Progress 06/15/14 to 02/14/17
Outputs
Target Audience:Introduction The goal of this Phase I SBIR proposal was to demonstrate the feasibility of developing all natural surfactants from non-food plants to replace many of today's potentially harmful synthetic chemical surfactants for both industrial and consumer applications. Current ongoing research has begun to reveal that the toxic chemical surfactants used for cleansing, household, personal care, and other industrial applications including oil cleanup not only affect sea inhabitants and the plant population, but they may also eventually affect the human food chain. Therefore, there is a serious need for the development of biocompatible benign surfactants to replace the toxic chemical surfactants. The proposed research aims to use renewable bio-based materials to derive environmentally benign, non-toxic, healthy actives, which will be proven to be ideal candidates for various surfactants applications, in addition to helping in reducing the synthetic chemical pollution. NSS plant derived surfactants have the potential to be used as benign substitute for many surfactants related applications. The surfactants discovered by NSS have been demonstrated to be much more effective than today's commercial surfactants in preliminary studies discussed below and are expected to be cost competitive. Results For Phase I, we have focused on the first of the four extracts with the strongest surface-active properties based on our preliminary studies. The soluble active components from the plant materials were Soxhlet-extracted using a methanol and water mixture (80:20). The solvent ratio was fixed based on preliminary research conducted by NSS. The solvent volume was reduced under low pressure at 50oC and the remaining solution was freeze-dried. The resultant dried powder was used for all studies. The recovered solvents has been recycled for the extraction purpose. Objective 1. Separation of surfactants from the most promising plant extracts Task 1. (1.i & 1.ii) Surfactants will be isolated from the most promising plant extract, (i) at different pH levels keeping the salt concentration constant and (ii) at different salt concentrations keeping the pH constant. Key Observations: · 70-74% surfactant separation · More active surfactants can be separated from the less active surfactants and non-surfactants in room temperature, in a neutral pH range and in the absence of any added salt · Separated surfactants have strong foaming properties and are better than commercial foaming agents · Foaming property of the surfactants is higher in pH 7 and pH 10 than pH 4 Objective 2. Study of the surface active properties of the isolated surfactants Task 2. (2.i, 2.ii & 2.iii) Surface active properties of the isolated surfactants will be studied (i) at different pH levels keeping the temperature and salt concentration constant, (ii) at different temperatures keeping pH and salt concentration constant, and (iii) at different salt concentrations keeping pH and temperature constant Key Observations: · It was found that the most surface active compounds come out in the first three fractionations. Hence, the surfactants collected in the first three stages were combined and was used for Toxicity (Objective 4) and Temperature effect on surface tension studies · pH 7.0 without any added salt found to be the right condition to separate the most surface active compounds from the non active compounds. The separation was observed to be done successfully in room temperature · It was found that the surfactants separated in the presence of salt have the similar behavior to that of the surfactants separated in the absence of added salt · It was observed that with increase in temperature from 25-60oC, the surface tension values were decreased by 4-5mN/m, which was expected · Addition of salt increases the surface tension values Objective 3. Exploration of surface active properties of the source extract under various environmental conditions Task 3. (3.i, 3.ii & 3.iii) Surface active properties of the first extract will be conducted (i) at different pH levels keeping temperature and salt concentration constant, (ii) at different temperatures keeping pH and salt concentration constant, and (iii) at different salt concentrations keeping pH and temperature constant. Key Observations: · It was found that soluble inactive do not have any significant impact in the surface activity of surfactants · It was observed that with increase in temperature from 25-60oC, the surface tension value was decreased by 4-5mN/m, which is expected · Surface tension values increased in the presence of salt Objective 4. Analysis of the toxicity of the isolated surfactants using both bacterial as well as mammalian cells Task 4.1 (4.1.i &4.1.ii) Antimicrobial assays will be conducted (i) at various surfactant concentrations keeping interaction time constant (ii) for different interaction periods, keeping the surfactant concentration constant. Key Observations: · Isolated surfactants (0.5%) did not show observable toxic impact in the growth of Gram negative "Escherichia coli" and Gram positive "Bacillus polymyxa" bacterial cells. · Mammalian cells (CHO-K1) were found to grow well in the presence of 0.5% surfactants (combined surfactants that was extracted from stage 1-3) · It was further demonstrated that the surfactant pre-exposed (previously grown in 0.5% surfactant for seven days) CHO-K1 cells growth pattern was similar to that of CHO-K1 normal cells, hence it was concluded that there is no noticeable toxic impact of surfactants in the cell viability of CHO-K1 mammalian cells Summary and Conclusions 1. We have successfully separated the more surface active compounds from the less surface active compounds and non-surface active compounds 2. Surface active compounds isolated here are found to posses strong foaming properties and are better than the available commercial foaming agents 3. Isolated surfactants are found to posses strong surface active properties and are better than commercial surfactants 4. More active surfactants could be isolated efficiently from the less active surfactants and non-active compounds in room temperature, in a neutral pH range and in absence of any added salt 4. Non-surface active compound do not interfere with the surface active properties of the surface active compounds and hence could be used as it is for many applications where color of the surfactants is not important 5. The isolated surfactants are found to be non-toxic (no noticeable toxicity was observed) to bacterial as well as mammalian cells Hence, it can be concluded that NSS has successfully demonstrated the feasibility of isolating benign natural surfactants for personal care, firefighting, cleansing, household, oil industries and other industrial applications including oil cleanup and recovery by successfully completing all the proposed four objectives of our Phase I research. Acknowledgements The financial support by USDA under SBIR Phase I grant to establish the feasibility is thankfully acknowledged. Changes/Problems:Current ongoing research has begun to reveal that the toxic chemical surfactants used for cleansing, household, personal care, and other industrial applications including oil cleanup not only affect sea inhabitants and the plant population, but they may also eventually affect the human food chain. Therefore, there is a serious need for the development of biocompatible benign surfactants to replace the toxic chemical surfactants. The proposed research aims to use renewable bio-based materials to derive environmentally benign, non-toxic, healthy actives, which will be proven to be ideal candidates for various surfactants applications, in addition to helping in reducing the synthetic chemical pollution. NSS plant derived surfactants have the potential to be used as benign substitute for many surfactants related applications. The surfactants discovered by NSS have been demonstrated to be much more effective than today's commercial surfactants in preliminary studies discussed below and are expected to be cost competitive. ? What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Prepare for commercialization of our products for different applications, especially in personal care, firefighting, cleansing, household, oil industries and other industrial applications including oil cleanup and recovery ?areas
Impacts
What was accomplished under these goals?
Summary and Conclusions 1. We have successfully separated the more surface active compounds from the less surface active compounds and non-surface active compounds 2. Surface active compounds isolated here are found to posses strong foaming properties and are better than the available commercial foaming agents 3. Isolated surfactants are found to posses strong surface active properties and are better than commercial surfactants 4. More active surfactants could be isolated efficiently from the less active surfactants and non-active compounds in room temperature, in a neutral pH range and in absence of any added salt 4. Non-surface active compound do not interfere with the surface active properties of the surface active compounds and hence could be used as it is for many applications where color of the surfactants is not important 5. The isolated surfactants are found to be non-toxic (no noticeable toxicity was observed) to bacterial as well as mammalian cells Hence, it can be concluded that NSS has successfully demonstrated the feasibility of isolating benign natural surfactants for personal care, firefighting, cleansing, household, oil industries and other industrial applications including oil cleanup and recovery by successfully completing all the proposed four objectives of our Phase I research. ?
Publications
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Progress 06/15/14 to 06/14/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Currently we are Working on the Following Objectives: Objective 4. Analysis of the toxicity of the isolated surfactants using both bacterial as well as mammalian cells Task 4.1 (4.1.i &4.1.ii) Antimicrobial assays will be conducted (i) at various surfactant concentrations keeping interaction time constant (ii) for different interaction periods, keeping the surfactant concentration constant. The antibacterial activity of the isolated surfactants will be studied against a panel of both Gram-positive and Gram-negative bacteria using the microbroth dilution technique. A known amount of isolated freeze dried phyto-surfactants will be dissolved in buffer solutions and the solutions will be passed through a sterile membrane filter (0.2 µm). Gentamicin will be used as a positive antibiotic control, and DMSO (dimethylsulfoxide) will be used as a negative control. Dilution of the compound solutions will be prepared in cation-adjusted Muller Hinton Broth (CAMHB) in 96-well plates. Task 4.1.i at various surfactant concentrations keeping interaction time constant 10ml of 1% w/w phyto-surfactants in CAMHB will be added to the first chamber of a 96-well cluster plate and will then be made 10 times serial dilutions. Bacteria at a concentration of approximately 1.5´107 colony-forming units (CFUs) ml-1 will be added to each well. The plates will then be incubated at 37oC for 24 h, and the final minimum inhibitory concentration (MIC) will be determined as the lowest concentration without turbidity. The compounds will be considered exhibiting antibacterial activity if the MIC of the samples is £ 19.5 mg.ml-1. The number of surviving cells will be plotted as a function of surfactant concentrations Task 4.1.ii. for different interaction periods, keeping the surfactant concentration constant. The concentration at which 50% bacterial inactivity is observed as determined by the above test will be used for this study. If no toxicity is evident the maximum concentration tested will be used. A 12-well cluster plate will be used for this study. Bacteria at a concentration of approximately 1.5´107 colony-forming units (CFUs) ml-1 will be added to each well. The cells will be incubated at 37°C for various time periods, for example, 12h, 24h, and 48 h. The turbidity of each well will be measured at the end of the stipulated time. The number of surviving cells will be plotted as a function of time. The results will be compared with the experimental results of a commercially available surfactant compound, for example SDS. All the experiments will be conducted in triplicate. Task 4.2 (4.2.i & 4.2.ii) Toxicity tests against mammalian cells will be performed: (i) at various surfactant concentrations keeping interaction time constant (ii) for different interaction periods keeping the surfactant concentration constant. The Chinese hamster cell line CHO-K1 cells will be used for this purpose. Cells will be grown in Ham's F12 medium supplemented with 10% (v/v) fetal-calf serum (Gibco) that has been heat inactivated for 30 minutes at 56oC. CHO-K1 cells will initially be allowed to grow near confluence in 10 ml of F12 medium in 100mm diameter glass dishes. The cells will then be trypsinized and re-suspended in fresh medium and will be used for the toxicity studies. Task 4.2.i at various surfactant concentrations keeping interaction time constant CHO-K1 cell survival will be evaluated by colony formation. Two million cells will be seeded into 100-mm dishes (3 replicates for each treated group) and cultured at 37?C for 6 h, which will allow the cells to attach to the dishes. The medium will then be changed to serum-free medium containing various concentrations of NSS surfactants as well as no surfactants. After 4 hours of cell exposure to NSS surfactants, the cells will be trypsinized, counted, and 200 cells from each surfactant concentration will be inoculated into individual 6-well dishes in regular medium and made 10 times serial dilutions. One week later, the colonies will be fixed in methanol, stained with crystal violet and counted. Cell survival will be expressed as a percentage of the negative control. All exposures will be carried out in triplicate. Task 4.2.ii for different interaction periods keeping the surfactant concentration constant Two million CHO-K1 cells will be inoculated in 100-mm dishes containing 10 ml of F12 medium with 10% FBS and incubated for 6 h. The medium will then be replaced with serum-free medium and a concentration of NSS surfactants that results in 50% killing as determined by the previous test. If no toxicity is evident the maximum concentration tested will be used. Each experiment will include a negative control (culture medium) and a positive control (ethyl methane sulfonate, a known mutagen). The cells will be exposed to surfactant solutions for various time periods, for example, 1h, 4h, 12h and 24 h. Following exposure, the cells will be trypsinized and tested for cell survival by the clonogenic assay following the method described in Task 4.2.i.. All exposures will be carried out in triplicate. Objective 5. Analyzethe results and write a detail final report
Impacts
What was accomplished under these goals?
Brief Update Request for 1 year No Cost Extension We are requesting a no cost extension of this SBIR project for one year. Because of PI's health problem we are still working on the proposed Objectives. The one year extension will give us time to complete the proposed work and properly analyze the data. Work Completed to Date Extraction of the soluble actives The soluble active components from the Centella asiatica plant materials have been Soxhlet-extracted using a methanol and water mixture (80:20). The solvent ratio was fixed based on preliminary research conducted by NSS. The solvent volume was reduced under low pressure at 50°C, and the remaining solution was freeze-dried and weight was taken. The resultant dried powders are being used to perform the proposed studies. The recovered solvents are reused for the extraction purpose. Objective 1. Separation of surfactants from the most promising plant extracts Task 1. (1.i & 1.ii) Surfactants will be isolated from the most promising plant extract, (i) at different pH levels keeping the salt concentration constant and (ii) at different salt concentrations keeping the pH constant. A foam fractionators (Figure 1) is used for this study. The air is flown through the fractionators at a fixed rate. The foamates were collected in three stages. In first stage, the foamate was collected for initial 15 minutes. In the second stage, the foamate was collected from 15-30 minutes and in the last stage the foamate was collected from 30-45 minutes. The collected foams were frozen and then freeze-dried. The weight of the freeze dried samples was taken and the dried samples were preserved for the experiments. The collapsed foamate and the bulk solution were analyzed for surfactant concentration. The column was thoroughly cleaned with distilled water before starting the next experiment. All the experiments were performed in triplicate. Task 1.i. The experiments were conducted at different pH levels (for example 4, 7-8, 10) keeping the salt concentration 10-2M. KNO3 was used as a salt. Task 1.ii. The experiments were conducted at different salt concentrations (for example 0, 10-2M, 10-1M) keeping the pH constant (pH 7-8). KNO3 was used as a salt. Figure 1 Foam fractionators Picture couldbe uploaded here Objective 2. Study of the surface active properties of the isolated surfactants Task 2.i. Surface active property of the isolated surfactants was measured at different pH levels, for example 4, 7-8, 10, keeping the temperature at 25oC and salt concentration at 10-2M. Task 2.ii. Surface active property of the isolated surfactants was measured at different temperatures (for example 25oC, 40oC and 60oC), keeping pH at 7-8 and salt concentration at 10-2M. Task 2.iii. Surface active property of the isolated surfactants was measured at different salt concentrations (for example 0, 10-2M, 10-1M) keeping pH 7-8 and temperature at 25oC. The micellization behavior of the isolated surfactants has been analyzed using the surface tension results. Objective 3. Exploration of surface active properties of the source extract under various environmental conditions Task 3. (3.i, 3.ii & 3.iii) Surface active properties of the first extract will be conducted (i) at different pH levels keeping temperature and salt concentration constant, (ii) at different temperatures keeping pH and salt concentration constant, and (iii) at different salt concentrations keeping pH and temperature constant. Task 2. i. Surface active property of the source extract was measured at different pH levels, for example 4, 7-8, 10, keeping the temperature at 25oC and salt concentration at 10-2M. Task 2.ii. Surface active property of the source extract was measured at different temperatures (for example 25oC, 40oC and 60oC), keeping pH at 7-8 and salt concentration at 10-2M. Task 2.iii. Surface active property of the source extract was measured at different salt concentrations (for example 0, 10-2M, 10-1M) keeping pH 7-8 and temperature at 25oC. The micellization behavior of the extract has been analyzed using the surface tension data. The impact of other soluble actives (non-surface active agents) on the surface active properties of the plant extract has been determined.
Publications
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