Novel food extracts

Abstract

Disclosed is a method of preparing microbial extracts, in particular protein extracts. The method involves the treatment of microbial biomass with ozone and optionally nucleases to obtain a protein extract with improved odour and taste. Also discloses are microbial extracts and methods for preparing such extracts, characterized in that the extracts contain trehalose.

Description

[0001] NOVEL FOOD EXTRACTS

[0002] FIELD

[0003] The disclosure relates to microbial protein and carbohydrate extracts and methods for their preparation. The disclosure also relates to the preparation of whole cell preparation of microbial extracts from edible microbes.

[0004] INTRODUCTION

[0005] The demand for proteins either from animal-based foods (meat, poultry, fish, eggs, and dairy foods) or plant-based foods (fruits, vegetable, grains, nuts, and seeds) is constantly increasing. This has driven researchers to investigate more sustainable food sources such as algae-based foods. The current protein demand is approximately 202 million tonnes globally, the reference nutrient intake (RNI) for protein for adults being about 0.75 g protein per kg body weight per day (Matos et al, Front Food Sci Tech, 2022, 2:989801 doi: 10.3389 / frfst.2022.989801). According to the Food and Agriculture Organization of the United Nations (FAO), the world’s population is projected to reach 9.7 billion by 2050. A deficiency in food sources is therefore likely to become a severe problem.

[0006] Algae and certain cyanobacteria represent a promising possibility to supply the growing need for protein demand as well as providing a source of various bioactive compounds. Like tofu, tempeh, lentils, chickpeas, and almonds, these microbes and their products can efficiently be used for the development of new foods, providing rich biomass as substitutes for plant-based meat alternatives. Microalgae are microscopic photoautotrophic microorganisms that use energy from sunlight to convert carbon dioxide and water into organic materials for cellular functions.

[0007] Several recent studies found in the literature have proposed the use of whole algae or algae extract for the development of new foods. Among the best-known microalgae species are Arthrospira platensis (Spirulina) and Chlorella vulgaris, which can act as a vegan protein source due to their high protein content (50%-60% of dry weight) with a complementary roll of essential amino acids and vitamin B12. These microalgae have been listed in the EU Novel food catalogue for food applications by the European Food Safety Authority (EFSA) and approved by the United States Food Drug Administration (FDA) as generally recognized as safe (GRAS). However, the addition of high concentration of algal biomass might result in negative effects on color and flavor of the final product, which depends on algae species and end product, decreasing consumers’ acceptance.

[0008] Minimizing and / or eliminating these palatability factors from microbial food products is therefore important for the development of food products for human consumption.

[0009] Uric acid is a product of purine metabolism generated during the breakdown of nucleic acids (DNA and RNA) and is in found in blood, where it passes through the kidneys and leaves the body in urine. Food products that are high in nucleic acids therefore result in increased levels of uric acid. If too much uric acid stays in the body, a condition called hyperuricemia will occur. Hyperuricemia can cause crystals of uric acid (or urate) to form. These crystals can settle in the joints and cause gout, a form of arthritis that can be very painful. They can also settle in the kidneys.

[0010] Serum uric acid levels vary greatly in humans and can range from 2.5 to 1 mg / dl or more. Those subjects with higher levels are at increased risk for developing gout and uric acid kidney stones. More importantly, an elevated uric acid also predicts the development of obesity, metabolic syndrome and diabetes, fatty liver, hypertension and cardiovascular and renal disease.

[0011] Human consumption of large quantities (>2 g / day) of isolated or (single-cell) yeast derived nucleotides has been shown to result in acute increases in circulating uric acid concentrations. Epidemiological and observational data have reported that circulating uric acid concentrations positively correlate with development of gout, hypertension, and metabolic syndrome, and are a predictor of type 2 diabetes (Coelho et al, Nutrients, 2020, 12:1115). Nevertheless, these findings informed the FAO / WHO / UNICEF (Food and Agriculture Organization / World Health Organization / United Nations Children’s Fund) Protein Advisory Group recommendation to limit the additional dietary nucleic acid load from single cell protein novel foods to no more than 2 g / day. Single cell organisms are inevitably rich in nucleic acids which means that consumption of these organisms or products derived from the organisms may in large quantities result in health issues. For this reason, some research has indicated that consumption of microalgae should be limited to about 20g per day for a human adult, or about 0.3g of algae per kg body weight.

[0012] SUMMARY

[0013] The present invention provides methods to overcome, eliminate or mitigate deficiencies of the prior art, for example the deficiencies described in the above.

[0014] In an aspect, the invention relates to a method of preparing protein extracts from microbes. The method comprises steps of (i) resuspending a microbial biomass in a suitable basic solution to obtain a soluble fraction and an insoluble fraction; (ii) separating the soluble fraction from the insoluble fraction; (iii) lowering the pH of the soluble fraction to a pH in the range of 7 to 9 using a suitable acid; (iv) treating the soluble fraction with ozone; (v) concentrating the solution and / or precipitating protein from the solution to obtain a protein extract; and (vi) adding trehalose to the ozone- treated solution.

[0015] The method can include an optional step of adding one or more nuclease to the treated soluble fraction following the ozone treatment. The method can further optionally include the addition of one or more preservative to the concentrated and / or precipitated protein.

[0016] In another aspect, the invention relates to a method of improving smell and / or flavor of a whole-cell preparation or cellular extract from an edible microbe comprising treating cells or cellular fragments from one or more microbe with ozone followed by the addition of trehalose to obtain a microbe preparation or extract having improved smell and / or flavor.

[0017] There can be an option drying step that precedes or follows the addition of trehalose, to obtain a dry whole-cell preparation or cellular extract.

[0018] In another aspect the invention relates to method of preparing nucleic-acid depleted protein and / or carbohydrate extract from one or more edible microbe, the method comprising steps of (i) preparing a liquid extract from one or more edible microbial species, the liquid extract comprising protein and / or carbohydrates; and (ii) adding one or more nuclease to the liquid extract from step (i) and incubating for a period of time sufficient to allow the one or more nuclease to digest nucleic acid in the extract. By this method, a nucleic-acid depleted protein and / or carbohydrate extract is obtained, i.e. a protein and / or carbohydrate extract that contains a reduced amount of nucleic acid.

[0019] In another aspect, the invention relates to a microbial extract characterized in that the extract comprises in the range of 0.5-20% by weight of trehalose.

[0020] In a further aspect, the invention relates to a microbial extract from one or more microalgae, characterized in that the extract contains less than about 500ppm chlorophyll.

[0021] Microbial extract useful for carrying out the invention can generally be any edible microbial extract. By the term “edible”, it is meant that the extract is intended for human or animal consumption. Preferably, the extract can be an edible extract for human consumption.

[0022] Microbes useful for carrying out the invention can generally be one or more edible bacterial, yeast, fungal or microalgal species. For example, the microbes can be from a species selected from the genus Chlorella, Arthrospira, Tetraselmis, Scenedesmus and Nannochloropsia. The species can be selected from Chlorella vulgaris, Arthrospira platensis, Tetraselmis chuii, Isochrysis galbana, Tetraselmis suecica, Scenedesmus almeriensis and Nannochloropsis gaditana.

[0023] Microbial extract can be a whole-cell microbial extract. The extract comprise protein and / or carbohydrates. For example, the extract can be a protein extract.

[0024] The extract can be depleted in nucleic acid content, i.e. the extract can contain a reduced amount of nucleic acid or a reduced content of nucleic acid.

[0025] For example, the depleted extract can contain 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of the original nucleic acid content of the extract (i.e., before treatment with one or more nuclease).

[0026] The extract can be depleted in RNA, DNA or both RNA and DNA. Accordingly, the one or more nuclease can be one or more RNase, one or more DNase or a combination of one or more RNase and one or more DNase. A basic solution useful in the methods disclosed herein can be an aqueous solution containing a suitable base, such as NaOH, KOH or other bases known in the art. A combination of one or more base can also be used. The solution can contain in the range of about 0.1 M to about 5 M base, such as about 0.1 M to about 4M, or about 0.2M to about 4M, or about 0.2M to about 3M, or about 0.2M to about 2M, or about 0.2M to about 1 M.

[0027] Treatment with ozone generally involves bubbling ozone gas through the solution. The ozone treatment can generally be done at a temperature in the range of about 0°C to about 40°C, such as about 5°C to about 30°C, about 5°C to about 25°C, or about 5°C to about 20°C.

[0028] The treatment may be performed for a period of time in the range of about 1 to 60 minutes, such as about 2 to 60 minutes, about 5 to about 60 minutes, about 10 to about 60 minutes, about 20 to about 60 minutes about 20 to about 40 minutes or about 20 to about 40 minutes.

[0029] Concentrating protein can be done by methods generally known in the art. For example, the concentrating can be done by removing solvent (i.e., water) by evaporation or distillation, by heating, or by filtration or precipitation. For example, the concentrating can be done by heating, for example at a temperature of about 50°C to 80C or 60 to 80°C for a period of time in the range of about 0.5 to 5 hours.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG.1 shows a schematic diagram of a protein extraction process from wet biomass.

[0032] FIG. 2 shows effects of ethanol treatment to remove chlorophyll on microbial suspensions. Suspension before (B) and after (A) ethanol treatment are shown.

[0033] FIG. 3 shows microbial cultures after (A) and before (B) ethanol treatment to remove chlorophyll. DESCRIPTION

[0034] The present invention relates to methods for providing edible microbial extracts that are free from adverse smell and / or taste. The invention also relates to production of microbial protein and / or carbohydrate extracts that have reduced quantity of nucleic acids or are free from, or depleted of, nucleic acid material. The combination of treatment of the biomass to both reduce nucleic acid content and minimize or eliminate foul taste and / or smell results in whole-cell products and extracted products such as protein and carbohydrates with advantageous properties for use in human food, food supplements or food alternatives.

[0035] Edible microbial biomass as an alternative to conventional sources of food and feed is a promising technology option to prevent habitat destruction caused by increases in food demand. Microorganisms such as bacteria, yeasts, algae and filamentous fungi have several beneficial properties including their rapid growth rate and their ability to assimilate simple organic substrates such as hydrocarbons, alcohols or organic acids. Microbial biomass has high protein content and often contains beneficial lipids and vitamins.

[0036] Microscopic algae can use photosynthesis to grow on CO2 and have therefore long been recognized as an alternative food source. Microalgae, including cyanobacteria, are photoautotrophic microorganisms, which use CO2 as carbon source and perform oxygenic photosynthesis to obtain energy from sunlight or artificial light. Cyanobacteria, in particular the Arthrospira and Nostoc species, have been used in Africa and Asia as food for centuries. Commercial microalgae production is well known using a variety of species, starting with Chlorella about sixty years ago.

[0037] Microalgae and cyanobacterial species known in food production include Arthrospira (including Arthrospira platensis and Aphanizomenon flos-aquae), Nostocales, Chlorella (including C. vulgaris, C. sorokinina, C. luteoviridis and C. pyrenoidosa), Arthrospira, Dunaliella, Haematococcus and Tetraselmis (including Tetraselmis chuii).

[0038] Commercial production of microalgae and cyanobacteria is generally carried out in open ponds under natural light, typically using dissolved CO2 as a carbon source. Such culturing can be Carbon neutral as it uses CO2 from ambient air. Biomass useful in the methods described herein can thus conveniently be obtained through open pond cultivation of microbes such as microalgae and / or cyanobacteria. The biomass is collected from the open pond culturing and used in subsequent extraction and purification to produce suitable food products or extracts useful in the preparation of food products.

[0039] Microalgae represent one source of single-cell-proteins (SCP), with other sources of SCP being fungi, yeast and bacteria. The term SCP has developed the meaning of biomass or protein extract that can be used in animal and human nutrition.

[0040] Accordingly, the whole-cell or cellular extracts disclosed herein, including protein extracts, can be used generally in human and animal nutrition, including in human food or food supplements, and in animal food, animal feed or animal food supplements.

[0041] Microbial biomass can be used as a food alternative, either as a whole-cell or cell fragment product, or as a source of valuable nutritional compounds. The biomass can supply proteins, lipids and carbohydrates, but also vitamins such as biotin, folic acid, niacin, pantothenic acid, pyridoxine, riboflavin and thiamine.

[0042] A process for the preparation of microbial extracts such as protein extracts starts with the cultivation of the microbes. Following cultivation, the microbes are harvested through a filtration and / or sedimentation process. This is followed by homogenization which is typically performed by sonication, use of microwaves, by enzymatic means, through electroporation or by other means known in the art. The homogenization involves rupturing the cells to obtain cellular fragments and thereby releasing intracellular material. Following homogenization, the desired substances (such as proteins) are extracted by one or more process steps including precipitation (by heating, lowering pH by addition of acid or other means), filtration, acidification and / or drying. This way, a dry substance (powder) or liquid paste is obtained.

[0043] Microalgae have an intense greenish color due to their high chlorophyll content. This coloring is not desirable in some food applications. As a consequence, it may be desirable to minimize or eliminate the chlorophyll content in extracts from microalgae, in particular wholecell and cell fragment extracts. For example, the microalgal biomass can be depigmented by use of ethanol. The extraction of chlorophyll pigments from the biomass can be done in one or in repeated extraction cycles, until the final extract chlorophyll content is below a desired value. For example, it may be desirable to have a chlorophyll content below about 500ppm, about 400ppm, about 300ppm, about 200 ppm or about 100ppm. The desired chlorophyll content can be obtained by two or more cycles of ethanol extraction of biomass, for example wet biomass.

[0044] Accordingly, the invention provides microalgal biomass having a reduced chlorophyll content that is preferably less than about 500ppm, such as less than about 200ppm or less than about 100ppm.

[0045] The extracted chlorophyll can be reused for other applications such as in food coloring, cosmetics, as food supplement and other nutraceutical applications. After removing the ethanol from the extracted biomiass, the depigmented biomass can be processed as whole cell product. Alternatively, the depigmented biomass can be used as a starting material for extracting nutrients such as protein and carbohydrate and used in various food applications. The extracting can be done by methods disclosed herein for obtaining microbial protein extracts.

[0046] The nucleic acid content of crude microbial biomass is generally too high for direct consumption by humans as well as many animals and can cause gout-like symptoms as a result of elevated levels of uric acid in the blood following ingestion. Crude microbial biomass is therefore preferably treated to remove nucleic acids. Conventionally done by heat treatment, the present invention provides an alternative and efficient manner in which microbial biomass or extracts from the biomass can be reduced in nucleic acid content using naturally occurring DNases or RNases.

[0047] Four classes of typical odor-active volatile chemicals in microbes such as algae are known: 1 ) fatty acid-derived volatiles compounds (aldehydes, alcohols and ketones) originated from the lipoxygenase activity or autoxidation of polyunsaturated fatty acids;

[0048] 2) sulfuric compounds such as dimethyl sulfuric (DMS, C2H6O4S), dimethyl disulfide (DMDS, CsHsSa) and methanethiol (CH4S); 3) nitrogen-containing compounds such as trimethylamine (TMA, C3H9N) originated from the action of microbial activity that reduces osmolyte trimethylamine oxide (TMAO) into TMA; and 4) umami flavor compounds related to the presence of specific free amino acids such as glutamate (Glu) and aspartate (Asp) as well as nucleotides, notably inosine monophosphate (IMP), guanosine monophosphate (GMP) and adenosine monophosphate (AMP). In this context it is important to highlight that high protein content (28%-71% dry weight) of certain microalgae could result in a high amount of free amino acids notably glutamic acid and aspartate, causing high umami flavor desirable for the imparting flavor in plantbased seafood alternative, whereas fishy off-flavors of microalgal biomass may be unwanted for certain food products (dairy foods, cookies, pasta, beverages, among others).

[0049] The disclosure provides a method of preparing protein extracts from microbes that have minimal issues with foul odour or flavor. By treating microbial biomass, in particular protein-containing fractions of microbial biomass, with ozone and the subsequently treated protein fraction with trehalose, it has been found that the resulting protein extract is free from odour and / or flavor issues. This is believed to be the result of the trehalose preventing the degradation and / or oxidation of proteins or other biomaterial that can result in the known odour and flavor issues commonly observed for microbial protein extract. Thus, while trehalose has been reported for use as an antifreeze and preservative, the function to prevent fouling as in the present context is hitherto unknown. The tremendous advantage of being able to prevent fouling with resulting smell and taste issues using a combination of trehalose and ozone is therefore unique.

[0050] Sensory assessment is a scientific field covering techniques for measuring, analyzing and interpreting human reaction to food. The assessment involves all five senses, i.e. taste, smell, touch, sight and hearing. Of these, taste and smell are generally considered most important since they are particularly related to ingestive behavior.

[0051] Odour determination can be performed by methods generally known in the art. Dynamic olfactometry is a sensorial analysis that involves human examiners for the determination of human sense of smell. In this method, samples of odorous air are collected at the source of the odour and presented to a panel of human examiners at increasing concentration using a dilution device called olfactometer. This method, which was introduced as a European standard in 2003, represents a valuable way to evaluate human exposure odour, in particular as instruments cannot determine smell in the same manner as humans.

[0052] Gas chromatography (GC) is a powerful technique for quantitative determination of volatile compounds present in odour and is therefore very useful for quantitative odor determination. Gas chromatography-olfactometry (GC-O) combines the sense of a human individual (examiner) and gas chromatography to obtain information on odour, linking particular odour to the molecular composition of the sample. This method therefore both detects a particular odour or aroma and simultaneously provides chemical information about the composition of the sample.

[0053] Tasting methods involve human participants but may also involve analytical methods. In monadic testing, human participants (examiners) are given a single product and are asked for their response with respect to palatability and whether the product is generally liked or disliked. Comparative testing involves the comparison of two or more products and the examiners asked to state which one they prefer after the tasting. In triangle tasting, three products are tasted, two of which are the same and one is different. This method represents a way to measure minor recipe modifications on existing products.

[0054] Tasting involves both flavor and texture. The former measures appearance, aftertaste and amplitude, while the latter measures parameters such as hardness, adhesiveness, viscosity and chewiness.

[0055] In Fig. 1 there is shown a schematic diagram of an exemplary protein extraction process in accordance with the invention. The process starts from a wet biomass that can be obtained through any cultivation means, such as by open pond cultivation or photobioreactor. The wet biomass (in the illustrated example the mass is C. vulgaris but can be any suitable microbial species such as microalgae) is suspended in a basic aqueous solution that contains any suitable base. For example, the base can be NaOH, KOH, Mg(OH)2or other suitable base. The treatment with base solubilizes proteins and lipids allowing separation of carbohydrates that remain largely insoluble. The carbohydrates can therefore be collected by separation techniques (e.g., filtration or sedimentation), used as is or purified further.

[0056] The soluble fraction containing dissolved protein is subsequently treated with ozone gas that is bubbled through the solution. Any suitable gas injector can be used for this purpose, including for example Venturi injectors or Mazzei injectors. Alternatively, bubble diffusers can be used. Prior to ozone treatment, the pH of the solution is preferably lowered to neutral or near-neutral, such as to a pH in the range of about 7 to 9.

[0057] The soluble fraction can be treated with one or more nucleases to remove nucleic acid in the fraction. The nuclease treatment can be performed prior to or following ozone treatment. The nuclease can be one or more DNase, one or more RNase or a combination of one or more DNase and one or more RNase. Since RNA represents a bulk of nucleic acid material, it may be sufficient to remove RNA only. Therefore, the treatment can preferably include treatment with one or more RNase only, such as RNaseA.

[0058] After the ozone and optional nuclease treatment, the protein can be isolated by filtration or precipitation. Any filtration means known in the art can be used, such as membrane filtration (e.g. tangential flow filtration). Protein precipitation can be done by heat treatment, where the solution is heated to certain temperature such as a temperature in the range of 60 to 80 °C to induce denaturation and precipitation of endogenous protein. Alternatively, protein precipitation can be initiated by addition of salt such as calcium, magnesium, manganese and ferrous salts, or by addition of polymeric materials such as dextran, polyethylene glycol, polyphosphate, alginate and / or carboxy methyl cellulose. The protein can also be precipitated by addition of acid (so-called isoelectric precipitation), for example using mineral acids such as hydrochloric acid or sulfuric acid. Yet another method of precipitating protein involves use of miscible solvents such as methanol or ethanol.

[0059] The final protein concentrate can be used as is, i.e. in a wet form. Alternatively, there can be one or more drying step to obtain a dried protein extract.

[0060] One or more preservatives can be added to obtained extracts, including protein extracts in dry or wet form. Any known preservative can be used for this purpose, including antibacterial and antioxidant preservatives. Exemplary preservatives include lactic acid, potassium or calcium sorbate, ascorbic acid, calcium nitrate, calcium propionate, sodium nitrite, sulfites, EDTA.

[0061] It should be appreciated that carbohydrate-containing fractions obtained through processing of microbes such as microalgae can also be treated with one or more nuclease to reduce the nucleic acid content of microbe-derived carbohydrate products. For example, the insoluble fraction obtained through treatment of wet biomass with an alkaline solution contains carbohydrates from the biomass. This insoluble fraction can be redissolved and treated with one or more nuclease as described in the above to obtain a carbohydrate-containing product that is at least partially depleted in nucleic acid. Protein extract obtained by the processes disclosed herein can be used as a source of protein to produce food products. The protein extract can used alone or in combination with other protein sources. For example, the protein extract can be used in the production of meat substitute products, either as the sole source of protein or in combination with one or more additional protein source. The protein extract can also be used in various food products, including crackers, bread or cake products, shakes and the like. The protein extract can also be used in food supplements, where the protein can be combined with suitable minerals, alternative protein sources, sugars, vitamins, plant extracts etc.

[0062] As should be appreciated from the foregoing, the improved palatability of the microbial products (e.g., microbial extracts such as protein and carbohydrate extracts) disclosed influences smell / odour and / or flavor of the microbial products (e.g., whole-cell products or protein and / or carbohydrate extracts). In other words, the microbial extracts and preparations described herein have improved smell and / or flavor compared with extracts and cellular preparations known in the art.

[0063] In particular, protein obtained by the process disclosed herein is found to be free of foul odour compared with protein obtained by processes known in the art. The protein and / or carbohydrate obtained through the disclosed processes is further depleted in nucleic acid content, rendering the protein more suitable for human consumption.

[0064] As used herein, including in the claims, singular forms of terms are to be construed as also including the plural form and vice versa, unless the context indicates otherwise. Thus, it should be noted that as used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0065] Throughout the description and claims, the terms “comprise”, “including”, “having”, and “contain” and their variations should be understood as meaning “including but not limited to” and are not intended to exclude other components.

[0066] Those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure are used. The present invention also covers the exact terms, features, values and ranges etc. in case these terms, features, values and ranges etc. are used in conjunction with terms such as about, around, generally, substantially, essentially, at least etc. (i.e., "about 3" shall also cover exactly 3 or "substantially constant" shall also cover exactly constant).

[0067] The term “at least one” should be understood as meaning “one or more”, and therefore includes both embodiments that include one or multiple components. Furthermore, dependent claims that refer to independent claims that describe features with “at least one” have the same meaning, both when the feature is referred to as “the” and “the at least one”.

[0068] Features disclosed in the specification, unless stated otherwise, can be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless stated otherwise, each feature disclosed represents one example of a generic series of equivalent or similar features.

[0069] Use of exemplary language, such as “for instance”, “such as”, “for example” and the like, is merely intended to better illustrate the invention and does not indicate a limitation on the scope of the invention unless so claimed. Any steps described in the specification may be performed in any order or simultaneously, unless the context clearly indicates otherwise.

[0070] All of the features and / or steps disclosed in the specification can be combined in any combination, except for combinations where at least some of the features and / or steps are mutually exclusive. In particular, preferred features of the invention are applicable to all aspects of the invention and may be used in any combination. The scope of the disclosure is only limited by the appended patent claims.

[0071] The invention is further described by the following non-limiting examples.

[0072] Example 1

[0073] 1 gram of wet Chlorella vulgaris biomass (water content about 70%, i.e. about 0.3g dry biomass) was resuspended in 4 to 10 mL of 0.01 to 1 M NaOH solution, and the resulting solution gentle mixed overnight at room temperature The next day, the soluble fraction was separated from the insoluble fraction by centrifugation, sedimentation or filtration. The pH of the soluble fraction was decreased to pH 7 to 9 and ozone gas bubbled through the solution for 1 - 10 min. After 30 minutes, nucleases (DNase and / or RNase) were added (3 p.g / mL of extracted solution).

[0074] The protein containing solution was concentrated by membrane filtration (Millipore, 10 kDa cutoff) and followed by drying overnight in 60 °C incubator. In an alternative process, the protein precipitated by heating (60 to 80 °C) for about 1 hour or by addition of acid (2M HCI) to lower the pH to about 3, followed by filtration or centrifugation to collect the precipitated protein. Finally, trehalose (1 %) and a preservative (lactic acid, 25 mg / g of wet extract) were added to the precipitated protein. The protein was prepared as a powder or as a paste / cake to be ready for subsequent use.

[0075] About 0.3 g of dry protein was obtained from 1 g (dry weight) of the biomass.

[0076] Example 2

[0077] Wet biomass (1g) was resuspended in NaOH as described under Example 1 , with the difference that there was neither treatment with ozone nor was there addition of trehalose.

[0078] The extracted protein thus obtained was compared to protein obtained by treatment of the biomass with ozone as described in Example 1 , without the addition of trehalose.

[0079] While the protein extracted without ozone / trehalose treatment had a distinct foul odour, the sample treated with ozone was free of the foul odor. Upon storage for 2 weeks, there was a gradual return of foul odor to the extracted protein.

[0080] Example 3

[0081] Extracted protein obtained through extraction using ozone treatment only was compared with extracted protein obtained through the process described in Example 1 . While the protein obtained through ozone treatment showed signs of foul smell appearing upon prolonged storage, the protein obtained according the process in Example 1 was found to be stable upon longer storage, with no appearance of foul smell following at least 3 months of storage.

[0082] Example 4

[0083] About 1 g wet Chlorella vulgaris biomass was resuspended in 20 ml ethanol for 30 min with gentle mixing. The ethanol treated biomass was centrifuged and the colored liquid was decanted. To the sedimented biomass another 20 ml ethanol was added and treated the same way as in the first round. After centrifugation, the sedimented biomass was washed with water. As shown in Fig 2. most of chlorophyll pigment is removed (the extractable chlorophyll content of the depigmented biomass is estimated as less than 200ppm).

[0084] Fig. 3 shows effects of depigmentation of algal biomass with ethanol. Samples on the left (A) are ethanol treated Chlorella vulgaris biomass and the one on the right (B) is untreated biomass. A clear difference of the ethanol treatment can be observed, with most of the pigment removed by the process.

[0085] Example 5

[0086] A sensory analysis was performed. Coded dried samples of whole cell extracts and protein extracts were provided to 30 adults to smell with reporting of which one of the samples were experienced as smelling bad.

[0087] As can be seen in the below tables, the treated samples had significantly less smell issues than untreated samples.

[0088] Table 1 . Bad smell removal from whole cell with ozone and trehalose treatment. Table 2. Bad smell removal from protein extract prepared using the process of Example 1.

Claims

CLAIMS1 . A method of preparing protein extract from one or more microbes comprising steps and order of: a. resuspending a microbial biomass in a suitable basic solution to obtain a soluble fraction and an insoluble fraction; b. separating the soluble fraction obtained in step (a) from the insoluble fraction; c. lowering the pH of the soluble fraction to a pH in the range of 7 to 9 using a suitable acid; d. treating the soluble fraction from step (c) with ozone and optionally add one or more nucleases to the ozone treated fraction; e. concentrating the solution from step (d) and / or precipitate protein from the solution to obtain a protein extract ; and f. adding trehalose and optionally one or more preservative to the protein extract.

2. The method of claim 1 , wherein the basic solution in step (a) is a solution containing in the range of 0.1 M to 5.0 M sodium hydroxide solution.

3. The method of any one of the previous claims, wherein the treating with ozone comprises bubbling ozone gas through the soluble fraction at temperature in the range of about 5-30°C for a period of time in the range of about 1-60 minutes.

4. The method of any one of the previous claims, wherein the concentrating in step (e) is performed by heating and / or filtration.

5. The method of the previous claim, wherein the heating is performed at a temperature of about 60 to 80°C for a period of time in the range of about 0.5 to 5 hours.

6. A method of improving smell and / or flavor of a whole-cell preparation or cellular extract from an edible microbe comprising treating cells or cellular fragments from one or more microbe with ozone followed by the addition of trehalose to obtain a microbe preparation or extract having improved smell and / or flavor.

7. The method of the previous claim wherein the treating with ozone comprises bubbling ozone gas a solution or suspension comprising microbial cells or cellular fragments at a temperature in the range of about 5-30°C for a period of time in the range of about 1 -60 minutes.

8. The method of any one of the previous two claims, wherein the whole cell preparation or cellular extract undergoes a further drying step.

9. The method of the previous claim, wherein trehalose is added to a final concentration in the range of 0.5-20% by weight of the dried cell preparation or cellular extract.

10. The method of any one of the previous claims, wherein the microbial biomass or microbial cells or cellular fragments are pretreated by one or more steps of extraction with ethanol.11 . A method of preparing nucleic-acid depleted protein and / or carbohydrate extract from one or more edible microbe, the method comprising i) preparing a liquid extract from one or more edible microbial species, the liquid extract comprising protein and / or carbohydrates; ii) adding one or more nuclease to the liquid extract from step (i) and incubating for a period of time sufficient to allow the one or more nuclease to digest nucleic acid in the extract; whereby a nucleic-acid depleted protein and / or carbohydrate extract is obtained.

12. The method of the previous claim, wherein the one or more nuclease is selected from at least one DNase and at least one RNase, such as DNasel and RNaseA.

13. The method of any one of the previous two claims, wherein the one or more nuclease is RNaseA, wherein treatment with the RNaseA results in a RNA-depleted protein and / or carbohydrate extract.

14. The method of any one of the previous three claims, comprising a further step of concentrating and / or drying the treated protein and / or carbohydrate extract.

15. The method of any one of the previous claims, wherein the one or more edible microbe is selected from one or more edible bacterial, yeast, fungal or microalgal species.

16. The method of the previous claim wherein the one or more edible microbe is selected from the genus Chlorella, Arthrospira, Tetraselmis, Scenedesmus and Nannochloropsis.

17. The method of the previous claim wherein the one or more edible microbe is selected from Chlorella vulgaris, Arthrospira platensis, Tetraselmis chuii, Isochrysis galbana, Tetraselmis suecica, Scenedesmus almeriensis and Nannochloropsis gaditana.

18. Microbial extract characterized in that the extract comprises in the range of 0.5-20% by weight of trehalose.

19. The microbial extract of the previous claim, wherein the extract is a protein extract.

20. The microbial extract of claim 18 or claim 19, wherein the extract is a whole cell extract.21 . The microbial extract of any one of the previous claims 18 - 20, wherein the extract is from one or more edible bacterial, yeast, fungal or microalgal species.

22. The microbial extract of the previous claim, wherein the extract is from a microalgal species and has a chlorophyll content that can be extracted by ethanol is less than about 200ppm.

23. The microbial extract of the previous claim wherein the species is selected from the genus Chlorella, Arthrospira, Tetraselmis, Scenedesmus and Nannochloropsia.

24. The microbial extract of the previous claim wherein the species is selected from Chlorella vulgaris, Arthrospira platensis, Tetraselmis chuii, Isochrysis galbana, Tetraselmis suecica, Scenedesmus almeriensis and Nannochloropsis gaditana.

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