Patent Application
20120258197
A first object of the invention relates to the development of a vineyard culture method resistant to meteorological and/or climatic roughness by developing deep and strong roots.
A second object of the invention relates to the development of a thorough fermentation method with wild grape yeasts reminiscing the soil and possessing its organoleptic properties as well as methods to obtain wines of high alcoholic grade.
A third object of the invention relates to the development of a method to obtain and use wild grape yeasts capable of fermenting sugars in low as well as high concentrations from sucrose solutions, commercial sugar, sugar cane molasses, beet molasses, fermentation of sugars from starch hydrolysis and fermentation of sugars from vegetal matter.
It is desirable for yeasts to complete fermentation of all sugar present in the grape juice during wine elaboration, thus a high alcohol degree, normally up to 14% in volume or not much higher, provides wine its final and aromatic structure.
Grape must fermentation in practice may be achieved through wild yeasts, inoculated yeasts or applying both procedures. Wild yeasts responsible for fermentation are naturally found in grape husks (generally as a fine white powder layer covering the grape's skin (vitis vinifera I.) named “pruina”. Specific cultured yeast strains yield specific fruitlike aromas, high alcohol degree, nose agreeable texture and other characteristics like fermenting at low temperatures or with relatively low pH, etc.
Most wine cellars normally use specific selected yeasts to turn sugar into alcohol; Saccharomyces cerevisiae being the best yeast species recommended for total alcohol fermentation.
Fermentation through wild or indigenous yeasts although not very extended, is mostly practiced by some European winemakers who traditionally ferment with wild yeasts with very good results. Likewise, Californian winemakers are also beginning to employ this spontaneous fermentation with very favorable results also. During vintage, grapes carry thousands of organisms, yeasts included, with the ensuing risk of infection. One of the most common characteristics of wild yeasts resides in its low resistance to alcohol and scarce resistance to infections.
However, many types of wild yeasts are incapable of acting once a 9% vol. alcohol level is reached, clogging fermentation and resulting in a wine without consistency, with a low immunity system and a great amount of residual unwanted sugar among other problems. Time needed for wild yeasts to establish colonies, wherein must remains exposed to infection by other organisms having a faster development or to oxidation, is an additional difficulty encountered when using wild yeasts in fermentation; besides, once fermentation begins, it is a lengthy and slow process with no guarantee of a satisfactory ending. The unpredictability of aromas and esters introduced by wild yeasts in wine is also a problem; however, selected or commercial laboratory yeasts enable attainment of such flavors and aromas as desired by winemakers' taste.
The fact that it takes longer to initiate fermentation, allowing more contact time with the grape's skin, thus translated into more body, depth of character, color and more fruitlike flavors (that is, much more variables than with industrial wines) is an advantage related to employment of wild yeasts. There is confirmation that many of the unpredictable aromas and esters conveyed by wild yeasts confer an interesting sophisticated nature to wine, resulting in a wine with complex flavors, good bouquet and very good alcohol.
A way to influence vintage is through irrigation; however, the effect of irrigation in relation to the wines' alcoholic grade redounds in a decrease in concentration of sugars due to the effect of dilution. The production increases when comparing irrigated and non irrigated grapevines, observing that phenol compounds and intensity of colorants decrease in wines from irrigated grapevines. The must shows an increase in malic acid in grapes from irrigated grapevines. The effect of water contributions at the end of maturation influences negatively in the composition and quality of final wine due to a decrease in concentration of elements, owing to dilution produced by water in the berry.
In known art there are very few works exploring in depth factors influencing wine quality, since factors influencing the fermentative glycolysis of sugars by wild yeasts are complex, due to existing interrelation among them and to the nature of parameters intervening during the fermentation process.
Object of paper FR 2.844.275 relates to a natural wine fermentation procedure with wild yeasts characterized by the following steps:
It does not employ pumps or tanks for reshuffling, only barrels.
Patent WO 2004.029.193 presents a production method for a fermentation product comprising a fermentation stage including contact with a microorganism in fermentation or fermentation means used with at least an esterase enzyme as for example: lipase, phitase, phospholipase and cutinase.
However, in known art there are no references to vineyard culture focusing on attainment of wild yeast strains to perform total sugar fermentation.
A method to obtain wild yeasts achieving total sugar fermentation and guaranteed to overcome wine infections would undoubtedly be of practical interest.
The present invention is related to a culture method of a vineyard resistant to meteorological and/or climatic roughness through development of deep and strong roots, a culture method through which wild yeasts achieving the wine fermentation process develop; and a method of thorough fermentation to obtain total transformation of sugar into alcohol, developing and multiplying these wild yeasts in grape juices and achieving a high alcohol degree, totally superior to that obtained through conventional fermentation practiced to date.
The natural culture method consists in obtaining a totally natural wine, starting by grapevine selection, such that the grapevine develops and produces fruit by itself and through its own yeasts. Human intervention focuses on the preparation of the soil, cultivating the herbs for further burying as organic yield to the soil “in situ”, with no addition of fertilizer whatsoever, the plant becoming strong in a natural way and adapting to the soil by itself. Following this procedure there is confirmation through the years that harvest could be delayed, thus obtaining grapes with more sugar proportion; collecting even in January and February, although in recent years it has been possible to advance it somewhat, the result remaining the same as to quality and production increase.
The method begins from the very moment of vine selection until yeast is obtained from these strains, to be genetically treated further in their case, achieving a faster multiplication and to be predominant in the fermentation process, that is, developing in such quantity and strength as to prevail in face of other yeasts that might appear, furthermore attaining total fermentation of sugars to obtain a wine with character and very good alcohol, rendering it longevity, aromas and flavors, that are not only reminiscences of the fruit but also of the soil and environment where the grapevines have been cultivated at, solving an actual problem consisting in that fermentation with a high degree of alcohol could not be obtained through wild or indigenous yeasts.
Essential characteristic stages of the natural culture method are:
The thorough fermentation method with wild grapes' yeasts reminisce the soil possessing his organoleptic properties; and more important, they ferment the totality of sugars contained in the grapes. The thorough fermentation method does not employ any type of additional yeast, fermenting only through wild yeasts of the own grapes, that is, fermentation is accomplished with yeasts obtained from grapes grown by the natural culture method. These wild yeasts of the own grapes whose properties greatly reminisce the soil are capable of fermentation even with high alcohol percentages. In wine cellars there is evidence of an evolution in time of wild yeasts yielded by grapes, wherein an enhancement and standardization of flavors, aromas and bouquet has taken place, as alcohol was ever much better until 2004, wherein its properties have stabilized, yielding since then wines more regular as to their properties.
Furthermore, the thorough fermentation method with wild grapes' yeasts enables obtainment of grape juices with ever more superior alcohol contents, allowing direct obtainment of alcoholic beverages as cognacs, etc. Finally, the method of the present invention creates conditions for industrial production of bio-ethanol with more effective energetic ratings, in comparison to those presently obtained in industrial production of bio-ethanol through fermentation of raw materials such as sugar cane or beet juice.
Essential characteristic stages of the thorough fermentation method are:
In
a). Culture Conditions:
Work initiated in 1989, selecting Bobal, Crujidera, Royal and Tardana autochthonous grapevines. Since the very beginning agriculture was totally natural, no chemical or organic fertilizer was employed except compost elaborated with herbs from the soil wherein the grapevines were planted. The former object corresponds to the First Object of the Invention, resistant vineyard culture method, consisting in achievement of resistant grapevines by themselves, able to be self-sufficient and relying on the soil's own nutrients, thereby developing deep and strong roots. Thus, the grape acquires flavors and aromas reminiscing the soil they are cultivated at. There was no irrigation though rainfall rate in the area was under 500 l/m2, typical of a continental dry climate (between 300 to 500 mm rainfall). This also corresponds to the First Object of the Invention for the culture type, furthermore enabling adaptation to this climatic condition of yeasts present in the grapes' skin.
b) Collection Chronology and Characteristics of Grapes:
According to the Second Object of the Invention, wine is obtained:
2.1 Production since 2002: In 2002 and 2003 juices were produced with 15.5° alcohol, obtaining in 2004 a concentration of alcohol between 15.5 and 18.98% volume.
In this manner a wine is obtained with body and very good alcohol, thus assuring its quality in time and with very good flavors, reminiscing the environment and the soil, said aromas reminiscing the fruit they came from and obtaining a characteristic bouquet and personality that, due to the longevity of the grapevines (old vineyards with more than 70 years), obtaining a greater quality in fermented juices.
2.2 Characterization of Autochthonous Wild Yeasts
According to the Third Object of the Invention, Method to obtain autochthonous wild yeasts, yeasts causing fermentation corresponding to the Second Object of the Invention were subjected to laboratory studies, extracting representative samples of the wine produced.
Samples: Collected samples belonged to the 2004 vintage and were:
Study of samples in plates:
The two samples were seeded in two plates in YPD medium observing occurrence of some filamentous fungi indicated in
2.3 Tests of Isolated Yeasts at Different Ethanol Concentrations
a.—Seeding of glycerinates from the three yeasts—Growth of yeasts at different ethanol and glucose concentrations.
Glycerinates of each of the three yeasts were carried out and seeded in media with different ethanol levels to see their development capability. Two types of media were prepared, one with glucose (1%) and another one without glucose. After some unsuccessful preparation intents in YPD medium with ethanol since elevated concentrations of ethanol fluidize in excess the YPD medium, an adequate procedure was carried out. The medium solidified by increasing the amount of agar depending on the amount of ethanol to be further added. The yeasts were seeded in these plates, carefully seeding the same amount of yeasts in all plates, obtaining the following results, see
1.—White yeast grows appropriately in all ethanol concentrations and in great amounts either in medium with glucose (1%) or in medium without glucose, for it can use as feeding source the carbon of ethanol as well as the peptone and the yeast extract, all of them components of the YPD medium.
2.—Yellow yeast presents a different morphology depending on the medium containing glucose or not. In glucose media it grows in big colonies, while in media without glucose it grows in smaller colonies, but in a much greater number of said colonies; producing in both cases a reduction of the quantity of yeast obtained by increasing the percentage of ethanol.
3.—Slow growth yeast as well as yellow yeast grows in a different manner depending on the medium containing glucose or not. In this case, the behavior is opposite to that of yellow yeast, thus colonies in medium without glucose present a bigger size than colonies in medium with glucose, although in smaller number of colonies. As to growth in different ethanol levels, at higher ethanol levels they grow with more difficulty.
b.—Yeasts growth at higher ethanol concentrations
In further tests ethanol concentration was increased to determine ethanol limit concentration resisted by each type of yeast. This test posed a problem since the amount of ethanol to be added was rather high and the medium could have problems in solidifying, so the decision was to increase concentration of agar in the medium from 2% to 3%. The amount of glucose employed in the medium was 1%.
The following results were obtained:
Therefore in a preliminary way as a conclusion, slow growth yeast does not resist ethanol concentration conditions over 25% volume, while white yeast resists very high concentrations of ethanol; as for yellow yeast it is very difficult to grow in all ethanol concentrations, thus growing in very small amounts.
c.—Yeast tests at even higher ethanol levels
Tested ethanol levels in these plates were: 40%, 45%, 50% and 60% volume. Results obtained (see
d.—Morphology of the different yeast types
The different types of yeasts were observed under the microscope.
A culture medium is prepared in flasks with different concentrations weight/volume of commercial sugar. The medium components are:
Sugar concentrations are: 1, 2, 10, 15, 20, 25, 30, 40 and 50%, all of them in weight/volume percentage.
Preparation of white yeast 1, yellow yeast 3, slow growth yeast 2 strains to be inoculated in different media:
The yeasts are seeded in liquid medium YPD (5 ml), colonies of the different strains grown in plates of YPD medium, each strain separately, and agitated during 24 hours at 240 rpm and 28° C. Once this time has elapsed and all the strains grown, 20 μl of each of them are placed in a test tube with 5 ml of the corresponding medium in each tube, agitating them at 240 rpm during 24 hours.
Once this time has elapsed each medium presents the following growth
Absorbance is measured in the tests to evaluate cellular growth in culture medium. Measurement of absorbance is directly related to the amount of yeast cells in a determined volume of culture medium. The difference measured by spectrophotometer between intensity of light emitted by the lamp and that reaching the detector once the sample is traversed, that is, the amount of light absorbed by the cells should be greater corresponding to a greater number of cells in the sample.
These growth values afford an idea that these yeasts have great resistance to high sugar concentrations.
The test tube content was transferred into a flask with culture medium at its corresponding concentration, leaving a total volume of 40 ml. It was agitated at 240 rpm during 4 hours, so inoculated cells would multiply. Once this time elapsed the agitation stopped leaving cultures at 31° C. in anaerobic conditions. A small agitation (22 rpm) followed so cells would be well spread in the whole culture. At this time the fermentation begun. The carbohydrates' concentration was determined the following week by the Phenol-Sulphuric Dubois Colorimetric Method obtaining the following percentages:
Therefore reduction of sugar contents is observed in all the flasks, indicating fermentation has been accomplished at all tested concentrations.
The amount of ethanol that could be found in these cases is theoretically described as follows: for example, for the initial 20% sugar concentration a final concentration of 9.5% sugar is obtained, this means that 10.5 g. of sugar are consumed in 100 ml of initial juice.
The reaction describing the alcoholic fermentation is as follows:
C5H12O6+2Pi+2 ADP→2CH3—CH2OH+2CO2+2 ATP
The stoichiometric relation expresses that by each mol of glucose two mol of ethanol are obtained.
100 ml of juice→10.5 grams glucose
10.5 grams glucose→0.058 mol. glucose (MP glucose=180.16 gr/mol)
0.058 mol glucose→0.116 mol ethanol (stechiometry 1:2)
0.116 mol ethanol→5.370 grams ethanol (MP ethanol=46.07 gr/mol)
5.370 grams ethanol→6.806 ml ethanol (ethanol density=0.789 gr/ml)
Summary: 100 ml of juice→6.806 ml ethanol. Considering real yield is 80% of the theoretical: 6.806×0.8=5.445 ml of ethanol. That is, in the example from an initial 20% concentration of sugar a final approximate concentration of alcohol of 5.4% is obtained.
Since it seems these yeast strains resist without difficulty sugar concentrations up to 50%, as following step the sugar concentration was increased. Tests were made with 60 and 70% (weight/volume) sugar concentrations, see
It is confirmed that strains grow even in medium with a 70% sugar concentration; at the end of fermentation of this juice theoretical alcoholic grade to be obtained would be 45.37°. Calculation algorithm of this datum is presented further.
The reaction describing alcoholic fermentation is as follows:
C6H12O6+2 Pi+2 ADP→2CH3—CH2OH+2CO2+2 ATP
The estechiometric relation shows that for each mol of glucose two mol of ethanol are obtained.
100 ml of juice→70 grams glucose
70 grams glucose→0.338 mol glucose (M.B. glucose=180.16 gr/mol)
0.338 mol glucose→0.777 mol ethanol (stoichiometric 1:2)
0.777 mol ethanol→35.8 grams ethanol (MP ethanol=46.07 g/mol)
35.8 grams ethanol→45.37 ml ethanol (ethanol density=0.789 g/ml)
Summary: 100 ml of juice→45.37 ml ethanol. Considering real yield is 80% of the theoretical: 45.37×0.8=36.30 ml of ethanol, or a concentration of 36.35 vol.
Sugar cane molasses were used as base for culture medium. From molasses different dilutions were made to test behavior of strains before different sugar levels. The starting molasses had 78.9° Brix and 49.9% sucrose content.
The following table shows prepared media:
Procedure for strain inoculation is as follows:
One colony of each strain, White Yeast 1, Slow Growth Yeast 2 and Yellow Yeast 3 is seeded in YPD medium re-suspending them together in 1 ml of water. A 5 ml aliquot of each prepared medium is placed in test tubes, inoculating 50 μl of the strains' mixture in each tube. It is agitated at 240 rpm and 28° C. At 66 hours the strains show the following growth.
OD600 values of each sample are independent from the others, it being proof of how yeasts have adapted to that medium.
The following step consists in transferring an aliquot of a 1/1000 dilution of the content of these tubes to its corresponding flask. Once the yeasts are inoculated to the flask, agitation is applied at 240 rpm and 28° C. At 16 hours they showed the following growth:
From 16 hours on the agitation is reduced to decrease oxygen supply allowing beginning of fermentation.
Another experiment was carried out with molasses from beets; these molasses had a 76.8° Brix concentration. Sucrose content of molasses was 47%. The following molasses dilutions were prepared for the preparation of media:
In these media studied strains of White Yeast 1, Slow Growth Yeast 2 and Yellow Yeast 3 were inoculated and agitated at 240 rpm and 28° C. Observed growth after a week of agitation was:
This table indicates how from 30.5% sucrose, yeasts find more resistance for reproduction, manifesting in a decrease in absorbance, although still at 42.7% persists formation of foam having enough capability to carry out alcoholic fermentation.
This can be seen in
The present embodiment is unquestionably of practical interest since starch is the origin of treatments for production of both, bread or beer. Different dissolutions of starch in water were prepared; starch is not soluble in water so it remains suspended. To hydrolyze it a method of acid hydrolysis was used consisting in lowering pH to 0.8 with sulphuric acid and agitation at 84° C. during 6 hours.
Starch concentrations used were: 10%, 20%, 40% and 50%, all of them referred as weight/volume percentage. Once the starch was hydrolyzed pH was neutralized with NaOH and strains from a saturated culture in YPD medium inoculated; a saturated culture is one in which the number of cells remains constant, that is cells die and are born in the same number. 170 μl of culture from each strain, the mixture of White Yeast 1, Slow Growth Yeast 2, and Yellow Yeast 3, were inoculated.
At 48 hours of agitation at 240 rpm and 28° C. the media presented the following growth:
Initial percentage of starch in the flask
As shown in the previous table, the lower OD600 correspond to flasks prepared with a higher starch concentration wherein yeasts find higher resistance to survive and thus their number is smaller. At 48 hours agitation stops so there is no oxygen supply, allowing end of fermentation.
Since flour contains starch and yeasts are capable of fermenting starch, the use of these three yeast strains simultaneously in the fermentation process may confer higher quality, whatever the flour fermentation product desired, since the coordinated action of the three yeasts will enhance in itself the fermentation process. Yeast that works better in glucose (Yellow yeast 3) can cover the other two in media with a high content of same (they must survive in 70% sucrose anyhow), Low Growth Yeast 2 can create a veil favoring oxygen absence, providing all the advantages to produce a better fermentation and lastly, White Yeast 1 can grow in medium of high ethanol concentrations and maybe protect the other yeast species present in the process. Consequently in any process where fermentation from fermentable sugars is present, these yeasts can presumably provide a higher quality to the product obtained.
To carry out this experiment the Nicotiana glauca species was selected as an example of vegetal matter from wild plants, taking samples from different parts of the plants as root, green stalk, woody stalk and leaves. To obtain the plants' sugars, these were crushed and the crushing mixed with water, so soluble sugars of the plants dissolved in water. At this point, the amounts (in percentage weight/volume) of sugar present in the media were:
The samples were inoculated with yeasts from a saturated culture in YPD medium. 35 μl of culture from each strain, White Yeast 1, Slow Growth Yeast 2 and Yellow Yeast 3, were inoculated. These media were agitated at 240 rpm and 28° C. for 48 hours; next cellular growth and amount of sugar in the samples was measured to determine sugar consumed.
Data show how yeasts consumed initial sugar. Agitation of media stopped to impede oxygenation thus allowing beginning of fermentation. As well as in previous figures, bubbles can be seen in
The experiment was repeated in media wherein sugar concentrations were higher. To increase content of sugars in media, an acid hydrolysis of vegetal matter was carried out thus obtaining the following sugar concentrations:
At this time strains from YPD medium saturated culture were inoculated with 35 μl of culture from each strain. These media were kept under agitation at 240 rpm and 28° C. for 48 hours measuring cellular growth once this time elapsed.
Therefore it can be confirmed that increasing sugar content does not affect yeasts.
Discussion
Different factors can be deduced from observed results previously presented. The simultaneous use of the three yeast strains of the present application in the fermentation process, that is in a coordinated manner can confer greater quality whatever the fermentation product to be obtained may be, since the coordinated action of the three yeasts will enhance in itself the fermentation process. As these three yeasts were obtained from casks, the three must be at the end of the fermentation process wherein ethanol concentrations approach 16-19 degrees. Better functioning yeast in glucose (Yellow Yeast 3) can cover the other two in media of high content of same (they must survive in 70% sucrose anyhow, confirmed by results obtained), Slow Growth Yeast 2 can create a veil favoring oxygen absence providing all the necessary advantages to produce a better fermentation and lastly White Yeast 1 can grow in a medium with very high ethanol concentrations, maybe protecting the other yeast species present in the process, so in this manner—protecting the set of yeasts—a further enhancement of obtained juices is achieved or whatever the obtained product may be. Consequently, in any process wherein fermentation is present, from fermentable sugars, these yeasts can presumably provide a higher quality to the product obtained. Therefore this would explain why a higher quality of the product obtained through different fermentable sources would be achieved (sucrose, molasses, starch, must, etc.).
On the other hand this coordinated action together with each strain's own characteristics (analyzed in the present Description) explains why solutions with a very elevated sucrose concentration (or succedaneums) can ferment and thus obtain products with a very high ethanol concentration (most yeast strains cannot endure ethanol concentrations over 13%.)
Coordinated action of these three yeasts together with initial presence of greater amounts of sugars makes the fermentation richer in secondary products. Diverse matters, acting as fermentation sources and covering a wide spectrum of representative products of the fermentation industry as sugar, starch, molasses and the rest of vegetables, etc. have been analyzed. Microorganisms need adequate conditions to grow, reproduce and develop. This is not always possible. Since the three yeasts studied in the present application are capable of fermenting in a simple medium containing starch, it can be inferred that they might be useful in those processes using starch as previous origin to the carbon source for fermentation. It is known that yeasts are capable of fermenting hydrolyzed starch and that yeasts are nevertheless necessary in the production of beer.
Thus it might be inferred from previous analysis herewith stated that from mixture of these yeasts the following can be obtained:
1.—greater ethanol concentrations
2.—higher quality products
3.—fermentation of non fermenting matters to date due to their high sugar content and ensuing high ethanol production.
Outlining Positive Results
Work of Yeasts Presented in the Present Application:
Once the invention has been sufficiently described, as well as some preferred embodiments of same, it should only be added that modifications to its constitution and materials used are possible without departing from its scope, defined in the ensuing claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| P200901997 | Oct 2009 | ES | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/ES2010/000421 | 10/15/2010 | WO | 00 | 6/28/2012 |
