DETECTION AND DECONTAMINATION OF ANTIMICROBIAL DRUG RESIDUES IN MILK AND MEAT

Abstract

Antibacterial drugs have been therapeutic, prophylactic or nutritive applications in both veterinary and human medicine. In most of the livestock and poultry farms at different divisions of Sindh province of Pakistan, quite a wide variety of antibiotics are being frequently and discriminately used for prevention and treatment of infectious diseases. This might be a cause of residual contamination in animal based food (milk, beef, chicken meat etc.). Thus in present study preliminary the information regarding the use of different antimicrobial drugs at commercial dairy and poultry farms at the vicinity of Hyderabad, Mirpurkhas, Karachi, Sukkur and Larkana Divisions was gathered. Thirty three brand names of antimicrobial drugs with their 14 number of generic names were found main drugs, and among these oxytetracycline, gentamicin, penicillin G and sulfonamide were abundant in the treatment of livestock and poultry suffering from bacterial diseases or disorders throughout Sindh province. Further, the samples (n=400) each of milk, beef and chicken meat from four talukas of each above said divisions of Sindh were examined to detect and estimate the levels of antimicrobial drug residues. Over 1200 number of animal based food samples examined, 43.83% (526 numbers) samples noted with residual contamination, and among these the contamination in milk samples (49.75%; 199 numbers) was high and in beef samples (38.33%; 152 numbers) the low, while it was intermediate (43.67%; 175 numbers) in chicken meat samples. Over residual positive samples of animal based food, the highest number was observed in the vicinity of Karachi followed by Hyderabad, Sukkur, Mirpurkhas and Larkana. Milk samples (n=175) positive to antimicrobial drugs screened through Twin SensorBT KIT020, confirmed the presence of β-lactam and tetracycline residues. The distribution of antimicrobial drug residues in raw milk, beef and chicken meat samples (n=40 of each) confirmed through HPLC method, revealed high occurrence of penicillin G followed by oxytetracycline, sulphadimidine and gentamicin except for chicken meat where gentamicin was observed high than sulphadimidine. In milk samples the average residual level (μg kg-1) of antimicrobial drugs quantified high for penicillin G (738.43±207.08) followed by sulphadimidine (392.06±76.70), oxytetracycline (293.72±79.93) and gentamicin (196.33±34.58); in beef and chicken meat, the concentration of gentamicin (5423±2403.00 and 2119.30±645.01μg kg-1, respectively) was high followed by penicillin G, oxytetracycline and sulphadimidine (2316.20±485.23 and 1019.80±312.71, 681.90±185.02 and 772.80±162.10 and 425.97±158.83 and 623.55±247.44μg kg-1, respectively). The residual concentration of penicillin G in milk samples (100%) and the gentamicin in beef and chicken meat samples (100%) found above the maximum residue limit (MRL) recommended by Codex Alimentarius Commission, while, between 40 and ~85% samples noted with level above the MRL of oxytetracycline, gentamicin and sulphadimidine in milk, and of penicillin G, oxytetracycline and sulphadimidine in beef and chicken. Approach for decontamination of spiked antimicrobial drug residues from milk and meat through heat treatments (i.e. for milk: pasteurization at 72°C for 15sec and sterilization at 1100C for 10min; for meat: boiling 1100C for 20min and microwaving under full power 900W microwave oven for 3min) found applicable. In pasteurized milk, the residues of sulphadimidine was reduced markedly high (48.32±0.43%) followed by penicillin G (39.44±2.50%), gentamicin (25.35±1.61%) and oxytetracycline (15.19±0.40%), while in sterilized milk it was decreased remarkably more for oxytetracycline (89.96±0.15%) compared to that of sulphadimidine (75.69±0.19%), penicillin G (48.79±4.55%) and gentamicin (38.52±1.59%). The sterilization process had significantly (P<0.05) reduced more residues of penicillin G, oxytetracycline, gentamicin and sulphadimidine in milk contrast to pasteurization process. Through boiling of beef the residual reduction of sulphadimidine (96.73±0.17%), gentamicin (66.66±0.60%) and penicillin G (64.00±0.82%) was noted significantly high compared to that of through microwaving (73.46±0.23, 48.15±5.69, 30.17±0.99%, respectively) except for oxytetracycline (57.11±0.12 and 54.18±0.17%, respectively) where the difference existed non-significant. In chicken meat, residual reduction of penicillin G, gentamicin and sulphadimidine was noticeably (P<0.05) high through boiling (52.53±0.40, 76.62±0.40 and 72.36±0.29%, respectively) in contrast to that of microwaving (41.46±0.57, 33.83±0.64 and 58.60±0.60%, respectively), whereas for oxytetracycline it was observed significantly low through boiling (52.46±1.19%) compared to microwaving (86.62±0.16%). Marked effect of antimicrobial drug residues on the fermentability trend of milk was noted. The control milk base achieved required pH values (4.7) within 3hr of time, and compared to this, milk base spiked with low level of antimicrobial drugs (150μg kg-1) acquired desired pH values (4.7) up to 6hr of time, and with addition of gelatin to milk base it reduced to 5hr. Milk base spiked with antimicrobials at higher concentration (500μg kg-1) with or without gelatin achieved the required pH (4.7) within 7hr of time. The curd quality of yoghurt in term of syneresis significantly (P<0.05) affected more by residues of spiked gentamicin, and the less by sulphadimidine, the influence of penicillin G and oxytetracycline residues on curd quality appeared at intermediate level. The syneresis in yoghurt prepared from milk base spiked (150 and/or 500μg kg-1) with antimicrobial drugs was significantly (P<0.05) improved with addition of gelatin in milk base. After fermentation the residual reduction in oxytetracycline (75.16±0.46%) and sulphadimidine spiked (75.75±0.05%) milk base was relatively similar (P>0.05) but it was significantly (P<0.05) high compared to that of spiked gentamicin (30.61±1.33%) and penicillin G (24.06±0.24%) milk base. The residues of spiked penicillin G, oxytetracycline, gentamicin and sulphadimidine were approached to decontaminate from milk through refrigeration [between 4 – 80C of pasteurized (720C for 15sec) and sterilized (1100C for 10min) milk up to 30days], and from meat (each of beef and chicken) through freezing at -10 to -200C for 6days found satisfactory. At the end of refrigeration of sterilized milk, the reduction level of penicillin G (97.01±0.43%) and oxytetracycline (98.64±0.17%) recoded relatively similar (P>0.05) but it was significantly high (P<0.05) compared to that of sulphadimidine (80.53±0.50%) and gentamicin (61.95±1.02%) sterilized milk. After refrigeration of pasteurized milk, the reduction of oxytetracycline and sulphadimidine (76.06±0.0.49 and 76.21±1.16%, respectively) found statistically similar (P>0.05) but comparatively (P<0.05) high than that of penicillin G (58.46±0.72%) and gentamicin (37.22±0.64%). The reduction level of penicillin G, oxytetracycline, gentamicin and sulphadimidine in sterilized milk refrigerated (30days) was noted significantly high (P<0.05) from that of corresponding drugs in refrigerated pasteurized milk (30days). In beef, considerably (P<0.05) high reduction level of penicillin G (53.39±0.33%) was recorded followed by gentamicin (23.64±2.01%), oxytetracycline (20.17±0.46%) and sulphadimidine (15.27±0.24%) at 6thday of freezing. In chicken meat, the remarkable reduction of oxytetracycline (96.18±0.13%) was found followed by gentamicin (59.58±0.16%), penicillin G (45.21±1.99%) and sulphadimidine (26.99±0.02 %) at 6th day of freezing. In general, it could be concluded that the food of animal origin like milk, beef and chicken meat at Sindh province found at a high risk of contamination of antimicrobial drug residues, and residual level of penicillin G, oxytetracycline, gentamicin and sulphadimidine exceeds the MRLs. The sterilization process significantly reduced the spiked residues in contrast to pasteurization process of milk, and in meat, the boiling process contrast to microwaving. The spiked antimicrobial drugs had significant effect on fermentability and curd quality of milk, although considerable reduction of residues was also noted after fermentation. Process refrigeration had considerably reduced high residual level of spiked antimicrobial drugs in sterilized milk compared to that of in pasteurized milk followed by freezing in chicken meat and beef. Above applied approaches found better for reduction of antimicrobial drug residues in milk, beef and chicken meat, particularly for oxytetracycline and sulphadimidine which appeared within range of MRL.