Microalgae-based products with improved nutrient bioavailability and oxidative stability

Abstract

The present invention relates to a method for making a product for human consumption comprising partially dissolved microalgae, the method comprising the steps of: a) preparing a suspension of microalgae, the microalgae belonging to a phylum selected from Chlorophyta, Ochrophyta, and Heterokontophyta; and b) applying a pulsed electric field to the suspension of microalgae, the pulsed electric field being between 25 and 150 kJ / kg suspension (kg sus). -1 c) optionally treating the microalgae with an enzyme; d) incubating the biomass suspension at 300 rpm and 4-37° C. for 6-48 hours; e) forming a partially lysed microalgal biomass; and f) adding the partially lysed microalgal biomass to a product for human consumption.

Description

Detailed Description of the Invention

[0001] [Introduction] Microalgae are increasingly attracting attention as an alternative source of omega-3-PUFAs and are the primary producers of such high-value compounds. Currently, the omega-3-PUFA-rich lipids of microalgae are mainly utilized through extraction. Meanwhile, dry powders of microalgae biomass, mainly Chlorella vulgaris and Arthospira platensis, commonly known as Spirulina, rich in proteins and omega-3-PUFAs, can already be found on the market. However, the use of whole cells limits the bioavailability of lipids. Moreover, omega-3-PUFAs are highly susceptible to oxidation due to the large number of double bonds. Oxidation leads to the formation of off-flavors and loss of nutritional value.

[0002] Maintaining the integrity of the whole cell structure in microalgae has been shown to protect omega-3 PUFAs from oxidation during post-harvest wet storage, however, in the presence of intact cell walls, indigestible polysaccharides can limit the bioavailability of omega-3 PUFAs.

[0003] There is a clear need to improve the bioavailability and oxidative stability of nutrients in microalgae-based products. Summary of the Invention

[0004] The present invention improves upon the prior art and addresses the above-mentioned needs by using pulsed electric fields and enzymatic treatment of cell walls.

[0005] The present invention relates broadly to a method for producing a product for human consumption comprising lysed microalgae, particularly partially lysed microalgae.

[0006] More specifically, the present invention relates to a method of making a product for human consumption comprising partially lysed microalgae, the method comprising applying a pulsed electric field to a suspension of microalgae and forming a partially lysed microalgae biomass.

[0007] More specifically, the present invention relates to a method of making a product for human consumption comprising partially lysed microalgae, the method comprising the steps of: a) preparing a suspension of microalgae; b) applying a pulsed electric field to the suspension of microalgae; and c) forming a partially lysed microalgae biomass.

[0008] More specifically, the present invention relates to a method of making a product for human consumption comprising partially lysed microalgae, the method comprising the steps of: a) preparing a suspension of microalgae; b) applying a pulsed electric field to the suspension of microalgae; c) forming a partially lysed microalgae biomass; and d) adding the partially lysed microalgae biomass as an ingredient in a product for human consumption.

[0009] More specifically, the present invention relates to a method for making a product for human consumption comprising partially lysed microalgae, the method comprising the steps of: a) preparing a suspension of microalgae, the microalgae belonging to a phylum selected from Chlorophyta, Ochrophyta, and Heterokonta; b) applying a pulsed electric field to the suspension of microalgae; c) forming a partially lysed microalgal biomass; and d) adding the partially lysed microalgal biomass as an ingredient of a product for human consumption.

[0010] More specifically, the present invention relates to a method for making a product for human consumption comprising partially lysed microalgae, the method comprising the steps of: a) preparing a suspension of microalgae, the microalgae belonging to a phylum selected from Chlorophyta, Ochrophyta, and Heterokontophyta; and b) applying a pulsed electric field to the suspension of microalgae, the pulsed electric field having a specific energy input (kJ / kg) of 25-150 kJ / kg suspension. sus -1 c) forming a partially lysed microalgal biomass; and d) adding the partially lysed microalgal biomass as an ingredient in a product for human consumption.

[0011] In one embodiment, the invention provides a method for making a product for human consumption comprising partially lysed microalgae, the method comprising the steps of: a) preparing a suspension of microalgae, the microalgae belonging to a phylum selected from Chlorophyta, Ochrophyta, and Heterokontophyta; and b) applying a pulsed electric field to the suspension of microalgae, the pulsed electric field having a specific energy input (EEE) of 25-150 kJ / kg suspension. sus -1 c) optionally adding an enzyme to the microalgae suspension; d) forming a partially lysed microalgae biomass; and e) adding the partially lysed microalgae biomass as an ingredient in a product for human consumption.

[0012] In one embodiment, the phylum is Chlorophyta. In one embodiment, the phylum is Ochrophyta. In one embodiment, the phylum is Heterokontophyta.

[0013] In one embodiment, the microalgae belongs to the species Chlorella. In one embodiment, the microalgae belongs to the species Auxenochlorella.

[0014] In one embodiment, the microalgae is Chlorella vulgaris, preferably CCALA 256.

[0015] In one embodiment, the enzyme is a galactanase, such as endo-1,4-β-galactanase.

[0016] In one embodiment the enzyme is a rhamnohydrolase, such as a rhamnogalacturonan rhamnohydrolase.

[0017] In one embodiment, the enzyme is chitinase.

[0018] In one embodiment, the enzymes are endo-1,4-β-galactanase and rhamnogalacturonan rhamnohydrolase.

[0019] In one embodiment the enzyme is endo-1,4-β-galactanase, rhamnogalacturonan rhamnohydrolase and / or chitinase.

[0020] In one embodiment, the temperature of the microalgae suspension is 2 to 30°C before the application of the pulsed electric field.

[0021] In one embodiment, the pulsed electric field is between 25 and 100 kJ kg sus -1 has a specific energy input of

[0022] In one embodiment, the pulsed electric field is 30 to 35 kJ kg sus -1 , for example, about 32 kJ kg sus -1 has a specific energy input of

[0023] In one embodiment, the pulsed electric field is 10 kV cm -1 ~45kV cm -1 , preferably 10 kV cm -1 ~35kV cm -1 , more preferably 20 kV cm -1 ~30kV cm -1 The electric field strength is

[0024] In one embodiment, the pulsed electric field is 20 to 25 kV cm -1 The electric field strength is

[0025] In one embodiment, the pulsed electric field has a pulse length of 5 μs to 25 μs.

[0026] In one embodiment, the pulsed electric field is 20 to 25 kV cm -1 and a pulse length of 5 μs.

[0027] In one embodiment, the pulsed electric field has between 5 and 30 pulses applied, preferably 10 pulses are applied.

[0028] In one embodiment, the pulsed electric field comprises a bipolar square wave electric pulse. In one embodiment, the pulsed electric field comprises a unipolar electric pulse. In one embodiment, the pulsed electric field comprises an exponentially decaying electric pulse. Preferably, the pulsed electric field comprises a bipolar square wave electric pulse.

[0029] In one embodiment, the microalgae is incubated at, for example, about 4°C, or about 25°C, or about 37°C after PEF treatment.

[0030] Typical incubation times can be up to about 72 hours, for example, 1 to 72 hours, or 6 to 72 hours, or 12 to 72 hours, or 18 to 72 hours, or 24 to 72 hours, or 48 to 72 hours. Typical incubation times can be about 1 hour, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 48 hours, or about 72 hours.

[0031] The suspension may be incubated with stirring, for example at about 300 rpm.

[0032] In one embodiment, the microalgae is incubated at about 4° C. for at least 24 hours, such as about 48 hours, after PEF treatment.

[0033] In one embodiment, the microalgae is incubated at about 25° C. or about 37° C. for at least 6 hours, such as about 12 hours, after PEF treatment.

[0034] In one embodiment, the microalgae are harvested, preferably by centrifugation, and resuspended in a buffer after application of a pulsed electric field, the buffer having a temperature between 4°C and 37°C, and the microalgae are incubated in the buffer for 6 to 72 hours, preferably 12 to 72 hours.

[0035] In one embodiment, the buffer is a phosphate buffer, preferably a 0.05 M potassium phosphate buffer at pH 6.

[0036] In one embodiment, the microalgae have an average particle size of 3 μm to 6 μm after application of a pulsed electric field or enzymatic treatment.

[0037] In one embodiment, the microalgae have an average particle size of 3 μm to 6 μm after application of a pulsed electric field and enzymatic treatment.

[0038] The present invention further provides a method for improving the bioaccessibility of lipids from microalgae for human consumption, comprising the steps of: a) preparing a suspension of microalgae, wherein the microalgae belong to a phylum selected from Chlorophyta, Ochrophyta, and Heterokontophyta; and b) applying a pulsed electric field to the suspension of microalgae, wherein the pulsed electric field has a specific energy input (kJ / kg) of 25-150 kJ / kg suspension. sus -1 and c) optionally adding an enzyme to the microalgae suspension, wherein the microalgae suspension is incubated after step b) and / or step c) at 4° C. to 37° C. for 6 to 48 hours.

[0039] In one embodiment, the microalgae suspension is incubated with stirring, for example at 300 rpm.

[0040] The present invention further provides a method for maintaining oxidative stability of lipids in microalgae for human consumption, comprising the steps of: a) preparing a suspension of microalgae, the microalgae belonging to a phylum selected from Chlorophyta, Ochrophyta, and Heterokontophyta; and b) providing a suspension of microalgae with a specific energy input (kg) of 25-150 kJ per kg of suspension. sus -1 a) applying a pulsed electric field having a voltage of 0.1 V to a suspension of microalgae; and c) optionally adding an enzyme to the suspension of microalgae.

[0041] The present invention further relates to a product for human consumption comprising a microalgal biomass, the product being made by the method according to the present invention.

[0042] In one embodiment, the product is for human consumption, for example as an RTD beverage. [Brief description of the drawings]

[0043] [Figure 1] Figure 1 shows the lipid extractability (%) of C. vulgaris biomass treated with 20 kV cm-1 and various pulse widths (5-25 μs), as well as biomass treated with 5 μs and various field strengths (20-30 kV cm-1). [Diagram 2] In Fig. 2, the effect of electric field strength was evaluated at a constant pulse width of 5 μs (20–30 kV cm−1). [Diagram 3] Figure 3 shows the lipid bioaccessibility (%) of C. vulgaris biomass after PEF treatment (20 kV cm-1 for 5 μs) followed by incubation at 4°C (●), 25°C (), and 37°C (▲). Lipid bioaccessibility of untreated biomass without and with incubation (4°C, 72 h) is also shown (■). [Figure 4] FIG. 4 shows that HPH resulted in the highest bioaccessibility of lipids. [Diagram 5]FIG. 5 shows the average particle size of C. vulgaris cells in untreated (control), PEF-treated, enzyme-treated (chitinase + rhamnohydrolase + galactanase), PEF + enzyme-treated, and 100 MPa HPH treatment. [Figure 6] Figure 6 shows the evolution of the secondary oxidation products A) (Z)-3-hexenal and B) hexanal during 12 weeks of storage at 40°C for C. vulgaris biomass in untreated C. vulgaris biomass (control), PEF-treated (◆), enzyme-treated (ET, chitinase + rhamnohydrolase + galactanase) (■), PEF + enzyme-treated (▲), and HPH-treated at 100 MPa (●). [Figure 7] Figure 7 shows particle size distribution (q3, μm-1) as volume density of C. vulgaris cells in untreated (control, ●), enzyme-treated (chitinase + rhamnohydrolase + galactanase, ■), PEF-treated (×), PEF + enzyme-treated (◆), and high-pressure homogenization treated (HPH, ▲). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Microalgae preparation Preferably, the microalgae are cultured in Bold's basal medium. The medium may be supplemented with about 20 g / L glucose. The microalgae may be grown in the dark, for example at about 25° C. Preferably, the microalgae are shaken while growing, for example at about 150 rpm in a shaking incubator. The microalgae may be harvested, for example, when they reach stationary phase, typically about 4 days after the start of the culture. The microalgae are then typically centrifuged, for example, at about 10000 g for about 10 minutes, at about 4° C. The resulting pellet is used for the pulsed electric field treatment. Preferably, the microalgae is C. vulgaris, for example CCALA256. The microalgae may be purchased from Culture Collection of Autotrophic Organisms (Trebon, Czech Republic).

[0045] Pulsed Electric Field Typically, the microalgae concentration is about 70 g / L before PEF treatment. The microalgae pellet is typically resuspended in, for example, a potassium phosphate buffer at about pH 6. The conductivity (σ) can be adjusted to about 2 mS cm-1. For PEF treatment of the microalgae suspension, a parallel plate-plate electroporation cuvette may be used. The electrode distance may be about 4 mm. The applied voltage may be varied between 8 kV and 12 kV. The resulting electric field strength may be 20 kV cm-1 to 30 kV cm-1. A pulse width of 5 μs to 25 μs may be applied. A pulse number of 10 may be applied. After PEF treatment, the microalgae may be incubated at different temperatures (e.g., about 4°C, 25°C, or 37°C) for different times (0 h, 1 h, 6 h, 12 h, 18 h, 24 h, 48 h, 72 h) at about 300 rpm. The microalgal biomass may then be flash frozen in liquid nitrogen.

[0046] Enzyme addition / enzyme treatment Prior to the enzymatic treatment, the microalgae are typically suspended in a potassium phosphate buffer. The buffer concentration is typically about 50 mM. The buffer is typically at a pH of about 6. The microalgae concentration may be about 20 g / L. Chitinase, rhamnogalacturonan rhamnohydrolase, and / or endo-1,4-β-galactanase may be added to the microalgae suspension. The endo-1,4-β-galactanase may be present in a potassium phosphate buffer at a pH of about 6. The suspension may then be incubated at 37° C. and about 300 rpm for about 24 hours. After incubation, the biomass may be flash frozen in liquid nitrogen.

[0047] PEF combined with enzyme treatment The microalgal biomass is typically treated with a pulse width of about 5 μs. The electric field strength may be about 20 kV cm-1. 10 pulses may be used. Enzymes may be added before diluting the suspension to about 20 g / L with potassium phosphate buffer, for example 50 mM potassium phosphate buffer at pH 6. Chitinase, rhamnogalacturonan rhamnohydrolase, and / or endo-1,4-β-galactanase may be added. After mixing, the sample is typically incubated at 37°C for about 24 hours at about 300 rpm. After incubation, the biomass may be immediately snap frozen.

[0048] definition When compositions are listed herein in terms of weight percent, they refer to mixtures of materials on a dry basis unless otherwise indicated.

[0049] As used herein, the term "about" should be understood to refer to a number within a numerical range, for example, within -30% to +30% of the referenced number, or within -20% to +20% of the referenced number, or within -10% to +10% of the referenced number, or within -5% to +5% of the referenced number, or within -1% to +1% of the referenced number. All numerical ranges herein should be understood to include all integers or fractions within that range.

[0050] Those skilled in the art will understand that all features of the present invention disclosed herein can be freely combined. In particular, features described for the composition of the present invention can be combined with the method or use of the present invention, and vice versa. Furthermore, features described for different embodiments of the present invention can be combined. Where known equivalents exist for a particular feature, such equivalents are incorporated herein as if specifically mentioned.

[0051] Further advantages and features of the invention are apparent from the drawings and non-limiting examples. EXAMPLES

[0052] The following examples are illustrative of some products and methods that also fall within the scope of the present invention.Changes and modifications can be made in the light of the present invention.Those skilled in the art will recognize that in these examples, reasonable adjustments can be made to the nutrients and other elements of the present invention in various applications, and there are a wide variety of variations in formulations, materials, processes, and mixtures.

[0053] Example 1 Microalgae Cultivation C. vulgaris (CCALA 256) was purchased from the Culture Collection of Autotrophic Organisms (Trebon, Czech Republic). C. vulgaris was cultivated in biological triplicates (duplicates for lipid extractability experiments) in 500 mL Erlenmeyer flasks (working volume 250 mL) in a shaking incubator (Multitron Pro, Infors AG, Bottmingen, Switzerland) in Bold's basal medium supplemented with 20 g / L glucose at 25 °C and 150 rpm in the dark. After 4 days of cultivation, the biomass was harvested when stationary phase was reached and centrifuged (10000g, 10 min, 4 °C). The supernatant was discarded and the pellet (fresh biomass) was used for further experiments.

[0054] The growth of C. vulgaris was monitored by optical density (OD) measurements at 750 nm (GENESYS™ 10S, Thermo Fischer Scientific Inc., Waltham, MA, USA). The correlation coefficient between OD and dry weight (DW) was calculated using the formula: 2 =0.9796). DW[gL -1 ]=0.5596·OD 750nm +0.4179

[0055] Example 2 Pulsed Electric Field Prior to electroporation of the cells, the concentration and conductivity of the microalgae cultures were standardized. For all PEF experiments, the microalgae concentration was set at 70 g / L.

[0056] Fresh biomass pellets were resuspended in potassium phosphate buffer at pH 6 and 2 mS cm -1 The conductivity (σ) was adjusted to 1.7 mS cm. The conductivity adjustment ensured suitable loading conditions for the subsequent PEF treatment. The conductivity was measured immediately after preparation of the microalgae suspension and was found to be 1.7 mS cm. -1 After vortexing, 1 mL aliquots of the microalgal suspension were transferred into parallel plate electroporation cuvettes (VWR International bvba., Leuven, Belgium) for PEF treatment. Samples were batch-treated with an electrode distance of 4 mm. The experimental setup consisted of a cuvette holder connected to a RUP6-15CL pulse generator (GBS-Elektronik, Radeberg, Germany). Pulse measurements were performed with a P6015A voltage probe (Tektronix Inc., Beaverton OR, USA) connected to a Wave Surfer 10 oscilloscope (Teledyne LeCroy GmbH, Heidelberg, Germany). The applied voltage was varied between 8 kV and 12 kV, respectively, at 20 kV cm -1 and 30 kV cm -1 A field strength of 100 kV cm was obtained. Pulse widths between 5 μs and 25 μs and a pulse number of 10 were applied. Various combinations were systematically tested to investigate their impact on lipid extraction capacity. After PEF treatment, biomasses from several cuvettes were combined and incubated at different temperatures (4 °C, 25 °C, 37 °C) for different times (0 h, 1 h, 6 h, 12 h, 18 h, 24 h, 48 h, 72 h) at 300 rpm. After incubation, the biomasses were immediately flash frozen in liquid nitrogen and stored at -20 °C until further analysis. Untreated samples (controls) were concentrated, standardized to the same conductivity, incubated and then flash frozen. Figure 1 shows the results of the experiments using a 20 kV cm -1 and C. vulgaris biomass treated with various pulse widths (5–25 μs) and 5 μs and various electric field strengths (20–30 kV cm -1 ) shows the lipid extractability (%) of biomass treated with

[0057] The effect of electric field strength was evaluated with a constant pulse width of 5 μs (20–30 kV cm -1 ) (Figure 2). In both experiments, the biomass suspension was incubated at 25 °C for 1 h after PEF treatment. Lipid extractability was enhanced by PEF treatment at 5 μs compared to the untreated control. Treatment with higher pulse widths, and therefore higher energy inputs, did not result in improved lipid extractability. Lipid extractability was evaluated at various electric field strengths and a constant pulse width of 5 μs. Biomass was subjected to a 20 kV cm -1 When treated with 25 kV cm, the lipid extraction capacity was 15.8% higher than that of the control. -1 The highest increase (+26.9%) was reached when treated with a higher electric field strength (30 kV cm -1 ), i.e., at higher energy inputs, lipid extraction capacity decreased. Notably, at 25 kV cm -1 Above 20 kV cm the system became unstable. -1 A 5μs pulse at 31.8kJ kg sus -1 represents the μsPEF processing with an energy input of

[0058] Example 3 Enzyme treatment: Fresh microalgal biomass was suspended at 20 g / L in 50 mM potassium phosphate buffer at pH 6. Chitinase solution (700 μL, 1 mg / mL in microalgal suspension, Sigma-Aldrich, Switzerland), rhamnogalacturonan rhamnohydrolase (35 μL, NZYTech, Portugal), endo-1,4-β-galactanase (1.75 μL, 750 U / mL in potassium phosphate buffer, pH 6, Megazyme, Ireland) were added to the microalgal suspension. After mixing, the samples were incubated for 24 h at 37 °C and 300 rpm on a stir plate. After incubation, the biomass was immediately flash frozen in liquid nitrogen and stored at -20 °C until further analysis. In the control (untreated biomass), the enzyme solution was replaced with potassium phosphate buffer (pH 6, 50 mM). All tubes were incubated for 24 h at 37 °C on a stir plate at 300 rpm. After incubation, samples were collected and immediately chilled in crushed ice.

[0059] Example 4 PEF combined with enzyme treatment Microalgae biomass was subjected to a 5 μs pulse width and 20 kV cm -1 The samples were treated with 10 pulses of electric field strength at 1000 rpm. Biomass from multiple electroporation cuvettes was combined and the following enzymes were added: chitinase (700 μL, 1 mg / mL in potassium phosphate buffer, pH 6), rhamnogalacturonan rhamnohydrolase (35 μL, NZYTech, Portugal), endo-1,4-β-galactanase (1.75 μL, 750 U / mL in potassium phosphate buffer, pH 6, Megazyme, Ireland), and then the suspension was diluted to 20 g / L in potassium phosphate buffer 50 mM, pH 6). After mixing, the samples were incubated at 37 °C for 24 h on a stir plate at 300 rpm. After incubation, the biomass was immediately flash frozen in liquid nitrogen and stored at -20 °C until further analysis.

[0060] Example 5 Lipid extractability Hexane:isopropanol (HI) extraction efficiency was measured to evaluate lipid extraction potential. HI extraction efficiency is expressed as the ratio of free lipids extracted by hexane:isopropanol (3:2 v / v) compared to total lipids measured by direct transesterification and expressed as a percentage. HI does not easily penetrate intact rigid microalgae cells, resulting in lower extraction yields.

[0061] Hexane:isopropanol (3:2 v / v, 1.5 mL) was added to 10 mg of freeze-dried PEF-treated microalgal biomass and the mixture was vortexed for 30 s. The sample was centrifuged (10 min, 750 g, 25 °C) and the solvent layer containing the extracted lipids was transferred to a weighed flask. These extraction steps were performed a total of four times. All solvent layers were combined and the solvent was subsequently removed by evaporation under a nitrogen stream. The extracted lipids and the total lipids in the dried biomass were quantified by gas chromatography (GC) upon transesterification to fatty acid methyl esters (FAMEs). Briefly, fatty acids were directly transesterified using a 1.5 N methanolic hydrochloric acid solution and analyzed by gas chromatography using an instrument equipped with a split injection port and flame ionization detection (FID) (7890A, Agilent Technologies, Basel, Switzerland). The following temperature-time program was used: 50 °C (0.2 min), 50–180 °C (120 °C min -1 , 180~220℃(6.7℃ min -1 , and 220 to 250°C (30°C min -1 ) was used on a 70% cyanopropyl polysilphenylene-siloxane column (BPX70; SGE Analytical Science, Milton Keynes, UK) with a length of 10 m, an inner diameter of 0.1 mm, and a film of 0.2 μm. Peak identification was performed by comparison of retention times with FAME standards (Nu-Chek Prep.Inc., Elysian, USA). Peak areas were quantified with OpenLab CDS VL software (Agilent Technologies, Basel, Switzerland).

[0062] Example 6 Lipid bioaccessibility Lipid bioaccessibility was measured by an in vitro digestion model according to a standardized protocol (INFOGEST2.0). Briefly, digestions were performed in triplicate (n=3) at 37 °C with stirring at 300 rpm. To simulate the oral phase (2 min, pH 7), the following solutions were added: water (1.13 mL), simulated saliva (SSF, 0.96 mL), and CaCl 2 (6 μL, 0.3 M) was mixed with the freeze-dried biomass (0.3 g). To initiate the gastric phase, simulated gastric fluid (SGF, 1.92 mL) and CaCl 2 (1.2 μL, 0.3 M) was added to the oral bolus and the pH was set to 3. Pepsin (0.12 mL, 80000 U / mL, Sigma-Aldrich, Buchs, Switzerland) and gastric lipase (0.12 mL, 2400 U / mL, Lipolytech, Marseille, France) were added and the final volume was brought to 4.8 mL with water. The pH was periodically adjusted to 3. After 2 h of incubation, the pH was set to 7 and simulated intestinal fluid (SIF, 2.04 mL), CaCl 2 (9.6 μL, 0.3 M), pancreatin (1.2 mL, 800 U / mL, Sigma-Aldrich), and bile salts (0.6 mL, 0.16 mM, Sigma-Aldrich) were added. Water was added to a final volume of 9.6 mL. After 2 h of incubation, an aliquot (2 mL) of the complete digest was frozen in liquid nitrogen and lyophilized. The remaining complete digest was centrifuged (30 min, 10000 g, 4 °C). The micellar phase (supernatant) and pellet were frozen separately in liquid nitrogen and lyophilized. An infant formula with expected overall bioaccessibility (Aptamil 1, Milupa, Dublin, Ireland) was used as a positive control and subjected to in vitro digestion. Water (1.2 mL) without microalgal biomass was digested as a blank to quantify fatty acids derived from the digesta and enzymes.

[0063] The lipid content of the whole digest and the micellar phase was analyzed. The lipid content was expressed as the total fatty acids measured. The lipid bioaccessibility was defined as the amount of lipid contained in the micellar phase (corrected by lipid in the micellar phase of the enzyme blank) divided by the amount of lipid in the whole digest (respectively corrected by lipid in the whole digest of the enzyme blank) and expressed as a percentage (%).

[0064] PEF treatment (20 kV cm -1 The lipid bioaccessibility was analyzed by an in vitro digestion model of C. vulgaris biomass, which was then subjected to incubation conditions at different temperatures (4 °C, 25 °C, 37 °C) for up to 72 h (Figure 3). After incubation, the algal suspension was immediately flash frozen in liquid nitrogen and lyophilized for the next in vitro digestion. This combination of parameters was selected as the one resulting in the highest lipid extraction capacity among the feasible ones previously tested in this study.

[0065] Figure 3 shows the results of PEF treatment (20 kV cm -1 Lipid bioaccessibility (%) of C. vulgaris biomass after 5 μs at 4°C (●), 25°C (), and 37°C (▲) is shown. Lipid bioaccessibility of untreated biomass without and with incubation (4°C, 72 h) is also shown (■). Error bars indicate standard deviation between biological triplicates (n=3).

[0066] Incubation without PEF treatment (4°C, 72 h) resulted in a lipid bioaccessibility of 10.4 ± 1.7%, which is lower than that obtained for samples treated with PEF followed by incubation at 25°C or 37°C for at least 6 h and at 4°C for at least 24 h. At both incubation temperatures, lipid bioaccessibility increased with increasing incubation time after PEF treatment. Figure 3 shows a plateau of lipid bioaccessibility with a maximum of 18%-19%. This value was reached after 12 h of incubation of biomass maintained at either 25°C or 37°C. A longer incubation at 4°C (48 h) was required to obtain a lipid bioaccessibility of 18.7 ± 1.6%. Without wishing to be bound by theory, the reason no further increase was obtained may be because aqueous incubation of PEF-treated algae led to the fusion of lipid droplets into large unique bodies that were difficult for digestive enzymes to react to.

[0067] The increase in lipid bioaccessibility with longer incubation is consistent with the theory that PEF induces the release of autolytic enzymes, which then degrade the cell wall, favoring increased lipid bioaccessibility. Moreover, the proportionality of the kinetic behavior of lipid bioaccessibility with temperature further suggests the role of endogenous enzymes in the mechanism of temperature dependence of enzyme activity. Such enzyme-facilitated processes may in fact be slowed down at non-physiological temperatures, i.e., 4°C.

[0068] Figure 4 shows that HPH resulted in the highest lipid bioaccessibility. Interestingly, incubation after this treatment did not have any effect on lipid bioaccessibility. HPH without incubation was 55.4 ± 2.4% and HPH with 24 h incubation was 56.5 ± 2.2%. The mechanical disruption of algal cells by HPH is intense and rapid, resulting in an almost instantaneous diffusion of intracellular compounds into the aqueous phase. The lipid fraction was already available without the need for additional incubation.

[0069] PEF treatment showed a 17% increase in lipid bioaccessibility compared to the control (4.0% ± 2.1%), whereas enzyme treatment did not have any relevant effect on lipid bioaccessibility (4.3% ± 3.6%).

[0070] Addition of enzymes to biomass in combination with PEF treatment showed no clear effect (24.4% ± 2.8%) compared to PEF-treated biomass (20.9% ± 3.2%). In this experiment, enzymes were added after PEF treatment because no differences were reported when enzymes were added before PEF treatment (data not shown).

[0071] Example 7 Particle size Cell integrity was assessed in triplicates (n = 3) by measuring the particle size of freshly treated microalgal cells with a LS 13 320 laser diffraction particle size analyzer (Beckman Coulter, Brea, Canada). Results were reported as the mean diameter of the volume-based droplet size distribution (d 43 ) are shown.

[0072] FIG. 5 shows the average particle size of C. vulgaris cells untreated (control), PEF-treated, enzyme-treated (chitinase+rhamnohydrolase+galactanase), PEF+enzyme-treated, and HPH-treated at 100 MPa. PEF (4.7±0.3 μm) and enzyme (5.0±0.1 μm) treatments resulted in only a slight decrease in particle size compared to the control (5.1±0.1 μm). PEF treatment maintains cell integrity resulting in visually intact microalgal cells. Cell intactness is maintained even when the autolysis process occurs after PEF treatment, as shown in yeast where autolysis induces cell wall degradation without complete cell destruction. In contrast, HPH treatment at 100 MPa reduced the average particle size to 2±0.1 μm and caused significant disintegration of the microalgal cells, with the average particle size of C. vulgaris being 2.22 μm after five passes at 150 MPa. The combination of PEF and enzyme reduced particle size to 4.5±0.1 μm, and thus PEF treatment appears to have enhanced the action of the enzyme on cell wall polysaccharides, resulting in slightly more cell wall disintegration than enzyme and PEF alone.

[0073] Figure 5 shows the mean diameter of the volumetric droplet size distribution (d43) of C. vulgaris cells in untreated (control), PEF-treated, enzyme-treated (chitinase + rhamnohydrolase + galactanase), PEF + enzyme-treated, and HPH-treated at 100 MPa. Error bars represent standard deviation of triplicates (n=3).

[0074] Figure 7 shows the particle size distribution (q3, μm) as a volume density of C. vulgaris cells in untreated (control, ●), enzyme-treated (chitinase + rhamnohydrolase + galactanase, ■), PEF-treated (×), PEF + enzyme-treated (◆), and high-pressure homogenization-treated (HPH, ▲). -1 ) is shown.

[0075] Example 8 Microbial growth Microbial growth (total viable count) of PEF-treated biomass (incubated at 4°C for 48 hours) was measured in biological triplicates. Biomass was diluted in duplicate (10 -1 , 10 -2 , 10 -3 ), 25 μL was plated onto general-purpose growth medium followed by incubation under cold (4°C) and warm (30°C) conditions. Colony forming units (CFU) were counted after 24 and 48 h, and plates with CFU counts between 20 and 200 were considered relevant.

[0076] In Table 1, microbial growth is given in colony forming units per mL of biomass (CFU / mL). Growth was well below the minimum limit considered satisfactory by the Swiss Federal Ministry of the Interior (≦100 CFU / g). The guidelines referred to are for the exemplary pathogenic mesophilic bacterium Escherichia coli and are therefore well suited for comparison.

[0077] Table 1 shows the microbial growth when PEF-treated and incubated (48 h, 4° C.) C. vulgaris suspensions were plated and incubated for up to 48 h at 4° C. and 30° C. Data are the average of biological triplicates and technical duplicates (n=6).

[0078] [Table 1]

[0079] The lack of microbial growth in treated and incubated algae ruled out the possibility of enzymatic activity originating from external bacteria that may be present in the suspension. This result further supports our hypothesis that enzymatic cell wall degrading activity is intrinsically present in C. vulgaris cells and is induced by PEF. Furthermore, these data suggested the relevance of the process presented here (PEF followed by incubation at 4°C) from a food safety perspective.

[0080] Example 9 Oxidative stability of lipids Lipid oxidation was investigated by measuring secondary lipid oxidation products. Briefly, treated biomass was freeze-dried, kept in amber vials (60 mg), and stored at 40 °C for 0, 2, 4, 6, 8, and 12 weeks. Secondary oxidation was evaluated in triplicates (n = 3). Biomass (60 mg) was dispersed in 1.5 mL of chloroform / methanol (1 / 2, v / v). Samples were mixed (2500 rpm, 10 min) in a multi-tube vortex mixer (DVX-2500, VWR, Switzerland). Sample supernatant (100 μL) was mixed with 100 μL of 7-(diethylamino)-2-oxochromene-3-carbohydrazide (CHH) and 5 μL of internal standard (ISTD, hexanal-d 12 , 10 μg / mL in acetonitrile). After 1.5 h of incubation at 1400 rpm and 37 °C using a thermomixer (Comfort, Eppendorf, Schonenbuch, Switzerland), the samples were diluted with acetonitrile (100 μL) and centrifuged (2500 × g, 20 °C). The supernatant was injected into an ultra-performance liquid chromatography (UPLC) (Dionex UltiMate 3000)-QExactive Plus (Thermo Scientific, Basel, Switzerland) system. (Z)-3-Hexenal and hexanal were selected as indicators of lipid oxidation as volatile substances derived from the ω-3 and ω-6 fatty acid degradation pathways, respectively. The response factor was calculated using the internal standard hexanal-d 12 The area was expressed as the ratio of the area of ​​volatile matter to the area of ​​nonvolatile matter.

[0081] In addition, the stored dry samples were coded, randomized, and evaluated by smelling by a panel of four members who ranked the samples and rated the odor by describing the perceived flavor notes.

[0082] Volatile production was investigated in untreated biomass (control), PEF treatment, enzyme treatment (chitinase + rhamnohydrolase + galactanase), PEF + enzyme treatment, and HPH treatment. The values ​​of hexanal and (Z)-3-hexenal as degradation products of interest for linoleic acid (C18:2, n6) and α-linolenic acid (C18:3, n3), respectively, the most abundant fatty acids in C. vulgaris, are shown in Figure 6.

[0083] FIG. 6 shows the evolution of the secondary oxidation products A) (Z)-3-hexenal and B) hexanal during 12 weeks of storage at 40° C. for C. vulgaris biomass in untreated (control), PEF-treated (◆), enzyme-treated (ET, chitinase + rhamnohydrolase + galactanase) (■), PEF + enzyme-treated (▲), and HPH-treated at 100 MPa (●). Signals are expressed as the area of ​​the compound of interest divided by the area of ​​the internal standard (ISTD) hexanal-d12. Error bars represent standard deviation of triplicates (n=3).

[0084] The (Z)-3-hexenal and hexanal signals in untreated, PEF-treated, ET-treated, and PEF+ET-treated biomass did not show any increase over 12 weeks of storage, indicating significant oxidative stability. These results indicate that both PEF and ET are gentle processes that preserve the quality of the lipid fraction. The signal intensities of hexanal and (Z)-3-hexenal in HPH-treated biomass were 4.1- and 7.3-fold higher than the control after 12 weeks, respectively, indicating that HPH has a harsh effect on biomass oxidative stability. The signals of both hexanal and (Z)-3-hexenal decreased from week 0 to week 4, which may indicate that extensive oxidation had already occurred due to the disruption treatment prior to storage. Some of the aldehydes produced may have been converted to alcohols and organic acids that were not detected in the present analysis.

[0085] The sensory results were in agreement with the analytical data. The HPH-treated biomass already exhibited rancid odor at time 0, whereas untreated and enzyme- and PEF-treated biomass showed only slight odor over the entire storage period. This result demonstrates the promise of the PEF treatment. Using this technique it is possible to improve the lipid bioaccessibility and maintain its oxidative stability in C. vulgaris biomass.

Claims

1. A method for producing a product for human consumption, comprising partially dissolved microalgae, comprising: a. preparing a suspension of microalgae, wherein the microalgae belong to a phylum selected from the phylum Chlorophyta, Ochrophyta, and Heterokonta; b. Applying a pulsed electric field to the suspension of the microalgae, wherein the pulsed electric field has a specific energy input of 25 to 150 kJ (kg ｓｕｓ －１ ) per kg of the suspension, and c. forming a biomass of partially dissolved microalgae; and d. adding the biomass of partially dissolved microalgae as a material for a product for human consumption.

2. The method according to claim 1, wherein the microalgae belong to the genus Chlorella or Auxenochlorella.

3. The method according to claim 1, wherein the microalgae are Chlorella vulgaris.

4. The method according to claim 1, wherein the temperature of the suspension of microalgae before applying the pulsed electric field is 2 to 30 °C.

5. The pulse electric field has a specific energy input of 25 to 100 kJ / kg ｓｕｓ －１ The method according to claim 1, having such specific energy input.

6. The pulsed electric field has an electric field strength of 10 kV / cm －１ to 45 kV / cm−1, the method according to claim 1.

7. The method according to claim 1, wherein the pulsed electric field has a pulse length of 5 μs to 25 μs.

8. The method according to claim 1, wherein the pulsed electric field has 5 to 30 pulses applied thereto.

9. The method according to claim 1, wherein the pulsed electric field comprises a bipolar rectangular wave electric pulse, a unipolar electric pulse, or an exponentially decaying electric pulse.

10. The method according to claim 1, wherein the microalgae are recovered after applying the pulsed electric field and resuspended in a buffer solution having a temperature of 4 to 37 °C, and the microalgae are incubated in the buffer solution for 6 to 72 hours.

11. The method according to claim 10, wherein the buffer solution is a phosphate buffer solution.

12. The method according to claim 1, wherein the microalgae have an average particle size of 3 to 6 μm after application of the pulsed electric field.

13. A method for improving the bioaccessibility of lipids of microalgae for human consumption, comprising: a. preparing a suspension of microalgae, wherein the microalgae belong to a phylum selected from the phylum Chlorophyta, Ochrophyta, and Heterokonta; and b. Applying a pulsed electric field to the suspension of the microalgae, wherein the pulsed electric field has a specific energy input of 25 to 150 kJ (kg ｓｕｓ －１ ) per kg of the suspension, and the step is included, The suspension of microalgae is incubated at 4 to 37 °C for 6 to 48 hours after step b).

14. A method for maintaining lipid oxidation stability in microalgae for human consumption, a. preparing a suspension of microalgae, wherein the microalgae belong to a phylum selected from the phyla Chlorophyta, Ochrophyta, and Heterokontophyta; b. Applying a pulsed electric field to the suspension of the microalgae, wherein the pulsed electric field has a specific energy input of 25 to 150 kJ (kg ｓｕｓ －１ per kg of the suspension), and the method comprises this step.

15. A product for human consumption, comprising a microalgal biomass, produced by the method according to any one of claims 1 to 12.

16. The product according to claim 15, wherein the product is an RTD beverage.

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