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National Dairy Research Institute, Karnal
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ThesisItem Open Access STUDIES ON THE DETECTION OF SORBITOL ADDITION IN MILK USING THIN-LAYER CHROMATOGRAPHIC APPROACH(ICAR-NDRI, KARNAL, 2022) RANJITH KUMAR N; VIVEK SHARMAMilk has long been known as a source of various nutrients essential for a proper growth in human beings. It is considered as a complete food due to its balanced nutritional value in terms of energy, protein, carbohydrate, fat, vitamins etc. In order to fulfil the ever-growing demand of milk, the suppliers have found it profitable to dilute milk with water and then adjust its SNF/ lactometer reading by addition of cheaper chemicals like urea, cane sugar, starch, saccharin, polyol (such as sorbitol), gelatin, etc. These chemicals are used for artificial sweetening, as a source of solid not fat (SNF) and milk fat replacers, etc. Sorbitol is the emerging adulterant in the recent times. Easy availability, low cost, and nonavailability of the sensitive methodology for its detection are the major factors which render it to be used as an adulterant in milk. Industry and academia are already working in this field and looking for the methodology to detect sorbitol in the presence of other commonly reported adulterants. In the present study, the copper impregnation time and solvent system optimization was carried out to resolve sugars and polyols on Cuimpregnated TLC plates. A TLC method using copper impregnated silica gel 60F plate (impregnation time of 90 sec), n-propanol: ethyl acetate: water (7:1:2) as solvent system and 0.5% potassium permanganate in 0.1 M NaOH as the detecting reagent was developed to detect sorbitol in both aqueous as well as in milk system. The minimum level of detection of sorbitol was 0.2%. Sorbitol in the Cu-impregnated TLC was detected as the dark yellow, short streak at the point of application whereas other sugars and polyols were found to be travelled to different distances from the point of application. The presence of glucose, lactose, sucrose, mannitol and maltitol did not interfere in the separation behavior of sorbitol on the standardized TLC methodology both in aqueous as well as milk system. The standardized method was also found to be effective in detecting the sorbitol in the presence formalin. The method was found to be suitable to detect sorbitol even in the presence of urea (0.2, 0.5 and 1%). Developed acidity and neutralization (with 0.1 N NaOH) of milk did not affect the efficiency of the developed TLC method. The developed method could also detect the presence of sorbitol in the diluted milk wherein SNF content was adjusted with sorbitol solution. Finally, the standardized method was applied in the branded (5) and unbranded (2) market milk samples. Results revealed that all the samples were negative for the sorbitol. These samples were also tested for sorbitol presence using color-based test (Karra, 2021) and the results were similar to the findings of developed TLC method. The advantage of the developed TLC based method was its better detection limit and specificity even in the presence of neutralizer and urea.ThesisItem Open Access DEVELOPMENT OF METHODS FOR THE DETECTION OF CELLULOSE IN MILK AND MILK PRODUCTS(ICAR-NDRI, KARNAL, 2022) SAPNA; RAJAN SHARMAMilk and milk products are the important source of nutrients like proteins, carbohydrate, fat, vitamins and minerals for humans. However, the safety of milk has always been challenging due to the use of adulterants by unscrupulous people for economic gain. Cellulose is one of the such adulterants which is used to increase the solid-not-fat content of milk. The present study was conducted to develop a rapid (strip-based & liquid-based) and instrument-based method for the detection of cellulose in milk. The concept used in the study include conversion of cellulose present in the adulterated milk sample to glucose by cellulase and detection of glucose produced using appropriate detection reagent. Cellulase from two different source (Trichoderma reesei and Aspergillus niger) was used and spiked cellulose (microcrystalline cellulose and carboxymethyl cellulose) in milk or buffer was hydrolysed into glucose using cellulase. Produced glucose was detected following the glucose oxidase- peroxidase coupled assay using prepared glucose strip and liquid-based method. In both strip-based as well as liquid-based method it was found that cellulase from Trichoderma reesei showed nil activity towards cellulose hydrolysis to produce glucose. During the experiments with cellulase from Aspergillus niger, it was found that glucose strip showed colour change in control samples also containing only cellulase. After conducing many experiments, it was concluded that cellulase itself contained glucose. So, efforts were made to remove glucose from cellulase using ultrafiltration/diafiltration and treatment of cellulase with glucose oxidase-peroxidase. The cellulase treated with glucose oxidase-peroxidase was used in the further study. Cellulose in MCC or CMC spiked buffer or milk samples was detected by both strip-based and liquid-based method. The optimized conditions include treatment of milk samples with treated cellulase at 50°C at pH 5.0 for 4 h. Limit of detection (LOD) of the method was 0.5% for MCC and CMC in milk. Cellulose detection strip was prepared but it did not work and this may be due to fact that cellulase action requires at least 4 h to hydrolyse cellulose into glucose. Instrument-based method of cellulose detection was developed using normal phase- high performance liquid chromatography. MCC/CMC were spiked (0.5%) in the pure raw milk (pH 5.0) followed by cellulose hydrolysis with treated cellulase for different incubation period at 50°C. An extra peak of glucose was observed in the milk samples spiked with MCC/CMC and hydrolysed with treated cellulase at the retention time of 4.699 ± 0.131 min followed by peak of lactose having retention time of 5.561 ± 0.32 min. It is proposed that the presence of extra peak of glucose as per the developed protocol may be used as test of presence of cellulose in milk.ThesisItem Open Access STUDIES ON ENHANCEMENT OF SHELF-LIFE OF PANEER USING CLOVE-OIL NANOEMULSIONS(ICAR-NDRI, KARNAL, 2022) DIKSHA BHARDWAJ; BIMLESH MANNPaneer is a popular dairy product in India and highly vulnerable to spoilage due to its high moisture and nutrient content. Current study was aimed for development of an easier and commercially viable method using clove-oil nanoemulsions to replace the conventional methods of chemical-additives and to enhance the shelf-life of paneer. For optimization of composition of clove-oil nanoemulsion, different combinations of nanoemulsions consisting of different concentration of tween 80 and clove-oil varying over the range of 0.1% to 3.0% simultaneously, were made using Ultraturrax T-25 homogenizer and were analyzed. The combinations containing tween 80 % x clove oil % as 0.25 x 0.10, 0.50 x 0.10, 0.75 x 0.25 and 3.00 x 0.10 were observed to be stable while those containing higher concentration of clove oil in comparison to surfactant such as 0.10 x 0.75, 0.25 x 0.50, 0.10 x 3.00 and 1.50 x 3.00 got destabilized when their stability was assessed. The combination consisting of 1% clove-oil, 1% tween 80 and 98% water was selected as the optimum combination and was prepared using pilot scale homogenizer followed by its characterization. The optimized combination was adjudged to be stable when subjected to different processing treatments like pH, moisture and ionic strength. Morphological characteristics were studied using TEM analysis and spherical shaped nanoparticles with mean particle-size 80.01 ± 27.85 nm varying over the range of 25.60 to 143.29 nm were observed. Relative cytotoxicity was observed as 98.46 ± 0.0931, 57.27 ± 0.0632, 25.87 ± 0.0856 , 14.29 ± 0.0703 and 5.23 ± 0.0913 % for the concentrations 10 mg/ ml, 2.5 mg/ ml, 0.625 mg/ ml, 0.156 mg/ ml and 0.039 mg/ ml respectively. Antimicrobial efficacy of the clove-oil nanoemulsion was studied against E.coli (ATCC 8739) and B.cereus (ATCC 35640) and reduction 1.98, 3.27 and 3.51 and 2.92, 3.65 and 4.05 log cycles at the concentration of 40 μg, 60 μg and 80 μg of clove oil in the nanoemulsion at 8th h was observed respectively. Minimum inhibitory concentration was calculated as 5000 μg/ ml by disc diffusion assay. Treatment of paneer samples and its packaging material with clove oil nanoemulsion resulted in enhancement of their shelf-life without having adverse effect on sensory characteristics. On 21st day of storage the moisture content, pH, free fatty acids and extent of proteolysis were ascertained to be 51.65 ± 0.13%, 5.65 ± 0.06, 13.33 ± 0.67 μeq./ g and 0.578 ± 0.003 mg/ ml for samples treated with clove oil nanoemulsion for 15 min while the values were 51.27 ± 0.13%, 5.29 ± 0.03, 32.67 ± 0.98 μeq./ g and 1.322 ± 0.763 mg/ ml in case of untreated control. Standard plate count (SPC) and yeast and mold count (Y & M) of paneer samples treated with clove oil nanoemulsion was observed to be 3.71 ± 0.08 and 2.76 ± 0.13 log cfu/ ml while in control samples it was 5.31 ± 0.03 and 4.55 ± 0.03 log cfu/ ml respectively. Paneer samples treated for 15 min showed reduction in SPC, coliform count and Y & M count by 2.01, 2.21 and 1.79 log cycles in comparison to control on 21st day of storage. Treated paneer samples elicited almost constant sensory scores throughout the storage period while drastic decrease was observed in paneer samples taken as control. Microwave treatment for 1 min resulted in reduction of eugenol content from 51.61 ± 2.30 to 11.11 ± 2.32 μg/ g for samples treated for 10 min and from 76.49 ± 1.52 to 24.78 ± 1.96 μg/ g of paneer for samples treated for 15 min and removed the flavour of clove-oil from paneer. On the basis of outcomes of the present study it could be interpreted that treatment of paneer with clove oil nanoemulsion enhanced its shelf-life to 21 days without having deteriorative effect on sensory characteristics. Therefore, it may be stated that clove oil, a natural antimicrobial essential oil in its nanoemulsion form may prove to be efficient in replacing the existing chemical methods of preservation of paneer.ThesisItem Open Access STUDY ON PHYSICO-CHEMICAL CHARACTERISTICS OF BUTTER PREPARED FROM INDIGENOUS AND CROSSBRED COW MILK(ICAR-NDRI, KARNAL, 2022) SATHIYA TAHERABBAS A.; RAMAN SETHThe milk production of India was 209.96 MT in year (2020-21), and increases at about 6.2 % compound annual growth rate. One third of worlds total milk production is utilised for manufacturing of butter. The present study was undertaken to find out effect of method of manufacture and season on butter prepared from indigenous and crossbred cow milk in relation to physico-chemical characteristics of butter. Animals in mid-lactation from indigenous breeds (Tharparkar, Sahiwal) and crossbred cow (Karan Fries) were selected for study after screening milk for somatic cell count. The results indicated that higher fat (%) and TS (%) was found in milk of indigenous breeds than crossbred. Fat and TS content of milk was higher in winter than summer season. Among the physicochemical characteristics of butter, FFA was higher in desi butter (0.28 ± 0.020) than conventional butter (0.14 ± 0.002). Non-significant differences was found in Fat (%) and moisture (%) among desi butter and conventional butter and average fat and moisture content was 82.13 ± 0.41% and 15.68 ± 0.60 %, respectively. β-carotene was significantly higher in summer season and average β-carotene content was highest in Tharparkar followed by conventional butter, Sahiwal and Karan-Fries butter. The conjugated linoleic acid was significantly increased during summer season and was found highest in Karan Fries followed by Tharparkar, Sahiwal and conventional butter. Indigenous cow butterfat had higher mono (28.99 ± 1.96%) and poly- unsaturated fatty acid content (2.07 ± 0.19%), whereas saturated fatty acid content was higher in Karan Fries (75.74 ± 0.25%) butterfat. Short (6.21 ± 0.80%) and medium chain fatty acid content (19.50 ± 2.83) was higher in crossbred. long chain fatty acid content (77.25 ± 2.51) was higher in indigenous cow milk butter. Among indigenous breed’s cow butter, poly-unsaturated fatty acid (2.1 ± 0.65%) and short chain fatty acid content (5.97 ± 0.29 %) was found to be higher in Tharparkar and Sahiwal cow milk butter, respectively and butter from Tharparkar milk contained higher saturated, medium chain, and long chain fatty acid. Butter from Sahiwal milk was high in mono-unsaturated fatty acid content. Short chain fatty acid content was significantly affected by manufacturing method, and it was higher in butter prepared by conventional butter than desi method. It can be concluded that indigenous cow breeds had slight edge over crossbred in terms of gross milk composition (higher fat and TS) and fatty acid profile of butter (higher MUFA and PUFA). The lightness, redness and yellowness of butter were significantly affected among breed as well as season, The butter prepared in summer season was more yellowish. The spreadability of conventional butter was lower than desi butter. Butter prepared during summer season especially from Sahiwal milk was found to be easily spreadable. Butter prepared by conventional method shows fracture point whereas, no fracturability was reported in butter prepared by desi method. Conventional butter was significantly harder than all butter whereas Sahiwal butter was lowest in hardness. During double bite test, conventional butter showed comparatively higher adhesiveness.ThesisItem Open Access PROFILING OF MAJOR MILK PROTEINS FROM SELECTED BREEDS OF INDIGENOUS CATTLE(ICAR-NDRI, KARNAL, 2022) NAVANEETHA G; RAJESH KUMAR BAJAJThe present study was aimed at the profiling of major milk proteins from indigenous breeds of cattle. The indigenous breeds of Tharparkar, Gir and Sahiwal and the cross-bred Karan Fries were studied. The milk samples collected from these animals were screened for mastitis and were grouped separately for further studies. The effect of mastitis on composition was also studied. Fat, SNF, Protein, lactose and ash content were higher in Tharparkar and lowest in Sahiwal among the indigenous breeds. Tharparkar was found to have the lowest milk yield among the breeds studied. Within the breeds, SNF, protein and lactose and total solids were lower in mastitic samples. Mastitis was also found to significantly lower (p<0.05) the milk yield in all the breeds. Tharparkar and Gir respectively had the highest and lowest crude protein %. A decline in crude protein, true protein, casein contents as well as casein to total protein ratio was observed in mastitic animals. Based on the protein profiling by RP-HPLC, the retention times of various casein fractions were as follows: κ-CN A (7.5-8.5 min), κ-CN b (9.5-10.5 min), αS2-CN (12-13 min), a peak of αS1-CN (18.5-19.5 min), αS1-CN B (20-20.5 min), β-CN A1 (23-24 min), β-CN A2 (24-25 min). Also, the amount of different fractions of milk proteins were found to be: αS1-CN (32-38%), αS2-CN (8-9.5%), β-CN (22.5-33.5%), κ-CN (10.5-14%) and whey proteins (15-21%).RP-HPLC results based on chromatograms of bovine milk casein standards were used for the identification of various casein fractions in milk and their corresponding genetic variants were identified based on literature. κ-CN AB variant was predominant in Tharparkar and Sahiwal breeds studied (77%), followed by κ-CN A, while the opposite was observed in Gir breed (72% κ-CN A and 28% κ-CN AB. αS1-CN B was dominant in indigenous breeds with a small population having an additional variant. The β-CN-A1A2 variant dominated in the studied population of Karan Fries (60%), while 40% of the herd still retained the A2 variant of its parent indigenous breed. Protein and Calcium content decreased significantly (p<0.05) with incidence of mastitis, accompanied by an increase in Rennet Coagulation Time (RCT). Protein, Calcium content and milk yield were significantly higher and RCT lower in milk samples having κ-CN AB variant in comparison to κ-CN A. Animals with β-CN A2 variant of cross-bred Karan Fries had significantly higher milk yield but longer RCT when compared with the β-CN A2 of Tharparkar. No significant differences were observed between compositional parameters in β-CN A1A2 and A2 population of Karan Fries. No significant effect of the variants of αS1-CN was found on the compositional parameters of indigenous breeds. The indigenous breeds were characterized for the genetic variants of different casein fractions. κ-CN AB was found to predominate in animals of Tharparkar and Sahiwal breeds in comparison to κ-CN A, which could possibly be related to better milk yield and rennetability.ThesisItem Open Access QUALITY EVALUATION OF COW BUTTER AND GHEE VALORISED WITH SELECT ANTIOXIDANTS OF NATURAL SOURCES(ICAR-SRS-NDRI, KARNAL, 2022) Chavhan Ankit Prakashrao; LAXMANA NAIK, M.Butter and ghee are major fat rich dairy products; these fat rich dairy products undergo spoilage during prolonged storage due to autoxidation. The end products formed from the lipid oxidation results in the production of objectionable flavour and odour; some of the secondary and tertiary end products of oxidation can cause diarrhoea and the free radicals formed can be of carcinogenic in nature. Since, the addition of synthetic antioxidant(s) is prohibited by the FSSAI, an attempt was made to prepare butter and ghee enriched with carotenoids as natural sources of antioxidants. The β-carotene is a natural yellow-red coloured carotenoid pigment. It is an important precursor for vitamin A and used as a colorant in the food industry under the numbers E-160. In this study, using Box Behnken Design methodology, optimization study was carried out for extraction of the β-carotene from carrot and microbial source, and in place of chemical solvents ghee was used as a solvent in the ultrasonication extraction process. Ultrasonication power, extraction time, and carrot powder to ghee ratio were optimized for extraction of β-carotene rich antioxidant ingredient. The optimized butter and ghee were prepared by subjecting the antioxidant rich extract during the working stage in butter and after clarification process in the ghee samples. The optimized butter and ghee product were found to be superior in terms of sensory properties and colour parameter than the control samples. The physico-chemical quality indicator was found to be unaltered and their values were on par with the specifications given by FSSAI. Sensory analysis and accelerated storage study was done till the sample became unacceptable. It was observed that the total phenolic content in the optimized ghee was found to be 88.36 gallic acid equivalent (GAE)/gm fat and for control ghee it was 9.61 GAE/gm fat and for butter it was 65.21 GAE/gm fat and for control butter it was 6.26 GAE/gm fat and also exhibited enhanced antioxidant activity. This study resulted in development of a green bio-refining method for extraction of β-carotene from natural source. The extracted beta-carotene rich antioxidant ingredient is also a potential alternative to Annato as a colourant in the butter. The study also resulted in creating an ingredient with 3 functionalities as a source of provitamin of A, colourant and natural antioxidant.ThesisItem Open Access EFFECT OF ALTERATION IN CALCIUM CONTENT ON PHYSICO – CHEMICAL PROPERTIES OF BUFFALO MILK(ICAR-NDRI, KARNAL, 2022) DESHMUKH UTKARSH PRAYASHCHIT; SUMIT ARORACalcium is the most abundant mineral present in milk and it plays a significant role in many functional characteristics of milk and milk products. It is responsible for maintaining the stability of casein micelles via CCP linkage of casein sub units and through the development of calcium bridges connecting negatively charged casein micelles. The presence of high amount of calcium in buffalo milk limits its application during the manufacture of products like cheese, chhana, milk protein concentrates (MPC), etc. However, addition of calcium is also practiced e.g. fortified milk products. Hence, alteration in calcium content could be useful for improving the functionality of buffalo milk during processing. In the present study, calcium content in buffalo milk was varied either by depleting calcium using strongly cation exchange resin (Amberlite IR 120) or by addition of calcium salt (CaCl2.2H2O) to milk. The alteration of calcium content in buffalo milk affects the different physico - chemical properties of milk. In the first phase of this study, 20-30 %, 40-50 % and 60-70 % calcium depletion of buffalo milk was attained using cation-exchange resin. Calcium removal from milk resulted in a progressive increase in pH; ethanol stability; zeta potential; soluble proteins; RCT; apparent viscosity. However, a decrease in firmness of acid and rennet gels; average size of casein micelles; whiteness of milk; buffering capacity of milk was observed. Depletion of 20-30 % calcium from buffalo milk resulted in an increase of HCT, whereas, 40-50 % and 60-70 % calcium depletion resulted in a decrease of HCT. Removal of calcium significantly affected (p<0.05) the concentration of major minerals such as Na, K and P in milk; whereas, non-significant affect (p>0.05) was observed on trace elements (Fe, Cu and Zn). In the second phase of this study, calcium was added to buffalo milk at three different concentrations i.e. 4.0, 8.0, and 12.0 mM/ kg. Addition of calcium resulted in a progressive decrease in pH; ethanol stability; RCT; HCT and zeta potential of buffalo milk. However, firmness of rennet and acid induced gels, buffering capacity increased in milk. Increasing the level of calcium addition had no influence on apparent viscosity; size of casein micelles; colour; soluble protein content; major elements i.e. Na, K and trace minerals (Fe, Cu and Zn) in buffalo milk. The outcomes from present work will be helpful in understanding the practical consequences of manipulating calcium levels during processing of buffalo milk. In addition, the results obtained from the present work can help in developing novel and functional dairy products with calcium adjusted buffalo milk.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.
