Effect of domestic cooking methods on protein digestibility and mineral bioaccessibility of wild harvested adult edible insects.

Wild harvested edible insects are characterised by high protein and mineral contents with potential to contribute substantially to nutrition security. However, nutritional content is only beneficial when proteins are digestible and minerals bioaccessible. This study determined the effects of domestic processing on protein digestibility and mineral bioaccessibility of two wild harvested insect species: Eulepida mashona (beetle) and Henicus whellani (cricket). Samples of both insects were subjected to boiling, roasting, or combined boiling and roasting, imitating the way insects are traditionally prepared in Zimbabwe. Moreover, they were in vitro digested according to INFOGEST protocol. Boiling of both insects resulted in loss of protein as it leached into the boiling water. The raw insects had a higher protein in vitro digestibility than the boiled and roasted insects, and the maximal decrease in protein digestibility was around 25% for twice boiling of the beetles and for boiled and roasted crickets. For both insect species, boiling resulted in non-significant loss of iron and zinc. Iron was the least bioaccessible mineral in both insects, based on the concentrations of soluble mineral measured by ICP-AES. However, beetles had a much higher iron bioaccessibility (30.7%) as compared to crickets (8.11%). Interestingly, boiling resulted in about 50% decrease in iron and zinc bioaccessibility in both species while roasting did not. The reduced protein digestibility and mineral accessibility with processing can be explained by protein modification and interactions of minerals with other food components, such as chitin and phytochemicals. Because of the reduction in protein digestibility and mineral accessibility during boiling, roasting should be favoured over boiling and in any case short boiling time is recommended.

Effect of endogenous phenoloxidase on protein solubility and digestibility after processing of Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens.

Upon extracting soluble proteins from insects as potential food ingredient, endogenous enzymes, such as phenoloxidases, are expected to negatively affect protein properties. The effect of phenoloxidases on solubility and digestibility of proteins was investigated for larvae of Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens. Phenoloxidase inhibition was done using blanching (50 s, 90 °C) before extraction or extracting in presence of sulfite. Similar soluble protein yields and compositions were found without and with sulfite addition, whereas blanching decreased soluble protein yield. Upon in-vitro hydrolysis by pepsin and trypsin, soluble proteins from H. illucens were more digestible than those of T. molitor and A. diaperinus. Phenoloxidase activity during grinding negatively affected in-vitro pepsin hydrolysis. Besides phenoloxidase activity, also endogenous proteases were shown to remain active at pH 8 in extracts containing sulfite and after blanching of larvae. This stresses that protease activity needs to be carefully controlled in the design of insect based ingredients.

Iron-polyphenol complexes cause blackening upon grinding Hermetia illucens (black soldier fly) larvae.

Insects are a promising alternative protein source. One of the bottlenecks in applying insects in food is the fast darkening initiated during grinding. Besides enzymatic browning, non-enzymatic factors can cause off-colour formation, which differs between species. This study investigates the impact of iron, phenoloxidase, and polyphenols on off-colour formation in insect larvae. Hermetia illucens showed a blackish colour, whereas Tenebrio molitor turned brown and Alphitobius diaperinus remained the lightest. This off-colour formation appeared correlated with the iron content in the larvae, which was 61 ± 9.71, 54 ± 1.72 and 221 ± 6.07 mg/kg dw for T. molitor, A. diaperinus and H. illucens, respectively. In model systems, the formation of iron-L-3,4-dihydroxyphenylalanine (L-DOPA) bis- and tris-complexes were evidenced by direct injection into ESI-TOF-MS, based on their charges combined with iron isotope patterns. The reversibility of the binding of iron to phenolics, and thereby loss of blackening, was confirmed by EDTA addition. Besides complex formation, oxidation of L-DOPA by redox reactions with iron occurred mainly at low pH, whereas auto-oxidation of L-DOPA mainly occurred at pH 10. Tyrosinase (i.e. phenoloxidase) activity did not change complex formation. The similarity in off-colour formation between the model system and insects indicated an important role for iron-phenolic complexation in blackening.

Correction: Involvement of phenoloxidase in browning during grinding of Tenebrio molitor larvae.

[This corrects the article DOI: 10.1371/journal.pone.0189685.].

Involvement of phenoloxidase in browning during grinding of Tenebrio molitor larvae.

Insects are investigated as alternative protein source to meet the increasing demand for proteins in the future. Enzymatic browning occurring during grinding of insect and subsequent extraction of proteins can influence the proteins' properties, but it is unclear which enzymes are responsible for this phenomenon. This study was performed on larvae of three commonly used insect species, namely Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens. Oxygen consumption measurements on protein extracts showed activity on L-tyrosine, L-3,4-di-hydroxy-phenylalanine (L-DOPA) and L-dopamine, indicating phenoloxidase as a key player in browning. Furthermore, no reaction on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) was observed, ruling out an important contribution of laccase to browning. The browning reaction was most prominent at pH 6 for T. molitor and A. diaperinus, and 7 for H. illucens. As the enzyme activity of H. illucens was the lowest with the darkest color formation, this was likely caused by another factor. The activity of phenoloxidase was confirmed for T. molitor and A. diaperinus by activity measurements after fractionation by anion-exchange chromatography. Color measurements showed the presence of activity on both L-DOPA and L-tyrosine in the same fractions. Both substrates were converted into dopachrome after incubation with enzyme-enriched fractions. No DOPA-decarboxylase, tyrosine hydroxylase and peroxidase activities were observed. By using native PAGE with L-DOPA as staining-solution, active T. molitor protein bands were resolved and characterized, identifying a tyrosinase/phenoloxidase as the active enzyme species. All together, these data confirmed that tyrosinase is an important enzyme in causing enzymatic browning in T. molitor and likely in A. diaperinus.

Nitrogen-to-Protein Conversion Factors for Three Edible Insects: Tenebrio molitor, Alphitobius diaperinus, and Hermetia illucens.

Insects are considered a nutritionally valuable source of alternative proteins, and their efficient protein extraction is a prerequisite for large-scale use. The protein content is usually calculated from total nitrogen using the nitrogen-to-protein conversion factor (Kp) of 6.25. This factor overestimates the protein content, due to the presence of nonprotein nitrogen in insects. In this paper, a specific Kp of 4.76 ± 0.09 was calculated for larvae from Tenebrio molitor, Alphitobius diaperinus, and Hermetia illucens, using amino acid analysis. After protein extraction and purification, a Kp factor of 5.60 ± 0.39 was found for the larvae of three insect species studied. We propose to adopt these Kp values for determining protein content of insects to avoid overestimation of the protein content.

Food Preference and Appetite after Switching between Sweet and Savoury Odours in Women.

BACKGROUND: Exposure to food odours increases the appetite for congruent foods and decreases the appetite for incongruent foods. However, the effect of exposure to a variety of food odours, as often occurs in daily life, is unknown. OBJECTIVE: Investigate how switching between sweet and savoury odours affects the appetite for sweet and savoury products. DESIGN: Thirty women (age: 18-45y; BMI: 18.5-25kg/m2) intensely smelled the contents of cups filled with banana, meat or water (no-odour) in a within-subject design with four combinations: no-odour/banana, no-odour/meat, meat/banana and banana/meat. Participants received one combination per test day. In each combination, two cups with different fillings were smelled for five minutes after each other. Treatment order was balanced as much as possible. The effects of previous exposure and current odour on the appetite for (in)congruent sweet and savoury products, and odour pleasantness were analysed. A change from meat to banana odour or banana to meat odour was referred to as switch, whereas a change from no-odour to meat odour or no-odour to banana odour was no-switch. RESULTS: The current odour (P<0.001), as opposed to the previous exposure (P = 0.71), determined the appetite for (in)congruent sweet and savoury products, already one minute after a switch between sweet and savoury odours. The pleasantness of the odour decreased during odour exposure (P = 0.005). CONCLUSIONS: After a switch, the appetite for specific products quickly adjusted to the new odour and followed the typical pattern as found during odour exposure in previous studies. Interestingly, the appetite for the smelled food remained elevated during odour exposure, known as sensory-specific appetite, whereas the pleasantness of the odour decreased over time, previously termed olfactory sensory-specific satiety. This seeming contradiction may result from different mechanisms underlying the odour-induced anticipation of food intake versus the decrease in hedonic value during prolonged sensory stimulation.

Sensory-specific appetite is affected by actively smelled food odors and remains stable over time in normal-weight women.

Understanding overconsumption starts with knowledge of how separate factors influence our eating behavior. Food cues such as food odors are known for their effect on general appetite and sensory-specific appetite (SSA). Active sniffing rather than passive exposure may induce satiation over time. The objective of this study was to investigate how actively sniffing banana odors affects general appetite, SSA, and subsequent food intake. In a crossover study, 61 women actively smelled cups containing natural banana, artificial banana odor, or water (no odor) for 10 min. Treatment order was randomly assigned as much as possible. General appetite and SSA were monitored by using 100-mm visual analog scales during the 10 min of active sniffing, followed by ad libitum intake of banana milkshake. Results showed that SSA was consistently high (+12 mm) during actively sniffing natural or artificial banana odors, with no decrease in SSA over time. Sniffing both banana odors increased the appetite for banana (+11 mm) and other sweet products (+4 mm), whereas the appetite for savory products decreased by 7 mm (all P < 0.01) compared with no odor. Actively sniffing banana odor did not significantly influence food intake (P = 0.68) or general appetite scores (P = 0.06). In conclusion, SSA scores during active sniffing were identical to the SSA found in a similar study that used passive smelling, suggesting that SSA is independent of the manner of sniffing and exposure time. Moreover, sweet/savory categorization may suggest that food odors communicate information about the nutrient composition of their associated foods. These data clearly show the appetizing effects of food odors.

Aroma exposure time and aroma concentration in relation to satiation.

The present study investigated the effect of aroma exposure time and aroma concentration on ad libitum intake and subjective satiation. In a within-subject study, thirty-eight unrestrained, healthy female participants (age: 18-39 years; BMI: 18·5-26·0 kg/m²) were asked to consume tomato soup during lunchtime, until they felt comfortably full. Every 30 s, the participants consumed 10 g of a bland soup base while tomato soup aroma was delivered separately through the nose via a retronasal tube that was attached to an olfactometer. This gave the impression of consuming real tomato soup. For each sip, the aroma varied in exposure time (3 and 18 s) and concentration (5 × ), resulting in four different test conditions. Ad libitum food intake and appetite profile parameters were measured. A 9% lower food intake was observed when the participants were exposed to the condition with 18 s exposure time and a high concentration than when exposed to the other three conditions. These results indicate that changing the retronasal aroma release by aroma concentration and aroma exposure time affects food intake.

Extraction and characterisation of protein fractions from five insect species.

Tenebrio molitor, Zophobas morio, Alphitobius diaperinus, Acheta domesticus and Blaptica dubia were evaluated for their potential as a future protein source. Crude protein content ranged from 19% to 22% (Dumas analysis). Essential amino acid levels in all insect species were comparable with soybean proteins, but lower than for casein. After aqueous extraction, next to a fat fraction, a supernatant, pellet, and residue were obtained, containing 17-23%, 33-39%, 31-47% of total protein, respectively. At 3% (w/v), supernatant fractions did not form stable foams and gels at pH 3, 5, 7, and 10, except for gelation for A. domesticus at pH 7. At 30% w/v, gels at pH 7 and pH 10 were formed, but not at pH 3 and pH 5. In conclusion, the insect species studied have potential to be used in foods due to: (1) absolute protein levels; (2) protein quality; (3) ability to form gels.

Consumption with large sip sizes increases food intake and leads to underestimation of the amount consumed.

BACKGROUND: A number of studies have shown that bite and sip sizes influence the amount of food intake. Consuming with small sips instead of large sips means relatively more sips for the same amount of food to be consumed; people may believe that intake is higher which leads to faster satiation. This effect may be disturbed when people are distracted. OBJECTIVE: The objective of the study is to assess the effects of sip size in a focused state and a distracted state on ad libitum intake and on the estimated amount consumed. DESIGN: In this 3×2 cross-over design, 53 healthy subjects consumed ad libitum soup with small sips (5 g, 60 g/min), large sips (15 g, 60 g/min), and free sips (where sip size was determined by subjects themselves), in both a distracted and focused state. Sips were administered via a pump. There were no visual cues toward consumption. Subjects then estimated how much they had consumed by filling soup in soup bowls. RESULTS: Intake in the small-sip condition was ∼30% lower than in both the large-sip and free-sip conditions (P<0.001). In addition, subjects underestimated how much they had consumed in the large-sip and free-sip conditions (P<0.03). Distraction led to a general increase in food intake (P = 0.003), independent of sip size. Distraction did not influence sip size or estimations. CONCLUSIONS: Consumption with large sips led to higher food intake, as expected. Large sips, that were either fixed or chosen by subjects themselves led to underestimations of the amount consumed. This may be a risk factor for over-consumption. Reducing sip or bite sizes may successfully lower food intake, even in a distracted state.

Effect of salt intensity in soup on ad libitum intake and on subsequent food choice.

The effect of salt intensity on ad libitum intake of tomato soup was investigated when soup was served as a first course and as a second course. Also the effect of salt intensity in soup on subsequent sweet vs. savory choice of sandwich fillings was investigated. Forty-three healthy subjects consumed ad libitum a low-salt (LS), ideal-salt (IS) and high-salt (HS) tomato soup in both meal settings. The salt concentrations were selected on an individual basis, in a way that IS was most pleasant and LS and HS were similar in pleasantness. The ad libitum intake of IS soup was higher than that of LS and HS soup, and the ad libitum intake of LS soup was higher than that of HS soup. The meal setting, soup as a first or as a second course, did not affect ad libitum intake. Salt intensity in soup did not predict sweet vs. savory choice of fillings in grams or energy, although most sodium from fillings was consumed after intake of HS soup. In conclusion, a higher salt intensity lead to lower ad libitum intake of soup similar in palatability (LS vs. HS). In addition, salt intensity in soup does not predict sweet vs. savory food choice.

Both longer oral sensory exposure to and higher intensity of saltiness decrease ad libitum food intake in healthy normal-weight men.

Orosensory exposure to sweetness has been shown to be important in satiation, whereas the effect of exposure to a salty taste on satiation is not known. The primary objective was to investigate the effect of orosensory exposure time to and intensity of saltiness in soup on ad libitum intake. The secondary objective was to investigate the effect of intensity on bite size. Fifty-five healthy men consumed ad libitum from both a low-salt (LS) and a high-salt (HS) creamy tomato soup in 2 exposure time conditions ("long" and "short") and a free condition ("free"). Bites were administered and controlled via a pump. In the long condition, bites of 5 g were administered in 2 s at intervals of 5 s (exposure time: 24 s/min). In the short condition, bites of 15 g were administered in 3 s at intervals of 15 s (exposure time: 12 s/min). The eating rate was equal in the long and short conditions (60 g/min). In the free condition, participants adjusted their bite sizes at intervals of 15 s. The short condition resulted in ~34% higher ad libitum intake compared to the long condition (P < 0.001); there was no interaction with intensity. Ad libitum intake of HS soup was ~9% lower than LS soup (P < 0.001). The free condition showed that HS soup was consumed with smaller bite sizes during the first half of the intake period (P < 0.05). Longer orosensory exposure and higher saltiness intensity both decreased food intake, although orosensory exposure had more impact than intensity. Prolonging the orosensory exposure per food unit may be helpful to reduce food intake.

Effect of salt intensity on ad libitum intake of tomato soup similar in palatability and on salt preference after consumption.

Sensory properties of food play an important role in satiation. Studies on the effect of taste intensity on satiation show conflicting results. This may be due to the notion that in these studies taste intensity and palatability were confounded. The objective of this study was to investigate the effect of salt intensity of tomato soup on ad libitum intake (satiation), while controlling for palatability on an individual basis. Forty-eight subjects consumed both a low-salt (LS) and high-salt (HS) soup ad libitum from a self-refilling bowl. The results showed no difference between LS and HS soup in ad libitum intake, eating rate, changes in appetite ratings, and changes in hedonic ratings after intake. After intake of HS soup, LS soup was perceived as more bland than before intake of HS soup. After intake of LS soup, HS soup was perceived as more salt intense than before intake of LS soup. In conclusion, this study found no effect of salt intensity on satiation of tomato soups that were similar in palatability. During consumption, subjects adapted quickly to the exposed salt intensity as contrasting salt intensities were rated further from the ideal salt intensity and therefore perceived as less pleasant after consumption.

Detection of conformational changes in immunoglobulin G using isothermal titration calorimetry with low-molecular-weight probes.

Proteins for therapeutic use may contain small amounts of partially misfolded monomeric precursors to postproduction aggregation. To detect these misfolded proteins in the presence of an excess of properly folded protein, fluorescent probes such as 8-anilino-1-naphthalene sulfonate (ANS) are commonly used. We investigated the possibility of using isothermal titration calorimetry (ITC) to improve the detection of this type of conformational change using hydrophobic probes. As a case study, conformational changes in human polyclonal immunoglobulin G (IgG) were monitored by measuring the enthalpies of binding of ANS using ITC. Results were compared with those using fluorescence spectroscopy. IgG heated at 63 degrees C was used as a model system for "damaged" IgG. Heat-treated IgG can be detected already at levels below 5% with both ITC and fluorescence. However, ITC allows a much wider molar probe-to-protein ratio to be sampled. In particular, using reverse titration experiments (allowing high probe-to-protein ratios not available to fluorescence spectroscopy), an increase in the number of binding sites with a K(d)>10 mM was observed for heat-treated IgG, reflecting subtle changes in structure. Both ITC and fluorescence spectroscopy showed low background signals for native IgG. The nature of the background signals was not clear from the fluorescence measurements. However, further analysis of the ITC background signals shows that a fraction (8%) binds ANS with a dissociation constant of approximately 0.2 mM. Measurements were also carried out at pH 4.5. Precipitation of IgG was induced by ANS at concentrations above 0.5 mM, interfering with the ITC measurements. Instead, with the nonfluorescent probes 4-amino-1-naphthalene sulfonate and 1-naphthalene sulfonate, no precipitation is observed. These probes yield differences in the enthalpies of binding to heated and nonheated IgG similar to ANS. The data illustrate that ITC with low-molecular-weight probes is a versatile tool to monitor conformational changes in proteins with a wider application potential than fluorescence measurements.

Detection of soy proteins in processed foods: literature overview and new experimental work.

Several tests for the detection of soy proteins in foods have been described in the literature, and some are commercially available. This article gives an overview of these methods and discusses the advantages and disadvantages of each individual method. Based on the conclusions of this inventory, an experimental approach was designed to improve the sensitivity of measuring soy protein in processed foods. The aimed sensitivity is 10 ppm (10 microg soy protein in 1 g solid sample), which is over 100-fold lower than presently available tests. The aimed sensitivity is this low because levels of food allergens at 10 ppm and above may provoke reactions in food allergic persons. Native soybean meal, soy protein isolate, soy protein concentrate, and textured soy flakes were used as test materials. Several extraction procedures were compared and a new method using high pH was selected. Polyclonal antibodies were raised in rabbits and goats, and immunopurified antibodies were used in sandwich and inhibition enzyme-linked immunosorbent assay (ELISA). Extraction at pH 12 resulted in good yields for all tested samples, both quantitatively (Bradford) and qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunopurified rabbit antibodies against this extract used in a competition ELISA format resulted in a sensitive test with a detection limit of 0.02 microg/mL, corresponding to 0.4 microg/g (0.4 ppm) in food samples. Cross-reactivity with some main food ingredients was measured and appeared to be negative in all cases. The presently developed test is applicable for soy ingredients and soy-containing foods that are processed in different ways. The limit of quantitation is 1 ppm, which is an enormous improvement over earlier described methods.

Differences in denaturation of genetic variants of soy glycinin.

In heat denaturation studies conducted in the past the genetic variants of glycinin have been considered as a homogeneous group of proteins. In this work the validity of this assumption was tested. It was found by calorimetric studies that glycinin denatures heterogeneously at pH 7.6. When the temperature of isothermal treatment is increased from 70 to 82 degrees C the proportion of glycinin remaining native progressively decreases from 95% to 5% while the denaturation temperature of the glycinin remaining native increases from 88.5 to 95 degrees C. Similar trends were found for pH 3.8. Fractionation and subsequent analysis (MALDI-TOF and CE) of isothermally treated samples demonstrated that at pH 7.6 the heterogeneous denaturation is caused by differences in thermal stability of the genetic variants of glycinin. The stability increases in the order G2/G3/G1< A(4)< G5 < G4.