TECHNICAL FIELD OF THE INVENTION
The invention relates to a screening method for predicting and identifying bacterial strains capable of producing high yields of galactooligosaccharides (GOS), by reverse enzyme reaction of β-galactosidases. The resultant GOS can be formulated as a selective prebiotic for the growth of a selected bacterial strain, species or genus.
BACKGROUND TO THE INVENTION
Probiotics are bacteria which confer health benefits to a host. Typically, cultures of probiotic bacterial strains are consumed or administered to individuals in order to add to and augment the naturally occurring bacteria population of the gut. A number of health benefits have been associated with probiotics, including reducing the incidence of cancer, traveler's diarrhoea, irritable bowel syndrome, and lactose intolerance to name a few. Preliminary studies also indicate that probiotics can be useful in reducing serum levels of cholesterol and blood pressure and help modulate diabetes.
Prebiotics are dietary ingredients which can selectively enhance the numbers and/or activity of beneficial indigenous gut microbiota, such as lactobacilli or bifidobacteria, and 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.
Fructo-oligosaccharides (FOS, inulin and oligofructose) and galactooligosaccharides (GOS) have been demonstrated to fulfil the criteria for prebiotic classification repeatedly in human intervention studies.
It is an object of the present invention to provide a method of predicting the likelihood a bacterial strain has of being able to produce prebiotics in relatively high yields. It is also an object of the present invention to provide a screening method for quickly identifying probiotic bacterial strains which are capable of producing and/or producing a high yield of GOS which could in turn be used as a selective growth prebiotic for that particular strain, species or genus. It would be advantageous if the screening method was high-through put.
SUMMARY OF THE INVENTION
In accordance with a first aspect of the present invention, there is provided a method of screening one or more Streptococcus, Lactobacillus or Propionibacterium bacterial strains for the ability to produce and/or produce a high yield of galactooligosaccharides (GOS) comprising assessing the β-galactosidase activity of a strain under growth conditions and identifying whether the activity has:
- a) Miller Units which are equal to or greater than about 60 for Streptococcus or Lactobacillus strains; or
- b) Miller Units which are equal to or greater than about 3 for Propionibacterium strains.
The method may comprise growing the one or more strains under standard growth conditions for a given incubation time and then lysing the cells and assessing the β-galactosidase activity in the lysate.
The method may further comprise:
- i) incubating the one or more strains at about 37° C. for up to 40 hours;
- ii) centrifuging the cells at a lower temperature than during incubation;
- iii) lysing the cells and removing a supernatant from the lysed cells; and
- iv) assessing β-galactosidase activity, expressed in Miller Units, in the supernatant.
If more than one strains are identified by the method as having the required β-galactosidase activity, the method may further comprise:
c) screening the strains at higher and lower temperatures (at least one of which will be different to the growth temperature) at a number of time points to assess which strains have the highest yield of GOS. The higher temperature may be about 50° C. and the lower temperature may be about 30° C.
The bacterial strains may comprise strains selected from: Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus buchneri Lactobacillus helveticus, Streptococcus thermophilus, Propionibacterium jensenii; Propionibacterium freudenreichii; Propionibacterium acidipropionici, or sub-species or mutant strain thereof.
In accordance with another related aspect, there is provided a method of screening a multiplicity of bacterial strains to identify a bacterial strain or strains, which would be suitable for high yield production of a prebiotic composition, the method comprising assessing the growth rate, enzyme production and enzyme activity of an enzyme utilised for the generation of the prebiotic composition by the bacterial strain for each strain and selecting those strains showing the highest growth rate, enzyme production and enzyme activities.
In accordance with a further aspect of the present invention, there is provided a prebiotic composition comprising a galactooligosaccharide (GOS) produced from one or more Streptococcus, Lactobacillus or Propionibacterium bacterial strains, wherein the GOS acts as a selective growth medium for the Streptococcus, Lactobacillus or Propionibacterium bacterial strains, the GOS being in substantially the same form as produced by reverse β-galactosidase reaction in the bacterial strains and the β-galactosidase activity of the Streptococcus, Lactobacillus or Propionibacterium bacterial strains having:
- a) a Miller Unit which is equal to or greater than about 60 for Streptococcus or Lactobacillus strains; or
- b) a Miller Unit which is equal to or greater than about 3 for Propionibacterium strains.
The GOS may be produced and/or is selective for one of more of the following bacterial strains: Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus buchneri Lactobacillus helveticus, Streptococcus thermophilus, Propionibacterium jensenii; Propionibacterium freudenreichii; Propionibacterium acidipropionici, or sub-species or mutant strain thereof.
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 of Lactobacillus or Propionibacterium probiotic bacterial strains 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, riboflavin 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, epigailocatechin gallate, theaflavins, thearubigins, proanthocyanins, flavonols, quercetin, kaempferol, myricetin, isorhamnetin, hesperetin, naringenin, eriodictyol, tangeretin, flavones, apigenin, luteolin, lignans, phytoestrogens, resveratrol, isoflavones, daidzein, genistein, glycitein, soy isoflavones, and combinations thereof.
The composition may be for use as a medicament and/or a dietary supplement and/or a nutraceutical or a functional food.
Preferably, the GOS of the composition is produced by a strain or strains identified in the screening method as herein above described.
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will now be described, by way of example only and with reference to the following Figures:
FIG. 1 is a graph showing the ratio between the most prevalent GOS species for P. jensenii synthesized 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 Lactobacillus and 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 microorganisms so as to produce a novel GOS. Ordinarily, β-galactosidases would hydrolyse lactose. However, by changing the reaction conditions, in terms of substrate and temperature, the enzyme acts reversibly and generates an oligosaccharide version of the lactose (GOS).
EXPERIMENTS
Experiments were conducted in two phases. The first phase screened 360 bacterial strains for the detection of β-galactosidase hydrolytic activity based on the breakdown of ortho-Nitrophenyl-β-galactoside (ONPG). The bacterial strains were selected from three bacterial genera (Streptococcus, Lactobacillus, Propionibacterium) and growth conditions were adjusted for each genus to attempt to improve the overall growth of each genus. β-galactosidase activity, expressed in Miller Units, was assessed and strains meeting the required activity were then put forward to the second phase. During the second phase, a feasibility study phase was conducted to screen the selected strains for their actual ability to synthesise GOS.
Experiment 1
Screening of 360 Streptococcus, Lactobacillus and Propionibacterium strains was conducted for the detection of β-galactosidase hydrolytic activity based on the breakdown of ONPG. Growth conditions were adjusted for each genus and the total β-galactosidase activity assessed in miller units.
β-Galactosidase Activity in Streptococcus thermophilus
S. thermophilus strains were pre-grown from a −80° C. stock for 22 hours at 37° C. in 200 μl GM17 medium supplemented with 1% glucose in a standard 96 wells-plates. Cultures were re-diluted 100 fold to 1600 μl GM17 supplied with 1% glucose in deep-well plates. Growth was performed in anaerobic conditions at 37° C. for 22 hours. OD600 was determined after a 10-fold dilution of the cultures. For the β-galactosidase activity, the cells were centrifuged at 5000×g at 4° C. and the pellets were subsequently 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 ONPG test protocol to assess the Miller Units. Table 1 below illustrates the results of those S. thermophilus strains which were screened using the above protocol.
TABLE 1
|
|
Total Bgal
Bgal activity
|
activity
Average OD600
(Miller Units)
|
Strain no.
Average
Stdev
Average
Stdev
Average
Stdev
|
no.
μmol/min/l
AU
μmol/min/OD-Unit
|
|
883
2690
299
1.37
0.13
1960
83
|
885
1020
20
0.70
0.06
1462
103
|
882
906
21
0.68
0.05
1347
135
|
886
883
35
0.71
0.16
1291
238
|
884
506
90
0.40
0.10
1267
86
|
114
1565
81
1.37
0.22
1155
126
|
121
1459
118
1.29
0.19
1140
75
|
2310
1250
88
1.28
0.04
976
68
|
106
864
119
0.91
0.16
954
36
|
2106
869
30
1.11
0.26
815
196
|
105
195
44
0.27
0.02
718
101
|
2113
1267
116
1.84
0.01
690
61
|
109
803
43
1.18
0.20
690
83
|
130
1410
223
2.07
0.18
678
48
|
113
926
74
1.43
0.33
665
101
|
2320
885
29
1.36
0.10
653
50
|
1796
1123
47
1.79
0.20
633
77
|
110
1443
103
2.40
0.01
602
46
|
132
1263
451
2.34
0.57
600
338
|
2271
382
106
1.62
1.37
598
570
|
117
1139
76
1.94
0.21
597
100
|
2305
842
37
1.42
0.16
596
44
|
2306
711
44
1.20
0.00
591
39
|
2304
702
51
1.20
0.05
583
23
|
131
892
20
1.54
0.09
580
29
|
2308
910
69
1.68
0.20
545
33
|
2112
1086
67
2.05
0.13
530
17
|
116
966
91
1.91
0.46
519
82
|
2290
1289
58
2.51
0.08
513
12
|
2105
716
33
1.47
0.14
490
35
|
2279
1100
80
2.27
0.02
485
38
|
2291
462
239
1.11
0.78
479
120
|
2312
640
170
1.34
0.17
471
69
|
2107
637
74
1.37
0.22
469
35
|
1122
407
55
0.89
0.22
467
53
|
107
820
79
1.76
0.01
465
42
|
2318
457
43
1.02
0.12
448
14
|
1794
881
87
2.01
0.02
438
48
|
2315
553
137
1.31
0.27
419
20
|
108
528
150
1.33
0.58
418
69
|
111
718
165
1.75
0.13
417
124
|
2314
617
162
1.48
0.34
416
19
|
2311
587
76
1.41
0.09
416
28
|
2280
615
52
1.48
0.08
415
13
|
2289
688
27
1.76
0.02
391
12
|
2319
473
30
1.23
0.17
389
31
|
2086
542
158
1.45
0.52
382
30
|
2321
628
43
1.75
0.08
359
11
|
104
429
97
1.49
0.81
335
116
|
2313
754
47
2.29
0.26
332
27
|
2109
743
3
2.39
0.16
311
21
|
2307
437
59
1.44
0.04
305
50
|
1128
440
30
1.55
0.17
284
12
|
1953
564
39
2.03
0.19
281
46
|
2309
551
53
1.98
0.08
279
38
|
2316
605
48
2.31
0.08
262
12
|
122
406
85
1.56
0.02
260
57
|
2108
557
10
2.14
0.04
260
9
|
112
483
46
1.92
0.03
252
28
|
124
235
36
0.93
0.11
251
10
|
2288
678
16
2.78
0.17
244
21
|
2272
636
34
2.67
0.39
240
22
|
2317
498
117
2.15
0.06
233
61
|
1797
478
28
2.07
0.20
232
10
|
2285
430
37
1.89
0.00
228
19
|
126
322
30
1.42
0.06
227
12
|
119
288
11
1.32
0.05
219
16
|
2104
262
7
1.36
0.26
199
43
|
2269
260
13
1.35
0.04
192
15
|
127
274
10
1.57
0.06
175
1
|
2292
405
19
2.33
0.10
174
2
|
125
304
172
2.10
0.35
158
108
|
1951
342
51
2.30
0.15
148
13
|
2111
289
13
2.00
0.32
146
16
|
2278
313
4
2.16
0.04
145
4
|
2273
333
5
2.31
0.03
144
2
|
2277
313
16
2.20
0.11
143
1
|
2287
377
102
2.76
0.10
136
32
|
2286
301
10
2.34
0.10
129
10
|
2282
340
17
2.65
0.08
129
10
|
1950
332
9
2.63
0.12
127
9
|
123
211
23
1.92
0.03
110
14
|
118
256
16
2.37
0.08
108
3
|
2110
220
6
2.21
0.11
100
3
|
128
98
22
0.99
0.23
99.2
0.5
|
2283
204
20
2.33
0.04
87.8
10.0
|
2274
107
118
1.18
1.22
68.4
29.6
|
2284
142
27
2.40
0.12
59.0
8.2
|
2270
97
3
2.20
0.05
44.1
1.0
|
2275
44
46
0.06
0.09
29.7
1.5
|
133
5
2
1.45
0.08
3.7
1.4
|
129
4
1
3.01
0.21
1.4
0.5
|
2276
7
2
0.01
0.00
0.0
0.0
|
2281
5
3
0.00
0.02
0.0
0.0
|
|
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 well-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 OD600 was 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 2 below illustrates the results of those Propionibacterium strains which were screened using the above protocol.
TABLE 2
|
|
Bgal activity
|
Total Bgal
Average
(Miller Units)
|
Strain
activity
OD600
Average
Stdev
|
no.
Average
Stdev
Average
Stdev
μmol/min/
|
no.
μmol/min/l
AU
OD-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
|
|
β-Galactosidase Activity in Lactobacillus
A range of Lactobacillus strains (including different species and sub-species) were pre-grown from a −80° C. stock for 48 hours at either 30° C. or 37° C. in 200 μl MRS medium in a standard 96 wells-plate in appropriate aerobiosis conditions. Cultures were re-diluted 100 fold to 1600 μl MRS medium in deep-well plates. Growth was performed in anaerobic conditions at 37° C. for 40 hours. OD600 was determined after a 10-fold dilution of the cultures. For the β-galactosidase activity, cells were centrifuged at 5000×g at 4° C. The pellets were subsequently lysed using 0.5 gram silicabeads (0.1 mm) in 800 μl 0.05M NaPi buffer pH=7.0. The supernatant was then used for determining the β-galactosidase activity at 30° C. using a standard protocol.
Table 3 below illustrates the results of those Lactobacillus strains which were screened using the above protocol.
TABLE 3
|
|
Bgal activity
|
Total Bgal
Average
(Miller Units)
|
Strain
activity
OD600
Average
Stdev
|
no.
Average
Stdev
Average
Stdev
μmol/min/
|
no.
μmol/min/l
AU
OD-Unit
Species
Subspecies
|
|
194
874
19
0.86
0.03
1019
59
Lactobacillus
bulgaricus
|
delbrueckii
|
191
1775
111
1.89
0.01
937
62
Lactobacillus
bulgaricus
|
delbrueckii
|
203
1525
96
1.90
0.16
804
16
Lactobacillus
bulgaricus
|
delbrueckii
|
192
1171
320
2.03
0.53
620
320
Lactobacillus
bulgaricus
|
delbrueckii
|
195
1157
256
2.14
0.02
541
114
Lactobacillus
bulgaricus
|
delbrueckii
|
202
1127
211
2.08
0.10
540
76
Lactobacillus
bulgaricus
|
delbrueckii
|
187
1084
197
2.51
0.79
442
61
Lactobacillus
bulgaricus
|
delbrueckii
|
189
1343
201
3.25
0.08
413
52
Lactobacillus
bulgaricus
|
delbrueckii
|
211
1205
37
3.06
0.48
398
51
Lactobacillus
|
helveticus
|
1456
1514
23
3.95
0.12
384
6
Lactobacillus
|
crispatus
|
204
1203
102
3.81
0.08
315
20
Lactobacillus
bulgaricus
|
delbrueckii
|
186
550
196
1.92
0.28
282
62
Lactobacillus
bulgaricus
|
delbrueckii
|
3273
866
7
3.40
0.17
255
14
Lactobacillus
|
helveticus
|
1795
391
185
1.79
0.38
213
58
Lactobacillus
|
delbrueckii
|
190
18
11
0.13
0.06
150
0
Lactobacillus
bulgaricus
|
delbrueckii
|
*301
500
37
3.48
0.04
144
12
Lactobacillus
|
buchneri
|
1572
207
254
1.38
1.97
139
Lactobacillus
|
fermentum
|
2954
286
2
2.58
0.02
111
0
Lactobacillus
|
reuteri
|
3416
261
0
2.49
0.11
105
5
Lactobacillus
|
reuteri
|
226
304
2
3.04
0.22
101
7
Lactobacillus
|
acidophilus
|
3909
310
6
3.29
0.11
94.0
1.2
Lactobacillus
|
reuteri
|
LR92
276
3
3.04
0.25
90.9
6.3
L. reuteri
|
2955
244
7
2.72
0.02
89.7
1.9
Lactobacillus
|
reuteri
|
LR1
306
5
3.68
0.70
84.5
14.5
L. rhamsnosus
|
2955
235
25
2.82
0.13
83.8
12.6
Lactobacillus
|
reuteri
|
3423
246
28
3.02
0.04
81.3
8.1
Lactobacillus
|
reuteri
|
3423
220
1
2.90
0.02
75.7
0.3
Lactobacillus
|
reuteri
|
3422
196
4
2.69
0.37
73.5
8.6
Lactobacillus
|
reuteri
|
LA1
294
12
4.07
0.18
72.3
6.2
L. acidophilus
|
11796
190
22
2.74
0.00
69.2
8.1
Lactobacillus
|
fermentum
|
634
183
10
2.96
0.16
61.9
0.1
Lactobacillus
|
acidophilus
|
3797
159
18
2.66
0.04
59.8
6.0
Lactobacillus
|
gasseri
|
227
14
14
0.26
0.29
52.0
0.0
Lactobacillus
|
acidophilus
|
3421
152
11
3.01
0.18
50.6
0.4
Lactobacillus
|
reuteri
|
3418
169
8
3.46
0.19
49.1
5.0
Lactobacillus
|
reuteri
|
197
59
1
1.25
0.12
47.2
3.6
Lactobacillus
bulgaricus
|
delbrueckii
|
D
119
3
2.86
0.15
41.5
1.1
Lactobacillus
|
3106
141
184
3.31
0.13
41.4
53.8
Lactobacillus
|
acidophilus
|
692
73
14
1.86
0.26
39.0
1.9
Lactobacillus
|
acidophilus
|
3117
131
180
2.26
1.73
38.6
50.0
Lactobacillus
|
amylolyticus
|
881
154
91
4.30
0.02
35.7
21.0
Lactobacillus
|
salivarius
|
*298
103
1
2.99
0.06
34.6
1.0
Lactobacillus
|
buchneri
|
205
33
15
1.04
0.45
31.7
1.0
Lactobacillus
bulgaricus
|
delbrueckii
|
1178
7
6
0.17
0.25
30.9
0.0
Lactobacillus
|
crispatus
|
3419
90
15
2.92
0.09
30.8
4.1
Lactobacillus
|
reuteri
|
3419
91
6
3.08
0.01
29.6
1.8
Lactobacillus
|
reuteri
|
2487
82
6
2.91
0.30
28.4
0.7
Lactobacillus
|
brevis
|
881
105
126
4.28
0.80
27.8
34.6
Lactobacillus
|
salivarius
|
2478
84
17
3.04
0.08
27.6
6.2
Lactobacillus
|
brevis
|
1178
19
16
0.85
0.82
25.8
5.9
Lactobacillus
|
crispatus
|
1688
75
4
2.92
0.17
25.8
3.0
Lactobacillus
|
fermentum
|
1177
60
7
2.42
0.36
25.0
0.8
Lactobacillus
|
helveticus
|
2472
73
2
3.08
0.06
23.7
0.3
Lactobacillus
|
brevis
|
1533
77
97
2.81
0.81
23.4
27.6
Lactobacillus
|
reuteri
|
2481
70
2
3.11
0.14
22.6
0.3
Lactobacillus
|
brevis
|
1229
89
123
4.72
0.13
18.4
25.6
Lactobacillus
|
jensenii
|
*297
50
5
2.78
0.02
17.9
1.6
Lactobacillus
|
buchneri
|
3417
38
2
2.16
0.04
17.8
1.2
Lactobacillus
|
reuteri
|
198
23
4
1.34
0.04
17.4
3.5
Lactobacillus
bulgaricus
|
delbrueckii
|
3420
53
5
3.14
0.24
17.0
0.3
Lactobacillus
|
reuteri
|
3473
54
53
3.11
0.36
16.4
15.0
Lactobacillus
|
helveticus
|
207
3
1
0.14
0.15
16.3
0.0
Lactobacillus
|
helveticus
|
*302
33
2
2.05
0.02
16.0
0.9
Lactobacillus
|
buchneri
|
3222
27
14
1.67
0.20
15.9
6.5
Lactobacillus
|
fermentum
|
2480
43
55
3.89
1.56
15.2
20.2
Lactobacillus
bulgaricus
|
brevis
|
196
41
3
2.73
0.09
15.0
0.6
Lactobacillus
bulgaricus
|
delbrueckii
|
3329
13
1
1.00
0.23
13.2
4.4
Lactobacillus
|
panis
|
695
45
16
3.55
0.09
12.8
4.7
Lactobacillus
|
crispatus
|
1457
44
6
3.90
0.73
11.4
0.7
Lactobacillus
|
crispatus
|
695
35
3
3.22
0.07
10.7
0.6
Lactobacillus
|
crispatus
|
30226
34
2
3.20
0.02
10.6
0.8
Lactobacillus
|
fermentum
|
1161
6
1
0.35
0.30
10.5
0.2
Lactobacillus
|
pentosus
|
216
31
5
2.94
0.40
10.4
0.4
Lactobacillus
|
helveticus
|
198
16
18
1.52
0.17
10.2
10.5
Lactobacillus
bulgaricus
|
delbrueckii
|
619
3
0
0.40
0.01
8.6
0.5
Lactobacillus
|
helveticus
|
*300
23
3
2.83
0.18
8.3
0.4
Lactobacillus
|
buchneri
|
212
26
0
3.46
0.46
7.7
1.0
Lactobacillus
|
helveticus
|
307
20
2
2.76
0.05
7.4
0.7
Lactobacillus
|
fermentum
|
1519
4
1
0.33
0.31
7.3
0.1
Lactobacillus
|
diolivorans
|
618
23
7
3.22
0.18
6.9
1.9
Lactobacillus
|
helveticus
|
3427
30
1
4.49
0.64
6.7
0.8
Lactobacillus
|
rhamnosus
|
285
2
1
0.25
0.18
6.5
1.0
Lactobacillus
|
pentosus
|
225
16
1
2.58
0.15
6.4
0.0
Lactobacillus
|
acidophilus
|
233
15
1
2.39
0.01
6.1
0.3
Lactobacillus
|
acidophilus
|
1518
3
1
0.35
0.29
5.9
0.1
Lactobacillus
|
diolivorans
|
206
15
3
2.66
0.49
5.8
0.1
Lactobacillus
|
helveticus
|
1307
3
0
0.28
0.31
5.8
0.0
Lactobacillus
|
buchneri
|
3191
20
19
3.46
0.31
5.7
5.1
Lactobacillus
|
crispatus
|
3098
14
0
2.60
0.15
5.3
0.3
Lactobacillus
|
acidophilus
|
223
12
0
2.31
0.14
5.2
0.2
Lactobacillus
|
acidophilus
|
3212
13
0
2.68
0.36
5.1
0.8
Lactobacillus
|
delbrueckii
|
199
8
1
1.77
0.16
4.4
0.9
Lactobacillus
bulgaricus
|
delbrueckii
|
267
10
0
2.77
0.30
3.7
0.5
Lactobacillus
|
acidophilus
|
294
10
1
2.91
0.01
3.5
0.2
Lactobacillus
|
fermentum
|
266
9
0
2.84
0.04
3.2
0.0
Lactobacillus
|
acidophilus
|
3118
3
1
0.93
0.03
3.0
0.7
Lactobacillus
|
amylolyticus
|
3119
3
0
1.10
0.08
2.9
0.5
Lactobacillus
|
amylolyticus
|
3114
4
0
1.23
0.15
2.9
0.3
Lactobacillus
|
amylolyticus
|
3115
4
1
1.35
0.03
2.8
0.6
Lactobacillus
|
amylolyticus
|
193
6
1
2.11
0.14
2.7
0.5
Lactobacillus
lactis
|
delbrueckii
|
3208
2
1
0.91
0.03
2.6
1.1
Lactobacillus
|
delbrueckii
|
241
3
0
1.13
0.01
2.2
0.2
Lactobacillus
|
casei
|
3116
4
1
1.72
0.01
2.2
0.6
Lactobacillus
|
amylolyticus
|
3234
7
0
3.27
0.25
2.1
0.2
Lactobacillus
|
fermentum
|
3117
2
1
1.00
0.00
2.0
0.7
Lactobacillus
|
amylolyticus
|
3122
3
1
1.81
0.03
1.9
0.6
Lactobacillus
|
amylolyticus
|
222
4
0
2.46
0.23
1.7
0.2
Lactobacillus
|
helveticus
|
871
3
1
1.82
0.09
1.7
0.3
Lactobacillus
|
crispatus
|
*3130
1
0
0.60
0.14
1.6
0.3
Lactobacillus
|
amylovorus
|
1356
5
0
3.03
0.05
1.5
0.1
Lactobacillus
|
graminus
|
3121
3
1
1.96
0.08
1.5
0.2
Lactobacillus
|
amylolyticus
|
C
4
1
2.58
0.12
1.4
0.5
Lactobacillus
|
3123
2
0
1.40
0.04
1.4
0.3
Lactobacillus
|
amylolyticus
|
3120
2
0
1.36
0.03
1.3
0.4
Lactobacillus
|
amylolyticus
|
3301
3
0
2.75
0.14
1.3
0.0
Lactobacillus
|
johnsonii
|
3299
3
1
2.47
0.11
1.1
0.5
Lactobacillus
|
johnsonii
|
*3245
1
0
0.61
0.26
1.1
0.1
Lactobacillus
|
gasseri
|
*3128
4
0
3.76
0.15
1.1
0.1
Lactobacillus
|
amylovorus
|
229
3
1
2.68
0.09
1.1
0.3
Lactobacillus
|
acidophilus
|
2828
3
1
3.06
0.19
1.0
0.3
Lactobacillus
|
plantarum
|
3114
2
1
2.53
1.62
1.0
0.9
Lactobacillus
|
amylolyticus
|
240
4
2
3.80
0.11
0.9
0.6
Lactobacillus
|
casei
|
3211
2
1
1.47
2.08
0.9
0.0
Lactobacillus
|
delbrueckii
|
3300
4
0
4.32
1.12
0.9
0.2
Lactobacillus
|
johnsonii
|
224
2
0
2.09
0.16
0.8
0.1
Lactobacillus
|
acidophilus
|
3431
1
0
1.01
0.07
0.8
0.0
Lactobacillus
|
rhamnosus
|
645
3
0
3.73
0.09
0.8
0.1
Lactobacillus
|
acidophilus
|
*3251
1
0
0.91
0.25
0.7
0.1
Lactobacillus
|
gasseri
|
265
4
3
5.10
0.00
0.7
0.5
Lactobacillus
|
crispatus
|
242
2
0
3.76
0.13
0.7
0.0
Lactobacillus
|
casei
|
3436
2
0
3.02
0.02
0.6
0.0
Lactobacillus
|
rhamnosus
|
2830
3
0
3.95
0.07
0.6
0.0
Lactobacillus
|
plantarum
|
1479
3
0
4.12
0.16
0.6
0.1
Lactobacillus
paracasei
|
paracasei
|
2691
1
0
2.51
0.30
0.5
0.1
Lactobacillus
|
plantarum
|
239
2
1
3.75
0.21
0.5
0.2
Lactobacillus
|
casei
|
645
2
1
4.04
0.17
0.5
0.3
Lactobacillus
|
acidophilus
|
880
2
1
3.67
0.13
0.5
0.2
Lactobacillus
|
salivarius
|
238
1
0
3.16
0.14
0.5
0.1
Lactobacillus
|
acidophilus
|
3350
2
1
3.52
0.21
0.4
0.2
Lactobacillus
|
paracasei
|
2523
2
1
3.57
0.21
0.4
0.3
Lactobacillus
|
helveticus
|
646
1
0
2.91
0.08
0.4
0.2
Lactobacillus
|
acidophilus
|
3440
1
0
3.34
0.92
0.4
0.2
Lactobacillus
|
rhamnosus
|
3429
1
0
3.70
0.05
0.4
0.0
Lactobacillus
|
rhamnosus
|
B
1
1
3.23
0.03
0.4
0.2
Lactobacillus
|
3428
2
0
4.34
0.37
0.4
0.1
Lactobacillus
|
rhamnosus
|
259
1
0
1.31
1.84
0.4
0.0
Lactobacillus
|
acidophilus
|
*870
1
0
2.40
0.01
0.4
0.2
Lactobacillus
|
amylovorus
|
3444
1
0
3.59
0.22
0.4
0.2
Lactobacillus
|
rhamnosus
|
3302
1
0
2.03
0.06
0.4
0.0
Lactobacillus
|
johnsonii
|
1353
1
0
2.89
0.02
0.4
0.1
Lactobacillus
paracasei
|
paracasei
|
101/37
2
1
5.15
0.04
0.3
0.2
L. paracasei
|
3445
1
0
3.79
0.37
0.3
0.1
Lactobacillus
|
rhamnosus
|
3443
2
0
4.65
0.30
0.3
0.0
Lactobacillus
|
rhamnosus
|
{circumflex over ( )}14D
2
0
4.88
0.33
0.3
0.0
L. plantarum
|
2937
1
0
4.50
0.08
0.3
0.0
Lactobacillus
|
rhamnosus
|
3439
1
0
3.86
0.15
0.3
0.1
Lactobacillus
|
rhamnosus
|
3434
1
0
4.39
0.26
0.3
0.1
Lactobacillus
|
rhamnosus
|
3426
1
0
4.67
0.53
0.3
0.1
Lactobacillus
|
rhamnosus
|
3303
1
0
2.42
0.08
0.3
0.1
Lactobacillus
|
johnsonii
|
*3246
1
0
2.46
0.05
0.3
0.1
Lactobacillus
|
gasseri
|
*1356
0
0
1.70
0.06
0.3
0.1
Lactobacillus
|
graminus
|
3438
1
0
4.13
0.06
0.3
0.0
Lactobacillus
|
rhamnosus
|
3442
1
0
5.01
0.34
0.3
0.1
Lactobacillus
|
rhamnosus
|
3430
1
0
4.65
0.05
0.3
0.0
Lactobacillus
|
rhamnosus
|
*3201
1
0
3.33
0.23
0.3
0.1
Lactobacillus
|
curvatus
|
3425
1
0
4.71
0.37
0.3
0.0
Lactobacillus
|
rhamnosus
|
1226
1
0
4.65
0.13
0.3
0.1
Lactobacillus
paracasei
|
paracasei
|
*3200
1
0
3.26
0.24
0.2
0.1
Lactobacillus
|
curvatus
|
3433
1
0
3.04
0.09
0.2
0.1
Lactobacillus
|
rhamnosus
|
3435
1
0
4.91
0.03
0.2
0.1
Lactobacillus
|
rhamnosus
|
E
1
1
4.39
0.30
0.2
0.2
Lactobacillus
|
2518
1
0
4.92
0.38
0.2
0.0
Lactobacillus
paracasei
|
paracasei
|
*3249
0
0
1.72
0.03
0.2
0.0
Lactobacillus
|
gasseri
|
636
1
0
4.34
0.05
0.2
0.0
Lactobacillus
|
salivarius
|
638
1
1
5.64
0.02
0.2
0.1
Lactobacillus
|
salivarius
|
3437
1
0
4.11
0.13
0.2
0.0
Lactobacillus
|
rhamnosus
|
A
1
1
5.08
0.12
0.2
0.1
Lactobacillus
|
*872
1
0
2.90
0.21
0.2
0.0
Lactobacillus
|
gasseri
|
*3196
1
0
3.32
0.07
0.2
0.0
Lactobacillus
|
curvatus
|
*1357
1
0
3.19
0.01
0.2
0.1
Lactobacillus
|
paralimentarius
|
3441
1
1
5.38
0.03
0.2
0.1
Lactobacillus
|
rhamnosus
|
*3202
1
0
3.25
0.12
0.2
0.1
Lactobacillus
|
curvatus
|
*3203
1
0
3.65
0.23
0.2
0.0
Lactobacillus
|
curvatus
|
3432
1
0
5.00
0.28
0.2
0.0
Lactobacillus
|
rhamnosus
|
*3195
1
0
3.75
0.07
0.2
0.0
Lactobacillus
|
curvatus
|
*1228
1
0
3.69
0.08
0.1
0.0
Lactobacillus
|
gasseri
|
3424
2
0
22.40
2.22
0.1
0.0
Lactobacillus
|
rhamnosus
|
1531
1
0
0.01
0.01
0.0
0.0
Lactobacillus
|
panis
|
*3129
1
0
−0.01
0.01
0.0
0.0
Lactobacillus
|
amylovorus
|
*3247
1
0
0.03
0.03
0.0
0.0
Lactobacillus
|
gasseri
|
*3248
0
0
0.07
0.02
0.0
0.0
Lactobacillus
|
gasseri
|
*3250
0
0
0.03
0.04
0.0
0.0
Lactobacillus
|
gasseri
|
*3252
1
0
0.65
0.93
0.0
0.0
Lactobacillus
|
gasseri
|
*3253
1
0
−0.01
0.01
0.0
0.0
Lactobacillus
|
gasseri
|
*3254
1
0
−0.01
0.00
0.0
0.0
Lactobacillus
|
gasseri
|
|
(Note: All strains were grown at 37° C. in anaerobic conditions, except those strains denoted “*” which were grown at 30° C. in aerobic conditions or “{circumflex over ( )}” which were grown at 37° C. in aerobic conditions).
|
Strains having β-galactosidase activity value of greater than 60 Miller Units were identified and put forward for further assessment for potential GOS synthesis and initial optimisation studies. Two S. thermophilus strains were selected, 4 lactobacilli (L. helveticus, L. reuters, L. delbrueckii, L. fermentum) with miller unit output above 60 and one Lactobacillus (L. plantarum 2830) with β-galactosidase activity bellow 60 miller units (for a control) were analysed. As none of the Propionibacterium screened gave β-galactosidase levels above 60, those with the highest β-galactosidase levels producers of each species was also included in the next phase of the study.
Analysis of GOS Production in the Chosen Strains
The following growth protocols were used for each species:
S. thermophilus—S. thermophilus strains were pre-grown from the −80° C. stock for 22 hours at 37° C. in 100 ml GM17 medium supplemented with 1% glucose in a closed 100 ml bottle. Cultures were then diluted 50, 200, 1000 and 4000-fold in a 1 litre bottle filled with GM17 medium supplemented with 1% glucose. Growth was performed at 37° 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.
Propionibacteria—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 calculated time that had been calculated to ensure a logarithmic culture and a stationary phase culture at the aimed time of harvesting.
Lactobacilli—Lactobacillus strains were pre-grown from the −80° C. stock for 48 hours at 37° C. in 100 ml MRS medium. Cultures were diluted 50, 200, 1000 and 4000-fold in a 1 litre bottle filled with MRS medium supplemented with 1% glucose. Growth was performed at 37° C. for a set calculated 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/I Na2HPO4.2H2O, 6.9 gram/I 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) at 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 4 below shows the predicted GOS formation rate at 30° C.
TABLE 4
|
|
Predicted
|
Bgal activity
GOS
|
(Miller Units)
formation
|
Average
Stdev
Used
rate
|
Strain
μmol/min/
conversion
Correction
mM/min/100
|
no.
Species
Subspecies
OD-Unit
factor
factor
OD units
|
|
883
Streptococcus
1960
83
26.5
5.0
20.0
|
thermophilus
|
885
Streptococcus
1462
103
26.5
5.0
14.9
|
thermophilus
|
882
Streptococcus
1347
135
26.5
5.0
13.7
|
thermophilus
|
886
Streptococcus
1291
238
26.5
5.0
13.2
|
thermophilus
|
884
Streptococcus
1267
86
26.5
5.0
12.9
|
thermophilus
|
114
Streptococcus
1155
126
26.5
5.0
11.8
|
thermophilus
|
121
Streptococcus
1140
75
26.5
5.0
11.6
|
thermophilus
|
194
Lactobacillus delbrueckii
bulgaricus
1019
59
4.1
5.0
1.6
|
2310
Streptococcus
976
68
26.5
5.0
10.0
|
thermophilus
|
106
Streptococcus
954
36
26.5
5.0
9.7
|
thermophilus
|
191
Lactobacillus delbrueckii
bulgaricus
937
62
4.1
5.0
1.5
|
2106
Streptococcus
815
196
26.5
5.0
8.3
|
thermophilus
|
203
Lactobacillus delbrueckii
bulgaricus
804
16
4.1
5.0
1.3
|
192
Lactobacillus delbrueckii
bulgaricus
620
320
4.1
5.0
1.0
|
195
Lactobacillus delbrueckii
bulgaricus
541
114
4.1
5.0
0.9
|
202
Lactobacillus delbrueckii
bulgaricus
540
76
4.1
5.0
0.9
|
187
Lactobacillus delbrueckii
bulgaricus
442
61
4.1
5.0
0.7
|
189
Lactobacillus delbrueckii
bulgaricus
413
52
4.1
5.0
0.7
|
211
Lactobacillus helveticus
398
51
4.1
5.0
0.6
|
1456
Lactobacillus crispatus
384
6
4.1
5.0
0.6
|
204
Lactobacillus delbrueckii
bulgaricus
315
20
4.1
5.0
0.5
|
186
Lactobacillus delbrueckii
bulgaricus
282
62
4.1
5.0
0.4
|
3273
Lactobacillus helveticus
255
14
4.1
5.0
0.4
|
1795
Lactobacillus delbrueckii
213
58
4.1
5.0
0.3
|
190
Lactobacillus delbrueckii
bulgaricus
150
4.1
5.0
0.2
|
1572
Lactobacillus fermentum
139
4.1
5.0
0.2
|
2954
Lactobacillus reuteri
111
0
4.1
5.0
0.2
|
3416
Lactobacillus reuteri
105
5
4.1
5.0
0.2
|
226
Lactobacillus
101
7
4.1
5.0
0.2
|
acidophilus
|
3909
Lactobacillus reuteri
94.0
1.2
4.1
5.0
0.1
|
LR92
L. reuteri
90.9
6.3
4.1
5.0
0.1
|
2955
Lactobacillus reuteri
89.7
1.9
4.1
5.0
0.1
|
LR1
L. rhamsnosus
84.5
14.5
4.1
5.0
0.1
|
LA1
L. acidophilus
72.3
6.2
4.1
5.0
0.1
|
11796
Lactobacillus fermentum
69.2
8.1
4.1
5.0
0.1
|
D
Lactobacillus
41.5
1.1
4.1
5.0
0.1
|
30226
Lactobacillus fermentum
10.6
0.8
4.1
5.0
0.017
|
C
Lactobacillus
1.4
0.5
4.1
5.0
0.002
|
2828
Lactobacillus plantarum
1.0
0.3
4.1
5.0
0.002
|
2830
Lactobacillus plantarum
0.6
0.0
4.1
5.0
0.001
|
2691
Lactobacillus plantarum
0.5
0.1
4.1
5.0
0.001
|
B
Lactobacillus
0.4
0.2
4.1
5.0
0.001
|
101/37
L. paracasei
0.3
0.2
4.1
5.0
0.001
|
14D
L. plantarum
0.3
0.0
4.1
5.0
0.000
|
E
Lactobacillus
0.2
0.2
4.1
5.0
0.000
|
A
Lactobacillus
0.2
0.1
4.1
5.0
0.000
|
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 5 below shows the predicted GOS formation rate at 50° C.
TABLE 5
|
|
Predicted
|
Bgal activity
GOS
|
(Miller Units)
formation
|
Average
Stdev
Used
rate
|
Strain
μmol/min/
conversion
Correction
mM/min/100
|
no.
Species
Subspecies
OD-Unit
factor
factor
OD units
|
|
883
Streptococcus
1960
83
26.5
5.0
20.0
|
thermophilus
|
885
Streptococcus
1462
103
26.5
5.0
14.9
|
thermophilus
|
882
Streptococcus
1347
135
26.5
5.0
13.7
|
thermophilus
|
886
Streptococcus
1291
238
26.5
5.0
13.2
|
thermophilus
|
884
Streptococcus
1267
86
26.5
5.0
12.9
|
thermophilus
|
114
Streptococcus
1155
126
26.5
5.0
11.8
|
thermophilus
|
121
Streptococcus
1140
75
26.5
5.0
11.6
|
thermophilus
|
194
Lactobacillus delbrueckii
bulgaricus
1019
59
4.1
5.0
1.6
|
2310
Streptococcus
976
68
26.5
5.0
10.0
|
thermophilus
|
106
Streptococcus
954
36
26.5
5.0
9.7
|
thermophilus
|
191
Lactobacillus delbrueckii
bulgaricus
937
62
4.1
5.0
1.5
|
2106
Streptococcus
815
196
26.5
5.0
8.3
|
thermophilus
|
203
Lactobacillus delbrueckii
bulgaricus
804
16
4.1
5.0
1.3
|
192
Lactobacillus delbrueckii
bulgaricus
620
320
4.1
5.0
1.0
|
195
Lactobacillus delbrueckii
bulgaricus
541
114
4.1
5.0
0.9
|
202
Lactobacillus delbrueckii
bulgaricus
540
76
4.1
5.0
0.9
|
187
Lactobacillus delbrueckii
bulgaricus
442
61
4.1
5.0
0.7
|
189
Lactobacillus delbrueckii
bulgaricus
413
52
4.1
5.0
0.7
|
211
Lactobacillus helveticus
398
51
4.1
5.0
0.6
|
1456
Lactobacillus crispatus
384
6
4.1
5.0
0.6
|
204
Lactobacillus delbrueckii
bulgaricus
315
20
4.1
5.0
0.5
|
186
Lactobacillus delbrueckii
bulgaricus
282
62
4.1
5.0
0.4
|
3273
Lactobacillus helveticus
255
14
4.1
5.0
0.4
|
1795
Lactobacillus delbrueckii
213
58
4.1
5.0
0.3
|
190
Lactobacillus delbrueckii
bulgaricus
150
4.1
5.0
0.2
|
1572
Lactobacillus fermentum
139
4.1
5.0
0.2
|
2954
Lactobacillus reuteri
111
0
4.1
5.0
0.2
|
3416
Lactobacillus reuteri
105
5
4.1
5.0
0.2
|
226
Lactobacillus
101
7
4.1
5.0
0.2
|
acidophilus
|
3909
Lactobacillus reuteri
94.0
1.2
4.1
5.0
0.1
|
LR92
L. reuteri
90.9
6.3
4.1
5.0
0.1
|
2955
Lactobacillus reuteri
89.7
1.9
4.1
5.0
0.1
|
LR1
L. rhamsnosus
84.5
14.5
4.1
5.0
0.1
|
LA1
L. acidophilus
72.3
6.2
4.1
5.0
0.1
|
11796
Lactobacillus fermentum
69.2
8.1
4.1
5.0
0.1
|
D
Lactobacillus
41.5
1.1
4.1
5.0
0.1
|
30226
Lactobacillus fermentum
10.6
0.8
4.1
5.0
0.017
|
C
Lactobacillus
1.4
0.5
4.1
5.0
0.002
|
2828
Lactobacillus plantarum
1.0
0.3
4.1
5.0
0.002
|
2830
Lactobacillus plantarum
0.6
0.0
4.1
5.0
0.001
|
2691
Lactobacillus plantarum
0.5
0.1
4.1
5.0
0.001
|
B
Lactobacillus
0.4
0.2
4.1
5.0
0.001
|
101/37
L. paracasei
0.3
0.2
4.1
5.0
0.001
|
14D
L. plantarum
0.3
0.0
4.1
5.0
0.000
|
E
Lactobacillus
0.2
0.2
4.1
5.0
0.000
|
A
Lactobacillus
0.2
0.1
4.1
5.0
0.000
|
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+100 mMNaOH, (B) 100 mMNaOH 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 6 below and illustrated in FIG. 1, it was found that the species ratio showed a strong temperature dependence and a small time dependence.
TABLE 6
|
|
Expected GOS
Expected GOS
β-D-Gal-
|
linkage type
linkage type
(1f4)-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 7 below and illustrated in FIG. 2, the two Lactobacillus strains showed a 3-4 fold in GOS formation rate at 50° C., for both Propionibacterium strains no significant increase in activity was detected.
TABLE 7
|
|
Strain
Temp
Total GOS
|
|
|
L. reuteri
30° C.
3.7
|
50° C.
13.9
|
L. fermentum
30° C.
1.4
|
50° C.
4.1
|
P. jensenii
30° C.
4.5
|
50° C.
4.1
|
P. freudenreichii
30° C.
5.3
|
50° C.
6.1
|
|
The theoretical GOS formation rate was calculated based on the β-galactosidase activity, expressed in Miller Units, measured in Phase 2 of the study. Table 8 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 8
|
|
Actual/Theoretical
Actual/Theoretical
|
Strain
No
GOS (30 C.)
GOS (50 C.)
Ratio
|
|
|
S.
thermophilus
883
28.3
|
S. thermophilus
883
11.3
|
S. thermophilus
114
39.9
|
L. helveticus
211
1.4
|
L. delbrueckii
191
3.1
|
L. reuteri
2954
1.5
5.5
3.8
|
L. fermentum
11796
0.9
2.7
2.9
|
P. jensenii
364
13.6
12.5
0.9
|
P. freudenreichii
1134
7.9
9.1
1.2
|
Average
26.5
|
streptococci
|
Average lactobacilli
1.7
4.1
2.4
|
Average propioni's
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 9 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 9
|
|
Strain no
Growth Phase
Ratio Phase 1/Phase 2
|
|
|
883
Mid-log
6.7
|
883
Stationary
4.5
|
114
Mid-log
27.7
|
114
Stationary
16.0
|
211
Mid-log
1.8
|
211
Stationary
Very High
|
191
Mid-log
9.1
|
191
Stationary
Very High
|
2954
Mid-log
5.1
|
2954
Mid-log
5.1
|
2954
Stationary
6.2
|
11796
Mid-log
3.8
|
11796
Mid-log
3.8
|
11796
Stationary
21.4
|
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 (as measured in Miller Units) was plotted for all strains. Table 10 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. With a few exceptions (strain 211 and 191) these differences are limited and it may be that the higher biomass yield in stationary phase off-sets the lower β-galactosidase activities.
TABLE 10
|
|
Ratio Log/Stat
|
|
|
Streptococcus thermophilus
883
0.7
|
Streptococcus thermophilus
114
0.6
|
Lactobacillus helveticus
211
Very high
|
Lactobacillus delbrueckii
191
12.5
|
Lactobacillus reuteri
2954
1.2
|
Lactobacillus fermentum
11796
5.6
|
Propionibacterium jensenii
364
1.8
|
Propionibacterium freudenreichii
1134
3.4
|
Propionibacterium acidipropionici
4204
4.9
|
|
CONCLUSIONS
GOS formation rates for the strains selected on the basis of the Miller Unit value were deemed as a good predictor for those strains showing good GOS production even when grown in non-optimised conditions. It was established that all Lactobacillus strains that gave β-galactosidase activity above 60 Miller Units in phase 1 produced GOS in the phase 2 feasibility study, whereas the one control strain that was below the 60 Miller Unit cut-off did not. Most of S. thermophilus showed Bgal activities significantly higher than 60 miller units and only those which appeared to be the best were selected for taking further to the phase 2 feasibility study. For Propionibacterium all were below the 60 miller unit cut off, but all strains selected produced GOS.
In general GOS formation rates were 3-4 fold higher at 50° C. as compared to 30° C. for the lactobacilli strains. The Propionibacterium 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. The studies established that Miller Unit activities translated well to potential GOS activity and proved to be a useful and accurate predictor of GOS production. For specific cases GOS production was up to 15 fold higher than ONPG hydrolysis activity had initially suggested. In general, the later GOS synthesis phase showed a 5-fold lower β-galactosidase activities as compared to the initial screening phase.
Using the described screening method, and Miller Unit cut-off of 60 (for Streptococcus or Lactobacillus) and 3 (for Propionibacterium), allows for a quick and reliable prediction of the likelihood of whether a bacteria can produce GOS in sufficient yields so as to allow purification and further testing of its prebiotic properties in vitro. It can also be used to help identify any potentially novel GOS structures. It advantageously provides a systematic methodology which permits screening of large numbers of bacteria for the potential to produce GOS, identify novel GOS, and scale up to test in in vitro models. Furthermore, as the Miller Unit is a composite of the growth rate, enzyme production and activity, then this parameter enables focus on only those strains which are most likely to be commercially viable.
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.