CPG MOTIFS - Vetpract

Veterinary Practitioner Vol. 14 No. 2 (Supple. 1)

December 2013

BACTERIAL DNA (CPG MOTIFS) SUBCUTANEOUSLY ADJUVANTED WITH RECOMBINANT OMP 28 (rOMP 28) OF BRUCELLA MELITENSIS PROTECTED MICE CHALLENGED WITH BRUCELLA ABORTUS 544 Ramya Kalaivanan 1, Pallab Chaudhury2, Tapas Kumar Goswami 1, Uddhav Chaudhuri 1, Sankar Palanisamy3 and G.C. Ram1 Education Cell, Veterinary College and Research Institute, Namakkal-2 ABSTRACT T he protective ef fic ienc y of rec ombinant outer memb erane protein 28 (rOMP28) of Brucrella melitensis administered subcutaneously with bacterial plasmid DNA as a source of CpG motifs was investigated in laboratory animal model mice. The mice were immunized on day 0, 14 and 21 except for the group 4 which was given as a single dose. Among the four groups, the humoral immune response in terms of immunoglobulins and cellular responses in terms of lymphocyte proliferation and nitric oxide production and cytokine production are high in the bacterial plasmid DNA administered group of animals. Moreover, the protective efficiency in terms of challenge study with virulent Brucella abortus 544 strongly validated the outcome of the study reinforcing the impact of bacterial plasmid DNA in provoking better immune response and protection. Key words: CpG, outer membrane protein (OMP), Brucella abortus, Brucella melitensis, immune response

Introduction Brucellosis is one of the most important bacterial zoonoses worldwide and particularly in developing countries the disease have important economic and public health consequences (Godfroid et al., 2005; Smits and Kadri, 2005; Pappas et al., 2006; Franco et al., 2007). From a public health perspective, studies have demonstrated that addressing the disease in domestic livestock is more cost-effective than treatment of human brucellosis (Roth et al., 2003 and Bernués et al., 1997). Vaccine improvement and development is an integral part of disease prevention and eradication. Economic constraints of vaccine affordability in developing world could be overcome by developing inexpensive, safe, and better vaccines (Ebensen et al., 2004). Vaccine cost effectiveness is also a major criterion for effective disease control (McLeod and Rushton., 2007). Brucellosis is caused by Gram negative bacteria of the genus Brucella, which are facultative intracellular coccobacilli that belong to the α2-Proteobacteriacea family (Garrity, 2001). Nine Brucella species are currently recognized, seven of them that affect terrestrial animals are: B. abortus, B. melitensis, B. suis, B. ovis, B. canis, B. neotomae and B. microti (Scholz et al., 2008; Verger et al., 1987) and two that affect marine mammals are: B. ceti and B. pinnipedialis (Foster et al., 2007). Importantly, there is approximately 94 per cent genetic similarity amongst the members of the genus (Verger et al., 1987; Delvecchio et al., 2002a), although specific genomic islands have been identified (Rajashekara et al., 2004). Brucella species are divided by their strong afliation to specific natural hosts but can infect heterogeneous hosts (Boschiroli et al., 2001). With the exception of B. ovis and B. neotomae, all other species are capable of infecting man (Hartigan, 1997). Although infection may occur through the skin, conjunctiva or respiratory mucosa by inhalation (Crawford et al.,1990; Ko and Splitter, 2003), the most common route of infection in cattle is the gastrointestinal tract (Payne, 1959; Crawford et al., 1990), from where infection 1

spreads to local lymph nodes where Brucella replicates intracellularly in phagocytes (Anderson et al., 1986a). Invasion of lymphatic vessels is followed by bacteremia leading to systemic infection, favoring colonization of the pregnant uterus, male genital organs, and mammary gland (Ko and Splitter, 2003). Importantly, the outcome of infection in cattle is dependent on age, reproductive and immunological status, natural resistance, route of infection, infectious challenge, and virulence of the infective strain (Nicoletti, 1980; Adams, 2002). Outbreaks of bovine Brucellosis are associated with abortion during the last trimester of gestation, and produces weak newborn calves, and infertility in cows and bulls (Enright et al., 1984; Poester et al., 2005; Xavier et al., 2008). In most cases, human infection is due to consumption of contaminated non-pasteurized milk and cheese or as an occupational exposure to infected animals or carcasses, uterine secretions or aborted foetuses. Less often accidental infection may occur due to manipulation of live vaccine strains or virulent Brucella in the laboratory (Young, 1983; Corbel, 1997). As human Brucellosis is essentially a zoonotic disease, control and prevention of Brucellosis in animals is essential for eradicating the disease in man. In an attempt to develop an inexpensive, safe, and better vaccine for Brucellosis, we investigated the use of bacterial genomic DNA as a source of CpG motifs to immunopotentiate the recombinant OMP 28 protein of Brucella melitensis in mice. Materials and Methods 2.1 Mice Swiss albino mice of either sex (4-6 weeks old) were brought from the Laboratory Animal Resource Centre, Indian Veterinary Research Institute, Izatnagar. They were housed and maintained in the experimental shed of Immunology section, Division of Biotechnology. The guidelines of Institutional animal ethics committee were followed for maintenance, handling

Veterinary College and Research Institute, Namakkal, 2Division of Biotechnology, Indian Veterinary Research Institute, Izaatnagar, Bareilly, U.P, India,3Veterinary College and Research Institute, Orathanadu. 399


and care of animals. Approval for the experimentation on the laboratory animals (mice) was taken from the Institutional Animal Ethics Committee. 2.2 Bacterial strains The virulent B. abortus Strain 544 and the attenuated B. abortus Strain19 were maintained in the Brucella Laboratory, Division of Veterinary Public Health, Indian Veterinary Research Institute (IVRI), Izatnagar, India. The B. abortus S19 and B. abortus 544 was sub cultured separately on tryptose phosphate agar slant in tubes. B. abortus S19 slants were incubated at 37ºC for 48 to 72 hours. However, B. abortus 544 slant cultures were incubated at 37ºC under 5% CO2 tension for 72 to 96 hours. Briefly, serial ten-fold dilution (10-1 to 10-10) of the bacterial suspension (B. abortus S19 and B. abortus 544 harvested from slants) were made in tryptose phosphate broth. 20 µl from each dilution starting from10-5 to 10-10 was taken and plated on tryptose phosphate agar in triplicate. The plates were incubated at 37ºC for 48-72 hours for B. abortus S19 and 7296 h at 37o C in 5% CO2 tension for B. abortus 544. Plates showing 30-300 CFU were selected for colony count. The mean of all the plates was multiplied by dilution factor and viable bacteria were counted as colony forming unit (cfu)/ml. Then 1x105 cfu/ml of B. abortus S19 was used for intraperitoneal immunization and 1x105 cfu/ml of B. abortus 544 were used for challenge study. 2.3 Purification of recombinant OMP28 of Brucella melitensis Recombinant E. coli, carrying 28 kDa OMP of B. melitensis in pPROC expression vector (provided by P. Chaudhuri, IVRI, Izatnagar) were grown on LB medium supplemented with required antibiotics. The recombinant clone was then grown in 6 ml of LB broth overnight in the presence of ampicillin (50µg/ml). Thereafter, 500 ml of LB broth was inoculated with 1 ml of the overnight culture having recombinant clone along with 500 µl of ampicillin and grown at 37o C in orbital shaker until O.D reaches 0.5 at 600 nm. Five hundred µl of 1M IPTG was then added to the culture to induce expression and kept at 37oC overnight. Polyhistidine tagged recombinant OMP28 was purified under denaturation condition by Ni-NTA affinity chromatography (Qiagen, USA). The presence of recombinant protein in the eluates was checked in 12.5% SDS PAGE. The concentration of the protein was determined by the method described by Lowry et al. (1951) as well as spectrophotometrically at 280 nm. 2.4 Isolation of bacterial genomic DNA As a source of CpG motifs, genomic DNA of Salmonella typhimurium were isolated by the procedure described in Current Protocols in Molecular Biology (Chapter 2.4.1) with minor modifications. Genomic DNA to be used as TLR agonist should be free from lipopolysaccharides. So the CTAB method was followed since it removes the contaminating

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lipopolysaccharides. In brief, one liter of LB medium supplemented with yeast extract was inoculated with Salmonella typhimurium and allowed to grow at 37 o C for overnight. The culture was spun at 10,000 rpm for 8 minutes at room temperature. Thereafter the supernatant was discarded and the pellet was resuspended in 80 ml of GTE buffer and vortexed gently. To that, 16 ml of 10% SDS and 600 µl of proteinase K (20 mg/ml) was added to the cell suspension and mixed thoroughly and incubated at 37oC for one hour. Then 40 ml of 5 M NaCl and 32 ml of CTAB (10% in 0.7 M Nacl) was added and mixed thoroughly and incubated at 65oC for 10 min. Equal volume of phenol : chloroform : isoamyl alcohol was added and mixed thoroughly and centrifuged at 10,000 rpm for 20 min at 4oC. A thick white layer of white proteins and lipopolysaccharides was formed at the interface. The aqueous layer was carefully transferred to a fresh 50 ml centrifuge tube and 0.1 volume of 3 M sodium acetate (pH 6.0) and 0.6 volume of isopropyl alcohol was added and mixed thoroughly. Chromosomal DNA appeared in the solution as small thread which was centrifuged at 10,000 rpm for 10 min at 4oC. The DNA pellet was washed with 5 ml of 70% ethanol at 10,000 rpm for 10 min at 4oC. The pellet was air dried and dissolved in 100 µl of nuclease free water. 2.5 Immunization of mice A total of 40 Swiss albino mice (4-6 weeks old) of either sex were randomly distributed into four groups and caged separately. All the groups were immunized on day 0, 14 and 21 as shown in the (Table 1) below. The Brucella abortus S19 was given as a single dose. 2.6. Collection of serum Six randomly selected mice /group were bled at weekly interval and same mice were followed up to 28th day. The blood was collected from retro orbital plexus using capillary tubes in sterile microfuge tube and allowed to clot at room temperature for 2 hrs. The blood was kept at 4oC for overnight for retraction. Finally, the serum was collected and centrifuged at 4,000 rpm for 10 min at 4°C to remove residual blood cells. The serum was collected in the fresh tubes and kept at -20oC until further use. 2.7. Isotype ELISA The presence of rOMP antigen specific total IgG and isotypes IgG1, IgG2a and IgG2b antibodies was measured in the serum samples collected from the immunized and control mice at different time intervals, by indirect ELISA. Briefly, 96 well ELISA plate (Nunc-Immunoplate with Maxisorp Surface) was coated overnight with 100 µl of purified rOMP antigen (2.5 µg/ml) in carbonate-bicarbonate buffer (0.05M, pH 9.6) at 4oC. The plates were washed thrice with PBS-T (phosphatebuffered saline containing 0.05% Tween-20). Blocking was done with 1% BSA in PBS-T and incubated for 2 h at 37 oC.

Table1: Immunization groups inoculated with different inoculums

Groups 1 2 3 4

Inoculum PBS rOMP 28 CpG + rOMP28 Brucella abortus strain 19 (Vaccine Strain)

Dose/mouse 0.1ml 50µg 50µg + 50µg 5 1x10 CFU

Route of administration S/C S/C S/C I/P 400


Plates were washed thrice with PBS-T and then 100 µl of diluted (1:400) test sera samples was added to duplicate wells and incubated at 37oC for 90 min. After three washing, the plates were further incubated with 1:10,000 dilutions of goat anti-mouse IgG, IgG1, IgG2a and IgG2b HRPO conjugates (100 µl/well) at 37 oC for 1 h. After the three washing steps, 100 µl of freshly prepared substrate solution (6mgOPD, 4µl 30% H 2O2/10 ml of substrate solution) was added to each well. The plates were incubated for 15 min in the dark and the reaction was stopped by addition of 50 µl of 1 M H2SO4 per well. Absorbance was measured at 492 nm in an ELISA reader. 2.8. Lymphocyte proliferation assay Spleen from immunized mice were taken after 28 days of first inoculation after sacrifice, splenocytes were collected by repeated perfusion with sterile cold PBS using insulin syringe. The splenocytes were isolated using histopaque. To 1.5 ml of histopaque 3 ml of splenocyte suspension was over layered and centrifuged at 1600 rpm for 40 min. The interface containing lymphocytes was collected in a fresh tube and two washings were done with sterile PBS and finally resuspended in RPMI 1640 growth medium containing antibiotics. The blastogenic response of splenocytes was assessed by MTT colorimetric method, 100 µl of cell suspension (2x106cells/ml) from each animal was added to a sterile 96-well flat bottom tissue culture plate (Griener) separately in triplicates. Then 100µl of RPMI-1640 growth medium containing either Con A (for positive control) @ 20 µg/ml or antigen (rOMP 28) @ 2µg/ ml was added in triplicate wells. For negative control, 100 µl of growth medium without rOMP 28 or Con A was added in triplicate wells. The plate was sealed properly with adhesive tape and incubated at 37°C in a humidified chamber with 5% CO2. After 96 hrs, 20 µl of MTT solution (5 mg/ml in PBS) was added to each well and further incubated at 37°C for 4 hrs. The plates were then centrifuged at 1,500 rpm for 10 min and 100 µl of culture supernatant was discarded from each well. Finally, 150 µl of dimethyl sulfoxide (DMSO) was added to each well and mixed thoroughly avoiding air bubbles. The absorbances of the stimulated and unstimulated cells were measured at 550nm to calculate the stimulation indices. Stimulation index (SI) was calculated using the following formula: S I(MTT) =

Mean OD of stimulated culture Mean OD of unstimulated culture

2.9 Nitric oxide production assay Splenocytes from the immunized groups were isolated as mentioned in the lymphocyte proliferation assay. In this assay the RPMI 1640 medium was supplemented with 5 mM L- arginine. One ml of cell suspension containing 1x106 cells was plated in triplicate in 96 well plates. Cells were activated rOMP28 @10 µg/ml. The plates were incubated at 37°C and 5% CO2 for 48 hrs. Culture supernatants were collected from all the wells at 24, 30 and 40 hrs intervals. The collected supernatants were stored at -20°C until NO estimation. For NO estimation, different concentration of NaNO2 (sodium nitrite) was used for preparing standard curve. In a 96 well ELISA plate to 50µl of the cell culture supernatant or standard, 50 µl of

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Griess reagent were added and incubated at 37°C for 30 min. Absorbance was measured at 550 nm. NO level in the sample was extrapolated using the standard curve (NaNO 2 concentration vs O.D at 550 nm) 2.10. Quantitative determination of cytokines by sandwich ELISA Splenocytes from the immunized groups were isolated as mentioned in the lymphocyte proliferation assay. 100 µl of cell suspension (2 x10 6cells/ml) was added to 96-well flat bottom tissue culture plate (Greiner) Then 100 µl of RPMI1640 growth medium containing antigen (rOMP 28) @ 2 µg / ml was added in all the wells. Plate was sealed properly with adhesive tape and incubated at 37°C in a humidified chamber with 5% CO2 for 48 hrs. The supernatant from all the groups were collected in sterile microfuge tubes for cytokine assay. Quantitation of murine IFN-γ, IL-2, and IL-4 was done by sandwich ELISA kit (Cytolab/Pepro Tech Inc.), as per manual instructions. Briefly, capture antibody of the three cytokines were diluted with PBS to a concentration of 1 µg/ml for IFN-γ and IL4 and 2µg/ml for IL-2. Immediately, 100 µl of diluted capture antibody was added to each well, plates were incubated overnight at room temperature. Then the plates were washed 4 times with PBS-T (PBSTween 0.05%), and blocked with 300 µl of 1% BSA in PBS and incubated for 2 h at room temperature. The plates were washed thrice. The two fold serial dilutions of the standard recombinant cytokine were made ranging from 2 µg /ml to zero in 0.1% BSA in PBST for IL-4 and IFN-γ and 3µg/ ml to zero for IL-2. 100µl of standard/sample (1:10 diluted) was added per well and incubated at room temperature for 2 hrs. The plates were washed and detection antibody (biotinylated anti-cytokine specific antibody) was diluted to a concentration of 0.5µg/ml for IL-4 and 0.25µg/ml for IL-2 and IFN-ã detection. Diluted detection antibody was added (100 µl/ well ) and plates were incubated at room temperature for 2 h. Thereafter, the plates were washed 4 times and 100 µl of avidin peroxidise conjugate was added per well in 1:1200 dilutions. The plates were incubated for 30 min at room temperature and then washed 4 times. 100µl of OPD substrate was added to each well and plates were incubated at room temperature for 15 min for colour development. Colour development was monitored with an ELISA reader at 405 nm. 2.11. Challenge infection All the groups of mice were challenged with 1 x 105 colony forming units (cfu) of Brucella abortus, strain 544, intraperitonially in 200 µl of sterile PBS, 45 days after primary immunization. Four weeks after challenge, three mice from each group were sacrificed for splenic clearance assay. Spleen from each mouse was removed, homogenized in 2 ml of TPB (tryptose phosphate broth). A tenfold serial dilution was made in TPB and 20 µl suspension from each dilution was plated on tryptose phosphate agar. B. abortus 544 colonies were counted after incubation for 72-96 h at 37 o C under 5% CO 2. The difference of log organism burdens in spleen of PBS control (unimmunized) mice and the vaccinated groups was considered as protection in terms of splenic clearance. 2.12. Statistical analysis The results of ELISA and lymphocyte proliferation assay were expressed as mean ± SE. The amount of cytokine 401


produced was determined by regression curve analysis. In protection studies, log values of total no. of Brucella per spleen of mice were calculated and protection was assessed by subtracting the log colony forming unit of immunized mice from those of PBS control. One way analysis of variance was used to test the statistical significance by SPSS 11.0. P value of less than 0.01 was considered statistically significant. Results 3.1. Purification of recombinant outer membrane protein 28(rOMP28) of Brucella melitensis The fractions collected from the Ni NTA affinity chromatography column (Qiagen, USA) was checked in 12.5% SDS PAGE and 28 kDa rOMP in different elutes could be noticed. The elutes containing high concentration of rOMP 28 were pooled and dialyzed (Fig.1) and used in the immunization protocols. 3.2. Isotypic ELISA The serum samples collected from the different group of animals on day 7, 14, 21 and 28 post immunizations was evaluated for the humoral immune responses. Anti rOMP 28 specific IgG isotypes were measured by indirect ELISA. The level of the different subclasses of antibodies in the test serum at dilution of 1:200 was expressed as absorbance (A492) of the colour complex developed in the assay (indirect ELISA). 3.2.1. Total IgG response Total IgG response against OMP 28 was found to be highly significant within groups (P95%, respectively in the different region while the lower percentage of milk adulteration with water was observed by Tasci (2011) as 30%. The adulteration of milk with water may be attributed to increased demand and short supply of milk in market which is directly linked and the physical nature of milk i.e. aqueous and opaque, which enhances the incidence by attracting unsocial persons to increase the bulk and make the profit. According to present study the urea adulterated milk samples were observed as 4% (Table 2), out of which 0% was rural samples and 8% were urban samples (Table 3). The higher percentage of milk adulteration with urea was reported by Lateef et al. (2009) as 86.66% in the different area. The urea might be added as a thickening agent to increase the solid not fat content of synthetic milk and to maintain the viscosity. During the present evaluation, it was observed that adulteration of milk with starch was recorded as 12% (Table 2), out of which 8% were rural samples and 16% were urban samples (Table 3). Earlier Ahmad (2009) reported a higher percentage of milk adulteration with starch as 35.50% in different region. Addition of starch may be attributed to increase the thickness of adulterated milk and to maintain the viscosity. The adulteration of milk with neutralizers was found as 18% (Table 2), out of which 8% were rural samples and 28% were urban samples (Table 3). It might be added to nullify the developed acidity in the milk. The adulteration of milk with detergent/shampoo/ washing powder was recorded as 16% (Table 2), out of which 4% were rural samples and 28% were urban samples (Table 3). The adulterant like detergent seems to be used to enhance the cosmetic nature of milk. When water is added in milk its foamy appearance diminishes, so to give milk a foamy appearance artificially, detergents are added in it (Afzal et al., 2011). The present analysis indicates adulteration of milk with sodium chloride/salt as 32% (Table 2), out of which 28% were rural samples and 36% were urban samples (Table 3). The presence of sodium chloride/salt in milk may be attributed to either addition of mastitis milk; Cl- content increases in case of mastitis or may be added as a thickening agent to maintain the physical state and density of milk. The increase in Cl- content (mastitis milk) can be overcome by use of organic practices for improvement of udder health (Mir et al., 2013). The present study showed adulteration of milk with skim milk powder as 22% (Table 2), out of which 12% were rural samples and 32% were urban samples (Table 3). Lower rate of adulteration with milk powder was reported by Tasci (2011) as 4% in different region. Addition of milk powder may be attributed to increase the thickness of adulterated milk and to maintain the viscosity. According to present study cane sugar/sucrose and glucose adulterated milk samples were observed as 20% and 4%,

Table 1: Showing area wise conformity of milk samples analysed in Malwa region

Area Rural Urban Total

Total number of sample analysed 25 25 50

Sample similar to legal standard 17 (68%) 7 (28%) 24 (48%)

Sample non-similar to legal standard 8 (32%) 18 (72%) 26 (52%) 537


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respectively (Table 2). Among cane sugar adulterated samples, 8% were from rural areas and 32% were from urban areas, the figure for glucose adulterated milk samples were found as 0% from rural areas and 8% from urban localities (Table 3). Earlier studies conducted by Lateef et al. (2009) reported a reasonably higher percentage of milk adulteration with sugar as 93.33% in different region. The sweeteners might be added to increase the solid not fat content of milk, which was decreased by dilution of milk with water. The present investigation indicates adulteration of milk with calcium carbonate/ chalk as 0% (Table 2), the chalk adulterated milk sample was not found in both of the studied area. Although it might be used as adulterant to maintain the colour and viscosity. During the present study formalin and hydrogen peroxide polluted milk samples were observed as 2% and 6%, respectively (Table 2). Among formalin polluted milk samples, 0% was from rural areas and 4% were from urban areas, the figure for hydrogen peroxide polluted milk samples were found as 0% from rural areas and 12% from urban localities (Table 3). Earlier studies conducted by Lateef et al. (2009) reported a reasonably higher percentage of milk adulteration with formalin and hydrogen peroxide as 46.66% and 13.33%, respectively in different regions. The preservatives like formalin, hydrogen peroxide might be added to increase the shelf life of milk (Tipu et al., 2007). Further hydrogen peroxide preservative of milk is usually used in summer season when environmental temperature is very high. This unethical activity is usually adapted to prevent the financial losses occurred due to the spoilage of milk during its transportation and sale (Naz, 2000). Milk adulteration and their public health significance Adding unhygienic water to pure milk can cause bacterial food poisoning syndrome, gastrointestinal complications and other waterborne illnesses in human. Urea adulteration in milk may causes pain in lower abdomen, irregular heartbeat, muscle cramps, numbness and weakness in hands and feet, chills and shivering fever. Urea also causes increase in bleeding from uterus. Appearance of unnecessary hairs on face especially of women and children (Baumgartner et al., 2005). Urea adulterated milk is very harmful to the girls as it hastens up the process of puberty (Tariq, 2001). It can affect the human body’s immune system and cause severe stomach complications (Kharat and Arak, 2013). High amounts of starch addition in milk may cause diarrhoea due to the effects of undigested starch in colon. Its accumulation in the body may

prove very fatal for the diabetic patients (Afzal et al., 2011). Addition of carbonates and bicarbonates as neutralizers in milk can potentially disrupt hormones signals in body that regulates development and reproduction process of man (Rideout et al., 2008). Adulteration of milk with detergents can cause breast cancer in women. They also decrease the sperm production from testicles (Ali et al., 2005). Salts in adulterated milk can causes serious illnesses like heart disease, problems of kidney, raised blood pressure, gastro-intestinal disturbances and allergies in human being. Sugars, dry skimmed milk powder and few other chemicals have serious health hazards on human body (Kharat and Arak, 2013). Addition of formalin in milk may cause vomiting, diarrhoea and abdominal pain in human. Larger doses may cause decreased body temp, shallow respiration, weak irregular pulse and unconscious. It also affects the optic nerve and cause blindness. It is one of the potent carcinogens (Gwin et al., 2009). Hydrogen peroxide used as milk preservative can damages the stomach cells, which can lead to gastritis and inflammation of the intestine and bloody diarrhoea in man (Murthy et al., 1981). Adulterated milk is a slow poison and children can get poisoned by taking such milk (Kharat and Arak, 2013). This adulterated milk increased the number of patients with kidney stones (Gale and Hu, 2007). Further this synthetic milk causes serious harms on human body like swelling in the eyes and complications of liver. It proves deadly for pregnant women and patients suffering from heart problems. Increasing number of human population is suffering from incurable diseases and research indicates that impure food is major reason responsible for these diseases. References Afzal, A. et al. (2011) Pak. J. Nutri. 10(12): 1195-1202. Ahmad, A. (2009) Pak. J. Nutri. 8: 439-440. Ali, N. et al. (2005) Corpse preservatives being used in unpacked milk. Daily Times, 4th Dec., 2005. Bansal, P. and Bansal N. (1997) Current Science. 73(11): 904. Baumgartner, M. et al. (2005) Acta Vet. Hung. 50: 263-271. Chand, S. et al. (2013) Vet. Pract. 14 (1): 23-25. Food Safety and Standards Authority of India, New Delhi (2012). Executive summary on national survey on milk adulteration, Prevention of food adulteration cases (FAC) including Food Safety & Standard act, rules, regulations cases 1972 to 2012 (2) (80 bound volumes). Gale, F. and Hu D. (2007). Supply Chain Issues in China’s milk adulteration incident. Economic Research Service U. S.

Table 2: Showing data regarding over all percentage of common adulterants found in milk of Malwa region

S.N.

Parameter / Adulterants

Water (Pond)/ Nitrate 1. . Urea 2. Starch 3. Neutralizers 4. Detergent / Shampoo 5. Sodium Chloride / salt 6. Skim milk powder 7. Cane sugar/ Sucrose 8. Glucose 9. Calcium Carbonate/chalk 10. Formalin 11. Hydrogen peroxide

Total number of sample tested 50 50 50 50 50 50 50 50 50 50 50 50

Total number of sample positive 26 2 6 9 8 16 11 10 2 0 1 3

Over all percentage of positive samples 52% 4% 12% 18% 16% 32% 22% 20% 4% 0% 2% 6% 538


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Table 3: Showing comparative data regarding proportion of common adulterants found in milk of rural and urban areas of Malwa region S.N.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Parameter / Adulterants Water (Pond)/ Nitrate Urea Starch Neutralizers Detergent / Shampoo Sodium Chloride/salt Skim milk powder Cane sugar/ Sucrose Glucose Calcium Carbonate/chalk Formalin Hydrogen peroxide

Positive samples in rural areas Number Frequency Percentage 8 8/25 32

Positive samples in urban areas Number Frequency Percentage 18 18/25 72

0 2 2 1

0/25 2/25 2/25 1/25

0 8 8 4

2 4 7 7

2/25 4/25 7/25 7/25

8 16 28 28

7

7/25

28

9

9/25

36

3

3/25

12

8

8/25

32

2

2/25

8

8

8/25

32

0 0

0/25 0/25

0 0

2 0

2/25 0/25

8 0

0 0

0/25 0/25

0 0

1 3

1/25 3/25

4 12

Fig. 1: Showing comparison of common milk adulterants found in rural and urban areas Department of Agriculture Washington, DC. Gupta, V.K. et al. (2012). Laboratory manual on Milk and Meat Hygiene, Food safety and Public Health. 1st ed., Deptt. of Vet. Public Hlth., M.P.P.C.V.V., C.V.Sc. & A.H., Mhow-453446, India. Pp. 18-19. Gwin, M.C. et al. (2009) Envrn. Hlth Perspect. 118: 313-317. Hundal, J.S. et al. (2013) Vet. Pract. 14 (2): 394-397. Kharat, G.T, and Arak V.D. (2013) Science J. 12 (1): 34-35. Kumar, H. et al. (2000) Indian J. of Chem. Tech. 7: 146-147. Lakshmi, V. (2012) Indian J. Sci. Invent. Today 1(2): 106-113. Lateef, M. et al. (2009) Pak. J. Zool. 9: 139-142. Mir, A. Q. et al. (2013) Vet. Pract. 14 (2): 308-310. Murthy, M.R. et al. (1981) J. Mol. Biol. 152: 465-99. Naz, W. (2000). Subject: The dairy sector. http://www.

Pakistaneconomist.com. Accessed Feb. 2011. Rideout, T.C. et al. (2008) Br. J. Nutr. 99: 984-92. Tariq, M.A. (2001) Subject: A close look at dietary patterns. http:// www.dawn.com/2001/ 11/05/ebr13. htm. Accessed Feb, 2011. Tasci, F. (2011) J. Vet. Adva. 10: 635-641. Tipu, M.S. et al. (2007) Monitoring of chemical adulterants and hygienic status of market milk. Hand book pub. by Quality control Lab., Uni. of Vet. and Ani. Sci., Lahore, Pakistan, pp: 7. Varley, H. (1969) Practical Clinical Biochem. 4th Ed., C.B.S. publishers, Delhi, India. Venkatasubramanian, V. et al. (2012) Vet. Pract. 13 (1): 121-125.

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December 2013

OPTIMIZATION OF THE BASIC FORMULATION AND PROCESSING CONDITIONS FOR THE PREPARATION OF CHICKEN PATTIES FROM MEAT OF SPENT HENS S. Gupta1, A. Kumar2, H.R. Hakeem1 and S. Shafat1 Division of LPT, F.V.Sc. & A.H., SKUAST-J, R.S. Pura-181102, Jammu and Kashmir, India ABSTRACT This study was aimed at the utilization of spent hen meat into convenience meat product viz chicken patties. Basic formulation and processing conditions were optimized for the preparation of chicken patties from spent hen meat. Incorporation of 9% refined vegetable oil and 4% refined wheat flour as binder were found to be optimum for chicken patties cooked to an internal temperature of 80±20C for a time of 25 minutes in hot air oven method of cooking. The chicken patties from meat of spent hens could be conveniently packed in LDPE for a period of 21 days in refrigerated (4±10C) condition without any marked loss of physico-chemical, microbial and sensory quality. Therefore, comparatively low cost chicken patties with good to very good acceptability were developed utilizing spent hen meat, which is otherwise known for its toughness and poor edible quality meat. Key words: Sensory attributes, chicken patties, oven cooking and spent hen meat

Introduction Poultry meat is the fastest growing component of global meat industry. However, due to the phenomenal expansion of poultry farming in India, availability of culled and spent hens has increased immensely and these culled stocks at the end of their active reproductive life are considered as poor meat or secondary grade meat because of more toughness and less juiciness attributed to high collagen content (Abe et al., 1996) and high degree of cross linkages Bailey (1984) in comparison to broilers. Thus the proper disposal of the layer stock at the end of their production is a real problem for the poultry farmer. Development of further processed convenient products are considered a potential solution to these concurrent problems which may show a promising outlet for such tough meats and is an ideal way of profitable disposal of spent hens whose demand is on decline due to increased availability of broilers. Thus, low cost of the spent hen’s can be advantageously used to stretch the access of these products or increasing the margins of the processed meat industry. Therefore, present study was aimed towards optimization of the basic formulation and processing conditions for the preparation of chicken patties from meat of spent hens. Materials and Methods Chicken meat: Spent layer hens of the age group of over 72 weeks were slaughtered by humane method. The lean meat was packed and kept at -18±2 0C until use. Methodology of preparation of nuggets from meat of spent hen: Lean meat was cut into smaller chunks and minced in a Sirman mincer with 6 mm plate. Meat emulsion for patties was prepared in Sirman Bowl Chopper as formulation having lean meat-68.7%, ice flakes-10.0%, vegetable oil- 8.0%, condiment mixture- 5.0%, refined wheat flour- 4.0%, spice mix2.0%, table salt-1.5%, monosodium glutamate-0.5%, sodium tripolyphosphate-0.3% and sodium nitrite-120 ppm (Ranjan, 2004). An accurately weighed (65 g) of meat emulsion, was spread accurately in the circular ring with moistened palm and fingers to shape it as patty. Steel plates with raw circular patties

on them were placed longitudinally in oven. The molded raw patties were placed on cooking trays and cooked in a preheated hot air oven at 180 oC for 25 minutes. After 15 minutes the patties were turned upside down and re-turned after 5 minutes. Following analytical techniques were followed to analyze various quality of the developed product. Sensory evaluation was determined by method of (Seman et al., 1987). Statistical analysis was as per Snedecor and Cochran (1989). Results and Discussion A series of experiments were conducted to standardize the formulation and processing of patties from meat of spent hens. The mean values for various sensory parameters are presented in Table 1. Standardization of level of added refined vegetable oil A slight improvement in scores for flavour, juiciness and texture was registered with an increase in added fat level. Overall acceptability of the product increased with increase in level of incorporation of refined vegetable oil. The sensory score at 8% level was significantly lower (P