METHOD FOR OBTAINING PROTEINS OR A RICH-PROTEIN EXTRACT FROM ALGAE, EXTRACTS AND USES THEREFORE

Abstract

The present disclosure relates to disrupting algae cell walls, in order to obtain protoplast with high nutrient digestibility for use as a food ingredient and/or as feed for farmed fish and shellfish species, which leads to the production of added value sea food items and to more sustainable and better performing food chains. The present disclosure described a method for obtaining proteins or a rich-protein extract from algae, comprising the steps of: disrupting a cell wall of algae by physical-mechanical means using a vibratory grinding mill with rings; submitting the disrupted algae to an enzymatic hydrolysis carried out with a mixture of enzymes; wherein the enzyme mixture is a mixture of at least two enzymes selected from the following list: lipase, pectinase, cellulase, hemicellullase, endo exo-arabanase, amylase, or mixtures thereof; provided that at least one of the enzymes is lipase, pectinase, amylase.

Claims

1. A method for obtaining proteins or a rich-protein extract from algae, comprising the steps of: disrupting a cell wall of algae by physical-mechanical means using a vibratory grinding mill with rings; and submitting the disrupted algae to an enzymatic hydrolysis carried out with a mixture of enzymes; wherein the enzyme mixture is a mixture of at least two enzymes selected from the group consisting of: lipase, pectinase, cellulase, hemicellullase, endo exo-arabanase, amylase, and mixtures thereof; provided that at least one of the enzymes is lipase, pectinase, amylase.

2. The method of claim 1, wherein the algae are microalgae or macroalgae.

3. The method of claim 1, wherein the enzyme mixture is: a pectinase, an endo exo-arabanase, a hemicellulase and a cellulase; a cellulase and a pectinase; or a lipase, a pectinase, a cellulase and an amylase.

4. The method of claim 1, wherein the enzyme mixture is a cellulase and a pectinase.

5. The method of claim 1, wherein the algae are selected from the group consisting of: Ulva sp., Gracilaria sp., Nannochloropsis sp., Tetraselmis sp., and mixtures thereof.

6. The method of claim 1, wherein the step of disrupting the cell wall by physical-mechanical means is carried out for 30 seconds-10 minutes.

7. The method of claim 6, wherein the step of disrupting the cell wall by physical-mechanical means is carried out for 1 minute-5 minutes.

8. The method of claim 1, wherein the enzymatic hydrolysis is performed at a temperature between 35-85 C.

9. The method of claim 1, wherein the enzymatic hydrolysis is performed at a pH between 5.5-9.5.

10. The method of claim 1, wherein the enzymatic hydrolysis is carried out for 30 minutes-5 hours.

11. The method of claim 1, wherein the enzymatic hydrolysis is carried out for 2 hours-4 hours.

12. The method of claim 1, wherein the enzymatic hydrolysis is carried out for 3 hours.

13. The method of claim 1, further comprising a step of separation or purification.

14. An algae extract obtainable by the method of claim 1, wherein the algae extract is a feed and food ingredient, and wherein at least 50% of the cell wall of algae was destructed.

15. An aquaculture feed comprising the algae extract of claim 14, wherein the aquaculture feed is a feed ingredient for aquatic organisms wherein at least 50% of the cell wall of algae was destructed.

16. The aquaculture feed of claim 15, wherein the aquaculture feed is a feed for fish and shellfish species.

17. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of disclosure.

[0055] FIG. 1Microscopy images (100) showing Ulva sp cell before (entire) and after wall disruption using ultra-turrax. AEntire and dry Ulva sp.; BEntire and hydrated Ulva sp.; CHydrated Ulva sp. after 1 minute of ultra-turrax action.

[0056] FIG. 2Microscopy images (100) showing Ulva sp cell wall disruption using ultra-turrax. AHydrated Ulva sp. after 1 minute of ultra-turrax action; BHydrated Ulva sp. after 5 minutes of ultra-turrax action.

[0057] FIG. 3Mills tested for algae cell wall disruption: Agravitational ball mill; Bballs used as aggressor agent; Cvibratory mill; DDish and rings used as aggressor agent.

[0058] FIG. 4Microscopy images (100) showing Ulva sp cell wall disruption by physical-mechanical means. AHydrated Ulva sp. after 5 minutes of ultra-turrax action; BHydrated Ulva sp. after 10 minutes of gravitational ball mill action; CHydrated Ulva sp. after 5 minutes of vibratory rings mill action.

[0059] FIG. 5Microscopy images (100) showing microalga cell wall before (entire) and after disruption using a vibratory rings mill. AHydrated entire Nannochloropsis sp; BHydrated Nannochloropsis sp after 5 minutes of vibratory rings mill action; CHydrated entire Tetraselmis sp; DHydrated Tetraselmis sp after 5 minutes of vibratory rings mill action.

DETAILED DESCRIPTION

[0060] Several tests were done at laboratory scale in order to select the best mechanical process and the best enzyme cocktail. Microscopy observation and the determination of soluble protein (Bradford method) of samples were used to verify and confirm the algae disruption.

[0061] In an embodiment, dry algae were obtained from the market and tested. The mechanical process was carried out both on dry algae and on hydrated (i.e. wet) algae, in order to simulate the fresh algae, before the drying process. For that the dry algae were soaked in distilled water for 10 minutes in order to warranty the algae hydration.

[0062] In an embodiment, the mechanical process was carried out by applying 1 minute of ultra-turrax into the dried algae and into hydrated algae.

[0063] In an embodiment, the impact of ultra-turrax action on algae disruption was evaluated over time (1-5 minutes as longer periods overheated the samples) to ascertain best duration time of action. Microscopy observation of samples was used to verify the level of algae disruption. By the observation of FIG. 2 it was concluded that the duration time of action of the mechanical process it is crucial to get protoplast with higher cell disruption.

[0064] In an embodiment, the ultra-turrax method is still limited to laboratorial scale not being possible to obtain large quantities of processed algae needed for industrial purposes (tonnes), being an unsuitable technological method to be applied. Thus, two different mills of possible industrial use were also tested (FIG. 3): a gravitational ball mill and; a vibratory rings mill. The results obtained are presented in Table 1.

TABLE-US-00001 TABLE 1 Soluble protein/Total Protein Available (%) obtained from Ulva sp. alga after the application of 3 different mechanical methods of disruption, in particular ultra-turrax, gravitational ball mill and vibratory rings mill, and enzymatic hydrolysis. Soluble Protein/ Total Protein Available (%) Ulva sp. hydrated and entire 0.00% Ulva sp. hydrated and after 10 0.00% minutes of gravitational ball mill action Ulva sp. hydrated and after 5 0.46% minutes of ultra-turrax action +2 h of enzymatic hydrolysis 1.00% +3 h of enzymatic hydrolysis 1.19% +4 h of enzymatic hydrolysis 0.91% Ulva sp. hydrated and after 5 0.38% minutes of vibratory rings mill action +2 h of enzymatic hydrolysis 1.14% +3 h of enzymatic hydrolysis 1.37% +4 h of enzymatic hydrolysis 1.22% Dry Ulva sp. after 5 minutes of 0.00% vibratory rings mill action +2 h of enzymatic hydrolysis 0.46% +3 h of enzymatic hydrolysis 0.77% +4 h of enzymatic hydrolysis 0.00%

[0065] In an embodiment, the results presented in Table 1, show that the application of gravitational ball mill was not efficient in disrupting the alga cell wall. Gravitational ball mill lack of vibration capacity to increase the impact between the sample and the balls (aggressor agent).

[0066] Furthermore, in an embodiment, the results presented in Table 1 also disclose that the vibratory rings mill was more efficient than the ultra-turrax when enzymatic hydrolysis is applied, which leads to an increase of soluble protein obtained. This vibratory mill also permits to process a large quantity of algae during the same 5 minutes of action, in particular, the ultra-turrax allows to process 5 g of algae whereas the vibratory mill allows to process 50 g of algae and can easily be adapted to industrial scale.

[0067] Furthermore, in an embodiment, the FIG. 4 confirms the results obtained for soluble protein, clearly showing the vibratory rings mill action that resulted in higher and almost total alga cell wall disruption.

[0068] In an embodiment, it was observed that milling during longer periods of time, in particular 10 minutes) was inappropriate, leading to increased temperature inside the mill dish. Therefore, it was considered that dish and disk refrigeration are essential to avoid the effect of high temperature over algae which might compromise its nutritional value.

[0069] In an embodiment, the considering the enzymatic hydrolysis, it is important to have a previous mechanical disruption of algae in order to facilitate the enzyme action. Therefore, algae were previously milled using the vibratory rings mill and then the enzyme cocktail was applied.

[0070] In an embodiment, the proposed enzyme cocktail is a low expensive mixture of lipase, pectinase, cellulase and amylase, already produced at large scale and used by a company for other purposes, and hence prone to further industrial applications. The optimal temperature for the action of the enzyme mixture is 60 C. (35-85 C.) at a pH of 6.5 (5.5-9.5), obtained by using a citrate-phosphate buffer solution. The optimal incubation period for the enzyme action in algae was tested and fixed in 3 hours (table 2).

[0071] In an embodiment, the enzyme mixture was used in a concentration of 360 APSU-CB/g.

[0072] In an embodiment, the besides the effects on macroalgae, microalgae were also used to test the effect of the vibratory rings mill and enzymatic mixture hydrolysis on the cell wall disruption and results are presented in table 3 and FIG. 5.

TABLE-US-00002 TABLE 3 Soluble protein/Total Protein Available (%) obtained from Nannochloropsis sp. and Tetraselmis sp. algae after the application of the vibratory rings mill (as mechanical method of disruption) and enzymatic hydrolysis. Soluble Protein/ Total Protein Available (%) Nannochloropsis sp hydrated and entire 0.00% +3 h of enzymatic hydrolysis 0.54% Nannochloropsis sp hydrated and 2.80% after 5 minutes of vibratory rings mill action +3 h of enzymatic hydrolysis 4.34% Tetraselmis sp hydrated and entire 0.00% +3 h of enzymatic hydrolysis 0.00% Tetraselmis sp hydrated and after 0.04% 5 minutes of vibratory rings mill action +3 h of enzymatic hydrolysis 0.29%

[0073] In an embodiment, the again, both disruption methods led to increased amount of the soluble protein fraction (%) and higher cell desegregation, permitting a better action of the enzymes. This processed algae biomass will hence have higher bio-availability to be used by fish when included in fish diets.

[0074] In an embodiment, some of these processed algae (micro and macroalgae) biomass were just included in sea bass dietary formulations as feed ingredients, replacing 30% of a commercial-based formula for this species. These diets were used to an in vivo digestibility trial where the capacity of sea bass to digest the algae was evaluated using a classical approach (collection of fish faeces to evaluate the amount of nutrients that were assimilated by the fish). The preliminary results of this trial showed a 14% increase in protein digestibility when Nannochloropsis was included in diets after the application of the vibratory rings mill; and a 36% increase in protein digestibility when the mechanical process was coupled with the enzymatic treatment. The protein digestibility of the mechanically processed Gracilaria also led to a 30% increase in protein digestibility by European sea bass.

[0075] The term comprising whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0076] It will be appreciated by those of ordinary skill in the art that unless otherwise indicated herein, the particular sequence of steps described is illustrative only and can be varied without departing from the disclosure. Thus, unless otherwise stated the steps described are so unordered meaning that, when possible, the steps can be performed in any convenient or desirable order.

[0077] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0078] The above described embodiments are combinable.

[0079] The following claims further set out particular embodiments of the disclosure.

[0080] The following reference are herewith incorporated by reference: [0081] [1] E. W. Becker, Micro-algae as a source of protein, Biotechnology Advances 25 (2) (2007) 207-210. [0082] [2] I. Lupatsch, C. Blake, Algae Alternative: Chlorella studied as protein source in tilapia feeds., Global Aquaculture Advocate May/june (2013) 78-79. [0083] [3] J. Rehacek, J. Schaefer, Disintegration of microorganisms in an industrial horizontal mill of novel design, Biotechnology and Bioengineering 19 (10) (1977) 1523-1534. [0084] [4] J. Limon-Lason, M. Hoare, C. B. Orsborn, D. J. Doyle, P. Dunnill, Reactor properties of a high-speed bead mill for microbial cell rupture, Biotechnology and Bioengineering 21 (5) (1979) 745-774. [0085] [5] P. Dunnill, M.D. Lilly, Protein Extraction from Microbial Cells, in: S. R. a. W. Tannenbaum, D. I. C. (Ed.), Single-Cell Protein II The MIT Press, Cambridge, 1975, p. 179. [0086] [6] H. Schtte, K. H. Kroner, H. Hustedt, M. R. Kula, Experiences with a 20 litre industrial bead mill for the disruption of microorganisms, Enzyme and Microbial Technology 5 (2) (1983) 143-148. [0087] [7] T. Becker, J. R. Ogez, S. E. Builder, Downstream processing of proteins, Biotechnology Advances 1 (2) (1983) 247-261. [0088] [8] H. Amano, H. Noda, Proteins of Protoplasts from Red Alga Porphyra yezoensis, NIPPON SUISAN GAKKAISHI 56 (11) (1990) 1859-1864.