Studies on the Color Development in Stored Plantation White Sugars

6

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Studies on the Color Development in Stored Plantation White Sugars HUNG-TSAI CHENG,

W A I - F U K L I N , and C H U N G - R E N

WANG

Taiwan Sugar Research Institute, 54 Sheng Chan Road, Tainan, Taiwan, Republic of China

Crude p l a n t a t i o n white sugars from sugar cane, manufactured by a carbonation or a s u l p h i t a t i o n process, developed c o l o r during storage l e a d i n g to degradation i n t h e i r c o l o r grade. This development of c o l o r or poor keeping q u a l i t y was more marked i n carbonation than i n s u l p h i t a t i o n sugars. Spectrophotometric and chromatographic s t u d i e s i n d i c a t e d that the c o l o r - b e a r i n g compounds r e s p o n s i b l e f o r the sugar q u a l i t y changes were humic a c i d s , caramel, 5-hydroxymethylfurfural, and melanoidins. In carbonation, a high percentage o f reducing sugar dest r u c t i o n i n the h i g h l y a l k a l i n e c o n d i t i o n of the first carbonation stage is b e l i e v e d r e s p o n s i b l e f o r the formation of c o l o r - b e a r i n g compounds of the 5-hydroxymethylfurfural and humic a c i d c a t e g o r i e s , and these reducing sugar degradation products play an important r o l e on the melanoidin and caramel f o r mation i n sugar c r y s t a l s during storage. Carbonates e v i d e n t l y c a t a l y z e the c a r a m e l i z a t i o n much f a s t e r than s u l p h i t e s , l e a d i n g to f a s t e r development o f c o l o r in carbonation sugars. Sulphitation inhibited the melanoidin formation, presumably by b l o c k i n g the carbonyl f u n c t i o n . Lowering o f the l e v e l of carbonates by r e p l a c i n g the second carbonation stage with s u l p h i t a t i o n or phosphatation, and improvement of the first carbonation technique f o r reducing sugar d e s t r u c t i o n , were recommended to improve the keeping q u a l i t y of the carbonation sugars.

The development of brown c o l o r i n sugar during storage i s one of the o l d e s t problems i n the sugar i n d u s t r y . In Taiwan, about 400,000 tons o f p l a n t a t i o n white sugar from sugar cane are

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Waller and Feather; The Maillard Reaction in Foods and Nutrition ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


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annually produced i n seven carbonation f a c t o r i e s and two s u l p h i t a t i o n f a c t o r i e s , during the p e r i o d of November-May. The sugar i s s t o r e d i n bags i n warehouses at ambient c o n d i t i o n s , i . e . , the r e l a t i v e humidity (RH) i s from 65 to 90% and the temperature i s between 5 and 40°C. Under these c o n d i t i o n s of storage, the p l a n t a t i o n white sugar d e t e r i o r a t e s i n the sense that some c o l o r i n g matter i s produced i n the sugar c r y s t a l s , l e a d i n g to degradation i n t h e i r c o l o r grade. This d e t e r i o r a t i o n i n c o l o r on storage i s more marked i n carbonation sugar than i n s u l p h i t a t i o n sugar, as shown i n Figure 1. Though much knowledge concerning the c o l o r a t i o n of sugar products was obtained from s t u d i e s i n v o l v i n g a l k a l i and heat treatment of sugars and the r e a c t i o n s of amino compounds and sugars (1,2), understanding of the nature of the c o l o r i n g matter formed during storage i s s t i l l very l i m i t e d . Most i n v e s t i g a t i o n s have concerned the browning of raw sugar. Tsuchida (3) reported that a d i r e c t r e l a t i o n s h i p e x i s t s between darkening of such sugar on storage and n i t r o g e n content, which i s probably r e l a t e d to the M a i l l a r d r e a c t i o n between the amino a c i d s and degradation products of reducing sugars. Chen (4) concluded that a sugar with h i g h o r i g i n a l c o l o r increases more than a sugar with low o r i g i n a l c o l o r . Monterde (5) s t a t e d that no r e l a t i o n s h i p e x i s t s between darkening and b a c t e r i a l content. According to C o r t i s - J o n e s (6), c o l o r development during raw sugar storage depends on o r i g i n a l c o l o r and the ambient c o n d i t i o n s . Ramon Samaniego (7) concluded that c o l o r i n g matter present i n raw sugars are simply c a r a m e l i z a t i o n products which i n t e r a c t with each other to give r i s e to the dark c o l o r developed during storage. G i l l e t t (2) reported that the c o l o r development of sugar i s dependent on s e v e r a l f a c t o r s such as v a r i e t y of cane, s o i l and season. P l a n t a t i o n white sugars contain ash, reducing sugars, and some amino a c i d s . These may i n t e r a c t during storage to give r i s e to colored products. This study was conducted to (a) develop a simple method of i s o l a t i n g sugar c o l o r a n t s , (b) determine the p o s s i b l e causes of c o l o r development during storage, and (c) f i n d methods to improve the keeping q u a l i t y of carbonation sugar i n particular. The present communication reports a summary of these studies. Experimental M a t e r i a l s and Methods I n v e s t i g a t i o n of f a c t o r s a f f e c t i n g the browning o c c u r r i n g during storage of p l a n t a t i o n white sugar Samples of sugar from the s u l p h i t a t i o n process and the carbonation process were s t o r e d at constant temperature of 70°C and RH 60% f o r 90 h. Changes i n terms of c o l o r v a r i a t i o n or development under such c o n d i t i o n s were found to be equal to those of sugar s t o r e d at about 20°C and RH 80% f o r one year (4).



CHENG ET AL.

Color

Development

in Sugars

90l-

80Γ

o

1

2

3

4

5

6

7

θ

9

10

1 1

12

Time (months) Figure L

Development of color in carbonation (Φ) and sulfitation (Ο) sugars during storage.



94

MAILLARD REACTIONS

Four p l a n t a t i o n white sugar samples from the same area e x h i b i t i n g various degrees of browning were c o l l e c t e d f o r t h i s experiment as follows : Color Value Whiteness (Lumetron) Samples Source Β A Β A 1 Sulphitation 60.08 69.75 0.723 0.675 2 Sulphitation 62.14 71.44 0.650 0.512 3 Carbonation 57.96 92.55 0.705 0.390 4 Carbonation 61.70 94.18 0.665 0.310 B: Before storage A: A f t e r storage These were analyzed f o r ash, reducing sugar, p o l , s t a r c h , p r o t e i n and moisture to determine i f any r e l a t i o n s h i p e x i s t s between these f a c t o r s and the c o l o r development of the sugar. Color values were determined with a Lumetron Spectrophotometer at 420 nm. Ash, reducing sugar, p o l , and moisture were determined i n accordance with the O f f i c i a l Methods of ICUMSA (8). " P o l " i s the value determined by d i r e c t or s i n g l e p o l a r i z a t i o n of the normal weight s o l u t i o n of a sugar product i n a saccharimeter; the f i g u r e i s used as the c o n c e n t r a t i o n of sucrose present i n the s o l u t i o n . Protein was determined c o l o r i m e t r i c a l l y by Lowry's F o l i n - p h e n o l method using bovine albumin (Sigma Chemical Co.) as a standard ( 9 ) . Starch was determined c o l o r i m e t r i c a l l y by W h i s t l e r ' s i o d i n e method using amylose from corn s t a r c h (G.R. TCI Chemical Co.) as a standard (10). I s o l a t i o n of c o l o r i n g matter produced i n p l a n t a t i o n white sugar during storage Fresh and s t o r e d p l a n t a t i o n white sugar samples from the carbonation process and the s u l p h i t a t i o n process were compared. A r a p i d method f o r i s o l a t i n g c o l o r a n t s from sugar samples was developed. A prepacked mini-column of SEP-PAK Cie of I.D. 0.8 χ 1.0 cm was used. Colorants of a sugar sample were i s o l a t e d from sucrose i n minutes by three simple steps. First, sample a f t e r d i l u t i o n with b u f f e r of pH 2.5 was pumped through the SEP-PAK Cie column. About 70% of the c o l o r a n t s were adsorbed on the column while sugar molecules passed through. Second, r e s i d u a l sugar i n the column was washed by pumping through p o r t i o n s of the same b u f f e r . F i n a l l y , the adsorbed c o l o r a n t s were e l u t e d with 2 ml of 50% aqueous CH CN c o n t a i n i n g 0.01 Ν NaOH, 1 ml of 50% CH CN c o n t a i n i n g 0.02 N HC£, and 2 ml of 0.1 M T r i s b u f f e r of pH 7.5 successively. The concentrated sugar-free colorant s o l u t i o n was analyzed by high performance g e l permeation chromatography (HPGPC), HPLC, UV, and IR. 3

3

Nature of the c o l o r i n g matter produced i n p l a n t a t i o n white sugar during storage The l i q u i d chromatograph used (Water A s s o c i a t e s ) c o n s i s t e d of a Model 6000A high pressure s o l v e n t d e l i v e r y system, a Model 440 dual wavelength detector, and a Model U6K i n j e c t o r . The column used f o r f r a c t i o n a t i o n was Bondagel E-125/3.9 mm I.D. x 30 cm length. I t i s an ether-bonded s i l i c a g e l column with nominal molecular weight s e p a r a t i o n range of 2000 to 50,000. The o p e r a t i n g c o n d i t i o n s f o r HPGPC were as f o l l o w s :


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95


Solvent: 0.1 M T r i s b u f f e r of pH 7.5 Flow r a t e : 0.4 ml/min I n j e c t i o n volume: 30 y£ Detection: UV detector with 436 nm wavelength f i l t e r S e n s i t i v i t y : 0.05 aufs (absorbance u n i t s f u l l s c a l e ) Chart Speed: 1.0 cm/min H i t a c h i Model 200-20 double-beam spectrophotometer with wavelength scanning was used f o r s p e c t r o p h o t o m e t r y study. The scanning covered the range 220 to 500 nm. Results and D i s c u s s i o n It has been observed that the c o l o r development of p l a n t a t i o n white sugar during storage i s more marked i n carbonation sugar than i n s u l f i t a t i o n sugar, as shown i n Figure 1. We have observed that (1) Sugars produced i n the e a r l y periods of the campaign (November-December) or the end of the campaign (April-May) developed c o l o r f a s t e r than those manufactured i n the periods of midseason (January-March), (2) The higher the temperature, the f a s t e r i s the c o l o r development; (3) Color development occurred even when sugar was kept out of contact with a i r and i n the dark. To help e x p l a i n these phenomena, chemical analyses were made to examine the r e l a t i o n s h i p of c o n s t i t u e n t s and the c o l o r development i n the sugar c r y s t a l s . Four p l a n t a t i o n white sugar samples from the same area e x h i b i t i n g v a r i o u s degrees of browning were analyzed. The r e s u l t s are shown i n Table 1. There are no c o r r e l a t i o n s between the f a c t o r s s t u d i e d (reducing sugar, p o l , s t a r c h , p r o t e i n and moisture) and the c o l o r development. The ash content of sugars v a r i e d between 0.037% and 0.071%. I t i s i n t e r e s t i n g to note that the sugars of darker c o l o r development had the higher ash content. Ash constituents present i n the c r y s t a l s c a t a l y z e the development of c o l o r i n sugars during storage. In order to understand the mechanism of c o l o r formation or the chemistry of the c o l o r a n t i t s e l f i n sugar products, a r a p i d chromatographic method f o r i s o l a t i o n of colorants from sugar samples was developed. The mini-column adopted, a SEP-PAK Cie c a r t r i d g e , i s able to i s o l a t e colorants from sugar products of very low c o l o r . By t h i s technique, h i g h l y concentrated and sugarfree c o l o r s o l u t i o n s can be obtained from 100 g of sugar sample; s u f f i c i e n t c o l o r a n t s were recovered on the SEP-PAK Cie column for HPGPC studies by t r e a t i n g 100 g of sugar samples. This high e f f i c i e n c y of c o l o r concentration and sugar removal of the c a r t r i d g e i s a consequence of i t s small column s i z e and w e l l placed nonpolar packing. The small s i z e of the c a r t r i d g e permits r a p i d o p e r a t i o n , and only a small amount of eluent i s needed f o r r e l e a s i n g the adsorbed c o l o r a n t s . Consequently, colorant i s concentrated a f t e r e l u t i o n . The nonpolar packing of the c a r t r i d g e can r e t a i n organics of moderate to low p o l a r i t y d i s o l v e d i n a p o l a r moving phase, yet r e j e c t very p o l a r compounds such as sugar completely. Because l i t t l e sugar colorant i s very p o l a r ,



Ash %

0.037

0.042

0.048

0.045

0.054

0.067

0.062

0.071

Sugar sample

1A

IB

2A

2B

3A

3B

4A

4B

0.046

0.039

0.045

0.044

0.036

0.041

0.038

0.032

Reducing Sugar %

99.84

99.83

99.82

99.81

99.83

99.80

99.82

99.81

50

44

24

20

36

30

28

20

Starch ppm

47

29

68

52

45

30

43

38

Protein ppm

0.038

0.037

0.031

0.032

0.023

0.036

0.044

0.035

Moisture %

Chemical Analyses of Sugars

Pol %

Table 1.

61.70

94.18

57.96

92.55

62.14

71.44

60.08

69.75

Color (Lumetron)



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Color

Development

in

Sugars

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s i g n i f i c a n t amounts of colorants can be i s o l a t e d free from sugar. Thus a n a l y s i s of colorant by methods such as HPGPC or TLC can be conducted with higher r e s o l u t i o n and l e s s e r d i f f i c u l t y . HPGPC f r a c t i o n a t i o n of sugar colorant i s compared with that of colored sugar i t s e l f i n Figure 2. This suggests that the p r i n c i p a l peak of colorant i s indeed present i n the o r i g i n a l sugar, and that the c o l o r a n t s adsorbed by the SEP-PAK Ci β c a r t r i d g e possessed the same range of molecular s i z e and p o l a r i t y as the o r i g i n a l sugar s o l u t i o n . Hence the treatment i s considered n o n s e l e c t i v e and gentle f o r i s o l a t i n g c o l o r a n t s . Figure 2 a l s o i l l u s t r a t e s the adverse e f f e c t of sugar i t s e l f , and the d i f f i c u l t y of f r a c t i o n a t ing very l i g h t - c o l o r e d samples, even when monitoring i s i n the UV region of the spectrum. The a p p l i c a t i o n of the SEP-PAK Cie c a r t r i d g e as a t o o l f o r sugar colorant i s o l a t i o n gives e x c e l l e n t e f f i c i e n c y and s i m p l i c i t y . F r a c t i o n a t i o n of sugar colorants based on molecular s i z e i s u s u a l l y conducted with Sephadex g e l . Many i n v e s t i g a t o r s (11,12) have employed t h i s method f o r sugar colorant s t u d i e s . Usually three to four peaks are obtained a f t e r at l e a s t 2 h of s e p a r a t i o n . In t h i s experiment, HPGPC was adopted f o r the f r a c t i o n a t i o n of i s o l a t e d c o l o r a n t s . R e p e a t a b i l i t y and e f f i c i e n c y of HPGPC were good. F r a c t i o n a t i n g one sample i n t o s i x peaks detected at 436 nm took only 20-40 min. HPGPC colorant molecular p r o f i l e s of f r e s h and stored p l a n t a t i o n white sugar samples from the carbonation process and the s u l p h i t a t i o n process showed s i g n i f i c a n t d i f f e r ences, as shown i n Figure 3. S u l f i t a t i o n sugar contained more high-molecular-weight c o l o r a n t s , and l e s s low-molecular-weight c o l o r a n t s , than carbonation sugar. F r a c t i o n s (2-3 ml and 4.5-5.5 ml) c o l l e c t e d and analyzed by UV absorption spectra also showed d i f f e r e n c e s as shown i n Figure 4. The carbonation sugar colorant of the 4.5-5.5-ml f r a c t i o n had an absorption maximum at 283 nm, and a stronger t o t a l absorbance around 280 nm. A f t e r storage, the low-molecular-weight c o l o r a n t s of carbonation sugar had become high-molecular-weight c o l o r a n t s , as shown i n Figure 5. Compared with Figure 3, these data i n d i c a t e d that the low-molecular-weight c o l o r a n t s undergo slow polymerization leading to the development of c o l o r i n sugar c r y s t a l s during storage, p a r t i c u l a r l y carameliz a t i o n . The calcium carbonate present i n the c r y s t a l s catalyzes the c a r a m e l i z a t i o n of the reducing sugar. UV spectra of the colorant f r a c t i o n s (2.5-3.5 ml) from HPGPC f o r s u l f i t a t i o n sugar and carbonation sugar a f t e r storage (Figure 6) show carbonation sugar colorant had stronger absorption at 280-285 nm, and more 5-HMF, caramel, humic a c i d s and melanoidins; f u r t h e r studies w i l l be done to c h a r a c t e r i z e these compounds. UV absorption maxima of these "compounds" are at 265 nm (humic acid), 282 nm (caramel), 285 nm (5-HMF) and 300 nm (melanoidins), as shown i n Figure 7. In carbona t i o n , a high percentage of reducing sugar d e s t r u c t i o n i n the h i g h l y a l k a l i n e c o n d i t i o n of the f i r s t carbonation stage helped cause formation of c o l o r - b e a r i n g compounds of the 5-HMF and humic c a t e g o r i e s , and these reducing sugar degradation products play an )



MAILLARD REACTIONS

2.0

Figure 3.

HPGPC

4.0 Retention volume (ml)

6.0

colorant molecular profiles of sulfitation sugar (—; and carbonation sugar ( ) before storage.



CHENG ET AL.


220

240

260

280

300

320

340

360

Wavelenqth (nm) Figure 4. UV spectra of colorant fractions from HPGPC for fresh sulfitation sugar and carbonation sugar. Key for 2-3-mL fraction: — · —, sulfitation sugar colorant; , carbonation sugar colorant. Key for 4.5-5.5-mL fraction: - · · -, carbonation sugar colorant; —, sulfitation sugar colorant.

Figure 5.

HPGPC

colorant molecular profiles of sulfitation sugar (—) and carbonation sugar ( ) after storage.


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MAILLARD REACTIONS

100Γ

90h


80|-

240

260

280

300

320

340

360

Wavelength (nm) Figure 6.

Figure 7.

UV spectra of colorant fraction (2.5-3.5-mL) from HPGPC for sulfi tation sugar (—) and carbonation sugar ( ) after storage.

UV spectra of main colorants. Key: - · · -, 5-HMF; , humic acids; and —, melanoidins.

— · —, caramel;



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101

important role in melanoidin and caramel formation in the sugar crystal during storage. They are formed by slow polymerization of reducing sugars present as impurities along with ash constituents, imbedded between the crystal layers. The nature of the ash constituents played an important role in the rate of deterioration of the sugar. Carbonates evidently catalyze the caramelization much faster than sulphites, leading to faster development of color in carbonation sugars. Sulphitation inhibited the melanoidin formation, presumably by blocking the carbonyl function. Reduction of carbonate content by replacing the second carbonation stage with sulphitation or phosphatation, and improvement of first carbonation technique so as to minimize reducing sugar destruction, were recommended to improve the keeping quality of the carbonation sugars. Conclusions A rapid method developed for sugar colorant isolation by adsorption column chromatography has been proven to possess excellent efficiency and simplicity. The mini-column adopted, a SEP-PAK Cie cartridge, is able to isolate colorants from crystal sugar of very low color. Colorants adsorbed by the column cover the same range of molecular size and polarity as the colorants before isolation. By using this technique, highly concentrated and sugar-free color solution can be obtained simultaneously from sugar products. For fractionation of the isolated colorants, a HPLC system for gel permeation chromatography was developed. The efficiency is considered higher than for the conventional gel filtration technique. Spectrophotometry and chromatographic studies indicated that the color-bearing compounds responsible for the sugar color development were 5-HMF, caramel, humic acids, and melanoidins. Reduction of these compounds by improving first carbonation technique to minimize reducing sugar destruction would improve color development of the carbonation sugar. Literature Cited 1. Keely, F.H.C.; Brown, D. W. "Sugar Technology Reviews"; Hutson, M.; McGinnis, R. Α., Eds.; Elsevier: Amsterdam, 1978; Vol. 6, p. 1-41. 2. Gillett, T. R. "Principles of Sugar Technology"; Honig, P., Ed.; Elsevier: Amsterdam, 1953; Vol. 1, p. 230-234. 3. Tsuchida, H.; Komoto, M. Proc. Res. Soc. Japan Sugar Refin. Technol. 1969, 21, p. 89-94. 4. Chen, W. Proc. 14th Congr. Intern. Soc. Sugar Cane Technol. 1971, p. 1564-1568. 5. Monterde, J.; Ruso, R.; Fajardo, R. Bol. Cienc. Tech. Univ. Central Las Villas 1970, 5 (6), 27-41. 6. Cortis-Jones, B. Intern. Sugar J . 73 (7),219.



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7. Ramon, S.; Salahuddin, S. Proc. 15th Congr. I n t e r n . Soc. Sugar Cane Technol. 1974, p. 1412-1424. 8. Payne, J . H. "Sugar Cane Factory A n a l y t i c a l C o n t r o l " ; E l s e v i e r : Amsterdam, 1969, p. 632-636. 9. Lowry, O. H.; Rosebrough, N. J . ; F a r r , A. L.; R a n d a l l , R. L. J . B i o l . Chem. 1951, 193, 265. 10. Colburn, C. R.; Schoch, T. J. "Methods i n Carbohydrate Chemistry"; W h i s t l e r , R. L., Ed.; Academic, New York, 1964, V o l . 4, p. 157-160. 11. Tu, J . C.; Kondo, Α.; Sloane, G. E. Proc. 15th Congr. I n t e r n . Soc. Sugar Cane Technol. 1974, p. 1393-1401. 12. L i n e c a r , D.; Paton, N. H.; Smith, P. Proc. 1978 Tech. Sess. Cane Sugar R e f i n . Res. 1979, p. 81-91. RECEIVED November 3, 1982


Studies on the Color Development in Stored Plantation White Sugars

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