FROM NON-REFINED MICRO ALGAE & USES THEREOF

Field of the invention

The present invention relates to a method for the preparation of microalgal for use as a foodgrade ingredient without the need of any further purification or separation step and resulting products therefrom.

Background

Microalgae are a source of protein and other nutrients for human diets (Caporgno & Mathys, 2018, Frontiers in Nutrition, 5. https://doi.org/10.3389/fnut.2018.00058). The biomass can be obtained via heterotrophic or phototrophic fermentation. Typically, biomass harvesting is done by centrifugation of fermentation broth, combined or not with a washing step. This concentrated wet biomass can be used for food manufacturing but is submitted to further downstream processing (DSP) to obtain purified extracts namely lipids, pigments, protein, and antioxidants (Grossmann et al., 2020, Critical Reviews in Food Science and Nutrition, 60(17), 2961-2989, https://doi.org/! 0.1080/10408398.2019.1672137; Nitsos et al., 2020, Biotechnology Advances, 45, 107650, https://doi.Org/10.1016/j.biotechadv.2020.107650}. DSP to obtain food-grade protein extracts from microalgae can be done by a purification approach or a less-refmement approach, involving cell disruption, solubilization (pH shifts, salt), fractionation by centrifugation, and drying (Fig. 1).

Among the cell disruption methods, the Pulsed Electric Fields (PEF) and the High Pressure Homogenization (HPH) treatments have emerged as promising methods for the mild and complete disruption of biological cells, respectively (Carullo et al, 2018, Algal Research 31 (2018) 60 69)

Mechanical cell disruption include the use of pulsed electric fields, high pressure processing and high pressure homogenization on microalgae for the aqueous extraction of intracellular compounds from those (Carullo et al, 2018, Algal Research 31 (2018) 60 69; Ahmed et al., 2022, Algal Research, 102617; Nunes, 2020, Algal Research, 45, 101749) at is used for maximizing the extraction yields of products from algae material such as proteins and lipids for further processing of those products. It is known that rheological properties of microalgal suspensions are affected by mechanical and thermal treatment (Bernaerts et al., 2017, Algal Research, 452-463) but the application potential of biomasses of C. vulgaris was limited to fluid food products which are mecanically and/or thermally processed such as vegetable based soups or several diary products. All the cell disruption techniques were developped with the aim of optimizing the yield of release of the of cellular constituents in microalgae wherein the cell wall rigidity as considered to be the bottleneck for an efficient extraction process.

In the field of plant-based products, in particular in meat alternatives such as plant-based chicken, burgers and bacon, several additives are needed to ensure an acceptable texture, taste and cooking behavior to nuggets and burgers, and then on cooking it gels to give very firm textures for meat alternatives like plant-based chicken, burgers and bacon. Among those additives, methyl cellulose, thickening agents and oils are currently the most frenquently used as an additive.

Since plant-based products are attracting more and more nterest from consumers, in particular as a source of protein as an alternative to meat, there is a need for methods rendering the preparation of those products “consumer friendly” by allowing the decrease or elimination of food additives.

Summary of the Invention

An object of this invention is to provide a method for the preparation of microalgae suspension which is directly suitable for use as a food-grade ingredient without the need of any further purification or separation step.

It is advantageous to provide a method which allows to prepare a microalgae material which uses microalgae as a whole (e.g. wet or in dry form) and which is suitable for further processing as a food-grade ingredient (e.g. by extrusion or spheronization or mixing).

It is advantageous to provide a method which allows the improvement of the rheological properties of microalgae material and which is suitable for further processing as a food-grade ingredient or additive without of the need of having recourse to chemical additives such as binders or thickeners into the final product.

It is advantageous to provide a method which allows the preparation of a texturing ingredient suitable for plant-based food products which improves the texture of plant-based food products such as meat alternative plant-based food product without the need of chemical additives.

Objects of this invention have been achieved by providing a method according to claim 1 and products thereof. Objects of this invention have been achieved by providing a food product according to claim 13.

Disclosed herein, according to a first aspect of the invention, is a method for the preparation of a food or a feed product or ingredient from non-refined microalgae, wherein said method comprises the following steps: a) Providing a microalgae biomass from Chlorella genus, in particular C. vulgaris,' b) Diluting the microalgae biomass with water at a concentration range of about 10 to about about 25 % w./w. to obtain a microalgae biomass suspension at a temperature from about 4 to about 15°C; c) Subjecting the microalgae biomass suspension to high pressure homogenization at a temperature lower than 55°C to obtain a homogenized microalgae biomass suspension; d) Isolating the said homogenized microalgae biomass suspension.

Disclosed herein, according to a further aspect of the invention, is a homogenized microalgae biomass suspension obtainable by a method according to the invention.

Disclosed herein, according to a further aspect of the invention, is the use of a homogenized microalgae biomass suspension according to claim 10 or 11 for the preparation of a food or a feed product.

Disclosed herein, according to a further aspect of the invention, is a composition for food or a food texturing ingredient, comprising a microalgae material according to the invention.

Disclosed herein according to a further aspect of the invention is meat alternative plant-based food product comprising a microalgae material according to the invention.

The invention is based on the unexpected finding that the method of the invention allows the provision of microalgae material with increased viscosity at a temperature from about 60 to about 95°C, increased binding ability, in particular increased gelling capacity upon heating. Those properties allow the preparation of food or feed products in various forms by incorporating concentrated microalgae material as a whole without the need of further prior purification/extraction from the microalgae material. The so-obtained food of feed products benefit from preserving all the initial components from the concentrated microalgae material, enriched nutritional benefits and from eliminating the need of using textural and binding chemical additives.

Other features and advantages of the invention will be apparent from the claims, detailed description, and figures. Description of the figures

Figure 1 illustrates production of food-grade protein extracts from microalgae/cyanobacteria by a purification approach (left) and a less-refmement approach (right) (Grossmann etal., 2020, supra).

Figure 2 illustrates the main steps a) to d) of a method according to the invention to obtain a homogenized microalgae biomass suspension. Optional step e) allows to obtain a dried homogenized microalgae biomass under step f) for use in the preparation of a food product or a texturing ingredient.

Figure 3 represents i) the apparent viscosity profile of A: GC cell (1), GC-900bar (2), GC- 1’200 bar (3) and GC-1500bar (4) at 13.2 % (w/w) dry matter content and of B: untreated microalgae cells at 25% w/w (1) and a sample (25 % w/w) treated by a method according to the invention (2) versus the temperature (T) measured as described in Example 2. Curves are average measurements done in triplicates; ii) the Rheologic behavior (C) of a sample at 25 % w.w. dry matter obtained by a method according to the invention at a high-pressure homogenization of 1’500 bar and before (left) and after (right) thermal treatment (90°C) and stored overnight at 4°C.

Figure 4 shows pictures of burger patties comprising a microalgae material treated according to a method of the invention as described in Example 3. A & B: before cooking; C & D: after cooking.

Detailed description

"Microalgae" refers to microscopic eukaryotic single cells composed of a nucleus, one or more chloroplasts, mitochondria, Golgi bodies, endoplasmic reticulum and other organelles.

Microalgae suitable in the context to the invention belong to the phylum Chlorophyta, preferably Chlorella genus, more preferably Chlorella vulgaris. Those include species such as Chlorella protothecoides, Chlorella ellipsoidea, Chlorella minutissima, Chlorella zofmienesi, Chlorella luteoviridis, Chlorella kessleri, Chlorella sorokiniana, Chlorella fusca var. vacuolata Chlorella sp., Chlorella cf. minutissima or Chlorella emersonii.

Other species of Chlorella can be selected from the group consisting of anitrata, Antarctica, aureoviridis, Candida, capsulate, desiccate, ellipsoidea (including strain CCAP 211/42), emersonii, fusca (including var. vacuolata), glucotropha, infusionum (including var. actophila and var. auxenophila), kessleri (including any of UTEX strains 397,2229,398), lobophora (including strain SAG 37.88), luteoviridis (including strain SAG 2203 and var. aureoviridis and lutescens), miniata, cf. minutissima, minutissima (including UTEX strain 2341), mutabilis, nocturna, ovalis, parva, photophila, pringsheimii, protothecoides (including any of UTEX strains 1806, 411 , 264, 256, 255, 250, 249, 31, 29, 25 or CCAP 211/8D, or CCAP 211/17 and var. acidicola), regularis (including var. minima, and umhricala , reisiglii (including strain CCP 11/8), saccharophila (including strain CCAP 211/31, CCAP 211/32 and var. ellipsoidea), salina, simplex, sorokiniana (including strain SAG 211.40B), sp. (including UTEX strain 2068 and CCAP 211/92), sphaerica, stigmatophora, trebouxioides, vanniellii, vulgaris (including strains CCAP 211/1 IK, CCAP 211/80 and f. tertia and var. autotrophica, viridis, vulgaris, vulgaris f. tertia, vulgaris f. viridis), xanthella, and zofingiensis.

The expression “heterotrophic fermentation conditions” refers to conditions for cell growth and propagation using an external carbon source under dark conditions. Several examples of microalgae growth under heterotrophic conditions, using different organic carbon sources, are summarized in the literature (Hu et al., 2018, Biotechnology Advances 36, 54 67).

A “microalgae culture medium" suitable for heterotrophic fermentation conditions comprises i) a carbon source and ii) nutrients for microalgal growth. According to the present invention, typical heterotrophic fermentation conditions comprise dark at 21 °C (and shaking in case of liquid cultures).

Nutrients for microalgae growth suitable for heterotrophic fermentation conditions are known to the skilled person and can be found for example under https://utex.org/pages/algal-culture- media (University of Texas at Austin for its algal culture collection (UTEX)).

The microalgal biomass is itself a finished food ingredient and may be used in foodstuffs without further, or with only minimal, modification. Alternatively, after concentration, microalgal biomass can be processed to produce different food or feed products containing microalgae as an ingredient aiming to increase the nutritional value (pasta, beverages, drinks, meat substitutes, etc.) and/or as food supplements (in form of capsules, pills, etc).

The expression “high pressure homogenization” (HPH) refers to a high-pressure treatment comprising forcing the biomass to pass through a narrow orifice at high pressure under a continuous flow of biomass as described in Soo Youn Lee et al., 2017, 244 (2), 1317-1328.

The expression “spheronization” is a process where extrudates (the output from an extruder) are shaped into small rounded or spherical granules. In practice, granules vary in size from 0.4 to about 3.0 mm. The microalgal material obtained by a method according to the invention can be further subjected to spheronization and the obtained granules can be used for the preparation of meat analogues such as burger patties.

The expression “binding ability” refers to the ability of gluing the minced particles such as texturized vegetable protein as described in Kyriakopoulou et a., 2021, Foods, 12; 10(3):600. doi: 10.3390/foodsl0030600 and can be measured by texture profile analysis such as hardness, and chewiness; and sensory evaluation such as mouthfeel and appearance of the end-product as described in d/wa et al., 2017, J. Food Process. Preserv., 41, el 3134.

Typically, good binding and texturizing abilities result in food products not falling apart while and also after cooking. Benchmark for the sensory and textural properties is the meat products (patties, nuggets, sausages, etc.).

The expression “gelling strength” or “gelling capacity” refers to the stiffness of a gel obtained upon heating and can be measured by rheological measurement. Typically, in food industry, preferred methylcellulose viscosity is approx. 4000 mPa.s or above (at 2% w.w. contents) for meat analogues. It is added into food formulas at a concentration from about 1 to about 2% w.w. contents.

Referring to the figures, in particular first to Figure 2, is provided an illustration of a method for the preparation of a food or a feed product or ingredient from non-refined microalgae. The illustrated method generally comprises the steps of subjecting an aqueous suspension of a microalgae biomass from Chlorella genus, in particular C. vulgaris at a concentration range of about 10 to about 25 % w./w. to high pressure homogenization at a pressure of about 150 to about 1’500 bar.

More specifically, the steps of the embodiment illustrated in Figure 2 comprise the following steps: a) Providing a microalgae biomass from Chlorella genus, in particular C. vulgaris; b) Diluting the microalgae biomass with water at a concentration range of about 10 to about 25 % w./w. to obtain a microalgae biomass suspension at a temperature from about 4 to about 15°C; c) Subjecting the microalgae biomass suspension to high pressure homogenization at a temperature lower than 55°C to obtain a homogenized microalgae biomass suspension d) Isolating the said homogenized microalgae biomass suspension.

According to another particular aspect of the invention, is provided a homogenized microalgae biomass suspension from Chlorella genus, in particular C. vulgaris wherein said microalgae biomass has a high protein content, from about 35 to about 50 % w.w..

According to another particular aspect of the invention, is provided a homogenized microalgae biomass suspension from Chlorella genus, in particular C. vulgaris wherein said microalgae biomass has a low lipid content, less than 30% w.w, typically from about 10% to about 30% w.w.. According to a particular aspect, the microalgae biomass suspension is at a pH from about 6 to about 7.

According to another particular aspect, microalgae biomass is diluted with water at a concentration range of about 10 to about 15 % w./w. (e.g. 14 w./w.) to obtain a microalgae biomass suspension.

According to a particular aspect, high pressure homogenization is carried out at a pressure from about from about 400 to about 1’500 bar such as from about 600 to about 1’500 bar.

According to a further particular aspect, high pressure homogenization is carried out at a pressure from about 900 to about 1’200 bars.

According to another particular aspect, high pressure homogenization is carried out under temperature control of the microalgae biomass suspension such that the temperature does not exceed 55°C.

In particular, the outlet of the homogenization chamber in which the microalgae biomass suspension is placed and where the high-pressure homogenization is applied is maintained at a temperature higher than 4°C but lower than 55°C, for example through the use of a heat exchanger at the exit of the homogenization chamber with water at about 12°C.

According to another particular aspect, high pressure homogenization is carried at a flow rate from about 45 to about 65 L/h, typically from 45 to about 50 L/h (e.g. 48 L/h).

According to another particular aspect of the invention, is provided a homogenized microalgae biomass suspension from Chlorella genus, in particular C. vulgaris, wherein said homogenized microalgae biomass suspension has a viscosity at 85-95°C increased by a factor 2 to about 5 compared to the initial microalgae biomass.

According to another particular aspect of the invention, is provided a homogenized microalgae biomass suspension from Chlorella genus, in particular C. vulgaris, wherein said homogenized microalgae biomass suspension at a concentration of about 14 % w/w has a viscosity at 85- 95°C increased by a factor 4 (e.g. from about 50 to 200 mPa.s) compared to the initial microalgae biomass.

According to another particular aspect of the invention, is provided a homogenized microalgae biomass suspension obtainable by a method according to the invention having a viscosity at 60- 70°C increased by a factor 4 (e.g. from about 50 to 200 mPa.s) compared to the initial microalgae biomass provided under step a).

According to another particular aspect of the invention, is provided a homogenized microalgae biomass suspension from Chlorella genus, in particular C. vulgaris at a concentration of about 10 to about 15 % w./w., having a high protein content, typically from about 35% to about 50% and a viscosity at 60-70°C increased from about 50 to 200 mPa.s.

According to another further aspect, the homogenized microalgae biomass suspension according to the invention, having a low lipid content, typically less than 30% w.w., typically from about 10% to about 30% w.w..

According to another particular aspect of the invention, the homogenized microalgae biomass suspension may be further subjected to extrusion or spheronization or direct mixing into a food product.

According to another particular aspect of the invention, the homogenized microalgae biomass may be used a texturing ingredient, a binder or a thickener, in particular in food products such as meat analogues such as burger patties, nuggets, fish sticks, etc., vegan dairy alternatives such as yogurts and creams.

According to another particular aspect of the invention, the homogenized microalgae biomass is further subjected to a heating step at a temperature from about 60 to about 95°C before being used.

According to another particular aspect of the invention, the homogenized microalgae biomass suspension is a food or a feed product or ingredient, in particular food or a feed product or ingredient that is intended to be subjected to a temperature from about 60 to about 95°C before being ingested.

According to another particular aspect of the invention, the composition is a cosmetic product or cosmetic ingredient.

According to another particular aspect of the invention, is provided a use of homogenized microalgae biomass suspension according to the invention for the preparation of a food or a feed product.

According to another particular aspect of the invention, is provided a food or a feed product comprising a microalgae material according to the invention.

According to another particular aspect of the invention, is provided a vegan food or a feed product such as a meat alternative product comprising a microalgae material according to the invention at a concentration from about 3 to about 10% w.w..

According to another particular aspect of the invention, is provided a meat alternative plantbased product or a vegan dairy alternative such as yogurt and cream comprising less than 50% w.w. of methyl cellulose. Examples illustrating the invention will be described hereinafter in a more detailed manner and by reference to the embodiments represented in the Figures.

EXAMPLES

Example 1: Method according to the invention

A method according to the invention was carried out as illustrated under Fig. 1 B follows:

Step 1: Provision of a microalgae biomass

Microalgae raw material (Chlorella vulgaris UTEX 30) was provided as a microalgae slurry in frozen state (stored in the freezer (-20 °C)). Their composition in dry basis (db.) and their initial dry matter content as determined by using a moisture analyzer (120°C, stop criteria Img in 50 seconds, initial weight of 2 grams) are summarized in Table 1 below:

Table 1

Step 2: Dilution of the microalgae biomass with water to obtain a microalgae biomass suspension

The frozen microalgae biomass was let thawing at room temperature for about 8 hours to 1 day and was then diluted at a concentration of 14% w./w. with demineralized water and the microalgae biomass suspension was stored overnight at 4°C before subjecting to the high- pressure homogenization.

Step 3: Subjecting the microalgae biomass suspension to high pressure homogenization (HPH)

GC was subjected to HPH treatment using NS3006L ARIETE Homogenizer (GEA Niro Soavi, n° 6892) at various pressures (900, 1’200 and 1’500 bar) and at various flow rates (64, 54 and 48 L/h) at an initial temperature of 5°C. Cold water (approx. 12°C) was used to minimize the biomass temperature increase during HPH.

Step 4: Isolating the obtained homogenized microalgae biomass suspension

The obtained homogenized microalgae biomass suspension can be mixed with other food ingredients directly or spray-dried for storage.

To obtain the spray dried product, the homogenized biomass was dried using a Mobile Minor MM-PSR Spray Dryer (GEA Niro Soavi, n° P42141.19.00551.01). The operating conditions were set at pressure of 1 bar, air flow of approx. 103 kg/h, inlet and outlet air temperatures of 180 and 103°C, respectively. The product was pumped using a peristaltic pump NEMA 4X IP66 (Watson Marlow, United Kingdom) at 9 rpm, corresponding to a product flow of 2 kg/h.

Example 2: Characterization of the microalgae biomass suspension obtained by a method according to the invention

The physicochemical properties of the resulting microalgae preparations from Example 1 were characterized as follows on the following batches: (i) initial GC biomass (GC cell), (ii) homogenized biomasses at 900, 1200 and 1’500 bar (GC_900, GC_1200 and GC_1500). pH, density, conductivity, and moisture content

The pH and conductivity of the different microalgae preparations were measured using a 913- pH meter (Metrohm). Density was done using a density meter (DMA 1001, Anton Paar). Moisture content was done using the rapid moisture analyzer (HC103 Mettler-Toledo) at the conditions of 120°C, initial mass of 2 grams and stop criteria of 1 mg/50 s.

Protein content and protein solubility

The calculation of the protein content of the microalgae preparations was done using a Total Organic Carbon/Total Nitrogen (TOC/TN) analyzer (Shimadzu, Japan). The samples were diluted 500 times using demineralized water. The auto dilution and injection volume during measurement were set at 2 and 100 pl, respectively. The total protein and total dry weight concentration were calculated using equations (1) and (2):

Total protein (mg/L) = f x TN (1)

Where TN is the total nitrogen concentration in mg/L and f is the nitrogen conversion factor, set at 4.21 (calculated based on the amino acid composition of the biomass).

Dry weight supernatant (mg/L = (1 — MC) * p * 10 ⁴ (2)

Where MC is the moisture content (% w/w) of the samples and p is the density (g/mL). The protein solubility was determined by the amount of protein remaining soluble in the supernatant after centrifugating the microalgae preparations at 9’000 rpm, for 30 min at 4°C. The respective supernatants were collected, diluted 50 times and TOC/TN analysis was performed. The protein solubility can afterwards be calculated using equation (3) 100 (3)

The Physicochemical characterization of the Chlorella samples is presented below under Table 2. Table 2

GC samples were slightly acidic (pH around 6.4). The protein solubility increased with the HPH treatment. Thermal properties of microalgae preparations

Viscosity-temperature profile

A viscosity -temperature profile was done for all the microalgae preparations using a modular compact rheometer (MCR 302, Anton Paar, Graz, Austria) fitted with a starch cell (C-ETD 160/ST, Anton Paar, Austria) to acquire information on gelling properties. Most of the biomasses were analysed at approx. 14 % w./w. dry matter. Analysis was then done on spray- dried HPH-treated powder which rediluted in water at a dry matter (DM) of about 30 % w./w. with the objective to obtained a gel.

The test consists of seven intervals: equilibrium, mixing, water uptake, heating, holding, cooling, and holding according to the protocol for the thermos-mechanical analysis in Table 3 below. The heating rate was 6°C/min. RheoCompass software and firmware (Anton Paar, Graz, Austria) provides the apparent viscosity.

Table 3 The viscosity profile of the different Chlorella preparations in function of temperature is shown in Figure 3A. Respective characterization values are displayed in Table 4.

Table 4

Those data indicates that peaks and final viscosities are linearly dependent on the pressure applied under the HPH treatment. At 900 bar, the peak viscosity increased by a factor 2; at 1’200 bar, by a factor 4 and at 1’500 bar, by a factor 5. Viscosity of the product obtained with a HPH treatment at 1’500 bar reached 4’000 mPa.s when viscosity analysis performed on the aqueous solution with 30% w/w concentration which compares with the preferred viscosity obtained at 2% w./w. content in methylcellulose). For the sake of comparison, the peak viscosity was increased by less than a factor 2 in Benaerts, 2017, supra.

Figure 3B shows that a gel formation is observed sample (25 % w./w.) treated by a method according to the invention which is not the case with the corresponding microalgae cells not treated with a method according to the invention. The behavior of a sample at 25 % w.w. dry matter obtained by a method according to the invention at 1500 bars as detailed above and before (left) and after (right) thermal treatment (95°C) and stored overnight at 4°C is shown under Figure 3C. Those data clearly support stronger gelling capacity of the microalgae material obtained by a method according to the invention upon heating compared to untreated. Final apparent viscosity increased by almost a factor 4 after the application of the method of preparation.

Example 3: Preparation of burger patties with homogenized microalgae material according to the invention

Burger patties were prepared as follows. The ingredients from Table 5 are mixed in a bowl except water and the homogenized microalgae. In a separate cup, the homogenized microalgae material which has been obtained by a method of the invention as described under Example 1 and is provided as spray dried material is solved in water with a spoon until the algae clumps disappear. Approximately after 3 mins of stirring the mixture, the obtained aqueous solution of homogenized microalgae material is added into the burger mix. Burger mix is kneaded and the patty shape is given with the help of a burger press and can be easily handled for cooking as standard meat patties (Figure 4A & B). Then, the burgers are fried for about 4 minutes each side on medium heat. To cook the patties in an oven, 120°C and 30 minutes each side (e.g. about 60 minutes total) is sufficient.

Table 5

The obtained cooked burger patties have a texture comparable to standard meat substitutes patties (Figure 4C & D). Altogether the present data support that a method according to the invention allows to achieve a homogenized microalgae biomass suspension with a gellig capacity that allows its use as a whole ingredient without any further purification, in wet or dry form, for different applications, in particular as a texturing ingredient suitable for plant-based food products intended to be subjected to heat treatment which improves the texture of plant-based food products such as meat alternative plant-based food product without the need of chemical additives.