Composition containing freeze-dried non-Saccharomyces yeast

Abstract

The present application is in the field of dried compositions of non-Saccharomyces yeasts, processes for preparing such compositions by freeze-drying, and compositions that can be prepared by such processes. The process is characterized in that the freeze-drying protective agent comprises maltodextrin, one or more antioxidants, and one or more disaccharides, and the dry weight ratio of the freeze-drying protective agent to the yeast biomass is 0.5:1 to 4:1, preferably 0.5:1 to 3:1.

Description

[Technical Field]

[0001] field This application generally relates to yeast, particularly freeze-dried yeast compositions, and their use in the preparation of fermented foods and beverage products. Furthermore, this application relates to the process for producing freeze-dried yeast. [Background technology]

[0002] background Microorganisms such as yeast are involved in numerous industrial processes. For example, yeast cultures are essential for the production of fermented foods, including fermented beverages, wine, beer, and fermented juices. In most cases, it is important that the microorganisms survive long-term storage so that they can produce beneficial effects.

[0003] The use of non-Saccharomyces yeasts in fermented foods has recently attracted attention and is gaining increasing interest. Since the 1990s, non-Saccharomyces yeasts have begun to draw attention, and 30 years later, they have transformed from undesirable yeasts associated with spoilage to microorganisms that can improve the aroma profile of products.

[0004] In wine fermentation, co-culturing of Saccharomyces yeasts with non-Saccharomyces yeasts of interest is often used for various purposes, such as producing other aromatic compounds through enzymatic reactions, reducing ethanol content, enhancing glycerol production, lowering acidity, and stabilizing color. In beer, another widely consumed alcoholic beverage, there is also growing interest in non-Saccharomyces yeasts, primarily for their aromatic depth. This interest is directly related to the development of the artisanal beer market, which seeks to develop products with novel and original aromatic characteristics. The potential use of non-Saccharomyces yeasts in other beverages derived from various fruits (particularly pineapple, lychee, papaya, and cranberry) has also been studied.

[0005] In other fermented foods such as dairy products or dairy-like products, as well as in foods or beverages prepared from cocoa beans or coffee beans, the use of starter cultures using non-Saccharomyces yeasts has attracted attention.

[0006] WO2011 / 134952 discloses a frozen starter culture containing non-Saccharomyces wine yeast for direct inoculation. However, the use of frozen cultures requires an uninterrupted cold chain during transport, which can be expensive and cumbersome. Freeze-drying, also known as lyophilization, is a dehydration method used to preserve cells in a dry state to give them suitability for storage at room temperature. Freeze-drying is a harsh process that negatively impacts both the viability and physiological state of yeast. Ice crystal formation leads to cell death during freezing and causes mechanical damage that affects viability. For example, the viability of Saccharomyces yeast was investigated after freeze-drying and storage under vacuum at 5°C. The viability of the yeast was less than 10%. Miyamoto-Shinohara, Yukie, et al. "Survival rate of microbes after freeze-drying and long-term storage." Cryobiology 41.3 (2000): 251-255.

[0007] To our knowledge, non-Saccharomyces yeasts have not yet been satisfactorily preserved using freeze-drying technology at an industrial level. Most of the described freeze-drying processes involving non-Saccharomyces yeasts have not been adapted for industrial use.

[0008] Therefore, as these microorganisms become increasingly popular for fermentation, freeze-dried yeast compositions, particularly non-Saccharomyces yeast compositions, are needed. It is especially necessary to provide a manufacturing process that can be adapted to industrial levels and that allows for good preservation and process viability of the yeast cells. Preferably, such compositions exhibit good stability and can be transported without requiring a cold chain. [Overview of the project] [Problems that the invention aims to solve]

[0009] Freeze-drying is a process known to cause temperature, osmotic pressure, and oxidative stress in yeast. The inventors have discovered that yeast cells can be effectively preserved with good process storage stability and viability by using a freeze-drying protectant containing maltodextrin, (one or more) antioxidants, and (one or more) disaccharides. The weight ratio of freeze-drying protectant to yeast (dry matter) in the composition is preferably in the range of 0.5 to 4. The prepared yeast was observed to have high viability throughout the freeze-drying step, and the resulting composition is storage-stable over long periods and can be transported at low temperatures or even at ambient temperatures. [Means for solving the problem]

[0010] This application provides freeze-dried yeast compositions, particularly freeze-dried non-Saccharomyces yeasts, such as Pichia species (Pichia spp.).

[0011] A first aspect of the present application provides a freeze-dried composition of non-Saccharomyces, preferably Pichia species, comprising a mixture of maltodextrin, (one or more) antioxidants, and (one or more) disaccharides as a freeze-dried protective agent to yeast biomass (dry matter) in a predetermined weight ratio of freeze-dried protective agent to yeast biomass (dry matter). This weight ratio is also referred to in the present application as the encapsulation index (EI). In a preferred embodiment, the weight ratio of freeze-dried protective agent to yeast biomass is 0.5:1 to 4:1, preferably 0.5:1 to 3:1 in the freeze-dried composition.

[0012] In some embodiments, the freeze-dried composition of non-Saccharomyces yeast according to the application is 10 7 ~10 12 Yeast cells, preferably 10 cfu / g 8 ~10 11cfu / g of yeast cells, more preferably 10 9 ~10 10 cfu / g of cells. In some embodiments, the lyophilized composition of non-Saccharomyces yeast according to the application may contain a non-reducing disaccharide which may be trehalose and / or sucrose. In some embodiments, the lyophilized composition of non-Saccharomyces yeast according to the application may contain a non-reducing disaccharide which may be trehalose, cellulose, and / or sucrose.

[0013] In some embodiments, the lyophilized composition of non-Saccharomyces yeast according to the application contains cellulose. In some preferred embodiments, the lyoprotectant further contains modified starch. In some preferred embodiments, the yeast is Pichia kluyveri. In a more preferred embodiment, Pichia kluyveri is DSM28484 or PK-KR1.

[0014] The second aspect of the application provides the use of a freeze-dried composition of non-Saccharomyces yeast for preparing fermented dairy products or non-dairy products, and fermented foods including beverages such as wine, beer, juice, cocoa, coffee, etc.

[0015] The third aspect of the application is a process for preparing a lyophilized non-Saccharomyces yeast composition, which includes preparing a yeast cell concentrate, adding a lyoprotectant to the yeast cell concentrate to form a suspension, and lyophilizing the suspension, wherein the lyoprotectant contains maltodextrin, (one or more) antioxidants, and (one or more) disaccharides, and the dry weight ratio of the lyoprotectant to the yeast biomass is 0.5:1 to 4:1, preferably 0.5:1 to 3:1, more preferably 0.7:1 to 2.5:1. The above process is provided.

[0016] In a preferred embodiment, the application is as follows: (1) Prepare non - Saccharomyces yeast cells, (2) Culture the yeast cells and harvest the yeast cells to obtain a yeast cell concentrate, where the concentrate contains 10 8 ~10 11 cfu / g of dry matter of yeast cells, (3) Add a lyoprotectant to the yeast cell concentrate to form a suspension, where the lyoprotectant contains maltodextrin, (one or more) antioxidants, and (one or more) disaccharides, (4) Freeze the suspension, (5) Dry the suspension, and (6) Obtain a freeze - dried non - Saccharomyces yeast composition, including, where the dry weight ratio of the lyoprotectant to the yeast biomass is 0.5:1 to 4:1, more preferably 0.5:1 to 3:1. Provide a process.

[0017] Preferably, the drying is carried out under a vacuum pressure of 0.01 - 2 mbar. Preferably, the temperature during drying is controlled so that the vacuum pressure is kept constant.

[0018] In some embodiments, the freeze - dried composition of non - Saccharomyces yeast contains 10 7 ~10 12 cfu / g of yeast cells, preferably 10 8 ~10 11 cfu / g of yeast cells, more preferably 10 9 ~10 10 cfu / g of yeast cells. In a preferred embodiment, the process further includes washing the cells after harvesting.

[0019] In some embodiments, the freeze - dried composition of non - Saccharomyces yeast contains a non - reducing disaccharide, and the non - reducing disaccharide is trehalose or sucrose. In some embodiments, the freeze-dried composition of non-Saccharomyces yeast contains a non-reducing disaccharide, which is trehalose, cellulose, or sucrose. In some embodiments, the freeze-dried composition of non-Saccharomyces yeast contains cellulose.

[0020] In some embodiments, the freeze-dried composition of non-Saccharomyces yeast contains antioxidants that may be ascorbic acid, citric acid, β-carotene, vitamin E, and glutathione and / or derivatives thereof, preferably trisodium citrate and sodium ascorbate.

[0021] In some preferred embodiments, the composition further comprises modified starch. In some preferred embodiments, the yeast is Pichia cruibergii. In more preferred embodiments, Pichia cruibergii is DSM28484 or PK-KR1. In other embodiments, the process further comprises adding (one or more) excipients to the resulting freeze-dried yeast composition.

[0022] Further embodiments provide yeast compositions obtained or obtainable by the process described in the application, and uses thereof.

[0023] Freeze-dried (FD) yeast compositions are particularly advantageous because, among other things, they offer the benefit of being able to be shipped and stored at temperatures ranging from -18°C to ambient temperature. Freeze-dried yeast can also be stored stably for extended periods at -18°C and may allow for adaptable dosing to suit producers of various scales.

[0024] This application provides a freeze-dried yeast composition having improved process viability or storage stability compared to other freeze-dried compositions known to date. Furthermore, the inventors also demonstrate, as shown in Example 6, that the freeze-dried yeast prepared by this process advantageously has similar or improved performance compared to frozen yeast compositions. [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 shows the 12-month stability data for compositions 1-7 at a storage temperature of -18°C. [Figure 2] Figure 2 shows the 12-month stability data for compositions 1-7 at a storage temperature of 4°C. [Figure 3] Figure 3 shows the 12-month stability data for compositions 8 and 10 at a storage temperature of -18°C. [Figure 4] Figure 4 shows the 12-month stability data for compositions 8 and 10 at a storage temperature of 4°C. [Figure 5] Figure 5 shows the 6-month stability data for compositions 1, 6, and 11 at a storage temperature of -18°C. [Figure 6] Figure 6 shows the 6-month stability data for compositions 1, 6, and 11 at a storage temperature of 4°C. [Figure 7] Figure 7 shows the 6-month stability data for composition 11 at a storage temperature of 25°C. [Figure 8] Figure 8 shows the 12-month stability data for composition 12 at a storage temperature of -18°C. [Figure 9] Figure 9 shows the 12-month stability data for composition 12 at a storage temperature of 4°C. [Figure 10] Figure 10 shows the 12-month stability data for compositions 13-17 at a storage temperature of 4°C. [Figure 11] Figure 11 shows the number of viable Pichia cruibergii bacteria analyzed one hour after inoculation into wine. [Figure 12] Figure 12 shows the production of 3-mercaptohexyl acetate (3-MHA) in fermented wine. [Figure 13]Figure 13 shows the 12-month stability data for compositions 1, 6, and 11 at a storage temperature of -18°C. [Figure 14] Figure 14 shows the 12-month stability data for compositions 1, 6, and 11 at a storage temperature of +4°C. [Figure 15] Figure 15 shows the 12-month stability data for composition 11 at a storage temperature of +25°C. [Modes for carrying out the invention]

[0026] Detailed explanation In general, the terms and phrases used herein have meanings recognized in the art, which can be found by referring to standard textbooks, journal references, and contexts known to those skilled in the art. The following definitions are provided to clarify their specific usage in relation to this disclosure.

[0027] As used herein, the singular forms "a," "an," and "the" are intended to include the plural form unless the context clearly indicates otherwise. As used herein, the term "and / or" means "and" or "or" or both. As used herein, "(s)" after a noun means the plural and / or singular form of that noun.

[0028] The terms "comprising," "having," "including," and "containing" should be interpreted as unrestricted unless otherwise specified (i.e., "comprising, but not limited to").

[0029] The inventors focused on a number of different protective agents for freeze-dried non-Saccharomyces yeasts and clarified a method for freeze-drying yeast cells that maintains a high viability after the process and exhibits good storage stability for the product. It was found that by using a mixture of maltodextrin, (one or more) antioxidants, and (one or more) disaccharides in specific amounts relative to the yeast cells, the yeast cells were freeze-dried in a protected manner with a high process viability. Favorable results were found to be achieved when the dry weight ratio of the freeze-drying protective agent to the yeast biomass was preferably in the range of 0.5:1 to 4:1, more preferably in the range of 0.5:1 to 3:1, and most preferably in the range of 0.7:1 to 2.5:1.

[0030] composition In a first embodiment, the present application provides a freeze-dried composition of a non-Saccharomyces yeast, preferably a Pichia species (such as Pichia cruibergii), comprising maltodextrin as a freeze-drying protective agent, one or more antioxidants, and one or more disaccharides (preferably non-reducing), wherein the dry weight ratio of the freeze-drying protective agent to the yeast biomass is preferably 0.5:1 to 4:1.

[0031] In a preferred embodiment, the application provides a freeze-dried composition in which yeast cells are present in the composition. 7 ~10 12 Yeast cells, preferably 10 cfu / g 8 ~10 11 Yeast cells with cfu / g, furt10 9 ~10 10 It exists at a cellular concentration of cfu / g.

[0032] In a preferred embodiment, the freeze-dried composition of a non-Saccharomyces yeast, preferably a Pichia species (such as Pichia cruiberi), contains maltodextrin, one or more antioxidants, and one or more non-reducing disaccharides as freeze-drying protectants, wherein the dry weight ratio of the freeze-drying protectants to the yeast biomass is 0.7:1 to 2.5:1.

[0033] The experimental results demonstrate that the freeze-dried yeast composition yields significantly better results compared to frozen yeast—the amount of aroma produced is comparable or even better, despite a smaller initial amount of cells.

[0034] Since the composition of this application is a freeze-dried composition, those skilled in the art will understand that the specified amounts of the individual components of the composition (e.g., yeast cells and protective agent) are measured as dry matter. In the context of this specification, the weight of the freeze-dried protective agent is given per 1 g of yeast cells in the composition. Those skilled in the art can prepare the composition using conventional methods so that the desired weight ratio is satisfied.

[0035] This application relates to freeze-dried yeast, particularly non-Saccharomyces yeast, and especially non-Saccharomyces yeast that can be used as a fermentation microorganism.

[0036] In one embodiment, non-Saccharomyces yeasts include Pichia cruiberi, Candida fructus, Citeromyces matritensis, Debaromyces hansenii, Hanseniaspora guillermondii, Hanseniaspora osmophila, Hanseniaspora uvarum, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Pichia fermentans, and Pichia membranifaciens. The non-Saccharomyces yeast is selected from Pichia cruibergii, Pichia membranifaciens, Lachansea thermotolerans, and Torulaspora delbrueckii.

[0037] In a preferred embodiment, the yeast is a species of the genus Pichia. The present application provides a particularly suitable method for formulating Pichia species, as this genus is known to have an unfavorable response to freeze-drying processes with low viability. Preferably, the Pichia species are Pichia fermentans, Pichia membranifasciens, and Pichia cruiberi. In a more preferred embodiment, the yeast is Pichia cruiberi.

[0038] In one embodiment, Pichia kruiberi is the strain deposited as DSM28484. In another embodiment, Pichia kruiberi is the PK-KR1 strain disclosed in Japanese Patent Publication WO2009110807A1, deposited as V06 / 022711.

[0039] Microorganisms are often preserved with the addition of protective agents (or “protectants” as used herein) that can help stabilize them in various ways. The term “protective agent(s) or protectants” (singular or plural) is understood herein to mean any agent that can help improve the viability during freeze-drying and storage stability with respect to yeast cells of interest. According to this application, a protective agent that acts to protect during freeze-drying is called a “freeze-drying protective agent.” Protection of viability from freeze-drying stress is called “freeze-drying protection.”

[0040] This application relates to the combined use of (one or more) antioxidants, (one or more) disaccharides, and maltodextrin as suitable freeze-drying protective agents.

[0041] One of the freeze-drying protective agents used in this application is an antioxidant. The term “antioxidant” refers to a compound that inhibits oxidation. Antioxidants may be industrial chemicals or natural compounds. As used herein, antioxidants include, but are not limited to, ascorbic acid, citric acid, β-carotene, vitamin E, glutathione, and their derivatives, particularly their salts, such as trisodium citrate and sodium ascorbate. The term “vitamin E,” whether used alone or in combination, is understood to encompass any and all variants of tocopherol and tocotrienol (α, β, γ, δ). In preferred embodiments, the antioxidant is ascorbic acid or a salt thereof.

[0042] Another (one or more) freeze-drying protective agent used in this application is a disaccharide. A disaccharide is formed from two monosaccharides and is classified as either reducing or non-reducing. According to this application, the disaccharide is preferably a non-reducing disaccharide. Preferably, the non-reducing disaccharide is trehalose, cellulose, or sucrose, more preferably trehalose or sucrose, and even more preferably trehalose. Preferably, the non-reducing disaccharide is trehalose or sucrose.

[0043] Another lyophilization protective agent used in this application is cellulose. Furthermore, the lyophilization protective agents used herein also include maltodextrin. Maltodextrin is a polysaccharide consisting of D-glucose units linked to chains of varying lengths, typically composed of a mixture of chains ranging in length from 3 to 17 glucose units. These glucose units are linked primarily by α(1→4) glycosidic bonds. Maltodextrin typically has a DE (dextrose equivalent) of 5 to 20. As used herein, maltodextrin may include those having a DE of 5 to 30, preferably 5 to 25, and more preferably 10 to 20. Higher DE values ​​correspond to shorter glucose chains, higher sweetness, higher solubility, and lower heat resistance.

[0044] High viability can be observed when the dry matter weight ratio of freeze-dried protective agent to yeast biomass is between 0.5:1 and 4:1. If the weight ratio is too low, adequate protection cannot be provided. On the other hand, if the weight ratio is too high, the benefits of the protective effect are reduced, incurring disadvantages such as increased production costs and cell dilution.

[0045] In a preferred embodiment, the present application provides a freeze-dried composition of a Pichia species (such as Pichia cruiberi) comprising maltodextrin, ascorbic acid or a salt thereof, and trehalose as a freeze-drying protective agent, wherein the dry weight ratio of the freeze-drying protective agent to the yeast biomass is 0.5:1 to 3:1.

[0046] In a preferred embodiment, the present application provides a freeze-dried composition of a Pichia species (such as Pichia cruiberi) comprising maltodextrin having 10 to 20 DE, sodium ascorbate, and trehalose as a freeze-drying protective agent, wherein the dry weight ratio of the freeze-drying protective agent to the yeast biomass is 0.7:1 to 2.5:1.

[0047] In a preferred embodiment, the composition further comprises modified starch as a freeze-drying protective agent. The term “modified starch” refers to starch that has been physically or chemically modified to improve its functional properties. Suitable modified starches include, but are not limited to, pregelatinized starch, low viscosity starch (e.g., dextrin, acid-modified starch, oxidized starch, enzyme-modified starch), stabilized starch (e.g., starch esters, starch ethers), cross-linked starch, starch sugar (e.g., glucose syrup, dextrose, isoglucose), and starch that has undergone a combination of treatments (e.g., cross-linking and gelatinization), as well as mixtures thereof. Modified starch made from starch substituted by non-chemical methods using hydrophobic residues is particularly preferred.

[0048] Freeze-dried composition, 10 7 ~10 12 Within the range of CFU / g, preferably at least 10 8 cfu / g, for example, at least 10 9 cfu / g, for example, at least 10 10 cfu / g, and more preferably at least 5.0 × 10 10 The cfu / g composition preferably contains a viable yeast concentration. As used herein, the term “viable” in relation to yeast cells refers to the ability to form colonies (CFU or colony-forming units) in a culture medium suitable for cell growth. Viable cells are alive and can be regenerated and / or proliferated.

[0049] Cell concentration is often assessed by counting colony-forming units (CFUs) per gram using standard methods known in the art, such as those specified in the Compendium of international methods of Analysis - OIV, particularly in paragraph 6, CASTELLUCCI, Federico. "Microbiological Analysis of Wines and Musts Detection, Differentiation and Counting of Micro-organisms." (2010).

[0050] For example, formulated and freeze-dried yeast is suspended in an isotonic solution (i.e., MRD, peptone-containing or non-peptone isotonic salt solution) that does not cause proliferation of the analytical cells. Mixing can be achieved using a stomacher or vortex mixer. This is repeated over several serial dilutions and reactivated in the solution for 30 minutes. The various diluted suspensions are then plate-cultured on nutrient medium agar plates (i.e., YM, YEPD, WL Nutrient, TRY, or YGC agar) using a sterile Drigalsky rod within 1-2 minutes before the liquid is absorbed onto the agar surface. Dilutions are selected that can yield a colony count in the range of 10-300 per plate. The plates are incubated under aerobic conditions at 25-28°C for 48-72 hours. During this period, the yeast forms colonies on the nutrient medium. These colonies are counted and each dilution is re-examined; the results are expressed as colony-forming units (CFU) per gram.

[0051] The freeze-dried composition can be used as a direct vat set (DVS) culture intended for direct inoculation into fermentation vessels or vats for the production of fermented food products, including fermented beverages.

[0052] The weight of the final product disclosed herein may range from 1 g to 50 kg. Therefore, the weight of the freeze-dried composition described herein is preferably 5 g to 30 kg, for example, 10 g to 40 kg, 50 g to 30 kg, or 100 g to 25 kg.

[0053] In a preferred embodiment, the composition further comprises, in addition to freeze-dried yeast, an excipient. Such excipient may be sucrose, maltodextrin, and / or dextrose. As used herein, “excipient” means an element added to the freeze-dried composition to act as a volume extender.

[0054] As illustrated in the examples herein, freeze-dried compositions such as those described herein exhibit high process viability and storage stability.

[0055] A “stable” composition is one in which the bioactive materials within it essentially maintain their physical stability, chemical stability, and / or biological activity during storage. Stability can be measured under selected temperature and humidity conditions over a given period. Trend analysis can be used to estimate the expected shelf life of a material before it is actually stored over that period. For yeast cells, for example, storage stability may be defined as the time it takes to lose 1 log CFU / g dry formulation under given temperature, humidity, and duration conditions.

[0056] Storage stability can be measured by analyzing how the number of viable microbial cells appears over time. The viability of cultured microorganisms is measured by CFU / g, as described herein. Therefore, the storage stability of freeze-dried yeast can be measured by placing a sample with a water activity (aw) of 0.15 in sealed laminated aluminum foil and evaluating the CFU / g of the yeast at time 0 (immediately after drying) and after 6 months of storage at 4°C.

[0057] Process viability, also referred to herein as viability, indicates the degree of cell survival after freeze-drying. This can be expressed as the logarithmic loss: the logarithmic difference between the 100% recovered CFU (theoretical value) and the actual measured CFU (measured value). Viability can also be expressed as a percentage decrease between the 100% recovered CFU and the actual measured CFU. A process viability of at least 30% can be achieved by the currently disclosed method.

[0058] Use of the composition In a further embodiment, the application also provides the use of freeze-dried prepared fermented food products. Any fermented food product in which yeast can be used may be included herein. In this application, the preparation of beverages such as wine, beer, juice (vegetable juice or fruit juice), cider, kefir, cocoa, and coffee is particularly relevant, for example. The yeast composition of this application may be rehydrated before inoculation. In another preferred embodiment, the yeast is not rehydrated before inoculation.

[0059] process One of the main embodiments of this application is a process for preparing a freeze-dried non-Saccharomyces yeast composition, comprising the following steps: (1) Prepare non-Saccharomyces yeast cells, preferably Pichia species, (2) Culture yeast cells and harvest the yeast cells to obtain a yeast cell concentrate, where the concentrate is 10 8 ~10 11 Contains yeast cells in cfu / g dry weight. (3) Add the freeze-dried protective agent to the yeast cell concentrate to form a suspension, wherein the freeze-dried protective agent comprises maltodextrin, (one or more) antioxidants, and (one or more) disaccharides. (4) Freeze the suspension, (5) Dry the suspension, and then, (6) To obtain a freeze-dried non-Saccharomyces yeast composition, Includes, Here, the dry weight ratio of the freeze-dried protective agent to the yeast biomass is 0.5:1 to 4:1, preferably 0.5:1 to 3:1. The aforementioned process is provided.

[0060] In a preferred embodiment, the application relates to a process for preparing a freeze-dried non-Saccharomyces yeast composition, comprising the following steps: (1) Prepare non-Saccharomyces yeast cells, (2) Culture yeast cells and harvest the yeast cells to obtain a yeast cell concentrate, where the concentrate is 10 8 ~10 11 Contains yeast cells in cfu / g dry weight. (3) Add the freeze-dried protective agent to the yeast cell concentrate to form a suspension, wherein the freeze-dried protective agent comprises maltodextrin, (one or more) antioxidants, and (one or more) (preferably non-reducing) disaccharides. (4) Freeze the suspension, (5) Dry the suspension, and then, (6) To obtain a freeze-dried non-Saccharomyces yeast composition, Includes, Here, the dry weight ratio of the freeze-dried protective agent to the yeast biomass is 0.5:1 to 3:1, preferably 0.7:1 to 2.5:1, and here, the antioxidant is ascorbic acid or a salt thereof, and the disaccharide is trehalose. The aforementioned process is provided.

[0061] To prepare the freeze-dried composition of the said application, non-Saccharomyces yeast is prepared to prepare a concentrate of yeast cells. In this process, the yeast cells are fermented and then harvested, and 10 8 ~10 11 This involves obtaining a yeast cell concentrate containing the dry matter amount of yeast cells at cfu / g.

[0062] Culturing non-Saccharomyces yeast cells of interest for large-scale propagation is a common practice for those skilled in the art. Propagation is typically carried out in a medium that will depend on the specific nutritional needs of the intended non-Saccharomyces yeast. As used herein, the terms “cultivate” or “culture” refer to the production of yeast biomass.

[0063] As is known in the art, cell harvesting typically involves removing a portion of the fermentation medium and then centrifuging to obtain a yeast cell concentrate. Alternative methods such as filtration are also possible.

[0064] The obtained yeast cell concentrate was 10 8 ~10 11 This may include the dry matter weight of a yeast cell concentrate at cfu / g. The term “dry matter weight” in the context of this specification, as a person skilled in the art would understand it, is to mean that the yeast concentrate contains a given amount of yeast cells relative to the dry weight of the yeast concentrate.

[0065] In one preferred embodiment, it may be preferable to include an additional washing step after harvesting the cells to remove fermentation medium components and to prepare the cells. Washing cells before drying is known in the art and is described, for example, in EP Patent No. 0189318. In a preferred embodiment, washing can be carried out by using an aqueous solution containing magnesium and calcium salts.

[0066] After obtaining a yeast cell concentrate, a lyophilization protectant is typically added as a liquid solution to form a suspension. As described herein, the lyophilization protectant comprises maltodextrin, (one or more) antioxidants, and (one or more) disaccharides.

[0067] The (one or more) disaccharides are preferably non-reducing disaccharides. Preferably, the non-reducing disaccharides are trehalose, cellulose, sucrose, more preferably trehalose or sucrose, and even more preferably trehalose. In some embodiments, the disaccharide is a (one or more) reducing disaccharide, such as lactose, isomaltose, maltose, palatinose, and cellobiose, and more preferably maltose.

[0068] Suitable antioxidants include, but are not limited to, ascorbic acid, citric acid, beta-carotene, vitamin E, glutathione, and their derivatives, particularly their salts, such as trisodium citrate and sodium ascorbate.

[0069] Suitable maltodextrins may have a DE of 5-30, 5-25, preferably 10-20. As previously described in this application, the dry weight ratio of the freeze-dried protective agent to the yeast biomass is preferably 0.5:1 to 4:1. Precisely formulating yeast cell concentrates to achieve the desired weight ratios described herein is a matter of common practice for those skilled in the art.

[0070] Other lyophilization protectants can also be added to the yeast cell concentrate. For example, modified starch may be included. Thus, in a preferred embodiment, the lyophilization protectant further comprises modified starch.

[0071] After a freeze-drying protective agent is added, the formulated yeast cells are subjected to freeze-drying. A typical freeze-drying process involves (1) freezing, where water is converted to ice, and (2) drying, where the ice is removed by sublimation and unfrozen water is removed by absorption. Freeze-drying is a well-known method in the art and is described, for example, in Labconco, I. "A Guide to Freeze Drying for the Laboratory." (2008).

[0072] Therefore, the freeze-dried compositions currently disclosed are prepared by first freezing yeast cells in a formulated suspension and then drying them. Preferably, the formulated yeast cells are transferred to a suitable container such as a tray before freezing. It is common practice for those skilled in the art to carry out the freezing step and to select suitable parameters in the process, such as pressure, temperature, and time. Freezing is typically carried out at below -10°C, preferably below -20°C, below -30°C, below -40°C, and below -50°C. As is known to those skilled in the art, this temperature should not exceed the glass transition temperature of the solution, otherwise thawing will occur which will affect yeast physiology. Freezing can be carried out in a freezer.

[0073] In some embodiments, a suspension containing yeast cells is frozen in liquid nitrogen to form pellets. This is also known as pelletization. The term "pellet" refers to small, solid granules. The term "pelletization" refers to the process for forming such solid granules. Pelletization methods are known in the art. One method may be performed by dropping droplets of the mixture into liquid nitrogen. Another method for forming pellets may involve spray cooling of the concentrate.

[0074] In a preferred embodiment, the formulated yeast cells are frozen in the trays. In large-scale industrial production, it is preferable to use multiple trays simultaneously, for example, more than 10 trays or more than 100 trays.

[0075] After freezing, the yeast cells are subjected to a second phase of freeze-drying. In this phase, dehydration of the yeast occurs by sublimation. Those skilled in the art know how to select suitable drying parameters in the freeze-drying process, which typically involve low temperature and reduced pressure.

[0076] In a preferred embodiment, drying is carried out at a temperature below -10°C, preferably below -20°C or below -30°C. Preferably, the temperature during drying is controlled to maintain a constant predetermined vacuum pressure.

[0077] In a preferred embodiment, drying is carried out under a vacuum pressure of 0.01 to 2 millibars (mbar). More preferably, it is carried out under a vacuum pressure of 0.5 to 2 mbar, for example, 0.7 to 2 mbar.

[0078] In a preferred embodiment, drying is carried out for a period of time sufficient to reduce the water activity (aw) to less than 0.20, preferably less than 0.15. Drying may be terminated when a constant weight is obtained. Water activity is a well-known parameter, obtained by dividing the partial pressure of water vapor in a substance by the standard partial pressure of water vapor. Hereinafter, the standard state is defined as the partial pressure of water vapor of pure water at the same temperature. Using this definition, pure distilled water has a water activity of exactly 1. Those skilled in the art know how to measure the water activity of a composition. For example, a good method for measuring aw is described in detail on the FDA website under the heading "Water Activity (aw) in Foods," published on August 27, 2014.

[0079] In a preferred embodiment, a device capable of performing both freezing and drying is used, such as a freeze dryer. The freeze-dried non-Saccharomyces yeast composition is available after the drying step. The freeze-dried yeast may be subjected to further processing that mechanically reduces its size before the formulation of the final product, such as milling or grinding it into smaller particles of 200-1000 μm.

[0080] As will be understood by those skilled in the art, after freeze-drying, the method may include optional steps to obtain the final product. As used herein, the term “product” may also, in some cases, include other components, including excipients or other microorganisms. In one preferred embodiment, the process further comprises adding an excipient to the freeze-dried yeast composition to obtain the final product. Such excipient may be sucrose.

[0081] An optional additional step relating to this specification would be, for example, appropriately packaging the resulting freeze-dried yeast composition. The packaging could be, for example, a bottle, box, or bag. The packaging must be able to keep the water activity of the composition low. In the context of this specification, the term “packaging” of a suitable amount of freeze-dried microorganisms in a suitable package refers to the final packaging to obtain a product that can be shipped to the buyer. Thus, a suitable package could be a container, bottle, or similar, and a suitable amount could be, for example, 10 g to 50 kg. Such a yeast composition is very useful. The composition may be inoculated directly into a fermentation medium without an intermediate growth step.

[0082] A further aspect of the present application is a composition obtained by the process of the application described herein. In one preferred embodiment, the composition obtained is 10 7 ~10 11 It contains yeast cells cfu / g. In another embodiment, the resulting composition is 10 8 ~10 11 It contains yeast cells cfu / g. In yet another embodiment, the resulting composition is 10 9 ~10 11 Contains cfu / g yeast cells.

[0083] Deposit and distribution of samples to specialists only. The applicant requires that, until the date the patent is granted, the applicant provide the following deposited microorganism samples only to experts, in accordance with the regulations set forth by the Industrial Property Office of the Budapest Convention Contracting Party. The applicant made the following deposit at the depositary institution that has acquired the status of an international depositary authority under the Budapest Convention for the International Recognition of Microbial Deposits in Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

[0084] [Table 1] [Examples]

[0085] This application has been described with reference to various embodiments, aspects, examples, or similars. These elements are not intended to be interpreted in isolation from one another. Thus, this disclosure provides two or more embodiments, aspects, examples, or similar combinations.

[0086] All embodiments described herein are intended to be within the scope of the disclosed application. These and other embodiments of the application will be readily apparent to those skilled in the art from the following detailed description of embodiments with reference to the entire description, and the application is not limited to any specific embodiments disclosed.

[0087] Several freeze-dried compositions of non-Saccharomyces species (Pichia cruiberi) were prepared using various formulations and amounts of freeze-drying protective agents.

[0088] Example 1: Freeze-dried Pichia kruiberi composition using PK-KR1 (V06 / 022711) This example shows several freeze-dried yeast compositions 1-7. The Pichia kruiberi yeast cells used were PK-KR1.

[0089] 1.1 Preparation of the composition yeast cell concentrate Yeast cells were cultured and harvested as follows: Yeast was cultured in a closed fermentation vessel using an immersion fermentation process with a liquid compound culture medium. Yeast biomass was extracted by release or continuous separation. The harvested and concentrated yeast biomass contained approximately 17% dry matter and 83% water.

[0090] Table 1 summarizes the total amount of wet biomass prescribed, the dry matter content of the wet biomass, the washing of the biomass before formulation, the total amount of freeze-drying protective agent used, and the encapsulation index.

[0091] [Table 2]

[0092] Composition formulation In short, three different freeze-dried protective agent stocks were prepared, each containing disaccharides, antioxidants, and maltodextrin, with or without modified starch. Solutions 1-3 all contained trehalose, sodium ascorbate, and maltodextrin, while solution 2 further contained modified starch. Different amounts of the solutions were added to Pichia cruiberi biomass to reach the inclusion index (ratio of dry matter of freeze-dried protective agent to dry matter of yeast).

[0093] Further details are provided below: Composition 1 Composition 1 served as a control, and no freeze-drying protective agent was added to the biomass. The moist biomass had a potency of 7.15E+09 CFU / g and was maintained at 5°C before being transferred to a small tray in a freeze-dryer.

[0094] Composition 2 Concentrated biomass was formulated using lyophilized protective agent solution 1, and 200 g of moist concentrated biomass was supplemented with a total of 34.4 g of trehalose, 15.1 g of sodium ascorbate, and 20 g of maltodextrin (Glucidex IT12, DE=12) to ensure that an inclusion index equivalent to 2 (total of 69.5 g of dried lyophilized protective agent and 34 g of dried biomass) was obtained. The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0095] Composition 3 Concentrated biomass was formulated using freeze-dried protective agent solution 1, and 200 g of moist concentrated biomass was supplemented with a total of 25.8 g of trehalose, 111.3 g of sodium ascorbate, and 14.9 g of maltodextrin to ensure an inclusion index equivalent to 1.5. The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0096] Composition 4 Concentrated biomass was formulated using freeze-dried protective agent solution 2, and 200 g of moist concentrated biomass was supplemented with a total of 31.4 g trehalose, 5.1 g sodium ascorbate, 2.4 g maltodextrin, and 30.5 g modified starch HICAP 100 (Ingredient, Westchester, IL) to ensure an inclusion index equivalent to 2. The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0097] Composition 5 The concentrated biomass was formulated using freeze-dried protective agent solution 2, and 200 g of moist concentrated biomass was supplemented with a total of 23.4 g of trehalose, 3.8 g of sodium ascorbate, 1.8 g of maltodextrin, and 22.8 g of modified starch HICAP100 to ensure an inclusion index equivalent to 1.5. The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0098] Composition 6 The concentrated biomass was formulated using lyophilized protective agent solution 3, and 200 g of moist concentrated biomass was supplemented with a total of 23.3 g of trehalose, 3.1 g of sodium ascorbate, and 42.9 g of maltodextrin to ensure that the inclusion index equivalent to 2 (total of 69.5 g of dried lyophilized protective agent and 34 g of dried biomass) was obtained. The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0099] Composition 7 The concentrated biomass was formulated using lyophilized protective agent solution 3, and 200 g of moist concentrated biomass was supplemented with a total of 17.5 g of trehalose, 2.3 g of sodium ascorbate, and 32.3 g of maltodextrin to ensure an inclusion index equivalent to 1.5 (total of 69.5 g of dried lyophilized protective agent and 34 g of dried biomass). The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0100] Freeze-drying The sample was transferred to a pre-cooled tray in a 10 kg freeze-dryer (HOF Sonderanlagenbau GmbH, Germany), slowly frozen to -50°C over 3 hours (<2K / min), and then maintained at -50°C for another 2 hours. Subsequently, the freeze-dryer chamber was adjusted to 0.01–2.00 mbar for approximately 45 minutes. The remaining primary drying was carried out over 30 hours, during which the temperature was slowly raised from -50°C to 0°C. Secondary drying was carried out at +25°C for 15 hours. The freeze-dried yeast was removed from the tray and ground into a powder.

[0101] The freeze-dried yeast concentration (CFU / g) was measured for each of the final products and is shown in Table 1.

[0102] 1.2 Evaluation of process viability and storage stability based on CFU / g Process survival rate The survival rate during freeze-drying indicates the degree of cell viability after freeze-drying. This is expressed as logarithmic loss: the logarithmic difference between the 100% recovered CFU (theoretical value) and the actual measured CFU (measured value). The predicted theoretical CFU value can be calculated by knowing the CFU / g of the concentrate and multiplying it by the dilution ratio obtained at the time of formulation and the concentration factor obtained during the freeze-drying process (FD). Survival rate can also be expressed as a percentage decrease between the 100% recovered CFU and the actually measured CFU.

[0103] Storage stability The long-term stability of freeze-dried cultures as a function of time is a critical characteristic for commercial use. Long-term stability was evaluated by placing freeze-dried cells in sealed laminated aluminum foil in a temperature-controlled chamber maintained at a predetermined temperature (25°C, 4°C, or -18°C) for a predetermined period (up to 12 months). The number of cells in the freeze-dried material was measured before and after exposure to the predetermined temperature and time. The difference in CFU was expressed as a logarithmic loss and calculated by subtracting the logarithm of the initially measured number of cells from the number of cells measured at the end of the stability test.

[0104] 1.3 Results Process survival rate results Table 2 shows the process viability calculated across the freeze-drying steps.

[0105] [Table 3]

[0106] Results of storage stability Table 3 shows the storage stability data for compositions 1-7 after 12 months of storage.

[0107] [Table 4]

[0108] In addition, Figures 1 and 2 show the stability data for the composition over a 12-month period at -18°C and 4°C, respectively.

[0109] 1.4 Conclusion Process survival rate As shown in the results from Table 2, using the tested lyophilization protective agent solutions reduces logarithmic losses over the freeze-drying step, thereby enhancing process viability. In addition, it was found that higher encapsulation indices resulted in lower logarithmic losses. For example, using lyophilization protective agent 2 and increasing the encapsulation indices from 1.5 to 2 (compositions 5 and 4) allowed for a further reduction of logarithmic losses from 0.42 to 0.28. Similarly, for compositions 7 and 6, increasing the encapsulation indices from 1.5 to 2 using lyophilization protective agent solution 3 allowed for a reduction of logarithmic losses from 0.3 to 0.13. The lowest viability during the freeze-drying step was observed in the control (composition 1) without the use of a lyophilization protective agent (EI=0). Thus, the viability during the freeze-drying step was approximately 25%.

[0110] Storage stability As shown in the results from Table 3 and Figures 1 and 2, the stability of freeze-dried Pichia cruiberi was significantly improved when using the tested lyophilizing protective agent. The poorest stability after 12 months of storage at -18°C and 4°C was observed for freeze-dried products without the lyophilizing protective agent (Composition 1). The logarithmic loss was 0.32 for products stored at -18°C and 1.33 for products stored at 4°C. No logarithmic loss was observed for freeze-dried products containing the lyophilizing protective agent stored at -18°C. Furthermore, compositions 2-7 all showed significantly lower logarithmic losses than composition 1 stored at the same temperature when stored at 4°C.

[0111] Example 2: Freeze-dried Pichia kruiberi composition using PK-KR1 (V06 / 022711) This example shows several freeze-dried non-Saccharomyces yeast Pichia kruiberi compositions 8-10. The Pichia kruiberi yeast cells used were PK-KR1.

[0112] 2.1 Preparation of the composition The compositions were prepared in substantially the same manner as described in Example 1, except that only stock solution 1 was used to prepare compositions 9 and 10. The harvested and concentrated yeast biomass contained approximately 17.4% dry matter and 82.6% water.

[0113] Table 4 summarizes the total amount of wet biomass prescribed, the dry matter content of the wet biomass, the washing of the biomass before formulation, the total amount of freeze-drying protective agent used, and the encapsulation index.

[0114] [Table 5]

[0115] Composition formulation Composition 8 Composition 8 served as a control, and no freeze-drying protective agent was added to the biomass. The concentrated moist biomass had a potency of 1.09E+10 CFU / g and was maintained at 5°C before being transferred to small trays in a freeze-dryer.

[0116] Composition 9 Concentrated biomass was formulated using lyophilized protective agent solution 1, and 200 g of moist concentrated biomass was supplemented with a total of 12.7 g of trehalose, 6 g of sodium ascorbate, and 7.5 g of maltodextrin to ensure an inclusion index equivalent to 0.75 (total of 26.2 g of dried lyophilized protective agent and 34.8 g of dried biomass). The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0117] Composition 10 Concentrated biomass was formulated using freeze-dried protective agent solution 1, and 200 g of moist concentrated biomass was supplemented with a total of 19.4 g of trehalose, 8.5 g of sodium ascorbate, and 11.2 g of maltodextrin to ensure an inclusion index equivalent to 1.1. The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0118] The freeze-dried yeast concentration (CFU / g) was measured for each of the final products and is shown in Table 4.

[0119] 2.2 Results Process viability and storage stability were evaluated as in Example 1. Process survival rate results Table 5 shows the process viability calculated across the freeze-drying steps for compositions 8-10.

[0120] [Table 6]

[0121] Results of storage stability Table 6 shows the storage stability data for compositions 8-10 after 12 months of storage. This data was not available for composition 9, as measurements were not taken for it.

[0122] [Table 7]

[0123] In addition, Figures 3 and 4 show the stability data for compositions 8 and 10 over a 12-month period at -18°C and 4°C, respectively.

[0124] 2.3 Conclusion Process survival rate As shown in the results from Table 2, using the tested lyophilization protective agent solution reduces logarithmic loss over the freeze-drying step, thereby enhancing process viability. In addition, it was found that increasing the encapsulation index from 0 to 0.75 and 1.1 could reduce logarithmic loss from 0.82 to 0.47 and 0.35, respectively. The lowest viability during the freeze-drying step was observed in the control (composition 8) without the use of a lyophilization protective agent. The viability over the freeze-drying step was approximately 15%, compared to 33% and 44% in compositions 9 and 10, respectively, with encapsulation indices of 0.75 and 1.1, respectively.

[0125] Storage stability As shown in the results from Table 6 and Figures 3 and 4, the stability of freeze-dried Pichia cruiberi was significantly improved when using the tested lyophilization protectants. The poorest stability after 12 months of storage at -18°C and 4°C was observed for freeze-dried products without the lyophilization protectant (composition 8). Here, the logarithmic loss was 0.47 for the product stored at -18°C and 1.51 for the product stored at 4°C. No logarithmic loss was observed for the freeze-dried product (composition 10) stored at -18°C using the highest encapsulation index. Composition 10, stored at 4°C, showed a significantly lower logarithmic loss than composition 8, stored at the same temperature.

[0126] Example 3: Freeze-dried Pichia kruiberi composition using PK-KR1 (V06 / 022711) with storage stability data at -18°C, 4°C, and 25°C. 3.1 Preparation of the composition The composition was prepared in substantially the same manner as composition 6 described in Example 1. A freeze-dried protective agent solution 3 was used. The harvested and concentrated yeast biomass contained 15.6% dry matter and 84.4% water.

[0127] Table 7 summarizes the total amount of wet biomass prescribed, the dry matter amount of the wet biomass, the washing of the biomass before formulation, the total amount of freeze-drying protective agent used, and the encapsulation index.

[0128] [Table 8]

[0129] Composition formulation Composition 11 The concentrated biomass was formulated using lyophilized protective agent solution 3, and 200 g of moist concentrated biomass was supplemented with a total of 23.3 g of trehalose, 3.1 g of sodium ascorbate, and 42.9 g of maltodextrin to ensure an inclusion index equivalent to 2.2 (total of 69.3 g of dried lyophilized protective agent and 31.2 g of dried biomass). The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0130] The freeze-dried yeast concentration (CFU / g) was measured and is shown in Table 7.

[0131] 3.2 Results Process viability and storage stability were evaluated as in Example 1. Process survival rate results Table 8 shows the process viability calculated across the freeze-drying steps for composition 11.

[0132] [Table 9]

[0133] Results of storage stability Table 9 shows the storage stability data for composition 11 after 6 months of storage at -18°C, 4°C, and 25°C.

[0134] [Table 10]

[0135] In addition, Figures 5, 6, and 7 show the stability data of composition 11 over a 6-month period stored at -18°C, 4°C, and 25°C, respectively. Composition 1 (control, no lyophilization protectant used) and composition 6 (same lyophilization protectant as composition 11 and nearly the same EI) are included in Figures 5 and 6 for comparison.

[0136] [Table 11]

[0137] In addition, Figures 13, 14, and 15 show the stability data of composition 11 over a 12-month period stored at -18°C, 4°C, and 25°C, respectively. Composition 1 (control, no lyophilization protectant used) and composition 6 (same lyophilization protectant as composition 11 and nearly the same EI) are included in Figures 13 and 14 for comparison.

[0138] 3.3 Conclusion Process survival rate As shown in the results from Table 8, it is possible to reduce the logarithmic loss over the freeze-drying step and thereby enhance the process viability compared to composition 1 (without the use of a freeze-drying protective agent, as described in Example 1). The process viability achieved through the freeze-drying step for composition 11 is more reproducible and comparable to the similar composition 6 described in Example 1. The process viability for composition 6 and composition 11 was approximately 0.13 and 0.14, respectively.

[0139] Storage stability As shown in the results from Table 9 and Figures 5-7, the stability of freeze-dried Pichia cruiberi was significantly improved when using the tested freeze-drying protective agents. For comparison, the stability data for compositions 1 and 6 are included in Figures 5 and 6. It was found that the stability profiles for both compositions 6 and 11 were very similar. Even at 25°C, the stability data shown in Figure 7 demonstrates that composition 11 has improved stability compared to composition 1, which was stored and measured at 4°C.

[0140] Example 4: Freeze-dried Pichia kruiberi composition using PK-KR1 (V06 / 022711) 4.1 Preparation of the composition Composition 12 was prepared using lyophilized protective agent solution 1 in substantially the same manner as composition 10 described in Example 2, except that the non-Saccharomyces yeast Pichia cruiberi biomass was washed with a solution containing magnesium and calcium salts before drying. The washed yeast biomass contained approximately 21.1% dry matter and 78.9% water.

[0141] Table 16 summarizes the total amount of wet biomass prescribed, the dry matter amount of wet biomass, the washing of the biomass before formulation, the total amount of freeze-drying protective agent used, and the encapsulation index. The biomass is pretreated with a solution of calcium chloride and magnesium chloride (79 g / L CaCl 2 × H2O, 10 9 The biomass was washed with g / L MgCl (6×H2O). Washing was performed for 30 minutes using 400 g of concentrated biomass in a 1.4:1 (biomass to salt solution ratio) mixture of aqueous solutions containing the magnesium and calcium salts described above. This was then concentrated by centrifugation, with an equal amount of supernatant removed along with the previously added salt solution. A freeze-drying protective agent was then added to the washed biomass before freeze-drying.

[0142] [Table 12]

[0143] Composition formulation Composition 12 The concentrated washed biomass was formulated using freeze-dried protective agent solution 1, and 200 g of moist concentrated biomass was supplemented with a total of 23 g of trehalose, 10 g of sodium ascorbate, and 13.3 g of maltodextrin to ensure an inclusion index equivalent to 1.1. The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0144] The freeze-dried yeast concentration (CFU / g) was measured and is shown in Table 16.

[0145] 4.2 Results Process viability and storage stability were evaluated as in Example 1. Process survival rate results Table 16 shows the process viability calculated across the freeze-drying steps for composition 12.

[0146] [Table 13]

[0147] Results of storage stability Table 16 shows the storage stability data for composition 12 after 12 months of storage at -18°C and 25°C.

[0148] [Table 14]

[0149] In addition, Figures 8 and 9 show the stability data of composition 12 over a 12-month period when stored at -18°C and 4°C, respectively.

[0150] 4.3 Conclusion Process survival rate The results from Table 16 demonstrate that washing the biomass significantly affects the viability of yeast through the freeze-drying step. The logarithmic loss was calculated to be 0.004, which corresponds to a viability of approximately 98%. This viability is significantly higher than that obtained using unwashed biomass tested with composition 10 in Example 2, where the viability was calculated to be 44%.

[0151] Storage stability As shown in the results from Table 16, Figures 8 and 9, it is also clear that the stability of freeze-dried Pichia kruiberi can be maintained after drying with freeze-drying protective agent solution 1 and pre-formulation biomass washing. There was no logarithmic loss of the freeze-dried Pichia kruiberi product stored at -18°C after 12 months of storage. The logarithmic loss after 12 months of storage at 4°C was measured at 0.12, which was slightly better than the results obtained for the unwashed biomass (composition 10) described in Example 2.

[0152] Example 5: Freeze-dried Pichia kruiberi composition containing DSM28484 This example shows several freeze-dried non-Saccharomyces yeast compositions 13-17 using different strains of Pichia cruiberi.

[0153] 5.1 Preparation of the composition The composition was prepared in substantially the same manner as described in Example 1, except that the prescribed yeast cells were DSM28484. The harvested and concentrated yeast biomass contained approximately 17.5% dry matter and 82.5% water. Table 16 summarizes the total amount of wet biomass prescribed, the dry matter amount of the wet biomass, the washing of the biomass before formulation, the total amount of freeze-drying protective agent used, and the encapsulation index.

[0154] [Table 15]

[0155] Composition formulation Composition 13 Composition 13 served as a control, and no freeze-drying protective agent was added to the biomass. The moist biomass had a potency of 1.37E+10 CFU / g and was maintained at 5°C before being transferred to a small tray in a freeze-dryer.

[0156] Composition 14 The concentrated biomass was formulated using lyophilized protective agent solution 4, and 200 g of moist concentrated biomass was supplemented with a total of 84 g of trehalose to ensure that the mixture yielded 2.4 (a total of 84 g of dry lyophilized protective agent and 35 g of dry biomass). The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0157] Composition 15 The concentrated biomass was formulated using freeze-dried protective agent solution 2, and 200 g of moist concentrated biomass was supplemented with a total of 32.7 g trehalose, 5.3 g sodium ascorbate, 2.5 g maltodextrin, and 31.8 g modified starch HICAP100 to ensure an inclusion index equivalent to 2.1. The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0158] Composition 16 Concentrated biomass was formulated using lyophilized protective agent solution 1, and 200 g of moist concentrated biomass was supplemented with a total of 34.7 g of trehalose, 15.6 g of sodium ascorbate, and 20.7 g of maltodextrin to ensure that the inclusion index equivalent to 2 (total of 69.5 g of dried lyophilized protective agent and 34 g of dried biomass) was obtained. The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0159] Composition 17 The concentrated biomass was formulated using lyophilized protective agent solution 3, and 200 g of moist concentrated biomass was supplemented with a total of 24.3 g of trehalose, 3.2 g of sodium ascorbate, and 44.7 g of maltodextrin to ensure an inclusion index equivalent to 2.1 (total of 69.5 g of dried lyophilized protective agent and 34 g of dried biomass). The formulated concentrate was maintained at 5°C and mixed for 20 minutes before being transferred to a tray in a freeze-dryer.

[0160] The freeze-dried yeast concentration (CFU / g) was measured for each of the final products and is shown in Table 16.

[0161] 5.2 Results Process viability and storage stability were measured as in Example 1. Process survival rate results Table 16 shows the process viability calculated across the freeze-drying steps for compositions 13-17.

[0162] [Table 16]

[0163] Results of storage stability Table 16 shows the stability data for compositions 13-17 after 12 months of storage at 4°C.

[0164] [Table 17]

[0165] 5.3 Conclusion Process survival rate As shown in the results from Table 16, using the tested lyophilization protective agent solutions reduces logarithmic loss over the freeze-drying step, thereby enhancing process viability, which supports the findings in Example 1. In addition, it was found that higher encapsulation indices resulted in lower logarithmic loss. For example, by using the encapsulation indices of lyophilization protective agent solutions 2 and 2.1 (composition 15), it was possible to reduce the logarithmic loss from 0.71 (control composition 13) to 0.22. Using trehalose alone (composition 14) also improved process viability. The poorest viability through the freeze-drying step was observed for Pichia cruiberi (composition 13) without the use of a lyophilization protective agent. The viability through the freeze-drying step was estimated at 20%.

[0166] Storage stability As shown in the results from Table 16 and Figure 10, the stability of freeze-dried Pichia kruiberi was significantly improved when all freeze-drying protective agents of this application were used. However, the stability of freeze-dried Pichia kruiberi was impaired by using trehalose alone (composition 14). The logarithmic loss was less than 1 unit after 12 months of storage at 4°C. It was observed that the use of trehalose alone resulted in a high degree of meringue formation in the freeze-dried final product compared to compositions 15-17, suggesting that a high degree of dissolution occurred during the freeze-drying process. The best stability was obtained by replacing part of the trehalose with an antioxidant and a large amount of modified starch (composition 15), or by adding an antioxidant and a large amount of maltodextrin (compositions 16 and 17).

[0167] Example 6: Use of freeze-dried non-Saccharomyces species for winemaking. 6.1 Winemaking The frozen or freeze-dried Pichia kruiberi prepared in Example 3 was used to prepare German Chardonnay, which requires continuous inoculation. Specifically, Pichia kruiberi (freeze-dried or frozen) was first inoculated, and two days later, the secondary yeast Saccharomyces cerevisiae was inoculated. The production of 3-mercaptohexyl acetate (3-MHA), a volatile thiol known to be produced by the yeast and which imparts passion fruit and tropical aromas, was measured.

[0168] Each fermentation was carried out in 5 liters of pasteurized German Chardonnay, but the grapes had to be harvested in 2022. Both freeze-dried and frozen Pichia kruiberi were planted directly without any pretreatment. The same Saccharomyces cerevisiae yeast was planted in all ferments at the same concentration two days later. All ferments were stabilized with 40 ppm SO2 and bottled after alcoholic fermentation was complete (total glucose / fructose < 1 g / L).

[0169] Viable cell count: The total yeast count was analyzed 1 hour after inoculation by spreading and plating the sample on YGC medium and incubating at 20°C for 72 hours.

[0170] Thiol concentration: The concentration of 3-mercaptohexyl acetate (3-MHA) in the final wine was measured by measuring polyfunctional mercaptans based on simultaneous derivatization and extraction using DCM, followed by LC-QqQ analysis, based on the results published in S. Vichi, N. Cortes-Francisco, J. Caixach. Analysis of volatile thiols in alcoholic beverages by simultaneous derivatization / extraction and liquid chromatography-high resolution mass spectrometry. Food Chemistry. Vol.175. May 2015, Pages 401-408.

[0171] 6.2 Results Wine fermented using freeze-dried Pichia cruiberg, despite initially detecting a lower cell count, had a higher concentration of 3-MHA than wine fermented using frozen Pichia cruiberg. Surprisingly, freeze-dried Pichia cruiberg led to greater 3-MHA production. This suggests that the freeze-drying method was able to produce wine of comparable quality to that produced from frozen yeast.

Claims

1. A freeze-dried composition of a non-Saccharomyces yeast, preferably a Pichia species or Pichia cruiberi, comprising maltodextrin as a freeze-drying protective agent, one or more antioxidants, and one or more disaccharides, wherein the dry weight ratio of the freeze-drying protective agent to the yeast biomass is 0.5:1 to 4:

1.

2. The freeze-dried composition according to claim 1, wherein the disaccharide is a non-reducing disaccharide, preferably trehalose or sucrose, and / or the antioxidant is ascorbic acid, citric acid, β-carotene, vitamin E, glutathione, and / or derivatives thereof.

3. A freeze-dried composition according to any one of claims 1 to 2, further comprising modified starch.

4. The freeze-dried composition according to any one of claims 1 to 3, wherein the maltodextrin has a dextrose equivalent (DE) of 5 to 30, preferably 10 to 20.

5. 10 ７ ~10 １２ A freeze-dried composition according to any one of claims 1 to 4, comprising yeast cells of cfu / g.

6. The freeze-dried composition according to any one of claims 1 to 5, wherein the yeast is a species of the genus Pichia, preferably Pichia cruiberi.

7. The freeze-dried composition according to claim 6, wherein the Pichia kruiberi is DSM28484 or PK-KR1.

8. A freeze-dried composition according to any one of claims 1 to 7, further comprising an excipient.

9. Use of the freeze-dried composition according to any one of claims 1 to 8 for preparing fermented food products, preferably beverages.

10. The following steps: (1) Prepare non-Saccharomyces yeast cells, (2) Culture yeast cells and harvest the yeast cells to obtain a yeast cell concentrate, where the concentrate is 10 ８ ~10 １１ Contains yeast cells in cfu / g dry weight. (3) Add the freeze-dried protective agent to the yeast cell concentrate to form a suspension, wherein the freeze-dried protective agent comprises maltodextrin, (one or more) antioxidants, and (one or more) disaccharides. (4) Freeze the suspension, (5) Dry the suspension, and then, (6) To obtain a freeze-dried non-Saccharomyces yeast composition, Includes, Here, the dry weight ratio of the freeze-dried protective agent to the yeast biomass is 0.5:1 to 4:1, preferably 0.5:1 to 3:1, and more preferably 0.7:1 to 2.5:

1. A process for preparing freeze-dried compositions of non-Saccharomyces yeasts.

11. The composition obtained is 10 ９ ~10 １３ The process according to claim 10, comprising yeast cells of cfu / g.

12. The process according to any one of claims 10 to 11, wherein the drying is carried out under a vacuum pressure of 0.01 to 2 millibars (mbar), and the temperature during drying is controlled so as to keep the vacuum pressure constant.

13. The process according to any one of claims 10 to 12, wherein in the freezing step (4), the suspension is frozen in liquid nitrogen to form pellets or frozen on a tray.

14. The process according to any one of claims 10 to 13, further comprising washing the cells after harvesting in step (2).

15. The process according to any one of claims 10 to 14, wherein the freeze-drying protective agent further comprises modified starch.

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