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Introduction
What we pretend in this workshop?
The field of biotechnology, in its broadest definition, includes activities related to the combined use of knowledge in applied biology and engineering. There are a number of activities in agriculture, engineering, environmental and medical sciences that are related to this field. In this workshop we will present an area of biotechnology that is widely known and used and yet has a vast potential for development: the use of microorganisms for the manufacture of food and beverages. We will focus on the manufacture of two alcoholic beverages (beer and rum) and three commonly known food products: yogurt, sauerkraut and vinegar.

Concepts and Definitions

There are a number of terms related to the fermentation and alcoholic beverage industries whose meaning is sometimes misunderstood. Some (such as fermentation) are words whose meaning has changed over time. Here we present some important definitions that would be used through the workshop. Microbiology - science of those living organisms we cannot see with the naked eye. Industrial Microbiology - application of microbiology in industrial activities such as fermentation, sterile products, food technology, etc. Fermentation (biology) - Anaerobic biochemical process that is used by some organisms to obtain energy of certain substrates. It differs from anaerobic respiration in that the final electron acceptor is an organic compound produced in the process itself and not an inorganic compound from the surroundings. Fermentation does not release as much energy as aerobic or anaerobic respiration. Fermentation (industry and engineering) - Any process by which chemical reactions occur due to the presence of microorganisms (or enzymes that they produce) and generate a good or service. Note that under this definition fermentations can be either aerobic or anaerobic.

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Distillation - A method of separating the compounds present in a mixture by virtue of their differences in boiling point temperatures. Brix degrees - Strictly speaking, it is the sugar mass content in a solution of sucrose and water, expressed as a percent. However, Brix degrees are more commonly referred as the mass of solids in a mixture expressed as a percent. This measurement is done using an hydrometer or a refractometer. Degrees proof - It is the volume content of ethanol (expressed as a percent) multiplied by 2.

The Use of Microorganisms

When we think about microorganisms, the first thing that comes to mind are the diseases caused by some of them. We always associate the smell of a garbage bag with food in a state of decomposition caused by the effect of bacteria or fungi. But as we will see, not everything is negative in the interaction between humans and microorganisms. Many products that are used by humans are produced by them. The goods that are produced using microorganisms include: Food and vitamins - examples are bread, cheese, yogurt, sour cabbage (sauerkraut), olives, sweet pickles (pickles), vinegar, chocolate, vanilla and soy sauce. Examples of vitamins are B12 and B2. Alcoholic beverages - examples are beer, wine, rum, brandy, vodka, whiskey and gin. Medicines and pharmaceutical products - examples are antibiotics, insulin and human growth hormone. Chemicals and fuels - examples are carbon dioxide (used to make dry ice and carbonated beverages), ethanol, glycerol, bio-diesel and methane. Enzyme Production - examples are amylase, invertase, cellulase, protease and pectinase. Biomass Production - examples are yeast and yeast extract.

Other uses of microorganisms include: The treatment of industrial, agricultural and municipal wastes - Many industrial, as well as agricultural and municipal wastes have a high load of BOD and COD which can be detrimental to life in rivers, lakes and oceans. In addition there are hazardous chemical compounds in some of these wastes. The use of microorganisms helps significantly to reduce the BOD and COD and some of these can degrade dangerous chemicals compounds.
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The environmental bioremediation - Processes have been developed with bacteria to degrade contaminants in soil such as gasoline, oil, plastic and metals. Biofiltration - Biofilters can remove certain toxic compounds or compounds that produce objectionable odors in the air. Unlike other filters, the biofilters trap not only the compounds to be separated in the air, but they degrade them too. Agricultural applications - Microorganisms are used for the treatment of compost, silage fermentation and the production of certain compounds that help control pests.

Many people are unaware of how microorganisms affect their lives. In the photos we see a tuna mayonnaise sandwich, a vanilla pudding and a cup of hot chocolate. Which of the ingredients used to prepare these delicacies are or may be prepared by using microorganisms?

Vinegar is a product that comes from the acetic fermentation of wine (1). Some seasonings like mustard and mayonnaise contain vinegar, although they are not fermented, can be considered as related products to fermentation.

SOME MICROORGANISMS AND THEIR USES IN FOOD FERMENTATION

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Microorganism Sacharomyces cerevisiae Zymomonas mobilis Lactobacillus plantarum Acetobacter aceti Lactobacillus bulgaricus S. lactis y S. cremoris Aspergillus oryzae Aspergillus niger

Raw material to ferment barley grape juice cane molasses corn cactus extract cabbage cucumber wine or distilled alcohol milk milk soy sucrose and glucose

It produces beer wine or brandy rum whiskey pulque sauerkraut pickles vinegar yogurt cheese soy sauce citric acid

(1) The term "acetic fermentation" is used here as the definition given for fermentation in engineering and industry (see page 1). In the fields of biology and biochemistry, the process of producing acetic acid is not considered a fermentation, because the microorganisms used require oxygen.

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History of Fermentation

The Early Years

The word fermentation comes from the Latin word fervere which means boiling. When grape juice is fermented to make wine, there is an emission of carbon dioxide bubbles that give the impression that the liquid is boiling. Possibly the first fermented product manufactured by mankind was beer. There is evidence that civilizations like the Babylonians knew the art of brewing 6,000 years before Christ. The Egyptians knew how to manufacture bread before 4,000 BC. It is believed that the wine was prepared around 3,000 BC in northern Iran, where certain varieties of wild grapes grew. For the year 2.400 BC, the production of beer and bread were common practices in these older civilizations. Many of these activities were made in a traditional way. The methodology for the manufacture of these products was in hands of a small group of people who passed the knowledge from generation to generation. It was totally unaware that the fermentation process was closely related to the presence of microorganisms that, through complex chemical reactions catalyzed by enzymes, changed the chemical composition of the raw material.

The Influence of Science and Technology

The mastery of craft techniques for the production of beer happened to be embodied in the brewer. As close as the past 20th century, the beer industry had brew masters in which the success of these industries was based, even though some of these masters were unaware that yeasts were actually living organisms. With the invention of the microscope by Zacharias Jensen in 1595 and its subsequent development, literally the eyes of mankind were opened into a new world. The first person that use the microscope to observe living organisms was Galileo Galilei (1564-1642), who studied about insect parts such as legs and eyes. The study of microscopic organisms (microbiology) probably began with the Dutch Robert Hooke (16351703) who use the term cellulae (small rooms) to describe their observations on what he saw under the microscope when he put into the bright thin cork. He had no awareness of what he saw were dead cells which were once a part of a living tree. Soon the word cell (a word whose root is cellula) began to be used by scientists.

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Observation drawings (1) made by Robert Hooke

Anthony van Leeuwenhoek (1632-1723) (2) Painting by Johannes Vercolje

Anthony van Leeuwenhoek (1632-1723) was probably the first person to observe bacteria. He also observed other microorganisms such as protozoa and spermatozoa. Robert Brown (1773-1858) was the first person to observe the nucleus of a cell. After many years of observation, Theodore Schwann (18101882) English botanist, reasoned that all plants and animals were composed of cells and the cells were themselves independent living organisms. Through the study of microorganisms and the progress in other areas of knowledge, scientist began to find evidence of a direct relationship between the field of biology and chemistry. Schwann showed in 1837 that microorganisms called yeasts were responsible for the formation of alcohol in wine and beer. This contradicted the statements of renowned German chemist Justus van Liebig (1803-1873) that the normal fermentation occurred as a result of chemical decomposition of organic matter. Other independent work related to the role of yeast in alcoholic fermentation was made by Cagniard-Latour and Ktzing.

(1) Image from Wikipedia: http://es.wikipedia.org/wiki/Archivo:RobertHookeMicrographia1665.jpg (2) Image from Wikimedia Commons: http://commons.wikimedia.org/wiki/File:Jan_Verkolje_- _Antonie_van_Leeuwenhoek.jpg

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Robert Brown (1655-1705)

(3)

Theodore Schwann (1810-1882)

(4)

Louis Pasteur (1822-1895) deepened in these studies and found that in fermentations where the wine had a bad taste, the presence of these rod-shaped bacteria (bacilli) was observed. Pasteur is most remembered for his famous experiments with gooseneck flask. This flask was designed to allow passage of air, trapping dust particles that could contaminate the contents with microorganisms. After boiling broth with the intension of killing microorganisms, he introduced it to the bottle and there was no bacterial growth and decomposition, contradicting the theory of spontaneous generation of life from inert matter. Pasteur was the first scientist to establish that some microbes were able to exist without the presence of oxygen, giving them the name of anaerobic to describe them. In honor of the many contributions made by Pasteur the field of microbiology, the term pasteurization was coined, which is related the process of heating a liquid to kill microorganisms. In 1877, Ferdinand Cohn (1828-1898) published his observations on the biology of a rod-shaped bacterium that produced structures known as endospores (or spores). The spores are intermediate forms of bacteria that are extremely resistant to heat and are easily identifiable under the microscope. With the method of heating to kill microorganisms proposed by Pasteur, spores could not be destroyed. A solution to this problem was proposed by the English physicist John Tyndall (1820-1893). Spores under appropriate temperature conditions, germinate into vegetative cells that can be destroyed by heat. So, a treatment of a heating and cooling in an alternating sequence implies a destruction of vegetative cells and the formation of them from the survivor spores. The process of eliminate all microorganisms, whether vegetative cells or spores, is known as sterilization. The sterilization technique developed by Tyndal is known of Tyndallization.
(3) Image from Wikimedia Commons: http://commons.wikimedia.org/wiki/File:Robert_Brown_memorial.png (4) Image from Wikipedia: http://en.wikipedia.org/wiki/File:Schwann_Theodore.jpg

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Louis Pasteur (1822-1895)

(5)

Ferdinand Cohn (1828-1898)

(6)

Continuing the study of microorganisms forced microbiologists to develop techniques for working with these organisms that cannot be seen with the naked eye. Among these techniques is the pasteurization and sterilization. We can also mention the isolation and propagation of strains, an area in which Emil Christian Hansen (1842-1909) was a pioneer for his work with yeast cells in the Carlsberg brewery. The development of microbiology and biology was a somewhat isolated from other fields of knowledge. For example, before the work of Pasteur, Cohn, and Tyndal, pasteurization and commercial sterilization had been made for food, without knowing that the process implied the killing of microorganisms. Nicholas Appert (1750-1841) an ingenious candy maker, chef, brewmaster, and manufacturer of sweet pickles (pickles), was probably the first person to think about packaged food and heat treatment to preserve it. His invention responded to an offer made by the government in 1795 of a reward of 12,000 francs to who ever developed new and efficient methods of food preservation. He worked on the concept for about 15 years and managed to preserve food in glass bottles normally used for wine packaging. Appert cooked foods introduced in these bottles and then tap with corks. After the package and cover, he heated the bottles in boiling water. These foods tested to maintain its integrity for about four months. Later in 1810, Peter Durand patented the concept in England but for canned food. In 1814, Bryan Donkin and John Hall had solved the technical difficulties to market the concept and opened the gates of a factory of canned goods, eight years before Pasteur born. In 1874, three years before Tyndall presented his publication about his method of sterilization, A. K. Shriver invented a closed kettle (retort), probably
(5) Image from Wikipedia: http://es.wikipedia.org/wiki/Archivo:Louis_Pasteur.jpg (6) Image from Wikipedia: http://es.wikipedia.org/wiki/Archivo:Ferdinand_Julius_Cohn_1828-1898.jpg

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John Tyndall (1820-1893)

)

(7)

Nicholas Appert (1750-1841)

(8)

the first system that used steam under pressure for commercial processing of canned foods. Although the system was designed for the treatment of foods products, the fact is that the autoclaves used now to sterilize samples in microbiology laboratories are more similar to the system invented by Shriver than to the process proposed by Tyndall. Into the 20th century, microbiology was already a discipline pretty well organized and structured. Other disciplines such as physics, chemistry and engineering had also developed impressively, and were part of the normal curricula of most universities. Now a day, the field of food fermentation and food technology is enriched by all the knowledge available in science and engineering.

(7) Photo from Wikipedia: http://es.wikipedia.org/wiki/Archivo:John_Tyndall_(scientist).jpg

(8) Photo from Wikipedia: http://en.wikipedia.org/wiki/File:Appert_Nicolas.jpg- Photo by Jean-Paul Barbier

Note: The photos and images used in Chapter 2 are in the public domain.

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Rum
Rum is an alcoholic beverage whose raw material comes from sugar cane. Although the juice of sugar cane may be used, the rum is actually made with sugar cane molasses. Molasses are remnants obtained from the refining process of sugar from cane.

Rum Manufacturing Process

The traditional process for preparing rum can be resumed in these simple steps: Yeast propagation Fermentation of the diluted sugar cane molasses with yeast Decantation or centrifugation of agitated fermentation Distillation of agitated fermentation Aging of the raw rum Final processing Yeast Propagation Usually the yeast strain used in a rum distilleries remains in recipients with a solid growth medium (with nutrients) that are preserved at low temperatures (4C). Periodically, the strains are transferred to new spots following aseptic processes. For the process of propagating yeast media, different volumes and concentrations are prepared with diluted molasses and a nitrogen source (such as ammonium sulfate or urea). The pH of this media is adjusted to 4.7 using sulfuric acid. The volume of each media is prepared in such way that is a predetermined fraction (in size) of the next media to be inoculated. This process of inoculation and transferring media to larger volumes continues until it arrives to the propagator, which is a type of fermenter that provide optimum conditions for the development of the cellular mass in the shortest time possible.

Schematic Diagram of Yeast Propagation Process

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Fermentation of Diluted Sugar Cane Molasses During fermentation, sugars in cane molasses (glucose, fructose and sucrose) are converted mainly into ethanol and carbon dioxide. Yeasts use their enzymes to hydrolyze sucrose into glucose and fructose. Fructose is changed to glucose by an enzyme (isomerase). The glucose is converted to ethanol. The ingredients and conditions for yeast fermentation

The yeast employed will determine the final taste and aroma profile. Distillers that make lighter rums prefer to use faster-working yeasts. Use of slower-working yeasts causes more esters to accumulate during fermentation, allowing for a fuller-tasting rum.

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Decantation (or Centrifugation) and Distillation Once the diluted sugarcane mixture is fermented, is recommended to remove the yeast and other organic material by decantation or centrifugation. After this, the process to separate the ethanol from the rest of the fermented product uses a series of distillation columns. The distillation is a separation method based on the difference of boiling point of substances; the substance with lower boiling points will be distillated first. The final product of distillation is known as raw rum. This is fundamentally a mixture of 94.85% (v/v) of ethanol (189.7 P), water and some traces of compound that provides aroma and the characteristic taste of distilled alcohol from cane. Aging of the Raw Rum Once is obtained the raw rum, it is stored in tanks before being diluted with deionized water. The diluted raw rum is introduced to white oak barrels which are stored for a period of time between one to eight years. During this process, the creation and transference of compounds between the rum and the wood gives a softer and agreeable flavor to the aged rum. Dilution of Raw Rum for Aging

Final Processing This process can involve the mixture of different aged rums following a particular recipe from each rum maker. Then the product is diluted with processed water and treated with activated carbon. During the later process, the rum loses its color. In the case of golden rums the lost of color is compensated with the addition of caramel. Finally, the rum is filtered and the bottled.

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Beer and Other Alcoholic Beverages

Beer
Beer is a fermented (not distilled or aged) beverage that comes from the fermentation of cereals, mostly malted barley. Although in many places of Europe barley is the only source of carbohydrates, in other parts of the world (like the United States) other sources of carbohydrates are grains such as rice and corn. The main ingredients for the production of beer are malted barley, adjuncts (aggregates), hops and water.

Beer Production Process

A. Digestion of raw material (mashing) The malted barley and adjuncts (other cereal sources of carbohydrates) are mashed and heated in water at over 60 C to remove its components. During this process the enzymes from the malt are activated and starch is degraded into a simple sugar (glucose).

B. The addition of hops Once the starch has been degraded, the resulting solution is exposed to boiling temperatures and then the hops are added. The hop contains flavor components that give the characteristic bitter taste to beer. During this process, part of the sugars obtained in the previous step is caramelized, giving the characteristic golden color to the solution.

C. Fermentation The yeast is added (usually freeze dried yeast), to begin the process of fermentation. The sugars present are converted into ethanol and carbon dioxide. A considerable amount of yeast is also formed. Beer fermentation usually takes place at lower temperatures than room temperature.

D. Sedimentation
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The fermented product is stored in cold for a period not exceeding two weeks. During this time, sedimentation of the yeast and other undesirable organic materials occurs. Also during this time other chemical reactions take place, which ultimately gave a better flavor to the beer.

E. Final Processing After sedimentation, beer may be treated with filter aid (bentonite or other similar compounds); this depends on the type of beer produced. In this process certain small organic particles add themselves to the larger filter aid particles and then they are removed by a traditional process of filtration. Filtration with filter aids makes the product translucent and with the particular brightness that characterizes it. The carbon dioxide is added to the product and then it is bottled and pasteurized. Some brewers substitute the process of pasteurization with a cold filtration with membranes with pore diameter less than 0.4 microns to remove all traces of yeast and other microorganisms that could alter the taste of the product.

Processing of Other Alcoholic Beverages

The manufacture of major fermented alcoholic beverages is characterized by some of the stages typically found in the manufacturing of beer and rum. Below is a summary of the manufacturing processes of the most common fermented beverages.

Vodka
Vodka is a drink that is characterized by the absence of flavors and aromas. It is assumed that a good vodka dont provide great changes in flavor of juices or other drinks that are mixed with. The raw material for the production of vodka can be cereals (grains) such as rice and corn, potato and also cane molasses. In the case of using sources of carbohydrates containing starch, it will be necessary to provide a process of digestion to reduce the starches to fermentable sugars, just like in the case of beer. Once this is done, it is proceed with the fermentation of glucose by yeast. After that, the yeast is removed by centrifugation or sedimentation and the fermented mixture is then distilled. Distillation process in the vodka is more rigorous (in terms of the separation of components) than in the rum. When using cane molasses, the obtained alcohol is usually redistilled. It is also very common to treat the distillate with an intense treatment of activated charcoal to reduce any undesirable flavor or aroma. Vodka is not an aged product.

Gin
The first steps to manufacture gin are identical to vodka. Technically speaking we can say that gin is vodka which flavor is imparted through the addition of extracts of plants and species, whether these come from commercial extracts or by an extractive distillation process using vodka alcohol and
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aromatic plants. The raw material for gin is the same as that used for the vodka. Like vodka, gin also is not aged.

Wine
Wine is a fermented fruit extract. Although technically speaking wine can be made from any fruit with high sugar content, we usually associate wines (especially quality wines) to grape juice as raw material. Those wines made from other fruits other than grapes are usually called "fruit wines". While the processes of propagation of yeast can be used in the case of wines, it is quite common that freeze-dried yeasts are used instead. Once the juice is fermented, the yeast is separated by sedimentation and then the fermented product can be stored in barrels for the aging process. Some wines like white wines are not aged.

Brandy
Brandy is a beverage made from fermented grape juice. Technically speaking, the brandy is a distilled grape wine. After distillation, the brandy is aged in oak barrels.

Champagne and other sparkling wines

Sparkling wines and champagnes (which are sparkling wines made in the Champagne province, France) are made from grape wine. The sparkling characteristic of these wines comes from the carbon dioxide produced in a second fermentation of the wine product. Sugar and yeast are added to the wines for these purposes. In the case of the most traditional methods, the second fermentation is done in the product bottles. Periodically, the bottles are inverted (upside down) and placed in chambers of low temperature. The yeast precipitates inside the bottles neck and it freezes due to the low temperatures. The rest of the wine does not freeze because of the alcohol content thereof. Periodically, the bottles are uncorked and the frozen sediments are removed. Some wine is added to compensate the lost in volume. This process is repeated until the sparkling wine is free of sediment and debris. There are industrial processes that allow the production of sparkling wines with their second fermentation in tanks and not inside the bottle.

Whiskey
In the United States, whiskey is a drink whose raw material is corn. In other places around the world, whiskey can be made of other grains. The production process involves a digestion of starch from cereals, fermentation of sugars to produce ethanol, distillation to separate and concentrate the ethanol, aging and final processing (mainly dilution and filtration).

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5
Adjust the solids of whole (or skim) milk to 12 Brix using powder milk. Heat the milk to a temperature between 85 to 90 C for period of 30 min. During this process, the microbial flora of milk is significantly reduced. Moreover, the increase in temperature helps to destabilize the casein enzyme. Cool to 42C and inoculate the milk with plain yogurt with live culture (use approximately 5 mL for every 95 mL of water). Pack the inoculated milk and add fruit or jelly. Incubate the medium at 40C until reaching a pH reading between 4.3 and 4.4 (around 24 hrs if live culture used was storage in the refrigerator). Remove from the incubator refrigerate at 4 C.

Yogurt, Sauerkraut and Vinegar

Yogurt
Yogurt is a product of the fermentation of milk by a combined cultivation of the bacteria Streptococcus thermophilus and Lactobacillus bulgaris. Yogurt is a semisolid food like a pure. Fruit or jelly can be added to improve flavor characteristics. A simple process of yogurt production is summarized below.

During the fermentation of yogurt, the bacterium S. thermophilus proliferates faster than L. bulgaris, leading to the production of diacetyl and acids (lactic, acetic and formic). The rapid growth of S. thermophilus is stimulated by peptides that are formed by the action of protease in milk proteins. Protease is an enzyme that is released by the bacterium L. bulgaris. As acid concentration increases, the pH of the medium decreases causing a decrease in the rate of growth of S. thermophilus, and an increase in the rate of growth of L. bulgaris. Most of the acidification and production of acetaldehyde comes from the L. bulgaris. These compounds are responsible for the aroma and flavor yogurt.

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Lactic acid also contributes to the texture of yogurt, because it helps to decrease the electric charge of casein micelles, causing the formation of flocs.

Sauerkraut
Sauerkraut is a product obtained by fermenting cabbage juice or extract. After fermentation, sauerkraut must have at least 1.5% (w/w) lactic acid. Although the bacterial flora comes from the same leaves of cabbage, you can get a consistent quality product using aseptic practices and control of fermentation conditions. Among the microorganisms involved in the process are Lactobacillus mesentoroides, Lactobacillus brevis and Lactobacillus plantarum. The participation of each of these species in the fermentation process depends on the concentration of added salt and pH. The procedure for the production of sauerkraut is as follows. Cabbage must be stored at room temperature for at least one day before being processed in a well ventilated area. Remove the outer and inner layers of the cabbage. Chop the cabbage into small pieces and extract the juice of cabbage with a press or similar apparatus. Add salt to the juice at a rate of 2 to 3% by weight of cabbage used. Mix the juice with the cabbage and keep the cabbage under the water extract surface using a plastic cover. The fermentation process must proceed at a temperature between 18 to 21 C until the product reaches the 1.5% (w/w) acid content. This can take 3 to 4 weeks. Finally, the cabbage is packed into clean glass jars and pasteurized.

The salt added in the process prevents the reproduction of bacteria that cause putrefaction. Osmotic pressure caused by the salt in the walls of the cabbage cells also helps to draw water from them. The presence of salt contributes to lactic acid fermentation, flavor and firmness of the final product.

Vinegar
Vinegar is a condiment or seasoning that is made by fermentation materials containing sugar or starch and subsequent fermentation of alcohol into acetic acid. Commercial vinegar usually has a concentration of acetic acid from 40 to

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70 grams per liter. The word vinegar comes from French and means literally sour wine. The composition of vinegar depends on the raw materials used and manufacturing conditions, aging and storage. For example, a typical apple vinegar has acetic acid and other compounds in trace amounts such as alcohol, glycerol, esters, salts, and some sugars. In the United States, the Food and Drug Administration (Food and Drug Administration) requires an acetic acid content of not less than 4g / L for products that are identified as vinegar. The process for the production of vinegar varies by commodity. With the exception of distilled vinegar, others vinegars require the fermentation of sugars from the raw material and subsequent acetic fermentation of ethyl alcohol. In the event that the raw material is cereals, a digestion of starches following with a similar procedure to the one used in the production of beer is required. Distilled vinegar is made from distilled ethanol obtained by the fermentation of molasses or grain. In this case, only diluted alcohol is fermented. The alcoholic fermentation of vinegar is usually done using strains of yeast Saccharomyces cerevisiae. Although there is a variety of microorganisms that produce acetic acid, not all are suitable for an acceptable flavor vinegar. The more appropriate include Acetobacter aceti, A. pasteurianus, A. peroxidans and Gluconobacter oxydans. The microorganisms used in fermentation of vinegar acetic require large amounts of oxygen. Specialized fermenting vessels (known as acetators) are designed to provide and uniformly distribute large amounts of air in the fermenting wort. Acetators also provide temperature control systems that remove heat that is produced from catabolic reactions of the process.

The general procedure to make vinegar from fruit is as follows: Extraction of fruit juice. Fermentation by yeast Saccharomyces cerevisiae. This fermentation is usually done with appropriate strains for wine. The fermentation temperature is usually 24 to 27 C. Sedimentation and filtration. Acetic fermentation by Acetobacter aceti or other microorganism. The fermentation temperature is usually between 26 to 29 C. Agitation and air injection is used to maintain large amount of oxygen dissolved. Storage and aging. At this stage the vinegar is stored in barrels or tanks and left there for about a year. This process improves the flavor of

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vinegar and enhances the clarity of it. The tanks or barrels must be filled out completely to avoid the presence of oxygen, as the bacteria could degrade the acetic acid oxidation. Clarification using filter aid. Bottling and pasteurization.

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