Impact of Cooking Time on the Physicochemical and Nutritional Properties of Macrotermes subhyalinus and Imbrasia obscura ()
1. Introduction
Due to the fact that edible insects are not included in national economic data on food consumption, the contribution of edible insects to food and nutrition security, as well as the battle against poverty, is underappreciated in Cameroon [1] [2] [3] . In most cases, insect usages and commercialisation remain informal. Though insect consumption seems to be innovative, it is rather a reintroduction of this food product in our feeding habits. Several studies show that edible insects were so popular at the time of our ancestors [2] [3] . A survey conducted in the Adamawa and East regions of Cameroon showed that insects such as I. obscura and M. subhyalinus are of great interest in the food habits of the local population [4] . These insects are mainly fried, toasted, grilled, scalded, and roasted. Many authors [3] [4] [5] reported that the cooking method affects proteins, fats, carbohydrates, total minerals, and in vitro digestibility of proteins of I. obscura and M. subhyalinus. It was reported that the method and cooking temperature considerably modify the content of certain nutrients in pork and beef meats [6] [7] [8] . Nevertheless, studies of the influence of culinary treatments on the content and quality of nutrients of edible insects are less known, especially for the two insects mentioned above. In the Adamawa and East regions of Cameroon, M. subhyalinus is generally consumed fried or toasted and I. obscura is either scalded or scalded and fried [5] . During various processing treatments, several chemical reactions happen [9] and can affect the texture of insects, and modify their physicochemical and nutritional properties [5] [10] found that the application of heat treatments to M. subhyalinus and I. obscura reduced the microbial load to the recommended values by AFNOR norm. Moreover, frying and toasting resulted in the reduction of nutritive and nutritional parameters from 5% to 64% for M. subhyalinus [5] . On the other hand, scalding, the combined process of scalding and frying reduces nutritional parameters from 5% to 30% for I. obscura [5] . However, according to data gathered by [5] from the populations of the East and Adamawa areas of Cameroon, the cooking time varies from person to person. These values range from 3 to 20 minutes. It is, therefore, known that for a given heating treatment, the breadth of its effect on the reduction of the nutritional parameters is a function of time. The study of the variations of the different parameters with respect to the cooking time of M. subhyalinus and I. obscura becomes a necessity. Therefore, the goal of this study is to determine how cooking time affects many factors, including protein digestibility, protein content, lipids, and total minerals.
2. Materials and Methods
Sources and Collection of Insects
I. obscura and M. subhyalinus (images are presented in Figure 1) were collected early in the morning in Garoua-Boulai (5˚53'00"N, 14˚33'00"E) East region of Cameroon in July 2020 for the first sample. The sample second was collected in Ngaoundere 3 subdivision (7˚19'39"N, 7˚19'39"E) Adamawa region, Cameroon, in the month of June of the same year.
Preparation of Insects
The different treatment methods applied (Figure 2 and Figure 3) on these insects are the most used, as mentioned by the population consuming the insects in the Adamawa and East regions of Cameroon [5] . Wings and other impurities of selected M. subhyalinus were removed while the digestive tract of I. obscura was emptied by exerting pressure using the fingers from the head to the abdomen end. These insects were then washed using distilled water before processing. A batch of insects weighing 250 g was used for each treatment. The cooking temperature for scalding was set at 93.5˚C ± 0.5˚C, and frying and grilling were done at 150˚C ± 0.5˚C. Sampling was done withing specific and regular time intervals of 0, 3, 6, 9, and 12 min for toasting and frying of M. subhyalinus, 0, 6, 9,
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(B)
Figure 1. Photograph of Imbrasia obscura (A) and Macrotermes subhyalinus (B).
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Figure 2. Cooking kinetic of M. subhyalinus.
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Figure 3. Cooking kinetics of I. obscura.
12, 15 min for scalding of I. obscura and 0, 3, 6, 9 and 12 min for the frying of I. obscura. The time used for the first sampling represents the minimum cooking time and the maximum cooking time is that of the last sampling The physicochemical analyses performed the so treated samples were the moisture, protein, carbohydrate, lipids and total mineral content whereas the proteins digestibility and the energy density as well as described below. Raw sample was considered as control.
Physicochemical and Nutritional Analyses
Moisture content was determined by the method described by [11] . Total mineral content were evaluated by [12] method; total fat content was done using Soxhlet as described by [13] , while total nitrogen was determined after mineralisation of samples according to the Kjeldahl method [14] and assessment was done by the colorimetric technique of [15] , protein content was calculated by multiplying the nitrogen content by the conversion factor of nitrogen to protein (6.25). The total carbohydrate content was determined by subtracting the water, proteins, lipids, and ashes from 100% of dry matter. The energetic density was calculated from the values of total proteins, fats and carbohydrate by applying the conversion into energy using the formula given by [16] . The method of [17] was used to determine the in vitro protein digestibility.
Statistical Analyses
Experiments were done in triplicate and data expressed as mean ± standard-deviation. Similarly, increasing or reducing percentage of different parameters with respect to the crude sample was calculated. Analysis of variance (ANOVA) was used to compare the means of the different treated samples. The Duncan multiple range test was applied to classify means when there was a significant difference at 5% using Statgraphics Centurion. The principal component analysis was investigated how variables related to one another and how samples varied. The plotting of curves was done using Sigma plot 11 software.
3. Results and Discussion
Table 1 shows the findings from the nutritional and physicochemical content of raw samples of M. subhyalinus and I. obscura.
The effects of cooking on total minerals, carbohydrates, moisture content, and in vitro protein digestibility on raw, fried, and roasted M. subhyalinus, scalded and roasted I. obscura were investigated.
Variation of Moisture Content of M. subhyalinus and I. obscura during Cooking
The moisture content significantly differed (p < 0.05) between raw samples of I. obsura (57.28%) and M. subhyalinus (52.47%). Results obtained while cooking the two insect samples are presented in Figure 4 and show that cooking has an influence (p < 0.05) on the moisture content.
Frying and toasting M. subhyalinus and the combined process of scalding and frying I. obscura significantly reduced the amount of water present during cooking. For fried and grilled M. subhyalinus, the correlation between cooking time and the outcome was negative (r = −0.91) and (r = −0.90), respectively. Additionally,
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Table 1. Physicochemical and nutritional composition of M. subhyalinus and I. obscura.
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Figure 4. Evolution of moisture content of M. subhyalinus and I. obscura with respect to the cooking method.
there was a significant difference (p < 0.05) between the water content and cooking time for all M. subhyalinus treatments. In contrast, scalding I. obscura allowed for a considerable (p < 0.05) increase in water during the first nine minutes of cooking, after which the moisture content became constant. The correlation (r = 0.89) between the moisture content and the scalding time of I. obscura is positive. The reduction of the moisture content at the end of 3, 6, 9 and 12 minutes for M. subhyalinus during frying (51.67%, 80.74%, 86.11% and 92.63%) is significantly (p < 0.05) higher than that of I. obscura (20.81%, 41.53%, 52.90% and 63.55%). These results show that insects obtained after cooking for 9 min and 12 min are good for long time storage. On the microbiological aspect, these low moisture contents could limit the development of microorganisms [18] . The effect of moisture content increase observed during scalding in this study is contrary to the works of [19] who found that boiling of rabbit meat for 5, 15 and 40 min enhanced the reduction of the water content by 12.03%, 11.66% and 12.98% respectively. This difference can be explained by the structure of proteins and their capacity to absorb and retain water. However, the reduction of the moisture content observed during frying and toasting is similar to the study of [19] in which frying of rabbit meat for 2, 4 and 6 min led to a reduction of humidity from 49.92%, 96.56% and 98.88%, respectively. These results show that boiling has less influence on the moisture content of insects compared to frying.
Variation of the Protein Content of M. subhyalinus and I. obscura during Cooking
The raw samples of I. obscura had higher protein content than the raw samples of M. subhyalinus (Table 1). The heat treatments by frying and toasting M. subhyalinus then fried and scalded I. obscura carried out in the course of this study enhanced an increase in protein content during the first 6 minutes of cooking, compared to the crude sample. After 6 min, a significant lost in protein content was observed. Figure 5 shows the differences in protein concentration depending on the type and length of treatment.
Frying and toasting resulted in an increase in protein content of M. subhyalinus and scalding I. obscura samples during the first 6 minutes of cooking of 3.45%, 5.64% and 5.77% respectively. These results contradict the findings of [19] who reported the reduction in protein content during rabbit meat scalding and frying. According to [19] , losses in protein content of boiled rabbit meat during 5, 15 and 40 min were 2.51%, 7.75% and 17.78%, respectively. Hydrolysis of proteins and the drainage of sarcoplasmic proteins could explain the higher protein losses of boiled meat for 15 to 40 min than meat boiled for 5 min compared to raw proteins. These differences may also be explained by the nature of the substrate and protein structure involved. The increase of protein losses is associated to the increase of frying time as described by [20] . The losses of protein content in this study happened with the increase of cooking time, and the loss due to frying is greater compared to boiling. Another possible reason is that, frying at high temperature (150˚C ± 3˚C) might have caused more important
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Figure 5. Evolution of the protein content for M. subhyalinus and I. obscura with respect to cooking time.
and rapid denaturation compared to boiling at low temperature, by so doing leading to losses of greater water molecules [21] . The oil absorption during frying or protein diffusion in oil used may be another reason of the slight reduction in protein content during frying as compared to boiling.
Variation of the Fat Content of M. subhyalinus and I. obscura during Cooking
Fat contents varied treatment type and cooking time, compared to raw insects in which fat values are 18.67 g/100 of DW and 54.24 g/100 DW respectively for I. obscura and for M. subhyalinus (Figure 6). Oil can be gained by absorption when frying. Fried M. subhyalinus and scalded/fried I. obscura after 12 min of cooking contributed to observe fat increase of 1.64% and 60.07% respectively. The different absorptions observed between the two insects may have resulted from the difference of hight and the structure (tissue matrix) of these insects which in turn is responsible of their abilities to retain absorbed oil. These results corroborate those of [22] , whose experiments with dry pork flesh demonstrated that a significant amount of oil can permeate meat during a straightforward frying. Meanwhile scalding I. obscura and toasting M. subhyalinus in different cooking times resulted to a significant reduction in the quality of lipids. The lipids loss varied from 2.67%, 8.78%, 12.37% and 13.81% after 6, 9, 12 and 15 min of scalding of I. obscura. This reduction may be explained by the melting of fat globules in boiling water. Similar results have been reported by [23] on beef meat. Still, grilling of M. subhyalinus revealed fat losses of 1.29%, 3.89% and 4.51% after 6, 9, and 12 min of cooking respectively. This loss may be due to fat
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Figure 6. Evolution of fat content for M. subhyalinus and I. obscura with respect to cooking mode and time.
melting and exudation after tissue contraction, while part of it was lost by thermal decomposition. Similar results have been presented in the works of [24] during grasshopper grilling.
Variation of Ash Content for M. subhyalinus and I. obscura during Cooking
Results presented in Figure 7 show that ash contents of insects are influenced by cooking methods and time. Ash content of raw M. subhyalinus was 5.87 g/100g DM and that of raw I. obscura was 7.82 g/100g DM.
A decrease in total mineral content of 9.46% was observed after 9 min of scalding of I. obscura and did no longer vary significantly (p < 0.05) up to 15 min of cooking. The cooking time was negatively correlated (r = −0.90) with the total mineral content. There were losses during scalding and this may be due to the dissolution of minerals in cooking water. These observations are dissimilar to those of [25] who mentioned an increase of ash content during boiling of three species of Nigerian fishes. This difference may be justified by the nature of the matrix substrate. Frying and roasting of M. subhyalinus and frying of I. obscura increases the amount of minerals in the dry matter. For fried and toasted M. subhyalinus and fried I. obscura, the correlations between ash contents and cooking duration are positive and, respectively, significant (r = 0.77, 0.88, and 0.97). Water lost observed during frying and toasting has favoured minerals concentration in dry matter. Thus, the increase in ash content observed with cooking time. These results are in accordance with those of [26] who found that ash content in raw fish (0.63%) is about twice or trice greater compared to that of fried fish (1.42%). Mineral contents of I. obscura and M. subhyalinus were equivalent compared to cooking effects on their variations. Mineral content of
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Figure 7. Variation of total mineral content for M. subhyalinus and I. obscura with respect to cooking method and time.
edible insects varies greatly with species and orders [3] and the applied heat treatments might have also affected the mineral composition from one insect species to another [27] .
Variation of the Carbohydrate Content for M. subhyalinus and I. obscura during Cooking
Results of the carbohydrate content for both studied insects are presented in Figure 8. Overall, the carbohydrate content decreases considerably during the first 6 minutes of processing. Losses were higher during frying and grilling of M. subhyalinus (up to 90% loss). Xiaoming et al. [28] found that the carbohydrate content of insects varies between 3.7 to 16.3 g/100g of DM with a mean value of 8.2 g/100g of DM. Comparing the present results with those of [28] , it shows that only the carbohydrate content of I. obscura falls within this intervalle (14.45 g/100g of DW). M. subhyalinus had low carbohydrate content of 3.04 g/100g of DW and the culinary treatments applied in this study contributed significantly (p < 0.05) to the reduction of the carbohydrate content with cooking time. Carbohydrates are involved in Maillard and caramelisation reactions [29] . This transforms carbohydrates into other products in particular the pyrazines responsible for the aroma and the melanoidins which give the brown color to these cooked insects. The decrease of carbohydrate content observed during scalding results from the losses of other constituents in particular by solubilization of free sugars and minerals which favour a concentration effect of other constituents. The cooking time considerably influenced the carbohydrate levels of M. subhyalinus and I. obscura. It is important to reduce the cooking time at 9 min.
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Figure 8. Evolution of carbohydrate content of M. subhyalinus and I. obscura with respect to the cooking mode and time.
Variation of Energetic Density of M. subhyalinus and I. obscura during Cooking
Energetic densities of insects vary with the cooking method and time (Figure 9). Significant differences (p < 0.05) were observed between the energetic density of scalded and fried I. obscura compared to the raw samples. The energetic density of scalded and fried insects increased after 12 min of cooking. This increase results from the loss of water during cooking and which contributed to concentrate the nutrients. Also, absorption of oil contributed to increase the quantity of lipids, and as such justifies the high energetic density. However, the energetic density of scalded I. obscura varied significatively and negatively (p < 0.05) after 15 min of cooking. A reduction of 2.17% was observed. This reduction results from water absorption during the treatment and the fats losses. This observation is in line with that of [2] . For M. subhyalinus, there was no significant difference between fried and raw samples after 12 min of cooking. Nevertheless, the reduction of energetic density was 2.66% after 12 min for the toasted sample. Despite the fact that the reduction is significant at 5% level, it has not influence on the energetic density.
Variation of Protein Digestibility for M. subhyalinus and I. obscura with Respect to Cooking Time
Based on results obtained in this study (Figure 10), the applied treatments lead to considerable loss of protein digestibility after a certain cooking time. The in vitro digestibility of proteins of raw samples is 85.20% and 86.65% respectively for I. obscura and M. subhyalinus.
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Figure 9. Evolution of energetic density for M. subhyalinus and I. obscura with respect to cooking time.
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Figure 10. In vitro digestibility of proteins for M. subhyalinus and I. obscura with respect to cooking methods and time.
Compared to the raw samples, digestibility increased with cooking temperature 70˚C to 90˚C and this corresponds to a 3 min of cooking time for fried and toasted M. subhyalinus and 6 min of cooking time for scalded I. obscura. This effect can be explained by a progressive denaturation of proteins, which exposed cleavage sites to digestive enzymes, meanwhile at higher temperature, oxidation lead to the degradation of proteins. This could hide the cleavage sites as explained by [30] , thus, the reduction of digestibility during treatments at high temperatures above the cooking temperature previously mentioned. Proteins undergo several technological treatments before being ingested. There exist typically three mechanical treatments which consist of product denaturation and its assembling; chemical treatment by which the action of solutes (salts, acids, spices, etc) modifies the structure and the composition of tissues; and thermal treatment for which the effects are variables with respect to temperatures and the target time. During these treatments, the macro and microstructures of the matter are modified by physicochemical changes involving proteins, lipids and micronutrients [31] . These structural modifications at the alimentary level are susceptible of either increasing or reducing the accessibility of the proteins to the site of enzyme digestion. The becoming of proteins is therefore susceptible of being modified by the nature of unit operation and notably the duration of heat treatments. In fact, digestibility of proteins is reduced by formation of disulfate bonds in the matrix of proteins or the formation of protein aggregates which limited the action of proteolytic enzymes. This observation may justify the reduction of digestibility of proteins of fried M. subhyalinus, toasted M. subhyalinus, scalded I. obscura, scalded and fried I. obscura after 6, 9, 9, 6 min of cooking time respectively. Also, peroxidation products of lipids increased with temperature above 100˚C. These products react with proteins notably to form Schiff bases [30] . These Schiff Bases constitute the first step in the aggregation at the molecular scale and new molecular interactions leading to hiding of cleavage sites. Authors [32] mentioned that digestibility is increase if the unfolding of polypeptide chain is favoured. In addition, structural modifications of proteins induced by the action of heating increase the inaccessibility of some digestive proteases. This observation can be elucidated by the increase of the digestibility of proteins at the end of 3, 3 and 6 min of cooking for fried M. subhyalinus, toasted M. subhyalinus and scalded I. obscura respectively. These events depend also for the intrinsic contributors to the conformation stability of proteins of different sources. Auto-oxidation reactions (that take place at high cooking temperatures like frying and toasting) improved the formation of covalent bonds that link polyphenols and proteins in a reversible mana, reducing as such the digestibility of proteins [33] . Based on results obtained, it is preferable to limit the cocking time at 6 min for scalding, 3 to 6 for toasting and frying, because these temperatures best preserve the digestibility of proteins. However, the issue will be the acceptability of these products by the consumers for these desired cooking durations.
Principal Component Analysis of Nutritional and Functional Properties of Cooking Duration
Principal component analysis (PCA) was done to group the cooking time that can have influence on the nutritional properties. The principal component analysis enabled the establishment of groups within constituents (dry matter, lipids, proteins, protein digestibility and total mineral). Figure 11 presents the bidimensional repartition of I. obscura and M. subhyalinus cooked at different times and their constituents that influenced the nutritional properties on the principal components coordinates (F1, F2).
Figure 11 reveals that the principal component F1 explains respectively 60.73%, 71.13%, 73.05%, 88.23% of total information. It is noted that contribution of variables for the formation of the principal axis F1 like the protein content, the protein digestibility and their fats content are highly associated to F1 axes. The distribution of constituents along F1 shows a distinction among them with respect to cooking time. It can be drawn from the graphs that:
- Fried M. subhyalinus for 3 and 6 min grouped a maximum of proteins, lipids and protein digestibility;
- Toasted M. subhyalinus for 3 and 6 min presented a maximum of protein content, lipids and protein digestibility;
- Scalded I. obscura for 6 min concentrated a maximum of the protein content, lipids, and protein digestibility;
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Figure 11. Two-dimensional distribution at different cooking times of the technological treatments (M. subhyalinus fried (A), M. subhyalinus roasted (B) and I. obscura scalded (C), I. obscura scalded fried (D)) and their components on the coordinates of the principal components (F1, F2). Legend: Io Eb: I. obscura scalded; Io Eb Fr: I. obscura scalded fried; Ms Fr: M. subhyalinus fried; Ms Gr: M. subhyalinus toasted; 0, 3, 6…: times.
- Scalded I. obscura for 6 min then fried for 3 min gave a maximum of the protein content, lipids, protein digestibility.
It clearly appears from Figure 11 that constituents like lipids, proteins, digestibility and a total mineral content which are elements of nutritional interest are well represented at the cooking time of 3 to 6 min for fried M. subhyalinus and toasted at 6 min for scalded I. obscura and 3 min of frying for previously scalded I. obscura for 9 min. These times are considered as best cooking times.
4. Conclusion
It was found from the present work that cooking induces the degradations of insect constituents. This study also revealed that the cooking time has influenced different parameters analysed. Ash and protein increased during frying and grilling, while carbohydrates decrease. The digestibility of M. subhyalinus and I. obscura proteins diminishes with the increase in the heat treatment cooking time compared to raw samples. The best treatment times for M. subhyalinus are 3 to 6 min for frying and 3 to 6 min for toasting and that of I. obscura are 6 min for the scalded treatment, and the combined processes of scalding (6 min) and frying (3 min). Based on our findings, M. subhyalinus and I. obscura should be incorporated into the daily diet to reduce mineral deficiencies.