Processing technology

Abstract

The invention relates to methods of downstream processing of a germ-free cell- cultured biomass of animal cells, the downstream processing method comprising aseptically harvesting an edible biomass of cells cultured in a culture medium under aseptic conditions, and processing and packaging the harvested edible biomass under aseptic conditions to form a packaged aseptic cell-cultured meat product. The invention also relates to packaged cell-cultured meat products when produced by the methods of the present invention. The invention also relates to methods of downstream processing for manufacturing aseptically-packaged germ-free cell- cultured meat, or aseptically-packaged germ-free food products containing such germ- free cell-cultured meat.

Description

[0001] PROCESSING TECHNOLOGY

[0002] INCORPORATION BY CROSS-REFERENCE

[0003] The present invention claims priority from Australian provisional patent application number 2023902900, filed on 8 September 2023, the entire content of which is incorporated herein by cross-reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to aseptic processing and packaging technologies. The invention has been developed primarily as a method of downstream processing of a germ-free animal cell-cultured biomass to manufacture an aseptic meat product and will be described hereinafter with reference to this application. The aseptically-packaged germ-free cell- cultured meat product may be either uncooked and / or not heat-treated. However, it will be appreciated that the invention is not limited to this particular field of use.

[0006] BACKGROUND OF THE INVENTION

[0007] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0008] Meat and fish are susceptible to spoilage during processing, packaging and storage due to the presence of bacteria. In order to reduce spoilage, it is standard practice to keep meat and fish products at reduced temperature to limit the growth of microbes during storage and transportation. This aspect of the supply chain is referred to as ‘cold chain logistics.’ However, the cold chain is costly, with an estimated cost of over USD230 billion in 2020 alone, and wasteful, with the shelf-life of packaged meat and fish being short (i.e. days to weeks). In order to extend the shelf-life of packaged food products, heat may be used to pasteurise or sterilise the product, either before or after the packaging step - however, this can adversely affect the meat product’s sensory or nutritional characteristics. For example, retort packaging methods can be used to eliminate the microbial load in the food product; however, this high temperature treatment can mar the quality of the food product (e.g., reduce vitamin content, alter flavour and texture due to modification of sulfhydryl groups and the Maillard reaction). Cell-cultured meat has emerged as a new alternative to meat harvested from slaughtered animals. There are significant advantages of cell-cultured meat such as no animal slaughtering, improved food safety and security, and a reduced environmental impact. For these reasons, it is expected the cultured meat industry will grow at 41% annually and reach USD600 billion global market by 2040 (Consultancy AT Kearney). By 2040, it is forecast that cultured meat will represent 35% of the global meat sales, whilst conventional meat will drop substantially to 40% of global sales (plant-based meat making up the remaining difference).

[0009] There are many scientific publications and resources available that outline the conventional culturing process, for example Reiss et al, 2021. One of the key requirements of the culturing technique is that the culturing needs to be aseptic to avoid microbes from contaminating the animal cells (which grow much slower than microbes). Small scale cultures in the laboratory may be kept aseptic through the addition of antibiotics to the culture medium; however, this would be unacceptable in food production.

[0010] Following the production of an animal cell biomass by culturing in a bioreactor, cell-cultured meat processing follows well-established methods which are familiar to those skilled in the art. Like conventional meat and fish, down-stream processing of cell-cultured meat occurs in a non-aseptic setting (typically a factory production line - such as those used for burger or sausage production), leading to the same problem of microbial contamination and resulting in short shelf-life, requiring either cold-chain implementation and / or heat sterilization to extend shelf life.

[0011] There would be a benefit to extending the shelf-life of cell cultured meat products without requiring cold-chain or heat sterilization, both of which increase manufacturing costs and / or energy requirements, and / or can compromise taste and texture. An extended product shelflife is also very important for the growth of the cell-cultured meat industry.

[0012] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0013] SUMMARY OF THE INVENTION In a first aspect, there is provided a method of downstream processing of a germ-free cell- cultured biomass of animal cells, the downstream processing method comprising: aseptically harvesting an edible biomass of cells cultured in a culture medium under aseptic conditions; and processing and packaging the harvested edible biomass under aseptic conditions to form a packaged aseptic cell-cultured meat product.

[0014] In an embodiment of the first aspect, the harvesting of the edible biomass comprises continuous-flow centrifugation.

[0015] In another embodiment of the first aspect, the harvesting of the edible biomass comprises filtration.

[0016] In another embodiment of the first aspect, the culture medium is reused or fortified and recycled for use in culturing other batches of cells after separation from the edible biomass during harvesting.

[0017] In another embodiment of the first aspect, the harvesting comprises aseptically washing the cells.

[0018] In another embodiment of the first aspect, the harvesting and / or processing is configured to achieve a moisture content of 40%-80% (w / w).

[0019] In another embodiment of the first aspect, the cultured cells or component(s) thereof are selected from the group consisting of muscle cells, adipocytes, hepatocytes, fibroblasts and combinations thereof.

[0020] In another embodiment of the first aspect, the processing of the harvested edible biomass comprises aseptically mixing or layering the edible biomass under aseptic conditions with at least one other aseptic edible biomass harvested from a separate batch of cells. In a further embodiment, the at least one other aseptic edible biomass comprises adipocytes, hepatocytes, fibroblasts, or muscle cells, or components thereof.

[0021] In another embodiment of the first aspect, the processing of the harvested edible biomass comprises aseptically mixing or layering the harvested edible biomass under aseptic conditions with another aseptic or sterilised food ingredient or food product. In a further embodiment, the food ingredient or food product is potato, pasta, rice, oil, vege-burger mix, flavouring and / or sauce.

[0022] In another embodiment of the first aspect, the germ-free cell-cultured meat product is uncooked and / or not heat-treated.

[0023] In another embodiment of the first aspect, the harvested edible biomass is:

[0024] - extruded through a nozzle during the processing step; or

[0025] - 3D printed during the processing step.

[0026] In another embodiment of the first aspect, a packaging material used to package the aseptic cell-cultured meat product is sterilized with heat, pressure, hydrogen peroxide, ionizing radiation, infrared radiation, ethylene oxide, or combinations thereof, prior to packaging.

[0027] In a second aspect, there is provided a packaged cell-cultured meat product when processed using the method according to the first aspect.

[0028] In a third aspect, there is provided a method of downstream processing for manufacturing aseptically-packaged germ-free cell-cultured meat, or aseptically-packaged germ-free food products containing such germ-free cell-cultured meat, the method comprising: aseptically harvesting an edible biomass of animal cells that have been cultured in a bioreactor under aseptic conditions; and processing and packaging the harvested edible biomass under aseptic conditions to form packaged germ-free cell-cultured meat, or germ-free food products that contain such germ-free cell-cultured meat.

[0029] In an embodiment of the third aspect, the germ-free cell-cultured meat is uncooked and / or not heat-treated.

[0030] SUMMARY OF THE ADVANTAGES OF THE INVENTION

[0031] The present invention provides the following advantages over the prior art. The person skilled in the art, after understanding the invention described herein, will appreciate that the invention may also provide other advantages not listed below. A person skilled in the art, after reading and understanding the invention described herein, will appreciate that the disclosed downstream processing methods provide a meat product that: -beneficially leverages, through an interworking relationship, the aseptic nature of the production of a mass of cultured cells for subsequent downstream processing steps (including harvesting, processing and packaging) conducted under aseptic conditions, therefore avoiding the need to remove or kill microbes from the meat product;

[0032] -is uncooked and where the germ-free cell-cultured meat can be cooked just prior to consumption for optimal texture, moisture content and flavour;

[0033] -does not need to be refrigerated during transportation or storage;

[0034] -does not need to be treated with heat or pressure before, during or after packaging as such processes may impair flavour, texture and / or nutritional profile of the meat (likewise, non-aseptically processed cell-cultured meat products may suffer from similar sensory and quality issues due to microbial activity over the product’s shelflife);

[0035] -may extend the shelf-life compared to cell-cultured meat processed under conventional (non-aseptic) conditions;

[0036] -may reduce transport and storage costs, as meat products manufactured according to the invention do not require refrigeration;

[0037] -may reduce the energy requirements for manufacturing cell-based meats (heat sterilisation or cold chain treatment), thereby reducing the environmental footprint from processing of the food product; and / or

[0038] -may be mixed with other sterilised food ingredients or other aseptic products and the resulting aseptic food products not requiring cold storage or heat treatment.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] A preferred embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0041] Figure 1 shows a conventional process for culturing cells and downstream processing for producing non-aseptic cell-cultured meat products (i.e. the prior art); and Figure 2 shows a method of downstream processing of a biomass of cell-cultured animal cells from a bioreactor to manufacture uncooked nor heat-treated germ-free cell-cultured meat product where all steps are performed under aseptic conditions according to the present invention.

[0042] DEFINITIONS

[0043] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.

[0045] Unless the context clearly requires otherwise, throughout the description and the claims, the terms “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process or method.

[0046] The transitional phrase "consisting of’ excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0047] The transitional phrase "consisting essentially of" is used to define a composition, process or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term "consisting essentially of" occupies a middle ground between "comprising" and "consisting of". Where the applicant has defined an invention or a portion thereof with an open-ended term such as "comprising", it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms "consisting essentially of" or "consisting of." In other words, with respect to the terms “comprising”, “consisting of’, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of’ or, alternatively, by “consisting essentially of’.

[0048] While reference may be made in this disclosure to the invention comprising a combination of a plurality of elements, it is also understood that this invention is regarded to comprise combinations which omit or exclude one or more of such elements, even if this omission or exclusion of an element or elements is not expressly stated herein, unless it is expressly stated herein that an element is essential to the applicant' s combination and cannot be omitted. It is further understood that the related prior art may include elements from which this invention may be distinguished by negative claim limitations, even without any express statement of such negative limitations herein. It is to be understood, between the positive statements of applicant's invention expressly stated herein, and the prior art and knowledge of the prior art by those of ordinary skill which is incorporated herein even if not expressly reproduced here for reasons of economy, that any and all such negative claim limitations supported by the prior art are also considered to be within the scope of this disclosure and its associated claims, even absent any express statement herein about any particular negative claim limitations.

[0049] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0050] The complete disclosures of the patents, patent documents and publications cited herein are incorporated by reference in their entirety as if each were individually incorporated.

[0051] Unless expressly stated to the contrary, "or" refers to an inclusive “or” and not to an exclusive “or”. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0052] The term "and / or" used in the context of "X and / or Y" should be interpreted as "X," or "Y," or "X and Y." Similarly, "at least one of X or Y" should be interpreted as "X," or "Y," or "both X and Y."

[0053] The indefinite articles "a" and "an" preceding an element or component of the invention are intended to be non-restrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0054] It will be understood that use of the term “between” herein when referring to a range of numerical values encompasses the numerical values at each endpoint of the range.

[0055] Various features of the embodiments of the invention disclosed herein are, for brevity, described in the context of a single embodiment, but may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the illustrative embodiments disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present compositions and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0056] As used herein “downstream processing” refers to steps in a method of processing and packaging a germ-free cell-cultured meat product that occur after a sufficient mass of cells have been produced aseptically in cell culture. The typical downstream processing includes harvesting an edible biomass of the aseptically cultured cells and then aseptically processing and packaging the resultant meat product.

[0057] As used herein “harvest” or “harvesting” refers to the step of selectively separating the cultured cells from the culture media and then continuing down-stream processes using the separated cultured cells. As used herein “germ-free” refers to a process or product where there is a complete absence of living microbes such as bacteria and yeasts. For example, germ- free refers to a process or product where there are no detectable microbes by analytic techniques such as PCR.

[0058] As used herein “aseptic” and “aseptically” refer to conditions or products where there is a complete absence of living microbes such as bacteria and yeasts. For example, aseptic refers to a process or product where there are no detectable microbes by analytic techniques such as PCR. Aseptic conditions include handling or processing samples in a hermetically-sealed controlled environment where all surfaces and the air have been sterilised to completely eliminate microbial contamination.

[0059] As used herein “cell-cultured” refers to a product that is produced by aseptically culturing animal cells in vitro in culture medium. Cell-cultured meat is also known as cell-based meat, in vitro meat, lab-grown meat, clean meat, cultured meat, cultivated meat, cell-cultivated meat.

[0060] As used herein “meat product” refers to a cell-cultured product, wherein the originating cells are derived from animal tissue. The animal tissue may be from a cow, pig, sheep, goat, chicken, fish, or any other animal. The animal tissue may include muscle, fat, liver or any other tissue from the animal.

[0061] As used herein “biomass” or “edible biomass” refers to a mass of animal cells, produced by culturing cells in vitro, that is used to create a meat product that is suitable for human consumption. As described in more detail in this specification, the mass of cells may be washed to prepare the mass of cells for inclusion in the meat product.

[0062] As used herein “animal cell(s)” refers to any type of cell derived from metazoan animals that can be used for cell culturing, including without limitation muscle cells, adipocytes, hepatocytes, or fibroblasts. The animal cells may be derived from, or modified from, any such animal whereby a cell-cultured meat is desired. Typically, this has included the likes of cows, pigs, sheep, goats, chicken, fish - however, a person skilled in the art would appreciate the same principals of cell-culturing may be applied to any such type of metazoan animal cell without limitation.

[0063] As used herein “muscle cells” refer to cells that are derived from the muscle tissue of an animal. Non-limiting examples of muscle cells include myoblasts, myocytes, and myotubes. As used herein “microbe(s)” or “microbial” refers to bacteria, viruses, yeasts or other microorganisms. The term “germ” is also used interchangeably with microbe(s).

[0064] As used herein “culture medium” and “culture media” refer to a solution that is used to culture animal cells. The terms may refer to solutions that have salts, glucose, vitamins, growth factors, fatty acids, serum, other cell culture media components, or a combination thereof.

[0065] As used herein “continuous-flow centrifugation” refers to a technique where a suspension of particulate in liquid is continuously pumped into a centrifuge while the centrifuge is operating. The centrifuge applies a centrifugal force to the suspension thereby separating the particulate from the liquid based on density. Particulate of different densities are separated from each other based on density in the centrifuge. In the context of the present invention, “continuous-flow centrifugation” is used to separate cultured cells from culture medium.

[0066] As used herein “filtration” refers to a process of separating particulate from a liquid by means of a physical barrier which allows liquid to pass through but does not allow particulate to pass through. In the context of the present invention, “filtration” is used to separate cultured cells from culture medium.

[0067] As used herein “fortified” refers to a process of adding components to a culture medium that have been depleted. This process may include adding vitamins, salts, growth factors, fatty acids, serum, other cell culture media components, or a combination thereof, to a culture medium. The process of fortifying a culture medium may also include adjusting the pH of the culture medium.

[0068] As used herein “wash” and “washing” refer to a technique where cultured cells are typically rinsed with a liquid solution such as saline to help remove residual media from the cultured cells. The liquid solution is then separated from the cultured cells, by a centrifugation or filtration step. A person skilled in the art will appreciate that these washing steps may be repeated any number of times.

[0069] As used herein "uncooked” refers to a meat product that has not been cooked. A person skilled in the art will appreciate that an uncooked meat product is a meat product that has not been cooked by means of temperature, pressure, altered pH or a combination thereof. The term “uncooked” also encompasses a meat product that is raw. As used herein "heat-treated” refers to a meat product that has been fully or at least partially cooked or sterilised by means of exposing the meat product to elevated temperatures with a primary goal to kill, destroy or slow the growth of microorganism(s) that spoil meat products.

[0070] As used herein "moisture content” refers to the amount of water (H2O) present in a product. Typically, moisture content is represented by the percentage of a product’ s total mass that is water.

[0071] As used herein "3D print” or “3D printed” refers to a technique where a three-dimensional product is constructed typically based on a digital three-dimensional model by depositing material onto a platform or substrate. The digital three-dimensional model may be a computer-aided design (CAD) model. The material may be deposited by layering onto the platform or substrate.

[0072] As used herein "vege-burger mix” refers to a product or mix of ingredients that are used to produce a plant-based food item that is in the form of a burger patty. Vege-burger mix may include chickpeas, beans, lentils, fungi or combinations thereof.

[0073] PREFERRED EMBODIMENT OF THE INVENTION

[0074] The present invention provides a method of downstream processing of an aseptic biomass of animal cells to manufacture a germ-free cell-cultured meat product that may be either preferably uncooked and / or not heat-treated. The downstream processing method comprises aseptically harvesting an edible biomass from cells cultured in a culture medium under aseptic conditions, then processing and packaging the harvested edible biomass under aseptic conditions to form a packaged germ-free cell-cultured meat product.

[0075] Optional pre-culturing

[0076] The current invention preferably relates to the post-culturing steps taken after conventional aseptic culturing (see Figure 1 for an overview). Therefore, the method does not necessarily require the actual culturing steps, such that the invention may be employed on pre-cultured aseptic cells provided by another contractor or third party.

[0077] Optionally, the method includes pre-culturing. Cells derived from an animal may be aseptically cultured in a culture medium. A person skilled in the art will appreciate that the manufacture of cultured I cultivated meat is a ‘scaled-up’ version of the well-established laboratory technique of cell culture I tissue culture (Broucke et al, 2023; Pajcin et al, 2022; Hong et al, 2021; Bryant & Barnett, 2018; Pakseresht et al, 2022; Chodkowska et al, 2022). This is a widely-practised method for growing eukaryotic cells (e.g., animal cells) under aseptic conditions in the laboratory. The cells are grown in a sterilized liquid medium that provides the necessary nutrients, which may be supplemented with growth factors (e.g., foetal bovine serum) using sterilized equipment and containers. This technique has been used to grow small amounts (grammes) of animal cells for biological / medical research for decades. Accordingly, a skilled person will be familiar with the techniques and procedures used to manufacture cultured / cultivated meat.

[0078] The production of cultured meat involves the growth of vast amounts of animal cells such as muscle cells, adipocytes, hepatocytes, and / or fibroblasts that are then formed into meat products. The cells must be grown under aseptic conditions (i.e., aseptically), as microbes generally grow quicker than the animal cells and would rapidly spoil the animal cell culture if there were any contamination.

[0079] In one embodiment, the animal cells are muscle cells (e.g. myoblasts, myocytes, or myotubes). In other embodiments, the animal cells are adipocytes, hepatocytes, or fibroblasts. It is envisioned that cells derived from a range of species may be used in the present invention. For example, the cells may be derived from cows, pigs, sheep, goats, chicken, fish or any other animal.

[0080] In one embodiment the animal cells are cultured in a bioreactor. Bioreactors are well known in the art, for example as described in Reiss et al, 2021. Other types of culturing vessels may be used, and those are also well known in the art.

[0081] When a suitable mass of cells has been produced, the culture is ready for aseptic harvesting, which is the first part of the downstream processing according to the present invention.

[0082] Harvesting

[0083] In one embodiment, an edible biomass is harvested from the culture medium using continuous-flow centrifugation. Using continuous-flow centrifugation, cultured cells and culture media are aseptically pumped from the bioreactor into a continuous-flow centrifuge where cultured cells are separated from culture media based on density. In another embodiment, an edible biomass is harvested from the culture medium using filtration. In a further embodiment, both continuous-flow centrifugation and filtration are used to harvest the edible biomass. One skilled in the art would appreciate other conventional options are available to effectively harvest the edible biomass from the culture medium without limitation, albeit understanding that according to the present invention the harvesting must be done under aseptic conditions to prevent microbial contamination of the already microbe-free cultured cells.

[0084] In one embodiment, the culture medium is aseptically collected after separation from the cultured cells during the harvesting of the edible biomass. The culture medium may be fortified and recycled for use in aseptic culturing other batches of cells. The culture medium may be fortified by adding vitamins, salts, growth factors, fatty acids, serum, other cell culture media components, or a combination thereof. The culture medium may have the pH adjusted using acids or bases, as appropriate, before its use to culture other batches of cells. Alternatively, the culture medium may be directly reused to culture other batches of cells.

[0085] Washing

[0086] In one embodiment, the cultured cells are aseptically washed before, during or after the harvesting of the edible biomass. Aseptic washing may comprise rinsing the cultured with a buffered salt solution. The buffered salt solution may be a solution comprising sodium chloride. After washing, the cultured cells can then be separated from the buffered salt solution through, for example, filtration and / or continuous -flow centrifugation.

[0087] Processing / Packaging

[0088] In one embodiment, during the harvesting and / or processing steps, the edible biomass is sampled and tested for quality control. For example, the edible biomass may be tested for moisture content and the moisture content can then be adjusted as desired - for example by further harvesting to remove excess moisture.

[0089] In one embodiment, the moisture content of the edible biomass is adjusted to between 30%- 90% (w / w), or more preferably between 40% -80% (w / w). Soren & Biswas, 2020 outlines the typical moisture content of various forms of meat based on the cut and animal.

[0090] In a further embodiment, the moisture content of the edible biomass is adjusted to approximately 75% (w / w). One skilled in the art would appreciate the edible biomass may also be tested and adjusted as desired for protein content, fat content, and ash content (to closely mimic or improve on the levels for conventional meat as discussed by Soren & Biswas, 2020).

[0091] In one embodiment, the edible biomass is aseptically mixed homogeneously or heterogeneously (for example layered) with at least one other aseptic edible biomass during the processing step. For instance, the at least one other aseptic edible biomass may be produced by harvesting an edible biomass from a separate batch of cells cultured in a culture medium. Other aseptic edible biomass(es) may comprise adipocytes, hepatocytes, fibroblasts, or muscle cells (e.g. myoblasts, myocytes, or myoblasts). The edible biomasses may be derived from the same species or a different species.

[0092] In another embodiment, the harvested edible biomass is aseptically mixed or layered with another aseptic or sterilised food ingredient or food product during the processing step.

[0093] For example, in preparing a cell-cultured burger patty, the edible biomass may be aseptically mixed with an oil (e.g. olive oil, peanut oil, canola oil, or sunflower oil), vege-burger mix, flavouring, vegetable, sauce or any other suitable food ingredient. Mixing or layering with these food ingredients may be used to adjust the flavour or texture of the germ-free cell- cultured meat product. A vege-burger mix may include chickpeas, beans, lentils, fungi or combinations thereof.

[0094] In one embodiment, the harvesting includes extruding the edible biomass through a nozzle prior to, or just before, the packaging step. The edible biomass may also be sliced to produce a food product with a desired shape. Alternatively, the edible biomass may be 3D printed during processing and / or packaging. The edible biomass may also be moulded or formed during processing and / or packaging.

[0095] The edible biomass may be aseptically mixed or layered with potato, pasta, rice, or another food product during the processing I packaging step. Mixing or layering with these food products may be used to manufacture a ready-to-cook meal preparation. The edible biomass may be aseptically mixed or layered with flavouring, sauce or a combination thereof during the packaging step. Mixing or layering with flavouring and / or sauce may be used to adjust the flavour of the germ-free cell-cultured meat product.

[0096] Packaging In an embodiment the packaging material used to package the germ-free cell-cultured meat product may be sterilized by pre-treatment with heat, pressure, hydrogen peroxide, ionizing radiation, infrared radiation, ethylene oxide, or combinations thereof, prior to packaging.

[0097] In an embodiment, the aseptic packaged germ- free cell-cultured meat product is uncooked and / or not heat-treated. An uncooked preparation then allows a consumer to cook the germ- free cell-cultured meat product at home. It is also envisioned that the aseptic packaged germ- free cell-cultured meat product may be cooked or partially cooked to reduce meal preparation time for a consumer. Similarly, it may be beneficial, albeit not required, to heat-treat the meat product before, during or after packaging for regulatory compliance, improved consumer confidence, or any other reason.

[0098] The present invention also provides an aseptic packaged cell-cultured meat product when processed using the methods described above.

[0099] Due to the aseptic down-stream processing according to the present invention, the packaged cell-cultured meat products of the present invention may be uncooked and stored at ambient temperature for extended periods of time compared to meat processed conventionally, without the disadvantages previously described - such as the cold chain or heat sterilisation.

[0100] For instances, it is envisioned that the sterile packaged aseptic cell-cultured meat may be stored at ambient temperature and unrefrigerated for several months, for example 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or a length of time therebetween.

[0101] During and after such storage, the aseptically processed and packaged cell-cultured meat may have better appearance, odour, acidity, meat texture, fibre stability, cell morphology, and / or protein stability as compared to a cell-based meat product that has been processed using conventional processing. This is supported by the fact that conventionally processed foods (under non-aseptic conditions) often have one or more of these undesirable effects even during their normal shelf-life due to background microbial activity.

[0102] It will be appreciated that the disclosed methods of downstream processing of a germ-free cell-cultured meat product have improved hygiene and safety while producing food products with an increased shelf-life that do not require refrigeration or retort packaging. BEST MODES

[0103] Referring to the drawings, Figure 1 shows a conventional method of collecting animal cells from a source, producing an expanded population of differentiated cells and downstream processing for manufacturing cell-cultured meat products. It should be appreciated that this Figure is provided as an example only and does not form an essential part of the invention. Merely, it is shown to illustrate the “up-stream” process of the cell culturing prior to downstream processing that forms the invention at hand.

[0104] The conventional method includes the following steps:

[0105] 1) A tissue biopsy is collected from an animal (e.g. cow, pig, sheep, goat, chicken, or fish) using a needle or scalpel. The tissue biopsy can be collected from any suitable tissue which may include muscle, fat, skin, liver, or reproductive tissue (blastocyst).

[0106] 2) The tissue biopsy is then dissociated to release the cells contained therein. The tissue biopsy can be dissociated through application of gentle mechanical forces by using, for example, a Dounce homogenizer or a Potter- Elvehjem homogenizer. The tissue biopsy can also be dissociated using sonication or enzymatic digestion (trypsin, Accutase®, or papain).

[0107] 3) If non-reproductive tissue is biopsied, a desired progenitor cell population can be isolated from the dissociated cells by selecting for cell surface markers (for example, Pax7, M-cadherin, Syndecan-4, CXCR4, a-7 integrin, VCAM-1, CD56, CD 105, CD73, CD90, Sca-1, or PDGFRa). If reproductive tissue is biopsied, a desired stem cell population can be isolated from the dissociated cells by selecting for cell surface markers (for example, Oct4, Sox2, Nanog, c-Myc, or Klf4). Isolation of the desired cell population can be achieved using fluorescence-activated cell sorting (FACS) or magnetic bead-based isolation and antibodies targeting a suitable cell surface marker. Alternatively, an induced pluripotent stem cell (iPSC) line may be used to produce an expanded population of differentiated cells. A person skilled in the art will be familiar with methods of reprogramming animal cells (e.g. fibroblasts) to derive an iPSC line.

[0108] 4) Once the desired progenitor or stem cell population is isolated the cells are differentiated into muscle cells (e.g. myoblasts, myocytes, or myotubes), adipocytes, hepatocytes, or fibroblasts. A person skilled in the art will be familiar with methods of differentiating progenitor or stem cells into a specific cell type using growth factors and other means. 5) The differentiated cells can then be expanded and cultured using a conventional bioreactor. Alternatively, an immortalised cell line can be used instead of a differentiated cell line. For example, an immortalise cell line of muscle cells (e.g. myoblasts, myocytes, or myotubes), adipocytes, hepatocytes, or fibroblasts can be expanded and cultured using a conventional bioreactor.

[0109] 6) The cultured cells are then harvested and a meat product is prepared under nonaseptic conditions. More specifically, a meat product is prepared with standard hygienic practices used at a meat packing plant which are not aseptic.

[0110] 7) The cell-cultured meat product is packaged in non-aseptic packaging and, in some cases, is heat-treated to reduce the viability of contaminating microbial organisms.

[0111] 8) The cell-cultured meat product is then transported and stored using the cold chain (i.e. at reduced temperature) to inhibit the growth of contaminating microbial organisms.

[0112] Referring now to Figure 2, and according to the present invention, the following steps are taken:

[0113] 1) (Optional) Animal cells are aseptically cultured in a bioreactor. Once enough cells have been cultured, an edible biomass is harvested of the cultured cells. (Note this preliminary step may be pre-completed and is not an essential step in the invention).

[0114] 2) The suspension of cells and culture medium are aseptically pumped into a continuous- flow centrifuge and the cultured cells are separated from the culture medium based on density. The culture medium is removed and may be re-used to culture another batch of animal cells, or it may be fortified and recycled to use when culturing another batch of animal cells (see Steps 2b 12c).

[0115] 3) To remove residual culture media, the cultured cells are then washed by resuspending in an aseptic buffered salt solution. The resuspended cells are centrifuged again and the aseptic buffered solution is removed. The washing step may be performed multiple times as necessary.

[0116] 4) The edible biomass is sampled and tested for moisture content, protein content, fat content, and ash content. The moisture content may then be adjusted to a suitable value typically within 40%-80% (w / w), if necessary.

[0117] 5) The edible biomass is then further aseptically processed to form a germ-free cell- cultured meat product. The edible biomass may be aseptically mixed with at least one other aseptic edible biomass. The other edible biomass may comprise muscle cells (e.g. myoblasts, myocytes, or myoblasts), adipocytes, hepatocytes, or fibroblasts. The edible biomasses may be derived from the same species or from different species.

[0118] 6) The edible biomass may then also be aseptically mixed with aseptic or sterilised food ingredients or food products such as olive oil, vege-burger mix, flavouring and / or sauce.

[0119] 7) The edible biomass may then be extruded through a nozzle or 3D printed to aseptically package the germ-free cell-cultured meat product. During the processing / packaging step, the edible biomass may be aseptically mixed or layered with at least one aseptic or sterilised food ingredient or food product (i.e. sterilized non-meat component) such as potato puree, pasta, rice, flavouring and / or sauce (see Step 7b).

[0120] 8) The germ-free cell-cultured meat product is then aseptically packaged into presterilised (heat, pressure, hydrogen peroxide, ionizing radiation, infrared radiation, ethylene oxide, or combinations thereof), pre-formed packaging. This germ-free cell- cultured meat product may be transported and stored at room temperature for extended periods of time. The germ-free cell-cultured meat may remain uncooked and / or not heat treated - although in some embodiments it may be desired to include a full or partial cooking or heat treatment step.

[0121] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, and in particular features of any one of the various described examples may be provided in any combination in any of the other described examples. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows. REFERENCES

[0122] Broucke et al. 2023. Cultured meat and challenges ahead: A review on nutritional, technofunctional and sensorial properties, safety and legislation. Meat Sci. 195: 109006.

[0123] Bryant & Barnett. 2018. Consumer acceptance of cultured meat: A systematic review. Meat Sci. 143: 8-17.

[0124] Chodkowska et al. 2022. Sustainable Future Protein Foods: The Challenges and the Future of Cultivated Meat. Foods. 11: 4008.

[0125] Hong et al. 2021. Current Issues and Technical Advances in Cultured Meat Production: A Review. Food Sci Anim Resour. 41: 355-372. Pajcin et al. 2022. Bioengineering Outlook on Cultivated Meat Production.

[0126] Micromachines (Basel). 13: 402.

[0127] Pakseresht et al. 2022. Review of factors affecting consumer acceptance of cultured meat. Appetite. 170: 105829.

[0128] Reiss et al. 2021. Cell Sources for Cultivated Meat: Applications and Considerations throughout the Production Workflow. Ini. J. Mol. Sci. 22: 7513.

[0129] Soren & Biswas. 2020. Methods for nutritional quality analysis of meat. Meat Quality Analysis. 21-36.

Claims

CLAIMS1. A method of downstream processing of a germ- free cell-cultured biomass of animal cells, the downstream processing method comprising: aseptically harvesting an edible biomass of cells cultured in a culture medium under aseptic conditions; and processing and packaging the harvested edible biomass under aseptic conditions to form a packaged aseptic cell-cultured meat product.

2. The method according to claim 1, wherein the harvesting of the edible biomass comprises continuous-flow centrifugation.

3. The method according to claim 1 or claim 2, wherein the harvesting of the edible biomass comprises filtration.

4. The method according to any one of the preceding claims, wherein the culture medium is reused or fortified and recycled for use in culturing other batches of cells after separation from the edible biomass during harvesting.

5. The method according to any one of the preceding claims, wherein the harvesting comprises aseptically washing the cells.

6. The method according to any one of the preceding claims, wherein the harvesting and / or processing is configured to achieve a moisture content of 40%-80% (w / w).

7. The method according to any one of the preceding claims, wherein the cultured cells or component(s) thereof are selected from the group consisting of muscle cells, adipocytes, hepatocytes, fibroblasts and combinations thereof.

8. The method according to any one of the preceding claims, wherein the processing of the harvested edible biomass comprises aseptically mixing or layering the edible biomass under aseptic conditions with at least one other aseptic edible biomass harvested from a separate batch of cells.

9. The method according to claim 8, wherein the at least one other aseptic edible biomass comprises adipocytes, hepatocytes, fibroblasts, or muscle cells, or components thereof.

10. The method according to any one of the preceding claims, wherein the processing of the harvested edible biomass comprises aseptically mixing or layering the harvested edible biomass under aseptic conditions with another aseptic or sterilised food ingredient or food product.

11. The method according to claim 10, wherein the food ingredient or food product is potato, pasta, rice, oil, vege-burger mix, flavouring and / or sauce.

12. The method according to any one of the preceding claims, wherein the germ-free cell- cultured meat product is uncooked and / or not heat-treated.

13. The method according to any one of the preceding claims, wherein the harvested edible biomass is: extruded through a nozzle during the processing step; or3D printed during the processing step.

14. The method according to any one of the preceding claims, wherein a packaging material used to package the aseptic cell-cultured meat product is sterilized with heat, pressure, hydrogen peroxide, ionizing radiation, infrared radiation, ethylene oxide, or combinations thereof, prior to packaging.

15. A packaged cell-cultured meat product when processed using the method according to any one of the preceding claims.

16. A method of downstream processing for manufacturing aseptically -packaged germ- free cell-cultured meat, or aseptically -packaged germ-free food products containing such germ- free cell-cultured meat, the method comprising: aseptically harvesting an edible biomass of animal cells that have been cultured in a bioreactor under aseptic conditions; and processing and packaging the harvested edible biomass under aseptic conditions to form packaged germ- free cell-cultured meat, or germ-free food products that contain such germ-free cell-cultured meat.

17. The method according to claim 16, wherein the germ-free cell-cultured meat is uncooked and / or not heat-treated.

Images