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UNI VERSI TY
(Estd. u/s 3 of UGC Act 1956)
Vellore 632 014, Tamil Nadu, India
LABORATORY CUM PRACTICAL MANUAL
Name Reg. No. Batch Semester Course code Programme : : : : : : III 09MSM513 L M.Sc. Applied Microbiology
Prepared and compiled by Prof. V.MOHANASRINIVASAN Prof. CHITRA KALAICHELVAN Mrs. M. THENMOZHI
School of Bio Sciences and Technology
(For Private circulation only)
VIT – A Place to learn; A chance to grow
UNI VERSI TY
(Estd. u/s 3 of UGC Act 1956)
School of Bio Sciences and Technology FERMENTATION TECHNOLOGY CERTIFICATE
This is to certify that this is a bonafide record of work done by ________________________________ (Reg. No.) ___________________, a student of M.Sc. Applied Microbiology during the III Semester of the Year 2010 – 11 at VIT University, Vellore – 632 014. This record is submitted for the practical examination held on
LIST OF EXPERIMENTS
TITLE OF THE EXPERIMENTS LABORATORY PRACTISES
BIOREACTOR AND ITS STRUCTURE
ISOLATION AND CHARACTERIZATION OF
Lactobacillus FROM FERMENTED MILK
ISOLATION AND CHARACTERIZATION OF
Leuconostoc FROM RICE FLOUR
ISOLATION AND CHARACTERIZATION OF 5.
Saccharomyces cerevisiae FROM GRAPE
JUICE PRODUCTION OF GRAPE WINE
ALCOHOL FERMENTATION BY YEAST
ISOLATION AND CHARACTERIZATION OF BACTERIAL PIGMENTS
ISOLATION AND CHARACTERIZATION OF Monascus FROM SOIL
PIGMENT EXTRACTION FROM
BIOCATALYSIS OF PRECURSORS TO AROMA AND FLAVOR COMPOUNDS USING
. peroxides. 5. phenols. All microbial cultures are handled and treated as potential biohazards and dispose them of organisms. Wrap a lyophilized culture vial with disinfectant wetted cotton before breaking. 6. 3. Do not eat. 7. organic solvents. Never mouth pipette any solution (acids. 2. Do not blow infectious materials out of pipettes. 4. drink or smoke inside the laboratory. etc) of after autoclaving or treating with 10% formalin for 30 min. culture Wear laboratory coat (use gloves when necessary). Aseptic techniques should be followed rigorously at all times.GENERAL INSTRUCTIONS FOR USING THE LABORATORY Good laboratory practices : 1.
2. batch No. Label all the reagents. Experiments and record keeping 1. correctly before starting the experiment. Note down the date of expiry. 3. 5. tubes. Make sure the glasswares. make in advance. Store all the sensitive reagents and solutions should be stored in brown/ dark bottles). and catalogue No. data sheets supplied along with fine chemicals and enzymes. 4. needed sterilize them previous day itself). amount of alkali or acid used to dissolve should be recorded then and there). etc. brand. 2. .. 4. gossiping and loud discussions inside the laboratory (especially in front of Do not store food materials and drinks in the labotatory refrigerator. (Weight of the substance. type of water used. Discuss the experiment with your Professor in advance. 1. Keep appropriate controls. date of preparation of the reagent and initials of the person who prepared. for labile substances. concentration (% molar). The label should contain the name of the solution reagent. If it requires prior permission and reservation. experiment. 9. Unlabelled solutions/reagents should be discarded. cultures. 6. (Light Keep record of the reagent preparation and experimental details correctly. of the substance. Store the reagents in appropriate bottles and at appropriate temperatures. Attach print outs of instruction sheets and supporting materials to record. solutions. media. 3. etc are ready for the experiment (if Make sure that you have studied and understood the principle and methodology of each corrosive solvents should be cleaned immediately and appropriately). Make sure that the balance is cleaned after your weighing. 5. Make sure the laboratory and laboratory benches are clean (all spillover of cultures. Make sure that the equipments are in working condition and available for the experiment. Preparation of reagents Store chemicals/ solvents at appropriate temperatures as mentioned on the label The highest purity chemicals and double distilled water should be used for reagents and laminar air flow) as it affects yours and colleagues work. Label all the plates. batch No. plasticwares. Copy the protocols of the experiment in the record notebook. No chatting.8.
Record all data directly into the lab note book. Bioreactor . Start new experiment on a new page. code names and code numbers in recording the data. enzymes. Avoid abbreviations. All graphs should be titled. And it is the control of the process that concerns chemical engineers first and foremost. standard graphs. (Relationship between two variables in experiments can be best represented using graphs rather than in tables. Make sure that you know the operations of the equipments correctly or get apt Microbial counts. printouts. Do not record data on loose sheets.. title and objective(s). it could be controlled. standard graphs.7. should be labelled to indicate what they are supposed to represent and then attach them directly on to the note book as soon as the experiment is over. BIOREACTOR AND ITS STRUCTURE Louis Pasteur’s work on fermentation of wine laid the foundation for bioreactors as we know them today. Use permanent ink in writing the record. Don’t rely on memory. All calculations should be done step by step and record neatly. Every experiment should have date. The scope of bioengineering has grown from simple wine-bottle microbiology to the industrialization of not only beer. wine. 8. because once the process is identified and understood. Do not take the record note book outside the laboratory.antibiotics. which passes through maximum number of points. plot all the points and draw the line. 9. dated and fixed in the record book on the same day. chromatograms. cheese and milk production. etc. enzyme assays should be done in triplicates. This has been possible due to the invention of bioreactors. For assistance. sugars and organic acids. Use a record notebook of permanent binding. vitamins. but also the production of biotechnology’s newer products . Photographs. steroidal hormones. Do not overwrite on mistakes. Number all the pages before using the record notebook.
6. singularly or in series. 1997). The bioreactors include mechanical vessels in which organisms are cultivated in a controlled manner and materials are converted or transformed through specific reactions. 2. 5. ebullized bed reactors and fluidized bed reactors (Kajiwara et al. 7. However. Adequate aeration and agitation should be provided. Sampling facilities should be provided. This lead to the eventual use of the first truly large scale aseptic fermentation vessels (Hastings. Minimum requirement for labor in operation. These were used in central Europe in the 1930’s for the production of compressed yeast. Bioreactor Configuration In designing and constructing a bioreactor the points to be considered are:1. Bioreactors are specifically designed to influence metabolic pathways. The bioreactors consisted of large cylindrical tanks with air introduced at the base via networks of perforated pipes. A bioreactor is a system in which a biological conversion is effected. 3. cleaning and maintenance. The models and designs that are used for bioreactions include continuous stirred-tank reactors. The vessel should be capable of being operated aseptically for a number of days. A system of temperature and pH control should be provided. harvesting. Evaporation losses should not be excessive. 4. since the organisms are more sensitive and less stable than chemicals.A research team led by Chaim Weizmann in Great Britain during the First World War developed a process for the production of acetone by a deep liquid fermentation using Clostridium acetobutylicum. 1978).. Bioreactors support and control biological entities and provide a higher degree of control over process upsets and contaminations. the mixing should not damage the cells. Power consumption should be as low as possible. . continuous flow stirred-tank reactors.
A schematic representation of a simple bioreactor is as shown in Figure 1. The design of a bioreactor involves the parameters such as temperature. water. pH. vitamins. There should be adequate service provisions for individual plants. 1996). Bioreactor Design A bioreactor is a vessel which provides a controlled environment for the growth of microbial cells to obtain a desired product.8. agitation and aseptic conditions is the most important requirement for any bioreaction. substrate concentration. The bioreactor to be designed should consider to promote the optimal morphology of the organism and eliminate or reduce contamination by unwanted organisms or mutation of the organism (Viktor Nedovic and Ronnie Willard. gas evolution and product removal. 9. . availability of salts. Of these. The cheapest material which enables satisfactory results to be achieved should be used. oxygen (for aerobic processes). maintenance of adequate aeration.
The structural components involved in this are: (i) (ii) (iii) (iv) Agitator or impeller Stirrer glands and bearings Baffles Aeration system Agitator or Impeller: Agitator helps in mixing objects either by any of the following types: a) d) Disc turbines Marine propellers Good mixing and aeration in high viscosity broth may be achieved by a dual impeller. Most of the large scale and pilot scale bioreactors are constructed from ISI grade 316 stainless steel which has 18% chromium and 10% nickel. b) Vaned discs c) Open turbines . b) Aeration and Agitation Systems: The type of aeration and agitation systems used for a particular bioreactor depends on characteristic fermentation conditions. On a bench scale.Figure : A stirred tank bioreactor with baffles and an agitator a) Body Construction: The material used for body construction should withstand repeated sterilization cycles. An effective seal is made between the bioreactor sides. The lower impeller in a bioreactor acts as the gas depressor and upper impeller area primarily acts as the device for circulating the vessel contents. The thickness of the construction material will increase with scale. The top and bottom of the seal can be made with a compressible gas fit or lip seal or V-ring seal. fine glass can be used.
through which circulation of water takes place to control the temperature. Baffles: A typical bioreactor has four baffles and they prevent vortexing and improve its aeration efficiency. c) Temperature probes and control: Temperature control is one of the most important parameter to be monitored and controlled in any type of bioreactor. Heat will be generated in the bioreactor due to agitation. The earlier seal being described by Rirch Johenson and Peterson in 1950 as a porous bronze bearing of 13 mm shaft fitted in the centre of the bioreactor top. acid or alkali to a small scale bioreactor is normally done in silicon tubes which are autoclaved separately and pumped by peristaltic pump often under aseptic conditions.Stirrer Glands and Bearings: There are a number of shafts and glands to obtain satisfactory aseptic seal. Aeration System: Aeration inside the bioreactor is provided with a sparger. N 1/10 of vessel’s diameter and are attached radially to the wall. Baffles are actually metal strips. e) Feed ports: Addition of nutrients. f) Valves: Valves are attached to the bioreactor and are used for controlling the flow of liquids and gases. each being used depending upon the type of bioreactor. . In a large scale bioreactor. it is essential to maintain it at a positive pressure and the sampling port is to be provided with steam supply. Spargers may be of porous type. heating coils or heating jackets are provided. There may be: (i) Sample on/off valves which are either fully opened or fully closed. d) Sampling ports: To prevent contamination when operating a bioreactor requiring GILSP. A sparger is defined as a device for introducing air or gas into the liquid phase of the bioreactor.
A typical steam trap has 2 elements. Valves which may be adjusted precisely so that flow rates are accurately controlled.(ii) (iii) (iv) Valves which provide coarse control of flow rates. This is achieved by steam traps which collect and remove automatically any condensate at appropriate points in steam line. one being the valve and steam assembly which provides an opening and the second valve for measuring some parameter of the condensate. Fermentation process and stages Figure: A generalized. schematic representation of a fermentation process . g) Steam traps: In steam lines it is essential that steam condensate which accumulates in the pipe be removed to ensure optimum porous conditions. Safety valves which are constructed in such a way that they allow the flow of liquid or gas only in one direction.
(Reproduced with permission from P. J. If they are hetero fermentative they produce appreciable amounts of volatile products including alcohol in addition to lactic acid. The homofermentative lacticacid bacteria ferment sugars chiefly to lactic acid with small amount of acetic acid. They are also used in the manufactures of industrial lactic acid. Lactobacilli are commonly used in fermented diary products and vegetables. ‘Principles of Fermentation Technology’. Pergamon Press. Stanbury. Oxford. A. F. 1995) ISOLATION AND CHARACTERIZATION OF Lactobacillus FROM FERMENTED MILK PRODUCTS AIM: To isolate and characterize Lactobacillus species from curd. carbon dioxide and some trace products. Hall. . Whitaker and S. BACKGROUND: The genus Lactobacillus consists of both hetero fermentative and homo fermentative lactic acid bacteria.
.004%. Sugars were added at the concentration of 1% and phenol red at the concentration of 0. Catalase test c. MRS broth without glucose and beef extract was prepared. Samples from last 3 dilutions were taken and pour plated. pairs or in short chains. microaerophilic and their nutritional requirements are complex as optimal growth is obtained only in media containing fermentable sugars and adequate growth factors.Lactobacilli are gram positive bacilli occurring in singles. Isolation of Lactobacillus a. lactose and sucrose Arginine PROCEDURE: i.glucose. REQUIREMENTS: Curd sample MRS agar MRS broth Sugars . non spore forming. Examined for large cream colored slightly mucoid colonies. Arginine hydrolysis Sugar fermentation test 1. Characterization Isolated colonies were characterized by a. 2. All the plates were incubated at room temperature d. Durham tubes were placed in inverted position to trap the gas production. They are non acid-fast. c. Growth at 15˚Cand 45oC f. ii. The curd sample was serially diluted in (10-4) to (10-6) b. Sugar fermentation test e. Oxidase test d. Gram staining b.
Arginine Hydrolysis test: 1. Arginine was added at the concentration of 0. Media Grams reaction /Shape Catalase Oxidase Sugar fermentation/gas production Dextrose Sucrose Lactose Arginine hydrolysis Growth at 15°C and 45°C MRS media Growth at 15oC and 45oC: 1. 2. After incubation few drops of Nesseler’s reagent was added to test presence of Ammonia (which will give brown colour with Nesseler’s reagent). sterilized and inoculated with the culture and incubated at 15˚C and 45oC separately for 3 – 4 days upto 1 week. MRS broth was prepared.5% after sterilization the tube was inoculated with the culture and incubated at room temperature for 1 – 2 days. 2. After incubation the tubes were examined for turbidity.3. OBSERVATION: RESULT: . After sterilization the tubes were inoculated with isolated culture and incubated for 2 days at room temperature and observed for acid and gas production. MRS broth without Ammonium citrate was prepared.
BACKGROUND: The genus Leuconostoc contain hetero fermentative lactic Streptococci which ferments sugar lactose to Lactic acid and considerable amount of acetic acid. carbon dioxide and other acids.ISOLATION AND CHARACTERIZATION OF Leuconostoc FROM RICE FLOUR AIM: To isolate and characterize Leuconostoc from fermented rice flour. has the ability to initiate lactic acid fermentation in food stuffs more rapidly than other lactic acid bacteria and enough acids to inhibit the growth of non lactic acid bacteria. The most common . Leuconostoc sp. plays major role in most of the food fermentation. therefore. Leuconostoc sp. They produce an important flavor compound called diacetyl. ethyl alcohol.
messenteroids and L. Catalase test c.cremoris. iii. Citrate utilization test e. They require nutrient rich medium. Gram staining b.The isolated culture on Leuconostoc agar are characterized by a.species are L. H2O2 Sucrose agar. Sugar fermentation broth. Sugar fermentation test f. PROCEDURE: ISOLATION: i. All the plates were incubated at room temperature iv. Leuconostoc sp are mostly used in production of fermented vegetables and some dairy products. After incubation the plates were examined for pale white slimy colonies. It is a gram positive cocci occurring in pairs (or) short chains. Leuconostoc agar. Esculin hydrolysis test g. CHARACTERIZATION: i. Samples from last 3 dilutions were taken and spread plated on leuconostoc agar in duplicates. Simmon citrate agar. VP test d. non motile and dextran producing organisms. Esculin hydrolysis agar MR-VP broth. They are non spore forming. . The sample (fermented rice flour) was serially diluted in (10-4) to (10-6) ii. Leuconostoc broth without beef extract was prepared. MATERIALS REQUIRED: Fermented rice flour (Sample). Dextran production SUGAR FERMENTATION TEST 1.
After incubation the slant was observed for colour change to dark brown to black. Sugars were added at the concentration of 1% and phenol red at the concentration of 0. After sterilization the tubes were inoculated with isolated culture and incubated for 2 days at room temperature and observed for acid and gas production.2.Leuconostoc agar . the plates were observed for gummy mucous whole irregular colonies. DEXTRAN PRODUCTION: The isolated culture was inoculated on Leuconostoc sucrose agar and incubated at 37oC for 1-2 days. OBSERVATION LA. 3. After incubation.004%. Durham tubes were placed in inverted position to trap Media Grams reaction/ Shape LA Catalase VP test Citrate utilization Sugar fermentation /gas production Dextrose Lactose sucrose Esculin hydrolysis Dextran production the gas production. ESCULIN HYDROLYSIS: The isolated culture are inoculated on esculin hydrolysis agar slant and incubated at 37oC for 24-48 hrs.
They reproduce by budding (or) ascospore formation.RESULT: ISOLATION AND CHARACTERIZATION OF Saccharomyces cerevisiae FROM GRAPE JUICE AIM: To isolate and characterize Saccharomyces cerevisiae from grape juice. oval (or) elongated) pseudomycelium. . Ascospores are formed either by conjugation of two cells (or) from diploid cells. BACKGROUND: Saccharomyces cerevisiae is a type of yeast. Four ascospores are seen in one ascus by process of meiosis. with many forms(round.
CHARACTERIZATION: LACTOPHENOL COTTON BLUE STAINING: A loopful of isolated culture was emulsified in a drop of LPCB stain on a glass slide. vii. The sample (grape juice) was serially diluted (10 fold dilution) upto 10-4 dilution. A coverslip was placed over the emulsion and the prepared wet mount was observed. production of distilled liquor in distilleries. Grape juice Potato dextrose agar (PDA) Ascospore agar Nitrate broth Christinson ‘s urea agar α – Napthyl amine Sulphanilic acid Lactophenol cotton blue PROCEDURE: ISOLATION: i. iv. After incubation the plates were examined for medium sized creamy colonies with yeasty odor. viii. oval budding yeast cells. NITRATE REDUCTION TEST: . ii. iv. It is also used to produce home made fermented foods like bread. ii. vi. under low power objective for round. v. production of invertase used in confectionaries etc. MATERIALS REQUIRED: i. wine. Incubated at room temperature for 72 h. cake and curd etc. It is also used to produce industrial alcohol and recombinant proteins. iii. Plates were prepared from all the dilutions using PDA agar by pour plate method.Saccharomyces cerevisiae is widely employed in many industries for leavening of bread in bakeries. production of wine in breweries. iii.
ASCOSPORE FERMENTATION: Ascospore agar plate was prepared and inoculated with isolated culture and incubated at room temperature for 3-4 days. The color change was observed (pink). Next day. inoculated with isolated culture and incubated at room temperature for 24 hrs. OBSERVATION: .Nitrate broth was prepared. After incubation few drops of α – Napthyl amine reagent and sulphanilic acid reagent was added. UREASE TEST: Christinson urea agar slant was prepared and inoculated with the isolated culture and incubated at room temperature. The color change in the slant was observed(pink). the tubes were observed for acid and gas production. After incubation a wet mount with LPCB was prepared and observed under low power objective for ascospores. inoculated with isolated culture and incubated at room temperature for 1-2 days. SUGAR FERMENTATION TEST: Sugar fermentation broth with 1% sugar was prepared.
Media LPCB Staining /Shape PDA Nitrate Urease Sugar fermentation/gas production Dextrose Sucrose Lactose Ascospore RESULT: PRODUCTION OF GRAPE WINE PRINCIPLE .
The resultant wine can contain alcohol up to 15%. . Incubate the flask for 21 days at room temp without shaking. 4. [As mentioned earlier. PREPARATION OF WINE 1. washed cells of Saccharomyces cerevisiae. Follow steps 1 to 4 above (procedure A) using 750 g grapes in order to obtain approx 500ml of must in a 100ml flask. Amount of sucrose should be 50g. the must may or may not be pasteurized]. However. in a constant temp shaker incubator at 25oC for 24 h or until a heavy inoculum is built. 3. Incubate the flask in shaking conditions. This is however optional]. The percent acidity is determined by titration against NaOH with phenolphthalein indicator. 5. 3. PROCEDURE A. 2. the contents may be gently mixed daily. Hygienically prepared grape juice (must) is fermented to alcoholic wine by Saccharomyces at 22oC by the end of 21 days. Wash with clean drinking water and crush in blender. into the freshly prepared must. clarity and alcohol content. Other parameters studied at regular intervals of the fermentation include aroma. 2. Select black grapes for red wine and white grapes (green) for white wine. Inoculate the must so prepared. (Peeled black grapes may also be used for white wine). Pour the entire contents of the inoculum prepared in ‘A’ above. Strain the juice with the help of a clean strainer and collect the juice in a sterile 250ml flask. Yeast ferment the sugars to acetaldehyde and then to alcohol. with 3ml of fresh. Add 5g glucose to the flask and dissolve.Fresh grape juice (must) contain up to 30% sugar (Fructose + Glucose). Pluck and remove the stalks of 100g of fresh grapes. [some labs prefer pasteurization of this juice at 60oC for 30 min. B. PREPARATION OF YEAST INOCULUM 1.
% tatarate (total acidity). 5. odour. Age the clarified wine in a refrigerator at 4 – 8o C for a month or two. and 21 days.1 x 7.4. ml of alkali x Normality of alkali x 7.0* % Acetate = Wt of sample in grams (1 ml = 1 g) . Mix and titrate to the first persistent pink colour with 0. Determination of total acidity (expressed as % tartarate) and volatile acidity (expressed as % acetic acid). 14. 12. and % alcohol.1 N NaOH. Volatile acidity: ml of alkali x Normality of alkali x 6. 1. Calculate the total acidity and volatile acidity. The prepared wine should be subjected to clarification on the 22 nd day by centrifugation followed by filteration. C. % acetic acid (volatile acidity).5 % Tartarate = 10 2. 10 ml fermenting wine sample + 10 ml d / w + 5 drops of 1% phenolphthalein solution. check the fermenting beverage for various parameters such as color. 7.5* % Tartarate = Wt of sample in grams (1 ml = 1 mg) ml of alkali x 0. clarity. 6. At intervals of 3.
% alcohol depends on various factors. No 1.6 respectively. 3.5 & 6. 5. 2. The fermented wine was clarified and stored on a refrigerator for further maturation and aging.1 x 6.ml of alkali x 0.0 % Tartarate = 10 (*7. Trouble shoot • Appearance of white cottony mycelium on the surface during fermentation indicates spoilage due to fungal contamination. 6. particularly the initial total sugar content. • The finished product is expected to have % tartarate & acetate around 0.0 are standardized calculation factors) OBSERVATION AND RESULTS S. . Parameter Colour Aroma pH % Tartarate % Acetate % Alcohol Raw must 7 days 14 days 21 days Conclude as: Red / White wine was prepared by fermenting grape juice with yeast inoculum and physical and chemical characteristics were studied.7 & 0. • Sour odor or flavor is indicative of bacterial contamination. 4.
Dichromate reagent. molasses. fructose and sucrose) into ethanol and carbon dioxide. Dilute molasses to 12 brix (1. Inoculate the slant culture of yeast S. b. For example Yeast Sucrose ethanol + CO2. YPD medium. Prepare the inoculum for fermentation as follows: a. conical flask.5 with 10 N H2SO4. BACKGROUND The yeast Saccharomyces cerevisiae converts the fermentable sugars (glucose. standard volumetric flask. Sterilize at 10 pounds pressure for 30 minutes and then cool. DNS reagent. PROCEDURE 1. MATERIALS REQUIRED Test tubes. by product in the sugar refinery industries. micro distillation unit. cerevisiae into a 50ml of YPD medium.1 kg of molasses in 8 lit of water). fermentation jar. urea. Sucrose is metabolized by yeast through Emden Meyerhoff Pathway (EMP) and alcohol and CO2 are the end products of fermentation. contains about 45 – 55 % wt / v of fermentable sugar as sucrose. . slant culture of yeast Saccharomyces cerevisiae.ALCOHOL FERMENTATION BY YEAST OBJECTIVE To demonstrate the production of ethanol by yeast and quantification of ethanol produced in yeast fermentation. Incubate at 30oC in a shaker. Adjust the pH to 5. In the large scale production of alcohol molasses is used as the substrate for yeast fermentation molasses. c. 3. 2.
Prepare the fermentation medium as follows: a. d. . ESTIMATION OF ETHANOL Reagent: Potassium dichromate Dissolve 34 gm of K2Cr2O7 in 500ml of distilled water in 1 litre flask. Add urea to the final concentration of 100 ppm. of molasses in 8 lit of water). Pasteurize this medium in 2 fermentation jars (10 lit capacity) and cool. Prepare 1 to 10% v/v Alcohol in various test tubes.1 kg. at 600nm. Dilute molasses to 22 brix (2. b. (add 400ml to inoculum 4 lit of the fermentation medium). Measure O. Add 325 ml of concentrate H2SO4 slowly keeping the flask in an ice bucket. Keep the alcohol – potassium dichromate complex at 60oC for 30 min. Take 1 ml of the various concentration of alcohol into 25ml of water in 100ml distillation flasks fitted with liebig condenser. PREPARATION OF STANDARD CURVE FOR ALCOHOL CONCENTRATIONS 1. 7. Inoculate with 10% v/v inoculum. Incubate the culture in a shaker for 12 hrs.D. 3. 4.5 with 10 N H2SO4. Fermentation conditions a. b. 2. Plot a graph with time on axis and alcohol and sugar concentration on Y axis. 5.d. 5. 4. 6. e. Make up the volume to 50ml. Distill and collect 15 ml of the distillate in a 50 ml volumetric flask containing 25ml of K2Cr2O7 solutions. Inoculate this 250 ml molasses medium (2 flasks) with 10% of the yeast culture grown in YPD. Incubate at 30oC for 48 hrs to 72 hrs. Withdraw 10 ml sample at every 12 hrs intervals and estimate alcohol and sugar concentration. c. Adjust the pH to 5.
at 550 nm. DETERMINATION OF CONCENTRATION OF ALCOHOL FROM THE FERMENTATION MEDIUM Take 1 ml of sample. Dilute this sample suitably and estimate the sugar as above. d. mix with 25ml of water and distill as above and measure O.D. PREPARATION OF STANDARD CURVE FOR REDUCING SUGAR a. To one ml of sample add 3ml of DNSA reagent.D. Plot a standard curve with concentration of alcohol on X – axis and O. c. To the clear supernatant add 5 ml of water and 1ml of 10 N HCl. On Y – axis. e. Find the alcohol concentration from the standard graph. Note: If molasses could not be obtained for laboratory exercise. and 600 nm. c. Boil it for 15 min. Prepare the glucose solution in varying concentrations (0.6. Plot the standard curve with concentration of glucose on X – axis and OD at 550 nm on Y axis. f. Find the concentration of sucrose interms of glucose equivalent from the standard graph. and neutralize with Na2CO3. Boil in a water bath for 20 min.1 mg to 1 mg/ml) b. use YPD medium with 50 g/l sucrose for inoculum development and with 150 g/l Sucrose for preparation of fermentation broth. Observation . ESTIMATION OF REDUCING SUGAR a. d. Take 5 ml of the sample from the fermented mash centrifuge at 5000 rpm to remove cells. Dilute the sample to 25 ml. b.D. Measure O.
organic reagents. petroleum ether. Resuspend the cells in 50 ml of methanol and transfer the suspension an Erlenmeyer flask. MATERIALS REQUIRED Erlenmayer flask with minimal medium. Corynebacterium poinsettiae). sodium sulfate. Methods for isolating and characterizing carotenoids pigments of gram positive and negative cells are known. 6. Resuspend the cells in 100ml of distilled water and recentrifuge. cultures (Pseudomonas aureginosa. xanthomonas compestris.000 rpm for 15 min. water bath. benzene.Result ISOLATION AND CHARACTERIZATION OF BACTERIAL PIGMENTS OBJECTIVE To demonstrate the production of different kinds of pigments by bacteria and to isolate and characterize the pigments. Serratia marcesens. Inoculate 100ml of minimal medium with 1 ml of log phase culture of bacteria known to produce pigments. 4. 3. PROCEDURE 1. 2. Incubate for 48 hrs at 30oC in a shaker. Harvest the cells by centrifugation at 10. 6% KOH in methanol ether. . Keep the flask containing the bacterial suspension in a boiling water for 5 -10 min. spectrophotometer. BACKGROUND Many soil bacteria are chromogenic and their pigmentation has been used as one of the key characters of identification. 5.
11. 14. then dehydrate by adding anhydrous solid sodium sulfate.7. 10. Gently shake the flask. 17. 8. 18. If you see the water. The bacterial pigment gets into the ether phases. 19. Transfer the supernatant to a flask and add equal volume of 6% KOH in methanol.1 filter paper. 12. OBSERVATION RESULT: . Cool the suspension. 13. Transfer the ether to flask evaporator and evaporate to dryness at 30oC. 9. Remove the ether phase. 15. centrifuge to remove the cells. Record the peaks of maximum absorbance. Redissolve the material in 10ml of petroleum ether and filter through Wattmann No. Intermittently swirl the flask to facilitate the release of bacterial pigment. Wash the ether phase gain with water then dehydrate by adding anhydrous solid sodium sulfate. 16. swirl the content intermittently. Allow to stand for few minutes for separation of two phases. Read the absorbance of the filtrate between 350 nm to 540 nm using a spectrophotometer. Transfer the content to a separating flask and add two volumes of diethyl ether and enough water to separate the layer. Warm the content at 40oC for 10 min.
sexually by ascospore formation. . chocolate etc. ice cream. MATERIALS REQUIRED Soil sample. These pigments possess antimicrobial and immunosuppressive activity. Standard glassware’s. fish. The pigment can be seen on the reverse side of the plate. PROCEDURE 1. 3. 2. Plates were prepared from all the dilutions using SDA agar by spread plate method. These pigments are used as coloring agent in poultry. 4. It is also used in cosmetics and textile industries as natural coloring agents.The common species of Monascus are Monascus purpureus. Incubated at room temperature for 7 days. Monascus pilosus and Monascus rubber.The pigments produced by various Monascus sp are monascin. LPCB stain. Soil sample was serially diluted (10 fold dilution) upto 10-4 dilution.Also used in color wine. SDA plates. During incubation the plates were examined for white color mycelium initially which becomes orange with age. They are filamentous fungi reproduce asexually by forming conidia.ISOLATION AND CHARACTERIZATION OF MONASCUS SP FROM SOIL AIM To isolate and characterize Monascus sp from soil BACKGROUND Monascus sp is a pigment producing fungi present in soil. monascorubin and monascotin. meat and other food products. pickle.
under low power objective. A coverslip was placed over the emulsion and the prepared wet mount was observed. OBSERVATION RESULT .CHARECTERIZATION LACTOPHENOL COTTON BLUE STAINING: A loopful of isolated culture was emulsified in a drop of LPCB stain on a glass slide.
PROCEDURE INOCULUM PREPARATION To fully sporulated agar slope culture of Monascus sp 5.MASS MULTIPLICATIONOF MONASCUS SP AND PIGMENT EXTRACTION AIM To mass multiply Monascus sp and to perform solid state fermentation for pigment extraction. 2. textiles and cosmetics. 40 g of substrate (raw rice) was taken in a 250 ml conical flask and mixed with approximately 15 ml of SDB. SDB. standard glasswares. Being a soil fungus Monascus sp can grow on readily available agro waste material. Production of pigment on industrial scale requires low cost procedure. The obtained spore suspension used as inoculum for fermentation. SOLID STATE FERMENTATION 1. BACKGROUND Red pigments produced by Monascus sp are used as coloring agent in food industries. ethanol.ml of distilled water added aseptically and the spores were scraped using inoculation needle. Standard glasswares. pH was adjusted to 4. MATERIALS REQUIRED Raw rice. The solid substrate provides nutrients as well as anchorage to the organism. Therefore agro industrial waste material can be used as substrate for solid state fermentation using Monascus sp to produce pigments.5 – 6 (preferably acid pH) .
4. The solvent was completely evaporated and the obtained pigment was emulsified in suitable emulsifier.3.D of the filtrate was measured at 510 nm against solvent blank. The mixture was kept in rotary mixture for 1h and allowed to stand for 15 min. OBSERVATION: RESULT: . Moisture content was adjusted to 60% using sterile water 5. PIGMENT EXTRACTION AND MEASUREMENT 1. The moisten substrate was steamed for 10 – 20 min and cooled at room temperature. 2. After incubation the pigmented substrate was steamed for 20 min to kill the fungi and dried completely at 45°C. 2 ml of spore suspension was transferred to the flask aseptically. powdered and the pigment was extracted using 90% ethanol. (5 ml of solvent for each gram of powdered substrate) 3. 1 and O. The dried substrate was taken. mixed well and incubated at room temperature for 7. 5. The extract was filtered through whatman filter paper No.10 days with daily examination and mixing. 4.
Therefore used as biocatalyst for the production of diphenyl ethanol. This compound was produced by many bacteria and yeast in food fermentation. Saccharomyces cerivisiae is the common producers of this aromatic alcohol. BIOSYNTHETIC PATHWAY PHENYLALANINE ά keto glutarate ά keto glutamate PHENYL PYRUVATE Décarboxylation PHENYL ACETALDEHYDE transamination .BIOCATALYSIS OF PRECURSORS TO AROMA AND FLAVOR COMPOUNDS USING Saccharomyces cerevisiae AIM To produce diphenyl ethanol using Saccharomyces cerevisiae BACKGROUND Diphenyl ethanol is an aromatic alcohol with rosy odor.
5. OBSERVATION RESULT . The inoculated broth was incubated at room temperature for 24 to 48 h 4. Saccharomyces cerevisiae culture (0.1 ml) was inoculated in 10 ml of YEPD medium 3.Dehydrogenase NADH+ + H+ NAD DIPHENYL ETHANOL MATERIALS REQUIRED Saccharomyces cerivisiae culture YEPD broth Phenyl alanine Ferric ammonium nitrate Standard glass wares PROCEDURE 1. YEPD medium with phenyl alanine is prepared and sterilized 2. Supernatant was taken and tested for presence of diphenyl ethanol by mixing 1 ml of ferric ammonium nitrate solution for observing red colorization within 2 min. After incubation the broth was centrifuged at 5000rpm for 15 min.
5 .4 YEPD BROTH Yeast extract Peptone Dextrose Phenyl alanine Distilled water pH – – – – – – 1g 2g 2g 0.APPENDIX MRS BROTH Yeast Extract Beef extract Peptone Glucose Tween 20 K2HPO4 Sodium acetate Diammonium citrate MgSO4 MnSo4 Distilled water pH – – – – – – – – – – – – 0.6.2 g 0.5 g 0.1 g Peptone – 0.5 g 0.2 – 6.5 g 1g 1g 2g 0.02g 0.25 g Beef extract – 0.2 .5 g 1000 ml 6.5 LEUCONOSTOC AGAR CaCO3 – 5g Malt extract – 5g NaCl – 0.1 g Agar – 2g Distilled water – 100 ml pH – 6.2 g 0.02g 100 ml 6.
v.1g Distilled water – 100 ml Dissolve the ingredients.5g NaCl 5.0g NH4Cl Stir until dissolved Adjust to 1000ml with distilled H2O Sterilize by autoclaving Measure ~700ml of distilled H2O (sterile) Add 200ml of M9 salts Add 2ml of 1M MgSO4 (sterile) Add 20 ml of 20% glucose (or other carbon source) Add 100ul of 1M CaCl2 (sterile) Adjust to 1000ml with distilled H2O NITRATE BROTH Beef extract – 0. vi.M9 MINIMAL MEDIUM Preparation of M9 Salts aliquot 800ml H2O and add i. 64g Na2HPO4-7H2O 15g KH2PO4 2.0. ii. distribute in 5 ml quantities in 15 X 125 mm tubes and autoclave at 121ºC for 15 min.5g Potassium nitrate – 0. iii. . iv. vii.3g Peptone .
5g D.100 ml Reagent B Sulfanilic acid – 0.100 ml Store the reagents in brown bottles.5 g Acetic acid (5N) 30% .0g Beef extract .0g NaCl – 0.Reagent A Alpha napthalamine – 0. at 121ºC. Base Peptone – 1.0g D.Indicator Phenol red – 1.Water – 100 ml B.8g Acetic acid (5N) 30% .Water – 100 ml C.1.6g Ethanol – 100 ml A = 900 ml B = 100 ml C = 1 ml Mix dispenses in 1 ml amounts in 12 X 100 mm test tubes and autoclave for 10 min. Carbohydrate fermentation A. .Carbohydrate solution Carbohydrate – 10.
H. Whitaker and S. F. and Ohtaguchi. Ohkani.2 – 9. Appl. J.3 . S.(2009). H. Energy Conversion and Management. 9. Viktor. J. Kajiwara. A. Microbiol. Applications of cell immobilization technology. Introdutory practical microbiology. N. 212-216. 38 (2). (1995). 44 (6). K. Yamada.REFERENCES Hasting.. (1997). N.. J. Oxford. (2002). 9 – 10 Jayababu mudili . Pergamon Press. Hall. 1-45. Stanbury P. Adv. 529-532. 16 (2). W. Development of the fermentation industries in Great Britain. Springer. Elsevier. (1971).‘Principles of Fermentation Technology’. and Ronie. Narosa publishers.
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