IDENTIFICATION OF ENDOSPORE-FORMING BACTERIA ISOLATED FROM WEANING DRIED FOODS الملخص العربي : أجريت الدراسة على 64 عينة من أغذية الفطام الجافة تم تجميعها من الأسواق المحلية والصيدليات في مدينة تعز بالجمهورية اليمنية. شملت تلک العينات 24 عينة من أغذية الفطام التجارية وأغذية الفطام المحضرة بالطريقة التقليدي

d from Weaning Dried Foods     

Consumed in Taiz City, Republic of Yemen

Fahd A. Alsharjabi* and Amani M. Al-Qadasi

Department of Applied Microbiology, Faculty of Applied Sciences, Taiz University, Taiz, Yemen

*Corresponding author: f_alsharjabi@yahoo.com

ABSTRACT:

The present work was carried out on 64 random weaning dried food samples collected from local markets and pharmacies in Taiz city, Republic of Yemen. These samples included 24 commercial (CWF) and traditional (TWF) weaning food samples, in addition to 40 cereals and legumes samples comprised the ingredients of traditional weaning dried foods. The total bacterial isolates resulting from identification of mesophilic aerobic endospore-forming bacteria were 77 isolates. These isolates represent about 40.10% of studied samples comprised 15 isolates from the CWF (41.7%), 18 isolates from the TWF (50%) and 44 isolates (36.67%) from cereals and legumes samples. Ten Bacillus species and one species of Brevibacillus, Paenibacillus and Virgibacillus were identified.

Key words: Endospore-forming bacteria, Bacillus species, weaning food, cereals, legumes.

1. INTRODUCTION

Weaning foods include commercial and traditional weaning foods products. Commercial weaning foods are easy to prepare, hygienic provided it is packaged, but expensive and not available everywhere locally. However, traditional weaning foods are cheaper, always available locally (Castel & Wijngaart, 2005). Weaning foods made from ingredients do not differ from these for adult foods so that the same types and levels of microorganisms would occur on these ingredients naturally (ACMSF, 2006). Soil, water, air, dust, insects, rodents, birds, animals, humans, storage and shipping containers, and handling and processing equipment are the most important sources of the contaminated cereal grains (Bullerman & Bianchini, 2009). The most important factors associated with contamination of weaning foods are the preparation of food several hours prior of consumption, inadequate storage conditions, and insufficient cooking or reheating of stored food before feeding (WHO, 1984). Traditional weaning foods in Yemen are cereals-legumes blending, mostly composed of wheat, maize, barley, red and white sorghum, millet and rice blending in equal quantities, besides adequate amount of one this legumes: black or red lentil or both and sometimes fewer quantities of beans. Traditional weaning foods in Yemen are cereals-legumes blending, usually prepared as composite soft gruels and sometimes baked as cookies. Many literatures pointed out that the most commonly bacteria found surviving on weaning foods and cereals, the major components of weaning foods, and their flours are species of the spore-former Bacillus (Barrell & Rowland, 1980; Singh et al., 1980; Michanie et al., 1987;Becker et al., 1994; Afifi et al., 1998; Ikah et al., 2001; Sheth & Arora, 2001; Berghofer et al., 2003; Amusa et al., 2005; Badau, 2006; Ifediora et al., 2006; Aydin et al., 2009; Abanno et al., 2012; Adebayo-Tayo et al., 2012; Nwogwugwu et al., 2012; Bintu et al., 2015; Degaga et al., 2015 andNworie et al., 2016).

This work was carried out to identify Bacillus spp. isolated from tested samples in an attempt to shed light upon the microbial status of traditionally weaning dried foods consumed in Taiz City, Republic of Yemen.

1. MATERIALS AND METHODS

2.1.   Samples collection

A total of 64 random weaning dried food samples (commercial, traditional and it's ingredients) were collected from local markets and pharmacies in Taiz city. These samples including 12 commercially weaning food samples, 12 traditionally weaning food samples and 40 cereal and legume samples (wheat, barley, corn, rice, millet, red sorghum, white sorghum, black lentil, red lentil and bean) as a total 4 samples from each kind of cereal and legume. Each sample was put in sterile polyethylene bag and transferred to the laboratory, where they were prepared for the microbiological examination.        

2.2.  Samples preparation

Twenty five grams of each sample were added to 225 ml of peptone water. Decimal dilution was prepared and spread on the appropriate plates (Harrigan & McCance, 1976).

2.3.  Isolation and identification of mesophilic aerobic endospore forming bacteria:

Mesophilic aerobic endospore forming bacterial count were determined according to method described by Priest (1989) as follow: the suitable dilution was subjected to 85^oC at 10 min and cold at 45^oC. After that,1 ml of the dilution was transferred to a petri dish with tryptone glucose extract (TGE) agar and incubated for 48-72hr. The second step was subculture on slant agar for identification process as mentioned by Priest and Alexander (1988).

The PIBWin (Probabilistic Identification of Bacteria for Windows) program software was applied to provides probabilistic identification of unknown bacterial isolates against identification matrices of known strains (Bryant, 2004) (Figure no.1), and data matrix described by Priest and Alexander (1988) (Figure no.2), were used for identification of mesophilic aerobic endospore-forming bacteria isolates.

Figure 1:The PIBWin program software that provides probabilistic identification of unknown bacterial isolates against identification matrices of known strains.

Figure 2:The data matrix described by Priest and Alexander (1988) used for identification of mesophilic aerobic endospore-forming bacteria isolates.

1. RESULTS AND DISCUSSION

The average ofmesophilic aerobic endospore forming bacterial counts were 2.44 ± 0.57 and 2.35 ± 0.46 log₁₀ CFU g^-1 for TWF and CWF, respectively. Such results are in general coinciding with those reported by Ikah et al. (2001); Ifediora et al. (2006) andBintu et al. (2015).

The total mesophilic aerobic endospore forming bacterial counts in cereals and legumes were 2.26 ± 0.48 log₁₀ CFU g^-1 that varied from 2.73 ± 0.92 log₁₀ CFU g^-1 in rice to 1.87 ± 0.27 log₁₀ CFU g^-1 in red sorghum samples. Berghofer et al. (2003) reported that the mesophilic aerobic spore counts were 10 and 1 CFU/g for Australian wheat and flour, respectively. These differences can be attributed to the differences in the storage conditions of these crops. The data presented in Tables No.1 & No.2 showed the results of mesophilic aerobic endospore-forming bacteria isolates identification by the PIBWin program software. Ten Bacillus species and one species of Brevibacillus, Paenibacillus and Virgibacillus in addition to three unnamed taxa i.e. Taxon 18, Taxon 27 and Taxon 28 were recorded from these investigation samples. A total of 77 isolates, 38 isolates of Bacillus spp., 10 isolates of Brevibacillus laterosporus (formerly Bacillus laterosporus),7 isolates of Virgibacillus pantothenticus (formerly Bacillus pantothenticus), 2 isolates of Paenibacillus alvei (formerly Bacillus alvei) and 20 isolates of unnamed taxa (3 of Taxon 18, 15 of Taxon 27 and 2 of Taxon 28) were identified in our study in 40.10% of studied samples comprising 15 isolates from CWF (41.7% of CWF total), 18 isolate from TWF (50% of TWF total) and 44 isolate (36.67%) from cereal & legume samples. Similar trend of results reported for isolation of Bacillus from different weaning food samples as these reported by Ikah et al. (2001); Ifediora et al. (2006); Abanno et al. (2012); Nwogwugwu et al. (2012); Bintu et al. (2015); Degaga et al. (2015)andNworie et al. (2016).

In depending on the number of isolate cases, B. coagulans was the most frequently species, it was isolated from 21.9% of the total samples comprising 41.7% of the TWF samples (in samples No.T2a, T2b, T3, T4a, T4_b, T6, T12) and 22.5% of cereal samples in samples No.2 (wheat), No.10 and 12 (barley), No.15 (millet), No.26 (rice), No.29 (red lentil), No.33a, 33b, 34 and 35 (black lentil). This species was not reported from CWF samples. Such results are in good accord with Badau (2006) who isolated Bacillus coagulans from pearl millet. The second rank in the number of isolation cases was occupied by Taxon 27 that occurred in 18.8% of samples contributing 16.7% of CWF samples (samples No.C8 & C9), 41.7% of TWF samples (samples No. T4a, T4_b, T7, T8, T9 & T11) and 12.5% of cereal samples in samples No.7 (maize), No.12 (barley), No.17a,17b,18a and 18b (white sorghum) and No.26 (red sorghum).

Bacillus cereus and Brevibacillus  laterosporus  came behind Taxon 27 in the number of cases of isolation. B. cereus was recorded in samples Nos. C4, C9, C10, C11 and C12 from the CWF samples (41.7%), also in samples No.4 (wheat), No.11 (barley), No.20 (white sorghum), No.21b (red sorghum) and No.29 (red lentil) in 12.5% of cereal samples but was not recorded in the TWF samples. Some studies mentioned to isolation of B. cereus such as those reported by Barrell & Rowland (1980); Singh et al. (1980); Michanie et al. (1987); Sheth & Arora (2001) and Amusa et al. (2005) from the weaning food samples, as well as Berghofer et al. (2003) and Aydin et al. (2009)  from wheat; Badau (2006) from  pearl millet.

Brevibacillus laterosporus was isolated from one sample (8.3%) of the CWF samples, 16.7% of  the TWF samples in samples Nos.T6a, T6b & T12 and from 15% of cereal samples in samples No.2 (wheat), No.15 (millet), No.18 (white sorghum),  No.30 (red lentil),  No.34 (black lentil) and  No.37 (bean).  The next species was Virgibacillus pantothenticus that isolated from 10.9% of samples comprising 16.7% of CWF samples in samples Nos.C9 and C11, also from 12.5% of cereal samples in samples No.1 (wheat), 14 (millet), 18 (white sorghum), 28 (rice),  and 35 (black lentil). This kind of Bacillus species was not recorded in the TWF samples.

Bacillus amyloliquefaciens was isolated from 9.4% of samples comprising  16.7% of the CWF samples in samples Nos.C1 & C2 and from 10% of the cereal samples in samples No.5 (maize), 11 (barley), 26 (rice) and 37 (bean).

Taxon 18 was isolated from 4.5% of the three types of samples in sample No.C10 (CWF), T8 (TWF) and 13 (millet).

Bacillus baduis, Paenibacillus alvei, and Taxon 28 were isolated from 3.1% of samples.  B. baduis was isolated from one sample of the CWF (sample No.C2) and one sample of cereal samples (sample No.26 in rice), also Paenibacillus alvei was isolated from one sample of CWF (sample No.C7) and one sample from cereal samples (sample No. 1 in wheat). Whereas, Taxon 28 was isolated from two samples of cereal (samples No.13 & No.21a in millet and red sorghum, respectively).

         The remaining species were isolated one time from one sample, B. pumils was recorded from TWF in sample No.T12, B. subtilis ssp. Subtilis from wheat in sample No.4, and B. stearothermophilus from red lentil in sample No.30. Amusa et al. (2005); Adebayo-Tayo et al. (2012) and Bintu et al. (2015) reported isolation of B. subtilis from weaning foods samples. As well as millet (Badau, 2006).

Table 1:Mesophilic Aerobic Endospore-forming Bacteria Isolates recorded from the Investigated Samples Identified by PIBWin program software.

Table 1:  Continued:

Table 2:Number of Cases of Isolation (NCI) of the Mesophilic Aerobic Endospore-forming Bacteria Recovered from Investigated Samples.

1. CONCLUSION:

From these results, it could be concluded that the total bacterial isolates of mesophilic aerobic endospore-forming bacteria from investigated samples were 77 isolates, and that were occurred in 40.10% of studied samples comprised 15 isolates from CWF (41.7%), 18 isolates from TWF (50%) and 44 isolates (36.67%) from cereals and legumes samples. Ten Bacillus species and one species of Brevibacillus, Paenibacillus and Virgibacillus were identified.

Consequently we recommended that the sufficient cooking for these foods is a critical point to reduce the microbial load to safe limit. As well as the important of preparation this kind of food under hygienic condition that include, hand washing, cleaned utensils, and  pure water.

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