Method for producing sheets, coatings, or molded bodies from yeast biomass

Abstract

The present invention relates to a method for producing sheets, coatings, or moldings from yeast biomass, in which the biomass is first crushed, homogenized, and then further processed into sheets, coatings, or moldings. The method is characterized in that the yeast used is an oleaginous yeast, in particular Y. lipolytica, and the homogenization involves a cell wall-lytic enzyme treatment. This method can be used to produce biobased, fully biodegradable packaging products that are based exclusively on renewable, natural raw materials and have good mechanical properties.

Description

[Technical Field]

[0001] The present invention relates to a method for producing sheets, coatings or moldings made of biodegradable material, in particular for packaging, in which a yeast biomass is provided and homogenized by mechanical treatment, and the biomass thus disrupted is processed directly or after the addition of one or more further substances into sheets, coatings or moldings.

[0002] There is a growing demand for biodegradable polymers to manufacture product packaging. Sustainable solutions include using renewable resources, such as plant biomass, as starting materials for polymer production. Bio-based polymers, in particular, offer the potential for significantly reducing the use of fossil resources. Bio-based polymers are characterized by being CO₂-neutral, i.e., they add no or at least very little CO₂ to the atmosphere throughout their entire life cycle. Currently established bio-based plastics on the market utilize renewable resources derived from agriculture. However, this often requires fertile arable land. In contrast, the use of microbial biomass offers the advantages of being a completely renewable polymer source, not directly requiring fertile arable land for biomass procurement, and not directly risking increased deforestation for biomass production. [Background technology]

[0003] Currently, renewable resources such as corn, wheat, potatoes, sugarcane, sugar beets, bamboo, or wood are used as starting materials for producing polymers. Compared to conventional plastics derived from fossil resources, these bioplastics can help reduce greenhouse gas emissions and the consumption of fossil raw materials. However, this often results in a worsening of the balance compared to the fossil basis in several other impact areas (e.g., land and water consumption, eutrophication of water bodies).

[0004] Another known strategy is the use of microbial biomass (e.g., yeast or fungi) for the production of sheets and coatings in the materials industry. Current research and development projects aim, inter alia, at using biomass from the baker's yeast Saccharomyces cerevisiae in combination with glycerol as a plasticizer. A corresponding method is described, for example, in Non-Patent Document 1. Glycerol improves the integrity, flexibility, and mechanical properties of the produced sheets by acting as a spacer and mechanical mediator between the biopolymer chains at the molecular level. However, a significant disadvantage of S. cerevisiae is its dependence on food-related raw materials, since the wild type is often cultivated on glucose.

[0005] To utilize yeast components, the cell wall must first be disrupted to release the cytoplasm. Delgado et al. (Non-Patent Document 1) produced sheets from S. cerevisiae biomass using a high-pressure homogenization technique at pressures exceeding 100 MPa. The exposed biopolymers then interact with each other and form a stable network. The next step involves heat treatment in a 90°C water bath to denature proteins and thus enhance protein-protein interactions. A second homogenization is applied to remove any aggregates that may have formed during the heat treatment. Finally, glycerol is added to the yeast dispersion, and a film is cast from the mixture. Finally, the water is removed by drying at 50°C. This further promotes interactions between the polymer chains and the formation of a film matrix.

[0006] Biopolymers derived from agriculturally produced biomass or traditionally used biopolymers derived from microbial sources (i.e., for example, baker's yeast, S. cerevisae) also typically interact strongly with water, and accordingly, the resulting sheets form a weaker water vapor barrier than synthetic polymers, leading to faster drying of the packaged items. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] JF Delgado et al., "Characterization of thermal, mechanical and hydration properties of novel films based on Saccharomyces cerevisiae biomass," Innovative Food Science & Emerging Technologies, 2018, 48, pp. 240-247 Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present invention is to provide a method for producing 100% biobased and biodegradable sheets, coatings or moldings, which can be produced from renewable natural raw materials and are suitable for a wide range of applications in the food and packaging industries, i.e., they also have barrier properties sufficient for food packaging. [Means for solving the problem]

[0009] The above problem is solved by a method according to claim 1. Advantageous embodiments of the method are the subject matter of the dependent claims or can be found in the following detailed description as well as in the exemplary embodiments.

[0010] The proposed method is based on the production of sheets, coatings, or molded bodies (hereinafter also referred to as products or manufactured articles) using yeast biomass. In this case, biomass should be understood as the intact wet cell mass of yeast cells that typically occurs in the fermentation process in which yeast is used. Yeast biomass is a cost-effective and readily available source of biopolymers with promising properties for the development of biodegradable materials. In the proposed method, yeast biomass is first prepared and homogenized, where at least one mechanical treatment is performed for homogenization. The correspondingly homogenized biomass is then processed into sheets, coatings, or molded bodies, optionally after further intermediate steps, such as heat treatment and further homogenization, either directly or after the addition of one or more additional substances. The proposed method is characterized in that the yeast used is an oleaginous yeast (hereinafter also referred to as oleaginous yeast), and the homogenization includes a cell wall-lytic enzyme treatment. This enzyme treatment is preferably a Zymolase treatment. Alternatively, other suitable enzymes such as lysozyme or papain can be used.

[0011] In this case, oleaginous yeast is understood to be a yeast capable of storing more than 20% of its dry cell weight as lipids or triacylglycerides, as is known. Examples of oleaginous yeasts that can be used in the proposed method are Schwanniomyces occidentalis, Rhodotorula toruloides, or Yarrowia lipolytica (Y. lipolytica).

[0012] To prepare the wet biomass of oleaginous yeast, at least one strain of oleaginous yeast is cultivated. In the proposed method, this cultivation is preferably carried out using crude glycerol as a substrate. For example, glycerol, which may contain impurities and is generated as a residue during the production of biodiesel from fats and oils, can be used as a substrate for culturing the oleaginous yeast used in the proposed method. Instead of crude glycerol, another substrate, such as (used) edible oils and fats, can be used, which is equivalent in terms of economic efficiency and resource conservation. This can significantly improve the economic efficiency of the production process, as it saves on the cost of refining the substrate. By utilizing by-products such as crude glycerol, they are not only returned to the material cycle but also added value (upcycling). The use of food-related substrates such as sugars is not necessary. The production of the product is resource-saving, and the preparation of yeast biomass as a raw material does not increase the use of fertile and ecologically valuable areas, thereby not negatively impacting biodiversity or increasing water consumption.

[0013] Therefore, the proposed method can be used to produce 100% biobased and biodegradable sheets, coatings, and moldings. These products have a wide range of applications in the food and packaging industries, are fully compostable, and are produced exclusively from renewable natural raw materials. This also allows for the production of packaging products with sufficient barrier properties, particularly against oxygen and water, as desired in the food packaging industry. A further advantage of using oleaginous yeast is that it results in improved sheet mechanical properties compared to comparable starting materials of this type. These improved mechanical properties are due to the high lipid and polysaccharide content of oleaginous yeast. Furthermore, proteins derived from the yeast cytoplasm and cell wall polymers (polysaccharides such as mannan, glucan, and chitin) are particularly suitable for sheet formation. Compared to the yeast S. cerevisiae mentioned at the beginning, oleaginous yeast generally has a higher polysaccharide content and a significantly higher chitin content.

[0014] Particularly advantageously, the proposed method uses the oleaginous yeast Yarrowia lipolytica (Y. lipolytica). This yeast is capable of synthesizing numerous valuable metabolic products. Important products include lipases, other hydrolases, lipids, citric acid, erythritol, and gamma-decalactone. Many commercial processes based on the fermentation of the yeast Y. lipolytica also have GRAS (Generally Recognized as Safe) status granted by the U.S. Food and Drug Administration. Furthermore, because Y. lipolytica yeast biomass has been approved as a new food, sheets produced therefrom are expected to be approved for use as food packaging materials without any problems. These packaging sheets are versatile membranes that may only require the use of additives to tailor their integrity and mechanical properties to their intended use.

[0015] In addition to functionality and recyclability, another important requirement for packaging materials is the protection of the packaged product. Particularly in the food industry, reducing the exchange of water between the protected food and its environment is essential. In this case, preventing the packaged item from drying out is a particularly important goal, requiring appropriate packaging materials. Biopolymers made from agriculturally produced raw materials, or even currently used microbial biopolymers, typically interact strongly with water, and sheets made from them form a weaker water vapor barrier than synthetic polymers. A weaker packaging water vapor barrier generally leads to more rapid drying of the packaged item. Oleaginous yeasts, such as Y. lipolytica, have a significantly higher cytoplasmic lipid content (between 20% and 30% of the dry mass, depending on the strain and growth conditions) than other microorganisms. While lipids alone are not suitable for forming matrices with acceptable mechanical properties, they have already been effectively used to reduce the strong interaction of sheets and coatings with water and improve the waterproof barrier properties of sheets. The premise is that the advantageous composition of biomass from oleaginous yeasts, particularly Y. lipolytica, with a high content of lipids and biopolymers reduces water solubility and therefore water permeability.

[0016] In the proposed method, homogenization or cell disruption is particularly advantageously carried out not by using a high-pressure homogenizer, but by using mechanical treatment with ultrasound in combination with an enzymatic treatment of the biomass before and / or during the sonication treatment. In this case, it has been found that the combination of enzymatic and ultrasonic treatment can achieve much better homogenization results than when high-pressure homogenization is performed. Furthermore, after this first homogenization step, it is advantageous to subject the biomass to heat treatment, for example in a water bath, to denature the proteins in the biomass. This is preferably followed by a further homogenization step, preferably also by ultrasonic treatment.

[0017] A further advantageous embodiment of the proposed method, also in combination with the embodiments already described above, consists in subjecting the biomass after cultivation, optionally after one or more washing steps, to freeze-drying, which allows for longer storage of the biomass before further processing according to the proposed method.

[0018] Finally, the oleaginous yeast biomass processed according to the proposed method is further processed into the desired product, i.e., a sheet, coating, or molding. For this purpose, one or more additional substances can be added to the biomass, which is present during the method in a liquid form, in particular as a dispersion in a solvent such as water. To influence the mechanical properties of the resulting product, this or one of these additional substances can be a suitable plasticizer, such as glycerol. It is also possible to add other substances, such as fillers suitable for the respective use. In the resulting product, i.e., for example, a sheet or coating, the oleaginous yeast, in particular Y. lipolytica, biomass still constitutes the main component, i.e., more than 50%, generally significantly more than 50%, and in particular 70% to 80% or more (by weight). In this case, the corresponding product can be produced in a known manner, for example, by simple casting and drying when producing sheets, by spraying and drying when producing coatings, or by injection molding when producing moldings.

[0019] The proposed method will be explained in more detail below on the basis of exemplary embodiments in conjunction with the drawings. [Brief explanation of the drawings]

[0020] [Figure 1] Figures 1a and 1b show the growth of two different strains (DSM3286 and PO1F) when cultured in different glycerol batches. [Figure 2] FIG. 1 shows measurements of sheets produced by the present method at various glycerol loadings. [Figure 3] FIG. 1 shows a chart of an exemplary process sequence for the production of sheets according to the proposed method. DETAILED DESCRIPTION OF THE INVENTION

[0021] The proposed method is described below again based on the beneficial use of the oleaginous yeast Y. lipolytica. This oleaginous yeast can utilize crude glycerol as the sole carbon source for biomass formation, resulting in the production of a high percentage of lipids and polysaccharides. In this example, two different Y. lipolytica strains (DSM3286 and PO1F) were cultured using substrates from crude glycerol of different qualities and pure glycerol as a reference. In this case, the crude glycerol was obtained from various biodiesel manufacturers. Glycerol 1 had a purity of over 99%, glycerol 2 had a purity of approximately 90%, glycerol 3 had a purity of approximately 82%, glycerol 4 had a purity of approximately 70%, and glycerol 5 had a purity of only approximately 50%.

[0022] Initially, both Y. lipolytica strains were cultivated in mineral salts medium containing the above-mentioned glycerol batch as substrate (each at a starting concentration of 20 g / L). The Y. lipolytica PO1F strain is a genetically modified version of the wild-type W29 strain, frequently used in laboratory or industrial processes. Here, genes for leucine synthesis and uracil synthesis were deleted, thus establishing auxotrophy as a selection marker for genetic engineering methods. The DSM3286 strain is a wild-type strain obtained from the culture collection at DSMZ. The crude glycerol was not specially treated for cultivation but was simply used directly after autoclaving. The cultivation was carried out at 200 rpm for 48 hours at 30°C. Subsequently, the biomass was harvested by centrifugation (8000 g, 10 min, 4°C).

[0023] Both strains tested were shown to be able to utilize all glycerol sources used, with strain PO1F achieving lower biomass yields than strain DSM3286. Depending on the strain and glycerol batch, maximum biomass concentrations could be achieved in a time frame between 24 and 36 hours under the conditions tested here. Biomass formation over time using both strains in mineral salts medium and each glycerol batch was recorded over a 48-hour period in parallel microbioreactors. Figure 1 shows the substrates glycerol 1 through glycerol 4 and the measured biomass concentrations as a function of time for both strains utilized, DSM3286 (Figure 1a) and PO1F (Figure 1b). Vigorous foaming occurred when glycerol 5 was used, which precluded reliable measurement of optical density to determine biomass concentration. However, both strains were able to utilize glycerol 5 as a growth source, despite its low purity.

[0024] As can be seen from Figure 1, the DSM3286 strain exhibits a significantly higher biomass yield than the PO1F strain, making it better suited for industrial use. Furthermore, DSM3286 is not a genetically modified organism (GVO) but naturally achieves a high lipid yield. Therefore, the following experiments were performed using biomass obtained from this strain, in which glycerol 2 and glycerol 3 were used as crude glycerol sources for cultivation.

[0025] During experiments to produce sheets from this biomass, it was confirmed that the homogeneity of the sheets could be improved by using the following disruption method on the biomass instead of high-pressure homogenization. To this end, instead of high-pressure homogenization, the biomass was homogenized in an ultrasonic sonotrode for 5 minutes each, with prior treatment with Zymolase at 37°C for 1 hour. Zymolase contains enzymes that, among other things, hydrolyze the β-1,3-glucan bonds in the cell walls, thereby ensuring improved degradation and disruption of the yeast cell walls. This measure produced sheets that were more homogeneous than those produced by the previously commonly used high-pressure homogenization method and no longer contained intact yeast cells. The use of ultrasound for homogenization is not necessary to achieve the objectives of the present invention. Other mechanical homogenization methods can also be used for this purpose. However, the use of ultrasound in combination with Zymolase treatment offers the additional advantage mentioned above of producing more uniform sheets. The entire process from cultivation to sheet or molded body production is shown as an example in the chart in Figure 3.

[0026] The sheets were subsequently examined for sheet properties, e.g., by dynamic differential calorimetry and determination of Young's modulus. The Young's modulus characterizes the degree to which a material deflects when stressed (its resistance to deformation). For the same load and geometry, a component made from a material with a high Young's modulus (e.g., steel) will be stiffer than the same component made from a material with a low Young's modulus (e.g., rubber). To this end, experiments were conducted with various glycerol contents in the sheets. Of the glycerol contents tested (0%, 5%, 10%, and 20%), the best Young's modulus was achieved with a 10% glycerol content, as can be seen in Figure 2. Dynamic differential calorimetry analysis revealed a melting point of approximately 90°C.

[0027] Furthermore, compacts (pressed bodies) were also produced from layered sheets. This demonstrates that the sheets can be processed into solid workpieces and are therefore suitable for the compaction process. For this purpose, multiple layers of sheets were cut to fit the compaction mold and stacked. Compacts were produced at a temperature of 120°C and for a time of 80 seconds with final cooling.

[0028] Products produced using the proposed method are also well compostable. This has been demonstrated experimentally. Depending on the conditions and thickness of the material, complete composting takes approximately 4 to 8 weeks. The key to the products' excellent compostability lies in their natural components. In contrast to many bioplastics, the sheets produced using this method are based on natural biopolymers (i.e., yeast biomass and glycerol). Here, natural microorganisms already possess the enzymes necessary to decompose these compounds and utilize them as substrates for their natural metabolism. Thus, biomass is regenerated in the environment, closing the carbon cycle.

[0029] The barrier properties of the sheets produced using this method are sufficient for use in the food industry, where a sheet produced with 10% glycerol according to the method of the present invention was measured to have a barrier resistance of 36 cm at 23°C / 50% relative humidity. 3 100μm / (m 2 ·d·bar)~41cm 3 100μm / (m 2 The normalized oxygen permeability in the range of g·100μm / (m ·d·bar) was measured and was 1409±227g·100μm / (m ·d·bar) at 23°C / 85% relative humidity. 2 ·d) The average normalized water vapor transmission rate was measured.

[0030] Natural polymers, such as proteins and polysaccharides, exhibit rather low barrier properties against water vapor but high barrier properties against oxygen due to their numerous hydrogen bridges. The opposite is often true for lipids, whose hydrophobic properties result in good barrier properties against water vapor but high permeability to oxygen. In sheets produced according to the proposed method, the polysaccharide and lipid content can be further controlled during fermentation, for example, by changing the medium composition, fermentation conditions (duration, temperature, shaking speed, etc.), or even by genetically modifying the yeast. The properties of products produced using this method can be further influenced through modification of the yeast biomass, for example, to improve packaging-related functionality. Here, the production rate and yield of yeast biomass are largely determined not only by the concentrations of carbon and nitrogen sources, but also by the aeration and incubation temperature of the culture medium. In particular, the composition of the medium has a decisive influence on the composition of the biomass, especially its lipid content. Therefore, by influencing these parameters, i.e., particularly the medium composition and fermentation conditions, the properties of the produced product can be further optimized.

Claims

1. At least the following steps: providing a yeast biomass; homogenizing the biomass, which comprises subjecting the biomass to at least one mechanical treatment; processing the homogenized biomass directly or after adding one or more additional substances into a sheet, coating, or formed body; 1. A method for producing a sheet, coating or molding made of a biodegradable material, in particular for packaging, comprising: A method characterized in that an oleaginous yeast is used as the yeast, and the homogenization further comprises a cell wall-lytic enzyme treatment.

2. 10. The method of claim 1, wherein the providing of the oleaginous yeast comprises culturing one or more strains of the yeast on crude glycerol as a substrate.

3. 3. The method according to claim 1 or 2, wherein Yarrowia lipolytica is used as the oil-producing yeast.

4. The method according to any one of claims 1 to 3, wherein the enzyme treatment is carried out by zymolase treatment.

5. 5. The method according to claim 1, wherein the mechanical treatment is carried out by ultrasound.

6. 6. The method according to any one of claims 1 to 5, characterized in that glycerol is added as the further substance or as one of the further substances.

7. 7. The method according to any one of claims 1 to 6, characterized in that the biomass, after cell disruption, is subjected to a heat treatment, preferably in a water bath, to denature proteins in the biomass.

8. 8. The method of claim 7, wherein the heat treatment is followed by a further homogenization of the biomass.

9. 9. The method according to any one of claims 1 to 8, characterized in that the biomass is freeze-dried and then homogenized.

10. Use of biomass of an oleaginous yeast, in particular Yarrowia lipolytica, for the production of sheets, coatings or moldings.

11. A sheet, coating, or molding formed from the biomass of an oleaginous yeast, particularly Yarrowia lipolytica, as a primary component.

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