Preservation of microorganisms by understanding the protective mechanisms of oligosaccharides

Project Information

PREMIUM

Grant agreement ID: 777657

DOI

10.3030/777657

Project closed

EC signature date 21 November 2017

Start date 1 January 2018

End date 30 June 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 634 500,00

EU contribution

€ 634 500,00

Coordinated by

INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULTURE, L'ALIMENTATION ET L'ENVIRONNEMENT
France

Periodic Reporting for period 2 - PREMIUM (Preservation of microorganisms by understanding the protective mechanisms of oligosaccharides)

Reporting period: 2020-01-01 to 2023-06-30

Summary of the context and overall objectives of the project

Lactic acid bacteria (LAB) are a family of micro-organisms widely used for producing fermented foods. The market of concentrated cultures (starters) is continuously growing due to the development of novel health products (probiotics) and the LAB’s ability to use plant proteins as fermentation substrate and convert by-products for green chemistry applications. The manufacturing of starters requires successive operations that generate stress, potential cellular damage, and loss of functionality, especially following the stabilisation processes: freezing, freeze-drying, or spray drying. Furthermore, the involved operations are usually energy-intensive and result in some negative impacts on the environment.
The aim of PREMIUM was to completely revisit LAB preservation processes to offer innovative alternatives benefitting companies and Society by considering three main dimensions: product quality, process efficiency and environmental impact.
PREMIUM activities targeted the understanding of LAB preservation mechanisms by oligosaccharides as a promising way for proposing different and more sustainable stabilisation processes relevant to industry and, more generally, for biodiversity preservation.
During the project we improved existing methods for producing oligosaccharides and stabilised bacteria. We also developed new ones, such as hydrolysis processes for producing oligosaccharides and layer-by-layer encapsulation for protecting LAB. We also progressed in understanding the mechanisms of protection/degradation of bacteria and in the transfer of knowledge and methods to improve the preservation of other cells (namely, mammalian cells). Moreover, we acquired better knowledge of the effect of protective molecules and the production process (particularly on stabilisation steps) on the environmental impacts, and we were able to propose some guidelines for reducing impacts.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The scientific objectives of PREMIUM were fulfilled thanks to secondments (141 PM in 5.5 years) and work performed outside secondments. More than 30 samples of oligosaccharides of original composition were produced either by synthesis from sugars (sucrose and lactose) or by hydrolysis of polysaccharides from natural substrates (yacon, chickpeas, lentils). Original protocols were setup to obtain oligosaccharides from wastewater of food industry. The OS were screened according to their physical properties, ability to protect LAB during freezing and freeze-drying, and the environmental impact of their production process. Two PREMIUM’s oligosaccharides and one commercial sample were selected to perform experiments at a pilot scale, which were necessary to conduct life cycle analysis and multicriteria analysis of the LAB production system. 18 scenarios were compared, including 3 processes (freezing, freeze-drying, spray-drying), 3 oligosaccharides' mixtures and 2 LAB. Relevant indicators and methods for evaluating the stabilization alternatives and identifying the most promising strategies for the eco-friendly preservation of LAB were defined at pilot and industrial scales.
An innovative process based on layer-by-layer encapsulation was developed to improve bacterial recovery after drying. This process was setup at the laboratory scale and then optimised to be scale-up at the pilot scale.
Vibrational spectroscopic techniques were developed and applied during the project. They were used as high throughput methods to determine the oligosaccharides compositions, their ability to protect LAB during the process, and to identify the main cellular damages induced by freeze-drying, spray-drying, and storage. Cellular markers of degradation, such as nucleic acids, cell proteins and cell walls, were identified. Microscopy was applied to obtain super resolution imaging of single LAB cells. Furthermore, experimental approaches and molecular dynamics simulations were developed to investigate potential interactions between oligosaccharides and LAB membranes. The influence of the physical properties, such as the glass transition temperature, on the long-term storage stability of dehydrated LAB, was also investigated.
Methods and knowledge acquired during the project were transferred to study other LAB of commercial interest (probiotics) and other cellular models, such as mammalian cells.
Information, experience, and skills were exchanged among all partners, both internationally and across sectors (47 secondments), thus expanding and enriching knowledge on the different topics and methods covered by PREMIUM. This was possible through 85 secondments and 141 PM, 4 face-to-face meetings, supervision of PhD students (10), and collaborative dissemination of results: 21 articles, 4 book chapters, 35 conferences participations and 4 invited lectures.
PREMIUM developed a visual identity and targeted actions to communicate and create awareness on PREMIUM’s activities and results (logo, flyer, website, presence on social media (Facebook, Twitter, Instagram), YouTube channel, 4 external newsletters). PREMIUM organised: 4 workshops, 14 training sessions and 4 winter schools involving around 450 people.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Progress beyond state of the art and results at the end the project:
-improvement of knowledge of cell preservation mechanisms (bacteria, mammalian cells)
-expansion of protective molecules sources
-development of high-throughput tools for the characterisation and screening of protective molecules
-development of novel preservation processes and environmental impact evaluation of the whole micro-organism production system, elaboration of a robust methodology for comparing stabilisation alternatives, from laboratory to industrial scale
-increase knowledge on the environmental impacts of the production of lactic acid bacteria
- introduces new challenges and perspectives to this field of study-development of processes to add value to co-products from the pulses' production

Potential impacts:
-improving the stability of dehydrated bacteria by defining adequate choice of protective formulation, process and storage conditions
-increase availability and exploitation of promising bacteria for Society (increased diversity and nutritional value of fermented foods and health products, improved preservation of raw food and reduced food spoilage, green chemistry application, biodiversity preservation)
-molecular modelling and simulation strategies could be increasingly applied in the study of functional foods
-add value to co-products (legumes wastewaters) and contribute to a circular economy
-future commercial exploitation of obtained results (new mixtures of oligosaccharides as protective compounds, better LAB stabilisation conditions, innovative analytical tools)
-raise the awareness among starters producers, stakeholders, and Society at large to include sustainability approaches in the design and/or improvement of process lines. LCA appeared as an interesting tool to inform about the impact of industrial production processes and could be a key for decision-making on process development.
-improve the public information and education on topics related to PREMIUM scientific domains (health and biopreservation benefits of LAB, sustainability, biodiversity preservation)
- enhance skills of partners and researcher interaction with industry
- boost career prospects of researchers, particularly of early-stage-stage researchers, through knowledge sharing and networking during inter-sectoral and international mobility.

PREMIUM project