A STUDY ON THE EFFECT OF GARLIC AND NIGELLA SATIVA ON SOME FOOD POISONING BACTERIA ISOLATED FROM READY-TO-EAT MEAT SANDWICHES IN ALEXANDRIA CITY

Dept. of Food Hygiene,

Animal Health Research Institute, Alex., Lab., Egypt.

A STUDY ON THE EFFECT OF GARLIC

AND NIGELLA SATIVA ON SOME FOOD POISONING BACTERIA ISOLATED FROM READY-TO-EAT MEAT SANDWICHES

IN ALEXANDRIA CITY

(With 7 Tables)

By
 HANAA H.A. EL-MOSSALAMI;

 A.A. ABD-EL-RAHMAN* and MAGDY, M.E.**
* Dept. of Bacteriology, Animal Health Research Institute, Alex., Lab., Egypt.

** Dept. of Bacteriology, Animal Health Research Institute,

El-Mini., Lab., Egypt.
(Received at 14/9/2008)

دراسة تأ ثير الثوم وحبة البرکه على بعض بکتيريا التسمم الغذائى المعزوله من سندوتشا ت اللحوم الجاهزه فى مدينة الأ سکندريه

هناء حسينى أحمد سليم المسلمى ، عبد الرحمن عبد المجيد عبد الرحمن ، ايهاب محمود مجدى

 تم تجميع مائة عينه عشوائيه من سندوتشات السجق, البيف برجر, الشاورمه والکبده الجاهزه للأکل (25 من کل نوع) من بعض المطاعم والباعه الجائلين بمدينة الأسکندريه وذلک بهدف التعرف على الحاله البکتيريه لها من حيث العد الکلى للبکتيريا الهوائيه والعد الکلى لکل من ميکروب عصيات سيريس, السبحى البرازى والمکورالعنقودى الذهبى حيث أظهرت النتائج ان متوسط العد الکلى فى سندوتشات السجق, البيف برجر, الشاورمه والکبده کان (3.2± 16 x 10⁴ ), (2.3 ± 1.2 × 10⁴ ) , ( 4.2 ± 2.1 × 10⁴ ) و ( 3.6 ± 1.8× 10³ ) خليه/ جم بالترتيب وقد ثبت وجود فروق معنويه بين هذه المتوسطات وکانت نسبة العينات الموجبه لميکروب عصيات سيريس (100 , 92 , 84 , 72 )  %           و بمتوسطات ( 2.75 ±  1.4 × 10⁴ ), ( 2.4 ± 1.2 × 10² ), ( 35.2 ± 17.6 )      و ( 3.7 ± 1.8 × 10³  ) خليه /جم على التوالى. وقد کانت هناک فروق معنويه بين سندوتشات السجق والکبده من ناحيه وکل من البيف برجر, الشاورمه. وکانت نسبة العينات الموجبه لميکروب السبحى البرازى ( 88 , 84 , 88 , 76 ) % بمتوسطات ( 45.7 ± 5.9 × 10³ ) ,  ( 28 ± 7 × 10² ) ,  ( 3.1 ± 1.5 × 10² ) و ( 2 ± 0.6 × 10³ ) خليه/جم. وقد کانت هناک فروق معنويه بين سندوتشات السجق والبيف برجر من ناحيه وکل من الکبده والشاورمه. وکانت نسبة العينات الموجبه لميکروب المکورالعنقودى الذهبى       ( 92 , 80 , 88 , 80  ) %  و بمتوسطات ( 3.25 ± 1.6× 10³  ), ( 2.8 ± 1.4× 10² ), ( 4.1 ± 2 × 10³ ) و ( 4.8 ± 2.4 × 10³ ) خليه/جم لکل من سندوتشات السجق, البيف برجر, الشاورمه والکبده على التوالى. وقد کانت هناک فروق معنويه بين سندوتشات الکبده والشاورمه من ناحيه وکل من البيف برجر والسجق. وقد تم کذلک دراسة ترکيزات مختلفه من الثوم المهروس طازجا بترکيز ( 2 , 4 , 6 ) % وکذلک حبة البرکه المطحونه بترکيز ( 1 , 3 , 5 ) % مع اللحم المفروم ضد الثلاث ميکروبات المذکوره سابقا عند درجة حرارة الغرفه بحيث لاتزيد عن 16°م.  وقد وجد هناک فروق معنويه کبيره فى التعداد اللوغاريتمى للميکروبات فى آخر أيام التخزين ( اليوم الرابع) بين العينات المعامله بالترکيزات المختلفه والعينات التى لم تعامل. وقد تم مناقشة الاهميه الصحيه للميکروبات المعزوله والاحتياطات الواجب اتخاذها لتقليل تواجدها والتخلص منها أثناء اعداد السندوتشات لحماية المستهلک.

SUMMARY

One hundred random samples of ready-to-eat sausage, beef burger, shawarma and liver sandwiches (25 of each) were obtained from food restaurants and street-vendors with different sanitation levels in AlexandriaCity. The samples were examined bacteriologically for aerobic plate count, Bacillus cereus, Streptococcus faecalis and Staphylococcus aureus count. The obtained results revealed that the mean values of aerobic plate count of sausage, beef burger, shawarma and liver were 3.2± 16x10⁴ , 2.3± 1.2x10⁴ , 4.2± 2.1x10⁴ and 3.6± 1.8 x10³ cfu /g, respectively. Significant differences were detected among the four different sandwiches. Positive Bacillus cereus samples were 100, 92, 84 and 72% with mean values of 2.75± 1.4 x10⁴ , 2.4± 1.2x10² , 35.2± 17.6 and 3.7± 1.8 x10³ cfu /g for sausage, beef burger, shawarma and liver, respectively. There was a significant difference between sausage, liver and the other two tested sandwiches. S. faecalis was isolated from 88, 84, 88 and 76%, with mean values of  45.7± 5.9 x10³ , 28± 7x10² , 3.1± 1.5 x10² and 2± 0.6x10³ cfu /g for sausage, beef burger, shawarma and liver, respectively. There was a significant difference between sausage, beef burger and the other tested sandwiches. S. aureus was isolated from 92, 80, 88 and 80 %, with mean values of 3.25 ± 1.6 x10³ , 2.8±1.4 x10² , 4.1± 2x10³ and 4.8± 2.4 x10³ cfu /g, respectively. There was a significant difference between liver, shawarma and the other tested sandwiches. The effect of different concentrations of both freshly crushed Garlic 2, 4 & 6 % and Nigella sativa (NS)1, 3 & 5 % were tested against the isolated B. cereus, S. faecalis and S. aureus using minced meat stored at room temperature (not more than 16°C). Highly significant differences were recorded between the control and the treated samples. Public health significance and the quality of ready-to-eat meat sandwiches to protect the consumer were given.

Key words: Street vended foods, meat sandwitches, garlic, Nigella sativa

Introduction

Street vended foods are ready-to-eat foods prepared and sold by vendors on street. Street foods provide a source of readily available, nutritional meals, while providing a source of income for the vendors (Bryan et al., 1992; Swanepoel et al., 1995; Ekanem, 1998; Mosupye and Holy, 1999). The vendors congregate mainly in the central business district, nearby new building under construction and at major point of transit, where large numbers of minibus taxi that are headed for different destination. In Egypt, as in other developing countries, street-food-vending has increased markedly because of the increased unemployment rate and limited work opportunities.

Foods are often held for several hours after cooking until sold. This included, for some foods, overnight at ambient temperature, although reheating could alleviate some hazards but this action has not always been done effectively at vending site (Bryan et al., 1988; Bryan et al., 1992). Inadequate reheating is a frequent contributing factor to foodborne outbreaks (Bryan, 1978; Bryan et al., 1988). In contrast to these potential benefits, concerns over the safety and quality of street-vended foods have been raised because the vendors lack an adequate appreciation of basic food safety issues (Moy et al., 1997).

Health risks are associated with initial contamination of raw foods with pathogenic bacteria and subsequent contamination by vendors during preparation from cross-contamination, survival of pathogens during preparation, and microbial proliferation during display (Abou-Zaid et al., 2001; Soliman et al., 2002). The World Health Organization statistics indicated that food-borne diseases may be 300 to 350 times more frequent than the reported cases reveal (Saucier, 1999).

In countries in which street-food vending is prevalent, there is commonly a lack of information on the incidence of food-borne diseases related to street-vended food (Bryan et al., 1988). However, microbiological studies on street-vended foods in America, Asian and African countries have revealed high bacterial counts and high incidences of food-borne bacterial pathogens in food (Bryan et al., 1992; Bryan et al., 1997; Ekanem, 1998). In some cases, street-vended foods have been implicated in outbreaks of food-borne diseases (Dawson and Canet, 1991).

 The most common ready-to-eat sandwiches sold by street vendors and fast food restaurants are liver, sausage, brain, spleen, El-hawawshy and shawarma which is a popular meat sandwich in Middle East Countries (Ayaz et al., 1985).

Bacillus cereus is widely distributed in nature and can be isolated readily from a wide variety of foods in which it may be present normally. Its presence is insignificant, however, unless it is able to grow. Consumption of food containing millions of viable B. cereus cells per gram has resulted in outbreaks of food poisoning. Foods incriminated in past outbreaks of B. cereus poisoning included vanilla pudding, cooked meat, and vegetable dishes, boiled and fried rice (Geopfert, 1976).

The psychrotrophic strains of B. cereus, some capable of producing toxins and evenly grow in foods held at refrigeration temperature have been detected (Buchat et al., 1979; Ahmed et al., 1983; Wood and Waites, 1988; Sutherland and Murdoch, 1994) and those of public health concern. B. cereus causes two types of food-poisoning outbreaks, emetic and diarrhea. Several kinds of toxins produced by B. cereus strains, such as hemolysin, cytotoxin, emetic toxin and enterotoxins, have been studied (Johnson, 1984; Fagerlund et al., 2007).

Group D streptococci includes, enterococci as S. faecalis and S. faecium and non enterococci. Enterococci were isolated from shawarma samples collected from fast-food restaurants in Assiut city (Refaie and Moustafa, 1990; Mohamed et al., 2004; Ismail, 2006).

Improper holding of meat products after cooking lead to growth of staphylococci reading without competition with other organisms which have been killed by heat treatment and produced enterotoxin resulting in a staphylococcal food poisoning (Brandly, 1977). The Staphylococcus aureus enterotoxins are produced during active growth of the microorganisms in the foods and often during storage. Each enterotoxin is a single poly-peptide chain which resist many proteolytic enzymes and with stands cooking for up to 30 minutes (heat stable) although the vegetative cells would not survive such conditions (Eley, 1992; Baeza etal., 2007).

Nigella sativa (black cumin, black seeds) is important as a cytoprotective herb due to its content of thymoquinone. It is found to increase hepatic GST, as antibacterial, anti-helmintic, antifungal (Toppozada et al., 1965; Agarwal et al., 1979; Hanafy and Hatem, 1991; Hosseinzadeh et al., 2007), immuno-stimulant (El-kadi et al., 1990; Abd-El-Moniem, 1999), anticarcinogenic (Salomi et al., 1992; EL-Gendy et al., 2008), antidiabetic (Abd-El-Aziz et al., 1995), antihistaminic (Chakravarty, 1983), anti-inflammatory, antioxidant and hepatoprotective (Houghton et al., 1995; Youssef and Ashry, 1999). NS crushed seeds supplement stimulate the thyroid gland directly and/or through the pituitary level hence anabolic effect is expected (Khodary et al., 1996). It is a spicy plant and used as flavouring agent for bakery products (Saleh et al., 2002). The antimicrobial activity of Nigella sativa seeds were reported by Mahmoud (1993), Sabreen (1996), Abdel-Kader et al., (2001) and  AL-Beitawi and EL-Ghousein, (2008).

Garlic (Allium sativum), family lilaceae is widely distributed and used in all parts of the world as a spice and herbal remedy for prevention and treatment of variety of diseases due to its active substance Allicin which possesses its characteristic odour (Kamal and Daoud, 2003). Allicin has a broad spectrum antimicrobial effect, it appears to inhibit sulfhydryl enzyme in wide variety of bacteria (Council for Agricultural Science and Technology, 1998; Aydin et al., 2007).

This study was aimed to: 

1- Determine the total bacterial count in some popular ready-to-eat sandwiches (sausage, beef burger, shawarma and liver) in Alexandria city.

2- Investigate the occurrence of B. cereus, S. faecalis and S. aureus in the examined sandwiches.

3- Study the effect of different concentrations of fresh minced garlic and black seed on the viability and survival of the isolated strains of B. cereus, S. faecalis and S.  aureus.

MATERIALS and METHODS

1- Collection of samples:

One hundred ready-to-eat sandwiches were obtained from fast food restaurants and street-vendors with different sanitation levels in AlexandriaCity. Sandwiches types evaluated were sausage, beef burger, shawarma and liver (25 of each). All samples were obtained aseptically in sterile polyethylene bags. All samples were analyzed immediately after transporting to the laboratory for enumeration of:

a- Aerobic plate count (ICMCF, 1978).

b- B.cereus count (Harmon and Goepfert, 1984), the technique of surface spread method was applied using Bacillus cereus selective agar media.

c- S. faecalis count (Deibel and Hartman, 1984).

d- S. aureus count  (ICMSF, 1978) the same technique of surface spread method was applied using Baird-Parker agar media.

2- Preparation of samples:

This was done as recommended by APHA, (1984).

3- Experimental test:

The effect of different concentration of garlic and N. sativa on the growth rate of the strains (B. cereus, S. faecalis and S. aureus isolated from ready-to-eat sandwiches) in minced meat stored at room temperature (not more than 16°C) for 4 days was conducted.

Minced meat samples were prepared according to Hefnawy et al., (1993). Meat samples were purchased from local supermarket aseptically in sterile polyethylene bags and divided aseptically into three equal parts, each part was transferred into a sterile glass blender jar. One part was inoculated with strain culture of B. cereus, the second part was inoculated with strain culture of S. faecalis and the third part was inoculated with strain culture of S. aureus as described below.

Test organism:

The organism was grown in 10 ml of nutrient broth at 37°C/ 24h. The culture was decimally diluted and plated to enumerate the organisms present. The culture was diluted to achieve an inoculum level of 10⁴/g for B. cereus and 10⁶/g for S. faecalis and S. aureus.

Each strain culture was thoroughly mixed with minced meat, then divided into 7parts each part (100g) in sterile beakers. The first one  was considered as a control, while the 2^nd , 3^rd and 4^th were mixed with 2, 4 and 6% of garlic (freshly crushed garlic) and 5^th , 6^th and 7^th beakers were mixed with 1, 3 and 5% of  N. sativa (freshly ground seeds), respectively. All beakers were covered with aluminium foil and stored at room temperature (not more than 16°C) and daily examined for B. cereus, S. faecalis and S. aureus.

RESULTS

 Table 1:  Statistical analysis of aerobic plate count /g of the examined ready- t0-eat sandwiches (n=25 of each).

* Means have the same letters are not significant (P<0.05).

** S.E. = Standard Error.
*** Average of trials

Table (2):  Statistical analysis of B. cereus count /g of the examined ready-to-eat sandwiches (n= 25 of each).

* Means have the same letters are not significant (P<0.05).

** S.E. = Standard Error.
*** Average of trials

Table 3:  Statistical analysis of S. faecalis count /g of the examined ready-to-eat sandwiches (n= 25 of each).

* Means have the same letters are not significant (P<0.05).

** S.E. = Standard Error.
*** Average of trials.

Table 4:  Statistical analysis of S. aureus count /g of the examined ready-to-eat sandwiches (n= 25 of each).

* Means have the same letters are not significant (P<0.05).

** S.E. = Standard Error.
*** Average of trials.

Table 5: Effect different concentrations of Garlic and  Nigella sativa on B. cereus.
Log₁₀  cfu /g havethe same letters are not significant (P<0.05).

Table 6: Effect different concentrations of Garlic and  Nigella sativa on S. faecalis.

Log₁₀  cfu /g havethe same letters are not significant (P<0.05).

Table 7: Effect different concentrations of Garlic and  Nigella sativaon S. aureus.

Log₁₀  cfu /g havethe same letters are not significant (P<0.05)

DISCUSSION

The results recorded in Table (1), reveal that the aerobic plate count of the examined ready-to-eat sausage sandwiches ranged from 3x10³  to  4.5 x 10⁵  with a mean value of 3.2x10⁴± 16 x 10⁴  cfu/g.Our results are lower than those of  Abou-Zaid et al., ( 2001) who recorded  3x 10⁸ cfu/g and higher than those of Soliman et al., (2002) who recorded 3.2x10² cfu/g.

As regarded to beef burger ready-to-eat sandwiches, the total aerobic count varied from 2.7x10³ to 3.3x10⁵ with a mean value of 2.3x10⁴± 1.2x10⁴ cfu/g. Nearly similar results were obtained by  Mohamed (1997), while lower results were obtained by Mohamed (1985), Gill et al., (1996 ) and Soliman et al., (2002).

Although the aerobic plate count of any food article is not a sure indicative of its safety for consumption, yet it is of supreme importance in judging the hygienic conditions under which it has been produced, handled and stored (Levine, 1961).

    Concerning shawarma sandwiches, the APC varied from 3.3x10³ to 5.8x10⁵ with a mean value of 4.2x10⁴± 2.1x10⁴ cfu/g. These results are lower than those reported by Ayaz et al., (1985), Refaie and Moustafa, (1990) and Ebraheem, (2001) who recorded a mean APC of 10² to 3.0x10⁶, 24.6x10⁷ and 67.88± 31.7x10⁶ cfu/g ready-to-eat shawarma sandwiches, respectively. On the other hand, Morshedy et al., (1986) and Mohamed et al., (2004) recorded higher results (38x10² and 29± 4.9x10³ cfu/g, respectively). 

The results recorded in Table (1), reveal that the aerobic plate count of the examined ready-to-eat liver sandwiches ranged from 2.4x10² to 4.0x10⁴ with a mean value of 3.6x10³± 1.8x10³ cfu/g. Nearly similar results were obtained by Mohamed et al., (2004) (27±18x10³ cfu/g). Higher counts were recorded by Hegazy (1999), Ebraheem (2001) and Abou-Zaid et al., (2001) were the last one recorded a mean value of 2x10⁷ cfu/g.

Table (1), also showed that there were significant differences     (P >0.05) among the four different sandwiches studied. The highest contaminated sandwiches were shawarma and sausage. Bacterial counts in the sandwiches analyzed in the present study were found to be lower than those reported by Refaie and Moustafa, (1990). However, the APC recorded by Mouspye and Holy, (1999) in meat samples collected from street-vendors in Johannesburg, South Africa were lower than ours  (3.1± 0.9 log cfu/g). However, in other countries as Pakistan, Zambia and Nigeria higher results were recorded for street-vended cooked meals (Bryan et al., 1992; Ekanem 1998; Mouspye and Holy, (1999).

According to the microbiological standard for hot meals recorded by Lufthansa and Swiss Air Service, (1986) it is clearly evident that the means of APC for sausage, beef burger, shawarma and liver were less than 10⁵ cfu/g which is considered within the permissible limits.

Table (2), shows that the minimum, maximum and means of B.cereus counts in sausage ready-to-eat sandwiches were 2.5x10³, 3.4x10⁵ and 2.75x10⁴± 1.4x10⁴ cfu/g, respectively. B.cereus was isolated from 100% of sausage sandwiches. Our results were higher than those of Abou-Zaid et al., (2001) who recorded 40% positive samples with a mean value of 7x10⁴cfu/g and Soliman et al., (2002) who reported 3.1x10² cfu/g of ready-to-eat sausage sandwiches.

The recorded data in Table (2), showed that 92% of the examined ready-to-eat beef burger sandwiches  were positive for B.cereus with count ranged from 2 x10 to 3.1x10³and a mean value of 2.4x10²± 1.2x10² cfu/g. Nearly similar results (2.51x10² cfu/g) were obtained by Soliman et al., (2002).

B.cereus was isolated from 84% of the examined shawarma sandwiches with count ranged from 2.7x10 to 4.0x10² with a mean value of 35.2± 17.6 cfu/g. However, higher incidence (88%) and count (2x10³± 4x10² and 28± 7x10² cfu/g) were reported by Nassar et al., (2003) and Mohamed et al., (2004).

In Table (2), the minimum, maximum and the means of B.cereus counts in liver ready-to-eat sandwiches were 2.8x10 to 4.2x10⁴ and 3.7x10³± 1.8x10³ cfu/g, respectively. Nearly similar results were obtained by Abou-Zaid et al., (2001) (10³cfu/g) and Mohamed et al., (2004) (2± 0.6x10³ cfu/g). Hegazy, (1999) recorded little higher count (10⁴± 6x10³ cfu/g), while Ebraheem, (2001) recorded 100% positive samples with a mean value of 99.6± 7.9 cfu/g.

The results obtained in this study showed significant differences (P>0.05) the in the mean values of B.cereus count among four different sandwiches studied. The highest contaminated sandwiches were sausage and liver (Table 2).

  Members of the genus Bacillus are ubiquitous soil microorganisms and are generally considered harmless contaminants. However, a few species are known as toxin producers, including the food borne pathogen, B.cereus.  It is observed from the achieved results that B.cereus contaminated very high percentage of the examined ready-to-eat sandwiches and this may due to the fact that B.cereus is widely distributed in nature. Kramer and Gilbert, (1989) stated that B.cereus population between 10⁵ and 10⁷cells/g of food is required to produce intoxication. Accordingly, the level of contamination with B.cereus appeared to be not significant to public health.

As recorded in Table (3), Streptococcus faecalis were existed in the examined ready-to-eat sausage sandwiches in numbers varied from 12x10³ to 12x10⁴ with a mean value of 45.7x10³± 5.9x10³ cfu/g, the count of S. faecalis in beef burger ranged from 10² to 11.4x10³ with a mean value of 28x10²± 7x10² cfu/g, In shawarma from 2.7x10 to 3.5x10³ with a mean value of 3.1x10²± 1.5x10² cfu/g and from 10 to 9x10³ with a mean value of 2x10³± 0.6x10³ cfu/g in the tested ready-to-
eat liver sandwiches, respectively.                                                                                                                        

A higher results in both count and percentage of positive samples were recorded by Elwi, (1988) who reported 5x10⁵ cfu/g and nearly similar results were obtained by Mohamed et al., (2004) with a mean value of 26.7± 6x10² cfu/g for cooked liver. Morshedy et al., (1986) and Ebraheem, (2001) recorded nearly similar count (6x10² cfu/g) in shawarma but, Mohamed et al., (2004) reported a mean value of 28± 7x10² cfu/g which seemed to be higher.

Significant differences in the means of S. faecalis count between sausage, and beef burger and both liver and shawarma sandwiches were observed. The highest contaminated sandwiches were sausage, and beef burger (Table 3)

Mohs (1972) reported a level of <10³ /g of S. faecalis as an acceptable bacteriological quality in cooked foods. The examined beef burger and shawarma sandwiches comply with this limit. The presence of S. faecalis in the examined ready-to-eat sandwiches may be due to post-processing contamination or heat resistant character of the organism.

Table (4),shows that the minimum, maximum and the means of Staphylococcus aureus counts in sausage ready-to-eat sandwiches were 2.7x10² , 4.15x10⁴ and 3.25x10³± 1.6x10³ cfu/g, respectively. Our results were higher than those of Abou-Zaid et al., (2001) who reported 4x10² cfu/g and Soliman et al., (2002) who recorded a mean value of 3.25x10² cfu/gof ready-to-eat sausage sandwiches. As for ready-to-eat beef burger sandwiches 80% were positive for S. aureus where the count ranged from 2.3x10 to 3.95x10³ with a mean value of 2.8x10²± 1.4x10² cfu/g. Nearly similar findings 2.1x10² cfu/g were obtained by Soliman et al., (2002), while higher results were reported by Mohamed, (1997).

Hoshyar et al., (1984) and Mohamed, (1985) obtained nearly similar results as regard S. aureus.  Growth of S. aureus can occur either before or during processing of meat so that the meat and its products have been implicated in several food poisoning outbreaks (Niskanen, 1977). While S. aureus in food indicates its contamination from the skin, mouth or nose of workers handling and inadequately cleaned equipments (Thatcher and Clark, 1978).  

It is worth to mention that 88% of the examined ready-to-eat shawarma sandwiches were positive for S. aureus with count ranged from 3.4x10² to 5.2x10⁴ and a mean value of 4.1x10³± 2x10³ cfu/g. Soliman et al., (2001) recorded low results 2x10² cfu/g (Table 4). However, ready-to-eat liver sandwiches (80%) contained S. aureus ranged from 3.7x10² to 6x10⁴ and a mean value of 4.8x10³± 2.4x10³ cfu/g. These results were lower than those of Soliman et al., (2001) who reported 10² cfu/g. Significant difference was observed in the means of  S. aureus count among beef burger and sausage, shawarma and liver.The highest contaminated sandwiches were liver and shawarma (Table4).

Tables (5,6&7), show the effect of different concentrations of Garlic and Nigella sativa on B. cereus, S. faecalis and  S. aureus isolated from ready-to-eat sandwiches, in minced meat stored at room temperature (not more than 16°C). The effect of 2, 4 and 6% Garlic decreased the numbers of B. cereus by 4.3 orders of magnitude in the 4^th, 3^rd and 3^rd days of storage comparable to zero time and 6.3 and 6.5 orders of magnitude in the 3^rd and 4^th days of storage comparable to control, respectively. While these concentrations decreased the numbers of S. faecalis 1.2, 1.5 and 1.9 orders of magnitude in the 4^th day of storage comparable to zero time and 2.9, 3.2 and 3.6 orders of magnitude comparable to control in the last day of storage, respectively (Table 6). On the other hand, these concentrations decreased the numbers of S. aureus 1.2, 1.6 and 1.8 orders of magnitude in the 4^th day of storage comparable to zero time 2.7, 3.1 and 3.3 orders of magnitude comparable to control in the last day of storage, respectively (Table 7).

No significant differences were observed between the different concentrations of N. sativa on the 3^rd day of storage on B. cereus (Table 5) and of the numbers of organisms in the last day of storage decreased by 4.3 orders of magnitude comparable to zero time and by 6.5 orders of magnitude comparable to the control regardless to the concentration of N. sativa used with B. cereus. Table (6), show that the numbers of S. faecalis decreased by 0.5, 1.2 and 1.6 log cycles at concentrations of 1, 3 and 5% of N. sativa comparable to zero time and by 2.2, 2.9 and 3.3 log cycles as compared with control in the last day of storage, respectively. Finally, Table (7) show that the numbers of S. aureus decreased by 0.5, 1.3 and 1.7 log cycles at concentrations of 1, 3 and 5% of N. sativa comparable to zero time and by 2, 2.8 and 3.2 log cycles as compared with control in the last day of storage, respectively. Our results are in agreement with those obtained by Allatif and Ibraheem, (1996). The antibacterial effect of both Garlic and N. sativa was recorded in many other studies (Mahmoud, 1993; Council for Agricultural Science and Technology, 1998; Abdel-Kader et al., 2001; Mohamed et al., 2004).

In these studies, high bacterial counts and high incidence of food-borne pathogens have been attributed to a process by which food are held for long period of time at temperature of 46°C. And also, it was reported that vendors cooked their foods on temperature lower than 95°C, but they then held the food at ambient temperature for more than 6 h. and sometimes even held it overnight without reheating (Bryan et al., 1997). They also added that the presence of contaminating bacteria in the food was attributed to cross-contamination from environmental sources and to handling by vendors during holding. High percentage of these samples contained high number of B. cereus, S. faecalis and S. aureus, it is indicated improper sanitation and neglected hygienic measures during preparation causing sandwiches of inferior quality, unfit for consumption, index of fecal contamination and possibility results of presence of enteric pathogens.

From the previous results we concluded that Garlic and N. sativa play a significant role in prevention of bacterial growth, so must be added to sandwiches to improve the quality of ready-to-eat sandwiches. Services the ten golden rules drawn by (WHO, 1989). Educational programs and training courses should be recommended to the meat handlers and workers. Periodical cleaning of clothes, utensils, hands especially after visiting toilet and protect foods from (dust, insects, avoid keeping of warm below 60°C and food may only be stored for a limited time). Consumers should be informed about the risk of foods contamination during the months of year (Gongal, 1998).

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