Preparation method of chickpea protein with high solubility and gelation capability

Abstract

The invention discloses a preparation method of chickpea protein with high solubility and gelling capacity, and belongs to the technical field of food processing. Pediococcus pentosaceus is inoculated into chickpea powder to ferment the chickpea powder, and the fermented chickpea powder is degreased, subjected to alkali dissolution and acid precipitation and redissolved to obtain the chickpea protein. The pediococcus pentosaceus fermentation treatment is utilized, so that the content of phytic acid can be effectively reduced, the change of a protein structure is caused, the solubility of chickpea protein is improved, the nutritional value is increased, the processing performance is improved, and the pediococcus pentosaceus fermentation method has certain guiding significance on research and development of chickpea products.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, in particular to a method for preparing chickpea protein with high solubility and gelling ability. Background Art

[0002] Chickpea is a legume with rich nutritional value and widespread distribution. It is a high-quality plant protein resource. At present, chickpea is mainly eaten in two ways: dried and fresh. In terms of application, chickpea protein is mainly used as a protein nutrition enhancement ingredient and as an additive in food processing, such as pasta, meat products, dairy products, beverages and other foods.

[0003] The most important properties that affect the application of chickpea protein in food include solubility and gelation. Among them, protein solubility is the decisive factor in determining its bioavailability, and it is also a prerequisite for various protein properties, such as gelation, emulsification, foaming, etc. The gelation properties are related to the formation and maintenance of the protein matrix. Under certain conditions, improving the functional properties of protein gel such as hardness, elasticity, cohesion and chewiness can effectively improve the quality of food, and the chickpea protein extraction method has a significant effect on its gelation. At present, commercially prepared chickpea protein usually undergoes extreme treatments such as acid precipitation and high-temperature drying, which leads to its structural destruction, denaturation and aggregation, and poor solubility, which seriously hinders the application of chickpea protein in the food industry.

[0004] At the same time, the low digestibility of bean protein restricts its bioaccessibility and utilization in the human body, which is related to the rigid and dense structure of its protein and the anti-nutritional factors contained in beans. Phytic acid is the main anti-nutritional factor in chickpea protein and is a secondary compound. Phytic acid can bind to nutrients such as protein, starch and essential minerals, reducing their bioaccessibility.

[0005] Therefore, the preparation of emerging chickpea protein with high added value and high technological content is an urgent problem to be solved in the industry. A method for preparing chickpea protein with improved solubility, good gel properties and low anti-nutritional factors is needed. Summary of the invention

[0006] [Technical issues]

[0007] The technical problem to be solved by the present invention is that the anti-nutritional factors in the existing chickpea protein affect the digestion and absorption of the chickpea protein, and the characteristics such as poor solubility and unstable gel performance limit the application of the chickpea protein.

[0008] [Technical solution]

[0009] In order to solve the shortcomings of the prior art, the present invention provides a method for preparing chickpea protein, wherein the chickpea protein has the characteristics of low anti-nutritional factor, high solubility and stable gel, wherein the low anti-nutritional factor means that the phytic acid content in the chickpea protein is 0.65g / 100g to 0.91g / 100g, the high solubility means that the solubility of the chickpea protein reaches 91.40% to 93.58%; and the stable gel means that a stable gel can be formed at a protein concentration of 10%.

[0010] The method mainly comprises the following steps:

[0011] (1) inoculating Pediococcus pentosaceus DSM 20336 into sterilized chickpea flour for solid-state fermentation;

[0012] (2) freeze-drying, defatting, alkali dissolution and acid precipitation to obtain the solid-state fermented chickpea protein.

[0013] In some embodiments of the present invention, in step (1), before solid-state fermentation, the moisture content of the chickpea fraction is adjusted to 50%-70%, preferably 65%, with water.

[0014] In some embodiments of the present invention, in step (1), the inoculation method and inoculation amount are as follows: inoculate the bacteria into 10 mL of MRS liquid culture medium and culture at 37° C. for 12 h, centrifuge 1 mL of the bacterial solution at 10,000 rpm for 5 min, discard the supernatant, wash once with 1 mL of saline, and centrifuge again under the same conditions, resuspend in saline after centrifugation, and inoculate into sterilized chickpea flour at an inoculation amount of 5% (volume of seed solution / volume of water added to chickpea flour when adjusting the moisture content of chickpea flour).

[0015] In some embodiments of the present invention, in step (1), the temperature of the solid-state fermentation is 25°C-37°C, preferably 37°C, and the fermentation time is 24h-48h.

[0016] In some embodiments of the present invention, in step (2), the freeze-drying conditions are drying at a temperature of -90 to -50°C and a vacuum degree of 1-5 Pa for 12 h to 24 h.

[0017] In some embodiments of the present invention, in step (2), the defatting is performed by mixing the fermented chickpea flour with petroleum ether in a ratio of 1:2 (m / v, g / mL) and stirring for 1 h-2 h, standing for stratification, and collecting the defatted chickpea flour.

[0018] In some embodiments of the present invention, in step (2), the alkali dissolution and acid precipitation comprises mixing defatted chickpea flour with water, adjusting the pH to 7.5-8.5, extracting with stirring at room temperature for 1h-2h, and obtaining a supernatant by centrifugation; adjusting the pH of the supernatant to 4.5-4.6, collecting a precipitate by centrifugation; dissolving the precipitate with water and adjusting the pH to 7.0, and obtaining chickpea protein by dialysis and freeze-drying.

[0019] The present invention provides chickpea protein prepared by the above-mentioned preparation method, which has the characteristics of low anti-nutritional factor, high solubility and stable gel, wherein the low anti-nutritional factor refers to that the phytic acid content in the chickpea protein is 0.65g / 100g to 0.91g / 100g, the high solubility refers to that the solubility of the chickpea protein reaches 91.40% to 93.58%; the stable gel refers to that a stable gel can be formed at a protein concentration of 10%. The chickpea protein prepared by the present invention can be used as a protein nutrition enhancing ingredient and as an additive raw material in food processing, such as in flour products, meat products, dairy products, beverages and other foods.

[0020] [Beneficial Effects]

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a method for removing anti-nutritional factors from chickpea protein and improving solubility and gel properties by solid-state fermentation. The method can not only improve the nutritional value, solubility and gel properties of chickpeas, but also play an important role in improving processing performance and edible quality. The chickpea protein prepared by solid-state fermentation can be used as an additive in food processing according to specific product requirements.

[0023] The invention selects microorganisms that can be screened from chickpeas as fermentation agents, performs controlled fermentation on chickpeas, and has better tolerance to bean fermentation substrates. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Growth of strains on medium supplemented with 2.5% raffinose;

[0025] Figure 2 Chickpea protein gel formation; A: 8% protein concentration; B: 10% protein concentration; C: 12% protein concentration; D: 14% protein concentration; UCF (Unfermented chickpea flour): control sample of comparative example 1 without fermentation treatment; P. pentosaceus: sample prepared in example 1;

[0026] Figure 3From left to right, they are the temperature scanning process and strain scanning of protein thermal gelation; from top to bottom, they are 8%, 10%, 12%, and 14% protein samples;

[0027] Figure 4 From left to right, the functional relationship between protein concentration and G', and the strain crossover points of protein gels at different concentrations. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. It should be noted that, under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0029] The detection indexes and test methods used in the present invention are as follows:

[0030] (1) Acid production capacity

[0031] A single colony was picked up and added to 10 mL of MRS liquid medium and cultured at 37°C for 24 h. The culture was centrifuged at 10,000 rpm for 10 min at 4°C. The pH of the fermentation supernatant was measured, and the acidity was titrated to pH 8.2 using 0.1 M NaOH. The result was expressed as the volume of NaOH consumed.

[0032] (2) Acid and salt resistance of strains

[0033] 500 μL of the bacterial solution inoculated in MRS medium was inoculated into 10 mL of MRS broth at pH 2.5 and pH 3.0 and cultured at 37°C for 3 h. The culture solution was taken at 0 h and 3 h, and the viable bacterial count was determined using the plate count technique. The bacterial survival rate was calculated as follows (1):

[0034]

[0035] The strain was inoculated into MRS broth and adjusted to 10 8 Then fresh MRS liquid medium containing 0.3% and 0.5% (w / v) bile salts was added and the survival rate was calculated using the same method.

[0036] (3) α-galactoside digestibility

[0037] The strain was inoculated into a medium containing 2.5% (w / v) raffinose and cultured for 72 h. The OD was measured at 60-min intervals. 600 , observe the growth curve.

[0038] (4) Determination of phytic acid content in fermented chickpea flour

[0039] The phytic acid content was determined using the Megazyme kit K-PHYT 05 / 07 kit according to the instructions.

[0040] (5) Protein solubility

[0041] ① Sample to be tested:

[0042] A 1% (w / v) chickpea protein solution was prepared, the pH was adjusted to 3.0-7.0 using 0.1 M NaOH and 0.1 M HCl, stirred at room temperature for 1 h, the solution was centrifuged at 8000 rpm for 10 min, and the supernatant was collected.

[0043] ②Control solution:

[0044] Chickpea protein was dissolved in 0.1 M NaOH to prepare a 1% (w / v) protein solution and centrifuged to collect the supernatant.

[0045] ③ Use the BCA method to determine the protein concentration in the supernatant obtained in ① and ②. Protein solubility is the ratio of the protein concentration in the supernatant of the sample to be tested to the protein concentration in the supernatant of the control solution.

[0046] (6) Protein gelation

[0047] The chickpea protein was dispersed in deionized water to prepare 8%, 10%, 12%, and 14% (w / v) chickpea protein solutions, respectively, stirred for 3 hours, fully hydrated overnight, and the pH of the solution was adjusted to 7.0. The solution was then heated in a 90°C water bath for 30 minutes, placed in a 4°C refrigerator overnight, and cooled to gel.

[0048] (7) Protein gel rheology determination

[0049] The thermally induced gelation process and rheological properties of the gels of different samples were studied using an MCR 302 rheometer (Anton Paar, Graz, Austria) and titanium sandblasted concentric cylinder geometry (CC17). Chickpea protein was prepared at four protein concentrations (8%, 10%, 12% and 14% w / w) and stirred overnight to ensure adequate hydration. The pH of the sample dispersions was adjusted to 7.0 by adding 2 mol / L NaOH or HCl.

[0050] Small amplitude oscillatory shear (SAOS) tests were performed at a fixed amplitude (1%) and frequency (1 Hz), with the sample first allowed to submerge at 20°C for 5 minutes and then the temperature was increased from 20°C to 95°C at 3°C / min. Afterwards, the temperature was kept at 95°C for 10 minutes and then cooled back to 20°C at the same rate (3°C / min). Finally, the resulting gel was kept at 20°C in the rheometer for 5 minutes before further testing. Throughout the experiment, G′, G″ and tan δ were recorded as a function of time.

[0051] Large amplitude oscillatory shear (LAOS) was conducted by subjecting the above thermosetting gel to a strain amplitude sweep from 0.1% to 1000% (logarithmic ramp mode) at a constant frequency and temperature of 1 Hz and 20°C. G′, G″ are functions of the inter-ring strain (γ0), and the shear stress is a function of the intra-ring strain and the strain rate.

[0052] (8) Analysis methods

[0053] Unless otherwise specified, all sample determinations in the examples were independently repeated at least three times. All experiments used freshly prepared samples, and each test was repeated three times. SPSS22.0 software was used for data analysis of variance (ANOVA), and Duncan's test was used for significance analysis among multiple groups of samples (p<0.05).

[0054] Example 1

[0055] This example uses P. pentosaceus DSM 20336, which can be isolated from chickpeas or from https: / / www.dsmz.de / collection / catalogue / details / culture / DSM-20336 Available for purchase.

[0056] (1) The bacterial strain was inoculated into 10 mL of MRS liquid medium and cultured at 37°C for 12 h. 1 mL of the bacterial culture was centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, the culture was washed once with 1 mL of saline, and centrifuged again under the same conditions. After centrifugation, the culture was resuspended in saline.

[0057] The moisture content of chickpea flour was adjusted to 65% with water and sterilized. Pediococcus pentosaceus (P. pentosaceus) DSM20336 was inoculated into the sterilized chickpea flour at an inoculum amount of 5% and fermented at 37°C for 48 hours.

[0058] (2) The fermented chickpea flour was freeze-dried at a temperature of -90 to -50 ° C and a vacuum degree of 1-5 Pa for 12 h to 24 h, and then the freeze-dried chickpea flour was mixed with petroleum ether at a ratio of 1:2 (m / v) and stirred for 1 h, and allowed to stand for stratification to obtain defatted chickpea flour. The defatted chickpea flour was mixed with deionized water, adjusted to pH 8.5 with 0.5 M NaOH, stirred and extracted for 2 h, centrifuged to obtain a supernatant, and then the pH of the supernatant was adjusted to 4.5 with 0.5 M HCl to precipitate protein. After the protein was redissolved, it was dialyzed at 4 ° C for 48 h and freeze-dried to obtain solid-state fermented chickpea protein.

[0059] Comparative Example 1

[0060] The preparation method of the chickpea protein in this comparative example is similar to that in Example 1, except that the fermentation time is 0 h.

[0061] Performance Testing

[0062] As a type of lactic acid bacteria, the acid production ability of P. pentosaceus is an important indicator for screening as a fermentation agent. The acid production ability, acid and salt resistance, and α-galactoside digestion ability of P. pentosaceus DSM 20336 were compared. The experimental results are shown in Table 1. Figure 1 .

[0063] Table 1 pH and titratable acidity of fermentation supernatant of strains

[0064]

[0065] Note: The experimental results are expressed as mean ± standard deviation (SD). Different letters in the same column indicate significant differences between the experimental results (P < 0.05), the same below.

[0066] In terms of acid resistance and bile salt resistance tests, at pH 3.0, the survival rate of DSM 20336 reached 72.81±4.46%; when it was further reduced to pH 2.5, the survival rate dropped significantly to 28.07±1.79%. In the culture medium with 0.3% bile salts added, the survival rate of DSM 20336 was 71.11±1.01%; when the bile salt concentration was further increased to 0.5%, the survival rate exceeded 50%.

[0067] Raffinose oligosaccharides have anti-nutritional properties. Since the human body lacks enzymes that can hydrolyze α-galactosidic bonds, they cannot be absorbed and utilized. They pass through the digestive system without being digested, resulting in gastrointestinal flatulence and bloating. In order to investigate the digestion ability of the strain for raffinose, the strain was grown on a medium containing 2.5% raffinose. The OD value changes during the process are shown in Figure 2. Figure 1 As shown, DSM 20336 has a fast growth rate.

[0068] The phytic acid content of the fermented chickpea flour of Example 1 and Comparative Example 1 was measured, and the protein solubility and gel property of the finally prepared chickpea protein were measured. The experimental results are shown in Table 2. Figure 2-Figure 4 .

[0069] Anti-nutritional factors are a series of secondary metabolites produced by plants for self-protection. Anti-nutritional factors in beans include trypsin inhibitors and phytic acid. Among them, phytic acid can combine with essential minerals, proteins, starch and other nutrients to reduce their bioaccessibility. Although beans have a high protein content and a balanced amino acid composition, the presence of anti-nutritional factors restricts the digestion and absorption of bean protein in the human body. The results of phytic acid content determination are shown in Table 2. Compared with unfermented chickpea flour, the phytic acid content in fermented chickpea flour has decreased, indicating that fermentation is an effective means to reduce the phytic acid content in beans. According to investigations, the solid-state fermentation process may increase the activity of inherent phytase, which can destroy insoluble organic complexes with minerals. In addition, the optimal pH value of most phytases is between 4.0-4.6. The decrease in pH after fermentation may activate phytase to reduce the content of phytic acid.

[0070] Table 2 Phytic acid content in chickpea flour fermented at different times

[0071] Comparative Example 1 Example 1 Fermentation time 0h 48h Phytic acid (g / 100g) <![CDATA[1.27±0.04 a ]]> <![CDATA[0.89±0.02 b ]]>

[0072] Protein solubility reflects the interaction between protein and water, and is closely related to the foaming, emulsifying, and gelling properties of the protein. P. pentosaceus is a chickpea protein sample that was solid-state fermented for 48 hours in Example 1, and UCF is a control sample of Example 1 that has not been fermented. The solubility of P. pentosaceus reached 93.58±1.19% at pH 7; compared with the solubility (46.13±0.44%) of the control group (Comparative Example 1) at the corresponding pH, the solubility was significantly improved. This may be due to the changes in protein structure caused by the fermentation process of Pediococcus pentosaceus, which proves that fermentation is an effective means to improve the properties of chickpea protein.

[0073] To investigate the gelling properties of proteins extracted from different samples, protein solutions with concentrations of 8%, 10%, 12%, and 14% were prepared from UCF and P. pentosaceus, respectively. Figure 3 As shown, according to the gel formation of proteins with different gradient concentrations, it can be observed that unfermented chickpea protein can form a relatively stable gel at a protein concentration of 14%, and P. pentosaceus can form a stable gel at a protein concentration of 8%.

[0074] The rheological study of thermal gelation was conducted on 8%, 10%, 12%, and 14% protein solutions. The temperature scanning process can reveal the specific changes in protein gel formation during the heating process, such as Figure 3 As shown in Figure 2, the thermal gelation curves of 8%, 10%, 12%, and 14% chickpea protein solutions were observed. As the temperature rose, due to the exposure of more sulfhydryl groups and hydrophobic groups in the protein, G' gradually became greater than G", resulting in enhanced protein interaction. At each concentration, P. pentosaceus showed higher G' during the heating process (20℃-95℃); in the cooling stage (95℃-20℃), the formation of intermolecular forces (such as hydrogen bonds) in the protein can promote the increase of sample G', which indicates the formation of a gel network. At the end of the cooling, the G' of P. pentosaceus was higher than UCF, indicating that the chickpea protein gel extracted after fermentation had stronger rigidity. Further analysis of the thermal gelation curves defined the temperature corresponding to G'=G" as the critical temperature for gel formation. It can be seen from the data in Table 3 that with the increase of protein concentration, T gel And gradually decreased. At 14% concentration, T gel The decrease from 87.83℃ of UCF to 62.23℃ of P.pentosaceus indicates that the fermented chickpea protein has an earlier gel point; comparing the average slope of G' changing with time under the temperature equilibrium state of 95℃, the average slope of P.pentosaceus is greater than that of UCF, indicating that the hardening process in the gel formation process is promoted. The functional relationship between G' and protein concentration in the linear viscoelastic region shows that the slope change of P.pentosaceu is similar to that of UCF, but the overall G' is higher than that of UCF, and at a concentration of 14%, the difference in G' between the two gradually decreases.

[0075] Table 3 Critical temperature of gel formation during temperature scanning and average slope of G' changing with time at equilibrium temperature

[0076]

[0077] like Figure 4 As shown in the figure, the results of amplitude scanning show that the gel of P. pentosaceus at different concentrations has a wider linear viscoelastic region compared with UCF. At low strain values, the modulus value remains basically unchanged, indicating that both are in a gel state dominated by elasticity. When the strain amplitude increases, the modulus curve will drop rapidly into the nonlinear viscoelastic region, and G" and G' will have a cross strain point at this time. It can be seen from the figure that the strain cross point of P. pentosaceus is higher than UCF, showing a strong ability to resist deformation, and has no strong dependence on protein concentration. It proves that the gel properties of chickpea protein extracted after fermentation are improved, which may be due to the change in protein structure causing the difference in gel strength.

[0078] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing chickpea protein, characterized in that, The main steps include: (1) inoculating Pediococcus pentosaceus into sterilized chickpea flour for solid-state fermentation; (2) The chickpea flour fermented in step (1) is freeze-dried, defatted, alkali-dissolved and acid-precipitated to obtain the solid-state fermented chickpea protein.

2. The method for preparing chickpea protein according to claim 1, characterized in that The Pediococcus pentosaceus is Pediococcus pentosaceus DSM 20336.

3. The method for preparing chickpea protein according to claim 1 or 2, characterized in that In step (1), before inoculation, the moisture content of the chickpea fraction is adjusted to 50%-70% with water.

4. The method for preparing chickpea protein according to claim 3, characterized in that In step (1), before inoculation, the moisture content of the chickpea fraction is adjusted to 65% with water.

5. The method for preparing chickpea protein according to any one of claims 1, 2 or 3, characterized in that: In step (1), the temperature of solid-state fermentation is 25°C-37°C, and the fermentation time is 24h-48h.

6. The method for preparing chickpea protein according to claim 5, characterized in that In step (1), the solid-state fermentation temperature is 37° C. and the fermentation time is 48 h.

7. The method for preparing chickpea protein according to claim 1, characterized in that: In step (1), Pediococcus pentosaceus is inoculated in the form of seed liquid.

8. The method for preparing chickpea protein according to claim 1, characterized in that: In step (2), the defatting is performed by mixing the fermented chickpea flour with petroleum ether, stirring the mixture, standing the mixture for stratification, and collecting the defatted chickpea flour.

9. The method for preparing chickpea protein according to claim 1, characterized in that: In step (2), the alkali dissolution and acid precipitation comprises mixing defatted chickpea flour with water, adjusting the pH to 7.5-8.5, extracting with stirring at room temperature for 1h-2h, and obtaining a supernatant by centrifugation; adjusting the pH of the supernatant to 4.5-4.6, collecting a precipitate by centrifugation; dissolving the precipitate with water and adjusting the pH to 7.0, and obtaining chickpea protein by dialysis and freeze-drying.

10. Chickpea protein prepared by the method according to any one of claims 1 to 9.

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