WO2014046543A1 - Procédé pour isoler des protéines à partir de biomasse solide contenant des protéines, sélectionnée parmi biomasse végétale, algues, varech et des combinaisons de ceux-ci - Google Patents

Procédé pour isoler des protéines à partir de biomasse solide contenant des protéines, sélectionnée parmi biomasse végétale, algues, varech et des combinaisons de ceux-ci Download PDF

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Publication number
WO2014046543A1
WO2014046543A1 PCT/NL2013/050676 NL2013050676W WO2014046543A1 WO 2014046543 A1 WO2014046543 A1 WO 2014046543A1 NL 2013050676 W NL2013050676 W NL 2013050676W WO 2014046543 A1 WO2014046543 A1 WO 2014046543A1
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WIPO (PCT)
Prior art keywords
protein
biomass
dispersion
process according
range
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PCT/NL2013/050676
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English (en)
Inventor
Zhang CHEN
Johan Pieter Marinus Sanders
Marieke Elisabeth BRUINS
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Wageningen Universiteit
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Publication of WO2014046543A1 publication Critical patent/WO2014046543A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/001—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste
    • A23J1/005—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste from vegetable waste materials
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/006—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/006—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials
    • A23J1/007—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials from leafy vegetables, e.g. alfalfa, clover, grass
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation

Definitions

  • the present invention relates to a process for isolating proteins from solid protein-containing biomass selected from vegetable biomass, algae, seaweed and combinations thereof.
  • Protein is present in a relatively high concentration in plant leaves.
  • the protein content in leaves can amount to 15 to 30 wt%, in particular in for example green tea leaves, Jatropha leaves, tobacco leaves, alfalfa leaves, or grass leaves.
  • Leaf protein concentrates have been proposed as food supplement since the
  • Isolation of proteins from leaves is typically carried out by first extracting the protein by extracting comminuted leaves with e.g. water, a phosphate buffer or an alkaline solution, separating the extract from the extracted leaves and then recovering protein from the extract.
  • alkaline extractions at moderate temperature (50 °C) and a pH of up to 10 are mentioned.
  • S. Chiesa and E. Gnansounou, Bioresource Technology 102 (2011), p. 427-436 it is mentioned that extraction is always carried out at relatively low temperatures in order to prevent extensive protein deterioration and that the combination of high temperature and alkaline conditions can irreparably damage the protein in biomass.
  • Protein extraction yields of alkaline extraction of leaf proteins at low or mild temperature are reported to be typically between 40 and 60 % of the protein present in the starting material.
  • the present invention relates to a process for isolating proteins from solid protein-containing biomass selected from vegetable biomass, algae, seaweed and combinations thereof, the process comprising the following steps:
  • pH of the dispersion during extraction step b) is in the range of from 12.3 to 13.5.
  • control of the pH of the dispersion during extraction step b) is important to obtain a high yield of proteins with a relatively high molecular weight.
  • a further advantage of the process according to the invention is that also coagulated protein can be extracted from biomass due to the relatively high alkaline conditions applied during the extraction. Therefore, the process can also suitably be applied for extraction of biomass wherein protein has been concentrated by means of a pretreatment that causes protein coagulation, such as for example biomass that has been subjected to a heat, acid or alkaline pretreatment step.
  • biomass with a relatively high protein content can be obtained from which protein can be suitably extracted by means of the process according to the invention.
  • An advantage of biomass with a relatively high protein content is that a protein extract with a higher protein content is obtained.
  • the pretreated biomass may be sun-dried without development of an unacceptable odour or excessive microbial degradation of the biomass or autolysis of proteins or other biomass compounds. This is particularly important for biomass with a high moisture content that is harvested and processed in dry and/or climates, such as for example algae, seaweed or leaves of tropical plants such as water hyacinth.
  • proteins are isolated from solid protein-containing biomass by first extracting proteins from the solid biomass by a solid-liquid extraction at a temperature in the range of from 70 to 150 °C and at a pH in the range of from 12.3 to 13.5 to obtain a protein-comprising extract and protein- depleted biomass and then recovering protein from the extract.
  • step a) of the process according to the invention a dispersion of the solid protein-containing biomass with a cell wall structure in an aqueous liquid is provided. Protein is then extracted from the biomass by keeping the dispersion at a temperature in the range of from 70 to 150 °C during an extraction time (extraction step b), wherein the pH of the dispersion during extraction step b) is in the range of from 12.3 to 13.5. After the extraction time, the dispersion is separated into a liquid protein-comprising extract and solid protein-depleted biomass (step c). This may be done by any suitable solid liquid separation known in the art, for example filtration, centrifugation or sedimentation. Protein is then recovered from the extract in a recovery step d).
  • any suitable protein-containing biomass selected from vegetable biomass, algae, seaweed and combinations thereof.
  • the biomass comprises plant leaves, straw, algae and/or sea weed, more preferably plant leaves and/or straw.
  • the protein-containing biomass is vegetable biomass, still more plant leaves.
  • suitable plant leaves are tea leaves, Jatropha leaves, water hyacinth leaves and other tropical leaves, sugar beet leaves, and grass leaves. It has been found that the process according to the invention is suitable to isolate proteins from biomass comprising intact cell walls.
  • the biomass therefore preferably is biomass comprising intact cell walls.
  • the biomass may be untreated or pretreated biomass.
  • the process according to the invention is particularly suitable for protein- and water-comprising biomass that has been subjected to a pretreatment wherein the protein has been concentrated by means of coagulation of at least part of the protein followed by removal of at least part of the water, prior to extraction steps a) and b).
  • Such pretreatment may comprise any suitable protein coagulation step and any suitable water removal step.
  • Such steps are well- known in the art.
  • Protein may for example be coagulated by means of a heat treatment, an acid or an alkaline treatment. Water may subsequently be removed by means of for example mechanical pressing, centrifugation and/or drying, for example sun-drying.
  • the biomass is obtained by a pre-treatment comprising heating protein and water-containing biomass at a temperature above 80 °C, preferably in the range of from 80 to 105 °C, to coagulate at least part of the protein.
  • a pre-treatment comprising heating protein and water-containing biomass at a temperature above 80 °C, preferably in the range of from 80 to 105 °C, to coagulate at least part of the protein.
  • An example of biomass pretreated by a heat treatment that causes protein coagulation are extracted tea leaves, also referred to as tea leaves residue.
  • the biomass may be pretreated biomass that has been subjected to a mild protein extraction, for example a mild heating step followed by a pressing step, an alkaline extraction at a mild temperature and/or a mild pH .
  • a mild protein extraction for example a mild heating step followed by a pressing step, an alkaline extraction at a mild temperature and/or a mild pH .
  • Biomass residue from biorefining processes such as bioethanol manufacturing processes wherein cellulose and hemicelluloses from the biomass are converted into ethanol, may suitably be used as the solid protein-containing biomass in the process according to the invention.
  • the protein-containing biomass is preferably chopped or otherwise comminuted prior to being extracted in step b). More preferably, the biomass is comminuted such that it has an average particle size in the range of from 1 to 50 mm. Reference herein to particle size is to the largest dimension of the biomass particle. It has been found that, unlike in some prior art protein extraction processes, milling of the biomass is not needed in order to obtain a high protein yield.
  • a dispersion of solid biomass in an aqueous liquid is provided.
  • the aqueous liquid serves as extraction liquid in alkaline extraction step b).
  • the dispersion is provided by providing an aqueous alkaline solution and then mixing the alkaline solution with the biomass to obtain the dispersion.
  • the dispersion may be provided by adding solid alkaline compound and water to the biomass. In case of biomass with a very high moisture content, for example above 50 wt% or above 80 wt%, the dispersion may even be provided by adding solid alkaline compound to the biomass without addition of water.
  • the pH of the dispersion during extraction step b) is in the range of from 12.3 to 13.5.
  • the alkaline solution may be any alkaline solution that, after mixing with the biomass, may provide a dispersion with such high pH.
  • the alkaline solution is a solution of a strong base in water, more preferably a solution of sodium hydroxide and/or potassium hydroxide in water.
  • the solution may further comprise a weaker base such as for example ammonia or calcium hydroxide.
  • step b) of the process according to the invention biomass compounds such as pigments and polyphenols are extracted and react with protein to form tannins or other compounds or complexes with a brown colour.
  • Calcium or magnesium ions can react or coagulate with some of the coloured/brown compound formed.
  • the dispersion of biomass in alkaline solution preferably comprises calcium and/or magnesium ions.
  • the presence of calcium or magnesium ions in the dispersion may for example be achieved by using biomass that has been pre-treated with a calcium or magnesium salt, for example biomass that has been subjected to a prior extraction with a solution comprising a calcium or magnesium salt.
  • the presence of calcium or magnesium ions in the dispersion is achieved by providing the dispersion by mixing an alkaline solution comprising a calcium or magnesium salt, with the solid biomass and/or by adding a calcium or magnesium salt to the dispersion during step b).
  • the calcium or magnesium salt preferably is calcium hydroxide and/or magnesium hydroxide, more preferably calcium hydroxide.
  • the dispersion provided in step a) is a dispersion of biomass in an aqueous solution of sodium hydroxide and/or potassium hydroxide that further comprises calcium hydroxide.
  • the pH of the dispersion during extraction step b) is in the range of from 12.3 to 13.5, preferably in the range of from 12.3 to 13.3, during the entire extraction step.
  • the pH of the dispersion may decrease due to biomass components with a buffering capacity, such as for example proteins, pectin or lignin. It is therefore preferred to monitor the pH of the dispersion during extraction step b) and add additional alkaline compound if needed to keep the pH in the range of from 12.3 to 13.5 or within any of the preferred pH ranges.
  • the concentration of hydroxide in the alkaline solution provided in step a) is not critical as long as the dispersion obtained by mixing the alkaline solution with the solid biomass has a pH of at least 12.3 and not above 13.5 in order to limit protein hydrolysis.
  • the ratio of volume of the liquid to dry weight of the solid protein-containing biomass in the dispersion may be any suitable ratio.
  • the ratio of volume of alkaline solution to weight of protein-containing biomass is in the range of from 0.5 to 50 litres per kilogram, more preferably of from 1 to 40 litres per kilogram, even more preferably of from 2 to 25 litres per kilogram, still more preferably of from 3 to 10 litres per kilogram.
  • the ratio of volume of the liquid to weight of solid biomass is not critical, in the sense that it does not importantly affect the extractability of proteins and the molecular weight of the proteins extracted.
  • a low ratio i.e. below 10 litres per kilogram is preferred. It will be appreciated that the optimum ratio inter alia depends on the protein concentration in the biomass.
  • Extraction step b) is carried out at a temperature in the range of from 70 to 150 °C, preferably of from 80 to 130 °C, more preferably of from 80 to 105 °C. At temperatures above 150 °C, substantial protein hydrolysis takes place and it is no longer possible to obtain proteins with a high molecular weight in acceptable amounts.
  • the extraction time will strongly depend on the extraction temperature and the pH during extraction step b). The lower the extraction temperature and the pH, the more extraction time is needed in order to achieve a higher protein yield.
  • the extraction time is in the range of from 15 minutes to 30 hours, more preferably of from 1 to 24 hours.
  • protein is recovered from the protein-comprising extract obtained in separation step c). Recovery may be done by any means known in the art, preferably by means of precipitation, more preferably acid precipitation, or membrane filtration, more preferably ultrafiltration, size exclusion or ion exchange. Preferably, protein is recovered from the extract by means of precipitation, more preferably acid precipitation.
  • a protein-depleted biomass is obtained.
  • Such protein- depleted biomass may suitably be used as raw material for the production of bio- ethanol and/or other bio-materials such as lactic acid or biogas.
  • Green tea leave residue was obtained by extracting dried green tea leaves during 45 minutes with water at 80 °C.
  • an amount of 0.5 grams of GTR was immersed in 20 ml of a solution of 0.1 N sodium hydroxide in water (volume to weight ratio of 40 ml alkaline solution per gram biomass; initial pH of 13).
  • Protein was extracted by placing the tubes in a water bath kept at the desired extraction temperature. During extraction the samples were shaken. The pH during extraction was monitored and did not decrease below a value of 12.3.
  • the samples were centrifuged (17,200 g for 10 minutes) and the supernatants (protein-comprising extracts) were stored at -20 °C prior to analysis.
  • the protein content of the supernatants was determined by means of Kjeldahl analysis. Extraction experiments as described above were carried out with GTR at temperatures of 40, 60, 80 and 95 °C. Extraction experiments with Oolong tea residue (dried Oolong tea leaves extracted with water for 45 minutes at 80 °C), Jatropha leaves (ground to 0.1 - 2 mm), road grass (ground to 0.2 - 4 mm), and barley straw (chopped to 0.5 - 5 mm) were carried out as described above at 95 °C for 4 hours. In Table 1, the extraction yields (wt% of protein extracted based on the weight of protein in the biomass) obtained in the different experiments are shown.
  • Table 1 Protein extractability for different types of biomass (volume/weight ratio of 40; 0.1 N NaOH). No NaOH addition during the extraction step.
  • the experiments show that at a temperature of 60 °C , almost 70 wt% of the proteins can be extracted, at a temperature of 80 °C or 95 °C even more than 90 wt%.
  • the molecular weight of the proteins extracted in experiment 8 was determined by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). It appeared that more than 90% of the proteins had a molecular weight above 14 kDa. Approximately 75 wt% of the proteins had a molecular weight of approximately 67 kDa.
  • Protein was recovered as follows from supernatants that were obtained by alkaline extraction of green tea residue carried out as described in Example 1.
  • the pH of the supernatants was adjusted to 3.5 by addition of IN HC1.
  • Example 3 Six extraction experiments were carried out wherein green tea residue was extracted at 95 °C during 2 hours. The concentration of sodium hydroxide in the initial alkaline solution and the ratio of alkaline solution to biomass were varied. In the first 5 experiments (experiments 21-25), no alkali was added during the extraction. In the last experiment (experiment 26) In the final experiment (experiment 26), a relatively low volume to biomass ratio was used (8 ml of alkaline solution per gram GTR) and the initial concentration of the alkaline solution was 0.1N NaOH. During the extraction step, additional NaOH was added after each 30 minutes extraction time (three additions in total; 1 mmol NaOH per gram GTR in each addition).
  • the samples were centrifuged (17,200 g for 10 minutes) and the supernatants (protein-comprising extracts) were stored at -20 °C prior to analysis.
  • the protein content of the supernatants was determined by means of Kjeldahl analysis. The absorbance of the supernatants at 400 and 680nm was determined.
  • experiment 27 an extraction with calcium hydroxide at 95 °C for two hours (experiment 27a) was followed by extraction of the proteins recovered with NaOH at 95 °C for two hours (experiment 27b).

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  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
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Abstract

L'invention concerne un procédé pour isoler des protéines à partir de biomasse solide contenant des protéines, sélectionnée parmi biomasse végétale, algues, varech et des combinaisons de ceux-ci, le procédé comprenant les étapes consistant à : a) fournir une dispersion de la biomasse solide contenant des protéines dans un liquide aqueux; b) extraire les protéines de la biomasse en maintenant la dispersion à une température dans une plage de 70 à 150 °C pendant la durée de l'extraction; c) séparer la dispersion en un extrait contenant des protéines et une biomasse appauvrie en protéines; et d) récupérer les protéines de l'extrait, le pH de la dispersion pendant l'étape d'extraction b) étant compris entre 12,3 et 13,5.
PCT/NL2013/050676 2012-09-21 2013-09-20 Procédé pour isoler des protéines à partir de biomasse solide contenant des protéines, sélectionnée parmi biomasse végétale, algues, varech et des combinaisons de ceux-ci WO2014046543A1 (fr)

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Cited By (15)

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WO2016015013A1 (fr) * 2014-07-25 2016-01-28 Synthetic Genomics, Inc. Ingrédient alimentaire riche en protéine provenant de biomasse et procédés de préparation
US9421477B2 (en) 2013-08-12 2016-08-23 Green Extraction Technologies Biomass fractionation and extraction apparatus
WO2016201379A1 (fr) * 2015-06-10 2016-12-15 Parabel Ltd. Procédés et systèmes pour l'extraction de produits riches en protéines et hydrates de carbone à partir d'une microculture et compositions de ceux-ci
WO2017019125A1 (fr) * 2015-07-24 2017-02-02 Synthetic Genomics, Inc. Ingrédient alimentaire riche en protéine provenant d'une biomasse et procédés de préparation
JP2018522922A (ja) * 2015-08-10 2018-08-16 パラベル リミテッド 水生生物種からシュウ酸が低減されたタンパク質を抽出するための方法及び系並びにその組成物。
US10596048B2 (en) 2015-06-10 2020-03-24 Parabel Ltd. Methods and systems for forming moisture absorbing products from a microcrop
US10856478B2 (en) 2015-06-10 2020-12-08 Parabel Nutrition, Inc. Apparatuses, methods, and systems for cultivating a microcrop involving a floating coupling device
US10961326B2 (en) 2015-07-06 2021-03-30 Parabel Nutrition, Inc. Methods and systems for extracting a polysaccharide product from a microcrop and compositions thereof
US10981083B2 (en) 2013-08-12 2021-04-20 Green Extraction Technologies Process for fractionation and extraction of herbal plant material to isolate extractives for pharmaceuticals and nutraceuticals
US11122817B2 (en) 2014-07-25 2021-09-21 Smallfood Inc. Protein rich food ingredient from biomass and methods of production
US11174355B2 (en) 2013-08-12 2021-11-16 Green Extraction Technologies Isolation method for water insoluble components of a biomass
US11213048B2 (en) 2014-07-25 2022-01-04 Smallfood, Inc. Protein rich food ingredient from biomass and methods of preparation
US11229171B2 (en) 2009-04-20 2022-01-25 Parabel Nutrition, Inc. Methods of culturing a floating aquatic species using an apparatus for fluid conveyance in a continuous loop
WO2022147184A1 (fr) * 2020-12-31 2022-07-07 Trophic Llc Concentré de protéines et procédé de préparation
US11452305B2 (en) 2015-09-10 2022-09-27 Lemnature AquaFars Corporation Methods and systems for processing a high-concentration protein product from a microcrop and compositions thereof

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US11229171B2 (en) 2009-04-20 2022-01-25 Parabel Nutrition, Inc. Methods of culturing a floating aquatic species using an apparatus for fluid conveyance in a continuous loop
US11174355B2 (en) 2013-08-12 2021-11-16 Green Extraction Technologies Isolation method for water insoluble components of a biomass
US9421477B2 (en) 2013-08-12 2016-08-23 Green Extraction Technologies Biomass fractionation and extraction apparatus
US10981083B2 (en) 2013-08-12 2021-04-20 Green Extraction Technologies Process for fractionation and extraction of herbal plant material to isolate extractives for pharmaceuticals and nutraceuticals
US9718001B2 (en) 2013-08-12 2017-08-01 Green Extraction Technologies Biomass fractionation and extraction methods
US10207197B2 (en) 2013-08-12 2019-02-19 Green Extraction Technologies Process for ambient temperature fractionation and extraction of various biomasses
US11213048B2 (en) 2014-07-25 2022-01-04 Smallfood, Inc. Protein rich food ingredient from biomass and methods of preparation
US11122817B2 (en) 2014-07-25 2021-09-21 Smallfood Inc. Protein rich food ingredient from biomass and methods of production
WO2016015013A1 (fr) * 2014-07-25 2016-01-28 Synthetic Genomics, Inc. Ingrédient alimentaire riche en protéine provenant de biomasse et procédés de préparation
JP2018516993A (ja) * 2015-06-10 2018-06-28 パラベル リミテッド マイクロクロップからタンパク質及び高炭水化物産生物を抽出するための方法及び系並びにその組成物
US10568343B2 (en) 2015-06-10 2020-02-25 Parabel Ltd. Methods and systems for extracting protein and carbohydrate rich products from a microcrop and compositions thereof
US10596048B2 (en) 2015-06-10 2020-03-24 Parabel Ltd. Methods and systems for forming moisture absorbing products from a microcrop
US10856478B2 (en) 2015-06-10 2020-12-08 Parabel Nutrition, Inc. Apparatuses, methods, and systems for cultivating a microcrop involving a floating coupling device
WO2016201379A1 (fr) * 2015-06-10 2016-12-15 Parabel Ltd. Procédés et systèmes pour l'extraction de produits riches en protéines et hydrates de carbone à partir d'une microculture et compositions de ceux-ci
US11166476B2 (en) 2015-06-10 2021-11-09 Parabel Nutrition, Inc. Methods and systems for extracting protein and carbohydrate rich products from a microcrop and compositions thereof
US10961326B2 (en) 2015-07-06 2021-03-30 Parabel Nutrition, Inc. Methods and systems for extracting a polysaccharide product from a microcrop and compositions thereof
WO2017019125A1 (fr) * 2015-07-24 2017-02-02 Synthetic Genomics, Inc. Ingrédient alimentaire riche en protéine provenant d'une biomasse et procédés de préparation
JP2021138757A (ja) * 2015-08-10 2021-09-16 パラベル ニュートリション インコーポレイテッド 水生生物種からシュウ酸が低減されたタンパク質を抽出するための方法及び系並びにその組成物。
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