EFFECT OF NISIN AND CINNAMON ON THE GROWTH AND SURVIVAL OF ENTEROTOXIGENIC METHICILLIN-RESISTANT STAPH. AUREUS IN STERILE MILK AND ICE CREAM

Dept. of Food Hygiene,

Faculty of Vet. Med., Assiut University.

Effect of nisin and cinnamon on the growth and survival of enterotoxigenic methicillin-resistant Staph. aureus in
sterile milk and ice cream

(With 4 Figures)

By

S. Nasr; Nagah M. Saad and Rania M. Ewida

(Received at 19/3/2009)

تأثير النيسين والقرفة على نمو وبقاء ميکروب المکور العنقودى الذهبى المفرز للسموم والمقاوم للميثيسيللين فى اللبن المعقم والآيس کريم

سعد الدين نصر ، نجاح محمد سعد ، رانيا محمد عويضه

تم دراسة تأثير ترکيزات مختلفة من النيسين (100، 200وحدة/مل) على نمو الميکروب المکور العنقودى الذهبى المفرز للسموم والمقاوم للميثيسيللين المحقون فى عينات اللبن المعقمة معمليا والمضاف إليها (1×⁷10/مل) من الميکروب والذى تم عزله مسبقاَ من عينات لبن الخام حيث حفظت العينات عند درجات حرارة الغرفة (20±2ºم) والثلاجة (4±2ºم) وقد تم فحص العينات بعد الحقن مباشرة وکل اثنتى عشرة ساعة. وقد أسفرت الدراسة على أن ترکيز النيسين ودرجة الحرارة هما العاملان المؤثران على بقاء هذا الميکروب ، ولم يتم عزل الميکروب بعد 72 ساعة فى الترکيز (200 وحدة/مل) بينما لم يتم عزله بعد 96 ساعة فى الترکيز (100وحدة/مل) من التخزين فى درجة حرارة الغرفة. وعلى الجانب الآخر لم يتم عزل الميکروب بعد 48و24 ساعة من التخزين فى درجة حرارة الثلاجة وعند ترکيزات (100، 200وحدة/مل) على الترتيب. وقد أشتملت الدراسة أيضا على دراسة تأثير القرفة بنسب 0.3و 0.6% على الميکروب المکور العنقودى الذهبى المقاوم للميثيسيللين والمفرز للسموم فى عينات الآيس الکريم  المصنع معمليا والتى تم حفظها تحت درجات تجميد -4±2 و -18±2ºم، وقد تم فحص العينات مباشرة بعد التصنيع واليوم الأول والثانى والثالث ثم أسبوعياًَ لمدة 4 أسابيع لعد ميکروب المکور العنقودى الذهبى. ولخصت الدراسة إلى أن درجة حرارة التجميد -18±2ºم مع الترکيز 0.6% کان لهما دور کبير فى تثبيط نمو الميکروب حيث أنه لم يتم عزله بعد أسبوع واحد من التخزين.

SUMMARY

The effect of nisin on the survival of enterotoxingenic methicillin-resistant Staph. aureus (MRSA) was evaluated using different concentrations of nisin (0.00, 100 and 200 IU/ml) in a laboratory prepared sterile milk and inoculated with the previously isolated and identified MRSA to yield a concentration of 1x10⁷ cfu/ml. The inoculated sterile milks were kept at room temperature (20±2ºC) and refrigerator temperature (4±2ºC). MRSAcounts were determined using Oxacillin Resistance Screen Agar Base (ORSAB) supplement with two antibiotics-oxacillin at (2 mg/L) and polymyxin B (50.000 IU/L). ORSAB and pH value were determined every twelve hours. MRSA strains couldn't be detected after 96 and 72 h in the samples of sterile milk containing nisin in concentrations of 100 and 200 IU/ml, while, in the control sample the MRSA survived till the end of the 96 h of storage at room temperature. Moreover, MRSA failed to be detected after 48 and 24 h in the samples of sterile milk containing nisin in concentrations of 100 and 200 IU/ml and storaed at refrigerator temperature, respectively. Ice cream was prepared at the laboratory to study the effect of ground cinnamon in concentrations of 0.3 and 0.6% on the growth and survival of enterotoxingenic MRSA a concentration of 2x10⁷ cfu/ml at freezing  (-4±2ºC) and deep freezing (-18±2ºC) temperatures. The obtained results showed that the advantage of using 0.6% is better than using of 0.3% of ground cinnamon.

Key words: Enterotoxigenic MRSA, milk, nisin, ice cream, cinnamon.

INTRODUCTION

MRSA infections are of the global health issues due to the severity of the illnesses that may occur. The roles and the sources of food contamination are still unclear since only few reports on the presence and possible origin of MRSA in foods are available. In his analysis of specimens from food-producing animals, including milk, Lee (2003) found 15 strains harbouring the mecA gene and most of the MRSA isolates were from milk. He concluded that contaminated foods of animal origin may represent a source of MRSA infection for humans. Kitai et al. (2005) suggested that food handlers were the source of contamination. This finding emphasized the role of humans as an important reservoir of MRSA. In the two foodborne acquired MRSA outbreaks, food handlers carrying the epidemic strains in their nostrils were identified to be the source of contamination of the incriminated food (Kluytmans et al., 1995 and Jones et al., 2002).

Food additive is a substance or mixture of substances, other than the basic food stuff, which are present in food as a result of any aspect of production, processing, storage, or packaging. Consequently, biopreservatives and natural preservatives are needed which posses antimicrobial activity and cause no problems to the handlers and consumers. In this respect, nisin and cinnamon were tested for their inhibitory activity towards the growth of some microorganisms and used as food additives.

Nisin is a natural antimicrobial peptide of 34 amino acids produced byLactococcus lactis subsp. lactis (Carr et al., 2002). The peptide is suggested to be effective against a wide range of Gram positive bacteria including Staph. aureus (Narasimhan et al., 1988 and Choi et al., 2000). In the U.S., nisin was confirmed to be "generally recognized as safe" in 1988 (F.D.A., 1988). Because of its high antibacterial activity and nontoxicity for humans, nisin has already been employed as food preservative for a long time and is licensed by 48 countries around the world (Deegan et al., 2006).

Cinnamon has been used since ancient times both as a culinary spice and for medicinal and other purposes. The medical properties of cinnamon were utilized by ancient Egyptians. Its mild anti-inflammatory, anti-spasmodic, and anti-clotting properties are believed to be due to its content of cinnamaldehyde. Cinnamon extracts have also inhibited the growth of cultured tumor cells(Craig, 2008). Moreover, cinnamon can be useful in the treatment of type 2 diabetes; as well as lowering triglyceride levels and serum cholesterol(Khan et al., 2003). Also, it is useful as a food preservative to inhibit the growth of common foodborne bacteria as Staph. aureus and MRSA (Chang et al., 2001; Yuste and Fung, 2003; Prabuseenivasan et al., 2006 and Ağaoğlu et al., 2007).

This work was undertaken to throw light on the effect of adding different concentrations of nisin and cinnamon on viability of enterotoxigenic MRSA in sterile milk and ice cream.

MATERIALS and METHODS

I- Effect of different concentrations of nisin on the growth and survival of enterotoxigenic MRSA in laboratory prepared sterile milk:

- The organism:

Enterotoxigenic MRSA strain used was previously isolated and well identified from the examined milk samples.

- Experimental procedure:

Raw milk was laboratory sterilized by autoclaving. The sterile milk inoculated with MRSA in concentration 1x10⁷cfu/ml and nisin was added in concentration of 0.00 (nisin-free), 100 and 200 IU/ml. Each sample was divided into two portions, the first portion was kept at room temperature (20±2ºC) and the other was stored at refrigerator temperature (4±2ºC). The effect of nisin on the growth and survival of MRSA was determined by using ORSAB after 3 h and then every 12 h and compared with nisin-free samples.

- Measurement of pH value:

The pH value of each sample was determined according to standard methods of A.P.H.A. (1992) with a pH meter (Orion Research model 3200 A/digital analyzer) previously standardized with buffer solutions of pH 3.0 and pH 7.0 buffer solutions.

II- Effect of different concentrations of ground cinnamon on the growth and survival of enterotoxigenic MRSA in laboratory made ice cream:

- Organism:

Enterotoxigenic MRSA strain used was previously isolated and well identified from the examined ice cream samples.

- Experimental procedure:

Ice cream samples were prepared in the laboratory according to the manufacture (Egyptian Dairy & Food Company) and then the mixture inoculated with MRSA culture to contain 2x10⁷ cfu/ml. After that, the ground cinnamon was added during manufacture of these samples in concentrations of 0.00 (cinnamon-free), 0.3 and 0.6%. Each sample was divided into two portions, the first portion was kept at freezing temperature (-4±2ºC) and the other was stored at deep freezing temperature (-18±2ºC).

Samples from the two portions were taken before and after addition of ground cinnamon to determine the initial count of the organism by using ORSAB, the count after hardening of ice cream samples, after the first, second and third day. Then, the samples were tested every week up to 4 weeks of storage for MRSAcount.

RESULTS

The obtained results were recorded in Figures 1-4.

Fig. 1: Effect of different concentration of added nisin on the growth and survival of MRSA in sterile milk stored at room temperature (20±2ºC).

Fig. 2: Effect of different concentration of added nisin on the growth and survival of MRSA in sterile milk stored at refrigerator temperature (4±2ºC).

Fig. 3: Effect of different concentration of added ground cinnamon on the growth and survival of MRSA in laboratory made ice cream stored at freezing temperature (-4±2ºC).

Fig. 4: Effect of different concentration of added ground cinnamon on the growth and survival of MRSA in laboratory made ice cream stored at deep freezing temperature (-18±2ºC).

Discussion

I- Effect of different concentrations of nisin on the growth and survival of enterotoxigenic MRSA in laboratery prepared sterile milk:

The data presented in Fig. 1showed that the count of MRSA in sterilized milk stored at room temperature containing nisin 100 IU/ml was increased to 4.0x10⁸ cfu/ml at the 3^rd h of the storage, then it reached 3.0x10⁷ at the 12^th h of the storage. Then at the end of first 24 h of the storage the count gradually decreased and reached 8.5x10⁵ cfu/ml. The count of MRSA decreased until the pathogen failed to be detected on the plate and could not be isolated at the end of 96 h. While, sterile milk containing nisin 200IU/ml, the count of MRSA increased 1.4x10⁸ cfu/ml after 3 h of storage. The count gradually decreased until the pathogen failed to be detected on the plate at the end of 72 h of the storage and failed to be isolated from the milk. On the other side, in control sample, MRSA increased from the initial count of 1x10⁷ to 4.5x10⁹ cfu/ml in 96 h. Gradual decrease of pH value of the sterile milk occurred from 6.40 to 5.62 in 0 time to the end of 96 h, respectively.

Result in Fig. 2depicted the effect of nisin on the survival of MRSA in sterile milk stored at refrigerator temperature (4±2ºC). The initial count of MRSA was 1.0x10⁷ cfu/ml decreased to 2.0x10⁶ and 3.0x10⁵ cfu/ml after 3^rd h after storage100 and 200 IU/ml nisin containing samples, respectively. The organism failed to be detected on the plate and could not be isolated from the samples containg 100 and 200 IU/ml nisin after 48 and 24 h storage at (4±2ºC), respectively. While, the pathogen survived in the sample free from nisin up to 48 h of the storage at refrigerator temperature. Slight decrease of pH of the sterile milk from 6.40 to 6.06 at the end of 48 h of the storage.

From the aforementioned results, it was evident that the increased concentration of nisin and the storage of the sterile milk at refrigerator temperature had an inhibitory effect on the survival of Staph. aureus. These results agree with the theory which pointed out that Staph. aureus is a robust bacterium and can survive for long period at low temperatures below those which permit growth. Yet, refrigeration at <4ºC may be considered the only viable method for control of growth and toxins production (I.C.M.S.F., 1996 and Ryser, 2001).

Also, the obtained results agree with thoserecorded by Rilla et al. (2004), who found the inhibitory effect of Lactococcus lactis subsp. lactisIPLA 729 on MRSA inoculated into milk in different inoculums (1.8x10⁴ and 7.2x10⁶ cfu/ml). While, MRSA when growing alone, it reached levels of 6.4x10⁸ and 1.9x10⁹ cfu/ml, respectively. In both cases, a slight growth of MRSA occurred within the first 3 h of incubation. However, complete disappearance of the pathogen occurred at the end of the incubation period.

Many investigators recorded the inhibitory effect of nisin on Staph. aureus as Aman and Ahmed (1997), they added nisin in concentration of 50, 100, 150, and 200 IU/ml of nisin in cheese milk had a highly bactericidal effect against Staph. aureus. Also, Narasimhan et al. (1988) and Choi et al. (2000) reported the inhibitory effect of nisin on Staph. aureus.

Numerous studies have been made on the mode of action of nisin on susceptible vegetative cells. The target of the inhibition effect of nisin is the cytoplasmic membrane where it depolarizes energized bacterial membranes (reduces transmembrane potential) and form voltage-dependent multistate pores. The result of a pore formation is the loss of accumulated amino acids and the inhibition of amino acid transport (Shehata, 1981). Delves (1990) indicated that nisin causes disruption, either resulting in leakage of essential cellular material such as adenosine triphosphate from the cell or in more severe cases lysis of the cell.

II- Effect of different concentrations of ground cinnamon on the growth and survival of enterotoxigenic MRSA in laboratory made ice cream:

In ice cream samples stored at freezing temperature (-4±2ºC) and containing 0.3% ground cinnamon (Fig. 3), the counts of MRSA diminished gradually from 1.1x10⁴ to 2.8x10² cfu/ml by the end of 3^rd week, and failed to recover from the samples by the end of the 4^th week. The ice cream samples containing 0.6% ground cinnamon, the counts of MRSA decreased gradually from 8.0x10³ to 3.0x10² cfu/ml by the end of the 2^nd week, and failed to be detected in the samples by the end of 3^rd week. Concerning cinnamon free ice cream, the number of MRSA decreased gradually during the storage period to reach its minimum level 6.0x10⁴ cfu/ml by the end of the 4^th week.

As presented in Fig. 4, the numbers of MRSA (4.7x10³ and 2.1x10³ cfu/ml) reduced in ice cream samples with 0.3 and 0.6% added ground cinnamon during the weeks of storage at the deep freezing temperature (-18±2ºC). No viable MRSA could be detected in the samples by the end of 3^rd and 1^st week, respectively.

These findings were in agreement with the theory that cinnamon had antibacterial activities against many of bacteria including methicillin resistant Staph. aureus (Chang et al., 2001). Also, many other investigators reported the inhibitory effect of cinnamon against Staph. aureusas Yuste and Fung (2003), Prabuseenivasan et al. (2006) and Ağaoğlu et al. (2007).

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 Figure 1. Effect of different concentration of added nisin on the growth and survival of MRSA in sterile milk stored at room temperature (20±2ºC).

 Figure 2. Effect of different concentration of added nisin on the growth and survival of MRSA in sterile milk stored at refrigerator temperature (4±2ºC).

 Figure 3. Effect of different concentration of added ground cinnamon on the growth and survival of MRSA in laboratory made ice cream stored at freezing temperature (-4±2ºC).

Figure 4. Effect of different concentration of added ground cinnamon on the growth and survival of MRSA in laboratory made ice cream stored at deep freezing temperature (-18±2ºC).