Christian Harrison: Bacterial cellulose scaffolds derived from brewing waste for cultivated meat applications

Methods: The composition of BSY and its ability to support BC growth were assessed through metabolic analysis and growth trials. Properties relevant to CM applications of BSY-derived BC were then investigated. Scanning electron microscopy was used to quantify surface porosity. Mechanical properties were measured using texture profile analysis. Thermal and chemical properties were assessed using differential scanning calorimetry and Fourier-transform infrared spectroscopy, and biocompatibility was evaluated through cell attachment assays.

Results: BSY supported BC production, yielding material with structural, thermal, and textural properties comparable to BC grown on conventional media and similar to conventional meat products. BSY-derived BC also supported L929 fibroblasts, with 35.9% ± 2.5% cell attachment after 24 h and evidence of continued proliferation.

Discussion: These findings demonstrate that BSY can be effectively valorized to produce BC scaffolds for CM. This approach offers a cost-effective and sustainable strategy to improve the scalability of cultivated meat, contributing to future sustainable food production.

Introduction

Significant concerns have been raised over the negative externalities of current animal agriculture practices, particularly factory farming, for the environment, animal welfare and human health (1–3). Nonetheless, global demand for meat products is forecast to rise substantially in future decades (4). In developed countries meat consumption is mostly static, while in developing countries it is growing fast (5). A variety of solutions to meet global protein demand sustainably have been proposed, including those aiming for more sustainable agricultural practices (6). Among these are alternative proteins, of which there are several types (7). For some applications, alternative proteins aim to satisfy protein needs while reducing or replacing conventionally produced animal products in supply chains. For other applications, the aim is to offer multiple sources of proteins to consumers with no competition between them. One form of alternative proteins, plant-based meat analogs, have had some success but may have plateaued in popularity due to their high cost and inability to replicate the taste and texture of meat (8, 9). Another subset of alternative protein food products, cultivated meat (CM), seeks to produce meat products by culturing animal cells in controlled environments, without using any animal-derived ingredients. This approach may be better able to deliver products that meet consumer expectations. CM also has potential to be a more sustainable alternative to conventional meat production through lower land use, water consumption and greenhouse gas emissions, and potential for more ethical and efficient food production (9). However, these benefits are currently uncertain, due to the difficulty of conducting life cycle assessments at this early stage of development and will depend heavily on the exact materials and energy sources used for CM production (10). Additionally, a number of significant technical hurdles remain before CM can become a viable process capable of reaching a mass market.

If they are to displace conventional meat products, alternative proteins must be competitive on price, texture, taste, nutrition, and convenience as well as safe. These factors are crucial for any potential consumer and regulatory acceptance of CM products (11, 12). Achieving parity with conventional meat has been challenging and alternative proteins have so far failed to gain widespread acceptance (7). Part of the problem is the difficulty of replicating the complex hierarchical structure of animal tissues that produces the taste and texture of meat (13). This cannot be achieved using cells alone, which are conventionally grown in 2D formats and further constrained by using non-edible substrate materials. Edible tissue scaffolds could solve this by providing a 3D framework to support and guide cell growth and differentiation and contributing additional desirable organoleptic properties (13). To be suitable for mass production, scaffold materials must be cheap, biocompatible, and safe for consumption, ideally porous to facilitate cell ingression and nutrient perfusion, and malleable to allow production of varied and aesthetically pleasing products. A variety of materials have been investigated as potential scaffolds for tissue engineering related to healthcare applications, such as synthetic polymers and decellularized plant material (13). However, many of these scaffolds are too expensive for mass food production at a competitive price point, inappropriate for human consumption or insufficiently biodegradable for human digestion.

Bacterial cellulose (BC) is an abundant alternative material, a biopolymer of glucose synthesized by several bacterial species, such as Komagataeibacter xylinus, for purposes of mechanical, chemical and biological protection in their environment (14). BC consists of a polysaccharide of glucose units linked by β(1 → 4) bonds, with intrachain hydrogen bonds that add strength to the structure (15). It is chemically identical to plant cellulose, but lacks the lignin, hemicellulose and pectin found in plants that necessitate chemical purification for CM applications which adds expense and may not be food-safe (16). In static cultures, BC is produced in the form of a pellicle covering the surface of the growth medium, while in agitated cultures BC may form in balls or strands (14). Several properties make it well-suited as a scaffold material for cell attachment and growth, including its web-like network structure, tensile strength, and high water-holding capacity (17). BC is structurally similar to the extracellular matrix and if processed correctly, has high purity and low cytotoxicity (18, 19). Its relatively high porosity also enables ingress of cells into the material and facilitates diffusion of essential nutrients and waste products through the scaffold (18). Consequently, BC has been shown to be at least as biocompatible as other scaffold materials for various biomedical applications, as well as more affordable (20). Furthermore, BC pellicles can be formed in almost any desired shape (21), allowing it to masquerade as conventional meat products. Due to these properties, BC has been suggested as a scaffold material for CM (22). Studies have succeeded in culturing both human and animal myoblasts on BC, showing promise for CM applications, though further optimisation of cell attachment may be needed.

BC production requires culture medium containing a carbon source, such as glucose, a nitrogen source, such as peptone or yeast extract, and other critical components such as phosphorus and magnesium (23). Hestrin-Schramm (HS) medium is one of the most widely used formulations (24). 105 media is another similar formulation recommended for cultivation of BC producing bacteria such as K. xylinus (25). The cost of conventional culture medium such as HS may be a large component of total production costs and is one obstacle to potential use of BC in CM (26). However, BC can be produced using a wide range of alternative carbon and nitrogen sources. For example, the main commercial edible BC product currently is the dessert “nata de coco”, made using coconut water (27). Growth media derived from food or agricultural waste products are of particular interest; they are cheap, and using waste products avoids competition for inputs with other industries (28). If production methods using waste streams can be developed, these materials can be valorised, while also enabling BC to be made cheaply, at scale and more sustainably. No study thus far is known to have proposed using BC derived from industrial or agricultural wastes for CM production.

One such waste stream is brewing waste produced in the beer-making process. There are several types of brewing waste; brewers spent grain (BSG), trub, and brewers spent yeast (BSY) (29). All three have great potential as sources of carbohydrates, proteins and micronutrients for edible applications (30–32). Global beer production stands at ~1.9 billion hectolitres annually, and per hL of beer, around 20 kg of BSG and 0.3 kg of BSY are generated, resulting in large volumes of waste (33). Some brewing waste is repurposed as animal feed or as food additives to enhance taste or nutrition (29, 32). Despite this, brewing waste is underutilized and much of it is disposed in landfill, which can lead to water pollution or greenhouse gas release (34). Even if managed correctly disposal can impose significant costs on brewers (29). Incorporating brewing wastes into the CM supply chain would valorise this waste product, simultaneously reducing costs for brewers, and providing a sustainable feedstock for food production. Brewing wastes have already been trialed as a growth medium for BC-producing bacteria (35). We propose using brewing waste to grow BC, which can in turn act as a cellular scaffold for CM production. In this study we aim to demonstrate proof of concept for this production process.

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