Low lipid pea protein isolate

Abstract

To provide a leguminous protein isolate, especially a pea protein isolate having a low lipid content, and a method for preparing the same.SOLUTION: Provided is a legume protein isolate characterized in that it contains 7 to 9 grams of total lipids per 100 grams of protein, wherein the legume is a pea.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of plant proteins, particularly legume protein isolates, and more particularly pea protein isolates with low lipid content. [Background technology]

[0002] Humans' daily protein requirement is 12-20% of their dietary intake, and these proteins can be obtained from both animal-based products (meat, fish, eggs, dairy products) and plant-based foods (cereals, legumes, seaweed).

[0003] However, in developed countries, protein intake is mainly provided by animal-derived proteins, but numerous studies have shown that excessive intake of animal-derived proteins and insufficient intake of plant proteins is one of the causes of increased cancer and cardiovascular diseases.

[0004] Furthermore, animal proteins have many drawbacks, both in terms of the allergenicity of proteins, especially those derived from milk or eggs, and the negative environmental impact of intensive farming.

[0005] Therefore, there is an increasing demand from manufacturers for plant-derived compounds that have beneficial nutritional and functional properties but without the drawbacks of animal-derived compounds.

[0006] Nevertheless, replacing animal proteins by plant proteins is not always easy, since their physical and chemical properties differ, which affect the sensory qualities of the foods in which these proteins are incorporated.

[0007] Since the 1970s, the development of pulse plants, particularly peas, has progressed dramatically in Europe, primarily in France, as an alternative protein source to animal protein for animal and human consumption. Peas contain approximately 27% protein by weight. The term "pea" is considered herein in its most widely accepted usage and specifically includes all wild varieties of "round peas" and all variants of "round peas" and "wrinkled peas," regardless of the variety's usual purpose (human food, animal feed, and / or other uses). These seeds, unlike soybeans, are non-GMO and do not require a de-oiling process using solvents.

[0008] A drawback of some plant proteins, particularly legume proteins, and more particularly pea proteins, is that they are tasteless. Therefore, they can impart off-flavors to foods into which they are incorporated. These flavors are frequently described by consumers as "beany," pea-like, or bitter.

[0009] A known solution to this problem is to mask these unpleasant flavors by introducing compounds such as flavors during the manufacturing process. Nevertheless, this solution is often unsatisfactory because it cannot hide the unpleasant flavors, but only slightly reduces them. A second drawback is that adding additional ingredients makes the food manufacturing process more expensive. In addition, more and more consumers are choosing healthier foods and turning away from products containing compounds.

[0010] A more advantageous solution would be to directly use vegetable protein isolates that have little or no unpleasant taste. Some examples of methods for obtaining such isolates have already been described. For example, WO 2015 / 071498 describes a wet-milling extraction method combined with lactic acid fermentation to extract a purified pea protein isolate. Another example in WO 2017 / 120597 describes a method of precipitation in salt form, combined with specific washing of the protein with a large amount of aqueous solution at neutral pH. Nevertheless, these methods are unsatisfactory because they result in a protein isolate that still has a pea-like taste and a bitter taste.

[0011] Since lipids are substrates for lipoxygenases and oxidation reactions that lead to the formation of volatile compounds that cause off-flavors in legume proteins, lipid extraction can be an efficient method for producing protein isolates that are free of these off-flavors and / or have a more stable flavor during storage, especially due to the oxidation of residual lipids. Indeed, it has been shown in the literature (Sessa and Rackis JA. Oil Chemists' Soc 1979, 56, 262-271) that the oxidation of unsaturated fatty acids, especially linoleic and linolenic acids, is the main cause of these off-flavors during harvesting, processing, and storage.

[0012] Among the routes investigated for extraction is the use of cyclodextrins, which are cyclic oligosaccharides composed of several glucopyranose units (C6H10O5) linked by α-(1,4) bonds. The most common are α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, each consisting of six, seven, and eight glucopyranose units, respectively. In the literature, the use of β-cyclodextrin has been experimented with to remove residual lipids and phospholipids from soy protein isolates derived from defatted meal. Because lipids are substrates for lipoxygenase and oxidation reactions that lead to the formation of volatile compounds responsible for off-flavors in legumes, lipid extraction may be an efficient method for producing protein isolates free of these off-flavors. In this area, reference can be made to Zhu et al., Food Chemistry, 264 (2018), which proposes the use of α-cyclodextrin, and Akshay Arora et al., Food Chemistry, 127, no. 3, 2011 and U.S. Patent Application Publication No. 20110045128(A1), which proposes the use of ultrasonic treatment in combination with the use of cyclodextrin.

[0013] It would therefore be advantageous to have legume proteins, in particular legume protein isolates, more particularly pea protein isolates, with a low lipid content. Summary of the Invention

[0014] According to a first aspect of the invention, a legume protein isolate is proposed, the legumes being in particular chosen from peas and faba beans, preferentially peas, and characterized in that it contains between 7g and 9g, preferentially between 7.5g and 8.5g, of total lipids per 100g of protein.

[0015] According to another aspect, there is provided a method for preparing a protein isolate according to the invention, characterized in that it comprises the following steps: 1) suspending in water a protein of a legume, preferentially pea, selected between pea and faba bean; 2) Adjust the pH to 8.5; 3) Heating to a temperature of 40-50°C, preferably 45°C; 4) adding an aqueous solution containing a mixture of phospholipase and β-cyclodextrin, characterized in that the ratio between the phospholipase A2 activity, expressed in units of PLA2 activity per gram of β-cyclodextrin, and the amount of β-cyclodextrin is between 10 and 100, preferentially between 20 and 80, and even more preferentially between 25 and 50; 5) Stirring for 160 to 200 minutes, preferably 180 minutes; 6) Adjusting the pH to 4.5; 7) Heat to a temperature of 50-70°C, preferably 60°C for 8-12 minutes, preferably 10 minutes; 8) Centrifuging, then optionally washing with demineralized water, then centrifuging a second time; 9) suspending the protein pellet in water and then adjusting the pH to 7; 10) Drying the resulting protein isolate.

[0016] According to a final aspect of the invention, the industrial use of a legume protein isolate selected from pea, prickly pear and fava bean, and even more preferentially a pea protein isolate according to the invention, is proposed, in particular in animal and human food.

[0017] The present invention will be better understood from the following examples. DETAILED DESCRIPTION OF THE INVENTION

[0018] Thus, according to a first aspect of the invention, a legume protein isolate is proposed, the legumes being in particular chosen from pea and broad bean, preferentially pea, and characterized in that it contains between 7g and 9g, preferably between 7.5g and 8.5g, of total lipids per 100g of protein. Preferentially, the legume protein composition is a pea protein isolate.

[0019] The term "protein isolate" is to be understood in the present application as a composition having a protein content of more than 70%, preferentially more than 80%, and even more preferentially more than 85%, this percentage being understood relative to the dry matter of the composition. The protein content is calculated by any technique known to those skilled in the art, in particular by assaying the total Kjeldahl nitrogen and multiplying the result by the factor 6.25. The composition therefore comprises proteins, which are macromolecules formed from one or more polypeptide chains consisting of a sequence of amino acid residues linked together by peptide bonds. In the specific context of pea proteins, the invention relates more particularly to globulins (approximately 50-60% of pea proteins). Pea globulins are classified into three main subfamilies: legumins, vicilins, and convicilins.

[0020] "Leguminous plants" or "legumes" will be understood in this application to mean the family of dicotyledonous plants of the order Fabaceae. Fabaceae is the third most numerous flowering plant family in terms of number of species after Orchidaceae and Asteraceae. Fabaceae includes approximately 765 genera and over 19,500 species. Several legumes, such as soybean, common bean, pea, chickpea, faba bean, peanut, cultivated lentil, cultivated alfalfa, various clovers, faba bean, carob, licorice, and prickly pear, are important crop plants.

[0021] In the present application, the term "pea" includes pea varieties belonging to the genus Pisum sativum and more particularly to the species Aestivum sativum. The mutant varieties are specifically "mutants r" and "mutants r" described in the paper by CL HEYDLEY et al., entitled "Developing novel pea starches," Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemical Society, 1996, pp. 77-87. They were named "mutants rug3", "mutants rug4", "mutants rug5" and "mutants lam".

[0022] In this application, the term "total lipids" is defined as all lipid molecules without distinction. These include triglycerides, phospholipids, and free fatty acids. Lipid determination is carried out by acid hydrolysis followed by extraction with hexane and specific determination of the lipids thus extracted, preferentially by gas chromatography. A preferred method is described below.

[0023] Preferentially, the legume of the protein isolate is pea.

[0024] Preferentially, the protein isolate according to the invention is characterized in that its linoleic acid content is reduced by 20% to 30%, preferentially by 25%, compared to the content present in legume seeds.

[0025] By "linoleic acid" is meant, according to the present invention, an omega-6 polyunsaturated fatty acid corresponding to the all-cis-Δ9,12 C18:2 n-6 acid, whose structural formula is: H3C—(CH2)4—CH=CH—CH2—CH=CH—(CH2)7—COOH.

[0026] For example, as stated in (Sessa and Rackis 1977), "The oxidation of polyunsaturated fatty acids (e.g., linoleic and linolenic acids) is the primary cause of off-flavors during harvesting, processing, and storage." It is noteworthy that, thanks to the present invention, the content of these acids is significantly reduced, as shown in the examples below.

[0027] Protein isolate can have the advantage of not containing traces of organic solvent, that is, based on the dry mass of isolate, it contains less than 100 ppm of solvent.Preferably, based on the dry mass of isolate, isolate contains less than 10 ppm, and preferentially does not contain any.Organic solvent means a solvent made of molecules that contain at least one carbon atom.Instead, isolate can contain inorganic solvent, typically water.This is an advantage over isolates made by a method that includes a lipid extraction step using organic solvents such as hexane.

[0028] The protein isolate of the present invention may have good functional properties due to good oil or water retention.

[0029] According to another aspect, there is provided a method for producing a legume protein composition according to the invention, characterized in that the method comprises the steps of: 1) suspending in water a protein of a legume, preferentially pea, selected between pea and faba bean; 2) Adjust the pH to 8.5; 3) Heating to a temperature of 40-50°C, preferably 45°C; 4) adding an aqueous solution containing a mixture of phospholipase and β-cyclodextrin, characterized in that the ratio between the phospholipase A2 activity, expressed in units of PLA2 activity per gram of β-cyclodextrin, and the amount of β-cyclodextrin is between 10 and 100, preferentially between 20 and 80, and even more preferentially between 25 and 50; 5) Stirring for 160 to 200 minutes, preferably 180 minutes; 6) Adjusting the pH to 4.5; 7) Heat to a temperature of 50-70°C, preferably 60°C for 8-12 minutes, preferably 10 minutes; 8) Centrifuging, then optionally washing with demineralized water, then centrifuging a second time; 9) suspending the protein pellet in water and then adjusting the pH to 7; 10) Drying the resulting protein isolate.

[0030] Preferentially, step 1 is carried out by suspending legume proteins, characterized in that the legume is composed of more than 50%, preferentially more than 70%, and even more preferentially more than 80% globulins. Such globulins can be easily obtained by grinding the seeds into a meal, suspending it in water, and separating the fiber and starch using a hydrocyclone and centrifugation. The protein-enriched supernatant solution is then adjusted to an isoelectric pH (about 4.5) and subjected to controlled heating to separate the globulins into a floc. Such a method is described in the applicant's EP 1 400 537.

[0031] In steps 1 and 8, "water" means any type of water suitable for protein extraction for food consumption. Preferably, decarbonated water, demineralized water, or drinking water is used.

[0032] For step 4, the ratio between the phospholipase A2 activity, expressed in units of PLA2 activity per g of β-cyclodextrin, and the amount of β-cyclodextrin is between 10 and 100, preferentially between 20 and 80, and even more preferentially between 25 and 50.

[0033] Preferably, the amount of β-cyclodextrin is calculated relative to the amount of total lipid in the protein isolate, which varies from 0.04 to 0.8 g per 1 g of lipid, and the amount of phospholipase is calculated according to the above ratio.

[0034] Phospholipases are enzymes that hydrolyze phospholipids. One phospholipase that can be used in the methods of the present invention is a type A2 phospholipase, i.e., PLA2 Nagase 10P / R, manufactured by Nagase ChemteX and derived from Streptomyces violaceum NBRC15146.

[0035] PLA2 activity is measured using soybean lecithin as a substrate. The substrate is kept at 37°C and pH 8.0, and activity is measured using, for example, the Wako NEFA-C Test Enzyme Kit (Wako Pure Chemical Industries). One unit of enzyme activity corresponds to the hydrolysis of 1 μmol of fatty acid per minute.

[0036] Therefore, the PLA2 Nagase 10P / R enzyme is 100,000 U / g.

[0037] Cyclodextrins consist of several glucopyranose units (CH) linked by α-(1,4) bonds. 10 O5). The most common are α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, which consist of six, seven, and eight glucopyranose units, respectively. One of the main advantages of cyclodextrins is their ability to form inclusion complexes with a variety of compounds due to their "conical cylinder" structure.

[0038] According to the invention, drying of the composition can be carried out by any method known per se, preferentially by freeze-drying, drum drying or spray-drying, in particular by freeze-drying.

[0039] The advantage of this method is that it allows bypassing the sonication step, known per se, in which ultrasound waves are applied to the material (meals, isolates, etc.) during the process.

[0040] According to a final aspect of the invention, the industrial use of the legume protein composition according to the invention, preferentially a legume protein isolate selected from pea and faba bean, and even more preferentially a pea protein isolate, is proposed, in particular in animal and human food.

[0041] The present invention will be better understood from the following non-limiting examples. [Example]

[0042] In these examples, yellow peas are used, the total lipid content of which is equal to 2.3% of the dry matter. Linoleic acid represents 53.3% of the total fatty acids in this meal.

[0043] The BCD (beta cyclodextrin) used is Kreptose® from Roquette.

[0044] The phospholipase used is Nagase PLA2 10P / R, diluted with demineralized water to a concentration of 1% by weight. The solution also contains 0.5% NaCl and 0.1% CaCl.

[0045] Example 1a: Production of a legume protein isolate according to the invention from previously extracted globulins

[0046] Globulins are extracted using conventional extraction methods. In this example, yellow pea seeds are used. After removing the outer fiber using a hammer mill, the pea seeds are crushed to produce a meal. The meal is then soaked in water at room temperature for 30 minutes at pH 6.5 to a final concentration of 16.5% solids by weight relative to the weight of the suspension. The 25% solids by weight meal suspension is then introduced into a series of hydrocyclones, where it is separated into a light phase consisting of a mixture of protein, internal fiber (pulp), and soluble matter, and a heavy phase containing starch. The light phase at the outlet of the hydrocyclones is then adjusted to a solids content of 11.2% relative to the weight of the suspension. Separation of the internal fiber is carried out by treatment in a WESTFALIA-type decanter centrifuge. The light phase at the outlet of the decanter centrifuge contains a mixture of protein and soluble matter, while the heavy phase contains pea fiber. The light phase at the outlet of the decanter centrifuge is adjusted to pH 4.6 and heated at 70°C for 4 minutes to coagulate the proteins at their isoelectric points. After protein coagulation, a protein floc consisting mainly of globulins is obtained.

[0047] The protein flocs were resuspended in demineralized water and then introduced into a reactor, where reagents (βCD and PLA2) were added under specific temperature (45°C) and pH (8.5) conditions. The amounts of βCD and PLA2 were calculated based on the residual amount of lipid in the assay, respectively, and were equivalent to 0.04 g of βCD per g of lipid and 0.002 g of phospholipase Nagase PLA2 10P / R in a 1% solution. After 180 min of reaction, the solution was heated to 60°C for 10 min to inhibit PLA2. The treated solution was then aggregated at 60°C for 10 min at pH 4.5, followed by centrifugation twice at 8000 g for 10 min to remove the βCD complex. Finally, the globulins were resuspended in demineralized water, and the pH was raised to 7 before lyophilization.

[0048] Example 1b: Production of legume protein isolate from meal

[0049] This example differs from Example 1a in that the injection points for βCD and phospholipase are upstream during the pea meal suspension step.

[0050] In this example, yellow pea seeds are used. After removing the outer fibers using a hammer mill, the pea seeds are crushed to produce a meal. This meal is then immersed in water in a reactor to a final concentration of 16.5% by weight solids relative to the weight of the suspension. The reagents (βCD and PLA2) are introduced into the reactor and the resulting solution is heated to a temperature (4 The suspension is then placed under specific conditions of 5°C and pH 8.5. The amounts of βCD and PLA2 are calculated relative to the residual lipid content in the assay and are equivalent to 0.04 g of βCD per gram of lipid and 0.002 g of phospholipase Nagase PLA2 10P / R per gram of lipid, respectively. After 180 minutes of reaction, the solution is heated to 60°C for 10 minutes to inhibit PLA2. The meal suspension is then introduced into a series of hydrocyclones, where it is separated into a light phase consisting of a mixture of proteins, internal fiber (pulp), and soluble substances, and a heavy phase containing starch. The light phase at the outlet of the hydrocyclones is then adjusted to a solids content of 11.2% by weight of the suspension. The internal fiber is separated by processing in a Westfalia decanter centrifuge. The light phase at the outlet of the decanter centrifuge contains a mixture of proteins and soluble materials, while the heavy phase contains pea fiber. The pH of the light phase at the outlet of the decanter centrifuge is adjusted to 4.6, and the solution is heated to 70°C for 4 minutes, causing the proteins to coagulate at their isoelectric point. After protein coagulation, a protein floc consisting mainly of globulins is obtained.

[0051] Example 1c: Production of a legume protein isolate according to the present invention from previously extracted globulins having a βCD / lipase ratio outside the present invention

[0052] The purpose of this example is to demonstrate the importance of the βCD / lipase ratio.

[0053] In this example, yellow pea seeds are used. After removing the outer fiber using a hammer mill, the pea seeds are crushed to produce a meal. The meal is then soaked in water at room temperature for 30 minutes at pH 6.5 to a final concentration of 16.5% solids by weight relative to the weight of the suspension. The 25% solids by weight meal suspension is then introduced into a series of hydrocyclones, where it is separated into a light phase consisting of a mixture of proteins, internal fiber (pulp), and soluble matter, and a heavy phase containing starch. The light phase at the outlet of the hydrocyclones is then adjusted to a solids content of 11.2% relative to the weight of the suspension. Separation of the internal fiber is carried out by treatment in a WESTFALIA-type decanter centrifuge. The light phase at the outlet of the decanter centrifuge contains a mixture of proteins and soluble matter, while the heavy phase contains pea fiber. The light phase at the outlet of the decanter centrifuge is adjusted to pH 4.6 and heated at 70°C for 4 minutes to coagulate the proteins at their isoelectric points. After protein coagulation, a protein floc consisting mainly of globulins is obtained.

[0054] The protein flocs were resuspended in demineralized water and then introduced into a reactor, where reagents (βCD and PLA2) were added under specific temperature (45°C) and pH (8.5) conditions. The amounts of βCD and PLA2 were calculated based on the residual amount of lipid in the assay, respectively, and were equivalent to 0.71 g of βCD per g of lipid and 0.002 g of phospholipase Nagase PLA2 10P / R in a 1% solution. After 180 min of reaction, the solution was heated to 60°C for 10 min to inhibit PLA2. The treated solution was then aggregated at 60°C for 10 min at pH 4.5, followed by centrifugation twice at 8000 g for 10 min to remove the βCD complex. Finally, the globulins were resuspended in demineralized water, and the pH was raised to 7 before lyophilization.

[0055] Example 2: Determination of total lipids in different isolates according to the invention Total lipids are analyzed as the content of the different fatty acids present. The procedure for analyzing total lipids is as follows. The procedure for analyzing the linoleic acid content is as follows. Both values ​​are expressed in terms of protein content in order to compare different samples, which is obtained by measuring the nitrogen content of the sample and multiplying it by a factor of 6.25.

[0056] The table below summarizes and compares the various tests. [Table 1] These assays demonstrate both the importance of the injection site of βCD and phospholipase, as well as the importance of the ratio of βCD to lipase. Indeed, only Example 1a according to the present invention exhibits the following: Significantly reduced fat content resulting in a ratio of less than 9g total fat per 100g protein; It is possible to obtain protein isolates with a significant reduction in linoleic acid content of more than 20% (23.6%).

Claims

1. 1. A legume protein isolate characterized in that it contains 7g to 9g of total lipids per 100g of protein, wherein said legume is pea.

2. A legume protein isolate as described in claim 1, characterized in that the total lipids are contained in an amount of 7.5 g to 8.5 g per 100 g of protein.

3. A legume protein isolate as described in claim 1 or 2, characterized in that the linoleic acid content is reduced by 20% to 30% compared to the content present in the legume.

4. A legume protein isolate described in any one of claims 1 to 3, characterized in that it does not contain an organic solvent.