substantially greater compared to un-supplemented control cultures. This effect was observed in at least 20 separate experiments. BF-supplemented cultures are also more viscous, as can be seen in the greater number of bubbles in the air-liquid interfaces.

(59) In terms of the explanation for the data in FIG. 10, it is possible that the increase in viscosity and surface attachment observed is mediated in part by increased exopolysaccharide production by the bacteria. This is a potentially important benefit additional to the improved viability observed in FIG. 9, since the contribution of lactic acid bacteria exopolysaccharide to yoghurt texture is well recognised. In addition, it is also likely that any augmentation in the ability of the lactic acid bacteria to adhere to surfaces will have a direct effect on the ability of these bacteria to attach to the gastrointestinal epithelia, and therefore be of benefit in the ability of the probiotic lactic acid bacteria to colonise the host gut.

(60) As well as enhancing growth, it now also appears from the data in FIGS. 9 and 10 that BF is additionally capable of resurrecting environmentally stressed and damaged lactic acid bacteria which were previously non-viable, giving a boost to their metabolism which renders them fully culturable as well as possibly increasing their potential for host colonisation.