A method for preparing a potato instant polypeptide powder

Abstract

The application discloses a preparation method of instant potato polypeptide powder and belongs to the field of food protein processing. The method comprises the following steps: dispersing potato crude protein powder in deionized water, stirring, adjusting pH to 9-10, heating and dissolving, collecting supernatant, adjusting pH to 4.5-5.5, centrifuging, washing, adjusting pH to 6.5-7.5, and drying to obtain purified potato protein powder; adding the potato protein powder into deionized water, adding alkaline protease into the dispersion liquid to hydrolyze; heating the reaction system to 80-90 DEG C to inactivate the alkaline protease, and removing impurities by centrifugation; collecting polypeptide liquid with a molecular weight less than 10 KD; precipitating the polypeptide liquid by adopting a pH gradient precipitation method; resuspending and washing the precipitate with deionized water; homogenizing, heating to 45-55 DEG C, and spray drying. The instant potato polypeptide powder is obtained by adopting the alkali dissolution and acid precipitation method combined with the enzymolysis method, and the instant potato polypeptide powder with a purity of 90%-95% is obtained.

Description

Technical Field

[0001] This invention belongs to the field of food protein processing, and specifically relates to a method for preparing instant potato polypeptide powder. Background Technology

[0002] Potato protein is a high-quality plant protein, mainly composed of patatin (40-45 kDa, accounting for 60-80%) and protease inhibitors (5-25 kDa, accounting for 20-40%). Potato protein provides essential amino acids, with a PDCAAS (Protein Digestibility Corrected Amino Acid Score) of 0.99-1.0, close to that of animal protein. It is particularly suitable as a protein source for vegetarians and people with lactose intolerance, and can be widely used in plant-based meat products, dairy substitutes, functional beverages, and protein bars. Furthermore, some components of potato protein possess certain physiological activities; for example, patatin has antioxidant and anti-inflammatory effects, showing potential applications in the pharmaceutical field and could be used to develop health products or drugs with specific functions. Naturally active potato protein has good emulsifying, gelling, and thermal stability, and can be used as a functional additive in food.

[0003] Potato protein, with its high nutritional value, low allergenicity, and multifunctionality, is an ideal choice for vegetarians and people with egg, dairy, or soy allergies. It can be widely used in plant-based meat products, dairy alternatives, functional beverages, and protein bars. Potato protein also possesses excellent technical functional properties, such as water solubility, gel formation, emulsification, and foaming properties.

[0004] Currently, thermal flocculation has significant advantages in the low-cost, large-scale recycling of potato protein (especially in low-value-added fields such as feed and fertilizer). However, the purity of potato protein extracted by thermal flocculation is low (50%-70%), and the high temperature destroys the functional activity of potato protein, which limits its application in high-end fields (such as functional foods, pharmaceuticals, and health products). Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing instant potato polypeptide powder to solve the problems of low purity and low solubility of potato protein extracted by thermal flocculation.

[0006] The technical solution of this invention is: a method for preparing instant potato polypeptide powder, comprising the following steps: A. Raw material processing: Disperse potato crude protein powder (purity 50-70%) in deionized water, stir, add NaOH or KOH, adjust pH to 9-10, heat and stir to dissolve, centrifuge to remove undissolved residue and particles, collect the supernatant, adjust the pH of the supernatant to 4.5-5.5, centrifuge again, wash repeatedly with deionized water 3-5 times, adjust pH to 6.5-7.5, and dry to obtain purified potato protein powder (purity up to 95%). B. Proteolytic hydrolysis: Add the potato protein powder obtained in step A to deionized water, disperse it evenly, and add alkaline protease to the dispersion for hydrolysis. C. Enzyme inactivation: Heat the reaction system in step B to 80-90℃ to inactivate the alkaline protease. Cool the inactivated reaction system to room temperature and centrifuge to remove impurities. Take the supernatant and centrifuge it in an ultrafiltration tube. Collect the peptide solution with <10 KD, or use an ultrafiltration membrane to perform ultrafiltration and collect the peptide solution with <10 KD. D. pH gradient precipitation: The polypeptide solution was precipitated using the pH gradient precipitation method, and the precipitate was collected by multiple centrifugations to obtain the enzymatic hydrolysate; E. Desalination: Resuspend the precipitate in deionized water, centrifuge, and repeat the washing process 2-3 times until the conductivity of the supernatant is <10 mS / cm, and collect the precipitate. F. Homogenization: Resuspend the precipitate in deionized water and homogenize to a size less than 80 mesh. The solid content after homogenization is usually 15%-20%, which yields potato protein hydrolysate. Heat the solution to 45-55℃ and stir continuously. G. Spray drying: The potato protein hydrolysate solution is dried using a spray dryer to obtain instant potato polypeptide powder.

[0007] Furthermore, in step A, the mass ratio of potato crude protein powder to deionized water is 1:3-5.

[0008] Furthermore, in step A, the heating temperature is 50-60℃.

[0009] Furthermore, in step B, the potato protein powder has a mass concentration of 4%, the alkaline protease activity is 200,000 U-300,000 U, and the enzyme-to-substrate volume ratio is 9:100.

[0010] Furthermore, in step B, the pH is kept stable at 9-10 during enzymatic hydrolysis, the enzymatic hydrolysis temperature is maintained at 50-55℃, and the enzymatic hydrolysis time is 6-8 hours.

[0011] Furthermore, in step B, ultrasonic-assisted dissolution is used during enzymatic hydrolysis at a frequency of 25-30 kHz and a power of 200-400 W.

[0012] Furthermore, in step B, the mass concentration of the dispersion is 6%-10%.

[0013] Further, in step C, the reaction system is heated to 85°C.

[0014] Furthermore, in step G, the inlet air temperature for spray drying is set to 140-150℃, and the outlet air temperature is set to 70-80℃.

[0015] The beneficial effects of this invention are: 1. This invention uses an alkaline dissolution and acid precipitation method combined with enzymatic hydrolysis to obtain potato instant polypeptide powder, achieving a purity of 90%-95%, with high yield, good hydrolysis effect, improved water dispersibility, enhanced industrial application value of potato protein, and improved economic utilization rate.

[0016] 2. The preparation method provided by this invention is simple, easy to scale up production, does not require desalination filtration steps such as filter membranes or dialysis, does not cause loss of active ingredients, the preparation process is scientific and reasonable, green and safe, and the preparation process conditions are controllable, which has good prospects for industrialization.

[0017] 3. The potato instant polypeptide powder prepared by the method of the present invention has antioxidant activity, ACE inhibitory activity, blood pressure lowering and other functions. It is a multifunctional peptide that can be used in functional foods, biopharmaceuticals or cosmetics. Attached Figure Description

[0018] Figure 1 This is a process flow diagram of the present invention; Figure 2 This is a total ion chromatogram of potato protein powder after hydrolysis in a specific embodiment of the present invention; Figure 3 This is a size distribution diagram of potato protein hydrolysate polypeptides in a specific embodiment of the present invention; Figure 4 This is a bar chart showing the C-terminal amino acid categories of potato proteinase-digested peptides in a specific embodiment of the present invention; Figure 5 This is a bar chart showing the N-terminal amino acid categories of potato protein-digested peptides in a specific embodiment of the present invention. Figure 6 This is a mass spectrum (I) of a polypeptide with both antioxidant and ACE inhibitory activity screened in a specific embodiment of the present invention. Figure 7 This is the mass spectrum (II) of the polypeptide with both antioxidant and ACE inhibitory activities screened in a specific embodiment of the present invention. Figure 8 This is a comparison diagram of the solubility of potato instant polypeptide powder prepared according to a specific embodiment of the present invention, potato thermal flocculation protein, and potato natural active protein. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Example 1 A method for preparing instant potato polypeptide powder includes the following steps: A. Raw material processing: Potato crude protein powder (purity <70%) is dispersed in deionized water at a mass ratio of 1:3 to deionized water. After stirring at 180 rpm for 1 hour, NaOH is added to adjust the pH to 9-10. The mixture is then heated in a water bath to 50-60℃ and stirred for 2 hours to dissolve. After centrifugation at 3000-5000 rpm for 30 minutes, undissolved residues and particles are removed. The supernatant is collected, and the pH is adjusted to 4.5-5.5 with 4M citric acid or lactic acid. The precipitate is collected by centrifugation, washed repeatedly with deionized water 5 times, and the pH is adjusted to 6.5-7.5. Finally, the mixture is spray-dried to obtain high-purity potato protein powder.

[0021] B. Proteolytic hydrolysis: The potato protein powder obtained in step A was added to deionized water and dispersed evenly to a dispersion concentration of 4%. Alkaline protease (activity 200,000 U-300,000 U) was added at an enzyme-to-protein ratio of 9:100 (v / v) for hydrolysis. Ultrasonic treatment was used with a frequency of 30 kHz and a power of 200 W. Hydrolysis was carried out at pH 9 and 55℃ for 6 hours. Intermittent ultrasound was employed to avoid continuous ultrasound causing a sudden temperature rise that could inactivate the protease.

[0022] C. Enzyme Inactivation: Transfer the reaction system from step B to an 85°C water bath and heat for 15 min to inactivate the alkaline protease. Cool the inactivated reaction system to room temperature and centrifuge at 3000 rpm to remove undigested protein and insoluble impurities. Transfer the supernatant to a 10 KD ultrafiltration tube and centrifuge at 12000 rpm for 10 min. Collect peptide solutions with a molecular weight cutoff of <10 KD, or use an ultrafiltration membrane with a molecular weight cutoff of 10 KD to perform ultrafiltration and collect peptide solutions with a molecular weight cutoff of <10 KD.

[0023] D. pH gradient precipitation: The collected peptide solution with <10 KD was adjusted to pH using a 4M citric acid or lactate gradient (pH adjusted to 6, 5.5, and 4.5 respectively) to precipitate the peptides. After centrifugation at 12000 rpm for 20 min, the precipitate was collected to obtain the enzymatic hydrolysate.

[0024] E. Desalination: Resuspend the precipitate in three times its volume of deionized water, centrifuge at 12,000 rpm for 20 min, and repeat the washing process 2-3 times until the conductivity of the supernatant is <10 mS / cm, and collect the precipitate.

[0025] F. Homogenization: Resuspend the precipitate with three times the volume of deionized water and homogenize to less than 80 mesh to obtain potato protein hydrolysate solution. Heat to 55°C and stir continuously to reduce its viscosity, which can improve the drying efficiency of potato instant polypeptide powder.

[0026] G. Spray drying: The potato protein hydrolysate was dried using a spray dryer. The inlet air temperature was set to 140-150℃, and the outlet air temperature was set to 70-80℃ to obtain instant potato polypeptide powder. The purity of the polypeptide powder was determined to be 95% using the Kjeldahl method.

[0027] (a) Total ion chromatogram analysis The potato protein powder obtained in step A was hydrolyzed by alkaline protease, and then analyzed by LC-MS. A total ion chromatogram (TIC) was plotted with ion intensity as the ordinate and time as the abscissa, providing an overall view of the separation of all ions in the sample. The total ion chromatogram of the alkaline protease hydrolysis products is shown below. Figure 2 As shown in the figure, the peaks exhibit a broad and continuous distribution throughout the chromatographic window (10–65 minutes). The signal intensity gradually increases after approximately 30 minutes, followed by a dense cluster of peaks between 35 and 60 minutes. Several high-intensity peaks were detected in the late elution region, including those at 50.6, 55.3, 57.6, 59.3, and 60.3 minutes. These characteristics indicate that alkaline protease digestion produces a highly complex mixture of peptides with significant differences in hydrophobicity and molecular size. These results demonstrate the high diversity of peptide groups produced by alkaline protease digestion, covering a broad elution range.

[0028] (II) Amino acid composition analysis of enzymatically hydrolyzed peptides Analysis showed that the alkaline protease hydrolysate contained 10,900 peptides, including 20 amino acids, covering all essential amino acids for humans (8 essential amino acids for adults + histidine, which is essential for infants). The amino acid composition of the potato protease hydrolysate peptides is shown in Table 1. Cystine (Cys), histidine (His), and tryptophan (Trp) were the lowest, accounting for 0.21%, 1%, and 0.97%, respectively; leucine (Leu), isoleucine (Ile), and proline (Pro) were the highest, accounting for 15.12%, 12.35%, and 9.23%, respectively. The 3.18% lysine content compensated for the lysine deficiency in wheat and rice, helping to promote calcium absorption and bone growth. The branched-chain amino acid (BCAA) content was high (leucine 15.12%, isoleucine 12.35%, valine 7.97%), close to whey protein levels, which is beneficial for muscle synthesis. Amino acid composition analysis revealed that the potato protein hydrolysates obtained through alkaline protease hydrolysis possess unique amino acid distribution characteristics and nutritional functional advantages. These are highly valuable functional protein sources suitable for various applications, including sports recovery, specific dietary supplementation, and targeted fortification in food formulations.

[0029] (III) Size distribution of enzymatically hydrolyzed peptides The molecular weight and length of peptides also reflect the properties of the peptide fragments. The molecular weight and length of hydrolyzed potato peptides treated with alkaline protease were analyzed. For example... Figure 3 As shown, ultrafiltration collected peptides with a molecular weight below 10 kDa. Among the peptides hydrolyzed by alkaline protease, peptides <1 kDa accounted for 93.7%, peptides 1-1.5 kDa accounted for 5.7%, and peptides 1.5-3 kDa accounted for 0.5%. The peptide lengths were mainly between 2 and 17 amino acids, with tripeptides, tetrapeptides, and pentapeptides accounting for the highest proportions, at 10.8%, 36.0%, and 31.5%, respectively.

[0030] (iv) Functional prediction of bioactive peptides A model was trained based on reported peptide information and related data to predict the activities of identified peptides. Table 2 shows the predicted bioactivities of peptides obtained from the hydrolysis of potato protein using alkaline protease. The predictions indicated that 7375 peptides exhibited ACE inhibitory activity and 7155 peptides exhibited antioxidant activity in the alkaline protease hydrolysis system. Among these, 1200 overlapping peptides possessed both ACE inhibitory and antioxidant activities. Furthermore, among these overlapping peptides, 31 sequences had recorded activity data retrieved from the BIOPEP-UWM database, providing additional validation for their functional potential.

[0031] (v) Identification and characterization of polypeptides The presence of tyrosine (Tyr) or tryptophan (Trp) residues in peptide molecules endows them with antioxidant activity. To further focus on peptides with potentially high antioxidant activity, peptides containing Tyr / Trp residues were preferentially screened. Based on bioactivity prediction results, high-quality peptides containing both Tyr or Trp residues and predicted to possess both angiotensin-converting enzyme (ACE) inhibitory and antioxidant activities were specifically selected from the alkaline protease system. The characteristic parameters of tyrosine / tryptophan residue-containing peptides obtained from alkaline protease hydrolysis of potato protein are shown in Table 3.

[0032] (vi) Analysis of antioxidant activity and ACE inhibitory activity of C-terminal and N-terminal amino acids like Figure 4 , Figure 5 As shown, ACE-inhibiting peptides containing Trp, Phe, Tyr, or Pro at the C-terminus and Leu, Ile, or Val at the N-terminus exhibit better ACE-inhibiting activity. Positively charged amino acids at the C-terminus (Lys and Arg) have a beneficial effect on enhancing ACE-inhibiting activity. N-terminal or C-terminal amino acids have a significant impact on the bioactivity of peptides. Potato peptide powder contains a large amount of this type of peptide, and prediction results indicate that it possesses strong antioxidant activity and ACE-inhibiting activity.

[0033] The presence of tyrosine (Tyr) or tryptophan (Trp) residues in peptide molecules endows them with antioxidant activity. In an alkaline protease hydrolysis system, 20 peptides containing tyrosine residues were identified, of which 45% originated from block Ptatin protein and 55% from protease inhibitors; only one peptide contained a tryptophan residue. Among the target peptides identified through the above screening, only one sequence had a matching record in the BIOPEP database. The remaining peptides were predicted novel peptides, previously unreported, and based on bioactivity prediction, they all possess potential antioxidant activity. Further screening identified 10 peptides with both antioxidant and ACE inhibitory activities, such as... Figure 6 , Figure 7 As shown.

[0034] (vii) Solubility of instant potato polypeptide powder Protein solubility affects its functional properties, including foaming ability, gelling ability, and emulsifying properties. Protein solubility is significantly influenced by structural changes caused by processing. Figure 8 This is a comparison diagram of the solubility of potato instant polypeptide powder (b) prepared according to a specific embodiment of the present invention, potato thermal flocculation protein (a), and potato natural active protein (c). Figure 8As shown, the natural active protein from potatoes exhibited the highest solubility (1.009 mg / mL), indicating its intact tertiary and quaternary structures and abundant exposure of hydrophilic residues. In contrast, the solubility of the potato thermally flocculant protein was negligible (0.003 mg / mL), likely due to protein denaturation caused by heat treatment, which reduced solubility. Thermal denaturation exposes hydrophobic regions, leading to the formation of numerous insoluble aggregates. Notably, the potato instant polypeptide powder in this embodiment significantly improved solubility (0.353 mg / mL), suggesting that enzymatic hydrolysis-induced disaggregation of thermally flocculant protein aggregates may partially disrupt hydrophobic interactions, improving hydrophilicity and thus enhancing solubility.

[0035] In summary, the potato instant polypeptide powder prepared by this invention exhibits good solubility, a more balanced amino acid composition, low allergenicity, and antioxidant and ACE inhibitory activities. This invention not only compensates for the nutritional deficiencies of traditional plant proteins but also avoids the allergenicity and environmental problems associated with animal proteins, while possessing unique functional activities, making it highly promising for applications in food, health care, medicine, and cosmetics. This invention effectively enhances the application value of potato protein, enabling industrial production and providing a new solution for the resource-efficient and high-value utilization of potato crude protein powder.

Claims

1. A method for preparing instant potato polypeptide powder, characterized in that, Includes the following steps: A. Raw material processing: Potato crude protein powder is dispersed in deionized water, stirred, and the pH is adjusted to 9-10. It is then heated and stirred to dissolve, centrifuged to remove undissolved residue and particles, and the supernatant is collected. The pH of the supernatant is adjusted to 4.5-5.5, then centrifuged, washed, and the pH is adjusted to 6.5-7.

5. Finally, it is dried to obtain purified potato protein powder. B. Proteolytic hydrolysis: Add the potato protein powder obtained in step A to deionized water, disperse it evenly, and add alkaline protease to the dispersion for hydrolysis. C. Enzyme inactivation: Heat the reaction system in step B to 80-90℃ to inactivate the alkaline protease. Cool the inactivated reaction system to room temperature and centrifuge to remove impurities. Take the supernatant and centrifuge it in an ultrafiltration tube. Collect the peptide solution with <10 KD, or use an ultrafiltration membrane to perform ultrafiltration and collect the peptide solution with <10 KD. D. pH gradient precipitation: The polypeptide solution was precipitated using the pH gradient precipitation method, and the precipitate was collected by multiple centrifugations to obtain the enzymatic hydrolysate; E. Desalination: Resuspend the precipitate in deionized water, centrifuge, and repeat the washing process 2-3 times until the conductivity of the supernatant is <10mS / cm, and collect the precipitate. F. Homogenization: Resuspend the precipitate in deionized water and homogenize to a size less than 80 mesh to obtain potato protein hydrolysate solution. Heat to 45-55℃ and stir continuously. G. Spray drying: The potato protein hydrolysate solution is dried using a spray dryer to obtain instant potato polypeptide powder.

2. The method for preparing instant potato polypeptide powder according to claim 1, characterized in that: In step A, the mass ratio of potato crude protein powder to deionized water is 1:3-5.

3. The method for preparing instant potato polypeptide powder according to claim 1, characterized in that: In step A, the heating temperature is 50-60℃.

4. The method for preparing instant potato polypeptide powder according to claim 1, characterized in that: In step B, the potato protein powder has a mass concentration of 4%, the alkaline protease activity is 200,000 U-300,000 U, and the enzyme-to-substrate volume ratio is 9:

100.

5. The method for preparing instant potato polypeptide powder according to claim 1, characterized in that: In step B, the pH is kept stable at 9-10 during enzymatic hydrolysis, the enzymatic hydrolysis temperature is maintained at 50-55℃, and the enzymatic hydrolysis time is 6-8 hours.

6. The method for preparing instant potato polypeptide powder according to claim 1, characterized in that: In step B, ultrasonic-assisted dissolution is used during the enzymatic hydrolysis process.

7. The method for preparing instant potato polypeptide powder according to claim 1, characterized in that: In step B, the mass concentration of the dispersion is 6%-10%.

8. The method for preparing instant potato polypeptide powder according to claim 1, characterized in that: In step C, the reaction system is heated to 85°C.

9. The method for preparing instant potato polypeptide powder according to claim 1, characterized in that: In step G, the inlet air temperature for spray drying is set to 140-150℃, and the outlet air temperature is set to 70-80℃.

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