| Whey proteins are important by-products obtained from cheese industry and casein manufacture,also known as the serum proteins of milk.Whey proteins are considered as a complete protein because it contains all 9 essential amino acids.Whey in liquid form cannot be used,so it is converted into various product with different processes and technologies such as whey protein concentrate,whey protein isolate,condensed whey,dried whey,dried modified whey,and lactose.Whey proteins are widely used in food products for their excellent nutritional quality and functional properties.Whey protein possesses excellent nutritional and health benefits such as hypotensive,immunomodulatory,anticancer,mineral binding,gut health-enhancing,psychomodulatory,insulinotropic,hypocholesterolemic,antagonist,and antimicrobial ability.Whey proteins products have received great attention in the last decade by researchers due to their emulsifying,thickening,gelation,foaming,and encapsulating properties.Thus,whey proteins are used in various food formulations because of their good nutritional,functional and biological properties such as amino acid composition,highly nutritional protein source,excellent sensory properties.These physical and functional properties can be enhanced by different methods including physical and chemical modifications of whey proteins,which will rise their applications.Several methods have been used to modify the physicochemical and functional properties of whey proteins such as heat-induced polymerization,enzymatic treatment and chemical modifications.Currently,novel technologies such as plasma technology,filtration,hydrostatic pressure,pulsed electric field,gamma irradiation and ultrasound are used to modify whey protein to improve the functional and physicochemical properties.Heat-induced polymerization is the main applied physical modification to whey proteins to improve functional properties.Whey proteins when heated above70°C begins thermal denaturation and form three-dimensional network and possible aggregation,this denatured or modified whey protein is also called polymerized whey protein(PWP),and are widely used in the food industry as a stabilizer,emulsifier agent,edible coating,and thickening agents.Polymerized whey proteins could be used as a microencapsulating agent for bioactive compounds to improve physicochemical properties and bioavailability;as well as to protect its chemical and physical properties in harsh environmental condition and improve texture and quality.Ultrasound is an emerging green novel non-thermal processing technology,extensively employed in dairy and related industry to modify physicochemical and functional properties of proteins.This method could be used to induce changes in the molecular characteristics of proteins,and hence modify their functional properties.Sonication could led to improvements in gelling ability,solubility,specific surface area and encapsulation properties of whey protein.These technologies are the best choice to protect the efficiency of bioactive compounds during harsh environmental conditions and enhance the oral bioavailability.3,3’-Diindolylmethane(DIM),a lipophilic natural compound found in Brassica family most notably kale,broccoli and cauliflower.DIM has shown to have beneficial effects on human health due to the strong therapeutic and medicinal values,such as anticancer,antioxidant,anti-inflammatory,and anti-bacterial.Most importantly,the antioxidant effect has shown their inhibitory effect on inflammation,tumor growth,oncogenesis,and progression;these properties suggest the use of DIM as a chemopreventive or chemotherapeutic agent in the cancer patients to improve their quality of life.However,its clinical advancements and broad-spectrum applications is hampered in food and pharmaceutical industry,because it is degraded easily when exposed to light and heat,and is unstable at low pH and susceptible to oxidative degradation.Moreover,its absorption is limited in the gastrointestinal tract due to its high lipophilicity and low solubility,which reduces its overall oral bioavailability.To overcome these limitations,there is a need to develop an enhanced bio-available orally active pharmaceutical dosage of DIM to prevent various diseases.In this context,whey protein nanoparticles-based microencapsulation is a promising approach to protect the hydrophobic bioactive compound against harmful conditions,due to the good emulsification,solubility,and film-forming properties.Whey protein-based nanoparticles are now generally acceptable due to the fact that various compounds can be successfully entrapped into whey protein-based nanoparticles,which can enhance the bioavailability along with an increase in chemical and physical stability during digestion and food storage.The purpose of this work is to prepare and characterize the polymerized whey protein nanoparticles,to modify and enhance its properties using ultrasound technology,and encapsulate the bioactive compound diindolylmethane(DIM)at different ratios with whey protein.Further,to explore the physicochemical,rheological and microstructure properties of polymerized whey protein-based nanoparticles as a microencapsulating agent for DIM;as well as its physical and chemical properties during storage at different harsh conditions.Also,to assess the effect of ultrasound treatment on whey protein-based nanoencapsulation on physicochemical and antioxidative properties of DIM.The whey protein-based nanoencapsulation technology not only provide a new method for the protection of bioactive compound with enhanced physicochemical properties,but also enhanced the bioavailability of diindolylmethane(DIM),which was reported in the human clinical trial by lowering PSA level with improved Quality of Life(QoL)in the castrate-resistance prostate cancer.This dissertation includes the following aspects:(1)Preparation and characterization of whey protein isolate-DIM nanoparticles(2)Physicochemical and microstructural properties of polymerized whey protein encapsulated 3,3’-diindolylmethane nanoparticles(3)Effect of ultrasound treatment on the physicochemical,microstructural and antioxidative properties of whey protein concentrate encapsulated 3,3’-diindolylmethane nanoparticles(4)Clinical study on oral whey protein encapsulated DIM in castrate-resistant metastatic prostate cancer.1.The hydrophobic biological active substance,DIM is limited in food and medical application due to its poor stability and light sensitivity.It can be effectively protected from adverse conditions by nanoencapsulation technology.This study aimed the preparation and characterization of WPI-based DIM nanoparticles with different formulations.Different DIM ratios were used with whey protein isolate(WPI)nanoparticles to prepare a stable formulation using the combined heating-ultrasound method,and were assessed regarding physicochemical,thermal,rheological,and micro-structural properties.Further,the nanoparticles were examined for pH stability and photostability.Results showed that,all the samples showed adequate physicochemical characteristics:the mean particle size values of DIM-encapsulated whey protein isolate nanoparticles were 157.47±8.09,141.43±1.62,140.00±2.16,and 142.83±13.23 nm for samples of WPI to DIM ratios at 2.5:1,5:1,7.5:1,10:1,respectively,which were found significantly higher as compared to native WPI(96.53±3.02 nm)(P<0.05),which showed that the mean particle size of the nanoparticles could be controlled down to 96-157 nm depending on the DIM to WPI ratio used in the preparation.The PDI value was increased when DIM was incorporated in WPI nanoparticles with volume ratio of 2.5:1(WPI:DIM),followed by a decrease in PDI value with higher concentration of DIM;however,the changes were not significant(P<0.05),but all nanoparticles were found with a low polydispersity index(<0.5).The zeta potential value of WPI only(-32.86±0.20 mV)significantly dropped to(-42.00±2.62--48.76±4.55 mV)after DIM was coated by WPI nanoparticles(P<0.05),indicating that DIM may be attached to WPI nanoparticles,which showed higher negative values of zeta potential(>-40 mV)for encapsulated DIM.All the samples encapsulation efficiency was found greater than80%.Flow behavior indices showed the shear-thinning Non-Newtonian or pseudoplastic(n<1)behavior,decreasing apparent viscosity with an increase in shear rate followed by constant viscosity at higher shear rate of the nanoparticles.The increasing DIM ratio with WPI nanoparticles had a significant effect(P<0.05)on the viscosity of DIM-encapsulated WPI nanoparticles.The thermal properties were characterized by differential scanning calorimetry(DSC).The DSC curve of WPI only showed a broad endothermic peak between 40 to 115°C centered at 87°C,and the endothermic peak of physical mixture of WPI and DIM existed between 70 to100°C.However,no melting point was investigated in native WPI.For the physical mixture of WPI and DIM nanoparticles both the endothermic peak of WPI and melting peak of DIM has been detected,which showed that DIM was successfully entrapped in WPI nanoparticles.The secondary structure of WPI was changed after DIM incorporation;electrostatic interaction and hydrogen bond were major facilitating forces for nanoparticles formation,confirmed by Fourier Transform Infrared Spectroscopy(FT-IR).Transmission electron microscopy(TEM)micrograph showed that all the samples had a smooth surface and spherical structure.The nanoparticles micrographs also illustrated the remarkable difference depending on DIM mass ratios in WPI system.It is clear that increased in DIM ratio with WPI resulted in larger particle size.The wall material(WPI)and encapsulation method provide effective protection to DIM against UV light and broad range of physiologically relevant pH(2.5,3.5,4.5,5.5,and 7).In conclusion,whey protein isolate(WPI)based nanoparticles is a promising approach to encapsulate DIM and overcome its physicochemical limitations with improved stability.These characteristics made WPI nanoparticles an attractive wall material for the encapsulation of bioactive components in the development of nutraceutical and food products.2.3,3?-Diindolylmethane(DIM),a natural phytochemical found in brassica vegetables,such as broccoli,cabbage,and brussels sprouts.The stability of this compound is a major challenge for its applications.Polymerized whey protein(PWP)-based DIM nanoparticles were developed and mixed with DIM at different mass ratios.All the formulation obtained were treated by ultrasound for 4 min.Then,the nanoparticles were studied for particle size,zeta potential,rheological and microstructural properties,and storage stability.Results revealed that mean particle size of PWP-based nanoparticles was significantly increased(from 241.33±14.82 to270.57±15.28 nm)(P<0.05)by the addition of DIM at different mass ratios.Zeta potential values of all nanoparticles were highly negative(greater than±30 mV),suggesting a stable solution due its electrostatic repulsive forces.All samples exhibited shear thinning behavior(n<1),fitted with Sisko model(R~2>0.997).Fourier Transform Infrared(FTIR)spectra revealed that secondary structure was changed and absorption intensity for hydrogen bonding getting stronger by further incorporating DIM into PWP.Transmission electronic microscopy(TEM)images showed spherical and smooth surface shape of the PWP based nanoparticles.DIM encapsulated by PWP showed enhanced stability at 4,37,and 55?C for 15 days evidenced by changes in mean particle size and color(a*-value and b*-value)compared with control(DIM only).In conclusion,the polymerized whey protein based 3,3?-diindolylmethane nanoparticles are stable and the encapsulation may protect the core material from oxidation.3.Whey proteins are considered to be a suitable wall material for the nano-encapsulation of bioactive compounds,and have been modified by various technology to improve the functional properties.Ultrasound is an emerging green novel non-thermal processing technology,extensively employed in dairy and related industry to modify physicochemical and functional properties of proteins.This study evaluated the impact of ultrasound duration on the encapsulation of 3,3’-diindolylmethane(DIM)using whey protein concentrate nanoparticles.Whey protein concentrate-based DIM nanoparticles were prepared and treated with different ultrasound times(0–20 min)with 30%amplitude.The results showed after ultrasound treatment of WPC–DIM nanoparticles,a considerable significant reduction(P<0.05)in particle size of WPC–DIM nanoparticles could be seen(from 265 nm to 218 nm),however,ultrasound treatment for 5 min the particle size significantly dropped(P<0.05)particle size of untreated nanoparticles from 265.96 to 255.86 nm.The PDI value after ultrasound treatment for 5,10,15,and 20 min was significantly decrease(P<0.05)to 0.48,0.46,0.43,and 0.43,respectively.After ultrasound treatment for 5 min,the zeta potential values of WPC–DIM nanoparticles was significantly increased from-27.96mV to-30 mV;however,ultrasonication for 10,and 15 minutes did not significantly affect the zeta potential of the nanoparticles,followed by a greater increase when sample was treated for 20 minutes(-32 mV)(P<0.05).The encapsulation efficiency(EE%)increased with increasing sonication time(0–20 min)from 76%to 77,79,81,and 88%,respectively.The ultrasound treatment had a significant effect on the apparent viscosity,and decrease in the viscosity as a function of shear rate was observed with increasing sonication time.The results suggest the shear thinning and pseudo-plastic flow behavior of the samples,and were remained pseudo-plastic after ultrasound treatments.The transmission electronic microscopy(TEM)micrographs demonstrated that all the formulations treated with different sonication times had a smooth and uniform spherical shape and ultrasound treatment led to the reduction of particle size,especially for 20 min of ultrasound.The thermal stability of the WPC–DIM nanoparticles were enhanced with increasing sonication time by increasing peak denaturation temperature and enthalpy.The Fourier transform infrared spectroscopy(FT-IR)spectra analysis revealed that ultrasound treatment had a remarkable effect on the secondary structure of WPC–DIM nanoparticles,and electrostatic interactions and hydrogen bonds between DIM and whey protein were strengthened.Color data showed no significant change(P<0.05)was caused by ultrasonication with different times on the a*-value and b*-value color parameter of the WPC–DIM nanoparticles,except for ultrasound treatment with 20 min where a significant change was noted for b*-value;the pH results showed that ultrasound treatment significantly increased the pH of WPC–DIM nanoparticles with increasing sonication time(P<0.05).Moreover,the length of ultrasound treatment exhibited a significant effect on the DPPH(2,2-diphenyl-2-picrylhydrazyl)scavenging activity(from 56%to 62%)and ABTS(2,2’-azinobis(2 ethylbenzothiazoline-6-sulfonate)scavenging activity(from 47%to 68%).In conclusion,the ultrasound treatment successfully improved the physicochemical,microstructural,and anti-oxidative properties of WPC–DIM nanoparticles,therefore it is considered an efficient method for the development of whey protein concentrate-based DIM nanoparticles to be used for medical and nutritional applications.4.3,3′-Diindolylmethane(DIM),is an anticancer compound,act as anti-androgen and modulates estrogens metabolism,which down regulates the prostate specific antigen(PSA)and androgen receptors with improved Quality of Life(QoL).Prostate cancer(PCa)is the most commonly detected malignancy among males in developed countries,it is a life-threatening illness.Therefore,a randomized,placebo-controlled,single-blind design study was carried out in two parallel groups to determine the effect of the encapsulated DIM with improved bioavailability on serum prostate specific antigen(PSA)level and Quality of Life(QoL)of patients with castrate-resistance prostate cancer.Among two parallel groups 20 patients,age 60-71 year,histologically confirmed castrate-resistance prostate cancer(CRPC)for 6 weeks duration were selected.Patients of group I(cases)were prescribed whey protein encapsulated DIM at a dose of 150 mg orally twice daily;group II(control)were administrated at a dose of 150 mg whey protein(placebo).All the patients’serum PSA level and Quality of Life(QoL)using EORTC QLQ-PR25 questionnaire were determined before and after study.Results showed that a significant decrease(P<0.05)in the symptoms scale over the time was observed in the urinary and hormonal-treatment-related symptoms of the group I(cases)after the active therapy as compared to group II(control).Further,a significant improvement over the time was observed in the sexual activity in the patients after the treatment;The cases achieved PSA decline during treatment(Median,10%;average,21%;range,1%-76%)as compared to control PSA level,where increase in PSA level was reported(Median,66%;average,169%;range,1-415%).No significant adverse effect of the study were reported by the patients during follow-up visits.Based on our clinical data,it is concluded that the whey protein encapsulated DIM helped to treat or delay the CRPC condition with improved Quality of Life(QoL),because of its antioxidant,anti-inflammatory,and anti-androgen activities in the metastatic CRPC patients.Through the above research whey proteins-based nanoparticles can be considered as an efficient and promising approach to encapsulate bioactive compounds(DIM)and overcome its physicochemical limitations with improved stability for medical and nutritional applications.Moreover,whey protein encapsulated DIM may have the ability to delay the progression of CRPC and drug resistance in prostate cancer by lowering PSA level with improved Quality of Life(QoL)in metastatic prostate cancer patients. |
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