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PROJECTS

RUPP aims to improve research activities under 5 supporting funds (1) Investment project of RUPP, (2) Bilateral of RUPP-SIDA project (3) HEIP-RUPP project (4) National Partnership project (5) International part. Bio-Engineering department has obtained some projects as follow:

  1. RUPP project: 8,000 USD (1918-2019)
  2. RUPP-SIDA project: 1,496,550.15 USD (2019-2023)
  3. HEIP-RUPP project: 1,351,636.56 USD (2021-2024)
  4. International partnership project: 25,000 USD (2019-2020)

There are 4 research projects currently conducted at Bio-Engineering department

Growth optimization of Haematococcus pluvialis for astaxanthin and algal powder supplements (Dr. Hangsak Huy)

Introduction: Microalgae accumulate large quantities of intracellular proteins and numerous secondary metabolites and represent as a promising source of renewable nutrition (1). Among the commercial microalgal products, astaxanthin is known as a super anti-oxidant and health beneficial compound (2). Haematococcus pluvialis is the most important microalga that richly synthesizes astaxanthin. However, only <1% of natural astaxanthin from H. pluvialis compared to its synthetic counterpart (>95%) are commercialized (2). It may be due to the low productivity and biomass yields that are influenced by the multiple factors and remain to be optimized. Therefore, this research aims to optimize the growth condition and enhance productivity of astaxanthin in H. pluvialis.

The specific objectives of this research are:

  1. To isolate and identify the novel and close-related microalgal strains to H. pluvialis from various freshwater bodies and to characterize the composition of cellular contents and fatty acids. The novel or close-related species might provide an optional source for astaxanthin and crude products. The bacteria that are associated with the microalgal culture will be also identified at genetic level and verified their growth influence on the microalgal growth and production.

  2. To optimize the multiple factors that critically influence the growth of microalgae and astaxanthin accumulation including cultural media, carbon (C) to nitrogen (N) sources and C/N ratios, micronutrient supplements, light irradiation, temperature, pH, or nitrogen limitation. In addition, various plant hormones will also be tested for the synthesis of astaxanthin by analyzing the expression of genes involving the astaxanthin synthetic pathway.

  3. To chemically mutate the microalgal strains and select the best clone that favor to grow in a stress induction and accumulate a higher astaxanthin levels.

  4. To cultivate the best strain or clone in a close bioreactor and small scale of indoor open-pool system. The crude biomass and astaxanthin will be extracted, analyzed and subjected for the test of their anti-oxidative activities using mammalian cell line, mouse embryonic fibroblast cells, or oxidative test kits, and other biochemical tests.

  5. To set up a large-scale cultivation and harvest system for production and commercialization at Sokkimmanima Group Co. Ltd. The finding in this research will be transferred to the partner company and further develop and innovate with the assisting of the research team and/or future collaborators.

Hypothesis: Haematococcus pluvialis shows variables in astaxanthin synthesis that range from 2 to 5% in aplanospore cell state depending on the environmental factors, nutritional stress, derivatives and plant hormones (2). To increase its concentration, various investigations are necessary. Therefore, we hypothesize that the growth of microalgae and enhancement of astaxanthin production can be achieved through the cultural optimization and genetic manipulation.

To achieve these hypotheses, several specific objectives shall be implemented as the following. 1) To isolate, identify and characterize the novel microalgae, closed-related strains to or sub-strains of H. pluvialis and compare their cellular contents with the commercial strain, H. pluvialis UTEX 2505. 2) To optimize the growth condition including cultural media, micronutrient supplement, carbon-to-nitrogen sources and ratios, light irradiation, temperature, pH, or nitrogen limitation. 3) To analyze the impact on the algal growth and astaxanthin synthesis caused by the microalgal associated-bacteria and plant hormones. 4) To chemically induce mutation in some strains and selected the best grown and stress tolerated microalgae that barring higher astaxanthin content.  

Methodology: Multiple methodologies will be conducted. First, to collect microalgae from the freshwater sample, identify their morphologies and genetics using the microscopic and 18S rDNA sequencing. The associated bacteria will be confirmed by 16S rRNA sequencing. The novel strains confirmed by sequencing will be further characterized. The growth and carotenoid accumulation in the isolates will be analyzed by cell counting and pigment reading. Second, to optimize the best growing medium, micronutrient supplements, C and N sources, C/N ratios, N limitation, and environmental conditions for the growth and carotenoid accumulation in the standard strain UTEX 2505 (3). The growth and carotenoids production from the isolate, UTEX 2505, and induced-mutant microalgae will be compared. The effect of associated-bacteria and several plant hormones such as abscisic acid, jasmonic acid, methyl jasmonate or growth regulators like gibberellic acid, salicylic acid or brassinosteroid-2,4-epibrassinolide on astaxanthin accumulation (2) will also be tested using real time (RT) qPCR amplification, cell counting method, and pigment analysis. Third, to induce the mutation in H. pluvialis using the chemical mutagen, N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (4, 5) and identify the best growth resistance under unfavorable growth conditions. Fourth, to extract and purify astaxanthin from the crude extract using the solvent base-extraction. The structure of astaxanthin will be analyzed by using NMR and HPLC. Fifth, compare the antioxidative activity of astaxanthin extracted from this study with the commercial counterpart using mammalian cell lines and anti-oxidative test kit. The expression of several antioxidant genes will also be confirmed by RT-qPCR amplification.

Expected Results: We expect to isolate various microalgal species that potentially produce astaxanthin and other valuable sources of nutrition. We also expect to identify novel species of bacteria that grow associated with microalgae and explore their influence on microalgal cultivation. The optimal growth condition, mutagenesis, and astaxanthin synthesis that will be discovered in this study will play an important protocol and fundamental knowledge toward the future development for large-scale production. Finally, these results will be published in the local or international journal. 

Analyzing bioactive compounds of marine algae in developing of moisturizing, hypermimentation, and anti-aging cosmeceutical cream (Dr. Solida Long)

Introduction: The increasing of toiletry products in Cambodia is notable. Cambodian people tend to use the chemical mercury added cream for skin-whitening and block the formation of melanin [1] leading to kidney damage and neurological toxicity and skin disorders. Tones of fake cosmetics costed million dollars for Cambodian users [2]. Marine algae have been reported for their benefits for diverse skin disorders including pigmentation, skin aging, atopic dermatitis and skin carcinogenesis [3]. Marine algae along the coastal area of Cambodia have shown less interest in these applications. This research project will target to produce a cream/lotion with sun-blocking, antioxidant, and moisturizing functions. The main objectives is to extract and characterization of bioactive compounds from brown, red and green algae and produce a formulation of cream products potentially commercialized in Cambodia. The specific objectives of this research are:

  1. To extract and purify small organic molecules including phenolic and non-phenolic compounds from brown, red, and green algae for a baseline analysis of bioactive compounds from marine sources.

  2. To identify the structure of the extracted bioactive compounds using physical and chemical methods and evaluate their anti-aging properties (antioxidant, anti-melanogenesis, anti-aging, photo protective properties) in order to obtain a lead compound and establish structure activity relationships.

  3. To determine the formulation of cosmetical products-face and body cream/lotions, potential commercial products, in order to create a brand name of RUPP cosmetic products.

  4. To evaluate anti-aging, moisturizing, and anti-melanogenic activity in vivo study with collaboration of beauty Clinique.

Hypothesis: The selective algae would present huge number of saccharides and phenolic compounds with stress oxidation and photoprotective properties. Compounds of interest would be known and/or include new structures. Two types of emulsions creams/lotions would be successfully formulated with the lead compound to possess wide range of antioxidant, anti-melanogenesis, moisturizing effects both in vitro and in vivo test which is potentially patentable.

Methodology: Different polarity solvents will be used for extraction and further purification using chromatography methods. Structure elucidation will include NMR spectroscopy and HPLC method will allow quantitative studies. Antioxidation properties will be conducted by DPPH, metal, hydroxy radical, and reducing power assays. UVA-UVB protection, anti-aging, anti-melanogenesis properties will be examined on HaCat and high expressing melanin cell lines. Cells viabilities will be examined using MTT assay. Type-1 pro-collagen and melanin contents, and expression of MMP-1 will be evaluated and confirmed by western blot assay.Tyrosinase, elastase assays also be included. Several types of day creams/lotions will be formulated by adding to the base formula of the algae ‘extracts and/or isolated compound. The most active compound/fraction will be selected in development process. Cream characteristic like rheology, chemical and physical stability, oxidation stability, and phase separation stability, pH, color, smoothness, spread ability, conductivity will be also determined. Volunteers will be selected and tested of final products for 60 days. The skin characteristic-epidermal water loss, capacitance, firmness, sebum, and wrinkle will be evaluated by using skin testing machine. The statistical analysis will be performed using ANNOVA analysis.

Expected results: We expect that the extraction and purifications will render significant amount of bioactive compounds -carbohydrates and phenolic compounds with high anti-aging, anti-pigmentation, antioxidant, and anti-UV radiation with potential for skin application. We strongly expected that the cream/lotion will show excellent properties toward skin care problems leading to issue a patent and potentially commercialized which may create a whole new algae farming and related cosmetic industries in Cambodia. We expect to produce 3 masters, 6 undergraduate students under this project. We will become the baseline research group for cosmeceutical products in RUPP and Cambodia.

Development of probiotic fermented vegetables in Cambodia and their effect on immunue system in vitro (Ms. Huoy Laingsun)

Introduction: Lactic acid bacteria (LAB) have been well documented as a good source for variety of fermented foods due to their ability to produce many active compounds as antimicrobial and as inducer for many white blood cells in epithelial of digestion system (Rhee et al., 2011; Arihara et al., 1996; Barefoot et al., 1983). The overall objective of this research aims to isolate and characterized good potential LAB from difference kind of fermented vegetable from urban local market in Cambodia. In vitro testing will be conducted on specific macrophage cell line to evaluate their role in immune system. The compromise strains which show high ability as antimicrobial and immune modulator will be kept to use as a starter culture to adapt from small scale to large scale industry production. This mean that after the complete project, our team will develop at least one best fermented formula, the technique to store fermented food product for longer shelf life, and these formulas maybe use by our industrial partner for producing new product that fit with both local and maybe international market. In recent years, LAB have attracted significant attention because of their generally recognized as safe (GRAS) status in food production industry. This research project aims to screen for significant LAB stains which have antimicrobial activity and immunological inducer properties to use as culture start up in large scale with the following specific objectives:

  1. To characterize LAB from local fermented vegetable and startup culture growth conditions by using culture technique to isolate the LAB strain from fermented food and characterize strains by biochemical test.

  2. To determine the inhibitory compounds such as bacteriocin and hydrogen peroxide (H2O2) production for stimulating effect on macrophage RAW 264.7 cells for TNF‐α, IL‐10, and nitrogen oxide (NO) production.

  3. To set up fermented vegetable formula and develop the shelf-life quality of the final product.

Best fermented food formula will be shared to private sector partner for production development from small scale to large scale to meet the market requirement

Hypothesis: In our research we hypothesized that:

  1. Local Cambodian fermented vegetable would contain many useful LAB strains that can be potential strains for apply as starter culture for future fermented food processing in industrial level.

  2. LABs isolated would also provide the antimicrobial compound (such as bacteriocin and hydrogen peroxide) that can be used to stimulate RAW 264.7 cell line to produce TNF‐α, IL‐10, and nitrogen oxide (NO).

  3. From survey study on tradition fermented formula and the application study of LABs in fermented food, Final unique fermented food formula will be set up with the optimum shelf-life quality for final fermented food product.

Methodology:

  1. Isolation and characterization: LAB strains will be isolated from various fermented vegetable using culture method on Man, Rogosa and Sharp (MRS) agar and M17 agar, and then confirmed by gram stain method and biochemical test kit API 50 CHL.

  2. Agar diffusion bioassay will be used for screening the bacteriocin production and Lactobacillus sakei and Listeria innocua will be used as indicator organisms. Then the production of H2O2 from LAB will be determined using the colorimetric method.

  3. Total isolated LAB strains will be screened for activation of macrophage cell line for TNF‐α, IL‐10, and nitrogen oxide (NO) production by ELISA technique and Griess reaction; respectively. In brief, macrophage RAW 264.7, one of model cell line for the study of the immune system in vitro, will be cultured and stimulated by the LAB that have been characterized in (step 1) to see whether, Lipoteichoic acid (LTA), an immune stimulatory protein found on the cell wall LAB, could induce the production TNF‐α, IL‐10, and nitrogen oxide (NO) production. To determine the TNF‐α and IL‐10, the supernatant from culture will be collected and tested for the presence of TNF‐α and IL‐10 by using the specific antibodies for these 2 substances by ELISA reader. To determine the NO production, the supernatant of the stimulation culture will be test by using Griess reagent kit. The absorbance of the mixture was measured at 540 nm on an automated EL800 plate reader

  4. LAB with antimicrobial and immunological stimulation criteria will be test for optimum growth range of pH, salt bile, and temperature to clarify that those LABs still have function during the production scale up. Shortly, the isolated LAB will be inoculated to medium culture with pH range of 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0, for the determination of the pH tolerant. Similarly, 0.1%,- 0.5% bile salts (w/v) will be supplemented with MRS broth for the investigation of bile salt concentration. Temperature 20, 37 and 44 °C were set up for incubation of LAB, to investigate whether LAB still function well when go through the digestive tract of human being.

  5. The standard formula for industry production will be conducted base on the preference of the tastiest healthy individual. Two target groups of people; 30 individuals with fermented food as their favorite and another 30 people who do not really like the fermented food will include in this study for the sensory test of the developed product in order to get the data for the taste analysis whether it is acceptable. Once the product has been developed, the microbiological test will be done for the safety quality of the product such as Culturable microorganisms (22 0C and 370C), Total Coliforms, Escherichia coli, Intestinal Enterococci, Spore of sulfite reducing anaerobes and physico-chemical tested like Turbidity, pH, Chloride, Ammonia, Nitrite, Nitrate, Hardness, and Iron will be conducted for measuring the final products shelf-life quality. In this step, the safety concern is taken in account before lunch the product to the customer. SPSS program will be used for the analysis in this study.

Expected outcome: We highly expect that via this project, we could isolate and characterize potential LAB from many vegetables fermented food to use as a starter for traditional Cambodian fermented food. Moreover, through this significant project, we hope to establish acceptable formula to transform the production of tradition fermented vegetable from family scale to proper packaging that meet the standard safety for the food level. This research outcome will also serve as literature reference for young researcher to develop their future research and cooperation with private company will be happened for the bring the final products to the local or oversea market in near future. Lastly, 1 article will be published in the peer review journal.

Watermelon variety improvement through conventional breeding and tissue culture (Dr. Phat Phanna)

Introduction: Despite having favorable conditions for the year-round production of watermelon and good demand in the world market, less efforts have been made to breed and produce high quality watermelon in Cambodia, especially the high cost seedless watermelon resulting from a crossing of tetraploid and diploid. This project intends to produce OP varieties of diploid and tetraploid watermelon with improved traits such as, high yield, desired rind colors, excellent flesh colors, and high soluble solids for commercial production in the high end market. The improved seeds from this project will be registered and disseminated to collaborative partners and watermelon growers for commercial production with a cultivating guide book. The success of this activity will lead to new era of watermelon seed industry in Cambodia and future seed supply in nationwide and exporting produces to the international market with the promise of premium quality and prices.

Because horticultural crops could contribute substantially to poverty alleviation, Royal Government of Cambodia has set a clear strategy on increasing productivity, diversification and commercialization of horticultural produces (MAFF, 2017). Achieving this strategy requires advanced horticultural research focusing on variety improvement, commercial seed production and safe production systems. Watermelon (Citrulus lanatus) is among horticultural crops widely grown in Cambodia in both uplands and lowlands as a cash crop and can be intercropped or grown before or after rain-fed lowland rice and suitable for incorporating in agroforestry. Up to now, watermelon varietal improvement in Cambodia have been initiated with two released varieties by Cambodian Agricultural Research and Development Institute (CARDI) in 2001 and ended in 2006, while more than 1200 varieties which are varied with shape and color have been grown in many regions of the world. And the two red flesh varieties have not been well adopted by the farmers as the markets preferred introduced hybrids with high yield, popular flesh color with high sugar content. This project aim to produce new diploid and tetraploid varieties with high yield and quality such as, high sugar content, good shelf life, excellent flesh colors (yellow and red) and good shapes and register as National New Varieties Release, and provide seeds to forest community, industrial partners for commercial production. The specific objectives of this sub-project are:

  1. To collect and characterize indigenous and introduced varieties collected for identification of genetic resources associated with beneficial traits for use in breeding programs

  2. To improve watermelon varieties through pedigree breeding for selection of high yield and high quality OP varieties and new parents for seeded hybrids

  3. To optimize tissue culture conditions using aqueous colchicine at different concentration levels for optimizing tetraploid induction, a prerequisite to the production of seedless hybrids, and screening for improved varieties according to somaclonal variations

  4. To share the outputs of the 5 OP varieties with forest community and industrial partner and develop diploid and triploid hybrids

Hypothesis: Within a wide collection of both indigenous and introduced varieties, the high quality and yield of introduced varieties and well adaptability of wild/indigenous varieties collected will be substantial for new varietal development. Five new open pollinated varieties with traits of interests such as, excellent flesh and rind color, long shelf life, high soluble solids and high yield would successfully produce through pedigree selection breeding and ten lines of tetraploids will produced through plant tissue culture with/without application of colchicine, a chemical agent induced chromosome doubling.

Methodology: 11 provinces include Kandal, Kampong cham, Kratie, Stoeng treng, Mondulkiri, Ratanakiri, Preah Vihear, Kampongthom, Siem Reap, Battambang, and Banteay Meanchey will be assigned for seed collections of both indigenous and introduced varieties. The examination of distinctness, uniformity and stability and production of harmonized variety descriptions according to UPOV guidelines will be conducted. Varieties with potential agronomic traits such as, high yield, long shelf life, high soluble solids with yellow, orange, or red flesh color, or adaptable to Cambodian climate will be used in pedigree selection breeding, whereby two parents are crossed to produce an F1 which later are repeated self-pollinating or sib mating for selection of the best line from generation to generation which ultimately at the advanced inbreeding, the best lines (F6/F7) are tested for potential release as new OP varieties or new parents for hybrids. Measurements of fruit yield and quality will be fruit weight, length, and diameter, hollowheart, rind pattern, flesh color and soluble solids. Uniform and homogenous varieties with high yield and quality according to the first screening and F4-F6 derived from pedigree breeding will be germinated in in vitro for excising of cotyledon for culturing on MS medium supplemented with 1mg/l BAP and 0.1mg/l IAA to initiate adventitious calli and treated without/with aqueous colchicine at different levels (0, 0.1%, 0.2%, 0.5%) to induce somaclonal variants showing superior or tetraploid traits. Morphological characteristics and number of chloroplasts in stomata will be used as indicators for discrimination between diploids and tetraploids. Diploid somaclonal variants, F5 and F6 generation derived from pedigree breeding which become uniform and homogeneous and show superior traits will be further evaluated in trials and do seed increase, register as new open pollinated varieties and disseminated to collaborative partners, whereas tetraploids will be proposed for producing triploid hybrids for production of seedless watermelon. The statistical analysis will be performed using analysis of variances (ANOVA).

Expected results: We expect to produce at least 5 open pollinated varieties with high yield, long shelf life, high soluble solids with yellow, orange, or red flesh color and adaptable to Cambodian climate, which potentially for registration as National New Variety Release and disseminate to forestry community and industrial partners. We expect to use tetraploids and diploids from this project for producing diploid and triploid hybrids which are premium quality with industrial partner and issue patents for commercialization. We expect to produce three graduate thesis, 4 undergraduate, 1 article in international peer-reviewed journal and 2 articles in national peer-reviewed journal, and one watermelon breeding book.

Contact Info

Department of Bio-Engineering

www.rupp.edu.kh
Tel: (+855)77 23 5040/78 78 4154
103 STEM Building, Campus I, Russian Confederation Bolvd., Toul Kok, Phnom Penh, Cambodia

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