WO2012082079A1

WO2012082079A1 - Procedure for the magnetic separation of yeast biomass from sparkling wine

Info

Publication number: WO2012082079A1
Application number: PCT/SI2011/000074
Authority: WO
Inventors: Marin Berovic; Darko Makovec; Suzana BOŠKOVIC
Original assignee: Univerza Ljubljana v Fakulteta za Farmazijo; Institut Jozef Stefan
Current assignee: Univerza Ljubljana v Fakulteta za Farmazijo; Institut Jozef Stefan
Priority date: 2010-12-13
Filing date: 2011-12-12
Publication date: 2012-06-21
Anticipated expiration: 2013-06-13
Also published as: SI23583A

Abstract

The topic of this invention is a procedure for the magnetic separation of yeast biomass from sparkling wine using magnetic particles. The procedure will replace the time-consuming and expensive classic procedure. The classic procedure is based on manual rotation and simultaneous tilting of the bottles from the horizontal to the vertical position with the bottle neck facing down (remuage). For the classic procedure to be completed, approximately 60 days of manual labour are needed to sediment the used yeast biomass in the bottle neck. When the sediment is in the bottle neck, it is removed by freezing and expelled from the bottle during disgorgement. The application of the magnetic particles, which are adsorbed onto the yeast cells needed for the secondary fermentation, and/or on the used yeast biomass after the secondary fermentation, makes the yeast biomass responsive to the magnetic field and thus enables the magnetic separation. The biomass with magnetic particles absorbed onto its surfaces is separated into the bottle neck using an external magnetic field in approximately 15 minutes, whereas the subsequent procedure of purification for the yeast biomass from the wine remains the same as in the classic champagne procedure, including the freezing of the sediment and its expulsion from the bottle during disgorgement.

Description

PROCEDURE FOR THE MAGNETIC SEPARATION OF YEAST BIOMASS FROM SPARKLING WINE

The topic of the invention is a procedure for the magnetic separation of yeast biomass after the secondary fermentation of wine in pressure bottles. The procedure is based on the application of magnetic particles. The classic champagne procedure is based on the secondary fermentation of still wine in a pressure bottle that is started by the addition of inoculum and sugar, resulting in the formation of carbon-dioxide bubbles. In approximately one month this secondary fermentation ferments the sugar to ethanol and carbon dioxide. This results in an increase of the pressure in the bottle to between 5· 10⁵ Pa and 6 · 10^s Pa. During the subsequent aging, the formed carbon dioxide dissolves in the sparkling wine. When the sparkling wine is opened and poured into the glass, the carbon dioxide shows as chains of bubbles.

The well-known procedure for the biomass separation from the bottle after secondary fermentation is based on rotation and simultaneous tilting of the bottles from the horizontal to the vertical position (remuage) followed by freezing of the used yeast in the bottle neck. The procedure was invented by Mme.Clicot from the homonymous champagnerie in the 18^th Century and is still in use today. At least 60 days of expensive manual labour is needed to separate the used yeast with this traditional procedure.

The technical problem that is solved with this patent in a satisfactory way is the separation of the used yeast biomass after the secondary fermentation in the classic production of the sparkling wine. Currently, the traditional procedure is employed, where the used biomass in the bottle of the sparkling wine is concentrated in the neck of the bottle using the time-consuming and expensive procedure of bottle rotation and its simultaneous tilting. The sediment in the bottle neck is frozen and expelled from the bottle when the plug is released. After expelling the sediment, the bottle is closed once again with a new, usually cork, plug reinforced with a wire basket. The task and the goal of this invention is avoiding the time-consuming and expensive manual labour and shortening the time needed for the sedimentation of the used yeast biomass into the bottle neck during the sparkling-wine production.

According to this invention the task is solved with a procedure that enables the separation of the used yeast biomass with magnetic particles. After the yeast revitalization before the secondary fermentation, or after the secondary fermentation, the yeast cells are exposed to the magnetic particles in a liquid medium. The magnetic particles are irreversibly absorbed onto the yeast cells' surfaces. The yeast cells with the magnetic particles, i.e., the used yeast biomass, are then separated from the sparkling wine using an external magnetic field.

In the procedure according to the innovation, nanoparticles or larger particles of different magnetic oxides or magnetic metals in the size range between 3 nm and 10 μιη are used, although superparamagnetic nanoparticles with sizes between 10 nm and 20 nm are preferred. These superparamagnetic nanoparticles of the magnetic iron oxides should be maghemite or magnetite. The nanoparticles of the iron oxides are believed to be non-toxic and were registered by the American Food and Drug Association for in vivo medical applications. The synthesis of the iron- oxide nanoparticles is relatively simple, based on the simple precipitation of the iron ions from their aqueous solutions. Superparamagnetic beads and other magnetic particles normally used in magnetic separation can also be employed as the magnetic particles for the separation of the used yeast biomass.

It is beneficial if the magnetic particles display a positive surface charge, enabling their adsorption onto the yeast cells, which display a negative surface charge. The positive surface charge can be ensured by grafting specific molecules, such as aminosilanes, onto the particles' surfaces. The procedure of the aminosilane grafting onto the particles' surfaces is described in the following literature: S. Campelj, D. Makovec, M. Drofenik, J. Mag. Mag. Mat. 321 (2009) 1346-1350.

The separation of the cells from their suspensions in the liquid medium using the magnetic particles and the external magnetic field is well known in biotechnology. The procedure of the cell separation using magnetic nanoparticles is described in the following literature: J.L. Corchero, A. Villaverde, Trends in Biotechnology, 27 (2009) 468-476. Description of the innovation

The innovation will be described using illustrative examples and figures showing the procedure of the yeast biomass separation from the bottles after the secondary fermentation of the sparkling wine using the addition of the superparamagnetic maghemite nanoparticles and an external magnetic field.

The figures show:

Figure 1 : Changing of the zeta-potential of the 3-(2-amino-ethil-amino) propyl silane (APMS)- modified magnetic maghemite nanoparticles and of the yeast cells Saccharomyces bayanus as a function of the pH value of the aqueous suspension.

Figure 2: Transmission electron micrograph of the yeast cells Saccharomyces bayanus with the magnetic nanoparticles absorbed at their surfaces.

Figure 3: Demonstration of the yeast separation on a permanent magnet. The magnetic nanoparticles were absorbed at the surfaces of the yeast cells.

The procedure of the magnetic yeast-biomass separation from the sparkling wine after the secondary fermentation in the pressure bottles occurs with the absorption of the magnetic particles of the magnetic oxide or magnetic metal, preferably superparamagnetic nanoparticles of the magnetic iron oxide, with sizes between 3 nm and 10 μπι, preferably sizes between 10 nm and 20 nm, onto the surfaces of the yeast cells added to the wine for secondary fermentation prior to the secondary fermentation and/or onto the surfaces of the used yeast biomass after the secondary fermentation, and the external magnetic field is used for the sedimentation of the yeast biomass with the magnetic particles absorbed on it, into the bottle neck, whereas the following procedure of the purification is based on freezing of the sediment in the bottle neck and its expulsion from the bottle during disgorgement.

The use of magnetic particles absorbed on the yeast cells employed for the secondary fermentation, or on the yeast biomass after the secondary fermentation, ensures that the biomass responds to the magnetic field and can be magnetically separated. The biomass with the magnetic particles absorbed at its surfaces is sedimented in the bottle neck in approximately 15 minutes using the external magnetic field, while the subsequent procedure for the yeast biomass purification remains the same as in the classic champagne procedure, including freezing of the sediment and its expulsion from the bottle during disgorgement.

Illustrative example 1: Use of the yeast cells on which the magnetic nanoparticles are absorbed prior to the secondary fermentation of the sparkling wine in the pressure bottles followed by the magnetic separation of the yeast biomass.

In the described illustrative example the following chemicals were used:

- FeS0₄ . 7 H₂0, ACS 99+%, 033316, Alfa Aesar, USA

- Fe₂(S0₄)₃ . x H₂0, Reagent Grade, 014498, Alfa Aesar, USA

- NH₄OH, p.a., 25%, Applichem, Germany

- Tetramethyl ammonium hydroxide (TMAH), 25%, Alfa Aesar, USA

- Citric acid, 99%, Alfa Aesar, USA

- Tetraethyl orthosilicate (TEOS), 99,9 %, Alfa Aesar, USA

- 3-(2-amino-ethyl-amino) propyl silane (APMS), 97%, Alfa Aesar, USA

Yeast cells Saccharomyces bayanus, 18-2007, Epernay, France

Physiological solution

Distilled water

Wine purified from wine stone

The synthesis of the superparamagnetic maghemite nanoparticles, which display a positive surface charge, can be divided into four steps. In the first step, the maghemite nanoparticles are synthesized. In the last, fourth step, the aminosilane APMS is grafted onto their surfaces. APMS bonds to the nanoparticle surfaces with the silane group, whereas the aliphatic chain terminated with the amino group is oriented outward and thus ensures the positive surface charge of the particle. The efficiency of the APMS grafting onto the surfaces of the maghemite nanoparticles is improved if the nanoparticles are coated with a thin silica layer prior to the grafting. The silica coating to the nanoparticles' surfaces takes place in the third step of the synthesis procedure. The silica is terminated with surface OH groups that enable bonding of the silane with the formation of strong Si-O-Si bonds. To coat the individual nanoparticle with a thin silica layer and not their agglomerates, the nanoparticles are de-agglomerated in their aqueous suspension using citric acid as the surfactant in the second step of the synthesis procedure. of Fe ⁺ equal to 0.027 mol/L and of Fe(UI) sulphate with the concentration of Fe³⁺ equal to 0.023 mol/L, 140 mL of the aqueous solution of NH₄OH with a concentration of 0.5 wt.% is added. The maghemite nanoparticles displaying a narrow size distribution centred at approximately 13 nm are precipitated. The nanoparticles are superparamagnetic with a saturation magnetization of approximately 65 Am /kg.

The nanoparticles are washed with distilled water and re-dispersed in 80 mL of distilled water. The procedure of precipitation takes place at room temperature.

Second step: The stable suspension is prepared from the synthesized nanoparticles. 2 g of the synthesized nanoparticles are mixed with 60 mL of an aqueous solution of citric acid with a concentration of 0.02 g/mL, and the pH value is set with the addition of NH₄OH to pH = 5.2. Then, the suspension is heated to 80°C while being intensively stirred. After 90 minutes, the pH value of the suspension is further increased to pH = 1 1 to form the stable suspension.

Third step: Coating of the thin silica layer onto the surfaces of the dispersed nanoparticles: Into 60 mL of the nanoparticle suspension, 2.5 mL of TEOS dissolved in 25.5 mL of ethanol is added. The mixture is stirred for 3 hours at room temperature to let the hydrolysis and polycondensation of the TEOS take place and a uniform, 1-2-nm-thick silica layer is formed on the surfaces of the nanoparticles. Finally, the coated nanoparticles are washed with distilled water.

Fourth step: APMS is grafted onto the surfaces of the silica-coated maghemite nanoparticles. In 100 mL of the suspension of the silica-coated nanoparticles with a concentration of 0.5 wt.%, TMAH is added to set the pH to 10.5. Then, 270 μΐ of APMS dissolved in 25 ml of ethanol is added and the suspension is mixed for 5 hours at 50°C. The stable suspension is washed with distilled water using ultrafiltration. The aqueous suspension of the APMS-modified nanoparticles shows a strongly positive zeta-potential at a pH below the isoelectric point at pH ~ 7.0. Thus, the surface charge of the APMS-modified nanoparticles is opposite to the surface charge of the yeast cells, which is negative throughout a broad region of pH. Figure 1 shows the changing of the zeta- potential of the aqueous suspensions of the APMS-modified nanoparticles and the yeast cells as a function of pH.

To enable rapid magnetic separation of the yeast biomass after the secondary fermentation of the sparkling wine in the pressure bottles, the yeast cells Saccharomyces bayanus are exposed to the magnetic particles displaying a positive surface charge in the liquid medium. First, 375 mg of the lyophilized yeast cells are revitalized in water for 30 minutes at a temperature of 38°C. After the cooling of the yeast-cells suspension to 18°C, the aqueous suspension of the magnetic nanoparticles with a concentration of 1 .0 wt. % is added. The mass ratio between the nanoparticles and the dry yeast cells is 1 : 10. The pH of the mixture is then set to pH = 3.4 and the suspension is mixed for 15 minutes to let the nanoparticles absorb at the yeast cells. Figure 2 is a transmission electron micrograph of the yeast cells after exposure to the magnetic nanoparticles. In Figure 2 the magnetic nanoparticles are clearly visible on the surfaces of the yeast cells. Because the magnetic nanoparticles are absorbed at the yeast cells, the yeast cells respond to the magnetic field. If a permanent magnet is brought close to the suspension of the yeast cells with the absorbed magnetic nanoparticles they concentrate at the areas with the largest magnetic field, so enabling their magnetic separation. The magnetic separation of the yeast cells is illustrated in Figure 3, showing how the sediment of the yeast cells Saccharomyces bayanus with the absorbed magnetic nanoparticles is accumulated at the wall of the container close to the permanent magnet.

In the following procedure, the pressure bottles for the sparkling wine are filled with the stabilized still wine, which was previously cooled to a temperature of 18 °C and the wine stone (tartar) was separated. In the wine in the bottle 18 g of glucose and 0.38 g of yeast cells with the absorbed magnetic nanoparticles are added. The pressure bottles are closed with the metal crown cap. In the pressure bottles the secondary fermentation, i.e., the formation of the sparkling wine, takes place. The fermentation takes place at a temperature of 18 °C and is finished in approximately 30 days. At the end of the fermentation the pressure in the bottles increases to 5.2 · 10^s Pa.

After 30 days the bottles are shaken. The suspension in the bottle is placed with the bottle neck and the metal crown cap in a magnetic field of 0.5 T. In a time shorter than 15 minutes, the yeast biomass is sedimented in the bottle neck. In the following procedure, the bottles are placed with the bottle neck down into a freezer, where the bottle neck is immersed into a freezing liquid to a depth of 2 cm. After a few minutes at a temperature of - 27 °C the sediment is frozen into the ice plug. The bottle is then retracted from the freezer and the frozen yeast biomass is disgorged when the metal crown cap is opened and the inner pressure forces the frozen yeast biomass from the bottle. Using this procedure, the sparkling wine is purified from the sediment. After the following standard procedure, some sparkling wine is added to replace the missing wine, along with the liqueur d'expedition. Finally, the bottle is closed using a cork plug reinforced with a wire basket.

The sparkling wine, purified from the yeast biomass using nanoparticles, was analysed using the ICP-AES method. It was found that the maghemite nanoparticles are effectively separated from the sparkling wine using the magnetic separation. The concentration of iron in the sparkling wine remained below the acceptable limit. Based on analyses, including a sensorial analysis of the sparkling wine prepared with use of the magnetic nanoparticles, no significant differences were found compared to the control sample, prepared with the classic procedure without the magnetic nanoparticles.

Illustraiive example 2: Magnetic separation of the yeast biomass with the addition of the magnetic nanoparticles in the sparkling wine in the pressure bottles after the secondary fermentation.

The APMS-modified magnetic maghemite nanoparticles displaying the positive surface charge in the acidic region of pH are prepared as described in the illustrative example 1. To ensure the magnetic separation of the yeast biomass, the pressure bottles after the secondary fermentation are opened, the magnetic nanoparticles are added, and the bottles are closed again with plugs for multiple uses. After the absorption of the magnetic nanoparticles onto the yeast cells representing the yeast biomass, the biomass is magnetically separated into the bottle neck. The stabilized still wine is filled in the pressure bottles and the yeast cells and the glucose are added. The bottles are closed with the metal crown caps, as required by the classic procedure for sparkling wine production. After the end of the secondary fermentation at a temperature of 18 °C, which is after approximately 30 days, the cooled bottle is carefully opened and 0.028 g of magnetic nanoparticles in the form of a suspension with a concentration of 2 wt.% are added to the sparkling wine. After the addition of the nanoparticles the bottle is quickly closed with the plug for multiple uses. The closed bottle is shaken. After a time of approximately 15 minutes, when the nanoparticles are absorbed onto the surfaces of the yeast biomass, the bottle is placed with the bottle neck down in a magnetic field of 0.5 T. In a time shorter than 15 minutes the yeast biomass is separated in the bottle neck. In the following procedure, the sediment is frozen, expelled and the bottle is closed with the cork plug, as described in the illustrative example 1.

An analysis of the sparkling wine using the ICP-AES method showed that the magnetic separation effectively separates all the maghemite nanoparticles from the sparkling wine. The content of the iron in the wine was below the acceptable limit. With analyses, including a sensorial analysis of the sparkling wine prepared with use of the magnetic nanoparticles, no significant differences were found compared to the control sample, prepared with the classic procedure without the magnetic nanoparticles.

Claims

PATENT CLAIMS

1 . A procedure for the magnetic purification of a sparkling wine from a yeast biomass after secondary fermentation in pressure bottles, characterised by the use of magnetic particles of magnetic oxide or magnetic metal, preferably superparamagnetic nanoparticles of magnetic iron oxide maghemite or magnetite in the size range from 3 nm to 10 μπι, preferably between 10 and 20 nm, absorbed onto the surfaces of yeast cells that have been added to the wine for the secondary fermentation, and/or to the surfaces of the used yeast biomass after the secondary fermentation.

2. The procedure according to claim 1 , characterised by the use of external magnetic field for the sedimentation of the yeast biomass into the bottle neck.

3. The procedure according to claims 1 to 2, characterised by the use of a following procedure of purification based on freezing of the sediment in the bottle neck and its expulsion from the bottle during disgorgement.