WO2018007491A1 - Isolat de protéines de colza natives de qualité alimentaire et procédé pour l'obtenir - Google Patents

Isolat de protéines de colza natives de qualité alimentaire et procédé pour l'obtenir Download PDF

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Publication number
WO2018007491A1
WO2018007491A1 PCT/EP2017/066870 EP2017066870W WO2018007491A1 WO 2018007491 A1 WO2018007491 A1 WO 2018007491A1 EP 2017066870 W EP2017066870 W EP 2017066870W WO 2018007491 A1 WO2018007491 A1 WO 2018007491A1
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WIPO (PCT)
Prior art keywords
protein isolate
rapeseed protein
aqueous liquid
native rapeseed
native
Prior art date
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PCT/EP2017/066870
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English (en)
Inventor
Johannes Hendrikus Maria WILLEMSEN
Gerardus Johannes Franciscus Smolders
Richard Zamolo
Original Assignee
Dsm Ip Assets B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dsm Ip Assets B.V. filed Critical Dsm Ip Assets B.V.
Priority to CA3026604A priority Critical patent/CA3026604A1/fr
Priority to EP17734755.6A priority patent/EP3481215A1/fr
Publication of WO2018007491A1 publication Critical patent/WO2018007491A1/fr

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Classifications

    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17Amino acids, peptides or proteins
    • A23L33/185Vegetable proteins
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23JPROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/14Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K20/00Accessory food factors for animal feeding-stuffs
    • A23K20/10Organic substances
    • A23K20/142Amino acids; Derivatives thereof
    • A23K20/147Polymeric derivatives, e.g. peptides or proteins
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to food grade soluble native rapeseed protein isolate, to a food grade soluble native rapeseed protein isolate that has a low microbe level, a process for obtaining food grade soluble native rapeseed protein isolate and use of the food grade soluble native rapeseed protein isolate in a food product.
  • WO 2008/094434 discloses the use of wheat protein isolates as an alternative to the use of egg yolk protein in compositions.
  • soy based protein has also been described for example in WO 2014/018922.
  • the use of wheat protein isolates may not be desirable for those with gluten allergies and there may also be intolerances to soy based proteins.
  • Soy protein is widely used, however in view of some intolerances to soy products there is a need to find other sources of vegetable proteins.
  • rapeseed seeds are rich in oil and contain considerable amounts of protein that accounts for 17 to 25% of seed dry weight. Processing rapeseed for oil for human consumption produces rapeseed meal (also referred to as cake, 60%) as a by-product which contains about 30 to 40% protein.
  • the rapeseed used for this purpose is usually of the varieties Brassica napus and Brassica juncea. These varieties contain only low levels of erucic acid and glucosinolates, and are also known as Canola.
  • Canola is a contraction of Canada and "ola" (for "oil low acid”), but is now a generic term defined as rapeseed oil comprising ⁇ 2% erucic acid and ⁇ 30 mmol/g glucosinolates.
  • the resultant rapeseed meal is currently used as a high-protein animal feed.
  • Hydrolysates are proteins that have been partially broken down by exposing the protein to heat, acid or enzymes that break apart the bonds linking amino acids. This makes it taste more bitter, but also allows it to be absorbed more rapidly during digestion than a native (non-hydrolyzed) protein. Isolates are purer than concentrates, meaning other non-protein components have been partially removed to "isolate" the protein. Many concentrates are around 80% protein, which means that on a dry basis, 80% of the total weight is protein. Isolates are typically around 90% protein (dry basis). This is calculated using the Kjeldahl method. The predominant storage proteins found in rapeseed are cruciferins and napins.
  • Cruciferins are globulins and are the major storage protein in the seed.
  • a cruciferin is composed of 6 subunits and has a total molecular weight of approximately 300 kDa.
  • Napins are albumins and are low molecular weight storage proteins with a molecular weight of approximately 14 kDa. Napins are more easily solubilized and in for example EP 1715752B1 a process is disclosed to separate out the more soluble napin fraction, preferably to at least 85 wt.%. Napins are primarily proposed for use in applications where solubility is key.
  • EP 1389921 B1 discloses a process of forming a food composition, which comprises extracting rapeseed oil seed meal with an aqueous food-grade salt solution at a temperature of at least 5°C to cause solubilization of protein in the rapeseed oil seed meal and to form an aqueous protein solution having a protein content of 5 to 30 g/l and a pH of 5 to 6.8, and subsequently two protein fractions are separated out via micelles. This is done to improve solubility as the cruciferin fraction is usually considered as less soluble over a wide pH range when not in the presence of a salt.
  • the resultant protein isolate is incorporated in said food composition in substitution for egg white, milk protein, whole egg, meat fibers, or gelatin.
  • DE 10 2014 005466 A1 also describes a process for obtaining purified cruciferin and napin fractions. During the process, also a protein mixture of the two with 55-60% napins and 40-45% cruciferins is obtained. The solubility of this protein mixture is approximately 75%.
  • a low microbe count is not only a requirement for the end-product but the microbial stability of the concentrate before drying is also important.
  • oilseed pressed meal has a relatively high oil content (typically >8%, for example >10%, on dry matter basis) and is an excellent source of proteins with preserved functionality. These proteins can be readily extracted from the meal by for instance an aqueous extraction (Rosenthal ei a/., Enzyme and Microbial Technology 19 (1996) 402-420, Rosenthal ei a/. , Trans iChemE, Part C, 76 (1998) 224-230 and Lawhon ei a/., J. Food Sci.
  • aqueous extraction Rossenthal ei a/., Enzyme and Microbial Technology 19 (1996) 402-420, Rosenthal ei a/. , Trans iChemE, Part C, 76 (1998) 224-230 and Lawhon ei a/., J. Food Sci.
  • microbe levels as low as less than 1 ,000 CFU/g.
  • the soluble native rapeseed protein isolate comprising both cruciferins and napins, obtained according to the present invention after mild extraction of rapeseed oil meal obtained using the cold-press method mentioned above, gave surprisingly low microbe levels.
  • a native rapeseed protein isolate comprising 40 to 65 wt.% cruciferins and 35 to 60 wt.% napins and having a solubility of at least 88% over a pH range from 3 to 10 at a temperature of 23 ⁇ 2°C with a microbe level of less than 1 ,000 CFU/g.
  • the microbe level is less than 500 CFU/g, more preferably from 200 CFU/g to 1 ,000 CFU/g.
  • a level of around 1 ,000 CFU/g such as from 800 to 1 ,200 CFU/g would be suitable for application in adults whereas a level of around 500 CFU/g, such as from 250 to 600 CFU/g would be suitable for application in children.
  • the native rapeseed protein isolate of the invention has a solubility of at least 88%, preferably at least 90%, more preferably at least 94%, and most preferably at least 96%, at a pH in the range of from 3 to 10 at a temperature of 23 ⁇ 2°C. This is also known as the soluble solids index (SSI).
  • SSI soluble solids index
  • the native rapeseed protein isolate preferably comprises a low level of salt. This is established by measuring the conductivity.
  • the conductivity of the native rapeseed protein isolate in a 2 wt.% aqueous solution is less than 9,000 ⁇ 3 ⁇ over a pH range of 2 to 12. More preferably the conductivity of the native rapeseed protein isolate in a 2 wt.% aqueous solution is less than 4,000 ⁇ 3/ ⁇ over a pH range of 2.5 to 1 1 .5.
  • the conductivity of an aqueous 5 g/l sodium chloride solution is around 9,400 ⁇ 3/ ⁇ .
  • the native rapeseed protein isolate has a phytate level of less than 0.4 wt.%, preferably of less than 0.25 wt.% and more preferably of less than 0.15 wt.%.
  • the native rapeseed protein isolate has a protein content of at least 90 wt.% (calculated as Kjeldahl N x 6.25) on a dry weight basis, more preferably at least 94 wt.%, most preferably at least 96 wt.% and especially at least 98 wt.%.
  • the native rapeseed protein isolate is substantially unhydrolyzed.
  • substantially unhydrolyzed is meant that the protein is not deliberately hydrolyzed.
  • a process for obtaining a native rapeseed protein isolate comprising the steps of: i) mixing cold-pressed rapeseed oil meal with an aqueous liquid at a temperature of from 45 to 65°C;
  • step iv) adjusting the pH of the decreamed aqueous liquid obtained in step iii) to neutral by adding acid or base, and mixing with a precipitant to obtain a precipitate; v) removing the precipitate obtained in step iv) to obtain an aqueous liquid;
  • step vii) isolating native rapeseed protein isolate from the concentrated and washed aqueous liquid obtained in step vi) by means of drying.
  • the rapeseed protein isolate is produced from cold pressed rapeseed press meal, the by-product of rapeseed oil production.
  • the process starts with an extraction step i), in which rapeseed meal is combined with an aqueous salt solution, for example 0 to 5% sodium chloride, at a temperature between 4 to 75°C, more preferably 20 to 75°C and most preferably 45 to 65°C.
  • an aqueous salt solution for example 0 to 5% sodium chloride
  • said mixing is carried out such that the ratio between said cold-pressed rapeseed oil meal and said aqueous liquid is from 1 :2 to 1 :30 (w/w).
  • the meal to water ratio is in the range of from 1 :5 to 1 :40, more preferably 1 :5 to 1 :20.
  • the protein rich solution is separated from the insoluble material in the separation step ii).
  • the protein rich solution is hereafter referred to as the extract.
  • the pH of the extract is preferably adjusted to neutral and the extract is further processed to clarify the material and remove non-protein substances.
  • the decreaming step iii) the residual fat and formed precipitates are removed via a solid/liquid separation step (e.g. filtration or centrifugation).
  • the decreaming in step iii) is carried out by means of centrifugation.
  • the extract is then concentrated and washed in an ultrafiltration/diafiltration (UF/DF) step vi).
  • the UF/DF step has the purpose of concentrating the protein and removing anti-nutritional factors (e.g. polyphenols, residual phytate, glucosinolates).
  • the concentrating and washing in step vi) is preferably carried out by means of ultrafiltration and diafiltration.
  • the washed concentrate may be dried in a suitable dryer, such as a spray drier (single or multistage) with an inlet temperature in the range of from 150 to 200°C and an outlet temperature in the range of from 50 to 100°C resulting in the rapeseed protein isolate.
  • a suitable dryer such as a spray drier (single or multistage) with an inlet temperature in the range of from 150 to 200°C and an outlet temperature in the range of from 50 to 100°C resulting in the rapeseed protein isolate.
  • the rapeseed protein isolate is obtained in a process without a fractionating step for separating out cruciferins and napins.
  • the rapeseed protein isolate is obtained in a process where the levels of napin and cruciferin are kept substantially constant (i.e. neither the napin or cruciferin levels are deliberately increased).
  • the concentrate showed good microbial stability.
  • the microbial levels of the material before the removal of non-protein substance by centrifugation were above 1*10 5 CFU/ml, after removal, preferably after precipitation and centrifugation to remove the precipitated material, the levels dropped below the 1*10 2 CFU/ml. This means that further processing to reduce the microbial count, for example by microfiltration is not required.
  • the process of the instant invention is characterized in that it is well-suited for large-scale application. Hence, in one embodiment the process is carried out at a scale of at least 500 kg, preferably of from 500 to 10,000 kg or from 1 ,000 to 5,000 kg in a period of from 2 to 10 hours.
  • the invention provides the use of the native protein isolate according to the first aspect of the invention in food products or pet food products.
  • the invention provides the use of an emulsion in pet food products that comprise from 5% to 35% of native rapeseed protein isolate by weight of the pet food product, preferably from 25% to 30%.
  • the native rapeseed protein isolate of the instant invention can be used as a germ-free ingredient in pet food, replacing e.g. wheat.
  • the term "pet food” means any composition intended to be consumed by a pet.
  • Meat or fish pet food can be a meat or fish emulsion product having a realistic meat- or fish-like image.
  • the rapeseed protein isolate can be added to the meat or fish material before and/or after the meat or fish material is emulsified as described in e.g. WO 2015/1 14543.
  • the pet can be any suitable animal, such as avian, bovine, canine, equine, feline, hircine, lupine, murine, ovine, or porcine animal.
  • Protein content was determined by the Kjeldahl method according to AOAC Official Method 991.20 Nitrogen (Total) in Milk, using a conversion factor of 6.25 was used to determine the amount of protein (% (w/w)).
  • Protein solubility (%) (protein in supernatant / protein in total dispersion) x 100.
  • Alternative methods for determining solubility are available and in some case use buffers, like borate-phosphate buffer in WO 201 1/057408. However, such as values are incomparable with the ones obtained in the instant application that are determined in the absence of buffer. MW determination by Blue Native PAGE
  • the protein charge has an impact on the electrophoretic mobility.
  • the Coomassie Brilliant Blue dye provides the necessary charges to the protein complexes for the electrophoretic separation.
  • the proteins were dissolved in 500 mM sodium chloride. As high salt concentrations are incompatible with electrophoretic separation, the sample was diluted 10-fold with water (final salt concentration: 50 mM).
  • Coomassie® G-250 (SimplyBlueTM, ThermoFischer Scientific) was used and gels were scanned with an ExQuestTM Spot Cutter (BioRad). Resultant bands after carrying out Blue Native PAGE were observed. It would be expected that bands around 14 kDa indicate 2S, around 150 kDa indicate 7S and around 300 kDa indicate 12S proteins.
  • the C/N ratio was determined by Size Exclusion Chromatography (SEC) analysis. Samples were dissolved in a 500 mM sodium chloride saline solution and analyzed by HP-SEC using the same solution as the mobile phase. Detection was done by measuring UV absorbance at 280 nm. The relative contribution of cruciferin and napin (%) was calculated as the ratio of the peak area of each protein with respect to the sum of both peak areas.
  • SEC Size Exclusion Chromatography
  • the rapeseed protein isolate was produced from cold-pressed rapeseed oil seed meal having an oil content of less than 15% on dry matter basis, cleaned and processed below 75°C.
  • the cold-pressed rapeseed oil seed meal was mixed with an aqueous salt solution (1 to 5% sodium chloride), at a temperature between 40 to 75°C.
  • the meal to aqueous salt solution ratio was in the range of from 1 :5 to 1 :20.
  • the protein rich solution (extract) was separated from the insoluble material.
  • the pH of the extract was adjusted to neutral and the extract was further processed to clarify the material and remove non-protein substances.
  • the residual fat was removed using centrifugation. Non-protein substances were removed by adjusting the pH of the material to neutral in the presence of a salt with which phytate precipitates (e.g. calcium chloride).
  • the formed precipitate is removed via a solid/liquid separation step (e.g. a membrane filter press or centrifugation) in which the impurities are removed in a solid salt form (e.g. calcium phytate).
  • a solid/liquid separation step e.g. a membrane filter press or centrifugation
  • the extract was then concentrated and washed in an ultrafiltration/diafiltration (UF/DF) step.
  • UF/DF ultrafiltration/diafiltration
  • the washed concentrate was dried in a spray drier with an inlet temperature in the range of from 150 to 200°C and an outlet temperature in the range of from 50 to 100°C resulting in the rapeseed protein isolate.
  • the microbial count was measured using the international FDA measuring standard after the separation step where it was found to be in the range of 10 6 CFU/ml. After precipitation and removal of the precipitate by centrifugation the microbial count was below the limit of detection of 10 2 CFU/ml. The microbial removal is also important for the stability of the concentrate after the UF/DF step.
  • the conductivity of the resultant native rapeseed protein isolates in a 2% solution was less than 4,000 ⁇ S/cm over a pH range of 2.5 to 1 1.5.
  • the resultant native rapeseed protein isolate comprised in the range of from 40 to 65% cruciferins and 35 to 60% napins.
  • the resultant native rapeseed protein isolate contained less than 0.26 wt.% phytate.
  • the resultant native rapeseed protein isolates had a solubility of at least 88% when measured over a pH range from 3 to 10 at a temperature of 23 ⁇ 2°C as shown for two batches in the below table.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
  • Zoology (AREA)
  • Mycology (AREA)
  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Biochemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Fodder In General (AREA)
  • Edible Oils And Fats (AREA)

Abstract

La présente invention concerne un isolat de protéines de colza natives comprenant de 40 à 65 % pds de cruciférines et de 35 à 60 % pds de napines et présentant une solubilité d'au moins 88 % sur une plage de pH de 3 à 10 à une température de 23 ± 2 °C avec une teneur en microbes inférieure à 1000 UFC/g. Elle concerne également l'utilisation de l'isolat de protéines de colza natives dans des produits alimentaires ou des produits alimentaires pour animaux.L'invention concerne également un procédé d'obtention d'un isolat de protéines de colza natives comprenant les étapes suivantes : i) mélange de farine d'huile de colza pressée à froid avec un liquide aqueux à une température de 45 à 65 °C ; ii) séparation du liquide aqueux du mélange obtenu à l'étape i) ; iii) écrémage du liquide aqueux obtenu à l'étape ii) ; iv) ajustement du pH du liquide aqueux écrémé obtenu à l'étape iii) à un pH neutre par addition d'acide ou de base, et mélange avec un agent de précipitation pour obtenir un précipité ; v) élimination du précipité obtenu à l'étape iv) pour obtenir un liquide aqueux ; vi) concentration et lavage du liquide aqueux obtenu à l'étape v) ; vii) isolement de l'isolat de protéines de colza natives à partir du liquide aqueux concentré et lavé obtenu à l'étape vi) au moyen d'un séchage.
PCT/EP2017/066870 2016-07-07 2017-07-06 Isolat de protéines de colza natives de qualité alimentaire et procédé pour l'obtenir WO2018007491A1 (fr)

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CA3026604A CA3026604A1 (fr) 2016-07-07 2017-07-06 Isolat de proteines de colza natives de qualite alimentaire et procede pour l'obtenir
EP17734755.6A EP3481215A1 (fr) 2016-07-07 2017-07-06 Isolat de protéines de colza natives de qualité alimentaire et procédé pour l'obtenir

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EP16178347.7 2016-07-07
EP16178347 2016-07-07
EP17166994.8 2017-04-19
EP17166994 2017-04-19

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
CN113993389A (zh) * 2019-06-21 2022-01-28 帝斯曼知识产权资产管理有限公司 热稳定的油菜籽蛋白组合物
US11457644B2 (en) 2016-07-07 2022-10-04 Dsm Ip Assets B.V. Emulsion comprising rapeseed protein isolate
US11564403B2 (en) 2016-07-07 2023-01-31 Dsm Ip Assets B.V. Soluble rapeseed protein isolate
US11844363B2 (en) 2015-12-17 2023-12-19 Dsm Ip Assets B.V. Gluten free native rapeseed protein isolate
US11903396B2 (en) 2016-07-07 2024-02-20 Dsm Ip Assets B.V. Process for making a soluble rapeseed protein isolate

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11844363B2 (en) 2015-12-17 2023-12-19 Dsm Ip Assets B.V. Gluten free native rapeseed protein isolate
US11457644B2 (en) 2016-07-07 2022-10-04 Dsm Ip Assets B.V. Emulsion comprising rapeseed protein isolate
US11564403B2 (en) 2016-07-07 2023-01-31 Dsm Ip Assets B.V. Soluble rapeseed protein isolate
US11903396B2 (en) 2016-07-07 2024-02-20 Dsm Ip Assets B.V. Process for making a soluble rapeseed protein isolate
CN113993389A (zh) * 2019-06-21 2022-01-28 帝斯曼知识产权资产管理有限公司 热稳定的油菜籽蛋白组合物

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EP3481215A1 (fr) 2019-05-15
CA3026604A1 (fr) 2018-01-11

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