Antioxidant and antimicrobial effects of condiments paste used as nitrite replacer in chicken mince

Aim: Materials and Methods:


Introduction
Nitrite is one of the food additives, which is widely used currently in the processed meat manufacturing.Nitrite is one of the important ingredients in curing of the meat products along with salt, sugar, ascorbate and polyphosphate.Nitrite contributes for the cured flavor and cured color due to the formation of nitric oxide myochrome [(NO) MC] in the cooked meat products and it also inhibits growth of several undesirable bacteria such as [1].However, the guidelines for the healthy nutrition published by the World Cancer Research Fund suggested to limit the intake of the meat and especially to avoid the processed meat because Nitrite forms N-nitrosamine compounds with the secondary amines in the acidic environment of the stomach, which are known to be potent carcinogens.It also causes conversion of hemoglobin to methmyoglobin which lacks oxygen transporting ability [2].
Several approaches have been tried to replace the nitrite in the meat products.They include the use of betalein, bacteria [3], citrus co products [4] and tea catechins [5] etc. However; nitrite is still widely used in the processed meats because of its potent antibotulinal action which enhances the food safety.Various phytochemicals obtained by the spices offer a promising alternative to the chemical additives for ensuring the meat safety.Inhibitory activity of the spices and their derivatives on the growth of the bacteria, yeasts, fungi and microbial toxin synthesis has been previously reported [6,7,8].Hence, they can be used for meat preservation as main or adjuvant antimicrobial compounds to ensure the production of microbiologically stable foods.Turmeric is one of the spices which is widely used in Indian culinary practices.Curcumin and hydroxyl curcumin are alkaloids present in turmeric which possesses antioxidant, antimicrobial and therapeutic properties [9].Recently, Sharma [10] reported that the turmeric when used in raw chicken mince was effective against and exhibited potent antioxidant properties 2

Staphylococcus aureus
The present study was conducted to evaluate the antioxidant and antimicrobial effects of 4% Ginger + 4% Garlic + Turmeric 1000ppm (GGT), nitrite 200ppm (N) and the control (C) on minced chicken stored at 4±1 C. Physico-chemical properties (pH, Thio Barbituric acid value (TBA), Peroxide value (PV) and Free Fatty acid (FFA) were evaluated on 0, 3, 6 and 9 day of the storage.The antimicrobial studies Total viable count (TVC), count count and count were carried out on 1, 4, 7 and 10 day of storage.
The Ginger garlic turmeric paste has an excellent potential to replace the nitrite as a natural antioxidant with other advantages.It is highly effective against It is also significantly effective against and TVC as compared to C but lesser than N. We recommend further research by replacing the nitrite with various combinations of the spices and the condiments.ginger, garlic, turmeric, nitrite.comparable or superior to the nitrite and ascorbic acid.
Huhtanen [11] reported that oleoresin content of the few spices including the turmeric was highly effective against in pure culture by disc diffusion method.Garlic is also used as a seasoning agent or condiment in various meat products.Garlic yields allicin on chopping which is a powerful antibiotic and also an anti-fungal compound (phytoncide).Ginger is also an integral part of Indian cooking methods both for vegetarian as well as for non-vegetarian products.Its active ingredients are gingerol and hexahydrocurcumin which prevent the oxidation of oils and fats [12].
Hence, the present investigation was carried out to compare the antioxidative and antimicrobial properties of the ginger garlic turmeric paste with the nitrite in chicken mince.
Permission was obtained from the University Ethics Committee, GABPUA&T, Pantnagar, Uttarakhand.
Live birds were procured from Instructional Poultry Farm, Nagla, Govind Ballabh Pant University ofAgriculture and Technology, Pantnagar and were brought to the Department of Livestock Products Technology.Slaughtering and dressing were carried out following the standard and humane procedures.The carcasses were kept in refrigerator at 4±1 C for 24 hours.The chilled carcasses were deboned on next day and lean meat was then stored at -20 C till further use.Turmeric powder of "Agmark" grade was purchased from the local market for use in the experiments.It contained 2.5% oleoresin content as determined by the ether extraction method.All the chemicals used in the study were of analytical grade and were procured from Hi Media laboratories (P) Ltd, Mumbai.The culture media used in the study were procured from Hi Media Laboratories (P) Ltd, Mumbai and Tulip Diagnostics (P) Ltd, Goa, India.The low density Polyethylene (LDPE) bags were obtained from the local market and sterilized by exposing to UV light for 30 minutes before use.
The following standard pure cultures (MTCC-1349), (MTCC-450) and (MTCC-1687) were procured from Microbial Type Culture Collection Centre, Institute of Microbial Technology, Chandigarh, India and were used in the study.These cultures were revived under the aseptic conditions as per the instructions.
Approximately 1.2 kg of boneless chicken was defrosted before use.The deboned chicken was minced by passing twice through 9mm and 4mm plates of presterilized meat mincer (Sirman® TC-32, Italy).Then minced meat was divided into approximately three equal portions and assigned as the following treatments: control (C), turmeric @1000ppm added along with 4%ginger and 4%garlic (GGT) and sodium nitrite added @ 200ppm (N).
The turmeric (1000ppm to chicken mince) was weighed accurately and dissolved in water (10% by weight of chicken mince) and then incorporated in the chicken mince thoroughly by kneading or massaging for uniform distribution.For the heated turmeric, 1000ppm of turmeric was calculated, accurately weighed and then dissolved in water (10% by weight of chicken mince) as above and then boiled for 15 minutes on a hot plate.The rate of heating was kept uniform between the replications.Each treatment (C, T and GGT) portion was further sub divided into two parts of approximately 300g; one part for studying the physicochemical properties and the second part for various microbiological studies.
The portion of minced chicken intended for the microbiological studies was further subdivided into four parts, accurately into 100 g portions; for TVC, , a n d count.
The broth containing known concentrations of and stored in refrigerator were serially diluted separately with normal saline solution to approximately 107 bacterial colonies per ml.1ml of each of the inoculums was added to 100 gram of the minced chicken (stored in sterilized LDPE bags) separately and then thoroughly mixed by the kneading so that a final concentration of approximately 105 CFU/gram of the meat mince could be obtained.The concentrations of bacterial cultures were kept deliberately high.
The LDPE bags containing inoculated chicken mince were sealed with the help of a sealer (Singhal®, HSP-200, India).The minced portions assigned for the antioxidant and physicochemical properties, were also sealed in LDPE bags and both were stored at refrigeration temperature (4±1 C) untill further analysis.A total of 6 replications were carried out with each analysis done in duplicate.
The present investigation was carried out to compare various antioxidant and antimicrobial properties of ginger garlic turmeric paste as a nitrite The physico-chemical properties were evaluated on 0, 3, 6, and 9th day of storage as per AOAC [13] where as antimicrobial studies were carried out on 1, 4, 7 and 10th day of storage as per APHA [14].The following abbreviations were used: Control-C; Turmeric 1000ppm + 4% Garlic + 4% Ginger-GGT and 200ppm Nitrite-N.

Results and Discussion
The data were analyzed using ANOVAtechnique by Snedecor and Cochran [15].

Statistical analysis:
replacer in chicken mince stored at 4±1 C upto 10 days of storage.
The analysis of variance indicated a highly significant difference (P<0.01) in pH between treatments and between storage periods (Table -1).However, the interaction between treatments and storage periods showed no significant difference (P>0.05).Addition of N did not have significant effect (P>0.05) on pH of chicken mince, where as GGT treated minced meat had significantly higher (P<0.01)mean pH values as compared to C and N. Mathew [16] found no significant difference in pH between control and 200ppm nitrite added buffalo meat chunks stored at 4±1 C for 20 days.
Reddy [17] also found significant increase (P<0.01) in pH of chicken meat balls with 6% ginger during storage at -20±2 C for 60 days.Krishnan [18] observed higher pH and enhanced shelf life in raw chicken meat with incorporation of spice mix under refrigeration storage.Ginger extract (2%) treated microwave oven cooked chevon patties had significantly higher pH than control samples [19].Therefore, it is highly possible that the ginger and garlic present in GGT treated mince samples might have increased the pH and not the T. Yumin [20] found increased pH values of stewed pork prepared with incorporation of ginger, onion and garlic under refrigerated storage.Overall, storage mean pH of chicken mince increased significantly (P<0.01) with the progression of the storage period.Kumar and Sharma [21] attributed the increase in pH during storage of low fat pork patties to proteolysis due to bacterial growth.Vijaykumar and Biswas [22] reported a significant increase in pH of enrobed duck cutlets stored at refrigeration temperature for 21 days.
The analysis of variance indicated highly significant difference (P<0.01) between treatments, between storage periods as well as for the interaction between treatments and storage period (Table -1).The overall mean TBA value for GGT and N were significantly lower (P<0.01)as compared to C.
Han [23] recorded that 100ppm nitrite treated ground pork samples stored at 5 C showed higher antioxidative effect than control.The antioxidant effect of the nitrite in cured meat is due to the formation of strong complex with haem Fe which in turn prevents the release of non haem Fe and helps in delaying of lipid oxidation [24].Curcuminoids in turmeric scavenge free radicals at the cost of becoming free radicals themselves.These second hand free radicals are unreactive products and are short lived, so the lipid oxidation reaction is terminated.Reema [19] evaluated the antioxidant properties of ginger and found that 2% ginger extract treated microwave oven cooked chevon patties had significantly lower TBA values than control samples when stored at 0-2 C for 28 days.Oyas [25] also reported that ethyl acetate extract of spices like ginger, garlic, turmeric and cinnamon have got profound antibacterial and antioxidant effect in fish meat preservation.The overall mean TBA values were significantly (P<0.01)increased during the refrigeration storage in all the treatments.Nayak and Tanwar [26] attributed the increase in the TBA values with the advancement of storage period which was due to increased lipid oxidation and the production of volatile metabolites in the presence of oxygen.
The overall mean PV for N and GGT were significantly lower (P<0.01)than C. Highly significant difference (P<0.01) was found in between GGT and N treated chicken mince (Table -1).It may be because of composition of the nitrite where one part of nitrogen is present in combination with two parts of oxygen.Chan [27] found that turmeric extract was an effective antioxidant in chicken mince stored at refrigerated temperature.Sreejayan [9] reported that curcumin is a potent inhibitor of lipid peroxidation catalyzed by Fe and its chelates in rat brain homogenate and rat liver microsomes.Dzudie [28] evaluated the antioxidant properties of ginger and found that the beef patties containing 0.2% level of ginger essential oils showed the best characteristics in relation to lipid oxidation as compared to ground oil and animal fat.
The analysis of variance indicated highly significant difference in FFA value between treatments, between storage periods and for the interaction between treatments and storage periods (Table -1).The overall mean FFA value for N and GGT were significantly lower than C. GGT had significantly lower overall mean FFA value than N. Mathew [16] observed that nitrite treated buffalo meat chunks had significantly lower FFA values than control and were acceptable up to 20 days when stored at refrigeration temperature.
Chan [27] observed that turmeric had significantly higher antioxidant effect in minced poultry meat as compared to control during storage at 3 C for 9 days.El-Ghorab [29] studied the antioxidant properties of cumin ginger and garlic and stated that exogenous addition of these natural spices in food reduced the lipid oxidation by reducing Fe+3 and free radicals scavenging activities.Isothiocynates present in ginger and garlic inactivated and modified several enzymes in meat such as 3-hydroxy-3-methyl glutaryl CoA reductase, glutathione-S-transferase and catalase, resulting in the prevention of lipid oxidation.Overall, storage mean FFA values were increased significantly during the whole storage period in all treatments.Kanatt [30] reported that lipolysis of meat during storage by the action of tissue enzymes and lipolytic enzymes, obtained from spoilage micro organisms led to the formation of FFA.
The overall mean TVC (log CFU/g) for N and GGT were significantly lower when compared to C. However, highly significant difference was observed between GGT and N (Table -2).The total viable count was found exceptionally high in C on 1st day due to less satisfactorily dressing and deboning procedure followed in the experiment.Ockerman [31] stated that Sodium nitrite inhibited growth of bacteria and delayed the microbial spoilage of cured meat products.
Khanna [6] reported that Curcumin and other curcuminoids inhibit the growth of various bacteria like    and in concentration varying from 1 in 20000 to 1 in 640000.Neogi [32] studied synergistic combination of various spices and reported that garlic and turmeric (70% ethanolic extract) were highly effective against and Sagufta [33] evaluated antimicrobial properties of different varieties of turmeric against and using agar well diffusion method and observed that the MIC values for different strains and varieties ranged from 3.0 to 20.6 mm in diameter. Sunilson [34] evaluated the antimicrobial properties of and and found that methanol extracts (100µg/ml) of these two had highly significant reduction in common food borne bacteria like and etc. Isothiocynates present in ginger and garlic can inactivate the extracellular enzymes through the oxidative cleavage of disulphide bonds necessary for survival of various bacteria [24].Overall, storage mean for TVC increased significantly on storage period for all treatments.
The analysis of variance indicated highly significant difference in count between treatments, between storage periods as well as for the interaction between treatments and storage periods (Table -2).Overall mean count (log CFU/g) were significantly lower for N and GGT when compared to C. However no significant difference was found between GGT and N. Nakajima [35] reported a significant reduction in count when added with nitrite as compared to control in chicken meat.Morita and Hiroshi [36] explained that the antibacterial activity of NO derived from sodium nitrite against 0157:H7 was due to the generation of dinitrosyl Fe complex from the reaction of Fe-S -protein (enzymes) with NO.Enzymes related to respiratory chain were inactivated resulting in decreased ATP levels in the cells.Neogi [8] reported that combination of garlic and turmeric (70% ethanolic extract) was synergistic and much effective against Sana and Ifra [37] evaluated antibacterial activity of aqueous and ethanol extracts of spices and reported that among the selected spices garlic had best inhibitory activity with a zone of 22mm against .Tajkarimi [38] observed anti microbial activity of garlic and turmeric against and averted herbs and spices have strong antimicrobial activity in various complex food systems.
The treatment mean of count for GGT and N were significantly lower when compared to C. N was found to be more effective than GGT (Table -2).The effectiveness of nitrite against was shown by Jaffery and Andrew [39] who stated that antibotulinal effect in thermally processed meat products takes place at two different stages in the life cycle of the microorganism.The first controlling effect of nitrite is the inhibition of vegetative cells emerging from surviving spores.The second controlling effect is preventing cell division in any vegetative cells In present study, GGT was found to be significantly more effective than C. Madhumita [40] found that paste of garlic turmeric and cinnamon with gum Arabic as binding agent enhanced the shelf life of minced meat when used as bio edible coating.Haiying [41] reported antimicrobial activity of various spices including turmeric rhizome, garlic and ginger rhizome against as a nitrite replacer.Storage mean of count increased significantly on all storage days.In the present investigation, was selected to study because it shares almost all the physiological properties with and produces more heat resistant spores than the later.The analysis of variance indicated highly significant difference in count between treatments, between storage periods and for the interaction between treatments and storage periods (Table -2).Treatment mean of count for GGT and N were significantly lower as compared to C. However, no significant difference was found between N and GGT.Mauricio [42] showed antimicrobial activity against on addition of sodium nitrite and erythrobate in ham.Storage mean of count increased significantly on all storage days.Zarringhalami [43] reported that nitrite could be partially replaced with annatto with better color values but antimicrobial properties of nitrite were better than annatto against .Goswami [44] observed that ginger garlic and turmeric paste and heat treated turmeric had comparable activity with nitrite against Hoi [45] reported that methanol extract of turmeric rhizomes had strong antibacterial activity against at a concentration of 5.0 mg/disc than control by disc diffusion method.Sunilson [33] evaluated the invitro antibacterial activity of and and observed that methanol extracts (100µg/ml) of these spices had highly significant reduction against We conclude from the present study that the ginger garlic and turmeric paste had better antioxidant properties than the nitrite in a complex food system like chicken mince.It can replace the nitrite as a strong natural antioxidant.It had very good antimicrobial count: count: count:

Escherichia coli Escherichia coli
Escherichia coli activity against the total viable count but the effect was lower than the nitrite.GGT had an antimicrobial effect equal to the nitrite against .GGT had a better antimicrobial effect against and the effect was slightly lower than the nitrite.However, the antimicrobial effect against was the same for both the nitrite and the GGT.These findings revealed that there is a great possibility for the replacement of the nitrite with the natural spices and the condiments like ginger, garlic, turmeric etc.However, we recommend further research work in this direction using new variations

W
Different superscripts between columns (A, B, C, D….) and between rows (W, X, Y, Z……) show significant (P<0.05)different in between storage and treatments respectively; Different superscripts (a, b, c, d…..) differ significantly (P<0.05)show interaction between different treatments and storage columns (A, B, C, D….) and between rows (W, X, Y, Z……) show significant (P<0.05)different in between storage and treatments respectively; Different superscripts (a, b, c, d…..) differ significantly (P<0.05)show interaction between different treatments and storage spices and condiments with well established antioxidant and antimicrobial properties.MG collected literature and prepared the initial version of manuscript.PPP and VKT helped for carried out research and revised manuscript for critical scientific corrections.All authors read and approved the final manuscript.The present research work is the part of MG's thesis work.Snedecor, G.W. and Cochran, G.W. (1989) Statistical methods, 8 edition.Iowa State University Press, Ames, Iowa.p88-92. th

Table 1 :
-Mean±S.E.values of various physico-chemical properties for different treatments and storage period

Table : - 2
Mean±S.E.values of various microbiological properties for different treatments and storage period