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EFFECTS OF ON PRESERVATION AND METABOLIC ACTIVITIES OF AND MICROFLORA

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Oranusi Solomon Abah Kingsley Covenant University Ota Ogun State, Nigeria Covenant University Ota Ogun State, Nigeria

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The user has requested enhancement of the downloaded file. EFFECTS OF SALTS ON PRESERVATION AND METABOLIC ACTIVITIES OF FISH AND MEAT MICROFLORA

ORANUSI, 5.*, ABAH, K. A. AND ANOSIKE S.O. Department of Biological Sciences, Covenant University, Ota, Nigeria

*Corresponding Author; Email: [email protected]; +2348065299155

Abstract usually carry a mixed population ofmicroorganisms derived from both the natural microfloro of the plant or animo/ and those introduced during handling, processing, and storage. is a widely used additive and , which, influences in different concentrations. This study aims to determine the effect of salts on food preservation and metabolic activities of food microfloro. Two food samples (row fish and raw lean meat) were investigated. (NaCI), {KCI} and Calcium chloride {CoCI,) were grouped into varying concentrations of2, 2.5 and 4.5% respectively. The food samples were pulverized and salted according to these concentrations and on unsalted group served os the control. Storage was for fifteen days with samples analysis on days 0, 7 and 15. The first category of food sample was preserved under room temperature (28 ± 2 ·c), with the second and third under (4 t 2 •C) and freezer (-18 t 2 •C) temperature respectively. Total and Lipid Peroxidation were assayed during the duration ofstor age. Organisms isolated from the samples were identified based on biochemical and cultural characteristics to include Staphylococcus oureus, Escherichia coli, and species of Pseudomonas, Klebsiella, Enterobocter, Aspergillus, Moulds, and , with Bacillus spp mostly predominant. There was a decrease in microbial population along concentration and temperature gradient, the population, however, increosed.along the duration of storage. The toto/ protein decreased while lipid peroxidotion increased along the duration ofstorage. NaCI showed the highest efficiency in preservation, with KCI showing the least efficacy. Freezer temperature (-18 t 2 •C) and 4.5% salt concentrations were the most effective. Salts alone at concentrations acceptable in food cannot effectively contra/ on initio/ high load of natural and cantominont micraflora in row foods. Use of salts in synergy with ather / methods like refrigeration will drastically reduce the activities offood microflora whilst enhancing the shelf-life ofthese foods.

Keywords: Salts, food preservation, metabolic activities, lipid peroxidation, Microflora

Introduction foods (Hall, 1997). Foods are rarely sterile Food is essential for the growth and because these organisms are present in the survival of man but becomes harmful air surrounding the food, water, and soil through the presence and action of from where the food plants and animals spoilage organisms and foodborne are harvested (ICMSF, 1996). Foods usually (ICMSF, 1996). Microorganisms carry a mixed populatio n of are known to be ubiquitous in nature; they microorganisms derived from both the can be found almost everywhere including natural microflora of the original plant or

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animal and those introduced during a/., (2010), salt is one of the most preferably harvest/slaughter and subsequent handling, used addit ives in food industries because of processing, and storage (Hall, 1997). Quality its low cost and varied properties. Its unique deterioration caused by th ese properties lie in the preservative and microorganisms may include a wide range of effect as a direct consequence types of spoilage that are not desirable of the capacity of sodium chloride to reduce commercially, because they limit or values (Albarracin et a/., lead to quality complaints, but are safe from a 2010). Salt, which interacts with other public health point of view (ICMSF, 1996). The physicochemical parameters, have been presence or growth of infectious or toxigenic used as a preservative to inhibit the growth microorganisms (foodborne pathogens) of microorganisms while maintaining the represents the worst forms of quality shelf-life of food products (Andres et a/., deterioration because they threaten the 2005). Furthermo re, sodium ch loride health ofthe consumer (ICMSF, 1996). ICMSF, influences the activity of different proteases (1996) observed that despite the extensive and such as (a-dependent scientific progress and technological protease; Cathepsin D and Cathepsin L developments achieved in recent years, food (Armenteros eta/., 2009). An increase in the safety problems continue to exist and may concentration of NaCI has been reported to actually increase in the future. Both intrinsic decrease protease activities and thus and extrinsic factors act synergistically to prevent the spoilage of meat (Armenteros et enhance or retard the growth of a/.,2009). microorganisms in food (Jay eta/., 2005).Salt is a widely used additive and preservative, Food-borne illnesses that occur from enhancing the flavour (Silva eta/., 2003) and consuming contaminated food with improving water adsorption in foods pathogenic have been of serious (Lawrence et a/., 2003). A high salt public health concern all over the world. concentration generates changes in cellular Food-borne illnesses associated with £. coli because of its osmotic effect, 0157:H7, , which, influences microorganisms in enteritidis, Salmonella typhi and different concentrations. Bautista et a/ monocytogenes, is a major concern (2007) and Blesta eta/ (2008) reported that (Beuchat 1996, Hall, 1997 and Farber, 2000). the salts, NaCI, KCI and CaCI2 or their The result also informs of the need to wash combination drastically reduced water raw food products in potable water to activity in meat. Addition of salts leads to a reduce microflora before preservation sudden onset of plasmolysis, which causes because, in the presence of a high initial inhibition of nutrient uptake, DNA microbial load, preservation may virtually be replication and triggers an increase in the impossible. This work aims to determine ATP levels of cells, which further leads to the effects of salts on metabolic activities of inhibition of macromolecular biosynthesis food (meat and fish) microflora as (Csonka, 1989). Accord ing to Albarracin et purchased from the market

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M aterials and M ethods Isolation and Enumeration of Bacteria and Sampling points Fungi Fish and Meat samples were collected from All inoculated plates were incubated at three different locations in a local market in 3rc for 24- 48 h to obtain viable bacterial Ota {Oja-Ota). Ota is located in Ogun state, counts, except, however, Saboraud Nigeria {7.9452"N, 4.7888°N). This market Dextrose plates that were incubated was chosen because it is the major one in at laboratory room temperature of 28 ± 2 town and many vendors patronize the oc for 72 h. Colonies were counted at the market for sales. Food grades of NaCI, KCI, expiration of incubation period using the colony counter (Gallenkamp, England). and CaCI were obtained from reputable 2 Counts were expressed as colony forming vendors. units per ml of sample homogenate (cfu/ml). Characteristic discrete colonies Collection of Samples on the different media were isolated and Major vendors of fresh fish and meat were purified by repeated sub-culturing on identified in the market. Samples were nutrient agar. Pure cultures were stored on randomly obtained from six vendors, three agar slants at 4°C for further (3) each for raw fish and meat samples. All characterization. the samples were collected in polyethylene bags as sold and transported in cold pack (2 Coliform Test ± 2"C) to the Laboratory of The standard method of Speck, (1976) as Covenant University, Ota for analysis described by Oranusi et a/., (2013) was within 30 minutes-1hour after col lection. adopted. One gram of each sample was transferred to a sterile test tube containing Sample Preparat ion lactose broth and inverted Durham tubes. A sterile knife was used to remove fatty Incubation was for 24-48 h at 3rc before portions of the meat to obtain lean meat. tubes were checked for gas production. Twenty-five grams (25g) portions of the This was the presumptive test. A loop full of samples were minced in a laboratory inoculum from gas positive tubes was blender (Model 400 EVO, Stomacher) and streaked onto Eosin methylene blue (EMB) homogenized in 225ml of sterile peptone agar plates and incubated at 3rC and 45°C water. The resultant homogenate was for 24 hrs. Following incubation, colonies 2 3 4 5 diluted 10· , 10· , 10· and 10 • From the which formed bluish black colonies with appropriate dilution, 0.2 ml were plated in green metallic sheen, and reddish/ brown duplicate onto the different media using colonies were noted and isolated on agar the spread plate technique. Nutrient agar, slants (confirmatory test). Also, colonies Eosin Methylene blue agar, and Potato showing metallic sheen on EMB were Dextrose agar were inoculated for isolation subcultured into tubes of lactose broth of bacteria and fungi and for Total aerobic (single strength) and incubated at 3rC. plate count, Coliform count and fungal The tubes were observed after 24 h for gas count respectively. Mannitol salt agar was production (completed test). The growth used for isolation and counting of of coliform at 45°C incubation was noted as Staphylococcus aureus. faecal coliform.

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Identification of Isolates Assay for Metabolic Activities and The bacterial isolates were identified based Microbial Profile during Storage on standard methods of Speck {1976); Metabolic activity was determined by total Cowan, (1985). Isolates were Gram stained protein and lipid peroxidation. The microbial profile was evaluated following the and specific biochemical tests performed to procedure earlier described in enumeration include Catalase activity, utilization, and identification of isolates. Determination Oxidase test, Indole test, Urease test, of protein was by biuret method following Methyl red and Voges Proskauer tests, the description of Sapan and Lundblad Coagulase activity, Citrate utilization and (2015). Egg albumin served as standard and Motility test. API bioMerieux© kit was the absorbance was read at 450nm using a employed for additional identification of the spectrophotometer (Model G105 UV-VIS bacterial isolates. Fungal isolates were Genesys, ThermoFisher Scientific). Lipid peroxidation was evaluated by TBARS assay identified based on their macroscopic and measuring thiobarbituric reactive microscopic characteristics employing substances (Ma londia ldehyde-M DA). cultural presentation, pigment production Aliquot of 1 ml of sample homogenate was on media, hyphal and spore characteristics combined with 2 ml of Thiobarbituric acid and with reference to standard (TBA) reagent and mixed thoroughly. The identification keys and atlas for fungal solution was incubated in water bath identification {Fawole, 1986; Tsuneo, 2010). for 15 minutes. After boiling, the tubes were immediately placed under a running tap to Preservation Study cool. The tubes were then centrifuged at 1000 rpm for 10 minutes and the The samples were minced with a laboratory absorbance of the clear supernatant was blender (Model400 EVO, Stomacher). Three read against a blank (all reagent minus different weights of 2g, 2.5g, and 4.Sg of the sample homogenate) at 535nm. TBARS salts NaCI; KCI and caq were well mixed content was calculated using 1.56 x 105 M with 100g of samples for preservation. The 1cm·1 which is the extinction coefficient of salted samples were transferred into sterile MDA-TBAcomplexat535 nm. tubes with lids and appropriately labeled. A group of unsalted samples served as the Results Table 1 shows the results of samples before control. Storage of the samples was under preservation. It reveals initial high microbial three varying conditions namely: load in all the sa mples. Table 2 presents the • Room Temperature at28±2°C microbial loads during storage. It shows an • Refrigeration Temperature at4±2°C increase along the duration of storage and • Freezing Temperature at -18±2°C along the salt concentration with more Samples were stored for fifteen days during increase at room temperature storage when which assay for microbial profile and compared to refrigeration and freezing metabolic activities was conducted on day 0, temperature. Table 3 shows the TBARS day 7 and day 15. values (moiMDA/g) of samples during

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storage. The table reveals an increase in shown to decrease along salt lipid peroxidation along the stora ge concentration levels and duration of duration, temperature and salt storage concentrations. The protein content s were

Table 1: Mean microbial load (cfu/ml), lipid peroxidation and protein of samples before preservation ----- Analysis Meat Samples Fish Samples (du/ml)

1 2 3 1 2 3

6 6 8 8 8 6 TAPC 7.2 X 10 7.8 X 10 1.1 X 10 2.0x 10 1.7 X 10 5.9 X 10

4 5 4 TCC 5.0 X 10 1.4 X 10 1.5 X 10 1.8 X lOS 4.1 X lOS 1.2 X lOs

6 4 5 TFC 1.6 X lOS 6.2 x to• 7.o x w• 5.9 X 10 8.0 X 10 1.4 X 10

Lipid peroxidation 0.011 0.099

(moiMDA/g)

Protein content 0.588 1.330

(mg/g)

Key: TAPC: Total Aerobic Plate Count; TCC: Total Coliform Count; TFC: Total Fungal Count

Journal of Industrial Research and Technology. .. JIRT Vol. 6, No 1, 2017 94 '- c:0 3 9.. .Q, 0 :;- Ol Cl.c: :> Table 2 : M e an total m icrobia l count (cfu/ ml) during preservation at 28:1:2°C ro o m temperature c: "'~ ~. [ ~ Concen- Meat Samples Fish Samples Q ::1) :-:- 111 I ration m 111 0"' ~ NaCI K CI CaCI1 Control NaCI KCI CaCI1 Control !l ~ ::r Day 7 Day IS Day 7 Day Day 7 Day Day Day Day Day Day Day Day 7 Day Day 7 Day "' 0 s. ::1 IS IS 7 IS 7 IS 15 15 15 VI Cl. TAPC 01 9 9 9 9 10 10 2.0 2.2 X J0 4.8 X JOQ J.3 X JOQ 4.3 X JO' ).) X J0 J.J X ).4 X 3.4 X 3.4 X 6.0 X 5.2 X 7.2 X JO' 5.0 X J0 5,9 X J0 2.0 X J0 4.2 x 10 ~ i;;i 0 <')::r JO' 1010 to'" to• ro• 1o ' :> ::1 ~ 0 9 9 9 ;;; 0 2.5 J.8 X 10 4.0 x ro• 9.8 X 10' 4.1 x to• R.2 X tO' ~.2 X 2.8 X 5.8 X 5.0. 6.3 X 10 4.8 X 10 5.5 x to• ., IQ 9 to' "' ";<: 10' 10 10' 9 8 9 ~ 4.5 6.0 X JO' 3.9x 10' R.9 x tn' 4,0 X )0 6.3 X 10 7.9 X 2.5 X 4.5 X 3.7 X 5.8 x ro• 4.3 x ro• 4.9 X 10 a· 8 9 9 to• :::1 10 10 10 01 :::1 Total Coliform Count Q. 1 ~ 2.0 1.9 x to' 3.2 x t o' 1.1 x to' 4.5 X 10 9.9 X JO) 2.5 X 2.) X 2.1 X 4.5• 4.5x 5.t X 7.9x 10' ) , ) X JO' 3.7 x t o' 2.0 x ro• 3.5 X JO' :!: ::1) ~ -i 108 10' ro• 10' ro' 108 01 CT ~ 7 8 8 2. :- 2.5 J.6 X 10 2.8 X IO' 6.6 x to• 4.0 x to' 9.2 x tn' 2.t X 4.1 X 4.3 X 4.9 X 7.5 X 10 2.1 x ro' 3.1 X 10 ;:r ,01 10' 10' 108 10' 01 ~ <: 7 7 8 c:· 0 4.5 5.o x to• 1.9 X tO' s.2 x ro• 3.4 X 10 7.7 X J0 J.9 X 3.2 X 3.8 X 4.4 X 6.8 X 10 2.4 x to' 2.1 x ro• ;:+ ,.... 7 8 ;;;· 10 to' JO' 10 "' .....""0 ..,s. Total Fungal Count ji;' 6 :::1' "' X 6 3.2 X 6 6.5 X X 2.0 5.9 X J0° 6.5 JO) 6.2 x ro• 7.3 x ro' 4.8• 10 5.1x !.I X 3.2 X 2.0x 2.0x 3.2 X JOj 8.2x 10 8.2x 10 JOg 6.5 JOB 01 7 1 7 :::1 10 10' ro tn' 10 to' Q.

7 7 7 :!: 2.5 5.2 x to• 6.2 X 10 5.R x 1o• 6.8x 10 4.5x to• 4.7x 9.J X 9.J X 2.8 X 2.8 X 10 7.3 x to• 7.3 x ro• ., 01 7 6 7 ,. 10 10 rn• t0 :!: 5 7 7 6 6 ;:;· 4.9x 10 5.9x tO' 5.3 x to• 4.t x to• X X 1.6 X X 6.8x 10 4.5 6.2x 10 4.2x 8.7 8.7 1.6 10 6.8x 10 0 tO' ro• to• JO' :::!! 0 Ol

10 V1 Table 3: Mean TSARS values ( moiMDA/g) of samples at days 7 and I 5

(--·~•1 Mrat Sampln fl~h Sampl" ..,, t\aCI KCI CaCl, Control N•CI KCI CaCil Concrol " 8 c " c " B c " c " 8 c A B c 8 c " D c 2.0 0.031 0029 0016 0.04S 003S 0031 0.021 0.031 0.028 0042 0036 0.04S 0.223 0211 0.202 0 250 0.230 0.223 0.221" 0 222 OJS 0.305 0.276 0269

2.5 0.021 0 024 0 026 0.039 0.032 0.021 0.027 O.Oll 0025 0.195 0.203 0.1 98 0.241 0 224 0 219 0.195 0.217 0207

4.5 0 024 0.019 0 018 0.028 0026 0.022 0.019 0.025 0.020 0.187 0. 198 0.193 0.212 0.217 0.208 0.187 Q211 0 199

TBARS n lut' of samptft a1 da) 15

2.0 0.061 0.048 0.03 1 0 075 0.062 0052 0.067 0.053 0.1144 0.0144 0.079 0.068 0.330 0 315 OJOS 0.5 17 0.387 0 ..154 0.298 0276 0.241 0.424 0.410 0397

2.5 0054 0042 0.039 0.062 0.051 0.049 OOS9 0.047 0.041 0.323 0.306 0 299 0 1Q4 0.326 0.348 0.274 0.253 0 230

4.5 0.036 0037 0 033 0048 0.046 0046 0.032 0041 0039 OJ II 0 297 0.282 0363 0.312 OJJ2 0 ..165 0.240 0221

Key: A= 28±2'C; B= 4±2'C; C= -18±2'C 0 OJ :I c: !!!. ~ ,.,.Q m Table 4: Mean total protein value (mglg) of samples at day 7 and 15 ~ ~ Ill Conn nl Meat Samples Fish Samples s. ration "'~ Ill NaCI KCI CaCI, Control IliaCI KCI CaCI2 Control 0 :I l>a) 7 "0 A B c A 8 c A 8 c ,\ B c A 11 c A B c A 8 c A 8 c ;; Ill II) 2.0 0.16 0.22 0.22 0.09 0.15 0.15 0.02 0.13 013 0.06 0.08 0.08 044 045 0.45 0.30 0.31 0.31 0.18 0.20 0.20 0.10 0 II 0 II ...< 4 9 2 2 "'a· :I 2.5 0.18 0.26 0.26 0.10 0.17 0.17 O.o7 0. 13 0.13 058 0.59 0.59 0.35 0.39 0.39 0.22 025 0.25 :I 3 "'Q. 0.1 9 0.32 0.32 0.25 0.29 0.29 0.08 0.31 0.31 0.74 0.75 0.75 0 76 0.78 0.78 0.72 0.75 0.75 3:: 4.5 II) 6 2 .. a Q."' Total protein value of samples at day 15 ;:;· 0.10 0.10 0.10 0.09 0.09 0.09 0.12 0.12 0.124 O.QJ O.QJ 0.04 0.28 028 0.29 0.27 0.27 0.27 0.15 0.16 0.16 005 0.0 0.065 2.0 "'~ 65 ~ 2.5 0.13 0.12 0.12 0.11 0.1 1 0.11 0.1 5 0.15 0.151 0.52 0.52 0.52 0.33 0.33 0.35 0.17 0.18 0.18 ; · Ill 7 4 0 9 I s..., 4.5 O.IS 0.1 4 0.14 0.13 0.13 0.13 0.22 0.22 0.223 0.63 0.63 0.63 0.34 0.34 0.34 0.26 0.26 0.26 ;;;· 0 6 8 8 ':r :I "'Q. Key: A = 28±2'C; 8= 4±2'C; C• -18±2'C 3:: II) ..."' 3:: ;:;· 0 :::!! 0 OJ Oranusi eta/: Effects of Salts on Preservation and Metabolic activities of Fish and Meat Microflora

Discussion and meat samples may be attributed to poor This study investigated the effects of salts on sanitary practices in slaughtering, food preservation and the metabolic processing, and packaging prior to purchase activities of food microflora. , like all (Famurewa et a/., 2017). E. coli are normal other preservation methods, aim to flora of the human and animal intestine, it is maintain the nutritional characteristics of an indicator organism of faecal the food and ensure a longer shelf life. The contamination, and pathogenic strains have different preservation methods applied to been identified as a leading cause of foods and the shelf life ultimately all over the world. E. coli determines their commercial viability. has previously been isolated from meat Results showed that the diverse microbial samples (Hussein, 2007). Its presence in the species isolated from both fish and meat samples could be associated with poor prior to addition of preservatives include sanitary practices in the handling of the Pseudomonas spp, Staphylococcus aureus, meat and fish samples, this call for public Proteus spp, Escherichia coli, Klebsiella spp, health concerns. Staphylococci are normal Enterobacter spp, Aspergillus spp and Mucor flora of man and animals, its presence in spp. Microbiological quality of food samples these samples collaborate with the food is often assessed by the total microbial load handlers and normal flora of the animals. determined using the total aerobic plate The presence of Staphylococcus aureus also count, total coliform count and total fungal raises some concerns as toxigenic strains count. The microbial counts gradually have been associated with foodborne increased with storage, this could be infection (Hennekinne eta/., 2012; Ji-Yeon et attributed to high moisture and nutrient a/.,2013). contents of the samples which could Results indicated that the TBARS value of promote the growth of the organisms the fish samples was higher than those of (Gandotra, 2017). the meat samples before treatment. The TBARS value of the samples increased as The presence of these diverse species of storage time increased. This was expected organisms on the food samples could be and corroborates findings by Farbod (2015) attributed to contamination from the who reported that TBARS values increase environment. Meat and fish are rich in with storage time. In the meat samples, nutrients required for growth and those treated with NaCI and stored at room proliferation of microorganisms. Proteus temperature potentially retarded the and Pseudomonas spp are common generation of the reactive species the environmental contaminants of food and most. The KCI treated meat samples food products. Species of Klebsiella, exhibited the highest increase in TBARS Enterobacter, and Escherichia coli are values between day 7 and 15. Although it members of Enterobacteriaceae associated has been earlier suggested that TBARS with the gastrointestinal tract, the value increases with storage time, there occurrence of these organisms in the fish was an unexpected increase among meat

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samples treated with 2% NaCI between day 7 values were generally higher than those of

and 15. In the fish samples, the CaC12 treated their respective control. This may be samples had minimal variation in TSARS attributed to strong salting out effects on the increment compared to the NaCI and KCL proteins (Thorarinsdottireta/., 2004). treated samples. Generally, the most Sodium Chloride (NaCI) exhibited the highest retardation of MDA generation was observed efficacy in the preservation of food samples in samples stored at freezing temperatures. overall, while Potassium Chloride (KCI) This corroborates findings by Aydin (2014) showed the least efficacy. who reported that low freezing temperatures result in low oxidation levels. Freezing is also a Conclusion good preservation method known to prevent remains an important microbial proliferation. The TSARS of the parameter for good health thus must be in samples were lower than those of the control check. To ensure freshness of foods, during the storage period which is indicative preservative methods like salting are often of the preservative potential of the salts. employed, this present study showed the However, the increase in the TSARS values presence of certain organisms associated during storage may also have bee~ mediated with fresh fish and meat. There was an by the sa lts themselves as reports have increase in the microbial population in the shown that salts such as NaCI possess selected foods along the duration of prooxidant activity and have been implicated preservation, with more increase at room in lipid oxidation in meat and temperature; the microbial counts, however, (Mariutti and Sragagnolo, 2017). fell within permissible limits for raw food. The 4.5% salt concentration and freezing was the Results showed that the total protein of the most effective preservation method. The samples decreased with storage after salt decrease in total protein along the duration of treatment, with the fish samples showing a preservation shows how essential nutrients higher rate of protein decrease compared to are depleted due to the metabolic activities of the meat samples. It has been reported that organisms. However, an increase in total this pattern of total protein decrease may be a protein along salt concentration and consequence of denaturation (Gupta, 2017) temperature gradient shows how the there was a sharp decrease in total protein and inhibition of certain organisms among sample stored under refrigeration and slightly maintain the nutrients in foods.The freezing conditions. This corroborates increase in lipid peroxidation along storage findings by Gandotra et al., (2014) that low duration could be indicative of microbial, storage temperatures enhance protein enzymatic and prooxidant activities in the denaturation. However, it was observed that salts. Storage temperature and salt

the KCI and CaCI2 treated meat samples stored concentration ultimately determine the at room temperature showed an increase in rate at which lipid peroxidation occurred. total protein value during storage. In both the In this study, NaCI showed the highest meat and fish samples, the total protein efficacy in preservation, while freezer

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temperature and 4.5% salt concentration Bautista, J., Arroyo, N., Duran, M. and were the most effective, Preserving Garrido, A. (2008). Individual effects food with a high initial level of of sodium, potassium, calcium, and microbial load, makes preservatives magnesium chloride salts on ineffective and preservation difficult. Lactobacillus pentosus and It is advanced that for effective Saccharomyces cerevisiae growth. preservation of raw fish and meat Journal of Food Protection, products, initial levels of microbial 71 :1412-1421. contaminants must be reduced to the barest minimum. Beuchat, L.R. (1996) . Pathogenic microorganisms associated with References fresh produce. Journal of Food Albaraccin, W., Ivan C., Raul, G. and Jose, M. Protection, 59:204-216. (2010).Salt in , usage, and reduction a Blesata, E., Alina, M., Barat, J., Toldra, F. and review.lnternational Journal of Food Pagan, M. J. (2008). Microbiology and Science and Technology, 46: 1329- physicochemical changes of dry­ 1336. cured during the post-salting stage as affected by partial Andres, A., Rodnguez-Barona, S., Barat, replacement of NaCI by other salts. J.M. and Fito, P. (2005). Salted cod , 78:135-142. : influence of salting procedure on process yield and Cowan, S.T. (1985). Cowan and Steel's product characteristics. Journal of manual for the identification of FoodEngineering, 69(4}: 467-471. medical bacteria. Cambridge University Press. ISBN: 381264537. Armenteros, M., Aristoy, M. and Toldra, F. (2009).Effects of sodium, potassium, Csonka, L.N. (1989). Physiological and calcium, and magnesium chloride genetic responses of bacteria to salts on porcine muscle osmotic stress.Microbiology proteases.European Food Research Review.Pp 34-40. 53:121-147. andTechnology,229: 93-98. Famurewa, J. A. V., Akise, 0. G. and Aydin, I. and Gokoglu, N. (2014). Effects of Ogunbodede, T. (2017).Effect of temperature and time of freezing on storage methods on the nutritional lipid oxidation in anchovy (Engravers qualities of African encrasicholus) during frozen Clariasgarepinus (Burchell, 1982), storage.European Journal of Science African Journal of . 11 and Technology, 16 (8): 996-1001. (7): 223-233

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