الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 158 |  |  |
| | | from | 158 | | |
Abstract
The scientific interest focused on the nutritional value and medicinal properties of edible oyster mushroom opened a new area to study their biologically active substances, where it’s used as a source of milk clotting enzyme (MCE) as well as study of their biochemical characteristics is still limited in order to investigate its technological suitability in cheese production. Therefore, the present work aimed to extract and purify MCE from edible oyster mushroom (Pleurotus florida 14 MICC) available in Egypt as a calf rennet substitute in cheese production. Also, the biochemical properties of mushroom MCE, and its effect on different milk species were investigated. Moreover, the influence of mushroom MCE as an alternative coagulant on chemical, textural, sensorial, and nutritional characteristics of soft cheese were investigated in comparison of both commercial calf rennet and microbial coagulant. Thus, plan of work included: 1) Isolation of MCE from edible oyster mushroom (Pleurotus florida 14 MICC). 2) Partial purification of isolated MCE by Ammonium Sulfate Precipitation (ASP). 3) Purification of MCE rich fraction by Size Exclusion chromatography (SEC) on Sephadex G-100 column. 4) Determination of molecular weight and purity of MCE fractions by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). 5) Assessment the biochemical characteristics of both crude and partial purified MCE of oyster mushroom as follow: • Optimum pH. • Optimum reaction temperature. • Thermal stability. • Effect of calcium chloride concentration on MCE activity. • Effect of sodium chloride concentration on MCE activity. • Determination of MCE activity on different milk types. 6) Soft cheese manufacture using MCE of mushroom as a coagulant in comparison of both commercial calf rennet and microbial coagulant. 7) Chemical characterization of cheese. English Summary 98 8) Texture profile analysis of cheese using universal testing machine. 9) Amino acids composition of cheese using HPLC. 10) Calculation of chemical score, protein efficiency ratio and biological value of cheese. 11) Microstructure profile of cheese using Scanning Electron Microscopy (SEM). 12) Organoleptic properties evaluation of cheese. 1. Isolation and Purification of MCE from oyster mushroom (Pleurotus florida 14 MICC): MCE is extracted and purified from an edible oyster mushroom with successive purification processes such as ASP and SEC. Moreover, the biochemical characteristics of MCE fractions, and it’s suitability towards different milk species were investigated. The briefly results are presented as follow: 1) MCE activity was determined on dried fruit bodies of oyster (Pleurotus florida 14 MICC) mushroom using different extraction buffers. The highest MCE activity (75.49 SU/ml) and its specific activity (356.08 SU/mg protein) was observed in oyster mushroom dissolved in 0.2 M sodium acetate buffer pH 5.0, followed by citrate phosphate pH 5.0 and distilled water. The MCE from oyster mushroom formed a strong clot with forming quite whey after adding the enzymatic extract in skim milk solution. 2) MCE was partially purified from the selected crude extract of oyster mushroom using ASP procedure. The highest total MCE activity (367.85 SU) was recorded at 20% saturation of ammonium sulfate with specific MCE activity of 343.79 SU mg-1 protein as well as the MCE was partially purified 1.01-fold from the crude extract of oyster mushroom with a yield of 24.36%. 3) Mushroom MCE was further purified using SEC on Sephadex G-100. MCE activity was observed in one peak among 40 collected fractions with maximal activity in fraction no. 18 (67.8 SU/ml). Also, the purification of MCE using Sephadex G-100 column recovered 17.96% with 3.46 as a purification fold where the total MCE activity was 271.20 SU with 1232.73 SU/mg protein as specific activity. 4) Electrophoretic profile of MCE fractions showed different bands in the crude fraction, which decreased in the partially purified fraction using ASP. Moreover, SDS-PAGE of the purified MCE from oyster Pleurotus florida 14 MICC) mushroom showed a single band with a molecular mass of about 45 kDa. 5) SDS-PAGE of casein proteolysis showed that mushroom MCE promoted extensive cleavage of k-CN, αs- and β-CN fractions within 120 min at the optimal reaction condition. 6) The proteolytic activity (PA) of crude MCE extracted from oyster (Pleurotus florida 14 MICC) mushroom was 2.39 U/ml with a ratio of MCA/PA of 31.59. Moreover, PA of partial purified MCE was 2.45 U/ml, while the purified mushroom MCE recorded PA of 2.82 U/ml with a ratio of MCA/PA of 30.03 and 24.04, respectively. 7) Oyster (Pleurotus florida 14 MICC) mushroom MCE exhibited the optimal activity at 55 °C for the crude and partial purified extracts, while the purified mushroom MCE recorded their maximal activity at 50 °C. 8) The optimal reaction pH for crude MCE of oyster mushroom was 5.0 while the purified and partial purified MCE was observed at pH 6.0. Also, neutral and alkaline pH reduces mushroom MCE for all resulted fractions. 9) The calcium chloride concentration (0.01%) recorded the maximal MCE activity for crude MCE extracted from oyster mushroom, followed by a gradual decrease in their activity. Moreover, purified and partially purified mushroom MCE activity increased until 0.02% and 0.04%, respectively. 10) The mushroom MCE activity gradually decreased as the level of sodium chloride (1-5%) increased for all enzyme fractions. 11) The thermal stability of all mushroom MCE fractions showed that the MCE activity decreased as both temperature and pre-incubation time increased. Also, the results indicated that the lowest reduced activity of mushroom MCE after pre-incubation at 40 °C for crude extract followed by partially purified and purified MCE fractions. Also, at 50 °C mushroom MCE activity were reduced about 60-65%, while the reduction of its activity reached about 70-95% at 60 °C for all mushroom fractions after 30-60 min. Moreover, all mushroom MCE fractions activities were quickly diminished after incubation at 70 °C. 12) The suitability of mushroom MCE fractions towards different milk types, including cow, buffalo, goat, and sheep milk. Mushroom MCE could be capable of coagulating all examined milk types with various coagulation times started with cow milk followed by buffalo, sheep, and goat milk for both crude and partially purified MCE fractions. Also, close MCE activities recorded for both partial purified and purified fractions of mushroom on buffalo milk 2. Influence of mushroom MCE as a coagulant in soft cheese production compared to both calf rennet and microbial coagulant: The technological suitability of MCE fractions (crude, and partial purified) from edible oyster mushroom (Pleurotus florida 14 MICC) as a calf rennet substitute in soft cheese production was examined in comparison of available commercial animal calf rennet and microbial coagulant. Thus, the chemical, textural, sensorial, and nutritional characteristics of the resulted cheese were investigated during its storage period at 5 °C for 4 weeks. The briefly results are presented as follow: 1) The moisture content of soft cheese coagulated with crude mushroom MCE were slightly lower than those of cheese coagulated by calf rennet or microbial coagulant without significantly (p≤0.05) differences during the storage period. Also, the moisture content of cheese made with partial purified MCE from mushroom was close to cheese made with commercial coagulants without significantly (p≤0.05) differences during storage. Moreover, the moisture content of all resulted cheese was decreased gradually with the storage period progressed with significantly (p≤0.05) differences after 2nd week of storage. 2) The protein content of soft cheese coagulated with crude mushroom MCE were close to resulted cheese made with commercial coagulants without significant (p≤0.05) differences. Also, cheese coagulated with partial purified MCE from mushroom had the lowest protein content among resulted cheese treatments. 3) No significant (p≤0.05) differences of fat content for soft cheese made with mushroom MCE fractions in comparison of those coagulated with both calf rennet and microbial coagulant. 4) The soft cheese coagulated with crude MCE of mushroom had the highest ash content among other cheese treatments with significantly (p≤0.05) differences during all storage period. 5) The WSN of cheese made with mushroom coagulant was higher than those coagulated with commercial coagulants with significantly (p≤0.05) differences after 2 weeks of storage period. Moreover, WSN ratio of cheese coagulated with partial purified MCE of mushroom were higher than cheese coagulated with crude MCE extract from mushroom with significantly (p≤0.05) differences after 2 weeks of storage period. 6) The acidity (%) with opposite trends in pH values of soft cheese coagulated by crude MCE from mushroom (Pleurotus florida 14 MICC) were higher than those coagulated with commercial coagulants without significantly (p≤0.05) differences except in 4th week of storage. Also, it could be noted that the cheese acidity made with partial purified MCE from mushroom were close to calf rennet and microbial coagulant without significantly (p≤0.05) differences during storage. 7) Amino acid content of soft cheese made with different coagulants showed that all resulted cheese contains all essential amino acids (EAA) with highest content in calf rennet made-cheese (lysine, valine, threonine, and histidine), followed by crude MCE from mushroom made-cheese (methionine, and isoleucine), and microbial coagulant made-cheese (leucine, and phenylalnine). Valine amino acid content of calf rennet made-cheese (43.86 mg/g protein) is close to both microbial (40.10 mg/g protein) and crude mushroom coagulants made-cheese (43.16 mg/g protein); while isoleucine of mushroom (crude MCE) made-cheese is close to commercial coagulants made-cheese. Lysine content of calf rennet made-cheese (78.49 mg/g protein) and leucine content of microbial coagulant made-cheese (38.06 mg/g protein) were close to partial purified MCE of mushroom made-cheese (70.92, and 35.06 mg/g protein, respectively). Meanwhile, sulfur amino acids including methionine and cysteine recorded the highest content in cheese made with crude MCE of mushroom for methionine content but cysteine content recorded in microbial coagulant made-cheese. Also, mushroom crude MCE made-cheese recorded the highest content of non-essential amino acids content including aspartic, glutamic, and tyrosine acids. Moreover, EAA and the total amino acids (TAA) content of cheese made with mushroom coagulant were lower than cheese made with calf rennet or microbial coagulant. Also, EAA/TAA ratio of mushroom cheese was close to cheese made with commercial coagulants. 8) The chemical score of soft cheese made with different coagulants. Leucine was the first limiting amino acid in cheese coagulated with crude MCE of mushroom but using of partial purified MCE of mushroom as coagulant raised the chemical score of leucine which close to cheese coagulated with commercial coagulants. Also, valine and isoleucine were the second limiting amino acids for the partial purified MCE of mushroom made-cheese but using of crude MCE of mushroom as coagulant raised the chemical score of valine and isoleucine which close to commercial coagulants made-cheese. 9) The calculated protein efficiency ratio (PER) of soft cheese showed that cheese made with microbial coagulant had the highest PER followed by cheese coagulated with partial purified MCE of mushroom, calf rennet, and crude extract of mushroom. 10) The calculated biological value (BV) cheese made with microbial coagulant had the highest BV followed by cheese coagulated with partial purified MCE of mushroom, calf rennet, and crude extract of mushroom. 11) Texture profile analysis of soft cheese made with different coagulants showed that no significantly (p≤0.05) differences in hardness, springiness, and cohesiveness of the resulted cheese using different coagulants. As regards gumminess, and chewiness of cheese coagulated with mushroom MCE had the highest significantly (p≤0.05) values compared to both animal rennet and microbial coagulant. 12) SEM micrographs of soft cheese coagulated with different coagulants showed that all cheese microstructures were clearly similar. 13) Total sensory attributes score including the collective scores of individual soft cheese properties such as flavor, appearance, body and texture which coagulated with different coagulants showed that no significantly (p≤0.05) differences in all sensorial characteristics of cheese made by different coagulants during storage with slightly higher scores for commercial coagulants compared to mushroom MCE fractions.