CHAPTER 2 REVIEW OF LITERATURE

Transcription

1 CHAPTER 2 REVIEW OF LITERATURE This chapter deals with the scientific work carried out by researchers related to composition of banana pulp and changes occurred in the pulp during ripening, enzymatic clarification of fruit juices in general and of banana pulp in particular and browning problems associated during clarification of fruit pulp and their inhibition. The chapter is divided into 4 sections namely 2.1 Composition of ripened banana pulp 2.2 Changes during ripening of banana 2.3 Enzymatic extraction and clarification of fruit juices 2.4 Browning during clarification of fruit pulp and its inhibition 2.1 Composition of Ripened Banana Pulp Banana fruit is strongly recommended by nutritionists (Chandler, 1995), and highly appreciated by consumers because of its flavour and sweetness. The biochemical composition of banana fruits depends on the cultivar, abiotic factors such as climate, cultivation method, soil type and storage conditions. The chemical composition of ripened banana pulp studied by various researchers is as shown in Table 2.1 According to data by various researchers presented in Table 2.1, the moisture and total sugars content in banana pulp ranges from per cent and per cent, respectively. Reducing sugars constitute the bulk of carbohydrate. The reducing sugars present in ripe bananas are mainly fructose and glucose whereas nonreducing sugar is sucrose. Banana contains per cent starch, per cent fibre while pectin content ranges from per cent. Pectin stabilizes and gives a viscous body to the pulp. Total carbohydrate in the pulp ranges from per cent while per cent protein occurs. The major amino acids present in over ripen banana pulp are aspartic acid, histidine, leucine and valine with total amino acid content of 3517 milligram per hundred gram (Askar, 1973). 7

2 Table 2.1 Chemical Composition of Ripened Banana Pulp Constituents Range References Moisture (%) ,2,3,4,5,7 Total sugars (%) Reducing sugars (%) ,7,8 Non reducing sugars (%) ,7,8 Starch (%) ,3 Fibre (%) ,5, Pectin (%) Tannins (%) ,7 Total carbohydrates (%) ,5 Protein (%) ,2,3,5 Fat (%) ,2,3,5 Ash (%) Potassium (mg %) ,5 Magnesium (mg %) ,5 Phosphorus (mg %) ,4,5 Calcium (mg %) ,4,5 Iron (mg %) ,4,5 Vitamin C (mg %) ,3,4,5,7 Niacin (mg %) ,4,5 Vitamin B6 (mg %) ,5 Riboflavin (mg %) ,3,5 β-carotene (mg %) ,4,5 Thiamine (mg %) ,4,5 1. Select Committee on Nutrition and Human Needs, U.S Senate (1977) 2. Ockerman (1978) 3. Paul and Southgate (1978) 4. Gopalan et al, (1987) 5. CIQUAL-CNEVA (1993) 6. Kotecha et al, (1994) 7. Yousaf et al, (2006) 8. Ramesh Kumar et al, (2008) 8

3 Fat content of banana pulp varies from per cent (Ockerman, 1978). The minerals present in the banana pulp include potassium, magnesium, phosphorus, calcium and iron. Potassium constitutes about per cent of total mineral matter. Banana pulp has also been reported to contain ascorbic acid, niacin, vitamin B 6, riboflavin, β-carotene in good amount (Paul and Southgate, 1978; Gopalan et al., 1987). 2.2 Changes during Ripening of Banana Fruit ripening is a genetically programmed, highly coordinated process of organ transformation from unripe to ripe stage, to yield an attractive edible fruit with an optimum blend of colour, taste, aroma and texture. In banana, compositional changes following harvest are important since banana is a climacteric fruit. Dramatic changes in banana peel colour and pulp texture occur during the rise in respiration during climacteric. In commercial trade, the ripening is initiated after transporting the green banana to locations where the fruits are treated with ethylene. The research work associated with changes during ripening of banana is as summarized below. Kheng et al, (2012) determined the optimum harvest maturity and physicochemical quality of Rastali banana (Musa AAB Rastali) during fruit ripening. Rastali banana fruit exhibited a climacteric rise with the peaks of both CO 2 and ethylene production occurring simultaneously at day 3 after ripening was initiated and declined at day 5 when fruits entered the senescence stage. De-greening was observed in both of the harvesting weeks i.e. 11 and 12 weeks after emergence of the first hand with peel turned from green to yellow, tissue softening, and fruits became more acidic and sweeter as ripening progressed. Sucrose, fructose and glucose were the main sugars found while malic, citric and succinic acids were the main organic acids found in the fruit. Rastali banana harvested at weeks 11 and 12 can be considered as commercial harvest period when the fruits have developed good organoleptic and quality attributes during ripening. Soltani et al, (2011) investigated the changes occurred in physical and mechanical properties of banana fruit (var. Cavendish) at different level of ripeness during ripening in an airtight ware house with ethylene gas control system. Relation between various stages of ripeness and these properties were determined and 9

4 correlation coefficients were calculated. The colour of the fruit skin was measured as L*, a* and b* in CIELAB system. The mechanical properties were extracted from plotted force-deformation curve. A significant difference was found between the level of ripeness and these properties except deformation. Results showed that changes in L*, b* and C was similar, also variation of colour index (CI) was similar to a*. The a* increased when banana fruits reached to a full-ripe stage. A positive correlation was observed between a* and various stages of ripeness. As it was noted, an increase in a* means a decrease in the degree of greenness. The firmness, rupture energy and hardness decreased as banana fruit ripened. The firmness degraded from 75.1 N at stage one to 27 N at stage seven. All measured physic-mechanical properties of banana fruit except deformation had high correlation with stage of ripeness. Result of deformation analysis showed no significant difference at various stages of ripeness. The correlation between deformation and stage of ripeness was obtained as 0.2. Kulkarni et al, (2010) studied the physico-chemical changes occurred during artificial ripening of banana. Banana fruits harvested at 75 80% maturity were dip treated with different concentrations of ethrel (250 1,000 ppm) solution for 5 min. Ethrel at 500 ppm induced uniform ripening without impairing taste and flavour of banana. Fruits treated with 500 ppm of ethrel ripened well in 6 days at 20±1 C. Changes in total soluble solids, acidity, total sugars and total carotenoids showed increasing trends during ripening whereas fruit shear force values, pulp ph and total chlorophyll in peel showed decreasing trends. Sensory quality of ethrel treated fully ripe banana fruits was excellent with respect to external colour, taste, flavour and overall quality. Adeyemi and Oladiji (2009) studied the compositional changes in banana (Musa spp) fruits during ripening. Banana fruits were collected, dried, ground and ashed. The moisture content and mineral elements composition was determined as ripening proceeds. The results showed that the nutritional composition of banana pulp was diversely affected by ripening. The moisture content increased with ripening. Changes in mineral composition varied and were not consistent with the stages of ripeness. The magnesium content of the banana kept decreasing with ripening, while increase in zinc and manganese reached a peak at the ripe stage and decreased thereafter. Bananas were considered a good source of Mg in the diet, and the data 10

5 obtained herein support these assertions. Zn and Mn were other minerals of nutritional importance in bananas and this study has shown that their average values are adequate to support its nutritive value at the various ripening stages. The result obtained in this study showed that banana fruits at any ripening stage (unripe, ripe or overripe) can be a potential source of mineral elements supplement in the diet especially for Mg. Duan et al, (2008) evaluated the changes in the pectin polysaccharide during ripening of banana fruit. Pectin is one of the major components of the primary cellular walls and middle lamella in plant tissues. In this study, water-soluble pectin (WSP) and acid-soluble pectin (ASP) fractions were isolated from pulp tissues of banana fruit at various ripening stages. Their monosaccharide compositions, glycosyl linkages and molecular mass distributions were evaluated. As ripening progressed, fruit firmness decreased rapidly, which was associated with the increase in the WSP content and the decrease in the ASP content. Meanwhile, the molecular mass distributions of WSP and ASP fractions exhibited a downshift tendency, indicating the disassembly of pectin polysaccharides. Moreover, galactose and galacturonic acid as the major monosaccharide compositions of pectin polysaccharides increased in WSP fraction but decreased in ASP fraction during fruit softening. GC MS analysis further revealed that pectin polysaccharide had a 1,4- linked galactan/galacturonan backbone with different types of branching and terminal linkages in WSP and ASP fractions. During banana fruit ripening, the amount of 1,4-linked Galp residues of ASP fraction decreased significantly whereas 1,3,6-linked Galp, 1,2-linked Manp and 4-linked Araf residues disappeared, which was related to depolymerization of pectin polysaccharides. Overall, the study indicated that the modifications in polysaccharide compositions and glycosyl linkages, reduced molecular mass distributions and enhanced depolymerization of pectin fraction during banana ripening were responsible for fruit softening. Tadakittisarn et al, (2007) investigated the changes occurred in activity of enzymes in bananas [Musa acuminate (AAA group) Gros Michel ] associated with the different ripening stages. Polygalacturonase (PG) and pectate lyase (PL) enzymes from ripening stages 2-8 were extracted and partially purified by ammonium sulphate fractionation. The results showed an increase in PG activity from 3.0±0.11unit/g fresh 11

6 banana in the 2 nd stage to 4.89 ±0.39 unit/g fresh banana in the 6 th stage. Furthermore, the PG activity decreased slightly to approximately 4.33±0.49 unit/g fresh banana as the ripening stage increased (the 8 th stage). PL enzyme activity also depended on the ripening stage of the banana. When the banana was ripening, the PL activity gradually increased from unit/g fresh banana in the 2 nd stage to unit/g fresh banana in the 8th Stage. When compared to the commercial pectinase enzyme from Aspergillus aculentus (Pectinex Ultra SP-L, Novozymes A/S, Denmark), the enzymes obtained from the banana demonstrated much lower activity. The PG and PL activities from the commercial pectinase were and 1709 unit/ml, respectively. The reducing sugar content also increased from 2.52 ±0.00 mg/g in the 2 nd stage to ±0.00 mg/g in the 8 th stage of ripening. Chen and Ramaswamy (2002) examined the kinetics of colour and texture changes in ripening bananas as a function of storage temperature (10, 16, 22, 28 C). Colour was evaluated in terms of L, a and b values as well as the total colour difference (ΔE) representing the residual deviations from the ripe stage. Puncture force (PF) was used to evaluate the texture properties of banana. The results indicated that the time dependence of L, ΔE and PF values followed a logistic model, while a and b values were well described by a simple zero-order and fraction conversion models, respectively. The Arrhenius equation adequately described the temperature dependence of the reaction rate constants for both colour and texture parameters, from which the activation energies and rate constant at reference temperature 15 C were obtained. There were significant linear correlations between colour parameters (L, a, b, ΔE) and texture parameter. Prabha and Bhagyalakshmi (1998) investigated a comprehensive picture of changes in carbohydrates, carbohydrate hydrolases, cell structure and texture in banana fruit during ripening. Softening in the pulp during ripening was significant as measured by the compression test on the pulp which decreased from 314 to 15N mm -2 from the raw to the ripe stage. The shear force decreased only to the extent of five fold (from 133 to 27 N mm -2 indicating a much lesser degree of textural softening in the peel. Starch (approx. 18%) had almost disappeared at the ripe stage. Total hemicelluloses content lowered considerably from 2.4 to 0.9% during ripening whereas pectin decreased from 1.1 to 0.8. There was no apparent change in cellulose. 12

7 More than 80% of the radio activity of starch was incorporated into soluble sugars viz. glucose, fructose and sucrose indicating active sugar inter conversions. The total content of these soluble sugars increased from 1.8 to 19% with a concomitant decrease in starch content during ripening. The various carbohydrate hydrolases viz. polygalacturonase, pectin methyl esterase, xylanase, laminarinase, alphamannosidase, beta-galactosidase, amylase, cellulase and hemicellulase registered a general increase in their activities. Microscopically loss of cell wall integrity, cell wall thinning, increased intercellular spaces, loosening of cells and disappearance of starch granules were evident. Wills et al, (1984) analyzed the proximate composition of `Cavendish banana pulp at different maturity stages during ethylene induced ripening. Results showed that the major changes in the pulp during ripening of the bananas were in the carbohydrates. About 21 g/100 g of the pulp of the unripe fruit was starch, which decreases continuously during ripening to reach 0.8 g/100 g in the fully ripe fruit (stage 7= completely yellow with brown spots). Sugars were at low level in the unripe fruit (1.3 g/100 g), but increase as a result of starch hydrolysis during ripening. Sucrose was always the major sugar present; the major increase in sucrose occurs early in the ripening process (stage 1= green to stage 3= green turning yellow) and reaches a maximum (about 11 g/100 g) in firm ripe fruit (stage 7= fully yellow). Fructose was always present at slightly lower levels than glucose. Both fructose and glucose increased continuously during ripening and reach their maximum level in fully ripe fruit (about 3 g/100 g and 4 g/100 g, respectively). Total carbohydrate decreases by 5% during ripening, presumably because sugars are utilized in respiration. The water content of the banana pulp increased during ripening from 72 g/100 g to 76 g/100 g. There was a decrease in dietary fibre early in the ripening process (stage 1 to 3) and again when the fruits were fully ripe (stage 7). This change was from 3.2 to 2.7 g/100 g as a result of hydrolysis of hemicelluloses and breakdown of pectic substances. Terra et al, (1983) observed that the accumulation of sucrose in banana fruit preceded the increase of glucose and fructose during starch degradation. The transformation of starch to sucrose was one of the possible mechanisms involved in 13

8 the ripening of bananas. They proposed the following reaction mechanisms leading to the formation of sucrose, the major sugar in banana. (1) Starch (n) + Pi Glucose-1-P + Starch (n-1) (2) Glucose-1-P + UTP UDP-glucose + PPi (3) UDP-glucose + Fructose Sucrose + UDP Reaction (1) is catalyzed by phosphorylase enzyme. Reaction (2) is catalyzed by UDP-glucose pyrophosphorylase. Reaction (3) is catalyzed by sucrose-synthetase. Lii et al, (1982) investigated changes during ripening of dessert bananas with respect to physical and chemical properties of their starch and their content of reducing sugars and sucrose. During ripening from maturity stage 1 starch content decreases gradually whereas reducing sugar and sucrose content increases from stage 1 to fully ripened stage. Wade and Bishop (1978) studied the changes in the lipid composition of ripening banana fruits. The content of total lipid of banana fruit pulp tissue remained constant during the climacteric rise induced by applied ethylene. The relative proportions of neutral lipid, glycolipid and phospholipid did not change. However, the fatty acid composition of the lipid did change during ripening. This change was confined largely to the phospholipids fraction, in which there was an increase in the proportion of linolenic acid and a decrease in the proportion of linoleic acid. The net result was an increase in total unsaturation of the fatty acids in the phospholipids fraction. The review of literature related to changes during ripening of banana is summarized in Table Enzymatic Extraction and Clarification of Fruit Juices Over the last two decades, great advances have been made in the technology of fruit juice processing. The advances have been towards the improvements in the existing conventional processing procedures and equipments as well as introducing new techniques to achieve maximum yield of juice with good quality. Enzymes catalyses various reactions involved in the preparation of different food products. 14

9 Table 2.2 Review on Changes during Ripening of Banana Sr. Author Year Important finding No. 1 Kheng et al 2012 Harvesting weeks i.e. 11 and 12 weeks after emergence of the first hand could be considered as a optimum harvesting period in Rastali banana 2 Soltani et al 2011 The surface colour of banana along with textural properties was correlated with degree of ripeness 3 Kulkarni et al 2010 Artificially ripened bananas with ethrel were at par in all qualities with naturally ripened bananas 4 Adeyemi and 2009 The nutritional composition of banana pulp was Oladiji diversely affected by ripening 5 Duan et al 2008 As ripening progressed, fruit firmness decreased rapidly, which was associated with the increase in the water soluble pectin content and the decrease in the acid soluble pectin content 6 Tadakittisarn et al 7 Chen and Ramaswamy 8 Prabha and Bhagyalakshmi 2007 Activity of polygalacturonase and pectate lyase enzyme increased upto 6 th stage of ripening and then slight decrease in 8 th stage Investigated the kinetics of colour and texture changes in ripening bananas as a function of storage temperature 1998 Investigated a comprehensive picture of changes in carbohydrates, carbohydrate hydrolases, cell structure and texture in banana fruit during ripening 9 Wills et al 1984 Major changes in the pulp during ripening of the bananas were in the carbohydrates 10 Terra et al 1983 Accumulation of sucrose in banana fruit preceded the increase of glucose and fructose during starch degradation 11 Lii et al 1982 Investigated changes during ripening of dessert bananas with respect to physical and chemical properties of their starch and their content of reducing sugars and sucrose 12 Wade and Bishop 1978 The relative proportions of neutral lipid, glycolipid and phospholipid did not change. However, the fatty acid composition of the lipid did change during ripening 15

10 It is one of the important tools in modern food industry because while processing many intermediate processes are simplified due to use of enzymes. Recently many types of commercial enzyme preparations have found application as processing aid in fruit juice processing industry too. A wide variety of enzymes are in use for different purposes in fruit juice industry, among them pectinases are the most important in extraction and clarification of fruit juices. In this section, the research work associated with enzymatic extraction and clarification of fruit juices has been summarized under following sub-topics: Pectic substances and pectolytic enzymes in fruit juice processing Extraction and clarification of fruit juices Clarification of banana pulp Pectic Substances and Pectolytic Enzymes in Fruit Juice Processing Pectic substances and pectolytic enzymes play an important role in fruit juice processing. Only in the 1960s did the chemical nature of plant tissues become apparent and with this knowledge, scientists began to use a greater range of enzymes more efficiently. Pectinolytic enzymes are one of the important groups of enzymes used in fruit processing industry. Several researchers have reported that depectinization using pectinase could effectively clarify fruit juices. Primarily, these enzymes are responsible for the degradation of the long and complex molecules in the fruit pulp called pectin that occur as structural polysaccharides and responsible for turbidity in pulp. Pectinases are now an integral part of fruit juice industries as well as having various biotechnological applications. Vaillant et al, (2001) worked on clarification study on six tropical fruit juices (mango, pineapple, naranjilla, castillas blackberry, passion fruit, tangerine). He observed that fruit juices contain colloids that are mainly polysaccharides (pectin, cellulose, hemicelluloses and starch), protein and tannin. One of the major problems encountered in preparation of fruit juices is cloudiness primarily due to the presence of pectin. Usually tropical fruits are too pulpy and pertinacious to yield juice by simple pressing. Pectin makes the fruit juices turbid and viscous and makes the clarification process harder due to their fibre like molecular structure. 16

11 Blanco et al, (1999) reviewed the classification of pectinases. Pectinases can be divided into two main groups, namely pectin esterases (PE), which de-esterify pectin by removing the methoxyl residues and depolymerases, which readily split the main chain. Pectin esterases remove methoxy groups from high methoxy pectin to give methanol and low methoxy pectin. The depolymerising enzymes can be classified according to their preferred substrate, whether cleavage is random or endwise, and if the enzyme acts by trans-elimination or hydrolysis. Polygalacturonases (PG) cleave the glycosidic bonds by hydrolysis and pectate lyases (PL) break the glycosidic bonds by β elimination. These enzymes can also be classified according to whether they exhibit a preferential hydrolytic power against pectin, pectic acid or oligogalacturonate as the substrate, and whether the mode of action is random (endo-) or terminal (exo-). The classification of pectinases is shown in Table 2.3. Be Miller (1986) describe the classification of pectic substances. Chemically, pectic substances are complex colloidal acid polysaccharides, with a backbone of galacturonic acid residues linked by α (1-4) linkage. The side chains of the pectin molecule consist of L-rhamnose, arabinose, galactose and xylose. The carboxyl groups of galacturonic acid are partially esterified by methyl groups and partially or completely neutralized by sodium, potassium or ammonium ions. Based on the type of modifications of the backbone chain, pectic substances are classified into protopectin, pectic acid, pectinic acid and pectin. Baumann (1981) observed that temperature has a significant effect on the activity of pectic enzymes. There was a close relationship between temperature and time during enzyme treatment of fruit juices. As temperature increases, the rate of pectin degradation was increases and the time of enzyme treatment decreases. Rombouts and Pilnik (1978) reported that most of commercial pectic enzyme preparations used in fruit juice extraction and clarification are of fungal origin since these enzymes have a low ph optimum and other characteristics suited for fruit juices. Pectic enzyme preparations mainly contain a mixture of pectolytic enzymes like pectin methyl esterase (PE) and poly galacturonase (PG).They also contain other non pectic enzyme activities such as cellulose, hemicelluloses, amylase, esterase etc. 17

12 Table 2.3 Classification of Pectinases Group Enzyme Substrate Action Polymethyl Pectin Hydrolysis galacturonases (PMG) Endo- PMG Random cleavage of α- 1,4glycosidic bonds Exo-PMG Sequential cleavage of α-1,4 glycosidic bonds from the non-reducing end Polygalacturonase Pectic acid Hydrolysis (PG) Endo-PG Random hydrolysis of α- 1,4glycosidic linkages Exo-PG Sequential cleavage of α- 1,4glycosidic linkages from the non-reducing end Polymethyl galacturonate Pectin Trans-eliminative cleavage lyases (PMGL) Endo- PMGL Random cleavage of α- 1,4glycosidic linkages Exo- PMGL Sequential cleavage of α- 1,4glycosidic linkages Polygalacturonate lyases (PGL) Pectic acid Trans-eliminative Cleavage Endo- PGL Random cleavage of α- 1,4glycosidic linkages Exo-PGL Sequential cleavage of α- 1,4glycosidic linkages 18

13 Neubeck (1975) reported the need for enzyme treatment varies with the type of fruits and associated difficulties during juice preparation. Some fruits require the enzyme treatment to expedite pressing for juice extraction. Other fruits may be readily pressed without the need of adding enzymes but the cloudy pressed juices would require enzyme treatment to improve their filtration to obtain clear juice. Such differences are due to the wide variation in the physical and chemical composition of different fruits particularly their pectic substances and the ratio of insoluble to soluble pectin content. He also reported that the time required for flock formation during clarification of apple juice was decreased by two folds for each 10 0 C rise in temperature in the range of C and by 1.5 fold at temp above 30 0 C. Endo (1965) studied the mechanism of enzymatic clarification of apple juice. Cloudy apple juice contained soluble pectin with a small amount of insoluble pectin bound to the suspended particles. Soluble pectin was found to act as a protective colloid for other particles suspended in the cloudy juice and partial hydrolysis of this pectin allowed other particles to flocculate and precipitate. The mechanism of enzymatic clarification process involved 3 stages: solubilization of insoluble pectin, decrease in viscosity of soluble pectin and finally flocculation of the suspended particles. The review of literature related to this section can be summarized as shown in Table Extraction and Clarification of Fruit Juices Bahramian et al, (2011) evaluated the effectiveness of enzymes pectinases and cellulases in sugar extraction process from date fruits. Kabkab, a date cultivar from Kerman province in Iran, which is industrially used for extraction of its sugar, was selected for enzymatic extraction. Comparison of samples, pretreated by either Pectinex Smash XXL or Cellubrix L with untreated date fruits, showed that amount of both extracted sugar and clarity of juices thus produced, were affected by enzymatic pre-treatment of fruits. Pre-treatment of fruits by each of the two enzymes caused equally about 18% increase in the amount of extracted sugars, while using a precisely determined mixture of two enzymes and a suitable condition, resulted in a further increase of sugar to about 46%, in relation to untreated samples. Regarding the clarity of the juice, the results indicated that increased sugar content of the extracts positively affects the clarity of juices, with some exceptions. 19

14 Table 2.4 Review on Pectic Substances and Pectolytic Enzymes in Fruit Juice Processing Sr. Author Year Important finding No. 1 Vaillant et al 2001 Fruit juices contain colloids that are mainly polysaccharides, protein and tannin. One of the major problems encountered in preparation of fruit juices is cloudiness primarily due to the presence of pectin. 2 Blanco et al 1999 Reviewed the classification of pectinases. 3 Be Miller 1986 Described the classification of pectic substances. 4 Baumann 1981 There was a close relationship between temperature and time during enzyme treatment of fruit juices. 5 Rombouts and Pilnik 1978 Most of commercial pectic enzyme preparations used in fruit juice extraction and clarification are of fungal origin since these enzymes have a low ph optimum and other characteristics suited for fruit juices. 6 Neubeck 1975 Enzyme treatment varies with the type of fruits and associated difficulties during juice preparation 7 Endo 1965 Soluble pectin was found to act as a protective colloid for other particles suspended in the cloudy juice and partial hydrolysis of this pectin allowed other particles to flocculate and precipitate. Joshi et al, (2011) extracted the pectin methyl esterase from apple pomace and evaluated its efficacy for extraction and clarification of plum, peach, pear and apricot juice. The juice recovery of enzymatically treated pulps increased significantly from 52-72% in plums, 38-63% in peach, 60-72% in pear and 50-80% in apricot. Addition of pectinase significantly increased the total soluble solid (TSS), titratable acidity and total sugar in the enzymatically treated juices. The ph, Brix acid ratio and relative viscosity of extracted juices were decreased. The ascorbic acid content remained unaffected with the increase in enzyme concentration. The overall sensory evaluation of extracted juices using hedonic scale showed a significant improvement in the colour and clarity scores. The flavour of extracted juices remained unaffected. For extraction of juice 2.5% enzyme concentration was found to 20

15 be best. However, for the clarification of apple and pear juice 1.0 and 0.5% enzyme concentration gave the optimum results. Karangwa et al, (2010) optimized the processing parameters for the clarification of blended carrot-orange juice using Response Surface Methodology (RSM) and to improve the carotene content. The blended carrot-orange juice was treated with Pectinex Ultra SP-L enzyme at different concentrations ( %), ph (2.5-5), reaction temperature (40-60ºC) and time ( min). The effect of these independent variables on clarity, turbidity, and viscosity of the carrot-orange juice was evaluated (coefficient of determination (R 2 ) greater than 0.9). From the RSM analysis, the optimum processing conditions were found as; 0.06% (w/v) enzyme concentration, 3.6 ph, 49ºC temperature, 91 min reaction time. Clarified carrotorange juice thus obtained favourably improved the nutritional content and consumer acceptance. Shailza et al, (2009) evaluated the effect of four different clarifying treatments viz. Sedimentation, Filtration, Pectinase and Filtration and Kaolin and Pectinase for clarification of Kiwi and Peach fruits. Higher juice content was obtained when the extraction was carried out in the presence of pectinase enzyme. The recovery was higher when clarification of juice was carried out using the combination of Kaolin and Pectinase in both Kiwi (85.40%) and Peach (84.10%) juice. Vaidya et al, (2009) studied the enzymatic treatment for fruit juice extraction from Kiwi fruit. Due to slimy pulp, extraction of pulp from Kiwi fruit was difficult. To overcome this problem a combination of enzymes (pectinase 0.025g/kg + amylase 0.025g/kg + mash enzyme 0.05g / kg) were used to macerate the pulp (2h at 50 0 C) and thus facilitating the juice extraction. The treatment enhanced the juice recovery (78.46%) compared to control (58.44%) and the treatment did not affect the TSS. Titratable acidity, ph, reducing and total sugar of clarified juice. Also a drastic decrease in the pectin content of the juice occurs. The outstanding feature of the juice was its high acidity and high concentration of ascorbic acid which however decreased by 21% after clarification. 21

16 Shah (2007) optimized the conditions for enzymatic extraction of Litchi pulp with various concentration levels of hydrolytic enzymes viz. pectinase ( %w/w), cellulase ( %w/w) and hemicellulase (0-0.20%w/w) for different durations ( min) at 45 C. Yield, clarity and TSS of juice were found to increase and apparent viscosity was found to decrease significantly by enzymatic treatment. The optimum conditions for enzymatic treatment of pulp obtained after a double sided desirability function with the responses juice yield, clarity and TSS to be maximized and viscosity to be minimized were 0.076% (w/w) pectinase, 0.138% (w/w) cellulase, 0.107% (w/w) hemicellulase and incubation time of min. The predicted values for juice yield, clarity, viscosity and TSS under optimized conditions were %, 93.53%, 1.359mPa s and brix which showed a good agreement with the experimental values under the same set of conditions. Sin et al, (2006) studied the enzymatic clarification of sapodilla juice. Sapodilla juice was treated with pectinase enzyme at different incubation times ( min), temperature (30 50 C) and enzyme concentration ( %). These three factors were used as independent variables whose effects on turbidity, clarity, viscosity and colour (L values) were evaluated. The results indicated that enzyme concentration was the most important factor affecting the characteristics of the juice as it exerted a significant influence on all the dependent variables. The recommended enzyme clarification condition was 0.1% enzyme concentration at 40 C for 120 min. Sorrivas et al, (2006) studied the mechanisms governing the enzymatic clarification of apple juice by electron microscopy techniques. Full ripe and unripe apple juice samples were treated with commercial pectinase (Solvay 5XLHA) and amylase (Rohalase HT) enzymes, respectively. Scanning electron microscopy studies revealed that commercial amylolytic enzymes quickly reduced starch content in unripe apple juice to undetectable values. It was also observed that after pasteurization of this juice (90 0 C, 5 min) all starch granules gelatinized. The effect of pectic enzyme to destroy the protective pectin colloid was also detected with this technique. It was speculated that the destruction of the weak pectin net by the action of the specific enzyme caused particle aggregation, followed by the collapse of aggregates, increasing the number of particles to less than 500 nm. 22

17 Lan Quin et al, (2005) investigated that the colour and cloud stability of cloudy carrot juice were improved by enzymatic hydrolysis and addition of hydrocolloids. Cellulytic and pectolytic enzyme preparations were used to prepare the carrot juice and their optimum dosages were 1.6 g kg -1 and g kg -1, respectively. Hydrocolloids including guar gum, pectin and flaxseed gum were each added to the carrot juice and assessed for their ability to stabilize the carrot juice. Singh et al, (2000) investigated the various physico-chemical changes during enzymatic liquefaction of mango pulp (cv. Keitt). Pulps were treated at 40 0 C for up to 2 h with a mixture of commercial enzymes, namely Pectinex Ultra SP-L, Celluclast and Rapidase C PE at concentration of 1.5:6:20 v/v. Apparent viscosity of pulp and serum reduced rapidly to 78 % and 93% respectively, in 30 min liquefaction. No marked changes in apparent viscosity pulp samples and percentage cell wall hydrolysis in the subsequent 60, 90 and 120 min were observed. The enzyme treated pulp showed 83 % and 84 % serum yields during 30 and 120 min reaction time respectively against 52% for untreated pulp. Slight increase in TSS ( 0 Brix), acidity, reducing and total sugars, and slight decrease in ph was found in the pulp as well as serum fractions as the incubation continued. No marked change in colour (yellowness) was observed in the enzyme-treated pulp, however, the corresponding serum showed less yellowness and colour saturation indicating retention of yellow pigments in the pulp fraction. About 9% loss in the total aroma components was observed during the liquefaction process. Brasil et al, (1995) prepared the clarified guava juice by treating the pulp with 600 ppm of pectic enzymes at 45 C for 120 min. The pulp so-treated was pressed to give an average juice yield of 84.70%. The pressed juice was cloudy and pink in colour but, after addition of fining agents and filtration, a clear juice with a light yellow colour was obtained. This clear juice was preserved by the Hot-pack method. During the extraction and clarification of the juice, some of the important physical and chemical changes were followed by measuring changes in total soluble solids ( o Brix), acidity, viscosity, total phenolics content, colour, turbidity and ascorbic acid retention. Chang et al, (1995) investigated the efficacy of five commercial pectinases for improvement of juice yield and quality from plums. Pectinases, to a varying 23

18 degree, improved the yield, color-assayed as release of anthocyanins, and clarity of the juice. A significant increase in the effectiveness of pectinases was observed as the concentration was increased from 0.01 to 0.60% v/w. However, at concentrations > 0.20% they tended to impart a bitter flavour in the juice. Among five pectinases, Clarex L at 0.20% produced higher yield and a sediment-free clear juice. Dawes et al, (1994) evaluated the effect of commercial fungal proteolytic enzyme from Aspergillus niger in kiwifruit juice as a replacement for conventional fining agents to produce a stable clarified juice. Reductions in detectable protein levels of 73% and 82% were achieved using 500 mg/kg of enzyme and incubating at 60 C for 20 and 60 min respectively. Concentrates prepared from proteolytic enzymetreated juice had reduced browning and haze formation compared to a control, without affecting ascorbic acid level. When stored at 20 C, proteolytic enzyme treated concentrates (60 min) remained clear up to 90 days and had minimal haze (A 650 nm = 0.047) and browning (A 420 nm = 0.93) after 6 months storage. Sreenath and Santhanam (1992) found that a commercial pectinase from Aspergillus niger containing various polysaccharases clarified the white grape juice to an extent of 98-99% and also degraded the grape mash by 25-30%. This was achieved by optimising the grape mash treatment with 0.048% of enzyme at C for 30 min without changing the mash ph. After pectinolytic juice clarification, both juice viscosity and total phenols were reduced by 25% and 32% respectively. The review of literature related to extraction and clarification of fruit juices can be summarized as shown in Table 2.5 Table 2.5 Review on Extraction and Clarification of Fruit Juices Sr. Author Year Important finding No. 1 Bahramian et 2011 Pectinase and cellulose enzyme treatments to date al fruit yields juice with more sugar and clarity as compared to untreated one. 2 Joshi et al 2011 Addition of pectinase significantly increased the total soluble solid (TSS), titratable acidity and total sugar in the enzymatically treated juices whereas the ph, Brix acid ratio and relative viscosity of extracted juices were decreased. 3 Karangwa et al 2010 Optimized the processing parameters for the 24

19 clarification of blended carrot-orange juice using Response Surface Methodology 4 Shailza et al 2009 Evaluated the effect of four different clarifying treatments viz. Sedimentation, Filtration, Pectinase and Filtration and Kaolin and Pectinase for clarification of Kiwi and Peach fruits 5 Vaidya et al 2009 Studied the enzymatic treatment for fruit juice extraction from Kiwi fruit. 6 Shah 2007 The effect of enzyme treatment conditions was studied on yield, clarity, apparent viscosity and total soluble solids of litchi juice obtained from the pulp. 7 Sin et al 2006 Enzyme concentration was the most important factor affecting the characteristics of the sapodilla juice as it exerted a significant influence on all the dependent variables. 8 Sorrivas et al 2006 Studied the effect of commercial pectinase and amylase enzyme on full ripe and unripe apple juice, respectively by using electron microscopy techniques. 9 Lan Quin et al 2005 The colour and cloud stability of cloudy carrot juice were improved by enzymatic hydrolysis and addition of hydrocolloids. 10 Singh et al 2000 About 30 min enzyme treatment was sufficient to bring down the pulp viscosity to a suitable level for clarification. Prolonged incubation beyond this time did not bring any additional desirable change; instead it has an effect on total aroma content 11 Brasil et al 1995 Studied the physic-chemical changes during enzymatic extraction and clarification of guava juice. 12 Chang et al 1995 Pectinases at concentrations > 0.20% impart a bitter flavor in the plum juice 13 Dawes et al 1994 A commercial fungal proteolytic enzyme from Aspergillus niger was used in kiwifruit juice as a replacement for conventional fining agents to produce a stable clarified juice. 14 Sreenath and Santhanam 1992 Commercial pectinase from Aspergillus niger containing various polysaccharases could clarified white grape juice to an extent of 98-99% and also degraded the grape mash by 25-30%. 25

20 2.3.3 Clarification of Banana Pulp Different researchers have used different commercial enzymes (especially those with pectinolytic activities) in banana pulp processing. However most of the researchers seem to have used only one stage i.e. ripened stage of banana pulp in their study. In this study, pulp of three different ripening stages was used for clarification study. Cheirsilp and Umsakul (2008) prepared banana wine by treating the banana must with pectinase and α-amylase to hydrolyze pectin and starch. The synergistic activities of the enzymes enhanced hydrolysis of the complex carbohydrates. A decrease of 55% in the viscosity and a 2.7-fold increase in the amount of extracted juice were obtained after incubating with 0.05% (w/w) of pectinase at 40 0 C for 2 h, followed by treating with 0.05% (w/w) of a-amylase at 50 0 C for 3 h. A 15 and 39% increase in total soluble sugars and reducing sugars in extracted juice were achieved, respectively. Enzyme-treated banana must was diluted with four volumes of water and then fermented by yeast to produce banana wine. The pre-treatment of banana with enzymes before wine fermentation resulted in a higher level of reducing sugars than that of the control (non enzyme-treated banana wine) during fermentation. The clarity of the enzyme-treated banana wine was also fourfold higher than that of the control at 25 days of fermentation. The concentrations of total soluble solids, total soluble sugars, and alcohol in the enzyme-treated banana wine and the control have no significant differences. Tadakittisarn et al, (2007) optimized the pectinase enzyme liquefaction of banana Gros Micheal pulp by response surface methodology (RSM). The effect of pectinase enzyme concentrations (0-0.2%) and incubation times ( min) on juice yield (%), total soluble solids (TSS), recovery soluble solids (RSS), clarity (%T670) and browning index (A420) of juice were studied using central composite design of experiments. Results showed that pectinase enzyme concentration played an important role that significantly (p 0.001) influenced most of dependent variables of banana juice. The coefficient of determination (R 2 ) of yield (%), recovery soluble solids (RSS), clarity (%T670) and browning index (A420) were 0.907, 0.924, and 0.793, respectively. The optimum condition for enzymatic extraction was 0.15% 26

21 of pectinase enzyme incubated for 120 min at 50 C. The yield was 62%, RSS 14, %T670 96% and A Lee et al, (2006) studied on optimization of conditions for the enzymatic clarification process of banana juice using response surface methodology. Banana juice was treated with pectinase at various enzyme concentrations ( %), temperatures ( C) and time ( min) of treatment. The effect of these enzyme treatments on filterability, clarity, turbidity and viscosity of the juice were studied by employing a second order central composite design. The coefficient of determination, R 2 values for filterability, clarity, turbidity and viscosity were greater than Statistical analysis showed that filterability, clarity, viscosity and turbidity were significantly (p<0.05) correlated to enzyme concentration, incubation temperature and incubation time. Enzyme concentration was the most important factor affecting the characteristics of the banana juice as it exerted a highly significant influence (p<0.01) on all the dependent variables. An increase in time and/or concentration of enzyme treatment was associated with an increase in filterability and clarity, and decrease in turbidity and viscosity. Based on response surface and contour plots, the optimum conditions for clarifying the banana juice were: 0.084% enzyme concentration, incubation temperature of C and incubation time of 80min. Shahadan and Abdullah (1995) determined the optimum conditions for extraction of banana juice. RSM with a central composite design was used for optimization. The effects of temperature (20-50ºC), ph ( ) and enzyme concentration ( %) on the yield of banana juice were studied after a 4h reaction time. The optimal conditions for the enzymatic extraction of banana juice were 0.42% enzyme at 35ºC with a ph of 3.4. Yunchand et al, (1995) prepared the clarified banana juice by using commercial enzyme pectinase and amyloglucosidase to increase juice yield. The optimum concentration was 0.03% Pectinex Ultra SP-L or Rohapex TF with 0.02% amyloglucosidase. Juice yield reached about 80% (base on pulp weight), when the banana puree was preheated to 90 0 C in steam blancher, followed by enzyme treatment at 45 0 C and maintained for 2 hrs. The effectiveness of enzyme on juice yield was greatly depending on pre-heating the pulp and the stage of banana maturity. 27

22 Kotecha et al, (1994) carried out preliminary studies to carry out banana juice extraction by using different levels of pectinase enzymes and different levels of incubation periods at C. Based on these studies a 0.2% pectinase addition and a 4h incubation time were selected for obtaining the juice from pulp. The juice was separated by centrifugation and the clear juice was used for preparation of juice. Pheantaveerat and Anprung (1993) studied the application of commercially available pectinases, cellulases and amylases for hydrolysis of ripe banana (Grade 7-8) pulp. He observed that the synergistic activities of enzymes in increasing degree of pulp hydrolysis expressed as percent decreased viscosity of juice obtained after the pulp was incubated with 0.06% by weight of cellulases and with 0.05% by weight of pectinases at 45 0 C for 2 h. Under such condition, the clear juice yield of 73% (base on pulp weight used) was obtained. Furthermore, amylases were not effective to the above activity. Koffi et al, (1991) conducted experiments to determine the effects of commercial enzyme preparations on viscosity reduction and filterability of banana juice and the effectiveness of various anti-browning treatments on clarified juice. Two different combinations of pectinase, cellulase and hemicellulase were more effective in reducing viscosity and improving filterability of both green and ripe banana purees than a pectinase, galactomannanase or cellulase after incubation periods of 3, 6 and 9 h. An alpha-amylase was not effective in reducing viscosity as compared to the control, even of green banana puree high in starch. Viquez et al, (1981) studied the pectinolytic enzyme treatments on banana pulp to increase the yield, reduce the viscosity and clarify the juice. Clear juice yields of between 55 and 60% (based on pulp weight used) are obtained from pulp incubated at 45 C for 1 hr with 0.01% w/w of enzyme by subsequent centrifugation at 2900 maximal relative centrifugal force for 20 min. This corresponds to a yield of total and reducing sugars present in the pulp of over 75%. Untreated control pulps yield less than 5% of juice under these conditions. Hydraulic pressing of the pulps at 16 kg/cm 2 gives similar juice yields to those obtained by centrifugation. The juice has an excellent flavour and aroma and provides a possible use for the large quantities of 28

23 reject bananas available in producer countries. The research related to clarification of banana pulp can be summarized as shown in Table 2.6 Table 2.6 Review on Clarification of Banana pulp Sr. No. 1 Cheirsilp and Umsakul Author Year Important finding 2 Tadakittisarn et al 2008 A decrease of 55% in the viscosity and a 2.7-fold increase in the amount of extracted juice were obtained after incubating banana pulp with 0.05% (w/w) of pectinase at 40 0 C for 2 h, followed by treating with 0.05% (w/w) of a- amylase at 50 0 C for 3 h Dependent variables viz. yield, recovery soluble solids, clarity and browning index are significantly influenced by enzyme concentration. 3 Lee et al 2006 An increase in time and/or concentration of enzyme treatment was associated with an increase in filterability and clarity, and decrease in turbidity and viscosity. 4 Shahadan and Abdullah 1995 The optimal conditions for the enzymatic extraction of banana juice were 0.42% enzyme at 35ºC with a ph of Yunchalad et al 1995 The effectiveness of enzyme on juice yield was greatly depending on pre-heating the pulp and the stage of banana maturity. 6 Kotecha et al 1994 At 28±20 0 C, 0.2% pectinase concentration and 4h incubation time yields maximum clarified banana juice. 7 Pheantaveerat and Anprung 1993 Amylases were not effective for the clarification of pulp 8 Koffi et al 1991 Two different combinations of pectinase, cellulase and hemicellulase were more effective in reducing viscosity and improving filterability of banana purees than alpha-amylase enzyme 9 Viquez et al 1981 Clear juice yields of between 55 and 60% were obtained from pulp incubated at 45 C for 1 hr with 0.01% w/w of enzyme concentration 29

24 2.4 Browning during Clarification of Fruit Pulp its Inhibition Three general situations may cause browning reactions in fruit viz. physiological changes associated with ripening, disorders associated with cold storage or chilling injury and thirdly operations associated with harvesting and processing of fruit where crushing, wounding or juice extraction occur. During preparation of juices, once the fruit structure is disintegrated by crushing enzymes which are normally associated with structural components would be brought into contact with substrates and oxygen which stimulate some enzymatic and non enzymatic reactions. These reactions may responsible to change in colour, flavour and appearance of the juice (Pollard and Timberlake, 1971). Banana pulp is highly susceptible to enzymatic browning during pulping. Browning reaction of banana fruit results from the enzymatic oxidation of dopamine (3,4 -dihydroxyphenyl ethylamine) by polyphenoloxidase (Griffiths, 1959) leading to the production of brown pigments. Various techniques and mechanisms have been developed over the years for the control of these undesirable enzyme activities. These techniques attempt to eliminate one or more of the essential components (oxygen, enzyme, copper, or substrate) from the reaction. Samanta et al, (2010) evaluated the anti-browning (inhibition of polyphenol oxidase activity) effect of cysteine (Cys), ascorbic acid (AA), citric acid (CA), sodium metabisulphite (SMB) alone or in combination, at three different ph (3.5, 4 and 4.5) in banana (Musa paradisiaca L. var. Kanthali), apple (Malus pumila Mill. var. Ambri kashmiri), and mushroom (Agaricus bisporus). All the samples were mixed with Cys (100, 200 and 300 mg/kg), AA (250, 500 and 1000 mg/kg), CA (250, 500 and 1000 mg/kg) and SMB (100, 200 and 300 mg/kg) to assess their effect on PPO. PPO activity was analyzed spectrophotometrically at 420 nm. The most effective PPO inhibitors were AA and SMB and in combination with CA and Cys in all the samples tested. No significant differences were observed for PPO activity among concentrations of Cys and CA when both anti-browning agents were used alone or in combination and mixed with the samples. Danyen et al, (2009) studied the interaction effects between ascorbic acid and calcium chloride in minimizing browning of fresh-cut green banana slices. Dwarf 30