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ThesisItem Embargo ELECTROSPINNING INTERVENTIONS FOR DRYING OF LACTIPLANTIBACILLUS PLANTARUM IN BIOPOLYMERIC NANOFIBRES(ICAR-NDRI , BENGALURU, 2023) SEETHU. B. G.; P. Heartwin AmaladhasProbiotics are defined as “live microorganisms that when administered in adequate amounts confer a health benefit on the host”. The Food Safety and Standards Authority of India (FSSAI) has approved the use of 31 probiotics in food with viable population ≥108 CFU in the recommended serving size per day. L. plantarum is a five-star rated probiotic (www.Probiotics.org) that could be used for the treatment of gastrointestinal disorders. In this study, electrospinning is attempted as an alternative encapsulation technique for L. plantarum to spray and freeze drying. Based on preliminary trials, 14% (w/w) pullulan was used as the base polymer for encapsulation of L. plantarum. Fibre formation was observed to occur only when the inequality conditions of Oh ≥ 1, De ≥ 1 and De ≥ Oh ≥ 1 were satisfied. To select the prebiotic, pullulan solutions containing inulin, sorbitol, isomalto-oligosaccharides (IMO) and polydextrose were electrospun at 18 kV. FESEM micrographs revealed that only polydextrose and IMO formed smooth beadfree fibres with pullulan. Based on mean fibre diameter, IMO was selected as the prebiotic for encapsulation of L. plantarum by electrospinning. Also, lyoprotectants such as dextran, lactose and trehalose at 10, 20 and 30% concentrations were evaluated to improve the viability of L. plantarum during electrospinning, which was carried out at 18, 21 and 24 kV. FESEM and AFM confirmed the presence of L. plantarum encapsulated within the electrospun fibres. The mean fibre diameter ranged from 90.32 to 192.50 nm, while the length and width of encapsulated L. plantarum ranged from 2.34 to 5.45 μm and 0.41 to 0.88 μm, respectively. Lyoprotectants helped in preserving the membrane integrity during electrospinning, which was confirmed by fluorescence microscopy. The highest survival rate of 91.51% was achieved at 18 kV with 20% trehalose. The electrospinning parameters were optimized using Taguchi orthogonal array design. Factorial analysis showed that voltage had the highest effect (p<0.05) on survival rate, followed by the type and concentration of lyoprotectants. The absence of characteristic peaks of proteins from the probiotic in the FTIR spectra confirmed its successful encapsulation. Also, the peak corresponding to the destruction of cell components of L. plantarum were absent in the DSC thermograms of electrospun fibres, which also indicated the successful encapsulation of probiotic within the electrospun fibres. The BET surface area of L. plantarum-loaded electrospun fibres was 3.81 m²/g, manifesting Type II adsorption isotherm. Electrospinning helped in maintaining the viability of L. plantarum above 8 log CFU/g under acid and bile salt conditions. The in-vitro digestion of electrospun L. plantarum showed the highest survival rate of 80.62% in 20% trehalose at 18 kV. After 60 days of storage at 4ºC, the viability reduced by 1.67 to 2.14 log CFU/g. L. plantarum was also spray-dried using pullulan and WPI in different ratios with IMO and thermal protectants at inlet and outlet temperatures of 170°C and 70°C. FESEM and AFM images revealed the fibrous structure of spraydried encapsulate with L. plantarum cells along the fibres. Pullulan and WPI combination with 30% IMO and 10% dextran provided the highest survival rate (p<005) of 89.30%. Similarly, L. plantarum was freeze-dried using pullulan with cryoprotectants at varying concentrations. The freeze-dried encapsulates were rough-textured with flaky and sheet structure. The randomly distributed encapsulated cells were seen as rod-shaped, and embedded in the wall material. Fluorescence microscopic images of spray- and freeze-dried L. plantarum had very high proportion of live probiotic. The freeze-dried encapsulates had the highest survival rate of 89.97% for L. plantarum with 30% lactose as cryoprotectant. The spray- and freeze-dried encapsulates were also characterized using FTIR, DSC and BET. The FTIR and DSC results of spray- and freeze-dried encapsulates were similar to that of electrospun fibres. As compared to electrospun encapsulates of L. plantarum, the BET surface area of spray- and freeze-dried encapsulates was much less at 1.49 m²/g and 0.23 m²/g, respectively, and exhibited Type I adsorption isotherm. The survival rate of L. plantarum in spray- and freeze-dried encapsulates after in-vitro digestion was also much less at 63 to 73% and 74 to 77%, respectively.ThesisItem Embargo ECONOMIC ANALYSIS OF MILK PRODUCTION IN NORTH-EASTERN STATES(ICAR-NDRI, KARNAL, 2023) POULAMI RAY; AJMER SINGHThe Indian government intends to bring about a paradigm change in the long-term planning of the country by concentrating its efforts on less developed regions like the North-Eastern States to enhance the agriculture and livestock sectors. Therefore, the current study "Economic Analysis of Milk Production in North-Eastern States," was undertaken to assess the yield gaps in milk production and factors contributing to these, decompose the effect of crossbreeding technology in milk yield, examine the economic efficiency of dairy farms in milk production and to identify and prioritize the constraints in milk production. The N-E area consists of 8 states, of which Assam, Tripura, and Meghalaya were chosen based on the highest milk production. The sampling units (i.e., households) were chosen using a multistage sampling procedure. A total of 300 dairy households were chosen for this study. Both primary and secondary data were collected from respondents and research stations on a pre-tested schedule. The study employed a combination of tabular analysis and econometric techniques in order to effectively accomplish its objectives. The study revealed that the Yield Gap-II in milk production was higher than Yield Gap-I in the region. Among the states, Meghalaya’s Yield Gap-II percentage (35.60 %) was lower than Assam (51.70 %) and Tripura (48.87 %). The percentage of total yield gap with respect to actual farm yield worked out to be 69.96 per cent, comprising YG I as 24.98 per cent and YG II as 44.47 per cent in the region. Thus, the percentage of YG II accounted for two-thirds of the total increase in actual milk yield. It was found that the experience in dairy farming, size of the animal shed, feed and fodder price, distance to the research station, training, labour allotted for dairy, dairy cooperative membership, and access to information significantly influenced the milk Yield Gap-II. The adoption of new dairy technology, i.e., crossbred cattle in the place of existing dairy technology, i.e., indigenous cow led to higher per day milk yield (total percentage gain estimated was 87.36 %). Around 67 per cent of the total change in milk production was due to the difference in the levels of technological efficiency (both neutral and non-neutral) of crossbred cow vis-à-vis indigenous cow and the remaining (nearly one-third) has been contributed by increased level of input use (20.42 %). The percentage change in milk output due to new technology was found to be the lowest in Tripura (82.81 %). This study indicated that the small farms were found to be more technically and economically efficient than the large and medium category dairy farms. Even though medium-category farmers were not the most technically efficient, it was found that they were allocatively more efficient. Large farmers were found more input redundant in the study area making them less technically efficient. Socio-economic factors i.e., non-farm annual income, access to information, herd size and membership of dairy cooperative society, and experience, were significantly influencing farmers’ technical efficiency in milk production. Among all the significant variables, non-farm income, access to information and membership in dairy cooperative society had a positive influence, while the herd size had negative effect on efficiency of dairy farms. The estimated loss due to the major problems was relatively higher in Tripura (Rs. 2.80 million) than in Assam (Rs. 2.65 million) and Meghalaya (Rs. 2.52 million). Study revealed that economic loss due to repeat breeding (Rs. 1.8 million) and abortion (Rs. 3.1 million) was the single most important problem in the region. Total estimated loss due to the affected animals in surveyed farming households was Rs. 8 million annually. Feed & fodder related constraints were found to be the major problem in the region with scaling factor of 0.288.ThesisItem Embargo DEVELOPMENT OF LACTOBIONIC ACID ENRICHED YOGHURT FOR ENHANCED CALCIUM ABSORPTION(ICAR-NDRI, KARNAL, 2023) SHAMIM HOSSAIN; YOGESH KHETRAThe current research was aimed to produce lactobionic acid (LBA) enriched yoghurt using Acetobacter orientalis and lactose oxidase, comparison of the developed yoghurts with the control yoghurt and validate the calcium absorption enhancing capability of the LBA-enriched yoghurts using in-vitro and in-vivo rat model. The three different market yoghurt samples were analysed for the presence of the LBA, but no LBA was found in those samples. For the development of LBA-enriched yoghurt, initially, the inoculation level of Acetobacter orientalis (10, 15, 20%) and incubation temperature (27, 30, 33°C) were optimized and later, the lactose oxidase level (10, 20, 30U) and incubation temperature (37, 40, 42°C) were optimized based on the maximum LBA production and sensorial acceptability. Various parameters like pH, acidity, acidification rate, lactose, lactic acid, LBA concentration and microbial count were analysed during incubation. The final product was analysed for sensorial acceptability and whey syneresis. From the first stage, the yoghurt with 20% Acetobacter orientalis inoculation with incubation at 30°C/16h was optimized as this sample had desirable acidity (0.72 %LA), highest LBA concentration (3.62 mg/100g), best overall sensorial acceptability and least whey syneresis. Among the yoghurt with lactose oxidase, the yoghurt with 30U LactoYIELD® addition and incubation at 40°C/8h was optimized as it had highest LBA concentration (16.88 mg/100g), best overall acceptability and least whey syneresis. In the second stage, these two optimized yoghurts were compared with the control yoghurt. The physicochemical, microbiological, rheological, textural, sensorial and microstructure parameters were analysed. The developed samples were comparable to the control sample, except for the presence of sweetness in the LBA-enriched yoghurts and increased hardness for the yoghurt with lactose oxidase. All the yoghurts were acceptable for up to 15 days under refrigerated storage. The cost of developed yoghurts was slightly higher than the control yoghurt. From the in-vitro analysis, it could be seen that the calcium bioavailability of the LBA-enriched yoghurt was significantly higher than the control yoghurt. In-vivo studies showed that the groups with LBA-enriched yoghurt had a 10-12% higher apparent digestibility coefficient compared to the control feed groups. The apparent digestibility coefficient was highest (81.04%) for the yoghurt with lactose oxidase group. From the organ indices, it can be proven that consumption of LBA had no adverse effect on any organs. The bone parameters of the groups fed with LBA-enriched yoghurt were better than the control feed group. Hence based on the above results, it is concluded that LBAenriched yoghurt could be made using Acetobacter orientalis and lactose oxidase, with similar properties compared to the control yoghurt and can improve the calcium bioavailability and bone health upon regular ingestion.ThesisItem Embargo SOLAR – ELECTRIC SYNERGY UNIT FOR PRODUCTION OF DAHI AND YOGURT(ICAR-NDRI, KARNAL, 2023) SHARANABASAVA; ChitranayakA low-cost batch fermentation unit for dahi and yogurt production was developed, utilizing renewable solar energy as the primary source of heating. The unit incorporated a hybrid system with an electric heater and phase change material (PCM) based heating. It consisted of a solar flat plate collector (FPC) with optional PCM containers, an electric heater and blower assembly, a fermentation cabinet with trays, and an evaporative cooling system. The system efficiently utilized outgoing hot air from the fermentation cabinet by connecting a recirculation pipe to the solar collector's air inlet. During sunshine hours, the collector absorbed heat, and the air passing through it was blown into the cabinet, maintaining a desired temperature. In the absence of sunlight, the hybrid heating system was employed. Computational fluid dynamics (CFD) simulations were conducted to analyse temperature distribution and airflow inside the fermentation cabinet, validating the results against experimental data. The system's performance was evaluated through experiments, demonstrating its ability to produce quality dahi and yogurt. The trials were conducted at different air velocities, with faster temperature rise observed at higher velocities. The optimal electrical wattage for heating was determined, and paraffin wax served as the PCM for thermal energy storage. The developed system achieved effective cup pre-cooling with varying air velocities. During summer, a significant temperature drop of 16 ℃ was achieved, while in peak winter, cups were cooled to 5.5 ℃. Comparative analyses showed no significant differences in product quality between the developed system and the conventional method. Energy consumption was a crucial consideration, with the solar system exhibiting significantly lower power consumption compared to the conventional electrical unit. Overall, the developed fermentation unit utilizing solar energy as the primary heating source demonstrated efficient performance, cost-effectiveness, and excellent product quality to conventional methods.ThesisItem Embargo EFFECT OF LATE GESTATION HEAT STRESS ON COLOSTRUM QUALITY OF SAHIWAL COWS IN RELATION TO THE IMMUNE SYSTEM AND GROWTH PERFORMANCE OF NEONATE CALVES(ICAR-NDRI, KARNAL, 2023) VISHWA RANJAN UPADHYAY; ASHUTOSHThe study was undertaken to evaluate the comparative seasonal effect of heat stress on late gestation in Sahiwal cows and their effect on the progenies. First objective was designed to unveil the effect of season and climatic chamber on physiological, haematological, biochemical, metabolic and specific hormone responses along with mineral profile of dam. The late gestation Sahiwal cows (n=48) were divided into 4 equal groups, viz. naturally heat stressed (NHS), cooling treated (CLT), spring and winter. The environmental variables were recorded and temperature-humidity index (THI) was calculated daily throughout the experimental period. Blood samples were taken during different seasons and the plasma was used for the estimation of haemato-biochemical and other indices. The animals were under thermal stress in NHS and spring groups as reflected from THI. A significantly higher physiological responses were observed in NHS group compared with the CLT group during summer. Haematological study indicated higher (P < 0.05) WBC and platelets count, and significantly lower RBC, haemoglobin and haematocrit level in NHS group as compared to other groups. The plasma levels of cortisol, IL-6, TNF-α and TBARS level were higher (P < 0.05) in the NHS compared to the CLT group. Conversely IGF-1, growth hormone, total antioxidant activity (TAC) and IgG level was high (P < 0.05) in CLT and winter groups. Second objective included the study of the neonate calves born on summer (IUHS-intra uterine heat stressed, IUCL-intra uterine cooled, HSCL-IUHS calves supplied colostrum from CLT cows and CLHS-IUCL calves supplied colostrum from NHS cows), spring and winter season as well as quality of colostrum and milk production. Season and climatic chamber had no significant (P<0.05) influence on overall mean of colostrum and milk constituents viz. fat, SNF, protein, lactose and milk yield during early lactation. The overall one-way ANOVA and post hoc-analysis of colostrum metabolites revealed 89 significant and 11 insignificant metabolites. Multivariate analysis of metabolome profiles using PLS-DA was demonstrated to distinguish between the colostrum samples of different groups at varying time intervals. The metabolites like oxalic acid, 3,4-dihydroxy-phenylglycol, methylamine, quinone, ribitol, arabinonic acid, glycerol 1-hexadecanoate, malonic acid, pectin, N-acetyl-D-glucosamine, hydroxypyruvic acid, gamma-aminobutyric acid, propylene glycol and phthalic acid were found in higher concentrations in the winter groups. The study demonstrated that the late gestation HS negatively alters the physiological responses and blood haematology of IUHS and HSCL calves. Additionally IUHS calves had significantly (P<0.05) lower mean levels of total cholesterol, glucose, total protein, globulin, sodium, potassium, calcium, zinc, iron and manganese. The overall mean value of plasma IGF-1, GH, thyroxine, TAC and IgG were found to be significantly (P<0.05) lower in heat stressed calves while the levels of inflammatory and oxidative biomarkers were significantly high. The IUHS calves had lowest dry matter intake and body weight and the lowest average daily gain during the experimental period. It is concluded that in-utero heat stress may lead the calves to have an altered physiological indices. The strategies to control heat stress during late gestation should be considered not only for the productivity and welfare of the pregnant dam but also for the developing of calf and their future performance.ThesisItem Open Access DEVELOPMENT AND EVALUATION OF LACTOSE HYDROLYSED SKIMMED MILK POWDER WITH REDUCED MAILLARD BROWNING(ICAR-NDRI, KARNAL, 2022) PAYAL SINGH; ARORA SUMITLactose hydrolysed milk powder (LHMP) with reduced Maillard browning and improved techno-functional properties was prepared. The source of β-galactosidase (K. lactis and B. lichenformis) was selected on the basis of their maximum hydrolytic and trasgalactosylation activity in a model system. β-galactosidase from K. lactis was more effective than B. lichenformis for hydrolysis of lactose (DH) and galactooligosaccharide (GOS) formation in model system (16 % lactose). Hence, β-galactosidase from K. lactis was utilised (2.50, 4.80, 7.14 and 9.53 U/mL) for hydrolysing concentrated skim milk (35-40 % total solids). DH and GOS formation were evaluated at different time intervals (0-24 h). The use of 9.53 U/mL enzyme resulted in significantly high DH (100 %) after 12 h with 9.45 % IL (initial lactose) GOS formation. Multi-enzyme approach was also followed besides a single enzyme for preparing lactose hydrolysed/free milk. Glucose oxidase (GOX) was used to oxidise the monosaccharide (glucose) produced during lactose hydrolysis in milk. The activity of GOX with and without catalase (CAT) was also evaluated for maximising its activity. The time interval at which GOX+CAT should be added after the β-galactosidase action in milk was also standardised. Further, the optimised concentration of GOX (G1, G2, G3 and G4 U/mL) incorporated alongwith CAT (C1 U/mL) resulted in a significant (p<0.05) reduction of glucose (~50 %) after t11 h of reaction with minimum changes in pH of milk. The multi enzyme-based lactose hydrolysed milk was then spray dried for the preparation of powder (P3) by optimising inlet air temperature (T1, T2 and T3 ºC) at feed flow rate of F mL/min based on physical and Maillard browning characteristics (moisture, water activity, solubility, colour, hydroxylmethylfurfural (HMF), available lysine and furosine). Powders P1 i.e. skim milk powder (SMP) and P2 (single enzyme-based LHMP) were also prepared using the optimised conditions. Among the different inlet air temperatures, P3 prepared at T2 °C resulted in lower moisture content, water activity and Maillard reaction indicators (HMF, furosine and browning index), whereas, higher solubility and available lysine content was observed. Powder P3 prepared under optimised conditions was further evaluated for compositional and techno-functional properties i.e., lactose (0.20 %), glucose (10.11 %), galactose (21.48 %), GOS (7.89 % IL), fat (1.48 %), protein (34.02 %), ash (7.43 %) and moisture (4.83 %). Loose bulk density, tapped bulk density, flowability, wettability and dispersibility were 0.45 g/mL, 0.55 g/mL, 36.20º, 11.07 s and 88.62 %, respectively. Powder P3 was comparable to P1 with respect to all the parameters evaluated including sensory properties and was superior in quality to the powder P2. Osmolality of reconstituted milk powders P2 and P3 was significantly higher (p<0.05) than powder P1 but were below the maximum limit. Powder P1, P2 and P3 were packed in aluminium laminate pouches and evaluated for physico-chemical changes during storage at 30 °C for 6 months. A significant increase (p<0.05) was observed in moisture content, water activity, HMF, browning index, furosine and titratable acidity, whereas, available lysine and solubility decreased on 6 months of storage for all the powders. Bulk density, tapped density, wettability, dispersibility and flowability of powders P2 and P3 changed significantly (p<0.05) upon 6 months of storage, whereas, for P1 a non-significant (p>0.05) change was observed. Significant decrease (p<0.05) in overall acceptability of powders P2 and P3 was evident after storage for 6 and 3 months, respectively, however, P1 reflected non-significant decrease (p>0.05) on storage. No change in crystallisation behaviour of powder P1 and P3 was observed upon storage. However, storage of powder P2 for 6 months led to change in its crystallisation behaviour. Glass transition temperature of milk powders P1, P2 and P3 decreased upon storage for 6 months. In vitro digestibility of proteins and available lysine of powders P1 and P3 were comparable, whereas, the digestibility values of powder P2 was comparatively lower. The present research was successful in overcoming the limitations of LHMP prepared using single enzyme approach with the use of multi enzyme-based approach which minimised the rate of Maillard related undesirable changes during processing and storage.ThesisItem Open Access MIGRATION STUDIES OF CHEMICAL ADDITIVES FROM FOOD PACKAGING POLYMERS INTO MILK AND GHEE(ICAR-NDRI, KARNAL, 2022) NISHI SINGH; BIMLESH MANNPolymers are commonly used in food packaging applications and varieties of chemical compounds, such as starting materials, additives, etc. are added during their manufacture. Migration of compounds from packaging materials into food could be a potential means of contamination which possess serious human health risk. In order to assess food contact materials safety, analytical methodologies to identify potential migrants are required. Hence, physico-chemical characterization of the secured 49 packaging materials was carried out; followed by optimization of solvent and analytical conditions to bring about an exhaustive and rapid extraction of chemical additives from packaging materials. Spectroscopic studies demonstrated that blend of LDPE: o-/b-LLDPE & Nylon 6: LDPE + o-/b LLDPE are widely used for packaging of milk and ghee respectively. Chloroform: methanol (1:1) was screened as suitable extraction solvent with focused ultra sound liquid solid extraction (FULSE) as the superior method for additives extraction from packaging films. Frequently occurring 10 chemical compounds with substantial relative abundance were picked for quantification in multiple reaction monitoring (MRM) mode. Thereafter, overall and specific migration studies of the procured packaging samples were carried out. Extraction methodologies for quantification of the identified migrants from milk and ghee were developed and validated in terms of linearity, % recovery, repeatability, and limits of detection and quantification. Spiked recoveries varied between 88.5 and 102.64%, and relative standard deviation (RSD) was in the range of 0.21–6.53%. Acetyl tributylcitrate was the highest detected compound in both milk and ghee at the concentration of 16 ppm and 56.57 ppm respectively, followed by Irganox®1076 and Irgafos®168. All quantified migrants were present below the prescribed SMLs by EU 10/2011. To understand the behaviour of additives and factors influencing migration, kinetics studies were carried at 4℃ and 25℃ in milk and at 25℃ in ghee. Diffusion coefficients (Dp) of migrants were calculated using Brandsch equation and FDA model, wherein significantly different values of Dp were observed.ThesisItem Open Access PREPARATION AND CHARACTERIZATION OF MILK PROTEIN HYDROLYSATE-IRON COMPLEX AND ITS APPLICATION IN MILK(ICAR-NDRI, KARNAL, 2022) SRISHTY; RAJESH KUMARIron is very important nutrient for human, but availability of iron from dietary source has limitations and its simple fortification in food causes adverse effect on quality of the food. Milk-derived bioactive peptides can be used to overcome these challenges because of their mineral binding abilities. In the present research milk protein concentrate was used as a precursor for generating iron-binding peptides. On hydrolysis using alcalase, and flavourzyme; alcalase showed high chelation (75.6%) and DH (35%) with 1150U/g protein. 10kDa fraction of hydrolysate showed maximum chelation activity (80%), than lower molecular weight fractions. Metal chelating affinity chromatography was used for further enrichment of iron binding peptides. Based on SEC, molecular weight distribution of hydrolysate showed presence of 43% low molecular weight peptide i.e., molecular weight less than 0.5KDa but after in-vitro digestion percentage of this peptide increased up to 77% due to degradation of peptides. Characterization of peptides based on LCMS showed the presence of phosphorylated peptides and other charged amino acid residues responsible for iron-binding (QSEEQQQTEDE (B-CNf(34-44)), KFQSEEQQQTEDELQDK (B-CN f(32- 48), SQSKVLPVSQ (B-CNf(66-75)), KTKLTEEE αS2f(150-157), VVRNANEEE αS2f(43-51), NSKKTVDMESTE αs2f(134-145), KNMAINPSKE αs2f(25-33), AMEDIKQM αs1f(53-60), KVPQLEIVPN αs1f(105-114)). For the preparation of the iron peptide complex alcalase hydrolysate under optimized condition (pH 8 at 50°C for 2H in the 1:0.2ratio (hydrolysate: Fe)), resulted in 43mg Fe/g of protein (corresponding to 60% of ref peptide-CPP in iron-binding capacity). Characterization of iron peptide complex by using particle size, Zeta potential, UV, Fluorescence, FTIR, SEM, EDX, VSM, and XRD techniques showed that complex between iron and hydrolysate is formed with high stability. The bioavailability of iron peptide complex was 2.3 times higher than free iron salt. The iron peptide complex was used in milk fortification (with iron conc. 25ppm based on sensory acceptability). Iron complex possessed high stability (resistance towards the release of bound iron) during different processing conditions such as pH (6-8) and heat treatment. Storage studies of fortified milk showed lesser changes in physicochemical properties and better oxidative stability. Thus, the methodology was standardized to prepare milk protein hydrolysate based functional ingredient that has high iron chelation activity, high iron bioavailability (81%), and stability under different milk processing conditions.ThesisItem Open Access EVALUATION OF NANOPARTICLES FOR DETECTION OF ORGANOPHOSPHORUS PESTICIDE RESIDUES IN MILK(ICAR-NDRI, Karnal, 2021) WANGDARE SACHIN SUBHASH; RAMAN SETHIn twenty-first-century, organophosphate pesticides are being extensively used in the agricultural practices to increase the crop yield. Immeasurable use of these pesticides leads to variety of short-term and chronic adverse health implications. Most of the countries have banned these pesticides for agricultural purposes but they are still permitted in India. In many parts of India, milk was found to be contaminated with organophosphate pesticides particularly, chlorpyrifos and malathion. Source of contamination of these pesticides in milk is through feed and fodder. The present research work was aimed to develop gold nanoparticles (GNPs) based assay for detection chlorpyrifos and malathion pesticide in milk. Conventional assays already available for the detection of organophosphate pesticides such as GC-MS, LC-MS, and HPLC have been widely used. These methods however, require highly expensive instruments, tedious steps for sample preparation, time-consuming pretreatments and well trained technicians which limit their applications for on-site screening of these pesticides in milk. The development of present pesticide (chlorpyrifos and malathion) detection assay, required salt functionalization of GNPs was carried out using different salts and sodium sulphate was found to be excellent for functionalization of GNPs. The qualitative detection of chlorpyrifos and malathion was achieved up to the 15 and 30 ppb, respectively, using functionalized GNPs in pure acetonitrile system. Furthermore, limit of detection (LOD) of chlorpyrifos and malathion was achieved as 10.99 and 23.90 ppb, respectively in acetonitrile system. For milk system, qualitative detection of chlorpyrifos and malathion was achieved up to the 20 and 35 ppb, respectively, using functionalized GNPs. The limit of detection (LOD) of chlorpyrifos and malathion was achieved as 14.76 and 24.53 ppb, respectively, in milk system. The percent recovery of chlorpyrifos and malathion was 106.08 and 105.48, respectively, in milk system with developed assay. The HPLC-MS instrumental validation was also carried out in order to assess the analytical performance in detection of chlorpyrifos and malathion in milk using the GNPs based developed method. The LOD of HPLC-MS method for chlorpyrifos and malathion was found to be 7.58 and 3.21 ppb, respectively. The percent recovery of HPLC-MS method for chlorpyrifos and malathion from spiked raw milk samples were obtained as 102.13 and 101.84, respectively. It was found that both the methods were excellent for detection of chlorpyrifos and malathion pesticide in milk. The GNPs based developed present assay is very simple when compared with sophisticated instrumental assays. The developed GNPs based assay is very rapid, cost effective and reliable for qualitative as well as quantitative detection of chlorpyrifos and malathion pesticides in milk system. However, an inference of other prevalently occurring pesticides provides progressive future scope in the development of multi-pesticide detection assay for milk system using GNPs.
