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Microbial Production of Casein

The MiProCas project has come to an end. A testimonial on the outcome of this project can be found BELOW or on our Testimonials and Collaboration Results Webpage. Scroll down to the section “Bridging the gap for start-ups and SMEs” and unfold “Those Vegan Cowboys (Ghent, BE): Scale-up of casein production for application testing”.  

Consumer research shows that current plant-based cheese products do not meet expectations in terms of taste, texture and mouthfeel. The lack therein of caseins, by far the most important milk protein and accounting for 80% of all protein in cow’s milk, is at the root of this problem. Caseins give the so-familiar cheese products their unique and indispensable properties. Founded in 2020, Those Vegan Cowboys – NewMilkLab is capitalizing on this unique opportunity. Casein proteins with exactly the same functional properties can be produced through fermentation, without the need for dairy cattle, and thus with positive consequences for the ecological and animal welfare impact of casein and dairy production. In this way, this project contributes to several pillars of the Flemish Protein Strategy: on the one hand, more product diversity will be created (more animal-free cheese alternatives) and on the other hand, this will lead to a sustainable protein consumption because animal caseins will be replaced by microbial proteins.

The MiProCas project aims to further fine-tune and scale up the fermentative production of one casein variant with the help of the Bio Base Europe Pilot Plant. The purification of the protein will also be scaled up at BBEPP in order to obtain it in a sufficient quantity and with sufficient purity (food grade) for formulation into animal-free cheese alternatives.

Results of the MiProCas project by December 2024

Within the MiProCas project, Those Vegan Cowboys – NewMilkLab screened several hosts/expression systems to produce different caseins. The strain showing the highest casein expression was selected and its performance validated through 1 L and 15 L cultivation. A fed-batch production process was optimized in terms of medium composition, temperature and feed profiles at lab scale and validated at 300 L pilot scale, allowing to develop and optimize a unique microbial casein downstream processing protocol at lab scale.

Both the fermentation process and downstream processing protocols were transferred to BBEPP and implemented at 150 L pilot scale for further optimization and comparison of different DSP techniques. The finetuned process was smoothly and successfully scaled to 1500 L fermentation scale. The titer was increased with 25% when moving from 150 L to 1500 L scale, while all runs showed reproducible growth (OD600, offgas, base consumption) and productivities (titer, yield on biomass and yield on carbon source). This allowed to produce the first kilograms of pure casein after downstream processing. Both fermentation and DSP were then evaluated by BBEPP’s quality department via a HACCP study, after which the entire process was performed in a food grade way at 1500 L scale. After spray drying, tens of kgs of pure, food grade microbial casein were obtained for application development in both ripened and unripened cheeses. Important learnings on how to improve the process further, were shared with NML.

Will van den Tweel (COO/CTO -Those Vegan Cowboys): Our collaboration with Bio Base Europe Pilot Plant (BBEPP) has been nothing short of transformative. From the outset, their team demonstrated expertise, professionalism, and an unwavering commitment to innovation. The state-of-the-art facilities and pilot-scale capabilities at BBEPP provided us with the ideal environment to scale up our biotechnological processes seamlessly.

Their hands-on approach and deep understanding of bioprocess development ensured that every stage of the project was executed efficiently, delivering results that met and exceeded our expectations. Beyond technical proficiency, BBEPP’s collaborative spirit and dedication to fostering sustainable solutions made them an invaluable partner in achieving our goals.

We highly recommend Bio Base Europe Pilot Plant to anyone seeking a reliable and forward-thinking partner in biotechnology and process development. It was a pleasure working with them, and we look forward to future collaborations.

Using the obtained casein powders, Those Vegan Cowboys tested the functionality of the casein in microscaled cheese models, as well as the possibility to transform the batches into applications similar to ripened dairy cheeses. 15 casein batches were compared for their emulsification, acidification, cheese consistency, melting and stretching capabilities. This evaluation revealed that clear progress was made in increasing functionality, and important conclusions were made linking the casein batch characteristics vs. functional properties. The casein performed extremely well in an unripened mozzarella-like food system: it reached over 1.7 meter stretch and showed a similar melted flowability as dairy Mozzarella. In parallel, food grade casein batches were used to optimize the production process of ripened cheeses like Gouda or Feta, in collaboration with an internal panel group. Successful prototypes were obtained.

To conclude, within the MiProCas project, casein proteins with exactly the same functional properties could be produced and scaled up, without the involvement of dairy cattle, and thus with positive consequences for the ecological and animal welfare impact of casein and dairy production. In this way, this project contributes to pillars 4, 5 and 6 of the Flemish Protein Strategy: on the one hand, there will be more product diversity (more animal-free cheese alternatives) and on the other hand, this will lead to sustainable protein consumption because animal casein will be replaced by alternative, fermentatively produced proteins. MiProCas focused on one microbial casein and its scale-up to TRL6, and application in cheese prototypes, as a preparation for further commercialization. >10 kg of casein were produced by fermentation, and unripened and ripened cheese prototypes were made. The protein and its production process through fermentation is highly innovative and hence requires regulatory approval through EFSA Novel Food before a potential launch on the EU market. Approval is foreseen for 2028. Commercialization is foreseen for 2029.

     

ACTIVITIES of the Bio Base Europe Pilot Plant

  • Project coordination 
  • Scale-up of casein fermentation process to 1500 L scale
  • Optimization and scale-up of downstream process for casein purification and delivery of food grade casein for formulation into dairy-free cheese prototypes
  • Communication & Dissemination of project results

PARTNERS

SUPPORTED by

Vlaams Departement Landbouw & Visserij – Projecten Relance 2021 – Realisatie Eiwitstrategie

The results of this project will be available after May 31, 2024 through this website. The results will be freely available to all chain actors involved in the production, processing and consumption of microbial protein, and alternative proteins in general, in food and feed products. The results will remain freely available on the internet for at least five years after the project closure.

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Microbiële productie van caseïne

Uit consumentenonderzoek blijkt dat de huidige plantaardige kaasproducten qua smaak, textuur en mondgevoel niet aan de verwachtingen voldoen. Het ontbreken van caseïne, veruit het belangrijkste melkeiwit en goed voor 80% van alle eiwitten in koemelk, ligt aan de basis van dit probleem. Caseïnes geven de zo vertrouwde kaasproducten hun unieke en onmisbare eigenschappen. Those Vegan Cowboys – NewMilkLab, opgericht in 2020, speelt in op deze unieke kans. Caseïne-eiwitten met exact dezelfde functionele eigenschappen kunnen via fermentatie worden geproduceerd, zonder dat er melkvee nodig is, en dus met positieve gevolgen voor de ecologische en dierenwelzijnsimpact van de caseïne- en zuivelproductie. Op die manier draagt dit project bij tot verschillende pijlers van de Vlaamse Eiwitstrategie: enerzijds wordt er meer productdiversiteit gecreëerd (meer dierproefvrije kaasalternatieven) en anderzijds leidt dit tot een duurzame eiwitconsumptie omdat dierlijke caseïnes vervangen worden door microbiële eiwitten.

Het MiProCas project beoogt de fermentatieve productie van één caseïnevariant verder te verfijnen en op te schalen met hulp van de Bio Base Europe Pilot Plant. De zuivering van het eiwit zal ook worden opgeschaald bij BBEPP om het in voldoende hoeveelheid en met voldoende zuiverheid (voedselkwaliteit) te verkrijgen voor de formulering in dierproefvrije kaasalternatieven.

ACTIVITEITEN van de Bio Base Europe Europe Pilot Plant

  • Coördinatie van het project
  • Opschaling van het caseïnefermentatieproces tot 1500 L schaal
  • Optimalisatie en opschaling van het downstream proces voor caseïnezuivering en gebruik van caseïne van voedselkwaliteit voor de formulering van prototypes van zuivelvrije kaas.
  • Communicatie en verspreiding van projectresultaten

PARTNERS

GESTEUND door

Vlaams Departement Landbouw & Visserij – Projecten Relance 2021 – Realisatie Eiwitstrategie

De resultaten van dit project zullen na 31 mei 2024 beschikbaar gesteld worden via deze website. De resultaten zullen vrij beschikbaar zijn voor alle ketenpartijen die betrokken zijn bij de productie, verwerking en consumptie van microbieel eiwit, en alternatieve eiwitten in het algemeen, in voedingsmiddelen en diervoeders. De resultaten zullen gedurende ten minste vijf jaar na de afsluiting van het project vrij beschikbaar blijven op het internet.


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