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Direct and indirect biorefinery technologies for conversion of organic side-streams into multiple marketable products

Periodic Reporting for period 2 - InDIRECT (Direct and indirect biorefinery technologies for conversion of organic side-streams into multiple marketable products)

Reporting period: 2018-05-01 to 2019-10-31

Diverse side-streams are generated at the farmers and post-harvesting level as well as in the associated retail sector. A part of these biomass streams are not exploited or under-exploited. The Indirect project targets biorefinery of under-spent side-streams as feedstock, aiming at ‘closing loops’ and ‘producing more with less’. Feedstocks considered are under-spent side streams/residues originating from the agro-sector (like biomass left on the field or removed after harvesting), processing sector (plant part, biomass that did not pass the quality control, press cakes, etc.) as well as from the retail sector (expired biomass, inferior quality products, etc.). Valuable compounds can be recycled from this biomass via different biorefinery approaches.
• Direct biorefinery of plant biomass is a biomass specific approach with adapted biorefinery procedures for each biomass type. As such a direct approach is challenging to scale for small biomass streams and is expected to be seasonally dependent and not operational all year. On the other hand, it is an approach that can generate a large variety of potentially high value compounds when applied in a cascading mode (proteins, carbohydrates, fibers, and multiple minor compounds like carotenoids, polyphenols).
• Indirect biorefinery converts side-streams into crude extracts via a two-step process consisting of (1) conversion of biomass by insects and subsequent (2) biorefinery of the insect biomass. The aim of the first step is to convert heterogenic feedstock into a homogenous biomass via insects. Insects are able to convert a variety of feedstock into a more homogenous biomass, being their own biomass. The indirect approach was shown to have potential for a year-round relatively stable production of mainly chitin, proteins, lipids and N-light compost (frass).
Can side-streams be used as feed ingredients for insect rearing?
- Plant based side-streams that are feed-grade and are free of animal by-products and contaminants (like agrochemicals, plastic waste) can be used as feed to rear insects within the current legal framework. Within InDIRECT different types of side-streams from the agri-food sector were evaluated as feed ingredient for Black soldier fly larvae and the Lesser mealworm, comprising green leaves, fruits, vegetables, grain derivatives and press cakes.
- Insect rearing trials within InDIRECT revealed that growth on multiple side-streams is possible, where side-stream mixtures perform generally better than single side-streams. The larvae are able to concentrate the proteins and lipids, and as such to upgrade these compounds.
- With respect to the impact of varying feed ingredients (side-streams), only a slight effect on the larval nutrient concentrations was observed within the tests performed. Cost estimations (based on lesser mealworm) indicated that side-stream inclusions in feed can be economically favorable in terms of cost per fresh larvae reared as well as cost per dry amount of protein.
- In conclusion, plant-based side-streams do have potential for insect rearing.

Do insect-based products have potential as marketable compounds?
- Within InDIRECT different cascading biorefinery approaches were elaborated and evaluated on lab and pilot scale. A first approach was applied on wet larvae (no drying process required) and included a mechanical separation step to dechitinize the majority of the insect biomass. This biomass was further fractionated into a lipid enriched and protein enriched fraction. The proteins in the chitin rich fraction that was generated, were recovered and the chitin was purified and converted to chitosan and chitosan (oligomers). Via another fractionation approach, involving enzymes, peptides was generated.
As such, insect-based products rich in proteins, lipids and chitin were produced with potential interesting properties towards feed and food application, as well as for other applications like chemicals, textiles and cosmetics. Based on feed/food related in vitro tests, interesting bioactive properties were identified.

What about safety issues of insect-derived food and feed ingredients?
- The potential risks related to the use of insects as food and feed has already been reviewed by EFSA in 2015 (“Risk profile related to production and consumption of insects as food and feed”, EFSA at that time already concluded that microbiological risks are expected to be comparable with other food or feed ingredients, if insects are fed with permitted feed materials. The documents concluded that, due to the lack of data, also further studies were needed about the fate of the chemical contaminants and allergen in insect-derived processed products (such as protein fractions or oils). Within InDIRECT, the presence of mycotoxins, agrochemicals, heavy metals, allergens and pathogens were considered, both on full insects and on ingredients derived from them.
- Mycotoxins and agrochemicals (and potentially also heavy metals), are transferred from the substrates used to feed insects to the insects themselves, but no indications have been found from the collected data suggesting that insect concentrate obnoxious compounds inside their body. Rather, the concentration has always been found lower in insects than in the feed substrates, when using naturally contaminated (into the legal limits) substrates.
- Pathogens can be a risk if the rearing conditions are not carefully checked, yielding microbiologically contaminated insects. This finding confirms the ruling out, as feeding substrates for insects meant for food and feed applications, naturally microbiologically contaminated materials, such as for example manure.
- Allergen risk has been confirmed to be present in insects and insect-derived preparation, both indirectly (proteins with high similarity with known allergens) and directly (IgG reactivity with tropomyosin 1 polyclonal antibody isolated from rabbit and reactive against mammals and IgE reactivity of insect preparations with sera of patients allergic to crustaceans)

For the direct biorefinery (WP2), three leaves (leek, sugar beet leaves & alfalfa) were considered for isolation of proteins and minor compounds. Four approaches for the wet fractionation process to extract the protein fraction from the reference material alfalfa were evaluated in order 1) to get mass and nitrogen balances all along the process and 2) to monitor the fate of the polyphenols. Two fractionation approaches were studied in detail for leek and sugar beet leaves.Protein extraction yields were generally below 15 % (on dry matter basis), while the purities varied between 37 % and 86 %. It was shown that polyphenols were distributed over all fractions and as such not separated from the proteins. Ensiling was shown to be a suitable process for preserving green leaves, although conversion processed do occur during ensiling. Because of the low yields, protein extraction from green leaves is estimated to be only economic feasible for very high end product.
The InDIRECT project aimed to contribute to lower barriers towards implementation of insect-based value chains in Europe. The 19 recommendations formulated are available at