Jae Sung Lee; Yun Hwan Kang; Kyoung Kon Kim; Yeong Kyeong Yun; Jun Gu Lim; Tae Woo Kim; Dae Jung Kim; Sang Yeon Won; Moo Hoan Bae; Han Seok Choi; Myeon Choe

doi:10.4163/jnh.2014.47.1.12

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Lee, Kang, Kim, Yun, Lim, Kim, Kim, Won, Bae, Choi, and Choe: Exploration of optimum conditions for production of saccharogenic mixed grain beverages and assessment of anti-diabetic activity∗

Research Article

J Nutr Health 2014;47(1):12-22.

Published online: 20 January 2014

DOI: https://doi.org/10.4163/jnh.2014.47.1.12

Exploration of optimum conditions for production of saccharogenic mixed grain beverages and assessment of anti-diabetic activity∗

Jae Sung Lee¹, Yun Hwan Kang², Kyoung Kon Kim¹, Yeong Kyeong Yun¹, Jun Gu Lim², Tae Woo Kim², Dae Jung Kim², Sang Yeon Won³, Moo Hoan Bae³, Han Seok Choi⁴, Myeon Choe^1,²

¹Department of Bio-Health Technology, Kangwon National University, Gangwon 200–701, Korea

²Well-Being Bioproducts RIC, Kangwon National University, Gangwon 200–701, Korea

³NS Mall, Seongnam-si 463–400, Korea

⁴Fermented Food Science Division, National Academy of Agricultural Science, RDA, Suwon 441–853, Korea

Received 2 December 2013 Revised 6 January 2014 Accepted 16 January 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

This study was conducted to establish the production conditions through optimization of the production process of beverages using Aspergillus oryzae CF1001, and to analyze volatile compounds and antidiabetic activity. Methods: The optimum condition was selected using the response surface methodology (RSM), through a regression analysis with the following independent variables gelatinization temperature (GT, X₁), saccharogenic time (ST, X₂), and dependent variable; ΔE value (y). The condition with the lowest ΔE value occurred with combined 45 min ST and 50℃ GT. The volatile compounds were analyzed quantitatively by GC-MS. Results: Assessment of antidiabetic activity of saccharogenic mixed grain beverage (SMGB) was determined by measurement of α-glucosidase inhibition activity, and glucose uptake activity and glucose metabolic protein expression by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. Results of volatile compounds analysis, 62 kinds of volatile compounds were detected in SMGB. Palmitic acid (9.534% ratio), benzaldehyde (8.948% ratio), benzyl ethyl ether (8.792% ratio), ethyl alcohol (8.35% ratio), and 2-amyl furan (4.826% ratio) were abundant in SMGB. We confirmed that α-glucosidase inhibition activity, glucose uptake activity, and glucose-metabolic proteins were upregulated by SMGB treatment with concentration dependent manner. Conclusion: Saccharogenic mixed grain beverage (SMGB) showed potential antidiabetic activity. Further studies will be needed in order to improve the taste and functionality of SMGB.

Keywords: response surface methodology, volatile compounds, saccharogenic mixed grain beverage, α, -glucosidase, anti-diabetic activity.

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Fig. 1.

Response surface plot of Brix° on the SMGB as functions of gelanization temperature and saccharogenic times.

Fig. 2.

Contour map of optimzied conditions for the brix° of SMGB as functions of gelanization temperature and saccharogenic

Fig. 3.

Effect of SMGB on α-glucosidase inhibition activity. Values are mean ± standard deviation of triplicate determination, different letters on the bars (a-c) indicate significant differences (p < 0.05) by Duncan's multiple range test. Acarbose Asp. CF 1001

Fig. 4.

Measurement of glucose uptake activity and GLUT-2, −4 expression in HepG2 cell. A: Effect of SMGB (Asp. CF1001) on glucose uptake. B: Effect of SMGB on GLUT-2, GLUT-4 mRNA expression. C: Effect of SMGB on GLUT-2, GLUT-4 protein expression. Values are mean ± standard deviation of triplicate determination, different letters on the bars (a-d) indicate significant differences (p < 0.05) by Duncan's multiple range test. GLUT: Glucose-transporter.

Fig. 5.

Measurement of glycolytic key enzyme expression in HepG2 cell. A: Effect of SMGB on GK and PDH mRNA expression. B: Effect of SMGB on GK and PDH protein expression. Values are mean ± standard deviation of triplicate determination, different letters on the bars (a-c) indicate significant differences (p < 0.05) by Duncan's multiple range test. GK: glucokinase, PDH: pyruvate dehydrogenase.

Fig. 6.

Effect of SMGB on ACL and ACC mRNA expression in HepG2 cell. Values are mean ± standard deviation of triplicate determination, different letters on the bars (a-c) indicate significant differences (p < 0.05) by Duncan's multiple range test. ACL: ATP-citrate lyase, ACC: Acetyl-CoA carboxylase.

Table 1.

Central composite design for brix˚ on sacchrogeni mixed grain beverage (SMGB) with gelatinzation temperatur and saccharogenic time

Sample	X₁	X₂
01	50	40
02	50	40
03	50	40
04	60	20
05	64.1	40
06	50	40
07	50	68.3
08	50	40
09	35.9	40
10	50	11.7
11	60	60
12	40	20
13	40	60

X₁: Gelatinzation temperature (℃), X₂: Saccharogenic time (min

Table 2.

PCR primer sequences

Gene	Primer	Sequence (5'→3')
GK	Forward	ACC GCA AGC AGA TCT ACA AC
GK	Reverse	TGG GGT GCA GCT TGT ACA
PDH	Forward	AAT CCA ACT GGT TAC TTT TGA AGA
PDH	Reverse	AAG AGC TGA GCA GCT GTG TAA
ACL	Forward	AAC TTG GTC TCG TTG GGG TC
ACL	Reverse	CGT GGT GGA ACA GGA CGT AG
GLUT-2	Forward	GAT GAA CTG CCC ACA ATC TC
GLUT-2	Reverse	CTG ATG AAA AGT GCC AAG TG
GLUT-4	Forward	GTT AAT CGG CAT TCT GAT CG
GLUT-4	Reverse	GTG AAG ACT GTG TTG ACC AC
ACC	Forward	GAG CCT GAG AAA CGG CTA C
ACC	Reverse	CCC ATT ATT CCT AGC TGC G
β-actin	Forward	ACA GGA AGT CCC TTG CCA TC
β-actin	Reverse	AGG GAG ACC AAA AGC CTT CA

Table 3.

PCR condition of each primer

Gene	Pre-denaturation	Denaturation	Annealing	Extension	Final extension
GK	94℃ 5 min	94℃ 30 sec	58℃ 30 sec	72℃ 30 sec	72℃ 5 min
GK	94℃ 5 min	38 cycle			72℃ 5 min
PDH	94℃ 5 min	94℃ 30 sec	50.8℃ 30 sec	72℃ 30 sec	72℃ 5 min
PDH	94℃ 5 min	28 cycle			72℃ 5 min
ACL	94℃ 5 min	94℃ 30 sec	55℃ 30 sec	72℃ 30 sec	72℃ 5 min
ACL	94℃ 5 min	23 cycle			72℃ 5 min
GLUT-2	94℃ 5 min	94℃ 30 sec	59℃ 30 sec	72℃ 30 sec	72℃ 5 min
GLUT-2	94℃ 5 min	26 cycle			72℃ 5 min
GLUT-4	94℃ 5 min	94℃ 30 sec	59℃ 30 sec	72℃ 30 sec	72℃ 5 min
GLUT-4	94℃ 5 min	30 cycle			72℃ 5 min
ACC	94℃ 5 min	94℃ 30 sec	47℃ 30 sec	72℃ 30 sec	72℃ 5 min
ACC	94℃ 5 min	25 cycle			72℃ 5 min
β-actin	94℃ 5 min	94℃ 30 sec	55℃ 30 sec	72℃ 30 sec	72℃ 5 min
β-actin	94℃ 5 min	18 cycle			72℃ 5 min

Table 4.

Central composite design for brix˚ on SMGB with gelat inzation temperature and saccharogenic time

Sample	X₁	X₂	Brix°
01	50	40	16.6
02	50	40	16.6
03	50	40	16.6
04	60	20	14.6
05	64.1	40	14.5
06	50	40	16.6
07	50	68.3	16.6
08	50	40	16.7
09	35.9	40	13.9
10	50	11.7	15.7
11	60	60	15.2
12	40	20	14.1
13	40	60	14.8

Table 5.

Polynomial equation calculated for Brix° by RSM program for SMGB

Responses	Polynomial equation	R²	p-value
Brix°	Y = 1 6.62 + 0.2186 X₁ + 0.3216 X₂-1.3350 X₁ ²-0.36 X₂ ²-0.250 X₁X₂	0.9646	0.001

X₁: Gelatinzation temperature (℃), X₂: Saccharogenic time (min)

Table 6.

Predicted levels of optimum conditions for the maximized and minimized responses of variables by the ridge analysis of their response surface

Response	Saccharogenic condition
Response	X₁	X₂	Estimated responses (Max)	Morphology
Brix°	50.71	45.12	16.6876	Maximum point

X1: Gelatinzation temperature (℃), X2: Saccharogenic time (min)

Table 7.

Regression analysis for regression model of Brix° in sac charogenic condition of SMGB

Extract condition	F-ratio
X₁	194.13
X₂	14.12

X₁: Gelatinzation temperature (℃), X₂: Saccharogenic time (min

Table 8.

Volatile compouds of SMGB

Compound	% peak area of flavor components in SMGBs
Palmitic acid	9.534
Benzaldehyde	8.948
Benzyl ethyl ether	8.792
Ethyl alcohol	8.350
2-Amyl furan	4.826
Aldehyde C-6 (Hexanal)	3.243
Acetoin	3.205
D-Limonene	2.828
Aldehyde C-9 (Nonanal)	2.687
Furfural	2.309
Myristic acid	2.131
Acetic acid	1.938
4-vinyl Guaiacol	1.506
2-Ethyl Hexanol	0.844
Germacrene-D	0.695
Salicylic aldehyde	0.664
Aldehyde C-7 (Heptanal)	0.598
Dihydro Benzofuran	0.541
Isovaleric acid	0.540
Methyl propyl ketone	0.473
2-Heptanone	0.473
Isoamyl acetate	0.468
2-Octanone	0.468
Hexanoic acid	0.434
2-Butyl furan	0.424
2-Ethyl furan	0.419
1-Octen-3-ol	0.372
P-cymene	0.371
Ethyl caprylate	0.358
Octanoic acid	0.326
Benzyl alcohol	0.256
Linalool	0.250
Nonanoic acid	0.216
Non identified components	30.763

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