Patent Description:
Urban solid waste management is considered one of the most immediate and serious environmental problems confronting urban governments. The severity of this challenge will increase in the future given the trends of rapid urbanisation and growth in urban population. At present recycling organic waste material is still fairly limited, although this is by far the largest fraction of all generated municipal waste.

A fairly novel approach to bio-waste conversion is by insect larvae, using for example the black soldier fly (Hermetia illucens), the house fly (Musca domestica), and the meal worm (Tenebrio molitor L. Their popularity links to the promising opportunities of using the harvested larvae as a source of protein and fat for animal feed or food products, thus providing a valuable alternative to other (animal) sources of protein and fat.

Even though many people dislike the consumption of whole insects, the use of insects as a source for separated fats and proteins, which are subsequently used in the preparation of food and feed is more acceptable. However, in order for insects to become a commercially viable nutrient source their production and processing should be carried out on a large scale and the nutrient streams obtained should be free from unwanted flavours and discolorations.

Although large scale insect production facilities are already in use, the economical preparation of nutrient streams of sufficient quality remains a challenge. In the international patent application <CIT> a general method has been described for obtaining nutrient streams from insects or worms. Despite the fact that the methods disclosed therein are able to provide nutrient streams of sufficient quality, a need remains for further increasing the yield of such methods and the quality of the nutrient streams obtained therewith.

Insects are commonly consumed as food in many cultures around the world. In European countries, insect proteins are gaining rapid acceptance as high value protein ingredients in animal diets. The European Union has already approved the inclusion of insect proteins in pet food and aquaculture feed formulations. Chicken meal and fish meal are common ingredients in pet food and aquaculture feed preparations, respectively. Insect proteins are increasingly being viewed as an alternative to chicken meal and fish meal in these markets. Amongst all the insect being produced on industrial scale, black soldier fly (Hermetia illucens) larvae has gained special attention due to its ability to grow on wide range of organic residues and unique nutritional composition. The nutritional suitability of black soldier fly larvae (BSF) proteins in aquaculture and pet diets is well established.

Pets develop wide range of health disorders with age. Aging can accelerate the free radical damage in a pet's body which might lead to cognitive and locomotor system malfunctioning. Similarly oxidative stress in fishes as a result of immune response could lead to compromised health. Neutrophils (white blood cells) are responsible for the primary defense mechanism of the body. Upon receiving the signal, neutrophils rush to the site of intrusion by pathogenic microbes. Neutrophils then inactivate the pathogens by phagocytosis and release of reactive oxygen species (ROS). Production of ROS is crucial for the host defense. However, in the long term, excessive ROS production by neutrophils could damage animal cells and might lead to cellular aging, cancer, reduced immunity, etc. Dietary interventions that can scavenge ROS may help in reducing oxidative damage in the animal body and resulting health conditions. The field is in need of such dietary interventions.

The present invention aims to further improve the yield and quality of the nutrient streams obtained from insects over methods known in the prior art.

A first aspect of the present invention relates to a method to convert black soldier fly larvae into nutrient streams, comprising the steps of:.

An aspect of the present invention relates to a method to convert insects into nutrient streams, comprising the steps of:.

thereby obtaining an black soldier fly larvae puree (pulp). The nutrient streams is a black soldier fly larvae puree obtained after the heating of the prepared pulp from fresh Black Soldier Fly (BSF, black soldier fly) larvae. The method comprises a further step c) after step b): subjecting the heated pulp to a physical separation step thereby obtaining a fat fraction, an aqueous protein fraction and a solid-containing fraction. Drying of the protein fraction reveals protein meal obtained from BSF larvae.

An aspect of the present invention relates to a method to convert black soldier fly larvae into nutrient streams, comprising the steps of:.

thereby obtaining a hydrolysed black soldier fly larvae puree. The nutrient streams is a hydrolysed black soldier fly larvae puree obtained after the heating of the hydrolysed pulp, the pulp obtained from fresh black soldier fly larvae. The method comprises a further step c) after step b): subjecting the heated and hydrolysed pulp to a physical separation step thereby obtaining a fat fraction, an aqueous protein fraction and a solid-containing fraction. Drying of the protein fraction reveals hydrolysed protein meal obtained from BSF larvae.

It has been found that with the method of the present invention it is possible to increase the yield of fat from the processed black soldier fly larvae. It has surprisingly been found that by using the present time-temperature combination less oil is entrapped with the protein fraction. Since the method of the invention results in less protein aggregation and granulate formation in step b) and step c) of the invention, when compared to methods applying heating times for longer than <NUM> seconds such as at least <NUM> minutes, less oil can become entrapped at and inside such protein aggregates (without wishing to be bound by any theory). It has also been found that discolouration of the pulp and the nutrient fractions is reduced. The method of the present invention also saves energy as considerably less time is needed to heat the pulp as has previously been suggested in the prior art. Furthermore, the inventors found that the heated black soldier fly larvae pulp and the heated hydrolysed black soldier fly larvae pulp have anti-oxidant properties.

In an embodiment, the method to convert black soldier fly larvae into nutrient streams, comprises the steps of:.

An aspect not part of the presently claimed invention relates to heated black soldier fly larvae puree, obtained with the method of the invention. Such heated black soldier fly larvae puree surprisingly has anti-oxidant properties and is suitable for use as an anti-oxidant feed or food or ingredient thereof.

An aspect not part of the presently claimed invention relates to hydrolysed black soldier fly larvae puree. Hydrolysis of black soldier fly larvae pure before the heating step, under influence of e.g. a peptidase, results in a puree with improved anti-oxidant activity. Moreover, protein separated from such hydrolysed puree comprises improvingly less fat compared to non-hydrolysed puree which is obtained according to the method of the invention, and compared to non-hydrolysed puree which is conventionally obtained by applying an extended heating step of e.g. <NUM> minutes.

An aspect not part of the presently claimed invention relates to heated black soldier fly larvae puree, obtained with the method of the invention. Such heated black soldier fly larvae puree surprisingly has anti-oxidant properties and is suitable for use as a health promoting feed or food or ingredient thereof.

An aspect not part of the presently claimed invention relates to hydrolysed black soldier fly larvae puree. Hydrolysis of black soldier fly larvae pure before the heating step, under influence of e.g. a peptidase, results in a puree with improved anti-oxidant properties. Moreover, protein separated from such hydrolysed puree comprises improvingly less fat compared to non-hydrolysed puree which is obtained according to the method of the invention, and compared to non-hydrolysed puree which is conventionally obtained by applying an extended heating step of e.g. <NUM> minutes.

An aspect not part of the presently claimed invention relates to black soldier fly larvae pulp obtainable by steps a) and b), or steps a), a1) and b), of the method according to the present invention.

An aspect not part of the presently claimed invention relates to the use of the hydrolysed black soldier fly larvae pulp as an antioxidant food ingredient or feed ingredient, wherein the (hydrolysed) pulp is obtainable with the method of the invention, the method either or not involving the enzymatic hydrolysis step a1) prior to step b) of the method.

An aspect not part of the presently claimed invention relates to the use of the hydrolysed black soldier fly larvae pulp as a health-promoting food ingredient or feed ingredient, wherein the (hydrolysed) pulp is obtainable with the method of the invention, the method either or not involving the enzymatic hydrolysis step a1) prior to step b) of the method.

An aspect not part of the presently claimed invention relates to the use of the hydrolysed black soldier fly larvae pulp as a food ingredient or as a feed ingredient, wherein the (hydrolysed) pulp is obtainable with the method of the invention, the method either or not involving the enzymatic hydrolysis step a1) prior to step b) of the method.

An aspect not part of the presently claimed invention relates to the black soldier fly larvae pulp or the hydrolysed black soldier fly larvae pulp or derivative thereof for use as feed or feed ingredient, in particular pet food.

An aspect not part of the presently claimed invention relates to the use of the black soldier fly larvae pulp or the hydrolysed black soldier fly larvae pulp or derivative thereof as a food or feed ingredient with health promoting potential.

An aspect not part of the presently claimed invention relates to the black soldier fly larvae pulp or the hydrolysed black soldier fly larvae pulp or derivative thereof for use in a method for the prevention and/or suppression of cellular oxidative damage.

The term "insects" as used herein refers to insects in any development stage, such as adult insects, insect larvae, insect prepupae and pupae.

The term "hatching" as used herein has its conventional meaning and refers to the process of young larvae emerging from the egg.

The term "larvae" as used herein has its conventional meaning and refers to the juvenile stadium of holometabolous insects, such as black soldier flies larvae.

The term "hatchling" or "neonate" as used herein has its conventional meaning and refers to larvae that have just hatched from the eggs.

The term "prepupae" as used herein refers to the last larval stage wherein the chitin content of the larvae has increased significantly.

The term "pupae" as used herein has its conventional meaning and refers to the stage of the insects life wherein the metamorphosis from larva to adult insect, such as black soldier flies.

The term "pulp" or "insect pulp" or "puree" or "larvae puree" as used herein all have the same meaning and refers to the product obtained after mechanical size reduction of the insects to a size of less than <NUM>.

The term "nutrient stream" as used herein has its conventional meaning and refers to streams that contain nutrients, such as fats, protein and protein-derived material, carbohydrates, minerals and/or chitin. Within the context of the present invention, chitin is also considered a nutrient.

The term "anti-oxidant property" has its regular scientific meaning and here refers to a compound or a composition that consists of or comprises an antioxidant with antioxidant activity, such as an anti-inflammatory response. Typically, such an anti-inflammatory response is a response to the inflammatory response induced by for example reactive oxygen species, e.g. in the cells of a mammal such as a pet or a human subject, or in the cells of a fish. Reference is made to for example book chapters <NUM>, <NUM> and <NUM> of "<NPL>). An anti-oxidant is a compound with anti-oxidant activity or a composition with anti-oxidant activity or a composition comprising a compound with anti-oxidant activity, such as activity against the oxidative damage resulting from host immune response. An anti-oxidant for example inhibits oxidation. Oxidation, e.g. reactive oxygen species, in a subject for example induces cellular (oxidative) damage.

The term "health promoting", such as in 'health promoting food', 'health promoting activity', 'health promoting property', and 'health promoting potential', has its regular scientific meaning and here refers to the effect of a compound or a composition on the health of an animal such as a mammal such as a pet animal or a human subject, when such compound or composition is consumed by the animal. Consumption of the compound or composition with health promoting potential contributes to or supports or promotes or increases or maintains the health status of the animal such as a mammal such as a pet animal or a human subject. Reference is made to for example book chapters <NUM>, <NUM> and <NUM> of "Antioxidants in<NPL>).

It has surprisingly been found that with the method of the present invention it is possible to increase the yield of fat from the processed black soldier fly larvae. It has further been found that by using the present time-temperature combination less oil is entrapped with the protein fraction. It has also been found that discolouration of the pulp and the nutrient fractions is reduced. The method of the present invention also saves energy as considerably less time is needed to heat the pulp, in contrast to time needed for methods previously suggested in the prior art.

thereby obtaining an black soldier fly larvae puree (pulp). The nutrient streams is an insect puree obtained after the heating of the prepared pulp from fresh black soldier fly , (BSF, black soldier fly) larvae. The method comprises a further step c) after step b): subjecting the heated pulp to a physical separation step thereby obtaining a fat fraction, an aqueous protein fraction and a solid-containing fraction. Drying of the protein fraction reveals protein meal obtained from BSF larvae.

The enzyme-treated black soldier fly larvaepulp such as minced larvae, and (control) black soldier fly larvaepulp without enzyme treatment (e.g. minced larvae that were not enzymatically hydrolysed), are for example heated to <NUM> and the pulp is for example kept at this temperature for <NUM> seconds. The obtained black soldier fly larvaepuree can be subjected to tests for measuring free amino acid content and digestibility. Protein meal can be obtained from the heated BSF larvae pulp, and from the heated pulp after enzyme treatment before the heating step. For example, protein meal is obtained when batches of heated BSF larvae pulp that was first subjected to enzymatic hydrolysis of proteins using either <NUM> wt%, or <NUM> wt% Flavourzyme prior to the heating step at <NUM>, e.g. by steps of protein separation from the pulp, evaporation, drying and grinding. It is part of the invention that fat is separated from the protein fraction to a higher extent when prior to heating the black soldier fly larvaepuree (BSF larvae puree) is enzymatically treated, compared to black soldier fly larvaepuree that is not enzymatically treated prior to heating. According to the invention, a dose dependent extent of fat separation from protein is obtained, when the amount of applied enzyme is considered. Thus, according to the invention, enzymatic treatment of e.g. minced larvae such as minced BSF larvae, using Flavourzyme prior to the heating step increases the free amino acid content and increases pepsin digestibility of the obtained hydrolysed larvae puree, and as a result, the obtained puree comprising hydrolysed protein has a better taste (free amino-acid content relates to attractive, appealable taste when animals and humans consume a product comprising free amino-acids), is highly digestible and has anti-oxidant properties (see test results, here below), according to the invention. In addition, enzymatic treatment of BSF larvae pulp improves the fat separation in the following separation step after enzymatic hydrolysis and heat-treatment of the enzymatically digested pulp, which increases the fat extraction from the protein meal, when compared to fat separation from the protein fraction obtained with larvae puree that was not subjected to an enzymatic hydrolysis step prior to heating at <NUM>, according to the invention.

Although in the prior art methods are described for separating nutrient streams from insect pulp it has thus far not been recognized that a relatively mild treatment, i.e. relatively low and short temperature/time treatments could result in an increase yield of fat. In this regard reference is made to <CIT> wherein a method for separating nutrient streams is described, but wherein it was considered that in order to increase the yield of fat obtained from the insect pulp relatively long and high temperature/time combinations should be used.

However, using long and high temperature/time combinations has a negative effect on the quality of the nutrient streams, in particular the fat fraction obtained. For example, if the insect pulp is heated for a relatively long time at a relatively high temperature a discolouration of the pulp occurs. Furthermore, since the fat fraction of the processed black soldier fly larvae contains a relatively high amount of unsaturated fatty acids, the fat becomes rancid. This leads to off-flavours of the fat fraction and limits its scope of use.

With the method of the present invention these problems are largely overcome. First of all, with the method of the present invention discolouration of the black soldier fly larvae pulp is greatly reduced. A further surprising advantage of the present method is that the fat fraction obtained from the black soldier fly larvae pulp is less rancid than fat fractions obtained with methods known in the art. Moreover, the yield of fat from the processed black soldier fly larvae is also considerably higher. Without wishing to be bound by any theory it is assumed that when using known methods a considerable part of fat is entrapped within denaturated proteins. By using milder treatment conditions this entrapment of fat can be avoided, resulting in a higher yield of fat from the black soldier fly larvae pulp. Moreover, Maillard reactions with the proteins is occurring to a lesser extent due to the short heating time at mild temperature.

An embodiment is the method according to the invention, wherein the black soldier fly larvae are between <NUM> and <NUM> days of age, preferably between <NUM> and <NUM> days, more preferably <NUM>-<NUM> days, most preferably <NUM> hours-<NUM> days before the larvae transform into prepupae, such as <NUM>-<NUM> days before transformation.

An embodiment is the method according to the invention, wherein the pulp is not enzymatically treated prior to heating.

An embodiment is the method according to the invention, wherein the method further comprises a step a1) of treating the pulp by an enzyme prior to the step b).

An embodiment is the method according to the invention, wherein the method further comprises a step a1) of treating the pulp by an enzyme prior to the step b) and wherein the pulp is treated by the enzyme for <NUM> to <NUM> hours, preferably <NUM> to <NUM> hours at a temperature of <NUM> to <NUM>, preferably at <NUM> to <NUM>, more preferably at a temperature of <NUM> ± <NUM>.

An embodiment is the method according to the invention, wherein the method further comprises a step a1) of treating the pulp by an enzyme prior to the step b) and wherein the enzyme is a protease such as a peptidase, preferably a mixture of at least one protease and at least one peptidase, such as Flavourzyme.

An embodiment is the method according to the invention, wherein in step a) a pulp is prepared by mincing the black soldier fly larvae.

An embodiment is the method according to the invention, wherein the average particle size of the remains of the black soldier fly larvae in the pulp of step a) range between <NUM> and <NUM> micron.

An embodiment is the method according to the invention, wherein in step b) the pulp is heated for <NUM>-<NUM> seconds at a temperature of <NUM> ± <NUM>.

An embodiment is the method according to the invention, wherein the physical separation step comprises decanting and/or centrifugation.

An embodiment is the method according to the invention, wherein the aqueous protein fraction and the solid containing fraction are dried together after step (c), preferably by spray-drying, more preferably fluidized bed drying.

An embodiment is the method according to the invention, wherein the aqueous protein fraction and the solid-containing fraction are dried separately after step c), wherein the aqueous protein fraction is preferably dried by spray drying, fluidized bed drying, lyophilisation or refractive drying, or a combination thereof.

As a result of the separation step, i.e. step c), a fat fraction, an aqueous protein fraction and a solid containing fraction are obtained. In this way, the method results directly in several nutrient streams.

The larvae are preferably made into a black soldier fly pulp before they become prepupae. Although it is possible to use prepupae for preparing insect pulp their chitin content is relatively high compared to 'normal' larvae before their transformation into prepupae, making them less suitable as a nutrient source for human or animal consumption. It is highly preferred to use larvae of the Black Soldier Fly, and most preferably the larvae have been cultivated but have not yet reached the prepupa stage. Typically they have been allowed to grow under normal growth conditions for <NUM>-<NUM> days, preferably <NUM>-<NUM> days, more preferably <NUM>-<NUM> days after hatching such as <NUM>, <NUM>, <NUM>, <NUM>, <NUM> days. For the aim of maximum yield of nutrient streams from the larvae, it is preferred that the larvae are subjected to the method of the invention shortly before the transformation of the larvae into the prepupae phase.

Therefore, it is advantageous to subject larvae to the method of the invention <NUM> hours to <NUM> days before the larvae start to transform into prepupae, preferably <NUM> day to <NUM> days before transformation into prepupae. Larvae such as Black soldier fly larvae, are at their largest size, containing maximum amounts of protein and fat compared to earlier and later stages in the larvae life cycle, when shortly before the transformation phase into prepupae. Moreover, larvae that start to transform intro prepupae discolour from light-yellow into gray/brown when larvae of Black soldier fly are considered, which is not desired when lightly coloured white/yellow protein is desired. Insect pulp and protein derived therefrom which has a darker, brownish/grayish colour due to application of prepupae in the method of the invention, is perceived as a product with inferior quality when compared to a lighter coloured product with a white/yellow appearance. In this regard it is noted that the options and preferences mentioned below are carried out with insect pulp derived from such Black Soldier Fly larvae.

The preparation of a pulp from the black soldier fly larvae- as referred to in step a) of the method of the present invention - may be done in different ways. One option is to squash the black soldier fly larvae to obtain a pulp. Preferably, mincing, cutting or milling is used for preparing the pulp. This results in a homogeneous starting material of viscous consistency. The reducing in size can conveniently be done in a micro-cutter mill, although other suitable techniques may also be used. During this step, the particle size of the black soldier fly larvae remaining in the pulp is preferably less than <NUM> micron, preferably between <NUM> and <NUM> micron (largest size determined by using a microscope). The particle size can be controlled by the selection of specific knife and plate combination and rotating speed in case of a micro-cutter mill. It is also possible to use a sieve (e.g. made of stainless steel) and push the black soldier fly larvae through it. In principle a small remaining particle size is advantageous as it facilitates the fat extraction from the pulp, however a too small particle size could create an emulsion making it more difficult to separate the fat and protein in the next process steps. Hence, the particle size is preferably larger than <NUM> micron. Preferably, the average particle size (d<NUM>; volume/average) ranges between <NUM> and <NUM> micron.

In the following step, step (b), the pulp is heated to a temperature of <NUM>-<NUM>. The heating assures that the majority of fats is liquefied in order to prepare a suitable mixture for the following separation step. Preferably the heating is effectuated under mixing conditions to promote separation of different phases, such as the watery phase and fat phase. In order to avoid discolouration (e.g. by formation of Maillard reaction products) and oxidation of the fat and to improve fat yield the pulp is only heated at said temperature for a relatively short time, i.e. for <NUM> to <NUM> seconds. Preferably the pulp is heated for <NUM>-<NUM> seconds at a temperature of <NUM> ± <NUM>.

After heating the pulp to the above mentioned temperature, the pulp is subjected to a physical separation step, i.e. step (c) of the method of the present invention. During this step the various nutrient streams (such as fat and protein) are at least partly separated from each other. However, as explained above, the yield of fat is considerably higher with the method of the present invention as with methods known in the art. Furthermore, also the quality of the nutrient streams has improved. The nutrient streams obtained are a fat containing fraction, an aqueous protein containing fraction and a solids containing fraction. The physical separation preferably encompasses decanting, centrifugation, or a combination of these methods. Preferably, the physical separation is performed at a normal (atmospheric) pressure.

The fat may be separated by means of decanting, and the remaining mixture is further separated into an aqueous protein fraction and a solid-containing fraction by decanting or centrifugation. However, it is also possible to obtain the fat, protein and solid-containing fractions in a different order, or simultaneously, e.g. by using a <NUM>-phase decanter. An advantage of such a simultaneous method is that the three nutrient streams are obtained with a minimum of steps and thus with minimal losses of the product. Reducing the number of separation steps has also advantages when used in a continuous process.

In an embodiment of the present invention, a fat fraction is separated first, e.g. by decanting, and the remaining mixture is not further separated but subjected to drying. The remaining mixture therefore combines both the solid fraction and the aqueous protein fraction. In this embodiment, the non-fat phases are preferably further dried to produce dried material. The dried material is protein rich and contains both the protein-rich material from the aqueous protein fraction and solid-containing fraction. Since it is possible with the method of the present invention to better separate the fat from the protein a higher quality product is obtained. Furthermore, with the method of the present invention discolouration (of protein) due to Maillard reactions is largely avoided, thereby improving the quality of the product obtained. Drying can be effected by different methods, such as air drying, fluidized bed drying, drum drying, disc drying, flash drying, lyophilisation, refractive drying or preferably by means of spray drying.

In a further embodiment the aqueous protein fraction and the solid-containing fraction are dried separately after step c). Due to the relatively mild heating conditions at least a part of the proteins present in the aqueous protein fraction will have retained their functionality. It would be beneficial not to lose such functionality during subsequent drying. Hence, fluidized bed drying or spray drying or freeze drying or refractive drying is preferred in such circumstances.

An aspect not part of the presently claimed invention relates to heated black soldier fly larvae puree, obtained with the method of the invention. Such heated insect puree surprisingly has anti-oxidant properties and is suitable for use as a health promoting feed or food or ingredient thereof.

The solid-containing fraction obtained by the method of claim <NUM> comprises a pulp having a dry matter content of <NUM>% by weight or more. This pulp can easily be distinguished and separated from the aqueous protein fraction. The pulp contains solids such as chitin and derivatives thereof. The solid-containing fraction may also comprise residual fat or protein, however, to a lesser extent than in known methods. This solid-containing fraction may be further dried and chitin may also be isolated therefrom. The chitin obtained may be used as an ingredient in food or feed. An aspect not part of the presently claimed invention relates to the use of the hydrolysed black soldier fly larvae pulp as a health-promoting food ingredient or feed ingredient, wherein the (hydrolysed) pulp is obtainable with the method of the invention, the method either or not involving the enzymatic hydrolysis step a1) prior to step b) of the method.

The inventors surprisingly established and analyzed in vitro anti-oxidant activity of black soldier fly proteins and protein hydrolysates using radical scavenging models (DPPH and ABTS assays), enzymatic models involving myeloperoxidases response (classical and SIEFED assays), and a cellular model involving neutrophil response. Commercial fish meal and chicken meal were used as industrial benchmarks in comparative examples. Outcomes of the tests and comparisons made by the inventors reveal that fish meal and chicken meal offer little to no advantage in terms of suppressing the oxidative damage occurring as a result of neutrophil and myeloperoxidase response. Moreover, fish meal and chicken meal also exhibit pro-oxidant behavior in some of the test assays and models used. Results show that black soldier fly proteins (as part of (hydrolysed) larvae puree) are effective in protecting against the cellular damage resulting from host neutrophil and myeloperoxidase response. Therefore, black soldier fly derivatives (puree, hydrolysed puree) tested by the inventors show advantages over chicken meal and fish meal for inclusion in pet food/feed and aquaculture feed formulations, such as use as a feed ingredient.

The inventors established that BSF proteins hydrolysates have a significant share of proteins <<NUM> Da. This includes a mixture of short chain peptides and free amino acids. Some short chain peptides and free amino acids are known to possess anti-oxidant activity. These molecules can actively scavenge ROS and free radicals. Firmansyah and Abduh [<NUM>] evaluated the DPPH (<NUM>,<NUM>-diphenyl-<NUM>-picrylhydrazyl) scavenging activity of BSF protein hydrolysates. [<NUM>] evaluated the DPPH, ABTS (<NUM>,<NUM>'-azino-bis(<NUM>-ethylbenzothiazoline-<NUM>-sulfonic acid), superoxide and hydroxyl radical scavenging activity of BSF protein hydrolysates. No studies have been realized till date, to the best of the knowledge of the inventors, that established the antioxidant activity of BSF protein hydrolysate using fundamental enzymatic and cellular models, showing the surprisingly high anti-oxidant activity and surprisingly high protection against oxidative stress and/or damage by the BSF larvae puree and the hydrolysed BSF larvae puree. Therefore, before the currently established invention, the antioxidant potential of BSF protein hydrolysates is poorly understood on a fundamental level and was not yet disclosed, nor anticipated, nor expected. According to the invention, detailed investigations on the in vitro antioxidant activity of BSF proteins and protein hydrolysates unlock new applications of these protein derivatives to improve animal health.

The inventors unraveled the surprisingly high antioxidant potential of BSF proteins and protein hydrolysates, using: (<NUM>) radical scavenging models involving DPPH and ABTS; (<NUM>) enzymatic models involving myeloperoxidases response; and (<NUM>). a cellular model involving neutrophil response. Chicken meal and fish meal were used as industrial benchmarks in comparative examples for comparison with the anti-oxidant activities of the (hydrolysed) BSF larvae puree.

The inventors surprisingly established that black soldier fly larvae puree heated at <NUM> for <NUM> has relatively high amounts for free fatty acids (approx. See for example Example <NUM>, relating to subjecting minced black soldier fly larvae to the method of the invention. Heating puree just for <NUM> is ineffective in the deactivation of lipase. Whereas, low amounts of free fatty acids are observed when puree is heated for <NUM>, indicating the sufficiency of this time for enzyme inactivation at <NUM>. At heating times of over <NUM> seconds, such as up to <NUM> seconds or <NUM> seconds, the fatty acid content increases when compared to the low fatty acid content obtained when the black soldier fly larvae puree is heated for <NUM> seconds. Therefore, for obtaining a protein fraction which comprises a relatively low free fatty acid content with the method of the invention, the heating time in the method of the invention should be longer than <NUM> seconds and shorter than <NUM> seconds, such as <NUM> - <NUM> seconds, or a heating time selected from <NUM> - <NUM> seconds, or <NUM> - <NUM> seconds, such as about <NUM> seconds. Preferred heating time is about <NUM> or <NUM> ± <NUM>.

Surprisingly, the inventors established that higher peroxide value is observed for heating duration of less than <NUM> seconds, such as about <NUM>, when minced black soldier fly larvae pulp such as puree obtained with mincing black soldier fly larvae, is subjected to the method steps of the invention, though with a heating step duration of less than <NUM> seconds (therewith deviating from the method of the invention). The high amounts of free fatty acids that could participate in enzyme oxidation leading to the production of hydroperoxides, obtained when the puree or pulp is heated for <NUM> seconds, is at the basis for the occurrence of the relatively high amount of peroxides. However, when insect pulp or puree as puree obtained from black soldier fly larvae, is subjected to the method of the invention, with a heating step of <NUM> treatment, a relatively lower peroxide value is obtained, compared with the value obtained when heating for shorter than <NUM> seconds. Heating the product (e.g. puree from larvae) for a longer period than <NUM> seconds or less according to the method of the invention, such has for <NUM> seconds or <NUM> seconds, has an effect on fat oxidation in that the peroxide value increases compared for example to the peroxide value when the puree or pulp is heated for a time of <NUM> seconds. Therefore, for obtaining a protein fraction which comprises a relatively low peroxide value using the method of the invention, the heating time in the method of the invention should be longer than <NUM> seconds and shorter than <NUM> seconds, such as <NUM> - <NUM> seconds, or a heating time selected from <NUM> - <NUM> seconds, or <NUM> - <NUM> seconds, such as about <NUM> seconds. Preferred heating time is about <NUM> or <NUM> ± <NUM>.

Surprisingly, the inventors established that the protein fraction obtained with the method of the invention has radical scavenging activity. The radical scavenging activity is higher than the radical scavenging activity obtainable with conventional fish feed fed to fish, when the oxidation state of fish flesh is assessed after feeding the fish feed comprising a series of doses of black soldier fly larvae protein obtained with the method of the invention (lowest dose: <NUM>% BSF larvae protein in the feed; highest dose: <NUM>% BSF larvae protein; <NUM>% and <NUM>% are also tested, based on the total mass of the fish feed fed to fish). See also the examples section, here below, Example <NUM>. ABTS radical scavenging activity of fish flesh after the fish were fed previously with feed comprising or consisting of protein obtained with the method of the invention (referred to as Water soluble extract powders (WSEP)) was compared with radical scavenging activity of fish flesh after the fish were fed previously with conventional feed lacking the black soldier fly larvae protein fraction obtained with the method of the invention. For all the samples increase in concentration resulted in an increase of mean inhibition values (showing the anti-oxidant behaviour of the fed feed when radical scavenging activity in the fish flesh is assessed). Emax (maximum inhibition obtained during the test) were in the following order: HI-<NUM> > HI-<NUM> > HI-<NUM> > HI-<NUM> (all obtained at <NUM>/ml, final concentration), referring to fish feed comprising <NUM>, <NUM>, <NUM> and <NUM> weight % BSF larvae protein obtained with the method of the invention, respectively, based on the total mass of the fed feed. This result show that at the dose of the HI-<NUM> sample, the highest ABTS radical scavenging activity is obtained. On the other hand, HI-<NUM> samples exhibit least ABTS radical scavenging activity. As assessed by using the ABTS assay, all the protein samples obtained by applying the method of the invention with black soldier fly larvae, exhibit anti-oxidant activity in a dose-dependent manner. HI-<NUM> samples show the highest anti-oxidant activity.

In summary, with the method of the present invention it has become possible to obtain valuable nutrient streams from black soldier fly larvae. These streams are not contaminated by foreign chemicals and have a purely biologic origin. The isolated nutrient streams may be used in the preparation of food, feed or pharmaceuticals. Moreover, depending on the intended application the nutrient streams obtained may be processed further, e.g. to isolate specific ingredients, such as hydrolysed protein, amino acids or specific fatty acids.

An embodiment is the method to convert black soldier fly larvae into nutrient streams according to the invention, wherein in step b) the pulp is at a temperature of <NUM>-<NUM>, preferably <NUM>-<NUM>. An embodiment is the method to convert black soldier fly larvae into nutrient streams according to the invention, wherein in step b) the pulp is heated for <NUM>-<NUM> seconds, particularly for <NUM>-<NUM> seconds, preferably <NUM>-<NUM> seconds such as <NUM> - <NUM> seconds. As said, the inventors established that by applying the (black soldier fly) larvae processing method of the invention, in step a) of the method, a nutrient stream comprising a stream of aqueous protein fraction obtained from black soldier fly larvae as the protein source, is obtained which aqueous protein fraction has surprisingly improved features and structural characteristics, when compared to protein obtained from such larvae by applying an alternative black soldier fly larvae processing method known in the art, such as the method outlined in <CIT>, in the Examples section thereof, in particular Example <NUM>, page <NUM>, line <NUM>-<NUM> and page <NUM>, line <NUM>-<NUM>. In particular, the relatively short heating time of <NUM> seconds or less than <NUM> seconds, in step b) of the method of the invention, is deemed essential for obtaining improved larvae pulp in step b) and subsequently for obtaining improved aqueous protein fraction in step c) of the method. It has been found by the inventors that by using the time-temperature combination less oil is entrapped within the aqueous protein fraction, therewith providing a more pure protein source in step c) of the method of the invention, compared with protein sources provided using alternative, current means and methods for isolating a protein fraction from black soldier fly larvae as the protein source. It has also been found that discolouration of the heated pulp (of protein) in step b) of the method, due to Maillard reactions, is largely avoided, thereby improving the quality of the product obtained, e.g. the heated pulp in step b) and the aqueous protein fraction in step c) of the method of the invention. For example, aqueous protein fraction with reduced fat content is obtained when minced black soldier fly larvae are heated for <NUM> seconds at <NUM>, or for <NUM>-<NUM> seconds at a temperature of <NUM> ± <NUM>. In <CIT>, heating time is at least <NUM> minutes and even <NUM> minutes. Furthermore, the relatively short heating time of at most <NUM> seconds, preferably about <NUM>-<NUM> seconds, of the minced black soldier fly larvae pulp (provided in step a) of the method) results in less granulated and less aggregated heated black soldier fly larvae pulp in step b) of the method compared to longer heating times applied in current methods known in the art. Less granulated larvae pulp after step b) of the method is beneficial for, for example, the enzymatic hydrolysis of the protein comprised by the pulp, and in general, a less granulated and aggregated black soldier fly larvae pulp and subsequent protein fraction contribute to improved separation results and improved ease of processing the pulp and protein. For example, granulation reduces enzyme efficiency since the particulate matter is less accessible for the enzyme molecules. By applying the larvae processing method steps as part of the method of the invention, the short heating time results in a protein composition consisting of smaller protein clusters, aggregates, particles, or even (mainly) of protein monomers and/or small multimers, compared with the larger protein aggregates and particles obtained when black soldier fly larvae pulp is subjected to heating for over <NUM> seconds such as for <NUM> minutes up to <NUM> minutes. In addition, fat is entrapped inside the relatively large protein pulp clusters and protein fraction aggregates after heating the black soldier fly larvae pulp for longer times than <NUM> seconds, for example at <NUM>.

The present invention will be illustrated further by means of the following non-limiting examples.

Live and washed larvae of <NUM> days old (<NUM>) were collected just before the mincer and stored at <NUM> until used. For each experiment <NUM> larvae were minced freshly. <NUM> of minced larvae were heated to <NUM> (using a hot plate and with continuously stirring) and the product was kept at this temperature for <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. Approximate <NUM> of heated larvae were collected in <NUM> centrifugable tubes and centrifuged at <NUM> for <NUM> minutes. The fat fraction was extracted after centrifugation and were collected using a Pasteur pipette and weighed to calculate the % fat separation (of total puree weight on wet basis).

The fat separation as a function of time is provided in table <NUM> below and <FIG>. From this it is clear that upon centrifugation of unheated insect pulp no fat was obtained, but that it steadily increased till a heating time of <NUM> seconds. Thereafter it remained stable until the heating time of <NUM> seconds, after which the fat separation started to decline. The surprising results show that different from what was expected in the art, relatively short heating times of <NUM> minutes or less may be used for the most efficient removal of fat from the heated insect pulp. Furthermore, due to the reduced heating times less discolouration of the heated insect pulp occurred, which leads to a product (and derived nutrient streams) with a higher quality. In <FIG> the influence of heating time on colour of the insect pulp is depicted. Moreover, the short heating time results in less granulated and less aggregated insect pulp compared to longer heating times of over <NUM> seconds (see e.g. <FIG>).

A puree of BSF larvae, also referred to as BSF PureeX™ (BSF-P), and hydrolyzed puree (BSF-HP) obtained by enzymatic hydrolysis of the puree of BSF larvae, were prepared by <NPL>. The puree and the enzymatically digested puree were obtained according to the following method. Live and washed Black Soldier Fly larvae of <NUM> days old were collected just before being subjected to the mincer for mincing the larvae (therewith providing larvae pulp), and subsequently stored at <NUM> until used. For each experiment, larvae were minced freshly. The minced larvae was treated with <NUM>% or <NUM>% Favourzyme, based on the mass of the minced larvae, for <NUM> - <NUM> hours, for example <NUM> to <NUM> hours, at <NUM> to <NUM> (± <NUM>) under continuous stirring. The batch of hydrolysed BSF larvae puree applied in Example <NUM> was obtained by enzymatic hydrolysis for <NUM> hr at <NUM> (± <NUM>). Flavourzyme (Novozymes, Denmark) is a combination of aminopeptidases which have endopeptidase and exopeptidase activities. The enzyme-treated minced larvae, and control larvae without enzyme treatment, were heated to <NUM> and the product was kept at this temperature for <NUM> seconds. The obtained insect puree were used for measuring free amino acid content (<FIG>: Free amino acid content (n=<NUM>)- Tested in puree) and pepsin digestibility (<FIG>: Digestibility (n=<NUM>)- Tested in puree). Protein meal was obtained from the heated BSF larvae pulp, and from the two batches of heated BSF larvae pulp that was first subjected to enzymatic hydrolysis of proteins using either <NUM> wt%, or <NUM> wt% Flavourzyme prior to the heating step at <NUM>, e.g. by steps of protein separation from the pulp, evaporation, drying and grinding. The percentage of fat separation was measured during the separation step (<FIG>: % fat separation (n=<NUM>)- Tested during separation step to make protein meal). In all experiments performed, a dose dependent effect was observed, when the amount of applied enzyme is considered. Thus, enzymatic treatment of minced larvae using Flavourzyme prior to the heating step increases the free amino acid content and increases pepsin digestibility of the obtained hydrolysed larvae puree, and as a result, the obtained puree comprising hydrolysed protein has a better taste (free amino-acid content relates to attractive, appealable taste when animals and humans consume a product comprising free amino-acids), is highly digestible and has anti-oxidant properties (see test results, here below). In addition, enzymatic treatment of BSF larvae pulp improves the fat separation in the following separation step after enzymatic hydrolysis and heat-treatment of the enzymatically digested pulp, which increases the fat extraction from the protein meal, when compared to fat separation from the protein fraction obtained with larvae puree that was not subjected to an enzymatic hydrolysis step prior to heating at <NUM>.

All the reagents were of analytical grade. Dimethylsulfoxide, methanol, ethanol, calcium chloride, potassium chloride, sodium chloride, hydrogen peroxide and Tween-<NUM> were purchased from Merck (VWR, Leuven, Belgium). Sodium nitrite, bovine serum albumin, phorbol <NUM>-myristate <NUM>-acetate and Percoll™ were purchased from Sigma (Bornem, Belgium). Aqueous extracts and solutions were made in Milli-Q water obtained using Milli-Q water system (Millipore, Bedford, USA). Bicinchoninic acid and copper (II) sulfate solutions were purchased from Sigma (Steinheim, Germany). Whatman filter paper grade <NUM> (<NUM>) was purchased from Amersham (Buckinghamshire, UK). Sterlip <NUM> disposable vacuum filter system was purchased from Millipore (Bedford, USA). <NUM>,<NUM>-Diphenyl-<NUM>-picrylhydrazyl and <NUM>'-azino-bis(<NUM>-ethylbenzthiazoline-<NUM>-sulfonic acid) were purchased from Aldrich (Darmstadt, Germany). <NUM>-amino-<NUM>-chloro-<NUM>-phenylpyrido[<NUM>,<NUM>-d]pyridazine-<NUM>,<NUM>(<NUM>,<NUM>)dione (L-<NUM>) was purchased from Wako Chemicals (Neuss, Germany).

Chicken meal (CM) and fish meal (FM) were purchased from an online webshop in September <NUM>. The chemical composition of both ingredients as declared by the supplier is indicated in table <NUM>.

BSF-P and hydrolyzed puree BSF-HP were prepared by<NPL>. BSF-P was pasteurized BSF minced 'meat' (puree, pulp) supplied frozen at -<NUM>. BSF-P is also the raw material to produce BSF protein meal (ProteinX™). BSF-HP was hydrolyzed and pasteurized BSF meat (puree) also supplied frozen at -<NUM>. The chemical composition of the two ingredients BSF-P and BSF-HP were as is indicated in table <NUM>.

Water soluble extracts were prepared for CM, FM, BSF-P and BSF-HP. These products (<NUM> each) were dissolved with six times volumes of Milli-Q water based on their respective dry matter contents (e.g. BSF-P had dry matter content of <NUM>% and was diluted <NUM> Milli-Q water) and stirred for <NUM> on a magnetic stirrer. Post centrifugation (<NUM> x g for <NUM> at <NUM>), the top fat layer was removed and the supernatant was filtered using Whatman Filter (grade <NUM>). The centrifugation and filtration step was repeated again to remove all non-soluble residues. Finally the supernatant was filtered using a Sterlip Filter (<NUM>, <NUM>) and freeze dried over a period of two days to obtain respective water soluble extract powders. All four water soluble extract were stored in a desiccator (at <NUM>) until further use.

Total protein content of the four water soluble extracts was analysed using Bicinchoninic acid (BCA) protein assay [<NUM>]. The calibration curve was obtained using bovine serum albumin (BSA) as standard at concentrations: <NUM>, <NUM>, <NUM>, <NUM> and <NUM>/ml. Stock solutions of <NUM>/ml water soluble extracts were used for analysis. A test solution was made by dissolving <NUM>µl BCA (<NUM>/<NUM>) and <NUM>µl copper (II) sulfate (<NUM>/<NUM>). Sample stock solutions (<NUM>µl) and test solution (<NUM>µl) were added in wells of <NUM>-well plate. This plate was incubated at <NUM> for <NUM> and absorbance was measured at <NUM> using a Multiscan Ascent (Fisher Scientific, Asse, Belgium).

DPPH radical scavenging activity was analysed according to protocol of Brand-Willams et al. [<NUM>], with some modifications. DPPH test solution was made by dissolving <NUM> DPPH in <NUM> ethanol. Test solution was made fresh and stored in dark until further use. DPPH working solution was made by diluting test solution with <NUM> times ethanol (to obtain absorbance of <NUM> to <NUM> at <NUM>). DPPH working solution (<NUM>µl) was mixed with <NUM>µl of samples dilutions (four water soluble extracts in Milli-Q water) to obtain final concentration of <NUM>, <NUM>, <NUM>, <NUM> and <NUM>/ml. The decrease in absorbance after <NUM> and <NUM> of incubation in dark was recorded at <NUM> using HP <NUM> UV-vis spectrophotometer (Agilent Technologies, Waldbronn, Germany). Instead of sample dilutions only Milli-Q water was used in case of control.

ABTS cation radical scavenging activity was analysed according to protocol of Arnao et al. [<NUM>], with some modifications. ABTS test solution was made by dissolving <NUM> mmol/l ABTS and <NUM> mmol/l potassium persulfate in Milli-Q water. The test solution was kept overnight in dark at room temperature. ABTS working solution was made by diluting with methanol to obtain the absorbance between <NUM> and <NUM> at <NUM>. ABTS working solution (<NUM>µl) was mixed with <NUM>µl of samples dilutions (four water soluble extracts in Milli-Q water) to obtain final concentration of <NUM>, <NUM>, <NUM>, <NUM> and <NUM>/ml. The decrease in absorbance after <NUM> of incubation in dark was recorded at <NUM> using HP <NUM> UV-vis spectrophotometer (Agilent Technologies, Waldbronn, Germany). Instead of sample dilutions only Milli-Q water was used in case of control.

SIEFED assay is a licensed method developed by Franck et al. [<NUM>] for specific detection of animal origin MPO. MPO solution was made by diluting human MPO in <NUM> phosphate buffer saline (at pH <NUM>), <NUM>/l BSA and <NUM>% Tween-<NUM>. Sample dilutions at final concentration of <NUM>, <NUM>, <NUM>, <NUM> and <NUM>/ml were incubated for <NUM> (at <NUM>) with MPO solution at a final concentration of <NUM> ng/ml. After incubation, the mixtures were loaded into the wells of a <NUM> wells microtitre plate coated with rabbit polyclonal antibodies (<NUM>µl/ml) against equine MPO and incubated for <NUM> at <NUM> in darkness. After washing up the wells, the activity of the enzymes captured by the antibodies was measured by adding hydrogen peroxide (<NUM>), NO<NUM>- (<NUM>) and Amplex™ Red (<NUM>). The oxidation of Amplex™ Red into the fluorescent adduct resorufin was monitored for <NUM> at <NUM> with Fluorosckan Ascent (Fisher Scientific, Asse, Belgium). Instead of sample dilutions only Milli-Q water was used in case of control.

MPO solution was prepared as mention in section <NUM>. Sample dilutions at final concentration of <NUM>, <NUM>, <NUM>, <NUM> and <NUM>/ml were incubated for <NUM> (at <NUM>) with MPO solution at a final concentration of <NUM> ng/ml. After incubation, the mixture (<NUM>µl) was immediately transferred into <NUM>-well microtitre plate. This was followed by addition of <NUM>µl NO<NUM>- (<NUM>) and <NUM>µl of Amplex™ Red and hydrogen peroxide mixture (at concentrations mentioned in section <NUM>). The oxidation of Amplex™ Red into the fluorescent adduct resorufin was monitored for <NUM> at <NUM> with Fluorosckan Ascent (Fisher Scientific, Asse, Belgium) immediately after addition of relevation mixture. Instead of sample dilutions only Milli-Q water was used in case of control.

Preparation of the neutrophil and phorbol <NUM>-myristate <NUM>-acetate (PMA) solutions were made according to Paul et al. Neutrophil response modulation activity of samples was analysed using the protocol of Tsumbu et al. Neutrophil suspension (<NUM> million cells/<NUM>µl PBS) was loaded in wells of <NUM>-wells microtite plate and incubated for <NUM> (at <NUM> in dark) with phospahte buffer saline solution of samples at final concentrations of <NUM>, <NUM>, <NUM>, <NUM> and <NUM>/ml. After incubation, <NUM>µl calcium chloride (<NUM>) and <NUM>µl L-<NUM> (<NUM>) were added in wells. The neutrophils were activated with <NUM>µl PMA (<NUM>) immediately before monitoring the chemiluminesence response of neutrophils during <NUM> at <NUM> using Fluorosckan Ascent (Fisher Scientific, Asse, Belgium). Instead of sample dilutions only phosphate buffer saline was used in case of control.

All the analyses were performed in triplicates. For protein quantification, the equation of a fitted line and R-square value were calculated using linear regression. The relationships between concentration and inhibition obtained for antioxidant assays were non-monotonic in nature. To address this, the locally estimated scatterpot smoothing (LOESS) regression technique was used to model the relationship [<NUM>]. Models were fitted using the R statistical software [<NUM>]. These models require a span parameter that defines the smoothing sensitivity of the local regressions. By visual inspection a span parameter value of <NUM> was found to be suitable for all concentration and inhibition relationship curves. Concentrations with a predicted inhibition percentrage of interest, such as IC<NUM> (concentration at which <NUM> % inhibition is reached), were found using the fitted models in combination with a numerical search routine.

The calibration curve obtained using BSA, equation of the line and R-square value are established. The optical density of samples and relative concentration of proteins (calculated using equation of line) are mentioned in table <NUM>. BSF-P extract solution (<NUM>/ml) exhibits the highest, on the other hand BSF-HP solution exhibits the lowest protein concentrations amongst the tested solutions using Bichinchoninic acid assay.

DPPH radical scavenging activity of all five samples after <NUM> and <NUM> minutes of incubation is indicated in <FIG> and <FIG>, respectively. The plot shows the measured values as well as fitted curves obtained from LOESS. CM exhibited pro-oxidant behavior at all tested concentrations after <NUM> as well as <NUM> minutes of incubation. Whereas, FM exhibited pro-oxidant behavior at four out of five tested concentrations after <NUM> of incubation and at all tested concentrations after <NUM> of incubation. The IC<NUM> of BSF-HP after <NUM> of incubation is indicated in table <NUM>. It was not possible to calculate IC<NUM> for other samples (after <NUM> or <NUM> of incubation) because the samples either exhibited pro-oxidant activity or <NUM>% inhibition was not achieved during the assay. The Emax (maximum inhibition achieved during the assay) of all the samples are also indicated in table <NUM> and are in following order: BSF-HP> BSF-P> FM and BSF-HP> BSF-P after <NUM> and <NUM> minutes of incubation, respectively.

ABTS cation radical scavenging activity of samples after <NUM> minutes of incubation is shown in <FIG> (measured values as well as fitted curves obtained from LOESS). All the samples exhibited a similar inhibition pattern i.e., % inhibition increased as a function of increasing concentration. The IC<NUM> of samples are mentioned in table <NUM> and are in following order: FM> CM> BSF-HP > BSF-P. Lower the IC<NUM>, higher is the ABTS cation radical scavenging activity. The Emax (maximum inhibition achieved during the assay) of all the samples are indicated in table <NUM> and are in following order: BSF-P> BSF-HP> FM> CM.

MPO response modulation activity of samples obtained using SIEFED assay is shown in <FIG> (measured values as well as fitted curves obtained from LOESS). BSF-HP exhibited strong inhibition behavior, with ><NUM> % inhibition at <NUM>/ml concentration. The IC<NUM> of samples are mentioned in table <NUM> and are in following order: BSF-HP. The Emax of samples are shown in table <NUM>, and are in following order: BSF-HP> BSF-P. FM and CM show pro-oxidant behavior at all tested concentrations. On the other hand Emaz for BSF-P was < <NUM> %.

MPO response modulation activity of samples obtained using classical assay is indicated in <FIG> (measured values as well as fitted curves obtained from LOESS). CM and FM exhibited pro-oxidant behavior at all tested concentrations. The Emax of all the samples tested are indicated in table <NUM>. BSF-P and BSF-HP exhibited Emax > <NUM> %. The IC<NUM> of samples are mentioned in table <NUM> and are in following order: BSF-P> BSF-HP.

Neutrophil response modulation activity (measured values as well as fitted curves obtained from LOESS) and Emax of samples are shown in <FIG> and table <NUM>, respectively. All the tested samples exhibited Emax > <NUM>%. FM and CM exhibited Emax < <NUM>%. CM exhibited pro-oxidant behavior at <NUM> out of <NUM> tested concentration. The IC<NUM> of samples are mentioned in table <NUM>. BSF-P and BSF-HP have the same numerical IC<NUM> values.

The protein concentration of the four water soluble extracts estimated using Bichinchoninic acid assay are displayed in table <NUM>. Considering the amino acid pattern similarities between black soldier fly proteins, FM and CM, the protein content of four water soluble extracts are in following order: BSF-P> CM> FM> <NUM> %> BSF-HP.

DPPH and ABTS assays are commonly used to analyze antioxidant potential of food and feed products. DPPH radical scavenging activity represents the ability of a sample to donate hydrogen atom (referred as hydrogen atom transfer) or electrons (referred as single electron transfer) to stabilize free radicals. DPPH assay IC<NUM> and Emax for all tested samples are mentioned in table <NUM> and table <NUM>, respectively. Post <NUM> of incubation, all the tested samples exhibit Emax < <NUM> % (with BSF-HP exhibiting highest Emax). On the other hand, after <NUM> of incubation only BSF-HP exhibit Emax > <NUM> %. BSF-HP is manufactured by controlled hydrolysis of black soldier fly proteins and contains at least <NUM> % of proteins <<NUM> Da (see table <NUM>). On the other hand BSF-P contains at least <NUM> % proteins <<NUM> Da. The inventors were not able to find any representative literature for molecular weight distribution of FM and CM. However, according to the literature, FM and CM contain <NUM> % and <NUM> % free amino acid (of total proteins), respectively [<NPL>. Which translates into FM and CM containing at least <NUM> % and <NUM> % proteins <<NUM> Da respectively. The capacity of proteinaceous materials to scavenge free radicals depends on the protein molecular weight distribution. Proteins with low molecular weight peptides could scavenge free radicals more efficiently. Free radical scavenging activity of proteinaceous molecules is also influenced by: (<NUM>). Amino acid composition: hydrophobic amino acids (for e.g. Tyr, Phe, Pro, Ala, His and Leu) have superior radical scavenging activity in comparison to hydrophilic amino acids; (<NUM>). Amino acid sequence: Peptides with amphiphilic nature could enhance radical scavenging activity of a sample. Chemical analyses have indicated that Tyr exhibit antioxidant behavior via hydrogen atom transfer mechanism. On the other hand, amino acids such as Cys, Trp and His exhibit antioxidant behavior via single electron transfer mechanism.

FM and CM exhibit pro-oxidant behavior at most concentrations tested after <NUM> as well as <NUM> of incubation (see <FIG> and <FIG>). This behavior mainly arises from the thermal processing. For both FM and CM, thermal processing commonly involves heating the raw product at high temperatures for <NUM> to <NUM>. In Norway, during fishmeal production, wild caught fishes are subjected to heating at temperatures ≥<NUM> for time ≥<NUM> in order to achieve <NUM> log<NUM> reductions of Enterobacteriaceae and Salmonella counts. Such strict thermal processing conditions may result in oxidation of fats and proteins. Fish meal contains lipids rich in polyunsaturated fatty acids that are more susceptible to thermal oxidation. Antioxidant are commonly added in fish meal to prevent the oxidation of polyunsaturated fatty acids (also visible in table <NUM>). Heat induced oxidation of amino acids lead to development of wide range oxidation products. The pro-oxidant behavior of amino acid oxidation by products is already known. They can result in a wide range of health conditions in animal body. All the black soldier fly protein derivatives (puree, hydrolysed puree) used and analyzed by the inventors were thermally processed at temperatures <<NUM> for time <. <NUM> (e.g. at <NUM> for <NUM> seconds). These thermal processing time-temperature combinations of the current invention were adopted to ensure minimum damage to nutrients (proteins and fat) and adequate inactivation of pathogenic microbiota. This implies that pro-oxidant behavior of FM and CM arises mainly due to stringent production method.

In one of the recent studies [<NUM>], researchers made BSF protein hydrolysate using bromelain enzyme. Bromelain derived protein hydrolysate was also tested for DPPH radical scavenging activity, which resulted into the IC<NUM> of <NUM>/ml. The DPPH radical scavenging activity of this bromelain derived protein hydrolysate was much lower when compared to the activity of products such as BSF-HP (IC<NUM> <NUM>/ml after <NUM> of incubation). The higher activity of BSF-HP as found by the inventors could arise from compositional attributes (as previously discussed in this section) and quality of raw material itself. Protix is reportedly producing insect proteins in GMP+ and SecureFeed certified facilities, under HACCP conditions.

ABTS cation radical scavenging denotes the ability of sample to donate electron and stabilize free radicals. ABTS assay IC<NUM> of all samples are indicated in table <NUM>. They are in following order: FM> CM> BSF-HP> BSF-P. The higher the IC<NUM>, the lower the antioxidant activity. In this assay even FM and CM exhibit antioxidant activity. It appears that FM and CM extracts may be efficient where free radical(s) could be stabilized using single electron transfer mechanism. However, they still exhibit lower scavenging activity in comparison to the surprisingly high scavenging activity of BSF derivatives BSF larvae puree and hydrolysed BSF larvae puree.

Dependence of radical scavenging activity on protein molecular weight is already explained in section <NUM>. At Protix the inventors established that BSF-P and BSF-HP have exactly the same amino acids composition. However, due to protein hydrolysis, the amount of proteins <<NUM> Da is higher in BSF-HP than in BSF-P. It is therefore somewhat surprising that the BSF-P IC<NUM> value was slightly lower in comparison to BSF-HP. This could be explained by the mechanism of hydrolysis. Enzymatic hydrolysis is achieved through exo- and endo- peptidase. Exopeptidase cleaves the terminal peptide bond, on the other hand endopeptidase cleaves the non-terminal peptide bond. In both cases the sequence of amino acids is altered. The radical scavenging ability of resulting peptides via single electron transfer is also dependent on the amphiphilic nature of proteinaceous molecules. It is possible that peptides in BSF-HP are less amphiphilic in nature that results into lower ABTS cation radical scavenging activity of BSF-HP compared to BSF-P.

[<NUM>] developed BSF protein hydrolysate using wide range of commercial enzymes. The hydrolysates were further fractionated into following group: group <NUM> (<<NUM> Da), group <NUM> (<NUM> to <NUM>,<NUM> Da) and group <NUM> (><NUM>,<NUM> Da) using ultrafiltration. The activity of these hydrolyzed fractionates were also investigated for ABTS cation radical scavenging activity. Ascorbic acid was used as the reference molecule in the example. Interestingly the best performing fractionate and ascorbic acid were able to inhibit <NUM> % and <NUM> % of ABTS cation radical at <NUM>/ml concentration, respectively. The current inventors now surprisingly established that BSF-P already exhibits ABTS cation radical scavenging Emax of as high as <NUM>, (at <NUM>/ml). This shows that fractioning BSF-P reveals fractions that have very strong antioxidant potential.

Strong free radical scavenging activities of BSF derivatives are evident from section <NUM> and <NUM>. Furthermore, all the samples were also tested for neutrophil response modulation activity. Neutrophils are white blood cells present in animal body (including humans, pets, fishes, poultry and swine). They are involved in the primary defense against pathogens. When pathogenic microbes enter the animal body, neutrophils rush to the site of infestation and initiate defense. During granulation, neutrophil release a wide range of oxidative enzymes including NADPH oxidase, which is responsible for production of superoxide anion and by product (e.g. hydrogen peroxide). Superoxide anion can further react with nitric oxide radical to produce peroxynitrite. This process also generates hydroxyl radical (by reaction of hydrogen peroxide with metal ion). This battery of oxidative reactions are crucial to the defense of the host animal. However, these ROS generated during host defense can react with enzymes, proteins, lipids, etc. of body cells and result in the development of different health conditions (for e.g. cellular ageing, cancer, etc.). The neutrophil assay conducted in this research determines the ability of proteinaceous molecules to scavenge ROS produced as a result of neutrophil activity. PMA was used to activate protein kinase C present in neutrophils, which results in production of NADPH oxidase responsible for catalyzing ROS production. ROS production in system is coupled with lucigenin amplified chemi-luminescence. Ability of proteinaceous sample to scavenge ROS (particularly superoxide anion) is marked by decreased chemi-luminescence.

To the inventors knowledge, this is the first analysis of in vitro neutrophil response modulation activity of BSF derivatives BSF larvae puree and hydrolysed BSF larvae puree. CM exhibited pro-oxidant behavior at <NUM> out of <NUM> tested concentrations, and Emax of only <NUM>% at <NUM>/ml (see <FIG> and table <NUM>). CM is commonly used in pet food preparations. However, the comparative test results in the examples and embodiments of the invention indicate that CM inclusion offers little or no benefits relating to scavenging the ROS produced by neutrophils. Moreover, CM inclusion could even result in inflammatory damage to host cells. Repetitive inflammatory damage of canine or feline cells could translate into conditions such as accelerated aging, slow cognitive function, etc..

On the other hand, FM exhibits mild anti-oxidant behavior in this assay, with Emax of <NUM>% (see table <NUM>). At <NUM>/ml, FM exhibits inhibition of <NUM>%. Aquaculture rearing media (i.e. water) offers a continuous buffer of pathogenic bacteria. Therefore, aquaculture organisms are at constant risk of pathogenic bacterial invasions. This results in wide range of health conditions, including reduced immunity, aging, etc. The comparative examples of the current inventors highlight the inadequacy of FM to suppress the inflammatory damage from repetitive neutrophil activity. This often translates into incremental cost occurring as a result of antibiotics and nutritional supplement usage. BSF-P exhibits Emax and IC<NUM> of <NUM>% and <NUM>/ml, respectively (see table <NUM>). Moreover, BSF-HP also exhibits neutrophil response modulation activity comparable to BSF-P (see table <NUM>).

The general mechanism of neutrophil response is known. The neutrophil extracellular trap contains several molecules required to inactivate pathogenic microbes. MPO enzyme present in neutrophil extracellular trap can produce hypochlorous acid from hydrogen peroxide and chloride ion. Additionally, MPO is capable of oxidizing tyrosine into the tyrosyl free radical. Both products of MPO oxidation (hypochlorous acid and tyrosyl free radical) are crucial to inactivate pathogens. Again, repetitive interaction of these molecules with animal cells result in inflammatory damage. In an animal body, MPO-Fe(lll) (active form) reacts with hydrogen peroxide to form oxoferryl π cation radical (Cpl form). Cpl form converts back into MPO-Fe(III) coupled with chloride ion transforming into hypochlorous acid. However, in the present experiment, back reduction of the Cp I form to MPO-Fe(III) was achieved in <NUM> stages. First reduction of Cpl to MPO-Fe(IV)=O via electron transfer through nitrite ions. Then, electron provisioning was done (via Amplex™ Red oxidation to resorufin reaction) which converts MPO-Fe(IV)=O to MPO-Fe(III) form. Proteinaceous molecules could prevent the oxidative damage resulting from MPO either by directly reacting with Cpl form and terminating the halogenation, or by donating hydrogen (hydrogen atom transfer) to ROS produced as a consequence of MPO activity. MPO response modulation activity was analyzed using the classical and SIEFED assay. The classical assay measures ability of sample to complex with Cpl form and stabilize ROS. Whereas in SIEFED assay, MPO is bound to rabbit polyclonal antibodies (and rest of the compounds are washed away), so it purely measures the ability of samples to complex with Cpl form.

As with neutrophil response modulation activity, MPO response modulation activity of BSF derivatives BSF larvae puree and hydrolysed BSF larvae puree is also being established by the inventors for the first time, after provision of the puree according to the method of the invention. FM and CM exhibit pro-oxidant behavior in both the assays (see <FIG>). Presence of oxidative reaction products in FM and CM (as a consequence of production process) that are capable of initiating prooxidative response has been already discussed in section <NUM>. Detailed in vitro investigations realized by the inventors indicate that inclusion of FM and CM in animal diets may result in inflammatory damage.

In classical assay, BSF derivatives exhibit surprisingly strong antioxidant potential, with IC<NUM> in following order: BSF-P> BSF-HP. In the SIEFED assay, BSF-P did not reach <NUM>% inhibition (even at highest concentration tested). Thus while, BSF-P is more effective in stabilizing ROS, BSF-HP has higher efficacy in complexing with Cpl form of MPO. These observations show that the two BSF derivatives are suitable for use as an ingredient in pet food and aquaculture formulations to effectively suppress inflammatory damages resulting from MPO activity.

BSF protein derivatives offer anti-oxidative advantage over FM and CM.

Fat oxidation as a consequence of puree heating times.

Pasteurization is crucial for production of processed insect protein. Optimal heating time and optimal heating temperature combinations is important for:.

With respect to fat quality (of insect proteins), pasteurization time x temperature combination is significant for:.

In this current Example <NUM> the extent of FFA formation in pasteurized puree (to analyze lipase inactivation) and development of hydroperoxides as a consequence of heating times, is established.

Approximately <NUM> live black soldier fly larvae were harvested from a rearing unit (Protix, The Netherlands). These larvae were washed thoroughly to remove all visible impurities. <NUM> of washed larvae were used per treatments. Larvae were minced to obtain a smooth slurry (puree) and pasteurized according to treatments indicated in Table <NUM>. The pasteurized larvae puree was immediately frozen (to - <NUM>). These frozen samples were later thawed and analysed for free fatty acid content and peroxide values. All the experiments were performed in duplicates.

The free fatty acid contents and peroxide values obtained for respective treatments are mentioned in Table <NUM>.

As visible from Table <NUM>, that insect puree heated at <NUM> for <NUM> has relatively high amounts for free fatty acids (approx. Heating puree just for <NUM> is ineffective in the deactivation of lipase. Whereas, low amounts of free fatty acids are observed when puree is heated for <NUM>, indicating the sufficiency of this time for enzyme inactivation at <NUM>. At heating times of over <NUM> seconds (treatment <NUM> and <NUM> in Table <NUM> and Table <NUM>), the fatty acid content increases when compared to the low fatty acid content obtained when the black soldier fly larvae puree is heated for <NUM> seconds. Therefore, for obtaining a protein fraction which comprises a relatively low free fatty acid content, the heating time in the method of the invention should be longer than <NUM> seconds and shorter than <NUM> seconds, such as <NUM> - <NUM> seconds, or a heating time selected from <NUM> - <NUM> seconds, or <NUM> - <NUM> seconds, such as about <NUM> seconds. Preferred heating time is about <NUM> or <NUM> ± <NUM>.

Also higher peroxide value is observed for heating duration of <NUM>. This could be explained by high amounts of free fatty acids that could participate in enzyme oxidation leading to the production of hydroperoxides. However, in case of <NUM> treatment, lower peroxide value is obtained. Heating the product further has an effect on fat oxidation in that the peroxide value for Treatment <NUM> and Treatment <NUM> is increased compared to the peroxide value for Treatment <NUM>, i.e. a heating time of <NUM> seconds. Therefore, for obtaining a protein fraction which comprises a relatively low peroxide value, the heating time in the method of the invention should be longer than <NUM> seconds and shorter than <NUM> seconds, such as <NUM> - <NUM> seconds, or a heating time selected from <NUM> - <NUM> seconds, or <NUM> - <NUM> seconds, such as about <NUM> seconds. Preferred heating time is about <NUM> or <NUM> ± <NUM>.

Heating the minced insect larvae at <NUM> for <NUM> is sufficient for enzyme inactivation, prevents free fatty acid formation, and prevents peroxide formation.

Antioxidant activity of Fish fillet samples was investigated using anti-radical (ABTS) technique. Water soluble black soldier fly larvae protein extracts, referred to as HI-<NUM>, HI-<NUM>, HI-<NUM> and HI-<NUM>, were prepared in distilled water and used at different concentrations. In the ABTS assay, the percentage inhibition at maximum tested concentration was in following order: HI-<NUM> > HI-<NUM> > HI-<NUM> > HI-<NUM>. In the ABTS assay, the HI-<NUM> filet sample reached the IC50 ≥<NUM>%. In conclusion, within the concentration series tested, the HI-<NUM> sample had the maximum ABTS radical scavenging activity among all the tested Fish fillet samples.

Using the ABTS assay, activity of fish flesh samples was assessed with regard to radical lowering activity of black soldier fly protein obtained with the method of the invention.

ProteinX (high protein meal of Hermetia illucens) used in this study was produced according to the method of the invention and was provided by Protix (The Netherlands). Two iso-nitrogenous, iso-lipidic and iso-energetic diets where formulated with either fish meal (<NUM>%) or ProteinX (<NUM>%) as the main protein source. Both diets where produced by Research Diet Services (the Netherlands). Diamol was added to both diets as inert marker for digestibility measurements. Four treatments where formed by feeding either one of both feeds, or mixing both feeds to a determined ratio.

The dietary treatments where referred to as:.

The proximate composition of all four dietary treatments is presented in Table <NUM>.

The proximate composition of ProteinX and of the four experimental dietary treatments was determined by DISAFA laboratories and is presented in Table <NUM>. Feed samples were ground using a cutting mill (MLI <NUM>; Bühler AG, Uzwil, Switzerland) and analysed for dry matter content (AOAC #<NUM>), crude protein content (AOAC #<NUM>), ash content (AOAC #<NUM>), and ether extract (AOAC #<NUM>) were analysed according to AOAC International. Crude protein content was calculated as: nitrogen * <NUM>.

The animal feeding trial was conducted at the Experimental Facility of the Department of Agricultural, Forest, and Food Sciences of the University of Torino (Italy). The experimental protocol was designed according to the guidelines of the current European and Italian laws on the care and use of experimental animals (European directive <NUM><NUM>/EEC, put into law in Italy with D. <NUM>/<NUM>). The experimental protocol was approved by the Ethical Committee of the University of Turin (protocol n° <NUM>). Juvenile rainbow trout were obtained from a private hatchery (Troticoltura Bassignana, Cuneo, Italy) and randomly distributed over <NUM> fiberglass tanks (n = <NUM>) (<NUM> fish per tank). Tanks were supplied artesian well water (<NUM> ±<NUM>) in a flow-through system at a flow rate of <NUM>/min. Dissolved oxygen was measured every fortnight and ranged between <NUM> and <NUM> / L. Fish were acclimatized to the experimental set up for four weeks during which a commercial diet was fed at <NUM>% of the total body weight per tank. After acclimatization, fish were restrictively fed a dietary treatment for <NUM> days of which <NUM> days at a fixed ratio of <NUM>% followed by <NUM> days at a fixed ratio of <NUM>%. Feed intake was monitored at each administration. Feed was distributed by hand twice a day, <NUM> days per week. The biomass of each tank was weighed in bulk every <NUM> days to correct the daily feeding rate. Mortality was checked daily.

At the end of the trial, fish were fasted for one day, and <NUM> fish/treatment (<NUM> fish/tank) fish per treatment where killed by overdose of anaesthesia (MS-<NUM> - PHARMAQ Ltd, UK; <NUM>/L) and individually weighed. The right fillets were collected of each fish and vacuum-sealed, frozen (-<NUM>°) and subsequently freeze dried.

Fish samples (in powdered form) were received. The samples were kept in a freezer (-<NUM>) until further processing. Water soluble extract powders (WSEP) of respective samples were made according to the protocol of Mouithys-Mickalad et al. WSEP were stored in a desiccator (at <NUM>) until further experimentations. The stock solutions of the WSEP were made by dissolving to a final concentration of <NUM>/mL in Milli-Q water, which stock solution will be then diluted by half to obtain <NUM>, <NUM>, <NUM> and <NUM>/mL, successively.

The radical scavenging activity of WSEP samples toward ABTS radical cation were analysed according to the method of Arnao et al. ABTS stock solution was made by completely dissolving <NUM> mmol/L ABTS and <NUM> mmol/L potassium persulfate in Milli-Q water. The test solution was kept overnight in dark (at <NUM>) for reaction to complete. ABTS working solution was made by diluting stock solution with methyl alcohol to obtain absorbance between <NUM> to <NUM> at <NUM>. ABTS working solution (<NUM>µL) was mixed with <NUM>µL of WSEP solutions (obtained by dissolving WSEP in Milli-Q water) to obtain a final concentration of <NUM>, <NUM>, <NUM>, <NUM> and <NUM>/ml. Decrease in absorbance post <NUM> of incubation in dark was measured at <NUM> using an HP <NUM> UV-vis spectrophotometer. Instead of WSEP dilution, only Milli-Q water was used in case of control. The repetitions with increasing concentrations of different samples was performed as described by Paul (<NUM>) and transferred into a multi-well device (<NUM>-well plate) with a final volume of <NUM>µL (<NUM>µL of solvent and <NUM>µL of WSEP). Likewise, after a <NUM>-min incubation period, the absorbance was read at <NUM> using a Multiskan Ascent reader (Thermo Fisher Scientific).

ABTS radical scavenging activity of WSEP samples is indicated in <FIG>: displayed is the ABTS radical scavenging activity of WSEP performed in methanol. Results are mean ± SD of three independent assays in triplicate. For all the samples increase in concentration resulted into an increase of mean inhibition values (indicating anti-oxidant behaviour). Emax (maximum inhibition obtained during the test) were in the following order: HI-<NUM> > HI-<NUM> > HI-<NUM> > HI-<NUM> (all obtained at <NUM>/ml, final concentration). This result indicates that HI-<NUM> samples exhibit highest ABTS radical scavenging activity. On the other hand, HI-<NUM> samples exhibit least ABTS radical scavenging activity. Eventually, HI-<NUM> samples exhibited mean inhibition values ≥ <NUM>% (IC<NUM> was achieved).

Claim 1:
Method to convert black soldier fly larvae into nutrient streams, comprising the steps of:
a) providing fresh black soldier fly larvae and preparing a pulp thereof;
b) heating the pulp for <NUM>-<NUM> seconds at a temperature of <NUM>-<NUM>;
c) subjecting the heated pulp to a physical separation step thereby obtaining a fat fraction, an aqueous protein fraction and a solid-containing fraction.