Enhancing sustainable and safe protein production in Insects through Next Generation Sequencing (InsectNGS)

Enhancing sustainable and safe protein production in Insects through Next Generation Sequencing (InsectNGS)






Landbouw, Water, Voedsel>D. Gewaardeerd, gezond en veilig voedsel>D4. Duurzame en veilige verwerking






In de komende decennia zal de wereldbevolking sterk toenemen. Om al die mensen te kunnen voeden, is er een sterk stijgende vraag naar eiwitten. Bekende eiwitbronnen voor voeding van de mens zijn planten en dieren. Tegelijk met een toenemende vraag naar eiwitten is er een maatschappelijke discussie over de ongunstige CO2 footprint van de productie van hoogwaardige dierlijke eiwitten (vlees van pluimvee, varkens en runderen) en dierwelzijnsaspecten. Een alternatief zijn eiwitten afkomstig van insecten. Deze productie heeft een gunstiger CO2 footprint en er zijn geen dierwelzijn discussies zoals bij hogere organismen. Deze nieuwe voedselproductie systemen staan uiteraard voor nieuwe uitdagingen: naast consumentenacceptatie zijn er technische vragen. De basis van dit voorstel is de veronderstelling dat de verschillende soorten micro-organismen die zich ín en óp de larven bevinden kunnen worden aangetoond, zoals bijvoorbeeld ook wordt gedaan bij andere complexe systemen zoals micro-organismen in de darm van mens en dier. Met moderne DNA technieken (‘metagenomics’) kan de samenstelling van deze populatie (‘flora’) worden bepaald voor bacteriën, schimmels, virussen en parasieten. Hiermee kunnen in de productie in een vroeg stadium (early warning) ziekteverwekkers voor de mens (voedselveiligheid) als ook ziekteverwekkers voor de larven worden aangetoond. Daarnaast wordt in dit project nagegaan hoe de flora is samengesteld in batches larven die met een hoge efficiency worden geproduceerd en hoe de flora afwijkt bij batches met lage efficiency. Met het ontwikkelen van een systeem waarbij geregeld gemeten wordt (b.v. iedere week), kan de producent tijdig een signaal krijgen of er bijgestuurd moet worden in het productieproces. In dit project worden de ervaringen van een bedrijf dat insecteneiwitten voor humane voeding produceert, een bedrijf dat gegevensverzameling en verwerking automatiseert en structureert, en een bedrijf dat de data kan verwerken en daarmee voorspellingen kan doen, gecombineerd met de ervaring van de universiteit op het gebied van (snelle) voorspellende DNA-gebaseerde methoden. Het project maakt een innovatieve techniek toepasbaar in een vernieuwend, toekomstbestendig eiwit productieproces.

Doel van het project

With the increasing demand for high value proteins in the human diet, insect-derived proteins are a sustainable alternative for the traditional animal- and plant-derived proteins. Science-based optimisation of the breeding process of insects is needed to keep a competitive position for the Dutch industry in the rapidly developing insect protein industry. With new technological developments, preparedness for new food safety risks is of utmost importance to produce a safe product for humans, and to meet the consumers requests for a reliable and healthy product. The production of insects at the scale of Protifarm is completely new, and so are the safety aspects for as well the insects as humans around the production are a novelty.
In general, the knowledge of insect pathogens in the production of insects is limited and the effect of different pathogens is yet unknown. Protifarm has experience with the Gregarina parasite. The effect of an infection could be limited to a lower growth rate or no signs, but with a severe infected population the effect could be dramatic, such as lower survival rate, lower yield per crate and less pupation. It could be very useful to be able to monitor other insect pathogens like Bacillus thuringiensis, to prevent serious infections. Furthermore, the total microbiome can be monitored with metagenomics and composition can be associated with production features. There is no metagenomics data available of the microbiome of A. diaperinus. This will be first study to do so and will set the standard. Although not a lot of specific information is known, this project is considered highly feasible as the laboratory techniques are available.
Furthermore, it is preferable to maintain a production-system free of human pathogens, as in animal and plant production systems. The insect production systems are closed but introduction of pathogens through humans working in the production area is a possible food hazard. Although the insect proteins are processed in the post-harvest phase with methods that inactivate microbial contaminants, for product integrity reasons it is highly desirable to screen for human pathogens in production. As part of the quality system, a continuous monitoring of the presence of human pathogens is recommended. If contamination is detected, the production can be destroyed and a systematic evaluation of routes of introduction of the pathogens will take place with consequently targeted interventions (e.g. regarding biosecurity). The fact that insect protein production for human consumption is relatively new, the introduction of (new) types of pathogens is rather unpredictable and therefore an unbiased method, detecting the broadest scope of potential pathogens and deviations should be used. Metagenomics meets these criteria.
In the project, Next Generation Sequencing (NGS) of larvae (intestine) samples will be used. This approach circumvents the limitations of traditional culturing approaches. The huge increased throughput and reduced cost of genome sequencing has made it possible to sequence not just single genomes, but all the species in a sample. This approach is called metagenomics: all DNA in a sample is sequenced. DNA of a sample is directly investigated for the presence of potential pathogens, whether they are bacteria, fungi, viruses or parasites. Costs of sequencing, but also the time required for sequencing a sample however has limited the use of this approach for SMEs.
Recently, inexpensive single molecule sequencing devices have been developed and are now entering a stage of maturity. These smartphone-sized devices, developed by Oxford Nanopore Technologies, of which the MinION is the most well-known, require very little sample preparation, and produce long sequence reads, directly from isolated DNA, addressing limitations which have thus far hampered uptake of rapid NGS-based sample investigation. Using the Oxford Nanopore Technologies sequencing platform, we will investigate and describe the microbiome of A. diaperinus larvae with different aims: i) investigate which species are often found associated with successful harvested and efficiently produced larvae, ii) which pathogens (for larvae) are detected that may affect production negatively and iii), rapidly detect the presence of potential pathogens that may pose a problem for human health.
The project has three main objectives:
i) the identification of the microbiome-composition associated with efficient larvae production;
ii) the quantification of (known) larvae-pathogens
iii) developing a detection system for human pathogens in the larvae production.
Combining these 3 aims will lead to an integrated early warning system (EWS) that detects deviations of an optimal production system and the presence of potential human pathogens. This is input for corrective measures in the management of the larvae production, and facilitates a safe production of insect protein sources.

Relatie met missie (Motivatie)

The societal impact is that the project supports Protifarm in the development of a reliable and sustainable production of insects and that food safety is secured. The economic impact is by having an EWS, Protifarm is able to take corrective actions before the production collapses and/or food safety risks become manifest. The production of insects at the scale of Protifarm is completely new, therefore all the aspects around the production have not been explored. Very little is known about bacteria and other pathogens (fungi, parasites) in the production.
For Hellebrekers it is the first project concerning complex metagenomics data. This method of detecting environmental microbes is rapidly developing and already used in a wide range of fields, e.g. in other agricultural businesses, engineering, medicine, sustainability and ecology. Metagenomics concerns highly complex bioinformatics and data analysis. Knowledge and software which will be obtained and developed during this project can be used in a wide range of other projects.
We consider this project highly feasible as the laboratory techniques are available. The only potential “setback” would be that all the crates in all the phases of the cycle will have the same production results. The experience of Protifarm so far, however, is that clear variation in production results have been observed. It is crucial to plan the place and moments of sampling correct, so we could correlate the production data with the sampling data.

Geplande acties

The project is successful when at least 3 of the following 5 criteria are met. Firstly, the project is successful when the microbiome approach is able to detect human pathogens with high sensitivity in the production process. Secondly, the microbiome of Alphitobius diaperinus should be described from the metagenomics data and variations within production cycles should be stable enough to analyse deviations from normal microbiome compositions. Thirdly, when variation in technical production characteristics, such as feed, temperature, moisture can be associated with differences in microbiome composition. Fourthly, when the presence of the known parasite Gregarina spp can be quantitatively associated with the production efficiency, and when this can act as prediction (signal is prior to negative effect in production efficacy). This allows corrective actions in the management to prevent production losses. Finally, when potentially other specific pathogens can be identified associated with impaired production.