Phosphorus-based composition for yeast fermentation, method for applying the composition and use of the composition

Abstract

The present invention refers to a phosphorous-based composition to be used in ethanol / alcohol production, specifically used in the fermentation step, more particularly, such composition is suitable for improving the yeast metabolism in a fermentation process, thereby, increasing the ethanol yield production, without resulting in increased costs or modifications in the production process. A method for applying said composition in an ethanol process and the use of the composition are also described.

Description

[0001] PHOSPHORUS-BASED COMPOSITION FOR YEAST FERMENTATION, METHOD FOR APPLYING THE COMPOSITION AND USE OF THE COMPOSITION TECHNICAL FIELD

[0002] The present invention refers to a phosphorous-based composition to be used in ethanol / alcohol production, specifically to be used in the fermentation step, more particularly, such composition is suitable for improving the yeast metabolism in a fermentation process, thereby, increasing the ethanol yield production, without resulting in increased costs or modifications in the production process. A method for applying said composition in an ethanol process and use of the composition are also described.

[0003] BACKGROUND OF THE INVENTION

[0004] According to a conventional ethanol / alcohol production process, the fermentation step requires the use of yeast that, as an independent living entity, ferments sugar with the aim of obtaining the chemical energy necessary for its survival, with ethanol being only a by-product of this process. Considering that human being intends to benefit from this metabolic ability, they must seek knowledge that allows to provide a yeast with ideal conditions to increase the yield in the production of ethanol.

[0005] The transformation of sugar (glucose) to ethanol and CO2 involves twelve reactions in an ordered sequence, each catalyzed by a specific enzyme. Such enzymes are acted upon by various factors (mineral nutrients, vitamins, inhibitors, substances involved in metabolism, pH, temperature, etc.) some that stimulate others that repress enzymatic action, thus, affecting the performance of the process.

[0006] The primary objective of the yeast, when anaerobically metabolizing sugar, is to generate a chemical energy (ATP) that will be used to carry out various physiological works and biosynthesis to maintain life, growth, and multiplication, thus, perpetuating the species.

[0007] The transformation of the raw material into alcohol is carried out by microorganisms, usually the Saccharomyces Cerevisiae species, through alcoholic fermentation. For fermentation to be successful, it is very important that an amount of yeast capable of converting sugars into alcohol and carbon dioxide is mixed into the wort. This group of microorganisms is usually called as initial inoculum.

[0008] The fermentative activity of the yeast is used in processes to produce fermented drinks (wines, beer, whiskey, rum, etc.), bioethanol and bread.

[0009] Especially in fermentation for bioethanol production, the yeast cells are used up to three times a day. After each fermentation cycle or during the continuous fermentation process, the cells are separated from the fermented medium by centrifugation (Melle-Boinot process) or decantation and reused in the process. In the Melle-Boinot process, the wine is pumped from the reaction tanks to be separated in a centrifuge as soon as the sugars in the fermentation are exhausted. Then, the yeast (milk) is separated from the yeast-free wine or centrifuged wine.

[0010] The main mineral nutrients for the yeast, to be used in a fermentation step, are the following: potassium, magnesium, calcium, zinc, manganese, iron, copper, molybdenum, cobalt, boron, nitrogen, sulfur, and phosphorus.

[0011] More specifically, the use of phosphorus as a mineral nutrient is well known in the state of the art. The phosphorus actively participates in the processes of transformation and transfer of chemical energy (glycolysis, fermentation, respiration, biosynthesis, etc.), in addition to integrating DNA and RNA molecules and structural molecules (phospholipid membrane). Phosphorus is, therefore, an essential nutrient for yeast multiplication as well as fermentation.

[0012] According to the literature, the yeast absorbs phosphorus in the form of the phosphate or H2PO4 ion. This feature can be reinforced in AMORIM, H.V. (1977)1which state that phosphorous is absorbed by yeast in monovalent form of anion H2PO . At a pH of 4.5 the phosphate anion is completely in the form of H2PO4 therefore, this pH is optimal for phosphorus absorption by yeast.

[0013] According to SANTOS, A. (2008)2, phosphorus, in the form of P2O5, is extremely important for the alcohol formation during fermentation. In addition to promoting the action of yeast, phosphorus also increases the alcoholic yield of fermentation.

[0014] The use of nutrients for yeasts in a fermentation process has already been widely explored in the prior art, in which several compositions containing mixtures of nutrients and appropriate concentrations were developed to optimize the ethanol production yield.

[0015] However, the inventors of the present invention sought for alternatives for yeast activity that could further improve the ethanol production yield, without

[0016] 1Amorim, H.V. - Introdugao a bioquimica da fermentagao alcodlica, Araras, IAA / PLANALSUCAR.COSUL, 1977.

[0017] 2Santos, Alessandra Marques dos.

[0018] Estudo da influencia da complementagao de nutrientes no mosto sobre o processo de fermentagao alcodlica em batelada-Maceid. 2008- Universidade Federal de Alagoas. resulting in increased costs or modifications in the production process. In this regard, the inventors of the present invention surprisingly found that the phosphorus source could be modified to further stimulate yeast and significantly increase the ethanol yield.

[0019] This development was even more surprising since it uses other forms of phosphorus ions, different from the phosphate already widely known in this technical field.

[0020] SUMMARY OF THE INVENTION

[0021] To achieve the objective of improving the yeast metabolism and consequently increasing the ethanol yield production, the present invention provides a composition comprising a phosphorus source, which is different from the state of the art and has different absorption rates.

[0022] In one embodiment, the phosphorous-based composition comprises the following ingredients:

[0023] (i) At least one phosphite source compound; and

[0024] (ii) An acceptable solvent selected from water.

[0025] The composition of the present invention can be applied directly to the yeast milk during the fermentation process in ethanol / alcohol production.

[0026] In one aspect of the present invention, the at least phosphite source compound can be any phosphite salt and mixtures thereof.

[0027] Non-limitative examples of phosphite salts include phosphite salts of alkali metals or alkaline-earth metals, such as sodium phosphite, potassium phosphite, calcium phosphite, magnesium phosphite, manganese phosphite, or mixture thereof.

[0028] Other examples of phosphite salts compounds include phosphorous acid (H3PO3), metaphosphoric acid (HPO3), or mixture thereof.

[0029] In a second embodiment, the phosphorous-based composition further comprises (iii) at least one phosphate source compound.

[0030] The at least one phosphate source compound is selected from phosphoric acid (H3PO4), dibasic ammonium phosphate (NH4)2HPO4), dipotassium hydrogen phosphate (K2HPO4), dibasic sodium phosphate (Na2HPO4), phosphorous pentoxide (P2O5), and mixtures thereof.

[0031] It was found that a composition comprising at least one phosphite salt with water is suitable for improving the yeast metabolism in a fermentation process, thereby, increasing the ethanol yield production. A method for applying said composition and uses thereof are also described. The composition of the present invention has some advantages, for example, improving the use of the raw materials rate, and improving the fermentation yield without increasing the general costs of alcohol fermentation. Moreover, the composition of the present invention can be applied in any conventional fermentation process, such as batch or continuous flow, as it is simple and easy to be used.

[0032] By using the composition of the present invention, it is possible to achieve about 2% to 14% higher of fermentation yield in ethanol production in comparison with a process without the composition from the invention.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 shows a batch type fermentation, which comprises an initial fermentation tank (2), a feed wort (1 ), a pre-fermenter tank (3) and centrifuges (4). The centrifuges provide the separation of yeast milk (5) and wine (6). The wine is directed to a distillation process (7) for separating the alcohol and the vinasse. The yeast milk (5) returns to a pre-fermenter tank (3). The composition of the invention (8) is then applied by means of a pump into the yeast milk (5) which is recovered from the centrifuges (4) and then fed to the pre-fermenter (3).

[0035] Figure 2 shows a continuous flow fermentation, the fermentation system comprises the following elements: a feed wort (10), a pre-fermenter (30), an initial rection tank (20), at least 4 additional tanks (21 ,22,23,24) and centrifuges (40). The centrifuges provide the separation of yeast milk (50) and wine (60). The wine is directed to a distillation process (70) for separating the alcohol and the vinasse. The yeast milk (50) returns to a pre-fermenter tank (30). The composition of the invention (80) is then applied by means of a pump into the yeast milk (50) which is recovered from the centrifuges (40) and then fed to the pre-fermenter (30).

[0036] Figure 3 shows a graph containing the samples 1 to 6 of one embodiment of the invention, as described in example 2, which were tested in a laboratory test to show the % variation of the fermentation yield in relation to a blank sample (without the composition of the invention).

[0037] Figure 4 shows a comparison of the % of variation of °GL / ART obtained in an industrial plant of (i) a composition of the invention comprising a mixture of phosphite and phosphate salts (H3PO3 + H3PO4), and (ii) a composition of the invention comprising phosphite salt (H3PO3), in relation to (iii) a composition comprising phosphate salt (H3PO4) alone, representing the state of the art, as described in example 4. Figure 5 shows a comparison of the % of variation of fermentation yield in an industrial plant of (i) a composition of the invention comprising a mixture of phosphite and phosphate salts (H3PO3 + H3PO4), (ii) a composition of the invention comprising phosphite salt (H3PO3), in relation to (iii) a composition comprising phosphate salt (H3PO4) alone, representing the state of the art, as described in example 5.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] The phosphorous-based composition of the present invention comprises:

[0040] (i) At least one phosphite source compound; and

[0041] (ii) An acceptable solvent selected from water.

[0042] In one aspect of the present invention, the at least one phosphite source compound (i) refers to any phosphite salt or mixture thereof. The at least one phosphite source compound (i) comprises any compound that releases the phosphite anion (HPOs2-) and / or the dihydrogen phosphite (H2PO3-) in an aqueous medium.

[0043] The phosphite source compound is a phosphite salt selected from: phosphorous acid (H3PO3), metaphosphoric acid (HPO3), alkali metal phosphite salt, alkaline-earth metal alkaline phosphite salt, or mixtures thereof.

[0044] In one embodiment, the alkali metals, or alkaline-earth metals phosphite salts are preferably selected from sodium phosphite, potassium phosphite, calcium phosphite, magnesium phosphite, manganese phosphite, or mixture thereof.

[0045] In one embodiment, the amount of phosphite source compound (i) is from 10% to 60% by weight, preferably from 20% to 50% by weight, more preferably from 30% to 40% by weight, even more preferably 40% by weight, based on the total weight of the composition.

[0046] The phosphite source compound should be diluted in an acceptable and inert solvent, such as water.

[0047] In one embodiment, the amount of solvent is from 20% to 80% by weight, preferably from 30% to 70% by weight, more preferably from 40% to 60% by weight, even more preferably 40% by weight, based on the total weight of the composition.

[0048] Preferably, (i) the amount of phosphite source compound is from 20 to 60%, and (ii) the amount of water is from 20% to 50%, based on the total weigh of the composition.

[0049] In other embodiment, the composition of the present invention may further comprise (iii) at least one phosphate source compound.

[0050] The at least one phosphate source compound (iii) refers to any compound that releases a phosphate anion in an aqueous medium. Such phosphate anion is, for example, a phosphoric acid (H3PO4), dihydrogen phosphate (H2PO4-), hydrogen phosphate (HPO42-), phosphate (PO43-), orthophosphate (-PO43-), and mixtures thereof.

[0051] The phosphate source (iii) is available commercially in form of salts or as phosphoric acid (also known as orthophosphoric acid).

[0052] Examples of phosphate source compound (iii) of the present invention can be selected from phosphoric acid (H3PO4), dibasic ammonium phosphate (NH4)2HPO4), dipotassium hydrogen phosphate (K2HPO4), dibasic sodium phosphate (Na2HPO4), phosphorous pentoxide (P2O5) and mixtures thereof.

[0053] In one embodiment, when the phosphate source compound (iii) is present, the amount of phosphate in the composition is from 0.1 % to 40% by weight, preferable from 10% to 40%, more preferably 5% to 30% by weight, even more preferably 10% to 20% by weight, based on the total weight of the composition.

[0054] In one embodiment of the invention, the composition of the invention comprises (i) 20 to 50% by weight of one phosphite source compound, (ii) 30 to 60% by weight of water and (iii) 10 to 40% by weight of phosphate compound.

[0055] In a more particular embodiment of the invention, the composition comprises:

[0056] (i) 40% by weight of one phosphite source compound,

[0057] (ii) 40% by weight of water as acceptable solvent, and

[0058] (iii) 20% by weight of one phosphate source compound, based on the total weight of the composition. Preferably, the phosphite source compound is phosphorous acid and the phosphate source compound is phosphoric acid.

[0059] In one aspect of the invention, the composition may comprise additional ingredients, preferably chelators or mineral nutrients (such as potassium, magnesium, calcium, manganese, iron, zinc, or copper).

[0060] The additional ingredients can be present in the composition in a range from 0.1 % to 10% by weight, based on the total weight of the composition.

[0061] In a preferred embodiment, the chelating agent is di-phosphonic hydroxyethylene acid, also known as HEDP.

[0062] In one aspect of the invention, the composition comprises: - from 20% to 50% by weight of a phosphite source compound selected from phosphorous acid (H3PO3);

[0063] - from 40% to 60% by weight of water;

[0064] - from 10% to 40% by weight of a phosphate source compound selected from phosphoric acid (H3PO4); and

[0065] - from 1 to 10% by weight of HEDP; based on the total weight of the composition.

[0066] The composition of the present invention has a liquid form and an average density of 1 ,226 Kg / L.

[0067] The composition of the present invention is mainly suitable for use in process involving fermentation steps such as wine or alcohol production, for which the yeast uses the sucrose, glucose, and fructose as carbon source. The sugar or sucrose (or glucose) containing raw materials can be any sugar source used in fermentation steps, such as sugar cane or molasses. Acceptable yeasts are from the species Saccharomyces cerevisiae.

[0068] The composition of the present invention can be used in batch or continuous flow fermentation.

[0069] In a classical batch fermentation, the fermentation system comprises the following elements: a fermentation tank (2), a feed wort (1 ), a pre-fermenter (3) and centrifuges (4). The feed wort (1 ) is responsible for introducing the sugar-containing wort into the fermentation tank. The centrifuges provide the separation of yeast milk (5) and wine (6). The wine (6) is directed to a distillation process (7) and the yeast milk (5) returns to the pre-fermenter tank (3). The pre-fermenter (3) consists of a tank that is responsible to prepare the new yeast along which the reused and recovered concentrated yeasts (5) obtained from the centrifuges (4). In the fermentation tank (2), the wort is mixed with yeasts, which allow the sugar to be converted into alcohol. The composition of the invention (8) is then applied by means of a pump into the yeast milk (5) which is recovered from the centrifuges (4) and then fed to the pre-fermenter tank (3).

[0070] In a continuous flow fermentation, the fermentation system comprises the following elements: a feed wort (10), a pre-fermenter (30), an initial fermentation rection tank (20), at least 4 additional tanks (21 ,22,23,24) and centrifuges (40). The centrifuges provide the yeast milk (50) and wine (60). The wine (60) is directed to a distillation process (70) and the yeast milk (50) returns to the process in a pre- fermenter tank (30). The feed wort (10) is responsible for introducing the sugar- containing wort into the initial fermentation reaction tank (20). The pre-fermenter (30) consists of a rector tank that is responsible to prepare the yeast in which the reused and recovered concentrated yeasts (50) obtained from the centrifuges (40) are also inserted. In the fermentation tanks (20,21 ,22,23,24), the wort is mixed with yeasts, which allow the sugar to be converted into alcohol. The composition of the invention (80) is then applied by means of a pump into the yeast milk (50) which is recovered from the centrifuges (40) and then fed to the pre-fermenter (30).

[0071] The composition is dosed in a fermentation process in a batch or continuous flow by means of a pump, in a concentration varying from 50 ppm to 200 ppm, based on the yeast milk or centrifuged yeast milk.

[0072] In a more preferred embodiment, the composition is applied in a concentration of about 100 ppm to about 150 ppm in relation to the yeast-free milk. In another preferred embodiment, the composition is applied in a concentration of about 100 ppm in relation to the yeast milk. Even more preferably, the composition is applied in a concentration of about 150 ppm in relation to the yeast milk.

[0073] Particularly, the composition is injected directly into the recovered yeast milk or centrifuged yeast milk (5,50).

[0074] After the application of the composition in the yeast milk, the yeasts will consume the composition and their metabolism will be accelerated by the injected composition, which will consequently increase the conversion of sugar into alcohol.

[0075] Thus, a further embodiment of the present invention consists of a method of applying the composition in a fermentation step for producing bioethanol. The method comprises:

[0076] (i) Injecting in a pump from 50 to 200 ppm of the composition (8,80); and

[0077] (ii) Adding the composition in the recovered yeast or in the yeast milk (5,50) obtained from the fermentation reactor (2,20); and

[0078] (iii) Feeding the concentrated ferment, containing the composition (8,80), in the pre-fermentation tank (3,30).

[0079] The invention also refers to the use of the composition for preparing a product that provides phosphorous nutrient for yeast in a batch or continuous flow fermentation for bioethanol production.

[0080] Terms

[0081] As used herein, the expression “at least one” means “one or more” and thus, includes individual components as well as mixtures / combinations.

[0082] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients and / or reaction conditions are to be understood as being modified in all instances by the term “about,” meaning within + / - 5% of the indicated number.

[0083] As used herein, all ranges provided are meant to include every specific range within, and combination of sub ranges between, the given ranges. Thus, a range from 1 -5, includes specifically 1 , 2, 3, 4 and 5, as well as sub ranges such as 2-5, 3-5, 2-3, 2-4, 1 -4, etc. All ranges and values disclosed herein are inclusive and combinable. For examples, any value or point described herein that falls within a range described herein can serve as a minimum or maximum value to derive a sub-range, etc.

[0084] Throughout the disclosure, the term “mixtures thereof” may be used following a list of elements as shown in the following example where letters A-F represent the elements: “one or more elements selected from the group consisting of

[0085] A, B, C, D, E, F, and a combination thereof.” The term, “a combination thereof” does not require that the combination include all of A, B, C, D, E, and F (although all of A,

[0086] B, C, D, E, and F may be included). Rather, it indicates that a combination of any two or more of A, B, C, D, E, and F can be included. In other words, it is equivalent to the phrase “one or more elements selected from the group consisting of A, B, C, D, E, F, and a combination of any two or more of A, B, C, D, E, and F.”

[0087] The cited chemical compounds contain synonyms, respective CAS number, IIIPAC names and should be considered therein.

[0088] Those skilled in the art should appreciated that the examples and embodiments described herein are for illustrate purposes only and not to limit the scope of the present invention. Various of modifications, combination and subcombinations and changes can be made, if necessary, all of modifications, combinations, sub-combinations, changes, and equivalents are fallen in the scope of the following claims.

[0089] By way of non-limiting illustration, the invention will now be described with reference to the following examples.

[0090] EXAMPLE 1: Composition comprising phosphite salt to be applied in alcohol fermentation and its performance in an industrial plant

[0091] An industrial scale test was carried out to verify the fermentation yield of the composition of the present invention containing one phosphite source compound and water and compared to a blank sample. To perform the comparison, the same raw materials were used (yeast and wort feed) and having the same reaction conditions.

[0092] A yeast type Fleishmann® Biological Yeast (Saccharomyces cerevisiae) was selected, simulating, on a laboratory scale, the dilution and concentration conventionally practiced in the process, in this case approximately 25% of cell mass.

[0093] The yeast was conditioned at 40°C (+ / -) for activation and the pH was adjusted with sulfuric acid at pH 2.50.

[0094] Then, the yeast was divided and diluted into 10 different containers containing 100mL each. After dosing, the containers containing the diluted yeast and the respective dosages were conditioned in an environment for a period of approximately 30 minutes for the action of the samples, microbial interaction, and metabolic activation of cells.

[0095] Then, we added 200mL of feed wort, which is prepared with exhausted molasses, and we measured the °Brix and corrected to approximately 15.00° (+ / -).

[0096] This mixture (treated yeast + feed wort) was duly identified and conditioned at 32°C in an incubation oven for a period of approximately 12 hours.

[0097] After the fermentation period, they had an aliquot extracted and analyzed, and the results are shown in the tables 1 , 2 and 3 below. The composition of the invention tested in this example comprises 40% of sodium phosphite and 60% of water.

[0098] Table 1 : Dosage used in the comparative test.

[0099] The dosage described in the table 1 above has the concentration of the Active dosed relative to the volume of Diluted Yeast (in this case under a volume of 10OmL).

[0100] Table 2: Results obtained for evaluating the relevant parameters to calculate the fermentation yield performance.

[0101] Table 3: Comparative variation of main parameters of the composition of the present invention in relation to blank.

[0102] From table 3 above it is possible to verify that the composition of the present invention provides a better performance in terms of °GL / ART and fermentation yield in relation to a blank sample.

[0103] EXAMPLE 2: Composition containing phosphite and phosphate salts applied in alcohol fermentation and its performance in a laboratory test

[0104] A laboratory assay was carried out for evaluating the fermentation yield of the composition of the present invention. The same raw materials were used (yeast and wort feed) and having the same reaction conditions.

[0105] A biological yeast known as Fleishmann® (Saccharomyces cerevisiae) was used, simulating, on a laboratory scale, the dilution and concentration conventionally used in the process. In this case, it was used approximately 25% of cell mass.

[0106] The yeast was conditioned at 40°C for activation, and we adjusted the pH with sulfuric acid at pH at 2.50. Then, the diluted yeast was divided into two different flasks containing 100 mL each.

[0107] After that, one of the diluted flasks was subjected to the dosage of the evaluated active ingredient, while the second flask was used for carrying out the comparative assessment. The second flask did not receive any component, as described in the table 1 shown below.

[0108] After dosing the first flask, both flasks containing the diluted ferments were conditioned in an environment temperature for a period of approximately 30 minutes to allow the action of the composition of the present invention, that is, the microbial interaction and metabolic activation of the yeast cells. Then, it was added 200m L of feed wort in each flask, which was prepared with 10% sucrose, 1 % glucose and 1 % fructose, which was prepared with “Grade P.A.” and diluted in distilled water. The feed wort was prepared simulating the concentrations of substrate compounds conventionally found in a natural process.

[0109] This mixture (treated yeast + feed wort) was duly identified and conditioned at 32°C in an incubation oven for a period of approximately 12 hours.

[0110] After the fermentation period, an aliquot was extracted and analyzed. This procedure was replicated in 6 different batches.

[0111] The composition of the present invention used in the present test comprises the following concentration: (i) 40% of sodium phosphite (ii) 50% of water and (iii) 10% of phosphoric acid. The dosages are described in table 4 below.

[0112] Table 4: Dosage used to perform 6 laboratory batches.

[0113] *The dosages described in table 4 above have the concentrations of the Actives dosed relative to the volume of Yeast (in this case under a volume of 10OmL).

[0114] The main parameters were measured in the batches performed above to calculate the fermentation yield obtained by the composition of the invention in comparison to a composition without the addition of the ingredients of the invention.

[0115] The results obtained for the evaluation of relevant parameters to calculate the fermentation yield performance is gathered in tables 5, 6 and 7.

[0116] Table 5: Results obtained for evaluating the relevant parameters to calculate the fermentation yield performance in a laboratory test.

[0117] Based on the table 5 above, it is possible to verify that the globa fermentation yield by using the composition of the invention, comprising a mixture of phosphite with phosphate salts, is higher in relation to the blank samples.

[0118] Table 6 - Comparative variation (%) of main parameters by using the composition of the invention in relation to blank samples:

[0119] From table 6 above it is possible to verify that the use of the composition of the invention provides an average of 7.308% higher in ethanol production in comparison with a blank sample (see % variation °GL-medium).

[0120] Table 7: Comparative variation (%) of fermentation yield in relation to blank samples:

[0121] From table 7 above and figure 3 it is possible to verify that the use of the composition of the invention provides an average of 7.309% higher of fermentation yield in comparison with a blank sample (see % variation fermentation yield-medium).

[0122] EXAMPLE 3: Composition containing phosphite and phosphate salts, applied in alcohol fermentation and its performance in an industrial plant.

[0123] An industrial evaluation was carried out to verify the fermentation yield of the composition of the present invention containing phosphite and phosphate salts. The same raw materials were used (yeast and wort feed) and having the same reaction conditions.

[0124] The present test consists of the application of the composition of the invention, which is applied to yeast after centrifugation and before conventional treatment. This product aims exclusively to increase the efficiency of converting sugars into ethanol, that is, to improve the alcohol content (°GL) ratio, whether in the final tank or in the yeast-raised wine, over the total reducing sugar (ART) of the wort consumed.

[0125] The analysis was performed in a High-Performance Liquid Chromatograph (HPLC) manufactured by Waters-USA. This HPLC specifically is equipped with a column that allows you to accurately measure ethanol, sugars separately (sucrose, glucose and fructose), the main “by-products” coming from the fermentation process, such as: organic acids (succinic, lactic, acetic and propionic), glycerol, higher alcohols, and biopolymers (dextran and starch).

[0126] The following aspects were considered in the test:

[0127] • Points analyzed: Wort, Pre-Fermenter (PF), Final Tank and yeast-free wine.

[0128] • Frequency of analysis: at each of these points we carried out three daily analyzes and, in the following day, it was performed one analysis per day; It was totalized twenty-four samples, one taken every hour and conditioned under refrigeration.

[0129] • Ethanol contained in the feed wort: In the conversion ratio of °GL / ART shown below, possible alcoholic content present in it (feed wort) was disregarded. Therefore, in the mentioned relationship (°GL / ART), it was present only the amount formed during the fermentation process; even being aware that in the official efficiency calculation methodology this data is incorporated and considered.

[0130] • Calculation of ART: as the instrument (HPLC) allows the separation of sucrose from reducing sugars (glucose + fructose), the calculation of ART is made by replacing apparent sucrose (Pol) with sucrose (real sucrose), which is added to the reducing sugars cited.

[0131] The results were obtained were described in table 8 below:

[0132] Table 8: Results of °GL / ART ratio, reduction in glycerol formation and ART of the composition of the invention in relation to blank

[0133] Based on table 8 above, the following advantages can be observed with the composition of the present invention:

[0134] • °GL / ART ratio - it was obtained an increase of 5.61 % in the composition of the invention in relation to a blank sample. Before application, we had an average of 0.41 and during application it was 0.433 (see the results of °GL / ART for yeast-free wine).

[0135] • Reduction in glycerol formation - the composition of the invention reduced this feature by 57%. The formation of glycerol (ppm) was 934.59 and decreased to 402.46 ppm (see the results of glycerol formation for yeast-free wine). It should be noted that the higher the glycerol production, the lower the ethanol production.

[0136] • Total residual reducing sugars - the reduction of residual sugars using the composition of the invention was 34.74%, i.e. , from 2967.54 Kg to 1937.10 Kg (see kg of ART for yeast-free wine). The smaller the among of residual sugars, the greater the ethanol conversion.

[0137] Therefore, the potential increase of 5.61 % in ethanol production is quite significant in a large scale.

[0138] To evaluate statistically with production numbers, based on the production unit in which the present composition was tested, it is possible to obtain an average production of 300,000 liters of ethanol per day (considering this average number, as part of the harvest was 400,000 and the other 200,000), for a harvest of approximately 180 days, we will have an effective increase in production by 3,030 m3.

[0139] Hypothetically, when considering the current price of R$3.50 per liter of anhydrous ethanol (U$$0.694 / L at the current price), an increase in revenue in the order of R$10,605,000.00 or U$$3,030,000.00 annually can be achieved.

[0140] Also hypothetically, we can convert this into sugar cane grinding. When considering the acquisition value of a ton of sugarcane at R$175.50 (with an average of 135 kg of ATR and having its current hypothetical value of R$1.30 / Kg of ATR, an increasing in the total crushing by an amount approximately 60,427 tons of sugarcane can be obtained.

[0141] For comparison purposes, it was also conducted the evaluation in the industrial unit according to the parameters obtained in weekly bulletins, i.e., in 2 weeks prior to the application of the composition of the invention versus 2 weeks of application period of the composition of the invention.

[0142] With the available data regarding grinding for ethanol, honey consumed for ethanol, the respective ARTs and ethanol production it was possible to obtain evaluate the fermentation yield in a large-scale industrial unit, as follows:

[0143] Table 9: Results obtained in large scale before the application of the composition and during the application of the composition.

[0144] From the table 9 above, the increase in ART conversion efficiency into ethanol was 6.05%.

[0145] Based on the tests results, it is indeed clear that the composition of the invention provides improved results. EXAMPLE 4: Comparative tests of the composition of the present invention in relation to the state of the art

[0146] A comparative test was carried out to verify the fermentation yield of the composition of the present invention in relation to the state of the art. Tests were carried out with two different embodiments of the invention: (i) one composition of the invention comprising phosphite salt and water; and (ii) one composition of the invention comprising a mixture of phosphite salt, water, and phosphate salt. The composition of the state of the art comprises phosphate salt alone.

[0147] The same raw materials were used (yeast and wort feed) and having the same reaction conditions. A yeast type Fleishmann® Biological Yeast (Saccharomyces cerevisiae) was selected, simulating, on a laboratory scale, the dilution and concentration conventionally practiced in the process, in this case approximately 25% of cell mass. The yeast was conditioned at 40°C (+ / -) for activation and the pH was adjusted with sulfuric acid at pH 2.50.

[0148] Then, the yeast was divided and diluted into 10 different containers containing 100mL each. After dosing, the containers containing the diluted yeast and the respective dosages were conditioned in an environment for a period of approximately 30 minutes for the action of the samples, microbial interaction, and metabolic activation of cells.

[0149] Then, we added 200mL of feed wort, which is prepared with exhausted molasses, and we measured the °Brix and corrected to approximately 15.00° (+ / -).

[0150] This mixture (treated yeast + feed wort) was duly identified and conditioned at 32°C in an incubation oven for a period of approximately 12 hours.

[0151] After the fermentation period, they had an aliquot extracted and analyzed, and the results are shown in the tables 10, 11 and 12 below.

[0152] In the present test, the following concentrations were used:

[0153] • Composition of the state of the art: 50% of phosphoric acid and 50% of water;

[0154] • Composition of the invention n°1 (phosphite): 40% of sodium phosphite and 60% of water; and

[0155] • Composition of the invention n°2 (phosphite and phosphate): 40% of sodium phosphite, 40% of water and 20% of phosphoric acid.

[0156] Table 10: Dosage used in the comparative test:

[0157] The dosages described in the table above have the concentrations of the Actives dosed relative to the volume of Diluted Yeast (in this case under a volume of 10OmL).

[0158] Table 11 : Results obtained for the evaluation of relevant parameters to calculate the fermentation yield performance. Table 12: Comparative variation of main parameters of the compositions of the present invention in relation to the state of the art.

[0159] From table 12 above, it is possible to verify that the compositions of the present invention provide a better performance in terms of °GL / ART and fermentation yield in relation to the use of phosphate alone (state of the art). These unexpected results are also shown in Figures 4 and 5.

Claims

SET OF CLAIMS1. A phosphorus-based composition for yeast fermentation, wherein it comprises:(i) At least one phosphite source compound; and(ii) An acceptable solvent selected from water.

2. Composition, according to claim 1 , wherein the at least one phosphite source compound (i) is selected from phosphite salts and mixtures thereof.

3. Composition, according to claim 2, wherein the at least one phosphite salt is selected from phosphorous acid (H3PO3), metaphosphoric acid (HPO3), alkali metal phosphite salt, alkaline-earth metal alkaline phosphite salt, and mixtures thereof.

4. Composition, according to claim 2, wherein the alkali metal phosphite salt or alkaline-earth metal phosphite salt is selected from sodium phosphite, potassium phosphite, calcium phosphite, magnesium phosphite, manganese phosphite, and mixtures thereof.

5. Composition, according to claim 1 , wherein the amount of phosphite source compound (i) is from 10% to 60% by weight, preferably from 20% to 50% by weight, more preferably from 30 to 40% by weight, based on the total weight of the composition.

6. Composition, according to claim 1 , wherein the amount of acceptable solvent water (ii) is from 20% to 80% by weight, preferably from 30% to 70%, more preferably from 40% to 60%, based on the total weight of the composition.

7. Composition, according to claim 1 , wherein it further comprises (iii) at least one phosphate source compound.

8. Composition, according to claim 7, wherein the phosphate source compound (iii) is selected from phosphoric acid (H3PO4), dibasic ammonium phosphate (NH4)2HPO4), dipotassium hydrogen phosphate (K2HPO4), dibasic sodium phosphate (Na2HPO4), phosphorous pentoxide (P2O5), and mixtures thereof.

9. Composition, according to claim 7, wherein the amount of phosphate source compound (iii) is from 0.1 % to 40% by weight, preferable from 10% to 40%, more preferably 5% to 30% by weight, even more preferably 10% to 20% by weight, based on the total weight of the composition.

10. Composition, according to claim 1 , wherein the composition further comprises additional ingredients, preferably, chelators or mineral nutrients.11 . Composition, according to claim 1 , wherein it comprises:- from 20% to 50% by weight of a phosphite source compound selected from phosphorous acid (H3PO3);- from 40% to 60% by weight of water; and- from 10% to 40% by weight of a phosphate source compound selected from phosphoric acid (H3PO4); based on the total weight of the composition.

12. Method for applying the composition in a pre-fermentation tank for producing bioethanol, wherein it comprises the following steps:- Injecting in a pump from 50 to 200 ppm of the composition (8,80), as defined in claim 1 ;- Adding the composition in the recovered yeast or in the yeast milk (5,50) obtained from the fermentation reactor (2,20); and- Feeding the concentrated ferment containing the composition (8,80), in the pre-fermentation tank (3,30).

13. Use of the composition, as defined in claim 1 , wherein said composition is for preparing a product that provides phosphorous nutrient for yeast in a batch or continuous flow fermentation for bioethanol production.

Images