ناهید مژگانی

 The food and water contamination with heavy metals is increasing due to the environmental pollutions. Heavy metals are the elements with the density of more than 5 g/cm3 and have become a serious problem as a result of the urbanization and industrialization. These toxic metals pollute water, soil, plants, and eventually foodstuffs and our bodies. Several methods exist to remediate heavy metal pollution in waters such as membrane filtration, ion exchange mechanisms, or by precipitation. Yet, these techniques are not cost effective, in some cases, and do produce wastes that need to be properly disposed of. Microbial bioremediation could be an alternative. The use of microbes for remediation of heavy metals has been well studied. Some microorganisms, especially soil bacteria, have the ability to tolerate these contaminants. In addition, certain bacterial strains are capable of binding to heavy metals or transforming them into less toxic forms. Low operating costs, usable in foodstuffs, selective removal for specific toxic metals, minimal use of chemicals (resulting in low sludge production) and high efficiencies at very low levels of heavy metals are some of the advantages of biosorption methods. In this regard, the purpose of this study was to investigate the ability of active and passive absorption of heavy metals by a number of Lactic Acid Bacteria (LAB) strains in laboratory environment and food. 

 Seven LAB isolates including Lacticaseibacillus casei (RTCC 1296-3), Lacticaseibacillus rhamnosus (RTCC 1293-2), Lactiplantibacillus plantarum (RTCC 1290), Limosilactobacillus fermentum (RTCC 1303), Enterococcus faecium (RTCC 2347), Lactobacillus helveticus (RTCC 1304) and Lactobacillus acidophilus (RTCC 1299) were obtained from Razi type culture collection (RTCC), located at Razi vaccine and Serum Research Institute, Iran. All isolates were cultured in MRS (Scharlau, Spain) broth medium, at 37 °C for 24 hours, under anaerobic conditions. Pure cultures were preserved for long term by freezing at -70°C with 20% Glycerol. Heavy metals including Nitrate of Pb (II), Cd (II) and Ni (II) were purchased from Merck (Darmstadt, Germany). All standard solutions were prepared from the stock solutions containing 1000 mgl-1 in distilled water. Other chemicals used in study including Nitric acid (65%) and Hydrogen peroxide (37%), were also purchased from Merck, Germany. This study was conducted in two in- vitro and in-vivo phases; in the in- vitro phase, seven strains of bacteria with probiotic properties (L. casei, L. rhamnosus, L. plantarum, L. fermentum, Ent. facium, L. helveticus and L. acidofilous) were screened and then their ability to bind to cadmium (Cd), Lead (Pb) and nickel (Ni) in aqueous solution was investigated. Then, in the in-vivo stage, three probiotic strains that had the highest biosorption efficiency in the previously stage were selected and their effect with a ratio of 1:1:1 and contact time of 15 and 30 minutes on the removal of these toxic metals in coriander, leek and parsley fresh vegetables was evaluated. The residual concentrations of heavy metals in solution were measured by Inductively Coupled Plasma Mass Spectrometer (ICP-MS; ELAN DRC-e, PerkinElmer SCIEX, Canada) and Morphology of bacteria cell surfaces incubated with metals were monitored by scanning electron microscopy (JEOL JSM 5400 LV, Japan). 

The results of the in vitro stage showed that the most ability to heavy metals adsorption was related to the Ent. Facium bacterium which were equal to 79.75±0.11, 75.28±0.05 and 83.99±0.10% for Pb, Cd and Ni, respectively.  In general, the removal efficiency of heavy metals by LAB bacteria in the inactive and killed state was significantly higher than the active removal efficiency of these bacteria, so that the highest percentage of passive absorption of lead, cadmium and nickel metals by inactive strains of L. casei, L. plantarum and Ent. Facium were 90.01, 81.98 and 86.56%, respectively. Electron microscopy observations and energy dispersive X-ray (EDX) analysis confirmed that the majority of these toxic metals significantly damage the surface of living cells by accumulating and binding on the surface of bacterial cells. A combination of three bacterial strains had a synergistic effect on the binding properties of toxic metals compared to the single state of these bacteria, so that in both active and inactive states, 90-99% of heavy metals from edible leafy vegetables were removed in less than 15 minutes. The results of this research generally showed that the binding capacity of dead biomass is significantly high and it is possible to dispose and reuse biomass in case of biological absorption.

In this study, the role of bacteriocin produced by Lactobacillus plantarum TA013 (Plantarsin which has the most effect against Listeria monocytogenes) in the persistence of lactic cheese (LCh) was investigated. Groups: 1) LCh without probiotic bacteria and bacteriocin as a control 2) Probiotic LCh containing L. plantarum 3) LCh containing Plantarsin and 4 (LCh containing Nisin were considered. Physicochemical and microbiological properties of cheese in each treatment were determined in terms of pH, dry matter, fat, protein, salt, aflatoxin M1, fatty acid profile, and the growth inhibition of L. monocytogenes during 60 days storage in the refrigerator. Statistical analysis of this design was performed in a completely randomized design. The results showed that the reduction of L. monocytogenes in the treatment of LCh containing nisin in comparison with the control group was 3.05 Log(CFU/g), which was significantly different from the control group. in other treatments containing probiotics and Plantarisin, the rate of reduction of this pathogen compared to the control treatment was 2.76 and 1.79 Log(CFU/g) respectively, but no significant difference was observed between the two samples. Also, the survival rate of probiotics at the end of storage was 107 CFU/ g in the refrigerator and only two Log(CFU/g) were reduced. The results showed that cheese containing probiotics had superior properties over other treatments. Therefore, environmental conditions such as pasteurization are not sufficient to completely eliminate L. monocytogenes in lactic cheese, and it is necessary to use natural additives to create favorable conditions in different stages of production and storage.

 

Lactic cheese is a highly consumed dairy product that is of great nutritional values. However, great concerns are reported regarding their safety, microbial quality and short shelf life. Addition of antimicrobial compound - producing lactic acid bacteria as protective cultures is known to improve safety and ensure quality of food products. The current study was carried out to assess antibacterial and several technological characteristics of autochthonous Lactic acid bacterial isolates for their use in goat milk lactic cheese to control Staphylococcus aureus and Listeria monocytogenes.

Antibacterial and other technological characteristics, including proteolytic activity, diacetyl production, autolytic activity and survival, in various NaCl concentrations and at suboptimal temperatures of several Lactic acid bacteria solates were assessed. The potent isolates were identified to species level by phenotypic and genotypic methods (16 srRNA gene sequencing) and finally th ey were assessed for their ability to inhibit Staphylococcus aureus and Listeria monocytogenes in prepared goat milk lactic cheese.

Lactic acid bacterial isolates, showing significant antibacterial action, proteolytic activity and diacetyl production, were identified as Lactiplantibacillus plantarum RTCC 1290 - 1, Lactobacillus acidophilus RTCC 1299 and Lacticaseibacillus casei RTCC 1296 - 1. These isolates survived at 25 and 45 ºC, tolerated up to 8% NaCl and 55 ºC for 10 min ( p ≤0.01). Lactobacillus acidophilus demonstrated the highest autolytic activity (38.69%) followed by Lacticaseibacillus casei (28.44%) and Lactiplantibacillus plantarum (18.38%), respectively. Cocultures of the selected lactic acid bacterial isolates in goat milk lactic cheese resulted in a 5 log CFU g - 1 decrease in Listeria monocytogenes , while, Staphylococcus aureus counts decreased by only 3 Log CFU g - 1 ( p ≤ 0.05) during a month of storage at room temperatures. However, lactic acid bacterial counts increased 2 – 4 Log CFU g - 1 from Day 15, which was reported stable up to Day 30. In conclusions, Lacticaseibacillus casei, Lactiplantibacillus plantarum and Lactobacillus acidophilus bears promising biopreservative effect for the control of foodborne pathogens.

The determination of bovine tuberculosis is made with different tests and with certain limitations. Antigenic proteins can be extracted from Mycobacterium bovis culture that can improve the sensitivity and specificity of diagnostic tests. One of the targets of this study is to design an indirect ELISA. For this reason, purified Mycobacterium bovis AN5 antigen were utilized to distinguish bovine tuberculosis. Another goal is to review and compare the results of the system designed to Interferon-ɣ (IFN-ɣ) assay. Mycobacterium bovis AN5 antigens were isolated and purified. The selected antigen was then used to design the indirect ELISA system. The sera of 230 cows from different farms were tested with designed ELISA. Their results were at that point compared with interferon-gamma test as a corroborative diagnostic test. Correlation and independent study between variables were performed by Pearson and T test. The sensitivity and specificity of the designed indirect ELISA system were 77.8% and 80.7%, respectively. The p value for Pearson and T test was zero percent. Noteworthy lightweight antigens were purified that might analyze the disease. Due to the high sensitivity and specificity gotten, the ELISA system outlined for starting screening within the diagnosis of bovine tuberculosis.

Tuberculosis is one of the major global health problems with high mortality. Establishing a rapid and low-cost identification method is significant, especially in developing countries. In this study, we aimed to design an indirect ELISA system with low molecular weight antigens secreted by Mycobacterium tuberculosis strain C, for identification of Mycobacterium tuberculosis Complex infection in human.

The secretory proteins from culture filtrate of Mtb strain C extracted by Sephadex-G50 were used as target antigen in designing an indirect ELISA system. Then, human sera with tuberculin skin test, culture, acid-fast bacilli smear, and PCR test results were selected to detect human serum antibodies against these low molecular weight proteins.

During gel chromatography and SDS-PAGE (12.5%) analysis, the purified protein F2 fractions with approximately 0.7 mg/ml and molecular weights in the range of 14-40 kDa were used as the target antigen in designing the indirect ELISA system. The ELISA system designed by F2 showed 78% sensitivity and 92% specificity in identifying tuberculosis.

The ELISA results based on low molecular weight proteins, with the sensitivity and specificity achieved in this study, might support the hypothesis that the Mtb culture filtrate antigens could be used as a rapid and sensitive assay in ELISA test for detection of pulmonary TB.

Burkholderia mallei, is the etiologic agent of the glanders disease. Clinical and bacteriological diagnosis of glanders is difficult in the early stages of the disease. Some methods such as Complement fixation test (CFT) due to false positive results and troublesome for veterinary authorities and cause financial losses to animal owners, The other one is Malleination test, which requires appropriate equipment and efficient laboratory personnel. Therefore, in order to quickly and accurately diagnose the disease, especially in areas that cannot be kept animals, new methods should be used to identify the disease. The Western blot is a serological diagnostic test and has been recommended by the World Organization for Animal Health (OIE). In this study, a Western blot assay making use of a purified lipopoly-saccharide (LPS) containing antigen of Burkholderia mallei was designated.

in this study, a total of 75 sera were collected from different horse populations from several geographical areas of Iran. Specificity and sensitivity of the Complement fixation test, ELISA and a Western blot were compared for serodiagnosis of glanders. ELISA test was based on B. mallei antigens and Western blot made use of a purified LPS-containing B. mallei-antigen.

The Western blot and ELISA, were more specific than the Complement fixation test. ELISA based on B. mallei antigens had more sensitivity compared to Complement fixation test and Western blot. Finally, sensitivity and specificity were obtained for Complement fixation test (92.31% and 98.38%), Western blot (92.31%, 100%) and (100% and 100%) respectively.

Complement fixation test for glanders is still the prescribed serological method for commercial purposes, which is used to confirm the health of animals. However, in order to maintain the biosafety of valuable and expensive horse colonies, it is important to implement a more accurate and intelligent disease control and eradication program. This enhances the laboratory diagnostic capabilities to better understand the cause of the disease and to use and optimize the diagnostic methods of glanders in the country. Therefore, efforts to further improve and optimize ELISA and Western blotting should be continue.

The bovine rumen is the suitable growth medium to many of the bacteria responsible for converting food into energy. In recent years, many rumen microbes have been identified and isolated using 16sRNA gene sequencing. Some microbes are suggested as food additives to promote the growth and production of animals. The use of probiotics as feed additive has been induced by stimulating the positive effects on the host by creating a favorable balance of intestinal microflora. Therefore, is study aimed to isolate, identify and select lactic acid bacteria to produce probiotics from the rumen and feces of Sistani cows.

In this experiment, samples were taken from the rumen of 12 and the feces of 4 Sistani cows in the industrial slaughterhouse of Zabol city. The samples were transferred to test tubes containing liquid MRS culture medium and incubated at 39 ° C for 48 hours. After growing from the mentioned medium, the plates containing MRS agar were linearly cultured and the plates were incubated in aerobic conditions at 39 ° C for 48 hours. The emerged colonies were examined for growth characteristics and morphology as well as purity. Gram-positive, spore-free, spherical negative catalase, and rod-shaped bacteria that did not produce beta hemolysis were tested for antimicrobial, acid, bile salt, and antibiotic susceptibility testing.

Of the initial 158 isolates of lactic acid bacteria, 84 isolates were tested for antimicrobial activity against four species of Salmonella and one species of E. coli. Then the isolates obtained from the antimicrobial test were examined for pH resistance test. Thirteen samples selected from this experiment were studied for bile resistance test, of which five isolates were examined for antibiotic susceptibility testing and entered the final stage for genetic and molecular testing.There were only two isolates that were obtained by molecular testing and after sequencing and using NCBI software had a high genetic affinity with two bacteria Streptoccos infantarius (isolate A) and Entrococcos faecium isolate B. These two isolates were observed as potential probiotics in Sistani cows.

The general results of the study showed that isolates A and B have suitable properties for the use of probiotics. . These characteristics are high survival rate at low pH and 3% bile concentration and inhibition of pathogenic bacteria such as E. coli and Salmonella, as well as antibiotic resistance. Therefore, isolates A and B will later be used as probiotics the ruminants feed.

European honey bees (Apis mellifera) are the most important pollinator insects that play vital role in maintenance of all most all life forms on earth. However, over the last decade major concerns have raised due to decline in the population of these insect species. A variety of factors have been responsible for these concerns of which the most important is honey bee bacterial diseases like Nosemosis (Nosema), American and European foulbrood diseases. While, overuse of antibiotics utilized for the treatment and control of these diseases has resulted in the emergence of antibiotic resistant strains of these pathogens. Probiotics have been considered a suitable substitute of antibiotics in human and animals. In last several years, lactobacillus species isolated from honeybees have been considered of significance in enhancing the life span of honeybees by reducing the incidence of bacterial and viral infections in these tiny insects. Among the isolated microbes in the gut of honeybees, Lactic Acid Bacteria (LAB) are of utmost importance showing direct impacts on the health of their host by modulating the gut microbial flora and are termed as Probiotic bacteria.The objective of this research was to isolate and identify LAB from different parts of the intestinal tract of honeybees Apis mellifera and to characterize their probiotic properties.

Twenty-four honeybees collected from the hives located in the city of karaj were analyzed for the presence of LAB species. The stomach contents of honeybees were inoculated into MRS broth, incubated at 37ºCfor 48 hrs. The obtained colonies were purified and identified to species levels phenotypically and geno-typically. Hemolytic activity and sugar fermentation reactions of the isolates were recorded and later subjected 16SrRNA sequencing using a pair of universal primers. The identified isolates were evaluated for their viability in acidic conditions at pH 2.5, 3.0, 4.0 and 6.5 at different time intervals. Bile resistance of the isolates was tested by culturing the isolates in the presence of different concentrations of the said salts (0.5, 0.7 and 1%). Survival of LAB isolates in simulated gastric and intestinal conditions containing different enzymes and bile salts, antibacterial spectrum against a number of gram positive and gram-negative pathogens by agar well diffusion assay, and their in vitro colonization ability (aggregation, co-aggregation and hydrophobicity percentages) were evaluated. The results were analyzed statistically.

Twenty nine gram positive, catalase negative and non-hemolytic colonies were isolated from 24 honeybee samples. Among these, only 7 colonies showed enhanced antibacterial activity and were selected for further studies. Based on phenotypic characteristics, sugar fermentation reactions and 16S rRNA sequencing the isolates were identified as Lactobacillus acidophilus (1), Lacticaseiobacillus casei, Lactiplantibacillus plantarum (2), Lactobacillus apis (1), Enterococcus faecium (1) and Pediococcus acidilactici (1). During probiotic characterizations, the identified isolates were shown to retain their viability in acidic conditions and resisted pH 2.5, 3 and 4 for more than 4 hrs. However, slight decrease in viability at pH 2.5 and 3.0 was observed, compared to pH 4.0 and above. All isolates appeared bile resistant and tolerated all used concentrations of bile salts during 8 hrs of incubation. Survival rate of the isolates in simulated intestinal conditions was significantly (p˂0.05) greater compared to simulated gastric conditions indicating greater stability of the isolates to alkaline conditions rather than to acidic conditions. L.acidophilus and E.faecium showed least resistance in gastric conditions and their growth rate was decreased more than 50% under said conditions. In contrast, the growth rate of these two isolates was highest in simulated intestinal conditions as they resisted these conditions for more than 24 hours.  The isolates demonstrated antibacterial affect against a number of tested pathogens including Listeria monocytogenes, Escherichia coli, Enterococcus faecalis and Streprtococcus mutans. The auto-aggregation, and cell surface hydrophobicity percentages of L.casei appeared highest compared to other tested Lactic Acid bacteria in study (p˂0.05), while, L.apis showed the highest co-aggregation with S.typhi strain. P.acidilactici possessed the least auto-aggregation (46%), co-aggregation (10%) and hydrophobicity (43%) percentage. Auto-aggregation ability appeared directly related to hydrophobicity percentages and isolates showing high aggregation ability also showed high hydrophobicity %.

In conclusion, honeybee gut appeared a reservoir of LAB with probiotic potential. It is suggested that further studies should be conducted in order to determine the health benefits of these LABs in honeybees, with especial emphasis on their ability to prevent honeybee diseases.

The aim of the research was to investigate the antibacterial potential of local isolates of probiotic bacteria in the presence of herbal extracts including Chamomile (Matricaria chamomilla), Fennel (Foeniculum vulgare), Peppermint (Mentha piperita) and thyme (thymus vulgaris). Lactobacillus casei(TA0021), L. plantarum(TA0028,TA0026), L.reuteri(TA0034), Enterococcus faecium(TA0033) and Pediococcus acidilactici(TA0049,TA00170) were used as probiotic bacteria, while standard strains of Escherichia coli, Salmonella typhimurium and Salmonella enteriditis were used as indicator organisms. The antibacterial activity of the mentioned probiotic isolates and the herbal extracts against the selected pathogens were examined by agar well diffusion method. The synergistic effect of the mix of probiotic bacteria and different herbal extracts(1:10,1:100 and 1:1000) were also studied. The viability and synergistic inhibitory effect of the selected probiotic bacteria in the presence of different concentrations of herbal extracts were determined after 24, 48 and 72 hours. In order to determine the minimum inhibitory concentrations (MIC), herbal extracts were serially diluted and their effect on fixed concentrations of the indicator organism was measured by microdilution assay. Similarly, the survival percentage of probiotic bacteria in the presence of different concentrations of the herbal extract was determined. MIC values were recorded as the lowest concentration of extract that was able to inhibit the growth of the pathogens. All experiments were performed in triplicate and results analyzed statistically(Using the SAS Software). According to the results the probiotic bacteria in the presence of different concentrations of the extracts was above 70%(P<0/05). Overall, the locally isolated probiotic bacteria with herbal extracts are strong antibacterial agents.

The pathogenesis of Mycobacterium tuberculosis (Mtb) is related to its low molecular weight proteins mainly ESAT6 and CFP10 that are highly specific and potentially useful for the diagnosis of tuberculosis. This research focused on isolation, purification, and characterization of low molecular weight proteins from Mtb. Cultures of Mtb were inactivated by heating at 68 °C for 90 min and 100 °C for 3 hrs, respectively. Inactivated cultures were filtered and the proteins in the supernatant fluid precipitated with two rounds of ammonium sulfate, at 4 °C. The collected precipitates were dialyzed and subjected to gel chromatography (G-50) and the obtained fractions were analyzed for protein concentrations and molecular weight. ESAT6 and CFP10 protein complex in the purified fraction was confirmed by Western blotting. Guinea pig sensitization assay was used for estimating the potency of the purified fraction compared to the standard PPD. The maximum amount of low molecular weight proteins were precipitated by 20% ammonium sulfate. SDS-PAGE analysis revealed protein bands of approximately 10-15 kDa. The purity of the proteins was ≥95%, as confirmed by SDS-PAGE. The presence of the ESAT-6/CFP10 complex was confirmed by Western blot analysis. The purified fractions showed no cross-reaction with BCG or M. avium strain. ESAT-6/CFP-10 purified by the ammonium sulfate method appeared to be suitable for the development of a diagnostic kit for the detection of Mtb.

Aflatoxins are mainly developed during the storage of feedstuffs, and their destruction is difficult after the occurrence. The most practical strategy to combat aflatoxins is the use of mycotoxin binders.

Comparison of the efficiency of traditional and lab-synthesized polymeric mycotoxin binder with gastrointestinal microflora modulating feed additives in amelioration of aflatoxin effects in broiler chicken.

A total of 240 1-day old broilers (Ross 308, straight forward) were examined in a completely randomized design with five treatments and four replicates of 12 birds for 24 days of study duration. Treatments were: 1. The negative control, feed without aflatoxin or any feed additive, 2. The positive control, aflatoxins contaminated feed (500 µg/kg), 3. Aflatoxins + probiotic (Hypro Tect), 4. Aflatoxins + molecular imprinted polymer (MIP), and 5. Aflatoxins + commercial toxin-binder (Zarin-binder). The growth performance of birds was measured during the experiment. At the end of the experiment, some biochemical and immunological analyses were performed on blood samples. Some bone characteristics were studied on tibia samples.

Supplementation of probiotics and toxin-binder in aflatoxin-contaminated feed improved the aflatoxin-induced reduction of feed intake and body weight gain in the first 10 days of the experiment (p < /em><0.05), compared to positive control group. Aflatoxin alone (the positive control) or with the feed additives did not affect feed conversion ratio. Aflatoxin reduced the levels of serum total protein, albumin, phosphorus, magnesium, and zinc (p < /em><0.05). Use of probiotic, MIP and commercial toxin-binder, in aflatoxin-contaminated feeds, has alleviated the adverse effects of aflatoxin on serum albumin (p < /em><0.05). The tibia weight increased in probiotic and MIP fed broilers compared to the birds fed aflatoxin-contaminated feed without additives-the positive control (p < /em><0.05). The highest tibia breaking strength was observed in probiotic fed birds, which was different from that of the positive control group. The tibia length was decreased by the aflatoxin compared to the negative control birds (p < /em><0.05). Anti-SRBC titers were decreased in aflatoxin contaminated group without feed additive supplementation-positive control (p < /em><0.05).

The tested feed additives in present study exerted just partial protection against some aflatoxicosis effects. The extent of effectiveness of studied feed additives in amelioration of aflatoxicosis affects on performance, immunological, skeletal and serum biochemical parameters could be ranked as probiotics, MIP and toxin binder, respectively.

To investigate the effect of oral administration of prepared specific egg yolk antibody (IgY) against enterotoxigenic Escherichia coli K99 on growth performance, health, the prevalence of diarrhea of neonatal calves, an experiment was conducted using 60 newborn calves in a completely randomized design with two treatments. Calves were kept individually. Calves in the control group received only milk or colostrum for 30 days and calves in the treatment group received milk or colostrum with IgY against inactivated K99. During the experiment, health parameters and the prevalence of diarrhea were recorded daily, and growth performance was evaluated weekly. The results showed that average daily gain, starter dry matter intake and feed and milk efficiency were higher in the IgY group than the control group (P<0.05). The general health score in the IgY group was higher than control group and fecal and ear scores in this group were significantly lower than the control group (P<0.05). The count of fecal coliforms in the IgY group was decreased by approximately one log compared to the control group (P<0.05). The results of the present study showed that oral administration of IgY against enterotoxigenic Escherichia coli K99 has beneficial effects on health and average daily gain of suckling Holstein calves and It can be a good way to replace antibiotics.

The purpose of current study was to investigate the synergistic effects of indigenous probiotic strains isolated from the gastrointestinal tract with herbal extracts on gastrointestinal mucosa health (intestinal microflora) and immunity of broiler chicks. In current study, the effect of two types of probiotics (Iranian and foreign products respectively), phytobiotic (thyme extract), profit (a mixture of thyme extract and probiotic) and antibiotics on broiler chicken’s performance were evaluated. Experimental diets provided from day one to end of the period. Statistical analysis results show that adding profits and both probiotics to the diet of chicks leads to a significant decrease in feed intake compared to the control and antibiotic treatment (p≤0.05). All supplements increased the relative weight of lymphatic tissues of the spleen and bursa. Probiotics and prophylaxis treatments had a significant higher weight gain than phytobiotic (Thyme) treatment (p≤0.05). Experimental treatments compared to control treatment significantly improved FCR (p≤ 0.05). Dietary additives can improve the immune system and adding them to broiler chicks diet lead to improving performance and in the future can be introduced as an alternative to antibiotics in poultry feeding.

A number of bacillus species have been recognized as effective and beneficial probiotics. However, some of the bacillus species are known to carry virulence genes which are the main cause of disease, hence, it is essential to evaluate their safety before considering them as probiotic. To date, in our country no studies have been performed on the genotypic virulence determinants of Bacillus species recognized as probiotics. In this study we aimed to assess the safety of indigenous bacillus species isolated from broiler chicken (Bacillus subtilis and B.leichniformis) and honey bee (B.subtilis and B.megaterium) samples. The selected bacillus species were identified based on biochemical and molecular, and evaluated for their probiotic properties such as acid and bile resistance, antimicrobial activity against Streptococcus agalactiae, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Listeria monocytogenes and Escherichia coli, and antibiotic susceptibility pattern. The hemolytic activity, DNase, lecithinase, lipase, gelatinase, and coagulase activity were also studied. The presence of virulence genes, including hbl, Nhe, cytK, bceT, lipA, ces, was analyzed by molecular methods in a PCR reaction. According to results, all 11 Bacillus species showed considerable resistance to acid and bile, and appeared sensitive to all tested antibiotics. Seven isolates were beta hemolytic and lecitinase positive, one isolate was DNase positive, four isolates were cytK positive, while TA0044 and TA0046 were LipA positive. Only two isolates of poultry origin lacked all tested virulence genes and were classified as safe probiotic.

In the present study, native fungi producing phytase from soil and other environmental samples (Includes: soil around the root of leguminous plants, Cows and goats excrement, Chicken floor, Vermicompost, Mineral phosphorus mine, Sewage water, Plants and fruits infected with fungi and Samples prepared from research centers of the country)were isolated and incubated for 5 days at 28 ° C on SDA(Saburad Dextrose Agar). Then a phytase screening medium (PSM) was used to identify and isolate fungi capable of producing phytase enzyme. Morphological characteristics of fungal strains were studied using optical microscopy.Samples which produced clear halo were chosen for enzymatic activity assay based on the release of a phosphorus nanomol in one minute. Based on the amount of enzyme and amount of biomass production, were evaluated by the respective isolates.The optimum pH and temperature for maximum production of enzyme through the selected isolates were 5.5 and 30 °C, respectively. Finally, the isolates were identified as Fusariumoxysporum and Aspergillus Niger using Genetic analyzer system. The maximum amount of enzyme production was recorded as 130.11 and 125.27 enzymatic units for Fusarium oxysporum and Aspergillus Niger, respectively. The results showed that from five to seven days during enzyme culture are the most suitable time for enzyme extraction and purification.The results of morphological studies were completely consistent with the molecular identification results

This experiment was conducted to compare the effect of some poultry probiotics produced in Iran on performance parameters, economic indicators and small intestinal morphology of broilers using 500 one-day-old Ross 308 broilers in a completely randomized design with 5 treatments and 5 replicates per each treatment. The experimental treatments were: 1- basal diet (control), 2- basal diet + Bacto-Gene® probiotic, 3- basal diet + Di-Pro® probiotic, 4- basal diet + Hypro-Tect® probiotic and 5- basal diet + Probitaz® probiotic. At 24 and 41 days of age, one male broiler chick was slaughtered from each experimental unit. Orthogonal contrasts were done to compare the effect of probiotics versus control. The results of this study showed that the treatments containing probiotic had no significant effect on body weight gain and feed conversion ratio. The utilization of probiotic Hypro-Tect® resulted in significant reduction in feed consumption in whole experimental period compare to other treatments containing probiotic (P≤0.05). The final body weight, European efficiency factor, economic indices and carcass percentage were not affected by experimental treatments. The utilization of probiotics had no significant effect on intestinal relative length and morphological indices of jejunum and ileum. In orthogonal contrast, the utilization of treatments containing probiotic increased feed cost as compared to control group (P<0.05). According to the results of this study, it seems that the utilization of studied probiotics had no significant effect on performance parameters, economic indices and small intestinal morphology of broilers reared in optimal condition as compared to control group.

In this study the antimicrobial potential of Lactobacillus plantarum strains isolated from goat,s milk was evaluated. Sixty seven goat,s milk samples were collected from Tehran and Karaj and screened for the presence of lactic acid bacteria (LAB). Among the isolated LAB, 18 strains were identified as Lactobacillus plantarum by their carbohydrate fermentation pattern and 16S rRNA sequencing. Antimicrobial activity of the isolatedLactobacillus plantarum strains against a number of pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Listeria monocytogenes and Escherichia coli was evaluated by agar spot and well diffusion method. Results indicated 10 of theLactobasillusplantarum strains to possess inhibitory activity towards the tested pathogens, while 6 of these strains were shown to have wide spectrum of inhibitory activity and were able to inhibit the growth of the entire tested gram positive and gram negative pathogens. The antibacterial activity demonstrated by these 6 strains was further analyzed by subjecting their supernatant fluid to various physico-chemical treatments. The supernatant fluid of these strains retained their antagonistic action even after neutralization, while only 2 strains (LP4 and LP14) retained their antimicrobial activity after treatment with 1 mg/ml catalase. The supernatant fluids of the respected isolates LP4 was resistant to enzyme Lysozyme and lipase, while proteolytic enzymes like pepsin, trypsin and proteinase K completely destroyed its antibacterial potential. The isolates were resistant to their own antagonistic action. The bacteriocin like substance produced by LP4 was pH and heat resistant as it resisted wide pH range of 3-8 and autoclave temperatures of 121ºC for 15 min. This substance after 4 weeks storage at -20 0C still retained its antimicrobial activity and after treating by concentration of 7 percent NaCl, it was still active.

Enterococcus plays an important role in various industries. Some Enterococci isolated from dairy and breast milk has been reported as probiotics. However, these species selected as starter cultures or as probiotics should be evaluated for their safety and the absence of different virulence traits including virulence genes and antibiotic resistance. The aim of this study was to determine the safety of locally isolated Enterococcus species biochemically and targeting essential virulence genes by PCR method. A number of suspected Enterococcus species isolated from ewe milk, traditional cheese and mothers' milk were identified to genus level by phenotypic characteristics and 16SrRNA sequencing. The selected isolates were screened for their probiotic properties by determining their acid and bile resistance, antibacterial activity against a number of pathogens and antibiotic resistance. Phenotypic virulence parameters including lipase, DNAse and hemolysis of red blood cells, was determined. The presence or absence of a number of virulence genes including asa1, hyl, esp, agg, gelE, cylA, cylB, cylM, efaAfm, efaAfs and vancomycin resistance genes including vanA, vanH, vanR and vanY was evaluated. The results of this study determined the probiotic properties of some of these species. Four isolates showed acid and bile salt tolerance and showed significant antimicrobial effects. All isolates from Breast milk, lacked virulence genes. In virulance phenotypic experiments, all strains were alpha hemolytic. All strains were susceptible to Tetracycline, Cephalothin Amoxicillin, Cefazolin and Doxycycline and all of them except the TA00154 strain, were resistant to Nalidixic acid and Trimethoprim sulfamethoxazole.

In this study, the effect of probiotics, extract powder of thyme (Thymus vulgaris L.) and licorice (Glycyrrhiza glabra L.) were investigated on blood parameters, immune response, carcass characteristics and performance of broilers. Three hundred and thirty six broiler chicks (Ross 308) were housed in a completely randomized design with six treatments, four replicates and 14 birds in each experimental unit and reared on litter for 42 days. The treatments consisted of 1) basal diet probiotic Hyprozyme, 2) basal diet Bactocell, 3) basal diet Prophyt (extract powder thyme Hyprozyme licorice), 4) basal diet Phytobiotic (extract powder thyme licorice), 5) control and 6) basal diet antibiotic. The experimental diets were fed from day one until end of the experiment (day 42). Statistical analysis of data showed significant difference (P≤0.05) of FCR in probiotic Hyprozyme group ratio compared to the control group. Feed consumption was lowest (P≤0.05) in treatment 1 compared to others. The treatments had no significant effect on carcass characteristics. The relative weight of bursa Fabricius in antibiotic supplemented group was significantly different with treatment 3 (P≤0.05). Statistical analysis of the results showed significant difference (P≤0.05) of serum cholesterol in Bactocell group compared to control group. Addition of Haprozym to basal diet significantly increased (P≤0.05) the total protein and triglyceride levels compared to control group. Serum globulin was statistically higher (P≤0.05) in treatments 1, 3 and 4 compared to control group. The highest antibody titer against SRBC was observed in Bactocell group compared to control group (P≤0.05). Overall, it can be stated that the use of resources such as hypro-zyme (732gr/ton) and phytobiotic can be introduced as antibiotic alternatives in poultry production.

This exprement was conducted to compare the effects of antibiotic and some Iranian and similar importedprobiotics on performance, economic indicators and intestine morphology of broilers. 540 1-day old broilers were used in a completly randomized design with 6 treatments and 5 riplicates per treatment. Pedi-Guard and Lacto-Feed were used as Iranian and Bactocell and Primalac probiotics as imported products. Treatments were: 1) basal diet (control) and basal diet with: 2) antibiotic, 3) Bactocell probiotic, 4) Pedi-Guard probiotic, 5) Primalac probiotic and 6) Lacto-Feed probiotic. Orthogonal contrasts were used to compare the effect of probiotics versus control and antibiotic, Iranian probiotics versus imported products and single strain probiotics versus multi strains. Primalac decreased feed intake and body weight gain, while antibiotic and Bactocell increased the feed intake and body weight gain in the starter period (P

Probiotics have to reach their site of action in certain numbers in order to exhibit positive health effects. Encapsulation has shown remarkable enhancing effects on probiotic survival in simulated gastric conditions compared to free bacteria. The purpose of this study was identification and evaluation of a potential probiotic strain using encapsulation process by new carriers in order to improve probiotic viability during in vitro simulated conditions.
Material and A native Lactobacillus was isolated from yogurt, identified as Lactobacillus casei PM01 (NCBI registered) and analyzed for probiotic properties alongside established probiotic strains of Lactobacillus acidophilus ATCC 43556, and Lactobacillus rhamnosus ATCC 7469. Acid and bile resistance, adhesion to Caco-2 cells and antibiotic resistance were evaluated. Lactobacillus casei PM01 was encapsulated with alginate, chitosan and natural branched polysaccharides (pectin, tragacanth gum and gum Arabic) by using extrusion technique. Encapsulation efficiency, acidification activity and viability of entrapped Lactobacillus casei PM01 in simulated gastric pH were determined.
Results and Based on the results, all the three strains could be considered as potential probiotics, and are good candidates for further in vitro and in vivo evaluation. The results showed that the survival of encapsulated Lactobacillus casei PM01 was significantly (p≤0.05) increased when it was incubated in simulated gastric pH. It can be concluded that indigenous Lactobacillus casei PM01 in encapsulated form is introduced as an efficient probiotic strain for using in dairy products.
Conflict of interest: The authors declare no conflict of interest.

Probiotic are live microbial food supplement which bestows beneficial effects on the host by creating intestinal microbial balance. In this research, we aimed to identify locally isolated LAB with probiotic potentials by phenotypic and genotypic methods.
Material and A number of locally isolated LAB identified by phenotypic characteristics were selected and screened for their probiotic properties. The isolates were tested for their acid and bile resistance, antibacterial activity, cholesterol reducing. The selected probiotic LAB were identified to species level by using universal primers and 16S rRNA sequencing.
Among the 20 LAB isolates, only 3 isolates resisted low pH value of 2.5, while 5 isolates resisted 0.3 to 1% bile salt. Among these 5 isolates, 3 isolates showed the ability to lower cholesterol significantly as they reduced 94.8, 95.73 and 97.82 % of cholesterol within 2 hours of incubation. All isolates except for 24SN hydrolyzed bile salt. Based on 16S rRNA sequencing these 5 isolates were identified as Lactobacillus plantarum (22SN, 24SN, NPN0022, 10SN), and Lactobacillus rhamnosus (30SN).
The LAB isolates in this study possessed significant probiotic properties and might be used as a probiotic in human, livestock, and poultry products in the future.