Method for pausing development of insect neonate larvae and neotenic mixture
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-02-02
- Publication Date
- 2026-08-13
Abstract
Description
METHOD FOR PAUSING DEVELOPMENT OF INSECT NEONATE LARVAE AND NEOTENIC MIXTUREField of the invention
[0001] The present invention is in the field of producing valuable nutrients from insect larvae, and specifically relates to a method and system for delaying the development of insect larvae.Background of the invention
[0002] The human population is expected to reach 9.1 billion by the year 2050, and at the same time, animal production is expected to rise, leading to an increasing requirement for protein rich fodder sources in the animal production sector. Moreover, the trend of an increasing population will also lead to an increased demand for alternative protein sources. Insect bioconversion of organic waste streams could provide a solution to these challenges by successfully recirculating valuable nutrients and energy that would otherwise be wasted, while providing a more sustainable protein source and thus contributing to food security. Among the most promising insect species to be used as an alternative protein source isHermetia illucens, also commonly known as the black soldier fly (BSF). The adult flies only serve reproductive purposes and live about 8-20 days, depending on temperature, sex, larval food quality and larval development time.H. illucenslarvae live in the wild in organic debris and have a short development time of about 20 days at 27 °C. The larvae go through six instar stages before they become prepupa. In the prepupa form, they migrate into dry areas such as ground vegetation where they pupate for about 18 days. The duration of pupation is highly influenced by environmental factors like temperature, humidity and nutrition.
[0003] The large-scale production of insect larvae is a delicate process and one of the challenging parts of it is to maintain a breeding colony in a good condition. Such large-scale production requires a delicate control of the environment and the stage of the insect life cycle and leaves little room for failures and limits flexibility in scheduling. Despite such strict control, a certain batch of insect could become unfit for further use in the process due to unforeseen circumstances. At present, such events cause a cascade of disruptions in the production process and huge losses when it comes to valuable insect strains. Also, peaks and dips in the production process are difficult to manage when working with batches of insects that may not be timely ready for direct use.
[0004] WO 2021 / 186428 discloses a method wherein a suspended animation state is induced in fly neonate larvae, wherein the larvae are kept in a feed composition in gel form comprising a gelling agent and a preservative. WO 2019 / 199309 discloses a method for storing black soldier fly larvae by placing the larvae between two layers of fermented nutrients. There remains a need for a better way to store a backup for a prolonged period of time, in case a batch of insects becomes unfit for further use. These conventional methods employ a storage medium which is relatively wet, which leads to increased weight of the storage composition, increased transportation costs and increased risk of microbial contamination. The present invention provides a solution for the above-mentioned drawbacks.Summary of the invention
[0005] The inventors surprisingly found that the development of larvae could be delayed or paused by storing the larvae in a particulate material in a climate-controlled environment. By storing the larvae at specific conditions in a mixture with a particulate material, the larvae remain in a neotenic stage where development is delayed or even completely halted. As such, a batch of larvae can be stored for a prolonged period of time and then used as deemed fit.
[0006] The present invention concerns a method for delaying the development of larvae, as well as a neotenic mixture wherein the larvae development is delayed. The method according to the invention comprises:
[0007] (a) providing a batch of insect larvae;
[0008] (b) combining the batch of larvae with a particulate material to obtain a neotenic mixture, wherein the particulate material is an animal feed filler;
[0009] (c) storing the neotenic mixture at a temperature below 15 °C and a relative humidity of at least 60 %.
[0010] The neotenic mixture according to the invention comprises a batch of larvae and a particulate material, wherein the particulate material is an animal feed filler. The neotenic mixture may be comprised in a container.Detailed description
[0011] The present invention concerns delaying the development of insect larvae. Delaying the development can also be referred to as inhibiting the growth, inhibiting the development, halting the development or bringing into a neotenic stage. As such, the development of the larvae is stopped and the larvae remain in the same developmental stage. In other words, the larvae are brought in a neotenic stage, wherein they remain in the then-current developmental stage for a prolonged period during the storage of step (c). In a preferred embodiment, the larvae remain in the same instar stage during the entire neotenic stage and do not undergo moulting. More preferably, the larvae remain in the same phase of an instar stage, typically the growth and feeding phase, during the entire neotenic stage. Even though growth is inhibited and feeding is stopped, this is still referred to as the growth and feeding phase in the context of the present invention.
[0012] Without being bound to a theory, it is believed that the particulate material provides a matrix wherein the larvae are protected and therefore able to survive the conditions during the storage of step (c). Surprisingly, the inventors found that the larvae that are brought back to growth conditions develop further with no or minimal losses due to premature death of the larvae. After the delay in development according to the present invention, the larvae were able to develop further and mature to the same extent as larvae that have not undergone a delay in development.
[0013] The present invention is typically implemented in a production process for the production of nutrients, wherein insect larvae are processed into various nutrient streams such as a fat fraction, a protein rich fraction or a puree comprising or consisting of minced larvae typically in their 5th-6thinstar of larval development. Unlike in the wild where the insects within the batches are at the different development stages, it is of importance for large-scale farming of insects that insects of production are essentially at the same predetermined developmental stage. This simplifies production as processes and resources specific to each developmental stage can be efficiently used to achieve a predictable and stable production output. Normally, this is done by recirculating a small part of the population into the breeding cycle to develop into flies which lay new eggs, that will eventually hatch into larvae. The remainder of the larvae are processed into various nutrient streams such as a fat fraction, a protein rich fraction or a minced-whole larvae puree. To mitigate such disruptions, different stages ofH. illucenslarvae are stored at a lower temperature for extended periods of time. This way it would in theory be possible to re-introduce them at the correct stage into the process, should a disruption occur. However, exposing black soldier fly larvae to reduced temperature has detrimental consequences on their lifecycle, in terms of development success, development rate, fitness and reproduction. In general, it is not possible to store live larvae (1stto 6thinstar) or live pupae ofH. illucensfor extended periods of time (> 2 weeks) at a reduced temperature (e.g.below ambient temperature). Under such conditions, most of the larvae fail to develop further while the pupae either fail to complete metamorphosis or those that do develop into flies die shortly thereafter, thus making the shelf-life of backup batches very short.
[0014] A production process for the production of nutrients, wherein insect larvae are processed into various nutrient streams such as a fat fraction, a protein rich fraction or puree of minced larvae, typically uses multiple batches of larvae of different developmental stages, wherein the larvae in a single batch are in the same developmental stage. With the present invention, it has become possible to buffer batches of larvae at the start of the production process. Thus, in one embodiment, the method according to the invention is used as buffering step at the start of the production process, wherein one or more batches of larvae are collected and stored for a specific period until the larvae are introduced into the production process. As such, the duration of the neotenic stage can be varied from batch to batch to optimize the timing of the introduction of the larvae into the production process. Further, the present invention enables to separate a batch of larvae and store them as backup, which can be used in case a disruption of the production process occurs that would render the batch of larvae that is used for production unfit for further processing. A batch of larvae in a neotenic stage, according to the present invention, can then be used as back-up to replace one of the batches in the production process. In such circumstances, the stored batch of larvae will be brought back into growth conditions by increasing the temperature, and can be entered at the start or the appropriate position in the production process. The process of bringing the larva back to growth conditions is also referred to as “arousal” in the context of the present invention. Hence, the method according to the invention may be for preparing one or more backup batches for larva batches that are used in a process for the production of nutrients.
[0015] It is beneficial to have batches of neotenic larvae in one of their earliest developmental stages. Such young larvae, herein also referred to as “neonates”, are aroused and introduced at the start of the production process, typically by subjecting them to one or more feeding steps to produce valuable nutrients. As such, the neotenic mixtures can be used to start a production process of valuable nutrients. Hence, the method according to the invention is preferentially used to prepare batches of larvae for the early instar stage of the insect, such as the 1stinstar stage ofH. illucens. The instar stages of insects, such asH. illucens, are known to the skilled person, who is able to detect the differences between the instar stages and to determine the instar stage of a particular larva or batch of larvae.
[0016] The present invention concerns a method for delaying the development of larvae, wherein they are comprised in a neotenic mixture, as well as the neotenic mixture comprising the larvae. The neotenic mixture can be comprised in a container. Anything defined for the method according to the invention equally applies to the neotenic mixture according to the invention andvice versa.Method for delaying the development of larvae
[0017] In a first aspect, the invention concerns a method for the delaying of the development of larvae. The method comprises:
[0018] (a) providing a batch of insect larvae;
[0019] (b) combining the batch of larvae with a particulate material to obtain a neotenic mixture, wherein the particulate material is an animal feed filler;
[0020] (c) storing the neotenic mixture at a temperature below 15 °C and a relative humidity of at least 60 %.
[0021] (d) optionally arousing the batch of larvae from the neotenic mixture after the storage of step (c) and introducing it in a process for the production of nutrients.
[0022] The method according to the invention typically finds application in the context of a production process for valuable nutrients. Hence, the method according to the invention can also be worded as a method for starting up a production process, or even a method for the production of valuable nutrients. In case step (d) is performed and the larvae are further developed, the method according to the invention may be worded as a method for rearing insect larvae. Alternatively, the method according to the invention may also be worded as a method for preparing and maintaining a back-up batch of larvae, wherein it is preferred that step (d) is not part of the method and remains optional. The method according to the invention can also be worded as a method for starting or re-starting a process for the production of nutrients, wherein step (d) is performed as part of the method according to the invention. The method according to the present invention is also beneficial for transporting the batch of larvae. Development can be delayed with the method according to the invention, and the neotenic mixture can be transported at climate-controlled conditions for as long as needed, and at their destination the larvae can easily be introduced in a production process or used as deemed fit.
[0023] Step (a) – Providing a batch of larvae
[0024] In step (a), a batch of larvae is provided that is subjected to the method according to the invention. Such a batch of larvae typically comprises a certain amount of larvae of the same species at the same developmental stage. Preferably, the size of the batches matches the size of the batches used in the production process. Suitable sizes include any number of larvae ranging between 500 to 1.500.000 larvae, preferably any number of larvae in the range of 1.000 – 800.000 larvae, such as 2.000 – 600.000, 4.000 – 500.000, 5.000 – 400.000, 10.000 – 300.000, 20.000 – 200.000, 30.000 – 100.000 larvae, preferably 40.000 ± 5.000 larvae, 60.000 ± 5.000, 80.000 ± 5.000, 100.000 ± 5.000, 120.000 ± 5.000, 140.000 ± 5.000, 175.000 ± 5.000, 250.000 ± 5.000, 300.000 ± 5.000, 400.000 ± 5.000, 600.000 ± 5.000, 800.000 ± 5.000 or 1.000.000 ± 5.000 larvae.
[0025] The larvae are of an insect species, preferably of a fly species. Excellent results have been obtained with larvae of the black soldier fly (BSF). Hence, in a preferred embodiment, the larvae are of the same level as the black soldier fly in the tree of life,i.e.of the orderDipteraand the familyStratiomyidae. Thus, in one embodiment, the larvae are of a species of the familyStratiomyidaeselected fromHermetia illucens,Stratiomys chamaeleon,Odontomyia cinctaandPtecticus tenebrifer. In one embodiment, the larvae are of a species of the genusHermetia, such theHermetia illucens,Hermetia comstocki orHermetia fenestrata. Most preferably, the larvae are of the speciesHermetia illucens, also known as the black soldier fly. The batch preferable comprises 90 – 100 % by weight of larvae of a single species, more preferably 92 – 98 wt%, 94 – 96 wt%, 95 ± 2 wt%, 95 ± 1 wt% or 95 ± 0.5 wt%, more preferably at least 95 wt%, such as 95, 96, 97, 98, 99, 99.5 or 100 wt%, most preferably about 100 wt% of larvae of the same species.
[0026] Preferably, the larva are at the beginning of their infancy, such as in the 1stor 2ndinstar, most preferably they are in the 1stinstar. Such newly born larvae are also referred to as “neonates”. In a preferred embodiment, the larvae are subjected to the method of the invention directly after birth (hatching), such as within 1 h, and have not fed yet. As such, there is certainty that the neotenic mixture is not contaminated with larvae feed. Such larvae are ideally suited as a back-up batch of larvae, as they are directly useable for the production of nutrient streams, and can be entered into a production process at the desired position without problem. Such larvae are also ideally suited to be introduced from a buffer at the start of the production process.
[0027] Also, in the context of the present invention, it is not relevant at which phase within an instar stage the larvae are. Typically, the larvae are in the phase of growth and feeding, but some or even all could be moulting. Preferably, the batch of larvae that is subjected to the method according to the invention are essentially in the same developmental stage. Typically, this means that the greater majority of the larvae are in the same instar stage, but it is not excluded that a subset of the larvae is moulting whereas another subset is in the growth and feeding phase. As development is delayed and resumed at a later moment, it is not relevant in which stage or phase the larvae are when subjected to the method according to the invention.
[0028] Advantageously, the larvae within a batch are essentially in the same developmental stage. Such batches are typically used in a production process, and ideally the back-up batch is of the same developmental stage. Hence, it is preferred that the larvae have an age difference of less than 24 hours. Preferably, the age difference within the batch is in the range of 10 seconds – 24 hours, such as 20 seconds – 10 hours, 30 seconds – 8 hours, 40 seconds – 6 hours, 50 seconds – 4 hours, 60 seconds – 3 hours, 90 seconds – 2 hours, 2 minutes – 90 minutes, 3 minutes – 75 minutes, 4 minutes – 60 minutes, 5 minutes – 50 minutes, 6 minutes – 45 minutes, 7 minutes – 40 minutes, 8 minutes – 30 minutes, 9 – 20 minutes or 10 – 15 minutes. More preferably, the larvae to larvae age difference within the batch is in the range of 10 seconds – 2 minutes. As it may be quite labour intensive to factually determine whether the larvae in a batch are indeed in substantially the same developmental stage, it is preferred that the uniformity of the batch is controlled by controlling the age difference between the larvae, which is easily determined when the batch is prepared and kept constant during the entire lifetime of the batch.
[0029] Step (b) – Combining
[0030] The batch of larvae is combined in step (b) with a particulate, which provides a solid matrix for the larvae to be protected and to survive a prolonged storage. The particulate can also be referred to as particulate material, dry material, solid matrix material, filler or texturizer. Without being bound to a theory, it is believed that the dry particulate material offers protection to the larvae, such that they do not stick to the container or to themselves. The inventors found that the formation of clumps or clusters of larvae during storage leads to a high mortality and low survival rates. Such protecting environment enables the larvae to survive the storage conditions of step (c) and are able to be removed from the container again at the end of the storage period. Also, the particulate nature is believed to be crucial, as it allows the larvae to still be in contact with the environment, such that they do not dry out during storage but can survive until they are aroused.
[0031] The particulate should be dry, in order to delay the development of the larvae. Preferably, the water content of the particulate material is at most 15 wt%, more preferably at most 10 wt%, such as 1 – 10 wt% or 2 – 10 wt%, most preferably at most 7 wt%, such as 1 – 7 wt% or 2 – 7 wt%, based on total weight of the particulate. Further, the particulate is preferably inert, meaning that it does not serve as food for the larvae. Since, the larvae are brought in a neotenic stage during step (c), they are not able to feed on the particulate material. Yet, to avoid feeding during step (b) or at the beginning of step (c), it is preferred that the particulate material does not normally serve as food for the larvae. It is further preferred that the particulate material is biodegradable, more preferably food- or feed-grade material. Especially preferred are particulate materials that are normally discarded as waste, such as the hulls or bran of seeds.
[0032] In order to ensure that the larvae are properly protected to survive the storage of step (c) and at the same time their development is sufficiently delayed, it is preferred that the particulate material has a particle size that is in more or less the same magnitude as the size of the larvae. As will be understood by the skilled person, the size of the larvae depends on the exact stage of their development, and the particle diameter of the particulate can be adjusted accordingly. In a preferred embodiment, the average particle diameter of the particulate is 0.1 – 20 times the average length of the larvae, such as 0.5 – 10 times the average length of the larvae, most preferably 2 – 20 times the average length of the larvae. In the context of black soldier fly larvae, the average length is typically in the range of 0.8 – 1.2 mm, mostly about 0.9 mm. Additionally or alternatively, it is preferred that the average particle diameter is in the range of 0.1 – 20 mm, preferably in the range of 0.2 – 10 mm, most preferably in the range of 0.3 – 5 mm. In one embodiment, the average particle diameter is at most 1 mm, such as 0.05 – 1 mm, preferably in the range of 0.1 – 1 mm, most preferably in the range of 0.2 – 1 mm. In one embodiment, the average particle diameter is at least 0.6 mm, such as 0.6 – 20 mm, preferably in the range of 0.6 – 10 mm, most preferably in the range of 0.6 – 5 mm. In an especially preferred embodiment, the average particle diameter is in the range of 0.6 – 1 mm. The inventors found that such particle sizes are ideally suited in the context of the present invention, as the larvae may fall through larger particles, and therefore end up in contact with the container beneath the particulate material, yet smaller particles may create a too dense layer in which the larvae are no longer in contact with the environment and risk drying out.
[0033] The particulate material can be any filler suitable for feed applications. Such fillers are known in the art and are suitable to provide bulk to animal feed. Fillers typically have a low nutritional value and are therefore ideally suited for protecting the larvae in an neotenic stage. The particulate material is not intended as feed for the larvae. To reflect the desired low nutritional value of the particulate material, it is preferred that the protein content thereof is low, such as at most 20 wt%, or even 0 – 15, more preferably 1 – 12 wt%, most preferably 1 – 8 wt%, based on dry weight of the particulate material. Even though the complete absence of protein is optimal, the particulate material may contain some protein for practical reasons. Some protein does not hamper the functioning of the particulate material and therefore does not need to be removed. As further advantage, the low protein content, and low water content as outlined above, of the particulate material greatly reduce the chance of microbial contamination, and therefore increase the survival rate of the larvae. In one embodiment, the particulate material is not fermented. Preferably, the particulate material is selected from hulls, bran and other by-products of seed processing, corn cobs, wood chips, straw chips and plant pulp. Preferably, the particulate material is selected from seed hulls, corn cobs, wood chips and straw chips. Seed hulls are especially preferred as they typically do not contain contaminants that are detrimental to the survival of the larvae. However, other fillers such wood chips are also suitable in case the level of contamination is sufficiently low. If needed, the particulate material can be decontaminated before it is used in step (b). Herein, the chips may be of any particle size, in line with the preferred particle sizes discussed above, and includes sawdust. The particulate material is typically in the form of chips, flakes or sawdust. If needed, the material can be chopped or shredded prior to being used in step (b), to ensure it has the desired particle size. In a preferred embodiment, the particulate material is a seed hull, preferably the hulls of sunflower, wheat, rice, corn, barley, cottonseed, peanut or oat. Excellent results have been obtained with sunflower hulls (SFH). Such hulls typically come in the form of flakes, which are ideally suited as particulate material.
[0034] In a preferred embodiment, the weight of the particulate material is about as much as the weight of the larvae. Preferred weight ratios of the batch of larvae to the particulate material are in the range of 0.5 – 100, such as in the range of 1 – 50 or even in the range of 1 – 25, preferably in the range of 2 – 50, such as in the range of 2 – 25, more preferably in the range of 5 – 50, most preferably in the range of 5 – 25. Advantageously, 1 gram of particulate material is used per 1 thousand – 50 thousand larvae, preferably per 5 thousand – 25 thousand larvae, most preferably per 10 thousand – 22 thousand larvae. Thus, it is preferred that the size of the batch of larvae determines the amount of particulate material that is used.
[0035] As the skilled person will appreciate, the larvae are carefully combined with the particulate, in such a way that the larvae are not damaged. Combining may be accomplished by a simple combining of the two streams, for example by depositing the batch of larvae on top of the particulate material. Excellent results have been obtained when the batch of larvae is placed on top of the particulate. In this respect, it is preferred that substantially all larvae of the batch are in contact with the particulate material, and not with the wall of the container in which the neotenic mixture is placed. This gives optimal results in terms of survival rate and also facilitates the arousal step, where the batch of larvae is typically removed from the container and introduced in a production process located elsewhere. It is not needed and even undesired to actively mix the larvae,e.g.by gentle stirring, with the particulate material. Being mobile, the larvae typically will crawl into the particulate material when placed on top. The crawling is stopped when the mixture is placed under the conditions of step (c), but some crawling during step (b) or the beginning of step (c) is not detrimental. In a preferred embodiment, the larvae are deposited on top of a first layer of the particulate material and a second layer of particulate material is placed on top of the deposited larvae. Such a second layer offers additional protection to the larvae, avoids excessive crawling and eliminates the chance that some larvae crawl out of the container before it can be placed under the conditions of step (c). Herein, the total amount of particulate material is typically split into two more or less equal parts, and one part forms the first layer and the second part forms the second layer.
[0036] Conventional systems to place the larvae into a container can be used. The container is defined in more detail below in context of the neotenic mixture according to the invention. Transferring the larvae to a container is known in the art, and can be done manually as well as automatically,e.g.using a robotic arm. Also, the inventors found that the larvae, even in a very early stage of development, could withstand a certain fall into the container. Typically, the dosing height form the larvae into the container can be anything in the range of 0 – 150 cm, such as 0 – 100 cm, or even 1 – 80 cm. Herein, a dosing height of 0 cm refers to no fall but gently placing the larvae into the container and a dosing height of 100 cm refers to the larvae falling from a height of 100 cm into the container. The dosing into the container may occur with only the batch of larvae, when the particulate material is already placed into the container, or the larvae may be pre-combined with the particulate material and the mixture is dosed into the container. Preferably, the batch of larvae is dosed into a container already comprising the particulate material, as that gives the least (mechanical) stress for the larvae and leads to a storage environment wherein the development of the larvae is suitably delayed during step (c).
[0037] Upon the combining of step (b), the neotenic mixture is obtained, which is subjected to step (c). Preferably, the neotenic mixture obtained in step (b) is subjected directly to step (c), without any intermediate step.
[0038] Step (c) – Storage
[0039] The neotenic mixture obtained in step (b) is subsequently stored in step (c). The conditions during the storage of step (c) are crucial, in order to sufficiently delay development on one hand and for sufficient survival of the larvae after arousal on the other hand. Therefore, the storage of step (c) should occur at a temperature below 15 °C, preferably at a temperature in the range of 3 – 15 °C, preferably in the range of 4 – 12 °C, more preferably in the range of 5 – 10.5 °C, most preferably at about 10 °C, such as 10 ± 2 °C, 10 ± 1 °C or even 10 ± 0.5 °C. Such temperatures are suitable to delay development, but also to sufficiently immobilize the larvae. Crawl-out of larvae from the neotenic mixture within the container should be avoided as much as possible. In case an open container is used, the temperature preferably does not exceed 12 °C, such as a temperature in the range of 3 – 11 °C, preferably in the range of 4 – 11 °C, more preferably in the range of 5 – 10.5 °C. Above these ranges, crawl-out of some larvae during step (c) could be observed, which lowers the survival rate of the larvae. Closed containers can be kept at a slightly higher temperature, as the container lid avoids crawl-out. In case a closed container is used, the temperature should not exceed 15 °C, such as a temperature in the range of 3 – 15 °C, preferably in the range of 4 – 12 °C, more preferably in the range of 5 – 10.5 °C. These temperatures are a marked improvement over methods for delaying the development of larvae that require much lower temperatures, such as around 4 °C or even lower, as the energy expenditure for such cooling is much higher.
[0040] Furthermore, the storage of step (c) should occur at a relative humidity of at least 60 %, preferably at least 65 %, more preferably at least 70 %. Such relative humidities ensure that the larvae do not dry out during the storage of step (c). On the other hand, too high relative humidities may lead to the larvae aggregating or clustering together, which may be detrimental to their survival. Thus, in one embodiment, the relative humidity is at most 90 %, preferably at most 85 %, more preferably at most 80 %. Thus, the relative humidity is preferably in the range of 60 – 90 %, more preferably in the range of 65 – 85 %, most preferably in the range of 70 – 80 %.
[0041] In order to ensure that the storage conditions are maintained for the entire duration of step (c), it is preferred that the storage of step (c) is done in a climate-controlled environment. The conditions that are controlled within the climate-controlled environment are selected from temperature, humidity, oxygen concentration, carbon dioxide concentration and pressure. Typically, at least the temperature and humidity is controlled. Furthermore, since most larvae are photophobic to some extent, it is preferred to keep the larvae in the dark during step (c). Conveniently, it is not needed to establish a flow of air over the neotenic larva. Contrary during developing larvae, where an inflow of oxygen and an outflow of waste gases is desired, the neotenic larvae survive without such an air flow. Thus, in one preferred embodiment, the climate-controlled environment during step (c) has no air flow, or at least an air flow below 1 m3 / h.
[0042] The inventors found that under such storage conditions, the development of the larvae is sufficiently delayed, and / or the growth of the larvae is sufficiently inhibited, such that the larvae remain in the same developmental stage. In other words, the development is halted in the stage at which they are subjected to the conditions of step (c). Moreover, the inventors found that because of the particulate material within the neotenic mixture, the delay in development and inhibition of growth is only temporarily, and is reversed upon arousal of the larvae. The skilled person can easily determine that the larvae remain in the same developmental stage, for example by microscope observation of the larvae, weight determination and size measurements. Such techniques are readily available to the skilled person, such as described by Gligorescuet al.,Development, growth and metabolic rate of Hermetia illucens larvaein J. Appl. Entomol. 2019; 00:1-7.
[0043] Typically, the storage of step (c) starts with placing the neotenic mixture in a climate-controlled environment having the appropriate temperature and relative humidity. Alternatively, the temperature and / or relative humidity may be actively changed,e.g.by cooling, to start the storage of step (c). Advantageously, the environment of the combining of step (b) already has the desired relative humidity for the storage of step (c), and only the temperature needs to be changed (lowered) to start the storage of step (c). In one embodiment, the combining of step (b) is done in an environment which is already at the conditions of step (c).
[0044] In one embodiment, the method according to the invention is used to transport a batch of larvae. Advantageously, the development of the larvae is delayed or stopped during transport. In this embodiment, the batch of larvae is transported during step (c), and typically aroused after arrival at the desired location. Hence, in one embodiment, the batch of larvae is transported during step (c). During such a transport, the batch of larvae is kept in the neotenic stage and development is halted. Hence, measures are taken to keep the conditions as defined herein, such as transport in a climatized container or a climatized vehicle. Especially for longer transports, it is beneficial to delay development of the larvae. Hence, the transport is preferably for over 1 km, such as 1 – 10.000 km or 1 – 5.000 km, more preferably at least 5 km, such as 5 – 10.000 km or 5 – 5.000 km, most preferably at least 10 km, such as 10 – 10.000 km or 10 – 5.000 km. Alternatively, the length of the transport is defined by the duration, such as at least 5 min, such as 5 min – 24 h or 5 min – 10 h, preferably at least 15 min, such as 15 min – 24 h or 15 min – 10 h, preferably at least 1 h, such as 1 – 24 h or 1 – 10 h. It is advantageous to imply larvae of BSF at their 1stinstar (preferably before their first feeding) in the method of the invention, for example, when the method of the invention is used to transport a batch of larvae. This way, the total weight of the neotenic mixture obtained in step (b) is the lowest that can be obtained, therewith saving on energy required for temperature- and humidity control, fuel for transportation, and volume, for example of a climatized reefer or container, required for transportation, to name a few advantages.
[0045] The duration of the storage of step (c) is not crucial in the context of the present invention. Typically, the stored batches of larvae serve as backup and are used whenever needed, and are therefore stored for a prolonged period of time. Typically, the neotenic mixture is stored for 0.1 day to 1 year, or for 0.5 – 140 days, or for 1 – 40 days or even for 1 – 7 days. In one embodiment, the storage is for at least 5 days, more preferably at least 2 weeks, most preferably at least 1 month. Furthermore, storage may last up to 1 year, preferably up to 140 days, or even up to 40 days. In step (c) of the method of the present invention, the larvae may be stored for a short period, such as at least 10 min, such as 10 min – 48 h or 10 min – 24 h, preferably at least 30 min, such as 30 min – 48 h or 30 min – 24 h, or even at least 1h, such as 1 – 48 h or 2 – 24 h. Alternatively, the batch may be stored for longer than 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 130 days or 140 days, and preferably stored for less than 250 days, 240 days, 230 days, 220 days, 210 days, 200 days, 190 days, 180 days, 170 days or 160 days. Preferred storage periods are in the range of 15 days – 48 months, more preferably in the range of 15 – 65 days, even more preferably in the range of 25 – 65 days, most preferably in the range of 25– 45 days.
[0046] The optimal duration of the storage of step (c) may vary from application to application. For example, when a batch of larvae is stored as back-up batch to be used when needed for pupation and development into adult flies, the survival percentage of the larvae is of lesser importance. After all, even if only a few larvae survive, these can be developed into adult flies, and used to produce a new batch of eggs and subsequently larvae. Thus, in one embodiment, the method according to the invention is for preparing and maintaining a back-up batch of larvae, and the duration of step (c) may last up to 1 year.
[0047] Alternatively, when the method is used as buffering step at the start of the production process, the survival rate of the larvae is of the utmost importance, as the entire production process depends on these larvae’s survival to produce the desired valuable nutrients. In the context of this embodiment, the duration of step (c) is typically in the range of 10 min – 48 h or 10 min – 24 h, preferably at least 30 min, such as 30 min – 48 h or 30 min – 24 h, or even at least 1h, such as 1 – 48 h or 1 – 24 h. In this context, the neotenic mixture is placed in a buffer during step (c), wherein several of such batches are collected, and then removed from that buffer regularly for arousal to start the production process. Such a buffer of neotenic mixtures improves the flexibility of the production process, as newly born neonate larvae are always available, even if some batches did not hatch properly. Furthermore, such a buffer allows to cope with peaks and dips in the production process.
[0048] Step (d) – Arousal
[0049] Whenever needed, for example in case of a disruption in the production process of nutrients or if the batch of larvae is needed for another reason, the batch of larvae in a neotenic stage can be aroused. Thus, in one embodiment, step (d) is performed in the method according to the invention. However, the backup batch of larvae may not be needed, in which case step (d) is not performed in the method according to the invention. Step (d) is thus an optional step. In one embodiment, step (d) is performed. In another embodiment, step (d) is not performed. When the method according to the invention is used to buffer the start of a production process, step (d) is performed and marks the start of the rearing of the larvae.
[0050] When aroused, the larvae continue their development at the stage they were when brought into the neotenic stage. Arousal is done by bringing the larvae under growth conditions, which typically involves transferring the larvae to another climate-controlled environment and / or changing the conditions within the climate-controlled environment. Bringing the larvae under growth conditions typically involves warming the batch of larvae to a temperature above 20 °C, such as in the range of 20 – 40 °C, preferably in the range 23 – 38 °C, most preferably in the range of 25 – 35 °C. The relative humidity may also change during arousal, but should at least be kept above 50 %, preferably above 55 %. Preferably, the growth conditions include a relative humidity in the range of 50 – 70 %, more preferably in the range of 51 – 65 %, most preferably in the range of 52 – 60 %. Additionally or alternatively, the specific humidity of the growth conditions may be controlled, for example in the range of 5 – 40 g / kg, preferably in the range of 8 – 5 g / kg, most preferably in the range of 18 – 33 g / kg. The skilled person is able to determine the optimal growth conditions for arousal of the larvae, based on the needs for the species and their developmental stage. In a preferred embodiment, the growth conditions within the climate-controlled environment also involve a specific air flow. Creating an air flow is beneficial for growth of the larvae, as it feeds oxygen towards the larvae and removes waste gases such as CO2and NH3from the larvae. The flow of air depends on the developmental stage of the larvae, as smaller larvae need less oxygen and produce less waste gases, a lower flow suffices. Typically, the flow of air is in the range of 1 – 25 m3 / h, preferably in the range of 2 – 15 m3 / h.
[0051] For a successful arousal, the larvae should be contacted with a suitable feed source, such that the feeding and growth phase can be re-initiated. The preferred conditions in terms of temperature and relative humidity, as well as the preferred nature of the feed, may depend on the developmental stage of the larvae. The skilled person is able to select the appropriate conditions and feed. To arouse the larvae the neotenic mixture can conveniently be combined with an appropriate larva feed, for example by placing the neotenic mixture on top of the feed. When the conditions are changed such that the larvae are able to develop further, the larvae will simply start feeding and continue their development where it was left before the storage of step (c). For example, the neotenic mixture may be placed or dropped in a feeding container or feeding crate containing the desired amount of larvae feed, suitable to feed the batch until the desired developmental stage.
[0052] Part of the arousal of step (d) may be the introduction of the batch of larvae in a production process, for example for starting up a new production process or for replacing a batch of larvae that has become unfit for further use. During such introduction, the requirements for a successful arousal are automatically met, as both the conditions and the feed are specifically selected for the specific developmental stage of the larvae that are introduced.Neotenic mixture of larvae and particulate
[0053] The present invention further concerns the neotenic mixture of the batch of insect larvae and the particulate material. Preferred embodiments of the batch of larvae and of the neotenic mixture are defined above and equally apply to the neotenic mixture of the invention. The neotenic mixture according to the invention typically consists of the batch of larvae as defined above and the particulate material as defined above. Preferably, the neotenic mixture is placed at storage conditions as defined above for step (c), yet also the uncooled mixture, prior to or directly after step (c), is encompassed within the present aspect of the invention.
[0054] The neotenic mixture of the invention is typically contained in a container, such as a carrier or a receptacle. The container may be a closed or an open container. A closed container may be used, which is advantageous as it may prevent spoilage or contamination of the stored larvae. The container may be provided with means for maintaining the desired temperature and humidity as defined above for step (c). The container may be thermally insulated, such that it is able to maintain the reduced temperature of the storage of step (c). However, it is also possible to place the container in a climatized room, wherein the temperature and humidity of the room can be controlled. In such circumstances the container may be open or closed. In one embodiment, the container is closed and provided with holes that are small enough that the larvae cannot crawl through and large enough to enable contact between the environment within the container and the environment outside the container, such that the container may be placed within a climatized room and the conditions in the room also apply within the container. The skilled person is able to determine the size of such holes. Ideally, the diameter of the holes is at most 0.5 × the average diameter of the larvae in the batch. Conveniently, such holes are provided in the lid of the container, but these may also be elsewhere. An open tray with sufficient ventilation and space may be used as a container to store the larvae, which trays may be placed in a climatized rooms. Hence, in one embodiment, the container with the neotenic mixture of the invention may be comprised in a climatized room, which preferably is placed under the conditions as defined above for step (c).
[0055] The container may be of any material suitable to carry insect larvae, such as plastic or metal. Typically, the metal is stainless steel, and the plastic may be selected from polyolefin (e.g.polyethylene, polypropylene, polystyrene or mixed polyolefins such as acrylonitrile butadiene styrene (ABS)), polyamide, polyester (e.g.polyethylene terephthalate) and polycarbonate. Preferably, a plastic container is used, which facilitates removing the neonates from the container, for example before or during arousal. Even though the use of the particulate material substantially avoids that the larvae stick to the surface of the container, which would hamper arousal and further use of the larvae, the inventors found that sticking of the larvae further reduced when a plastic container was used. The loss in larvae yield due to the sticking to the container was minimized with a plastic material. Whenever the neotenic mixture is to be used as backup for a batch in a production process, typically the mixture is removed from the container into the processing system, for example in a crate where the larvae are allowed to further develop. The inventors found that such transfer of the mixture from one container to another is more efficiently performed with a plastic container, with less loss of larvae during the transfer.Examples
[0056] The following examples are intended to illustrate the invention.Example 1
[0057] Batches of 12k, 65k or 100k of neonates of the black soldier fly (larvae of 1stinstar within 10 min after birth) were dosed by larvae weight into a carrier comprising storage medium (the particulate material). A further layer of storage medium was placed on top of the neonates, and the layered mixtures were placed for a specific duration at a temperature of 10 °C. Both layers of storage medium weighted 2 g for the 65k larvae batches, and the corresponding amounts for the 12k and 100k batches. During storage, the larvae did not feed and did not develop further. After the duration of the storage, the larvae were further developed by a conventional feeding regime for 5 days. Survival rates were determined at the end of the feeding regime by taking six samples per experiment, and counting all larvae in these samples. The results were extrapolated to the entire batch, and are averages of at least two tests done in parallel. Details of each experiment and survival rate results are given in Table 1 below.
[0058] Table 1: Survival experiments and resultsExpBatch sizeCarrier materialMediumParticle sizeDurationSurvivalMedium comparison165kstainless steelSFHmix4 h97.1 %265kstainless steelsawdust> 0.6 mm4 h90.4 %365kpolyamide (white)SFHmix4 h93.5 %465kpolyamide (white)sawdust> 0.6 mm4 h95.8 %Particle size comparison565kstainless steelSFHmix4 h97.1 %665kstainless steelSFH< 0.6 mm4 h98.4 %765kstainless steelSFH0.6-1 mm4 h99.4 %865kstainless steelSFH> 1 mm4 h95.7 %Carrier comparison965kstainless steelSFHmix4 h97.1 %1065kABSSFH0.6-1 mm4 h96.6 %1165kpolyamide (white)SFHmix4 h93.5 %1265kpolyamide (black)SFHmix4 h99.8 %1365kpolypropyleneSFHmix4 h100.1 %Batch size comparison1412kstainless steelSFHmix4 h108.4 %1565kstainless steelSFHmix4 h97.1 %16100kstainless steelSFHmix4 h102 %Duration test (1)1765kstainless steelSFHmix1 h102.1 %1865kstainless steelSFHmix2 h100.1 %1965kstainless steelSFHmix3 h100.2 %2065kstainless steelSFHmix4 h97.1 %2165kstainless steelSFHmix20 h105.2 %2265kstainless steelSFHmix24 h103 %2365kstainless steelSFHmix48 h84.4 %Duration test (2)2465kpolyamide (white)SFHmix4 h93.5 %2565kpolyamide (white)SFHmix24 h95.3 %2665kpolyamide (white)SFHmix29 h86.5 %Duration test (3)2765kABSSFH0.6-1 mm4 h96.6 %2865kABSSFH0.6-1 mm19 h101.4 %2965kABSSFH0.6-1 mm24 h96 %
[0059] The experiments show that survival of the neonates was not significantly affected by prolonged storage at 10 °C. Both sunflower husk (SFH) and sawdust as particulate material for storage gave good survival rates. Sawdust was less preferred than SFH because of its fine and dusty nature, which hampered downstream processing and gave potential problems with apparatus mechanics. SFH of all particle sizes gave good neonate survival, however the intermediate particles of 0.6 – 1 mm gave the best results. Even the worst performing larger particles (> 1 mm) gave a survival rate of 95.7 %.
[0060] Several carrier materials have been tested, with similar results. It was thus concluded that the type of carrier had no influence on the survival of the neonates. Neonates in stainless steel carriers had a greater tendency to stick to the carrier when transferred after the duration of the experiment to determine the survival rate.
[0061] The results show that the size of the batch does not affect survival rates. Excellent survival rates were obtained with batches of 12k, 65k or 100k neonates. In view of the low amounts of larvae, the survival results of the 12k batch are slightly overestimated.
[0062] Similar experiments have been performed at 11 °C (data not shown), which gave similar survival rates but showed some neonate crawl-out. Hence, some neonates had still sufficient activity to move and crawl out of the carrier, which slightly reduced the yield of neonates at the end of the experiment.Example 2
[0063] Batches of 65k of neonates of the black soldier fly (larvae of 1stinstar within 10 min after birth) were dosed into a carrier comprising 2 g of the storage medium (the particulate material). A further layer of 2 g of storage medium was placed on top of the neonates, and the layered mixture was placed at a temperature of 12 °C for 3 h. During storage, the larvae did not feed and did not develop further. After the duration of the storage, the larvae were further developed by a conventional feeding regime for 5 days. Survival rates were determined at the end of the feeding regime by taking six samples per experiment, and counting all larvae in these samples. The results were extrapolated to the entire batch, and are averages of at least two tests done in parallel. Survival rate results are given in Table 2 below.
[0064] Table 2: Survival rate resultsStorage mediumAverage particle sizeSurvivalSFH< 0.6 mm96.6 %SFH0.6 – 1.0 mm94.9 %SFH> 1.0 mm98.9 %Dried frass0.5 – 2.0 mm98.4 %Wheat bran (untreated)0.5 – 2.0 mm98.2 %Wheat bran (milled)0.2 – 1.0 mm96.2 %Rapeseed (milled)0.5 – 2.0 mm86.2 %Example 3 – comparative
[0065] Batches of 65k of neonates of the black soldier fly (larvae of 1stinstar within 10 min after birth) were dosed into a carrier comprising water at a temperature of 14 °C for 3 h. During storage, the larvae did not feed and did not develop further. After the duration of the storage, the larvae were further developed by a conventional feeding regime for 5 days. Survival rates were determined at the end of the feeding regime by taking six samples per experiment, and counting all larvae in these samples. The results were extrapolated to the entire batch, and are averages of at least two tests done in parallel. Survival rates after 24 h of storage was not consistent and varied between 66 % and 90 %. Survival dropped further after 48 h of storage in the ranges of 45 – 65 %. The survival rates at 24 h were considered unsatisfactory for using that batch as back-up or to start an economically viable production process of valuable nutrients.
Claims
A method for delaying the development of insect larvae, comprising:(a) providing a batch of insect larvae;(b) combining the batch of larvae with a particulate material to obtain a neotenic mixture, wherein the particulate material is an animal feed filler;(c) storing the neotenic mixture at a temperature below 15 °C and a relative humidity of at least 60 %.The method according to claim 1, wherein the larvae areHermetia illucenslarvae, preferably in their 1stinstar stage.The method according to claim 1 or 2, wherein the storing of step (c) is for a prolonged period of time, preferably at least 10 min, more preferably 10 min – 48 h.The method according to any of the previous claims, which further comprises:(d) arousing the batch of larvae from the neotenic mixture after the storage of step (c) and introducing it in a process for the production of nutrients.The method according to any of the previous claims, which is for preparing one or more backup batches for larva batches that are used in a process for the production of nutrients or for providing a buffer at the start of a process for the production of nutrients.The method according to any of the previous claims, wherein the batch of insect larvae contain 500 to 1.500.000 larvae, of which 90 – 100 % by weight are of a single species, and the age difference between the larvae is less than 24 hours.The method according to any of the previous claims, wherein the particulate material has a protein content of at most 20 wt%, based on dry weight.The method according to any of the previous claims, wherein the particulate material has an average particle diameter which is 0.1 – 20 times the average length of the larvae.The method according to any of the previous claims, wherein the particulate material is selected from seed hulls, corn cobs, wood chips and straw chips.The method according to any of the previous claims, wherein the combining of step (b) employs 1 gram of particulate material per 1 thousand – 50 thousand larvae in the batch, preferably per 5 thousand – 25 thousand larvae, most preferably per 10 thousand – 22 thousand larvae.A neotenic mixture comprising a batch of insect larvae and a particulate material, wherein the particulate material is an animal feed filler.The neotenic mixture according to claim 11, wherein the batch of insect larvae contains 500 to 1.500.000 larvae, of which 90 – 100 % by weight areHermetia illucenslarvae, preferably in their 1stinstar stage, and the age difference between the larvae is less than 24 hours.The neotenic mixture according to claim 11 or 12, wherein the particulate material is selected from seed hulls, corn cobs, wood chips and straw chips.The neotenic mixture according to any one of claims 11 – 13, wherein the neotenic mixture is comprised in a container at a temperature below 15 °C and a relative humidity of at least 60 %.The neotenic mixture according to claim 14, wherein the container is placed in a climate room.