الفهرس 
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Abstract
The use of milk permeate as low cost medium for Phaffia rhodozyma growth and total carotenoids production:
Phaffia rhodozyma (sexual state: Xanthophyllomyces dendrorhous) is a basidiomycetous yeast. This yeast produces astaxanthin, a carotenoid pigment with biotechnological importance because it is used in aquaculture for fish pigmentation. We hypothesize that the possibility for using milk permeate as growth medium for Phaffia rhodozyma, (NRRL-Y 10922) was obtained from National Center for Radiation Research and Technology (NCRRT) was used throughout this work as a main source of Astaxanthin pigment.
Cell viability, mass and total carotenoids production under incubation conditions (22°C for 120 h and inoculation rate 5.0%) were studied as follows:.
1. Growth and viability of Phaffia rhodozyma in sweet whey and milk permeate as compared with basal medium YM:
The growth and viability of Phaffia rhodozyma in different four media YM, YM fortified with lactose, milk permeate and sweet whey media along the incubation period of 120 h at 22 °C were done and results show that:
1. Growth and viability of Phaffia rhodozyma were higher in YM than in milk permeate and sweet whey media.
2. Viability increased as the incubation period increased till the 4th day of the storage followed by a slight remarkable decrease till the end of the incubation period.
3. Sweet whey couldn’t be used as culture medium for growth of Phaffia rhodozyma as compared with both YM and milk permeate media, and, milk permeate was less favorable than YM and YM with lactose media.
4. The data conducted that the Yeast growth of Xanthophyllomyces dendrorhous depends on the culture type and its conditions.
5. The basal medium (YM) was an optimum medium for Phaffia rhodozyma growth as compared with sweet whey and milk permeate media under agitated condition.
6. Cell growth was greatest in the YM medium followed by the growth in milk permeate. On the contrary, the least growth was noticed in sweet whey.
7. Agitated condition enhanced the growth of Phaffia rhodozyma as compared with static conditions at the same degree 22°C and period 120 h.
It could be concluded that, milk permeate could be use as growth medium for Phaffia rhodozyma and production of total carotenoids. Also, agitated conditions are better than static condition for Phaffia rhodozyma growth, viability, cell mass and total carotenoids production.
2. Effect of pH values on growth of Phaffia rhodozyma in milk permeate and basal YM medium:
The effect of different pH values (4.0, 4.5, 5.0, 5.5 and 6.0) on growth and total carotenoids production of Phaffia rhodozyma was investigated along the incubation at 22°C for 120 h. Both viability (log CFU/mL) and cell growth (OD600 nm) were studied at the beginning and during the incubation period results indicated that:
1. The greatest remarkable growth and viability of Phaffia rhodozyma was at pH 6.0.
2. The least growth and viability of Phaffia rhodozyma was at pH value 4.0.
3. Dry cell weight increased as the pH value increased and total carotenoids produced were pH value dependant.
4. The greatest dry cell weight (g/L) and total carotenoids produced (mg/L) were in milk permeate medium with pH value 6.0.
5. pH value 6.0 was the most suitable for Phaffia rhodozyma growth and total carotenoids production in agitated conditions.
3. Fortification of milk permeate with nitrogen sources:
3.1. Effect of ammonium sulfate concentration on growth and viability of Phaffia rhodozyma in milk permeate and basal YM medium:
Influence of (0.1, 0.2, 0.3, 0.4 and 0.5% w/v) ammonium sulfate (NH4)2SO4 as nitrogen source was studied on cell growth and subsequently carotenoids formation.
1. Ammonium sulfate 0.4% showed the greatest effect on cell growth followed by 0.3% compared with 0.5% which showed the least effect.
2. Growth of Phaffia rhodozyma increased by increasing the ammonium sulfate concentration till 0.4% (w/v).
3. Effects of nitrogen sources on the growth of Phaffia. Rhodozyma and yield of total carotenoids and subsequently astaxanthin, in single using (NH4)2SO4 as nitrogen source enhanced the Phaffia rhodozyma growth and viability.
4. Total carotenoids and cell mass were significantly high in milk permeate with 0.4% (w/v) ammonium sulfate as compared with control samples, in agitated conditions.
3.2. Effect of ammonium chloride concentrations on growth and viability of Phaffia rhodozyma in milk permeate and basal YM medium:
To investigate the effect of ammonium chloride supplementation (0.1, 0.2, 0.3, 0.4 and 0.5% of N) to milk permeate medium on the growth of Phaffia rhodozyma were studied during the incubation period at 22°C for 120 h.
1. The growth and viability increased by increasing the incubation period for 3rd day of the incubation followed by slight decrease as the storage period progressed in all samples.
2. The greatest remarkable growth was in 0.2% (w/v) ammonium chloride milk permeate.
3. Total carotenoids and cell mass were significantly high in milk permeate with 0.2% (w/v) ammonium chloride as compared with control samples, in agitate condition.
4. Ammonium chloride supplementation decreased the growth and viability of Phaffia rhodozyma in milk permeate medium as compared with the effect of supplementation by ammonium sulfate.
3.3. Effect of potassium nitrate concentrations on the growth and viability of Phaffia rhodozyma in milk permeate and basal YM media:
The use of potassium nitrate concentrations (0.1, 0.2, 0.3, 0.4 and 0.5% of N) as nitrogen sources for milk permeate supplementation were investigated for growth of Phaffia rhodozyma.
1. High growth rate of Phaffia rhodozyma have been obtained using 0.1% of N as potassium nitrate.
2. High nitrogen concentrations (as potassium nitrate) have decreased the growth and viability of Phaffia rhodozyma during the incubation period as compared with the traditional growth rate under agitate condition.
3. Supplementation of milk permeate by 0.5% potassium nitrate as N source decreased the viability as the storage period progressed.
4. Growth rate was about 1 log cycle during 72 h of incubation with the supplementation rate 0.1 and 0.2% of as a source of N.
5. Potassium nitrate as a source of N more than 0.2% (w/v) must be excluded.
It could be recommended that, milk permeate could be use as basal medium for Phaffia rhodozyma growth and subsequently production of total carotenoids and cell mass production using (NH4)2SO4 at 0.4%N , pH value of 6.0 and agitat condition.
Part II: Effect of some technological treatments on stability of total carotenoids produced from Phaffia rhodozyma.
1. Effect of pH value on stability of total carotenoids extract:
The effect of certain different pH values (3.5, 4.0, 4.5, 5.0, 5.5 and 6.0) on stability of total carotenoid extract produced by Phaffia rhodozyma was stored at (5.0 ±1.0°C) for 15 days.
1. Stability of Phaffia rhodozyma extract was pH value and storage period dependent.
2. Stability of Phaffia rhodozyma extract decreased with the decrease of pH value.
3. The highest stability was at pH value 6.0 and 5.5 when fresh and during the first day of the storage followed by a slight increase with the increase of the storage period.
4. The least significant stability of total carotenoids extracts of Phaffia rhodozyma was observed at pH values 3.5.
2. Effect of NaCl concentration on stability of total carotenoids:
Effect of certain NaCl concentration(1.0, 2.0, 3.0, 5.0 and 7.0% w/v) on the stability of total carotenoids extracted from Phaffia rhodozyma cells during storage at 5.0 ±1.0°C for 28 days were studied:
1. The stability of total carotenoids extracted from Phaffia rhodozyma was NaCl concentration and storage period dependant.
2. During the first 15 days of the storage, stability of total carotenoids extracted from Phaffia rhodozyma greatly decreased followed by a slightly gradual decrease in all NaCl concentration treatments.
3. At the end of the storage period, NaCl concentration was significantly affected the total carotenoids extract stability.
4. The addition of 1% and 2% NaCl had the lowest significant effect on the total carotenoids stability during the 28 days of the storage.
3. Effect of incubation temperature on stability of total carotenoids:
Effect of different incubation temperatures 30°C, 37 and 45°C on stability of total carotenoids were studied during 4 h of incubation.
1. Stability of total carotenoids decreased as the incubation period progressed
2. The incubation temperature 45°C caused the highest decrease in stability of total carotenoids as compared with incubation at degrees 30 and 37°C.
3. The effect of incubation temperatures on stability of total carotenoids produced from Phaffia rhodozyma was lower than all other technological treatments.
4. Effect of storage temperature on stability of total carotenoids:
Effect of storage temperature; frozen storage (-20) °C, refrigeration (5) °C and room temperature (20) °C on the total carotenoids stability were studied during 15 day of storage. The data indicated that:
1. The highest decrease in stability of total carotenoids was in treatments stored at (20) °C.
2. Stability of total carotenoids was increased during 15 days of the freezing storage at (-20) °C followed by a slight decreased till the end of freezing storage.
3. Frozen storage at (-20) °C had the lowest effect on stability of total carotenoids which slightly increased during 15 days of the freezing storage.
5. Effect of thermal treatment on stability of total carotenoids:
Effect of heat treatments on stability of total carotenoids produced from Phaffia rhodozyma was conducted using the following treatments: 1. Traditional pasteurization (63°C/30 min), 2. HTST treatment (72°C/15 sec), 3. High thermal treatments (85°C/10 min, 85°C/30 min and 90°C/5 min) and 4. Autoclaving at (121°C/10 min). The data indicated that:
1. Heat treatment Traditional pasteurization process (63°C/30 min); HTST treatment (72°C/15 sec) and the autoclaving process (121°C/10 min) had no effect on total carotenoids stability.
2. Thermal Treatments, 85°C/10 min, 85°C/30 min and 90°C/5 min showed the higher total carotenoids stability.
3. The autoclaving process (121°C/10 min) had no effect on total carotenoids stability as pasteurization process.
Inculcation, the data indicated that total carotenoids stability had thermal stability.
Part III: Use of total carotenoids extract produced from Phaffia rhodozyma as natural antioxidants in butter and samna production:
1. Butter:
Butter was produced from cow’s milk and divided into 8 portions. The first portion was treated as control samples without adding any antioxidants. The antioxidant effect of total carotenoids produced from Phaffia rhodozyma was used at a rat of (0.5, 1.0, 1.5, 2.0 and 2.5) mL total carotenoids extract and /100 mL butter. BHT was used in two portions 0.5 and 1.0/100 mL butter as comparison treatments and subsequently incubation at 60±2.0°C for 21 days Acid, peroxide and TBA values were determined weekly along acceleration process at 60±2.0°C for 21 days.
1. The highest increase in acid value was in control samples.
2. Butter sample containing 1.5 mL of total carotenoids extract has the least acid value followed by butter samples containing 1.0 mL of BHT solution.
3. Peroxide value gradually increased as the acceleration process increased in all samples.
4. Butter containing 1.5 mL followed by 1.0 mL of total carotenoids solution and 1.0 mL of BHT solution had the least peroxide value.
5. The highest peroxide value was recorded in control butter samples when fresh and throughout acceleration process for 21 days.
6. The peroxide value increased as the acceleration process time increased.
7. Butter containing 1.5 mL of total carotenoids solution followed by 1.0 mL from the same solution had the lowest BHT value in butter at the end of storage period.
8. The highest TBA values were in control butter samples when fresh and along acceleration process.
9. TBA values increased as the acceleration process time increased till the end of storage period.
2. Samna (anhydrous milk fat):
Melted samna was divided into 8 portions. The first portion was treated as control without adding antioxidant the next five portions were fortified by (0.5, 1.0, 1.5, 2.0 and 2.5) mL of total carotenoids extract. The last two portions were fortified by (0.5 and 1.0) mL of BHT antioxidant substrate. All samples were tested for fat deterioration during acceleration process at 60 ±2 °C for 21 days. Acid, peroxide and TBA values were determined weekly along acceleration process.
1. The acid value in all samples slightly increased in the first 15 days of acceleration process followed by gradual increase till the end of acceleration process for 21 days.
2. The highest increase in acid value was in control samples.
3. Samna samples containing 1.5 mL of total carotenoids extract had the lowest acid value during acceleration process followed by samna samples containing 1.0 mL of BHT solution.
4. Peroxide value gradually increased by increasing of acceleration process time till the end of the storage period in all Samna samples.
5. Samna containing 1.5 mL followed by 2.5 mL of total carotenoids extract and samna containing 1.0 mL of BHT solution had the lowest peroxide value during all acceleration process times.
6. Control samna samples had the highest peroxide values when fresh and along acceleration process till the end of acceleration for 21 days.
7. TBA value significantly increased in all samna samples as the acceleration process progressed.
8. The highest increase in TBA value was in control samna sample.
9. the least significantly TBA value was recorded in samna sample containing 1.5 mL of total carotenoids solution during acceleration process times followed by samna samples containing 1.0 mL of BHT solution as artificial antioxidant.
It could be recommended that, the addition of total carotenoids extract produced by Phaffia rhodozyma had antioxidant effect for prevention of fat oxidation and rancidity in butter along shelf life. Also, this extracts had effect than the BHT
Generally, it can be recommended the use of milk permeate as growth medium for Phaffia rhodozyma to produce carotenoids which have high antioxidant effect against oxidative rancidity of fat and therapeutic effects.