Production of Commercially Suitable

CHAPTER 5

MATERIALS AND METHODS

5.1. Materials

Oranges (cultivar Valencia from Antalya, Turkey) were purchased from a local market in İzmir. In the laboratory, the juices of oranges were extracted with a home type manual extractor and the peels obtained were frozen and kept at –18 ⁰ C until used in the experiments. The commercially cultivated mushrooms (Agaricus biosporus) were purchased from a local market in İzmir, Turkey. In the laboratory, the stems of mushrooms, cut approximately 1 cm below their caps, were collected and processed to acetone powder immediately. The citrus pectin (Galacturonic acid content 79 %, methoxy content 8 %), dialysis tubing (prepared as described in the product information), insoluble PVPP (polyvinylpolypyrrolidone), and dextran were purchased from Sigma Chem. Co. (St Louis, Mo., USA). Ammonium sulphate (for biochemistry), and L-tyrosine (for biochemistry) were purchased from Merck (Darmstadt, Germany). 3-(3,4 Dihydroxyphenyl)–L-alanine and phloridzin dihydrate were purchased from Fluka (Steinheim, Germany). Saccharose was purchased from Pancreac (Barcelona, Spain) and sodium benzoate and potassium sorbate were purchased from AppliChem (Darmstadt, Germany). All the other chemicals were reagent grade.

5.2. Methods

5.2.1. Methods related to PME enzyme
5.2.1.1. PME extraction
For the extraction of PME enzyme five different extraction procedures were tested;
Extraction procedure # 1: Homogenization of orange peels (30 g) with cold distilled water (90 mL) / Filtration from cheese-cloth (4 layers) to collect the enzyme extract (the pellet was discarded) / Centrifugation of enzyme extract (at 3500 g for 15 min at 4 ^oC)
Extraction procedure # 2: Homogenization of orange peels (30 g) with 1 M NaCl solution (90 mL) / Filtration from cheese-cloth (4 layers) to collect the enzyme extract (the pellet was discarded) / Centrifugation of enzyme extract (at 3500 g for 15 min at 4 ^oC)
Extraction procedure # 3: Homogenization of orange peels (30-50 g) with cold distilled water (150 mL) / Filtration from cheese-cloth (2 layers) to collect the pellet (the water extract was discarded) / The pellet was once more homogenized with cold water (150 mL) / Filtration from cheese-cloth (2 layers) to collect the pellet (the water extract was discarded) / The pellet was mixed with 6 or 10 g NaCl and the final weight of mixture was made up to 100 g with distilled water / The mixture was mixed for 30 or 45 min for the extraction of enzyme / Filtration from cheese-cloth (4 layers) to collect the enzyme extract (the pellet was discarded) / Centrifugation of enzyme extract (at 3500 g for 15 min at 4 ^oC).
Extraction procedure # 4: Homogenization of orange peels (30 g) with cold distilled water (150 mL) / Filtration from cheese-cloth (2 layers) to collect the pellet (the water extract was discarded) / The pellet was once more homogenized with cold distilled water (150 mL) / Filtration from cheese-cloth (2 layers) to collect the pellet (the water extract was discarded) / The pellet was mixed with 6 g NaCl and the final weight of mixture was made up to 100 g with distilled water / 2 g PVPP was added to medium and the mixture was mixed for 45 min for the extraction of enzyme / Filtration from cheese-cloth (4 layers) to collect the enzyme extract (the pellet was discarded) / Centrifugation of enzyme extract (at 3500 g for 15 min at 4 ^oC)
Extraction procedure # 5: Homogenization of orange peels (30 g) with cold acetone at -18 ^oC (200 mL) / Filtration from a Buncher funnel, containing Whatman No:1, under vacuum to collect the pellet (the acetone extract was discarded) / The pellet was once more homogenized with cold distilled water (150 mL) / Filtration from cheese-cloth (2 layers) to collect the pellet (the water extract was discarded) / Pellet was mixed with 6 g NaCl and the final weight was made up to 100 g with distilled water / The mixture was mixed for 45 min for the extraction of enzyme / Filtration from cheese-cloth (4 layers) to collect the enzyme extract (the pellet was discarded) / Centrifugation of enzyme extract (at 3500 g for 15 min at 4 ^oC).

5.2.1.2. Determination of PME activity
For the determination of PME enzyme activity titrimetric and spectrophotometric methods were used. In the titrimetric assays, the method given in Yemenicioğlu (2002) was used with slight modifications. The reaction mixture contained 1 or 3 mL of enzyme extract and 20 mL of 0.5 % pectin solution prepared in 0.1 M NaCl. The pH of the reaction mixture was brought to 7.5 with 0.1 N NaOH and kept constant for 5 or 10 min by titrating with 0.01 N NaOH. The titrations were performed in a double walled magnetically stirred cell connected to a circulating water bath working at 30 ^oC. The enzyme activity was expressed as percent initial activity or amount of 0.01 N NaOH spend in titrations per minute per mL of enzyme extract (mL 0.01 N NaOH/min/mL). All activity measurements were performed for three times and averages were calculated.

In spectrophotometric assays the method given in Hagerman and Austin (1986) was used with slight modifications. The reaction mixture was formed by mixing 2.3 mL of 0.3 % pectin solution prepared in 0.1 M NaCl, 0.5 mL of 0.01 % (w/v) bromothymol blue prepared in 0.003 M sodium phosphate buffer at pH 7.5 and 0.1 mL crude enzyme. The decrease in absorbance at 620 nm was monitored by using a Shimadzu (Model 2450) spectrophotometer, equipped with a constant temperature cell holder working at 30 ^oC. The enzyme activity was determined from the slope of the initial linear portion of abs versus time curve and expressed as Unit. One Unit was defined as that amount of enzyme that caused 0.001 changes in absorbance in 1 min.

5.2.1.3. Effect of mild heating on PME activity
To determine the effects of mild heating on PME, pieces of orange peels (obtained from 16 peel halves from different oranges) were put into sacks made from cheese-cloth and incubated between 30 ^o and 55 ^oC for 30 min in a circulating water bath. At the end of the incubation period the PME was extracted from the peels by the extraction procedure # 3 (by using 10 g NaCl and 30 min mixing for enzyme extraction) and tested for enzyme activity by the titrimetric method.


5.2.1.4. Effect of CaCl₂ on PME activity
To determine the effect of CaCl₂ on PME activity, the activity of enzyme was determined in the presence of 0.75-50 mM CaCl₂ (The final concentrations in reaction mixture). The enzyme extract used in these experiments was obtained with the extraction procedure # 3 (by using 10 g NaCl and 30 min mixing for enzyme extraction). Enzyme activities were determined by the spectrophotometric method by including 0.1 mL CaCl₂ (at varying concentrations) to the reaction mixture.
5.2.1.5. Preparation of a commercial PME preparation and test of its stability
In order to obtain a commercial PME enzyme preparation, the PME was extracted by extraction procedure # 3 (by using 10 g NaCl and 30 min mixing for enzyme extraction). To clarify the preparation, the enzyme extract was incubated for 1 week and the precipitate formed was collected by applying centrifugation at 3500 g for 15 min at 4 ^oC. To prevent the microbiological spoilage, 0.1 % K-sorbate and 0.1 % Na-benzoate were then added to the clear enzyme extract that was at pH 3.8. The storage stability of the enzyme was determined by monitoring enzyme activity at 4 ^oC with or without the presence of 1 mM CaCl₂. The activity of enzyme was tested by the titrimetric method.
5.2.1.6. Test of obtained PME in the preparation of edible pectin films
For the preparation of pectin films, the following steps were followed; (1) 18 mL 2 % pectin solution was demethylized by 1 mL PME preparation containing 1.0 mL 0.01 N NaOH/min/mL enzyme activity. The demethylation was conducted at room temperature until 2.1 mL, 1 M NaOH was spent in the titration; (2) 10 g demethylized PME solution was pipetted onto a 9.8 cm diameter glass petri dish and left 1 day at room temperature to dry; (3) The cross-linking of film was conducted by adding 5 mL of 1 M CaCl₂ onto the dried pectin film and by applying further drying for 5-6 h at room temperature. At the end of the drying period, the film was peeled from the petri dish and wetted to check whether insoluble Ca-pectate structure is formed by the action of PME.

5.2.2. Methods related to PPO enzyme
5.2.2.1. Acetone powder preparation
To obtain the acetone powder, 100 g mushroom stems were homogenized in a Waring blender for 3 min with 200 mL cold acetone at -18 ^oC and 2 g insoluble PVPP. The slurry obtained was filtered under vacuum from a Buncher funnel containing a Whatman No:1 filter paper and the solid residue remained on the filter paper was collected. The homogenization with 200 mL cold acetone and filtration were then repeated for two more times for the collected residue without using PVPP and the light brown colored powder, left overnight to evaporate the acetone, was stored at – 18 ^oC until used for enzyme extraction.
5.2.2.2. PPO extraction
The enzyme extraction from acetone powder was conducted by mixing 3 g acetone powder, 3 g insoluble PVPP, 50 mL 0.05 M, pH 7 Na-phosphate buffer and stirring for 30 min at + 4 ^oC with a magnetic stirrer. The extract was then filtered from a four layers of cheese-cloth and clarified by centrifuging 15 min at 10000 x g and + 4 ^oC.
5.2.2.3. Ammonium sulfate precipitation
For ammonium sulphate precipitation, solid (NH₄)₂SO₄ was slowly added to crude enzyme extracts up to 90 % saturation. The mixture was stirred slowly at + 4 ^oC for 2 h and the precipitate was collected by applying 45 min centrifugation at 15000 x g and + 4 ^oC. The precipitate collected was then dissolved in 20 mL of 0.05 M Na-phosphate buffer at pH 7 and dialyzed 24 h at + 4 ^oC against 2000 mL distilled water by three changes.
5.2.2.4. Acetone precipitation
When PPO was partially purified with acetone, two volume of cold acetone at -18 ^oC was added to one volume of crude enzyme extract. After 10 min stirring at + 4 ^oC, the precipitate formed was collected by 45 min centrifugation at 15000 x g and 0 ^oC, and dissolved in 15 mL of 0.05 M Na-phosphate buffer at pH 7. The enzyme was then dialyzed for 24 h at + 4 ^oC against 2000 mL distilled water by three changes.
5.2.2.5. Determination of PPO activity
The PPO activities were determined at + 30 ^oC by using a Shimadzu (Model 2450) spectrophotometer equipped with a constant temperature cell holder. The monophenolase activity of PPO was determined by forming the following reaction mixture; 0.5 mL enzyme extract, 1.5 mL, 0.05 M Na-phosphate buffer at pH 7 and 1 mL, 0.25 or 2 mM tyrosine. The increase in absorbance was recorded at 280 nm and enzyme activity was determined from the initial linear portion of absorbance vs. time curve coming after the initial lag period. The catechol oxidase activity of PPO, on the other hand, was determined by mixing 0.1 mL enzyme extract, 0.1 mL 10 mM L-DOPA and 2.8 mL, 0.05 M Na-phosphate buffer at pH 7. The increase in absorbance was determined at 475 nm, and enzyme activity was calculated form the slope of the initial linear portions of absorbance vs. time curve. The activities of enzymes were expressed as percent initial activity or Unit, which gives the amount of enzyme that causes 0.001 absorbance change in one minute.
5.2.2.6. Characterization studies
The temperature profiles of PPO were determined by 30 min incubation of enzyme extracts in TIT (Thermal Inactivation Time) tubes (i.d., 9 mm; wall thickness, 1 mm) between 35 ^o and 60 ^oC.
The optimum temperature was determined by measuring enzyme activities between 25 and 40 ^oC.
The optimum pH was determined by changing the standard reaction mixture to 0.2 mL enzyme extract, 2 mL, 0.1 M acetate (at pH 4 and 5), Na-phosphate (at pH 6.0, 6.6 and 7.0) or Tris-HCl (at pH 8.0) buffers and 0.8 mL, 2 mM tyrosine.
The pH stabilities were determined by mixing 0.2 (or 0.5) mL enzyme extract and 0.4 (or 1) mL of 0.1 M buffer (suitable buffer as given in optimum pH determination) at pH 4.0, 5.0, 6.0, 6.6, 7.0 or 8.0. The enzyme-buffer mixtures formed were incubated at 4 ^oC for 24 h and their remaining activities were determined at pH 7.0 by using the following reaction mixture; 0.5 mL enzyme-buffer mixture, 1.5 mL, 0.5 M Na-phosphate buffer at pH 7.0 and 1 mL, 2 mM tyrosine prepared in 0.5 M Na-phosphate buffer at pH 7.0.
5.2.2.7. Storage stability of PPO in acetone powders
In order to investigate the storage stability of PPO in acetone powders, 0.5 g acetone powder was distributed to different test tubes. Some of these tubes were incubated in a refrigerator at almost + 4 ^oC, whereas the others were incubated in a deep-freezer working at –18 ^oC. The monophenolase activity of samples was determined by using 0.25 mM tyrosine in the reaction mixture.