Добірка наукової літератури з теми "Submerged culturing"

Surface-associated multicellular assemblage is an important bacterial trait to withstand harsh environmental conditions. Bacillus subtilis is one of the most studied Gram-positive bacteria, serving as a model for the study of genetic pathways involved in the different steps of 3D biofilm formation. B. subtilis biofilm studies have mainly focused on pellicle formation at the air-liquid interface or complex macrocolonies formed on nutritive agar. However, only few studies focus on the genetic features of B. subtilis submerged biofilm formation and their link with other multicellular models at the air interface. NDmed, an undomesticated B. subtilis strain isolated from a hospital, has demonstrated the ability to produce highly structured immersed biofilms when compared to strains classically used for studying B. subtilis biofilms. In this contribution, we have conducted a multi-culturing comparison (between macrocolony, swarming, pellicle, and submerged biofilm) of B. subtilis multicellular communities using the NDmed strain and mutated derivatives for genes shown to be required for motility and biofilm formation in pellicle and macrocolony models. For the 15 mutated NDmed strains studied, all showed an altered phenotype for at least one of the different culture laboratory assays. Mutation of genes involved in matrix production (i.e., tasA, epsA-O, cap, ypqP) caused a negative impact on all biofilm phenotypes but favored swarming motility on semi-solid surfaces. Mutation of bslA, a gene coding for an amphiphilic protein, affected the stability of the pellicle at the air-liquid interface with no impact on the submerged biofilm model. Moreover, mutation of lytF, an autolysin gene required for cell separation, had a greater effect on the submerged biofilm model than that formed at aerial level, opposite to the observation for lytABC mutant. In addition, B. subtilis NDmed with sinR mutation formed wrinkled macrocolony, less than that formed by the wild type, but was unable to form neither thick pellicle nor structured submerged biofilm. The results are discussed in terms of the relevancy to determine whether genes involved in colony and pellicle formation also govern submerged biofilm formation, by regarding the specificities in each model.

Culturing airway epithelial cells with most of the apical media removed (air-liquid interface) has been shown to enhance cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl− secretory current. Thus we hypothesized that cellular oxygenation may modulate CFTR expression. We tested this notion using type I Madin-Darby canine kidney cells that endogenously express low levels of CFTR. Growing monolayers of these cells for 4 to 5 days with an air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl− current, compared with conventional (submerged) controls. Assaying for possible changes in CFTR by immunoprecipitation and immunocytochemical localization revealed that CFTR appeared as an immature 140-kDa form intracellularly in conventional cultures. In contrast, monolayers grown with an air-liquid interface possessed more CFTR protein, accompanied by increases toward the mature 170-kDa form and apical membrane staining. Culturing submerged monolayers with 95% O2 produced similar improvements in Cl− current and CFTR protein as air-liquid interface culture, while increasing Po 2 from 2.5% to 20% in air-liquid interface cultures yielded graded enhancements. Together, our data indicate that improved cellular oxygenation can increase endogenous CFTR maturation and/or trafficking.

The objective of the present study was to get better production of FPase from Bacillus aerius (MG597041) by optimizing different process parameters under submerged fermentation through the statistical approach. Optimization of physical factors of culturing medium by one factor at a time (OFAT) revealed optimum incubation time of 24 h, inoculum size of 1%, pH 5.5, and substrate concentration of 4%. Plackett-Burman design (PBD) was performed to identify the significant nutritional influence of cellulase production. Among the nine parameters screened, peptone, yeast extract, FeSO4 and K2HPO4 were found significant. CCD of significant parameters revealed maximum FPase activity (127.4 IU/mL/min) at the optimum concentration of yeast extract of 0.5 g/L, peptone of 0.5 g/L, FeSO4 of 0.2 g/L, and K2HPO4 of 0.02 g/L. ANOVA was used to analyze these results. The analysis of the results showed an F-value of 8.74 and a p-value 0.00. Maximum hydrolysis of 10% of raw Bombax ceiba seed pods using this indigenous cellulase was obtained after 24 h. Also, the study explored the potential of the obtained cellulase to be applied in denim biostoning finishing. The findings demonstrated the efficient use of the obtained enzyme in saccharification of raw Bombax ceiba seed pods, which can be of interest for production of biofuel, and in biostoning treatment of denim fabrics.

Submerged culturing of mycelium is an efficient technique used to increase biomass yields, more so when employed with naturally slow-growing species of mushrooms. This paper is concerned with optimizing nutrient broth components used in Inonotus obliquus cultures for achieving high biomass yields. We modeled the effect of seven biotechnological parameters (six broth ingredients and the initial pH of nutritive broth) on mycelial biomass and predicted an optimum broth formula using response surface methodology. An analysis of variance showed that the elaborated model is significant (F-value of 2.76 and p-value of 0.0316). We used bioreactor cultures to confirm the model’s optimum prediction and to compare these results with a general-purpose mycology medium, namely potato dextrose broth (PDB). The optimized bioreactor culture yielded 4.37 g/L (93.36% of the dry weight prediction), while the PDB bioreactor culture yielded 2.084 g/L, after 15 days of cultivation. The optimized formula was: 2.15299 g malt extract, 3.99296 g yeast extract, 11.0041 g fructose, 17.4 g soluble starch, 0.1 g MgSO4, and 0.05 g CaCl2 per liter of broth.

Phellinus linteus is known to be a medicinal mushroom which is very good to human health. They are used as medicinal mushroom in many Asian countries such as Japan, Korea, China, Thailand and Vietnam. Because of long time growth and over-havest by humans in natural conditions, they are artificially cultured in these countries. There were advances of liquid culture of mushroom such as time saving and scaleable in culture in comparison with culturing of mushroom in decaying wood. In this study, Phellinus linteus is cultured in optimum liquid medium, lyophilized, ground into fine powder and evaluated for microbiological and total of Asen and Lead to ensure the biomass product will meet safety standards for users. Results are as follows: Total of anaerobic bacteria 2,2 × 10­4 CFU/g, Coliforms< 1,0 × 101 CFU/g, Coagulase positive with Staphylococci <1,0×101 CFU/g, total amount of yeast-mold < 1,0 × 101 CFU/g, not detected of E.coli, Clostridium perfringens and Salmonella spp.. Furthermore, we did not find any Asen and Lead which are supposed to remain in our sample.

Robust Vallisneria americana was cultured for restoration purposes. Preliminary studies, with various iron treatments, were conducted to ascertain the amount of phosphorous release into the water column from sediments. There was a significant difference in the amount of phosphorous released if commercial sediment was used with a low iron amendment or without an iron amendment. The second study consisted of planting V. americana on two different sediment types while supplying half of the plants with additional CO2. Plants grown on pond sediment with additional CO2 had significantly more biomass. In the third study all plants were grown on pond sediment, and half were treated with CO2. All plants that were treated with additional CO2 had significantly more biomass than those that were aerated.

Відомим мікроорганізмом–продуцентом рибофлавіну є аскоміцет Eremothecium ashbyi, який використовується у промисловості. Крім надсинтезу рибофлавіну E. ashbyi здійснює синтез флавінаденіндинуклеотиду (ФАД). За допомогою E. ashbyi можна отримувати як кормовий рибофлавін, що використовується в якості кормової добавки для тварин, так і, при застосуванні певних методів виділення та очистки, рибофлавін медичного призначення. Одночасно з синтезом рибофлавіну E. ashbyi здійснює синтез ефірної олії, яка за ароматом та своїми властивостями ідентична ефірній олії, отриманій з пелюсток троянди. У своєму складі вона містить такі ароматичні речовини, як гераніол (69,5–84,5%), нерол, лінаноол та β-фенілетанол (12,7–27,7%). Це дає можливість розглядати E. ashbyi як перспективний продуцент ароматичних речовин, що є необхідними для парфюмерно-косметичної промисловості. Біотехнологія трояндової ефірної олії, однієї з найцінніших олій в світі, досі не розроблена. На даний момент в Україні не налагоджено виробництво рибофлавіну за допомогою біотехнології, а можливість одночасного отримання ще й ефірної олії робить тему дисертаційної роботи актуальною, своєчасною та важливою. В ході виконання роботи досліджено морфологічні та культуральні особливості штаму Eremothecium ashbyi F-340. Він відноситься до аскоміцетів, що не утворюють плодові тіла, має справжній дихотомічний розгалужений міцелій яскраво-жовтого кольору, який складається з багатоядерних клітин. Колір міцелію обумовлений присутністю рибофлавіну, який накопичується в такій кількості, що випадає у вигляді кристалів в вакуолях. Показана природна мінливість штаму. Гриб утворює пігментовані жовті та жовтогарячі колонії з високою здатністю до біосинтезу рибофлавіну, та білі колонії, з низькою. Найчастіше колонії білого кольору з’являються при відновленні музейної культури і майже не з’являються при регулярних пересівах культури та чергуванні рідких та агаризованих поживних середовищ. Досліджено умови зберігання штаму. Встановлено, що короткотривале зберігання E. ashbyi F-340 у активному стані можливе на агаризованих глюкозопептонно-дріжджовому та соєвому середовищах за температури зберігання 5°C. Довготривале зберігання культури E. ashbyi (протягом 7 місяців) можливе лише за кімнатної температури. Досліджено вплив температури на життєдіяльність міцелію Eremothecium ashbyi F-340. Нижня гранична температура для E. ashbyi становить 4°С. Верхня гранична температура дорівнює 38°С. За цієї температури ще спостерігається незначний ріст гриба, а вже при 39°С ріст міцелію не спостерігається та відновлення росту при 28°С не відбувається. Динаміка росту штаму E. ashbyi F-340 у глибинній культурі підкоряється відомим закономірностям для періодичних культур. Фаза експоненціального росту триває протягом 2 діб, потім спостерігається уповільнення росту та перехід культури у стаціонарну фазу росту, яка триває до 5 доби культивування, після чого культура переходить у фазу відмирання або автолізу. Встановлено, що під час інтенсивного росту штаму відбувається зниження рН до 5,2, а інтенсивне накопичення рибофлавіну у культуральній рідині та у біомасі пов’язане з підвищенням рН до 7,8. Найбільш інтенсивно накопичення рибофлавіну в культуральній рідині відбувається у стаціонарній фазі росту на 3–4 добу культивування та його концентрація досягає 341,6 мг/дм3. Другий етап накопичення рибофлавіну відбувається на 5-7 добу та пов'язаний з автолізом культури, вміст рибофлавіну досягає 55,22,7 мг/дм3. Рибофлавін спочатку накопичується у міцелії E.ashbyi, де досягає рівня 8,1-10,7 мг/г сухої біомаси і залишається на такому рівні протягом 4–5 доби культивування. Незважаючи на постійну підтримуючу селекцію при культивуванні штаму у лабораторних умовах протягом 3 років спостерігалося поступове значне зниження рівня накопичення рибофлавіну та відповідне збільшення рівня накопичення біомаси. З даних літератури відомо, що надсинтез рибофлавіну грибом E. ashbyi у природних умовах здійснюється як захисна реакція на дію сонячних ультрафіолетових променів. Тому нами було запропоновано здійснювати УФопромінення продуценту для підвищення синтезу рибофлавіну. Опромінення культуральної рідини продуценту призводить до збільшення біосинтезу рибофлавіну на 72–74%, опромінення водної суспензії міцелію штамупродуценту – до збільшення синтезу на 80%. Встановлено, що найбільшому виходу рибофлавіну сприяє використання посівного матеріалу у віці 3-4 діб та у кількості 1%. На наступному етапі досліджено вплив умов культивування на біосинтетичну здатність продуценту. Показано, що початковий рівень рН середовищ, призначених для отримання біомаси та рибофлавіну, має бути різним. Для отримання максимальної кількості біомаси, а також посівного матеріалу, доцільно створювати у середовищі рН на рівні 5,5–6,0, а от для максимального накопичення рибофлавіну початкове рН середовища має становити 7,5. Встановлено, що за умов аерації на качалці при 180 об/хв синтезується на 70 % більше рибофлавіну, ніж при 70 об/хв. E. ashbyi здатен рости в широкому діапазоні температур від 20 до 38С. Оптимальною температурою для максимального виходу цільового продукту є 27–29С. Досліджено вплив різних джерел карбону на накопичення біомаси та синтез рибофлавіну штамом E. ashbyi F-340. Для синтезу рибофлавіну краще підходять моносахариди (фруктоза, галактоза) та шестиатомний спирт сорбіт, а біомаса краще накопичується при наявності в середовищі фруктози, сахарози та гліцерину. Кращим джерелом нітрогену для E. ashbyi F-340 виявився дріжджовий екстракт, кількість рибофлавіну, що синтезована на середовищі з дріжджовим екстрактом на 54% більша, ніж на інших джерелах нітрогену. Однак досі не було запропоновано жодного середовища для культивування Eremothecium ashbyi, яке б містило у складі наведені вуглеводи та було досить дешевим та технологічним. Для вирішення даної проблеми запропоновано використовувати таке перспективне натуральне джерело карбону, як глюкозо-фруктозний сироп (ГФС), який виробляють з кукурудзяного крохмалю ферментативним гідролізом його до глюкози з наступною ізомеризацією частини глюкози у фруктозу та подальшим очищенням. Показано, що найбільша кількість вітаміну синтезується при використанні ГФС-10 (140 мг/дм3), що у 7 разів більше, ніж на середовищі з глюкозою, та у 3,8 разів більше, ніж на середовищі з фруктозою. З метою здійснення оптимізації поживного середовища був запланований повний факторний експеримент на двох рівнях для 3 факторів, матрицю планування доповнили «зірковими» точками та отримали ортогональний центрально-композиційний план 2-го порядку для 3-х факторного експерименту. В результаті розрахунків отримано рівняння регресії другого порядку. Статистичну значимість коефіцієнтів рівняння перевіряли за критерієм Стьюдента, адекватність отриманого рівняння за критерієм Фішера. В результаті математичної обробки експериментальних даних отримано рівняння регресії залежності концентрації рибофлавіну в культуральній рідині від концентрації ГФС-10 (m), дріжджового екстракту (w) та пептону (v): Y1= –758,483+41,029·m+9,959·w+5,777·v+0,693·m·w–0,472·m·v–3,51·w·v– –0,547·m2+5,701·v2 Аналізуючи поверхні відгуку встановлено склад модифікованого середовища: оптимальна концентрація ГФС-10 для максимального накопичення рибофлавіну становить 40 г/дм3, концентрації дріжджового екстракту та пептону у середовищі становлять відповідно 10 та 1 г/дм3. Концентрація рибофлавіну, що спостерігалася при культивуванні на модифікованому середовищі у культуральній рідині становить 350,4 мг/дм3, що у 17 раз більше, ніж на ГПД середовищі, та у 2,5 рази більше, ніж на початковому середовищі з ГФС-10. Визначення вмісту у культуральній рідини ефірної олії проводили трьохкратною екстракцією гексаном з наступним видаленням розчинника. Показаний широкий діапазон варіювання кількості ефірної олії. Найбільша кількість спостерігається на середовищі, що містить в якості джерела карбону ГФС-10 (273…453 мг/дм3). Кількість ефірної олії збільшується зі збільшенням концентрації ГФС у середовищі. Наведено технологічну схему одночасного отримання рибофлавіну та ефірної олії методом гідродистиляції з подальшим розділенням потоків виділення рибофлавіну та ефірної олії. Наукова новизна одержаних результатів: - досліджено динаміку росту, виходу біомаси, накопичення рибофлавіну та ефірної олії обраним штамом-продуцентом Eremothecium ashbyi на середовищах з різними джерелами живлення; - визначено склад та кислотність середовищ, які є сприятливими для росту штаму-продуценту в глибинній культурі; - знайдено раціональні біотехнологічні параметри для отримання максимального виходу рибофлавіну та ефірної олії: температура культивування 27-29С, початкове рН середовища 7,5, перемішування 180 об/хв; - за допомогою методів планування експерименту оптимізовано поживне середовище для накопичення рибофлавіну та ефірної олії, що складається з ГФС-10, дріжджового екстракту та пептону, та перевірена можливість одночасного отримання цих продуктів; - вперше науково обґрунтовано та створено біотехнологію отримання рибофлавіну та ефірної олії з вітчизняної відновлюваної сировини – глюкозофруктозного сиропу, що виготовляється з кукурудзи.
A well-known microorganism-producer of riboflavin is ascomycete Eremothecium ashbyi used in industry. Besides overexpression of riboflavin, E. ashbyi also performs synthesis of flavinadeninedinucleotide (FAD). Using E. ashbyi, one can obtain either forage riboflavin used as a feed additive for livestock, or, using certain isolation and purification methods, riboflavin of medical purpose. Concomitantly with riboflavin synthesis, E. ashbyi performs synthesis of essential oil, identical by its aroma and properties to essential oil derived from rose petals. It contains such aromatic substances as geraniol (69.5–84.5%), nerol, linalool, and β-phenylethanol (12.7–27.7%). This allows viewing E. ashbyi as a promising producer of aromatic substances, which are necessary for perfume and toiletry industry. Biotechnology of rose essential oil, one of the most valuable in the world, has not been developed so far. At present, manufacture of riboflavin using biotechnology is not established in Ukraine, and the potential of concomitant production of essential oil as well makes the topic of this thesis urgent, timely, and important. During the work, morphological and cultural peculiarities of the strain Eremothecium ashbyi F-340 have been investigated. It belongs to ascomycetes not generating ascocarps, has true dichotomic branched bright-yellow mycelium composed of multinucleate cells. Mycelium color is due to the presence of riboflavin, which is accumulated in such quantities that it is precipitated in vacuoles as crystals. Natural variability of the strain has been shown. The fungus forms pigmented yellow and orange colonies with high ability to riboflavin biosynthesis, and white colonies with low biosynthetic ability. Most frequently, white colonies develop upon archive culture reactivation and almost do not appear upon regular reinoculations and alterations of liquid and agar nutrient media. The strain storage conditions have been investigated. It has been established that short-term storage of E. ashbyi F-340 in the active state is possible on agar glucose-peptone-yeast and soybean media at storage temperature 5°C. Long-term storage of E. ashbyi culture (for 7 months) is possible only at room temperature. Temperature effects on viability of Eremothecium ashbyi F-340 mycelium have been investigated. The lower limit temperature for E. ashbyi is 4°С. The upper limit temperature is equal to 38°С. Minor growth of the fungus is still observed at this temperature, and at 39°С no mycelium growth is observed, and growth restoration at 28°С is not observed. Growth dynamics of E. ashbyi strain F-340 in submerged culture follows the known regularities for periodic cultures. Exponential growth phase lasts for about 2 days; after this, growth deceleration and culture switch into stationery growth phase are observed; the latter one lasts for about 5 days of culturing, after which the culture is switched into die-off or autolysis phase. It has been established that pH decrease to 5.2 occurs during intensive strain growth; intensive riboflavin accumulation in cultural fluid and biomass is associated with pH increase to 7.8. The most intensive riboflavin accumulation occurs in stationery growth phase on culturing day 3–4, and its concentration reaches 341,6 mg/dm3. The second stage of riboflavin accumulation occurs on day 5-7 and is associated with culture autolysis; riboflavin content reaches 55,22,7 mg/dm3. Riboflavin is accumulated at the beginning in E.ashbyi mycelium, where it reaches the level of 8.1-10.7 mg/g of dry biomass and remains at that level until completion of culturing. Despite continuous maintenance selection during the strain culturing under laboratory conditions for 3 years, gradual considerable decrease in riboflavin accumulation and relevant increase in biomass accumulation level has been observed. It is known from literature data that riboflavin overexpression by fungus E. ashbyi in natural conditions occurs as a defense reaction on the effect of sun ultraviolet radiation. That is why we suggested to perform UV irradiation of the producer in order to increase riboflavin synthesis. Irradiation of the producer cultural fluid results in increase of riboflavin biosynthesis by 72-74%, and irradiation of aqueous suspension of mycelium of the producer strain results in synthesis increase by 80%. It has been established that the highest riboflavin yield is achieved when the inoculum aged 3-4 days in quantity 1% is used. The effect of culturing conditions on biosynthetic ability of the producer has been investigated at the following stage. It has been shown that the initial pH level of media intended for biomass and riboflavin production has to be different. In order to obtain maximum quantities of biomass and inoculum, it is expedient to adjust the medium pH to the level 5.5–6.0, and for maximum riboflavin accumulation, initial medium рН has to be 7.5. It has been established that, under aeration conditions on a rocker at 180 rpm, 70 % more riboflavin is synthesized compared to 70 rpm. E. ashbyi is capable to growth in a wide range of temperatures from 20 to 38С. The optimal temperature for maximum target product yield is 27-29С. The effect of various carbon sources on biomass accumulation and riboflavin synthesis by E. ashbyi strain F-340 has been studied. Monosaccharides (fructose, galactose) and hexatomic alcohol sorbitol are better suitable for riboflavin synthesis, and biomass is accumulated better in the presence of fructose, sucrose, and glycerol in the medium. The best nitrogen source for E.ashbyi F-340 turned out to be yeast extract; riboflavin quantity synthesized in a medium with yeast extract was 54% more compared to other nitrogen sources. Nevertheless, no medium for Eremothecium ashbyi culturing containing the said carbohydrates and being cheap and technological enough has been suggested yet. In order to solve this problem, we have suggested to use such a promising natural carbon source as glucose-fructose syrup (GFS), manufactured from corn starch via its enzymatic hydrolysis to glucose with following isomerization of glucose parts into fructose and further purification. It has been shown that the highest vitamin quantity is synthesized with the use of GFS-10 (140 mg/dm3), which is 7 times as high as in a medium with glucose, and 3.8 times as high as in a medium with fructose. For the nutrient medium optimization, we have planned a complete factorial experiment at two levels for 3 factors; the planning matrix was supplemented with “star” points, and orthographic central composite design of second order for 3-factor experiment has been obtained. As a result of calculations, regression equation of the second order has been obtained. Statistical significance of the equation coefficients was verified according to Student’s criterion, and adequacy of the obtained equation was verified according to Fisher’s criterion. As a result of mathematical processing of experimental data, we have obtained the regression equation of relation between riboflavin concentration in cultural fluid and concentrations of GFS-10 (m), yeast extract (w) and peptone (v): Y1= –758.483+41.029·m+9.959·w+5.777·v+0.693·m·w–0.472·m·v–3.51·w·v– –0.547·m2+5.701·v2 Analyzing the response surfaces, we have established the composition of modified medium: optimal GFS-10 concentration for maximum riboflavin accumulation is 40 g/dm3, and concentrations of yeast extract and peptone in the medium are 10 and 1 g/dm3, respectively. Riboflavin concentration observed during culturing on modified medium in cultural fluid is 350.4 mg/dm3, which is 17 times higher than on GPY medium and 2.5 times higher than on initial medium with GFS- 10. Testing of essential oil content in cultural medium was performed via triple extraction with hexane with further removal of the solvent. Wide range of variation of essential oil content has been shown. The highest quantity is observed in the medium containing GFS-10 (273…420 mg/dm3) as carbon source. Essential oil quantity is increased with increase of GSF concentration in the medium. Technological flow chart for concomitant production of riboflavin and essential oil production by hydrodistillation with further separation of riboflavin and essential oil isolation flows is provided. Scientific novelty of the obtained results: - growth dynamics, biomass yield, riboflavin and essential oil accumulation by the selected producer strain Eremothecium ashbyi have been investigated in media with different nutrition sources; - composition and acid content of media, favorable for the growth of the producer strain in submerged culture, have been determined; - rational biotechnological parameters for achievement of maximum riboflavin and essential oil yield have been determined: culturing temperature 27-29С, initial medium pH 7,5, stirring 180 rpm; - nutrient medium for riboflavin and essential oil accumulation has been optimized using experiment planning methods (such medium includes GFS-10, yeast extract and peptone), and possibility of concomitant production of these products has been verified; - biotechnology for riboflavin and essential oil production from domestic renewable raw material – glucose-fructose syrup manufactured from corn – has been scientifically justified are developed for the first time.

碩士
東海大學
食品科學系
91
Antrodia camphorata is a valuable herb, and only grown in Taiwan. It can strengthen the immune system of human body. At the present time, the cultivation of fructification of A. camphorata has not been successful. The purpose of this study is by using submerged cultures of A. camphorata, to exam culture conditions for the mycelium growth and its polysaccharide formation. The results show that, the uptimaml condition in shake flask cultivations of A. camphorat mycelium and polysaccharide formation is at 28℃, with initial pH 5, at rotating speed 100 rpm and inoculum size 5% (v/v). The 500 ml Erlenmeyer flask without baffle and containing 100 ml medium, after 14 days cultivation, the maximal mycelium dry weigh is 448 mg per 100 ml, exopolysaccharide is 0.837 mg per ml. Culture conditions were adapted to cultivate A. camphorat by different fermentors, at 28℃, agitation speed 100 rpm, and aeration rate 0.5 vvm. After 14 days fermentation, the mycelium dry weight for 7 L Airlift fermentor (AL) was 7.09 g/L; for 5 L stirred tank fetmentor with Marine impeller (MST) was 5.99 g/L; and for 5 L stirred tank fetmentor with turbine disk impeller (TDS) was 5.68 g/L, in compare with 5.48 g/L of the shake flasks. After 14 day fermentation, the microbial polysaccharide formation in the TDS fermentor was low, as compared to the other two fermentors. The concentration of exopolysaccharide and endopolysaccharide in TDS was 0.763 mg/ml and 0.144 mg/ml respectively, while in MST fermentor was 0.873 mg/ml and 0.246 mg/ml; and in AL fermentor, which was the highest, was 1.016 mg/ml and 0.398 mg/ml respectively. The improvement on the TDS fermentor in mycelium growth and polysaccharide production by using MST and AL fermentor would be beneficial in reducing the production cost of the submerged cultures of A. camphorata mycelium.

碩士
東海大學
食品科學系
93
Abstract Wolfiporia cocos is an edibles fungi, it has long been used as traditional Chinese herb with curing incontinence, edema, sputum, palpitations and sleeplessness. Pharmacology studies have proven that its anti-tumor, anti-emetic, ant-inflammation, anti- nephritis, diabetes. The potential markets for W.cocos as neutraceutial and pharmaceutical products will stimulate the research for methods of its cultivation. The results indicated that in shake flask fermentation at 26ºC, 100rpm, a 5% olive oil addition in the medium is the best among different concentrations for the mycelium growth, while the 4% olive favors the exo-polysaccharide formation. In fermentor studies, the 7 liter bubble column fermentor (BCF) with 2 vvm aeration is the best for exo-polysaccharide production, and has a concentration of 0.39mg/ml in 7 days,as compared to 0.23mg/ml by 5 liter stirred tank fermentor(STF) at 2 vvm . At 1 vvm, the polysaccharide is 0.17 mg/ml and 0.21 mg/ml for BCF and STF, respectively.Lower aeration rate resulted in poor polysaccharide production which is caused by the aggregation of the mycelium which accumulated at the bottom of fermentor, and interfered the air supply. The fermentation condition affect the molecular weight (MW) of the polysaccharides greatly. MW distribution of polysaccharides, as determined by GPC, from STF 1vvm, BCF 2vvm, and BCF 0.5vvm, were in the rang of 103~108Da, and during the 7 days fermentation, the MW changed from one single distribution in early stage into two MW distribution in the late stage. STF 2vvm and BCF 0.5 vvm was in the range of 103~109Da, while the MW was unchanged throught the cultivation period.

碩士
東海大學
食品科學系
92
Abstract North-Cordyceps, also known as “Pupa-Cordyceps” is a popular and precious Chinese folk medicine. It growes from pupa with fruiting body by fungus Cordyceps militaris in nature. Due to the environmental impact, production of natural Cordyceps militaris is limited. Fermentation of C.militaris using submerged cultures to produce the mycelium and its useful components as the supply of market need is already on the way by the food and pharmaceutical industries. We found that in shake flask study, a 2% sunflower oil addition in the medium improved the mycelium growth and formation of useful components. Batch fermentation in the 7L airlift fermentor with 2vvm aeration rate resulted in 37.0mg/ml mycelium dry weight, 415μg/ml adenosine, 665μg/ml cordycepin and 2.31mg/ml polysaccharide, which is the hest as compared to 1vvm or 0.5vvm in the 11days fermentation. The airlift fermentor, in general, is better than stirred tank fermentor in regard to C.militaris cultivation. When C.militaris fermentation run by the fed-batch method, where the half of sugar substrate were added at the 3rd day of the fermentation higher cell concentration, polysaccharide, adenosine and cordycepin were found both in airlift and stirred tank fermentors as compared to batch process. However, controlling pH at 4.5 or 6.0 in the fermentor showed poor effect on mycelium growth and the formation of adenosine, cordycepin and polysaccharide as compared to uncontrolled pH where the initial fermentation started at pH 6.0. Key word : C.militaris, adenosine, cordycepin, stirred tank fermentor, airlift fermentor, fed-batch culture

碩士
東海大學
食品科學系
95
Abstract Agaricus blazei Murrill, an edible and medicinal mushroom originally grown in Brazil, was recently developed as a functional food. The metabolites of this mushroom from submerged culture were proved to have unique biological activity such as antitumor, antioxidative and immunity-stimulating capabilities. This research was aimed to study the effects of submerged-culturing conditions and different types of fermentors on the production and functional properties (molecular weight, antibacterial and antioxidative activities) of exopolysaccharide (EPS) by A. blazei. Results showed that the optimal submerged-culturing conditions were : incubation temperature of 25℃, initial pH of 5.4, culture volumes of 50ml, corn steep liquor at 0.3% (w/v), fructose at 4% (w/v), yeast extract at 0.4% (w/v), and a C/N ratio (fructose / yeast extract) of 10. The maximal yields of EPS and mycelial biomass, 0.302 and 25.2 g/l, respectively, were reached on the 14th day of incubation from shake flask culture under the optimized culturing conditions. A shorter incubation time for maximal EPS and mycelial production (7~10 vs. 14 days), greater EPS yield (3.20~6.96 vs. 0.302 g/l) and higher mycelial biomass (42.01~46.70 vs. 25.22 g/l) were obtained from cultures of fermentors as compared with those from shake flask culture. Among the three types of fermentors, the agitated fermentor showed the highest specific growth rate (μ=0.476 day-1) and the lowest EPS formation rate (Qp=0.46 g/l/day). On the contrary, the air lift fermentor demonstrated the lowest specific growth rate (μ=0.365 day-1) and the highest EPS formation rate (Qp=0.77 g/l/day). As for specific EPS yield (Yp/x), EPS yield (Yp/s) and biomass yield (Yx/s), results showed an increasing tendency of air lift fermentor > air bubble fermentor > agitated fermentor. Cultures of fermentors had higher and broader spectrum of antibacterial activies than shake flask culture. The inhibitory capability also demonstrated the same order as : air lift fermentor > air bubble fermentor > agitated fermentor. The bacterial strains susceptible to fermented cultures were : Bacillus cereus, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Listeria monocytogenes, Shigella dysenteriae and Pseudomonas aeruginosa. Among the ten strains, B. cereus was the most susceptible one. This finding could be applied to use the culture broth of A. blazei as a potential biopreservative. Same major molecular weights (2.5×105 and 3.0×103 Da) and molecular weight distribution (7.4×104~2.1×106 Da) were observed for EPS from the three batch cultures of fermentors. The EPS from culture of air lift fermentor had higher content of molecular weight of 2.5×105 Da. The antioxidative activity (DPPH scavenging effect) of the three fermented cultures was equal or higher than that of control antioxidants. Under the same concentration, the fermented culture of air lift fermentor presented the highest antioxidative activity. As far as the EPS yield, the mycelial biomass production, the formation rates of EPS and mycelial biomass, and the antibacterial and antioxidative activities of fermented cultures were concerned, all above results showed a trend of air lift fermentor > air bubble fermentor > agitated fermentor. In conclusion, using the air lift fermentor, which has lower shearing force and better convective circulation as compared with agitated and air bubble fermentors, could produce higher yield of EPS from A. blazei with better functional properties.

碩士
東海大學
食品科學系
97
The exopolysaccharides (EPS) from many mushrooms have been reported to possess important biological functions including anti-tumor, immuno-stimulating, hypoglycemic and antioxidative capacities. Phellinus igniarius PI, a potently medicinal and EPS-producing mushroom, was selected in this research to study the effect of submerged culturing condition and different types of fermentors on the production and the bioactivity (molecular weight, relative β-(1→3)-D-glucan content, antimicrobial and antioxidative activities) of EPS by P. igniarius PI. Results showed that factors for optimal submerged-culturing condition were : incubation temperature of 30℃, glucose at 1% (w/v), yeast extract at 1% (w/v), corn steep liquor at 0.3% (w/v), initial pH of 5.4, and a C/N ratio (glucose / yeast extract) of 1.0. Under this optimized culturing condition, maximal yields of mycelial biomass (9.39 g/l) and EPS (1.93 g/l) were reached on the 8th day of incubation from shake flask culture and increased 2.4 and 10.1 times, respectively, as compared with those of the basal medium. Among the three types of fermentors, the stirred-tank fermentor showed the highest specific growth rate (μ=0.88 day-1). The air bubble fermentor showed the highest biomass yield (Yx/s=122.29 mg/g) and EPS formation rate (QP=0.39 g/l/day). As for specific EPS yield (Yp/x) and EPS yield (Yp/s), results showed an increasing tendency of air bubble fermentor > stirred-tank fermentor > air lift fermentor. The EPS of P. igniarius PI demonstrated no antibacterial activies against the six bacterial strains (Bacillus cereus BCRC 10250, Escherichia coli BCRC 10239, Micrococcus luteus BCRC 10449, Pseudomonas aeruginosa BCRC 10261, Staphylococcus aureus BCRC 10451 and Salmonella typhimurium BCRC 10241) tested. Same molecular weight (4000 Da) was found in EPS from the shake flask and three different types of fermentors. The EPS from culture of air bubble fermentor had higher content of molecular weight of 4000 Da. Under same concentration (10g/l), the fermented cultures from stirred-tank and air bubble fermentors showed the highest DPPH scavenging effect (86.94%, IC50=4.83g/l) and total antioxidant capacity (93.26%, IC50=1.52g/l), respectively, among those from shake flask and three different types of fermentors.The EPS from air bubble fermentor also had the highest relative β-(1→3)-D-glucan contents. As mentioned above, using air bubble fermentor, which has lower shearing force and better mixing effect as compared with stirred-tank and air bubble fermentors, could produce higher yield of EPS from Phellinus igniarius PI with better bioactivity. Replacing reagent-grade medium components with food-grade components not only reduced the production cost of EPS but also increased the yields of mycelial biomass (5.76 g/l) and exopolysaccharide (3.74 g/l) 2.4 and 10.1 times, respectively. In addition, the food-grade medium components offered a better buffering effect.