FR2550221A1 - Process for the manufacture of alcohol with the aid of a destructive alcoholic yeast - Google Patents

Abstract

Process for the production of alcohol with the aid of a destructive alcoholic yeast. The process employs the strain of Saccharomyces cerevisiae K2-M111 as destructive alcoholic yeast which has the same destructive characteristics as a K2 destructive yeast.

Description

PROCESS FOR PRODUCING ALCOHOL USING A
ALCOHOLIC YEAST DESTRUCTIVE
The invention relates to a process for producing alcohol and more particularly to a process for producing alcohol from sugar solutions using a new destructive alcoholic yeast.

 The processes commonly used for the production of alcohol are batch fermentation processes, reseeding processes, and continuous fermentation processes, all using so-called alcoholic yeast. However, in these processes, since the sugar solution is generally used for fermentation in a non-sterilized form, there is a possibility of contamination of the yeast by the microorganisms or wild yeast existing in the sugar solution. the possibility of this contamination being higher in the reseeding processes and in the continuous fermentation processes, the discontinuous fermentation codec where the possibility of contamination is lower, are currently more commonly used. In the manufacture of alcohol by reseeding or continuous fermentation processes, a need therefore exists to prevent contamination by microorganisms or wild yeasts. Various measures have been taken for this purpose. For example, in the so-called Melle Boinot method, according to which the yeast is recovered from a stirring mixture using a separator by centrifugation, and this recovery yeast is recycled. for re-use it is possible to prevent contamination by microorganisms by lowering the pH of the fermentation system or adding an antibiotic, but effective protection against contamination by wild yeasts can not be achieved. The method of alcoholic fermentation by the use of immobilized yeast, which is an improved version of the continuous fermentation process, makes it possible to continue the fermentation for a long time without contamination by the microorganisms or the wild yeasts, provided that the sugar solution of which the fermentation system is fed completely sterilized. But when the sugar solution is not completely sterilized, for example by heat, the fermentation system allows the different wild yeasts to grow gradually and possibly contaminate even the interior of the plants. Yeast grains immobilized thereby preventing it from performing its inherent function of alcoholic fermentation. This results in a problem of decreasing the yield of alcohol and the speed of the alcoholic fermentation. There is therefore an acute need in the continuous fermentation processes for the production of alcohol with a high yield from sugar solutions having no slurries of destruction of wild yeasts unduly growing in sugar solutions. It is therefore desirable to use a destructive alcoholic yeast having an inherent power to destroy these wild yeasts.

It has recently been demonstrated that species of the genus Saccharomyces are capable of destroying opposing yeasts. They are referred to as destructive yeasts. The practical use of a destructive yeast has been described by Antonie van Leeuwenhoek 44,59-77 (1978)
The use of destructive yeast K1 has since been applied to the production of sauté or wine, but never to the production of alcohol from sugar solutions.

This results from the alcoholic fermentation of sugar solutions at temperatures above 30 ° C, higher than the fermentation of sake or wine, and at these higher temperatures the toxic protein secreted by the destructive yeast
K1 is no longer active.

 The present invention makes it possible to obtain a destructive alcoholic yeast K2 having a high alcoholic fermentation power and possessing the same destructive character as the destructive yeast K2.

The subject of the invention is therefore a process for producing alcohol with a high yield, from sugar solutions, using destructive alcoholic yeast.
K2. The invention also relates to a method for producing alcohol using the strain Saccharomyces cerevisiae K2-M11V as destructive alcoholic yeast
K2 supra. The invention also relates to a process for producing alcohol with a high yield in a continuous fermentation operation using an unboiled sugar solution.

 The use of Saccharomyces cerevisiae, a destructive alcoholic yeast, with a high alcoholic fermentation power and the same destructive character as the destructive yeast K2, makes it possible to produce alcohol with a high yield from sugar solutions, without contamination. by wild yeasts, in wider ranges of fermentation conditions than those made possible until now.When Saccharomyces cerevisiae K2-Mlll strain is used as a destructive alcoholic yeast among other strains of the same species, the alcoholic fermentation of unboiled sugar solutions in a pH range wider than that of 4.0 to 5.0 heretofore considered optimum for alcoholic fermentation, and at temperatures higher than 30 ° C. Thus, the fermentation process using this particular strain has proved particularly effective in the processes reseeding or continuous fermentation processes that presume a high probability of contamination by wild yeasts.

The invention will be better understood on reading the following detailed description of a preferred embodiment of the invention and on examining the appended drawings, which represent, in a non-limiting manner, various embodiments of the invention. invention.On these drawings
FIG. 1 is a diagram showing the comparison of the fermentation capacity of the prepared K2-M III strain with that of the parental strains (M111 receptor strain), in molasses culture media, under conditions with a total sugar content. 22.5%, pH 5.1 and 30 ° C;
FIG. 2 is a diagram showing the variation of the number of surviving wild yeasts in a batch fermentation, using destructive alcoholic yeast;
FIG. 3 is a diagram showing the variation of the number of surviving wild yeasts in a continuous fermentation using a permanent, alcoholic, destructive yeast; ;
Figure 4 is a diagram showing the effect of pH on the destructive toxin in a continuous fermentation at 30 ° C;
Figure 5 is a diagram showing the effect of the destructive toxin on continuous fermentation at 35 ° C, and
FIG. 6 is a diagram showing the relationship between the contamination rate caused by wild yeasts in the continuous production of alcohol from unsterilized molasses, the yield of ethanol produced and the yield of the fermentation.

 The destructive yeast K2, previously described by Antonie van Leeuwenhoek, 44, 59-77 - (1978) and known for its tolerance to higher temperatures and a wider range of pH than the destructive yeast K1, was tested. Its alcoholic fermentation power has been studied, and this destructive alcoholic yeast has been studied with respect to the persistence of the destructive activity exhibited by the yeast when used in fermentation alcohol production. These studies have led to the present invention of a destructive alcoholic yeast K2, possessing a destructive toxin K2, capable of destroying a wide range of wild yeasts and, in addition, having a high alcoholic fermentation potency.

The destructive alcoholic yeast K2 is selected by cell fusion of an alcoholic yeast and the destructive yeast K2. Among the alcoholic yeasts useful as receptors for the destructive plasmid K2, mention may be made, for example, of the strains of Saccharomyces cerevisiae IFO 0224 and
IFO 0215 and their M111 halotolerant variant strains.

More particularly, the strain M111 excels as a receiver having a high fermentation power and a high halo concentration.

Among the destructive yeasts K2 acting as donors of the destructive plasmid K2, mention may be made of the strains Saccharomyces cerevisiae NCYC 738, 1001, 1385, 1387 and 1428 and Saccharomyces diastaticus
NCYC 713. More particularly the strain Saccharomyces cerevisiae 5170, which is produced from the aforementioned strain 1387, a destructive yeast K2, and the strain Saccharomyces cerevisiae 1020, and which involves a variation of caryogamic fusion deficiency, is effective for the selective introduction of the destructive plasmid K2 into the alcoholic yeast. The destructive alcoholic yeast, produced from the aforementioned alcoholic yeast of the strain Miii and the destructive yeast K2 of the strain 5170, was deposited under the designation of yeast destructive alcoholic
Saccharomyces cerevisiae K2-M111 strain (FERM-P No.7071) from the Fermentation Research Institute of the
Agency of Industrial Science and Technology ".

 Figure 1 and Table 1 for conventional yeast alcoholic fermentation and destructive alcoholic yeast K2 of the invention represent the amount of carbon dioxide released and the amounts of acetaldehyde and ethyl acetate by-products produced. in alcoholic fermentation. It can be seen that the destructive alcoholic yeast of the invention and the conventional alcoholic yeast show no discernible difference in their alcoholic fermentation power and the by-products formed.

TABLE I
Comparison of yields of alcohol and fermentation by-products obtained with the selected strain
K2-M111 and parental strains

<tb><SEP> Strains <SEP> tried
<tb><SEP> Products <SEP> of <SEP> fermentation <SEP> K2-Mlil <SEP> Miii <SEP> 5170
<tb> Alcohol <SEP> (V / V <SEP>%) <SEP> 12.1 <SEP> 12.1 <SEP> 2,3
<tb> Sugar <SEP> total <SEP> (W / V <SEP>%) <SEP> 2.59 <SEP> 2.57 <SEP> 18.2
<tb> Acidity <SEP> (ml <SEP> 0.1N <SEP> NaOH / 10 <SEP> mi) <SEP> 9.9 <SEP> 10.0 <SEP> 8.7
<tb> pH <SEP> 5.2 <SEP> 5.2 <SEP> 5.1
<tb> Yield <SEP> of <SEP><SEP> Fermentation <SEP> (%) <SEP> 83.5 <SEP> 83.5 <SEP> 15.9
<tb> Acétaidehyde <SEP> (ppm) <SEP><SEP> 175 <SEP> 157 <SEP> - <SEP>
<tb> Acetate <SEP> Ethyl <SEP> (ppm) <SEP> 11.7 <SEP> 13.4 <SEP><SEP> - <SEP>
<tb> Alcohol <SEP> Methyl <SEP> (ppm) <SEP> 8.5 <SEP> 10.0 <SEP>
<tb> n-Propyl alcohol <SEP><SEP> (ppm) <SEP> 67 <SEP> 71 <SEP>
<tb><SEP> i-butyl alcohol <SEP> (ppm) <SEP> 55 <SEP> 57 <SEP>
<tb> Alcohol <SEP> i-amylate <SEP> (ppm) <SEP> 138 <SEP> 151 <SEP>
The destructive yeast K2 is then tested in a sugar solution to determine its destructive power of wild yeasts. The results of this test are as follows (A) To determine the change in the number of cells
survivors of wild yeasts in a fermenta
discontinuous use with the use of alcoholic yeast
that destructive, one innoculates a quantity of 107
cells / ml, respectively of the alcoholic levurer
Destructive strain 14111 and yeast
destructive alcoholic strain K-M111 to a
a mixture of molasses adjusted in advance to a certain
sugar total of 15% and a p14 of 4.2 and the
MCA wild yeast, made tolerant
with chemical cyclohexymide, is innoculated in
variables of 105 cells / ml, 106 cells and 107 cel
lules / ml. Culture is carried out at 30 ° C. in the medium and
obtained containing different yeasts and during the
culture, the culture broth is analyzed at different
intervals to determine how many cells survived
of the MCA wild yeast. In the sys
mixed themes of alcoholic yeast strain M111 and
wild MCA strain yeast, wild yeast
MCA has spread to all concentrations
undermined. In mixed systems of alcoholic yeast
destructive strain K2-M111 and wild yeast
strain MCA, the growth of wild yeast strain
MCA decreases at all concentrations tested. In
the latter mixed systems the effect of the toxin of the
This is obvious (see Figure 2).

(B) Variation in the number of surviving yeast cells
fermentation in continuous fermentation is carried out with
use of destructive alcoholic immobilized yeast. The
alcoholic yeast strain M1lfit the alcoholic yeast
destructive strain K2-M111, both immobilized
aluminum alginate, are introduced into a
reactor at a rate of 40% by volume, the reactor is fed with
with a sugar solution adjusted to a certain
total of 15% sugar and pH 4.2 and allowed to reach 30 ° C. with a fixed residence time of 10 hours; ;
when the fermentation stabilizes, the
wild strain of MCA strain in the fermenta system
in varying quantities of 104, 105 or 106 cells /
ml, and the effluent of the fermentation system is analyzed
to determine the variation in the number of surviving cells
vines of wild MCA strain yeast. In the reactor
using the immobilized alcoholic yeast strain M111,
we find that the wild yeast strain MCA, innocu
In the reactor using the immobilized destructive alcoholic yeast, of strain K2-M111, no wild yeast was detected 24 hours after the addition of 10 4 cells / ml. innoculation of the wild yeast MCA strain in an amount of 104 cells / ml, 48 hours after innoculation of the same yeast in amount of cells / ml, or 144 hours after innoculation of the same yeast in an amount of 106 cells / ml, respectively. (see Figure 3).

 Comparison of these test results with those obtained in batch fermentation shows that in continuous fermentation, which promotes the continuous growth of yeast, the effect of the destructive toxin is made more apparent.

(C) To determine the effects of pH and temperature
on the destructive toxin in fermentation con
tinue using destructive alcoholic yeast
immobilized, we use solutions of molasses
pre-adjusted to, oH 3.2, 4.2, and 5.2 and to a
total fixed sugar of 15%, which is subject to
continuous fermentation at 30 C; we are innoculating the yeast
wild MCA strain with fermentation systems
and effluents from fermen systems are analyzed
to measure the variation in the number of cells
the survivors of yeast.When one feeds with the
molasses solution of pH 5.2, the decrease in
number of surviving cells of wild yeast
of strain MCA is more manifest in the reactor
using immobilized yeast strain K2-M111
that in the case of the reactor using the yeast immo
bilized strain Mini. Even at low value
pH of 3.2, no surviving cell is detected
MCA strain of wild yeast 48 hrs. of fermen
retaliation. At all pH values tested, yeast
immobilized strain K2-M111 invariably reveals itself
effective to destroy the wild yeast strain
MCA, although the time required for the manifesta
This effect varies with the pH value of the
molasses feed solution (see Figure 4).

The same fermentations are then carried out
continuously at 35 C. During fermentation,
innocule 106 cells / ml of wild yeast strain
MCA and effluents from iron systems are analyzed
to measure changes in the number of
surviving cells of wild yeast strain MCA. The
immobilized yeast of K2-M111 manifests its effect
even at 35au. (see Figure 5).

The same phenomena are observed with
K2 yeasts destructive to wild yeasts other than
the previously mentioned alcoholic yeast, the
alcoholic destructive K2 and the yeast of the
strain 5170.

 From the above results it can be seen that the destructive alcoholic yeast K2 of the present invention permits alcoholic fermentation to be carried out in a larger range of pH values than 4.0 to 5.0 and at higher temperatures. only 30 "C, which are the optimal conditions for a conventional alcohol fermentation, and is effective in a continuous fermentation that presents a greater possibility of contamination by wild yeasts.

 The present invention relates to a process for the manufacture of alcohol from sugar solutions by the use of a destructive alcoholic yeast which has a high alcoholic fermentation power, which is capable of tolerating the destructive toxin K2 and producing it. in short, which possesses the same destructive characteristic as the destructive strain.

 The method of the invention can be advantageously applied to any batch fermentation system, reseeding systems and continuous fermentation systems. It is particularly effective in reseeding and continuous fermentation processes that have a greater potential for contamination by wild yeasts. The process of the invention allows the fermentation to be carried out at elevated temperatures of 25 to 38 ° C, preferably 30 to 35 ° C, and in a wide pH range of 3.0 to 5.2, to produce the Destructive toxin capable of destroying a wide variety of wild yeasts and allowing alcoholic fermentation to continue for long periods of time.

 These data are milestones in the field of alcoholic fermentation of sugar solutions. In continuous fermentation, the destructive alcoholic yeast of the invention can be used in the immobilized form with Al alginate, carrageenan sum, polyacrylic amide and glutaraldehyde.

 The fermented sugar solutions according to the invention comprise sugar solutions obtained by saccharification of starch by amylase or an acid, and sugar solutions obtained by saccarification of woody substances by cellulase or an acid.

 The invention is further illustrated by the following practical examples.

Example No. 1 of reference. Production of a destructive alcoholic yeast.

(A) Production of a destructive plasmid donor K2.

In the introduction of a K2 destructive plasmid into a Ml11 receptor by cell fusion, it is necessary to suppress the karyogamy of both yeasts as far as possible, and to ensure the production of a strain possessing a destructive plasmid K2 and containing a core formed only of Pf111, of high fermentation power. For this, the production of a destructive plasmid donor K2, the strain 5170 (genotype: a karl-1, his 4) is carried out as follows.
[KIL-K2] [NEX-O]), which has a caryogamic defect variation and a genetic marker distinguishable from the donor.First, to obtain a strain of the type of conjugation-a, possessing at the same time the destruction properties K7 ([KIL-K2]) and histidine claimant (his 4), Saccharomyces cerevisiae 1020 is innoculated (genotype: a, his 4, karl-1, [KIL-O] [EXL-O] and Saccharomyces cerevisiae 1387 (genotype: a ura 1 [KIL-K2] [NEX-O]) each at 5 mi of a YEPD culture medium (containing 2% glucose, 2% polypeptone, and 1% extract of yeast) which is subjected to a stationary culture at 30 ° C. for 1 day 5 ml of the YEPD culture medium are added all at once, portions of 100 μl of the various culture broths. in the media combined with a stationary culture at 30 ° C. for 1 day The resulting culture broth is washed twice with sterile water and spread on a minimum medium (0.67% nitrogen-based yeast). W Amino acid, 2% glucose and 2% agaragar. The colony, which then spreads on the minimum medium, is split as a hybrid strain spread on a spore-forming medium (0.5% potassium acetate and 2% agar) and cultured at 300C for three months. days for spore formation. An asque is then divided into four spores with a micromanipulator into four molecules and allowed to germinate on a YEPD plate. From this plate, the strains which are unable to propagate on the minimum culture medium and which can be propagated on a medium obtained by adding histidine to the minimum medium, are selected as histidine-seeking strains. From these strains, cells of the type of conjugation-a are selected.

To select a strain with
a variation of caryogamic (Karl-1) deficiency among
strains of the type of conjugation-a, possessing the pro
K2 destruction and histidine claimants,
selected as indicated above, we cross these penny
with a strain of type a, owning the property
leucine claimant, canavanine tolerance
and of respiratory deficiency. From these crossed strains, a colony-producing strain is selected with a high frequency on a medium obtained by adding leucine, canavanine and glycerin in place of glucose, at the minimum medium, and producing colonies with a very low frequency on the minimum medium, as the deficient caryogamic variant Saccharomyces cerevisiae 5170 (genotype: a karl-1 his 4 I KIL-K2] [NEX-O]). This strain is streaked on a medium
YEPD containing 0.003% methylene blue (pH 4.2), rubbed with a K2 destructive receptor, to confirm that the strain has destructive activity.

(B) Production of destructive alcoholic yeast K2-MIII
It is grown independently, on media
YEPD, the strain Saccharomyces cerevisiae M111, a recipient alcoholic yeast, and the strain Saccharomyces cerevisiae 5170, a destructive donor yeast of K2.

The cells produced during the logarithmic growth phase are collected and washed with water. The two cell masses were independently suspended in tris-sorbitol buffer (1 M sorbitol, 10 mM tris-HCl, pH 7.4 max, hereinafter referred to as ST solution) and, after addition of 0.1 ml of 0.5M methylene diamine ttracetic acid (EDTA) and 50 μl of 2-mercaptoethanol, incubated at 30 ° C for 10 min, washed with 2.5 ml of ST solution and resuspended in ST solution. These 50 μl cells of 0.5 M EDTA (pH 7.5) and 100 μl of Zymolyase 6000 (2.5 mg / lxl) were incubated at 30 ° C for 150 min to produce the protoplasts. respective, that we recover. One part of protoplast M111 of the strain Saccharomyces cerevisiae M111 is suspended and ten parts of the protoplast Saccharomyces cerevisiae 5170, prepared as described above, in 0.5 ml of a solution containing 1M sorbitol, 10 mM tris-HCL, and 10 mM of potassium chloride (hereinafter referred to as "STC" solution). The two strains are incubated in the suspension at 30 ° C. for 15 minutes. The cells of the resulting culture broth are collected and suspended in 0.5 ml of PTC solution (35% of PEG 4000). mM tris-HCl, 10 mM CaCl 2, pH 7.4), incubated at 30 ° C. for 20 minutes, washed once with STC solution and resuspended in 0.2 ml STC solution.

 The solution is spread on the minimum agar medium containing 1M sorbitol and cultured at 30 ° C. for one week.

The minimum agar medium on which a colony appears is milled in liquid YEPD medium (pH 4.2) and the cells are cultured in liquid medium for 2 days. 1 ml of the resulting culture broth is added to 100 ml of a culture medium.
YEPD liquid (pH 4.2), as well as 0.1 ml of the destructive yeast culture broth K2, strain Saccharomyces cerevisiae 5170. The resulting mixture is cultured at 25 ° C for three days. In 5 ml of YEPD medium (pH 4.2), 50 ul of the resulting culture broth was cultured at 25 ° C for two days. 0.1 ml of the produced culture broth is spread on the minimum agar-agar medium and cultured at 30 ° C. for two days. The strain which is propagated is subjected to the replica test on YEPD medium (pH 4.2). containing 0.003% of methylene blue and spread on a sensitive strain. A strain with destructive activity such as the destructive alcoholic yeast strain Saccharomyces cerevisiae K2-M111 is therefore selected.

The properties of the strain created and parental strains are compared to Table II. Since the strain created namely the strain Saccharomy these cerevisiae-K2-M possesses the destructive plasmid
K2 (L-ds RNA, M-ds RNA) and exhibits destructive activity at 30 ° C, similar to that of the destructive yeast
K2 donor, strain Saccharomyces cerevisiae 5170, it manifests an effect of primary importance in alcoholic fermentation, particularly in continuous fermentation where the possibility of contamination by wild yeasts is great.

This strain was deposited under the microorganic designation "Killer alcohol yeast Saccharomyces cerevisiae K2-M111 strain" (FERM-P No. 7071) with the "Fermentation Research Institute of the
Industrial Science and Technology ".

By electrophoretic analysis on an agarose gel, it is confirmed that the recipient alcoholic yeast, strain Saccharomyces cerevisiae Mu1, lacks L-ds RNA, M-ds RNA bands and that the strain created
Saccharomyces cerevisiae K2-Mt possesses L-ds RNA,
M RNA with the same molecular weight as the destructive donor yeast, strain Saccharomyces cerevisiae 5170.

TABLE 2
Comparison of properties of strain K2 M111 creates and properties of parental strains

<SEP> Strain <SEP> Strains
<tb><SEP> Created <SEP> Parenting
<tb><SEP> K2-M111 <SEP> M111 <SEP> 5170
<tb> Destructive <SEP> Activity <SEP> (30 C) <SEP> + <SEP> - <SEP>
<tb> Col <SEP> orati <SEP> on <SEP> TTC <SEP> Red <SEP> Red <SEP> Red
<tb> Potency <SEP> of <SEP> fermentation <SEP> of <SEP> sugar
<tb><SEP> G1 <SEP> ucose <SEP> + <SEP> + <SEP><SEP> + <SEP>
<tb><SEP> Galactose <SEP> + <SEP> + <SEP> +
<tb><SEP> Sucrose <SEP> + <SEP> + <SEP> +
<tb><SEP> Maltose <SEP> + <SEP> + <SEP> +
<tb><SEP> Lactose
<tb><SEP> Property of <SEP> Applicant <SEP> of Histidine <SEP> - <SEP><SEP> - <SEP> +
<Tb>
Example 2. Continuous alcoholic fermentation of molasses
not sterilized.

 Alcoholic yeast strain Saccharomyces cerevisiae M111 and an alcoholic yeast destructive strain Saccharomyces cerevisiae K2-M 111 are cultured separately to prepare two types of immobilized yeasts. Two bioreactors, with a volume of 110 liters, are separately filled with the two types of immobilized yeast. A solution of molasses, pre-adjusted to a total sugar content of 15% and at pH 3.7, not subjected to thermal sterilization, is passed through the reactors at a flow rate of 11 I / h ( T = 10 h) to perform a continuous alcoholic fermentation at 30 C for 100 days. Figure 6 shows the results obtained. In the bio-reactor containing the alcoholic yeast, the number of wild yeast cells reaches 20% of the total number of yeast cells on the 40th day, 40% on the 60th day and 90% on the 80th day; the yield of the fermentation, which was 83% on the 20th day, gradually decreases to 77% on the 6th day, and 69% on the 80th day, respectively, of continuous fermentation. In the effluent of the bio-reactor containing the destructive alcoholic yeast strain K2-M111, no wild yeast is detected during the entire fermentation period and the high fermentation yield of 83% remains intact for a long time.

 Thus, alcohol production must be interrupted on the 60th day of continuous fermentation when the bio-reactor uses the M111 alcoholic yeast, while the same fermentation may be continued for more than 100 days when the bio-reactor uses destructive alcoholic yeast strain K2-M111.

 Of course, the invention is not limited to the examples described and shown, it is capable of numerous variants accessible to those skilled in the art, depending on the intended applications and without departing from the scope of the invention. .

Claims (2)

 1. Process for the manufacture of alcohol from a sugar solution, characterized by the use of Saccharomyces cerevisiae, a destructive alcoholic yeast having a high alcoholic fermentation power and possessing the same destructive properties as a destructive yeast K2.  2. Process according to claim 1, characterized in that said destructive alcoholic yeast is the strain Saccharomyces cerevisiae K2-M111 K2-K11