I would like to express my sincere gratitude to my advisor Assoc. Prof. Ahmet Yemenicioğlu for his guidance, supervision, encouragement, and support at all steps of this study.
I am also grateful to my family for their support, encouragement, and understanding.
ABSTRACT
In this study, some simple and effective extraction and/or partial purification procedures were developed to obtain pectin methylesterase (PME) and polyphenol oxidase (PPO) enzymes from orange peels and mushroom stems, respectively. Also, some characteristics of enzymes were investigated and their stable preparations were obtained in liquid or lyophilized forms. Valencia orange peels contain considerable PME activity (300-350 mL NaOH/min/100 g) that is quite stable during season for at least 5 months. The enzyme was ionically bound to cell walls and can not be extracted by homogenization with water. However, the addition of suitable amounts of NaCl (10 g /100 g extraction mixture) to pellet, obtained by homogenization of peels several times with water, and 30 min mixing (at 200 rpm) may be effectively used to extract the enzyme. The PME in orange peels contains almost the same amount of heat stable and heat labile fractions and the enzyme can not be activated by mild heating. A slight activation (almost 20 %) may be achieved by adding 1 mM CaCl2 to enzyme extracts. However, at higher concentrations the addition of CaCl2 was inhibitory. The PME activity in extracts, stabilized by use of 0.1 % Na-benzoate and 0.1 % K-sorbate, is stable almost 5 months at + 4 oC (maintains > 90 % of its activity). Thus, the commercial preparations of enzyme may be obtained in liquid form. The extracted PME was successfully used to prepare edible films from citrus pectin.
Diğer yandan, PPO’nun ekstraksiyonu için öncelikle mantar sapları aseton tozuna işlenmiştir. Aseton tozları daha sonra Na-fosfat tamponu ile ekstrakte edilmiş ve % 90 amonyum sülfat çöktürmesi ya da 2–kat aseton çöktürmesi ve bunu takip eden diyaliz işlemi ile kısmi olarak saflaştırılmıştır. Aynı aseton tozundaki PPO’nun monofenolaz aktivitesinin, amonyum sülfat veya asetonla çöktürme yoluyla kısmi saflaştırılması ile elde edilen geri kazanım ve saflık katsayıları sırasıyla % 74-86 ve 3.4-4.3 ve % 55-67 ve 5.4-6.2’dir. Buna göre amonyum sülfat çöktürmesinin daha yüksek verime karşın daha düşük saflık sağladığı görülmektedir. Kısmi olarak saflaştırılmış PPO enziminin monofenolaz aktivitesinin ısıl direnci düşük olup inaktivasyonu 45 oC’ın üzerinde başlamaktadır. Enzim, pH 6.0 ve 8.0 arasında optimum aktivite göstermiş olup pH 7.0 ve 8.0’deki stabilitesi maksimumdur. Ancak buna karşın enzim, pH 4.0’te 24 saat inkübasyon sonucunda aktivitesinin büyük kısmını kaybetmiştir. Enzimin optimum sıcaklığı 40 oC olarak belirlenmiştir. PPO enziminin monofenolaz aktivitesi, aseton tozu halinde + 4 oC’de stabilite göstermemiş, ancak buna karşın - 18 oC’de iki ay süreyle % 60-70 oranında aktivitesini korumuştur. Ayrıca amonyum sülfat ve diyaliz ile kısmi saflaştırılmış ve destek maddesi olarak dekstran ve sakkaroz eklenerek liyofilize edilmiş PPO, –18 oC’de 3 ay süresince monofenolaz ve difenolaz aktivitesini korumuştur. Dekstranla liyofilizasyonun enzimin ısıl stabilitesi üzerinde herhangi bir etkisi belirlenememiştir. Ancak buna karşın dekstranla liyofilizasyon, pH 4.0’deki monofenolaz aktivitesinin stabilitesini kısmen azaltmıştır. Tirozin üzerindeki monofenolaz aktivitesi ve L-DOPA üzerindeki difenolaz aktivitesinin yanısıra dekstranla liyofilize edilmiş PPO, floridzin’i de substrat olarak kullanabilmektedir. Bu sonuç enzimin antioksidan ve renk maddelerinin üretimi, proteinlerin modifikasyonu, kakao ve siyah çayın fermantasyonu gibi farklı gıda uygulamalarında kullanılabileceğini göstermektedir.
LIST OF FIGURES
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xii
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LIST OF TABLES
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xiv
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CHAPTER 1. INTRODUCTION
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1
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CHAPTER 2. ENZYME PRODUCTION FOR INDUSTRIAL APPLICATIONS
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3
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2.1. Extraction of Enzymes
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3
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2.1.1. Cell disruption methods
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5
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2.1.1.1. Mechanical methods
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5
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2.1.1.2. Nonmechanical methods
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5
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2.2. Clarification of Enzyme Extracts
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6
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2.2.1. Centrifugation
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6
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2.2.2. Filtration
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6
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2.2.3. Flocculation and flotation
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7
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2.3. Concentration of Enzymes
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7
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2.3.1. Addition of a dry matrix polymer
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7
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2.3.2. Freeze-drying (Lyophilization)
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7
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2.3.3. Ultrafiltration
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8
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2.3.4. Precipitation
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9
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2.3.4.1. Precipitation by increasing the ionic strength (salting out)
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9
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2.3.4.2. Precipitation by decreasing the ionic strength (salting in)
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10
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2.3.4.3. Precipitation by organic solvents
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10
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2.3.4.4. Precipitation by alteration of pH
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11
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2.3.4.5. Precipitation by organic polymers
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11
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2.3.4.6. Precipitation by denaturation
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11
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2.3.5. Aqueous two-phase partitioning
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12
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2.3.6. Removal of salts and exchange of buffers
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12
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2.3.6.1. Dialysis
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12
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2.3.6.2. Diafiltration
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13
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2.3.6.3. Gel filtration
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13
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2.4. Purification
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13
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2.5. Product Formulation
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14
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CHAPTER 3. PECTIN METHYLESTERASE
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16
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3.1. Pectin methylesterase and Other Pectinases
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16
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3.2. Sources of PME
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16
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3.3. The Effects of PME on Food Quality
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17
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3.4. Industrial Applications of PME
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19
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3.4.1. Clarification of fruit juices
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19
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3.4.2. Firming of fruits and vegetables before processing
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21
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3.4.3. Modification of pectin
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21
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3.4.4. Production of low sugar jams, jellies, and compotes
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22
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3.4.5. Other applications
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22
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CHAPTER 4. POLYPHENOL OXIDASES
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24
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4.1. Polyphenol oxidases
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24
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4.2. Substrates of PPO
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26
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4.3. Sources and Some Characteristics of PPO
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27
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4.4. The Effects of PPO on Food Quality
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29
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4.5. Industrial Applications of PPO
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29
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4.5.1. Enzymatic cross-linking of proteins or polysaccharides |
30
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4.5.2. Production of flavonoid-derived colorants and antioxidants
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30
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4.5.3. The removal of haze forming polyphenols from beverages
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31
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4.5.4. Oxygen scavenging and removal of undesirable phenolics from food
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32
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4.5.5. Removal of undesirable phenolics from wastewaters
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32
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4.5.6. Analytical and clinical applications of PPO
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33
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4.5.6.1. Production of biosensors
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33
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4.5.6.2. Clinical applications
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33
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CHAPTER 5. MATERIALS AND METHODS
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35
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5.1. Materials
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35
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5.2. Methods
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35
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5.2.1. Methods related to PME enzyme
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35
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5.2.1.1. PME extraction
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35
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5.2.1.2. Determination of PME activity
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37
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5.2.1.3. Effect of mild heating on PME activity
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37
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5.2.1.4. Effect of CaCl2 on PME activity
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38
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5.2.1.5. Preparation of a commercial PME preparation and test of its
stability
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38
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5.2.1.6. Test of obtained PME in the preparation of edible pectin
films
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38
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5.2.2. Methods related to PPO enzyme
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39
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5.2.2.1. Acetone powder preparation
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39
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5.2.2.2. PPO extraction
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39
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5.2.2.3. Ammonium sulphate precipitation
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39
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5.2.2.4. Acetone precipitation
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39
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5.2.2.5. Determination of PPO activity
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40
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5.2.2.6. Characterization studies
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40
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5.2.2.7. Storage stability of PPO in acetone powders
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41
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5.2.2.8. Preparation of commercial PPO preparations and test of
their storage stabilities
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41
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5.2.2.9. The effect of lyophilization with dextran on temperature
and pH stability of PPO
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41
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5.2.3. Determination of protein content
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42
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CHAPTER 6. RESULTS AND DISCUSSION
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43
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6.1. The Results Obtained for PME Enzyme
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43
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6.1.1. Change of PME activity in orange peels during season
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43
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6.1.2. Effect of different extraction procedures on PME activity
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44
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6.1.3. Effect of NaCl concentration on PME activity extracted from
orange peels
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45
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6.1.4. Effect of extraction period on PME activity extracted from orange
peels
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46
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6.1.5. Effect of mild heating on PME activity
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47
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6.1.6. Effect of CaCl2 on PME activity
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48
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6.1.7. Stability of the prepared PME during storage
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49
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6.1.8. Test of obtained PME in the preparation of edible pectin films
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50
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6.2. The Results Obtained for PPO Enzyme
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52
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6.2.1. Monophenolase and diphenolase activities of PPO
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52
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6.2.2. Distribution of PPO in mushrooms
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53
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6.2.3. Partial purification of PPO
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54
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6.2.4. Characterization of monophenolase activity
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56
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6.2.5. Stability of monophenolase activity in acetone powders
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60
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6.2.6. Stability of the prepared PPO during storage
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61
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6.2.7. The effect of lyophilization with dextran on temperature and pH
stability of PPO
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63
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6.2.8. The ability of the prepared PPO to oxidize phloridzin
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64
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CHAPTER 7. CONCLUSIONS
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66
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REFERENCES
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68
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