Effects of Chemical Hurdles and Packaging Materials on Microbial Load and Bacterial Distribution in Kilishi under Ambient Storage

Effects of Chemical Hurdles and Packaging Materials on Microbial Load and Bacterial Distribution in Kilishi under Ambient Storage

1Abdullahi Nura,

1Department of Food Science and Technology, Kano University of Science and Technology, Wudil, Kano State Nigeria (P.M.B 3244)

2Araihu C. C. and 2Abu J. O.

2 Department of Food Science and Technology, University of Agriculture Makurdi, Benue State Nigeria (P.M.B 2373)

Abstract – The research work studied combine effects of chemical hurdles and packaging materials on microbial load and bacterial distribution in Kilishi under ambient storage (30±80C). Sucrose, citric acid and sodium benzoate were applied in different combination and concentrations. High Density Polyethylene (HDPE), aluminium foil and brown paper were used as packaging materials during twelve weeks ambient storage. The laboratory prepared samples were studied alongside a market sample which was collected from Agadasawa in Kano State Nigeria. Determination of total microbial load, and isolation and identification of bacterial group conducted at two weeks interval in the market sample and laboratory prepared samples. Increased in microbial counts was observed during storage with highest counts recorded in market sample throughout the storage time. Among the packaging materials, samples packaged in brown paper were found to have highest microbial load, and that packaged in HDPE were found to have least counts. Streptococcus spp., Staphylococcus spp., Salmonella spp., Pseudomonas spp. and Bacillus spp. were identified in all the samples. Escherichia coli was identified only in market sample. Growth of Salmonella spp. and Staphylococcus spp. was found to be subsiding during storage with total elimination occurred at week 8 and week 10 respectively from the treatment samples. Combination of 4% sucrose, 0.1% citric acid, 0.1% sodium benzoate and use of HDPE as packaging material provide best microbial quality.

Key Words; Hurdle, Kilishi, Microorganism, Bacteria, Ambient storage

INTRODUCTION

Kilishi is a sun dried traditional meat product made principally from beef. It is an intermediate moisture or semidry meat product. The product appears to have developed as a means of preserving meat in the absence of refrigeration facilities by the early Fulani and Hausa herdsmen of Northern Nigeria and the Sahelian Africa. As a ready-to-eat convenience meat product, Kilishi possess an excellent shelf life (Isah and Okubanjo, 2012). Keeping quality of Kilishi is greatly affected by the season and location of production (Fonkem et. al., 2010).

Food preservation implies putting microorganisms in a hostile environment in order to cause their death (Oladapo et al., 2014). Preservative agents are required to ensure that manufactured foods remain safe and unspoiled (Brul and Coote, 1999). When food is to be stored for a prolonged period, use of preservatives is essential 'in order to maintain its quality and flavour. Their use prevents spoilage of foods

due to the growth of bacteria and fungi. They also maintain the quality and consistency of the foods, along with its palatability and wholesomeness. Preservatives also maintain nutritional value, control appropriate pH and enhance flavour (Arora et al., 2014).

Chemical preservatives may be injurious when used in higher concentrations. Arora et al. (2014) reported that at higher concentration benzoates can trigger allergies such as skin rashes, asthma and can also causing brain damage. He also reported that sodium chloride when used in high amount in' meats and fish can lead to high blood pressure, kidney failure, stroke and heart attack.

External animal surfaces, as well as their feces and the environment, may serve as sources of contamination for carcasses during the slaughtering, dressing and cutting processes. Panagiotis et al. (2010) reported that majority of these microorganisms consist of nonpathogenic spoilage bacteria and indicator microorganisms, such as coliforms and Escherichia coli (at levels 10 to 107 cfu/cm2 or higher). However, there is also potential for contamination by pathogenic microorganisms such as Escherichia coli, Salmonella, Campylobacter spp. etc. These presents challenging problems to the meat industry and low bacteriological quality raise concern about its potential for the transmission of foodborne infections. (Agwu and Chisom, 2014).

Joerg et al. (2007) reported that Kilishi suffers contamination from various sources, example; Slaughtering of animal and carcass processing take place under unhygienic conditions, meat transport and marketing are done without refrigeration and no protection against sun and dust. Kilishi production and storage under conditions free of microbial activity has been seen as a process usually difficult to achieve because of its nutritious nature that attract agents of microbiological spoilage (Ogbonnaya and Linus, 2009). Kilishi is preserved by drying which in traditional production is achieved through sun-drying. Drying is not lethal and many types of microorganisms may be recovered from dried foods, especially if poor-quality foods were used for drying and if proper practices were not followed in the drying steps (James, 2000). Fonkem et al. (2010) reported that some spices used in Kilishi production

play a key role in inhibiting the growth and proliferation of some micro-organisms, this contradict the opinion of Ogbonnaya and Linus (2009) who reported that condiment used in the production of Kilishi can serve as a source of microbial contamination.

METHODOLOGY

Sample collection

Freshly prepared Kilishi was collected from Agadasawa in Kano metropolis, Kano State-Nigeria. To avoid contamination, the collected sample was wrapped in HDPE and aseptically transported to laboratory. The collected sample was divided in three, each of the portions was packaged into HDPE, aluminium foil and brown paper, and stored under ambient temperature (30±80C) for period of

twelve weeks. Samples were withdrawn from market and laboratory prepared samples and subjected to microbiological analyses at two weeks interval.

Procurement of raw material for Kilishi production

Beef was purchased from Kano central abattoir. Ginger, Cloves, Black Pepper, Hot Pepper, Sweet Pepper, onion, curry, salt, seasoning and peanut cake were purchased from Kurmi Market in Kano.

Production of Kilishi

Recipe for condiment production

The table below 2 provides recipe for the production of

Kilishi condiment.

Table 1: Recipe for Production of Kilishi Condiment

Ingredients Quantity (g)

Ginger	17.9
Cloves	1.3
Black Pepper	2.5
Hot Pepper	5.3
Sweet Pepper	11.0
Onion	12.5
Curry	3.7
Salt	23
Seasoning (Maggi)	53.5

Peanut cake 469.3 Source; Badau et al. (1997).

Fresh meat Wet cleaning

Trimming fat and connective tissues Slicing

Drying

Condiment bath (immersion/dipping) Drying

Light roasting Packaging

Kilishi

Figure 1: Tradition Kilishi Production Process

Source; Okonkwo et al. (2013)

Table 2; Percentages of Hurdles and Condiment used in Kilishi

Sample Code	Sucrose	Citric	Sodium Acid	Condiment Benzoate	Meat
001	0.0	0.0	0.0	45.0	55
014	2.0	0.1	0.1	42.8	55
017	2.0	0.2	0.1	42.7	55
023	4.0	0.1	0.1	40.8	55

Microbiological analyses

Total microbial load was performed on nutrient agar using serial dilution method described by the American Public Health Association (APHA, 1992). Isolation and identification of bacterial groups was achieved through the following biochemical tests; gram reaction, coagulase test, acid production from sugar, methyl red test, voges proskauer test (Robert and Greenwood, 2003) motility test, indole test, catalase test, oxidase test, citrate test (Chessbrough, 2000). The results of biochemical tests were compared with characteristics of taxa described by Bergeys Manual for Determinative Bacteriology (Buchanan and Gribbons, 1974).

RESULTS

Table 3: Effect of hurdles and packaging materials on microbial load of Kilishi samples under ambient storage (30±80c)

Sample Code

Packaging Materials Storage Time (weeks)

0 2 4 6 8 10 12

Total Plate Count (×103 cfu/g)

	001	HDPE	2.40	2.48	2.51	2.58	2.66	2.72	2.76
		Aluminium foil	2.40	2.53	2.64	2.72	2.85	2.92	3.10
		Brown paper	2.40	2.60	2.74	2.80	2.98	3.20	3.38
	014	HDPE	1.82	1.88	1.92	1.97	2.04	2.12	2.24
		Aluminium foil	1.82	1.90	1.97	2.12	2.18	2.26	2.32
		Brown paper	1.82	1.96	2.10	2.26	2.30	2.38	2.42
	017	HDPE	1.77	1.82	1.88	1.94	1.96	2.10	2.25
		Aluminium foil	1.77	1.83	1.92	2.11	2.23	2.29	2.34
		Brown paper	1.77	1.89	2.10	2.22	2.28	2.40	2.60
	023	HDPE	1.56	1.60	1.66	1.71	1.78	1.84	1.92
		Aluminium foil	1.56	1.63	1.70	1.79	1.85	1.90	1.97
		Brown paper	1.56	1.68	1.76	1.82	1.90	2.12	2.24
	MS	HDPE	7.32	7.56	7.62	7.84	8.10	8.27	8.62
		Aluminium foil	7.32	7.60	7.69	7.98	8.22	8.96	9.26
1		Brown paper	7.32	7.64	7.73	8.20	8.63	9.50	10.32

Table 4: bacterial distribution in Kilishi samples treated with different hurdles under ambient storage (30±80c) (Week 0)

Sample codes 001 014 017 023 MS

%Hurdles

Sucrose	0 2 2	4	0
Citric acid	0 0.1 0.2	0.1	0
Sodium benzoate	0 0.1 0.1	0.1	0
Bacterial group	Bacterial Distribution (%)
Streptococcus spp.	20 25 25	15	15
Staphylococcus spp.	20 18 14	21	20
Salmonella spp.	08 06 06	04	14
Pseudomonas spp.	22 26 25	28	11
Bacillus spp.	30 25 30	32	30
E. coli	00 00 00	00	10
Total	100 100 100	100	100
Sample code Sucrose (%)	Citric acid (%) Sodium benzoate (%)
001 0	0 0
014 2	0.1 0.1
017 2	0.2 0.1
023 4	0.1 0.1
MS-Market sample

Table 5: Bacterial distribution in Kilishi samples treated with different hurdles under ambient storage (30±80c) using various

packaging materials (Week 2)

Sample codes			001	014	017	023	MS
%Hurdles
Sucrose			0	2	2	4	0
Citric acid			0	0.1	0.2	0.1	0
Sodium benzoate			0	0.1	0.1	0.1	0
Packaging Bacterial Bacterial Distribution (%) Material group
	HDPE	Streptococcus spp.	22	27	20	12	15
		Staphylococcus spp.	18	16	13	19	15
		Salmonella spp.	12	06	05	05	15
		Pseudomonas spp.	20	25	28	32	18
		Bacillus spp.	28	26	34	32	27
		E. coli	00	00	00	00	10
		Total	100	100	100	100	100
	Aluminium Foil	Streptococcus spp.	18	25	25	15	15
		Staphylococcus spp.	20	18	14	21	20
		Salmonella spp.	14	05	06	05	10
		Pseudomonas spp.	20	26	25	27	15
		Bacillus spp.	28	26	30	32	30
		E. coli	00	00	00	00	10
		Total	100	100	100	100	100
	Brown Paper	Streptococcus spp.	23	26	25	16	15
		Staphylococcus spp.	15	17	14	20	20
		Salmonella spp.	16	06	06	04	11
		Pseudomonas spp.	20	24	27	30	15
		Bacillus spp.	26	27	28	30	27
		E. coli	00	00	00	00	12
		Total	100	100	100	100	100

Sample codes		001 %Hurdles	014	017	023	MS
	Sucrose	0	2	2	4	0
	Citric acid	0	0.1	0.2	0.1	0
	Sodium benzoate	0	0.1	0.1	0.1	0
Packaging	Bacterial	Bacterial Distribution (%)
Material	group
HDPE Streptococcus spp.			20	25	20	13	10
Staphylococcus spp.			20	18	15	20	15
Salmonella spp.			14	05	05	05	15
Pseudomonas spp.			22	30	30	31	22
Bacillus spp.			24	22	30	31	28
E. coli			00	00	00	00	00
Total			100	100	100	100	100
Staphylococcus spp.			22	20	14	22	20
Salmonella spp.			14	05	05	06	08
Pseudomonas spp.			22	27	27	27	20
Bacillus spp.			22	28	30	28	20
E. coli			00	00	00	00	12
Total			100	100	100	100	100
Staphylococcus spp.			17	17	17	18	15
Salmonella spp.			15	06	05	04	10
Pseudomonas spp.			18	30	26	30	25
Bacillus spp.			28	23	30	32	23
E. coli			00	00	00	00	12
Total			100	100	100	100	100

Sample codes		001 %Hurdles	014	017	023	MS
	Sucrose	0	2	2	4	0
	Citric acid	0	0.1	0.2	0.1	0
	Sodium benzoate	0	0.1	0.1	0.1	0
Packaging	Bacterial	Bacterial Distribution (%)
Material	group
HDPE Streptococcus spp.			20	25	20	13	10
Staphylococcus spp.			20	18	15	20	15
Salmonella spp.			14	05	05	05	15
Pseudomonas spp.			22	30	30	31	22
Bacillus spp.			24	22	30	31	28
E. coli			00	00	00	00	00
Total			100	100	100	100	100
Staphylococcus spp.			22	20	14	22	20
Salmonella spp.			14	05	05	06	08
Pseudomonas spp.			22	27	27	27	20
Bacillus spp.			22	28	30	28	20
E. coli			00	00	00	00	12
Total			100	100	100	100	100
Staphylococcus spp.			17	17	17	18	15
Salmonella spp.			15	06	05	04	10
Pseudomonas spp.			18	30	26	30	25
Bacillus spp.			28	23	30	32	23
E. coli			00	00	00	00	12
Total			100	100	100	100	100

Table 6: Bacterial distribution in Kilishi samples treated with different hurdles under ambient storage (30±80c) using various packaging materials (Week 4)

Aluminium Foil Streptococcus spp. 20 20 24 17 20

Brown Paper Streptococcus spp. 22 24 22 16 15

Table 7: Bacterial distribution in Kilishi samples treated with different hurdles under ambient storage (30±80c) using various packaging materials (Week 6)

Sample codes		001	014	017	023	MS
		%Hurdles
	Sucrose	0	2	2	4	0
	Citric acid	0	0.1	0.2	0.1	0
	Sodium benzoate	0	0.1	0.1	0.1	0

Packaging Bacterial Bacterial Distribution (%) Material group

	Streptococcus spp.	22	26	20	18	15
HDPE	Staphylococcus spp.	18	11	08	10	16
	Salmonella spp.	15	04	05	04	14
	Pseudomonas spp.	20	34	32	35	20
	Bacillus spp.	25	25	35	33	25
	E. coli	00	00	00	00	10
	Total	100	100	100	100	100
Aluminium Foil	Streptococcus spp.	22	24	28	25	22
	Staphylococcus spp.	22	10	03	08	18
	Salmonella spp.	12	10	03	08	10
	Pseudomonas spp.	20	30	31	30	20
	Bacillus spp.	24	32	34	32	18
	E. coli	00	00	00	00	12
	Total	100	100	100	100	100
Brown Paper	Streptococcus spp.	20	29	26	20	18
	Staphylococcus spp.	22	07	06	08	17
	Salmonella spp.	16	04	04	04	12
	Pseudomonas spp.	14	35	30	32	18
	Bacillus spp.	28	25	34	36	24
	E. coli	00	00	00	00	11
	Total	100	100	100	100	100

Table 8: Bacterial distribution in Kilishi samples treated with different hurdles under ambient storage (30±80c) using various packaging materials (Week 8)

Sample codes 001 014 017 023 MS

%Hurdles

Sucrose 0 2 2 4 0

Citric acid 0 0.1 0.2 0.1 0

Sodium benzoate 0 0.1 0.1 0.1 0

Packaging Bacterial Bacterial Distribution (%) Material group

	HDPE	Streptococcus spp.	24	26	24	22	16
		Staphylococcus spp.	16	05	04	05	15
		Salmonella spp.	18	00	00	00	15
		Pseudomonas spp.	17	39	35	37	21
		Bacillus spp.	25	30	37	36	22
		E. coli	00	00	00	00	11
		Total	100	100	100	100	100
	Aluminium Foil	Streptococcus spp.	23	22	24	25	22
		Staphylococcus spp.	22	10	00	00	16
		Salmonella spp.	14	00	00	00	12
		Pseudomonas spp.	19	38	38	39	23
		Bacillus spp.	22	30	38	36	15
		E. coli	00	00	00	00	12
		Total	100	100	100	100	100
	Brown Paper	Streptococcus spp.	22	32	26	24	22
		Staphylococcus spp.	18	00	06	04	16
		Salmonella spp.	18	00	00	00	14
		Pseudomonas spp.	15	39	34	36	16
		Bacillus spp.	27	29	34	36	22
		E. coli	00	00	00	00	10
		Total	100	100	100	100	100

Table 9: Bacterial distribution in Kilishi samples treated with different hurdles under ambient storage (30±80c) using various packaging materials (Week 10)

Sample codes 001 014 017 023 MS

%Hurdles

Sucrose		0	2	2	4	0
Citric acid		0	0.1	0.2	0.1	0
Sodium benzoate		0	0.1	0.1	0.1	0
Packaging Material	Bacterial group	Bacterial Distribution (%)
HDPE	Streptococcus spp.	22	27	24	24	18
	Staphylococcus spp.	18	00	00	00	16
	Salmonella spp.	18	00	00	00	13
	Pseudomonas spp.	20	41	40	40	24
	Bacillus spp.	22	32	36	36	19
	E. coli	00	00	00	00	10
	Total	100	100	100	100	100
Aluminium Foil	Streptococcus spp.	22	26	22	24	22
	Staphylococcus spp.	24	00	00	00	15
	Salmonella spp.	16	00	00	00	14
	Pseudomonas spp.	20	38	38	37	22
	Bacillus spp.	18	36	40	39	15
	E. coli	00	00	00	00	12
	Total	100	100	100	100	100
Brown Paper	Streptococcus spp.	24	30	24	25	22
	Staphylococcus spp.	16	00	00	00	15
	Salmonella spp.	20	00	00	00	15
	Pseudomonas spp.	16	35	36	35	18
	Bacillus spp.	24	35	40	40	20
	E. coli	00	00	00	00	10
	Total	100	100	100	100	100

Table 10: Bacterial distribution in Kilishi samples treated with different hurdles under ambient storage (30±80c) using various

packaging materials (Week 12)

Sample codes 001 014 017 023 MS

%Hurdles

Sucrose	0	2	2	4	0
Citric acid	0	0.1	0.2	0.1	0
Sodium benzoat e	0	0.1	0.1	0.1	0

Packaging Bacterial Bacterial Distribution (%) Material group

HDPE	Streptococcus spp.	22	24	22	24	18
	Staphylococcus spp.	20	00	00	00	15
	Salmonella spp.	20	00	00	00	14
	Pseudomonas spp.	18	40	38	38	24
	Bacillus spp.	20	36	40	38	18
	E. coli	00	00	00	00	11
	Total	100	100	100	100	100
Aluminium Foil	Streptococcus spp.	24	22	20	24	20
	Staphylococcus spp.	20	00	00	00	16
	Salmonella spp.	18	00	00	00	15
	Pseudomonas spp.	18	40	40	34	20
	Bacillus spp.	20	38	40	42	19
	E. coli	00	00	00	00	10
	Total	100	100	100	100	100

Brown Paper	Streptococcus spp.	22	25	20	20	20
	Staphylococcus spp.	20	00	00	00	14
	Salmonella spp.	18	00	00	00	17
	Pseudomonas spp.	18	35	35	30	18
	Bacillus spp.	22	40	45	50	21
	E. coli	00	00	00	00	10
	Total	100	100	100	100	100

Stated in Table 3 above are the microbial loads in Kilishi samples treated with sucrose, citric acid and sodium benzoate at different levels and combinations and packaged into HDPE, aluminium foil and brown paper.

Sample 001 (control) and Market Sample (MS) contained no preservatives. Samples 014, 017 and 023 were treated with different hurdle combinations as stated in Table 2 above. At the start, the microbial loads for Sample 001, 014, 017 and 023 were found to be 2.40×103, 1.82×103, 1.77×103, 1.56×103 and 7.32×103 cfu/g respectively. Highest microbial counts were recorded in commercial sample throughout the storage time and least counts were recorded in Sample 023. Among the packaging materials used HDPE was found to be more efficient with low microbial counts in all the treatments. Hurdle pattern in Sample 023 with combination of HDPE was found to be the best treatment in retaining the microbial quality of Kilishi.

Percentage distribution

Percentage distribution

Table 4 to 10 above presented the percentage distribution of bacterial groups in Kilishi treated with sucrose, citric acid and sodium benzoate at different levels and combinations over twelve weeks ambient storage (30±80C). Six bacterial groups were isolated from Market Sample these include;

Streptococcus spp., Staphylococcus spp., Salmonella spp., Pseudomonas spp., Bacillus spp. and Escherichia coli. All the above groups with exception of E. coli were also found to be presence in laboratory prepared samples.

Bacterial successions were observed in all the treatments and the packaging system. Percentage distribution of Pseudomonas spp. and Bacillus spp. were found to be increasing in the treatment samples (014, 017 and 023) during storage. While that of Streptococcus spp., Staphylococcus spp. and Salmonella spp. were found to be increasing at the start then dropped drastically at different points (depending on the packaging material and hurdle combination) before the end of the storage time.

E. coli which serves as indicator organism for faecal contamination was only found to be presence in commercial sample and it is distribution was found to be fairly stable in all the packaging materials used during the twelve weeks ambient storage (30±80C).

A graphical representations of the bacterial succession in different packaging materials were presented in Fig 2 to 4 below.

45

40

35

30

25

20

15

10

5

0

45

40

35

30

25

20

15

10

5

0

S01

S14

S17

S23

MS

S01

S14

S17

S23

MS

Bacterial isolates and sample codes

Wk0 Wk2 Wk4 Wk6 Wk8 Wk10 Wk12

Bacterial isolates and sample codes

Wk0 Wk2 Wk4 Wk6 Wk8 Wk10 Wk12

Streptococcus spp.

Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli

Streptococcus spp.

Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli

Fig 2: Bacterial succession in Kilishi samples packaged in HDPE over 12 weeks ambient storage (30±80C)

Published by :

International Journal of Engineering Research & Technology

International Journal of Engineering Research & Technology (IJERT)

ISSN: 2278-0181

Vol. 5 Issue 08, August-2016

45

40

35

30

25

20

15

10

5

0

S01

S14

S17

S23

MS

Bacterial isolates and sample codes

Wk0

Wk2

Wk4

Wk6

Wk8

Wk10

Wk12

Fig 3: Bacterial succession in Kilishi samples packaged in Aluminium Foil over 12 weeks ambient storage (30±80c)

50

45

40

35

30

25

20

15

10

5

0

S01

S14

S17

S23

MS

Bacterial isolates and sample codes

Wk0

Wk2

Wk4

Wk6

Wk8

Wk10

Wk12

Fig 4: Bacterial succession in Kilishi samples packaged in Brown Paper over 12 weeks ambient storage (30±80c)

313

IJERTV5IS080256

www.ijert.org

(This work is licensed under a Creative Commons Attribution 4.0 International License.)

www.ijert.org

(This work is licensed under a Creative Commons Attribution 4.0 International License.)

IJERTV5IS080256

313

Fig 4: Bacterial succession in Kilishi samples packaged in Brown Paper over 12 weeks ambient storage (30±80c)

Wk12

Wk10

Wk8

Wk6

Wk4

Wk2

Wk0

Bacterial isolates and sample codes

MS

S23

S17

S14

S01

50

45

40

35

30

25

20

15

10

5

0

Fig 3: Bacterial succession in Kilishi samples packaged in Aluminium Foil over 12 weeks ambient storage (30±80c)

Wk12

Wk10

Wk8

Wk6

Wk4

Wk2

Wk0

Bacterial isolates and sample codes

MS

S23

S17

S14

S01

45

40

35

30

25

20

15

10

5

0

International Journal of Engineering Research & Technology (IJERT)

ISSN: 2278-0181

Vol. 5 Issue 08, August-2016

Published by :

International Journal of Engineering Research & Technology

Percentage distribution

Streptococcus spp. Staphylococcus spp. Salmonella spp.

Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp.

Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp.

Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp.

Salmonella spp.

Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli

Percentage distribution

Streptococcus spp. Staphylococcus spp. Salmonella spp.

Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp.

Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli

Percentage distribution

Streptococcus spp. Staphylococcus spp. Salmonella spp.

Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp.

Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp.

Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp.

Salmonella spp.

Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli

Percentage distribution

Streptococcus spp. Staphylococcus spp. Salmonella spp.

Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp.

Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli Streptococcus spp. Staphylococcus spp. Salmonella spp. Pseudomonas spp.

Bacillus spp.

E. coli

Microbial load

DISCUSSION

Basillus spp were the most frequently isolated organisms in

Kilishi. Okonko et. al., (2013) isolate Bacillus species,

The microbial loads of Kilishi were found to be increasing in both commercial and laboratory prepared samples during ambient storage (30±80C). The microbial loads increased from 2.40×103 cfu/g to 3.38×103 cfu/g in sample 001,

1.82×103 cfu/g to 2.42×103 cfu/g in sample 014, 1.77×103 cfu/g to 2.60×103 cfu/g in sample 017, 1.56×103 cfu/g to 2.24×103 cfu/g in sample 023 and 7.32×103 cfu/g to 10.32×103 cfu/g in market sample. This is in agreement with finding of Jones et al. (2001) who reported increase in microbial counts and changes in chemical composition during ambient storage (310C) of Kilishi treated with potassium sorbet. The results contradict the finding of Ogbonnaya and Linus (2009) who reported decreased in microbial load during ambient storage of Kilishi treated with potassium sorbet.

Highest microbial counts were recorded in Kilishi samples packaged in brown paper in all the treatments throughout the storage period this agreed the finding of Jones et al. (2001) who reported significance difference between Kilishi samples packaged in brown paper and that packaged in polythene bag with highest counts recoded in samples packaged in brown paper. Moisture absorption characteristic of brown paper contribute to higher microbial loads in both laboratory and commercial samples during ambient storage (Okonkwo et. al., 2013)

The microbial loads for market sample were found to be within the range reported by Ogbonnaya and Linus (2009) in freshly prepared commercial Kilishi. The results for microbial loads in the laboratory prepared samples were below the range reported by these researchers in laboratory prepared Kilishi treated with potassium sorbet, but above that reported by Okonkwo et. al., (2013) in industrially produced Kilishi.

Commercial Kilishi samples collected from Port Harcourt (Okonkwo et. al., 2013), FCT (Abuja) (Daminabo et al. 2013) and that collected from Calabar, (Odey et al. 2013) were found to have better microbial quality than that collected from Kano. The variation in the three Nigerian cities may result from variation in meat handling practices, Kilishi manufacturing process and ingredients, and also variation in environmental factors such as temperature and humidity in the locations. This match with finding of Fonkem et al. (2010) who reported variation in microbial load in Kilishi samples collected from three locations (Garoua, Maroua and Ngaoundere) in Cameroun.

Six bacterial groups were isolated from Market Sample these include; Streptococcus spp., Staphylococcus spp., Salmonella spp., Pseudomonas spp., Bacillus spp. and Escherichia coli. All the above groups with exception of E. coli were also found to be presence in laboratory prepared samples. These isolates were very similar to that reported by many researchers; Odey et al. (2013) isolated Staphylococcus aureus, Escherichia coli, Streptococcus spp, Salmonella spp, Bacillus spp, Pseudomonas Spp and Proteus spp from selected Kilishi samples collected from Calabar, Cross River State-Nigeria, the researchers also concluded that Staphylococcus spp, Escherichia coli and

Staphylococcus aureus and Escherichia coli in Kilishi samples collected from Port Harcourt, Rivers State-Nigeria. Edema et al. (2008) isolated Bacillus cereus, Staphylococcus aureus and Salmonella spp in Kilishi samples collected from 6 selected cities within south western part of Nigeria. Fonkem et al. (2010) isolate E. coli and Staphylococcus aureus in Cameroonian Kilishi.

As the storage time progresses the condition in the treatment samples became critical for some bacterial species to survive. The growth of Salmonella spp, and Staphylococcus spp was arrested before the end of the twelve weeks ambient storage. Percentage distributions of this organisms were found to be decreasing during storage. At week 8, Salmonella spp, was totally eliminated from all the treatment samples and Staphylococcus spp was eliminated after 10 weeks. These revealed that the concentrations of the hurdles used in the treatment samples is beyond the range for optimal growth of these microorganisms and this result to their elimination. Streptococcus spp., Bacillus spp. and Pseudomonas spp. were found to succeed the growth Salmonella spp, and Staphylococcus spp during storage. Lee (2004) reported that hurdles used in food preservation could provide varying results depending on bacterial stress reactions such as the synthesis of protective proteins. These resistance organisms may likely synthesise these protective proteins during the storage time.

E. coli was found to be presences only in market sample throughout the storage time. The organism may source from unhygienic water used by local Kilishi producers during processing. Beney et al. (2003) reported that drying at lower temperatures enhances cell survival of E. coli after dehydration. Oladapo et al. (2014) reported that in an agar diffusion technique the minimum inhibitory concentration (MIC) of sodium benzoate and citric acid against growth of Staphyloccocus aureus, Pseudomonas aeruginosa and E. coli is 1.5mg/ml. Stanojevic et al. (2009) reported that combination of sodium nitrite and sodium benzoate at 10mg/ml of growth medium was found to eliminate Bacillus spp and Staphylococcus aureus. Same combination at 5mg/ml was found to eliminate E. coli and Pseudomonas spp. Bibek (2005) reported that the spores of Bacillus spp can withstand roasting. It was also reported by Leistner (2011) that the heat resistance of bacteria increases at low aw.

There has been a debate concerning the acceptability limit for the total viable counts in ready-to-eat meat. London Health Protection Agency (2009) put <106cfu/g as satisfactory limit, and 106 to <107cfu/g as acceptable range. Public Health Laboratory Service (2000) put <105cfu/g as satisfactory limit, 105 to <106cfu/g as acceptable range and

>106cfu/g as unsatisfactory limit. The limits set by both London Health Protection Agency (2009) and Public Health Laboratory Service (2000) render the all the samples acceptable for consumption twelve weeks after storage.

CONCLUSION

Microbial loads in Kilishi samples treated with sucrose, citric acid and sodium benzoate were found to be increasing during ambient storage. Highest counts were recorded in samples packaged in brown paper and least counts were recorded in samples packaged in HDPE. Commercial sample recorded poor microbial quality compared to laboratory prepared samples. Sample with 4% sucrose, 0.1% citric acid and 0.1% sodium benzoate was found to be the best among the treatment samples. At the end of the twelve weeks storage the microbial loads were found to be within the acceptable limits set by London Health Protection Agency and Public Health Laboratory Service. The results of the research also revealed that combination of sucrose, citric acid and sodium benzoate has the potential of eliminating the growth of Salmonella spp and Staphylococcus spp in Kilishi during ambient storage. The packaging materials used in this research has no effect on the bacterial group in the treatment samples.

REFERENCES

1. Agwu U. N. and Chisom O. U. (2014). Microbial Quality of Raw Meat Sold in Onitsha, Anambra State, Nigeria. International Journal of Science and Research (IJSR) Volume 3 Issue 2.

2. American Public Health Association (APHA, 1992). Compendium of Method for the Microbiological Examination of Foods 3rd Edition. Pp112-135.

3. Arora A. K., Shashi M. P.. and Sonney S.K. (2014). Is chemical safety to food hazardous? Dangers .of food preservatives. J Indian Acad Forensic Med, 3.1(4).

4. Badau MH, Igene JO, Collison EK, Nkama I (1997). Studies on Production, Physicochemical and Sensory Properties of a Standard Kilishi Ingredient Mix Powder. Int. J. Food Sci. Nutri., 48: 165-168.

5. Beney L., Perrier-Cornet J.M., Fine F. and Gervais P. (2003). Combining heat treatment, control of water activity and pressure to preserve foods. In; Peter Z. and Leif B. (Ed) Food preservation techniques. Woodhead Publishing Limited Cambridge England. Pp 179-198.

6. Bibek R. (2005). Fundamental Food Microbiology 3rdEdition. CRC Press London. Pp 498-500.

7. Brul S. and Coote P. (1997). Preservative Agents in Foods Mode of Action and Microbial Resistance Mechanisms. International Journal of Food Microbiology 50 (1999) 117

8. Buchanan R. E. and Gribbons N. E. (1974). Bergeys Manual of Determinative Bacteriology (8th edition). Williams & Wilkins Co.

   Baltimore USA

9. Chessbrough M. (2000). District Laboratory Practice in Tropical Country, Part 2. Cambridge University Press, Cambridge. Pp 64-70.

10. Daminabo V., Isu N. R. and Agarry O. O. (2013). Antibiotic Resistance Profile of Enterococcal Isolated from Dried Beef Crackers (Kilishi). Sky Journal of Microbiology Research Vol. 1(5), pp. 35 – 39, June, 2013.

11. Edema M. O., Osho A. T. and Diala C. I. (2008). Evaluation of Microbial Hazards Associated with the Processing of Suya (a grilled meat product). Scientific Research and Essay Vol. 3 (12), pp. 621- 626.

12. Fonkem D.N., Tanya V.N. and Ebangi A.L. (2010). Effects of Season on the Microbiological Quality of Kilishi, a Traditional Cameroonian Dried Beef Product. TROPICULTURA, 2010, 28, 1, 10-15

13. Isah O.A. and Okubanjo A.O. (2012). Effect of Various Additives on Proximate Composition and Acceptability of Kilishi made from Semitendinosus Muscle of White Fulani Cattle. The Pacific Journal of Science and Technology, Volume 13. Number 1. May 2012 (Spring).

14. James M. J. (2000). Modern Food Microbiology 6th Edition. Aspen Publishers, Inc. USA. P264.

15. Joerg S., Eva S., Andreas B. and Oliver H. (2007). Assessment of Meat Production and Meat Processing in Niamey/Niger: Hygienic Quality of Fresh Meat and of the Dry Meat Product Kilishi.Tropentag,

    October 9-11, 2007, Witzenhausen

16. Jones M. J., Tanya V. N., Mbofung C. M., Fonkem D. N. and Silverside D. E. (2001). A Microbiological and Nutritional Evaluation of the West African Dried Meat Product, Kilishi. The Journal of Food Technology in Africa. Vol. 6 no. 4.

17. Lee S. Y. (2004). Microbial Safety of Pickled Fruits and Vegetables and Hurdle Technology. Internet Journal of Food Safety, Vol. 4, 2004, p. 21-32.

18. Leistner L. (2011). Hurdle Technology. Encyclopedia of Life Support Systems (EOLSS), Food Engineering-Vol III

19. London Health Protection Agency (2009). Guidelines for Assessing the Microbiological Safety of Ready-to-Eat Foods. Placed on the Market. P 26.

20. Odey M. O., Mboso E. O., Ujong U. P., Johnson J. T., Gauje B. and Ategwu M. A. (2013). Microflora Analysis of Selected Meat and Meat Products from Calabar, Cross River State-Nigeria. Archives of Applied Science Research, 2013, 5 (3):50-56.

21. Ogbonnaya C. and Linus I. I. (2009). Influence of Storage Conditions on Shelf-Life of Dried Beef Product (Kilishi). World Journal of Agricultural Sciences 5 (1): 34-39, 2009.

22. Okonkow I. O., Odu N. N. and IGBOH I. E. (2013). Microbiological Analysis of Kilishi Sold In Port Harcourt, Nigeria. New York Science Journal 2013;6(7)

23. Oladapo A. S., Akinyosoye F. A. and Abiodun O. A. (2014). The Inhibitory effect of different Chemical Food Preservatives on the Growth of Selected Food Borne Pathogenic Bacteria. African Journal of Microbiology Research. Vol 8(14), Pp 1510-1515.

24. Panagiotis N. Skandamis G., John E. N., and John N. S. (2010). Meat Decontamination. In: Fidel T. (Ed) Handbook of Meat Processing.

    Blackwell Publishing. Pp 43-42

25. Public Health Laboratory Service (2000). Guidelines for the microbiological quality of some ready-to-eat foods sampled at the point of sale. Advisory Committee for Food and Dairy Products. (Comm Dis Pub Health 3(3):163-7, 2000).

26. Robert D. and Greenwood M. (2003). Practical Food Microbiology 3rd Edition. Blackwell Publishing Ltd Massachusetts, USA. Pp 243- 255.

27. Stanojevic D., Comic L., Stefanovic O. and Solujic-Sukdolak Sl. (2009). Antimicrobial Effects of Sodium Benzoate, Sodium Nitrite and Potassium Sorbate and their Synergistic Action In Vitro. Bulgarian Journal of Agricultural Science, 15 (No 4) 2009, 307-311.