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Suitability of Lactobacillus strains as components of probiotics
Food Biotechnology S. Bielecki, J. Tramper and J. Polak (Editors) 9 2000 Elsevier Science B.V. All rights reserved.
257
Suitability of Lactobacillus strains as components of probiotics J. Moneta and Z. Libudzisz Institute of Fermentation Technology and Microbiology, Technical University of L6d~, Poland, Stefanowskiego 4/10, 90-924 Lrd~, Poland.
The production of organic acids, acetaldehyde, 13-galactosidase, proteolytic activity, carbohydrate metabolism and resistance to low pH of 2.5 as well as bile of 4% salt concentration were used as selection criteria for the assessment of 19 strains of Lactobacillus acidophilus, with respect to their probiotic properties. Their activity was compared with the activity of reference strains. Most of the strains revealed the tested features at the same or higher level as the reference strains.
1. INTRODUCTION In recent years there has been an increasing interest in consumption of natural food, especially fermented dairy products containing specific strains of lactic acid bacteria known for their health-promoting effects. There are, however, only a few strains of clearly defined probiotic features. In addition these specific properties usually do not exist together in the same strain. Thus, the level of probiotic features must be checked carefully. It is necessary, when composing the starters, to include the cultures that are complementary with regards to their probiotic features.
2. MATERIALS AND METHODS 2.1. Microorganisms The aim of the study was to determine the range of differences in physiological properties of 19 strains of Lactobacillus acidophilus and of a strain Lactobacillus delbrueckii ssp. lactis L30. We used cultures from various collections, i.e. from the collection of pure cultures of Biolacta-Texel in Olsztyn, Poland: -336 (2), In3 (3), lndl (4), Ros (5), 172 (6), Czl (7), 20T1 (8), 343 (9), HI (10), V/74 (11) & CH-2 (12) strains; from the collection of the Institute of Chemical Technology in Prague, Czech Republic: - CH-5 (13), Nestle (14), Bauer (15), Diat (16), A92 (17) & L30 (1) strains; from Institute of Fermentation Technology & Microbiology, Technical University ofLrd~: - B (18) strain; from the National Collection of Agricultural and Industrial Microorganisms in Hungary - NCAIMB 1075 (19) & NCAIMB 1152 (20) strains. Moreover, 8 reference strains from the collection of Collegium Medicum, Jagiellonian University in Cracow, i.e. Lb. acidophilus ATCC 4356 (A), Lb. thermophilus 094 11.78-
258 NCDO 489 (B), Lb. casei-Sb.irota (C), Lb. acidophilus LC1 (D), Lb. rhamnosus GG ATCC 53105 (E), Lb. crispatus NCFB 2752 (F), Lb. casei-NCDO 206 (G) & Lb. gasseri (H) were tested. The final goal was to select a strain with probiotic activity higher or comparable to the activity of the reference strains. 2.2. Analytical Methods As a selection criteria the following features were examined: growth, lactic acid production and the level of the L (+) lactic acid in milk after 24 h incubation, production of acetic acid in milk after 24 h incubation, 13-galactosidase, tolerance to low pH (2.5), survival in the presence of 4% bile salt concentration, proteolytic activity, biochemical characteristic (carbohydrate fermentation) and acetaldehyde production (a compound responsible for desirable flavour of acidophilus milks). 2.2.1. Number of bacteria was estimated using MRS agar pour plates incubated at 37~ for 48 h, under anaerobic conditions. 2.2.2. Production of acid was measured by titration with 0.1 N NaOH. Results were expressed as grams of lactic acid per 100 ml of milk. L-isomer and acetic acid were determined enzymatically (Boehringer Mannheim, Germany) 2.2.3. 13-galactosidase activity of bacteria was estimated using o-nitrophenyl-Dgalactopyranoside (ONPG) as substrate. Bacteria were incubated in MRS medium at 37~ for 20 h and then cells of each strain were treated with SDS (sodium dodecyl sulfate)-chloroform [1,2]. The activity of 13-galactosidase was expressed as Iamoles of o-nitrophenol (ONP) liberated from ONPG per miligram of cell dry weight per minute. 2.2.4. Proteolytic activity of bacteria in skim milk incubated at 37~ for 48 h was assayed using Anson's method with Folin-Ciocalteu's reagent. Results were expressed as an amount of tyrosine (mg) released by bacteria per ml of trichloroacetic acid filtrate 2.2.5. Carbohydrate metabolism was checked on the basis of API 50 CHL test system (BioMerieux). 2.2.6. Acetaldehyde was assayed by method of Lindsay and Day [3,4] and enzymatically using the test ofBoehringer Mannheim. 2.2.7. Tolerance to low pH 2.5 and survival in the presence of 4% bile salt concentration. The cells, cultured in MRS medium were collected by centrifugation and suspended in sodium chloride solution (0.85 g/100 ml) to obtain an initial count of 108 CFU/ml. The solution was either adjusted to pH 2.5 by addition of sterile HC1 or suspended in a bile salt concentration of 4% w/v. All samples were incubated at 37~ for 8 h or 74 h, respectively. The viable cell counts of each strain were enumerated at different intervals using MRS agar.
3. RESULTS We found that the tested Lactobacillus strains showed different acidifying activity, accumulating from 0.6% (CH-5, Nestle strains) to 2.3% lactic acid (A92 strain) in milk, after 24 h of cultivation at 37~ (Fig. 1). In the same conditions the reference strains produced from 0.68% lactic acid (NCFB 2752 strain) to 1.1% (ATCC 4356 strain) (Fig. 2). The percent of L(+) lactic acid in total lactic acid ranged for tested strains from 19% (A92 strain) to 89% (NCAIMB 1152 strain) and for most of them (85%) was from 34% to 66%. In the case of
259
reference strains the percent of L(+) isomer ranged from 26% (NCFB 2752 strain) to 63-65% (LC1 and 094 11.78-NCDO 489 strains) (Fig.3).
a)
,
2.5
.
---O--1 --I--2 -* 31
"-" ~ 2-0~-~ I~ O1"5
X ~
<
r,.) 1.0 ~_ (9 0.5
4 5
~ 6 t
7
= 8 ------'9
0.0
----0-- 10i 0
3
6
9
12
15
18
21
24
TIME OF INCUBATION (h)
b)
~I
2.s o~~ 2.0 1.5
X
0.0
I
0
3
~
6
9
12
.........
15
18
~
21
1 4
71
24
TIME OF INCUBATION (h)
Figure 1. Acidifying activity of Lactobacillus strains; a)-strains 1-10, b)-strains 11-20
2.5
~Al
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a~' 2.0
--I-- B I
El
---Jr--C I ---I-- D 1
W O 1.5 < 0
1.0
.
~.....T........-.~.
O 5 0.5
+ E J
]
0.0 0
3
6
9
12
TIME
OF INCUBATION
15 (h)
Figure 2. Acidifying activity of Lactobacillus reference strains.
18
,
!
21
24
---'t--- G I "-"--'~ '
260
Figure 3. Production of L (+) lactic acid by Lactobacillus strains after 24 h incubation. After 24 hours of incubation in milk the tested strains produced from 0.4 g/1 (H-l, NCAIMB 1152 strains) to 1.4 g/1 (B, L30 strains) of acetic acid. 50% of strains produced this metabolite in the amount above 1.2 g/1. The reference strains produced acetic acid at the same level, accumulating from 0.44 g/l (Shirota strain) to 1.38 g/1 (ATCC4356 strain) (Fig. 4).
Figure 4. Production of acetic acid by Lactobacillus strains after 24 h incubation. The activity of 13-galactosidase of the tested bacteria ranged from 0.031 U/mg d.w. (NCAIMB 1075 strain) to 0.605 U/mg d.w. (CH-2 strain). The strains of similar activities in the range from 0.300 to 0.456 U/mg d.w. constituted the biggest group (50%). In contrast, the reference strains revealed weaker 13-galactosidase activity, i.e. from 0.136 U/mg d.w. (NCFB 2752 strain) to 0.0217 U/mg d.w. (094 11.78-NCDO 489 strain) (Fig. 5).
261
Figure 5.13-galactosidase activity of Lactobacillus strains. There were significant differences in bacterial viability at pH 2.5. After 8 hours of incubation the observed decrease in the number of living cells ranged from 1.2x 10S-l.1 x 10 9 CFU/ml to 3.5 - 2.6x 106 CFU/ml, depending on the strain. Twenty percent of strains revealed viability from 74% to 77%, while half of them viability from 36% to 66%. At the same conditions the reference strains survived at the level comparable with most tested bacteria (34-71%) (Fig. 6).
Figure 6. Survival of Lactobacillus strains at pH 2.5 after 8 h. Additionally, the tested bacteria showed various sensitivity to 4% of bile salt present in the medium. After 74 hours of incubation 5% of strains (B strain) survived at the level 86%, 75% of strains expressed viability from 35% to 66% and 20% of strains from 27% to 32%. Reference strains also showed various sensitivity to 4% bile salt and their viability ranged from 28% (NCFB2752 strain) to 67% (094 11.78-NCDO 489 strain) (Fig.7).
262
Figure 7. Survival of Lactobacillus strains in the presence of 4% bile salts alter 74 h. Significant differences in the proteolytic activity of tested strains and dynamics of protein degradation specific for a particular strain were observed. An increase in tyrosine level among the tested strains after 24 h of incubation in milk ranged from about 17 (NCAIMB 1152 strain) to 135 (172 strain) mg tyr./dm 3 Reference strains showed weaker proteolytic activity. Prolongation of the incubation time to 48 h caused a further increase in protein digestion for tested strains from 38 (NCAIMB 1152 strain) to 190 mg tyr./dm 3 (lndl strain). In the same conditions the reference strains showed an increase in tyrosine level from 26 (GG ATCC 53105 strain)to 81 mg tyr./dm 3 (ATCC 4356 strain)(Fig. 8).
Figure 8. Proteolytic activity of Lactobacillus strains (increase in tyrosine level). On the basis of API 50 CHL tests it was found that all examined strains were able to ferment glucose, galactose, fructose, mannose, maltose, lactose, saccharose and trehalose. The ability to metabolise other sugars was a feature of individual strains. Additionally, different diagnostic matching with the species pattern in the API system and according to Bergey (1986) was reported. The amount of acetaldehyde formed in milk after 24 hours of incubation ranged from 0.8 mg/1 (Bauer strain) to 16 mg/l (In3 strain) (Fig.9).
263
Figure 9. Production of acetaldehyde by Lactobacillus strains. The tested bacteria expressed a various ability to adhere to a specified line of intestinal epithelium cells. The strongest adherence was showed by the strains of Lb. acidophilus 172, Lb. acidophilus 336, Lb. acidophilus Nestle, Lb. delbrueckii ssp. lactis L30, Lb. acidophilus A92. (studies carried out by P. Heczko, E. Marewicz, G. Kukla, Institute of Microbiology, Collegium Medicum Jagiellonian University, Cracow) (not published data).
4. CONCLUSIONS Significant heterogeneity of the tested strains, with respect to their probiotic properties was found as a result of these investigations. Most strains were found to be able to form the tested metabolites on the same or higher level as compared to the reference strains. The most suitable appeared to be the cultures of Lb. acidophilus B, Lb. acidophilus lndl, Lb. acidophilus 172, Lb. acidophilus Ros, Lb. acidophilus 20T1, Lb. acidophilus Czl, Lb. acidophilus CH-2, Lb. acidophilus CH-5, Lb. acidophilus A92, Lb. acidophilus Diat, Lb. delbruecki ssp. lactis L30, Lb. acidophilus NCAIMB 1152. These strains can be used in the production of probiotics or dairy products.
REFERENCES
1. 2. 3. 4.
J.F. Citti, W.E. Sandine, P.R. Elliker, J. Bacteriol., 4 (1965) 937. N. Shah, P. Jelen, J. Food Sci., 55 (1990) 506. R.C. Lindsay, E.A. Day, J. Dairy Sci., 48 (1965) 665. T.M. Cogan, J. Dairy Sci., 55 (1972) 382.
257
Suitability of Lactobacillus strains as components of probiotics J. Moneta and Z. Libudzisz Institute of Fermentation Technology and Microbiology, Technical University of L6d~, Poland, Stefanowskiego 4/10, 90-924 Lrd~, Poland.
The production of organic acids, acetaldehyde, 13-galactosidase, proteolytic activity, carbohydrate metabolism and resistance to low pH of 2.5 as well as bile of 4% salt concentration were used as selection criteria for the assessment of 19 strains of Lactobacillus acidophilus, with respect to their probiotic properties. Their activity was compared with the activity of reference strains. Most of the strains revealed the tested features at the same or higher level as the reference strains.
1. INTRODUCTION In recent years there has been an increasing interest in consumption of natural food, especially fermented dairy products containing specific strains of lactic acid bacteria known for their health-promoting effects. There are, however, only a few strains of clearly defined probiotic features. In addition these specific properties usually do not exist together in the same strain. Thus, the level of probiotic features must be checked carefully. It is necessary, when composing the starters, to include the cultures that are complementary with regards to their probiotic features.
2. MATERIALS AND METHODS 2.1. Microorganisms The aim of the study was to determine the range of differences in physiological properties of 19 strains of Lactobacillus acidophilus and of a strain Lactobacillus delbrueckii ssp. lactis L30. We used cultures from various collections, i.e. from the collection of pure cultures of Biolacta-Texel in Olsztyn, Poland: -336 (2), In3 (3), lndl (4), Ros (5), 172 (6), Czl (7), 20T1 (8), 343 (9), HI (10), V/74 (11) & CH-2 (12) strains; from the collection of the Institute of Chemical Technology in Prague, Czech Republic: - CH-5 (13), Nestle (14), Bauer (15), Diat (16), A92 (17) & L30 (1) strains; from Institute of Fermentation Technology & Microbiology, Technical University ofLrd~: - B (18) strain; from the National Collection of Agricultural and Industrial Microorganisms in Hungary - NCAIMB 1075 (19) & NCAIMB 1152 (20) strains. Moreover, 8 reference strains from the collection of Collegium Medicum, Jagiellonian University in Cracow, i.e. Lb. acidophilus ATCC 4356 (A), Lb. thermophilus 094 11.78-
258 NCDO 489 (B), Lb. casei-Sb.irota (C), Lb. acidophilus LC1 (D), Lb. rhamnosus GG ATCC 53105 (E), Lb. crispatus NCFB 2752 (F), Lb. casei-NCDO 206 (G) & Lb. gasseri (H) were tested. The final goal was to select a strain with probiotic activity higher or comparable to the activity of the reference strains. 2.2. Analytical Methods As a selection criteria the following features were examined: growth, lactic acid production and the level of the L (+) lactic acid in milk after 24 h incubation, production of acetic acid in milk after 24 h incubation, 13-galactosidase, tolerance to low pH (2.5), survival in the presence of 4% bile salt concentration, proteolytic activity, biochemical characteristic (carbohydrate fermentation) and acetaldehyde production (a compound responsible for desirable flavour of acidophilus milks). 2.2.1. Number of bacteria was estimated using MRS agar pour plates incubated at 37~ for 48 h, under anaerobic conditions. 2.2.2. Production of acid was measured by titration with 0.1 N NaOH. Results were expressed as grams of lactic acid per 100 ml of milk. L-isomer and acetic acid were determined enzymatically (Boehringer Mannheim, Germany) 2.2.3. 13-galactosidase activity of bacteria was estimated using o-nitrophenyl-Dgalactopyranoside (ONPG) as substrate. Bacteria were incubated in MRS medium at 37~ for 20 h and then cells of each strain were treated with SDS (sodium dodecyl sulfate)-chloroform [1,2]. The activity of 13-galactosidase was expressed as Iamoles of o-nitrophenol (ONP) liberated from ONPG per miligram of cell dry weight per minute. 2.2.4. Proteolytic activity of bacteria in skim milk incubated at 37~ for 48 h was assayed using Anson's method with Folin-Ciocalteu's reagent. Results were expressed as an amount of tyrosine (mg) released by bacteria per ml of trichloroacetic acid filtrate 2.2.5. Carbohydrate metabolism was checked on the basis of API 50 CHL test system (BioMerieux). 2.2.6. Acetaldehyde was assayed by method of Lindsay and Day [3,4] and enzymatically using the test ofBoehringer Mannheim. 2.2.7. Tolerance to low pH 2.5 and survival in the presence of 4% bile salt concentration. The cells, cultured in MRS medium were collected by centrifugation and suspended in sodium chloride solution (0.85 g/100 ml) to obtain an initial count of 108 CFU/ml. The solution was either adjusted to pH 2.5 by addition of sterile HC1 or suspended in a bile salt concentration of 4% w/v. All samples were incubated at 37~ for 8 h or 74 h, respectively. The viable cell counts of each strain were enumerated at different intervals using MRS agar.
3. RESULTS We found that the tested Lactobacillus strains showed different acidifying activity, accumulating from 0.6% (CH-5, Nestle strains) to 2.3% lactic acid (A92 strain) in milk, after 24 h of cultivation at 37~ (Fig. 1). In the same conditions the reference strains produced from 0.68% lactic acid (NCFB 2752 strain) to 1.1% (ATCC 4356 strain) (Fig. 2). The percent of L(+) lactic acid in total lactic acid ranged for tested strains from 19% (A92 strain) to 89% (NCAIMB 1152 strain) and for most of them (85%) was from 34% to 66%. In the case of
259
reference strains the percent of L(+) isomer ranged from 26% (NCFB 2752 strain) to 63-65% (LC1 and 094 11.78-NCDO 489 strains) (Fig.3).
a)
,
2.5
.
---O--1 --I--2 -* 31
"-" ~ 2-0~-~ I~ O1"5
X ~
<
r,.) 1.0 ~_ (9 0.5
4 5
~ 6 t
7
= 8 ------'9
0.0
----0-- 10i 0
3
6
9
12
15
18
21
24
TIME OF INCUBATION (h)
b)
~I
2.s o~~ 2.0 1.5
X
0.0
I
0
3
~
6
9
12
.........
15
18
~
21
1 4
71
24
TIME OF INCUBATION (h)
Figure 1. Acidifying activity of Lactobacillus strains; a)-strains 1-10, b)-strains 11-20
2.5
~Al
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a~' 2.0
--I-- B I
El
---Jr--C I ---I-- D 1
W O 1.5 < 0
1.0
.
~.....T........-.~.
O 5 0.5
+ E J
]
0.0 0
3
6
9
12
TIME
OF INCUBATION
15 (h)
Figure 2. Acidifying activity of Lactobacillus reference strains.
18
,
!
21
24
---'t--- G I "-"--'~ '
260
Figure 3. Production of L (+) lactic acid by Lactobacillus strains after 24 h incubation. After 24 hours of incubation in milk the tested strains produced from 0.4 g/1 (H-l, NCAIMB 1152 strains) to 1.4 g/1 (B, L30 strains) of acetic acid. 50% of strains produced this metabolite in the amount above 1.2 g/1. The reference strains produced acetic acid at the same level, accumulating from 0.44 g/l (Shirota strain) to 1.38 g/1 (ATCC4356 strain) (Fig. 4).
Figure 4. Production of acetic acid by Lactobacillus strains after 24 h incubation. The activity of 13-galactosidase of the tested bacteria ranged from 0.031 U/mg d.w. (NCAIMB 1075 strain) to 0.605 U/mg d.w. (CH-2 strain). The strains of similar activities in the range from 0.300 to 0.456 U/mg d.w. constituted the biggest group (50%). In contrast, the reference strains revealed weaker 13-galactosidase activity, i.e. from 0.136 U/mg d.w. (NCFB 2752 strain) to 0.0217 U/mg d.w. (094 11.78-NCDO 489 strain) (Fig. 5).
261
Figure 5.13-galactosidase activity of Lactobacillus strains. There were significant differences in bacterial viability at pH 2.5. After 8 hours of incubation the observed decrease in the number of living cells ranged from 1.2x 10S-l.1 x 10 9 CFU/ml to 3.5 - 2.6x 106 CFU/ml, depending on the strain. Twenty percent of strains revealed viability from 74% to 77%, while half of them viability from 36% to 66%. At the same conditions the reference strains survived at the level comparable with most tested bacteria (34-71%) (Fig. 6).
Figure 6. Survival of Lactobacillus strains at pH 2.5 after 8 h. Additionally, the tested bacteria showed various sensitivity to 4% of bile salt present in the medium. After 74 hours of incubation 5% of strains (B strain) survived at the level 86%, 75% of strains expressed viability from 35% to 66% and 20% of strains from 27% to 32%. Reference strains also showed various sensitivity to 4% bile salt and their viability ranged from 28% (NCFB2752 strain) to 67% (094 11.78-NCDO 489 strain) (Fig.7).
262
Figure 7. Survival of Lactobacillus strains in the presence of 4% bile salts alter 74 h. Significant differences in the proteolytic activity of tested strains and dynamics of protein degradation specific for a particular strain were observed. An increase in tyrosine level among the tested strains after 24 h of incubation in milk ranged from about 17 (NCAIMB 1152 strain) to 135 (172 strain) mg tyr./dm 3 Reference strains showed weaker proteolytic activity. Prolongation of the incubation time to 48 h caused a further increase in protein digestion for tested strains from 38 (NCAIMB 1152 strain) to 190 mg tyr./dm 3 (lndl strain). In the same conditions the reference strains showed an increase in tyrosine level from 26 (GG ATCC 53105 strain)to 81 mg tyr./dm 3 (ATCC 4356 strain)(Fig. 8).
Figure 8. Proteolytic activity of Lactobacillus strains (increase in tyrosine level). On the basis of API 50 CHL tests it was found that all examined strains were able to ferment glucose, galactose, fructose, mannose, maltose, lactose, saccharose and trehalose. The ability to metabolise other sugars was a feature of individual strains. Additionally, different diagnostic matching with the species pattern in the API system and according to Bergey (1986) was reported. The amount of acetaldehyde formed in milk after 24 hours of incubation ranged from 0.8 mg/1 (Bauer strain) to 16 mg/l (In3 strain) (Fig.9).
263
Figure 9. Production of acetaldehyde by Lactobacillus strains. The tested bacteria expressed a various ability to adhere to a specified line of intestinal epithelium cells. The strongest adherence was showed by the strains of Lb. acidophilus 172, Lb. acidophilus 336, Lb. acidophilus Nestle, Lb. delbrueckii ssp. lactis L30, Lb. acidophilus A92. (studies carried out by P. Heczko, E. Marewicz, G. Kukla, Institute of Microbiology, Collegium Medicum Jagiellonian University, Cracow) (not published data).
4. CONCLUSIONS Significant heterogeneity of the tested strains, with respect to their probiotic properties was found as a result of these investigations. Most strains were found to be able to form the tested metabolites on the same or higher level as compared to the reference strains. The most suitable appeared to be the cultures of Lb. acidophilus B, Lb. acidophilus lndl, Lb. acidophilus 172, Lb. acidophilus Ros, Lb. acidophilus 20T1, Lb. acidophilus Czl, Lb. acidophilus CH-2, Lb. acidophilus CH-5, Lb. acidophilus A92, Lb. acidophilus Diat, Lb. delbruecki ssp. lactis L30, Lb. acidophilus NCAIMB 1152. These strains can be used in the production of probiotics or dairy products.
REFERENCES
1. 2. 3. 4.
J.F. Citti, W.E. Sandine, P.R. Elliker, J. Bacteriol., 4 (1965) 937. N. Shah, P. Jelen, J. Food Sci., 55 (1990) 506. R.C. Lindsay, E.A. Day, J. Dairy Sci., 48 (1965) 665. T.M. Cogan, J. Dairy Sci., 55 (1972) 382.