CONCENTRATED PROTEIN MATERIALS FROM DE-CHLOROPHYLLIZED AQUATIC PLANT BIOMASS

Abstract

A method for producing a water-soluble protein concentrate from plant biomass is disclosed, comprising: obtaining plant matter from said plant biomass; drying said plant matter; grinding said dry plant matter; de-chlorophyllizing said ground dry plant matter; treating said ground dry de-chlorophyllized plant matter with water, thereby at least partially dissolving water-soluble protein content of said dry de-chlorophyllized plant matter and preparing an aqueous suspension of said dry de-chlorophyllized plant matter; separating said aqueous suspension of said dry de-chlorophyllized plant matter into a first neutral extract and a wet solid; and, drying said first neutral extract, thereby yielding a water-soluble protein concentrate. Also disclosed is a water-soluble protein concentrate produced by the method.

Claims

1.-20. (canceled)

21. A method for producing a water-soluble protein concentrate from plant biomass, comprising: obtaining from said plant biomass dry de-chlorophyllized plant matter comprising water-soluble protein content; treating said dry de-chlorophyllized plant matter with water, thereby yielding: an aqueous solution comprising at least part of said water-soluble protein content of said dry de-chlorophyllized plant matter; and, a suspension of de-chlorophyllized plant matter in said aqueous solution; separating said suspension into a first neutral extract and a wet solid; and, drying said first neutral extract, thereby yielding a water-soluble protein concentrate.

22. The method of claim 21, comprising: washing said wet solid with water, thereby producing a second neutral extract; concentrating said second neutral extract; and, drying said second neutral extract, thereby yielding additional water-soluble protein concentrate.

23. The method of claim 21, comprising: washing said wet solid with water, thereby producing a second neutral extract; combining said first neutral extract and said second neutral extract prior to said step of drying said first neutral extract, thereby producing a combined neutral extract; wherein said step of drying said first neutral extract comprises drying said combined neutral extract, thereby yielding a water-soluble protein concentrate.

24. The method of claim 21, wherein said plant biomass does not comprise seeds.

25. The method of claim 21, comprising obtaining plant biomass from aquatic plants selected from the group consisting of algae, microalgae, and duckweed.

26. The method of claim 25, wherein said step of obtaining plant biomass comprises obtaining plant biomass from duckweed.

27. The method of claim 21, wherein said method does not comprise any step in which said plant biomass contacts a solvent that is not approved for use in food production.

28. The method of claim 21, wherein said method does not comprise any step in which a chemical lysis agent is used.

29. The method of claim 21, wherein said step of obtaining dry de-chlorophyllized plant matter comprises: drying said plant biomass, thereby producing dried plant biomass; grinding said dried plant biomass, thereby producing ground dried plant biomass; extracting chlorophyll from said ground dried plant biomass, thereby producing de-chlorophyllized plant matter; and, drying said de-chlorophyllized plant matter, thereby obtaining dry de-chlorophyllized plant matter.

30. The method of claim 29, wherein at least one of the following is true: said step of drying said plant biomass comprises drying said plant biomass in the absence of light at a temperature not exceeding 40 C.; and, said step of grinding said dried plant biomass comprises grinding said dried plant biomass to a powder characterized by a maximum particle diameter of 100 m.

31. The method of claim 21, wherein said step of treating said dry de-chlorophyllized plant matter with water comprises at least one step selected from the group consisting of: treating dry de-chlorophyllized plant matter with water in a plant matter/water ratio selected from the group consisting of: 5:95 by weight on a dry matter basis; 10:90 by weight on a dry matter basis; and, 20:80 by weight on a dry matter basis; treating dry de-chlorophyllized plant matter with water at a temperature of in a range selected from the group consisting of: 20-80 C.; 30-70 C.; and, 40-60 C.; and, treating dry de-chlorophyllized plant matter with water for a time period selected from the group consisting of: 2-12 hours; 3-8 hours; and, 4-6 hours.

32. The method of claim 22, wherein said step of concentrating said second neutral extract comprises a step selected from the group consisting of: concentrating said second neutral extract until said neutral extract is characterized by a dissolved solid content of not less than 10%; concentrating said second neutral extract until said neutral extract is characterized by a dissolved solid content of not less than 5%; and, concentrating said second neutral extract until said neutral extract is characterized by a dissolved solid content of not less than 3%.

33. The method of claim 22, wherein said step of washing said wet solid with water comprises at least one step selected from the group consisting of: washing said wet solid with water at a solid:water ratio of 1:3 by volume; washing at a temperature of 10 C.; and, washing with successive aliquots of water until an aliquot is produced that is characterized by a concentration of dissolved material of less than 0.1% by weight.

34. The method of claim 23, wherein said step of washing said wet solid with water comprises at least one step selected from the group consisting of: washing said wet solid with water at a solid:water ratio of 1:3 by volume; washing at a temperature of 10 C.; and, washing with successive aliquots of water until an aliquot is produced that is characterized by a concentration of dissolved material of less than 0.1% by weight.

35. The method of claim 21, comprising drying said wet solid after all steps of producing neutral extracts have been completed, thereby producing dry fibrous material.

36. The method of claim 34, wherein said step of drying said wet solid is followed by a step of grinding said dry fibrous material.

37. A water-soluble protein concentrate produced from plant biomass, wherein said plant biomass does not comprise seeds, and said water-soluble protein concentrate is characterized by at least one characteristic selected from the group consisting of: said water-soluble protein concentrate comprises proteins having an average molecular weight of less than 8,000 Da; said water-soluble protein concentrate comprises a crude protein content of 40-70% on an organic matter dry basis; said water-soluble protein concentrate comprises a moisture content of between 3% and 6% by weight; said water-soluble protein concentrate comprises an ash content of between 3% and 5% by weight; and, said water-soluble protein concentrate comprises a carbohydrate content, calculated as organic compounds without nitrogen, of less than 50% on an organic matter dry basis.

38. The water-soluble protein concentrate of claim 37, wherein said water-soluble protein concentrate is partially water-soluble.

39. The water-soluble protein concentrate of claim 37, wherein said water-soluble protein concentrate is entirely water-soluble.

40. A water-soluble protein concentrate produced from plant biomass, wherein: said plant biomass does not comprise seeds; said water-soluble protein concentrate is characterized by at least one characteristic selected from the group consisting of: said water-soluble protein concentrate comprises proteins having an average molecular weight of less than 8,000 Da; said water-soluble protein concentrate comprises a crude protein content of 40-70% on an organic matter dry basis; said water-soluble protein concentrate comprises a moisture content of between 3% and 6% by weight; said water-soluble protein concentrate comprises an ash content of between 3% and 5% by weight; and, said water-soluble protein concentrate comprises a carbohydrate content, calculated as organic compounds without nitrogen, of less than 50% on an organic matter dry basis; and said water-soluble protein concentrate is produced according to the method of claim 21.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The invention will now be described with reference to the drawings, wherein

[0056] FIGS. 1A and 1B present schematic illustrations of methods for derivatizing and activating, respectively, protein concentrates herein disclosed;

[0057] FIG. 2 presents a schematic illustration of chemical processing of protein concentrates herein disclosed to form non-food materials;

[0058] FIG. 3 presents results of tangential flow filtration of a neutral extract solution prepared by water treatment of dry de-chlorophyllized plant material at 50 C. for 4 hours; and,

[0059] FIG. 4 presents the relationship between the solution concentration and the reduced viscosity for a solution of the protein concentrate of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] In the following description, various aspects of the invention will be described. For the purposes of explanation, specific details are set forth in order to provide a thorough understanding of the invention. It will be apparent to one skilled in the art that there are other embodiments of the invention that differ in details without affecting the essential nature thereof. Therefore the figures and examples provided in the invention are to be considered exemplary and not limiting, and the invention is to be understood as limited only as indicated in the accompanying claims, with the proper scope determined only by the broadest interpretation of said claims.

[0061] Unless otherwise noted, all concentrations disclosed herein are given on a w/w basis.

[0062] The concentrated protein plant materials of the instant invention can be produced from any kind of green plant biomass. In preferred embodiments, the plant biomass is harvested from aquatic environments (marine or fresh water). In more preferred embodiments, the biomass is harvested from algae or duckweeds. In yet more preferred embodiments, duckweeds are used as the source of the plant biomass. In even more preferred embodiments, duckweed of genus Wolffia is used, and in the most preferred embodiments, the source of the biomass is Wolffia globosa.

[0063] Typical proximate analyses of the chemical composition of some common duckweed species are given in Table 1. All concentrations are given as percentages. The crude protein content was calculated as 6.25 the nitrogen content, and the carbohydrate content as 100 minus the sum of the moisture, fat, fiber, and ash.

TABLE-US-00001 TABLE 1 Fat Mois- Crude (Ether Crude Carbohy- Species ture Protein Extract) Fiber Ash drate L. gibba 4.6 25.2 4.7 9.4 14.1 46.6 S. punctata 5.2 28.7 5.5 9.2 13.7 42.9 S. polyrrhiza 5.1 29.1 4.5 8.8 15.2 42.4 W. columbiana 4.8 36.5 6.6 11 17.1 28.8 W. arrhiza 5.3 20.4 4.6 11.6 17.6 45.8

[0064] The inventive process uses dry de-chlorophyllized plant matter, which can be prepared by any method known in the art.

[0065] In preferred embodiments of the invention, the dry de-chlorophyllized plant matter is prepared according to the following protocol. First, raw plant biomass (preferably fresh) is washed to remove any dirt or foreign material and then dried. Any method for drying the biomass known in the art can be used. Preferably, the drying is done in the dark. In order to prevent thermal degradation of plant pigments, the drying is done at a fairly low temperature, preferably below 50 C., more preferably below 45 C., and most preferably below 40 C.

[0066] The dried raw plant biomass is then ground. The grinding is preferably performed in a ball mill, and preferably below 30 C. In preferred embodiments of the invention, the dried raw biomass is ground to a powder having a maximum particle diameter of 200 m. In more preferred embodiments of the invention, the dried raw biomass is ground to a powder having a maximum particle diameter of 150 m. In the most preferred embodiments of the invention, the dried raw biomass is ground to a powder having a maximum particle diameter of 100 m.

[0067] The chlorophyll can be removed from the ground dried raw biomass by any method known in the art. In preferred embodiments of the invention, it is extracted by Soxhlet extraction under vacuum using a water-miscible organic solvent. In more preferred embodiments of the invention, the organic solvent is one that is not poisonous to humans. In yet more preferred embodiments of the invention, a food-grade solvent is used. In the most preferred embodiments of the invention, the chlorophyll is extracted using ethanol as the solvent.

[0068] Following the extraction of the chlorophyll, the de-chlorophyllized plant material is dried to remove the solvent used to extract the chlorophyll.

[0069] The dried de-chlorophyllized plant material is treated with water, preferably demineralized water with a conductance of less than 4 S. Enough water is added to the de-chlorophyllized plant matter to produce an aqueous suspension. In some preferred embodiments of the invention, the suspension comprises a plant matter/water ratio of 5:95 by weight on a dry matter basis. In some more preferred embodiments of the invention, the suspension comprises a plant matter/water ratio of 10:90 by weight on a dry matter basis. In the most preferred embodiments of the invention, the suspension comprises a plant matter/water ratio of 20:80 by weight on a dry matter basis. The plant material is kept in contact with the water for a predetermined time. In some preferred embodiments of the invention, this treatment lasts between 2 and 12 hours. In some more preferred embodiments of the invention, this treatment lasts between 3 and 8 hours. In the most preferred embodiments of the invention, this treatment lasts between 4 and 6 hours. In typical embodiments of the invention, the temperature of the suspension is maintained between 20 C. and 80 C. during the treatment. In more preferred embodiments of the invention, the temperature of the suspension is maintained between 30 C. and 70 C. during the treatment. In the most preferred embodiments of the invention, the temperature of the suspension is maintained between 40 C. and 60 C. during the treatment.

[0070] After the de-chlorophyllized plant material has been treated with water, the suspension is separated, preferably by centrifugation, most preferably by 5000 g centrifugation, into a liquid fraction and a wet solid fraction.

[0071] The liquid fraction, also known as the neutral extract, contains water soluble protein (RPPRM-WS) extracted from the plant material during the treatment with water. In some embodiments of the invention, the solid fraction of this first neutral extract is in the range 0.5-2.5%. In some embodiments of the invention, the solid fraction of the neutral extract is in the range 1-3%. In some embodiments of the invention, the solid fraction of the neutral extract is in the range 5-10%.

[0072] The wet solid fraction remaining after removal of the liquid fraction, also known as the crude neutral fraction, comprises de-chlorophyllized fiber. In some preferred embodiments of the invention, the wet solid is washed with water and the washing water separated from the wet solid fraction (e.g. by 5000 g centrifugation at 10 C.) to produce a second neutral extract. In some embodiments of the invention, the washing is performed three times at a fiber:water ratio of 1:3 by volume. In some preferred embodiments of the invention, the washing is performed with multiple aliquots of water applied in succession until the supernatant washing water has a dissolved solids content of less than 0.1%.

[0073] In preferred embodiments of the invention, the neutral extracts are concentrated, preferably by evaporation in vacuo (typically at a pressure of 40 mbar and a temperature of 40 C.). In some embodiments of the invention, the first and second neutral extracts are combined prior to the step of concentrating them. The neutral extracts are concentrated until the dissolved solids reach a predetermined minimum concentration. In some embodiments of the invention, this concentration is 3%. In some preferred embodiments of the invention, it is 5%. In some more preferred embodiments of the invention, it is 10%.

[0074] The concentrated neutral extract is then dried. Any method of drying known in the art can be used. In preferred embodiments, spray drying or freeze drying is used. The resulting solid mass is generally in powder form and comprises a concentrate of the water-soluble protein from the de-chlorophyllized plant material.

[0075] In some embodiments of the invention, the wet solid fraction, comprising fibrous material from which water-soluble protein has been extracted, is dried following the washing with water. In typical embodiments, it is dried in a hot air oven at a temperature of about 75 C.-85 C. In preferred embodiments, the drying is performed until the moisture content falls below a predetermined level. In particularly preferred embodiments, the wet solid fraction is dried until the moisture content is less than 15%. The dried fibrous material can then be ground, preferably to a granular mass with particles having a maximum diameter of less than 1 mm, and stored for other uses.

[0076] The protein concentrate produced by this method is a water-soluble composition comprising a mixture of substances. Typically, the average molecular weight is less than 12,000 Dalton; in preferred embodiments, the average molecular weight is less than 8,000 Dalton.

[0077] Moreover, the protein in the concentrate can be seen to have at least partially undergone a conformational transition from random coil to rod when a dilute solution (0.5-1.5% protein concentrate) is prepared at 25 C.

[0078] The water-soluble protein concentrate can be used as raw materials for obtaining end products with novel three-dimensional configurations that can be based on covalent or non-covalent bonds. Reference is now made to FIGS. 1A, 1B, and 2, which present schematic illustrations of method of derivatizing, activating, and chemical processing, respectively, of wholly water-soluble protein-rich plant-containing raw materials (RPPRM-WS). The process illustrated in FIG. 2 produces non-food items such as cross-linked hydrogels.

[0079] Chemical processing of the protein concentrates herein disclosed may be performed in a variety of environments. Non-limiting examples include aqueous environments and organic solvents at temperatures that are typically between 20 C. and 80 C. Non-limiting examples chemical transformations that can be performed on the protein concentrates of the instant invention include nucleophilic substitution, addition reactions, and free radical polymerization.

[0080] The following non-limiting examples are presented in order to assist a person of ordinary skill in the art in understanding how to make and use the invention herein disclosed.

[0081] In all of the examples presented, the biomass starting material used was obtained from the duckweed species Wolffia globosa cultivated by Hino-man Ltd. (Israel). The plants were harvested, washed with demineralized water to remove dirt and foreign materials, and dried in a current of warm (40 C.) air using an Ezidri Ultra FD 1000 air dryer obtained from Food Dehydrators (Israel).

[0082] The dry green plant material was then de-chlorophyllized by extraction by ethanol according to the procedure disclosed in International (PCT) Pat. Appl. Pub. No. WO2015/145431. The crude de-chlorophyllized plant material obtained after the ethanol extraction was then dried using a Buchi rotary evaporator operated at 200 mbar pressure and 80 C.

[0083] A proximate analysis of the chemical composition of the de-chlorophyllized plant material is given in Table 2. All amounts are given in percent by weight.

TABLE-US-00002 TABLE 2 Component Amount Moisture 4.53 Ash 8.11 Crude protein 72.3 Fats 0 Carbohydrate 15.06

Example 1

[0084] 10 grams of de-chlorophyllized dried Wolffia globosa prepared as described above suspended in 50 ml demineralized water and 450 ml demineralized water (0.4 S) preheated to 50 C. were added under stirring at a speed of 200 rpm to a 1 liter glass double jacketed reactor (extraction reactor) equipped with an anchor type Teflon stirrer, an overhead stirrer, and a thermometer. A condenser and thermostatic water bath with recirculation were added to the extraction reactor. The resulting suspension was mixed for 4 hours at 50 C., cooled to room temperature, and discharged from the extraction reactor, after which it was separated by vacuum filtration using a Buchner funnel and polyester net with a pore diameter of 100 microns. The separation produced 360 ml of extract solution (neutral extract) and 146 g of insoluble wet solid. The neutral extract solution was found to contain 1.92 g of dissolved solids, as evaluated by a gravimetric method performed on 10 ml aliquots of the solution (average of 3 replicates). The solution was dried at 105 C. for 4 hours using an oven with forced-air convection. The remaining solution of extract was freeze-dried by using a lyophilizer (FreeZone, Labconco). 1.89 g of solid Rich Plant Protein Raw Material-Water Soluble (RPPRM-WS-1) was obtained.

[0085] A proximate chemical analysis was performed on this material. The ash (inorganic material) content was determined by the ignition method [Santisteban J. I. 2004]. Crude protein (CP) was determined as 6.25%the nitrogen content. Carbohydrate (CH) content was determined from the formula 100=Ash+M+Cp+Fat+CH. Bradford protein (BP) was determined by UV spectroscopy using the calibration curve obtained from treatment of samples of the solutions prepared with human serum albumin (BSA) and treated with reactive Bradford. The results obtained from the analysis are presented in Table 3. All concentrations are given in percent by weight.

TABLE-US-00003 TABLE 3 Component Amount Moisture 3 Ash 4.3 Fats 0 Crude protein 69.2 Bradford protein 38.6 Carbohydrate 23.5

[0086] Sufficient RPPRM-WS-1 was added to water buffered to a predetermined pH to make up a 0.5% solution. The components were centrifuged to 5,000 g at a temperature of 20 C. The RPPRM-WS-1 completely dissolved over the entire pH range 2-12.

[0087] The average molecular mass of the protein concentrate was determined using tangential flow filtration (TFF) by dilution at constant volume using a Minimate TFF apparatus obtained from Pall. For this purpose has been prepared a solution of RPPRM-WS-1 of 0.5% concentration in demineralized water using as medium a filter membrane of 12 kDa. Reference is now made to FIG. 3, which presents the results obtained from the application of TFF. The TFF results indicate the protein concentrate comprises water soluble compounds with average molecular mass lower than 12,000 Da.

[0088] A viscosimetric method was used to confirm the coil-rod conformational transition [Tsujita Y. et al 1979., Mark J. E. 2007]. The viscosity of a 1.89% solution of RPPRM-WS-1 in demineralized water (0.4 S) was determined by using an Ubbelohde viscometer with 1 A capillary (time for demineralized water=90 s) held at 25 C. by using thermostatic viscometer bath VB-1423 (J.P.Selecta, Spain). The results obtained for the variation of reduced viscosity .sub.red function on concentration are shown in FIG. 4. These results demonstrated that the coil to rod conformational transition had indeed occurred.

Example 2

[0089] The influence of the temperature and extraction time on the chemical composition of the protein concentrate was investigated. The results are summarized in Table 4.

TABLE-US-00004 TABLE 4 RPPRM-WS Protein Organic by Insoluble Soluble Ash matter Kjeldahl Protein Extraction factors %; %; %; %; %; by Carbohy- Non- T t g/100 g g/100 g g/100 g g/100 g g/100 g Bradford drate protein Sample code C. hours de-chl. de-chl. extr. de-chl. extr % % % RP-WS-2 80 4 75.60 24.40 4.10 23.40 68.15 19.23 7.45 48.92 RP-WS-3 60 2 74.58 25.42 4.00 24.40 48.31 30.87 12.61 17.46 RP-WS-4 40 6 75.65 24.35 3.80 23.42 76.37 33.61 5.54 42.77 RP-WS-5 60 4 76.27 23.73 3.90 22.80 69.38 35.10 6.98 34.29 RP-WS-6 20 12 81.52 18.48 4.20 17.70 65.16 23.62 6.17 41.54