Evaluation of Sugarcane Juice Quality As Influenced by Cane Treatment and Separn Concentrations

EVALUATION OF SUGARCANE JUICE QUALITY AS INFLUENCED BY CANE TREATMENT AND SEPARN CONCENTRATIONS

By Ghada A/Rahman A/Razig El Sheikh B.Sc. (Science) Department of Rural Education, Extension and Development University of Ahfad

A thesis submitted to University of Khartoum in partial fulfilment for the requirement of the degree of Master of Science in Agriculture

Supervisor Prof. Elfadil Elfadl Babiker

Department of Food Science and Technology Faculty of Agriculture University of Khartoum

January 2009

DEDICATION To my husband To my parents To my sisters and brothers To Abbass family To those whom I will never forget

ACKNOWLEDGEMENT

First I thank Allah with his wills this work completed.

Thank my family, who were ready to render much assistance, I asked for to complete this work.

Many people made great efforts and support me during study. My sincere gratitude to:

* The study supervisor, Professor Elfadil Elfadl Babiker, Faculty of Agriculture, University of Khartoum, for extending research works and to the final writings of the thesis, that allowed this study to reach conclusion.

* Syd/Mohamed Ahmed Fadlased, Kenana Human Resource General Manager. * Dr. Elbashir Ali Hamad, Former Kenana Ex-Training Manager, for invaluable guidance throughout the study which gave confidence to execute it. * Syd/Ibrahim Mustafa, Former Kenana Sugar Factory Manager. * Dr. Makawi Awad A/Rahman, Kenana Sugarcane Researcher, for extending research works to cover essential areas and helping the final writing of the thesis. * Dr.Kamal Sliman, Food engineering and Technology, University of Gezira, for follow-up and thesis revision. * Dr. Ibrahim Doka, Kenana Sugarcane Researcher, for research analysis. * Syd/Dafalla Hashim, Kenana Quality Control Manager, for providing research's requirements. * Kenana Sugarcane Research and Development Department * Kenana Quality Control Department. * My colleagues in Kenana Training Centre.

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LIST OF CONTENTS Page DEDICATION i ACKNOWLEDGEMENT ii LIST OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi ABSTRACT vii ARABIC ABSTRACT viii CHAPTER ONE: INTRODUCTION 1 CHAPTER TWO: LIETERATURE REVIEW 3 2.1. Sugarcane and Cane Juice Composition 3 2.1.1 Sugarcane 3 2.1.2 Cane Juice from the Mills 4 2.2 Characteristics of Cane Juice 4 2.3 Chemistry of coloured Non-sugars 4 2.4 Coloured non-sugar originally existing in sugarcane 5 2.4.1 Chlorophyll 6 2.4.2 Anthocyanin 6 2.4.3 Saccharetin 6 2.4.4 Tannins 7 2.5 None coluored in cane which may develop colour 7 2.5.1 Polyphenols 8 2.5.2 Amino compounds 8 2.6 Coloured non-sugars from sugar decomposition products 8 2.6.1 Caramel 9 2.7 Sugar decomposition products 9 2.8 Reaction products between reducing sugars and amino compounds 9 2.9 Physical and chemical properties of coloured non-sugars 10 2.9.1 Inversion 10 2.9.2 Reaction with phenols 10 2.9.3 Reaction with amines 11 2.9.4 Reaction with reducing agents 12 2.9.5 Reactions with oxidizing agents 12 2.9.6 Reaction with aldehydes 12 2.9.7 Effect of pH on colour 12 2.10 Colour developments 13 2.10.1 Raw sugar colour 14 2.10.2 Coluor development in processing raw cane sugar 15 2.10.3 Colour development in white sugar 15 2.11 Removal of colour by precipitate and adsorbents 15 2.11.1 Lime 16 2.11.2 Phosphoric acid 17 2.11.3 Flock conditioners 19 2.12 Definition 19 2.12.1 Primary juice (crushed juice) 19 2.12.2 Secondary juice (mixed juice) 19

2.12.3 Clarified juice 19 2.12.4 Imbibitions 20 2.12.5 pH 20 2.12.6 The sucrose (POL) 20 2,12,7 The dry matter content (brix) 29 2.12.8 Reducing sugar (R.S) 20 2.12.9 Purity 21 2.12.10 Turbidity 21 2.12.11 Colour 21 CHAPTER THREE: MATERIALS AND METHODS 22 3.1 Materials 22 3.2 Methods 22 3.2.1 Determination of pH 22 3.2.2 Determination of the sucrose content (pol) 24 3.2.3 Determination the dry matter content (BRIX) 25 3.2.4 Determination of reducing sugar (RS) 25 3.2.5 Determination of the colour value 26 3.2.6 Turbidity determination 27 3.2.7 Tannin determination 28 3.2.8 Total polyphenols determination 29 3.2.9 Saparan dose experiment 30 3.3 Statistical analysis 32 CHAPTER FOUR: RESULTS AND DICUSSIONS 33 4.1 Effect of milling on the proximate composition (%) of green and burned cane 33 4.2 Effect of processing on quality parameters of burned cane juice 36 4.3 Colouring substances in the raw materials and processed 38 4.4 Effect of different doses of separan on juice quality parameter 41 4.5 Effect of different doses of separan on polyphenols and tannins levels 42 CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS 45 5.1 Conclusion 45 5.2 Recommendations 46 REFERENCES 47

LIST OF TABLES

Table Page

1 Effect of milling on the proximate composition (%) of green and burned cane 34

2 Effect of processing on quality parameter of burned cane juice 37

3 Colouring substances in the raw materials and processed

4 Effect of different doses of Separan on juice quality parameters 42

5 Effect of different doses of Separan on polyphenols and tannins levels 45

LIST OF FIGURES

Figure Page

1 Cane samples 23

2 Sedimentation study apparatus in operation 31

3 Sample of green and burned cane 35

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EVALUATION OF SUGARCANE JUICE QUALITY AS INFLUENCED BY CANE TREATMENT AND SEPARN CONCENTRATIONS M.Sc. (Thesis) By Ghada A/Rahman A/Razig El Sheikh Abstract: The study is conducted to evaluate juice quality parameters ((percentage of cane )pol, brix, … etc) and the level of colouring materials in crushed cane (green or burned) crushed, mixed and clarified juice and to evaluate the effect of low doses of separan on juice quality and colour. The range of pol (sucrose) for juice treated 10.18 to 15.75%, brix ranged from 12.86 to 20.93%, purity ranged from 82.18 to 95.18%, pH ranged from 5.40 to 7.60, reducing sugar was ranged from 0.58 to 1.10%, turbidity was ranged from 4.96 to 9.60 NTU(nephlo turbidity unit) and colour was ranged from 2600 to 14692 ICUMCA. The study revealed that both the green cane and clarified juices and lime (temperature treatment) had significant (P≤0.05) higher colour readings compared to that of the burned cane, mixed and crushed juice. Likewise, the highest concentrations of colouring materials (polyphenols and tannins) were recorded in green 0.216, crushed 1.189 and final molasses 0.218. Addition of separan at very low concentration (0.015 ppm) was observed to reduce the colouring matter compared to the standard (3 ppm) concentration applied. The results obtained indicated that the juice colouring matter (polyphenols and tannins) levels had been greatly reduced during treatment.

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ﺘﻘﻴﻴﻡ ﺠﻭﺩﺓ ﻋﺼﻴﺭ ﺍﻟﻘﺼﺏ ﺍﻟﻤﻌﺎﻤل ﺒﺘﺭﺍﻜﻴﺯ ﺍﻟﺴﺒﺭﺍﻥ (ﺃﻁﺭﻭﺤﺔ ﻤﺎﺠﺴﺘﻴﺭ) ﻏﺎﺩﺓ ﻋﺒﺩ ﺍﻟﺭﺤﻤﻥ ﻋﺒﺩ ﺍﻟﺭﺍﺯﻕ

ﺍﻟﻤﺴﺘﺨﻠﺹ: ﺃﺠﺭﻴﺕ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻟﺘﻘﻴﻴﻡ ﺍﻟﻌﺼﻴﺭ ﻤـﻥ ﺤﻴـﺙ ﺍﻟﺒـﻭل (ﻨـﺴﺒﻪ ﺍﻟﺴﻜﺭﻴﺎﺕ) ﻭﺍﻟﺒﺭﻜﺱ (ﻨﺴﺒﻪ ﺍﻟﻤﻭﺍﺩ ﺍﻟﺼﻠﺒﻪ ) ﻭﻏﻴﺭﻫﺎ ﻤﻥ ﺍﻟﺘﺤﺎﻟﻴل ﻭ ﺃ ﻴ ﻀ ﺎﹰ ﺍﻟﻤﻭﺍﺩ ﺍﻟﻠﻭﻨﻴﺔ ﻓﻲ ﻋﺼﻴﺭ ﺍﻟﻘﺼﺏ ﺍﻷﺨﻀﺭ ﻭﺍ ﻟﻤﺤﺭﻭﻕ ﻭﺍﻟﻌﺼﻴﺭ ﺍﻷﻭﻟـﻲ ﻟﻠﻁـﻭﺍﺤﻴﻥ ﻭﺍﻟﻤﺨﻠﻭﻁ ﻭﺍﻟﻨﻘﻲ، ﻭﺘ ﻘﻴﻴﻡ ﺍﺜﺭ ﺍﻗل ﺠﺭﻋﺔ ﻤﻥ ﺍﻟﺴﺒﺭﺍﻥ ﻋﻠـﻲ ﺠـﻭﺩﺓ ﺍﻟﻌـﺼﻴﺭ ﻭﺍﻟﻠﻭﻥ. ﺍﻟﺘﺤﺎﻟﻴل ﻟﻌﺼﻴﺭ ﺍﻟﻘﺼﺏ ﺃﻭﺠﺩ ﺍﻟﺒﻭل ﻓـﻲ ﻤـﺩﻱ ﻤـﻥ 10.18 ﺇﻟـﻲ 15.75%، ﺒﺭﻜﺱ 12.86 ﺇﻟﻲ 20.93%، ﺩﺭﺠﺔ ﺍﻟﻨﻘﺎﺀ 82.18 ﺇﻟﻲ %95.18، ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻥ ﺍﻴﻭﻥ ﻤﻥ 5.40 ﺇﻟﻲ 7.60،ﺍﻟﺴﻜﺭ ﺍﻟﻤﺨﺘﺯل ﻤﻥ 58. ﺇﻟﻲ %1.10 ، ﻭﺍﻟﻠـﻭﻥ ﻤـﻥ 2600 ﺇﻟـﻲ NTD 14692 ﺍﻟﻌﻜﺎﺭﺓ ﻤﻥ 4.94 ﺇﻟـﻲ 9.60 ICUMCA.

ﺍﻟﻨﺘﺎﺌﺞ ﺘﻭﻀﺢ ﺍﻟﻌﻼﻗﺔ ﺒﻴﻥ ﺍﻟﻘﺼﺏ ﺍﻷﺨﻀﺭ ﻭﺍﻟﻌﺼﻴﺭ ﺍﻟﻨﻘﻲ ﺤﻴﺙ ﻭﺠﺩ ﺃﻨﻬﻤﺎ ﻴﺤﺘﻭﻴﺎﻥ ﻋﻠﻰ ﻨﺴﺒﺔ ﻋﺎﻟﻴﺔ ﻤﻥ ﻗ ﺭﺍﺀﺓ ﺍﻟﻠﻭﻥ ﻤﻘﺎﺭﻨـﺔ ﺒﺎﻟﻘـﺼﺏ ﺍﻟﻤﺤـﺭﻭﻕ ﻭﺍﻟﻌﺼﻴﺭ ﺍﻟﻤﺨﻠﻭﻁ ﻭﺍﻟﻌﺼﻴﺭ ﺍﻷﻭﻟﻲ.

ﻋﻠﻰ ﻨﻔﺱ ﺍﻟﻨﻤﻁ ﻨﺠﺩ ﺃﻋﻠﻰ ﺘﺭﻜﻴﺯ ﻟﻠﻤـﻭﺍﺩ ﺍﻟﻠﻭﻨﻴـﺔ ﺍﻟﺒـﻭﻟﻲ ﻓﻴﻨـﻭﻻﺕ ﻭﺍﻟﺘﺎﻨﻴﻨﻴﺎﺕ ﺴﺠﻠﺕ ﻓﻲ ﺍﻟﻘﺼﺏ ﺍﻷﺨﻀﺭ (0.216) ﻭﺍﻟﻌﺼﻴﺭ ﺍﻷﻭﻟـﻲ (1.189) ﻭﺍﻟﻤﻭﻻﺱ ﺍﻟﻨﻬﺎﺌﻲ (0.218). ﺍﻟﺩﺭﺍﺴﺔ ﺘﺅﻜﺩ ﺃﻥ ﺍﻟﻤﻭﺍﺩ ﺍﻟﻤﻠﻭﻨﺔ ﻟﻠﻌﺼﻴﺭ ﺘﻨﺨﻔﺽ ﺘﺩﺭﻴﺠﻴﺎﹰ ﺍﺜﻨﺎﺀ ﺍﻟﻤﻌﺎﻤﻼﺕ.

ﻨﺘﻴﺠﺔ ﺍﻀﺎﻓﺔ ﺍﻟﺴﺒﺭﺍﻥ ﻋﻨﺩ ﺃﺩﻨﻲ ﺘﺭﻜﻴﺯ (ppm 0.015) ﻤﻘﺎﺭﻨﺔ ﺒﺎﻟﻤﻘﻴﺎﺱ ﺍﻟﻤﻌﻴﺎﺭﻱ (ppm 3) ﺍﻟﻤﺴﺘﺨﺩﻡ ﻓﻲ ﺍﻟﻤﺼﻨﻊ ﻗﻠﻠﺕ ﻤﻥ ﻤﺴﺘﻭﻱ ﺍﻟﻤﻭﺍﺩ ﺍﻟﻠﻭﻨﻴﺔ ﻓﻲ ﺍﻟﺒﻭﻟﻲ ﻓﻴﻨﻭﻻﺕ ﻭﺍﻟﺘﺎﻨﻴﻨﺎﺕ ﻭﺼﺎﺤﺏ ﺫﻟﻙ ﺠﻭﺩﺓ ﻋﺎﻟﻴﺔ ﻤﻥ ﻨﻘﺎﺀ ﺍﻟﻌﺼﻴﺭ ﻭﻗـﺭﺍﺀﺓ ﺃﻗل ﻟﻠﻭﻥ.

CHAPTER ONE

INTRODUCTION

Colour of the sugar crystals is an important factor that determines its value in the market and acceptability for various uses. Formation of colour takes place in the juice syrup, and final products because of caramalisation and melunoidius formation. Besides, these juices contain a series of natural colouring compounds and other constituents such as polyphenols, amino acids, etc. All these constituents can not be eliminated during the process of clarification and generate colour during the post-clarification process up to the raw cane.

Mathur (1993) reported that polyphenols formed brown iron complexes where as others generate colour by polymerization due to the effect of high temperatures used in the processing operations. Although these colouring factors and constituents can not be eliminated during processing, efforts can be made to reduce the formation of brown iron complexes and polymerization of colouring matters by suitable measures.

Colouring matters are found in sugarcane, as in all growing plants. In milling, they are extracted with the cane juice and constitute a portion of the non-sugars to be contended with in subsequent processing of the sugar. Somasekhar (2001) stated that in addition, other colouring materials are formed in the manufacturing and refining operations as a result of chemical reactions between certain non-sugar materials present or developed during the process. The presence of such materials depend on type of cane, soil, and growing

1 conditions, geographical area, and the milling and refining process employed.

The substances responsible for the colour of the sugar are normally classified as non-sugar impurities. The production of both raw and refined sugars and the removal of the coloured impurities become extremely important; particularly in view of the increasing demands for exceptionally high quality white refined sugar (Srivastava, 2006).

Objectives:

The present study was conducted to meet the following objectives:

* To determine parameters that characterizes the sugar juice from the initial stage to the clarification stage.

* To study the effect of using different concentrations of Separan on the colour of the juice.

* To compare different parameters of green and burned cane juice.

CHAPTER TWO LITERATURE REVIEW

2.1 Sugarcane and cane juice composition: 2.1.1 Sugarcane: Basically the sugarcane stalk consists of the outer rind of thick walled cells and the softer parenchyma tissue and vascular bondless within. The rind and vascular bundles constitute the fibrous portion of the fibre whilst the parenchyma constitutes the pith. The materials together make up the fibre of the cane as determined by analytical methods (George et al., 1997). The parenchyma cells contain sugar rich juice and since these cells are easily ruptured the is liquid from these cells which is first expressed when the cane stalk is crushed. This is then the origin of the high purity first expressed juice. The remaining juice is found in the relatively sturdy vascular bundles carrying nutrients between the roots and leaves of the plant. The juice is dilute, of low purity and of variable composition. It is difficult to express, hence the last juice expressed is always of low purity. Also, non-sugars from the parenchyma cells are of such a nature that they are easily removed during clarification whereas non-sugars in the juice from the vascular bundles are not. The percentage of sugar (commonly called "polarization") in the cane varies from 8 to 16% and depends to a great extent on the variety of cane, its maturity, the condition of the soil, the climate and agricultural practices followed (Blackburn, 1984).

2.1.2 Cane juice from the mills: Cane juice is an easily foaming turbid liquid, ranging in colour from light grey to dark green. Fresh cane juice is slightly acidic and because of the colloidal matter content is not easily filterable. The green colouration is due to a combination of dyes from cane ferric salts in the juice. Ferric salts in turn react with tannins in the juice producing the green colouration. Chen et al. (1993) stated that cane Juice contains sucrose, reducing sugars, inorganic-salts, organic- salts, organic acids, pectin, gums, proteins, dyes, tannin and iron compounds in solution in add rife it contains bagasse, sand, clay, chlorophyll, wax, albumen, air and soil in suspension. 2.2 Characteristics of cane juice: Cane juice has an acidic reaction with the pH range 4.9 to 5.5. The cane juice is opaque owing to the presence of silica and colloidal substances such as waxes, proteins, gums, starch and they impart turbidity to the juice. These colloids do not settle ordinarily unless conditions are altered. The application of heat or addition of chemicals (electrolytes) brings about flocculation or coagulation (Perry's, 1988). Finar (1998) stated that the rind cells of sugarcane stalks contain a mixture of two colouring matters, the chlorophyll and the anthocyanin. The fibre of the cane contains saccharetin and the tops and buds of the plant contain tannins and several other colouring matters but very little are known about them. These pass with the juice on extraction. 2.3 Chemistry of coloured non-sugars: The general nature of the organic non-sugar substances responsible for the colouring in sugarcane and in raw cane juice has been reported in the literature to some degree during the past several

4 decades. Kul (1989) discussed that the colour of the cane juice may have two origins: (a) Colouring matters from the cane itself. This may have four origins:

(1) Chlorophyll, (2) anthocyanin, (3) saccharetin and (4) tannins.

(b) Chemical decomposition. This may have three origins:

(1) Colouration of the juice due to the decomposition of its constituents by the action of lime or of heat or of both. (2) colouration of the juice due to the presence of soluble iron salt (ferric) from equipment, because of reaction with polyphenols, and (3) colouration of the juice due to reaction of non-sugars with other substances.

2.4 Coloured non-sugar originally existing in sugarcane: While the literature refers to the colour substances in cane by a variety of names, there appear to be about six principal types, i.e. chlorophylls, xanthophylls, carotenes, sachharetin, tannins and Anthcyanins.

The colour produced by the pigments is dependent upon the hydrogen ion concentration of the cell sap. When the sap is acidic the colour is red and when it is alkaline the colour is blue (Srivastava, 2006).

Chlorophyll, carotene and saccharetin are harmless colouring matter in white sugar manufacturing. They are insoluble in sugar solutions and therefore are easily removed by clarification of the juice (John, 1988).

2.4.1 Chlorophyll: This is present in every green plant. It is a harmless colouring matter in white sugar manufacture since it does not react with lime aid and acids have no reaction with it. It is insoluble in water and in sugar solutions but soluble in alcohol, ether... etc. It is in colloidal nature and exists as suspension in cane juice, removed by filtration after clarification of the juice is done without affecting the colour of the sugar subsequently produced.

2.4.2 Anthocyanin: This is present only in certain dark varieties of cane. Unlike chlorophyll, it is readily soluble in water and cane juice and also during the process of milling, it passes almost completely into the cane juice giving a dark colouration to the juice. The purple colour of anthocyanin solution is changed into dark green by the addition of lime. The pigment, if present in small amounts, is precipitated by a small amount of calcium hydroxide (lime) used for defecation. If the pigment is of the amount normally found in dark varieties, then the quantity of lime used in defecation is insufficient for the elimination of the colouring bodies. More lime has to be added to precipitate these pigments completely (John, 1988). 2.4.3 Saccharetin: The saccharetin is found impregnated in the fiber of the cane. This colouring substance cannot be extracted with water or sugar solution from the fiber, but when these media are rendered alkaline with calcium hydroxide (lime) or with any other alkaline body, the hitherto colourless saccharetin becomes yellow and extracted by the liquid. In the raw juice, there are fine particles of bagasse in

6 suspension and when lime is added the yellow pigment is extracted. It is, therefore, important to remove as much of 'cush-cush' (fine bagasse) from juice as possible, before the juice is limed so that the saccharetin is prevented from entering the clarified juice. Saccharetin is comparatively a harmless pigment, as it becomes colourless again in neutral or acidic media below pH 7.0 (John, 1988).

2.4.4 Tannins: These bodies are located in the actively vegetative portion of the cells, especially of the 'tops' and the buds. It is soluble in juice. It is green but when reacts with iron salts present in the juice it becomes dark in colour. On heating it decomposes with the formation of catechol and combines with alkalis to form protocatechnic acid. Heating in acid solution produces pholobaphene and protocatechnic acid which is similar to saccharetin. John (1988) stated that with tannin is normally removed by the addition of Saparan to the juice in the clarification stage.Saparan enhances the formation of flogs containing tannins and those eventually settle at the bottom of the clarifier. The flocks are then pumped out to a rotary vacuum filter.

2.5 None coloured in cane which may develop colour Numerous non sugar materials have been described in the literature as being present in the cane in colourless form but when combined or reacted with other substances form colouring matter. Probably the most significant of such materials can be classified in two general groups, i.e. polyphenols and amino compounds (Saharia, 1997).

2.5.1 Polyphenols: Polyphenols react with iron (ferric) and oxygen particularly in alkaline solutions and form dark coloured products. In the raw juice it is attached to the bagasse particles and can therefore be separated during clarification.

2.5.2 Amino compounds: Cane juice contains nitrogenous bodies such as albuminods, ammonia, amino acids and amides. These compounds can react with reducing sugars and form coloured compounds.

2.6 Coloured non-sugars from sugar decomposition products: Rein et al. (2007) stated that with the processing of cane juices, both the sugars and non-sugars materials are subjected to heat, varying pH, air, iron "from equipment", added chemical compounds "such as lime", etc. All these factors have a distinct effect on the development of colour. Some of this colour may be developed from the chemical reaction with non-sugars component of the cane juice, and some may occur as a result of decomposition products formed. These coluored compounds vary in type and include products from caramalisation, decomposition of the sugars and the products subsequently formed with other compounds. Because these reactions are so inter-related, it is some what difficult to make any separation into particular groups. For example, sucrose solutions invert when becoming acid, the reducing sugars formed decompose with heat and in alkaline solutions, reactions occur with other non-sugar materials such as poly phenols, amino compounds, etc. The nature and extent of these various reactions and the final colour compounds formed are dependent upon the conditions involved.

2.6.1 Caramel: When heated sugar juice temperature is about 200C°a dark colored material is formed. This is apparently due to hydration and condensation reactions of the heated sugars. Caramels are formed from sucrose as well as glucose and fructose. The composition of the caramel depends upon temperature, pH and time of heating.

2.7 Sugar decomposition products: When sucrose in solution is subjected to high temperatures and acid conditions it hydrolyzes to form the reducing sugars, D-glucose and D-fructose. With prolonged heating and under strongly alkaline conditions, these hexoses decompose. The resulting products are brown colored and acids, causing further inversion of sucrose. However, in strongly alkaline solutions, dark coloured products are formed from decomposition of the reducing sugars. At low temperature the colour formation is much less than at high temperature. In the later case, the brown reaction products have the disadvantage of breaking up into acidic secondary products permitting further inversion (Hunsigi, 1993).

2.8 Reaction products between reducing sugars and amino compounds: Mathur (1993) stated that amino acids react or condense with dextrose dehydration products such as hydroxymethyl, furfural, laevulin acid and hydroxymethyl fluoric acid to form coluored bodies and this reaction is not prevented by the neutralization of the amino acids.

The colour development is distinguishable in heated solutions of sugar but is much more apparent when amino compounds, such as asparagines or aspartic acid are present with reducing sugars. The importance of those compounds in cane products will only appear to be pronounced where large amounts of reducing sugars are formed and subsequently destroyed. 2.9 Physical and chemical properties of coloured non-sugars: 2.9.1 Inversion: Joachim et al. (2000) stated that when sucrose is inverted, by means of an acid or enzyme, the molecule is broken up to give glucose and fructose.

C12H22O11 + H2O C6H12O6 + C6H12O6 Sucrose water glucose fructose

Inverted sugars This reaction is known as hydrolysis or inversion and proceeds at varying rates depending on conditions such as temperature, time and pH. These two reducing sugars develop colour when subjected to heat and alkaline conditions. If such conditions are extreme the reducing sugars will be destroyed and various reaction products are formed. These reaction products unite readily with other compounds that may be present or occur in process, and the resulting substances are often the source of the colouring materials found in cane juice and raw sugar products. 2.9.2 Reaction with phenols: Very strong colour reactions are obtained between sugars and various phenols, when concentrated mineral acids are present. This is

due to the combination of the sugar decomposition products, such as furfural, with the condensation products from the phenol derivatives (Joachim et al., 2000). Reducing sugars combine with different polyphenols to form amorphous condensation products. An example of such a reaction with a rabinose is as follows:

C5H10O5 + C6H10O6 C11H14O6 + H2O Arabinose Resorcinol Arabinose Resorcinol Water

The colour obtained with different sugars may be found upon examination of the decomposition products obtained when heating in acid solutions. It is mentioned that true glycoside of various phenols and polyphenols are found in nature.

2.9.3 Reaction with amines: Sugars combine readily with amino compounds such as the aromatic amines, aniline, xylonite, and diphenylamine, in the presence of concentrated hydrochloric acid. The colour reaction is not as significant as in the case of polyphenols. They result from the uniting of the sugar decomposition products, furfural, methyl furfural, and hydroxymethyl furfural with the amines. =Examples of the reaction in the case of glucose are as follows:

H-C = O + N2NC6H5 H-C = N – C6H5 + H2O

(HCOH)4 ( HCOH)4

CH2OH CH2OH Glucose Aniline Glucose Aniline water

Various colours are obtained in such reactions. Glucose gives a green colour, fructose a pale yellow, etc. Reducing sugars also react easily with other nitrogen compounds such as phenyl hydrazine (in the hydrazones osazone reactions), hydroxyl-amine (oxide reaction) and urea "uried reaction", etc (Joachim et al., 2000).

2.9.4 Reaction with reducing agents: Reducing agents change the reducing sugars to alcohols. For example, sodium amalgam reduces mannose to the alcohol mannitol. When maintained slightly acidic sorbet is formed.

2.9.5 Reactions with oxidizing agents: Oxidizing agents such as nitric acid change reducing sugars into dibasic acids. Ketoses sugars decompose into acids such as oxalic and formic acids. With weak oxidizing agents, monobasic acids are obtained. Some of these acids react with ferric salt to form dark coluored compounds (Steindl, 2005).

2.9.6 Reaction with aldehydes: Reducing sugars react with various aldehydes, such as formaldehyde, benzaldehyde, furfural, etc. to form various condensation products. Some of these compounds are unstable and decompose into different acids. Coluored compounds can result from any number of these reactions.

2.9.7 Effect of pH on colour: It is well recognized that the colour of sugar products is greatly dependent upon the pH of the sugar solution. In general, the colour is lighter in acid solutions than in alkaline solutions. This has been observed in many decolourization processes. It has also been noted in

12 investigation on method of colour measurement. In the latter case, the practice often used is of adjusting solutions to a standard pH of 7 prior to colour determination in order to avoid the variable effect caused by different pH values (Griffiths, 1991).

2.10. Colour developments: The processing of sugar is generally carried out in two stages, the crystallization of the clarified, concentrated cane juice in the raw sugar and then refining of the raw sugar to produce white sugar. In clarifying the raw sugar juice, heat and lime increase the colour due to decomposition of the reducing sugars. The presence of iron from equipment tends to increase the colour further because of reaction with polyphenols. In the evaporation and crystallization steps colour may be formed from caramalisation and decomposition products due to overheating discussed by (Hugot, 1990).

Care is needed in the process to avoid colour formation, as far as possible. Various steps are usually adopted to minimize the formation of colour. The solutions are maintained close to pH 7 and excessive temperature is avoided. Clarification with lime, phosphoric acid and separan and decolourization with bone char, or comparable methods, are used to remove colour. In the latter cases temperature and pH are carefully controlled to prevent additional colour formation.

Zerban (2000) discussed colour development in processing of raw cane juice at one length. He pointed out the colour formation in liming raw cane juice and the general practice of not adding any more lime than necessary to secure a good precipitate. He stated that most of the colouring matter formed from the reducing sugars is removed with the precipitate but the clarified juice may be darker than the raw

13 juice due primarily to polyphenol- iron compounds. If an excess of lime is used, neutralization is necessary by means of carbonation or sulphitation and the precipitates formed strongly adsorb colour. In regard to colour changes during evaporation and crystallization of the juice, the colour development is largely due to caramalisation and Melonoids resulting from heating of the juice. Dissolving of iron also tends to increase the colour.

From the preceding it is evident that cane juices develop colour in processing. The extent of darkening depending upon the nature of the juice and the conditions of processing. It is apparent that excessive heat and alkalinity must be avoided to prevent excessive colour development. Juice Colour increased by 50 and 100% with trash and trash plus tops, respectively (Reid and Lionnet 1989).

2.10.1 Raw sugar colour: In general, most raw or intermediate sugar products have a yellow amber, or dark reddish brown colour. The amount and nature of this depends on the type of original colouring matter and the reactions occurred during processing. The various factors involved are the source of raw, operation conditions (pH, temperature, etc.); adsorbents used in processing and other such variables. The problem of colour in processing raw sugar is referred to by Halverson and Bollaert (1987). Particular note is made of the importance of colour quality and mentioned is made of two types of raw cane sugar, i.e. the grey and the red variety. He pointed out that the calmed grey raw as more common than red raw. The red colour is apparently caused by excessive liming.

The amount and nature of colour in a raw sugar is of extreme importance in refining operations. As indicated previously, the colour is dependent on many factors such as variety of cane, soil conditions, method of processing, etc. while the total amount of colour will vary considerably. The general colour character of present day raw is similar, this is observed from spectrophotometer analysis made by heath .

2.10.2 Coluor development in processing raw cane sugar: In general, some factors that are involved in the darkening of cane juice and raw sugar are also involved apply in refining operations. In the usual processes, the raw sugar is first washed melted, limed, filtered, and finally decolorized. Decolourization is accomplished by several means such as phosphoric acid, lime defecation, carbonation, bone char, decolorizing carbons. It is undoubtedly apparent that even though one of the primary objectives of the refining process is to eliminate colour, complete elimination is not all together practical. Furthermore, the colour formed during the process so condition must be carefully controlled to avoid excessive colour development (Hugot, 1986).

2.10.3 Colour development in white sugar: In general, the white sugars develop colour very slowly if stored in cool areas (Nelson, 2005). This is to be due expected as the amount of their low coluored of the non-sugars material. 2.11 Removal of colour by precipitate and adsorbents: The coluored non-sugars material present in cane juice cans be removed by various chemical or physical processes. This may involve

removal by precipitating agents such as lime, phosphoric acid and separan that are used in clarification, or adsorbents such as bone char that are used in purification (Spri, 2001).

While precipitating or adsorbing agents are used to remove colour, there is danger of forming more colour if they are not used under proper conditions. For example, excessive low pH and temperature can form highly coluored complexes from decomposition products resulting from destruction of reducing sugars, as previously indicated. It is therefore essential to maintain conditions that helped reducing sugars neither formed nor destroyed. In commercial practice, every effort is normally made to avoid such conditions that lead to formation of dark coluored compounds.

2.11.1 Lime: Lime is the most commonly used material in clarifying cane juice and is effective in removing insoluble colouring compounds. It is only slightly soluble in water but sucrose greatly increases its solubility. Spencer and Meade (2001) described the purification in cane juice as follow; lime is added in the range of 450-750g CaO per ton of cane to neutralize the organic acids originally contained in the juice, they depending upon conditions. This treatment forms a heavy complex precipitate, In addition to other non-sugar materials such as waxes and gums, the insoluble coluored compounds, some in combination with calcium, are precipitated and subsequently removed.

This is a very important process affecting the colour and quality of raw sugar produced.

The method of clarification has a considerable effect on the amount of soluble lime salts remaining in the clarified juice. Pieter (1995) stated that with increasing pH, the lime content increases, particularly above pH8. It is the P2O5 content at the juice has a significant effect, by lowering the pH due to combination of lime and phosphate.

Excessive lime addition must be avoided to minimize colour formation and prevent poor quality dark coluored sugars. This is usually accomplished by controlling the pH at 8.0 – 8.5.

The principle of using lime clarification of raw cane juices is to precipitate the impurities. The lime combines with both organic and in organic compounds present to form numerous insoluble calcium salts. Among the various substances precipitated may be some of the coluored compounds, although simple lime defecation is not as effective as phosphate in colour removal.

2.11.2 Phosphoric acid: For many years the effectiveness of phosphates in clarification and removal of colour has been recognized. It has been noted that cane juice having a high P2O5 content, clarify much more readily and are lighter in colour than those with a low P2O5 content. Usually, the amount of phosphate in cane juices is quite small, and the precipitate will be developed when adding phosphoric acid in conjunction with lime, particularly in the refining process. The particular reactions involved related to the formation of heavy tri-calcium phosphate (Ca3

(PO4)2) flocculent precipitate which not only occludes the impurities but also adsorbs much of the colouring matter Pieter (1995).

For good clarification it has been stated that the P2O5 content of the cane juice should be above 300-350ppm. However, some juices high P2O5 content do not always clarify readily, presumably due to the excessive colloidal matter present. The nature of the flock formation depends greatly on the pH and calcium content of the juice, although the effect of pH is less above pH7. Additions of P2O5 have been found to be effective in lowering the colour of juice and or sugar (Honig, 1995). In the use of phosphoric acid – lime defecation in refining raw cane sugar, the colouring material removed consists of most of the colloidal polyphenol iron compounds which give a greenish brown colour to the sugar liquor. They found that affination syrup treated with 0.1% P2O5 as compared to filtered syrup, both at pH 7.2, gave 20% more colour removal at 459mµ and 28% at 620mµ. It is obvious of that the degree of colour removal will vary widely, depending on the character of the raw product being treated.

Halverson and Bollaert (1987) commented on the effectiveness of phosphoric acid-lime in the defecation of refining syrup. Similar comment was made by Franken (2007) in pressure filtration of defecated liquors. the following colour removal on using different concentration of p2O5. % P2O5 % colour removal 0 0 0.1% 47% 0.2% 50% 0.3% 55% 0.5% 56%

2.11.3 Flock conditioners: Flock formation can be improved by the use of high molecular weight water soluble synthetic resins like "Separan". The use of polymers in the treatment of refrectometer juices has become normal practice and less expensive than correcting phosphate deficient juice by the addition of phosphates.

Separan is usually added to the limed juice at the flash tank at a concentration of 2 to 3 parts per million. However, the method of the addition of Separan is of great importance for achieving the required, clarification. Normally a stock solution of 0.5% is prepared, diluted further to a working solution of 0.05% and fed to the flash tank via a metering pump.

Certain functional groups in the Separan molecule ionize in water, to an anionic or cationic molecule on certain conditions such as the pH of the solution, presence of electrolytes etc.

Segments of the long molecule absorbed on to flock particles forming a molecular linkage between flock particles. The larger particles so formed are thus more readily precipitated (Konkani, 1998).

2.12 Definition: 2.12.1 Primary juice (crushed juice): All juice obtained from cane prior to dilution. 2.12.2 Secondary juice (mixed juice): Total juice out put of milling tan demes in clouding imbibitions water. 2.12.3 Clarified juice: The juice obtained as a result of the clarification process.

2.12.4 Imbibitions: Addition of dilution water or juice to bagasse being milled (Kenana Technical Manual, 1984). 2.12.5 PH: Hydrogen-ion concentration by a logarithmic scale (Saharia 1997) 2.12.6 The sucrose (POL): As defined by ICUMSA (2005) the pol (polarization) of solution, is defined as the concentration (in grams of solute per 100g of solution) of a solution of pure sucrose in water having optical rotation as the sample at the same temperature .For solution containing only pure sucrose in water, pol is measure of the concentration of sucrose present, for solution, for solution containing sucrose and optically active substances,pol represent the algebraic sum of the rotation of the constituents present.

2.12.7 The dry matter content (brix): According to ICUMSA methods (1998) the unit, brix is intended to represent the dry substance content% mass by mass. Chen et al. (1993) Stated that the brix is the percentage by weight of sucrose in pure sugar solution. As they mentioned, the percentage of solid dissolved could be determined by refrectometer either by direct sugar scale or refractive indices and percentage sucrose.

2.12.8 Reducing sugar (R.S): Reduced sugars (monosaccharide) are formed by dissociation of sucrose.

Sucrose ∆ Glucose + Fructose

-OH2 Reduced sugar

2.12.9 Purity: Purity is the percentage of sugar in brix (Chen and Chou, 1993). Blackburn (1984) mentioned that the purity of a solution containing sucrose is the proportion by weight of sucrose to all dissolved solids, expressed a percentage. Determination of reducing sugar (RS) in practice sucrose is estimated as POL and solid as brix. Pol % Apparent purity = X 100 Brix %

2.12.10 Turbidity: The method applies to the determination of turbidity in clarified juice and it an indicative of the efficiency of the clarification process (Perry's 1985). The method measures absorbance due to suspended solid in clarified juice. The turbidity index, (S), is defined as: S=A/B Where the effect of light absorption is assumed to be zero. A=Abs over at 420nµ B= the cell length in cm 2.13.11 Colour Colour= brix × factor × absorbance

CHAPTER THREE MATERIALS AND METHODS 3.1 Materials: Four types of sugarcane juice were collected and analyzed for different parameters of the juice e.g. pol (sucrose), brix (dry matter), colour, turbidly, pH, etc. Out of 3 samples, uses collected from the factory of crushed, mixed, and clarified juice were as the first sample was collected from milling of clean cane from Labourites of the Sugarcane Research Department. Sugarcane variety CO997 was used. The raw materials of all types were carefully prepared before analysis. Unless otherwise stated all chemicals and reagents used in this study are of reagent grade. Cane sample collection is shown in Figure 1. 3.2 Methods: A specific method for detetermination of each parameter for all types of juice is standardized. 3.2.1 Determination of pH: Material: 1. Sugarcane juice. 2. Distilled water. Apparatus: 1. pH-Meter. 2. Thermometer. 3. Beaker. Reagents: Buffer solution

Fig. (1): Cane samples

Procedure: PH-Meter was standardized with the buffer solution of different ranges of PH from 7 to 9, Electrode was rinsed with apportion of the sample to be tested. A beaker was filled by the juice solution to be tested to depth covering the bulbs of the electrodes. The temperature of the solution read and the pH-Meter was adjusted for temperature correction. The system was allowed to be in equilibrium. The PH of the juice was then read. Electrodes were washed and stored in distilled water before dales reach reading.

3.2.2 Determination of the sucrose content (Pol): Material: 1. Sugarcane juice. 2. Distilled water. 3. Poly meter. Apparatus: Balance, volumetric flasks 100ml, filter paper (Whatman 19), polar meter, funnel. Reagents Lead Acetate (1.24g/ml). Procedure: The sugarcane juice (26.00g) was transferred to 100ml volumetric flask. Distilled water was added to obtain 100 ml of solution. Five drops of lead acetate was then added to the solution. The solution was shacked well, transferred to funnel lined with filter paper Whatman 19 and filtered. The filter ate was collected, and its polarization was recorded.

3.2.3 Determination of (soluble solid) the dry matter content (BRIX): Material: 1. Sugarcane juice. 2. Distilled water. Apparatus: Refrectometer, calibrated at 20°c and having water-jacketed prism. Procedure: The prism faces of the refrectometer was cleaned and dried. A drop of distilled water is transferred to the refrectometer prism to standardize the zero reading. A drop of the juice solution (sugarcane juice) was transferred to the prism and the reading was recorded, and corrected (ICUMSA 2005) 3.2.4 Determination of reducing sugar (RS): Material: 1. Sugarcane juice. 2. Distilled water. Apparatus: Plate, pipette 5 ml, burette 50ml, flasks 100ml, stand for the burette, funnel, filter papers. Reagents:

1) Fehling solution (A): 34,639g of cuso4.5H2O diluted in 500ml distilled water and filtrate. Fehling solution (B): 173g of Rochelle or signet.

2) Salt (Na-K-tart rate) and 50g of Na (oH) 2 diluted to 500ml by distilled water and filtrated.

3) Di-sodium oxalate powder 4) Methylene blue indicator. Procedures: Twenty five gram of juice was diluted by distilled water to 100ml in a flask. The solution was shaken well and filtered through a filter paper. The filtrate was transferred into burette. A mixture of Fehling solution (A) and (B) was prepared by mixing 5mls of each and few amount of distilled water in a flask and transferred to a hot plate. Before the mixture became hot 15ml of the sugar juice in the burette was drained in to the mixture of Fehling solution. Thereafter, addition of juice was continued drop wise till the colour changed to tricky. At this Point two drops of methylene blue were added to confirm no further change in colour. The volume of the juice filtrate used was recorded (ICUMSA, 2005). RS was then calculated using the following formula: Calculations: Fehling Factor RS = X 100 Volume of titration

Fehling factor (from table)

3.2.5 Determination of the colour value: Material: 1. Sugarcane juice. 2. Distilled water. Reagent: 1. One ml hydrochloric acid solution (HCL), 1ml sodium hydroxide solution (NAOH) for pH correction.

Apparatus: 1. Refrectometer (for measuring brix). 2. Telemeter (for measuring absorbance) with wave length 420nm. 3. Buchrer funnel. 4. Sucking pump. 5. 0.45mm membrane filter paper. Procedure: Fifty gram of the juice sample was weighted diluted according to the need of the experiment and filtered using bunchier funnel with 0.45mm membrane filter. The PH of filtrate was raised to pH 7. Brix and absorbance degree of the filtrate was recorded using the refrectometer, and telemeter receptively. Colour value= Reading (Telemeter) x brix factor = Mau. (ICUMSA 2005) 3.2.6 Turbidity determination Material: 1. Clarified juice. 2. Distilled water. 3. Juice sample pipes and containers. Apparatus Spectrophotometer- suitable for the measurement of absorbance at 900 nm with matched 1cm cells. Procedure The juice sample pipe and the sample containers were flushed and rinsed with the hot juice immediately before taking the sample. The hot samples taken were cooled under running cold water to room temperature (15-25°C). Then the nester tube filled by the sample that

27 to be determined and read, the reading is in, NTU, units (Icumsa, 2005). 3.2.7 Tannin determination: Apparatus: flask (100) ml. Test tube, Shaker, Centrifugal, Incubated and conical flask Material: Sugarcane juice. Reagent: Methanol. 1% HCL. Vanillin. Procedure: Quantitative estimation of tannin for each sample was carried out using modified vanillin –HCL in methanol method as described by Price et al. (1978). About 0.2g of the juice sample was placed in a100ml conical flask. Ten millilitres of 1% HCL in methanol (v/v) were added, shaken for 20min, and centrifuged at 2500rpm for min. One millilitre of the supernatant was pipetted into a test tube and 5ml of vanillin –HCL reagent were added. The optical density was read using Spectrophotometer (JENWAY 6305 UV/3V) at 500nm after 20 minutes incubation at 30ºC. A standard curve was prepared expressing the results as catching equivalent, i.e. amount of catching (mg per ml) which gives a colour intensity equivalent to that given by tannins after correcting for blank.

Calculations: Tannin concentration was expressed as catching equivalent (C.E) as follows: c x 10 x 100 C.E% = 200

Where: C = Concentration corresponding to the optical density 10 = Volume of extract (ml) 200= Sample weight (mg)

3.2.8 Total polyphenols determination: Material: 1. Sugarcane juice. 2. Distilled water. Apparatus Test tube, filter papers, constant shaking, funnel and spectrophotometer. Regent:

Methanol, 0.1ml Fecl3, 0.1 N HCL and 0.008MK3Fe (CN)6. Procedure: Polyphenols content was determined according to method described by price and Butlur (1977). Juice sample (0.06 gm) was extracted with 3ml absolute methanol in a test tube, by constant shaking for one minute, and then poured in to a filter paper. The tube was quickly rinsed with an additional 3 ml of methanol and the contents poured at once in to the filter paper. The filtrate was diluted to 50 ml with distilled water, mixed with 3 ml 0.1M Fe Cl3 in 0.1 N HCL for minutes, followed by the timed addition of 3 ml 0.008M

K3Fe (CN)6. The absorption was read after 10 minutes at 720nm on spectrophotometer (corning, 259). In all cases, tannic acid was used as a reference standard. DF--- Dilute Factor c x 56 x 100 DF = 60

3.2.9 Separan dose experiment: Material: Mixed juice. Apparatus: 1. Sedimentation Study Apparatus. 2. Beakers (2000 ml). 3. Cylinder (500 ml) 4. Pipes. 5. Distiller Water. Reagent: Separan. Procedure: Three samples of Separan weighing 1, 2, and 3 gram were taken in different conical flasks (1000ml). Each flask was then filled with distilled water to the 1000ml marks, and shacked to obtain even solution. Samples of 1, 2, and 3 ml from the above were taken and again diluted in 1000mls conical flasks to obtain Separan solution with concentration of 1, 2 and 3ppm…etc (Fig. 2). A sample of mixed juice was obtained from the factory before addition of Separan, and heated to 90°C. The heated mixed juice was divided into five Sedimentation Study Apparatus (1000ml size). Separan either at 1, 2, 3, 4 and 5 ppm was added to each Cylinder, to obtain a clarified juice. The parameters of clarified juice obtained from each cylinder were the recorded as determined before, and compared with parameter of the clarified juice in the factory.

Fig. (2): Sedimentation study apparatus in operation

3.3 Statistical analysis: Data generated was subjected to analysis of variance using randomized complete block design with five replications – Data analyzed using MSTATC program, means were compared using the Duncan's Multiple Ranges Test (DMRT), with a probability (P≤0.05).

CHAPTER FOUR RESULTS AND DICUSSIONS

4.1 Effect of milling on physicochemical properties of green and burned cane: Juice physicochemical parameters of both green and burned cane are presented in Table (1) and Fig. (3).

Cane juice pol (sucrose), brix, reducing sugar (RS), purity and pH of both green and burned cane were found to be similar and did not differ significantly (P≤0.05). However there was a significant (P≤0.05) difference between juice colour of burned (2600) and green cane (53600) was observed.

Milling of green cane (53600) resulted in significant higher colour reading than of burned cane (2600). This significant difference in colour reading between burned and green cane could be due to presence of chlorophyll.

The results obtained are in agreement with those obtained by Steindl (2005) who stated that the amount and nature of colour depends on the variety of sugarcane and also agree with those reported by John (1988) who declared that chlorophyll is present in every green plant.

It is noticeable that the reducing sugar level was found to be higher in burned (1.10) cane than the green one (0.79). This could be due to sucrose inversion in the presence of microbes in burned cane. This matches with the findings of Joachim et al. (2000) who reported

Table (1): Effect of milling on physicochemical properties of green and burned cane.

Color/ Treatment Pol % Brix% Purity R.S pH Icumsa

Green cane 18.15 20.56 88.28 5360* 0.79 5.40 (±1.27) (±1.12) (±4.70) (±0.49) (±0.52) (±0.09)

Burned cane 18.14 20.93 86.67 2600 1.10 5.40 (±0.94) (±0.66) (±1.88) (±0.32) (±0.36) (±0.20)

Values are means (±SD) of five replicates. Means sharing star superscript are significantly different at (P≤0.05)

Green cane

Burned cane

Fig. (3): Sample of green and burned cane

That, when sucrose is inverted, as for example by means of an acid, enzyme and micro organism. The molecule is broken up to give glucose and fructose, i.e.

C12H22O11 + H2O C6H12O6 + C6H12O6 Sucrose water glucose fructose

Inverted sugars

4.2 Effect of processing on physicochemical parameters of burned cane juice: Physicochemical parameters of crushed, mixed and clarified juice are presented in Table (2). The results obtained showed that pol, brix and purity of crushed juice is higher than those of the clarified and mixed juice.

The above quality parameters of both mixed and clarified juice were comparable and not different significantly. The higher Brix reading in the crushed juice (18.26) is due to the presence of high amount and not easily tolerable colloidal matters as stated by Chen. et al. (1993).

The results also showed that, the colour reading of both the crushed (5569) and clarified juices (13009) were significantly (P>0.05) higher compared to that of the mixed juice (3445). The high colour value of the crushed juice could be due to the presence of combinations of dyes in cane and ferric salts present in the crushed juice as stated by Chen et al. (1993).

Table (2): Effect of processing on physicochemical parameters of burned cane juice.

Treatment Pol % Brix % Purity pH Color/ R.S Turbidity Icumsa A. Crushed juice 15.75* 18.26* 84.26 5.42 55.69* 1.04 14.00 (untreated) (±1.82) (±1.85) (±2.56) (±0.81) (±0.81) (±0.50) (±1.04)

B. Mixed juice 12.24 12.86 95.18* 5.36 3445* 0.80 12.09 (imbibitions water) (±2.90) (±1.27) (±3.55) (±0.11) (±0.83) (±0.32) (±0.75)

C. Clarified juice 12.99 12.99 94.96* 6.80* 13009* 0.83 9.47 (Saparan added) (±1.87) (±1.87) (±2.80) (±0.20) (±0.76) (±0.32) (±0.73)

Value are means ( SD) of five replicates Means values having different superscript. Star in columns differ significantly (P≤0.05) Pol = Sucrose RS = Reducing sugar

The very high colour reading of the clarified juice could be due to the decomposition of its constituents by the action of added lime (Kulk, 1998), iron salts present in the juice (Griffith, 1988), and or subjection of both sugars and non sugars to heat, varying pH, iron from equipments, added chemicals such as lime (Rein et al., 2007). The application of heat or addition of chemicals (electrolytes) during clarification process brings about flocculation or coagulation of the colloidal matters and reduced clarified brix reading. The results obtained in this study agree with the result obtained by Perry’s (1988), who stated that cane juice has an acidic reaction at pH 5.5. According to Table (2) there is a significant difference (P<0.05) between mixed (5.36) and clarified juices (6.80) regarding pH and also there is a significant difference between crushed (5.42) and clarified juice (6.80) in pH values. It is clear that, there is no significant (P<0.05) difference of pH between crushed (5.42) and mixed juice (5.63). The change in pH from acidic in mixed juice to nearly neutral in clarified juice could be due to the addition of lime. Spencer and Meade (2001) described the purification in cane juice and stated that, the lime added to neutralize the organic acids originally contained in the juice.

4.3 Colouring substances in raw and processed materials: The levels of polyphenols and tannin of different juice types are presented in Table (3). The percentage of polyphenol is significantly higher in green cane (0.216) compared to that of burned cane (0.117). This is due to

Table (3): Colouring substances in the raw and processed materials.

Anti-nutrient Samples Polyphenols Tannin

Green cane 0.216c 0.006d Burned cane 0.117d 0.008d Crushed juice 1.189a 0.005d Mixed juice 0.094de 0.011d Clarified juice 0.006f 0.008d Syrup 0.037ef 0.008d Final molasses 0.218c 2.146a

Overall mean 0.334 0.467 SE± 0.025 0.006 CV% 16.84 3.00 LSD 0.075 0.018

Juice colour which increased in green may be with trash and trash tops. Reid and Lionnet (1989) stated that, the coloration in the juice increased by 50 and 100% with trash and trash plus tops respect.

Regarding Tannin its percentage is higher in burned cane (0.008) but not significantly compared to green cane.

The levels of polyphenols differ according to different types of juice. The highest concentration of polyphenols was recorded in the crushed juice (1.189), and the lowest level was found in clarified juice (0.006), this colour increase may be due to equipment like juice pump.

Kul (1989) stated that the high level of polyphenols in crushed juice is due to the presence of soluble iron salt (ferric) from equipment. The level of polyphenols in the final molasses (0.218) was found to be significantly (P≤0.05) higher than that of clarified juice (0.006), but at the same time is significantly lower than that of the crushed juice (1.189).

Regarding tannin, the highest level was recorded in the final molasses (2.146) flow by Syrup (0.468). The level of tannin in both molasses and syrup is significantly higher than that in crushed (0.005), mixed (0.011) and clarified juice (0.068). The level of tannin recorded, in the three types of juices did not differ significantly. The higher level of polyphenols and tannin in the syrup (0.468) and final molasses (2.146) was expected, as the addition of separan to the mixed juice enhances the formation of flogs containing tannins, which eventually settle at the bottom of clarifier (John, 1988).

4.4 Effect of different doses of separan on juice quality parameter: The effect of different doses of separan on juice quality is presented in Table (4). The factory sample was taken as control (3 ppm). As shown in Table (4) the concentration of pol decreases with increase in separan concentration (ppm). Moreover all parameters (except purity) were affected by separan concentration.

The highest pol (14.69) was obtained at 0.001 ppm dose of separan. The highest brix reading (18.05) was recorded at the dose of 3 ppm (standard), which is significantly (P>0.05) higher than all other samples. Different concentrations of separan showed no significant effect on purity values.

The highest pH (7.60) was obtained at 0.006 ppm dose of separan, but this not significantly different from other doses of separan except the control sample, at which the pH value was 6.91.

The highest colour value (14692 ICUMSA) was obtained when 3 ppm of separan was applied and the lowest colour value (10894 ICUMSA) was obtained at 0.001 ppm of separan.

A technical report by Narspri (2006) stated that coluored non- sugars that are present in cane juice can be removed by various chemical or physical processes. This may involve removal by precipitating agents such as lime, phosphoric acid and separan that are used in clarification.

The highest RS content (0.96) was obtained when 3 ppm of separan (standard) was used. This level is significantly higher than that obtained with the rest of separation concentrations. This could be due to hydrolysis due to the higher temperature in heaters at

Table (4): Effect of different doses of separan (ppm) on juice quality parameters Separan concentration (ppm) Parameter 0.001 0.003 0.005 0.006 0.008 0.010 0.009 0.012 0.015 3* Mean SE± CV% LSD Pol 14.69a 13.58ab 13.53ab 13.55ab 13.50ab 12.47ab 13.12ab 13.07ab 10.18b 12.19ab 13.06 0.86 14.65 2.58 Purity 87.11a 86.17a 82.18a 83.81a 88.08a 85.57a 85.90a 87.43a 85.01a 84.87a 85.61 2.13 5.56 6.39 Turbidity 9.60a 8.53a 9.02a 8.47a 7.92ab 7.56ab 7.87ab 5.10b 4.96b 5.10b 7.358 0.11 3.29 0.33 pH 7.49a 7.43a 7.38a 7.60a 7.57a 7.32a 7.34a 7.29a 7.25a 6.91b 7.36 0.11 3.29 0.33 colour 10894b 12191ab 13068ab 13538ab 13417ab 13497ab 13120ab 13532ab 12570ab 14692a 13068 0.86 14.69 2.58 brix 14.69c 15.952bc 15.62bc 15.62bc 16.62abc 16.03abc 16.38abc 16.68abc 16.94ab 18.05a 16.36 0.62 8.48 1.86 R.S 0.6b 0.58b 0.62b 0.66b 0.70b 0.64b 0.68b 0.64b 0.61b 0.96a 0.66 0.051 16.90 0.15

Values showing different superscript in a row are significantly different at (P≥0.05) • Control sample (factory sample)

42 the factory. This level is significantly higher than that of the vestige of separation concentrations.

4.5 Effect of different doses of Saparan (ppm) on polyphenols and tannins level: The level of polyphenols and tannins of different doses of separan (ppm) are presented in Table (5).

The level of polyphenols decreased as the concentration of separan was increased. The highest level of polyphenols (0.009) is recorded when 0.001 ppm separan is added, and when the concentration of separan increased to 3 ppm (control), polyphenol significantly (P>0.05) deceased to 0.006%. This is due to colour reaction between sugars and various phenols, when concentrated mineral acids are present.

Joachim et al. (2000) Stated that the colorization of the clarified juice is due to the combination of the sugar decomposition products, such as furfural, and the condensation products from the phenol derivatives. Tannin content was found to decrease with increase in separan concentration.

The lowest tannin content (0.01) was found at separan concentration of 0.010, 0.015 and 3 ppm.

Separan with 0.001, 0.003 and 0.005 ppm resulted in significant (P>0.05) higher levels of tannins compared, with other separan levels.

The higher reading of tannin in the clarified juice is due to the

Reaction with iron salts. Therefore, the higher the concentration of separan the lower tannin content. Colouring substances removal to a great extent is dependent on proper addition of lime and separan. This

43 indicates that the higher the separan, concentration level the lower the tannins levels in the juice. This in agreement with John (1988) who stated that, tannin is normally removed by proper addition of separan to the juice during clarification process.

Table (5): Effect of different doses of Separan (ppm) on polyphenols and tannins level.

Separan Anti-nutrient (%) concentration Polyphenols Tannin

0.001 0.009a 0.08a 0.003 0.008ab 0.06b 0.005 0.007bc 0.06b 0.006 0.007bc 0.05bc 0.008 0.006cd 0.04c 0.009 0.005dc 0.02d 0.010 0.005de 0.01de 0.012 0.004ef 0.02de 0.015 0.033f 0.01c 3.000* 0.006cd 0.01c

Means 0.006 0.03

SE± 0.0002 0.01 CV% 9.39 30.18 LSD 0.0006 0.03

Means of similar letter(s) are significantly different * Control sample

CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusion: Use of separan at very low concentration rations (0.015ppm) resulted in a significantly better quality clarified juice as compared to the standard or factory practice (3 ppm). This indicates the possibilities to obtain high quality clarified juice with cheap less cost method.

4.2 Recommendation: * The results achieved encourage utilization of lower concentration (0.015 ppm) of separan instead of standard practices applied (3 ppm) for production of clarified juice.

* Utilization of (0.015) ppm for better sugar quality.

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