MICROBIOLOGICAL ANALYSIS OF BIOMASS OF THE FOOD INSECT Tenebrio molitor

ABSTRACT

The biomass of Tenebrio molitor (mealworm) plays a significant role in the production of food and feed. This article examines the microbiological state of Tenebrio molitor biomass and studies the composition of microorganisms present in it. Data are presented on the bacterial flora isolated from biomass derived from dried Tenebrio molitor larvae. As a result of the research, bacterial isolates such as Staphylococcus warneri, Pseudomonas aeruginosa, and Enterobacter cloacae were identified from the biomass of Tenebrio molitor larvae. The study primarily determined the total count of mesophilic aerobic bacteria (MAB), the Enterobacteriaceae family, and the total count of molds. Colony-forming units (CFU/ml) were calculated, and the morphological and biochemical characteristics of the isolates were studied.

АННОТАЦИЯ

Биомасса Tenebrio molitor (мучной червь) имеет большое значение в производстве продуктов питания и кормов. В данной статье анализируется микробиологическое состояние биомассы Tenebrio molitor, изучается состав содержащихся в ней микроорганизмов. Представлены данные о бактериальной флоре, выделенной из биомассы, основанной на высушенных личинках Tenebrio molitor. В результате исследований из биомассы личинок Tenebrio molitor были выделены бактериальные изоляты, такие как Staphylococcus warneri, Pseudomonas aeruginosa, Enterobacter cloacae. B первую очередь были определены общее количество мезофильных аэробных бактерий (МАФАМ), семейство Enterobacteriaceae, а также общее количество плесневых грибков, подсчитаны колонии (КОЕ/мл), и изучены морфологические и биохимические свойства изолятов.

Keywords: Tenebrio molitor, Zophobas atratus, Staphylococcus, Escherichia, Pseudomonas, Salmonella, Enterobacter, MAFAM, Musca domestica.

Ключевые слова: Tenebrio molitor, Zophobas atratus, Staphylococcus, Escherichia, Pseudomonas, Salmonella, Enterobacter, MAFAM, Musca Domestica.

Introduction. As the world population grows, the demand for quality food sources increases. Currently, food needs are met by plant-based foods, organic foods, and regional and seasonal products. However, there are some difficulties in producing plant-based foods and animal-based foods, and in view of these challenges, new sources are being sought to address the food supply problem. In this context, edible insects are attracting increasing attention as a sustainable food source that provides quality protein [6,8]. Although there are an estimated 1900 species of food-eating insects on Earth [4], the species most studied in food production worldwide are Musca domestica, Hermetia illucens, Tenebrio molitor, Zophobas atratus, Alphitobus diaperinus, Galleria mellonella, Achroia grisella, Bombyx mori, Acheta domesticus, Gryllodes sigillatus, Locusta migratora migratorioides and Schistocerca americana [5]. It has been shown that insects can be used as an alternative option for producing food with high nutritional value by rearing them on a very large scale on an industrial basis [7].

T.molitor is known to be one of the most commonly used food insects worldwide [9,10]. As with traditionally produced food products, microbiological safety is important for insect-derived food products. Potential food hazards associated with edible insects include contamination with toxins (especially mycotoxins and bacterial toxins), heavy metals and chemicals (pesticides). There is a theory that in many cases, in addition to the bacteria in the insects, dangerous microflora may be microflora that enters the insects during their cultivation (naturally or accidentally in buildings and structures, naturally or accidentally in food products, etc.), as well as during processing or storage [3]. Microbiological studies of biomass samples of the food insect T.molitor have been carried out by several authors, studying the effects of various feed substrates and maintenance conditions. Therefore, the scientific community recommends a processing step to reduce the number of microorganisms [1]. Edible insects are gaining increasing interest as a sustainable and protein-rich alternative source. Tenebrio molitor is considered promising, in particular for the food and animal feed industries. However, ensuring the microbiological safety of the biomass remains an important issue. The aim of this study was to evaluate the microbiological composition of this insect.

Materials and methods. The biomass secreted by T.molitor larvae was used as the object of the study. The studies were conducted in the scientific laboratory of the Department of Biotechnology of the Tashkent Chemical-Technological Institute. All studies used traditional microbiological methods such as microscopy, Gram staining and CFU (colony forming units). T.molitor larvae were grown in plastic boxes under optimal conditions (25°C/75% RH per hour) in a growth chamber using the Blumberg method [2].

The biomass excreted by T.molitor larvae was obtained using aseptic procedures. The total mesophilic aerobic bacteria (MAFAM), enterobacteria (ITBG) and total molds were first counted from the T.molitor larval biomass and the colony count (CFU/ml) was determined by counting the total colony count (CFU/ml). For this purpose, the tubes and culture media were sterilized in an autoclave. The T.molitor biomass was homogenized in a laboratory homogenizer twice for 30 seconds in a test tube in 50 ml of sterile peptone water. The resulting suspensions were diluted 109 times and cultured on special nutrient media for each sample, and the total number of mesophilic aerobic bacteria (MAFAM), enterobacteria (ITBG) and the total number of mold fungi were determined by counting colonies (CFU/ml). Sabour's medium was used to detect mold, and Endo's medium was used to determine the total number of Enterobaceriaceae bacteria. Mueller-Hinton medium was used to estimate the total number of mesophilic aerobic bacteria. Microbiological analyses were repeated four times.

Results and discussion. Mealworm biomass was obtained using aseptic procedures. Mealworm biomass was homogenized twice in 20 ml of sterile peptone water for 30 seconds in a test tube. The resulting suspensions were diluted 10 times and the bacteria were seeded on special nutrient media to determine the total number of colonies (CFU/ml). The seeded samples were kept in a thermostat at 37±10 ℃ for 48–72 hours. To count the colonies, Petri dishes were viewed in transmitted light and the counted colonies were marked with ink or chalk. Their average number in 1 cm² was found and multiplied by the surface area of ​​the medium on the plate and found by calculation using the MAFAnM formula.

MAFAnM (10-1) = N·C/ gram. (см3) (N=10);

MAFAnM (10-2) = N·C/ gram. (см3) (N=100);

N= sample dilution rate (1:10 = 10);

C = number of colonies on both plates: 2 (arithmetic mean).

Table 1.

MAFAM counts and morphological characteristics of bacterial isolates isolated from Tenebrio molitor larval biomass

During our study, the total number of bacteria from the biomass of T.molitor larvae, MAFAM, was estimated by the standard microbiological method CFU (Colony Forming Unit). Fungi and molds were grown on Saburo and Chapeka nutrient media. The presence of pathogenic microorganisms Salmonella and E.coli was examined, and the morphological and biochemical characteristics of bacterial isolates such as Staphylococcus warneri, Enterobacter cloacae, Pseudomonas aerugino in the biomass were determined. The microscopy method was used to determine the cell shape of the studied isolates. When smears were prepared from colonies grown in nutrient media, they were stained using the Gram method and examined under a microscope, it was determined that the cell shape of Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, Salmonella bacteria was rod-shaped, and Staphylococcus warneri bacteria was cocci-shaped. When the colony suspected of being E.coli was cultured on the selective nutrient medium Endo, the colony color was red, Enterobacter cloacae formed white colonies on simple peptone agar, Pseudomonas aeruginosa formed white colonies on meat-peptone agar, Staphylococcus warneri formed yellow colonies on the selective nutrient medium MSA, and the colony suspected of being Salmonella formed white colonies when cultured on Bismuth nutrient medium. When we analyzed the data presented in Table 1, it was noted that the isolates were found at different levels, including the presence of bacteria belonging to the genus Enterobacter up to 2.11×103 (CFU/ml). In this case, Pseudomonas aeruginosa and bacterial species were found at 1.92×102, and Staphylococcus warneri was found at 1.88×104 (CFU/ml), while E.coli and Salmonella bacterial species were found to be the least abundant in biomass, although no significant differences were observed in their abundance.

Table 2.

Biochemical  properties of isolates

Note: + - positive; – - negative;: + - weak reaction; ++ - strong reaction; +++- very strong reaction.

During the studies, it was noted that among the isolates presented in Table 2, the oxidase activity of the isolates belonging to the cloacae species of the Enterobacter genus was negative, whereas the Pseudomonas aeruginosa isolate showed positive activity. It was also found that all the isolates, namely Pseudomonas aeruginosa bacteria, showed positive results for catalase activity. It was noted that the sulfur-forming species of the Enterobacter genus and Pseudomonas aeruginosa gave a negative reaction. Also, when observing the motility of the isolates, it was noted that the cells of the Enterobacter cloacae isolates were highly motile. The biochemical properties of  E.coli bacteria are determined using special microbiological tests. E.coli bacteria can break down lactose and produce acid and gas. It ferments glucose, producing acid and gas. It ferments mannitol, producing acid. E.coli is an oxidase-negative bacterium, meaning it lacks the enzyme cytochrome oxidase. E.coli usually cannot use citrate as a carbon source and does not produce hydrogen sulfide. The catalase test is positive, meaning it breaks down hydrogen peroxide into oxygen and water. The results (Table 2) show the biochemical properties of the bacterial isolates obtained from the biomass of T.molitor larvae. In particular, when analyzing the bacterial isolates obtained from the biomass of T.molitor larvae, it was found that no E.coli bacterial isolates were detected in the biomass. During the analysis of the isolates, it was found that the colonies presumably belonging to E.coli showed positive oxidase activity and negative catalase activity.

Table 3.

Morphocultural characteristics of Staphylococcus warneri bacteria

Note: + - positive; – - negative; (+ ) weak reaction; ++ - strong reaction;

d - positive from 11% to 89%, S - antibiotic sensitivity.

Bacteria of the Staphylococcus genus differ in morphological and biochemical properties from bacteria of the ITBG, Pseudomonas aeruginosa, Salmonella group. Staphylococcus warneri belongs to the group of coagulase-negative staphylococci. It is one of the cocci-like microorganisms that live on the skin and mucous membranes of humans and animals, and is usually not pathogenic. The Staphylococcus warneri bacterial group can damage food products if sanitary and hygienic requirements are violated during the processing of food products. The biochemical properties of the Staphylococcus warneri species from the isolates during the studies are shown in Table 3. It was found that the coagulase test showed a negative result in Staphylococcus warneri isolates. Based on biochemical analyses, it was found that the Staphylococcus warneri species showed a negative reaction in terms of nitrate reduction properties. It was also noted that bacteria belonging to the Staphylococcus warneri genus are resistant to standard heat treatment.

Conclusion. As a result of our experiments, we came to the following conclusion: T.molitor biomass has high nutritional value and meets the requirements of microbiological safety. However, it is important to comply with hygienic standards in biomass production. Microorganisms in T.molitor biomass are mainly beneficial and can have a positive effect on human health. However, proper storage and processing are necessary to maintain the microbiological safety of the product. Microbiological analysis of T.molitor biomass is of important scientific and practical importance in preventing future damage to organisms consuming this biomass (humans, livestock, poultry, fish, etc.). During the research, microbiological analysis of T.molitor biomass was carried out and the following was determined. No fungi or molds were detected in the composition of T.molitor biomass. Salmonella and E.coli bacteria belonging to the group of pathogenic microorganisms were also not detected in the biomass, which indicates that the biomass meets hygienic requirements. Staphylococcus warneri was found in an amount of 1.88×104 (CFU/ml), Pseudomonas aeruginosa and the bacterial species up to 1.92×102, and Enterobacter cloacae bacterial isolates up to 2.11×103 (CFU/ml), i.e. in very small quantities.

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