After the juice extraction, it is filtered through cloth and mixed with the sugar syrup in equal amounts inside a clean and dry plastic bucket. Due to the amount of wine they produce, the mixture is made in large buckets with approximately 50 liters of capacity.
Filtering the Orange juice
The biggest concern of Mr. Zé and Mrs. Ná is that the containers are cleaned and dried perfectly. According to them, “the science to make the wine is this and cannot change, the thing already came with this requisite, my mother learned this way and we do so as well”. The mixture intended for alcoholic fermentation (tecnically know by mosto) “is susceptible to acquire the smell and the taste that by chance may exist in the containers. Those which were in contact with fatty, oily or derived substances such as soap, kerosene, lard, grease, etc., transmit to the mosto its organoleptic properties, i.e., color, smell, taste” (Lima et al, 1975).
After they mixed the juice with the syrup, they put it into glass demijohns of five or 30 liters, the biggest externally coated with cement paste.
Mixture of Orange juice with the sugar syrup
The demijohns containing the mixture are then capped with cork stoppers that receive a beeswax layer around. Regarding the sealing of the demijohns Mr. Zé said: “The air is very important thing, it cannot enter, if it enters weakens the wine”, it prejudices the fermentation, but for Mrs. Ná: “If the air enters it’s possible that doesn’t ferment”. Dr. Rosane Freitas Schwan, microbiologista expert in yeasts from the Federal University of Lavras in Brazil, told us that fruit wines fermentation is carried out under anaerobic conditions, in the absence of oxygen, but that the presence of this gas in the initial step of the process is beneficial for the microorganisms’ growth. She even suggested for Mr. Zé and Mrs. Ná to use a small air pump as the type commonly used in aquariums, for the medium initial oxygenation.
The microorganism that is predominantly responsible for fermentation in the orange wine brewing is the Saccharomyces cerevisiae, a unicellular fungus widely distributed in nature that is originally present in orange and sugar cane juices, which is able to tolerate the medium conditions: high initial sugar concentration, acidity and temperature variations. These fungi survive in both aerobic and anaerobic conditions, but as already mentioned it is under anaerobiose that they make the fermentation, the process of converting sugar into ethanol (alcohol) with energy production in the form of ATP (adenosine triphosphate) that will be used by the Saccharomyces to fulfill many physiological activities as absorption and excretion, for instance, and to the required biosynthesis for life sustaining, such as growth and multiplication. Mrs. Ná said: “it is the ferment that makes it” and Mr. Zé: “it is the bitterness that is there inside”, the yeast Saccharomyces cerevisiae.
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He said that “it boils inside the demijohn to turn wine. It releases a gas that bubbles like when water boils to make coffee”. The fermentation produces also the carbon dioxide (carbonic gas, CO2) and “when it´s cold it boils slower”. At lower temperature the fermentative activity decreases producing lesser amount of gas. The word fermentation derives from Latin fermentare and was given by Louis Pasteur due the boiling aspect caused by the release of carbon dioxide. Pasteur was also who showed that fermentation is a vital process for some microorganisms. Soon, it became the general name for the anaerobic degradation of glucose or other organic nutrients with energy generation and conservation in the form of ATP (Nelson & Cox, 2006). The expression anaerobic glycolysis is also known (from the Greek glykys = sweet and lysis = break).
An aspect of alcoholic fermentation is the mechanism of sugars transportation into the Saccharomyces cerevisiae cell, where happens the conversion to ethanol. Due to its size, sucrose cannot penetrate through the cell membrane and is hydrolyzed in the external side with the aid of an enzyme produced by the yeast, the invertase. That produces glucose and fructose:
The glucose and fructose have the same chemical composition, differentiating on the positions of their atoms according the representations below:
They either do not cross the cell membrane easily, requiring the action of carrier proteins: the permeases, which, however, have a higher affinity for glucose. Thus, this molecule is captured faster from the medium and is carried into the cell by means of what is called facilitated diffusion (Zastrow & Stambuk, 2000).
The process of converting glucose in ethanol, the alcoholic fermentation, involves a sequence of 12 steps and enzymes. Its clearance involved enormous amount of research and at least 50 scientists during the first half of the twentieth century, with the development of methods, equipment, isolation and purification of substances and discussions that put the science of Biochemistry forward. The research followed two investigation routes: one involving the fermentation of yeast and other directed to the understanding of the lactic fermentation in the muscles, with similarities and interconnections.
Some scientists involved were refugees from Nazi Germany and developed their research in England and the United States. Six of them were awarded with Nobel Prizes for their contributions to the glycolysis steps elucidation, which was completely established in 1940 after a little more than 30 years of studies (Barnett, 2003). The equation showed below displays in a simplified manner the starting reactant (glucose), the enzymes participation and the reaction products: ethanol and carbon dioxide, but does not allow to realize the details, the complexity of the overall process and the intermediaries involved:
As Mr. Zé mentioned: “it doesn’t have any drop of alcohol in the wine. It’s on to ferment that gives the alcohol. We don’t put alcohol in the wine. It turns there inside. Its fermentation is that creates this alcohol”. When he said “fermentation”, he is using the same expression given to the process by the scientist Louis Pasteur. How did this word came to them? The verb “to ferment” and what Mrs. Ná said: “it is the ferment that makes it” and “If the air enters it’s possible that doesn’t ferment” are derived.
The final products of the alcoholic fermentation, the carbon dioxide (CO2) and ethanol (C2H5OH), have no use for the yeast (Lima et al, 2005). Because they are small molecules that interact properly with the lipid layer of the membrane cell they cross it by simple diffusion and are released out (Yuan et al, 2000; Zastrow and Stambuk, 2000) as Mr. Zé said: “it boils inside the demijohn to turn wine”, “it releases a gas that burbles like when water boils to make coffee”.
The mixture remains isolated inside the demijohns for eight days. Why this time? After this period, the cork stoppers are removed and replaced by small bamboo cylinders, measuring four to eight centimeters long, with a diameter that fits the demijohns´mouths. On these, they adjust the end of a plastic little hose of approximately 40 centimeters long and pass beeswax around to seal. They also use pierced cork stoppers rather the bamboo cylinders, adapt the little hose on them and insert in the demijohn’s mouth to cover it.
The free end of the little hoses is placed into a small container with water where the gas produced on fermentation bubbles: “have to put to ferment, if doesn’t put it doesn’t ferment” (Mrs. Ná), “have to put it into the glass of water to do not bring the air inside, the air has only to come out”. “The air is very important thing. It cannot come into. If it does it weakens the wine” (Mr. Zé). There is the requirement to ensure anaerobic condition to do not prejudice the fermentation. Therefore, they seal the demijohns’ mouth with stoppers containing hoses whose free ends are placed inside a glass with water to the gas exit. The fermentation releases carbon dioxide, but requires the oxygen absence.
The fermentation ends after around two months, where there is substantial decrease in the amount of the gas that bubbles in the container with water. In this step, the quantity of the ethanol produced affects the metabolism of the yeast. The yeast die when the alcohol content is above 16%. In 6% and 15% only 0.25% and 0.05% of the yeasts survive, respectively (Peixoto, 2002, p.302).
As the fermentation ends, the bamboo cylinders or the cork stoppers and the hoses are removed and the demijohns are again capped with normal cork stoppers. The wine is almost ready, but remains in the demijohns for more two months. A dreg is deposited in the bottom of them, which according to Mr. Zé is the “orange dirt or acid” probably composed by dead yeast or solids that were not properly separated by the filtration method used. After this time, they remove the wine by suction of the supernatant liquid using a hose. The wine prepared like that has a honey color with light orange taste. It is stored in glass bottles and it is ready to be tasted during meals and especially in festive days or family celebrations.
It is interesting to observe that the orange wine making by Mr. Zé and Mrs. Ná does not involve any explicit scientific knowledge or advanced technological resource. Surely that Ronaldo, their son, know a little more about what is involved in the production of the wine of their parents and should now see it otherwise. However, will this change the way his family make the orange wine? Will help them to produce a better wine? Will place the orange wine on the supermarket shelves? Does science and technology may alter these knowledge and practices?
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Barnett, J.A. (2003). A history of research on yeast 5: the fermentation pathway. Yeast, 20, p. 509 – 543.
Lima, U.A.; Aquarone, E.; Borzani, W. (1975). Tecnologia das fermentações. São Paulo: Edgard Blücher.
Lima, U.A.; Aquarone, E.; Borzani, W.; Schmidell, W. (2005). Biotecnologia Industrial. V. 3. São Paulo: Edgard Blücher.
Peixoto, A.M. (Coord.) (2002). Enciclopédia Agrícola Brasileira. São Paulo: Editora da Universidade de São Paulo.
Nelson, D.L.; Cox, M.M. Lehninger princípios de Bioquímica. 4.ed. São Paulo: Sarvier, 2006.
Yuan, Y.J.; Obuchi, K; Kuriyama, H. (2000). Dynamics of ethanol translocation in Saccharomyces cerevisae as detected by 13C-NMR. Biochimica et Biophysica Acta, 1474, p. 269 – 272.
Zastrow, C.R.; Stambuk, B.U. (2000). Transporte e fermentação de açúcares por leveduras da indústria cervejeira. Revista Univille, 5(1), p. 39 – 44.