Pediococcus urinaeequi IFO1217. Tetragenococcus halophila IAM. 100. 100 ... Lactobacillus johnsonii ATCC33. Lactobacillus delbrueckii subs. Lactobacillus ...
SCREENING AND CHARACTERIZATION OF BENEFICIAL PROBIOTICS FERMENTING GERMINATED GRAINS SULHEE LEE, HEESOO CHO, SO HYUN KIM, AND YOUNG-SEO PARK DEPARTMENT OF FOOD SCIENCE AND BIOTECHNOLOGY, GACHON UNIVERSITY
INTRODUCTION
ABSTRACT
GERMINATION
The germinated grains contain high molecular weight carbohydrates, and this makes it difficult in fermentation by beneficial probiotics, caused by lack of available carbon sources. In this study, probiotics fermenting germinated black bean, germinated brown rice, Cordyceps militaris grown on strains grown on each germinated grains were selected. Among them, 32 strains showed high
GRAINS
FERMENTATION
germinated soybean, and Phellinus linteus grown on germinated brown rice were screened. First, 217
GERMINATED GRAINS
antioxidant activity. The highest antioxidant activity and titratable acidity was measured 99.40% and 0.32% by Pediococcus pentosaceus ON-89A and Lactobacillus pentosus SC65, respectively. Eight strains were chosen by considering the titratable acidity and sensory property. When viable cells of 8 strains grown on each germinated grains were measured, L. pentosus SC65 had the highest viability of 8.08 log CFU/mL in 5% (w/v) germinated black bean medium. Consequently, L. pentosus SC65, P.
TRADITIONAL FOODS
pentosaceus SP-024, ON-89A, and SP-041 were chosen as starter strains for germinated black bean, germinated brown rice, C. militaris grown on germinated soybean, and P. linteus grown on
RESULTS
60 40 20 0 SC30
SC63
SC65
S.Pum8
Y-KC6
Control S-Per.s12
SC30
SC63
SC65
S.Pum8
Y-KC6
Viable cell count (log CFU/mL)
8
6
4
2
0 SC65
ON-81A
SC61
SP-024
SC64
ON-89A
ON-42A
SP-041
Fig. 3. Viable cell count of germinated grains fermented with isolated probiotics.
Reducing sugar
Control S-Per.s12
Glycerol Erythritol D-Arabinose L-Arabinose D-Ribose D-Xylose L-Xylose D-Adonitol Methyl-β-D-Xylopyranoside D-Galactose D-Glucose D-Fructose D-Mannose L-Sorbose L-Rhamnose Dulcitol Inositol D-Mannitol D-Sorbitol Methyl-α-D-Mannopyranoside Methyl-α-D-Glucopyranoside N-Acetyl Glucosamine Amygdalin Arbutin Esculin ferric citrate
10 5 0
20%
+ + + + + + + + + +
30%
40%
1%
5%
10%
20%
30%
10
8
8
6
6 4
2
0
0
40%
1%
5%
10%
20%
30%
40%
0 25℃
30℃
37℃
25℃
30℃
37℃
3 2 1 25℃
40℃
10
5
8
4
30℃
37℃
6
pH
4
0
0
40℃
SC64
ON89A
ON42A
SP041
Titratable acidity (%)
Carbon source
SC65
ON81A
SP024
SC61
ON89A
SC64
ON42A
46
SP041
62 100 83
+ + + + + + + + + + +
+ + + + + + + + + + +
+ + + + + + + + + + + + +
+ + + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + + +
Salicin D-Cellobiose D-Maltose D-Lactose (bovine origin) D-Melibiose D-Saccharose (sucrose) D-Trehalose Inulin D-Melezitose D-Raffinose Amidon (starch) Glycogen Xylitol Gentiobiose D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol Potassium Gluconate Potassium 2-ketogluconate Potassium 5-ketogluconate
+ + + + + + + -
+ + + + + + + + + + -
+ + + + + + + + + + + -
+ + + + + + + + + + -
+ + + + + + + + + + + + + -
+ + + + + + + + + + -
+ + + + + + + + + + -
4
0.05
0.1
0.125
24h
48h
72h
2 1
0
0 25℃
30℃
37℃
25℃
40℃
Lactobacillus oeni 59b
Pediococcus claussenii DSM 148 Pediococcus argentinicus
100
Lactobacillus casei ATCC393
100
26
Pediococcus parvulus JCM5889
Lactobacillus paracasei JCM813 Lactobacillus dextrinicus JCM5
12
97
Enterococcus durans DSM20633
12
Lactobacillus sakei DSM20017
36
100
99
Pediococcus ethanolidurans Z 9
59
Lactobacillus ingluviei KR3
100
Pediococcus cellicola Z 8
82
Lactobacillus gasseri ATCC3332
100
Pediococcus damnosus DSM 20331 Pediococcus inopinatus DSM 202
100
Lactobacillus reuteri DSM20016 Lactobacillus fermentum ATCC14
Pediococcus siamensis MCH3 2
65
Lactobacillus johnsonii ATCC33
Lactobacillus dextrinicus JCM
Lactobacillus delbrueckii subs
100
Lactobacillus amylolyticus
77
Pediococcus urinaeequi IFO1217
Lactobacillus acidophilus BCRC
100
Tetragenococcus halophila IAM
Lactobacillus helveticus DSM 2
99
10
0.01 100
Pediococcus pentosaceus DSM 20
100 53
Pediococcus stilesii LMG 23082
95
Pediococcus stilesii LMG 23082
Pediococcus claussenii DSM 148
Pediococcus claussenii DSM 148 Pediococcus argentinicus
100
Pediococcus argentinicus
100
Pediococcus parvulus JCM5889
Pediococcus parvulus JCM5889 97 100
98 100
Pediococcus damnosus DSM 20331
86
Pediococcus cellicola Z 8
83
Pediococcus cellicola Z 8 Pediococcus siamensis MCH3 2
64
Pediococcus siamensis MCH3 2
63
Pediococcus ethanolidurans Z 9
58
Pediococcus ethanolidurans Z 9
58
Pediococcus damnosus DSM 20331 Pediococcus inopinatus DSM 202
100
Pediococcus inopinatus DSM 202
100
Pediococcus lolii NGRI 0510Q
100 98
Pediococcus lolii NGRI 0510Q
100 99
Pediococcu acidilactici DSM 20
59
Pediococcu acidilactici DSM 20
56
Pediococcus pentosaceus DSM 20 90246 SP-041
53
90176 ON-89A
96
Lactobacillus dextrinicus JCM
Lactobacillus dextrinicus JCM
Pediococcus urinaeequi IFO1217
Pediococcus urinaeequi IFO1217
Tetragenococcus halophila IAM
Tetragenococcus halophila IAM 0.01
0.01
10
3 0.1 0.05
2 1
0
0 0.05
0.1
0.125
0.15
0.01
0.05
0.1
0.125
8 6 4 2 0
0.15
0.01
30℃
37℃
0.1
3
0.08 0.06 0.04 0.02
1 0
0
40℃
2
25℃
30℃
37℃
25℃
40℃
30℃
37℃
0.05
0.1
0.125
0.15
72h
4
6
3
2
0h
24h
48h
72h
4
2
2
1
0
0 0h
24h
48h
72h
0h
24h
48h
72h
3
0.12
Glucose mM
8
pH
4
0 48h
5
0.08 0.04 0
2 1 0
0h
24h
48h
72h
0h
24h
48h
8 6 4 2 0
40℃
Viable cell count (log CFU/mL)
0 24h
Pediococcus lolii NGRI 0510Q
100
Lactobacillus animalis NBRC158
0.15
0.01
Titratable acidity (%)
0.4
10
Viable cell count (log CFU/mL)
0.8
0h
Pediococcu acidilactici DSM 20
56 99
Fig. 4. Phylogenetic trees of selected strains.
0.15
3
2
40℃
6
1.2
Glucose mM
Titratable acidity (%)
pH
0h
Pediococcus stilesii LMG 23082
96
Lactobacillus plantarum JCM 11
78
FERMENTATION TIME: Germinated brown rice
0
Pediococcus pentosaceus DSM 20 90232 SP-024
52
Lactobacillus arizonensis NRRL
Lactobacillus kefiri LMG9480T
67
96
SP-041
100
SC65
SC65
Lactobacillus brevis ATCC14869
+ + + + + + + -
FERMENTATION TIME: Germinated black bean
2
ON-42A
Lactobacillus kimchii AP1077
Fig. 8. Effect of fermentation temperature on the pH, titratable acidity, reducing sugar, and viable cell count during the fermentation.
4
Bro22
Lactobacillus paraplantarum DS
Fig. 7. Effect of fermentation temperature on the pH, titratable acidity, reducing sugar, and viable cell count during the fermentation.
6
SC2
Viable cell count (log CFU/mL)
0.4
1
Control
Fig. 6. Effect of germinated brown rice concentration on the pH, titratable acidity, reducing sugar, and viable cell count during the fermentation.
Viable cell count (log CFU/mL)
Glucose mM
Titratable acidity (%)
pH
5
0.8
2
0
TEMPERATURE: Germinated brown rice
1.2
3
0.02
Lactobacillus pentosus JCM 155
024
0.01
TEMPERATURE: Germinated black bean 4
0.05
0.04
0
Bro14 S.Pum21 ON-30A GO-008 SP-024
4
2
Fig. 5. Effect of germinated black bean concentration on the pH, titratable acidity, reducing sugar, and viable cell count during the fermentation.
5
SP-041
0.06
Viable cell count (log CFU/mL)
15
0 10%
0.1
Glucose mM
0.5
5%
SC61
Titratable acidity (%)
1
1%
+ + + + + + + + + + + +
pH
20
Viable cell count (log CFU/mL)
pH
1.5
40%
ON-42A
0.08
Glucose mM
30%
SC7
Titratable acidity (%)
20%
Bro22
CONCENTRATION: Germinated brown rice Glucose mM
Titratable acidity (%) 10%
SC2
D. Phellinus linteus grown on germinated brown rice
0.15
CONCENTRATION: Germinated black bean
5%
Control
52
Viable cell count
1%
20 0
C. Cordyceps militaris grown on germinated soybean
81A
FERMENTATION TIME
0
40
SP-024
0.05
sal.cla9 S.Pum21 ON-184 ON-81A SP-038
10
2
Bro14 S.Pum21 ON-30A GO-008
Fig. 2. Titratable acidity of germinated grains fermented with isolated Table 1. Carbon source utilization of selected strains ONSPprobiotics. Carbon source SC65 SC61
TEMPERATURE
4
SC61
0 Control
6
SC7
Titratable acidity (%)
0.2
0
8
20
0.1
Titratable acidity (%)
Titratable acidity (%)
CONCENTRATION
•
40
60
0
B. Germinated brown rice
A. Germinated black bean
Optimal fermentation condition
•
20
sal.cla9 S.Pum21 ON-184 ON-81A SP-038
0.4
pH
40
60
80
Fig. 1. DPPH radical scavenging activity of germinated grains fermented with isolated probiotics.
Antioxidant activity
•
60
80
0 Control
Titratable acidity
80
DPPH radical scavenging activity (%)
16S rRNA gene PCR
DPPH radical scavenging activity (%)
DPPH radical scavenging activity (%)
API 50CHL
80
100
100
100
100
D. Phellinus linteus grown on germinated brown rice
C. Cordyceps militaris grown on germinated soybean
B. Germinated brown rice
A. Germinated black bean
DPPH radical scavenging activity (%)
Characterization of isolated strains
•
PROBIOTICS
ISOLATION
germinated brown rice, respectively.
MATERIALS AND METHODS
PROBIOTICS FERMENTING GERMINATED GRAINS
72h
25℃
30℃
37℃
40℃
0h
24h
48h
72h
8 6 4 2 0
Fig. 10. Effect of fermentation time on the pH, titratable acidity, reducing sugar, and viable cell count during the fermentation.
Fig. 9. Effect of fermentation time on the pH, titratable acidity, reducing sugar, and viable cell count during the fermentation.
CONCLUSION I.D. of STRAINS Lacobacillus pentosus SC65 Pediococcus pentosaceus SP-024 Pediococcus pentosaceus ON-89A Pediococcus pentosaceus SP-041
CONCENTRATION
20%
10%
TEMPERATURE
30℃
30℃
FERMENTATION TIME
24 h
24 h
