Sucrose Concentration In Medium Research Articles

Effect of Temperature and Sucrose on In Vitro Seed Germination and Bulblet Production of Pancratium maritimum L.

Sea daffodil is a perennial medicinal plant with several beneficial health properties that is enlisted as an endangered and protected species. For the exploitation and conservation of such valuable species, sustainable propagation is a key factor. For this purpose, the establishment of in vitro cultures using botanical seeds is an effective way to produce a large number of microplants which can be used as propagating material and/or bulblets with further uses for the isolation–production of biocactive substances. In this work, we performed two separate experiments where we studied (a) the effect of temperature (10 °C, 15 °C, 20 °C, 25 °C, and 30 °C) on seed germination in vitro and (b) the effect of temperature (10 °C, 20 °C, and 30 °C) and sucrose concentration (40 and 80 g/L) in growth medium on microplant development and the production of bulblets. Seed germination rates were high (over 70%) at a wide range of temperatures (15–30 °C), although the fastest and the most uniform germination was observed at 20–25 °C. Regarding the effect of temperature on microplant development and bulblet production, low temperatures (10 °C) had a negative effect on the number of leaves, bulb diameter, and fresh weight (F.W.) and dry weight (D.W.) of leaves, roots, and bulblets. The best growth of microplants (number of leaves, F.W. and D.W. of leaves, roots) and bulblet production (diameter, F.W., and D.W.) were observed in culture media with sucrose concentration 40 g/L and temperature of 30 °C. In culture medium with a sucrose concentration of 80 g/L the production of bulbs (diameter, F.W., and D.W.) was not inferior to culture media with a sucrose concentration of 40 g/L, although microplants had less root growth. It is concluded that the best treatment for the production of well-rooted microplants with large bulblets that could be used as propagating material or for the production of bulblets as a source for twin scales explants or pharmaceutical substances was the culture at 30 °C in culture medium with sucrose concentration 40 g/L. On the other hand, culture medium with a sucrose concentration of 80 g/L at 30 °C or 20 °C could be also used for the production of bulblets.

Ice recrystallization inhibition effect of cellulose nanocrystals: Influence of sucrose concentration

Stabilizers are commonly used to inhibit the ice recrystallization process that leads to the formation of large ice crystals, decreasing consumer acceptance of ice cream. However, the ice recrystallization inhibition (IRI) effect of stabilizers is very dependent on measurement conditions. The cause of this dependence is not clear, which imposes problems in comparing the IRI activity and understanding the IRI mechanism of stabilizers. This study investigated the influence of sucrose concentration on the IRI effect of cellulose nanocrystals (CNCs), which are newly identified IRI active materials. A splat assay and a sandwich assay were used to determine IRI activities of CNCs at sucrose concentrations from 2.0% to 49.0% w/w and incubation temperature of −8 °C for 30 min. The IRI activities of CNCs were higher at low sucrose concentrations, almost disappeared at medium sucrose concentrations, and restored slightly at high sucrose concentrations. The IRI effect of CNCs is correlated with the ratio between CNCs concentration in the unfrozen phase and total surface area of ice crystals. This surface area-related IRI effect was also observed with guar gum, locust bean gum, and poly(vinyl alcohol). It is a strong evidence of an ice-binding mechanism for the IRI effect of stabilizers. These research findings offer a new perspective in comparing and understanding the measurement condition-dependent IRI effect of stabilizers, which could potentially benefit the ice cream industry in developing new materials and recipes to control ice recrystallization.

Production of potato (Solanum tuberosum, L.) microtubers using plastic culture bags.

We have developed a system using plastic culture bags with forced aeration system for both liquid medium and gaseous phase to produce microtubers of potato (Solanum tuberosum L.). The production of microtubers under sterile conditions is a good way to produce disease-free materials for crop production, and bioreactors have been used for this purpose. However, bioreactors are expensive and difficult to handle. The plastic culture bags are relatively inexpensive and are easy to store and sterilize because they can be flattened. Microtuber production involves two stages: plant proliferation in one medium, followed by microtuber production in a different medium. Both steps are carried out using the same culture bag. Using this system, we produced 100 to 300 microtubers per 8 l culture bag, depending on the potato cultivar. We varied the nutrient concentrations in the media and found that a lower sucrose concentration in the plant proliferation medium and lower nitrogen concentration in the microtuber production medium both increased the total numbers of microtubers per bag. Notably, a higher concentration of potassium phosphate increased the numbers of larger microtubers. This is beneficial because larger microtubers are much more tolerant to field conditions. We produced about 250,000 microtubers per year in a 66 m2 tissue culture room using the culture bag system. These microtubers have been planted directly in the field and utilized for seed potato production.

Sweetness Enhancement by Aromas: Measured by Descriptive Sensory Analysis and Relative to Reference Scaling.

Sweetness enhancement by aromas has been suggested as a strategy to mitigate sugar reduction in food products, but enhancement is dependent on type of aroma and sugar level. A careful screening of aromas across sugar levels is thus required. Screening results might, however, depend on the method employed. Both descriptive sensory analysis and relative to reference scaling were therefore used to screen 5 aromas across 3 sucrose concentrations for their sweetness-enhancing effects in aqueous solutions. In the descriptive analysis, samples with added vanilla, honey, and banana aroma were rated as significantly sweeter than samples with added elderflower or raspberry aroma at all sucrose concentrations. In relative to reference scaling, honey aroma significantly increased the sweet taste compared with samples with added elderflower or no aroma at low and medium sucrose concentrations. Banana and raspberry aromas also increased the sweet taste significantly compared with the sample with added elderflower aroma at medium sucrose concentration in the relative to reference scaling. This demonstrates that the cross-modal effects observed by the 2 methods were different. In terms of the methods applied, relative to reference scaling was generally found to result in a decrease in the measured sweetness enhancement by aromas. In the descriptive analysis, the cross-modal effect of aromas on sweet taste perception was found to be significantly higher at 2.5% and 5.0% w/w sucrose compared with 7.5% w/w sucrose. These results highlight the importance of considering how references are employed in sensory analysis and how they affect cross-modal interactions.

Micropropagation of Piper crassinervium: an improved protocol for faster growth and augmented production of phenolic compounds

The medicinal plant Piper crassinervium is a source of bioactive compounds with potential use in agrochemical and pharmaceutical industries. However, its propagation is slow and influenced by the composition of the culture medium and growth conditions. In the genus Piper, tissue oxidation limits the biomass and recovery of bioactive compounds such as phenols. We evaluated the effect of medium formulations, sucrose, growth regulators, medium consistency, explant type, gas exchange and irradiance levels on growth and phenols and total flavonoids content in P. crassinervium in vitro plants. Murashige and Skoog (MS) and DKW/Juglans (DKW/J) media induced the highest production of chlorophyll and phenolic compounds; however, the regeneration rate of the explants in the DKW/J medium was the lowest (~75%) in comparison with the other media (90%). The maximum biomass production was achieved when half-strength MS (½MS) liquid medium was used; while the optimum sucrose level depended on the medium salt concentration used. Notably, the irradiance altered the accumulation of biomass and phenolic compounds. The presence of one remaining leaf in the nodal segments, used as explants, produced taller plants in ½MS liquid medium, combined or not with 2-iP. The optimal growth conditions for biomass and phenolic compound production in P. crassinervium in vitro plants established were: nodal explant with a preexisting leaf cultured on ½MS stationary liquid medium, 15 g L−1 sucrose, using lids with one PTFE 0.45-µm-pore size membrane and an irradiance of 100 µmol m−2 s−1. Medium formulation, sucrose concentration, irradiance levels, gas exchange and explants with preexisting leaf improved the in vitro propagation and phenolic compounds production in Piper crassinervium.

Sucrose Concentration, Light Intensity, and CO<sub>2</sub> Concentration Affect Growth and Development of Micropropagated Carnation

Carnation (Dianthus caryophyllus L.) ‘Purple Beauty’ at micropropagation stage 3 was cultured under two levels each of medium sucrose concentration (0 and 30 g·L-1), photosynthetic photon flux density (50 and 200 μmol·m-2·s-1 PPFD), and CO2 concentration (350 and 1,000 μmol·mol-1), in a factorial experimental design, consisting of all eight possible treatment combinations. Shoot tip explants, obtained from in vitro-grown plantlets, were cultured on 50 mL agar-solidified Murashige and Skoog medium per container. Each culture container was sealed with a rigid lid vented by a high-efficiency particulate air filter attached to a 10 mm diameter hole made in the lid center, with an estimated number of 2.8 air exchanges per hour. All cultures were maintained for four weeks at 24°C/22°C (day/night) temperatures, 70%–80% relative humidity, and a 16 h/8 h (day/night) photoperiod was provided by white light-emitting diodes. The treatment combining high light intensity (200 μmol·m-2·s-1), high CO2 concentration (1,000 μmol·mol-1), and without supplementation of sucrose to the medium (i.e., photo-autotrophic conditions) resulted in better plantlet growth and development, with higher values being observed in terms of total fresh weight, tissue water content, leaf length, leaf width, and total chlorophyll content than in plantlets grown under the other treatments.

Impacto de las concentraciones de ANA y sacarosa en el enraizamiento in vitro de Butia odorata (Barb. Rodr.) Noblick

The objective of this study was to evaluate the culture medium seeking the development and root formation of pindo palm seedlings for acclimatization. The MS medium were used at a concentration of 70% ammonium nitrate and potassium nitrate, and 0.8% of agar. In the first experiment, concentrations of naphthaleneacetic acid (ANA) added to the medium were tested. In the second, the rooting and growth of the seedlings were evaluated after varying the sucrose concentration (15, 30 and 45 g.L-1) in medium with 0.6 mg.L-1 of ANA. A third experiment was carried out to adjust the ANA concentration, based on the results of the fi rst two tests. The rooting of Butia odorata seedlings were optimized in vitro in medium with 0.3 to 0.9 mg.L-1 of ANA, combined with concentrations of sucrose between 30 and 45 g.L-1.

Synthetic Seed Preparation, Germination and Plantlet Regeneration of Litchi (<i>Litchi chinensis</i> Sonn.)

Litchi chinensis sonn.) ranks second after mango amongst the most important fruit crops cultivated worldwide. Litchi is a very valuable crop throughout the world because it is a table fruit and wines are also produced from it. The existing cultivars are highly polyploidy and heterozygous in nature. It is propagated through air layering and marcottage methods and storability is very low. Synthetic seeds can be stored for a long time and its genetic constitution could remain the same. For germplasm maintenance and clonal propagation, synthetic seeds can be used. Somatic embryogenesis has been reported from anther or embryogenic suspension culture in various species of litchi. Regeneration via organogenesis and somatic embryogenesis from zygotic embryos has also been reported in certain species. Developing a methodology for getting somatic embryogenesis with a high frequency from zygotic embryos which is available once in a year, would be particularly useful for genetic improvement of litchi. Cotyledonary stage somatic embryos developed from zygotic embryos were encapsulated in 2% alginate gel. The encapsulated somatic embryos (ESEs) germinated successfully on 0.7% agar medium containing 3% sucrose concentration in NN basal medium (half strength of major and minor salts) with 1 mg·l-1 of gibbrellic acid. Percentage germination and plantlet development for ESEs was higher than that of non encapsulated embryos (NSEs). In comparison to different hormones, gibberellic acid has a significant influence on the germination rate of ESEs after one week of dehydration was seen maximum at 9% sucrose and abscisic acid (1 mg·l-1) in half strength of major and minor salts in Nitsch and Nitsch medium resulting in extended storage up to 90 days without loss in germination potential and capability to regenerate into plantlets. Normally developed plantlets regenerated from ESEs were successfully adapted to soil to obtain a full grown plant.

Detection and Quantitation of Aflatoxin for the Diagnosis of Aspergillus flavus

Aflatoxins are the potent mycotoxins produced by Aspergillus flavus, which is hepatotoxic causing hepatocellular carcinoma. A. flavus produces sufficient amount of Aflatoxin B1 under favourable environments. Inhalation of spores and use of Aflatoxin B1, contaminated food by Aspergillus spp., could transfuse the toxins in the blood streams. The presence of these toxins in body fluid can be detected by immunological assays and which provides an effective technique for the diagnosis of the disease caused by A. flavus. Aflatoxins producing strain of A. flavus were screened in Aflatoxin Producing Medium. Production of Aflatoxin B1 by A. flavus was studied in different parameters such as incubation periods, temperatures, pH variations, sucrose concentration in Yeast Extract Sucrose medium and different natural media such as par-boiled rice, corn and groundnuts. The detection of toxins was done by TLC using silica gel (Merk) coated plates and confirmative test was done by Association of Official Analytical Chemists (AOAC) method. Presence and quantization was done by Enzyme Linked Immunosorbent Assay (ELISA) technique. Highest amount of Aflatoxin B1 was reported 68.56 ng/ml by ELISA in synthetic medium (Yeast Extract Sucrose) with 2% sucrose, pH 5.5, on 14th days of incubation, at 28±1°C (p-value 0.05). Similarly, highest amount was recorded in groundnuts (121.20ng/g) by ELISA and (500ng/kg) by TLC methods. ELISA is one of the most efficient methods used for detection and diagnosis of human diseases cause due to exposure of Aflatoxin B1 and A. flavus.Nepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1: 6-9

B-type cyclin modulation in response to carbon balance in callus of Populus alba

In plants, sucrose is the principal transported carbon compound. Sucrose and/or glucose and fructose are relevant in the regulation of meristematic cell proliferation. The aim was to define the sugar balance and the relationship between the osmoregulation processes and the cell division patterns in callus of Populus alba in response to different sucrose medium concentrations. Callus proliferation and metabolism were assessed through biometric measures, non-structural soluble carbohydrates quantification, osmotic potential determination, as well as by quantification of mRNA accumulation of B-type cyclin genes. Calli were cultured on proliferation medium without sucrose or supplemented with 10, 20 or 30 g l−1 of sucrose. A large amount of non-structural soluble carbohydrates was used to restore the osmotic balance between callus and medium; upon reaching the equilibrium, carbon was then used for cell division. The initial investment of carbon for osmoregulation processes can explain the differences in the lag phase duration in response to the decrease of medium sucrose concentration. However, calli cultured on medium added with 30 g l−1 of sucrose used carbon both to adjust their osmotic potential and to restore the growth whilst the calli on sucrose free medium entered in a quiescent state. The growth rates compared to the transcript accumulation trends suggested that a threshold effect, rather than a quantitative regulation model, governed the relation between CycB gene transcription and cell division. The findings showed that poplar calli used the carbon following a “hierarchical” model based on their physiological state and the sugar concentration available in the medium.

Yield enhancement strategies for the production of picroliv from hairy root culture of Picrorhiza kurroa Royle ex Benth.

Fast-growing hairy root cultures of Picrorhiza kurroa induced by Agrobacterium rhizogenes offers a potential production system for iridoid glycosides. In present study we have investigated the effects of various nutrient medium formulations viz B5, MS, WP and NN, and sucrose concentrations (1–8%) on the biomass and glycoside production of selected clone (14-P) of P. kurroa hairy root. Full strength B5 medium was found to be most suitable for maximum biomass yield on the 40th day of culture (GI = 32.72 ± 0.44) followed by the NN medium of the same strength (GI = 22.9 ± 0.43). Secondary metabolite production was 1.1 and 1.3 times higher in half strength B5 medium respectively in comparison to MS medium. Maximum biomass accumulation along with the maximum picroliv content was achieved with 4% sucrose concentration in basal medium. RT vitamin and Thiamine-HCl effected the growth and secondary metabolite production of hairy roots growing on MS medium but did not show any effect on other media. The pH of the medium played significant role in growth and secondary metabolite production and was found to be highest at pH 6.0 while lowest at pH 3.0 and pH 8.0. To enhance the production of biomass and Picroliv 5 liter working capacity bioreactor was used, 27-fold (324 g FW) higher growth was observed in bioreactor than shake flask and secondary metabolite production was similarly enhanced.

Comparison of growth, yield and related traits of resistant Hevea genotypes under high South American leaf blight pressure

Most natural rubber (NR) comes from the cultivation of Hevea brasiliensis in tropical areas. However, South American leaf blight (SALB), caused by Microcyclus ulei (P. Henn.) Arx, is the main factor limiting Hevea cultivation in the American humid tropics. Based on a preliminary selection from among more than 960 accessions, the aim of this work was to propose new SALB-resistant genotypes for South American plantations. Agronomic traits (SALB resistance, growth, production) and characters related to latex production, such as sucrose and inorganic phosphorus, along with structural traits of the bark of nine Hevea genotypes resistant to SALB (TP875, FDR5788, MDX608, PMB1, CDC429, CDC312, FDR4461, MDF180, SIAL893) were evaluated during the course of a 12-year trial located in the Michelin plantation (Bahia, Brazil). These genotypes presented a quantitative resistance to SALB characterized by partially sporulating lesions (anomorph stage). No sexual form of M. ulei (stromata, teleomorph stage) was found in FDR5788, MDF180, CDC312, PMB1. Only FDR5788 genotypes presented a higher NR yield than genotype TP875 used as a control. For FDR5788, MDX608, CDC312 and PMB1, the estimated yields of 12-year-old trees were, respectively 2.6, 2.0, 1.8 and 1.2tyear−1ha−1. In all genotypes, medium sucrose and inorganic phosphorus concentrations were related to intermediate metabolism for cis-polyisoprene synthesis. The low yield of genotypes SIAL893 and MDF180 is related to the limited number of vessel rings, limited translocation of sucrose in the latex, and a slow metabolism indicated by low average concentrations of inorganic phosphorus in the latex. For the south east of the Bahia state, FDR5788 and CDC312 are recommended for new planting areas because of their capacity to prevent teleomorph formation, combined with high NR yields.

Refinement of Cost Reduction Technology in Sugarcane Micro propagation

In the present study, an efficient ex-vitro rooting technique was developed for sugarcane commercial cultivar CoS 96268 based on the assessment of several factors. Axillary bud cultures of nodal segments were used in this experiment. The parameters optimized for ex vitro rooting includes the effects of auxin concentration, propagule size, shoot size, duration of auxin pulse treatment, sucrose concentration in multiplication medium, ex-vitro rooting medium were studied. The results showed that the auxin concentration @ 20 mg/l gave maximum rooting percentage. Among 0h, 6h,12h and 24h treatment duration with auxin, 24h treatment duration developed better roots. 90 to 95% rooting was observed in three shoot propagule with 5 to 6 cm length of in vitro micro shoots. It has been observed that sucrose concentration in multiplication medium also influenced the ex vitro rooting. Sucrose at 45 g/l gave good results. Sand was best rooting medium among the peat, peat + soil and vermiculite for the development of ex vitro roots in green house conditions. Ex vitro rooted plants were transplanted in field and 9098% survival percent was found same as in vitro propagated plants of sugarcane.

Sugarcane micropropagation using light emitting diodes and adjustment in growth-medium sucrose concentration

The aim of this research was to evaluate the use of light emitting diodes (LEDs) instead of white fluorescent lamps as light source and adequate growth-medium sucrose concentration for sugarcane micropropagation (Saccharum officinarum L.). Sugarcane (RB 872552 variety) bud explants were evaluated during the multiplication and rooting phases under controlled growth-room conditions. Different light sources (blue, red and green LEDs; Growlux and white fluorescent lamps) and different medium sucrose concentrations (0; 15; 30 and 45g L-1) were used, maintaining constant light intensity (20µmol m-2 s-1), photoperiod (16h) and temperature (25+2°C). The experiment was a completely randomized design, and treatments were arranged in a 5x4 factorial (five light sources and four medium sucrose concentrations) with six replications. Sugarcane bud growth was satisfactory under the three LED types studied. The presence of sucrose in growth media was essential for bud multiplication and rooting. Nevertheless, each light source requires the respective medium sucrose concentration adjustment for best results. Red LEDs provided a significantly high multiplication rate (although not the highest) with 8.5 buds per sub-culture and 34.9g L-1 of sucrose; also, the highest bud length (33.3mm) and the best plantlet acclimatization. Therefore, LED sources can advantageously substitute fluorescent lamps in laboratories of sugarcane micropropagation.

Establishment of in vitro culture, plant regeneration, and genetic transformation of Camelina sativa

The results on in vitro culture establishment, plantlet regeneration and rooting of Camelina sativa cultivar sample Peremozhets and cultivar Mirazh are presented. Effective concentrations of sterilizing agents and duration of plant material treatment were estimated. Phytohormone ratio, sucrose concentration in nutrient medium that induce effective formation of C. sativa shoots and NAA concentration for plantlet rooting have been established. The method of Agrobacterium-mediated transformation of Camelina by using binary vector pGH217 carrying reporter beta-glucoronidase (gus) gene driven under 35S CaMV promoter and nos-terminator, and selective marker hpt gene conferring hygromycin-resistance in transgenic plant was elaborated.

희귀.멸종위기 산개나리(Forsythia saxatilis) 화분의 최적 발아 조건

산개나리(Forsythia saxatilis Nakai)의 개화와 결실 특성을 구명하기 위하여, 개화 생리의 기초 연구로서 산개나리 화분의 적정 발아 조건을 제시하고자 본 연구를 실시하였다. 산개나리의 화분 시료는 3월말부터 4월 초 개화시기에 맞춰 채취하였다. 산개나리 화분의 적정 발아 조건을 결정하기 위하여, 최적 배양 온도, 배지 설탕 농도 및 배양 시간을 먼저 선정한 후, 가장 적당한 배지 pH를 결정하였다. 화분 발아율과 화분관 신장은 발아 온도, 배지 설탕 농도 및 발아 시간에 따라 뚜렷한 차이가 있었으며, 발아 온도와 발아 시간, 발아 온도와 배지 설탕 농도 및 발아 시간과 배지 설탕 농도 간에 상호 효과가 나타났다. 산개나리의 화분 발아율은 <TEX>$10^{\circ}C$</TEX>에서 가장 높았으며, 배지 설탕 농도가 높을수록 발아율은 증가하였으나, 발아 시간과는 상관이 없었다. 또한, 15% 이상의 설탕 농도에서는 화분 발아율과 화분관 신장이 큰 차이를 나타내지 않았고, 화분 살포 후 24시간 이상이 경과한 후에도 화분 발아율이 큰 차이를 보이지 않았다. 화분 발아율은 pH 5에서 20.8%로 가장 높았으며, pH 6에서 3.8%로 가장 낮았다. 따라서 산개나리 화분의 적정 발아 조건은 발아 온도 <TEX>$10^{\circ}C$</TEX>, 배지 설탕 농도 15% 및 배지 pH 5인 것으로 나타났다. 또한, 화분 발아 조사는 화분 살포 24시간 후에 실시하는 것이 가장 바람직한 것으로 판단된다. Optimal condition for pollen germination was suggested as a basic research of flowering physiology in order to identify the characteristics of flowering and seed setting of rare and endangered Forsythia saxatilis Nakai. Pollen samples were collected during flowering time from the end of March to the beginning of April. First, a suitable germination temperature, medium sucrose concentration and germination time were determined for pollen germination and pollen tube elongation in vitro, and then an optimal pH of culture medium. Pollen germination and tube elongation were significantly different among the levels of germination temperature, sucrose concentration and germination time. Interactive effects were observed between germination temperature and time, germination temperature and sucrose concentration, germination time and sucrose concentration. Pollen germination was the highest at <TEX>$10^{\circ}C$</TEX> and increased with the increase of sucrose concentration, whereas it had no relation with germination time. In addition, pollen germination and tube elongation did not increase at more than 15% of sucrose concentration and 24 hours later. Pollen germination was the highest at pH 5 (20.8%) and the lowest at pH 6 (3.8%). In conclusion, <TEX>$10^{\circ}C$</TEX>, 15% sucrose and pH 5 were proposed as the optimal condition for pollen germination 24 hours later of pollen culture.

Statistical optimization of levansucrase production from Bacillus subtilis ATCC 6633 using response surface methodology

Response surface methodology (RSM) was applied to optimize the conditions of levansucrase production by Bacillus subtilis ATCC 6633, an uninvestigated strain ofBacillus. The effects of the medium sucrose concentrations (100 to 350 g/L), pH (4.0 to 7.0), fermentation time (24 to 48 h) and agitation (100 to 200 rpm) were studied simultaneously using factorial analysis. The results showed that the optimum condition for levansucrase production was 300 g/L sucrose concentration, pH 6.0, 24 h of fermentation and 180 rpm agitation, reaching 6.57 UA activity. Analysis of variance (ANOVA) showed a high coefficient of determination (R2: 0.9486) ensuring a satisfactory adjustment of quadratic model with the experimental data. The variables: sucrose concentration, pH and agitation were significant at the level of 5%. A high similarity was observed between the predicted and experimental results, which reflected the accuracy and applicability of RSM to optimize the process for levansucrase production. This is the first report on levansucrase production by B. subtilis ATCC 6633 using response surface methodology in sucrose fermentation. Key words: Statistical methodology, bacterial enzyme, sucrose, fermentation time, pH.

Efficiency of neural network-based combinatorial model predicting optimal culture conditions for maximum biomass yields in hairy root cultures

KEY MESSAGE : ANN-based combinatorial model is proposed and its efficiency is assessed for the prediction of optimal culture conditions to achieve maximum productivity in a bioprocess in terms of high biomass. A neural network approach is utilized in combination with Hidden Markov concept to assess the optimal values of different environmental factors that result in maximum biomass productivity of cultured tissues after definite culture duration. Five hidden Markov models (HMMs) were derived for five test culture conditions, i.e. pH of liquid growth medium, volume of medium per culture vessel, sucrose concentration (%w/v) in growth medium, nitrate concentration (g/l) in the medium and finally the density of initial inoculum (g fresh weight) per culture vessel and their corresponding fresh weight biomass. The artificial neural network (ANN) model was represented as the function of these five Markov models, and the overall simulation of fresh weight biomass was done with this combinatorial ANN-HMM. The empirical results of Rauwolfia serpentina hairy roots were taken as model and compared with simulated results obtained from pure ANN and ANN-HMMs. The stochastic testing and Cronbach's α-value of pure and combinatorial model revealed more internal consistency and skewed character (0.4635) in histogram of ANN-HMM compared to pure ANN (0.3804). The simulated results for optimal conditions of maximum fresh weight production obtained from ANN-HMM and ANN model closely resemble the experimentally optimized culture conditions based on which highest fresh weight was obtained. However, only 2.99% deviation from the experimental values could be observed in the values obtained from combinatorial model when compared to the pure ANN model (5.44%). This comparison showed 45% better potential of combinatorial model for the prediction of optimal culture conditions for the best growth of hairy root cultures.

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA.

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.

Effects of ozone exposure on the xerophilic fungus, Eurotium amstelodami IS-SAB-01, isolated from naan bread

Xerophilic moulds cause contamination and spoilage of low moisture foods. This study examined the effect of ozone fumigation on growth of a Eurotium species isolated from naan bread. Two ozone treatments were used — a low-level long-term exposure (0.4μmol/mol for 21days) and high-level short-term exposure (300μmol/mol for 5 to 120min). For the low level exposure the combination of different media sucrose concentrations (0, 5, 10 and 20% w/v) with ozone treatment was also assessed. The growth of the isolate was found to be sensitive to low-level ozone fumigation depending on the media sucrose concentration and duration of the exposure. Low-level ozone exposure significantly (p<0.05) reduced the number of asexual spores formed in media with no added sucrose, an effect not observed in media with higher sucrose levels. Electron microscope observations of colonies indicated that ozone exposed cultures produced lower numbers of cleistothecia. High-level ozone exposure for short durations reduced spore viability although 100% reduction in viability was achieved only after 120min exposure. This work demonstrates that ozone may be used to reduce spore production in Eurotium but that the ozone effect can be mediated by sucrose levels in the growth medium.