ETHNO-PHARMACOLOGICAL USE AND ANTIMICROBIAL ACTIVITY OF TRADITIONAL DATE VINEGAR OF GHARDAÏA
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CHERIF Brahim1,BOURAS Noureddine2*, OUMOUNA Mustapha3, OULD EL HADJ Mohamed Didi4, HOLTZ Michael D.5 and SABAOU Nasserdine2

(1)Université Saâd Dahleb de Blida, Ouled Yaiche, Blida 09000, Algeria

(2)Laboratoire de Biologie des Systèmes Microbiens (LBSM),

Ecole Normale Supérieure de Kouba, 16050, Vieux-Kouba, Algiers, Algeria

(3)Université Dr. Yahia Fares de Médéa, Ain D’heb, Médéa 26000, Algeria

(4)Laboratoire de protection des écosystèmes en zones arides et semi-arides,

Université de Ouargla, 30000 Ouargla, Algeria

(5)Field Crop Development Centre, Alberta Agriculture and Rural Development, 6000 C & E Trail, Lacombe, Alberta, T4L 1W1, Canada,

E-mail: Cette adresse e-mail est protégée contre les robots spammeurs. Vous devez activer le JavaScript pour la visualiser.

 

 

Abstract.- This study aims to demonstrate the therapeutic advantage of traditional date vinegar. We carried out an ethno-pharmacological survey on the therapeutic use of date vinegar by the population of Ghardaïa. After we verified the quality of two vinegars made from Deglet-Nour and Temjouhart date varieties, we tested their antimicrobial activity on Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. These microorganisms are involved in skin infections treated with date vinegar. The ethno-pharmacological survey showed that the traditional date vinegar is used in the treatment of 30 afflictions. The treatment seems to be effective (75% of only positive effects compared to 14% of positive and also adverse effects). The antimicrobial activity was present, in spite of the weak concentration of acetic acid in the tested vinegars. The highest antimicrobial effect was observed on S. aureus. The inhibition diameter of this microorganism was 49 mm in Deglet-Nour vinegar and 33 mm in Temjouhart vinegar. In conclusion, date vinegar is used by the population of Ghardaïa to treat various illnesses and it was proven that it is effective in the treatment of skin infections caused by microorganisms mentioned previously.

Key words: date vinegar, ethno-pharmacology, antimicrobial activity, skin infections, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans.

USAGE ETHNO-PHARMACOLOGIQUE ET ACTIVITÉ ANTIMICROBIENNE DU VINAIGRE DE DATTES TRADITIONNEL DE GHARDAÏA

Résumé.- Le but de cette étude est de démontrer l’intérêt thérapeutique du vinaigre traditionnel de dattes. Nous avons mené une étude ethno-pharmacologique sur le traitement par le vinaigre de deux variétés de dattes auprès de la population de Ghardaïa. En suite, nous avons vérifié la qualité de deux vinaigres fabriqués à partir des dattes de Deglet-Nour et de Temjouhart, puis testé leur activité antimicrobienne contre Staphylococcus aureus, Pseudomonas aeruginosa et Candida albicans. Ces germes sont impliqués dans les infections de la peau traitées par le vinaigre de dattes. Les résultats ont montré que le vinaigre traditionnel de dattes est utilisé dans le traitement de 30 affections. Le traitement semble être efficace (75% de résultats positif et sans aucun effet indésirable par rapport à 14% de résultats positifs mais avec des effets indésirables). L’activité antimicrobienne était présente, malgré la concentration faible en acide acétique dans les deux vinaigres. L’effet antimicrobien le plus fort a été observé contre S. aureus avec un diamètre d’inhibition de 49 mm chez le vinaigre de Deglet-Nour et de 33 mm chez le vinaigre de Temjouhart. On a conclu que le vinaigre de dattes est utilisé par la population de Ghardaïa pour traiter diverses maladies et il s’est avéré efficace dans le traitement des infections de la peau causées par les germes précités.

Mots clés: vinaigre de dattes, ethno-pharmacologie, activité antimicrobienne, infections de la peau, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans.

Introduction

The number of date palm (Phoenix dactylifera L.) in Maghreb is estimated at 177000 trees. Date palm provides a wide range of products with multiple uses. The most important are dates which the total production reaches 426.000 tonnes a year, then derivative products used in the traditional pharmacopeia. These products kept their importance in the oasis populations of Maghreb, in spite of the progress of medicine. This is due to their proven effectiveness for many years [1]. The dates have been used for the production of traditional vinegar in the oasis for a long time. It is an inherited practice with traditional material that provides a vinegar with therapeutic and flavor advantages which are absent in the industrial vinegar [2]. Indeed, the vinegar has been recognized for a long time to have astonishing therapeutic properties and recently, studies showed that the acetic acid, a vinegar component, is effective in a diagnosis of cervix cancer [3]. The objective of this study is to show the ethno-pharmacological uses of date vinegar, to investigate the vinegar quality and to prove its effectiveness in treating some infections by testing its in vitro antimicrobial activity.

1.- Materials and Methods

We collected, from Ghardaïa region, two vinegars produced from Deglet-Nour and Temjouhart dates. These vinegars were produced traditionally by washing and putting dates in a clean plastic barrel then filling two thirds of its volume with tap water. Old date vinegar and some salt were also added. Finally, the barrel was closed at least for forty days.

1.1.- The ethno-pharmacological survey

The ethno-pharmacological survey questionnaire was divided into three main questions about the knowledge of traditional medicine, date vinegar and what kind of medicine is preferred. The questionnaire was distributed on three categories of people: herbalists, traditional healers and the general population in order to obtain a maximum of information about the uses of date vinegar in traditional medicine in Ghardaïa oasis.

1.2.- Determination of vinegars quality

The pH of the vinegar samples was determined by using a pre-calibrated pH-meter. The rate of soluble solids, expressed in Brix degrees (°B), is the percentage of sucrose (w/w) in a pure solution [4]. It was determined by refractometer method. The dry matter was obtained by desiccation at 105 °C until a constant weight [4]. The concentration of the acetic acid was determined by titration with NaOH solution (0.5 N). The concentration of acetic acid is the concentration of NaOH multiplied by its volume and divided on the vinegar volume used in titration. The alcohol (v/v) remaining after making vinegar was deduced by comparing the acetic acid concentration found in vinegar to alcohol standard concentration (0.80 g in a volume of 1 ml). Thus, it is the acetic acid (w/v) multiplied by 1 ml and divided on 0.80 g. The alcohol is expressed in GL (ml of alcohol in a volume of 100 ml of vinegar) [5]. The total microflora was determined by colony counter method on nutrient medium incubated at 30°C for 48 hours [6].

All these analyses were duplicated and the results are presented as a mean.

1.3.- Test of the antimicrobial activity

The microorganisms tested were Pseudomonas aeruginosa (ATCC 27853) and Candida albicans (IPA 200) which were conserved in ISP2 (International Streptomyces project 2) medium, and Staphylococcus aureus (ATCC 25923) which was maintained in conservation medium. The bacterium Staphylococcus aureus (ATCC 25923) was received from the university hospital of Blida. However, the bacterium Pseudomonas aeruginosa (ATCC 27853) and the yeast Candida albicans (IPA 200) were obtained from the collection of the Laboratoire de Biologie des Systèmes Microbiens (LBSM). All these microorganisms were stored at 4°C.

Microorganisms were plated in ISP2 medium (18 g agar /l) and incubated at 30°C [7]. After 24 hours, three colonies of each microorganism were homogenized in 9 ml sterile salt water. The antimicrobial activity of the non-diluted (pure) vinegars of Deglet-Nour and Temjouhart was realized. Furthermore, the antimicrobial effect of diluted two varieties of vinegars to 1/2, 1/4 and 1/8 with water, to determine the maximum dilution that should be employed in traditional mixtures, was also studied. In addition, the pure and diluted vinegars were tested in sterile (S) and non-sterile (NS) state with well and disc diffusion methods to highlight the probable antagonistic effect.

1.3.1.- Well diffusion method

A volume of 100 ml of ISP2 medium (12 g agar/l) was liquefied and inoculated with 80 µl of the microorganism suspension, then poured into Petri plates. After solidification of the medium, wells of 10 mm in diameter were punched and 100 µL of both sterile (S) and non-sterile (NS) vinegar dilutions was aseptically poured into the wells. The plates were kept for 4 hours at 4°C to allow the diffusion of the active substances while temporarily stopping the growth of the microorganisms. The plates were then incubated at 30°C. The diameters of the inhibition areas around the wells were measured after 24 hours of incubation for bacteria, and 48 hours for yeast [7].

1.3.2.- Paper disc diffusion method

Paper discs of 6 mm in diameter were sterilized by exposure to 254 nm ultra violet radiation for 45 min. After, they were soaked in the various dilutions of sterile (S) and non-sterile (NS) vinegars and deposited on the surface of ISP2 medium (12 g agar/l) which was previously inoculated with 80 µl of the microorganism suspension and poured into Petri plates. The plates were put at 4°C for 4 hours then incubated at 30°C. The diameters of the inhibition areas were measured after 24 hours of incubation for bacteria, and 48 hours for yeast [7].

The antimicrobial test experiments were duplicated and the results are presented as a mean.

2.- Results and Discussion

2.1.- The ethno-pharmacological use of date vinegar

The traditional date vinegar has 30 therapeutic virtues as shown in Table I, and it is used by 88% of the studied population sample, 7 herbalists and 2 traditional healers. The treatment has shown positive effects on 75% of people who used date vinegar. However, it has shown positive and also adverse effects on 14% (fig. 1).

Table I.- The therapeutic virtues of date vinegar

Disease

Vinegar (date variety)

Mode of use

Sunstrokes

All varieties

Compress, massage, washing (water and/or vinegar), syrup (vinegar and honey)

Cough

Temjouhart, Takernennayt

Syrup (vinegar and honey or vinegar and liquefied sugar)

Fever

Temjouhart,Takernennayt

Massage, compress on the head and the belly

Migraine and headaches

Deglet-Nour, Temjouhart, Tafezwine, Ghars, Tazeggwaght

Compress, massage, drink, inhalation

Flu and cold

Deglet-Nour,

Syrup, fumigation

Skin diseases

All varieties

Massage

Vitiligo

All varieties

Massage

Fungal skin infections

Deglet-Nour

Massage

Dandruff and alopecia

Deglet-Nour, Takernennayt

Washing

Indigestion

Deglet-Nour

Vinaigrette, drink (vinegar and water)

Obesity

All varieties

Drink (vinegar and water)

Hypercholesterolemia

Deglet-Nour, Temjouhart

Drink (vinegar and water)

Respiratory allergies

Deglet-Nour

Drink (vinegar and water), vinaigrette

Feet fatigue

All varieties

Bath

Angina

Deglet-Nour

Syrup (vinegar and honey), gargling (vinegar and water)

Dizziness

Deglet-Nour

Drink (vinegar and water)

Flatulence

All varieties

Compress, drink (vinegar and water)

Rheumatoid arthritis

Deglet-Nour

Massage, compress

Tear gas damage

Deglet-Nour

Face wash

Hypertension

Deglet-Nour, Temjouhart

Massage

Eczema

Deglet-Nour, Temjouhart

Cataplasm (vinegar and henna)

Cutaneous abscesses, furuncle and wound inflammation

Deglet-Nour, Takernennayt, Tafezwine

Compress, bath

Tonic

Deglet-Nour, Temjouhart

Drink (vinegar and water)

Inflammation and infection of mouth and gum

Deglet-Nour, Temjouhart

Gargling (vinegar and water)

Burns (reduce pain and infection)

Deglet-Nour

Massage, washing

Fungal infection of toes

Deglet-Nour

Cataplasm (vinegar and henna)

Several therapeutic virtues shown by this study were also revealed by Baliga et al. [8] on dates which are used in traditional medicine to treat hypertension, diabetes, cough, rheumatism, burning sensation, bronchitis, gastroenteritis, respiratory diseases, asthma, fever and fatigue. The fruit pulp is regarded to be antitussive, expectorant and tonic. Studies on rats showed that the defatted date seed flour reduced the plasma triglyceride, cholesterol and LDL. Other studies showed that the extract of date pulp and date seeds possess anti-inflammatory activity in the rat adjuvant arthritic model [8]. Moreover, the date vinegar has beneficial effects similar to those of other types of vinegars. Indeed, there is a close link between the risk to develop diabetes type 2 and excess weight. The vinegar has proven to be interesting in the prevention and the treatment of diabetes, because it decreases the presence of glucose and insulin in blood [9,10,11,12]. The acetic acid present in the vinegar is at the origin of the observed effects [11]. Moreover, the acetic acid increases the use of glucose by the human body [13]. Moreover, the vinegar increases satiety after the meal [9,10,11,12], and its daily consumption decreases blood pressure [14].

2.2.- Vinegars quality

The physico-chemical characteristics and the total microflora are presented in Table II and III. The results obtained in the analysis of our vinegars were compared with the studies of Ould El Hadj et al. (2001) [2] and Bouaziz and Ould El Hadj (2010) [15].

Table II.-The physico-chemical characteristics

   

Deglet-Nour

Temjouhart

pH

3.44

3.28

Dry Matter (%)

13.31

12.26

soluble solids (°B)

13.75

9.65

Alcohol degree (°GL)

6. 29

2.22

Acetic acid (g/l)

50.3

17.8

Table III. The number of total microflora

Vinegars

Total microflora (CFU/ml)

 

Dilution 10-3

Dilution 10-2

Deglet-Nour

7×103

1.8×103

Temjouhart

6×103

1.1×103

The pH of Deglet-Nour and Temjouhart vinegars was slightly higher than the pH in H’Chef of Deglet-Nour (3.12) and in Harchaya (3.25) vinegars, but lower than the pH in Hamraya and Tinissine vinegars (3.65) [2,15]. Moreover, the dry matter rate obtained in our study was higher compared to 11.26% in Hamraya, 10% in H’Chef of Deglet-Nour, 6.59% in Harchaya and 6.33% in Tinissine [2,15]. In addition, the soluble solids rate was higher than 8.30 B in the H’Chef of Deglet-Nour, 10.00 B in Harchaya and 7.00 B in Hamraya [2]. However, our obtained values were lower than 16.53 °B obtained in H’Chef of Deglet-Nour [15]. The high soluble solids rate reveals a higher amount of unfermented sucrose. The Degree of alcohol in Deglet-Nour was much higher than 3.61 GL in H’Chef of Deglet-Nour, 3.75 GL in Harchaya, but lower than 4.90 GL registered in Hamraya [2]. However, the alcohol degree measured in Temjouhart vinegar was lower than other studied vinegars. Also, it was much higher than 0.99% (0.99°GL) in H’Chef of Deglet-Nour and Harchaya and 0.98% in Tachrwit and Tinissine [15]. The acetic acid concentration was higher in Deglet-Nour but lower in Temjouhart comparing to 25.94 g/l in H’Chef of Deglet-Nour and 30.38 g/l in Harchaya, but both vinegar concentrations were higher than 15.31 g/L noticed in Hamraya [2]. In addition, the acetic acid concentration of Deglet-Nour vinegar was higher than 0.9% (9 g/l) in Tinissine and 2.7% in H’Chef of Deglet-Nour [15]. In addition, the acetic acid concentration of Temjouhart was higher than 0.9% in Tinissine but lower than 2.7% in H’Chef of Deglet-Nour [15]. We should notice that among date vinegar quality characteristics found in this study, only the acetic acid concentration of Deglet-Nour follows the official Algerian standards for vinegar. Furthermore, the properties and the quality of traditional production of vinegar depend strictly on the conditions and varieties of dates used in the process.

2.3.- The antimicrobial activity

The obtained results showed that the bacteria S. aureus and P. aeruginosa were susceptible to Deglet-Nour and Temjouhart vinegars (figures 2, 3 and 4). However, the yeast C. albicans was susceptible only to Deglet-Nour vinegar (fig. 5). In addition, all the three studied microorganisms were more susceptible to Deglet-Nour by comparison to Temjouhart vinegar. Correspondingly, the studied microorganisms were more susceptible to non-diluted vinegar compared to diluted (1/2, 1/4 and 1/8) vinegar.

The bacterium S. aureus was susceptible to vinegar tested by both diffusion methods (wells and discs) as shown in figure 2 and 3. In addition, the similar results were obtained for P. aeruginosa and C. albicans. Only the results obtained by the disc diffusion method (for P. aeroginosa) and by well diffusion method (for C. albicans) were shown (figure 4 and 5), respectively.

E0401102

E0401103

In general, Deglet-Nour and Temjouhart vinegars exhibited more antibacterial than antifungal effect. Likewise, both varieties of vinegars showed less effect in weak concentration. Consistently, the commercial vinegar (35 g/l of acetic acid) revealed more antimicrobial effect on S. aureus and Escherichia coli than Aspergillus niger and A. flavus, and no antifungal effect was observed at low concentration [16]. Besides, vegetables washed by 30% and 20% vinegar solutions reduced significantly the microbial load to 1.10×107 CFU/ml, but a very low effect was observed by using vinegar solutions of 10% [17]. Similar results were noticed in our case by using 1/8 vinegar dilution. The result of our study suggests using 1/4 as the maximum dilution of date vinegar in traditional mixtures.

Furthermore, Rutala et al. (2000) showed that white vinegar has antimicrobial activity against P. aeruginosa, both methicillin resistant and susceptible S. aureus, S. choleraesius, E. coli and against both vancomycin resistant and susceptible Enterococcus [18]. The vinegar has also an antibacterial activity against Salmonella typhimurium [19], and bamboo vinegar has an antimicrobial effect on fecal bacteria [20]. Moreover, Thorp et al. (1998) showed that acetic acid was effective against the growth of bacteria that cause the most frequent earaches (Pseudomonas, Staphylococcus and Proteus) [21]. This proves that the acetic acid is responsible for the antimicrobial effect. We can explain this by the fact that organic acids diffuse passively through bacteria cell wall and dissociate into anions and protons. The release of protonsdecreases theinternal pH which in turn provokes an inhibitory effect [22]. However, Baliga et al. (2011) showed that C. albicans treated with high concentration of Barhi dates extract caused strong damage in the form of cell lysis, leakage of cytoplasmic material and eventual cell death and that flavonoids presents in the extract may have been responsible for the observed effect [8]. Besides, Alabbasy et al. (2013) showed that Zahdi date, Thompson seedless grape and maize malt vinegars contain flavonoids, vitamins and phenols [23]. One of the Zahdi date vinegar vitamins is the ascorbic acid [23] which Kallio et al. (2012) found that it inhibited completely the growth of S. aureus and suggested that it affects the metabolism of S. aureus [24]. It increases the oxidative stress on bacterial cells when it is metabolized in aerobic conditions [24]. These results suggest that these phytochemicals could be present in Deglet-Nour and Temjouhart vinegars and were responsible for the observed antimicrobial effect.

S. aureus was more susceptible to Deglet-Nour non sterile vinegar. This may be due to the vinegar microflora antagonistic activity. Rajkowska et al. (2012) observed that Saccharomyces cerevisiae var. boulardii has an antagonistic activity only against S. aureus rather than Escherichia coli, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium and Campylobacter jejuni [25]. However, Vohra and Syal (2013) showed that Saccharomyces cerevisiae has an antagonistic activity against S. aureus, Pseudomonas sp. and Salmonella sp. [26]. Moreover, S. cerevisiae has an antagonistic activity against C. albicans, Protues, Shegilla and E. coli in vitro, and antagonistic activity against C. albicans, C. krusei and C. pseuditropicalis in vivo which disappeared when the S. cerevisae was killed by heating [27]. Although S. cerevisiae is likely present in date vinegar, we recommend further studies to identify and isolate its microorganisms then test their antagonistic activity.

Conclusion

Date vinegar is largely used by the population of oases in Maghreb for its various therapeutic benefits, and each date variety provides vinegar with particular therapeutic effects. Date vinegar has a strong antimicrobial activity on S. aureus, P. aeruginosa and C. albicans. Therefore, it is effective in traditional medicine for treating skin infections caused by these microorganisms. Moreover, optimizing the traditional date vinegar quality will increase its effectiveness.

Acknowledgments

We would like to thank Dr. Atika Meklat, Dr. Amina Kanane and Dr. Mohamed Nadjib Boukhatem for their valuable suggestions.

Bibliographicreferences

[1].- Guerradi M., Hamdouni N., Outlioua K., 2005.- Usage des produits et sous produits du palmier dattier dans la pharmacopée traditionnelle dans les oasis du Maghreb. Séminaire international des plantes médicinales dans les zones arides, Université de Ouargla.

[2].- Ould El Hadj M. D., Sebihi A.H., Siboukeur O., 2001.- Qualité hygiénique et caractéristiques physico-chimiques du vinaigre traditionnel de quelques variétés de dattes de la cuvette de Ouargla. Revue des Energie Renouvelables, Production et valorisation-biomasse (NS): 87-92.

[3].- LD., 2007.- Du vinaigre pour sauver des vies. Revue de BioTrubine: 23: 4.

[4].- Audigié C., Fegarelle J., Zonszain F., 1984.- Manipulation d'analyse biochimique. Techniques et Documentations, Paris, 274 p.

[5].- Cherif B., 2012.- Mémoire de fin d’études: Usage du vinaigre de dattes dans la médecine traditionnelle à Ghardaïa et mise en évidence de son activité contre certains germes infectieux de la peu. Université Saad Dahlab, Blida, 50 p.

[6].- Roché Y., Niel P., 2012.- Analyses en microbiologie-produits non stériles. In : Qualité et sécurité au laboratoire. Ed. Les Sélections Techniques de l’Ingénieur, Paris : Pp3352-1 - Pp3352-9.

[7].- Merrouche R., Bouras N., Coppel Y., Mathieu F., Monje M. C., Sabaou N., Lebrihi A., 2010.- Dithiolopyrrolone antibiotic formation induced by adding valeric acid to the culture broth of Saccharothrix algeriensis. Journal of Natural Products, Copublished with the American Society of Pharmacognosy, 73: 1164-1166.

[8].- Baliga M. S., Baliga B. R. V., Kandathil S. M., Bhat H. P., Vayalil P. K., 2011.- A review of the chemistry and pharmacology of the date fruits (Phoenix dactylifera L.). Food Research International, 44: 1812-1822.

[9].- Johnston C. S., Kim C. M., Buller A. J., 2004.- Vinegar improves insulin sensitivity to a high-carbohydrate meal in subjects with insulin resistance or type 2 diabetes. Diabetes Care, 27: 281-282.

[10].- Johnston C. S., 2005.- Strategies for healthy weight loss : from vitamin C to the glycemic response. Jouuranl of the American College of Nutrition, 24: 158-165.

[11].- Leeman M., Ostman E., Bjorck I., 2005.- Vinegar dressing and cold storage of potatoes lowers postprandial glycaemic and insulinaemic responses in healthy subjects. Europen Journal of Clinical Nutrition, 59: 1266-1271.

[12].- Ostman E., Granfeldt Y., Persson L., Bjorck I., 2005.- Vinegar supplementation lowers glucose and insulin responses and increases satiety after a bread meal in healthy subjects. European Journal of Clininical Nutrition, 59: 983-988.

[13].- Johnston C. S., Buller A. J., 2005.- Vinegar and peanut products as complementary foods to reduce postprandial glycemia. Journal of American Dietetic Association, 105: 1939-1942.

[14].- Kondo S., Tayama K., Tsukamoto Y., Ikeda K., Yamori Y., 2001.- Antihypertensive effects of acetic acid and vinegar on spontaneously hypertensive rats. Bioscience Biotechnology and Biochemistry, 65: 2690-2694.

[15].- Bouaziz S., Ould El Hadj M. D., 2010.- Contribution à l'étude des caractéristiques physico-chimiques et biochimiques de quelques types de vinaigres traditionnels de dattes obtenues à partir de quelques variétes de la région de Ouargla. Annales des Sciences et technologie, 2: 80-86.

[16].- Yang Z. Q., Wei H., Cao W., Zhao Y. J., Wang W., 2008.- Antimicrobial activity of vinegar and garlic extracts. Food Science, 29: 69-73.

[17].- Tagoe D. N. A., Aning O.A., 2011.- Effect of increasing concentration of antimicrobial agent on microbial load and antibiotic sensitivity pattern of  bacterial isolates from vegetables. Europian Journal of Experimental Biology, 1: 12-23.

[18].- Rutala W. A., Barbee S. L., Aguiar N. C., Sobsey M. D., Weber D. J., 2000.- Antimicrobial activity of home disinfectants and natural products against potential human pathogens. Infection Control and Hospital Epidemiology, 21: 33-38.

[19].- Sengun I. Y., Karapinar M., 2004.- Effectiveness of lemon juice, vinegar and their mixture in the elimination of Salmonella typhimurium on carrots (Daucus carota L.). International Journal of Food Microbiology, 96: 301-305.

[20].- Wang H. F., Wang J. L., Wang C., Zhang W. M., Liu J. X., Dai B., 2012.- Effect of bamboo vinegar as an antibiotic alternative on growth performance and fecal bacterial communities of weaned piglets. Livestock Science, 144: 173-180.

[21].- Thorp M. A., Kruger J., Oliver S., Nilssen E. L., Prescott C. A., 1998.- The antibacterial activity of acetic acid and Burow's solution as topical otological preparations. Journal of Laryngology & Otology, 112: 925-928.

[22].- Hindi N. K., 2013.- In vitro antibacterial activity of aquatic garlic extract, apple vinegar and apple vinegar-garlic extract combination. American Journal of  Phytomedicine and Clinical Therapeutics, 1: 42-51.

[23].- Alabbasy H. M., Abdul Razak R. K., Al Abdullah B. Y., 2013.- Determination of vitamins, phenolic compounds and flavonoids in vinegars produced from various sources. Journal of Tikrit University For Agriculture Sciences, 13: 81-89.

[24].-Kallio J., Jaakkola M., Mäki M., Kilpeläinen P., Virtanen V., 2012.- Vitamin C inhibits staphylococcus aureus growth and enhances the inhibitory effect of quercetin on growth of Escherichia coli in vitro. Planta Medica, 78: 1824-1830.

[25].- Rajkowska K., Kunicka-Styczynska A., Rygala A., 2012.- Probiotic activity of Saccharomyces cerevisiae var. boulardii against human pathogens. Food Technology and Biotechnology, 50: 230-236.

[26].-Syal P., Vohra A., 2013.- Probiotic potential of yeasts isolated from traditional indian fermented foods. International Journal of Microbiology Research, 5: 390-398.

[27].- Auclair E., 2001.- Yeast as an example of the mode of action of probiotics in monogastric and ruminant species. In : Brufau J., eds. Feed manufacturing in the Mediterranean region. Improving safety: From feed to food. Zaragoza, CIHEAM, Cahiers Options Méditerranéennes; n. 54: 45-53.