Antibacterial effects of Mustard, Garlic and Acetic acid against Salmonella typhimurium in ground beef

Antibacterial effects of Mustard, Garlic and Acetic acid against Salmonella typhimurium in ground beef

Amany M. Shalaby

Animal Health Research Institute

Dokki, Giza

Port said Lab of Food Hygiene

Summary

This study was designed to evaluate the effect of mustard flour, garlic powder and acetic acid either individually or in combined effects on Salmonella typhimurium. Samples treatments were prepared to achieve various concentration by addition of 5% and 10% of mustard flour and garlic powder, while acetic acid was added by 0.5% and 1%. The treatments were added to ground beef samples after inoculation with Salmonella typhimurium and stored at 4^° C.Samples were followed up after 0, 24, 48 and 72 h. The obtained result indicated that garlic powder showed the greatest antibacterial activity followed by acetic acid then mustard flour which showed lowest and slowest reduction rate of the tested organism. All combining effect showed remarkable reduction rate where salmonella typhi can't be detected after 24 h of inoculation in combining effect of mustard 5 %, garlic 5 % and acetic acid 0.5% also in mustard 10%, garlic 10% and acetic acid 1%.

Introduction

        Few studies have been addressed the use of spices and some organic acids against pathogens associated with meat. Since Salmonella typhimurium (S.typhi) is consider as one of food borne pathogens which are found in a wide variety of foods specially meat and meat products. The organism is the most commonly isolated Salmonella serotype, accounting for 23% of Laboratory confirmed Salmonella cases that occur in over 1.4 million each year in the United States (Centers for Disease Control and Prevention, 2001).                                                            

        In recent years, consumers become more concern about the processed food they eat. Spices and herbs have been added since ancient times not only as flavoring agents, but also as flok medicine and food preservatives (Beuchat, 1994; Nakatani, 1994; Cutler 1995). However there has been increasing interest to replace synthetic preservative,with natural, effective and nontoxic compounds (Smid and Gorris 1999). Allyl isothiocyanate (AITC), a component of mustard oil has potential for use as antimicrobial agent in a variety of food because of its natural origin (Lin et al 2000a, Park et al. 2000; Weissinger et al. 2001).Garlic is widely used in meat products. It's one of the herbal products consumed world wide because of its beneficial effects on health such as antidiabetic, hypochloesterolemic,antioxidant and cancer preventive (Ali et al. 2007). Garlic extracts have also been to have antibacterial active in different vivo models. The component responsible for the remarkable antibacterial activity of garlic is an oxygenated sulpher compound which termed Allicin from the Latin name (Allium sativuon) of garlic plant (Al Magboul et al.1988).acetic acid has also been widely tested as food preservative for killing of food borne pathogenic bacteria (Berry and Cutter 2000; Samelis et al. 2001).                                                        

      However many questions still raised about formulation, the best preservation and microbiological safety of various food to modified what we have been doing to minimize hazards that might exist in various food.

 The present study was designed to evaluate the effect of mustard, garlic and acetic acid on reduction the numbers of S. typhi in ground beef.                                                                                                                                 

Materials and Methods

Bacterial strain:                                                                                   

     The strain of Salmonella typhimurium was obtained from the Bacteriological laboratory Department, Animal Health Research Institute, Ministry of Agriculture. The stock cultures was maintained on Typticase soya agar slants (Difico) at 4° C                                                               

Cell suspension and Inoculation:                                                         

     The strain of S.typhi was cultured on Trypticase soya broth (Difico), for 24 h. at 37 ºC before use in experiments. The cell suspension was serially diluted with 0.1% sterile peptone water to an approximate concentration calculated to yield 10⁶- 10⁷  CFU/ml of sample according to Min_Suk et al. (2003). This working solution was used to inoculate all       groups in this experiment                                                                          .    Sample treatment:   

          Mustard flour, garlic powder and commercial vingar (containing 5% acetic acid) were obtained from localsupermarket. Samples treatments were prepared to achieve various concentrations of 5 % and 10 %of mustard flour and garlic powder. (In aqueous solution). The concentration of acetic acid was added to achieve 0.5% and 1% (vol/wt) by adding 10 and 20 ml of the commercial vingar.                                    

 Ground beef samples:                                                                           

          About 2 Kg of ground beef were purchased from local supermarket and transferred to the Laboratory in an ice box with minimal of delay, then prove to be free from the tested pathogenic organism according to   the method carried out by ISO 6579 (2002).                                           

Experimental design:

    Two main studies were carried out on 12 groups of ground beef samples (each of 100 gm) All groups were inoculated with 1 ml of the bacterial inoculm (working solution).                                                        

      1-Study of Antimicrobial effect of individual treatment on S. typhi  inoculated in ground beef samples:                                                        

       Seven groups of ground meat sample were used:                                  First and second groups were inoculated with 5 % and 10 % of mustard flour respectively .Third and fourth groups were inoculated with 5% and 10% of garlic powder respectively. Fifth and sixth groups were inoculated with 0.5% and 1% of acetic acid respectively. Seventh group was kept as control without treatment.                                                    

.                                                                             

2-Study of Antimicrobial effect of combining treatment on s. typhi  inoculated in ground beef samples:

Five group of ground meat samples were used:

 First groupwas inoculated with mustard 5% and acetic acid 1%. Secondgroupwas inoculated with mustard 10% and acetic acid 0.5%. Third group was inoculated with mustard 5%, garlic 5% and acetic acid 0.5%.    Fourth group was inoculated with mustard 10%, garlic 10% and acetic acid 1%. The last group was kept as control without any addition.                                                                                                     

        Both bacterial inoculum and treatments were distributed in the ground meat by hand Kneading under complete aseptic condition with application of all hygienic measures required for 5 min. All samples and control were stored at 4 ºC. Samples were followed up on xylose lysine desoxycholate agar (oxoid) plate at 0 h, 24 h, 48h and 72 hr. of storage for enumeration of S. typhi as recommended by Nottingham et al. (1975). The average of duplicate plates counts was converted to log10                                              

Results

Table (1): Antimicrobial effect of mustard flour, garlic and acetic  acid on Salmonella typhi at (4 ºC )

Table (2): Antimicrobial effect of combining treatment on Salmonella typhi at (4 ºC)

Discussion

     The survivals of S. typhi in various treatments at 4 ºC either in individual or in combining treatments are presented in Table (1) and Table (2). S. typhi was reduced in all treatments expect for control.                                   

     Regarding to Table (1) The most effective treatments on the population of S. typhi was toward acetic acid 1% and garlic powder 10% ,thus  S.typhi was undetectable at 48h of inoculation in both of them.On the other hand mustard flour treatment showed the slowest and least effect on growth of S.typhi in comparative to the effect of garlic powder and acetic acid .There is a reduction by 1.62 log CFU/ g and 1.71 log CFU/ g in mustard flour at 5% and 10% respectively from 0 time to 72h . In this aspect Lin et al. (2000b) demonstrated that S. typhi was less resistant to mustard than other type of gram negative bacteria.                        In concerning to the antibacterial effect of garlic powder as shown in Table (1) S. typhi can't be detected at 48 h of treatment with garlic 10% and at 72 h of 5% treatment of garlic. There is a reduction by 1.52 log CFU/g at 5% treatment between 0 h and 48 h and by 1.54 log CFU/g at garlic 10% between 0 time and 24h of inoculation. Our result of garlic treatment was supported by Uhart et al. (2006) and Marija and Nevena (2009). However the anti bacterial effect of garlic is due to the principal antimicrobial component of garlic which is allicin, garlic bulbs do not contain allicin in its active form, but contain its precursor, upon crushing garlic bulbs,alliin is enzymatically hyrolsed to allicin which is an inhibitor of respiratory SH^_group enzymes (Beuchat and Golden  1989)and acetyl_COA synthetase, Focke et al. (1990).On the other hand Ali  et al. (2000) recorded in their study that the chopped garlic has a slow down effect on microbial growth of Salmonella typhi in ground meat on the garlic concentration , but they explained that this effect was not at an expected level even at the highest concentration because potential antimicrobial agent in chopped garlic were probably                     insufficiently  extracted.                                                                             

      Regarding to the antimicrobial effect of acetic acid as shown in Table (1), S. typhi was undetectable at 48 h of treatment with 1 % and at 72 h of treatment with 0.5%. The level of S. typhi dropped by 1.1 log CFU/g at 5% treatment between 0 time and 48 h and by 0.92 log CFU/g at 1% treatment between 0 time and 24 h of inoculation .Such results are nearly inagreement with those recorded by Min_Suk et al.(2003) and Cosansu and Ayhan (2010). acetic acid antimicrobial activity is based primarily on its pH_loweing effect, and it exhibits this antimicrobial activity only in an undissociated form (Sofos et al. 1998)

         The result tabulated in Table (2) show strongly reduction of S.tyhi in all combining treatment except for control, this result was nearly

inagreement with those recorded by Min_Suk et al. (2003) and Uhart et al. (2006) S. typhi can 't be detected after 24 h of inoculation in the combining effect of (mustard 5 % , garlic 5 % and acetic acid 0.5%) also in (mustard 10%, garlic 10% and acetic acid 1% ). Combined treatment of mustard 5% and acetic acid 1% resulted in reduction of 1.16 log CFU/g between 0 time and 24 h, while combined treatment of mustard 10% and acetic acid 0.5% resulted in reduction by 1.21 log CFU/g between 0 time and 24 h of inoculation. Uhart et al. (2006) reported that combination of other interventions with spices may help inhibit S. typhi in food systems such as ground beef.

    The combining effect of: Allyl isothiocyante component of mustard which is used as an antimicrobial agent (Ward et al. 1998) acetic acid which is widly used as food preservative for killing food borne pathogenic bacteria (StratFord and Anslow 1996) and garlic which is responsible for remarkable antibacterial activity (Almagboul et al. 1988; Kumar and Berwal 1998), all of them make strong reduction against S. typhi better than each component individual in different ratio used .

              Comparatively both control samples showed slightly and slowest decrease through out the storage period and this may be due to the effect of low storage temp at 4 ºC and possible presence of competitive psychrotrophic microorganism                                                              

         However different antibacterial studies have revealed varying magnitudes of its effects depending on the organism and treatment used.   From the achieved result, it could be concluded that S. typhi,is more sensitive to garlic powder and acetic acid than mustard flour. Therefore research should be focused on addition of spices and acetic acid in certain concentration to obtain effective antimicrobial activity without adversely influence flavors and odours.                            .                                                                

References

Ali, A., Kamil, B., Mehmet, E. E. and, Baris B.  (2007): The antimicriobial effects of chopped garlic in ground beef and meat ball. J. of Medicinal Food 10, 1:203-207                                                            

Almagboul A.Z. , Basho A.K. , Karim, Salibm TA , Khalid SA (1988) : Antimicroial activity of certain Sudanese plants used in Folklore medicine. Screening for antifungal activity VI, Fitoterapia 59: 393-396                                                                                   

Berry, E. D. and Cutter, C.N (2000): Effects of acid adaptation    of E. coli O157:H7 on efficacy of acetic acid spray washes to           decontaminate beef carcass tissue. Appl. Environ. Microbiol. 66: 1493-1498.                                                                                                     

Beuchat, L.R. and Golden, D.A (1989): Antimicrobials occurring naturally in foods. Food Technology 43: 134-142.                                   

Beuchat,L.R (1994): Antimicrobial properties of spices and their essential oils , in Natural Antimicrobial system and food preservation Eds. Y.M. Dillon and R.G. Board , CAB International , Oxon PP. 167-179                

Centers for Disease Control prevention (2001): Outbreaks of multidrug-resistant Salmonella typhimurium associated with veterinary facilities. Idaho, Minneota, and Washington. 1999 Morb. Mortal.Wkly.Rep. 50:701-704                                                                                              

Cosansu, S. and Ahyhan, K. (2010): Effect of lactic and acetic acid on survival of Salmonella enteritidis during refrigerated and frozen storage of chiken and meat. Food Bioprocess Technol , Dol 10.1007/s 11947-009-0320-x                                                                                             

Cutler H.G (1995): Natural product flavor compounds as potential antimicrobials, insecticides, and medicinal Agro_Food Ind. Hi-Tech 19-23                                                           

Focke, M., Feld, A. and Linchtenthaler , H.K. (1990): Allicin , anaturally occurring antibiotic from Garlic , specifically inhibits acetyl-COA synthetase. European J. of Biochemistry 261: 106-108.                             

Iso 6579 (2002): Microbiolgy of food and animal feeding stuff                Horizontal method for the dectection of Salmonella species.4^th Ed. International organization for standardization.                                            

Kumar, M. and Berwal, J.S. (1998): sentsitivity of food pathogens to Garlic (Allium sativum) J. of Appl. Microbiol. 84:213-215                              

Lin C.M. Perston, J. F and Wei, C.I. (2000a): Antibacterial mechanism of allyl isothiocyanate J. Food Port. 63:727-734                                        

Lin, C.M., Kim, J.M,Du,W.X. and Wei C.I (2000b) : Bactericidal  activity of isothiocyanate  against pathogens on fresh produce.  J. Food Prot. 63:25-30                                                                                         

Marija M.S. and Nevena T. N. (2009): Antimicrobial effect of spices and herbs essential oils APTEFF, 40: 1-220                                                    

Min-Suk, R., Sun-Young, L., Richard H.D. and Dong-Hyun, K. (2003): Antimicrobial Effects of mustard flour and acetic acid against E.coli o157:H7, L. monocytogenes , and Salmonella enterica serovar     Typhimurium. Appl .and Environ. Microbiol. 69,5:2959-2963.                                    

Nakatani, M. (1994): Antioxidative and antimicrobial constituents of herbs spices in spices, Herbs and Edible Fungi. Ed. G. Charalam bous, Elsevier science, New York, PP. 251-271.                                               

Nottingham, P.M., Rushbrook, A.J. and Jung, K.E (1975): The effect of planting technique and incubation temperature on bacterial count food Technol., 10:273                                                                                     

Park, C.M, Taormina, P.J. and Beuchat L.R (2000): Efficacy of allyl isothiocyanate in killing enterohemorrhagic E.coli O157: H7 on alfalfa seeds Int. J. food Microbial 56: 13-20                                                    

Samelis, J., Sofos,N. J. Kendal P. A., and Smith, G.C. (2001): influence of  the natural the microbial flora on the Acid tolerance response of Listeria monocytogenes in a model system of fresh meat decontamination fluids.Appl. Environ. Microbiol. 67:2410-2420.                                      

Safos, J.N., Beuchat L.R. Davidosn , P.M. and Johnson , E.A. (1998) : Naturally occurring antimicrobials in food , P. 15-24 . Council for Agricultural Science and Technology AmesIowa.                                    

Smid, E.J. and Gorris L.G.M. (1999): Natural antimicrobials for food preservation, in Hand book of food preservation. Ed. M.S. Rahman, Marcel Dekker, New York PP. 283-308.                                                

Startford M. and Anslow, P.A (1996): Comparison of the inhibitory action of weak-acid preservatves, un couplers, and medium-chain fatty acids. FEMS Microbiol Lett 142:53-58.                                                  

Uhart , M., Maks, N. and Ravishankar, S. (2006) : Effect pf spices on growth and survival of S. typhi D 104 in ground beef stored at 4 and 8 c . J. of Food Safety: 26(2) 115:125 .                                                           

Ward, S.M. Delaquis, P.J., Holley, R.A. and Mazza, G. (1998): Inhibition of spoilage and pathogenic cacteria on agar and precooked roast beef by volatile horsera dish distillates. J of food Res. Int. 31-19-26.                                                                                                          

Weissinger, W.R., McWatters, K.H. and Beuchat, L.R. (2001): Evalution of volatile chemiced treatments for Lethality to Salmonella on alfalfa seeds and sprouts. J. FoodPort. 64: 442-450.                                         

التأثير المضاد للبکتريا لکل من المستردة والثوم وحمض الخليک على السالمونيلا تيفى ميوريم فى اللحوم المفرومة

الملخص العربى

أجريت هذة الدراسة لأستبيان تأتثر کل من المستردة والثوم وحمض الخليک کل على حدة او مجمعة على ميکروب السالمونيلا تيفى ميوريم المحقون فى اللحوم المفرومة  وقد جهزت هذة المواد بنسب مختلفة وذلک لتکون نسبة اضافة المستردة والثوم 5% و 10 %   اما حمض الخليک فقد اضيف بنسبة 0.5% و 1% وقد تم اضافة النسب المذکورة بعض حقنها بميکروب السالمونبلا تيفى ميوريوم وتقسيمها الى  مجموعات مختلفة وخفظها عند 4º م وقد تم متابعة هذا التأثير عند صفر و 24 و48 و72ساعة  من الحقن وقد أظهرت النتائج ان الثوم له تأثير کبير على الميکروب يلية حمض الخليک ثم المستردة التى أظهرت التأثير الأقل والأبطأ اذا ماقورنت بالثوم وحمض الخليک أما بالنسبة للتأثير المجمع للمواد المذکورة فقد أظهرت النتائج ان ميکروب السالمونيلا لم يکن له وجود بعد 24 ساعة من الحقن فى مجموعة (المستردة 0.5 % والثوم 5 % وحمض الخليک 0.5 % وکذلک فى مجموعة المستردة 10 % والثوم 10 %  وحمض الخليک 1 % )  0