Optimization of Subcritical Water Hydrolysis of Microalgae Biomass for Clean Meat Production

Team: Marshall Borrows, Lukas Keller, Bradley Lawson, Sam Perkins, and Emery Wheeler

Sponsor: Upside Foods Media

Abstract

Subcritical water hydrolysis (SWH) is a developing method of processing organic materials/waste without the use of acids and alkali materials. SWH lyses peptides differently than acid hydrolysis and can produce Maillard products, which have various effects in cell culture. In this study, a batch-mode pressure vessel with a semi-continuous sampling apparatus will be used to optimize temperature and retention time for the hydrolysis of Chlorella vulgaris and Arthrospira platensis biomass for the development of serum-free (or low-serum) cell culture media. Hydrolysis will be done at temperatures from 140°C to 300°C and retention times from 5 minutes to 30 minutes. The SWH products will be assessed for their general protein content, lipid content, bioactivity, and carbohydrate content. These efforts will be of use to Upside Foods in its efforts to create sustainable and ethical cultured meat products.

Introduction

  • Cultured meat is the in vitro cultivation of animal skeletal muscle and adipose tissue
  • Growth of animal tissue typically requires basal media supplemented with animal serum or recombinant factors [1]
  • Media is a major cost in meat culture, and animal serum constitutes the majority of media cost [2]
  • Hydrolysis products from plants (e.g. Algae) can contain nutrients present in basal media, as well as Maillard products [3]
  • Products from SWH on Chlorella vulgaris and Arthrospira platensis will be tested for use in cell culture
Figure demonstrating the chemical process of breaking down peptides during SWH [4]

Figure 1: Figure demonstrating the chemical process of breaking down peptides during SWH [4]

Methods

Figure 2: Process flow diagram showing project progression from algae to use in meat culture (made with biorender.com). Contains the following text: "Generally Recognized as Safe (GRAS) algal and cyanobacteria species. -> Subcritical water hydrolysis performed in a stirred pressure vessel. Optimization testing performed at 140-300°C and retention time from 5-30 min. -> Characterization of hydrolysis product for protein, amino acid, and Maillard product content -> Substitution of Fetal Bovine Serum and media with hydrolysis products for meat culture -> Chew test of cultured product and transfer to Upside Foods"

Figure 2: Process flow diagram showing project progression from algae to use in meat culture (made with biorender.com)

  • A Bradford assay, HPLC, SPV assay, and a DNS assay will be used to characterize products of SWH
  • Optimal SWH temperature, retention time, and pH will be determined through analysis of products
  • Chicken myoblasts will be cultured using SWH products as supplements to basal media and fetal bovine serum

Results

Sample Protein Content (ug/mL) Time (min), Temp (°C), pH
Run 1 7427.11 17.5, 140, 6.0
Run 3 11147.26 17.5, 220, 7.0
Run 5 4634.67 17.5, 300, 6.0
Run 7 12214.14 24.93, 172, 6.6
Run 8 19561.68 17.5, 220, 6.0

Figure 3: A table comparing the effects of different parameters on the protein content of our hydrolysate products

  • Optimal Temperature for protein production is 220°C
  • Hydrolysates at a pH of 6 produce the highest yields of proteins

Future Work

  • Conduct more runs on the stirred pressure vessel to build a larger basis for the characterization of SWH products
  • Test the effects of pretreatment on the algae and how this impacts the formation of proteins
  • Determine if different additives can reduce the formation of Maillard products
  • Measure the effectiveness of our hydrolysis products on the culturing of chicken cell
  • Create a cell culture media for the use in a bioreactor for the culturing of suspended chicken cells

References

  1. Lee, DY,. et al. Review of the Current Research on Fetal Bovine Serum and the Development of Cultured Meat. 2022.
  2. Specht, L. An Analysis of Culture Medium Costs and Production Volumes for Cell-Based Meat. 2020.
  3. Soto-Sierra, L., et al. Process development of enzymatically-generated algal protein hydrolysates for specialty food applications. 2021.
  4. Vela, C, et al. Protein Hydrolysis by Subcritical Water: A New Perspective on Obtaining Bioactive Peptides. 2021