Insect biorefinery for generating high-added-value products such as protein and chitin

Abstract

Over the last decade, insects have been introduced to the market as new livestock and since they are rich in protein and lipids, they can potentially decrease future food and feed shortages. In addition, insects can be reared on a large range of organic material, including underspent waste streams. This way, they can be employed to recycle side-stream nutrients back into the market as insect biomass. Academic research is growing exponentially, especially on larvae of the black soldier fly (for feed applications) and the yellow mealworm (for food applications), and an increasing amount of commercial insect rearing facilities are being established in Europa. Next to the yellow mealworm, another member of the Tenebrionidae family, the lesser mealworm is also commercially produced for human consumption but published data on this insect is scarce. Even though the insects' nutritional value is ideal for feed/food applications, the composition of the insect biomass is not necessarily so and fractions may be useful to tailor the composition of a diet/food product. In addition, studies have pointed out that insect-based food products are more likely to be accepted if the insect is unrecognizable and incorporated into a familiar food product. For these reasons, the fractionation, characterization, and valorization of insect fractions are gaining more attention. However, published data on insect-based fractionation processes are not covering all aspects, for instance (1) the focus is on extracting one compound, neglecting the impact of the extraction method on other compounds (proteins, lipids or chitin), (2) data is often limited to analytical fractionation and (3) it is scattered over different insect species. Next to a nutritional value, insect fractions can also be employed for other purposes. A protein fraction, for example, can have techno-functional properties that could be an added value to a food product. This aspect is still an unstudied domain for insect proteins, especially for the lesser mealworm. Moreover, insects also contain chitin which is a valuable biopolymer that can be applied in many applications and the market is expected to grow rapidly in the near future.

The general aim of the thesis is to increase (academic) knowledge on different aspects of the insect-based value chain with a special focus on scalability for easy transition of lab-scale findings to the industry. Two insect species were studied i.e the larvae of the black soldier fly and the lesser mealworm. The main focus was put on engineering a biorefinery approach with a focus on the recovery of multiple compounds (chitin, proteins, and lipids) that could be used as raw materials for feed, food, and technical applications. This approach started with a mechanical treatment to obtain a dechitinized fraction and a chitin-rich fraction. Next, the two subfractions were further fractionated in protein, lipid, and chitin enriched samples. The characteristics and possible functionalities of these fractions were evaluated in the study.

Firstly, reported data on larvae of the black soldier fly reared on side-streams have indicated that the composition of the larvae can change depending on the diet composition. These findings were cross-checked for the lesser mealworm. The mealworms were reared on 18 different diets composed of side-streams to (1) determine the nutritional composition of the larvae and (2) study the effect of dietary changes on the larval nutrient composition. The lesser mealworm proved to be of good nutritional value with essential amino acid profiles comparable with that of beef. The side-stream based diets varied on dry matter basis in protein (16 % to 34 %) and lipid content (2 % to 19 %). The nutrient content of the larvae reared on diets that supported good growth ranged between 35 % and 45 % of protein, 22 % and 26 % of lipid and 4 % to 6 % of chitin on a dry matter basis. No significant correlations were identified between the larval protein or lipid content and that of the diet, but it was found between the diet nutrients and larval growth. Based on larval growth data and economic considerations, diets composed of wheat middlings with a 10 % to 15 % inclusion of rapeseed meal were identified as suitable feed for LM.

Secondly, a fractionation approach was studied for dechitinized insect biomass of the larvae of the black soldier fly. The study aimed at the development of a robust fractionation process for wet insect biomass, targeting enriched protein fractions as well as enriched lipid fractions. The added value of different organic acids for an acid isoelectric point precipitation (AIPP) was evaluated and compared with an inorganic acid (HCl). A beneficial effect of organic acids on the lipid extraction yield (shift from 35 % to 45 %) was observed, which did not negatively influence the protein extraction efficiency. One organic acid, in particular, lactic acid, increased lipid purity from 75 % to 85 %. Protein fractions with a purity of 60 % proteins were achieved. In conclusion, the use of some organic acids at low pH (pH 2) resulted in (1) a higher lipid purity in the lipid-enriched fraction and (2) had similar results in protein fractionation as the inorganic acid.

This new fractionation approach was compared to different traditional defatting methods. Solvent-based extraction, supercritical CO2 extraction (SFE) and heat treatment, were applied on dechitinized lesser mealworm biomass and compared based on extraction yield, purity, and impact on protein solubility. AIPP was most successful in generating a protein-enriched fraction (71 % protein), followed by SFE (67 %), heat extraction (62 %) and solvent extraction (60 %). However, a lower yield and protein recovery were observed for AIPP and heat treatment. All techniques decreased total protein solubility. The most soluble protein-enriched fraction was SFE defatted meal (100 % soluble), then the supernatant of the heat treatment (89 %), followed by a hexane-defatted meal (73 %) and the AIPP generated fraction (30 %). SFE and AIPP treatments were able to recover most of the lipid (85 %), followed by the hexane extraction (61 %) and heat treatment (59 %). In conclusion, SFE is suggested as the best method for obtaining a high degree of defatting as well as good preservation of the protein solubility, whereas the AIPP treatment is more promising considering high protein content fractions and easy upscaling.

A new fractionation approach was also developed for the chitin-rich black soldier fly biomass for the extraction of chitin and its further conversion into high molecular weight chitosan. The aim was (1) to tailor the traditional methods (based on crustacea) towards insect chitin, (2) decrease the amount of process waste which would be beneficial towards upscaling, (3) to recuperate proteins and (4) to produce chitosan with different degrees of deacetylation (DDA). The result proved that a process with less chemical and/or milder process parameters was feasible for larvae of the black soldier fly and that pure chitin and chitosan fractions could be achieved with a DDA ranging between 30 % to 90 %. In addition, the alternative method allowed the recovery of proteins.

The effect of these fractionation approaches on the techno-functional properties of the lesser mealworm proteins was studied. Techno-functional properties of the native proteins (before fractionation) were measured and compared to the properties of the protein-enriched fraction generated by the AIPP approach, an SFE, and heat treatment. The study confirmed that proteins derived from the lesser mealworm contained techno-functional properties and the native proteins had a good solubility and gelling ability which illustrates the potential of lesser mealworm proteins as a functional ingredient in food/feed applications. In addition, the oil binding capacity was higher compared to reference proteins caseinate, egg white and albumin. Depending on the fractionation approach, the techno-functional properties were either increased or decreased. The gelling ability and oil binding capacity were increased by SFE and the defatted meal from the SFE was able to form a foam. The AIPP treatment decreased most TFP expect for the WBC and OBC which were increased whereas the heat treatment resulted in a protein fraction that had lost all its techno-functional properties. These findings are a first step towards tailoring the techno-functional properties of proteins to specific product-based needs.

Lastly, chitin samples were collected along different stages the lifecycle of the black soldier fly (larvae, prepupae, pupae, flies, shedding & cocoons) and examined for differences. The chitin content in the collected biomass ranged between 8 % and 24 %, with sheddings and cocoons being most rich in chitin. Purified chitin was subjected to a physicochemical evaluation based on FTIR, XRD, and TGA as well as a deacetylation step. The data indicated that BSF chitin was α-chitin with FTIR profiles matching closely to shrimp chitin and showing some differences compared to squid pen chitin (ß-chitin). Small physicochemical differences were observed among the different BSF samples. Prepupae and cocoon chitin was more crystalline while chitin from larvae and sheddings had a lower thermal degradation temperature. In addition, sheddings were more difficult to purify. Further processing to chitosan showed that a deacetylation degree of 89 % could be obtained for all samples after 3 hours, although prepupae and pupae chitin was found to be more reactive in the deacetylation process. Overall, the small differences in physicochemical properties that were detected between the BSF chitin samples did not prevent further processing of chitin to chitosan with the same degree of deacetylation via the same treatment.

In general, within this thesis, data were generated that contribute to close the knowledge gap related to the insect-based value chain. Data indicated that the lesser mealworm could be reared on side-streams without changing the larval composition and thus alterations the side-stream based diet throughout the year (for example due to seasonal availability) are possible. Furthermore, the thesis illustrates a new, two-step insect fractionation approach where 76 % of all proteins, 73 % all lipids and, 75 % of the BSF chitin could be recovered. Lastly, the thesis describes the techno-functional properties of the lesser mealworm and points out that different chitin containing waste-streams originating from the black soldier fly rearing industry could be processed to a homogenous chitosan product and valorized.