COMPARATIVE STUDY of Rate of Fermentation of Fruit Juices

COMPARATIVE STUDY OF RATE OF FERMENTATION OF FRUIT/VEGETABLE JUICES A CHEMISTRY PROJECT REPORT SUBMITTED BY ANUSHA PRASAD IN PARTIAL FULFILMENT OF THE CBSE GRADE XII IN CHEMISTRY AT

AECS MAGNOLIA MARUTI PUBLIC SCHOOL #36/909, ARAKERE, BANNERGHATTA ROAD, BANGALORE- 560076. 2013-2014

CERTIFICATE

This is to certify that ANUSHA PRASAD of Grade XII, AECS MAGNOLIA MAARUTI PUBLIC SCHOOL, BANGALORE with register number ____________________ has satisfactorily completed the project in Chemistry on COMPARATIVE STUDY OF RATE OF FERMENTATION OF FRUIT/VEGETABLE JUICES in partial fulfillment of the requirements of All India Secondary School Certificate Examination (AISSCE) as prescribed by CBSE in the year 2013-2014.

Signature of the Candidate

Signature of the Principal

Signature of the Teacher In-Charge

Signature of the External Examiner

Table of Contents

INTRODUCTION........................................................................................................................ 1 OBJECTIVE................................................................................................................................ 4 SCOPE AND LIMITATION......................................................................................................... 7 PRINCIPLE/THEORY............................................................................................................... 10 EXPERIMENT.......................................................................................................................... 13 AIM:.......................................................................................................................................... 13 REQUIREMENT:...................................................................................................................... 13 PROCEDURE............................................................................................................................ 16 OBSERVATION......................................................................................................................... 19 RESULT.................................................................................................................................... 21 BIBLIOGRAPHY...................................................................................................................... 22

ACKNOWLEDGEMENT I would like to thank my teachers, Mrs. Neelam and Mrs. Sowmya for guiding me through this project and for their valuable inputs which provided me with a constant nudge for improvement. It is imperative to thank our Principal, Mrs. Seema Goel for providing me the opportunity to work on this project. It goes without saying that my classmates, especially Rochana Ramakrishna, Pratusha Dinesh and Rahul for their help in due course of this project. My parents have also played a part in helping me in this project. My thanks goes out to them also. This project and reading-up on the same has provided me with an in depth understanding of the topic. It has nurtured my scientific temperament and curiosity.

Signature of the Candidate

ABBREVIATION Sl.No 1 2 3 4 5

Abbreviation C g ml O C

Expansion Centigrade gram milliliter Oxygen Carbon

INTRODUCTION Fermentation is typically the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using yeasts, bacteria, or a combination thereof, under anaerobic conditions (absence of oxygen) by the action of enzymes. Enzymes are complex organic compounds, generally proteins. They are highly specific with regard to their substrates. Fermentation in simple terms is the chemical conversion of sugars into ethanol. Ethanol fermentation, also referred to as alcoholic fermentation is the biological process in which sugars such as glucose, fructose, and sucrose are converted into cellular energy and thereby produce ethanol and carbon dioxide as metabolic waste products. All ethanol contained in alcoholic beverages is produced by means of fermentation induced by yeast. Wine is produced by fermentation of the natural sugars present in grapes and other kinds of fruit. Ethanol fermentation occurs in the production of alcoholic beverages and ethanol fuel, and in the leavening of bread dough. Fermentation is used in preservation techniques and in production of foods such as yogurt, cottage cheese (paneer), dhokla, idli, chocolates, cheese etc. ‘Fermentation’ has been derived from the Latin word ferver, which means ‘to boil’, as during fermentation, there is a lot of frothing in the liquid due to evolution of carbon dioxide. This gives it the appearance as if it is boiling! Yeasts are unicellular eukaryotic microorganisms classified in the kingdom Fungi, Yeast size can vary greatly depending on the species, typically measuring 3-4 µm in diameter, although some yeasts can reach over 40 µm. Most yeasts reproduce asexually by mitosis, and many do so by an asymmetric division process called budding. Yeasts do not form a single taxonomic or phylogenetic grouping. The term yeast is often taken as a synonym for Saccharomyces cerevisiae. Natural fermentation precedes human history. The earliest evidence of winemaking dates from eight thousand years ago, in Georgia, in the Caucasus area. Seven-thousand-year- old jars containing the remains of wine have been excavated in the Zagros Mountains in Iran. There is strong 1

evidence that people were fermenting beverages in Babylon circa 3000 BC, ancient Egypt circa 3150 BC, pre-Hispanic Mexico circa 2000 BC, and Sudan circa 1500 BC. Ancient fermented food processes were developed long before man had any knowledge of the existence of the microorganisms involved.

When studying the fermentation of sugar to alcohol by yeast, Louis Pasteur concluded that the fermentation was catalyzed by a vital force, called “ferments”, within the yeast cells. The “ferments” were thought to function only within the yeast cells. The “ferments” were thought to function only within living organisms. Nevertheless, it was known that yeast extracts (Yeast extract is the name given to processed yeast products made by extracting the cell contents (removing the cell walls)) can ferment sugar even in the absence of living yeast cells. While studying this process in 1897, Eduard Buchner found that sugar was fermented even when there were no living yeast cells in the mixture; by a yeast secretion that he termed zymase, i.e., fermenting activity of yeast is due to active catalyst of biochemical origin. In 1907 he received the Nobel Prize in Chemistry for his research and discovery of “cell-free fermentation.”

Main uses of fermentation The primary benefit of fermentation is the conversion of sugars and other carbohydrates, e.g., converting juice into wine, grains into beer, carbohydrates into carbon dioxide to leaven bread, and sugars in vegetables into preservative organic acids. Food fermentation has been said to serve five main purposes:  Enrichment of the diet through development of a diversity of flavors, aromas, and textures in food substrates.  Preservation of substantial amounts of foods through lactic acid, alcohol, acetic acid, and alkaline fermentations

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 Biological enrichment of food substrates with protein, essential amino acids, essential fatty acids, and vitamins  Elimination of antinutrients  A decrease in cooking time and fuel requirement

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OBJECTIVE In this project, time taken for fermentation of various fruit / vegetable juices had to be compared. Fermentation is one of the oldest methods of processing food into a form that is suitable for preservation. In fermentation technology, we stress in understanding the various process in fermentor and how various intrinsic factors influence the fermentation process. Fermentation technology being an industrial microbiology subject are geared in producing maximum amount of high economical fermentation products. The objective of this project is to compare the rates of fermentation of different fruit and vegetable juices. The information gained from this experiment may be used by wineries to determine which fruit juice ferments best. But it is difficult to understand and control the fermentation process as it involves various components such as effect of substrates, products inhibition, conditions and complex microbial interactions. Fermentation is affected by several factors including the temperature, salt concentration, pH, oxygen availability and nutrient availability. The rate of fermentation can be controlled by manipulating any of these factors. Temperature Different yeasts tolerate different temperatures. For Saccharomyces cerevisiae, it is around 35-400C. A variation of just a few degrees from this temperature alters the activity of the microbes and affects the quality of the final product. Nutrients i.e. Sugar content All bacteria require a source of nutrients for metabolism. The fermenters require carbohydrates, in this case sugars glucose and fructose. The energy requirements of microbes are very high. Limiting the amount of substrate available can reduce the rate of fermentation.

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Effect of oxygen If oxygen is present, some species of yeast will oxidize pyruvate completely to carbon dioxide and water. Thus, these species of yeast will produce ethanol only in an anaerobic environment. However, many yeasts such as the baker’s yeast Saccharomyces cerevisiae, or fission yeast Schizosaccharomyces pombe, prefer fermentation to respiration. These yeasts will produce ethanol even under aerobic conditions. Hence the rate of fermentation varies. The fermentation process is not only complex but always in a state of flux. Process, we are therefore in a situation to always be adaptive and reactive to these changes so that throughout the fermentation process we are always sustaining the conditions in a narrow window of optimal fermentation conditions. In order to help us do this we need to know fermentation kinetics. When we talk about fermentation kinetics we are talking about fermentation models. Kinetics and modellings are very useful to us as tools to make fermentation predictions and enhancing our experimental designs to be more focused to the specific problems such as the rate limiting steps or product inhibition. The study of fermentation kinetics helps us by providing clear quantitative data for us to understand the process and improve the process accordingly. Peering into observation ports might be good advertising gimmick for fermentation technology but do not really help much in understanding the process or even to control and predict the fermentation outcome. Subjective observations will rarely help in producing optimum fermentation process and thus affect profitability studies and making decisions. Its numbers that count! Thus the importance of the study of fermentation kinetics or models. 5

The first step in the study of fermentation kinetics is to understand the various processes involved in the whole process. Such questions such as inputs and outputs, the metabolic pathways involved and type of products or side products formed. The various individual reactions involved and what factors control the metabolite levels. Then only after all the relevant data are obtained do we start formulating the models.

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SCOPE AND LIMITATION SCOPE The scope of this project is as wide as the scope of process of fermentation. This project aspires to explore one of the innumerable applications of the biochemical concept of breakage of highly ordered large molecules into smaller ones by the action of microorganisms or enzymes. Some of the applications include: THE PRODUCTION OF ALCOHOL Beers, wines and spirits are all produced by fermenting various carbohydrates. Yeasts do this naturally to sugars; a property that has been utilized by humans for thousands of years. Ethanol is also produced industrially on a large scale for use as a biofuel. This has traditionally involved a two step fermentation procedure using aerated tanks containing the yeast Saccharomyces cerevisciae and substrate carbohydrates. THE PRODUCTION OF CITRIC ACID Citric acid is a useful product in both the food and pharmaceutical industries; it is used in food as a preservative and to produce an acidic, sour taste in soft drinks and other beverages. In the pharmaceutical industry it can be used as buffering agent and to clean equipment. Citric acid is formed by the fermentation of a molasses substrate by the fungus Aspergillus Niger. The biochemical pathway involved includes the production of pyruvate in glycolysis, followed by its conversion to citric acid via the condensation of acetyl co-enzyme A and oxaloaecetate.

ACETIC ACID PRODUCTION 7

In the presence of the Acetobacter bacterium and oxygen, fermented carbohydrates, ciders or wines can be converted to vinegar (acetic acid). The result is usually is usually a 5 % solution of acetic acid. Acetic acid is used in diluted form in the food industry as a condiment and pickling agent. It is also employed in industry as a solvent and an important reagent in many organic synthesis reactions.

A VERSATILE REACTION Fermentation certainly produces a diverse range of chemicals and is obviously a key reaction in many industries. The one thing all these processes have in common is an initial culture containing carbohydrates and a particular species of microorganism. LIMITATIONS One of the limitations of fermentation as a process is its requirement for multiple reagents. Secondly, in many cases the time taken is quite long and this creates a need for catalyst. Without catalysts, the reaction is extremely slow. The limitation of our project is the slight error in the result and the project is limited to the fermentation of the juices with Baker’s yeast and not under normal conditions i.e. without adding Baker’s yeast. Owing to the different criterion on which the rate of fermentation depends, if the experiment is not carried out in the optimal temperature range, the rates will turn out to be different than the actual rates of the juices that have been taken. It is not possible to get the exact theoretically estimated value due to impurities in the reagents as well as the compounds. Another point to be noted is that the rates calculated from this experiment is just one case and this can’t actually access the rate of fermentation of the fruit. An average needs to be taken to access its actual value.

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PRINCIPLE/THEORY Fermentation is the slow decomposition of complex organic compounds into simpler compounds by the action of enzymes. Enzymes are biological molecules that catalyze (i.e, increase the rates of) chemical reactions. Fruit and vegetable juices contain sugar such as sucrose, glucose and fructose. The chemical equations below summarize the fermentation of sucrose, whose chemical formula is C12 H22 O11. One mole of sucrose is converted into four moles of ethanol and four moles of carbon dioxide: C12H22O11 + H2O + Invertase  2 C6H12O6 Glucose + Fructose C6H12O6 + Zymase  2 C2H5OH + 2CO2 Glucose + Fructose Sucrose is hence first converted to glucose and fructose with the enzyme invertase, while enzyme zymase converts glucose and fructose to ethyl alcohol. Invertase Invertase (systematic name: beta-fructofuranosidase) is an enzyme that catalyzes the hydrolysis (breakdown) of sucrose. Related to invertases are sucrases. Invertases and sucrases hydrolyze sucrose to give the same mixture of glucose and fructose. Invertases cleave the O-C (fructose) bond, whereas sucrases cleave the O-C (glucose) bond. For industrial use, invertase is usually derived from yeast. It is also synthesized by bees, who use it to make honey from nectar. Optimum temperature at which the rate of reaction is at its greatest is 60 0 C and an optimum pH of 4.5.

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Invertase C12H22O11 + H2O Sucrose

C6H12O6 + C6H12O6 Glucose Fructose

Zymase Zymase is an enzyme complex (“mixture”) which catalyzes the fermentation of sugar into ethanol and carbon dioxide. They occur naturally in yeasts. Zymase activity varies among yeast strains. Zymase C6H12O6 + C6H12O6 Glucose Fructose

2C2H5OH + 2CO2 Ethanol

Chemical test: Fehling’s solution To test for the presence reducing sugars to the juice, a small amount of Fehling’s solution is added and boiled in a water bath. During a water bath, the solution progresses in the colors of blue (with no glucose present), green, yellow, orange, red, and then brick red or brown (with high glucose present). A colour change would signify and the presence of glucose. Sucrose (table sugar) contains two sugars (fructose and glucose) joined by their glycosidic bond in such a way as to prevent the glucose isomerizing to aldehyde, or the fructose to alpha-hydroxy-ketone form. Sucrose is thus a non-reducing sugar which does not react with Fehling’s solution.(Sucrose indirectly produces a positive result with Benedict’s reagent if heated with dilute hydrochloric acid prior to the test, although after this treatment it is no longer sucrose.) The products of sucrose decomposition are glucose and fructose, both of which can be detected by Fehling’s as described above. By comparing the time required for completion of fermentation of equal amounts of different substances containing starch the rates of fermentation can be compared. 11

Addition of yeast In wine making, yeast is normally already present on grape skins. Fermentation can be done with this endogenous “wild yeast,” but this procedure gives unpredictable results, which depend upon the exact types of yeast species present. For this reason, a pure yeast culture is usually added, this yeast quickly dominates the fermentation. Baker’s yeast is the common name for the strains of yeast commonly used as a leavening agent in baking bread and bakery products, where it converts the fermentable sugars present in the dough into carbon dioxide and ethanol. Baker’s yeast is of the species Saccharomyces cerevisiae, which is the same species commonly used in alcoholic fermentation, and so is also called brewer’s yeast. Pasteur’s salt Pasteur’s salt solution is prepared by dissolving ammonium tartarate, 10.0 g; potassium phosphate, 2.0 g; calcium phosphate, 0.2 g; and magnesium sulphate, 0.2 g dissolved in 860 ml of water. The Pasteur’s salts in solution act as a buffer to any acids the yeast may create. Since yeast only converts sugar (most likely sucrose or glucose) to ethanol under anaerobic conditions, and it is unreasonable to assume that there will be no oxygen present in the laboratory, some acetic acid is created as a result. The Pasteur salts act as buffers to the acidity so that the proteins in the yeast do not become denatured.

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EXPERIMENT Aim: To compare the rates of fermentation of some fruit/vegetable juices and determine the substance which has the highest rate of fermentation amongst the various samples taken.

Requirement: a.

Chemical Requirement  Pasteur’s salts  Yeast

 Fehling’s reagent

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b.

Apparatus Requirement  Conical flasks

 Test tubes

 Beaker

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 Bunsen burner, tripod stand and watch glass

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PROCEDURE 1. 5.0 ml of apple juice was taken in a clean 250 ml conical flask and diluted with 50 ml of distilled water.

2. 2.0 gram of Baker’s yeast and 5.0 ml of solution of Pasteur’s salts were added to the above conical flask. 3. The contents of the flask were shaken well and the temperature of the reaction mixture was maintained between 35-400C.

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4. After 10 minutes 5 drops of the reaction mixture were taken from the flask and added to a test tube containing 2 ml of Fehling reagent. The test tube was placed in a boiling water bath for about 2 minutes. The colour of the solution or precipitate was then noted.

5. Step 4 was repeated after every 10 minutes until the reaction mixture stopped giving any red colour or precipitate.

6. This time taken, i.e. time taken for the completion of fermentation was noted.

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7. All the above steps were repeated by taking 5 ml each of grape juice, black grape juice, sweet lime juice, orange juice and carrot juice.

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Precautions:  All apparatus should be clean and washed properly.  The flask should not be rinsed with any of the solution.

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OBSERVATION Volume of fruit juice taken = 5.0 ml Volume of distilled water added = 50.0 ml Weight of baker’s yeast added = 2.0 g Volume of solution of Pasteur’s salts = 5.0 ml Time ( in minutes ) 10 20 30 40 50 60 70

Colour of reaction mixture on reaction with Fehling solution Apple Juice Red Red

Sweet lime Juice Red Red

Carrot Juice Red Red

Orange Juice Red Red

Red Red Brownish Red Brown No Change

Red Red Greenish Brown No Change No Change

No Change No Change No Change

Red Brown No Change

Tomato Juice Red Brownish Red Brown Dark Brown No Change

No Change No Change

No Change No Change

No Change No Change

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Graph

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RESULT The time taken for fermentation of carrot juice was well before the rest of the juices, it’s recorded time being 30 minutes. This means that carrot juice has the highest sucrose content from the various samples taken. After 50 minutes orange and tomato juices gave positive test for fermentation with Fehling’s solution. For sweet lime juice time taken for fermentation was 60 minutes and for apple juice it was 70 minutes.

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BIBLIOGRAPHY Wikipedia - The free encyclopedia - (http://en.wikipedia.org) Comprehensive Practical Chemistry

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