METHOD FOR DEODORIZING SEAWEED

Abstract

The present invention relates to a process for deodorizing a Nannochloropsis algae biomass, comprising a) contacting the biomass with an adduct-forming compound selected from sodium bisulfite in an amount of 0.5-30% by weight, b) stirring or shaking, and c) collecting a solid from the suspension.

Description

METHOD FOR DEODORIZING SEAWEED FIELD OF INVENTION The present invention relates to a process for deodorizing algae. The invention relates particularly to a process for removing odoriferous substances, especially those responsible for the fishy odor found in algae, from algae of the genus Nannochloropsis. BACKGROUND OF THE INVENTION Algae are single-celled organisms that grow in water. They use photosynthesis to convert light, carbon dioxide, and nutrients into oils, carbohydrates, and proteins. Their cultivation does not require arable land, and in fact, they can be grown in brackish water, freshwater, or seawater at a high growth rate. Algae in the form of dry powder can be consumed alone, or can be used as a food additive in functional and mass-market healthy foods, such as food products and beverages, i.e. bread, dairy products, meat, fruit juices, nutrition bars, baby food, etc., long shelf-life microalgae-based nutraceutical products, such as tablets or simple powder for health supplements, in personal care products and as a fish feed ingredient in aquaculture. Recent studies indicate that the world's population is growing and along with this, food requirements are increasing, while at the same time resources and arable land are becoming progressively limited. Microalgae can be induced to produce specific lipids and fatty acids through relatively simple manipulations of the physical and chemical properties of their culture medium. They can produce and accumulate substantial amounts of lipids, up to 20–50% of their dry weight. Lipid accumulation in microalgae is attributed to the consumption of sugars at a rate exceeding the rate of cell regeneration, which promotes the conversion of excess sugar into lipids. Polyunsaturated fatty acids (PUFAs), such as omega-3 fatty acids, are vital for daily life and function. The beneficial effects of omega-3 fatty acids in reducing serum triglycerides are now well established. These compounds are also known for other cardioprotective benefits, such as lowering cholesterol levels, protecting against coronary heart disease, and suppressing platelet aggregation. Other benefits of PUFAs include those related to the prevention and / or treatment of inflammation, neurodegenerative diseases, and cognitive development. Nannochloropsis is one of the algae genera used to produce high-value oil containing PUFAs, among which eicosapentaenoic acid (EPA) is of particular value and interest. More than two-thirds of the fatty acids produced by Nannochloropsis consist of EPA, palmitic acid, and palmitoleic acid. Microalgae are a rich and sustainable source of plant protein, with protein content varying among the many species. Arthrospira platensis (Spirulina) and Chlorella are the most widely used commercially in this category, and can contain up to 70% protein by weight. Proteins derived from algae can be consumed as a nutritional supplement in tablet or powder form, or as additives in foods such as pasta, bread, dairy products, sports nutrition bars, and so on. Microalgae produce a wide range of volatile compounds that are frequently responsible for unpleasant tastes and / or odors. The PUFAs present in algae are highly susceptible to oxidation due to their high degree of unsaturation, which results in the breakdown of PUFAs and the formation of primary and secondary oxidation products known to have unpleasant tastes and / or odors. This can be attributed to the formation of aldehydes, ketones, and alcohols. In many of the algae applications mentioned above, such as in dairy products, it is desirable to eliminate these tastes and odors as much as possible and provide a product that is not only of superior nutritional value but also appealing to the consumer. The methods used to deodorize algae or algae-derived products are, in most cases, applied to the oils derived from them. Vacuum steam distillation at high temperatures and other optional steps, such as contacting the oil with adsorbents like silica, and adding antioxidants like ascorbyl palmitate, tocopherol, and lecithin, are commonly used methods for deodorizing algae and, more generally, marine oils. In some cases, rosemary or sage extracts are used as antioxidants, occasionally altering the odor and flavor. Very few methods are reported that describe the deodorization of algae in their raw form, without specific reference to Nannochloropsis, which is the species encountered by the inventors of this specification. A group of teams from a few institutions published a study in the Journal of the Science of Food and Agriculture, 2017, vol. 97, 5123-5130, which focuses on the removal of odorous compounds from Arthrospira platensis by solvent extraction and preforming tests using three different solvents: ethanol, hexane, and acetone. GC-MS analysis indicated that the ethanol extract removed most of the odorous and undesirable compounds. According to Japanese patent application JPH06311882, the algae *Crypthecodinium cohnii* are cultivated to produce DHA for medical use, and odors are removed by supercritical CO2 extraction. The efficiency of this method is highly dependent on the extraction temperature, pressure, and reaction time, although it is described as having a low percentage loss of DHA by weight. It can be efficient in extracting odorous compounds only if the algal biomass has less than 20% water by weight. A method for deodorizing seaweed is described in KR 101753224, in which seaweed is immersed in a carbonic acid buffer solution filled with carbon dioxide and subjected to high pressure (50–250 MPa). The carbon dioxide bubbles are said to selectively remove odor compounds from the seaweed, and by adjusting the reaction parameters—pressure, temperature, pH, CO2 production rate, etc.—the physical properties, texture, flavor, and shape of the product remain intact. However, this multi-parameter adjustment is what makes the method impractical, and the desired product is not easily achieved. Furthermore, the high pressure required can lead to alterations in overall cell integrity. Therefore, there is a need for a simple process to deodorize algae, and the Nannochloropsis genus in particular. Within the biorefinery concept for the sustainable manufacture of bio-based products, this process would maintain the functionality of the resulting fractions and allow for their subsequent use, thus enabling the maximum possible valorization of all production streams. BRIEF DESCRIPTION OF THE FIGURES Figure 1 and Figure 2 illustrate the removal trend of aldehydes and ketones from the algal biomass of Nannochloropsis Oculata according to the present invention. See also Table 1, where the respective values ​​are shown. DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, “microalgae” are microscopic algae, commonly found in freshwater and marine systems, that live in both water and sediment. “Microalgal” can be defined analogously. As used herein, the term “biomass” refers to carbon-containing materials resulting from algal growth, but may also include material from other growing organisms. The terms “microalgal biomass” and “algal biomass” are used interchangeably. As used herein, an “adduct” is a chemical species AB, each molecular entity of which is formed by direct combination of two separate molecular entities A and B such that there is a change in connectivity, but no loss, of atoms within the A and B portions. According to a first embodiment of the present invention, a process is provided for deodorizing a microalgal biomass of Nannochloropsis algae, comprising a) contacting the biomass with an adduct-forming compound selected from metal sulfites, metal bisulfites, ammonium bisulfite, metal metabisulfites, SO2 or Λ / a / zuzz / uuu 1 1 / mixtures of the same; b) stirring or agitation; c) collect a solid from the suspension. According to a second modality, the Nannochloropsis algae are selected from the group consisting of N. Gaditana, N. Granulata, N. Limnetica, N. Oceanica, N. Oculata, and N. Salina. According to a preferred modality, the Nannochloropsis alga is Nannochloropsis Oculata. Algal biomass is considered to be in dry form when it contains less water by weight than harvested algal biomass. The Nannochloropsis microalgal biomass provided in step (a) can be sun-dried, oven-dried, air-dried, freeze-dried, spray-dried, or processed according to other standard food-drying techniques known in the art and can contain 0 to 7% water by weight, preferably 0.5-6%, more preferably 1-5%; more preferably, the water content of the dry biomass used in the present invention is 4% water by weight. Alternatively, the algal biomass used in the present invention can be wet, with a water content of 7-99.95% by weight, preferably 7-98%, more preferably 7-85%, even more preferably 7-70% water by weight, and more preferably 7-50%. In another embodiment, the wet algae biomass used in the present invention has a water content of 15-99.95% by weight, preferably 15-98%, more preferably 15-85%, even more preferably 15-70% by weight of water, more preferably 15-50%. Drying microalgal biomass is advantageous for facilitating further processing. Drying refers to the removal of free surface moisture / water from predominantly intact biomass or the removal of surface water from a thick suspension of homogenized biomass (e.g., by micronization). In some cases, drying the biomass can facilitate a more efficient microalgal oil extraction process. The deodorization method of the present invention can be performed before or after the drying procedure on dry or wet biomass, respectively. The algal biomass is brought into contact with an aqueous solution of the adduct-forming compound(s) according to methods known to the person skilled in the art, and a suspension is formed according to step (a). An advantage of the method is that the use of an organic solvent is not required. According to step (b), metal sulfites or SO2 react with aldehydes and / or ketones and bisulfite adducts, which are subsequently removed from the algal biomass. The metal sulfites are preferably selected from sodium sulfite, potassium sulfite, and lithium sulfite; the metal bisulfites are preferably selected from sodium bisulfite, potassium bisulfite, and lithium bisulfite; and the metal metabisulfites are preferably selected from sodium metabisulfite, potassium metabisulfite, and lithium metabisulfite. Other materials may also be used. Λ / a / zuzz / uuu 1 1 / mixtures thereof. More preferably, sodium metabisulfite is used. The formation of bisulfite adducts depends strongly on the reactivity of the carbonyl group of aldehydes and ketones. The inventors found that spherically free-structured aldehydes and methyl ketones were efficiently removed from algal biomass. In some cases, a pH adjustment step may be required, depending on the adduct-forming compound used. It has been observed that at very acidic pH values, the color of the algae in the suspension changes, which is undesirable in most cases. The pH of the suspension can range from 2 to 12, preferably 3.5–8, more preferably 4–7, even more preferably 4–6, and most preferably 4–5. The collection of a solid, according to step c of the present invention, is carried out by methods known to the skilled synthetic chemist for separating solids from liquids, such as filtration, or decanting the supernatant and collecting the sediment. According to another approach, the process also includes: d) wash the solid with water and e) optionally dry. The washing, according to step (d), is carried out with a selected aqueous solution of water, aqueous solutions of NaCl, H2O2, phosphate, acetate or citric acid. Water is the preferred medium. The drying, according to step (e), can be carried out by any method known to the expert. The inventors of this specification found that freeze-drying is the optimal method in this case, since the organoleptic properties of the deodorized seaweed biomass remain intact. It was observed that oven drying resulted in a crumbly texture and a dark green-brown color, making it unattractive to the consumer. Free-space solid-phase microextraction (HS-SPME) with gas chromatography-mass spectrometry (GC-MS) was used to analyze deodorized algae samples reconstituted in DM water and to measure the content of odorous volatile compounds. GC-MS analysis was performed on a Shimadzu GC-2010 coupled with a GCMS-Q2010PLUS mass spectrometer. One-cm divinylbenzene / carboxene / polydimethylsiloxane (DVB / CAR / PDMS) SPME fibers were used for extraction. Table 1 shows the effect of sodium metabisulfite concentration on the removal of aldehydes and ketones. For the purpose of these measurements, samples were treated with aqueous solutions of MBSF or water (control) for 10 minutes, followed by washing with DM water and lyophilization. Table 1 0% MBSF (eg. lag. 7) 0.5% MBSF (eg. lag. 6) 1.0% MBSF (eg. lag. 5) 5.0% MBSF (eg. lag. 4) 10.0% MBSF (eg. lag. 3) 20.0% MBSF (eg. ref. 2) Analyte Name Area Similarity1 Area Area Area Area Area Aldehydes Isopentanal 1752183 89% 281610 272720 60207 ND ND 2methylbutanal 273731 84% ND ND ND ND ND Rentan al 1574983 90% ND ND ND ND ND trans-2-methyl2-butenal 265148 93% 84891 54392 ND ND ND trans-2Penten-1-al 746731 89% 202921 168191 143385 ND ND H exanal 10681018 97% 1152529 636175 315941 290714 2-Hexenal 1132379 91% 120636 6810 8707 ND ND cis-4-Heptene1-al 1492530 92% 55678 ND ND ND ND Heptanal 3635617 97% 946459 350191 234851 ND ND Benzaldehyde 1878233 96% 486352 415371 176306 73539 81960 (E)-2-Octen- 1-al 323309 94% 33573 35554 60452 28597 42087 Nonanal 833685 95% 468292 359572 258248 71750 49862 Ketones Isopropyl ketone 1383025 93% 103264 ND ND ND ND 6-methyl-5heptene-2one 13430122 93% 13320616 10350409 5585488 6665486 5272939 3,5Octadiene-2one 1463160 91% 1008530 970487 696176 659840 575853 6-methyl-3,5heptadien-2one 385529 89% 289636 269243 187707 194345 162380 2-Heptanone 481184 84% 412050 299070 287153 252852 161082 trans-betaionone 3248251 94% 2490068 2584660 2479652 2389275 2363725, 1. Similarity of the mass-to-charge ratio (m / z) for ions of known m / z. One of the approaches commonly used for mass spectra annotation is the search for similarity in a database of theoretical spectra generated from a database of substances. The content of odorous compounds in the deodorized algae biomass is reduced to either undetectable levels or significantly reduced, and this reduction is observed to improve with increasing sodium metabisulfite concentration. The sodium metabisulfite concentration can range from 0.5% to 30%, preferably 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, or 30% by weight, and most preferably 20% by weight. Another key advantage of the deodorization method described herein is that no significant loss of EPA was observed. Minimal loss of other fatty acids present in the algal biomass was also found. The process is even more non-destructive and reversible, maintaining the functionality of the products. The adducts formed can be further treated to regenerate the compounds initially removed from the algal biomass, which may possess valuable properties. The simple yet highly efficient method for deodorizing Nannochloropsis algal biomass, which selectively removes odorous compounds while leaving the nutritional components largely intact, is illustrated with examples. EXAMPLES The Nanocloropsis used in the examples of the present invention was spray-dried after harvest and contained up to 7% water by weight. Example 1 A 250 mL round-bottom flask fitted with a magnetic stir bar is charged with 20 g of N. oculata powder followed by 100 mL of 20% w / v sodium metabisulfite solution. The suspension is stirred under an inert atmosphere for 2.5 hours at room temperature, and then the solid is collected by filtration under reduced pressure. The filter cake is spray-washed with 2 × 100 mL of DM water, vacuum-dried for 30 minutes, and then vacuum-dried in an oven for 2 hours at 45°C to yield 17.6 g of dry powder. Example 2 A 250 mL round-bottom flask fitted with a magnetic stir bar is charged with 20 g of N. oculata powder followed by 100 mL of 20% w / v sodium metabisulfite solution. The suspension is stirred under an inert atmosphere for 2.5 hours at room temperature, and then the solid is collected by filtration under reduced pressure. The filter cake is spray-washed with 2 × 100 mL of DM water, vacuum-dried for 30 minutes, and then lyophilized for 18 hours to yield 17.2 g of dry powder. Example 3 A 250 mL round-bottom flask fitted with a magnetic stir bar is charged with 20 g of N. oculata powder followed by 100 mL of 10% w / v sodium metabisulfite solution. The suspension is stirred under an inert atmosphere for 2.5 hours at room temperature, and then the solid is collected by filtration under reduced pressure. The filter cake is spray-washed with 2 x 100 mL of DM water, vacuum-dried for 30 minutes, and then lyophilized for 18 hours to give 17.4 g of dry powder. Example 4 A 250 mL round-bottom flask fitted with a magnetic stir bar is charged with 20 g of N. oculata powder followed by 100 mL of 5% w / v sodium metabisulfite solution. The suspension is stirred under an inert atmosphere for 2.5 hours at room temperature, and then the solid is collected by filtration under reduced pressure. The filter cake is spray-washed with 2 × 100 mL of DM water, vacuum-dried for 30 minutes, and then lyophilized for 18 hours to yield 17.0 g of dry powder. Example 5 A 250 mL round-bottom flask fitted with a magnetic stir bar is charged with 20 g of N. oculata powder followed by 100 mL of 1% w / v sodium metabisulfite solution. The suspension is stirred under an inert atmosphere for 2.5 hours at room temperature, and then the solid is collected by filtration under reduced pressure. The filter cake is spray-washed with 2 × 100 mL of DM water, vacuum-dried for 30 minutes, and then lyophilized for 18 hours to yield 16.9 g of dry powder. Example 6 A 250 mL round-bottom flask fitted with a magnetic stir bar is charged with 20 g of N. oculata powder followed by 100 mL of 0.5% w / v sodium metabisulfite solution. The suspension is stirred under an inert atmosphere for 2.5 hours at room temperature, and then the solid is collected by filtration under reduced pressure. The filter cake is spray-washed with 2 × 100 mL of DM water, vacuum-dried for 30 minutes, and then lyophilized for 18 hours to yield 17.4 g of dry powder. Example 7 A 250 mL round-bottom flask fitted with a magnetic stir bar is charged with 20 g of N. oculata powder followed by 100 mL of DM water. The suspension is stirred under an inert atmosphere for 2.5 hours at room temperature, and then the solid is collected by filtration under reduced pressure. The filter cake is spray-washed with 2 x 100 mL of DM water, vacuum-dried for 30 minutes, and then lyophilized for 18 hours to yield 17.3 g of dry powder. Example 8 A 250 mL round-bottom flask fitted with a magnetic stir bar is charged with 20 g of N. oculata powder, followed by 100 mL of 20% w / v sodium bisulfite solution, pH 4–5, adjusted with dibasic sodium phosphate. The suspension is stirred under an inert atmosphere for 2.5 hours at room temperature, and then the solid is collected by filtration under reduced pressure. The filter cake is spray-washed with 2 × 100 mL of DM water, vacuum-dried for 30 minutes, and then lyophilized for 18 hours to yield 17.0 g of dry powder. Example 9 A 250 mL round-bottom flask fitted with a magnetic stir bar is charged with 20 g of N. oculata powder, followed by 100 mL of 5% w / v sodium bisulfite solution, pH 4–5, adjusted with dibasic sodium phosphate. The suspension is stirred under an inert atmosphere for 2.5 hours at room temperature, and then the solid is collected by filtration under reduced pressure. The filter cake is spray-washed with 2 × 100 mL of DM water, vacuum-dried for 30 minutes, and then lyophilized for 18 hours to yield 17.2 g of dry powder. NOVELTY OF THE INVENTION Having described the present invention, it is considered a novelty and, therefore, the contents of the following are claimed as property.

Claims

1. A process for deodorizing a Nannochloropsis algae biomass, comprising a) contacting the biomass with an adduct-forming compound selected from metal sulfites, metal bisulfites, ammonium bisulfite, metal metabisulfites, SO2, or mixtures thereof, b) stirring or shaking, and c) collecting a solid from the suspension.

2. A process according to claim 1, wherein Nannocloropsis is selected from the group consisting of N. Gaditana, N. Granulate, N. Limnetica, N. Oceanica, N. Oculata and N. Salina.

3. A process according to claim 2, wherein Nannochloropsis is Nannochloropsis Oculata.

4. The process according to any preceding claim, wherein the adduct-forming compound in step (a) is either a metal sulfite selected from sodium, potassium and mixtures thereof, a metal bisulfite selected from sodium bisulfite, potassium bisulfite and mixtures thereof, or a metal metabisulfite selected from sodium and potassium metabisulfite and mixtures thereof.

5. The process according to any of the preceding claims, wherein step (b) is performed for approximately 30 minutes to approximately 6 hours.

6. The process according to any of the preceding claims, wherein step (c) is performed by filtration or by decantation of the supernatant and collection of the sediment.

7. The process according to any of the preceding claims, further comprising d) washing the solid with an aqueous solution and e) optionally drying.

8. The process according to claim 7, wherein the solid is washed with an aqueous solution selected from water, NaCl, H2O2, phosphate or citric acid solution.

9. The process of claim 7, wherein the solid is washed with water.

10. The process according to any of the preceding claims, wherein the Nannochloropsis algae biomass is in dry form.

11. The process according to any preceding claim, wherein the Nannochloropsis algae biomass is in sun-dried, oven-dried, air-dried, spray-dried, or freeze-dried form.

12. The process according to claims 1-9, wherein the Nannochloropsis algal biomass is in wet form. A / a / zuzz / uuu 1 1 f