Search results for: probiotic yeast

Authors: Chang-Hui Shen, Paulina Konarzewska

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Cell homeostasis is regulated by vacuolar activity and it has been shown that lipid composition of the vacuole plays an important role in vacuolar function. The major phosphoinositide species present in the vacuolar membrane include phosphatidylinositol 3,5-biphosphate (PI(3,5)P₂) which is generated from PI(3)P controlled by Fab1p. Deletion of FAB1 gene reduce the synthesis of PI(3,5)P₂ and thus result in enlarged or fragmented vacuoles, with neutral vacuolar pH due to reduced vacuolar H⁺-ATPase activity. These mutants also exhibited poor growth at high extracellular pH and in the presence of CaCl₂. Conversely, VPS34 regulates the synthesis of PI(3)P from phosphatidylinositol (PI), and the lack of Vps34p results in the reduction of vacuolar activity. Although the cellular observations are clear, it is still unknown about the molecular mechanism between the phospholipid biosynthesis pathway and vacuolar activity. Since both VPS34 and FAB1 are important in vacuolar activity, we hypothesize that the molecular mechanism of vacuolar function might be regulated by the transcriptional regulators of phospholipid biosynthesis. In this study, we study the role of the major phospholipid biosynthesis transcription factor, INO2, in the regulation of vacuolar activity. We first performed qRT-PCR to examine the effect of Ino2p on the expression of VPS34 and FAB1. Our results showed that VPS34 was upregulated in the presence of inositol for both WT and ino2Δ cells. However, FAB1 was only upregulated significantly in ino2Δ cells. This indicated that Ino2p might be the negative regulator for FAB1 expression. Next, growth sensitivity experiment showed that WT, vma3Δ, and ino2Δ grew well in growth medium buffered to pH 5.5 containing 10 mM CaCl₂. As cells were switched to growth medium buffered to pH 7 containing CaCl₂ WT, ino2Δ and opi1Δ showed growth reduction, whereas vma3Δ was completely nonviable. As the concentration of CaCl₂ was increased to 60 mM, ino2Δ cells showed moderate growth reduction compared to WT. This result suggests that ino2Δ cells have better vacuolar activity. Microscopic analysis and vacuolar acidification were employed to further elucidate the importance of INO2 in vacuolar homeostasis. Analysis of vacuolar morphology indicated that WT and vma3Δ cells displayed vacuoles that occupied a small area of the cell when grown in media buffered to pH 5.5. Whereas, ino2Δ displayed fragmented vacuoles. On the other hand, all strains grown in media buffered to pH 7, exhibited enlarged vacuoles that occupied most of the cell’s surface. This indicated that the presence of INO2 may play negative effect in vacuolar morphology when cells are grown in media buffered to pH 5.5. Furthermore, vacuolar acidification assay showed that only vma3Δ cells displayed notably less acidic vacuoles as cells were grown in media buffered to pH 5.5 and pH 7. Whereas, ino2Δ cells displayed more acidic pH compared to WT at pH7. Taken together, our results demonstrated the molecular mechanism of the vacuolar activity regulated by the phospholipid biosynthesis transcription factors Ino2p. Ino2p negatively regulates vacuolar activity through the expression of FAB1.

Keywords: vacuole, phospholipid, homeostasis, Ino2p, FAB1

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Authors: Monika Trzaskowska, Dorota Zielinska, Anna Lepecka, Katarzyna Neffe-Skocinska, Beata Bilska, Marzena Tomaszewska, Danuta Kolozyn-Krajewska

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Microbiological quality and food safety are important food characteristics. Regulation (EU) No 1169/2011 of the European Parliament and of the Council on the provision of food information to consumers introduces the obligation to provide information on the 'use-by' date or the date of minimum durability (DMD). The second term is the date until which the properly stored or transported foodstuff retains its physical, chemical, microbiological and organoleptic properties. The date should be preceded by 'best before'. It is used for durable products, e.g., pasta. In relation to reducing food losses, the question may be asked whether products with the date of minimum durability currently declared retain quality and safety beyond this. The aim of the study was to assess the sensory quality and microbiological safety of selected cereal products, i.e., pasta and millet after DMD. The scope of the study was to determine the markers of microbiological quality, i.e., the total viable count (TVC), the number of bacteria from the Enterobacteriaceae family and the number of yeast and mold (TYMC) on the last day of DMD and after 1 and 3 months of storage. In addition, the presence of Salmonella and Listeria monocytogenes was examined on the last day of DMD. The sensory quality of products was assessed by quantitative descriptive analysis (QDA), the intensity of 14 differentiators and overall quality were defined and determined. In the tested samples of millet and pasta, no pathogenic bacteria Salmonella and Listeria monocytogenes were found. The value of the distinguishing features of selected quality and microbiological safety indicators on the last DMD day was in the range of about 3-1 log cfu/g. This demonstrates the good microbiological quality of the tested food. Comparing the products, a higher number of microorganisms was found in the samples of millet. After 3 months of storage, TVC decreased in millet, while in pasta, it was found to increase in value. In both products, the number of bacteria from the Enterobacretiaceae family decreased. In contrast, the number of TYMCs increased in samples of millet, and in pasta decreased. The intensity of sensory characteristic in the studied period varied. It remained at a similar level or increased. Millet was found to increase the intensity and flavor of 'cooked porridge' 3 months after DMD. Similarly, in the pasta, the smell and taste of 'cooked pasta' was more intense. To sum up, the researched products on the last day of the minimum durability date were characterized by very good microbiological and sensory quality, which was maintained for 3 months after this date. Based on these results, the date of minimum durability of tested products could be extended. The publication was financed on the basis of an agreement with the National Center for Research and Development No. Gospostrateg 1/385753/1/NCBR/2018 for the implementation and financing of the project under the strategic research and development program 'social and economic development of Poland in the conditions of globalizing markets – GOSPOSTRATEG - acronym PROM'.

Keywords: date of minimum durability, food losses, food quality and safety, millet, pasta

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Authors: Antònio Inês, Davide Silva, Filipa Carvalho, Luís Filipe-Riberiro, Fernando M. Nunes, Luís Abrunhosa, Fernanda Cosme

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The presence of mycotoxins in foodstuff is a matter of concern for food safety. Mycotoxins are toxic secondary metabolites produced by certain molds, being ochratoxin A (OTA) one of the most relevant. Wines can also be contaminated with these toxicants. Several authors have demonstrated the presence of mycotoxins in wine, especially ochratoxin A. Its chemical structure is a dihydro-isocoumarin connected at the 7-carboxy group to a molecule of L-β-phenylalanine via an amide bond. As these toxicants can never be completely removed from the food chain, many countries have defined levels in food in order to attend health concerns. OTA contamination of wines might be a risk to consumer health, thus requiring treatments to achieve acceptable standards for human consumption. The maximum acceptable level of OTA in wines is 2.0 μg/kg according to the Commission regulation No. 1881/2006. Therefore, the aim of this work was to reduce OTA to safer levels using different fining agents, as well as their impact on white wine physicochemical characteristics. To evaluate their efficiency, 11 commercial fining agents (mineral, synthetic, animal and vegetable proteins) were used to get new approaches on OTA removal from white wine. Trials (including a control without addition of a fining agent) were performed in white wine artificially supplemented with OTA (10 µg/L). OTA analyses were performed after wine fining. Wine was centrifuged at 4000 rpm for 10 min and 1 mL of the supernatant was collected and added of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v). Also, the solid fractions obtained after fining, were centrifuged (4000 rpm, 15 min), the resulting supernatant discarded, and the pellet extracted with 1 mL of the above solution and 1 mL of H2O. OTA analysis was performed by HPLC with fluorescence detection. The most effective fining agent in removing OTA (80%) from white wine was a commercial formulation that contains gelatin, bentonite and activated carbon. Removals between 10-30% were obtained with potassium caseinate, yeast cell walls and pea protein. With bentonites, carboxymethylcellulose, polyvinylpolypyrrolidone and chitosan no considerable OTA removal was verified. Following, the effectiveness of seven commercial activated carbons was also evaluated and compared with the commercial formulation that contains gelatin, bentonite and activated carbon. The different activated carbons were applied at the concentration recommended by the manufacturer in order to evaluate their efficiency in reducing OTA levels. Trial and OTA analysis were performed as explained previously. The results showed that in white wine all activated carbons except one reduced 100% of OTA. The commercial formulation that contains gelatin, bentonite and activated carbon reduced only 73% of OTA concentration. These results may provide useful information for winemakers, namely for the selection of the most appropriate oenological product for OTA removal, reducing wine toxicity and simultaneously enhancing food safety and wine quality.

Keywords: wine, ota removal, food safety, fining

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Authors: Kristina Karapetyan, Flora Tkhruni, Tsovinar Balabekyan, Arevik Israyelyan, Tatyana Khachatryan

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The ability of the lactic acid bacteria (LAB) to prevent and cure a variety of diseases, their protective role against infections and colonization of pathogenic microorganisms in the digestive tract, has lead to the coining of the term probiotics or pro-life. LAB inhibiting the growth of pathogenic and food spoilage microorganisms, maintaining the nutritive quality and improving the shelf life of foods. They have also been used as flavor and texture producers. Enterococcus strains have been used for treatment of diseases such as diarrhea or antibiotic associated diarrhea, inflammatory pathologies that affect colon such as irritable bowel syndrome, or immune regulation, diarrhea caused by antibiotic treatments. The obtaining and investigation of biological properties of proteinoceous antibiotics, on the basis of probiotic LAB shown, that bacteriocins, metabiotics, and peptides of LAB represent bactericides have a broad range of activity and are excellent candidates for development of new prophylactic and therapeutic substances to complement or replace conventional antibiotics. The genotyping by 16S rRNA sequencing for LAB were used. Cell free culture broth (CFC) broth was purified by the Gel filtration method on the Sephadex Superfine G 25 resin. Antimicrobial activity was determined by spot-on-lawn method and expressed in arbitrary units (AU/ml). The diversity of multidrug-resistance (MDR) of pathogenic strains to antibiotics, most widely used for treatment of human diseases in the Republics of Armenia and Nagorno Karabakh were examined. It was shown, that difference of resistance of pathogens to antibiotics depends on their isolation sources. The influences of partially purified antimicrobial preparations (AMP), obtained from the different strains of Enterococcus genus on the growth of MDR pathogenic bacteria were investigated. It was shown, that bacteriocin containing partially purified preparations, obtained from different strains of Enterococcus faecium and durans species, possess bactericidal or bacteriostatic activity against antibiotic resistant intestinal, spoilage and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, E. coli, and Salmonella. Endemic strains of LAB, isolated from Matsoni made from donkey, buffalo and goat milk, shown broad spectrum of activity against food spoiling microorganisms, moulds and fungi, such as Salmonella sp., Esherichia coli, Aspergillus and Penicillium species. Highest activity against MDR pathogens shown bacteria, isolated from goat milk products. High stability of the investigated strains of the genus Enerococcus, isolated from samples of matsun from different regions of Nagorno-Karabakh (NKR) to the antibiotics was shown. The obtained data show high stability of the investigated different strains of the genus Enerococcus. The high genetic diversity in Enterococcus group suggests adaptations for specific mutations in different environments. Thus, endemic strains of LAB are able to produce bacteriocins with high and different inhibitory activity against broad spectrum of microorganisms isolated from different sources and belong to different taxonomic group. Prospect of the use of certain antimicrobial preparations against pathogenic strains is obvious. These AMP can be applied for long term use against different etiology antibiotic resistant pathogens for prevention or treatment of infectional diseases as an alternative to antibiotics.

Keywords: antimicrobial biopreparation, endemic lactic acid bacteria, intra-species diversity, multidrug resistance of pathogens

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Authors: Cibele F. Oliveira, Debora P. Jaeschke, Rodrigo R. Laurino, Amanda R. Andrade, Ligia D. F. Marczak

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Raspberry is well-known as a good source of phenolic compounds, mainly anthocyanin. Some studies pointed out the importance of these bioactive compounds consumption, which is related to the decrease of the risk of cancer and cardiovascular diseases. The most consumed raspberry products are juices, yogurts, ice creams and jellies and, to ensure the safety of these products, raspberry is commonly pasteurized, for enzyme and microorganisms inactivation. Despite being efficient, the pasteurization process can lead to degradation reactions of the bioactive compounds, decreasing the products healthy benefits. Therefore, the aim of the present work was to evaluate moderate electric field (MEF) and ultrasound (US) technologies application on the pasteurization process of raspberry juice and compare the results with conventional pasteurization process. For this, phenolic compounds, anthocyanin content and physical-chemical parameters (pH, color changes, titratable acidity) of the juice were evaluated before and after the treatments. Moreover, microbiological analyses of aerobic mesophiles microorganisms, molds and yeast were performed in the samples before and after the treatments, to verify the potential of these technologies to inactivate microorganisms. All the pasteurization processes were performed in triplicate for 10 min, using a cylindrical Pyrex® vessel with a water jacket. The conventional pasteurization was performed at 90 °C using a hot water bath connected to the extraction cell. The US assisted pasteurization was performed using 423 and 508 W cm-2 (75 and 90 % of ultrasound intensity). It is important to mention that during US application the temperature was kept below 35 °C; for this, the water jacket of the extraction cell was connected to a water bath with cold water. MEF assisted pasteurization experiments were performed similarly to US experiments, using 25 and 50 V. Control experiments were performed at the maximum temperature of US and MEF experiments (35 °C) to evaluate only the effect of the aforementioned technologies on the pasteurization. The results showed that phenolic compounds concentration in the juice was not affected by US and MEF application. However, it was observed that the US assisted pasteurization, performed at the highest intensity, decreased anthocyanin content in 33 % (compared to in natura juice). This result was possibly due to the cavitation phenomena, which can lead to free radicals formation and accumulation on the medium; these radicals can react with anthocyanin decreasing the content of these antioxidant compounds in the juice. Physical-chemical parameters did not present statistical differences for samples before and after the treatments. Microbiological analyses results showed that all the pasteurization treatments decreased the microorganism content in two logarithmic cycles. However, as values were lower than 1000 CFU mL-1 it was not possible to verify the efficacy of each treatment. Thus, MEF and US were considered as potential alternative technologies for pasteurization process, once in the right conditions the application of the technologies decreased microorganism content in the juice and did not affected phenolic and anthocyanin content, as well as physical-chemical parameters. However, more studies are needed regarding the influence of MEF and US processes on microorganisms’ inactivation.

Keywords: MEF, microorganism inactivation, anthocyanin, phenolic compounds

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Authors: Marcin Debowski, Marcin Zielinski, Miroslaw Krzemieniewski, Agata Glowacka-Gil, Paulina Rusanowska, Magdalena Zielinska, Agnieszka Cydzik-Kwiatkowska

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Technologies of biogas upgrading are now perceived as competitive solution combustion and production of electricity and heat. Biomethane production will ensure broader application as energy carrier than biogas. Biomethane can be used as fuel in internal combustion engines or introduced into the natural gas transmission network. Therefore, there is a need to search for innovative, economically and technically justified methods for biogas enrichment. The aim of this paper is to present a technology solution for biogas upgrading with immobilized algae biomass. Reactor for biogas upgrading with immobilized algae biomass can be used for removing CO₂ from the biogas, flue gases and the waste gases especially coming from different industry sectors, e.g. from the food industry from yeast production process, biogas production systems, liquid and gaseous fuels combustion systems, hydrocarbon processing technology. The basis for the technological assumptions of presented technology were laboratory works and analyses that tested technological variants of biogas upgrading. The enrichment of biogas with a methane content of 90-97% pointed to technological assumptions for installation on a technical scale. Reactor for biogas upgrading with algae biomass is characterized by a significantly lower cubature in relation to the currently used solutions which use CO₂ removal processes. The invention, by its structure, assumes achieving a very high concentration of biomass of algae through its immobilization in capsules. This eliminates the phenomenon of lowering the pH value, i.e. acidification of the environment in which algae grow, resulting from the introduction of waste gases at a high CO₂ concentration. The system for introducing light into algae capsules is characterized by a higher degree of its use, due to lower losses resulting from the phenomenon of absorption of light energy by water. The light from the light source is continuously supplied to the formed biomass of algae or cyanobacteria in capsules by the light tubes. The light source may be sunlight or a light generator of a different wavelength of light from 300 nm to 800 nm. A portion of gas containing CO₂, accumulated in the tank and conveyed by the pump is periodically introduced into the housing of the photobioreactor tank. When conveying the gas that contains CO₂, it penetrates the algal biomass in capsules through the outer envelope, displacing, from the algal biomass, gaseous metabolic products which are discharged by the outlet duct for gases. It contributes to eliminating the negative impact of this factor on CO₂ binding processes. As a result of the cyclic dosing of gases containing carbon dioxide, gaseous metabolic products of algae are displaced and removed outside the technological system. Technology for biogas upgrading with immobilized algae biomass is suitable for the small biogas plant. The advantages of this technology are high efficiency as well as useful algae biomass which can be used mainly as animal feed, fertilizers and in the power industry. The construction of the device allows effective removal of carbon dioxide from gases at a high CO₂ concentration.

Keywords: biogas, carbon dioxide, immobilised biomass, microalgae, upgrading

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Authors: Jong H. Kim, Kathleen L. Chan, Luisa W. Cheng

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Control of fungal pathogens, such as foodborne mycotoxin producers, is problematic as effective antimycotic agents are often very limited. Mycotoxin contamination significantly interferes with the safe production of foods or crops worldwide. Moreover, expansion of fungal resistance to commercial drugs or fungicides is a global human health concern. Therefore, there is a persistent need to enhance the efficacy of commercial antimycotic agents or to develop new intervention strategies. Disruption of the cellular antioxidant system should be an effective method for pathogen control. Such disruption can be achieved with safe, redox-active compounds. Natural phenolic derivatives are potent redox cyclers that inhibit fungal growth through destabilization of the cellular antioxidant system. The goal of this study is to identify novel, redox-active compounds that disrupt the fungal antioxidant system. The identified compounds could also function as sensitizing agents to conventional antimycotics (i.e., chemosensitization) to improve antifungal efficacy. Various benzo derivatives were tested against fungal pathogens. Gene deletion mutants of the yeast Saccharomyces cerevisiae were used as model systems for identifying molecular targets of benzo analogs. The efficacy of identified compounds as potent antifungal agents or as chemosensitizing agents to commercial drugs or fungicides was examined with methods outlined by the Clinical Laboratory Standards Institute or the European Committee on Antimicrobial Susceptibility Testing. Selected benzo derivatives possessed potent antifungal or antimycotoxigenic activity. Molecular analyses by using S. cerevisiae mutants indicated antifungal activity of benzo derivatives was through disruption of cellular antioxidant or cell wall integrity system. Certain benzo analogs screened overcame tolerance of Aspergillus signaling mutants, namely mitogen-activated protein kinase mutants, to fludioxonil fungicide. Synergistic antifungal chemosensitization greatly lowered minimum inhibitory or fungicidal concentrations of test compounds, including inhibitors of mitochondrial respiration. Of note, salicylaldehyde is a potent antimycotic volatile that has some practical application as a fumigant. Altogether, benzo derivatives targeting cellular antioxidant system of fungi (along with cell wall integrity system) effectively suppress fungal growth. Candidate compounds possess the antifungal, antimycotoxigenic or chemosensitizing capacity to augment the efficacy of commercial antifungals. Therefore, chemogenetic approaches can lead to the development of novel antifungal intervention strategies, which enhance the efficacy of established microbe intervention practices and overcome drug/fungicide resistance. Chemosensitization further reduces costs and alleviates negative side effects associated with current antifungal treatments.

Keywords: antifungals, antioxidant system, benzo derivatives, chemosensitization

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Authors: Vita Krungleviciute, Rasa Zelvyte, Ingrida Monkeviciene, Jone Kantautaite, Rolandas Stankevicius, Modestas Ruzauskas, Elena Bartkiene

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The environmental impact of agricultural bioproducts from the processing of food crops is an increasing concern worldwide. Currently, cereal bran has been used as a low-value ingredient for both human consumption and animal feed. The most popular bioprocessing technologies for cereal bran nutritional and technological functionality increasing are enzymatic processing and fermentation, and the most popular starters in fermented feed production are lactic acid bacteria (LAB) including pediococci. However, the ruminant digestive system is unique, there are billions of microorganisms which help the cow to digest and utilize nutrients in the feed. To achieve efficient feed utilization and high milk yield, the microorganisms must have optimal conditions, and the disbalance of this system is highly undesirable. Pediococcus strains Pediococcus acidilactici BaltBio01 and Pediococcus pentosaceus BaltBio02 from spontaneous fermented rye were isolated (by rep – PCR method), identified, and characterized by their growth (by Thermo Bioscreen C automatic turbidometer), acidification rate (2 hours in 2.5 pH), gas production (Durham method), and carbohydrate metabolism (by API 50 CH test ). Antimicrobial activities of isolated pediococcus against variety of pathogenic and opportunistic bacterial strains previously isolated from diseased cattle, and their resistance to antibiotics were evaluated (EFSA-FEEDAP method). The isolated pediococcus strains were cultivated in barley/wheat bran (90 / 10, m / m) substrate, and developed supplements, with high content of valuable pediococcus, were used for Lithuanian black and white dairy cows feeding. In addition, the influence of supplements on milk production and composition was determined. Milk composition was evaluated by the LactoScope FTIR” FT1.0. 2001 (Delta Instruments, Holland). P. acidilactici BaltBio01 and P. pentosaceus BaltBio02 demonstrated versatile carbohydrate metabolism, grown at 30°C and 37°C temperatures, and acidic tolerance. Isolated pediococcus strains showed to be non resistant to antibiotics, and having antimicrobial activity against undesirable microorganisms. By barley/wheat bran utilisation using fermentation with selected pediococcus strains, it is possible to produce safer (reduced Enterobacteriaceae, total aerobic bacteria, yeast and mold count) feed stock with high content of pediococcus. Significantly higher milk yield (after 33 days) by using pediococcus supplements mix for dairy cows feeding could be obtained, while similar effect by using separate strains after 66 days of feeding could be achieved. It can be stated that barley/wheat bran could be used for higher value feed production in order to increase milk production. Therefore, further research is needed to identify what is the main mechanism of the positive action.

Keywords: barley/wheat bran, dairy cattle, fermented feed, milk, pediococcus

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Authors: Mubashar Iqbal, Iqra Anjum, Muhammad Dildar Gogi, Muhammad Jalal Arif

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The present study was carried to screen out the effective entomopathogenic fungi (EPF) inoculation method in maize and to evaluate pathogenicity and oviposition-choice in C. partellus. Three entomopathogenic fungi (EPF) formulations Pacer® (Metarhizium anisopliae), Racer® (Beauveria bassiana) and Meailkil® (Verticillium lecanii) were evaluated at three concentrations (5000, 10000 and 20000 ppm) for their endophytism in maize and pathogenicity in C. partellus. The stock solution of the highest concentration (20,000 ppm) was prepared and next lower from stock solution. In the first experiment, three EPF was inoculated in maize plant by four methods, i.e., leaf-inoculation (LI), whorl-inoculation (WI), shoot-inoculation (SI) and root-inoculation (RI). Leaf-discs and stem-cutting were sampled in all four inoculation methods and placed on fungus growth media in Petri dishes. In the second experiment, pathogenicity, pupal formation, adult emergence, sex ratio, oviposition-choice, and growth index of C. partellus were calculated. The leaves and stem of the inoculated plants were given to the counted number of larvae of C. Partellus. The mortality of larvae was recorded on daily basis till the pupation. The result shows that maximum percent mortality (86.67%) was recorded at high concentration (20000ppm) of Beauveria bassiana by leaf inoculation method. For oviposition choice bioassay, the newly emerged adults were fed on diet (water, honey and yeast in 9:1:1) for 48 hours. One pair of C. Partellus were aspirated from the rearing cages and were detained in large test tube plugged with diet soaked cotton. A set of four plants for each treatment were prepared and randomized inside the large oviposition chamber. The test tubes were opened and fitted in the hole made in the wall of oviposition chamber in front of each treatment. The oviposition chamber was placed in a completely dark laboratory to eliminate the effect of light on moth’s behavior. The plants were removed from the oviposition chamber after the death of adults. The number of eggs deposited on the plant was counted. The results of 2nd experiment revealed that in all EPF and inoculation methods, the fecundity, egg fertility and growth index of C. partellus decreased with the increase in concentration being significantly higher at low concentration (5000ppm) and lower at higher concentration (20000ppm). Application of B. bassiana demonstrated that minimum fecundity (126.83), egg fertility (119.52) and growth index (15%) in C. partellus followed by M. anisopliae with fecundity (135.93), egg fertility (132.29) and growth index (17.50%) while V. lecanii show higher values of fecundity (137.37), egg fertility (1135.42) and growth index (20%). Overall leaf inoculation method showed least fecundity (123.89) with egg fertility (115.36) and growth index (14%) followed by whorl, shoot inoculation method and root inoculation method show higher values of fecundity, egg fertility and growth index.

Keywords: Beauveria bassiana, Chilo partellus, entomopathoganic, Metarhizium anisopliae, Verticillium lecanii

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Authors: Abirami Baleswaran, Christel Couderc, Loubnah Belahcen, Jean Dayde, Hélène Tormo, Gwénaëlle Jard

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Cheeses are often reported to be spoiled by Pseudomonas spp., responsible for defects in appearance, texture, taste, and smell, leading to their non-marketing and even their destruction. Despite preventive actions, problems linked to Pseudomonas spp. are difficult to control by the lack of knowledge and control of these contaminants during the cheese manufacturing. Lactic goat cheese producers are not spared by this problem and are looking for solutions to decrease the number of spoiled cheeses. To explore different hypotheses, experiments are needed. However, cheese-making experiments at the pilot scale are expensive and time consuming. Thus, there is a real need to develop a miniature cheeses model system under controlled conditions. In a previous study, several miniature cheese models corresponding to different type of commercial cheeses have been developed for different purposes. The models were, for example, used to study the influence of milk, starters cultures, pathogen inhibiting additives, enzymatic reactions, microflora, freezing process on cheese. Nevertheless, no miniature model was described on the lactic goat cheese. The aim of this work was to develop a miniature cheese model system under controlled laboratory conditions which resembles commercial lactic goat cheese to study Pseudomonas spp. spoilage during the manufacturing and ripening process. First, a protocol for the preparation of miniature cheeses (3.5 times smaller than a commercial one) was designed based on the cheese factorymanufacturing process. The process was adapted from “Rocamadour” technology and involves maturation of pasteurized milk, coagulation, removal of whey by centrifugation, moulding, and ripening in a little scale cellar. Microbiological (total bacterial count, yeast, molds) and physicochemical (pH, saltinmoisture, moisture in fat-free)analyses were performed on four key stages of the process (before salting, after salting, 1st day of ripening, and end of ripening). Factory and miniature cheeses volatilomewere also obtained after full scan Sift-MS cheese analysis. Then, Pseudomonas spp. strains isolated from contaminated cheeses were selected on their origin, their ability to produce pigments, and their enzymatic activities (proteolytic, lecithinasic, and lipolytic). Factory and miniature curds were inoculated by spotting selected strains on the cheese surface. The expression of cheese spoilage was evaluated by counting the level of Pseudomonas spp. during the ripening and by visual observation and under UVlamp. The physicochemical and microbiological compositions of miniature cheeses permitted to assess that miniature process resembles factory process. As expected, differences involatilomes were observed, probably due to the fact that miniature cheeses are made usingpasteurized milk to better control the microbiological conditions and also because the little format of cheese induced probably a difference during the ripening even if the humidity and temperature in the cellar were quite similar. The spoilage expression of Pseudomonas spp. was observed in miniature and factory cheeses. It confirms that the proposed model is suitable for the preparation of miniature cheese specimens in the spoilage study of Pseudomonas spp. in lactic cheeses. This kind of model could be deployed for other applications and other type of cheese.

Keywords: cheese, miniature, model, pseudomonas spp, spoilage

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Authors: M. Kazou, A. Gavriil, O. Kalagkatsi, T. Paschos, E. Tsakalidou

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Kopanisti cheese is a Greek soft Protected Designation of Origin (PDO) cheese made of raw cow, sheep or goat milk, or mixtures of them, with similar organoleptic characteristics to that of Roquefort cheese. Traditional manufacturing of Kopanisti cheese is limited in small-scale dairies, without the addition of starter cultures. Instead, an amount of over-mature Kopanisti cheese, called Mana Kopanisti, is used to initiate ripening. Therefore, the selection of proper starter cultures and the understanding of the contribution of various microbial groups to its overall quality is crucial for the production of a high-quality final product with standardized organoleptic and physicochemical characteristics. Taking the above into account, the aim of the present study was the investigation of Kopanisti cheese microbiota and its role in cheese quality. For this purpose, four different types of Kopanisti were produced in triplicates, all with pasteurized cow milk, with the addition of (A) the typical mesophilic species Lactococcus lactis and Lactobacillus paracasei used as starters in the production of soft spread cheeses, (B) strains of Lactobacillus acidipiscis and Lactobacillus rennini previously isolated from Kopanisti and Mana Kopanisti, (C) all the species from (A) and (B) as inoculum, and finally (D) the species from (A) and Mana Kopanisti. Physicochemical and microbiological analysis was performed for milk and cheese samples during ripening. Enumeration was performed for major groups of lactic acid bacteria (LAB), total mesophilic bacteria, yeasts as well as hygiene indicator microorganisms. Bacterial isolates from all the different LAB groups, apart from enterococci, alongside yeasts isolates, were initially grouped using repetitive sequence-based polymerase chain reaction (rep-PCR) and then identified at the species level using 16S rRNA gene and internal transcribed spacer (ITS) DNA region sequencing, respectively. Sensory evaluation was also performed for final cheese samples at the end of the ripening period (35 days). Based on the results of the classical microbiological analysis, the average counts of the total mesophilic bacteria and LAB, apart from enterococci, ranged between 7 and 10 log colony forming unit (CFU) g⁻¹, phychrotrophic bacteria, and yeast extract glucose chloramphenicol (YGC) isolates between 4 and 8 log CFU g⁻¹, while coliforms and enterococci up to 2 log CFU g⁻¹ throughout ripening in cheese samples A, C and D. In contrast, in cheese sample B, the average counts of the total mesophilic bacteria and LAB, apart from enterococci, phychrotrophic bacteria, and YGC isolates ranged between 0 and 10 log CFU g⁻¹ and coliforms and enterococci up to 2 log CFU g⁻¹. Although the microbial counts were not that different among samples, identification of the bacterial and yeasts isolates revealed the complex microbial community structure present in each cheese sample. Differences in the physicochemical characteristics among the cheese samples were also observed, with pH ranging from 4.3 to 5.3 and moisture from 49.6 to 58.0 % in the final cheese products. Interestingly, the sensory evaluation also revealed differences among samples, with cheese sample B ranking first based on the total score. Overall, the combination of these analyses highlighted the impact of different starter cultures on the Kopanisti microbiota as well as on the physicochemical and sensory characteristics of the final product.

Keywords: Kopanisti cheese, microbiota, classical microbiological analysis, physicochemical analysis

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Authors: Akram Abi, Mohammad Hossein Sarrafzadeh

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Because of their several advantages over chemical surfactants, biosurfactants have given rise to a growing interest in the past decades. Advantages such as lower toxicity, higher biodegradability, higher selectivity and applicable at extreme temperature and pH which enables them to be used in a variety of applications such as: enhanced oil recovery, environmental and pharmaceutical applications, etc. Bacillus subtilis produces a cyclic lipopeptide, called surfactin, which is one of the most powerful biosurfactants with ability to decrease surface tension of water from 72 mN/m to 27 mN/m. In addition to its biosurfactant character, surfactin exhibits interesting biological activities such as: inhibition of fibrin clot formation, lyses of erythrocytes and several bacterial spheroplasts, antiviral, anti-tumoral and antibacterial properties. Surfactin is an antibiotic substance and has been shown recently to possess anti-HIV activity. However, application of biosurfactants is limited by their high production cost. The cost can be reduced by optimizing biosurfactant production using cheap feed stock. Utilization of inexpensive substrates and unconventional carbon sources like urban or agro-industrial wastes is a promising strategy to decrease the production cost of biosurfactants. With suitable engineering optimization and microbiological modifications, these wastes can be used as substrates for large-scale production of biosurfactants. As an effort to fulfill this purpose, in this work we have tried to utilize olive oil as second carbon source and also yeast extract as second nitrogen source to investigate the effect on both biomass and biosurfactant production improvement in Bacillus subtilis cultures. Since the turbidity of the culture was affected by presence of the oil, optical density was compromised and no longer could be used as an index of growth and biomass concentration. Therefore, cell Dry Weight measurements with applying necessary tactics for removing oil drops to prevent interference with biomass weight were carried out to monitor biomass concentration during the growth of the bacterium. The surface tension and critical micelle dilutions (CMD-1, CMD-2) were considered as an indirect measurement of biosurfactant production. Distinctive and promising results were obtained in the cultures containing olive oil compared to cultures without it: more than two fold increase in biomass production (from 2 g/l to 5 g/l) and considerable reduction in surface tension, down to 40 mN/m at surprisingly early hours of culture time (only 5hr after inoculation). This early onset of biosurfactant production in this culture is specially interesting when compared to the conventional cultures at which this reduction in surface tension is not obtained until 30 hour of culture time. Reducing the production time is a very prominent result to be considered for large scale process development. Furthermore, these results can be used to develop strategies for utilization of agro-industrial wastes (such as olive oil mill residue, molasses, etc.) as cheap and easily accessible feed stocks to decrease the high costs of biosurfactant production.

Keywords: agro-industrial waste, bacillus subtilis, biosurfactant, fermentation, second carbon and nitrogen source, surfactin

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Authors: Zanele D. Ngwenya, Mustapha Mohammed, Felix D. Dakora

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Legumes are a major source of biological nitrogen, and therefore play a crucial role in maintaining soil productivity in smallholder agriculture in southern Africa. Through their ability to fix atmospheric nitrogen in root nodules, legumes are a better option for sustainable nitrogen supply in cropping systems than chemical fertilisers. For decades, farmers have been highly receptive to the use of rhizobial inoculants as a source of nitrogen due mainly to the availability of elite rhizobial strains at a much lower compared to chemical fertilisers. To improve the efficiency of the legume-rhizobia symbiosis in African soils would require the use of highly effective rhizobia capable of nodulating a wide range of host plants. This study assessed the morphogenetic diversity, photosynthetic functioning and relative symbiotic effectiveness (RSE) of groundnut, jack bean and soybean microsymbionts in Eswatini soils as a first step to identifying superior isolates for inoculant production. According to the manufacturer's instructions, rhizobial isolates were cultured in yeast-mannitol (YM) broth until the late log phase and the bacterial genomic DNA was extracted using GenElute bacterial genomic DNA kit. The extracted DNA was subjected to enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and a dendrogram constructed from the band patterns to assess rhizobial diversity. To assess the N2-fixing efficiency of the authenticated rhizobia, photosynthetic rates (A), stomatal conductance (gs), and transpiration rates (E) were measured at flowering for plants inoculated with the test isolates. The plants were then harvested for nodulation assessment and measurement of plant growth as shoot biomass. The results of ERIC-PCR fingerprinting revealed the presence of high genetic diversity among the microsymbionts nodulating each of the three test legumes, with many of them showing less than 70% ERIC-PCR relatedness. The dendrogram generated from ERIC-PCR profiles grouped the groundnut isolates into 5 major clusters, while the jack bean and soybean isolates were grouped into 6 and 7 major clusters, respectively. Furthermore, the isolates also elicited variable nodule number per plant, nodule dry matter, shoot biomass and photosynthetic rates in their respective host plants under glasshouse conditions. Of the groundnut isolates tested, 38% recorded high relative symbiotic effectiveness (RSE >80), while 55% of the jack bean isolates and 93% of the soybean isolates recorded high RSE (>80) compared to the commercial Bradyrhizobium strains. About 13%, 27% and 83% of the top N₂-fixing groundnut, jack bean and soybean isolates, respectively, elicited much higher relative symbiotic efficiency (RSE) than the commercial strain, suggesting their potential for use in inoculant production after field testing. There was a tendency for both low and high N₂-fixing isolates to group together in the dendrogram from ERIC-PCR profiles, which suggests that RSE can differ significantly among closely related microsymbionts.

Keywords: genetic diversity, relative symbiotic effectiveness, inoculant, N₂-fixing

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Authors: K. Premakumar, D. S. Rushani, H. N. Hettiarachchi

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The production and consumption of preserved foods have gained much importance due to globalization, and they provide a health benefit apart from the basic nutritional functions. Therefore, a study was conducted to develop a jam incorporating carrot, banana, and lime. Considering the findings of several preliminary studies, five formulations of the jam were prepared by blending different percentages of carrot and banana including control (where the only carrot was added). The freshly prepared formulations were subjected to physicochemical and sensory analysis.Physico-Chemical parameters such as pH, TSS, titrable acidity, ascorbic acid content, total sugar and non-reducing sugar and organoleptic qualities such as colour, aroma, taste, spread ability and overall acceptability and microbial analysis (total plate count) were analyzed after formulations. Physico-Chemical Analysis of the freshly prepared Carrot –Banana Blend jam showed increasing trend in titrable acidity (from 0.8 to 0.96, as % of citric acid), TSS (from 70.05 to 67.5 0Brix), ascorbic acid content (from 0.83 to 11.465 mg/100ml), reducing sugar (from 15.64 to 20.553%) with increase in carrot pulp from 50 to 100%. pH, total sugar, and non-reducing sugar were also reduced when carrot concentration is increased. Five points hedonic scale was used to evaluate the organoleptic characters. According to Duncan's Multiple Range Test, the mean scores for all the assessed sensory characters varied significantly (p<0.05) in the freshly made carrot-banana blend jam formulations. Based on the physicochemical and sensory analysis, the most preferred carrot: banana combinations of 50:50, 100:0 and 80:20 (T1, T2, and T5) were selected for storage studies.The formulations were stored at 300 °C room temperature and 70-75% of RH for 12 weeks. The physicochemical characteristics were measured at two weeks interval during storage. The decreasing trends in pH and ascorbic acid and an increasing trend in TSS, titrable acidity, total sugar, reducing sugar and non-reducing sugar were noted with advancement of storage periods of 12 weeks. The results of the chemical analysis showed that there were significance differences (p<0.05) between the tested formulations. Sensory evaluation was done for carrot –banana blends jam after a period of 12 weeks through a panel of 16 semi-trained panelists. The sensory analysis showed that there were significant differences (p<0.05) for organoleptic characters between carrot-banana blend jam formulations. The highest overall acceptability was observed in formulation with 80% carrot and 20% banana pulp. Microbiological Analysis was carried out on the day of preparation, 1 month, 2 months and 3 months after preparation. No bacterial growth was observed in the freshly made carrot -banana blend jam. There were no counts of yeast and moulds and coliforms in all treatments after the heat treatments and during the storage period. Only the bacterial counts (Total Plate Counts) were observed after three months of storage below the critical level, and all formulations were microbiologically safe for consumption. Based on the results of physio-chemical characteristics, sensory attributes, and microbial test, the carrot –banana blend jam with 80% carrot and 20% banana (T2) was selected as best formulation and could be stored up to 12 weeks without any significant changes in the quality characteristics.

Keywords: formulations, physicochemical parameters, microbiological analysis, sensory evaluation

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Authors: Mabel Y. C. Yau, Roy C. F. Lai, Hugo Y. H. Or

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This study is a pilot surveillance of hygiene compliance and food microbial safety of both licensed and mobile vendors selling Chinese ready–to-eat snack foods in Hong Kong. The study reflects similar situations in running mobile food vending business on trucks. Hong Kong is about to launch the Food Truck Pilot Scheme by the end of 2016 or early 2017. Technically, selling food on the vehicle is no different from hawking food on the street or vending food on the street. Each type of business bears similar food safety issues and cast the same impact on public health. Present findings demonstrate exemplarily situations that also apply to food trucks. 9 types of Cantonese style snacks of 32 samples in total were selected for microbial screening. A total of 16 vending sites including supermarkets, street markets, and snack stores were visited. The study finally focused on a traditional snack, the steamed rice cake with red beans called Put Chai Ko (PCK). PCK is a type of classical Cantonese pastry sold on push carts on the street. It used to be sold at room temperature and served with bamboo sticks in the old days. Some shops would have them sold steam fresh. Microbial examinations on aerobic counts, yeast, and mould, coliform, salmonella as well as Staphylococcus aureus detections were carried out. Salmonella was not detected in all samples. Since PCK does not contain ingredients of beef, poultry, eggs or dairy products, the risk of the presence of Salmonella in PCK was relatively lower although other source of contamination might be possible. Coagulase positive Staphylococcus aureus was found in 6 of the 14 samples sold at room temperature. Among these 6 samples, 3 were PCK. One of the samples was in an unacceptable range of total colony forming units higher than 105. The rest were only satisfactory. Observational evaluations were made with checklists on personal hygiene, premises hygiene, food safety control, food storage, cleaning and sanitization as well as waste disposals. The maximum score was 25 if total compliance were obtained. The highest score among vendors was 20. Three stores were below average, and two of these stores were selling PCK. Most of the non-compliances were on food processing facilities, sanitization conditions and waste disposal. In conclusion, although no food poisoning outbreaks happened during the time of the investigation, the risk of food hazard existed in these stores, especially among street vendors. Attention is needed in the traditional practice of food selling, and that food handlers might not have sufficient knowledge to properly handle food products. Variations in food qualities existed among supply chains or franchise eateries or shops. It was commonly observed that packaging and storage conditions are not properly enforced in the retails. The same situation could be reflected across the food business. It did indicate need of food safety training in the industry and loopholes in quality control among business.

Keywords: cantonese snacks, food safety, microbial, hygiene, street food

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Authors: Cynthia K. Burzell

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Aequor's Founder, a Marine and Medical Microbiologist, discovered novel, non-toxic chemicals in the ocean that uniquely remove biofilm in minutes and prevent its formation for days. These chemicals and over 70 synthesized analogs that Aequor developed can replace thousands of toxic biocides used in consumer and industrial products and, as new drug candidates, kill biofilm-forming bacteria and fungi Superbugs -the antimicrobial-resistant (AMR) pathogens for which there is no cure. Cynthia Burzell, PhD., is a Marine and Medical Microbiologist studying natural mechanisms that inhibit biofilm formation on surfaces in contact with water. In 2002, she discovered a new genus and several new species of marine microbes that produce small molecules that remove biofilm in minutes and prevent its formation for days. The molecules include new antimicrobials that can replace thousands of toxic biocides used in consumer and industrial products and can be developed into new drug candidates to kill the biofilm-forming bacteria and fungi -- including the antimicrobial-resistant (AMR) Superbugs for which there is no cure. Today, Aequor has over 70 chemicals that are divided into categories: (1) Novel natural chemicals. Lonza validated that the primary natural chemical removed biofilm in minutes and stated: "Nothing else known can do this at non-toxic doses." (2) Specialty chemicals. 25 of these structural analogs are already approved under the U.S. Environmental Protection Agency (EPA)'s Toxic Substances Control Act, certified as "green" and available for immediate sale. These have been validated for the following agro-industrial verticals: (a) Surface cleaners: The U.S. Department of Agriculture validated that low concentrations of Aequor's formulations provide deep cleaning of inert, nano and organic surfaces and materials; (b) Water treatments: NASA validated that one dose of Aequor's treatment in the International Space Station's water reuse/recycling system lasted 15 months without replenishment. DOE validated that our treatments lower energy consumption by over 10% in buildings and industrial processes. Future validations include pilot projects with the EPA to test efficacy in hospital plumbing systems. (c) Algae cultivation and yeast fermentation: The U.S. Department of Energy (DOE) validated that Aequor's treatment boosted biomass of renewable feedstocks by 40% in half the time -- increasing the profitability of biofuels and biobased co-products. DOE also validated increased yields and crop protection of algae under cultivation in open ponds. A private oil and gas company validated decontamination of oilfield water. (3) New structural analogs. These kill Gram-negative and Gram-positive bacteria and fungi alone, in combinations with each other, and in combination with low doses of existing, ineffective antibiotics (including Penicillin), "potentiating" them to kill AMR pathogens at doses too low to trigger resistance. Both the U.S. National Institutes for Health (NIH) and Department of Defense (DOD) has executed contracts with Aequor to provide the pre-clinical trials needed for these new drug candidates to enter the regulatory approval pipelines. Aequor seeks partners/licensees to commercialize its specialty chemicals and support to evaluate the optimal methods to scale-up of several new structural analogs via activity-guided fractionation and/or biosynthesis in order to initiate the NIH and DOD pre-clinical trials.

Keywords: biofilm, potentiation, prevention, removal

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Authors: Thembelihle Portia Lubisi, Malusi Ntandoyenkosi Mkhize, Jonas Kalebe Johakimu

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Fertilizers play an important role in maintaining the productivity and quality of plants. Inorganic fertilizers (containing nitrogen, phosphorus, and potassium) are largely used in South Africa as they are considered inexpensive and highly productive. When applied, a portion of the excess fertilizer will be retained in the soil, a portion enters water streams due to surface runoff or the irrigation system adopted. Excess nutrient from the fertilizers entering the water stream eventually results harmful algal blooms (HABs) in freshwater systems, which not only disrupt wildlife but can also produce toxins harmful to humans. Use of agro-chemicals such as pesticides and herbicides has been associated with increased antimicrobial resistance (AMR) in humans as the plants are consumed by humans. This resistance of bacterial poses a threat as it prevents the Health sector from being able to treat infectious disease. Archaeological studies have found that pyrolysis liquids were already used in the time of the Neanderthal as a biocide and plant protection product. Pyrolysis is thermal degradation process of plant biomass or organic material under anaerobic conditions leading to production of char, bio-oils and syn gases. Bio-oil constituents can be categorized as water soluble (wood vinegar) and water insoluble fractions (tar and light oils). Wood vinegar (pyro-ligneous acid) is said to contain contains highly oxygenated compounds including acids, alcohols, aldehydes, ketones, phenols, esters, furans, and other multifunctional compounds with various molecular weights and compositions depending on the biomass material derived from and pyrolysis operating conditions. Various researchers have found the wood vinegar to be efficient in the eradication of termites, effective in plant protection and plant growth, has antibacterial characteristics and was found effective in inhibiting the micro-organisms such as candida yeast, E-coli, etc. This study investigated characterisation of South African forestry product processing waste with intention of evaluating the potential of using the respective biomass waste as feedstock for boil oil production via pyrolysis process. Ability to use biomass waste materials in production of wood-vinegar has advantages that it does not only allows for reduction of environmental pollution and landfill requirement, but it also does not negatively affect food security. The biomass wastes investigated were from the popular tree types in KZN, which are, pine saw dust (PSD), pine bark (PB), eucalyptus saw dust (ESD) and eucalyptus bark (EB). Furthermore, the research investigates the possibility of mixing the different wastes with an aim to lessen the cost of raw material separation prior to feeding into pyrolysis process and mixing also increases the amount of biomass material available for beneficiation. A 50/50 mixture of PSD and ESD (EPSD) and mixture containing pine saw dust; eucalyptus saw dust, pine bark and eucalyptus bark (EPSDB). Characterisation of the biomass waste will look at analysis such as proximate (volatiles, ash, fixed carbon), ultimate (carbon, hydrogen, nitrogen, oxygen, sulphur), high heating value, structural (cellulose, hemicellulose and lignin) and thermogravimetric analysis.

Keywords: characterisation, biomass waste, saw dust, wood waste

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Authors: Ipsita Pujari, Abitha Thomas, Vidhu S. Babu, K. Satyamoorthy

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Orchids are esteemed as celebrities in cut flower industry globally, due to their long-lasting fragrance and freshness. Apart from splendor, the unique metabolites endowed with pharmaceutical potency have made them one of the most hunted in plant kingdom. This had led to their trafficking, resulting in habitat loss, subsequently making them occupiers of IUCN red list as RET species. Many of the orchids especially wild varieties still remain undiscovered. In view to protect and conserve the wild germplasm, researchers have been inventing novel micropropagation protocols; thereby conserving Orchids. India is overflowing with exclusive wild cultivars of Orchids, whose pharmaceutical properties remain untapped and are not marketed owing to relatively small flowers. However, their germplasm is quite pertinent to be preserved for making unusual hybrids. Dendrobium genus is the second largest among Orchids exists in India and has highest demand attributable to enduring cut flowers and significant therapeutic uses in traditional medicinal system. Though the genus is quite endemic in Western Ghat regions of the country, many species are still anonymous with their unknown curative properties. A standard breeding cycle in Orchids usually takes five to seven years (Dendrobium hybrids taking a long juvenile phase of two to five years reaching maturity and flowering stage) and this extensive life cycle has always hindered the development of Dendrobium breeding. Dendrobium is reported with essential therapeutic plant bio-chemicals and ‘Moscatilin’ is one, found exclusive to this famous Dendrobium genus. Moscatilin is reported to have anti-mutagenic and anti-cancer properties, whose positive action has very recently been demonstrated against a range of cancers. Our preliminary study here established a simple and economic small-scale propagation protocol of Dendrobium ovatum describing in vitro production of Moscatilin. Subsequently for enhancing the content of Moscatilin, an efficient experimental related to the organization of transgenic (hairy) D. ovatum root cultures through infection of Agrobacterium rhizogenes 2364 strain on MS basal medium is being reported in the present study. Hairy roots generated on almost half of the explants used (spherules, in vitro plantlets and calli) maintained through suspension cultures, after 8 weeks of co-cultivation with Agrobacterium rhizogenes. GFP assay performed with isolated hairy roots has confirmed the integrative transformation which was further positively confirmed by PCR using rolB gene specific primers. Reverse phase-high performance liquid chromatography and mass spectrometry techniques were used for quantification and accurate identification of Moscatilin respectively from transgenic systems. A noticeable ~3 fold increase in contents were observed in transformed D. ovatum root cultures as compared to the simple in vitro culture, callus culture and callus regeneration plantlets. Role of elicitors e.g., Methyl jasmonate, Salicylic acid, Yeast extract and Chitosan were tested for elevating the Moscatilin content to obtain a comprehensive optimized protocol facilitating the in vitro production of valuable Moscatilin with larger yield. This study would provide evidence towards the in vitro assembly of Moscatilin within a short time-period through not a so-expensive technology for the first time. It also serves as an appropriate basis for bioreactor scale-up resulting in commercial bioproduction of Moscatilin.

Keywords: bioproduction, Dendrobium ovatum, hairy root culture, moscatilin

Procedia PDF Downloads 235