Food Biotechnology
Fatemeh Rahmani; Ali Moayedi; Marteza Khomeiri; Mahboobeh Kashiri
Abstract
Nowadays, plant-based dairy alternatives have gained considerable attention. However, the textural and sensorial characteristics of plant-based products limit their acceptance. The exploitation of lactic acid bacteria has been proposed as a promising approach to developing plant-based dairy analogs. ...
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Nowadays, plant-based dairy alternatives have gained considerable attention. However, the textural and sensorial characteristics of plant-based products limit their acceptance. The exploitation of lactic acid bacteria has been proposed as a promising approach to developing plant-based dairy analogs. In this study, the performance of three proteolytic lactic acid bacteria in the induction of soymilk gelation was compared and their effects on the physicochemical properties of resulting gels were investigated. Lactiplantibacillus plantarum MCM4, Streptococcus thermophilus, and Weissella confusa MDM8) were inoculated to the soy milk matrix, and incubated at 37 °C until reaching pH 4.7. To understand the effects of acidifying and proteolytic activity of starter culture, syneresis, cell counts, free amino acid content (O-phthalaldehyde method), evaluation of proteolysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and textural parameters of soymilk gels during fermentation were investigated. There was a significant difference among the strains in terms of viable cell counts and proteolytic activity during fermentation (p < 0.05). The amount of syneresis was also different among the resulted gels as it was in the range from 61% (sample fermented with S. thermophilus) to 69.5% (fermented with L. plantarum MCM4). The main soy proteins were degraded to different extents as a function of fermentation time. Texture analysis showed that fermentation of soymilk with W. confusa MDM8 resulted in soy gel with higher firmness and consistency, while the sample fermented with L. plantarum MCM4 had higher adhesiveness and viscosity index. Overall, it can be concluded that L. plantarum MCM4, W. confusa MDM8, and S. thermophilus can be introduced as starter cultures for the production of novel soymilk gels with reasonable properties.
Food Biotechnology
Shadi Atashgahi; Ali Moayedi; Alireza Sadeghi Mahoonak; Hoda Shahiri Tabarestani; Alireza Sadeghi
Abstract
Soy whey (SW) is a byproduct from tofu and soy protein isolate (SPI) production that contains various nutrients such as protein, amino acids, minerals, carbohydrates, isoflavones. In this study, SW was fermented with lactic acid bacteria (LAB) with the aim to enhance total phenolic contents (TPC), Gamma ...
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Soy whey (SW) is a byproduct from tofu and soy protein isolate (SPI) production that contains various nutrients such as protein, amino acids, minerals, carbohydrates, isoflavones. In this study, SW was fermented with lactic acid bacteria (LAB) with the aim to enhance total phenolic contents (TPC), Gamma amino butyric acid (GABA) and antioxidant activity. Eight different LAB strains were selected and the activity and cell counts of the most potent strains were investigated during fermentation. The results showed that all the isolates were able to grow in SW and the increase in incubation time led to significantly (p<0.05) decrease the pH of all samples from 5.75 to 4.5. Among eight LAB isolates, Lactiplantibacillus plantarum MCM4 and Weissella confusa MDM8 showed higher activity in terms of acid production, increase in TPC content and proteolytic activity. The sample fermented by L. plantarum MCM4 had the highest content of free amino acids (1.73 mg/ml) and the unfermented sample with 0.9 mg/ml had the lowest content. GABA concentration varied from 6.15 mg/mL (unfermented) to 24.175 mg/100 mL (SW fermented with L. plantarum MCM4). In this research, it was found that fermentation increased the antioxidant capacity of SW in such a way that the highest amount was observed in sample fermented with Lactiplantibacillus plantarum MCM4. A positive correlation (R2= +0.72) was found between viable cell counts and proteolysis. It can be concluded that, fermentation with L. plantarum MCM4 and W. confusa MDM8 can be applied as an approach to valorize SW.
Food Engineering
Maryam Azizkhani; Rafat Karbakhsh Ravari
Abstract
The objective of this study was to improve the survival of lactic acid bacteria (LAB) in Tarhana soup as a non-dairy matrix. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophiles were encapsulated in electrospun nanofiber mats fabricated from corn starch (CS) and sodium alginate ...
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The objective of this study was to improve the survival of lactic acid bacteria (LAB) in Tarhana soup as a non-dairy matrix. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophiles were encapsulated in electrospun nanofiber mats fabricated from corn starch (CS) and sodium alginate (SA) and the protective effect of the nanofibers were investigated on the cells during the preparation of Tarhana and in the gastrointestinal tract. The moisture content of the control and nanofiber- loaded dried Tarhana samples was 8.75 and 8.71%, respectively; therefore, using nanofiber mats in the formulation had no significant effect
on the moisture content of the samples. A negative zeta potential value of -15.1 mV was found for LAB- loaded nanofibers. The nanofibers mats prepared from SA and CS mix showed a bead- free and clean structure with uniformity in size. The diameter size of most of the fibers ranged from 175- 338 with an average of 265 nm. Loading nanofiber mats with L. delbrueckii subsp. bulgaricus and S. thermophilus cells led to a uniform distributed beaded structure and the average diameter enhanced to approximately 763 nm. The viability of L. delbrueckii and S. thermophilus at the end of the electrospinning process was 92.82% and 95.83%, respectively, which indicating a slight loss in their population. Survival of nanoencapsulated S. thermophilus and L. delbrueckii was 93.50% and 89.16% respectively, while for free cells it was 85.3 and 76.4% that showed considerable protective effect of CS/SA fibers on the cells against dehydration of Tarhana medium. Nanofiber mats improved the stability of the cells against ordinary heat treatment used in preparing Tarhana soup. The survival rate of S. thermophilus was higher than L. delbrueckii subsp. bulgaricus and a significant difference was observed between the viability of free and nanoencapsulated bacteria. The survival of CS/SA nanoencapsulated S. thermophilus and L. delbrueckii subsp. bulgaricus was 83.25% and 80.21%, respectively, which is indicative of the significant protective effect of fibers on the cells against the heating process. The nanofibers also provided good stability for the cells in the gastrointestinal tract as 106 to 107 CFUg-1 of the cells were survived which is within the recommended level of potential probiotic dose to be effective. There was no significant difference in the color of all samples. Nanoencapsulation in CS/ SA nanofiber mats improved the protection of both LAB strains in simulated fluids of the stomach and intestine (Table 4). After continuous exposure to simulated gastrointestinal fluid, a significant loss of viable free LAB cells (higher than 4 log CFU/ml) was found while the population of S. thermophilus and L. delbrueckii subsp. bulgaricus encapsulated in CS/ SA nanofibers decreased only 0.45 and 0.37 log CFU after 120 min (p> 0.01), 0.93 and 0.80 log CFU after 180 min (p< 0.01), respectively. Tarhana soup prepared with probiotic– loaded nanofibers gained higher scores in terms of consistency, mouth feel, odor, taste, flavor, and overall acceptability attributes. Tarhana soup with nanofibers possessed much sour taste and flavor than samples prepared with free cells of probiotics. The results of the present study indicated that the protection obtained from CS/ SA capsules secured around106 to 107 CFU/g of the probiotic cells which are within the recommended level of probiotic dose to be functional in consumers’ body. Therefore, this product can be used by the consumers like vegetarians and lactose or milk peptide intolerants who do not consume dairy products but need potential fermented probiotic food.
Food Engineering
Aliakbar Gholamhosseinpour; Mostafa Mazaheri Tehrani; Seyed Mohammad Ali Razavi
Abstract
UF- Feta cheese is mostly produced from bovine milk and is usually consumed fresh or only after a short period of ripening (60 days). In this research, the influence of commercial starter cultures (SafeIT 2, FRC- 65 and R- 704) and ripening time (0- 60 days) on chemical (total solids, fat, protein, ash, ...
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UF- Feta cheese is mostly produced from bovine milk and is usually consumed fresh or only after a short period of ripening (60 days). In this research, the influence of commercial starter cultures (SafeIT 2, FRC- 65 and R- 704) and ripening time (0- 60 days) on chemical (total solids, fat, protein, ash, salt, acidity, pH), biochemical (pH 4.6, TCA, PTA-soluble nitrogen, acid degree value) and sensory (color and appearance, aroma, texture, flavor and total acceptance) characteristics of UF- Feta cheese analogues was investigated. According to our results, the starter culture types were known to have a significant effect (P≤ 0.05) on pH, %salt, %protein, and pH 4.6- soluble nitrogen of cheeses, whereas the other chemical properties were not affected by them. Ripening time only significantly (P≤ 0.05) influenced %acidity, pH, %salt, acid degree value (meq acid 100 g-1 fat), %protein and %proteolysis products of samples. Also, the starter culture and ripening time did not affect the sensory properties significantly, excluding color and appearance, however, the produced cheeses from SafeIT 2 had higher sensory scores compared with the others containing FRC- 65 and R- 704 cultures.
Food Biotechnology
Nafiseh Davati; Sahar Bahrami
Abstract
Introduction: The consumption of local and traditional dairy products have increased in recent years and some local cheeses as functional foods with desirable organoleptic attributes have positive effects on human health. However, there is concern that consumption of these products may increase the risk ...
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Introduction: The consumption of local and traditional dairy products have increased in recent years and some local cheeses as functional foods with desirable organoleptic attributes have positive effects on human health. However, there is concern that consumption of these products may increase the risk of exposure to food born bacteria such as Enterobacteriaceae family, Staphylococcus aureus, and Listeria monocytogenes. The microbiome of fermented products such as cheese is one of the most powerful and important parameters in flavor development and ripening. Furthermore, cheese flavor formation as a dynamic biochemical process is related to environmental conditions including milk source, ripening time, and temperature of storage. These parameters affect the microbial community structures and metabolic pathways. Materials and Methods: In this study, a local cheese made from cow milk was prepared based on a local recipe. The traditional cheese was manufactured using mesophilic starter culture. The cheese was ripened at 10°C for 3 months. The samples were collected from the surface of the cheese. The serial dilution was performed until 10-10 dilution in sterile ringer. For isolation and phenotypic identification of lactic acid bacteria, a 100 µl of diluted sample was cultured on MRS agar and M17 agar, followed by incubation at 37°C for 48 h under anaerobic conditions with Gas-Pak A. After purification of colonies, the Gram-positive and catalase-negative isolates were phenotypically identified at genus level using physiological tests including capacity of gas production, growth at different pHs (9.6 and 4.4), salt tolerance (%6.5 and 18%), and different growth temperatures (10°C and 45°C). DNA extraction was performed with DNeasy®Blood & Tissue Kit. The microbial population of the cheese and its functional potential for ripening were investigated by whole-metagenome sequencing. The prepared library using Nextera™ DNA approach was sequenced by using the Illumina HiSeq® 2000, 2×100 bp paired- end reads. The metagenomics data of cheese microbiome were analyzed for taxonomic profiling and functional potential by De Novo Assemble Metagenome and Bin Pangenomes. The metabolic pathways were extracted from the KEGGdatabase. Results and Discussion: The results of phenotypic identification showed that most of the lactic acid bacteria strains belonged to Streptococcus, Lactococcus, and Lactobacillus. Also, the results of metagenomics analysis showed that there were various genera including Streptococcus, Lactococcus, Lactobacillus, Acinetobacter, Enterococcus, Glutamicibacter, and Weissella in cheese. Streptococcus thermophilus, Lactococcus lactis, and Lactobacillus helveticus were identified as dominant species. Pathogenic bacteria such as Enterobacter, Listeria, and Staphylococcuswere also slightly found and therefore there is nearly no concern for consumers and human health. The microbiome of this cheese showed the metabolic potential for the biosynthesis of a wide range of aroma compounds and associated with flavor development that related with the metabolism and biosynthesis of methane, branched chain amino acids (isoleucine, valine, and leucine), aromatic amino acids (tyrosine, tryptophan, and phenylalanine), other amino acids (beta-alanine, L-lysine), fatty acids (arachidonate, palmitate, stearate), and monosaccharides. The enzymes related to biosynthesis and metabolism of amino acids were found during ripening of this cheese. These enzymes included 4-hydroxy-tetrahydrodipicolinate reductase, 2-isopropylmalate synthase, 3-dehydroquinate dehydratase, 3-hydroxyisobutyryl-CoA hydrolase, 5-carboxymethyl-2-hydroxymuconate delta-isomerase, and 3-hydroxyacyl-CoA dehydrogenase. Based on the results of KAAS (KEGG Automatic Annotation Server), proteins involved in metabolic pathways of microbial community on the surface of the traditional cheese included Cytochrome P450 Photosynthesis Proteins, Peptidases & Inhibitors, Glycosyltransferases, Lipopolysaccharide Biosynthesis Proteins, Peptidoglycan Biosynthesis and Degradation Proteins, Lipid Biosynthesis Proteins, Protein Kinases, Polyketide Biosynthesis Proteins Prenyltransferases, Protein Phosphatases & Associated Proteins, and Amino Acid Related Enzymes. The cheese under our study as a functional food showed health benefits for consumers due to the presence of probiotic bacteria and genes encoded for biosynthesis of valuable compounds including antibiotics, drugs, and antioxidants.
Seyed Mohsen Mortazavi; Hossein Jalali; Seyed Hamidreza Ziaolhagh
Abstract
In this study, the probiotic bacterium Lactobacillus acidophilus with different percentages of pomegranate peel powder (0, 0.5, 1, 1.5, and 2%) were used to produce a functional camel milk-based beverage. The physicochemical, antioxidant and sensory properties of the resulting drinks were evaluated. ...
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In this study, the probiotic bacterium Lactobacillus acidophilus with different percentages of pomegranate peel powder (0, 0.5, 1, 1.5, and 2%) were used to produce a functional camel milk-based beverage. The physicochemical, antioxidant and sensory properties of the resulting drinks were evaluated. The results showed that enrichment of milk with pomegranate peel powder improved the survival of probiotic bacteria from 6.95 to 7.35 Log CFU/ml. Addition of pomegranate peel to beverages increased their antioxidant activity from 7 to 85.33, 9.13 to 93.66 and 0.126 to 0.435 as measured by DPPH free radical scavenging, ABTS+ free radical scavenging and reduction potency tests, respectively. Rheological studies also showed that the addition of pomegranate peel powder to beverages increased their viscosity from 5.65 to 21.5 mPa. Adding pomegranate peel powder to beverages also changed the color factors (L*, a* and b*) so that increasing the level of pomegranate peel powder increased the red and yellow color in the samples. Also, the results of the sensory evaluation, including taste, appearance, smell and general acceptance indicated that the produced beverages were well-liked by consumers. However, the results of sensory evaluation showed that adding high percentages of pomegranate peel powder to beverages could reduce the sensory acceptance of the final product.
Mohammad Ebrahim Goharjoo; Mohammad Reza Edalatian Dovom; Fakhri Shahidi; Farideh Tabatabaei Yazdi; Mohammad Javad Varidi
Abstract
Introduction: Carrot products such as carrot juice and fermented carrot products possess high nutritional value and they are considered as a major source of β-carotene. Carotenoids because of containing conjugated double bonds, have antioxidant properties and provide the natural yellow, orange and red ...
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Introduction: Carrot products such as carrot juice and fermented carrot products possess high nutritional value and they are considered as a major source of β-carotene. Carotenoids because of containing conjugated double bonds, have antioxidant properties and provide the natural yellow, orange and red colors in fruits and vegetables. Due to the outbreak of some problems such as lactose-intolerance and high blood cholesterol especially in dairy products’ consumption, great attention has been drawn toward fermented vegetable products. Lactic acid bacteria (LAB) including important genera: leuconostocs, lactobacilli, streptococci and pediococci are wide-spread and have been divided according to morphological features and fermentation pathway, which utilize glucose. Current knowledge regarding involved microorganisms in vegetable fermentation is still dependent on biochemical and classical data. Nowadays, application of molecular methods in the field of microbial identification has been provided better understanding from fermented foods ecology. Since local starter cultures are considered as precious genetic resources in each country and also they play an important role in production and creation of organoleptic characteristics in fermented products, therefore, the objective of present study was the isolation and identification of lactic flora from fermented carrot with the help of conventional (biochemical) and molecular methods and determination of phylogenetic relationships.
Materials and methods: Following the production of fermented carrot samples, they were packed in plastic container and stored at ambient temperatures (25-27°C). In the next step, total LAB count was performed according to Iranian standard of 5484. Isolation and selection of LAB was done during 32 days with the intervals of 0, 4, 8, 16, 24 and 32. For initial identification of LAB, isolated were subjected to gram staining and catalase tests. Also biochemical tests including growth at 15 and 45C, at NaCl 6.5% and 18%, pH=4.4 and 9.6, were done in order to identify and classify at genus level. Carbohydrate fermentation profiles were obtained for isolates with the aid of 10 sugars. Molecular identification was done with DNA extraction followed by amplification of 16S gene with universal primers (27 F and 1492 R). For sequencing of resulted PCR-products, they were sent to Macrogen Company, South Korea. Phylogenetic tree was plotted with Clustal Omega and Fig. Tree soft wares.
Results and discussion: In the first step, 144 gram positive, catalase negative isolates were screened and selected as presumptive LAB according to gram staining and catalase test and morphological characteristics. Among them, 48 representative isolates were chosen and identified up to genus level according to biochemical tests. Five distinct genera were identified as Pediococci (4.08%), homofermentative lactobacilli (34.69%), hetero fermentative lactobacilli (36.74%), Leuoconostocs (20.41%) and enterococci (4.08%). Carbohydrate fermentation profiles revealed Lactobacilli constitute the highest percent among other genera and also some species like Lb. kimchi and Lb. parakefiri were detected. Growth of lactic acid bacteria experienced increasing trend up to day-16 but thereafter showed decline trend until the end of storage time (day-32). 26 out of 48 isolates were subjected to molecular analysis. Results of sequencing revealed following species: Lb.plantarum (9), Lb. brevis (8), Leu. mesenteroides (4), Lb. casei (1), Lb. paracasei (1), and Lb, pantheris (1). Changes and variation of lactic flora during fermentation stages revealed that at initial stages of fermentation (0- day-8) Leuconostocs sp. were predominant species but disappeared then. In the next stages of fermentation Leuconostocs sp. were replaced by homo-fermentative strains such as Lb. plantarum which was present from the first day up to day-24 but constituted the majority of species on day-16. In the final stage, Lb. brevis dominated the others due to better survival and resistance of this bacterium at the increased acidity level. Phylogenetic tree results revealed three clusters including cluster I (composed of three sub-clusters), cluster II (three sub-clusters) and cluster III (two sub-clusters). Cluster I included two genera: Leuconostocs sp. (mesenteroides) and Lactobacillus (pantheris, casei and paracasei). Cluster II included Lb. brevis and finally cluster III composed of Lb. plantarum.
Hoda Ghayomi; Mohammad Ali Najafi; Mohammad Rahnama; Mohammad Soltani
Abstract
Introduction: The environmental problems of plastic materials have increased the importance of renewable edible films. In the manufacture of edible films, hydrocolloid compounds such as methyl cellulose and sodium caseinate are usually employed. Methyl cellulose is a biopolymer, soluble in water, and ...
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Introduction: The environmental problems of plastic materials have increased the importance of renewable edible films. In the manufacture of edible films, hydrocolloid compounds such as methyl cellulose and sodium caseinate are usually employed. Methyl cellulose is a biopolymer, soluble in water, and has substantial inhibitory properties against oxygen and lipids. Sodium caseinate has favorable mechanical properties and a high inhibitory effect on air transmission. Furthermore, it possesses good nutritional value. One of the most notorious pathogenic bacteria in food is the Listeria spp. which hastens food decay. Listeria monocytogenes is one of the most harmful species of bacteria that causes disease in humans. Lactic acid bacteria can inhibit the growth of cells that cause decay in food. The use of lactic acid bacteria in food has been made legal by the Food and Drug Administration of the United States of America (FDA). In this study, Lactobacillus casei PTCC1608 (L. casei 1608) was added directly to the film forming solution. The primary effect of L. casei 1608 and film type, i.e. sodium caseinate and methylcellulose polymers, were evaluated based on mechanical properties, water vapor permeability (WVP) and the opacity of films. Anti-listeria features of the films were studied under experimental conditions as the films were prepared against the growth of Listeria innocua IBRC-M10799 (L. innocua 10799). The L. innocua 10799 was selected because of its non-pathogenicity and its physiological similarity with Listeria monocytogenes.
Materials and methods: L. casei 1608 and L. innocua 10799 were purchased from credible organizations. The bacteria were prepared as lyophilization form. In order to propagate and prepare bacterial suspensions of L. casei 1608 and L. innocua 10799, the cells were inoculated in the de Man Rogosa Sharpe Broth (MRS Broth, Liofilchem, Italy) and Blood Heat Infusion broth medium (BHI, Liofilchem, Italy), respectively. The cultures were then incubated at temperatures between 30 and 37°C, respectively, for 24 hour. The cells were harvested after centrifugation for 15 minutes at 3500 rpm (Eppendorf 5810 model, Germany). The collected medium was washed twice with a saline buffer solution. The population growth curve of L. casei 1608 and L. innocua 10799 were obtained using the pour plate method and by using the UV-VIS spectrophotometer device (Cecil model, England) at OD= 600nm. In order to prepare the film formation solution of methyl cellulose (CAS 9004-67-5, Sigma-Aldrich, Madrid, Spain), four grams of powder and 1 g of glycerol were mixed with 100 ml sterilized distilled water. After homogenization and degassing, the film forming solution was transferred to the plastic container. To prepare the film formation solution of sodium caseinate, 5 g of sodium caseinate powder (CAS 9005-46-3, Sigma-Aldrich, Madrid, Spain) was added to distilled water along with 1.5 g of glycerol. The solution was planned to reach 100 ml and, after complete dissolution, the film formation solution warmed up to 85°C and then cooled. To prepare the bio-film, L. casei 1608 was added to the film forming solution until the number of L. casei 1608 in the dried film reached 106 CFU/cm2. The film formation solutions were transferred to plastic containers and were dried at 25°C for 24 hours. All films were placed in a desiccator at 25°C and 75% RH for one week. In order to evaluate the anti-listeria activity, the bio-films were put on tryptic soya agar (TSA) medium, inoculated with L. casei 1608 (102 CFU / cm2) for 12 days at 5°C. The number of pathogenic bacteria and L. casei 1608 were counted on days 0, 4, 8 and 12. Also, the prepared films were evaluated according to the ASTM D-882 (ASTM, 2001) as per the ASTM D-882 instruction manual (Testometric, Model M350-10CT, UK). WVP was determined according to the ASTM-96-95. The opacity of the films were assessed by using the spectrophotometric apparatus according to the method described by Nu nez-Flores et al. (2012). Comparing the mean values of data was based on a factorial experiment in a completely randomized design (CRD) with three replications, by the SAS software version 9.1. The chemical and microbial mean values were calculated using Duncan’s and Tukey’s tests, respectively.
Results & Discussion: Data analysis showed that the bio-caseinate film hosted the highest survival rate of L. casei 1608 on the eighth day of culture (123.8%) when L. casei was concurrently present with L. innocua 10799. Analysis of data by the bio-methyl cellulose film showed that the strongest inhibitory effect on the growth rate of L. innocua 10799 was observed on the fourth day of the 12-day experimental duration (P0.05). Laboratory evaluations showed that the presence of lactic acid bacteria in films significantly increased the transparency, WVP and amount of elasticity. Nonetheless, a significant decrease in tensile strength and modulus of elasticity was observed. The type of matrix had a significant effect on mechanical properties. By comparing the methyl cellulose matrix and the sodium caseinate, it was observed that the methyl cellulose matrix showed significantly greater elasticity, tensile strength and higher modulus of elasticity while having less WVP and less opacity (p
Mohammad Reza Edalatian Dovom; Masoud Yavarmanesh; Fariba Ghiamati Yazdi; Morteza Khomeiri; Neda Nayyeri
Abstract
Introduction : Lactic acid bacteria (LAB) are a group of gram positive, catalase negative bacteria which possess unique metabolic, physiological and morphological characteristics. In addition, lactic acid bacteria are considered as a valuable source of antimicrobial agents including bacteriocins. Bacteriocins ...
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Introduction : Lactic acid bacteria (LAB) are a group of gram positive, catalase negative bacteria which possess unique metabolic, physiological and morphological characteristics. In addition, lactic acid bacteria are considered as a valuable source of antimicrobial agents including bacteriocins. Bacteriocins are defined as non-toxic peptides produced by LAB. "Maskeh" as an Iranian traditional butter carries indigenous LAB which secrete and harbor bacteriocins. Our objective in this study was to evaluate the influence of antimicrobial compoundsproduced by isolated LAB from Maskeh, a local traditional butter, against some pathogenic bacteria suchas: staphylococcus aureus, Listeria innocoa and E. coli using culture-based methods. In the following step, influence of some technological parameters including different temperatures, pHs and proteinase K were determined on stability of antimicrobial compounds in bacteriocins.Materials and methods: Three samples of milk, yoghurt and local butter known as Maskeh were collected from different regions in the south ofKhorasanRazavi, Gonabadcity.Indicator microorganisms and culture condition: In order to activate the indicators, following the cultivation in corresponding and suitable media, they were incubated for 24 hours in suitable temperatures.Survey of antimicrobial activities: In Agar Spot, Antagonistic activities of isolates were evaluated in solid media. Finally clear zone of inhibition was measured in mm. In Well- Diffusion Assay (WDA), positive isolates from previous step, were selected for this assay. In this method, cell-free supernatant (CFS) of isolates were examined for their antagonistic activities. Finally clear zone of inhibition were evaluated around the wells.Determination of antimicrobial compound nature: Effect of proteinase K on CFS: In order to evaluate the enzyme sensitivity of bacteriocin like compounds produced by LAB, CFS of isolates were subjected to proteinase K (final concentration 0.5 mg/ml). Mixture of enzyme- CFS was incubated in 37C for four hours. Finally, the remaining inhibitory activity was determined using WDA.Influence of different temperatures on CFS: Heat sensitivity of bacteriocin like compounds was evaluated with subjecting the CFS of isolates to various temperatures. Again remaining activity of isolates was evaluated with the help of WDA.Influence of different levels of pH on CFS: CFS of isolates was adjusted at different pHs and their inhibitory effects were determined using WDA.Discussion & Results: Following the sequencing, 51 isolates were identified from different steps of Maskeh production. Results showed that 44 out of 51 isolates were effective at least against one indicator. It was clear that E.coli, Staph.aureus, Listeria. innocoa, Lb.plantarum, Lb.sake, Lac.lactis ssp. lactis,Lac.lactis ssp. cremoris were inhibited by 33, 7,11,12,7,35 and 7 isolates, respectively. Among pathogenic indicator bacteria, E.coli was inhibited maximally and regarding the non-pathogenic indicators, Lac.lactis ssp. Lactis was inhibited by the most of the isolates. Among the isolates, Ent.faecium B161 and Str.thermophilus B258 presented the highest inhibitory effect against Listeria.innocoa.Interestingly both these strains had been isolated from Maskeh, implying that they originated the same source. Formation of clear inhibition zone in agar spot method is related to colony-associated antimicrobial compounds like H2O2, lactic acid and other organic acids. Also, the strongest clear zone of inhibition was against Listeria.innocoa. Lb.plantarum B120 showed inhibitory effect against 6 out of 7 indicator bacteria. This strain has been isolated from Maskeh, and only did not affect on Lac.lactis ssp. lactis.In the next step, in WDA, CFS obtained from isolates were subjected to inhibition activity evaluation. In this assay, 39 isolates showed inhibitory activity against at least one indicator and 12 isolates had no inhibition against indicators. E.coli was inhibited by the most of the isolates (11), followed by Listeria.innocoa by 8 and Staph.aureus by 2 isolates. But noticeable point is that the strongest influence was seen against Listeria. In the last step, influence of technological parameters such as : temperature, pH and enzyme were determined on antimicrobial and inhibitory activity of CFS. In this survey, only those isolates were chosen which showed inhibitory effect against Listeria innocoa. Regarding the temperature, with increasing of this factor, the diameter of clear zone had decreasing trend. This means that bacteriocin- like compounds are sensitive to heat. Among analyzed isolates, CFS of two isolates namely Aerococcusviridans M156 and M141 possess the highest sensitivity. In contrast, the highest resistance was related to Aerococcusviridans M165. Evaluation of antagonistic activity of CFS of isolates at different pHsexperienced, in pH between 3-5, growing trend of inhibitory activity which is due to produced lactic acid. Maximum of antagonistic activity was seen at pH=3 and along with pH increase toward the alkaline condition, it decreased.Aerococcusviridans M165 and M141 showed clear zone of inhibition equal to 6.5 and 6, respectively at pH=7.Proteniase K as a proteolytic enzyme, exhibited decomposing influence on antimicrobial effects of all isolates, except two of them. This phenomenon implies the proteinaceous nature of antimicrobial compound in CFS, because of decomposition as a result of proteolytic enzyme. But regarding two isolates, the clear zone did mot destroyed even after enzyme treatment.Conclusion: Some CFS or their producing isolates can be exploited as bio-preservatives; CFS of Aerococcusviridans M 165 can be applied in foods subjected to high heat treatment. In acidic and low pH food, we can use those isolates which theirCFS remain their activity at low pH.
