COMPOSITIONS AND METHODS OF PRODUCTION THEREOF

Description

TECHNICAL FIELD OF THE INVENTION

The invention relates to a prebiotic composition which is selective for the growth of Propionibacterium bacterial strain(s), for use in, but not limited to, promoting propionate production in the gut so as to regulate appetite in an individual.

BACKGROUND TO THE INVENTION

Prebiotics are dietary ingredients which can selectively enhance the levels and/or activity of beneficial indigenous gut microbiota, such as lactobacilli or bifidobacteria, and they are finding much increased application in the food sector. Prebiotics are non-digestible food ingredients that are selectively metabolised by colonic bacteria which contribute to improved health. As such, their use can promote beneficial changes within the indigenous gut microbial milieu and they can therefore help survivability of probiotics. They are distinct from most dietary fibres like pectin, celluloses, xylan, which are not selectively metabolised in the gut. Criteria for classification as a prebiotic is that it must resist gastric acidity, hydrolysis by mammalian enzymes and absorption in the upper gastrointestinal tract, it is fermented by intestinal microflora and selectively stimulates the growth and/or activity of intestinal bacteria associated with health and well-being.

Increasing colonic levels of propionate is believed to help regulate appetite in an individual. However it is difficult to administer propionate directly to the large intestine due to the destructive digestive environment and the absorptive capacity of the upper gastrointestinal tract.

Fructo-oligosaccharides (FOS, inulin and oligofructose) and galactooligosaccharides (GOS) have been demonstrated to fulfil the criteria for prebiotic classification repeatedly in human intervention studies. Currently available fructooligosaccharides and galactooligosaccharides target the growth and/or activity of bifidobacteria and lactobacilli, neither of which can produce propionate. Currently, there is no known selective prebiotic for Propionibacterium.

It is an object of the present invention to provide a prebiotic composition which allows for the specific growth of a propionate producing bacteria. It would also be desirable if the prebiotic targeted a beneficial species or strain of Propionibacterium.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, there is provided a prebiotic composition comprising a galacto oligosaccharide (GOS) produced from one or more Propionibacterium bacterial strains, wherein the GOS acts as a selective growth medium for Propionibacterium bacterial strains, and wherein the GOS is substantially the same as the form produced by reverse β-galactosidase reaction from Propionibacterium bacterial strains.

Preferably, the GOS is produced and/or is selective for one of more of the following bacterial strains: Propionibacterium jensenii; Propionibacterium freudenreichii; Propionibacterium acidipropionici, or sub-species or mutant strains thereof.

The GOS may be produced from the selected Propionibacterium bacterial genera or strains and the GOS may act as a selective growth medium for said selected Propionibacterium bacterial genera or strain.

The prebiotic composition will preferably be present in the composition in an effective amount so as to elicit a positive and gradual change in the proportions and activity of Propionibacterium in the gut. Higher amounts may be utilised if change in the microbiota is required quickly or if the composition is being used to help seed the gut with a new bacterial strain not currently present.

The prebiotic composition may be encapsulated. Many encapsulation techniques will be apparent to the skilled addressee and the one employed will be tailored to the required stability of the prebiotic growth medium during digestive transit.

The prebiotic composition may further comprise an excipient or carrier compound to enable it to pass through at least part of the gastrointestinal environment of the body and be efficiently delivered to, and released in the lower gut. The prebiotic may be concentrated and/or freeze dried. The composition may be in a number of formats, such as in the form of a liquid (which may be drinkable) and/or powder which can be mixed with a solid or liquid food stuff.

The prebiotic composition may be combined with one or more active ingredients, such as vitamins, minerals, phytochemicals, antioxidants, probiotic bacterial strains and combinations thereof.

Vitamins may include fat soluble vitamins such as vitamin A, vitamin D, vitamin E, and vitamin and combinations thereof. In some embodiments, vitamins can include water soluble vitamins such as vitamin C (ascorbic acid), the B vitamins (thiamine or B1, riboflavoin or B25 niacin or B3, pyridoxine or B6, folic acid or B9, cyanocobalamin or B12, pantothenic acid, biotin), and combinations thereof.

Minerals may include but are not limited to sodium, magnesium, chromium, iodine, iron, manganese, calcium, copper, fluoride, potassium, phosphorous, molybdenum, selenium, zinc, and combinations thereof.

Antioxidants may include but are not limited to ascorbic acid, citric acid, rosemary oil, vitamin A, vitamin E, vitamin E phosphate, tocopherols, di-alpha-tocopheryl phosphate, tocotrienols, alpha lipoic acid, dihydrolipoic acid, xanthophylls, beta cryptoxanthin, lycopene, lutein, zeaxanthin, astaxanthin, beta-carotene, carotenes, mixed carotenoids, polyphenols, flavonoids, and combinations thereof.

Phytochemicals may include but are not limited to cartotenoids, chlorophyll, chlorophyllin, fiber, flavanoids, anthocyanins, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, flavanols, catechin, epicatechin, epigallocatechin, epigallocatechin gallate, theaflavins, thearubigins, proanthocyanins, flavonols, quercetin, kaempferol, myricetin, isorhamnetin, flavonones hesperidin, naringenin, eriodictyol, tangeretin, flavones, apigenin, luteolin, lignans, phytoestrogens, resveratrol, isoflavones, daidzein, genistein, glycitein, soy isoflavones, and combinations thereof.

Probiotic strains may also be incorporated into the composition. It is preferred that the probiotic strains comprise Propionibacterium bacterial strains. It is most preferred that probiotic strain or strains comprise the Propionibacterium bacterial strain or strains used to initially produce the GOS.

In accordance with a further aspect of the present invention, there is provided a prebiotic composition for use in the regulation and/or modulation of appetite. Alternatively or additionally, the composition may be for use in the management or treatment of obesity and/or weight management. The composition comprising a galactooligosaccharide (GOS) produced from one or more Propionibacterium bacterial strains, wherein the GOS acts as a selective growth medium for Propionibacterium bacterial strains, and wherein the GOS is substantially the same as the form produced by reverse β-galactosidase reaction from Propionibacterium bacterial strains may be for use as a medicament or pharmaceutical and/or a dietary supplement.

In accordance with a further aspect of the present invention, there is provided a prebiotic composition for use in the treatment of obesity, the composition comprising a galactooligosaccharide (GOS) produced from one or more Propionibacterium bacterial strains, wherein the GOS acts as a selective growth medium for Propionibacterium bacterial strains, and wherein the GOS is substantially the same as the form produced by reverse β-galactosidase reaction for Propionibacterium bacterial strains.

In a yet further aspect of the present invention, there is provided a use of a prebiotic composition, in the manufacture of a medicament for use in the treatment or management of obesity, the composition comprising a microbially produced oligosaccharide, wherein the composition comprises a galactooligosaccharide (GOS) produced from one or more Propionibacterium bacterial strains, wherein the GOS acts as a selective growth medium for Propionibacterium bacterial strains, and wherein the GOS is substantially the same as the form produced by reverse β-galactosidase reaction for Propionibacterium bacterial strains.

Alternative (or additionally) to a pharmaceutical or medicament, the composition may be used as a dietary supplement, a nutraceutical or a functional food. A yet further aspect of the present invention may be a prebiotic composition for use as a dietary supplement, a nutraceutical or a functional food, the composition comprising a galacto oligosaccharide (GOS) produced from one or more Propionibacterium bacterial strains, wherein the GOS acts as a selective growth medium for Propionibacterium bacterial strains, and wherein the GOS is substantially the same as the form produced by reverse β-galactosidase reaction for Propionibacterium bacterial strains.

It will be apparent to the skilled addressee that the features of the prebiotic composition in connection with the first aspect of the invention will also be applicable and interchangeable for the composition for use as a pharmaceutical, medicament, dietary supplement, nutraceutical or functional food.

Furthermore, the composition could be incorporated into an existing food, such as yoghurt or as a powder which can be easily blended with foodstuffs or made into a liquid drink.

In a further aspect of the present invention, there is provided a method of increasing propionate levels in the lower gut of an individual by administering a composition as herein above described so as to promote the growth of propionate secreting bacteria.

In another aspect of the present invention, there is provided a method of producing galactooligosaccharide (GOS) comprising the steps of growing one or more Propionibacterium strains in a growth medium comprising up to 50% lactose at a temperature of up to 55° C. for up to 24 hours under anaerobic conditions and harvesting GOS from the Propionibacterium cells.

Preferably, the one or more the one or more Propionibacterium strains are grown in a growth medium comprising up to 40% lactose at a temperature of up to 50° C. for up to 24 hours. The one or more Propionibacterium strains may be grown in a growth medium comprising in the range of about 20 to about 40% lactose at a temperature in the range of about 35 to about 50° C. for about 10 to about 14 hours.

The GOS may be harvested by a number of methods, but it is preferred that is harvested from the cells by lysis. Such a lysis may involve one or more freeze-thawing steps.

The Propionibacterium strains may be selected from one or more of the following: Propionibacterium jensenii; Propionibacterium freudenreichii; Propionibacterium acidipropionici, or sub-species or mutant strains thereof.

Preferably, the method of producing GOS in a selected Propionibacterium strain(s) is optimised.

The method as hereinabove described, may be used to produce GOS for use in a compositional aspects of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

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

FIG. 1 is a graph showing the ratio between the most prevalent GOS species for P. jensenii calculated at different temperatures and timepoints;

FIG. 2 is a graph showing the two GOS formation rates at 30° C. and 50° C. of the selected two Propionibacterium strains;

FIG. 3 is a graph showing the ratio of the actual GOS formation rate over the theoretical GOS formation rate at 30° C. and 50° C. for the selected strains;

FIG. 4 is a graph showing the analysis of the difference in β-galactosidase activity from the initial screening experiments and the later GOS synthesis experiments in the selected strains; and

FIG. 5 shows the analysis (by means of the Log/Stat ratio) of the dependency of expression of β-galactosidase of the selected strains.

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.

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.

The basis of the present experiments was to reversibly use β-galactosidases in Propionibacterium strains so as to produce a novel GOS. Ordinarily, β-galactosidases would hydrolyse lactose. However, by changing the reaction conditions, in terms of substrate concentration and temperature, the enzyme acts reversibly and generates an oligosaccharide version of the lactose (GOS).

EXPERIMENTS

Experiments were conducted in two phases. The first phase screened 77 strains for the detection of β-galactosidase hydrolytic activity based on the breakdown of ortho-Nitrophenyl-β-galactoside (ONPG). Growth conditions were adjusted to attempt to improve the overall growth characteristics. Total β-galactosidase activity was assessed and strains exhibiting the highest activity were then put forward to the second phase. During the second phase, a feasibility study was conducted to screen the selected strains for their actual ability to synthesise GOS.

Screening of 77 Propionibacterium strains was conducted for the detection of β-galactosidase hydrolytic activity based on the breakdown of ONPG. The total β-galactosidase activity was assessed in miller units.

β-Galactosidase Activity in Propionibacterium

A range of Propionibacterium strains (including different species and sub-species) were pre-grown from a −80° C. stock for 72 hours at 30° C. in 200 μl LB medium supplemented with 1% glucose in a standard 96 wells-plate. Cultures were re-diluted 100 fold to 1600 μl LB supplied with 1% glucose deep-well plates. Growth was performed in anaerobic conditions at 37° C. for 96 hours OD₅₀₀was determined after a 10-fold dilution of the cultures. To assess β-galactosidase activity, cells were first centrifuged at 5000×g at 4° C. Then the pellets were lysed using 0.5 gram silicabeads (0.1 mm) in 800 μl 0.05M NaPi buffer pH=7.0. The supernatant was used for determining the β-galactosidase activity at 30° C. using a standard protocol.

Table 1 below illustrates the results of those Propionibacterium strains which were screened using the above protocol.

TABLE 1

Bgal activity

Total Bgal
Average
(Miller Units)

Strain
activity
OD600
Average
Stdev

no.
Average
Stdev
Average
Stdev
μmol/min/OD-

no.
μmol/min/l
AU
Unit
Species
SubSpecies

4204
17.6
3.9
2.94
0.10
6.0
1.5

Propionibacterium

acidipropionici

380
15.8
0.7
2.86
0.34
5.6
0.4

Propionibacterium sp.

2166
0.9
0.3
0.18
0.03
4.8
0.7

Propionibacterium

freudenreichii

freudenreichii

359
7.9
4.0
1.77
0.90
4.5
0.1

Propionibacterium sp.

1134
10.6
1.0
2.49
0.07
4.2
0.3

Propionibacterium

shermanii

freudenreichii

364
10.8
1.3
2.91
0.03
3.7
0.4

Propionibacterium

jensenii

2060
8.7
1.1
2.45
0.22
3.5
0.1

Propionibacterium

shermanii

freudenreichii

4199
5.9
1.4
1.69
0.44
3.5
0.1

Propionibacterium

acidipropionici

4201
6.4
0.3
1.92
0.02
3.3
0.1

Propionibacterium

acidipropionici

2175
6.8
0.2
2.04
0.06
3.3
0.2

Propionibacterium

freudenreichii

freudenreichii

2145
8.0
0.6
2.39
0.01
3.3
0.2

Propionibacterium

freudenreichii

freudenreichii

2168
7.3
1.2
2.36
0.07
3.1
0.6

Propionibacterium

freudenreichii

freudenreichii

2174
4.5
2.9
1.33
0.68
3.1
0.6

Propionibacterium

freudenreichii

freudenreichii

2173
6.4
0.4
2.06
0.08
3.1
0.3

Propionibacterium

freudenreichii

freudenreichii

384
1.5
0.3
0.53
0.11
2.9
1.1

Propionibacterium

freudenreichii

freudenreichii

2171
7.7
3.5
3.00
0.00
2.6
1.2

Propionibacterium

freudenreichii

freudenreichii

362
5.7
0.6
2.38
0.08
2.4
0.3

Propionibacterium

acidipropionici

2172
4.4
2.2
1.83
0.34
2.3
0.8

Propionibacterium

freudenreichii

freudenreichii

360
0.6
0.2
0.14
0.22
2.2
0.3

Propionibacterium

shermanii

freudenreichii

2156
4.4
1.8
2.01
0.46
2.1
0.4

Propionibacterium

freudenreichii

freudenreichii

2541
3.2
2.7
1.65
0.18
2.1
1.9

Propionibacterium sp.

2149
5.4
3.0
2.70
0.04
2.0
1.1

Propionibacterium

freudenreichii

freudenreichii

375
5.4
0.5
3.00
0.00
1.8
0.2

Propionibacterium

acidipropionici

2169
4.7
0.6
2.68
0.05
1.8
0.2

Propionibacterium

freudenreichii

freudenreichii

2146
3.4
0.2
2.37
0.33
1.4
0.1

Propionibacterium

freudenreichii

freudenreichii

374
3.2
5.4
2.42
0.01
1.3
2.2

Propionibacterium

shermanii

freudenreichii

2167
2.9
0.6
2.23
0.06
1.3
0.2

Propionibacterium

freudenreichii

freudenreichii

2160
3.2
1.1
2.63
0.29
1.2
0.3

Propionibacterium

freudenreichii

freudenreichii

2150
0.9
0.5
1.24
1.04
1.1
0.7

Propionibacterium

freudenreichii

freudenreichii

2159
2.7
1.1
2.33
0.30
1.1
0.3

Propionibacterium

freudenreichii

freudenreichii

2543
2.1
0.3
1.88
0.01
1.1
0.2

Propionibacterium sp.

371
1.2
0.0
1.10
0.13
1.1
0.1

Propionibacterium

shermanii

freudenreichii

4200
2.6
0.4
2.37
0.11
1.1
0.1

Propionibacterium

acidipropionici

2178
1.2
0.2
1.33
0.54
1.1
0.6

Propionibacterium

freudenreichii

freudenreichii

2164
2.4
0.5
2.28
0.04
1.1
0.3

Propionibacterium

freudenreichii

freudenreichii

2162
2.4
1.9
2.04
1.23
1.0
0.3

Propionibacterium

freudenreichii

freudenreichii

367
2.0
0.5
1.93
0.08
1.0
0.2

Propionibacterium

shermanii

freudenreichii

2068
2.2
0.3
2.13
0.12
1.0
0.1

Propionibacterium

shermanii

freudenreichii

2177
1.9
0.3
1.86
0.12
1.0
0.1

Propionibacterium

freudenreichii

freudenreichii

2165
2.4
1.0
2.36
0.02
1.0
0.4

Propionibacterium

freudenreichii

freudenreichii

2155
2.7
0.1
2.65
0.11
1.0
0.1

Propionibacterium

freudenreichii

freudenreichii

2069
1.9
0.7
1.86
0.69
1.0
0.1

Propionibacterium

shermanii

freudenreichii

2161
2.2
1.3
2.17
0.31
1.0
0.5

Propionibacterium

freudenreichii

freudenreichii

2066
2.4
0.2
2.47
0.15
1.0
0.0

Propionibacterium

freudenreichii

freudenreichii

365
2.4
0.2
2.50
0.03
0.9
0.1

Propionibacterium

shermanii

freudenreichii

2544
2.2
0.4
2.32
0.02
0.9
0.2

Propionibacterium sp.

2158
2.1
1.2
2.24
0.06
0.9
0.5

Propionibacterium

freudenreichii

freudenreichii

361
2.7
2.1
2.96
0.09
0.9
0.7

Propionibacterium

shermanii

thoenni

2163
1.9
0.9
2.11
0.20
0.9
0.3

Propionibacterium

freudenreichii

freudenreichii

2154
2.2
0.2
2.56
0.16
0.9
0.0

Propionibacterium

freudenreichii

freudenreichii

382
2.1
0.9
2.41
0.83
0.8
0.1

Propionibacterium sp.

379
0.9
0.4
1.09
0.07
0.8
0.4

Propionibacterium

freudenreichii

freudenreichii

2542
1.9
1.2
2.32
0.05
0.8
0.5

Propionibacterium sp.

372
1.6
0.4
2.25
0.04
0.7
0.2

Propionibacterium

shermanii

freudenreichii

2157
2.0
1.3
2.61
0.38
0.7
0.4

Propionibacterium

freudenreichii

freudenreichii

369
1.4
0.1
2.04
0.04
0.7
0.1

Propionibacterium

shermanii

freudenreichii

1256
1.7
0.9
2.40
0.29
0.7
0.3

Propionibacterium sp.

2144
1.0
0.2
0.93
1.11
0.6
0.0

Propionibacterium

freudenreichii

freudenreichii

2179
1.3
0.3
2.12
0.03
0.6
0.1

Propionibacterium

freudenreichii

freudenreichii

2170
1.4
0.2
2.33
0.04
0.6
0.1

Propionibacterium

freudenreichii

freudenreichii

2336
1.4
0.8
2.48
0.05
0.6
0.3

Propionibacterium

shermanii

freudenreichii

2181
1.0
0.2
1.82
0.36
0.5
0.0

Propionibacterium

freudenreichii

freudenreichii

2176
1.6
0.1
3.13
0.15
0.5
0.0

Propionibacterium

freudenreichii

freudenreichii

2147
1.1
0.5
2.10
0.67
0.5
0.1

Propionibacterium

freudenreichii

freudenreichii

370
1.0
0.2
2.05
0.06
0.5
0.1

Propionibacterium

shermanii

freudenreichii

383
0.8
0.3
1.87
0.80
0.5
0.3

Propionibacterium

freudenreichii

freudenreichii

2663
1.2
0.7
2.60
0.04
0.5
0.3

Propionibacterium

shermanii

freudenreichii

2182
1.2
0.8
2.62
0.09
0.5
0.3

Propionibacterium

freudenreichii

freudenreichii

2151
1.0
0.1
2.28
0.26
0.4
0.1

Propionibacterium

freudenreichii

freudenreichii

2143
0.5
0.0
1.14
0.06
0.4
0.0

Propionibacterium

freudenreichii

freudenreichii

2152
0.9
0.1
2.45
0.19
0.4
0.1

Propionibacterium

freudenreichii

freudenreichii

2007
0.9
0.2
2.40
0.04
0.4
0.1

Propionibacterium

shermanii

freudenreichii

2065
0.9
0.1
2.39
0.11
0.4
0.0

Propionibacterium

shermanii

freudenreichii

363
0.6
0.1
1.66
0.07
0.3
0.1

Propionibacterium

freudenreichii

freudenreichii

2148
0.7
0.2
1.97
0.07
0.3
0.1

Propionibacterium

freudenreichii

freudenreichii

2180
1.0
0.1
3.00
0.00
0.3
0.0

Propionibacterium

freudenreichii

freudenreichii

2067
0.6
0.1
1.92
0.01
0.3
0.0

Propionibacterium

shermanii

freudenreichii

1219
0.4
0.2
2.27
0.06
0.2
0.1

Propionibacterium

freudenreichii

freudenreichii

The highest β-galactosidase expressing strains were then included in the next phase of the study.

Analysis of GOS Production in the Chosen Strains

The following growth protocols were used:

Propionibacterium strains were pre-grown from the −80° C. stock for 72 hours at 30° C. in 100 ml LB medium supplied with 1% glucose. Cultures were diluted 50, 200, 1000 and 4000-fold in a 1 litre bottle filled with LB medium supplied with 1% glucose. Growth was performed at 30° C. for a set time that had been calculated to ensure a logarithmic culture and a stationary phase culture at the aimed time of harvesting.

Analysis of β-Galactosidase Activity in the Chosen Strains

To analyse the β-galactosidase activity, cells were centrifuged at 5000×g at 4° C. for 15 minutes. Pellets were re-dissolved in 1% of the original volume using a phosphate buffer B (50 mM Na2HPO4.2H2O, 1 mM MgCl2) and then eight 1250 μl aliquots of each cell-free extract transferred to a deep well plate.

The pellets were subsequently lysed using 0.5 gram silicabeads (0.1 mm) in 800 μl 0.05M NaPi buffer pH=7.0 and 4 repetitions of 30 second bursts in a cell disruptor. The lysed pellets of the same cell-free extract were then recombined in a single 15 ml Geiner-tube. Cultures were centrifuged for 10 minutes at 5000×g after the indicated time-period using a 96-well plate centrifuge. 20 μl of supernatant of the cell lysate was dissolved in 180 μl phosphate buffer A (8.9 gr/l Na2HPO4.2H2O, 6.9 gram/l Na2HPO4.H2O, 1 mM DTT).

Additionally 10, 100 and 100 fold dilutions of the cell lysate phosphate buffer mix were prepared, to which an ONPG stock solution (20 mM in phopshate buffer) to a starting concentration of 1 mM was added. The absorbance at 420 nm was observed over time using a Pharmacia Biotech Ultrospec 2000 UV/visible spectrophotometer using Swift II Application software and the Miller Units were calculated using the above indicated dilutions.

GOS Synthesis Protocol

Activity was normalized to 2 mM/min in a total volume of 10 ml by dilution using phsopahe buffer B. 15 ml Greiner tubes were pre-warmed which contained 13.5 ml phosphate buffer B at 30°, 50°, and 60° C. The reaction was started by the addition of 1.5 ml cell-free extract (2 mM/min β-galactosidase activity) to the pre-warmed Greiner tubes. The reactions proceeded with a 30 second time interval. 1 ml samples were then transferred to an Eppendorf tube at 0, 30, 60, 90, 120, 180, 240, 300, and 1440 minute intervals. The GOS formation reaction was then stopped by incubation at 100° C. for 5 minutes and the samples immediately stored at −80° C.

Based on the activities of the β-galactosidases found, the actual activity for the GOS formation rate could be predicted. Conversion factors were calculated for each species.

Table 2 below shows the predicted GOS formation rate at 30° C.

TABLE 2

Predicted

GOS

Bgal activity

formation

(Miller Units)
Used

rate

Strain

Average
Stdev
conversion
Correction
mM/min/100

no.
Species
Subspecies
μmol/min/OD-Unit
factor
factor
OD units

4204

Propionibacterium

6.0
1.5
10.8
5.0
0.025

acidipropionici

380

Propionibacterium sp.

5.6
0.4
10.8
5.0
0.023

2166

Propionibacterium

freudenreichii

4.8
0.7
10.8
5.0
0.020

freudenreichii

359

Propionibacterium sp.

4.5
0.1
10.8
5.0
0.019

1134

Propionibacterium

shermanii

4.2
0.3
10.8
5.0
0.018

freudenreichii

364

Propionibacterium

3.7
0.4
10.8
5.0
0.015

jensenii

2060

Propionibacterium

shermanii

3.5
0.1
10.8
5.0
0.015

freudenreichii

4199

Propionibacterium

3.5
0.1
10.8
5.0
0.015

acidipropionici

4201

Propionibacterium

3.3
0.1
10.8
5.0
0.014

acidipropionici

2175

Propionibacterium

freudenreichii

3.3
0.2
10.8
5.0
0.014

freudenreichii

2145

Propionibacterium

freudenreichii

3.3
0.2
10.8
5.0
0.014

freudenreichii

2168

Propionibacterium

freudenreichii

3.1
0.6
10.8
5.0
0.013

freudenreichii

2174

Propionibacterium

freudenreichii

3.1
0.6
10.8
5.0
0.013

freudenreichii

Table 3 below shows the predicted GOS formation rate at 50° C.

TABLE 3

Predicted

GOS

Bgal activity

formation

(Miller Units)
Used

rate

Strain

Average
Stdev
conversion
Correction
mM/min/100

no.
Species
Subspecies
μmol/min/OD-Unit
factor
factor
OD units

4204

Propionibacterium

6.0
1.5
10.8
5.0
0.025

acidipropionici

380

Propionibacterium sp.

5.6
0.4
10.8
5.0
0.023

2166

Propionibacterium

freudenreichii

4.8
0.7
10.8
5.0
0.020

freudenreichii

359

Propionibacterium sp.

4.5
0.1
10.8
5.0
0.019

1134

Propionibacterium

shermanii

4.2
0.3
10.8
5.0
0.018

freudenreichii

364

Propionibacterium

3.7
0.4
10.8
5.0
0.015

jensenii

2060

Propionibacterium

shermanii

3.5
0.1
10.8
5.0
0.015

freudenreichii

4199

Propionibacterium

3.5
0.1
10.8
5.0
0.015

acidipropionici

4201

Propionibacterium

3.3
0.1
10.8
5.0
0.014

acidipropionici

2175

Propionibacterium

freudenreichii

3.3
0.2
10.8
5.0
0.014

freudenreichii

2145

Propionibacterium

freudenreichii

3.3
0.2
10.8
5.0
0.014

freudenreichii

2168

Propionibacterium

freudenreichii

3.1
0.6
10.8
5.0
0.013

freudenreichii

2174

Propionibacterium

freudenreichii

3.1
0.6
10.8
5.0
0.013

freudenreichii

GOS Analysis Protocol

High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) was used to undertake the GOS analysis. HPAEC-PAD analyses were performed on a DX-500 BIO-LCsystem (Dionex) equipped with a PAD. Galactooligosaccharide fractions were separated on CarboPac PA1 column with dimensions 250 mm*4 mm t a flow rate of 1 mL/min at 22° C. A CarboPac PA1 guard column with dimensions 50*4 mm i.d. (Dionex) was used for column protection. The eluents used for the analysis were (A) 500 mM NaOAc+100mMNaOH, (B) 100mMNaOH and (C) Milli-Q water.

Eluents A and B were mixed to form the following gradient 100% B from 0 to 5 min followed by 0-26% A in 73 min. After each run, the column was washed with 100% A for 6 min and re-equilibrated for 10 min at 100% B. Peak identification occurred on the basis of comparison of peak distribution of the HPLC chromatogram described in J. Agric. Food Chem. 2009, 57, 8488-8495. Lactose was used as a standard for elution time normalization.

Results

To determine the ratio between highly formed GOS species the most prevalent GOS species for P. jensenii were quantified and the ratio between the two species calculated at different temperatures and time points. As shown in Table 4 below and illustrated in FIG. 1, it was found that the species ratio showed a strong temperature dependence and a small time dependence.

TABLE 4

Expected
Expected
β-D-

GOS
GOS
Gal-(1f4)-

linkage type
linkage type
β-D-Gal-
Ratio

Strain
Temp
Unknown
(1f4)-D-Glc
2:1

Propionibacterium

Temp = 30 C.,
0.2
4.2
18.3

jensenii

Time 5 H

Propionibacterium

Temp = 50 C.,
1.2
2.9
2.5

jensenii

Time 5 H

Propionibacterium

Temp = 30 C.,
1.0
12.0
11.8

jensenii

Time 24 H

Propionibacterium

Temp = 50 C.,
4.4
6.2
1.4

jensenii

Time 24 H

Based on standard thermodynamics it was assumed that at 50° C. the β-galactosidase reaction occurs at a 4-8 times higher rate than at 30° C. For tested samples where the GOS formation rate was at a stage where this was expected to be linear the GOS formation rates were plotted. As shown in Table 5 below and illustrated in FIG. 2, no significant increase in activity was detected in either Propionibacterium strains.

TABLE 5

Strain
Temp
Total GOS

P. jensenii

30° C.
4.5

50° C.
4.1

The theoretical GOS formation rate was calculated based on the β-galactosidase activity measured in Miller Units in Phase 2 of the study. Table 6 below shows the ratio of actual GOS formation rate over theoretical GOS formation rate and FIG. 3 shows this plotted for both 30° C. and 50° C. Surprisingly, and advantageously, GOS formation rates were always found to be higher than the theoretical GOS formation rates.

TABLE 6

Actual/

Theoretical
Actual/Theoretical

Strain
No
GOS (30 C.)
GOS (50 C.)
Ratio

P. jensenii

364
13.6
12.5
0.9

P. freudenreichii

1134
7.9
9.1
1.2

Average

10.8
10.8
1.0

The β-galactosidase activity analysed in the initial phase of experiments in general appeared to be higher than those activities determined in the later phase. To find out whether there is a consistent error in the methodology the ratios of the activities in phase 1 and 2 were calculated (and shown in Table 7 below) and plotted on a graph shown in FIG. 4. FIG. 4 shows that for most samples a 5-fold difference is detected. Some samples clearly show much higher differences, and this is expected that these differences are mainly due to the differences in the growth phase of the cells.

TABLE 7

Strain no
Growth Phase
Ratio Phase 1/Phase 2

364
Mid-log
7.9

364
Mid-log
7.9

364
Stationary
14.4

1134
Mid-log
1.5

1134
Stationary
5.0

1134
Stationary
5.0

4204
Mid-log
1.2

4204
Stationary
6.1

To assess whether the expression of β-galactosidase was dependent on the growth phase of the organism, the activity (measured in Miller Units) was plotted for all strains. Table 8 and FIG. 5 show the data and plot respectively. It was found that for most strains a Log:Stat ratio ≥1 was found indicating that the activity of β-galactosidase is higher in the Log phase than in the stationary phase. These differences are limited and it may be that the higher biomass yield in stationary phase off-sets the lower β-galactosidase activities.

TABLE 8

Ratio Log/Stat

Propionibacterium jensenii

364
1.8

Propionibacterium freudenreichii

1134
3.4

Propionibacterium acidipropionici

4204
4.9

CONCLUSIONS

All Propionibacterium strains produced GOS and the cell-free extracts showed approximately similar GOS formation rates at 30° C. and 50° C. All samples show a different GOS profile than the GOS produced by Apergillus Oryzea enzyme. Specifically strain 364 (P. jensenii) showed significant GOS production yields. In general, the later GOS synthesis phase showed a 5-fold lower β-galactosidase activities as compared to the initial screening phase.

These experiments showed that it was possible for Propionibacterium strains to produce GOS, which could potentially be used as a selective growth medium for a chosen Propionibacterium probiotic bacterial strain so to promote growth in the lower gut so as help modulate appetite.

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.

Claims

1. A prebiotic composition comprising a galacto oligosaccharide (GOS) produced from one or more selected Propionibacterium bacterial strain wherein the GOS is substantially the same as the form produced by reverse β-galactosidase reaction for Propionibacterium bacterial strains.
2. The prebiotic composition as claimed in claim 1, wherein the GOS is produced and/or is selective for one of more of the following Propionibacterium bacterial strains: Propionibacterium jensenii; Propionibacterium freudenreichii; Propionibacterium acidipropionici, sub-species thereof or a mutant strain thereof.
3. The prebiotic composition as claimed in claim 1, wherein the GOS is produced from a selected Propionibacterium bacterial genus or strain and the GOS acts as a selective growth medium for said selected Propionibacterium bacterial genus or strain.
4. The prebiotic composition as claimed in claim 1, wherein the composition is encapsulated.
5. The prebiotic composition as claimed in claim 1, wherein the composition further comprises an excipient or carrier compound, wherein said excipient or carrier compound permits the prebiotic composition to pass through a gastrointestinal environment with preserved functional properties.
6. The prebiotic composition as claimed in claim 1, wherein the composition is in the form of a liquid, powder or form that can be mixed with a solid or liquid food stuff.
7. A medicament comprising the prebiotic composition as claimed in claim 1.
8. The prebiotic composition of claim 1 comprising a medicament for treating and/or managing obesity.
9. The prebiotic composition of claim 1 comprising a dietary supplement.
10. The prebiotic composition of claim 1 comprising a composition for regulating or modulating appetite.
11. The prebiotic composition of claim 1 comprising a composition for weight management.
12. A method of increasing propionate levels in a lower gut region comprising providing the prebiotic composition of claim 1 in an amount sufficient to promote growth of propionate secreting bacteria in the gut region.
13. A method of producing galactooligosaccharide (GOS) comprising the steps of growing one or more Propionibacterium strains in a growth medium comprising up to 50% lactose at a temperature of up to about 55° C. for up to 24 hours under anaerobic conditions and harvesting GOS from the Propionibacterium cells.
14. The method as claimed in claim 13, wherein the one or more Propionibacterium strains are grown in a growth medium comprising up to 40% lactose at a temperature of up to 50° C. for up to 24 hours.
15. The method as claimed in claim 13, wherein the GOS is harvested from a lysate of Propionibacterium cells.
16. The method as claimed in claim 13, wherein the Propionibacterium strains are selected from one or more of the following: Propionibacterium jensenii; Propionibacterium freudenreichii; Propionibacterium acidipropionici, a sub-species thereof or a mutant strain thereof.
17. The method as claimed in claim 13, wherein GOS is used in a medicament for treating or managing obesity, a dietary supplement, a medicament for regulating or modulating appetite, or in a medicament for weight management.
18. The method of claim 17 wherein the GOS is produced by a Propionibacterium strain selected from one or more of the following: Propionibacterium jensenii; Propionibacterium freudenreichii; Propionibacterium acidipropionici, a sub-species thereof or a mutant strain thereof.
19. The method of claim 17 wherein the GOS is produced from a selected Propionibacterium bacterial genus or strain and the GOS acts as a selective growth medium for said selected Propionibacterium bacterial genus or strain.
20. The method of claim 17 wherein the medicament or dietary supplement comprises an excipient or carrier compound, wherein said excipient or carrier compound permits the passage of the medicament or dietary supplement through a gastrointestinal environment with retained functional properties.
21. The method of claim 17 wherein the medicament or dietary supplement is encapsulated.
22. The method of claim 17 wherein the medicament or dietary supplement is in the form of a liquid, a powder or a form that can be mixed with a solid or liquid food stuff.

Priority Claims (1)

Number	Date	Country	Kind
1509023.6	May 2015	GB	national

PCT Information

Filing Document	Filing Date	Country	Kind
PCT/GB2016/051516	5/25/2016	WO	00

COMPOSITIONS AND METHODS OF PRODUCTION THEREOF

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Abstract

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Priority Claims (1)

PCT Information