Methods for improved air classification separation of pulse protein, fibre and starches

Abstract

Described herein are methods for pre-treating pulses prior to separation of the starch and protein fractions of the pulses by air classification. Specifically, the pre-treatments weaken the structural integrity of pulse cotyledons and physically separate protein and starch inside the cotyledons, which facilitates the separation between protein and starch during subsequent milling and air classification to achieve better purity in both protein-rich and starch-rich fractions. These higher purity fractions have improved functional properties, nutritional properties, and sensory profiles, such as stronger water-holding capacity and oil-absorption capacity, emulsifying capacity and emulsion stability, and foaming properties and foam stability as well as substantially reduced beany flavor and bitter taste.

Description

[0001] METHODS FOR IMPROVED AIR CLASSIFICATION SEPARATION OF PULSE PROTEIN, FIBRE AND STARCHES

[0002] PRIOR APPLICATION INFORMATION

[0003] The instant application claims the benefit of US Provisional Patent Application 63 / 517,527, filed August 3, 2023 and entitled “METHODS FOR IMPROVED AIR CLASSIFICATION SEPARATION OF PULSE PROTEIN, FIBRE AND STARCHES”, the entire contents of which are incorporated herein by reference for all purposes.

[0004] BACKGROUND OF THE INVENTION

[0005] Pulses are dry edible seeds of crops in the Fabaceae family, which include but are not limited to peas, Faba beans, beans, lentils, chickpeas, lupins, and cowpeas. Pulses are an important source of protein and starch, and can be fractionated into different ingredients for a broad range of industrial applications (Boye, Zare, & Pletch, 2010; Ren, Yuan, Chigwedere, & Ai, 2021 ). The production of functional ingredients from pulses can add significant economic value to this group of crops.

[0006] Air classification is a dry fractionation technology that separates protein and starch components in finely ground pulse, cereal, and pseudocereal flours based on their different densities and particle sizes. Specifically, the protein-rich flour (i.e., protein concentrate) is finer and lighter and the starch-rich flour is coarser and denser (Boye et al., 2010). Because the dry method has a significantly lower operating cost and uses less water (Sosulski & Sosulski, 1986) than a typical wet fractionation process, it is the method broadly used for the fractionation processing of pulses in Western Canada and many other parts of the world. However, air classification can only generate protein-enriched flours / protein concentrates that have about 45-60% protein at an industrial scale, which is noticeably lower than that of commercial pulse protein isolates obtained from wet fractionation method (75- 90%). The poor purity of pulse protein concentrates lowers its application value and market value.

[0007] Described herein is the use of different pre-treatment methods on pulse seeds to improve the separation efficiency of protein and starch during the subsequent air classification. These pre-treatment methods include germination, soaking in water, and soaking in salt solutions.

[0008] Germination of pulses, cereals, pseudocereals, and other grains is a natural, simple, clean-label, and efficient method that has been utilized to improve the functional properties, nutritional quality, and sensory profiles of the different grains. The effects of germination on the techno-functional attributes of pulse grains and flours have been comprehensively summarized (Setia et al., 2019; Xu et al., 2019). Soaking in water and soaking in salt solutions have been applied to shorten the cooking time and reduce the levels of anti-nutritional factors (e.g., phenolic compounds, phytic acid) of pulses (Coskuner & Karababa, 2003; Vijayakumari, Siddhuraju, Pugalenthi, & Janardhanan, 1998). However, no prior art has used these pre-treatment methods to enhance the separation efficiency of protein and starch during air classification.

[0009] SUMMARY OF THE INVENTION

[0010] According to a first aspect of the invention, there is provided a method for separating starch and protein fractions from pulse seeds comprising: subjecting a quantity of pulse seeds to a structural integrity weakening treatment for a period of time; reducing the size of the treated pulse seeds; and separating the starch fraction and the protein fraction of the size-reduced, treated pulse seeds by air classification.

[0011] In some embodiments, the pulse seeds are subjected to the structural integrity weakening treatment until the seeds are of a suitable softness for further processing.

[0012] The structural integrity weakening treatment may be germination, for example, by germinating the pulse seeds for 12-96 hours at a temperature between 20-50°C or for 24-96 hours.

[0013] In some embodiments, the structural integrity weakening treatment comprises soaking the pulse seeds in a salt solution at a temperature between 10-60°C or at a temperature between 22-55°C for 0.5-48 h.

[0014] In some embodiments, the salt is selected from the group consisting of potassium carbonate (K2CO3), potassium bicarbonate (KHCOj), sodium carbonate (Na^COj), sodium bicarbonate (NaHCOj), potassium chloride (KCI), sodium chloride (NaCl), magnesium chloride (MgCb), calcium chloride (CaCh), calcium hydroxide [CafOH),] and mixtures thereof.

[0015] In some embodiments, the salt is a pH increasing salt that increases the pH of a solution to a relatively high level, for example, to a pH of about 1 1.

[0016] In some embodiments, the pH increasing salt is selected from the group consisting of potassium carbonate (K2CO3), potassium bicarbonate (KHCO3), sodium carbonate (Na2COj) and sodium bicarbonate (NaHCO3).

[0017] In some embodiments, the salt is added at a concentration of 0.5- 10%.

[0018] In some embodiments, the structural integrity weakening treatment comprises soaking the pulse seeds in water at a temperature between 10-60°C or at a temperature between 22-55°C for 0.5-48 h.

[0019] In some embodiments, the pulse is selected from the group consisting of peas, lentils, Faba beans, chickpeas, dry beans, lupins, and cowpeas.

[0020] The pulse seeds may be reduced in size by milling or grinding, for example, by pin milling, for example, at a speed between 1,500 to 30,000 rpm.

[0021] The air classification may be carried out at a speed of approximately 1,200 to 25,000 rpm and an air flow rate between 20 to 30,000 m3 / h.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. I . Scanning electron microscopy (SEM) images of the cross section of cut cotyledons of round pea (RP), Faba bean (FB) and wrinkled pea (WP). R: raw; S: soaked; and G: germinated. Magnification at which the image was captured is shown in parentheses. Plus (+) symbols indicate starch granules; arrows indicate spaces in the protein and fiber matrix; and ovals indicate unruptured cotyledon matrix. FIG. 2. Pasting profiles of coarse stream of round pea and Faba bean samples determined using a Rapid Visco-Analyzer with 10.6 % (w / w, dry solids) flour suspension of 28.5 g total weight.

[0024] FIG. 3. Gelling ability of coarse stream of round pea and Faba bean samples (control, soaked, and germinated) determined using a Texture Analyser with 10.6 % (w / w, dry solids) flour suspension of 28.5 g total weight at 95 °C.

[0025] DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

[0027] The agri-food sector is continuously exploring natural, clean-label, simple, and cost-effective approaches to: (1 ) enhance the separation efficiency of protein and starch from pulses (e.g., peas, lentils, Faba beans, chickpeas, dry beans); and (2) improve the functional properties and nutritional value of the resulting plant-based protein and starch ingredients.

[0028] Presently, the economic value of pulse processing is largely from the protein ingredients. Current practices for producing air-classified pulse protein concentrates generate ingredients with up to a 65% protein maximum; however, as discussed herein, our process yields are remarkably higher (70.2-73.7% for pea protein concentrates). Furthermore, the ingredients produced by our invention can be used directly in extrusion to produce fibrous meat analogs, plant protein powder as a supplement, and other food products because of the remarkably higher purity. In contrast, existing pulse protein concentrates require further purification using a wet method in order to be used for these high-end products or they can only be used for low-end products with significantly lower value.

[0029] Described herein are methods for pre-treating pulses prior to separation of the fractions of the pulses by air classification. Specifically, as discussed herein, the pre-treatments weaken the structural integrity of pulse cotyledons and physically separate protein and starch inside cotyledons (Fig. I ), which facilitates the separation between protein and starch during subsequent pin milling and air classification to achieve better purity in both protein-rich and starch-rich fractions. As discussed herein, these fractions have improved functional properties, nutritional properties, and sensory profiles, such as stronger water-holding capacity and oil-absorption capacity, emulsifying capacity and emulsion stability, and foaming properties and foam stability. The improvement can be attributed to the higher purity of the fractions and the structural changes occurring to the protein and starch molecules.

[0030] Morphologies of the cross sections of seed cotyledons of raw, soaked and geminated (48 h) pea and Faba bean are shown in Fig. 1- 1 and 1-2. The SEM images of raw pea and Faba bean revealed that the starch granules (highlighted by plus symbols) were compactly embedded in the protein and fiber matrix in intact cells (highlighted by ovals). After soaking, the structure of the protein and fiber matrix became loose, and some starch granules were liberated from the matrix and there were voids (highlighted by arrows) found between in the pulse cotyledons. During soaking, the pulse seeds imbibed water and swelled, physically causing disruption of the matrix structure. Germination further loosened the matrix of pea and Faba bean as indicated by arrows and thus more starch granules were released from the structure (P-R and FB-R). The additional rupture of the matrix structure might be attributed to the breakdown of protein and fiber (such as cellulose, hemicellulose and pectin) by their respective hydrolytic enzymes. However, the hydrolysis of granular starch did not seem to take place during germination because there was no obvious change in the surface structure and morphology of starch granules. Because of the weakened association between starch and protein / flber caused by the soaking and germination pre-treatments, there was significant improvement in the separation efficiency between starch and protein in the subsequent air classification.

[0031] In some embodiments of the invention, the pulse seeds are subjected to the structural integrity weakening treatment until the seeds are of a suitable softness for processing. As will be appreciated by one of skill in the art, this suitable softness can be determined by various means known in the art, if necessary, for example, by use of a Texture Analyzer to determine the hardness of the treated seeds.

[0032] In some embodiments of the invention, the structural integrity weakening treatment prior to milling and air classification to improve separation of the protein and starch inside cotyledons comprises germination for 12- 96 hours at a temperature between 20-50°C or by soaking in a salt solution at a temperature between IO-6O°C, for example, between 22-55°C for about 0.5-48 h.

[0033] In one embodiment of the invention, the pulse seeds are pre-treated to modify the structural features, that is, to weaken the structural integrity of the pulses, by germinating the pulses for 12-96 hours at a temperature between 20-50°C, or for 24-96 hours at approximately room temperature, or by soaking the seeds in water or in a salt solution.

[0034] As will be apparent to those of skill in the art, germination at a higher temperature will shorten the required germination time.

[0035] Specifically, if the germination time is not sufficiently long, there will be insufficient disruption of structural integrity of pulse cotyledons and consequently insufficient separation of protein and starch inside the cotyledons. For example, as discussed in greater detail below, 24-hour germination at a lower temperature is not as good as 48-hour germination at a lower temperature (see Table 3).

[0036] Furthermore, if germination is too long, an undesirable amount of the protein and starch will be broken down by enzymes in the germinated seeds, which will make it more challenging to separate protein and starch during air classification. This can be seen in Table 3, which shows that 96-hour germination is not as good as 48- hour germination, as discussed below. As such, excess germination will decrease the production efficiency. Furthermore, there is also the risk that microorganisms will grow and cause spoilage in the seeds, leading to food safety concerns over the protein and starch ingredients.

[0037] In the examples below, the use of germination as a pre-treatment of pea and Faba bean prior to air classification is described. In comparison with the raw pea and Faba bean samples, the germination treatment for 24 to 96 hours increased the protein content of the pea protein concentrate from 61.6% to 67.3% and the protein content of the Faba bean protein concentrate from 74.2% to 77.7%%.

[0038] In other words, germination increases the protein retention from 60.9% to 68.8% for pea and from 59.7% to 77.2% for Faba bean.

[0039] This means that ( I ) the novel ingredients in our invention could be used directly in extrusion to produce fibrous meat analogs, plant protein powder as a supplement, and other food products; and (2) we can produce more protein concentrates from the same amount of pulses.

[0040] As discussed below, the pea seeds showed lower germination percentages but faster radicle growth (i.e., germination rate) than the Faba bean seeds; a longer germination time led to lower dry mass recovery rates in both pea and Faba bean; after 96-h germination, pea displayed a lower dry mass recovery rate than Faba bean (86.3% versus 92.0%), which could be linked to the faster radicle growth (i.e., germination rate) of the former (Table 3).

[0041] Table 1 shows the germination percentages, radicle length, dry mass recovery, and dehulling efficiency of round pea, Faba bean, and wrinkled pea seeds. As can be seen therein, germination for 48 hours increased the dehulling efficiency of pea, Faba bean, and wrinkled pea. This is desirable because we can obtain more dehulled seeds for subsequent pin milling and air classification. Specifically, pea increased from 84.2% to 86.0% while Faba bean increased from 79.0% to 79.8% and wrinkled pea increased from 80.2% to 83.7%.

[0042] However, Table 1 also shows that extending the germination time to 96 hours decreased the dehulling efficiency of pea, Faba bean, and wrinkled pea, thereby demonstrating that germination for too long is undesirable. During these germination periods, the germination percentages of round pea, Faba bean and wrinkled pea ranges from 89.2 to 91.2%, 93.6 to 95.4% and 91 .5% to 94.0%, respectively, indicating the high viability of the used seeds.

[0043] In summary, Table 1 shows that Faba bean seeds show a higher germination percentage as compared to round pea and Faba bean; that round pea and wrinkled pea seeds show faster radicle length growth as compared to Faba bean seeds; that soaking and germination for 24-48 h can improve the dehulling efficiency and that the optimal duration for dty mass recovery and dehulling yield is 48 h of germination.

[0044] Table 2 shows the fine and coarse stream yields of air classified round pea, Faba bean, and wrinkled pea flours. As can be seen, germination for 48 and 96 hours increased the yields of fine stream (protein-rich stream) from air classification of pea, Faba bean, and wrinkled pea. Specifically, pea increased from 25. 1% to 27.7% after 48 hours and from 25.1% to 29.5% after 96 hours; Faba bean increased from 27.4% to 37.3% after 48 hours and from 27.4% to 36.4% after 96 hours; and wrinkled pea increased from 29.5% to 47.3% after 48 hour and from 29.5% to 51.0% after 96 hours.

[0045] Table 3 shows the protein content of pin-milled flours and air-classified fine and coarse streams and percentages of protein retention in the fine streams of round pea. Faba bean, and wrinkled pea. As can be seen from Table 3, germination for 48 hours increased the protein contents of fine stream (protein-rich stream) from air classification of pea and Faba bean. Specifically, round pea increased from 61.6% to 65.9% and Faba bean increased from 74.2% to 76.6%.

[0046] Furthermore, germination for 48 hours increased the percentages of protein retention of fine stream (protein-rich stream) from air classification of pea, Faba bean, and wrinkled pea. Specifically, pea increased from 62.0% to 68.6%, Faba bean increased from 70.2% to 88.2% and wrinkled pea increased from 57.8% to 66.2%.

[0047] However, extending the germination time to 96 hours decreased the protein contents and the percentages of protein retention of fine stream (protein-rich stream) from air classification of pea and Faba bean.

[0048] In summary. Table 3 shows that protein contents of the fine stream of soaked, 24-h and 48-h germinated pea and Faba bean shows a gradual increase with increasing germination duration; that there is a decrease in the protein content for longer germination duration of 96 h; that prolonged germination duration (96 h) can cause a significant reduction in the protein contents of two varieties of pulse flours, mainly due to proteolytic enzyme activity and that there is a 6.6%, 18% and 8.4% increase in protein retention in pea, Faba bean and wrinkled pea, respectively, after 48 h of germination.

[0049] Table 4 shows the functional properties of air-classified fine streams of round pea, Faba bean, and wrinkled pea. Germination for 48 hours increased the emulsifying activity, emulsion stability, foaming capacity, and foam stability of fine stream (protein-rich stream) from air classification of pea, Faba bean, and wrinkled pea; however, extending the germination time to 96 hours decreased these properties. Specifically, the starch content in the coarse stream of soaked, 24-h, 48-h and 96-h germinated pea and Faba bean shows a gradual increase with increasing germination duration. Furthermore, air classification increases the starch content of pulse flours in the coarse stream and decreases the starch content in the fine stream.

[0050] As can be seen from Table 5, the pin-milled samples of pea and Faba bean show that protein content increases gradually with increasing germination time-period. The protein content increased slightly in pea, from 25.0% to 27.3% and from 26.8% to 30.1% in wrinkled pea, respectively. This increase is more significant in Faba bean, from 28.9% to 34.5%. Furthermore, the crude fat contents of the pin-milled samples slightly increase proportionally, and the ash content decreases slightly in the soaked and germinated samples.

[0051] As can be seen from Table 6, the protein content of coarse stream of round pea and wrinkled pea does not change significantly after soaking and germination; however, the protein content of coarse stream of Faba bean decreases after soaking and germination. This shows better separation of protein during air classification and higher purity of the resultant streams. Furthermore, the starch content of the coarse streams (coarse streams) of pea and Faba bean increases with soaking and increasing germination period indicating better separation of starch from protein-fiber matrix during air classification. Moreover, the separation of protein and starch improves with increasing germination period, hence this increase in starch content with increasing germination period. Germination tends to loosen the protein-fiber matrix (as also seen in the SEM images) that facilitates improved protein and starch separation during air-classification. The ash content of the course stream of round pea, Faba bean and wrinkled pea slightly changes during germination period. The observed variations in ash content could be attributed to changes in the biochemical composition during germination. Overall, germination did not appear to considerably alter the damaged starch content and amylose content of pea and Faba bean coarse stream, however it alters the damaged starch content and amylose content of wrinkled pea.

[0052] As can be seen from Table 7, the protein content of fine stream increases from 61.6% to 65.9% for round pea and from 74.2% to 76.6% for Faba bean, respectively. This increase in protein content can be explained by the fact that 48 h of germination may represent the optimal time for separation of starch and protein particles during air classification. However, longer germination duration for 96 h did not increase the protein content, which may indicate that the optimal separation of protein and starch particles may have already occurs during the first 48 h of germination. Ash content of the fine streams decreases with soaking and germination for these three pulse types. This could be attributed to the leaching of minerals during soaking and germination.

[0053] As can be seen from Table 8, the results show that soaking and germination can lead to a significant increase in water-holding capacity and slight increase in oil-absorption capacity (OAC). There is also a slight increase in emulsion activity and noticeable increase in emulsion stability is observed after 48 h. Foaming capacity did not change drastically; however, foaming stability has improved significantly.

[0054] As can be seen from Table 9, soaking tends to significantly increase surface hydrophobicity in round pea and wrinkled pea. The increase in hydrophobicity could be attributed to the breakdown of native protein into smaller peptides and thereby exposing more hydrophobic groups. Surface hydrophobicity of germinated pea and Faba bean decreased significantly compared to the soaked samples. This may be due to a greater number of hydrophobic sites being exposed initially through hydrolysis. Significant hydrophobic interactions then occur, which consequently mask the hydrophobic patches. As a result, this gives lower surface hydrophobicity values. Surface charge of the soaked and germinated samples change slightly but not significantly as compared to the control samples. In addition, zeta potential values were lower than -30 mV, which created strong repulsive forces to have a stable protein system.

[0055] As can be seen from Table I O, solubility values range from 81.8% to 84.4% for round pea and from 88.9% to 92.5% for Faba bean. The highest solubility is observed in 48-h germinated samples of round pea and Faba bean. Zeta potential values for all extracted samples show negative values suggesting favorable proteinwater interactions, and therefore high solubilities.

[0056] The majority of the thermal transition peaks shown in Table 1 1 corresponded to protein denaturation. These endothermic peaks could be attributed to the occurrence of protein denaturation during the process. In general, soaking and germination does not lead to considerable changes in the thermal properties of these samples.

[0057] As can be seen from Table 12, the majority of these thermal transition peaks correspond to starch gelatinization. The Tc of control pea and Faba bean samples (82.9-81 .9°C) is markedly higher than those of corresponding isolated pulse starches (72.1 -77.7°C). The extension of this endothermic peak of pulse flours to a higher temperature range could be attributed to the occurrence of protein denaturation during heating that partly overlapped with the starch gelatinization process. In general, soaking and germination does not lead to considerable changes in the thermal properties of the pulse samples.

[0058] As can be seen from Table 13, when analyzed under the heating temperature of 95°C, pea and Faba bean samples show significant viscosities. It is interesting to observe that soaking and germination up to 48 h show an increasing effect on the pasting viscosities of the pea and Faba bean flours. Soaking and germination loosens the protein and fiber matrix of pea and Faba bean cotyledons. Protein and fiber components are known to restrict the swelling of starch granules in pulse flours to reduce the viscosity. After soaking and germination, protein and fiber components could be more easily separated from starch granules during milling. Consequently, starch granules in soaked samples are able to swell to a greater extent during pasting to provide a higher viscosity when compared with the raw samples.

[0059] As can be seen from Table 14, different pre-treatments of seeds affect the dehulling yield of seeds. Soaking treatment with potassium carbonate at 55°C significantly improves the dehulling yield.

[0060] Table 15 shows that the total yield percentage for all the treatments is more than 95% revealing that both the streams are efficiently recovered.

[0061] Table 16 shows that the pin-milled samples of different treatments display a gradual increase in protein content as compared to control. Soaking with and without salts at various temperatures shows an increase in protein content in the fine stream compared to control samples.

[0062] Table 17 shows that different pre-treatments of seeds affect the dehulling yield of seeds. In no salt soaking, higher dehulling yield is observed at 35°C (73.0% and 85.5%) as compared to room temperature (76.8% and 70.2%) in both pea and Faba bean seeds, respectively. Furthermore, the dehulling yield considerably decreases with different salt treatments for both pea and Faba bean. This could be attributed to the fact that when seeds are treated with salts, seeds get more intact with the hulls and hence, they are harder to dehull. The effect is more pronounced in Faba bean seeds as compared to round pea seeds.

[0063] Table 18 shows that the total yield percentage for all the treatments is more than 97% revealing that both the streams are efficiently recovered. In pea, for no salt soaking, the fine stream yield decreases from 23.0% to 22.4% at room temperature and to 21.6% at 35°C. Further, among the salt treatments in pea, lowest fine stream yield is observed when seeds are soaked in potassium carbonate (19.3%). This could be connected to the higher purity in the fine stream samples. In corresponds to this, the coarse stream yield shows an increasing trend in pea. These results display that soaking at different temperatures and with different salts can enhance the separations efficiency and effect yield data of the fine and coarse stream during air classification.

[0064] Table 19 shows that the pin-milled pea samples of different treatments display a gradual increase in protein content as compared to control. The protein content in fine stream after air classification completely aligns with pin-milled samples results. Soaking with no salt and soaking with salts show an increase in protein content in comparison with control samples. However, it is observed that the protein content was increased from 60% in control to 70% after soaking with potassium carbonate. These effects could be attributed to the partial disruption of the cotyledon structure during salt soaking leading to release of starch granules from the protein-cell wall fiber matrix. However, protein retention did not show any notable increase in treated samples as compared to control. The fine stream of Faba bean shows a similar trend. The protein content increased from 74.5% (control) to 79.9% in potassium carbonate treated seeds. Interestingly, magnesium chloride displayed a decrease in protein content. In contrast to pea, protein retention in Faba bean increased from 52.0% (control) to 60.4% (potassium carbonate). Overall, a significant increase in protein content of fine stream is observed by potassium carbonate in both pea and Faba bean.

[0065] As can be seen in Table 20, both pea and Faba bean show changes in the functional properties upon different treatments as compared to control samples. The soaking treatments were able to affect the seed structural features, thereby impacting water retention ability. It is observed that both no salt soaking and salt soaking improved water-holding capacity (WHC) of pulse flours. However, potassium carbonate soaking in both pea and Faba bean shows no increase in WHC. Salt soaking and no salt soaking slightly increases oil-absorption capacity (OAC) of pulse flours. Salt soaking and no salt soaking slightly improves the emulsion activity (EA) by enhancing the surface activity of pulse proteins. The most stable emulsions are formed when peas are soaked in water at 35°C and Faba bean in calcium chloride. Overall, both EA and ES are slightly improved with these treatments in both pea and Faba bean. In both pea and Faba bean, it is observed that some treatments slightly improve the foaming capacity and foaming stability. In Faba bean, the fine stream yield increases after soaking in water and different salts. The lowest fine stream yield is observed in both potassium carbonate and potassium bicarbonate.

[0066] In one embodiment of the invention, the pulse seeds are pre-treated to weaken the structural integrity of the pulses by soaking in a salt solution at a temperature between l 0-60°C, for example, between 22-55°C. The pulse seeds may be soaked for about 0.5-48 h or for 0.5-48 h at a salt concentration of 0.5-10% (w / w).

[0067] In some embodiments of the invention, the salt is selected from the group consisting of potassium carbonate (K2CO3), potassium bicarbonate (KHCO3), sodium carbonate (Na^COj), sodium bicarbonate (NaHCOj), potassium chloride (K.C1), sodium chloride (NaCl), magnesium chloride (MgCh), calcium chloride (CaCh), calcium hydroxide [Ca(OH)i] and mixtures thereof.

[0068] In some embodiments of the invention, the salt is a salt that increases the pH of the solution to a relatively high level, such as for example, pH 1 1. As will be appreciated by one of skill in the art, the soaking at elevated pH (and elevated temperature) will disrupt and separate protein from starch during soaking.

[0069] In some embodiments of the invention, the pH increasing salt is selected from the group consisting of potassium carbonate (K2CO3), potassium bicarbonate (KHCO3), sodium carbonate (Na2COs) and sodium bicarbonate (NaHCOs).

[0070] As discussed above, the salt may be at a concentration of between 0.5-10%, for example, 1-3% or 1 .5- 3% or 1.5-2.5% or 2%.

[0071] As discussed above, the pulse seeds are soaked in the salt solution at a temperature between IO-6O°C. As will be appreciated by one of skill in the art, at lower soaking temperatures, the water and salts will be slower to penetrate the interior of the seeds, meaning that a longer time is needed to achieve a similar effect compared to soaking at a higher temperature.

[0072] Furthermore, soaking at a temperature above 60°C will result in some cooking and / or gelatinization of the starch. If gelatinization occurs, the starch will stick to protein in the seeds which will in turn make it more difficult to separate protein and starch during air classification.

[0073] As discussed above, the soaking time may be 0.5-48 h, for example, 4-48 h or 6-48 hours or for at least 4-6 hours.

[0074] Specifically, if soaked too long, the seed structure will become too soft, causing protein and starch to get mixed, which will in turn make separation of protein and starch during air classification more difficult. Furthermore, longer soaking increases the risk of microorganism growth which will cause spoilage in the seeds, leading to food safety concerns.

[0075] Specifically, as discussed below, pea, lentil, and Faba bean seeds were soaked in different salt solutions (each at 2% concentration), including potassium chloride (KC1), potassium bicarbonate (KHCO3), sodium carbonate (Na2CO3), potassium carbonate (K.2CO3), magnesium chloride (MgCl2), and calcium chloride (CaC12) over 22-55°C as pre-treatments of grains. After diying, the raw (untreated) and treated pea, lentil, and Faba bean seeds were air classified to produce protein concentrates and starch-rich flours. The separation efficiency and yields of protein concentrates and starch-rich flours were calculated, as discussed below.

[0076] In another embodiment of the invention, the pulse seeds are pre-treated to weaken the structural integrity of the pulses by soaking in water at a temperature between 10-60°C, for example, between 22-55°C. The pulse seeds may be soaked in water for about 0.5-48 h or for 0.5-48 h or for at least 4-5 hours.

[0077] Table 5 shows the dehulling efficiency of pulse seeds from the soaking treatment.

[0078] Table 6 shows yields of air-classified fine and coarse fractions from pin-milled pulse flours from the control and soaking treatment.

[0079] Table 7 shows protein content of air-classified fine (protein-rich) and coarse (starch-rich) fractions from pin-milled pulse flours (Control and soaking treatments)

[0080] As can be seen from Table 7, the following conditions increased the protein content of the fine stream (protein-rich stream) from air classification of pea:

[0081] Soaking in water at 35°C increased protein content of peas from 64.0% to 69.7%

[0082] Soaking in 2% potassium carbonate at room temperature increased protein content from 64.0% to 73.7% Soaking in 2% potassium carbonate at 35°C increased protein content from 64.0% to 72.8% Soaking in 2% potassium bicarbonate increased protein content from 64.0% to 70.2%

[0083] As will be appreciated by one of skill in the art, with a protein content higher than 70%, the fine stream (protein-rich stream) from air classification of pea will be useful for producing fibrous meat analogs, plant protein powder as a supplement, and other food products. Following the structural integrity weakening treatment, the treated pulse seeds are then reduced in size, for example, by milling or grinding, so as to be of a suitable particle size.

[0084] While pin milling is specifically used in the examples, it is worth noting that other milling and grinding methods, such as centrifugal milling, hammer milling and the like can be used within the invention. The ability to control the particle size ensures that the resulting flour is ideally suited for the subsequent air classification process, leading to superior outcomes in terms of the invention's objectives.

[0085] In some embodiments of the invention, the treated pulse seeds are subjected to pin milling at a speed between 1 ,500 to 30,000 rpm.

[0086] As will be appreciated by one of skill in the art, in general, the finer the flour, the better it is for air classification.

[0087] As will be appreciated by one of skill in the art, the air classification may be carried out under any suitable conditions, for example, at a speed of approximately 1,200 to 25,000 rpm and 20 to 30,000 m3 / h for the air-flow rate.

[0088] As discussed in the examples, we conducted air classification at speeds ranging from 5,000 to 15,000 rpm, with an air-flow rate of 40-1 ,200 m3 / h. However, it is important to note that for different types of air classifier machines, the optimal conditions will vary too.

[0089] While the air-classification process typically takes around 45 minutes to 1 hour to complete, we observed that the duration of the process does not appear to significantly influence the overall effectiveness of the air classification.

[0090] Pasting curves for coarse streams of raw, soaked and germinated samples are given in Figure 2. The curves include initial peak (initial peak in viscosity; between 400-600 sec), breakdown viscosity (difference in viscosity between peak and trough), final viscosity (viscosity at 1380 sec, cool temperature) and setback (difference between final viscosity and trough) viscosities, as well as time to reach the peak viscosity and pasting temperatures. When pulse flour is heated in excess water with stirring, the mixture starts developing viscosity at and beyond the gelatinization temperature of starch. After being gelatinized, starch granules swell and some starch molecules (e.g., amylose and small amylopectin molecules) leach out from the swollen granules, both contributing to a rise in viscosity. The changes in viscosity while heating is related to the swelling and rigidity of starch granules.

[0091] When analyzed under the heating temperature of 95°C, pea and Faba bean samples provided significant viscosities: raw pea and Faba bean samples showing pasting temperatures of 77.3°C and 76.3°C and peak viscosities of 1420.0 cP and 1217.5 cP, respectively. It is interesting to observe that soaking and germination up to 48 h showed an increasing effect on the pasting viscosities of the pea and Faba bean flours. Soaking was demonstrated to loosen the protein and fiber matrix of yellow pea and Faba bean cotyledons. Protein and fiber components are known to restrict the swelling of starch granules in pulse flours to reduce the viscosity. After soaking and germination, protein and fiber components could be more easily separated from starch granules during milling. Consequently, starch granules in soaked samples were able to swell to a greater extent during pasting to provide a higher viscosity when compared with the raw samples. After germination for 24 h and 48 h, the viscosities of both pea and Faba bean samples were further enhanced, which could be attributed to the fact that the abovementioned matrix structure was destroyed progressively. At 96 h, the viscosities of pea samples, however, were diminished, which could be due to their increased activity of a-amylase and possibly other enzymes, such as 0-amylase and amyloglucosidase. The endogenous amylase(s) in the flours could efficiently degrade starch molecules to promptly lower their pasting viscosities in the early stage of heating in RVA (i.e., before they were inactivated by heat at a temperature above 60°C). Coarse fraction from Faba bean gave the highest peak viscosity after 96-h germination (Figure 3). The highest peak viscosity after 96-h germination time period can be attributed to the composition of Faba bean flour and less alpha-amylase activity as compared to pea. Faba bean starch is composed of amylose and amylopectin. The ratio and structural integrity of these components can significantly influence the peak viscosity. Amylopectin, with its high molecular weight and branched structure, contributes to higher peak viscosity due to its ability to form a more extensive network that traps water.

[0092] The hardness of the gels produced after 95°C heating and subsequently cooling at room temperature for 2 h, exhibited an overall increasing trend with germination up to 48 h as shown in Fig 3. Specifically, the gelling ability of pea and Faba bean flours improved as the germination period extended to 48 h. However, with a longer germination period of 96 h, the gelling ability decreased. The gel hardness of the control (ungerminated) pea and Faba bean was measured at 234.8 g and 222.6 g, respectively. After 48 h of germination, the gel hardness significantly increased to 339.9 g for pea and 327.4 g for Faba bean. Conversely, after 96 h of germination, the gel hardness decreased to 264.4 g for pea and 258.8 g for Faba bean. This indicates that while a moderate germination period enhances the gel-forming properties of these pulse flours, excessively long germination periods may lead to a reduction in gel hardness.

[0093] Yet further, significantly, after the different pre-treatments of pulse seeds, we observed improvement in the flavor profiles of resultant protein-rich and starch-rich streams. Specifically, there is a significant reduction in beany flavor and bitter taste and there is significant reduction in aftertaste overall. While not wishing to be bound to a particular theory or hypothesis, the inventors believe that the germination and soaking in salts pre-treatments can remove different classes of volatile compounds responsible for the beany flavor, bitter taste, and strong aftertaste, such as aromatic hydrocarbons, aldehydes, alcohols, alkanes, ketones (furans) and esters while the germination and soaking in salts pre-treatments can inactive lipoxygenase and other enzymes that can partly cause the beany flavor, bitter taste, and strong aftertaste. For example, lipoxygenase catalyzes the oxidation of polyunsaturated fatty acids to produce different compounds responsible for the off-flavor and off-taste in pulse ingredients.

[0094] The invention will now be further explained and / or elucidated by way of examples; however, the invention is not necessarily limited to or by the examples.

[0095] EXAMPLE 1 - Germination

[0096] Materials Pulse seeds of pea (CDC Meadow variety) and Tabu bean ( DL Rico variety) were used in this study.

[0097] Soaking and germination of seeds

[0098] '[ lie pulse seeds (—600 g each batch ) were firstly w ashed w ith tap water to remove dust and oilier impurities. After washing, the seeds were soaked in distilled water ( I L) contain ing 0.07% (w v) of sodium hypochlorite (NaCIO ) for 30 m in. I his step served as a sterilization treatment prior to germination. The sterilized seeds were removed from the aqueous NaCIO solution and w ashed with distil led water for 4-5 times to remove NaCIO residue. The seeds were then soaked isi I L of distilled water 1 8 h (overnight) at room temperature ( --22 C ). I he next morning, the soaked seeds were removed from the water and labelled as soaked (0-h ) seeds in this study , and the other batches w ere used for subsequent germination experiment. The soaked seeds were placed on a flat tray to form an evenly th in layer. The seeds were then covered w ith a w et cheese cloth and placed in the dark for germination of 24. 48 and 96 h. During the germination, the seeds were evenly spray ed w ith 50 g of distilled witter once a day al around I O am to maintain adequate moisture for the germination to continue. At the end of the indicated germination periods, the germ inated seeds were collected and label led as 24-h. 48-h. and 96- h germinated samples. To determ ine the germination percentage, the number of germinated seeds w as div ided by the total number of seeds included in the batch. Radicle length of I 0 seeds randomly selected from one batch were measured using a Vernier caliper after each germ ination period. I he resulting seeds from the soaked (0-h ) batches and the germ inated batches were dried in a convection oven at 50 C for 24-36 h until the moisture contents fell into the range of 7-9%), The control samples are raw seeds without any treatment

[0099] Deh tilling of control, soaked, and germinated pulse seeds

[0100] Dehulling of the control (raw), soaked, and germ inated (24. 48 and 96 It ) seeds was carried out using a bench-scale dehuller ( Model I 06B, Canadian General Electric. Peterborough. ON. Canada). I he yield of dehulled seeds was calculated using the following equation :

[0101] Pin milling and air classification of deh tilled control, soaked, and germinated pulse flours

[0102] I'he dehulied control (raw ), soaked, and germinated (24. 48 and 96 11 ) pulse seeds were m illed into fine flours using a two-step process: ( 1 ) a "pre-break'' step: the seeds were milled into coarse grits using a laboratory disc m ill ( Model 33 1 (1. Perten Instruments AB, Sweden): and (2) a "pin-mill” step: the coarse grits w ere further milled using a pin m ill (Model 100 LJPZ, 1 losokawa Alpine, Augsburg. Germany ) at a rotor speed of 22.000 rpm to prepare fine Hours. A portion of the derived fine pulse Hour ( -50 g) was collected and labelled as "pin-milled fiour” for chem ical composition analy sis, and the remaining pin-mil led Hour w as fract ionated using an air classifier (Model 100 MZR, I losokawa Alpine. Augsburg. Germany).

[0103] Approximately 500 g of the pin-m illed flour was air classi fied every hour at an air classifier speed of 1 2.000 rpm and a flow rate of 46 nf h. A fter the air classification, two fractions w ere obtained, i.e.. fine (proteinrich ) and coarse (starch-rich ) fractions. The yields of the two fractions were calculated as follows:

[0104] %¥ield of fine fraction from air classification W eight of collected coarse- fraction

[0105] 5'iYield of coarse fraction from air classification — - x 100%

[0106] Initial weight of used pin milled flour

[0107] Chemical compositions of pin-milled and air-classified fine and coarse fractions of pulse flours

[0108] Moisture contents of the pin-m illed and air-classi fled fine and coarse fractions of pulse flours were measured using AOAC Method 925. 10. Nitrogen contents were quantitated in accordance w ith the Dumas combustion method using a Nitrogen Protein Analyzer (CN628. LEC'O Corp., St. Joseph. M l. U.S.A. ). which w as converted to protein content (%N • 6.25 ) follow ing AACC Method 46 .30.01 .

[0109] EXAMPLE 2 Salt Soaking

[0110] Materials

[0111] Pulse seeds of pea (CDC Meadow variety ) w ere used in this study.

[0112] Treatments and methods

[0113] All the samples (both control and treated ) were prepared in duplicates.

[0114] Soaking treatments a) Treatment 1 (No salt soaking)

[0115] I he pulse seeds ( -650 g each batch ) w ere w ashed with lap waler to remove dust and other impurities. After washing, the seeds were soaked in I 1. of disti lled water for 16 h (overnight) at three different temperatures ( RT. 35"C. 55"C) in a water bath Two batches for each temperature were prepared. T he following morn ing, the soaked seeds w ere removed from the w ater and rinsed with the waler at the same respective temperature. The temperature of the water w hich was used to rinse the seeds was maintained with the help of a water bath. Further, the seeds were dried al 5()°C to reach 7-9% moisture to achieve desirable results. h) Treatment 2 (Potassium carbonate soaking)

[0116] The pulse seeds ( - 650 g each batch ) w ere w ashed with tap water to remove dust and other impurities. After washing, the seeds w ere treated with 0.5. 1. 1.5. and 2% (w w) of potassium carbonate ( KzCOO solution. The pH of all the solutions was measured, and a concentration of 2% (w7w) K,CO-, was selected. Then, the treated seeds were soaked in 1 L of distilled w ater for 16 h (overnight ) at three different temperatures ( RT. 35*'C. 55°C) in a water bath. T w o batches for each temperature w ere prepared. The following morn ing, the soaked seeds were remov ed from the water and rinsed w ith the water at the same respective temperature. The temperature of the waler w hich was used to rinse the seeds was maintained with the help of a water bath. Further, the seeds were dried al 50°C to reach 7-9% moisture to achieve desirable results. c) Treatment 3 (Potassium bicarbonate soaking)

[0117] The pulse seeds (-650 g each batch) were washed with tap water to remove dust and other impurities. After w ashing, the seeds w ere treated w ith 0.5. I . 1.5. and 2% ( w v. i of potassium bicarbonate ( K1 ICO3 ) solution. T he pH of all the solutions w as measured, and a concentration of J .5% (w w ) KHCO3 was selected. Then, the treated seeds were soaked in I 1. of distilled water for 16 h (overnight) at three different temperatures (RT. 35"C. 55°C ) in a w ater bath. T w o batches for each temperature w ere prepared. T he following morning, the soaked seeds w ere removed from the waler and rinsed with the witter at the same respective temperature. The temperature of the water which was used to rinse the seeds was maintained with the help of a water bath. f urther. the seeds were dried at 5O‘’C to reach 7-9‘fo moisture to achieve desirable results.

[0118] Deh tilling of control and treated samples

[0119] Dehulling of the control ( raw) and treated seeds was carried out using a bench-scale dehuller ( Model 106B. Canadian General Electric, Peterborough. ON. Canada). The yield of dehulled seeds was calculated using the following equation:

[0120] Pin milling and air classification of dehulled control and treated pulse flours

[0121] The dehulled control (raw ) and treated pulse seeds were milled into fine Hours using a tw o-step process: ( I ) a "pre-break" step: the seeds w ere mil led into coarse grits using a laboratory disc mil l ( Model 33 10. I’erlen Instruments AB. Sweden ): and (2 ) a "pin-m ill" step: the coarse grits were further mi l led using a pin mill ( Model 100 UPZ. Hosokawa Alpine. Augsburg. Germany) at a rotor speed of 22.000 rpm to prepare line flours. A portion of the derived fine pulse flour ( • 30 g) w as collected and labelled as "pin-m illed Hour" lor chemical composition analysis, and the remaining pin-milled Hour w as fractionated using an air classifier ( Model 100 MZR, Hosokawa Alpine. Augsburg, German} ).

[0122] Approximately 500 g of the pin-milled Hour w as air classified every hour at an air classi fier speed of 14.000 rpm and a flow rale of 46 m”h. A fter the air classification, tw o fractions were obtained, / .e , fine ( proteinrich ) and coarse (starch-rich) fractions. The v ields of the two fractions were calculated as follow s:

[0123] % Yield of fine fraction from air classification g p

[0124] % Yield of coarse fraction from air classification

[0125] While the preferred embodiments of the invention have been described above, it w ill be recognized and understood that v arious modifications may be made therein, and the appended claims are intended to cov er all such modifications wh ich max tall within the spun and scope of the invention.

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[0137] REFERENCES

[0138] ■ Boye, J., Zare, F., & Pletch, A. (2010). Pulse proteins: Processing, characterization, functional properties and applications in food and feed. Food Research International, 43(2), 414-431.

[0139] ■ Coskuner, Y., & Karababa, K. (2003). Effect of location and soaking treatments on the cooking quality of some chickpea breeding lines. International Journal of Food Science and Technology, 38(7), 751- 757.

[0140] ■ Ren, Y., Yuan, T. Z., Chigwedere, C. M., & Ai, Y. (2021). A current review of structure, functional properties, and industrial applications of pulse starches for value-added utilization. Comprehensive Reviews in Food Science and Food Safety, 20(3), 3061-3092.

[0141] ■ Setia, R., Dai, Z., Nickerson, M. T., Sopiwnyk, E., Malcolmson, L., & Ai, Y. (2019). Impacts of shortterm germination on the chemical compositions, technological characteristics and nutritional quality of yellow pea and Faba bean flours. Food Research International, 122, 263-272.

[0142] ■ Sosulski, F., & Sosulski, K. ( 1986). Composition and functionality of protein, starch, and fiber from wet and dry processing of grain legumes. ACS Publications. 176-189.

[0143] • Vijayakumari, K., Siddhuraju, P., Pugalenthi, M„ & Janardhanan, K. ( 1998). Effect of soaking and heat processing on the levels of antinutrients and digestible proteins in seeds of Vigna aconitifolia and Vigna sinensis. Food Chemistry, 63(2), 259-264.

[0144] ■ Xu, M„ Jin, Z., Simsek, S., Hall, C„ Rao, J., & Chen, B. (2019). Effect of germination on the chemical composition, thermal, pasting, and moisture sorption properties of flours from chickpea, lentil, and yellow pea. Food Chemistry, 295, 579-587.

Claims

CLAIMS1. A method for separating starch and protein fractions from pulse seeds comprising: subjecting a quantity of pulse seeds to a structural integrity weakening treatment for a period of time; reducing the size of the treated pulse seeds; and separating the starch fraction and the protein fraction of the size-reduced, treated pulse seeds by air classification.

2. The method according to claim 1 wherein the pulse seeds are subjected to the structural integrity weakening treatment until the seeds are of a suitable softness for further processing.

3. The method according to claim 1 wherein the structural integrity weakening treatment is germination.

4. The method according to claim 3 wherein the pulse seeds are germinated for 12-96 hours at a temperature between 20-50°C.

5. The method according to claim 3 wherein the pulse seeds are germinated for 24-96 hours.

6. The method according to claim 1 wherein the structural integrity weakening treatment comprises soaking the pulse seeds in a salt solution at a temperature between 10-60°C for 0.5-48 h.

7. The method according to claim 6 wherein the temperature is between 22-55°C.

8. The method according to claim 6 wherein the salt is a pH increasing salt that increases the pH of a solution to a relatively high level.

9. The method according to claim 8 wherein the pH increasing salt increases the pH of the solution to about 1 1.

10. The method according to claim 9 wherein the pH increasing salt is selected from the group consisting of potassium carbonate (K2CO3), potassium bicarbonate (KHCO3), sodium carbonate (NajCCh) and sodium bicarbonate (NaHCCh).1 1. The method according to claim 6 wherein the pH increasing salt is selected from the group consisting of potassium carbonate (K2CO3), potassium bicarbonate (KHCO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCCh), potassium chloride (KC1), sodium chloride (NaCI), magnesium chloride (MgCh), calcium chloride (CaCh), calcium hydroxide [CafOHh] and mixtures thereof.

12. The method according to claim 6 wherein the salt is added at a concentration of 0.5- 10%.

13. The method according to claim 1 wherein the pulse is selected from the group consisting of peas, lentils, Faba beans, chickpeas, dry beans, lupins, and cowpeas.

14. The method according to claim 1 wherein the structural integrity weakening treatment comprises soaking the pulse seeds in water at a temperature between 10-60°C for 0.5-48 h.

15. The method according to claim 1 wherein the pulse seeds are reduced in size bymilling or grinding,16. The method according to claim 15 wherein the pulse seeds are milled by pin milling.

17. The method according to claim 16 wherein the treated pulse seeds are subjected to pin milling at a speed between 1,500 to 30,000 rpm.

18. The method according to claim 1 wherein the air classification is carried out at a speed of approximately 1,200 to 25,000 rpm and an air flow rate between 20 to 30,000 m3 / h.

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