METHOD FOR THE PRODUCTION OF MICROBIAL BIOCAPSULES, MICROBIAL BIOCAPSULES OBTAINED BY SAID METHOD AND USES THEREOF

TECHNICAL FIELD

The present invention relates to the technical field of microbiology, food technology, and bioengineering. In particular, it refers to a new method for the preparation of microbial biocapsules, to the microbial biocapsules obtained by said method and to the uses thereof.

BACKGROUND OF THE INVENTION

Yeast biocapsules are spherical-shaped yeast immobilization systems whereby yeast cells are attached to or entrapped in the hyphae of filamentous fungus (Figure 1, Garcia- Martinez, T. et al. “Co-culture of Penicillium chrysogenum and Saccharomyces cerevisiae leading to the immobilization of yeast", Journal of chemical technology and biotechnology, 2011, 86(6)).

Cell immobilization systems aim to physically confine intact cells to a certain region of space with the preservation of their biological activity. Their use offers numerous advantages over freely suspended cells in fermentation processes, such as facilitating yeast recovery and reusability, increasing ethanol tolerance and other inhibitors, and allowing easier product recovery (Mohd Azhar, S.H. et al. “Yeasts in sustainable bioethanol production: A review", Biochemistry and biophysics reports, 2017, 10, p. 52- 61).

In comparison with conventional immobilization technologies, yeast biocapsules use culturable, natural food-grade, and biodegradable filamentous fungal biomaterial as carriers. The porous and flexible fungal hyphae network allows it to withstand high mechanical force in bioreactors while other systems (e.g. alginate) cannot, provoking the release of yeast cells and carrier particles to the medium that can clog the filters (Duarte, J.C. et al., “Effect of immobilized cells in calcium alginate beads in alcoholic fermentation", AMB Express, 2013, 3(1), p. 31). Further, the porosity of the filamentous fungal pellet facilitates diffusion of nutrients/products which leads to higher product (i.e. ethanol) yields (Lopez-Menchero, J.R. et al., “Effect of calcium alginate coating on the cell retention and fermentation of a fungus-yeast immobilization system", LWT, 2021, 144, p. 111250).

When using yeast biocapsules, it is possible to keep the fungus alive or inactive depending on the application. In some cases, like winemaking or brewing, the filamentous fungus must be inactivated to inhibit filamentous fungus metabolism and competition with immobilized yeast cells. In some other circumstances, it is preferable to keep the filamentous fungus active in order to take advantage of its metabolism, which can be synergistic with the yeast’s metabolism. For instance, the fungus (unlike yeasts) are able to hydrolyze starch or lignocellulose from agricultural residues. This allows the simultaneous and coordinated fermentation of both yeast and fungus to produce advanced materials and products from polysaccharides, which is critical in biorefineries and food industry (e.g. beer and sake making). Moreover, the fungal biomass may be specifically cultured to express characteristics of interest or genetically modified.

The current method of forming yeast biocapsules consists of the co-inoculation of yeast and filamentous fungal spores to grow both organisms simultaneously, promote yeast cell entrapment in the filamentous fungus pellet, and permit yeast-hyphae attachment. This procedure is optimal for yeast cell attachment on the filamentous fungal hyphae but restricts the growth of microorganisms, which leads to production limitations, high risk of contamination, and scalability constraints. Certain yeast and filamentous fungal strains are not compatible for the production of yeast biocapsules because they cannot co-culture in the same medium or do not attach. In what follows, the spore-yeast co-inoculation methodology to produce yeast biocapsules will be also referred to as CMYB.

In comparison to the CMYB, the method for the production of yeast biocapsules, object of the invention, is a spore-yeast separated inoculation and vacuum infusion method referred to as SVMYB. As it will be further detailed hereinbelow, the new method offers several advantages over the CMYB process, providing a solution to the problems of scalability, contamination risk and low yeast biocapsule production of the current CMYB process, among other particularities.

Advantageously, the invention is not only limited to the production of yeast biocapsules, but it could be also applied for the production of biocapsules based on a different type of microorganism, such as bacteria or microalgae.

DESCRIPTION OF THE INVENTION

It is a first object of the invention, a method for the production of microbial biocapsules comprising at least one type of microbial cells of a microorganism selected from the group consisting of eukaryotes (e.g., yeasts and microalgae) and prokaryote (e.g., bacteria), attached to or entrapped in a carrier of at least one type of biodegradable filamentous fungus, characterized in that the method comprises: a) cultivating the filamentous fungus by a method comprising applying vortex agitation (preferably at a speed between 2000 and 2700 rpm) and sonication (preferably for a period of time comprised between 2 and 10 minutes and more preferably for 5 minutes) to filamentous fungal spore suspension in water, followed by an inoculation step to reach a concentration of between 0.1 x10 ⁶ and 1x10 ⁸ spores/mL in a filamentous fungal broth medium and incubation under high agitation (understanding for high agitation, a speed comprised between 175 and 250 rpm) and a temperature comprised between 25 and 35 °C, obtaining cultured filamentous fungal pellets (understanding for pellets a matrix of intertwining hyphae that have a non-dense or hollow interior core) and separately cultivating the microbial cells, obtaining cultured microbial cells. In particular, the microbial cells and the filamentous fungus are cultivated in separate growth media; each media optimized to maximize the microorganism (i.e., yeast, bacteria or microalgae) and filamentous fungal growth, respectively. Although not limiting, in a particular embodiment wherein the microbial cells are yeast cells, they can be cultivated at a temperature between 20 and 35°C, under agitation at 150 to 200 rpm for 12 to 72 h in a YPD (Yeast extract Peptone Dextrose) medium, YMB (Yeast Malt Broth), SBD (Sabouraud Dextrose Broth) or sterile grape must. In the case of bacteria cells, they can be cultivated at a temperature between 20 and 40°C, under agitation at 150 to 200 rpm for 12 to 24 h in a NB (Nutrient Broth) or MRS broth (medium for the cultivation and enumeration of Lactobacillus spp). In the case of microalgal cells, they can be cultivated at a temperature between 20 and 30°C and 0 to 200 rpm, under continuous light in greenhouse (between 30 and 60 PPFD), for 2 to 3 weeks in a BG11 (Blue Green algae) broth or any commercial growth media (e.g. MLA, TAP, etc.); b) preparing a suspension of the cultured filamentous fungal pellets and the cultured microbial cells in water, preferably in a proportion of 1 :1 to 1 :3 wet weight and subjecting the suspension to at least one vacuum infusion cycle, forcing the microbial cells inside the tight hyphae matrix of the filamentous fungual pellets; and c) incorporating the microbial cells entrapped in the filamentous fungus matrix into a growth medium (preferably a YPD, YMB or SBD broth or a sterile grape must, in the case of yeasts; a NB or MRS broth in the case of bacteria; or a BG11 , MLA or TAP broth in the case of microalgae), and incubating it at a temperature between 20 and 40 °C (preferably the microorganism optimal temperature), so that the microbial cells are attached to or entrapped in the filamentous fungus, forming the microbial biocapsules. This step is followed by a thorough rinsing procedure, preferably with deionized water, to remove the cells located in the surface of the biocapsules that can lead to cell leakage during the subsequent uses of the biocapsules. In a particular embodiment of the invention, after the rinsing process, in order to inactivate the microorganisms located in the periphery of the microbial biocapsules, said biocapsules can be submerged in a 2.5 to 7.5% (v/v) formol solution for three to six minutes under gentle shaking (from 25 to 75 rpm) to inactivate the yeasts, bacteria or microalgae located in the periphery of the biocapsules. Alternatively, in another particular embodiment of the invention, after the rinsing process the biocapsules can be coated with at least one layer of at least one biopolymer preferably selected from the group consisting of alginate, cellulose acetate, chitosan, pectin and gelatin, collagen or other plant proteins; in a percentage of between 0.2 and 4% (w/v). In still another embodiment of the invention, both processes can be carried out, i.e. , after the rinsing process, the biocapsules can be first submerged in a formol solution, rinsed with water and subsequently coated with at least one biopolymer layer.

In a particular embodiment of the invention, the process can additionally comprise a preliminary step, previous to cultivating the filamentous fungus, of preparation of the suspension comprising the spores of the filamentous fungus, preferably by pre-growing the filamentous fungus on a filamentous fungus sporulation media for 7 days at a temperature between 25 and 35 °C (preferably at 28 °C or 30 °C). This step can be carried out by any conventional process, not limiting for the invention as claimed.

One of the advantages of the invention is that, due to the application of vortex agitation and sonication of the suspension comprising the spores of at least one type of filamentous fungus, the spores are maintained separated, allowing the subsequent formation of filamentous fungal pellets. Otherwise, the spores would form amorphous agglomerations, which are advantageously avoided in the present method.

In a particular embodiment of the invention, before preparing a solution of the cultured filamentous fungal pellets and the cultured microbial cells in water (step (b)), the cultured filamentous fungal pellets can be inactivated immediately after pellet formation is complete. This inactivation process can be carried out, for example, by applying a physical or a chemical treatment.

In a further particular embodiment of the invention, the filamentous fungus can be edible (e.g., filamentous fungus used in the food industry).

It is also an object of the invention that the microbial biocapsules obtained by said process are characterized by being spherical-shaped microorganism immobilization systems whereby the microbial cells are attached to or entrapped in the hyphae matrix of filamentous fungus. In particular, these microbial biocapsules are capable of immobilizing millions of microbial cells. For example, in a particular embodiment of the invention wherein the microbial biocapsules are yeast biocapsules, it has been demonstrated that each biocapsule of between 0.01 and 0.1 mg dry weight is able to immobilize 0.85 to 8.5 millions of yeast cells.

In a particular embodiment of the invention, the microbial biocapsules can have a diameter from 1 to 20 mm and from 5 to 30% dry weight in relation to the total weight.

Finally, it is an object of the invention the use of the microbial biocapsules in fermentation processes, preferably in the production of wine, beer or biofuels such as bioethanol, biodiesel or biomethane; in food or health sectors (e.g., for the immobilization of probiotics in edible fungi), for encapsulation and delivery of microbes (e.g., beneficial plant microbes such as bacteria that can be used as biocontrol agents or beneficial bacteria such as nitrogen-fixing bacteria), in bioremediation, in pharmacy (e.g., in the synthesis of pharmaceutical drugs by immobilizing antibiotics, hormones, or drug-producing cells and/or for increasing the production by synergy with a specific type of filamentous fungus).

BRIEF DESCRIPTION OF THE FIGURES The following figures are given to show preferred embodiments of the invention as claimed:

FIG. 1 (State of the art). Macroscopic picture and scanning electron micrographs (SEM) of yeast biocapsules illustrating the immobilization of yeast cells to fungal hyphae.

FIG. 2. Methods of obtaining yeast biocapsules: FIG. 2A shows a state of the art sporeyeast co-inoculation methodology (CMYB), and FIG. 2B shows a spore-yeast separated inoculation and vacuum infusion methodology (SVMYB).

FIG. 3. Use of yeast biocapsules to restart stuck fermentations that comprises: preadaptation of yeast biocapsules (held in a mesh) in a container with arrested wine (FIG. 3A), transfer to the tank with arrested wine (FIG. 3B), and yeast reutilization (FIG. 3C).

FIG. 4. Use of free yeast in a conventional method (FIG. 4A), as compared to a process that uses yeast biocapsules inocula to ease clarification of fermentation products (FIG. 4B). In particular, both methods comprise: the inoculation of yeast (FIG. 4A1) or yeast immobilized in biocapsules (FIG. 4B1); the fermentation of must (FIG. 4A2 and FIG. 4B2), wherein it can be observed that the method inoculated with yeast biocapsules leaves less precipitated yeast cells (FIG. 4B2); yeast removal and product recovery (must) (FIG. 4A3 and FIG. 4B3), wherein it is observed that the method inoculated with yeast biocapsules implies less product loss (FIG. 4B3); and the possibility of easy yeast reutilization (FIG. 4B4).

FIG. 5. Use of yeast biocapsules in sequential fermentations: a first stage comprising inoculation, fermentation and removal of biocapsules with a specific yeast species/strain (FIG. 5A); a second stage comprising inoculation with biocapsules with another yeast species/strain, fermentation and removal of biocapsules (FIG. 5B); and a third stage comprising inoculation with yeast biocapsules with another yeast species/strain, fermentation, and removal of biocapsules with yeast (FIG. 5C).

FIG. 6. Use of yeast biocapsules in sparkling wine elaboration that comprises grape harvesting (FIG. 6A), alcoholic fermentation in tank (FIG. 6B), prise de mousse in bottles (FIG. 6C) (comprising inoculation of yeast biocapsules and addition of tirage liqueur (FIG. 6C1), second alcoholic fermentation and aging (FIG. 6C2) and riddling (FIG. 6C3)), disgorging (FIG. 6D) and labeling (FIG. 6E).

FIG.7. Production (FIG. 7A) and productivity (FIG. 7B) of yeast biocapsules using the state of the art method of generating yeast biocapsules (CMYB) and the new assembly of yeast biocapsules using the separate inoculation and vacuum infusion method as claimed (SVMYB) (Caption: DW: dry weight).

FIG. 8. Fig. 8A shows the cell immobilization yield using the state of the art CMYB method as compared with the SVMYB method as claimed. Fig. 8B and Fig 8C show SEM images wherein it can be observed yeast cells that are immobilized both in the filamentous fungus pellet surface and core.

FIG. 9. Major volatile compounds (Fig. 9A) and ethanol concentration (Fig. 9B) in wines made with yeast biocapsules using the state of the art method of generating yeast biocapsules (CMYB), the new assembly of yeast biocapsules using the separate inoculation and vacuum infusion (SVMYB) method as claimed, and free yeast cells (FY).

FIG. 10. Yeast cell retention after alcoholic fermentation in yeast biocapsules expressed in total immobilized cells after fermentation (Fig. 10A) and total non-immobilized or released cells after fermentation (Fig. 10B) using the state of the art method of generating yeast biocapsules (CMYB) and the new assembly of yeast biocapsules using the separate inoculation and vacuum infusion (SVMYB) method as claimed. Fig. 10B also includes a comparison of the results with a fermentation conducted with free yeast cells (FY).

FIG. 11. Fig. 11A shows Lacticaseibacillus casei cell immobilization in biocapsules using the separate inoculation and vacuum infusion (SVMYB) as claimed. Fig. 11 B and Fig. 11C show SEM images wherein it can be observed that bacterial cells are attached to or entrapped in the filamentous fungus hyphae both on the surface of the pellet and in the core.

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment of the invention, the method for the production of microbial biocapsules can consist of a method for the production of yeast biocapsules that can comprise: a) cultivate the filamentous fungus and the yeast cells separately. In a preferred embodiment, the yeast cells are inoculated in a YPD liquid medium (comprising yeast extract 1% w/v, peptone 2% w/v and dextrose 2% w/v) for 12 h at a temperature comprised between 20 and 35°C (preferably at 28 °C) and under an agitation speed comprised between 150 and 200 rpm (preferably of 175 rpm), obtaining cultured yeast cells.

On the other hand, filamentous fungus spores are grown on a filamentous fungus sporulation media (preferably comprising 1.7% w/v corn meal agar, 0.1% w/v yeast extract, 0.2% w/v glucose and 2% w/v bacteriological agar) for 7 days at a temperature comprised between 25 and 35 °C (preferably at 30 °C). Next, the filamentous fungus spores are suspended in sterile deionized water. The suspension is then vortexed and sonicated for a period of time comprised between 2 and 10 minutes (preferably 5 minutes), and inoculated to reach a final concentration of between 0.1 x10 ⁶ and 1x10 ⁸ spores/mL in a filamentous fungus broth medium (preferably comprising 6% w/v glucose, 0.3% w/v yeast extract, 0.3% w/v NaNCh, 0.1% w/v K2HPO4, 0.05% w/v MgSC , 0.05% w/v KCI, 0.001 % w/v FeSC ; adjusting the pH to 5.5, preferably with HCI), being after that incubated under high agitation (preferably between 175 and 250 rpm, and more preferably at 250 rpm at a temperature preferably between 25 and 35 °C and more preferably of 30 °C). After a period of time (preferably of 3 days) the filamentous fungus pellets are harvested; b) preparing a suspension of the cultured filamentous fungus pellets and the cultured yeast cells in water in a proportion of 1 :1 to 1 :3, preferably of 1 :1 wet weight and subjecting the suspension to a vacuum infusion process, forcing the yeast cells inside the tight hyphae matrix of the filamentous fungus pellets; and c) incorporating the yeast cells entrapped in the filamentous fungus matrix into a growth medium (preferably a YPD liquid medium), incubating it at a temperature between 20 and 40 °C (preferably at 28 °C), so that the yeast cells are attached to or entrapped in the filamentous fungus, forming the yeast biocapsules. After this procedure, the biocapsule surface is thoroughly rinsed with deionized water to remove the cells that could cause cell leakage when the biocapsule is used. For the same purpose, yeasts located in the periphery of the biocapsules can be inactivated by submerging the biocapsules in a 5% (v/v) formol solution for three minutes under moderate shaking (preferably at 50 rpm) and/or coated with at least one biopolymer layer, wherein the biopolymer can be selected from a group consisting of alginate, cellulose acetate, chitosan, pectin and gelatin, collagen or other plant proteins, preferably at a concentration of 1% (w/v). In case both processes are carried out, the biocapsules are rinsed with water after the formol treatment and before applying the coating layer.

In a preferred embodiment of the invention, the process can comprise an additional step of inactivation of the cultured filamentous fungus pellets previous to the incorporation of the culture yeast cells. This inactivation process can be carried out applying a physical treatment including pulsed, high voltage electric fields like pulsed electric field, high- moderate hydrostatic pressure, high shear pressure, and heat; more preferably, autoclaving the cultured filamentous fungus pellets at a temperature comprised between 100 and 150°C (preferably of 121 °C), 1 atm overpressure and during a period of time comprised between 15 and 25 min (preferably of 20 min); or chemically, submerging the cultured filamentous fungus pellets in 70% (v/v) ethanol and maintaining the solution under agitation during a period of time comprised between 1 and 5 h (preferably for 2 h) at a speed between 50 and 150 rpm (preferably of 100 rpm). Finally, the solution can be washed with sterile deionized water.

Figure 2 shows a comparison between the state of the art method used for the production of yeast biocapsules based on spore-yeast co-inoculation (CMYB) (FIG. 2A) and the method as claimed, based on spore-yeast separated inoculation and vacuum infusion methodology (SVMYB) (FIG. 2B).

In particular, FIG. 2A consists of the co-inoculation of yeast and filamentous fungus spores to grow both organisms simultaneously during 7 days at a temperature of 28°C and under agitation at 150 rpm in a BFM (Biocapsule Formation Medium) broth.

FIG. 2B shows a particular embodiment of the process as claimed that comprises: a) obtaining cultured filamentous fungus pellets by a method comprising subjecting a fungal spore suspension to vortex and sonication, followed by an inoculation step in a filamentous fungus broth, reaching a concentration of 1x10 ⁶ spores/mL, and an incubation step during 3 days at a temperature of 30°C and under high agitation (250 rpm) in a FPM broth; b) separately cultivating the yeast cells, c) preparing a suspension of the cultured filamentous fungus pellets and the cultured microbial cells in water in a proportion of 1:1 wet weight and subjecting the suspension to a vacuum infusion process, forcing the yeast cells inside the tight hyphae matrix of the filamentous fungus pellets; d) incorporating the microbial cells entrapped in the filamentous fungus matrix into a growth medium (YPD), and incubating it at a temperature of 28 °C during 1 day, forming the yeast biocapsules; and e) rinsing with deionized water to remove microbial cells located in the surface of biocapsules that could cause cell leakage when the biocapsules are used. Optionally, biocapsules can be immersed in a 5% (v/v) formol solution in a vessel for three minutes under gentle shacking to inactivate the yeasts from the periphery and/or coated with a biopolymer layer such as an alginate layer at a percentage comprised between 0.2 and 2% (w/v).

As it can be observed from Figure 2, the method object of the invention entails several distinctive features and steps versus the CMYB method:

• about 3-33 time larger filamentous fungus spore inoculation size,

• vortex and sonication steps previous to fungus inoculation to disperse spores and promote the formation of individual pellets over filamentous fungus amorphous clumps,

• culture yeast cells and filamentous fungus pellets in separate growth media; each media optimized to maximize yeast and filamentous fungus growth, respectively,

• about 1.7 higher agitation speed in the filamentous fungus culture to stimulate the filamentous fungus aerobic metabolism and growth,

• combination of yeast with fungus post pellet formation,

• vacuum-induced infusion of yeast cells into the filamentous fungus pellets that allow cell entrapment and subsequent yeast-hyphae attachment,

• washing step of biocapsules to remove microbial cells in the surface of the biocapsules that could cause cell leakage when the biocapsules are used,

• optionally inactivating the yeasts through the immersion of biocapsules in a 5% (v/v) formol solution for three minutes under gentle shacking, followed by a subsequent washing step, and optional biocapsule coating step with a biopolymer layer such as an alginate layer at a percentage of between 0.2 and 2% (w/v).

In addition, although it is not shown in Figure 2, the process as claimed allows the utilization of edible filamentous fungus (if required) that are traditionally used in the food industry.

All of these steps provide advantages over the state of the art CMYB process. Table 1 summarizes them. In particular, the SVMYB process as claimed:

1) is scalable from a laboratory to an industrial bioreactor scale,

2) increases the range of applications of the biocapsules, particularly, the use of edible filamentous fungus traditionally used in the food industry increases the range of applications within the food and pharma industries,

3) is customizable, by adjusting the spore inoculum size and agitation speed to customize the size and biomass production of the biocapsules,

4) has low risk of contamination,

5) allows to increase the production of the biocapsules,

6) achieves a higher cell immobilization yield,

7) decreases processing time,

8) provides lower t cell leakage and high mass transfer during fermentation processes,

9) allows unlimited fungal-cell combinations, since the method is not only restricted to yeasts with certain adhesive properties and allows the encapsulation of other microorganisms such as bacteria or microalgae.

Table 1. Main differences between the state of the art method of generating yeast biocapsules (CMYB) and the method as claimed (SVMYB).

In a particular embodiment of the invention, the yeast biocapsules obtained by the process as claimed can be used to restart stuck fermentations, understanding for them fermentations that have ceased prematurely or the rate of fermentation is considered too low for practical purposes, leaving a higher residual sugar content (>2 g/L) than desired in the alcoholic product at the end of the fermentation. Among other problems, stuck fermentations result in alcoholic fermentation delays, off-aroma development, and a significant probability of microbiological contamination, all of which can result in important financial losses for large wineries (Bisson, L.F. “Stuck and Sluggish Fermentations”, American journal of enology and viticulture, 1999, 50(1), pp. 107-119).

Winemakers usually resolve this problem by the addition of: (i) 50:50 juice:arrested wine, (ii) new fresh yeast with high alcohol tolerance and high fermentation capacity, (iii) yeast hulls that are able to absorb any toxic compounds that have led to the stuck ferment or (iv) a combination thereof (Bisson, L.F. “Stuck and Sluggish Fermentations”, American journal of enology and viticulture, 1999, 50(1), pp. 107-119). FIG. 3 shows that yeast biocapsules, loaded with millions of active yeasts inside, reactivates and finishes fermentations. In this process, yeast biocapsules (i.e. , the immobilized yeast cells) can be firstly pretreated with the arrested wine, allowing an easier adaptation of the yeasts, and subsequently be added to the tank with arrested wine to efficiently restart the fermentation process. In this particular use, the yeast biocapsules can be employed inside a promesh bag for tanks to facilitate their handling and reutilization.

In a further particular embodiment of the invention, the yeast biocapsules can be used in an alcoholic fermentation and, once the fermentation is finished and before the product bottling, the yeast biocapsules can be recovered and reutilized in new fermentation processes. This is an important advantage as compared to current processes wherein, once the yeast are removed, they are frequently discarded using inefficient and expensive filtration equipment that often results in product loss. As compared to the current processes, the yeast biocapsules as claimed allow to eliminate yeasts without product losses or filter clogging in a simple, cost-effective, safe, and quick manner, reducing fermentation failures and the associated economic losses. Further, the yeast biocapsules can be reused without losing their fermentative efficiency (up to 14 times), saving time and money on a crucial and fundamental step in the alcoholic product manufacturing process.

FIG. 4 shows a comparison between the use of free yeasts (FIG. 4A) and the use of yeast biocapsules (FIG. 4B), which reduces the loss of precipitated yeasts and allows the reutilization of the yeast biocapsules in further fermentation processes.

Furthermore, the yeast biocapsules as claimed can be used to customize wine chemical profiles through sequential fermentations with different immobilized yeasts. In particular, sequential fermentations are those where non- Saccharomyces yeasts are inoculated first, before Saccharomyces cerevisiae yeasts are added to take over the fermentation (Escribano-Viana, R. et al. “Wine aroma evolution throughout alcoholic fermentation sequentially inoculated with non- Saccharomyces/ Saccharomyces yeasts", Food research international, 2018, 112, p. 17-24). However, although this is considered a useful tool to modulate wine characteristics (Escribano-Viana, R. et al. “Wine aroma evolution throughout alcoholic fermentation sequentially inoculated with non- Saccharomyces/Saccharomyces yeasts”, Food research international, 2018, 112, p. 17- 24) or to reduce ethanol content to adapt to market trends, this practice has some flaws. For instance, if some of these yeasts remain in the tank for long periods, they provide negative fermentation products like ethyl acetate or lead to competition between yeast species (Canonico, L. et al. “Sequential Fermentation with Selected Immobilized Non- Saccharomyces Yeast for Reduction of Ethanol Content in Wine”, Frontiers in microbiology, 2016, 7, p. 278).

The yeast biocapsules as claimed can be used to solve this problem by immobilizing different types of yeast species that can provide a variety of characters to the wine. FIG. 5 shows this particular use of the yeast biocapsules. In particular, it is possible to design sequential fermentations wherein the immobilized yeasts are quickly removed once they have made their contribution but before they can lead to off-characters and competition with other yeasts.

In the process of winemaking, any strain of yeast may be selected and immobilized onto the yeast biocapsules. These may be strains which winemakers believe to be key to ferment their signature wines, the so-called “house strain”, or indiginous yeasts native to the winery. A mixture of indigenous yeast species and strains can be used as starter cultures tailored to reflect the yeast biodiversity of a given region. These fermentations, on the other hand, are difficult to manage, have low wine reproducibility from year to year, and may result in a stopped or sluggish fermentation, which raises the risk of wine spoilage. To prevent these issues, winemakers frequently conduct fermentations with native yeasts and later with a commercial yeast starter or carry out the fermentations with starters but at lower than recommended inoculum levels (Pretorius, I.S., “Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking’’, Yeast , 200, 16(8), pp. 675-729).

In this way, the yeast biocapsules as claimed offer an important advantage since they allow to "customize" the wines. In particular, winemakers will have the opportunity to creatively choose any combination of microorganisms to formulate a personalized yeast biocapsule that reflect the winery needs. Rather than creating undesired byproducts, the yeast can be inoculated and removed when the required metabolites are released.

In a further particular embodiment of the invention, the yeast biocapsules can be used to facilitate the riddling process in the elaboration of sparkling wine. In this relation, the traditional method (Champenoise) of sparkling wine elaboration involves a process after fermentation known as “prise de mousse" which entails a second fermentation and a period of aging in closed bottles. This process is followed by lees (dead yeast cells) riddling and disgorging before corking and marketing. The purpose of the riddling step is to slowly collect the lees into the neck of the bottle. It involves gradually tilting the bottle neck down while twisting it clockwise and anti-clockwise in small increments. Gravitational forces pull the sediment into the neck as the angle of tilt increases. Lees removal is a very labour-intensive and time-consuming process that lasts for about 24 days. Winemakers use automated removal systems. However, they take up a significant amount of physical space in the warehouse. As an alternative, some winemakers use yeast immobilized in alginate beads. However, this system increases calcium and sodium ions in the finished wine that can cause crystal precipitation (Lopez de Lerma, N. et al., “Influence of two yeast strains in free, bioimmobilized or immobilized with alginate forms on the aromatic profile of long aged sparkling wines’’, 2018, Food chemistry, 250, pp. 22-29).

The use of the yeast biocapsules as claimed as yeast immobilization systems allows complete riddling in less than two minutes. It has been also demonstrated that the obtained wines show slight differences (chemical/sensorial) and better foaming properties versus wines produced with free cells (Lopez de Lerma, N. et al., 2018, Food chemistry, 250). When inverted, the yeast biocapsules have a higher density than the wine and will swiftly fall to the neck of the bottle. Because the yeast biocapsules bind yeast together more firmly than traditional riddling, less wine is lost during the disgorging process. For this use, yeast biocapsules are added directly to the empty bottles, followed by the tirage liqueur (wine made from a first fermentation) and, after that, the bottles are capped. Then, the second fermentation and aging takes place. Preferably, the bottles are then stored on their sides for maximum contact between the wine and the yeast biocapsules.

FIG. 6 shows a comparison between the traditional sparkling wine elaboration process and the modifications introduced with the method as claimed.

Examples

The following examples show the results of the experiments carried out to demonstrate that the method as claimed outperforms the conventional method for the production of yeast biocapsules:

Example 1. Demonstration that the method as claimed increases production and productivity.

As previously described, the traditional technique of producing yeast biocapsules, which involves inoculating yeast and filamentous fungus spores in the same medium, has a production limit. In order to confirm the increase in yeast biocapsules production, yeast biocapsules were produced using the CMYB and SVMYB methodology. To quantify dry weight, yeast biocapsules were dried in an oven at a constant temperature of 80°C overnight, and then referred to the volume of culture medium. Figure 7 shows a notable increase in production and productivity when using the new method as claimed versus the current CMYB process. This result can be explained by the fact that in the SVMYB process, the organisms do not compete with one another, and the medium composition is geared towards biomass development rather than filamentous fungus-yeast attachment.

Example 2. Demonstration that the method as claimed increases biocapsule cell immobilization yield.

The methodology to produce microbial biocapsules as claimed entails a novel, non- obvious step, namely, the microbial cells vacuum infusion process. This technique forces microbial cells inside the tight hyphae matrix and fill the “air pockets” in the pellets thus, increasing the microbial cell population in the pellet core over the pellet surface. Furthermore, the washing step at the end of the procedure reduces the possibility of cell detachment from the pellet. In this way, the possibility of cell leakage from the pellet surface is reduced and immobilization yield is improved.

In order to analyze the cell immobilization with the new method as claimed, some experiments were carried out to measure the immobilized yeast cells per gram of yeast biocapsules when using the SVMYB method as compared with the results obtained when using the CMYB method. Cells were quantified by using the same protocol as described in Lopez-Menchero, J.R. et al., “Effect of calcium alginate coating on the cell retention and fermentation of a fungus-yeast immobilization system", LWT, 2021, 144, p. 111250. Further, the results were imaged using Scan Electron Microscopy (SEM) to visualize the yeast cells in the core and the surface of the pellets following the protocol described in Garcia-Martinez, T. et aL, “Potential use of wine yeasts immobilized on Penicillium chrysogenum for ethanol production", Journal of chemical technology and biotechnology, 2012, 87(3), pp. 351-359. The resulting samples were examined and photographed with a Thermo Fisher Quattro S Environmental SEM.

Figure 8A shows that there is a significant increase in the immobilized population within the filamentous fungus pellets, as expected. An explanation to this result is that yeast cells reach regions within the hypha framework that otherwise could not, i.e., if using the CMYB technique. Yeast cells attached to or entrapped in the pellet surface tend to leak from the carrier. After infusion, during the YPD culture step, yeast cells continue growing from the deepest part of the filamentous fungus pellet, thus, resulting in higher immobilization yields. Yeast cells are observed in the filamentous fungus pellet surface and core (Figures 8B and 8C).

Example 3. Demonstration that the yeast biocapsules produced with the method as claimed are functional, not affecting significantly the chemical and organoleptic profile of wines obtained and retaining a high yeast population.

In order to test the functionality of the yeast biocapsules using the method as claimed, three parallel alcoholic fermentations of grape must were conducted, inoculating with CMYB, SVMYB, and free yeast cells (FY) as the conventional winemaking practice. Fermentations were stopped at day 15 when all residual sugars were consumed. The wines obtained were subjected to a chemical analyses (gas chromatography and potassium dichromate assay for quantification of volatile compounds and ethanol, respectively) using the same protocols as described in Lopez-Menchero, J.R. et al., “Effect of calcium alginate coating on the cell retention and fermentation of a fungus-yeast immobilization system”, LWT, 2021, 144, p. 111250.

Figure 9 shows the main chemical compounds that influence wine sensory properties. It is observed that those compounds that overpassed the odorthreshold value or the concentration above which the compound is olfactory perceived, are the same in all inoculation formats except for ethyl lactate. This compound provides pineapple, varnish, balsamic aromas; and was not perceived in FY wines while it is in those wines inoculated with immobilized formats. Furthermore, an organoleptic analysis was also conducted. The tasting panel could not distinguish between SVMYB and FY wines (27.78% of judges could distinguish the sample), and higher general scores were given to the wines produced with biocapsules (6.96/10 versus 6.63/10 in free yeast wine). The fact that SVMYB and FY wines present overally similar chemical and organoleptic profiles (and even better scores) is a promising result taking into account that the use of yeast immobilization systems in industrial settings provides numerous advantages over free yeasts, as previously described.

After fermentation, the number of yeast cells immobilized and released to the wine was quantified using the same protocols as in example 2. It is observed in figure 10 that SVMYB retained more yeast cells than CMYB. This provides an important advantage for clarifications, avoiding product loss or clogging of filtration systems; as well as yeast cell reutilization in the wine industry.

Example 4. Demonstration that the new method as claimed allows encapsulation and immobilization of other types of cells and not only yeast.

As it has been previously described, the SVMYB method as claimed allows the encapsulation of other types of cells whose size allows their infusion through the hyphal matrix. This overcomes the restriction in the previous CMYB methodology in which only specific types of yeast species and strains can be used to produce the yeast biocapsules. This fact widens the number of applications depending on the immobilized cells. To study the capacity to encapsulate other types of cells, two bacterial species, Lacticaseibacillus casei and Lacticaseibacillus rhamnosus, were encapsulated in filamentous fungus pellets using the same methodology described in example 1 , but replacing YPD culture medium by MRS broth (medium for the cultivation and enumeration of Lactobacillus spp). Both species are considered probiotics, whilst the first one has also applications in the dairy and medical sector. Immobilized bacterial cells were quantified and visualized under widefield microscope.

Figure 11 shows how L. casei cells are attached both on the surface and inside the fungal pellets. Like yeast cells, bacterial cells can penetrate through the pores of the hyphal net and grow after the culture step. This fact allows the possibility of encapsulating and immobilizing other types of cells, thus bringing the chance to apply biocapsules to other purposes.

The above experiments demonstrate that the new methodology of assembling biocapsules brings three main advantages over the current CMYB methodology: i) it increases the production and productivity of biocapsules, ii) it increases cell immobilization, and iii) it allows the immobilization of other types of microbial cells, such as bacterial cells.