Statistical analysis of browining in relation to "Wasanbon" sugar-香川大学学術情報リポジトリ

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STATISTICAL ANALYSIS OF BROWNING IN

RELATION TO "WASANBON" SUGAR

Toshiyuki

MATSUI

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The statistical analysis was made by means of three way classification method (TWC) in order to examine the interaction among asparagine, fructose and pH on the basis of browning of model system in comparable concentra- tions as in sugarcane

In the case of 3-deoxyglucosone (3DG) product as indication, all the main effects such as asparagine, fructose and pH were significantly different a t 1% level On the other hand, in the case of 0 D value as indication, the main effect of asparagine and pH were significantly different a t 1% level The ability of detecting browning by use of O D value as indication seemed to be lower than use of 3DG production

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The color of "Wasanbon" sugar, owing to the browning mainly produced by the amino-carbonyl reaction is affected significantly under manufacturing conditions and varieties of sugarcane Furthermore, it is difficult to investigate the relationship between the manufacturing conditions and chemical components without using model system In the previous.paper'), the stepwise regression analysis (SRA) was carried out in order to select the number of important variables The selected variables for 3DG production as indication were fructose, temperature, pH, glucose, time and asparagine and those for O D value were temperature, asparagine and time Here, the author used three way classifi- cation method in order to know the interrelationships among asparagine, fructose and pH in this study

Methods

1 Measurement of 3-deoxyglucosone (3DG) and 0 D 440 nm In a 400 ml beaker amino acid and reducing sugar

were dissolved a t a certain concentration in 50 ml distilled water A few drops of 1 N sodium hydroxide was added to adjust pH Subsequently, the liquid inside the beaker was maintained a t 8Z°C by external heating with occasional stirring for 120 min The reaction solution was cooled to room temperature and then filled up to the mark with distilled water The 3DG contents of the solution were determined by 2,4-dinitrophenyl hydrazine method2), and the absorbance values were read a t 440 nm

2 Three way class~ficat~on method (TWC) When three factors consisting of chemical components and reaction con-

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elJkl, where u represents the overall mean, (ab)lJ --- ( a b ~ ) ~ ~ ~ stand for the interactions between factors, and elJkl stands for errors of the ex- periment The selected manufacturing conditions are adopted a s three factors and the relation on browning between these conditions was examined by the TWC3) The data obtained were caluculated from the program on the basis of "Bulletin of Computing Center"4) When some interactions between variables were found, the author tried to estimate comparisons among means on the basis of the least significant difference (LSD)5)

Results and Discussion

1 Determinat~on of variables used f o r model system in TWC Of 11 variables (6 amino acids, 2 reducing sugars and 3 conditions of reaction), asparagine, fructose, glucose, temperature and pH for 3DG production a s indication were selected by the stepwise regression analysis1), whereas asparagine and pH or time for 0 D value a s brown- ing indication were selected by the method1)

The advantage of the multiple way classification including TWC is not only in organizing experiments freely without depending on the number of variables but also in detecting several interactions between pairs of variables6) On the other hand, the disadvantage of the method increased prominently the size of experiment6)

In order to reduce variables, asparagine, fructose and pH were used a s variables of TWC Only asparagine was chosen by SRA and therefore it was used as variables of TWC. Although glucose and fructose were chosen by SRA in reducing sugars, fructose was used as variable of TWC I t was entered first into regression and was the highest correlation coefficient against 3DG production a s dependent variable1)

Two levels of asparagine represented average and the lower contents of 'N:CO 310' and ' C h i k ~ s h a ' ~ ) Three lev- els of fructose showed average, the higher and lower contents of both varieties The condensation processes of "Wasan- bon" sugar did not show drastic fluctuation of pHs)g), and therefore the pH values of 6 5 and 7 5 were adopted Other conditions except fructose, pH and asparagine were fixed a s follows; reaction time 2hr, internal reaction tem- perature 82"C, histidine 11 8 mg, threonine 22 2 mg, alanine 75.5 mg, valine 51 6 mg, leucine 9 1 mg per 25 ml and glucose 1 5 g per 50 ml Table 1 shows 3DG production and 0 D value in model system for TWC

Table 1 3-Deoxyglucosone production and 0 D. value in model systems for three way classification

System of reaction* 3DG production (mgl50 ml) 0.D value (440 nm)

Fir s t Second First Second

* Symbols in the Table indicate the following conditions (Al: Asn 440 0, A2: Asn 880 0 mg125 ml)

(Bl: Fru 1 00, B2: Fru 1 25, B3: Fru 1 50 g150 ml) (Cl: pH 6 5, C2: pH 7 5)

Other conditions: Reaction time 2 hr ,'Internal reaction temperature 8Z°C, His 11 8, Thr 22 2, Ala 75 5, Val 5) 6, Leu 9 1 mg125 ml and Glc 1 50 gl50 ml

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Table 2 Analysis of variances of 3-deoxyglucosone production in model systems

Factors s s d f m s F-r atio

Asparagine (Main effect A) Fructose (Main effect B) PH (Main effect C) Interaction (A x B) Interaction (A x C) Interaction (B x C) Interaction (A x B x C) Error Total * P C 0 05; F(1, 12; 0 05) = 4 75, * * P<O 01; F(1, 12; 0 01) = 9 33,

Table 3 Analysis of variances of 0 D value in model systems

Factors s s d f m s F-ratio

Asparagine (Main

effect^)

1 0 5 x 10V4 1 1 0 5 x 20.21 * * Fructose (Main effect B) 2.91 x 2 1 4 6 x 2 79 -

PH (Main effect C) 3 15 x 1 3 15 x 6 50 * * Interaction (A x B) 1 2 7 x 2 6 36 x l o A 5 12 21 * * Interaction (A x C) 1 5 0 x 1 1 5 0 x 0 2 9

-

Interaction (B x C) 1 3 3 x 2 6 65 x 1 2 8

-

Interaction (A x B x C) 1 2 1 x 2 6 10 x 1 1 6

-

Error 6 26 x 12 5 2 0 x - Total 3 83 x * * P<O 01; F(l, 12; 0 01) = 9 33, * * P<O 01; F(2, 12; 0 01) = 6 93 Table 4 Means of factors of 3-deoxyglucosone productions and 0 D value

3DG production 0 D value C1 C2 Mean C 1 C2 Mean A1 B1 1 8 4 3 2 594 2 218 0 022 0 037 0 030 A1 B2 2 490 2 674 2 582 0 048 0 050 0 049 A1 B3 3 308 3 193 3 251 0 050 0 049 0 050 A1 Mean 2 547 2 821

2.684

0 040 0 046

0.043

C l C2 Mean C 1 C2 Mean A2 B1 2 155 3 124 2 640 0 058 0 066 0 062 A2 B2 2 547 3 331 2 939 0 039 0 055 0 047 A2 B3 2 709 3 723 3 216 0 058 0 060 0 059 A2 Mean 2 471 3 393

2.932

0 052 0 061

0.056

C l C2 Mean C 1 C2 Mean B1 Mean 1 999 2 859

2.429

0 040 0 052

0.046

B2 Mean 2 519 3 003

2.761

0.044 0 053

0.048

B3 Mean 3 009 3 458

3.234

0 054 0 055

0.054

C1 C2 C1 C2 C Mean 2.589 3.107

0.046

0.053

Symbols in the Table indicate the following conditions (Al: Asn. 440 0, A2: Asn 880 0 m d 2 5 ml)

(Bl: Fru 1 00, B1: Fru 125, B3: Fru 1 5 0 g/50 ml) (Cl: pH 6 5, C2: pH 7 5)

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Table 5 Least significant difference among means on 3-deoxyglucosone production and 0.D value 3DG production 0 D 440 nm (0 05)* (0 01)** (0 05)* (0 01)- A 0 102 0 143 0 006 0 009 B 0 125 0 176 0 008 0 011 C 0 102 0 143 0 006 0 009 A x B 0 177 0 248 0 011 0 016 A x C 0 145 0 203 0 009 0 013 B x C 0 177 0 248 0 011 0 016

* and ** show significant difference level at 5 and 1%, respectively Symbols show in Table 1

2 Analysis b y the three way classifFcation

(TWC)

The analysis of variance of 3DG production is shown in Table 2, and that of absorbance value a t 440 nm is shown in Table 3 All the main effects on 3DG production were significantly d i e r e n t a t 1% level, namely the variations of asparagine, fructose and pH had effects upon the 3DG production When there were significant differences among several effects, the author intended to estimate the differences, and inspect- ed all the differences between pairs of the mean The mean values of 3DG production and 0 D values for each system in Table 1 is shown in Table 4 In this Table, C's of the uppermost row show 2 levels of pH and the values of column give the average of first and second values in Table 1 The standard error of the difference between the two means corresponded to square root of error of the m s in Table 2, and the degree of freedom5) was 12 The standard error multiplied by the 5%value of

t

of 12 d f makes the quantity which serves as a criterion This quantity is called the least significant difference (LSD)~) The LSD is shown in Table 5

3 The least s~gnificant difference of 3DGproduction When asparagine contents increased from 440 (Al) to 880 mg

(A2), the difference between the pair of mean was calculated by deduction of A2 mean from A1 mean in Table 4 in order to estimate LSD The difference between A2 mean and A1 mean was 0 248, whereas the LSD between A1 and A2 was 0 143 a t 1% level It means significant difference a t 1% level that 0 248 was larger than 0 143

B1 B2 B3 B1 B2 B3 C1 C2

Fructose contents (g150 ml) PH

Fig 1 Interactions between asparagine and fructose contents, fructose content and pH, and asparagine content and pH on the basis of the least significant difference A l ; Asparagine content (440 mg125 ml),

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When fructose content increases from 1 00 to 1 25 g and from 1 25 to 1 50 g, the difference between the pair of mean showed 0 332 and 0 473, respectively Both values show significant diierence a t 1% level In the case of pH, the differ- ence between the pair of means was 0 598, and the diierence was significant a t 1% level The interactions between asparagine and fructose, fructose and pH, and asparagine and pH on 3DG production showed significant difference a t 1% level, and therefore the interaction on the basis of LSD was estimated Fig 1 shows the interactions between A and B, B and C, and A and C on 3DG production When fructose and asparagine contents are higher, fructose contents and pH value higher, and pH value and asparagine contents higher, each of 3DG production is apt to increase 4 The least of signficant difference

of

0 D. value When the asparagine content increased from 440 (Al) to 880 mg (AZ),

the difference between the pair of mean (0 09) is significant a t 1% level I t means the significant difference in 0

D

440 nm of 0 013 (deduction of A2 mean of 0 056 from A1 mean of 0.043) When pH of the solution changed from 6 5 (Cl) to 7 5 (C2), the value of 0 D 440 nm showed the average absorbance increase of 0 007 and it means signifi- cant difference a t 5% level (0 006 in Table 5) The interaction between asparagine and fructose of 0 D value was significantly different a t 1% level, and next the interaction between A and B on the basis of LSD was estimated Fig 2 shows the interaction between A and B of 0 D value

I

A and B

1 0 0 1 2 5 1.50

B 1 B2 B3

Fructose contents (gl50 ml) Fig 2 Interaction between asparagine and

fructose contents on the basis of least significant difference

Symbols as shown in Fig 1

In the case of 440 nm asparagine (Al), there was no significant difference between 1 25 (B2) and 1 50 g (B3) of fructose content, whereas the differences between A1 and B1 and between A1 and B2 were recognized a t 1% level In the case of 880 mg of asparagine (A2), there was no significant difference between 1 00 (Bl) and 1 50 g/ 50 ml of aqueous solution (B3) of fructose content, whereas the difference between A2 and B l and between A2 and B2, and the difference between A2 and B2 and between A2 and B3 were recognized a t 5% level

In this study, the use of 3DG production as indication on browning gave often significant difference in faint browning solution, but the use of 0 D value did not Therefore, it is advisable to determine browning by 3DG production instead of 0 D value as indication

The author wishes to thank Prof Sin'itir6 Kawamura of Kagawa-ken Meizen Jounior Colledge for his sugges- tions He also thanks Mr Seiichi Miyoshi of the Agricultural Association of Onsen Seika for technical assistance

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References

1 ) T MATSUI, Nippon N6geikagaku Gakkaishi, in

contribution

2 ) T MATSUI, Nippon Shokuhm Kogyo Gakkalsh~, 23, 339 (1976).

3 ) K ISHIKAWA, T FUJIMORI and H KUME, "Jikken Keikaku How, 143, Tokyo Kagaku Dojin K K

,

Tokyo (1979)

4 ) S NONAKA, "Bulletin of the Computing Center for Research in Agriculture and Foresty", Norin Suisan Gijutsu Kaigi, Seris A, No 2, 53 (1968) 5 ) G W. SNEDECOR and W. G COCHRAN, "Statistical

Methods", 271, sixth Edition, The Iowa State Univ Press, Iowa (1978)

6 ) T KIMURA, "Jikken Keikakuho no Tehodoki", 7, Nankodo, Tokyo, (1976)

7 ) T MATSUI, Njppon Shokuhzn Kogyo Gakkalshz, 24, 487 (1977)

8 ) T MATSUI, Tech Bull Fac Agric Kagawa Unzv

,

33, 157 (1982)

9 ) K YAMANAKA, Njppon Shokuhin Kogyo Gak- k a ~ s h ~ , 21, 438 (1974)

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