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ThesisItem Open Access OPTIMIZATION OF SPRAY DRYING CONDITIONS FOR PREPARATION OF PROBIOTIC DIRECT VAT SET STARTERS(ICAR-NDRI, KARNAL, 2023) WALDE NEHA RAVI; CHAND RAMDirect vat set (DVS) starters are concentrated form (1011-1014 CFU/g), available in both freezedried and frozen state. Freeze drying is most commonly used for the production of probiotic DVS on a commercial scale which is a costly and time-consuming process. On the other hand, spray drying is a low-cost, high-yield technology being explored in the food sector to prepare large amounts of dried probiotics. The present study was aimed to select protective agents and optimize spray drying conditions to prepare DVS starters of probiotic Lactiplantibacillus plantarum strains. Two probiotic strains i.e. Lactiplantibacillus plantarum CRD7 and Lp. plantarum HD48 were selected based on their health-promoting features and evaluated for techno-functional attributes in milk for preparation of dahi. Significance difference (p<0.05) was observed w.r.t. techno-functional (i.e. curd setting time (14.33±0.02 h), pH (4.26±0.01), titratable acidity (0.7±0.01 % LA) and total probiotic counts (8.71±0.01 Log CFU/mL), overall acceptability sensory score (9.0±0.0) and texture profile (firmness 2.30±0.0 N), of probiotic dahi prepared with Lp. plantarum CRD7 compared to Lp. plantarum HD48 respectively. Based on better techno functional performance, two Lp. plantarum strains were subjected for heat challenge experiment using to optimize spray drying parameters i.e. protective agents and inlet temperature to determine their heat tolerance for the better survivability during spray drying of probiotic for probiotic DVS powder preparation. Three protective agents i.e. lactose, maltodextrin and sorbitol were assessed at varied concentrations and temperature combinations for their heat tolerance to enhance the survivability of selected probiotic Lp. plantarum strains. Evaluation of probiotic DVS prepared with maltodextrin @ 2.5% concentration showed better survivability rate exposed to 1 min (95.27±0.03) and 5 min (95.15±0.03) at 55°C compared to sorbitol and lactose. The cell survivability of selected probiotic Lactobacillus strains was found to be affected by the type and concentration of the protective agents. Inoculum levels @ 0.002% and 0.004% (w/v) of three different spray dried probiotic DVS was optimized for preparation of dahi. Observations on storage stability of probiotic DVS packed in three packaging (aluminium laminate, LDPE and EVOH) materials and stored at -20°C and 4°C exhibited no significant difference in techno-functional performance up to two months of storage. It is concluded that optimized spray drying conditions for preparation of probiotic DVS starters of Lp. plantarum CRD7 could be utilized for preparation of health-promoting fermented dairy foods such as dahi, lassi etc.ThesisItem Open Access EVALUATION OF IMMUNOMODULATORY EFFECTS OF BIOACTIVE PEPTIDES RICH WHEY DRINK IN ANIMAL MODEL(ICAR-NDRI, KARNAL, 2022) SALINI, S. V.; SHILPA VIJAccording to WHO estimates released on May 20, 2021, the COVID-19 pandemic has resulted in more than 120 million confirmed cases and 3.4 million deaths. The pathophysiology of SARS-Cov-2 infection revealed that hyper inflammation and immunological dysregulation were the most important factors in causing multi-organ damage. In this covid-19 scenario, Immunomodulation have a very great significance. Even though so many immunomodulatory drugs have been in use, they are not suitable for long term therapy owing to its high cost and inevitable side effects. BAP fragments released from proteins interact with specific receptors and stimulate or inhibit certain effects on the immune system. The present study investigated the immunomodulatory effects of bioactive peptides rich whey drink in animal model. Colostrum whey, which is rich in protein, was fermented with Lacticaseibacillus rhamnosus C-25 to produce bioactive peptides. During fermentation, the protein content of whey was decreased from 6.97 ± 0.50 mg/ml to 5.33 ± 0.64 mg/ml and peptide content increased from 0.959 ± 0.04 mg/ml to 1.238 ± 0.06 mg/ml. Peptides of less than10 kDa molecular cut-off was separated through ultrafiltration. Peptide content of 10kDa Permeate was found to be 0.860 ± 0.02 mg/ml. Bioactive peptide ingredient (BAPI) showed good antimicrobial activity against pathogens. B. cereus showed lowest MIC of 4.4 ± 0.29 mg/ml and S. enterica showed highest MIC of 66.5 ± 0.17 mg/ml. Two types of fermented whey drinks- Paneer-whey drink (PWD) with added BAPI (L. acidophilus 195 @ 2%, 37ºC/24h) and Colostrum-whey drink (CWD) with in-situ production of BAPI (195+C-25, 2:1, 37ºC/48 h) was prepared and checked its biofunctional properties. All the whey drinks showed good antioxidant activity. Among which CWD showed highest % scavenging of 85.5 ± 90.4. 10KDa BAPI and prepared whey drinks (PWD 1 (10% BAPI), PWD 2 (20% BAPI), CWD 1(10 times MIC), CWD 2 (20 times MIC), were checked for immuno modulatory activities in mice models against one normal control and infected model group. Mice were given treatments for a period of 7 days via oral gavage. The mice were challenged with E. coli ATTC 723 for 3 days and sacrificed on 15th day. Both CWD showed good phagocytotic activity of 46.88% and 46.89%. Blood serum collected from animals of each group were analysed for ALT/AST, Blood cholesterol, Triglycerides, IgA, IL-10 and TNF-α. CWD groups increased anti-inflamatory cytokines such as IgA (534.5 ± 1.00 μg/ml) and IL-10 (968 ± 0.01 pg/ml) levels and decreased pro-inflamatory TNF- α levels compared with model group. CWD 2 fed mice group significantly reduced ALT/AST levels in serum (12.80 ± 0.64 and 4.66 ± 0.58 U/L respectively. CWD 2 showed good lactobacillus translocation in large intestine and significantly decreased E. coli levels in the same. Protein-rich fermented colostrum whey drink with in-situ production of BAP as well as BAP ingredients may be used for immune modulation.ThesisItem Open Access ISOLATION AND CHARACTERIZATION OF ANTIBIOTIC RESISTANT BACTERIA FROM ORGANIZED DAIRY PRODUCTION(ICAR-NDRI, KARNAL, 2022) AKASH; RASHMI, H.M.Antibiotic resistance is a global health concern as many of the clinical pathogens are becoming resistant to the commonly available antibiotics. Agri-Food chain is one of the key drivers of antibiotic resistance, where environmental factors promote the dissemination of resistance amongst the human and animal sphere. Hence, the present study was designed with the objectives to find out the prevalence of antibiotic resistant bacteria in the dairy production environment, followed by their genotypic identification and characterization for antibiotic resistance. A total of 15 samples were collected from the organized dairy production system, comprising three samples each of milk, faeces, waste water, soil, and hand swab. Culturable antibiotic resistant bacteria were enumerated and isolated by employing spread plate technique using three different media i.e., PCA (Plate Count Agar), NA (Nutrient Agar) and VRBA (Violet Red Bile Agar) supplemented with 8 target antibiotics (amoxicillin, cefotaxime, chloramphenicol, ciprofloxacin, ceftriaxone, doripenem, gentamicin ,and tetracyclines) at sub-lethal concentration. Higher prevalence of suspected resistant bacteria were found against amoxicillin and tetracycline with highest numbers in dairy waste water. A total of 295 isolates suspected to be resistant were isolated, and preliminary identification was carried out by staining techniques besides biochemical characterization. Excluding the yeast isolates, chromosomal DNA was extracted from 277 isolates and the 16S rRNA gene product was successfully amplified from 247 isolates. Finally, 202 bacterial 16S rRNA PCR products were identified by Sanger sequencing. Total six genera viz Staphylococcus spp. Escherichia spp, Pseudomonas spp, Enterococcus spp, Shigellla spp, and Acinetobacter spp. were shortlisted for antibiotic disk susceptibility testing based on the WHO (World Health Organization) critical and high priority pathogen list. All selected genus were tested against different antibiotics given in CLSI (Clinical and Laboratory Standard Institute) guidelines for respective genus. A total of six Staphylococcus spp. were tested against 14 different antibiotics of which highest resistance was observed against the oxacillin and tetracycline. Twenty Escherichia spp. were tested against 26 antibiotics where highest resistance was observed against the tetracycline, ampicillin, piperacillin, cefotaxime, ciproloxacin, norfloxacin and trimethoprim. Four Enterococcus isolates were tested against 14 antibiotics of which highest resistance was observed against ampicillin, norfloxacin, tetracycline and erythromycin. Nine Shigella isolates were tested against 26 antibiotics and highest resistance was observed against piperacilli/tazobactam, Cefotaxime, Ertapenem, Amikacin, Trimethoprim, and Tetracycline. Fourteen Pseudomonas isolates were tested against 10 antibiotics of which highest resistance was observed against Piperacillin/Tazobactam, Piperacillin, Cefepime. Nine Acinetobater isolates were tested against 15 antibiotics, where resistance was observed against tetracycline, piperacillin, ampicillin/Sulbactam and Imipenem. Further, 20 Escherichia spp. isolates were screened for the presence of ESBL, in which 17 isolates were positive for ESBL. Similarly, 8 isolates of Acinetobacter spp. and 12 isolates of Pseudomonas spp. were screened positive for Carbapenemase production. The results of this study highlighted the presence of resistant bacteria against clinically relevant antibiotics in the dairy production system with a higher incidence in dairy wastewaters. In order to minimize the dispersion of resistances through dairy waste water, it is necessary to implement effective methods of wastewater treatment and disinfection. Further, tetracycline is the major concern in dairy sector and future stewardship measures should be directed towards the restricted use of tetracycline and alternative drug discovery should aim at replacing tetracycline.ThesisItem Open Access PROPIDIUM MONOAZIDE ASSISTED REAL TIME PCR-BASED ASSAY FOR RAPID QUANTIFICATION OF PROBIOTIC LACTOBACILLUS SPP.(ICAR-NDRI, KARNAL, 2022) RUSHIKESH R. DESHMUKH; RAGHU, H. V.In addition to their fundamental nutritional worth, probiotics are live bacteria that, when ingested in certain concentrations, have health advantages. "Live bacteria that, when fed in suitable amounts, impose a health advantage on the host," according to FAO/WHO (2001), are probiotics. To be qualified as a probiotic product, it must have defined probiotic bacteria in appropriate viable count at end of shelf life. The conventional plating methods are laborious, time-consuming and fails in discrimination of different species and detection of viable-but-not-culturable state of cells. In this regard, real- time PCR (qPCR) has been able to overcome above limitations to detect and quantify specific bacterial species in diverse food matrices including fermented probiotic dairy products. However, one of the limitations of qPCR being not able to distinguish between DNA from live cell and dead cell can be overcome by coupling it with propidium monoazide (PMA). Hence, the current research was focussed on development of PMA-qPCR assay to evaluate the viability of probiotic Lactobacillus spp. cells, including Lactiplantibacillus plantarum 91, Limosilactobacillus fermentum 1, and Lacticaseibacillus rhamnosus GG. In the development and evaluation of efficacy of PMA-qPCR assay in probiotic dairy products, the concentration of propidium monoazide (PMA), light exposure time and distance from light source were optimized (X, Y, Z) for quantification of aforesaid bacterial in saline system for live bacteria. Besides its efficacy in enumeration of viable probiotic bacteria in a mixture of live and dead probiotic bacteria was also evaluated. The developed assay was able to quantify live probiotic lactobacillus spp. living cells from the assortment of live and dead probiotic bacteria. The developed assay was also evaluated for its ability to quantify live probiotic Lactobacillus spp. from dairy matrices like Dahi and fermented milk. With the standardized DNA extraction procedure, the developed assay was able to yield very similar results with that of conventional method. On comparison, we found Developed PMA–qPCR-based assay was sensitive, accurate, precise, less biased, repeatable, reproducible, and user-friendly method for the enumeration/quantification of probiotic Lactobacillus spp. in probiotic dairy products. Hence, the developed PMA assisted q-PCR based assay could be an appropriate alternative for the existing time-consuming and laborious conventional methods for routine monitoring of probiotic count in probiotic dairy products at the designated threshold level prescribed by FSSR (2016)ThesisItem Open Access OPTIMIZATION OF LYOPHILIZATION CONDITIONS FOR PREPARATION OF PROBIOTIC DIRECT VAT SET STARTERS(ICAR-NDRI, KARNAL, 2022) BAKEERU LAKSHMAIAH; CHAND RAMDirect vat set (DVS) starters are concentrated form (1011-1014 CFU/g), available in freezedried and frozen state. Present study was aimed to investigate compatibility in milk and optimize lyophilization conditions for preparation of freeze dried DVS starters of probiotic Lactobacillus strains. Eight probiotic Lactobacillus strains were selected based on their health promoting features and evaluated for their compatibility in milk for preparation of dahi. Significance difference (p<0.05) was observed w.r.t. techno-functional, sensory and texture attributes of probiotic dahi prepared with co-culturing technique as compared to single cultures. Combinations of Lactiplantibacillus plantarum CRD7, Lp. plantarum HD48, Lp. plantarum HD51 used with each other showed best compatibility w.r.t. technofunctional i.e., curd setting time, pH, titratable acidity(% LA) and total probiotic counts (Log CFU/mL)sensory and textural parameters for probiotic dahi. Significance difference (p<0.05) was observed in probiotic dahi quality parameters prepared from mixed cultures as compared to single strain. Curd setting time (h), pH and titratable acidity (% LA) were found to be 14.33±0.02, 4.26±0.0, 0.72±0.00 and 8.71±0.01, respectively. Based on better compatibility of listed three Lactobacillus strains were selected for optimization of lyophilization conditions for probiotic DVS powder preparation. Six cryoprotective agents i.e. lactose, dextrose, mannitol, sorbitol, whey protein concentrate (WPC) and horse serum albumin (HSA) were assessed for their cryoprotectant ability to improve survivability of probiotic lactobacilli. Evaluation of probiotic DVS prepared with sugars at varied concentrations showed better techno-functional performance as compared to WPC and HSA. The cell survivability of probiotic selected Lactobacillus strains was found to be affected by the type and concentration of the cryoprotective agents. Inoculum level @0.25% (w/v) of three different lyophilized probiotic DVS was optimized for preparation of dahi. Observations on storage stability of probiotic DVS packed in three packaging (Aluminium laminate, LDPE and EVOH) and stored at -20°C and -4°C showed no significant difference in techno-functional performance upto two months. It is concluded that optimized lyophilized conditions for preparation of probiotic DVS starters using Lactobacillus strains Lp. plantarum CRD7, Lp. plantarum HD48 and Lp. plantarum HD51 could be utilized for preparation of health promoting fermented dairy foods such as dahi, lassi etc.ThesisItem Open Access Development of Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) assay as a point-of-care diagnostics for SARS-CoV-2(ICAR-NDRI, KARNAL, 2021) KELKAR, SHWETA; RASHMI, H.M.The present study aimed to develop a Reverse-transcription Loop-Mediated Isothermal Amplification (RT-LAMP) assay as a “Point-of-care” (PoC) diagnostic solution which can be useful for the detection of SARS CoV-2. To develop this assay, the complete genome sequences of SARS CoV-2 reported from different States and Union Territories of India were downloaded from GISAID (global initiative on sharing avian influenza data) database. The sequences were aligned to identify conserved regions for designing RT-LAMP primers. After literature search, three conserved regions with no previously reported primers for detection of SARS CoV-2 were selected and the primers were designed using Primer Explorer V5. A total of six sets (A, B, C, D, E, and F) of primers, two each from the single conserved region were designed. The specificity of these primers were again checked in silico with BLAST tool in the NCBI server. The designed primers did not yield any similarity matches with the other human coronavirus except MERS-CoV and Bat SARS-CoV. On the other hand for the development of LAMP assay with these designed primers, the target conserved regions were got synthesized and analysed for quality parameters prior to their use in the LAMP assay. Later, the primers (type, concentration and ratio) and LAMP reaction temperature were optimized in the development of LAMP assay using synthetic gene constructs. Further, the performance of developed assay was evaluated at KCGMC using cDNA synthesized from RNA of SARS-CoV-2 and developed LAMP techniques was found effective in the detection of SARS-CoV-2. The developed LAMP technique was further explored for development of colorimetric/fluorometric LAMP assay for rapid visual detection of SARS-CoV-2. The performance evaluation of developed colorimetric and fluorometric LAMP assays yielded satisfactory results in the detection of SARs-CoV-2 RNA. Hence, the developed Colorimetric and fluorometric LAMP assays can be used as PoC diagnostic test for the detection of SARSCoV- 2. However, its performance directly with RNA or samples needs to be evaluated to make it as an RT-LAMP assay and further validation is required with a larger sample size as per ICMR guidelines.ThesisItem Open Access IN VITRO IMMUNOMODULATORY ACTIVITY OF CUSTOM SYNTHESIZED PEPTIDES DERIVED FROM Lactobacillus rhamnosus NCDC24 FERMENTED MILK(ICAR-NDRI, KARNAL, 2021) SRIVASTAVA, UMANG; BEHARE, PRADIPThe current study was undertaken to investigate the immunomodulatory activity of four synthesized peptides viz., VLPVPQKA, ALPMHIR, AGWNIPM, and YLGYLEQLLR. These peptides were selected from 1-3 KDa of peptide fraction from peptide rich fermented dairy beverage that was prepared with a proteolytic strain L. rhamnosus NCDC 24, by in silico approach using BIOPEP-UWM database. AGWNIPM, ALPMHIR, VLPVPQKA, and YLGYLEQLLR scavenge 2,2’-azinobis (3-ethylbenzothiazoline-6- sulfonic acid) (ABTS) free radical, with half maximal inhibitory concentration (IC50) value of 69.49±0.43 μg/mL, 281.54±4.15 μg/mL, 336.17±1.04 μg/mL, and 87.62±0.63 μg/mL, respectively. Further, the immunomodulation potential of strong antioxidant peptides (AGWNIPM and YLGYLEQLLR) was evaluated in mouse peritoneal macrophages challenged with lipopolysaccharide (LPS), a potent pro-inflammatory stimulus. Prior to investigating the immunomodulation potential of the peptides in LPS challenged macrophages, the cytotoxicity of peptides and LPS against murine macrophages was evaluated by an MTT assay. The concentrations of 12.5, 25, 50, 100, and 200 μM for peptides AGWNIPM and YLGYLEQLLR and 1μg/mL for LPS were used. All the peptides concentration did not show cytotoxic effects as compared to the control cells. The treatment of AGWNIPM in LPS-stimulated mouse macrophages resulted into 43.09 to 90.93% reduction in IL-6, 22.78% to 63.26% reduction in IL-1β, and 35.44% to 75.83% reduction in TNF-α. Whereas YLGYLEQLLR treatment resulted into 26.37% to 89.22% reduction in IL-6, 13.32%, to 61.55% reduction in IL-1β and 22.44% to 74.38% decrease for TNF-α, in a concentration-dependent manner. Both peptide AGWNIPM and YLGYLEQLLR significantly increased (P < 0.001) the antiinflammatory cytokine (IL-10) and reduced the nitric oxide (NO) generation in LPSstimulated mouse macrophages in a concentration dependent manner. The phagocytic activities of macrophages treated with peptides (AGWNIPM and YLGYLEQLLR) increased significantly (P < 0.001) with increase in concentration of peptides (P < 0.001). Therefore, the findings support the potential use of multifunctional peptides in the development of new antioxidant and therapeutic agents for immunomodulation.ThesisItem Open Access DEVELOPMENT OF COLOSTRUM WHEY-DERIVED BIOACTIVE PEPTIDE INGREDIENTS FOR PREPARATION OF BIOFUNCTIONAL BEVERAGE(ICAR-NDRI, KARNAL, 2021) LIKITH, H. D.; VIJ, SHILPAIn this investigation, our main aim was to optimize the method for development of colostrum whey derived bioactive peptides through Lactobacillus fermentation, in order to improve biofunctionality of dairy beverages. Two cultures, Lactobacillus rhamnosus C25 and Lactobacillus plantarum C2 were selected as they are known for their proteolytic activity. Colostrum whey was fermented with the above-mentioned cultures, separately @ 2% inoculums levels for 48 hours and fermentate were separated into six fractions - retentates and permeates of 10kDa, 5kDa and 3kDa. These peptide fractions were evaluated for biofunctional properties. All the fractions had good antimicrobial activity. 10kDa P of both C25 and C2 showed significantly higher activity with MIC of 0.21 – 0.32 mg/ml and 0.46 – 0.8 mg/ml, respectively. All the fractions of C25 and C2 fermentate exhibited good anti-oxidative activity. However, 10kDa P exhibited highest activity of 91.44 ±0.56 and 86.45 ±0.06% ABTS radical scavenging activity, respectively. ACE Inhibitory activity was observed in all the fractions, however, C 25 10kDa P showed highest activity of 78.82±0.03% inhibition. Immunomodulatory activity (% macrophage phagocytosis) was highest in C25 10kDa peptides (45.32 ±0.27%), followed by C2 10kDa peptide (31.25±0.25%). The peptides were stable at different conditions like temperature, pH and digestive enzymes. C25 peptides exhibited higher antimicrobial activity, ranging from 15-20mm zone of inhibition and ABTS % scavenging activity of 80.58± 0.86 %, corresponding to 1963.54±0.83 μmol/ml TEAC even after 20 days of storage at 40C. C 25 peptides were selected on the basis of higher activity and stability to be used as ingredient and added @ 10% and 15% concentrations into flavored milk. The beverage was found to be biofunctional, and peptides added @ 15% concentration exhibited higher activity like, antimicrobial, antihypertensive and antioxidative properties even after 7th day of storage. At the end of 7th day of storage, there was significant decrease in the microbial counts. Thus, bioactive peptide derived from colostrum whey by microbial fermentation may be used as ingredients for the development of biofunctional dairy beverages.ThesisItem Open Access ENZYME STRIP FOR EARLY DETECTION OF SUB-CLINICAL AND CLINICAL MASTITIS(ICAR-NDRI, KARNAL, 2021) BHAVANI, N; KUMAR, NARESHIn current research program “Mastitis Strip” was developed for early detection of clinical and sub-clinical mastitis that could be used in dairy farms. During mastitis infection, somatic cells synthesis different enzymes to fight against the causative microorganisms like Lactate dehydrogenase, alkaline phosphatase, β-glucoronidase, esterase, β-galactosidase, β- glucosidase etc. An increase in the number of somatic cells leads to an increase in the amount of marker enzymes that could be targeted in the detection of mastitis infection. In current study, 4 enzymes were explored to develop diagnostic test clinical and sub clinical mastitis in milk. Paper strip sensor was prepared using watman filter paper-3 which is anchored on the plastic sheet. Chromogenic substrates solution was immobilized on the strips and was dried for 30 min in incubator at 55⁰C. Enzymes were screened by taking 300μl of milk sample in Eppendorf tube followed by addition of functionalized strip and incubation at 37⁰C for 30 min. Enzyme(s) produced colour on strip and marker enzyme was selected based on colour intensity and time. Subsequently, activators i.e. tween 80, triton X-100, EDTA, Sodium lauryl sulphate and chloroform were screened for their ability to enhance enzyme-substrate reaction on the strip. The assay was performed using selected activator (0.5%) solution and intensity of color developing on strip was found increased. The assay protocol was further optimized for incubation temperature 45⁰C, substrate volume 5μl, sample volume 250μl, activator volume 20μl and incubation time 15 & 30 min. The optimized protocol for screening milk samples for mastitis includes 250μL milk and 20μl of activator added in 2 different tubes, paper strip was inserted and incubated at 45⁰C for 15 & 30 min. Normal milk doesn’t produce color on both the strip after 15 /30 min; Sub-clinical mastitis detected based on color development within 30 min. Clinical sample produces a good color in after 15 &30 min of incubation. The developed assay was evaluated with 653 milk samples collected from health complex at NDRI. Out of 653 milk samples 78% were normal, 12% with sub-clinical and 10% clinical mastitis. Mastitis strip was evaluated with SCC counter which is considered as reference method and 100% correlation was established. Based on these results , working of kit was classified as normal milk with SCC < 5 lakhs, sub-clinical with counts of 5-10 lakhs and clinical stage with counts of 10-20 lakhs or more . These results were validated with CMT where 89.9 % correlation was established. Kit performance was correlated with average cell count on EMCCD microscope with correlation of 98.6 %. Milk samples with average counts of 0-75 indicates normal milk, 76-200 with Sub-clinical and clinical samples shows >200. Subsequently, developed kit was validated with Porta SCC quick mastitis test by analyzing 225 milk samples with correlation of 90%. The developed kit demonstrated precision of 0.96, 0.99 and 0.95, sensitivity 66.8%, 94.6% and 76.8%, and specificity of 98.9%, 99.8% & 98% with CMT, somatic cell counts on EMCCD and PortaSCC kit respectively. The strips were stable at room temperature for up to 135 days without vacuum packing and up to 210 days when vacuum packaged and maintained at room temperature. When stored under refrigeration without vacuum packaging and stored under refrigeration with a vacuum package the strips were stable for up to 240 days. At refrigeration conditions the shelf-stability study was still in progress. Based on above results the mastitis strip can be used under field condition for screening of clinical and sub-clinical mastitis leading to significant saving in losses due to mastitis, reduction in usage of antibiotics and development of Antimicrobial resistance in pathogenic bacteria in dairy sector.
