Changes in the level of consolidation of the fatty acid profile of Hermetia illucens larvae grown on a substrate contaminated with heavy metals

We conducted a comparative investigation to examine the alterations in the composition and content of the fatty acid complex in the larvae of the Black Lion fly ( Hermetia illucens ) as they were reared under different concentrations and combinations of heavy metals. The use of the method of mass spectrometric analysis of the obtained biomass showed that linoleic, lauric and oleic fatty acids predominated in the composition of the larvae. The use of the mathematical method of fractal analysis based on the data on the profile distribution of fatty acid components in the insect body according to the experimental variants showed that samples with metal concentrations of 20 mg of cadmium, 800 mg of cobalt and Mix (200 mg of copper, 20 mg cadmium, 200 mg cobalt, 20 mg aluminium and 50 mg lead) per kilogram of dry food. The variation in the values of the indices of the biosystemic consolidation of acids, based on the conversion to their molar masses, ranged from 0.41 to 0.82.


INTRODUCTION
One of the topical issues in the development of society at the moment is the world's hunger problem. In 2050 the world population is expected to grow from 7 to 10 billion. However, out of 800 million people who are already malnourished, 650 million are in developing third-world countries. Overall, two billion people suffer from nutritional deficiencies. The problem of producing and using cheap feed additives is also felt in the livestock industry when grazing and keeping livestock. One of the possible solutions here is to introduce non-traditional sources of fodder protein into the diet of humans and animals, which can be larval forms of edible insects.
The most frequently produced species of forage insects is the Black soldier fly (Hermetia illucens), from the lionfish family (Stratiomyia chamaeleon) [1], [2]. Individuals of this species in natural conditions are mainly distributed in tropical and subtropical countries. In Russia, the insect is called the "Black Lion", abroad -"Black Soldier" [3]. The first experiments on the cultivation of Hermetia illucens began in the 90s of the twentieth century in search of an effective way to utilize organic waste by converting it into biomass rich in proteins and fats. The biomass obtained from their larvae or pupae is a rich source of protein with essential amino acids (in particular, arginine, histidine, leucine and isoleucine, lysine, phenylalanine, tyrosine, valine and others), fatty acids (lauric, myristic, palmitic, stearic, oleic, linoleic acid, etc.), vitamins, macro-and microelements, and other biologically active substances

Scientific Hypothesis
Insects can be a new protein source in feed and food production upon risk assessment. But today, the presence of the availability of accurate data raises the suspicion of the danger of the risk associated with the presence of toxic elements in stable biomasses of insects. The aim of our work was to investigate the alleged detection of metals in the larvae of one of the most popular objects shortly for proposal as a source of protein -the Black soldier fly (Hermetia illucens). We are seeing an increase in the structure of acidic fat organization for permanent inclusion in the trophic food chain.

MATERIAL AND METHODOLOGY Insect
The eggs of the black soldier fly were selected from the brood colony of insects kept in the insectarium of the laboratory for the structural processing of biomass of the All-Russian Research Institute of Food Additives (St. Petersburg, Russia).

Samples
Before the current experiment, newborn larvae were grown in a light chamber (insectarium), 60×50×50 cm in size. The front wall of the chamber was a glass window, and the rest were made of chipboard. The temperature in the working area for breeding flies was maintained at 30 ±2 °C, relative humidity was 65 ±5% Testo 174H (Testo SE & Co. KGaA, Germany) ( Figure 1). Small ventilation holes were made on the cover of the insectarium. On the ceiling of the incubator, two fluorescent lamps with a power of 30 watts each were installed, connected to the network. The colour temperature of both lamps was 6500 K. The length of the day was 12 hours.

Feed
Initially, the larvae were fed wheat bran moistened with deionized water (water content 75%), then switched to standardized chicken feed (PC-2 compound feed, Gatchinsky Feed Mill JSC, Leningrad region, Russia).

Chemicals
To contaminate the substrate with heavy metals, the chicken feed was thoroughly mixed with the appropriate volume of the salt solution of the toxicant until the desired concentration was reached. Copper salts (CuSO4) were , [20], [27]. In addition, the same volume of deionized water without adding heavy metals was mixed with chicken feed as a control (40, 60 and 80 mg/kg).

Instruments
The content of fatty acids in insect biomass was determined using gas chromatography with mass spectrometric detection, a Varian 450-GC gas chromatograph with CP-Wax 58 FFAP CB, 50 m x 0.32 mm х 0.50 µm column coupled with a Varian 240-MS mass spectrometric detector (Varian, USA).

Analysis conditions
Carrier gas flow (Helium) rate 1 ml/min, injector temperature 250 °C, split 1:15, the start of chromatogram registration from 9 minutes. The temperature program is presented in Table 1.

Description of the Experiment
Sample preparation: Each experiment was carried out in triplicate. For each repetition, 150 larvae were collected. Insects were placed in plastic containers (12×10×8 cm) located inside the insectarium and fed daily with wheat bran with or without the addition of metals. Feeding was stopped after 8 days. At the end of the experiment, the larvae and their faeces were separated by manual sieving using 3 mm sieves and then ground in a laboratory mill IKA A11 basic (IKA-Werke GmbH & Co. KG, Germany) for further analysis (Figure 2).
The resulting ground samples were evaporated to dryness in JEIO TECH vacuum oven OV-12 with cold trap bath CTB-10 coupled with Woosung Vacuum Co vacuum pump MVP 6 600 µl of a 15% sulfuric acid solution in methanol was added to the evaporated samples, and 600 µl of chloroform was added. Eppendorf was carefully sealed with Parafilm and placed in a heater for 1 hour at 65 °C. After the sample was cooled, 200 μl of deionized water was added and thoroughly mixed. The organic layer was analysed and injected directly into the chromatograph at 1 µl using a CPAL autosampler and a 10 µl Hamilton chromatographic syringe.

Statistical Analysis
The experimental data were subjected to statistical analysis using the R program (version 4.1.0, https://cran.rproject.org/bin/windows/base/) for Windows [21], [22]. Statistical processing of the results of determining the profile of fatty acids in the samples was carried out using ANOVA analysis of variance and Fisher's test (F). Differences were considered significant, and the presence of a relationship between the indicators was recognized at a probability level not exceeding 0.05. Fractal portraits and indices of biosystematic determination (IF) of fatty acids were calculated based on the mathematical algorithm incorporated in the original computer program [23].
The initial values of fatty acids from percentages were converted into molar masses to approximate the obtained data and construct fractal portraits.
The visual difference between the data in the quantitative composition of the acid profiles for different experimentexperiment variants is shown on the heat map ( Figure 3).
Further, based on these data, two-dimensional coordinate planes were constructed, on which point represents each acid represents each acid represents each acid represents each acid with the x-coordinate equal to the fractional part of log2 (ei/emax), and the y-coordinate equal to log2 (ei/emax), where ei, emax is an acid with a nominal, established serial number (i) and with a maximum intensity of synthesis. The entire field of the fractal portrait is divided into rectangular sectors by horizontal and vertical lines. The sectors highlighted in color contain dotted images of fatty acids inside. Further, on the basis of a decreasing power series of fatty acid indicators  Table 3.   So far, only few data have been published on contaminants identified in commercially available insects and insect-based products for human consumption or animal feed. It seems clear that the substrate has an effect on the fatty acid composition, which are the source of energy in insects [40], [41]. A study by a fully accredited laboratory in the UK studying the chemical safety of four different species of fly larvae, including Hermetia illucens, as a protein source for animal feed showed that only cadmium was above the maximum EC limit in animal feed of 0.5 mg/kg (three of nine samples analyzed) Our results showed that the composition of the larvae was dominated by linoleic, lauric and oleic fatty acids. Insect hemolymph composition is known to change with developmental stage and within one stage [50]. Hence, the interpretation of lipid and fatty acid composition in the insects may be difference. In spite of that, this study lends further support to the observation that Hermetia illucens a is sensitive to Cd and these results suggest that the whole body lipid concentration are affected directly by Cd concentrarion in nutrient substrate.