IJFS#244_Marhamatizadeh_bozza   Ital. J. Food Sci., vol 28, 2016 - 517 PAPER THE COMBINED EFFECT OF THYMUS VULGARIS EXTRACT AND PROBIOTIC BACTERIA (LACTOBACILLUS ACIDOPHILUS AND BIFIDOBACTERIUM BIFIDUM) ON AFLATOXIN M1 CONCENTRATION IN KEFIR BEVERAGE MOHAMMAD HOSSEIN MARHAMATIZADEH1* and SEDIGHEH RAMEZANIAN GOOSHEH2 1Department of Food Hygiene, Faculty of Veterinary Medicine, Kazerun Branch, Islamic Azad University, Kazerun, Iran 2Department of Microbiology, Kazerun Branch, Islamic Azad University, Kazerun, Iran *Corresponding author. Tel.: +98 9177210645 E-mail address: drmarhamati@gmail.com ABSTRACT The current study was conducted to evaluate the effect of different doses of Thymus extracts (0, 2, 4 and 6) gr/L and various values of lactobacillus acidophilus and bifidobacterium bifidum on decreasing of fixed amount of aflatoxin in kefir beverage. The results showed that Thyme extracts reduced the amount of Aflatoxin M1. Thyme extract in combination with probiotic bacteria decreased Aflatoxin M1 more than Thymus alone. The most reduction of Aflatoxin M1 levels was detected by using 4 gr/L Thymus extract and 1×108 Lactobacillus acidophilus. Moreover, results revealed that Thymus extracts and probiotic bacteria could reduce AFM1 in kefir drink. Keywords: aflatoxin M1 reduction, Bifiobacterium bifidum, Lactobacillus acidophilus, Thymus extracts, Kefir   Ital. J. Food Sci., vol 28, 2016 - 518 1. INTRODUCTION Thymus is a small aromatic perennial herbaceous plant that cultivated in frequency due to their wide use in the food, cosmetic, and pharmaceutical industries (NABAVI et al., 2015). The genus Thymus is a taxonomically complex group of aromatic plants traditionally used for medicinal purposes because of their antiseptic, antispasmodic and antitussive properties (PINA-VAZ et al., 2004, NABAVI et al., 2015). Several researchers demonstrated that extracts and essential oils of some Thymus spp. have antiviral, antibacterial and antifungal activities. (FAZELI, et al., 2007, BEHNIA, 2008, SHARAFZADEH and ALIZADEH, 2012). Probiotics are used in dairy products as well as in food supplements and in agriculture as feed additives because of their beneficial health effects (NAGPAL et al., 2012). According to the currently international FAO/WHO definition (2001), probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Probiotics improve the health of both animals and humans through the improvement of its intestinal health by the regulation of microflora, stimulation and development of the immune system, synthesizing and enhancing the bioavailability of nutrients, reducing symptoms of lactose intolerance and reducing the risk of certain other diseases (KUMAR et al., 2011; NAGPAL et al., 2012). At present, probiotics are introduced as suitable replacing of antibiotics in order to confront with pathogens in animals and human being, and consumption of probiotic food products and medicines have considerable vogue. Several investigations suggested that using probiotic is associated with reducing the risk of antibiotic-associated diarrhea (32). The foods which contain probiotic bacteria are put in the group of special products, and according to the dairy products International Federation (IDF) recommendation, these probiotic products should have a minimum concentration of 106 CFU/g probiotic bacteria and consumer a total of some 108 to 109 probiotic microorganisms should be consumed daily for the suitable probiotic effects (15). The most commonly important probiotics belong to Lactobacillus and Bifidobacterium genus. There are many well-characterized strains of Lactobacilli and Bifidobacteria available for human use (HUSSAIN et al., 2009; KECHAGIA et al., 2012). Some of the beneficial properties of probiotics are anticarcinogenic, immunologic enhancement, antiatherogenic, cholesterol-lowering, anti-obesity and antidiabetic characteristics (NAGPAL et al., 2012). Kephir (kefir) is a viscous, highly acidic beverage produced from cow, goat, sheep or mare milks. The fermentation is initiated by “kefir grains” (clusters of yeast and bacteria), which are added to raw, pasteurized or UHT-treated milk (SARKAR, 2007; RIBEIRO and RIBEIRO, 2010). Kefir contains high contents of thiamine, riboflavin, pantothenic acid, vitamin C, protein and minerals; hence, kefir has both therapeutic and nutritional attributes (SARKAR, 2007). Kefir also has greater amounts of threonine, serine, alanine and lysine than milk (SARKAR, 2007). Kefir also has medical effects in order to treat hypertension, body skin fineness, stress and depression, cholesterolemia and arthritis (XIAO et al., 2003; NINANE el al., 2005). Aflatoxins are naturally occurring mycotoxins that are produced by some species of fungi like Aspergillus flavus and Aspergillus parasiticus. Aflatoxin M1 (AFM1) is one of the metabolites of Aflatoxin B1 that is excreted into milk when lactating animals are given feed with aflatoxin contaminated food (10). Contamination of milk or milk products with AFM1 is considered as a potential risk for public health (11, IARC, 2002). International Agency for Research on Cancer (IRAC) classified AFM1 as a group 2B agent (possibly carcinogenic to humans) and exposing to chronic aflatoxin causes acute liver problems, mutation and liver cancer (BEHFAR et al., 2012). Aflatoxin M1 is relatively stable during pasteurization, sterilization and preparation of dairy products (FALLAH,   Ital. J. Food Sci., vol 28, 2016 - 519 2010). Therefore, industries sustain irretrievable economic losses, if it is not controlled, and its potential risks for human health especially for children (ELKHOURY et al., 2011). The aim of this research was to produce kefir and reviewing Thymus extract effects in combined with Lactobacillus acidophilus and Bifidobacterium bifidum on the AFM1 concentration in kefir drink. 2. MATERIALS AND METHODS 2.1. Chemicals and instrumentation Lyophilized Lactobacillus acidophilus and Bifidobacterium bifidum (CHR Hansen Company, Denmark) were used as probiotic bacteria. The pasteurized low fat milk (1.5% fat) and kefir grain (Iran) were used to produce kefir drink. Thymus extracts (Zard band Company, Iran) (were prepared at 3 different concentrations including 2, 4, 6 g/L. AFM1 was procured from Merk Company, Iran) and 200 ppb concentration was used to add the kefir drink as follow explanation. 2.2. Producing kefir drink In order to produce kefir drink, 1 gram kefir grain was added to 1 litter pasteurized low fat milk (1.5% fat), and this was incubated at 38°C and acidity measurements were performed at different times until reaching 42°C (Standard and industrial search of Iran). At least this product was kept in refrigerator at 4°C, subsequently (MOATTER and SHAMS KASHANI, 1378; Iran standard and industry research institute, 1385). 2.3. Adding aflatoxin M1 and Thymus extracts to produced kefir Ten mL of produced kefir was poured equally in 4 tubes and 10 mL of contaminated milk with 200 ppb of AFM1 was added to each tube. Finally, different doses of Thymus extract (2, 4, 6 g/L) were added to the samples (OTLES and CAGINDI, 2003; MARHAMATIZADEH et al., 2011; MARHAMATIZADEH et al., 2012). 2.4. Adding aflatoxin M1, Thymus extract and Lactobacillus acidophilus or Bifidobacterium bifidum to kefir Four g/L fixed Thymus with 1 × 108, 3 × 108 and 6 × 108 Bifiodobacterium bifidum or Lactobacillus acidophilus were poured in 4 tubes and 10 mL of contaminated milk by 200 ppb of AFM1 was added to them, subsequently (OTLES and CAGINDI, 2005; MARHAMATIZADEH et al., 2011; MARHAMATIZADEH et al., 2012). Finally, all samples were kept into the incubator for 24 hours at 24°C. After 24 hours, the coagulation was separated from liquid by using a cloth filter, and remained liquid was incubated at 14°C for 24 hours. It was kept for 48 hours in refrigerator at 4°C and all samples were analyzed with ELISA reader (Europroxima Company), subsequently. 2.5. ELISA test Samples were centrifuged at 2,000 rpm for 10 min at 7°C and the upper oil layer was removed. Then, 100 mL of these samples were used for ELISA test. ELISA reader device in three repetitions and distinguished three optical densities for every sample was done. And every of three optical densities were put in Excel program (collected by Euro- Proxima   Ital. J. Food Sci., vol 28, 2016 - 520 Company) and three concentrations were got that the average of these three shows AFM1 reminder in every samples. 2.6. Statistical analyses The data was analyzed by using Kruskal-Wallis test in a meaningful surface at P< 0.05 by SPSS software (SPSS for Windows, version 20, SPSS Inc, Chicago, IL, USA). 3. RESULTS 3.1. Evaluating primary milk contamination with AFM1 The results showed that primary milk contains 32 ppb AFM1 and then 200 ppb of AFM1 were added to milk that totally AFM1 becomes 232 ppb. Table 1 shows the changes of aflatoxin level in the present of Thymus extracts, Bifidobacterium bifidum and Lactobacillus acidophilus bacteria in various groups. Table 1: Comparison of AFM1 concentration in the presence of Bifidobacterium bifidum and Lactobacillus acidophilus bacteria and Thyme extracts. Sample tubes Added Aflatoxin (ppb) AFM1 value added in the form of (ppb) Initial Aflatoxin (ppb) AFM1 value primary The remaining amount of aflatoxin (ppb) AFM1 value Reduced aflatoxin (ppb) AFM1value Decreased (ppb) Reduced aflatoxin (%) AFM1value decreased (%) 40 g kefir 200 232 174.3 57.7 24.87 2 g/l Thyme extract 200 232 154.6 77.4 33.36 4 g/l Thyme extract 200 232 155.9 76.1 32.80 6 g/l Thyme extract 200 232 162 69.8 30 4 g Thyme extract and 1×108 Bifidobacterium bifidum 200 232 169.5 62.5 26.93 4 g/l Thyme extrac and 3×108 Bifidobacterium bifidum 200 232 175.3 56.7 24.43 4 g/l Thyme extract and 6×108 Bifidobacterium bifidum 200 232 148.3 83.7 36.07 4 g/l Thymus extract and 1×108 Lactobacillus acidophilus 200 232 125.8 106.2 45.77 4 g/l thymus extrac and 3×108 Lactobacillus acidophilus 200 232 186.1 45.9 19.78 4 g/l thymus extracts and 6×108 Lactobacillus acidophilus 200 232 169.1 63 27.15 3.2. Evaluating kefir containing Thymus in detecting AFM1 The results indicated in Table 1 showed that Thymus extracts in kefir caused AFM1 reduction largely. The reduction percent of AFM1 in the kefir samples containing different concentrations of Thymus extracts 2, 4 and 6 gr/L were 33.36%, 32.80% and 30%,   Ital. J. Food Sci., vol 28, 2016 - 521 respectively. The most reduction of AFM1 was detected by 2 gr of Thymus and Thymus extract with 6 g/L concentration showed the least percent of AFM1 reduction. 3.3. Evaluating probiotic kefir containing Thymus and Lactobacillus acidophilus in AFM1 reduction The data in Table 1 shows that fixed quantity of Thymus (4 gr/L) with different amount of Lactobacillus acidophilus declined Aflatoxin. Reduction of AFM1 in the presence of Thymus extract and different value of Lactobacillus acidophilus 1×108, 3×108 and 6×108 were 45.77%, 19.78% and 27.15, respectively. The most decrease of Aflatoxin was detected with 1×108 level of Lactobacillus acidophilus and the amount 3×108 of bacteria has the least reduction of aflatoxin. 3.4. Evaluating probiotic kefir containing Thymus and Bifidobacterium bifidum in deleting AFM1 The results showed that Bifidobacterium bifidum bacteria decrease the amount of AFM1 in the samples. The most decline percent of AFM1 in the presence of 6×108 of Bifidobacterium bifidum occurred with 36.07%; and 1×108 and 3×108 of bacteria with 26.93% and 24.43%, respectively, had the least AFM1 reduction. 4. CONCLUSIONS The aim of the present study was the evaluation of the effect of Thymus extract and Lactobacillus acidophilus and Bifidobacterium bifidum bacteria on the reduction of Aflatoxin amount in kefir beverage. Furthermore, the possibility of producing a new probiotic food based on kefir and Thymus was assessed. The results of present study indicated that Thymus extract has anti-aflatoxin activity and capability for aflatoxin reduction. The Thymus extract in combination with probiotic bacteria has the more ability for aflatoxin decline. The results of the current research showed that Thymus extract with 2 gr/L concentration in combination with 6×108 Bifidobacterium bifidum have the most effect in reduction of AFM1 and the strongest anti- aflatoxin activity was shown by the Thymus extract with 4 gr/L concentration in combination with 1×108 Lactobacillus acidophilus. Our results are agreed with other studies (OTLES and CAGINDI, 2003; LEE et al., 2003; TRATNIK et al., 2006). Probiotic foods have been produced in order to treat intestinal infections as well as genital diseases. In three decades ago, commercial probiotic products have been supplied to the world market grew. Kefir is a fermented dairy product which originates from the Caucasus Mountains in Eastern Europe (TRATNIK et al., 2006). Kefir is the most popular probiotic product in Europe. Kefir has beneficial effects in healing and homeostasis due to its vitamins, minerals and essential amino acids (OTLES and CAGINDI, 2003). The vitamin content of kefir affects both type of milk and microbiological flora (SARKAR, 2007, ARSLAN 2014). Aflatoxins are a group of fungal secondary metabolites which are recognized as being of economic and health importance (10). AFB1 is currently of great interest due to their toxic, carcinogenic and mutagenic nature on human and animal health (11). A number of studies reported that many micro-organisms, including bacteria, yeasts, moulds, actinomycetes and algae are able to remove or degrade small amounts of aflatoxin in foods and feeds (LEE et al., 2003).   Ital. J. Food Sci., vol 28, 2016 - 522 Several lactic acid bacteria strains have shown different capabilities for binding AFM1 in solutions and in milk (HASKARD, et al., 2001). The various studies have reported that certain lactobacilli and Bifidobacteria are capable of removing AFB1 from liquid solutions by binding the toxin (PELTONEN et al., 2001; HASKARD et al., 2001). Some researchers have suggested that aflatoxin binds predominantly to polysaccharides and peptidoglycans of the bacterial cell wall (LAHTINEN et al., 2004). LOPEZ et al., (2003) showed that Saccharomyces yeast reduced 90% AFM1 in the milk. Powders and extracts of many herbs, plants and spices have been reported to inhibit the production of aflatoxin (PARANAGAMA et al., 2003). A recent study showed that the essential oils of T. daenensis and Thymus spp. (Elam ecotype) flowers exhibited antibacterial activities against L. monocytogenes from chicken meat (GHASEMI PIRBALOUTI et al., 2009). LIXANDRU et al., (2010) evaluated antimicrobial activity of some plant essential oils against bacterial and fungal species. The results showed Thymus, Coriander and Basil oils proved the best antibacterial activity, while Thymus and Spearmint oils better inhibited the fungal species. Thymus vulgaris extracts with 0.01 and 1% concentration decreased the AFB2 production by 83 and 91 % (FOUAD et al., 2013). HAMZAWY et al., (2012) reported that ethanolic and aqueous extracts of Thymus vulgaris has potential hepatorenoprotective effects against aflatoxins because of antioxidant properties and radical scavenging activity. In conclusion, the results of the current study revealed that Thymus extracts and probiotic bacteria can reduce AFM1 in kefir drink. Our results are agreed with other researches that showed probiotic bacteria and Thymus extract have anti-aflatoxin activity. Moreover; it may be stated that aflatoxins are not only a big problem at crop production level, but also it has become a global health topic due to the consequences following their consumption in animals and human being. 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