Method for the production of a film, a coating or a moulded body from yeast biomass

Abstract

The invention relates to a method for producing a film, a coating or a moulded body from biomass of a yeast, in which the biomass is first disintegrated and homogenised and then further processed to form the film, coating or moulded body. The method is characterised in that the yeast used is an oil-forming yeast, in particular Y. lipolytica, and the homogenisation involves a cell wall-dissolving enzyme treatment. The method can be used to produce bio-based and fully biodegradable packaging products that are based solely on renewable and natural raw materials and have good mechanical properties.

Description

[0001] Process for producing a film, a

[0002] Coating or a molded body made of yeast biomass

[0003] Technical application area

[0004] The present invention relates to a process for producing a film, a coating or a shaped body from a biodegradable material, in particular for packaging, in which biomass of a yeast is provided and homogenized by mechanical treatment and the biomass thus broken down is processed directly or after addition of one or more further substances into a film, a coating or a shaped body.

[0005] There is a growing demand for biodegradable polymers for the manufacture of product packaging. One sustainable solution is the use of renewable resources, such as plant biomass as a starting material for polymer production. In particular, bio-based polymers offer the potential to significantly reduce the use of fossil resources. They are characterized by being CO2-neutral, meaning they add little or no CO2 to the atmosphere over their entire life cycle. The bio-based plastics currently established on the market use renewable resources from agriculture. However, this often requires fertile land for cultivation.In contrast, the use of microbial biomass offers the advantage that it is a fully renewable source of polymers, does not directly require fertile agricultural land for the provision of the biomass, and there is no immediate risk of increased deforestation for biomass production.

[0006] State of the art

[0007] Currently, renewable resources such as corn, wheat, potatoes, sugar cane, sugar beets, bamboo, and wood are primarily used as raw materials for the production of polymers. Compared to conventional, fossil-based plastics, these bioplastics support the reduction of greenhouse gas emissions and can lower the consumption of fossil raw materials. However, in some other impact categories (e.g., land and water use, eutrophication of water bodies), their impacts are generally worse than those of the fossil reference.

[0008] Another well-known possibility is the use of microbial biomass (e.g. yeasts or fungi) for the production of films and coatings in the materials industry. Current research and development projects aim, among other things, at the use of the biomass of the baker's yeast Saccharomyces cerevisiae in combination with glycerol as a plasticizer. A corresponding process is described, for example, in 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, pages 240-247. Glycerol improves the integrity, flexibility and mechanical properties of the produced films by acting as a spacer and mechanical mediator between the biopolymer chains at the molecular level. A crucial disadvantage of S .cerevi siae is, however, that the wild type is mostly cultivated on glucose and thus depends on a food-relevant raw material.

[0009] To use the yeast components, the cell wall must first be broken down to release the cytoplasm. Delagdo et al. used the technique of high pressure homogenization at a pressure of more than 100 MPa to produce films from the biomass of S. cerevisiae. The exposed biopolymers can then interact with each other and form a stable network. The next step is thermal treatment in a water bath at 90 °C to denature the proteins and thus increase their interactions with each other. A second homogenization is used to remove any aggregates (clumps) that may have formed during the heat treatment. Finally, glycerol is added to the yeast dispersion, a film is cast from the mixture and finally the water is removed by drying at 50 °C, which further promotes the interaction between the polymer chains and the formation of the film matrix.

[0010] Biopolymers from agriculturally produced biomass or the commonly used microbial sources (e.g., baker's yeast S. cerevisiae) typically interact strongly with water. Therefore, the correspondingly manufactured films form weaker water vapor barriers than synthetic polymers, leading to faster drying of the packaged goods.

[0011] The object of the present invention is to provide a process for producing a film, a coating, or a molded article that is 100% bio-based and biodegradable. The resulting product should be made from renewable and natural raw materials and be suitable for a wide range of applications in the food and packaging industries, thus also exhibiting sufficient barrier properties for food packaging.

[0012] Description of the invention

[0013] This object is achieved by the method according to claim 1. Advantageous embodiments of the method are the subject of the dependent claims or can be found in the following description and the exemplary embodiment.

[0014] The proposed process is based on the use of yeast biomass to produce the film, coating or shaped body, hereinafter also referred to as product or manufactured product. Biomass is understood to mean the complete moist cell mass of the yeast cells that typically arises in a fermentation process in which this 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 process, yeast biomass is first provided and homogenized, with at least one mechanical treatment being carried out for the purpose of homogenization.The appropriately homogenized biomass is then, if necessary after further intermediate steps such as heat treatment and further homogenization, processed directly or after the addition of one or more other substances into a film, a coating or a shaped body. The proposed process is characterized in that an oil-forming yeast, also referred to below as oleaginous yeast, is used as the yeast, and the homogenization comprises a cell wall-dissolving enzyme treatment. This enzyme treatment is preferably a zymolase treatment. The use of other suitable enzymes such as lysozyme or papain is also possible.

[0015] Oil-forming yeasts are understood to be yeasts that can accumulate more than 20% of their dry cell weight as lipids or triacylglycerides. Examples of oil-forming yeasts that can be used in the proposed process are Schwann! omyces occidentalis, Rhodotorula torul oides, or Yarrowia lipolytica (Y. lipolytica).

[0016] To provide the moist biomass of the oleaginous yeast, at least one strain of oleaginous yeast is cultivated. In the proposed process, this cultivation is preferably carried out on crude glycerol as a substrate. For example, the glycerol which is a residue from biodiesel production from fats and oils and which may contain impurities can be used as a substrate for the cultivation of the oleaginous yeast used in the proposed process. Instead of crude glycerol, other substrates which are comparable in terms of economic efficiency and resource conservation, such as (used) cooking oils and fats, can be used. This can significantly improve the economic efficiency of the production process, as purification costs for the substrate can be saved. By using side streams such as crude glycerol, these are not only returned to the material cycle, but their value is also added (upcycling).The use of food-related substrates such as sugars is not required. The production of the product is resource-efficient; the provision of yeast biomass as a raw material does not result in increased use of fertile and ecologically valuable land and therefore has no negative impact on biodiversity and is not associated with increased water consumption.

[0017] The proposed process can be used to produce films, coatings, and molded articles that are 100% bio-based and biodegradable. These products have a wide range of applications in the food and packaging industries, are fully compostable, and are made only from renewable, natural raw materials. They can also be used to manufacture packaging products that have sufficient barrier properties, particularly against oxygen and water, as desired in the food packaging industry. A further advantage of using oleaginous yeasts is that, in contrast to comparable starting materials of this type, they result in improved mechanical properties of the films. These improved mechanical properties are due to the high lipid and polysaccharide content of the oleaginous yeast.In addition, proteins from the yeast cytoplasm and cell wall polymers (polysaccharides such as mannan, glucan, and chitin) are particularly relevant for film formation. Compared to the yeast S. cerevisiae mentioned above, oleaginous yeast generally has a higher polysaccharide content and also a significantly higher chitin content.

[0018] The proposed process makes particularly advantageous use of the oleaginous yeast Yarrowia lipolyti ca (Y. lipolyti ca). This yeast is capable of synthesizing a large group of valuable metabolites. Important products include lipases and other hydrolytic enzymes, lipids, citric acid, erythritol, and γ-decalactone. Many commercial processes based on the fermentation of the yeast Y. lipolyti ca also have GRAS status ("generally recognized as safe"), which is awarded by the Food and Drug Administration. Furthermore, the yeast biomass of Y. lipolyti ca has been approved as a novel food, so that films and similar materials made from it can be expected to be approved for use as food packaging without any problems.These packaging films are versatile membranes that may only require the use of additives to adapt their integrity and mechanical properties to the specific application.

[0019] Another important requirement for packaging materials, alongside functionality and recyclability, is the protection of the product to be packaged. In the food industry in particular, reducing the water exchange between the food to be protected and its environment is essential. Preventing packaged goods from drying out is a particularly important goal, and suitable packaging materials are needed for this. Biopolymers from agriculturally produced raw materials or the microbial biopolymers currently used typically interact strongly with water, and the films made from them form weaker water vapor barriers than are the case with synthetic polymers. A weak water vapor barrier in packaging generally leads to faster drying out of the packaged goods. Oil-forming yeasts such as Y.lipolyti ca exhibit a significantly higher lipid content in the cytoplasm (between 20-30% of the dry mass, depending on the strain and growth conditions) than other microorganisms. Although lipids alone are unsuitable for forming matrices with acceptable mechanical properties, they have already been used successfully to reduce the strong interaction of films and coatings with water and to improve the water barrier properties of the films. The advantageous composition of biomass from an oleaginous yeast, in particular Y. lipolyti ca, with its high content of lipids and biopolymers, creates the prerequisite for reduced water solubility and thus reduced water permeability.

[0020] In the proposed method, it is particularly advantageous not to use a high-pressure homogenizer for homogenization or cell disruption, but rather a mechanical treatment using ultrasound in conjunction with enzyme treatment of the biomass before and / or during the ultrasound treatment. It has been found that the combination of enzyme treatment and ultrasound treatment produces significantly better homogenization results than high-pressure homogenization. Furthermore, it is advantageous to subject the biomass to a thermal treatment, for example in a water bath, after this first homogenization step in order to denature proteins in the biomass. This is preferably followed by a further homogenization step, preferably also using ultrasound treatment.

[0021] A further advantageous embodiment of the proposed method, also in conjunction with the embodiments already explained above, consists in subjecting the biomass to lyophilization after cultivation and optionally one or more washing steps. Freeze-drying allows the biomass to be stored for longer before further processing according to the proposed method. The oleaginous yeast biomass processed according to the proposed method is finally further processed into the desired product, i.e. the film, coating or shaped body. For this purpose, one or more further substances can be added to the biomass, which in the process is present in a correspondingly liquid form, in particular as a dispersion in a solvent such as water.This or one of these further substances can be a suitable plasticizer, for example glycerol, in order to influence the mechanical properties of the manufactured product. The addition of other substances, for example fillers suitable for the respective application, is also possible. The biomass of the oleaginous yeast, in particular of Y. lipolytica, still forms the main component of the manufactured product, for example the film or the coating, i.e. a proportion (in percent by weight) of over 50%, generally well over 50%, in particular 70-80% or more. The corresponding product can be finished in a known manner, for example by simply casting and drying when manufacturing a film, for example by spraying and drying when manufacturing a coating, or by injection molding when manufacturing a molded body.

[0022] Short description of the drawings

[0023] The proposed method is explained in more detail below using an example embodiment in conjunction with the drawings. Figures 1a and 1b show the growth of two different strains (DSM3286 and P01 F) when cultivated on different glycerol batches;

[0024] Fig. 2 Measurements on films produced by the process with different additions of glycerol; and

[0025] Fig. 3 is a diagram of an exemplary

[0026] Process sequence for producing a film according to the proposed method.

[0027] Ways to implement the invention

[0028] The proposed process is explained again below using the preferred oleaginous yeast Y. lipolyti ca . This oleaginous yeast can use crude glycerol as the sole carbon source for the formation of biomass, producing a high proportion of lipids and polysaccharides . For cultivation, in this example, substrates made from crude glycerol of different qualities and a pure glycerol as a reference were used in conjunction with two different Y. lipolyti ca strains (DSM3286 and PO1 F). The crude glycerol came from different biodiesel producers. Glycerol 1 had a purity of > 99%, glycerol 2 a purity of around 90%, glycerol 3 a purity of around 82%, glycerol 4 a purity of around 70% and glycerol 5 a purity of only around 50%.

[0029] First, the two Y. lipolyti ca strains were cultivated in a mineral salt medium with the aforementioned glycerol batches (using a starting concentration of 20 g / L in each case) as substrates. The Y. lipolyti ca strain PO1 F is a genetically modified version of the wild-type strain W29, which is frequently used in the laboratory or in industrial processes. Here, genes for leucine and uracil synthesis were deleted in order to establish auxotrophies as selection markers for genetic engineering processes. The strain DSM3286 is a wild-type strain that comes from the culture collection of the DSMZ. The crude glycerols were not specially prepared for cultivation, but simply sterile autoclaved and then used directly. Cultivation was carried out at a temperature of 30 °C and 200 rpm for 48 hours. Subsequently, the biomass was harvested by centrifugation (8,000g, 10 min, 4 °C).

[0030] It was shown that both strains tested can utilize all glycerol sources used, although strain PO1 F achieved lower biomass yields than strain DSM3286. Depending on the strain and glycerol batch, the maximum biomass concentration could be reached under the conditions tested here within a time window between 24 hours and 36 hours. Biomass formation over time with the two strains in the mineral salt medium and the respective glycerol batches was recorded over 48 hours in a parallel microbioreactor. Figure 1 shows the measured biomass concentrations as a function of time for the substrates glycerol 1 to glycerol 4 and the two strains used, DSM3286 (Fig. 1a) and PO1 F (Fig. 1b). The use of glycerol 5 resulted in severe foam formation, which made a reliable measurement of the optical density to determine the biomass concentration impossible.Both strains were able to use glycerol 5 as a growth source despite its low purity. As can be seen from Figure 1, strain DSM3286 shows a significantly higher biomass yield than strain PO1 F and is therefore more suitable for industrial use. Furthermore, DSM3286 is not a genetically modified organism (GMO) and naturally achieves a high lipid yield. The following experiments were therefore conducted with biomass obtained from this strain, using glycerol 2 and glycerol 3 as the crude glycerol source for cultivation.

[0031] When conducting experiments on the production of films from this biomass it was found that the homogeneity of the film can be improved if the following digestion process for the biomass is used instead of high pressure homogenization. For this purpose the biomass was homogenized for 5 minutes in an ultrasonic sonotrode instead of high pressure homogenization, after being treated with Zymolase for one hour at 37°C. Zymolase contains a group of enzymes which, among other things, hydrolyze the ß-1,3-glucan bonds contained in the cell wall and thus ensure improved degradation and thus digestion of the yeast cell wall. This procedure made it possible to produce more uniform films which no longer contained any intact yeast cells than with the high pressure homogenization generally used to date.The use of ultrasound for homogenization is not absolutely necessary to solve this problem. Other mechanical homogenization methods can also be used. However, the use of ultrasound in combination with Zymolase treatment offers the additional advantage of producing more uniform films. The entire process, from cultivation to the production of films or a molded body, is illustrated by way of example in the diagram in Figure 3.

[0032] The films were then examined to determine their properties, for example by differential calorimetry and determining their modulus of elasticity. The modulus of elasticity (E-modulus) is a parameter that indicates how much a material gives way when subjected to force (resistance to deformation). For the same load and geometry, a component made from a material with a high modulus of elasticity (such as steel) is stiffer than the same component made from a material with a low modulus of elasticity (such as rubber). For this purpose, tests were carried out with different glycerol contents in the films. Of the glycerol contents tested (0%, 5%, 10% and 20%), the best modulus of elasticity was achieved with 10% glycerol, as can be seen from Figure 2. Differential calorimetry analyses showed a melting point of approximately 90°C.

[0033] Furthermore, compacts were produced from layered foils. This demonstrates that the foils can be processed into a tightly bonded workpiece and are therefore suitable for the pressing process. For this purpose, several layers of foil were cut and stacked according to the pressing mold. The compacts were produced at a temperature of 120 °C for a time of 80 seconds, with a final cooling.

[0034] The products manufactured using the proposed process are also easy to compost. This has been proven in tests. Depending on the conditions and the thickness of the material, complete composting takes approximately 4 to 8 weeks. The key to the product's excellent compostability lies in its natural ingredients. Unlike many bioplastics, the films produced using the process are based on natural biopolymers (i.e., yeast biomass and glycerin). Thus, the microorganisms in nature already possess the necessary enzymes to break down these compounds and use them as substrates for their natural metabolism. In this way, biomass is once again built up in the environment, and the carbon cycle is closed.

[0035] The barrier properties of the films produced using this process are also sufficient for applications in the food industry. Measurements of films produced with 10% glycerol according to this process showed a standardized oxygen permeability at 23 °C / 50% r. H. in the range of 36 to 41 cm 3 100 pm / (m 2 d bar ) and an averaged normalized water vapor permeability at 23 ° C / 85% r . F . of 1409 ± 227 g 100 pm / (m 2 d) measured .

[0036] Natural polymers such as proteins and polysaccharides, due to their high number of hydrogen bonds, tend to have a low barrier to water vapor, but exhibit a high barrier effect against oxygen. With lipids, it is usually the other way around: the hydrophobicity results in a good barrier to water vapor, but displays high permeability to oxygen. In the film produced according to the proposed process, the polysaccharide and lipid contents can be additionally controlled during fermentation, e.g. by media composition, fermentation conditions (duration, temperature, shaking rate, etc.) or by genetic modification of the yeast. The properties of the products manufactured using the process can be further influenced by modifying the yeast biomass, for example to improve packaging-relevant functionalities.The production rate and yield of yeast biomass depend heavily on the concentration of carbon and nitrogen sources, as well as on the aeration of the culture medium and the culture temperature. The composition of the medium, in particular, significantly influences the composition of the biomass, particularly its lipid content. Thus, the properties of the produced products can be further optimized by influencing these parameters, particularly the composition of the medium and the fermentation conditions.

Claims

Patent claims 1 . A process for producing a film, a coating or a shaped body from a biodegradable material, in particular for packaging, comprising at least the following steps: - Providing biomass of a yeast, - homogenisation of the biomass , which comprises at least one mechanical treatment of the biomass , and - processing of the homogenized biomass directly or after addition of one or more further substances to form a film, a coating or a shaped body, characterized in that an oil-forming yeast is used as yeast and the homogenization also comprises a cell wall-dissolving enzyme treatment. 2 . A method according to claim 1 , characterized in that the provision of the oil-forming yeast comprises cultivating one or more strains of this yeast on crude glycerol as a substrate. 3 . Process according to claim 1 or 2, characterized in that Yarrowia lipolyti ca is used as the oil-forming yeast.

4. Process according to one of claims 1 to 3, characterized in that the enzyme treatment is carried out by a zymolase treatment.

5. Method according to one of claims 1 to 4, characterized in that the mechanical treatment is carried out by means of ultrasound.

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

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

8. Process according to claim 7, characterized in that following the thermal treatment, further homogenization of the biomass takes place.

9. Process according to one of claims 1 to 8, characterized in that the biomass is freeze-dried before homogenization.

10. Use of the biomass of an oil-forming yeast, in particular of Yarrowia lipolytica, for producing a film, a coating or a shaped article.

11. Film, coating or shaped article which is formed as a main component from the biomass of an oil-producing yeast, in particular Yarrowia lipolytica.

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