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ThesisItem Open Access ATTENUATED TOTAL REFLECTANCE-FOURIER TRANSFORM INFRARED SPECTROSCOPY COUPLED WITH CHEMOMETRICS TO DETECT SELECTED ANIMAL BODY FATS, VEGETABLE OILS AND THEIR ADMIXTURE IN GHEE(ICAR-NDRI, KARNAL, 2022) VIVEK SONVANSHI; KAMAL GANDHIGhee is a popular traditional dairy product of India. Due to its high demand and insufficient supply during lean season, it becomes prone to adulteration by unscrupulous traders in the market. The present study was conducted to develop suitable models using ATR-FTIR coupled with chemometrics for detection of selected animal body fats, vegetable oils and their admixture in ghee. Milk samples were procured and ghee samples were prepared using creamery butter method. Vegetable oils from five reputable brands and animal body adipose tissues were purchased from local market of Karnal. Animal body fats were extracted from the respective adipose tissues of the animal using dry rendering process. GC-analysis revealed that the short chain fatty acids were only present in pure ghee and not in adulterants targeted in the study. Linoleic acid concentration was significantly higher in soybean oil as compared to that in other oils and fat studied. Palmitic acid was the major fatty acid in vanaspati and trans fatty acids (eladic acid) was only present in vanaspati. Goat body fat contained higher amount of stearic acid as compared to that in other oils/ fats studied. Oleic acid concentration was higher in palmolein oil and pig body fat as compared to that in other oils and fats studied. Prepared cow and buffalo ghee were mixed in equal proportions to obtained pure mixed ghee (PMG). Adulterants, viz. soybean oil (SO), vanaspati (VG), palmolein oil (PO), goat body fat (GBF), pig body fat (PBF), sheep body fat (SBF) were added individually at 1, 3, 5, 10, 15 and 20 as well as in combinations, viz. SO+GBF, VG+GBF, PO+GBF, SO+PBF, VG+PBF, PO+PBF, SO+SBF, VG+SBF and PO+SBF in PMG in the ratios of 1:2.3, 2:4.6, 3:7, 4:9.3 and 5:11.6, respectively. FTIR spectra analysis of all the samples were performed and the data obtained was subjected to chemometric analysis. Functional group regions of the mixed ghee and adulterants were almost similar while slight differences in their finger print regions were observed. Wavenumber regions which were found useful for detecting adulteration of ghee with SO, VG, PO, GBF, PBF and SBF were 727-702, 1120-1080 and 985-955, 1167-1137, 1760-1730, 1190-1140 and 1100-970, 1190-1140 and 1120-970 and 732-710 cm-1, respectively. Wavenumber regions which were found useful for detecting adulteration of ghee with admixture of SO+GBF, VG+GBF, PO+GBF, SO+PBF, VG+PBF, PO+PBF, SO+SBF, VG+SBF and PO+SBF were 1180-1140 and 1120-1098, 1170-1145 and 1120-1087, 1175-1135 and 1125-1080, 1170-1140 and 1130-1090, 1180-1140 and 1120-1090, 1200-1130 and 1123-1093, 730-710, 1125-1085 and 740-700 cm-1, respectively. PCA applied in the selected regions showed separate clusters from PMG even for the lowest level of spiking of each adulterant and their admixture and as the level of spiking of adulterants increased, clusters shifted towards pure adulterants. PLS and PCR models applied in the selected regions of the FTIR spectra were equally efficient in detecting the selected adulterants in ghee as indicated from the R2, RMSEC, RMSEV and Bias values. Calibration curves between the actual and predicted levels of all the individual adulterants and their admixture in ghee were linear with a slope of 45° and no x or y intercepts indicating the suitability of the models in detecting them in ghee. SIMCA approach in conjunction with the established PLS models applied in the selected wavenumber regions showed the classification efficiency for pure mixed ghee, pure body fats and pure vegetable oils as 100% indicating that models were effectively developed for their detection in ghee. Classification efficiencies for ghee samples containing individual adulterants and their admixture at all the levels studied were never fell down below 85 and 73%, respectively. Using ATR-FTIR, we can detect up to 1% level of selected individual adulterants and 3.3% level of admixture of animal body fat and vegetable oil in ghee. Rapid, non-destructive, low cost and accurate analytical protocol involving a combined use of FTIR spectroscopy and chemometrics for rapid and accurate determination of soybean oil, vanaspati, palmolein oil, goat, pig, sheep body fat and admixture of SO+GBF, VG+GBF, PO+GBF, SO+PBF, VG+PBF, PO+PBF, SO+SBF, VG+SBF and PO+SBF in ghee is now available which the industries can adopt for regular testing of their samples.ThesisItem Open Access PREPARATION OF NANOCAPSULES OF CURCUMIN USING NATIVE CASEIN MICELLES AND THEIR CHARACTERIZATION(ICAR-NDRI, KARNAL, 2022) ANKITA HOODA; BIMLESH MANNFood enrichment through functionalizing it by using milk proteins especially casein (CN) is a way to healthier world as demand for low calorie functional food is increasing. Nanoencapsulation has been a trending technology over others, in the past decade, due to advantages that it offers like increasing effectiveness and efficiency of delivery as well as maintaining product texture and consistency. This study focused on the fabrication of stable nanocapsules of curcumin in native casein micelles i.e. micellar casein concentrate (MCC) and skim milk casein micelles. This way all the native physicochemical and functional properties of casein micelles could be preserves and also the curcumin could be prevented from degradation through light, processing conditions. MCC and buffalo skim milk were subjected to pH variation to nanoencapsulateethanol solublizedcurcumin and then spray dried. Curcumin was dissolved in ethanol (10mg/ ml) and added dropwise with stirring at a rate of 80 mg/ 100 ml and 100 mg/ 100 ml in nanocapsules from MCC and buffalo skim milk respectively. Encapsulation efficiency (EE) was good for both wet and dry forms i.e. 97.00 ± 0.24% and 96.74 ± 0.39 % of MCC respectively. The EE of wet as well as dried sample of buffalo skim milk nanoencapsulated samples were excellent i.e. 98.81 ± 0.11 % & 98.34 ± 0.10 % respectively.There was no significant variation in EE and unencapsulatedcurcumin contents on variation under normal conditions of temperature (30˚C to boiling temperature), sucrose concentration (2 to 10 %) and ionic strength (0.1 to 1.0 M) for MCC as well as skim milk nanoencapsulated solution and spray dried formulation. The particle size, zeta potential and PDI values at these conditions indicated stability of solutions. The only drawback was dispersability of solutions which was <2 % in case of MCC based nanocapsules. Hence, further studies were done on buffalo skim milk nanocapsules of curcumin.During gastric digestion > 90 % of encapsulated curcumin was retained (5.23 ± 1.28 % & 6.12 ± 0.24 % in wet and dried samples). In the intestinal phase as the time progressed the sample clarified indicating total breakdown of casein protein chain to peptides of smaller length (98.43 ± 1.38 & 98.12 ± 1.49 % in wet and dried samples). It can be clearly seen that most of the characteristic peaks of curcumin disappeared in the secondary derivative spectra after its NE in skim milk which is due to reduced stretching and bending of curcumin bonds due to nanoencapsulation. Blood urea nitrogen levels increased in nanoencapsulated powder treated groups as compared to paracetamol only treated group whereas creatinine, glutamate pyruvatetransaminase and alkaline phosphatase levels decreased in blood serum of mice treated with nanoencapsulated powder after/ before paracetamol treatment.One serving (4 g powder formulation in 200 ml milk) having curcumin concentration (30-40 mg) equivalent to house hold preparation of HaldiDoodh [prepared using 500 mg haldi (equivalent to 15 mg curcumin) in 200 ml milk] and with 20% more milk proteins together with enhancement of SNF level by 18%.The kesar flavoured milk and mango lassi had no significant variation in sensory characteristics from market counterparts of the same products. Hence, curcumin was delivered successfully with high bioactivity. The nanocapsules were formed in form of a spray dried powder that is readily dispersible in dairy beverages.ThesisItem Open Access MILK PROTEIN BASED PICKERING EMULSION FOR STABILIZATION OF LUTEIN AND ITS APPLICATION IN YOGHURT(ICAR-NDRI, KARNAL, 2022) PAYAL KARMAKAR; RAJESH BAJAJLutein can be used in food products as a nutraceutical due to its potentially health benefits. However, its incorporation into foods is limited due to low water solubility, chemical instability, and poor oral bioavailability. Milk proteins when heated tend to aggregate through hydrophobic and covalent interactions, demonstrating the great potential of stabilizing emulsions via a Pickering mechanism. In this study, casein (CS) and whey proteins (WP) were combined with polysaccharides (sodium alginate [SA], gum Arabic, [GA], pectin, [PC]) to form composite gels (WP+GA, WP+SA, CS+GA, CS+SA) and used to stabilize o/w emulsion containing lutein (in oil phase). The protein-polysaccharide gels at various ratios were optimized based on rheological properties, particle size, zeta potential and interfacial contact angles (WP+GA[1:1], WP+SA[9:1], CS+GA[2:3], CS+PC[2:1]) which were further used in o/w emulsions. Particle size increases due to addition of polysaccharide. Negative zeta potential indicates effective coating of polysaccharide on to the surface of protein gel. WP+GA[1:1], WP+SA[9:1], CS+GA[2:3], and CS+PC[2:1] showed contact angle of 90°. The emulsifying conditions were optimized based on emulsifying, capacity, emulsion activity, emulsion stability indices, lutein encapsulation efficiency, physical, chemical, light stabilities (WP+GA[1:1], CS+GA[2:3]). Emulsifying activity of CS based coating matrix is better than WP based coating matrix. Emulsion stability is however better in case of Pickering emulsion prepared from WP based gel than CS based gel. WP-GA and CS-GA can encapsulate lutein up to 1% with efficiency of 95% and 92% respectively. Lutein encapsulated with protein-GA coating matrices have better stability (in terms of lutein retention) and light stability. The optimized emulsion formulations were used to fortify yoghurt (@ 10-50 mg Lutein/100 g), which were subjected to chemical analysis, microbiological enumeration, rheology and textural evaluation, oxidative stability, sensory evaluation and lutein stability. Sensory properties of yoghurt fortified with encapsulated lutein did not show significant variation from negative control sample. Significant increase in acidity and syneresis in fortified yoghurt is observed after 14 days of storage. The lutein retention during storage is better in case of lutein encapsulated with WP+GA coating matrix. However, significant reduction is observed after 21 days of storage. Encapsulated lutein, when fortified in yoghurt retards the change in colorimetric parameters as compared to non-encapsulated one. Pickering emulsion prepared using WP+GA ratio of 1:1 having 30% oil content induced maximum stability to the encapsulated lutein. The encapsulated lutein remained stable for 14 days.ThesisItem Open Access INDIGENOUS PROTEASES ACTIVITY OF BUFFALO MILK AND THEIR RAPID ESTIMATION WITH SPECIAL REFERENCE TO PLASMIN(ICAR-NDRI, KARNAL, 2022) PRIYAE BRATH GAUTAM; RAJAN SHARMAProteases are the class of enzymes which have the ability to depolymerize the proteins into peptides and amino acids. Although, this action of proteases in milk products like cheese and yogurt is desirable for the formation of flavor compounds, however, in fluid milk or ultra-high temperature (UHT) milk, proteolysis is undesirable resulting in gelation and precipitation of proteins. The present study was done to evaluate the enzymatic activity of indigenous milk proteases in buffalo milk, impact of processing treatments on their activity and developing a rapid assay to quantify the protease activity in milk. The milk samples were collected from mastitis free milch animals such as buffaloes (Murrah breed), cows (Sahiwal and Gir breeds) and goats (Alpine x Beetal and Sanen x Beetal breeds) during different stages of lactation. The enzymatic activity of plasmin, plasminogen and cathepsin D were determined. The activities of plasmin and plasminogen in buffalo milk did not change significantly during the early and mid lactation i.e. 4.20-4.24 U/ mL but increased during late lactation milk i.e. 5.49 U/ mL. The cathepsin D activity remained unchanged (2.76-3.19 mU/ mL) throughout the lactation period. In case of cow milk, the plasmin, plasminogen derived and cathepsin D activity increased during late lactation i.e. from 4.46 U/ mL to 6.63 U/ mL; 27.64 U/ mL to 36.80 U/ mL and 5.10 mU/ mL to 5.91 mU/ mL, respectively. The activity of plasminogen was 5-6 times higher in buffalo and cow milk vis-à-vis plasmin activity while goat milk exhibited a higher plasmin activity (6.82-11.23 U/ mL) than plasminogen derived activity (3.09-3.37 U/ mL). Among the three species, the cathepsin D activity was found to be highest for goat milk (6.39-8.47 mU/ mL) and lowest in buffalo milk. The correlation coefficients of the enzyme activity with stage of lactation, somatic cell counts were found to be positively correlated while for protein content, a negative correlation was observed. The thermal inactivation kinetics of plasmin, plasminogen and plasminogen-activators was found to follow first order kinetics, with plasminogen being more susceptible to heat than plasmin. The plasminogenactivators were found to be more heat stable than plasmin. pH of the milk sample did not affect the plasmin activity, as it remained stable between pH 4.5 to 7. Sonication when applied in conjunction with the thermal treatment was found to be more effective in inactivating the plasmin system in comparison to the either treatment when applied alone. Attempt was made to develop rapid assay to evaluate the protease activity in milk by using three different substrates. It was found that plasmin was unable to hydrolyze the two substrates which were specific for trypsin as no change in the rate of change of absorbance with time was observed. Azocasein, when used as a substrate to evaluate the protease activity (indigenous and bacterial) in milk was found to be a better substrate and the developed assay was validated for its linearity, sensitivity and precision. The method was found to exhibit a good linearity with the R2 value of 0.988 with good repeatability having an RSD < 2%. The developed method was also validated using the fluorescamine method by spiking the boiled milk sample with different levels of plasmin and bacterial protease and the values for both the methods were found to be correlated. The assay was also applied to the market milk samples i.e. pasteurized milk, UHT and gelled UHT milk. The developed protocol can be used to evaluate the protease activity in raw milk.ThesisItem Open Access DEVELOPMENT OF CALCIUM ENRICHED HIGH MILK PROTEIN POWDER FOR PANEER FORMULATION(ICAR-NDRI, KARNAL, 2020) GAWANDE HEMANT MURLIDHAR; SUMIT ARORAThe research was targeted to prepare calcium enriched high milk protein powder (HMPP) using buffalo skim milk for paneer formulation. Coagulation and spray drying conditions for preparation of HMPP were optimized. Gel points for coagulation of buffalo skim milk were determined at different concentration of coagulants i.e. calcium chloride (CaCl2; 05, 10, 15 mM) and glucono-delta-lactone (GDL; 0.05, 0.10, 0.15 %) in combinations using oscillatory shear rheology. The combination with 10 mM CaCl2, 0.05 % GDL, gel point of 80 ºC and coagulation time 13 min exhibited minimum loss of protein (0.39 %) and calcium (282 ppm) in whey. The dilution 1:1.4 (curd : water) was selected to prepare feed solution for efficient atomization during spray drying. Spray drying conditions were optimized for preparation of powder (with high solubility and protein content) by varying outlet temperature (70, 80 and 90 ºC) with constant inlet temperature (180 ºC) for feed solution of varying pH (6.6, 6.8 and 7.0) adjusted using disodium phosphate (DSP) and trisodium citrate (TSC). Among the different combinations, pH 6.8 and 6.6 with outlet air temperature 80 ºC and 90 ºC exhibited maximum solubility (31.48 % and 41.93 %) of the powders prepared using DSP and TSC as well as protein content (80.62 % and 76.30 %), respectively. HMPP prepared using DSP and TSC under optimized conditions consisted of protein (80.53 and 78.29 %), lactose (1.19 and 1.46 %), fat (1.81 and 1.68 %), ash (10.58 and 9.57 %), moisture (4.06 and 5.50 %) and calcium (21033 and 20941 ppm), respectively. Technofunctional properties i.e. bulk density; flowability, wettability, dispersibility, emulsifying capacity, emulsifying stability, foam capacity, foam stability, viscosity and zeta potential of the HMPP prepared using DSP were 0.45 g/ml, 33.90 º, 7.0 s, 47.20 %, 89.83 %, 90.73 %, 30.21 %, 30.56 %, 65.3 cP and -16.07 mV, respectively and that of the HMPP prepared using TSC were 0.55 g/ml, 34.05 º, 5.0 s, 50.68 %, 90.00 %, 89.63 %, 34.77 %, 31.21 %, 70.7 cP and -6.12 mV, respectively. Highest solubility of 32.06 % was observed for HMPP prepared with DSP at 50 °C, whereas, highest solubility of 39.71 % was observed for HMPP with TSC at 20 °C. HMPP prepared using optimized conditions were stored in aluminum laminate pouches for 6 months at 37 °C and 4 °C. These samples were analyzed for solubility, 5-hydroxy methyl furfural (HMF) content and titratable acidity at a regular time interval of month. During storage, loss in solubility was observed which was more pronounced after 5 months. HMF content of powder increased upto 4 months of storage and decreased thereafter. Increase in acidity of powder was evident only after 4 months of storage. Methods for preparation of paneer were standardized with two approaches i.e. using HMPP (method 1) and using HMPP along with cream (method 2). Paneer prepared by using HMPP with DSP (method 1) resembled control in all the sensory as well as textural attributes, whereas, paneer prepared using HMPP with TSC ranked significantly lower (p<0.05). Paneer prepared by using method 2 ranked lower in sensory as well as textural attributes. Recovery of TS in control paneer, paneer with DSP and TSC (method 1) was found to be 55.04, 61.84, 57.27 %, respectively. Similarly, recovery of TS in control paneer, paneer with DSP and TSC (method 2) was found to be 56.37, 69.33, 64.91 %, respectively. Paneer prepared (method 1 and 2) was significantly higher (p<0.05) in protein, ash and calcium content as compared to control paneer. Paneer samples prepared from both the methods using HMPP with DSP and TSC withstood the frying conditions and were found to possess desired softness as compared to control. The paneer prepared using optimized conditions were stored in low density polyethylene (LDPE) pouches and were analyzed for moisture, acidity, sensory and textural changes on 0th, 3rd and 7th day of storage at 4±1 °C. Microstructure of paneer samples prepared using HMPP revealed that the inclusion of HMPP resulted in enlargement of protein chains in network due to fusion of powder particles with proteins. Paneer prepared from HMPP (method 1) mimicked paneer in all sensory as well as textural attributes, whereas, paneer prepared using HMPP and cream (method 2) ranked lower due to acidic flavor and comparatively soft texture of the product.ThesisItem Open Access INSITU GENERATION OF HYDROGEN PEROXIDE TO ACTIVATE LACTOPEROXIDASE SYSTEM IN RAW MILK(ICAR-NDRI, KARNAL, 2020) ALKA PARMAR; VIVEK SHARMALactoperoxidase system is an alternative to chilling of milk in a situation where adequate chilling facilities are not available. Milk preserved through LP system remains fresh due to the antibacterial agent, hypothiocyanate ions. Raw milk contains only two of its components i.e. lactoperoxidase and thiocyanate in natural form. Hydrogen peroxide is the third component of the LP system which is not normally present in raw milk and added extraneously to activate Lactoperoxidase system. Extraneous addition of hydrogen peroxide is difficult to control at farm level/collection sites due to its corrosive nature and at the same time commercially available hydrogen peroxide cause rapid decomposition due to metallic impurities. In the present investigation the conditions for insitu generation of hydrogen peroxide was optimized using response surface methodology. Two approaches i.e. enzymes (glucose oxidase and lactase) with added substrate (glucose) and without added substrate were evaluated. Quadratic model was best suited for optimizing the conditions. Optimized conditions observed in the study were i) 3450 mg glucose with 586 U of glucose oxidase ii) 488 U of lactase with 547 U of glucose oxidase to generate 20 mg/l hydrogen peroxide at 37°C in 3 to 5 h. Conditions were also optimized to activate LP system using insitu generation of hydrogen peroxide approach where i) 3000 mg glucose with 450 U of glucose oxidase and ii) 400 U of Lactase with 350 U of glucose oxidase with 50 mg thiocyanate per liter of milk were used. The shelf life of such LP activated milk was 9-10 h as determined in terms titratable acidity, COB, alcohol test and hypothiocyanate ions. Effectiveness of optimal conditions at 25°C showed significant (p<0.05) effect on shelf life enhancement up to 15 h. Optimized formulation of enzyme and substrate were then stored at 37°C and in another study only the substrate was stored at 37°C and enzyme was stored at 8°C. The LP system activation using the designed formulation, revealed that the formulation stored at 37°C were active only till one month of storage on the contrary when substrate was stored at 37°C and enzyme stored at 8°C, used for activation of LP system, it remained effective till 3 months of storage. This revealed that best suited storage temperature for enzyme was 8°C whereas substrate can be stored at 37°C. It was also observed that for the effective LP system activation the milk should have initial good microbiological quality because the maximum shelf life was observed if LP system was activated within one hour of milking at 37°C. The formulation optimized for one liter milk can be used in higher quantities of milk by increasing the proportionate quantity of formulation. Standardized formulation was also effective in the raw milk collected from local dairy farms with the condition that LP system activation carried out within one hour of milking at 37°C. The milk preserved by using the standardized formulation to activate LP system did not show any variation in Physico-chemical parameters of milk generally used to check the quality of milk except lactose content, measured using HPLC method. However the routine method i.e. lane eynon method used in dairy industry to estimate lactose did not show any change in the lactose content of milk where LP system was activated without substrate. Pasteurization of LP-system activated milk did not show any loss in the thiocyanate content which remained almost similar to the content determined before pasteurization of milk. LP system activated milk samples showed lower microbial counts i.e. mesophilic count (6.397±0.94 log10 cfu/ ml) and coliform count (4.0413±0.93 log10 cfu/ ml) compared to control where the count was too numerous to count (TNTC) at the end of 9 h at 37°C .Starter culture activity was least in milk samples where LP system was activated without added substrate approach. In such samples after inoculating with starter culture the titratable acidity was increased to 0.20% LA after 3⅟2 h storage at 37°C. On the contrary in samples where LP system was activated with added substrate, activity of starter culture was at par with the control. The study concluded that the formulation standardized in present investigation was effective for activation of LP system to enhance the shelf life at 37°C and standardized formulation was also free from the risks associated with extraneously added hydrogen peroxide.ThesisItem Open Access PREPARATION AND CHARACTERIZATION OF CATECHIN LOADED NIOSOMES FOR FORTIFICATION OF MILK(ICAR-SRS-NDRI, KARNAL, 2020) G.SWARNALATHA; B. SURENDRA NATHCatechins, belonging to polyphenols, have received a great attention because of their health benefits. But, they are met with limited efficacy in food applications due to several reasons such as poor aqueous solubility, poor stability, and low bioavailability in GI conditions. Nanoencapsulation in the form of niosomes is expected to provide efficient delivery of these bioactive components. In this study, processing parameters for preparation of catechin loaded niosomes were optimized, and the optimized niosomes were characterized and used for fortification of milk, dahi and yoghurt. Concentration of EGCG, the most potent catechin, as measured by UV spectroscopy was 58.14%, which was also validated by HPLC method. Different parameters such as type and concentration of non-ionic surfactant (NIS), stabilizer, ratio of NIS:stabilizer and method of preparation were optimized to prepare catechin loaded niosomes. Among all the optimized formulations, the ones prepared with Tween 60 and lauryl alcohol (1.5:1) and Tween 80 and lauryl alcohol (1:0.5) by thin film hydration and high shear homogenization carried out at a speed of 15000 rpm for 15 min, showed desired particle size of 58.48 and 60.69 nm and entrapment efficiency of 85.82 and 85.69%, respectively. They also exhibited uniform size distribution and stability as measured by polydispersity index and zeta potential. SEM, TEM and AFM analysis revealed that the catechin loaded niosomes were monodisperse with spherical morphology with a size < 60 nm. SEM-EDS results showed the incorporation of catechins into niosomes. FTIR analysis indicated the formation of niosomes and the encapsulation of catechins into them. HPLC analysis of catechins before and after their encapsulation confirmed that they were encapsulated in the niosomes. Aqueous solubility of catechins improved after their nanoencapsulation. Free catechins were highly photosensitive, whereas the nanoencapsulated form exhibited good photo stability. Catechins from their niosomal form exhibited a sustained release under simulated GI conditions compared to the free forms. Antioxidant activity of the catechins, measured as DPPH free radical scavenging activity, was preserved well in the niosomal form. The niosomes were stable up to 30 days at 30 oC, and 90 and 20 days at 5oC for CNT60 and CNT80, respectively. Milk samples (3% fat and 8.5% SNF), fortified with 20% of catechin loaded niosomes and pasteurized were to be accepted well and comparable to control samples on sensory evaluation. Total solids, fat, protein and carbohydrate contents of the fortified samples reduced marginally due to dilution effect. pH and titratable acidity of fortified milk samples were not significantly affected but, viscosity decreased slightly. The catechin level was not affected significantly when the fortified milk samples were subjected to pasteurization and boiling but, decreased significantly due to sterilization. Catechin from its niosomal form demonstrated controlled and sustained release in the fortified milk. Both control and fortified milk samples kept well for 5 days during storage at 5oC; the level of catechin was not affected during storage. Sensory properties, pH and acidity of dahi and yoghurt samples prepared using fortified milk were comparable to those of the control samples. However, the textural properties i.e., firmness and consistency of dahi and yoghurt samples prepared with fortified milk were significantly lower than those of the control samples, probably due to decreased level of casein. The antioxidant activity increased significantly in all the fortified samples. This study demonstrated that catechin loaded niosomes could be successfully prepared using Tween 60 and lauryl alcohol as stabilizer, which served as delivery vehicles for fortification of milk.ThesisItem Open Access Bioactive Peptide Loaded Niosomes: Preparation, Characterization and Fortification of Milk(ICAR-SRS-NDRI, KARNAL, 2020) SONIA MOR; B.SURENDRA NATHBioactive peptides are defined as food derived peptides with a specific amino acid sequence of about 3-20 residues that exert a physiological effect in the body in addition to their nutritional value. Therapeutic use of peptides has remained limited due to their high instability in biological environment as they could encounter over 40 different enzymes during their passage through the small intestine. Encapsulation is an ideal delivery mechanism that can be used to improve the bioavailability and to maintain the organoleptic properties of the vehicle being fortified. In this regard, nanoencapsulation in the form of niosomes is expected to provide an efficient delivery of the biopeptides. In the present study, preparation and characterization of bioactive peptide loaded niosomes and their utilization for fortification was carried out. Casein was hydrolyzed with flavourzyme and the hydrolysates were subjected to ultrafiltration to obtain 10, 5 and 3 kDa permeate. Degree of hydrolysis of casein using flavourzyme (E:S=1:25, pH: 7.0, Temp.: 45-50ºC and duration: 8 h) was 33.53%. Of the several conditions attempted, the niosomes loaded with 10 kDa peptide, and prepared with 1.5:1 molar ratio of Tween 60 and lauryl alcohol at a homogenization speed of 15,000 rpm for 15 min was considered as the optimized formulation with respect to size, PDI, zeta potential and encapsulation efficiency. Both the methods, namely thin layer hydration + high shear homogenization (TLH+HSH) and high shear homogenization (HSH) gave comparable results. SEM, TEM and AFM images demonstrated that the biopeptide loaded niosomes were well dispersed without any apparent aggregation or precipitation and had spherical morphology with smooth surface and particle size of less than 100 nm. SEM-EDS spectrum of peptide loaded niosomes showed higher nitrogen content than blank niosomes, which confirmed the encapsulation of peptides. HPLC spectrum of peptide in the niosomes showed similar peaks as those of free peptides, indicating their encapsulation into the niosomes. Peptide sequences obtained by LC-MS/MS analyses were found consistent with the previously reported sequences and functional properties. Some peptides identified in the present study were reported to possess multifunctional properties, thereby showing that 10 kDa fraction used for nanoencapsulation has bioactive properties. The antioxidant activity of the biopeptides, measured by in vitro (DPPH method) was preserved in the niosomes. It was observed that 99% of peptide in its free form was released within 6 h whereas, nanoencapsulated peptide showed controlled and sustained release under simulated GI conditions. Niosomes stored at 30ºC were stable up to 8 days whereas, at 5ºC, they were stable up to 1 month. Sensorial characteristics of milk fortified with 20% peptide loaded niosomes were not significantly different from that of control sample on a 9 point hedonic scale. Fortified milk exhibited significantly enhanced antioxidant activity. Release profile of nanoencapsulated peptides in milk demonstrated sustained release of peptides under simulated GI conditions. Nanoencapsulation protected the peptides against heat degradation, when milk was subjected to pasteurization and boiling. Fortified milk was observed to be acceptable during storage period at 4ºC for 6 days along with that of control milk. Sensory and physico-chemical properties of dahi and yoghurt prepared with fortified milk were not affected. Fortified milk, dahi and yoghurt exhibited significantly enhanced antioxidant activity. The study demonstrated that niosomes loaded with biopeptides could be successfully prepared using Tween 60 as surfactant and lauryl alcohol as stabilizer by TLH+HSH and HSH methods. Biopeptide loaded niosomes served as potential delivery vehicles for fortification of milk and fermented products.ThesisItem Open Access PREPARATION, CHARACTERIZATION AND APPLICATION OF SPRAY DRIED MILK PROTEINVITAMIN A AND D COMPLEXES(ICAR-NDRI, KARNAL, 2020) BODEMALA HARISHA; ARORA, SUMITMicronutrient malnutrition is estimated to affect about 2 billion people across the world and Vitamin A and D deficiency is one of the most conspicuous forms of it. Vitamin fortification is one of the most viable strategies to address their deficiency in human beings. The major limitations in use of vitamins A and D in free (oily) form are their stability and bioavailability. Complexation of these vitamins with milk proteins has the potential to overcome these limitations. Spray dried vitamin A and D complexes were prepared using sodium caseinate (NaCas), and reassembled succinylated sodium caseinate (RSNaCas). Spray drying conditions were optimized for preparation of milk protein-vitamin complexes [sodium caseinate-vitamin A (NaCas-VA), sodium caseinatevitamin D (NaCas-VD), reassembled succinylated sodium caseinate-vitamin A (RSNaCas-VA) and reassembled succinylated sodium caseinate-vitamin D (RSNaCas- VD)] as follows: inlet air temperature: 170°C, feed flow rate: 4ml/min and total solids concentration: 14% with minimum moisture content and maximum solubility. Analytical conditions were optimized for quantification of total and unbound VA and VD in spray dried protein-vitamin complexes. Structural modification of proteins upon complexation with vitamins was ascertained with particle size, zeta potential, turbidity and tryptophan intensity analysis. The structure of vitamin complexes was evaluated by visualizing the microstructure of milk protein–vitamin complexes with confocal laser scanning microscopy, scanning electron and transmission electron microcopy. Milk proteinvitamin complexes exhibited significantly higher (p<0.05) stability in aluminium laminate pouches during storage at -20˚C, than at 4˚C. All the milk protein complexes were stable at pH 7.0, while RSNaCas-VA and VD complexes displayed significantly higher stability at pH 5.0 as well. Addition of milk protein-vitamin complexes and free vitamins to milk did not modify the sensory and physico-chemical properties (pH, titratable acidity, curd tension, viscosity, rennet coagulation time, alcohol stability, heat stability, colour profile and texture parameters) of milk. Milk protein-vitamin complexes and free vitamins displayed maximum stability in pasteurized milk, followed by boiled and sterilized milk. Higher recovery of vitamins during HPLC analysis was observed for pasteurized fortified milk packed in LDPE pouches than transparent glass bottles at 4- 7˚C for 48 h. Milk samples fortified with these complexes invariably exhibited higher stability than free from during exposure to 1485 than 4455 lux at 4˚C for 7 days. Among the different milk protein-vitamin complexes fortified milk, it was RSNaCas-VA and VD complex fortified milk which yielded higher in-vitro bioaccessibility of vitamins than NaCas-VA and VD complexes.
