Esquemas de Control Neuro Difuso Basados en Modelación

Research on alternative proteins sources usually focussed on the nutritional value of the proteins (Kinsella & Melachouris 1976). However, the application of insects as alternative protein source depends not only on the nutritional value of the proteins, but also on their functional properties. Endogenous enzymes are present in insects and can be active upon extraction, potentially hampering protein functionality. We showed that endogenous phenoloxidases are important for browning upon grinding (Chapter 3) and endogenous proteases remained active after protein extraction (Chapter 5). Thus, the implications of endogenous enzymes are important to consider, and the effect on functional properties will be discussed. Besides endogenous enzymes, the pH is known to largely affect protein properties, like solubility, and is therefore also taken into account.

6.2.1. Solubility of insect proteins

Solubility is an important aspect of proteins as it is necessary for many techno-functional properties, like foaming, emulsification and gelation (Boland et al. 2013; Kinsella & Melachouris 1976). The pH is known to have a large effect on protein solubility (Aalbersberg et al. 2003). The zeta potential was determined to predict the effect of pH on protein solubility as shown in Figure 6.1A. From this graph, the apparent pI of all proteins together was determined at zero net charge. The zeta potential for A. diaperinus and H. illucens

General discussion

107 reached zero charge around pH 4.9, whereas for T. molitor the zeta potential was zero around pH 4. Similar results were found for dried defatted T. molitor by Sipponen et al. (2017) , whereas a zero zeta potential around pH 3.5 for soluble T. molitor protein was found by Azagoh et al. (2016).

Figure 6.1. A: Effect pH on zeta potential (ζ) of Tenebrio molitor (triangle), Alphitobius diaperinus (square) and Hermetia illucens (circle). The point where the line crosses the x-axis represents the apparent pI of the protein solution (prepared in MilliQ water). B: Effect pH on solubility of proteins. The highest solubility was considered 100% of the respective species. The solubility test was based on Schwenzfeier et al. (2011). Error bars represent standard deviation (n=3).

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Zeta-potential values (either positive or negative) at the extremities of the curve indicate in general more electrostatic repulsion, thereby increasing thereby the solubility of the proteins. On the other hand, the solubility of proteins around the apparent pI was the lowest as little repulsion occurs. This is in agreement with the solubility curve shown in

Figure 6.1B. Minimal solubility of the proteins was found between pH 4 and 5. Highest

solubility was found at lower and higher pH.

Phenoloxidases did not significantly change the solubility of the extracts (Chapter 5) at pH 6, where the highest enzyme activity was found (Chapter 3). Higher solubility was found at increasing pH values, which supports the use of alkaline pH during extraction and/ or processing. On the other hand, also endogenous proteases are active at high pH (Chapter 5). Hydrolysis of proteins can change the solubility. The solubility of soy proteins was for example enhanced after hydrolysis (Tsumura et al. 2005).

So, the highest solubility as desired for techno-functional properties can be achieved at alkaline conditions, whereas phenoloxidase or the origin of the proteins did not affect this property (Chapter 5). Proteases have an effect at alkaline pH, but whether this is positive or negative should be studied.

6.2.2. Gelation

Besides solubility, gelation is an important protein functionality that plays a role in many dairy products like yoghurt, as well as eggs, sausages and tofu. For feed, gelation is for example important in wet pet food. Preliminary experiments investigating gelation characteristics of proteins from T. molitor, A. diaperinus and H. illucens at different pH values showed that gelation ability at all pH values was lost after freeze-drying protein extracts (data not shown). Hence, only frozen larvae were used for gelation experiments. Different extracts were prepared from larvae blended in MilliQ water adjusted to the desired pH: (i) Mixture was used completely including for example chitin particles, (ii) Mixture was sieved through a cheesecloth to remove larger chitin particles and (iii) soluble part after centrifugation. Insect extracts with a similar protein content were heated at 86 °C for 30 minutes in a tube, then turned upside down to determine if a gel had been formed (Yi et al. 2013) as shown in Table 6.5.

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Table 6.5. Gel formation of protein fractions from Tenebrio molitor, Alphitobius diaperinus and

Hermetia illucens at different pH values based on visual observation. Whole larvae were blended in

MilliQ, and from this three extracts were prepared: (i) Whole larvae were used completely including for example chitin particles. (ii) Mixture was sieved through a cheesecloth to remove larger chitin particles, and (iii) soluble part after centrifugation. The protein concentrations were adjusted to 2.6% w/w and gelation was determined after heating for 30 min at 86 °C. G = gel, WG = weak gel and X = no gel formation.

pH 3 pH 5 pH 7 _{+ HSO}pH 7 3- pH 7 + PPO pH 10

Colour _yellow Light _brown Light _brown Dark _yellow Light _brown Dark _brown Dark

T. m ol ito r Soluble protein G X X X X X Filtrate cheesecloth WG n.d. G n.d. n.d. n.d. Whole larvae G X G G n.d. X A. d ia pe ri nu s Soluble _protein G X X X G X Filtrate cheesecloth WG n.d. G n.d. n.d. n.d. Whole larvae G X WG WG n.d. X H. i llu ce n s Soluble _protein X X X X n.d. X Whole larvae X X X X n.d. X n.d. not determined.

H. illucens did not form a gel under any of the conditions tested, also not when the protein

concentration was doubled (data not shown). Gels were formed at pH 3 and pH 7 under certain conditions for T. molitor and A. diaperinus. A different effect of pH on gelation of soluble proteins was shown before, as Yi et al. (2013) found gelation at pH 7 and 10 when 30% w/v soluble fractions were used. It is hypothesized that this difference in gelation could be due to proteolytic activity upon storage. Preliminary results showed that proteolytic activity led to visually weaker gels when mixing a protein solution at pH 7 for longer time before gelation, as well as when extra trypsin was added (data not shown).

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Phenoloxidase activity on the other hand seems to strengthen gels in some cases based on visual observation, likely due to crosslinking of proteins. This was shown for example by addition of PO to soluble proteins from A. diaperinus at pH 7.