FEED COMPOSITION FOR TENEBRIO MOLITOR

Abstract

According to an aspect, there is provided a feed composition for Tenebrio molitor, comprising water, gelatin and alloferon peptide as effective components. The alloferon peptide is preferably included 1 ppm or more in the feed composition. The alloferon peptide can act as a growth promotor for Tenebrio molitor. Also, the alloferon peptide can act to enhance the survival rate of Tenebrio molitor. Also, the alloferon peptide can act to shorten the development time of Tenebrio molitor.

Claims

1. A feed composition for Tenebrio molitor comprising water, gelatin and alloferon peptide as effective components.

2. A feed composition for Tenebrio molitor of claim 1, wherein the alloferon peptide is included in 1 ppm or more in the feed composition.

3. A feed composition for Tenebrio molitor of claim 2, wherein the alloferon peptide acts as a growth promotor for Tenebrio molitor.

4. A feed composition for Tenebrio molitor of claim 2, wherein the alloferon peptide enhances the survival rate of Tenebrio molitor.

5. A feed composition for Tenebrio molitor of claim 2, wherein the alloferon peptide shortens the development time of Tenebrio molitor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 shows a body weight of mealworms (T. Molitor) after gelatin and alloferon-containing gelatin diet.

[0018] FIGS. 2A and 2B show a survival rate and time required for larvae-pupae transformation of mealworm.

[0019] FIG. 3. shows a cell proliferation of Sf9 cells after 300 nM of the alloferon treatment.

[0020] FIG. 4 shows a test result, in which PO activity is analyzed in mealworm body tissue after alloferon treatment.

[0021] FIGS. 5A and 5B show a test result related to a large-scale mealworm production.

DETAILED DESCRIPTION

[0022] In the following detailed description of the present disclosure, references are made to the accompanying drawings that show, by way of illustration, specific example embodiments in which the present disclosure may be practiced. These example embodiments are described in sufficient detail to enable those skilled in the art to practice the present disclosure. It is to be understood that the various example embodiments of the present disclosure, although different from each other, are not necessarily mutually exclusive. For example, specific shapes, structures and characteristics described herein may be implemented as modified from one example embodiment to another without departing from the spirit and scope of the present disclosure. Furthermore, it shall be understood that the positions or arrangements of individual elements within each of the example embodiments may also be modified without departing from the spirit and scope of the present disclosure. Therefore, the following detailed description is not to be taken in a limiting sense, and the scope of the present disclosure is to be taken as encompassing the scope of the appended claims and all equivalents thereof.

[0023] Hereinafter, various preferable example embodiments of the present disclosure will be described in detail with reference to the accompanying drawings to enable those skilled in the art to easily implement the present disclosure.

<Materials and Methods>

1. Fabrication of Feeding Gelatin

[0024] The feeding gelatin was made with combining 200 g of distilled water and 4 g of paper gelatin (Gelita H G, Eberbach, Germany), and solidified in the refrigerator using an ice cubic stick tray with the lid closed. The same process was performed for the experimental group of gelatin containing alloferon peptide (Biomatik, Del., USA) of 1 ppm. The tray had 10 slots to make each gelatin of approximately 20 g gelatin cubic and kept in the refrigerator with the lids closed to minimize loss of moisture.

2. Mealworms Larvae Feeding

[0025] Yellow mealworms (Tenebrio molitor) were provided by a private local insect breeder (Mealworm village, Seoul, Korea). Mealworms larvae were divided into three different dietary groups: (1) About 500 larvae were placed in a plastic container (18.5×12×5.5 cm) with 30 g wheat bran only (control group), (2) About 500 larvae with wheat bran supplemented with 20 g gelatin three times per week (positive control group), (3) About 500 larvae with wheat bran supplemented with 20 g gelatin containing alloferon peptide of 1 ppm three times per week (experimental group).

[0026] Two replicated containers per each dietary group were set up, after which the containers were placed in a climate chamber at 25° C. with a relative humidity of 60%.

[0027] In addition, the assessment of developmental time with large-scale was performed to confirm the result from the former laboratory-scale experiment. For the experimental group, 15,000 larvae with wheat bran supplemented with gelatin containing 10 ppm alloferon were prepared similar with lab-scale feeding protocol. For the control group, the same amount of larvae was fed with wheat bran only until larvae-pupae transformation.

3. Mealworms Larvae Growth, Survival Rate and Development (Larvae-Pupae Transformation) Analysis

[0028] Mealworms pupae and dead larvae were counted and removed daily from each experimental group, and the numbers were recorded for the evaluation of survival rate. The average weight of the mealworm larvae was recorded every week with 20 randomly selected mealworms.

[0029] Development time of mealworms was considered to be the number of days until 50% of mealworms larvae changed into pupae stage.

[0030] For various analysis of biologic response, harvested mealworms larvae were killed by freezing and then all mealworms stored at −20° C.

4. Phenoloxidase Activity Analysis

[0031] Penoloxidase activity in homogenized larvae tissue was analyzed with a 96 well microplate reader using catechol as the substrate. After the sample was incubated for 37° C. for 10 min, the 420 nm absorbance was measured with a microplate spectrophotometer (Bio-Rad, CA, USA). The essay was performed with three replicates and each experiment was repeated three times. The penoloxidase activity was expressed as units of enzyme activity per mg protein.

5. In Vitro Cell Proliferation Analysis by PrestoBlue Assay

[0032] The proliferation of Sf9 insect cells was analyzed by the PrestoBlue assay (Invitrogen, CA, USA). 8,000 cells/well were prepared in 96 well plates (Sigma-Aldrich, MI, USA). After cells were seeded, the cells were incubated with PBS with alloferon (300 nM). The control group received PBS without aloferon. At 0, 2, 4, and 6 days after applying 10% PrestoBlue, the color switchover of resazurin to resorufin (Absorbance 570 nm/600 nm) was measured with the microplate reader (BioTeck, Vt., USA).

6. Large-Scale Validation of Mealworms Larvae Development Time (Larvae-Pupae Transformation)

[0033] The assessment of developmental time with large-scale was performed using the same method with the former laboratory-scale experiment. The time point at which the total weight of the mealworms in each feeding tray reached 3 kg was set as the development time.

7. Statistical Test

[0034] Unpaired t-test was used for all statistical analysis using GraphPad Prism 8 (GraphPad, CA, USA). Statistical significance was set at p<0.05.

<Results>

1. Effect of the Gelatin and Alloferon-Containing Gelatin Diet on Body Weight of the Mealworms (T. Molitor).

[0035] For the three groups of mealworms, average weight of wheat bran diet group increased 140.2% over 10 weeks. Gelatin diet supplementation group improved the growth rate to 189.4% and Alloferon-containing gelatin supplementation group improved the growth rate to 267.3% (FIG. 1). In week 10, mealworms larvae fed on gelatin and alloferon supplement were 39.5%-90% heavier than those fed on wheat bran only (p<0.05).

[0036] (A) Gelatin contained with allfoeron supplement was provided to the mealworms (T. Molitor). (B) Effect of gelatin and alloferon-containing gelatin on body weight change of mealworms was analyzed over 10 weeks of larvae development. (C) Body weight of mealworms was increased after gelatin and alloferon-containing gelatin diet. Duplicate samples were analyzed in each condition. Two-way ANOVA with Tukey multiple comparisons test was used to analyze the statistical significance; p<0.001(***) and p<0.0001(****).

2. Effect of the Gelatin and Alloferon-Containing Gelatin Diet on Survival Rate and Development Time (Larvae-Pupae Transformation) of the Mealworms (T. Molitor).

[0037] The average survival rates of pupated mealworms fed on wheat bran diet, gelatin and alloferon-containing supplement diet were 56.4%, 68.5% and 91.3% respectively (FIG. 2A).

In this study, mealworms larvae transformed into pupae in 9 to 17 weeks. Development time was defined as the number of days until 50% of mealworm larvae changed into pupae stage. The development time of mealworms in the experimental group was shortened up to 20.6%-39.6% on gelatin and alloferon-containing gelatin diet compared to the control group with wheat bran diet.

[0038] (A) Survival rate of mealworms fed on wheat bran, gelatin supplement and alloferon-containing gelatin. (B) Development time of mealworms fed on wheat bran, gelatin supplement and alloferon-containing gelatin. Duplicate samples were analyzed in each condition. Unpaired t-test was used to analyze the statistical significance; p>0.05 (ns) and p<0.05 (*).

3. Effect of the Alloferon Treatment on Sf9 Insect Cell Proliferation.

[0039] To test whether alloferon treatment increases cell proliferation in insect cell, in vitro proliferation assay was performed on Sf9 insect cell. Sf9, which is originally established from ovarian tissue, is commonly used in insect cell culture for in vitro assay. Presto blue assay reveals that 300 nM alloferon significantly boosted cell proliferation of Sf9 cells only after 6 days of the treatment (FIG. 3). This result suggests that alloferon increase insect cell proliferation. Triplicate samples were analyzed in this assay. Two-way ANOVA with Tukey multiple comparisons test was used to analyze the statistical significance; p>0.01(ns) and p<0.01(***).

4. Effect of the Alloferon Diet on Phenoloxidase Activity in Mealworms (T. molitor) Tissue.

[0040] FIG. 4 shows a test result, in which PO activity is analyzed in mealworm body tissue after alloferon treatment. Duplicate samples were analyzed in this assay. Unpaired t-test was used to analyze the statistical significance; p<0.05 (*).

5. Validation of the Alloferon Diet on Shortening Development Time (Larvae-Pupae Transformation) of the Mealworms (T. Molitor) in Large-Scale Insect Farming

[0041] To confirm the effect of alloferon enhancing the growth rate of mealworms, large-scale experiment was performed with ˜15,000 mealworms (FIG. 5A). In this large-scale experiment, the development time of mealworms required to reach 3 kg in weight (n=15,000) was analyzed. The development time of the 1 ppm alloferon gelatin diet group significantly shortened up to 28.7% compared to the control group with wheat bran diet group (FIG. 5B). Unpaired t-test was used to analyze the statistical significance; p<0.01 (**).

[0042] Considering world population increases, food shortages, and environmental pollution, the insect proteins are expected as a favorable alternative. The edible insects have nutritional advantages because it is composed of high protein (50-60%), considerable amount of fat, fiber, vitamins, and minerals. Mealworms (Tenebrio molitor) used in this study are also known to contain a substantial amount of protein and unsaturated fatty acid. While the attempt for developing and applying mealworms for protein supply to patient diet has been continued, a recent study reported that mealworm diet not only increased the muscle mass and body fat, but also activated the immune cells in cancer patients. The nutrient contents of mealworms are considered valuable as a good source of nutrition for patients who need high nourishment even if with small amounts.

[0043] The antibacterial effects of alloferon on mealworms have been reported in the previous study. In particular, the correlation with the increased activity of the penoloxidase and the innate immunity of insects was evaluated. The present study is the first up to our knowledge to evaluate that alloferon shortened development time as well as increased survival rate of mealworms by increasing the expression of penoloxidase. This result suggested the possibility of application of alloferon to invertebrates including other insects. The study of Bai P P et al. supported our study that penoloxidase played an important role in melanin production, which is related in invertebrate immune mechanism and insect development process [Bai P P, Xie Y F, Shen G M, Wei D D, Wang J J. Phenoloxidase and its zymogen are required for the larval-pulpal transition in Bactrocera dorsalis (Diptera: Tephritidae). J Insect Physiol 2014; 71:137-4625].

[0044] Mealworms can be fed on wheat bran to obtain all required nutrients for growth, development, and reproduction. The previous studies showed that larval survival could be improved when additional ingredients were provided. However, most of the studies have focused on balance and replenishment of macronutrients including protein, fat, and starch. The present study is comparable with other studies in that it suggested a biological mechanism rather than a nutritional method for promoting mealworm growth. Large-scale feeding showed the parallel developmental time compared to the laboratory-scale.

[0045] In order to prevent cannibalism and increase productivity in mealworm breeding, sufficient moisture supply is an important factor. Previously, supplements such as vegetables and fruits were mainly used for hydration. In this study, growth disturbance was not detected when moisture supply was provided with a gelatin containing alloferon peptide without any conventional supplements such as vegetables. This might be attributable to the composition of gelatin containing hydrolyzed collagen peptide and water. Thus, it is expected that this type of gelatin might be applied to the insect farming industry as vehicles to supply hydrophilic peptide, alloferon and moisture simultaneously.

[0046] This study showed alloferon peptide of 1 ppm composed of 16 amino acid decreased the development time of mealworms by 40.3% and increased survival rate over 61.8% (FIGS. 1, 2A, and 2B). This result can be applicable for the increase of productivity required from a large-scale smart insect farming system, and used as an important basis for providing economic value to the insect production industry. As a conclusion, alloferon promoted the growth performance of mealworms as reducing the development time and survival rate, and it might lead to a favorable solution for the insect breeding industry. The effect of alloferon was confirmed in the large-scale feeding.