A method for extracting proteins from the cells of kluyveromyces lactis

Abstract

The application discloses a method for extracting Kluyveromyces lactis cell protein and belongs to the field of protein extraction processes. First, the single-factor conditions of high-pressure homogenization of Kluyveromyces lactis cell crushing are optimized to obtain the best solid-liquid ratio, crushing time and buffer pH condition, and then the response surface design and optimization are used to obtain the best Kluyveromyces lactis cell protein cell crushing condition, i.e. the solid-liquid ratio is 0.041 g / mL, the high-pressure homogenization time is 46 s, and the pH is 7.37. Then, the isoelectric point of the Kluyveromyces lactis cell protein is determined to be 2.5. The developed Kluyveromyces lactis protein extraction method based on high-pressure homogenization and alkali dissolution and acid precipitation is evaluated. The extraction rate of the Kluyveromyces lactis protein obtained by using the method is 40.6%, which provides a new efficient way for yeast cell protein extraction production and has important practical application value and significance.

Description

Technical Field

[0001] This invention relates to a method for extracting protein from Kluyveromyces lactis cells, belonging to the field of protein extraction technology. Background Technology

[0002] Yeast protein is a typical single-cell protein, characterized by a balanced amino acid composition, high digestibility, and rich in various essential amino acids, vitamins, and bioactive components, thus possessing broad application prospects in food, feed, health products, and pharmaceuticals. Among them, *Kluyveromyces lactis*, as an important food-grade yeast, is not only widely used in the dairy industry, but its intracellular protein is also a high-quality protein resource. However, due to the complex cell wall structure of *Kluyveromyces lactis*, mainly composed of polysaccharide complexes such as chitin, dextran, and mannan, which are dense and robust, the intracellular protein is difficult to release directly. Therefore, how to achieve efficient cell wall disruption and obtain high-purity yeast protein is a key issue for the industrialization of *Kluyveromyces lactis* protein. Existing yeast protein extraction methods mainly include three categories: mechanical methods, chemical methods, and enzymatic methods. 1) Mechanical disruption methods: such as bead milling and ultrasonic disruption, can achieve cell disruption, but the equipment consumes high energy, the operation is complex, and it easily causes thermal denaturation and degradation of the protein, which is not conducive to maintaining protein quality; 2) Chemical methods: including alkali dissolution and acid precipitation and salting out. Alkali dissolution and acid precipitation processes are simple to operate, but improper control of reaction conditions can easily lead to protein denaturation or a decrease in nutritional value. Salting out, while improving protein dissolution rates, requires large amounts of salt, increasing post-processing difficulty and cost, and is environmentally unfriendly. Enzymatic hydrolysis releases proteins by breaking down cell walls using cellulase, dextranase, or complex enzyme systems. This method is mild, but enzyme preparations are expensive and the reaction cycle is long, making it difficult to meet the needs of industrial-scale production. In summary, existing yeast protein extraction processes generally suffer from problems such as complex operation, high energy consumption, high cost, or low yield. A highly efficient, economical, mild method suitable for large-scale production of Kluyveromyces lactis protein extraction is still lacking. Therefore, developing a novel, highly efficient yeast cell wall disruption and protein extraction process has significant practical application value and industrialization implications. Summary of the Invention

[0003] The purpose of this invention is to provide a highly efficient, economical, and mild extraction process for Kluyveromyces lactis protein. This process combines alkali dissolution with acid precipitation and high-pressure homogenization to address the problems of low cell disruption efficiency, insufficient protein release rate, and high energy consumption in existing yeast protein extraction processes. Through systematic optimization of process parameters, efficient disruption of yeast cells and full release of intracellular proteins are achieved, thereby significantly improving the protein extraction rate and providing reliable technical support for the industrial development of Kluyveromyces lactis protein.

[0004] This invention provides a method for extracting Kluyveromyces lactis cell protein, comprising: culturing Kluyveromyces lactis, collecting cell cells, preparing a cell suspension with a material-to-liquid ratio of 0.025~0.05 g / mL, and subjecting the cell suspension to high-pressure homogenization.

[0005] In one embodiment, the bacterial suspension is homogenized under high pressure for 30-50 seconds.

[0006] In one embodiment, the method involves high-pressure homogenization for 30 seconds in a buffer system with a pH of 7-8.

[0007] In one embodiment, the bacterial suspension is prepared using deionized water.

[0008] In one embodiment, the feed-to-liquid ratio is controlled at 0.041 g / mL, the high-pressure homogenization time is 46 seconds, and the resuspension buffer pH is 7.37.

[0009] In one embodiment, the cultivation includes: culturing *Kluyveromyces lactis* in YPD medium at 28–30 °C for 16–24 h to obtain a primary seed culture; then inoculating the primary seed culture into YPD medium and culturing at 28–30 °C for 8–12 h to obtain a secondary seed culture; inoculating the secondary seed culture into a fermentation medium and fermenting at 28–30 °C, pH 5.0–5.5, DO 20 ± 0.1%, stirring speed 300–600 rpm, and aeration rate of 2 vvm; when the glucose concentration is below 1 g / L, fermenting at 5 g / L·h. - ¹Fed at a rate of 10-28 h, and collect the cells after fermentation.

[0010] In one embodiment, the fermented cells are weighed by wet weight, resuspended in deionized water, and the solid-liquid ratio is controlled at 0.025~0.05 g / mL. The cell suspension is homogenized under high pressure at a buffer pH of 7.0~8.0 for 30~50 s. The supernatant of the lysed product is centrifuged and the pH is adjusted to 9.0 with NaOH while stirring. The product is extracted in a 70 ℃ water bath for 70 min. After centrifugation, the supernatant is adjusted to pH 2.5 with HCl for acid precipitation. The supernatant is discarded, the precipitate is dissolved and the pH is adjusted to neutral. The product is then freeze-dried to obtain Kluyveromyces lactis protein.

[0011] In one embodiment, the Kluyveromyces lactis is Kluyveromyces lactis CICC 1772.

[0012] In one embodiment, the method further includes centrifuging the cell lysate after high-pressure homogenization to obtain the supernatant, adjusting the pH of the supernatant to 2.5, allowing it to stand for a period of time, centrifuging again, and then freeze-drying the precipitate.

[0013] In one embodiment, the method involves centrifuging the cell lysate after high-pressure homogenization to obtain the supernatant, adjusting the pH of the supernatant to 2.5, allowing it to stand for 1 hour, centrifuging again, and then freeze-drying the precipitate.

[0014] The present invention also provides the application of the method in the preparation of products containing yeast protein.

[0015] In one embodiment, the product includes, but is not limited to, a culture medium.

[0016] Beneficial effects: (1) This invention achieves efficient disruption of Kluyveromyces lactis cells and full release of intracellular proteins through the synergistic effect of high-pressure homogenization and alkali dissolution and acid precipitation, overcoming the defects of incomplete cell disruption by traditional mechanical methods, long cycle of enzymatic hydrolysis, and protein denaturation by chemical methods.

[0017] (2) The optimized combination of material-liquid ratio, high-pressure homogenization time and pH conditions of the present invention achieves a protein extraction rate of 40.8%, which is significantly better than the 15-25% of the traditional extraction method, and the obtained protein has a complete structure and good solubility.

[0018] (3) The present invention uses a low-temperature short-time homogenization and neutral buffer system (with deionized water as buffer) to avoid protein degradation caused by high temperature and strong acid and alkali treatment, and significantly reduce energy consumption and environmental burden.

[0019] (4) The process of this invention is simple, the equipment requirements are low, and it is easy to scale up. It does not rely on expensive enzyme preparations or a large amount of chemical reagents, and has good economic efficiency and sustainability. It is suitable for promotion and use in the food, feed and functional protein product industries.

[0020] (5) The present invention establishes a mathematical relationship between parameters and extraction rate through the Box-Behnken response surface optimization model. The model prediction results are highly consistent with the actual experiment, indicating that the parameter optimization of this method is scientific and reproducible.

[0021] (6) This invention determined the isoelectric point (pH 2.5) of Kluyveromyces lactis protein, providing a theoretical basis for subsequent protein separation and purification using the isoelectric point precipitation method, and expanding its application potential in high-value utilization. Attached Figure Description

[0022] Figure 1 This describes the effect of different material-to-liquid ratios on the extraction of Kluyveromyces lactis cell protein in Example 2.

[0023] Figure 2 This illustrates the effect of different high-pressure homogenization times on the extraction of Kluyveromyces lactis cell protein in Example 3.

[0024] Figure 3This illustrates the effect of different resuspension buffer pH values ​​on the extraction of Kluyveromyces lactis cell protein in Example 4.

[0025] Figure 4 The effect of response surface optimization design of fragmentation conditions on the extraction of Kluyveromyces lactis cell protein in Example 5.

[0026] Figure 5 The isoelectric point of the Kluyveromyces lactis cell protein in Example 6 was determined.

[0027] Figure 6 This is the standard curve determination for the standard bovine serum albumin in Example 7.

[0028] Figure 7 This shows the relationship between the dry and wet weights of Kluyveromyces lactis cells in Example 7. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available or can be prepared by known methods. The culture media involved in the following examples are also mentioned.

[0031] Fermentation medium: 40 g / L -1 Glucose, 10 g / L -1 Yeast extract, 10 g / L (NH4)2SO4, 5 g / L MgSO4·7H2O, 10 g / L KH2PO4, 10 g / L CaSO4·2H2O, 0.7% 500× trace element stock solution, and 0.2% 1000× vitamin stock solution.

[0032] 500× Trace element stock solution: 5 g / L KCl, 7.5 g / L EDTA, 2.25 g / L ZnSO4·7H2O, 0.5 g / L MnCl2·7H2O, 0.15 g / L CoCl2·6H2O, 0.15 g / L CuSO4·5H2O, 0.2 g / L Na2MoO4·2H2O, 2.25 g / L CaCl2·2H2O, 1.5 g / L FeSO4·7H2O, 0.5 g / L H3BO3, 0.05 g / L KI; 1000× Vitamin Stock Solution: 0.1 g / L Biotin, 25 mg / L Inositol (Vitamin B8), 1 mg / L Thiamine (Vitamin B1), 1 mg / L Pyridoxine (Vitamin B6), 25 mg / L Para-aminobenzoic acid (PABA), 15 mg / L Choline, 5 mg / L Riboflavin (Vitamin B2), 0.1 g / L Calcium Pantothenate, 0.1 g / L Niacin.

[0033] Method for determining bacterial protein content: Collect bacterial cells by centrifugation of the fermentation broth, wash three times with distilled water, treat the cells at 72℃ for 30 min to remove RNA and other genetic material, freeze-dry, pulverize, and filter through an 80-mesh cell sieve. Weigh 0.5 g of the above powder and add 3 times the volume (V / M) of n-hexane, treat at 300 rpm at room temperature for 10 min, filter and air-dry the obtained cells at room temperature in a fume hood for 3 h, mix thoroughly with 10 times the volume of distilled water, adjust the pH to 9 with 2 M NaOH while stirring, extract in a 70℃ water bath for 70 min, centrifuge at 4℃, 9000×g for 20 min, collect the supernatant, adjust the pH to 5 with 2 M HCl while stirring, react at 300 rpm for 30 min, discard the supernatant, dissolve the precipitate in distilled water and adjust the pH to neutral, freeze-dry and weigh. Extraction rate = (amount of protein obtained by separation, purification and freeze-drying / bacterial protein content) × 100%.

[0034] Example 1: Fermentation culture of Kluyveromyces lactis Fermentation medium: 40 g / L -1 Glucose, 10 g / L -1 Yeast extract, 10 g / L (NH4)2SO4, 5 g / L MgSO4·7H2O, 10 g / L KH2PO4, 10 g / L CaSO4·2H2O, 0.7% (v / v) 500× trace element stock solution, 0.2% (v / v) 1000× vitamin stock solution; 500× Trace element stock solution: 5 g / L KCl, 7.5 g / L EDTA, 2.25 g / L ZnSO4·7H2O, 0.5 g / L MnCl2·7H2O, 0.15 g / L CoCl2·6H2O, 0.15 g / L CuSO4·5H2O, 0.2 g / L Na2MoO4·2H2O, 2.25 g / L CaCl2·2H2O, 1.5 g / L FeSO4·7H2O, 0.5 g / L H3BO3, 0.05 g / L KI.

[0035] 1000× Vitamin Stock Solution: 0.1 g / L Biotin, 25 mg / L Inositol (Vitamin B8), 1 mg / L Thiamine (Vitamin B1), 1 mg / L Pyridoxine (Vitamin B6), 25 mg / L Para-aminobenzoic acid (PABA), 15 mg / L Choline, 5 mg / L Riboflavin (Vitamin B2), 0.1 g / L Calcium Pantothenate, 0.1 g / L Niacin.

[0036] A single colony of *Kluyveromyces lactis* CICC 1772 (obtained from CICC) was inoculated into 15 mL of YPD medium and cultured for 20 h at 30°C and 220 rpm on a shaker to obtain a primary seed culture. Then, the primary seed culture was transferred at a volume ratio of 5% to a shake flask containing 300 mL of YPD medium and cultured for 10 h at 30°C and 220 rpm on a shaker (OD200). 600 A secondary seed culture was obtained by inoculating the secondary seed culture at a volume ratio of 10% into a fermenter containing 2.7 L of fermentation medium. Fermentation was carried out under the following conditions: DO 20%, pH 5.5, 30℃, 300-600 rpm, and aeration rate of 2 vvm. When the glucose concentration was below 1 g / L, the feed was added at a constant rate of 5 g / L / h. After 24 h of fermentation, the culture was removed from the fermenter, and the OD was measured. 600 Regarding protein content, the fermentation broth was centrifuged and washed to obtain cell precipitate, which was then freeze-dried for 24 hours to obtain cell powder. Next, 0.5 g of the cell powder was weighed, and the protein content was determined and calculated according to the cell protein content determination method. 0.11 g of protein was obtained from 0.5 g of dry cell powder. Therefore, the ratio of protein content to cell dry weight is the total cell protein content, which is 22%.

[0037] Example 2: Effect of different solid-liquid ratios on the extraction of protein from Kluyveromyces lactis cells. The bacterial cells collected from fermentation in Example 1 were weighed to the same wet weight. Different volumes of deionized water were added to resuspend the bacterial cells, and the final solid-liquid ratio (cellular mass: deionized water) was set to 0.01825 g / mL, 0.02 g / mL, 0.025 g / mL, 0.0375 g / mL, and 0.05 g / mL, respectively. The bacterial cells were lysed under conditions of buffer pH 7 and high-pressure homogenization time of 30 seconds. The protein concentration in the supernatant of the lysed solution was determined using a Nanodrop micro-UV spectrophotometer. The results showed that the concentration of extracellular protein obtained from lysation increased with increasing solid-liquid ratio, and the protein concentration tended to stabilize between 0.0375 g / mL and 0.05 g / mL, reaching 2.2 mg / mL. Figure 1 ).

[0038] Example 3: Effect of different high-pressure homogenization times on the extraction of proteins from Kluyveromyces lactis cells. The bacterial cells collected from fermentation in Example 1 were weighed to the same wet weight. The same volume of deionized water was added to resuspend the bacterial cells, maintaining a fixed material-to-liquid ratio of 0.025 g / mL. The cells were disrupted using different high-pressure homogenization times (20, 30, 40, 50, 60 seconds) at a buffer pH of 7. The results showed that the protein concentration obtained from disruption first increased and then decreased with increasing disruption time; the highest soluble protein concentration, reaching 2.14 mg / mL, was achieved at a disruption time of 40 s. Figure 2 ).

[0039] Example 4: Effect of different disruption buffer pH on the extraction of Kluyveromyces lactis cell protein The bacterial cells collected from fermentation in Example 1 were weighed to the same wet weight. The same volume of buffer solution was added to resuspend the bacterial cells, maintaining a fixed solid-liquid ratio of 0.025 mg / mL. The pH of the resuspension buffer was adjusted to 7, 8, 9, 10, and 11, respectively, and the cells were lysed under high pressure homogenization for 30 seconds. The results showed that excessively high pH of the resuspension buffer affected cell disruption and protein extraction efficiency. When the buffer pH was 7-8, the concentration of soluble protein obtained from disruption reached 2.03-2.14 mg / mL. Figure 3 ).

[0040] Example 5: Effect of Response Surface Design of Cell Disruption Conditions on Protein Extraction from Kluyveromyces lactis Cells Based on the single-factor experiments, the material-to-liquid ratio, high-pressure homogenization time, and resuspension buffer pH were used as experimental factors. A three-factor, three-level orthogonal experiment was conducted to determine the optimal protein extraction process conditions. The factor levels are shown in Table 1.

[0041] Table 1. Factor levels in the orthogonal experiment for protein extraction from Kluyveromyces lactis cells.

[0042] The Box-Behnken experimental design and results are shown in Table 2. Using Design-Expert 8.0, a quadratic multiple regression was performed to obtain the relationship between the material-to-liquid ratio (A), homogenization time (B), and pH (C) and the response value extraction rate (Y). The quadratic multiple regression equation is: Y = 7.71 + 1.95A - 1.18B + 0.0042C - 0.0748AB - 0.2285AC - 0.1354BC - 2.69A 2 -0.3882B 2 -0.7178C 2 .

[0043] Table 2 Box-Behnken Experimental Design and Results

[0044] According to the analysis of variance results in Table 3, the p-value of the equation model is <0.01, indicating that the equation model is significant. The lack-of-fit term p-value is >0.05 and is not significant. The p-values ​​indicate that the linear terms A and B, and the quadratic term A... 2 The effect on protein extraction is extremely significant, with the quadratic term B. 2 C 2 The effects on protein extraction were significant, but the p-values ​​for the interaction terms AB, AC, and BC were greater than 0.05, indicating that the interactions were not significant. Therefore, the effects of each factor on protein extraction are not a simple linear relationship.

[0045] Table 3. Analysis of Variance of Regression Equations

[0046] Contour plots and response surface plots can reflect the influence of the interaction between two factors on protein extraction. The more rounded the contour plot, the smaller the influence; the flatter it is, the greater the influence. Similarly, the steeper the surface of the response surface, the greater the influence. Contour plots and response surface plots illustrating the pairwise interactions of various factors are shown below. Figure 4 As shown. Data analysis using Design-Expert 8.0.6 software revealed the optimal conditions for Kluyveromyces lactis cell protein disruption as follows: a material-to-liquid ratio of 0.041 g / mL, a high-pressure homogenization time of 46 seconds, and a pH of 7.37.

[0047] Example 6: Determination of the isoelectric point of Kluyveromyces lactis cell protein The cells were treated under the optimal cell disruption conditions described in Example 5. After centrifugation, the supernatant was collected, and the pH of the supernatant was adjusted to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5, respectively, and allowed to stand. The absorbance at different pH values ​​was measured to determine the isoelectric point. The experimental results showed that the absorbance was lowest at pH 2.5. Therefore, this pH was determined as the isoelectric point of Kluyveromyces lactis cell protein for subsequent separation and extraction (Figure 5).

[0048] Example 7 Evaluation of a method for extracting proteins from Kluyveromyces lactis based on a combination of alkali dissolution, acid precipitation, and high-pressure homogenization First, a standard curve was plotted using the Bradford method with standard bovine serum albumin (BSA). Specifically: First, a series of BSA standard solutions with concentration gradients (0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL) were precisely prepared. Then, equal volumes of each standard and the sample to be tested were mixed with Coomassie Brilliant Blue G-250 dye working solution and reacted at room temperature in the dark for 5-10 minutes. After the reaction, the absorbance of each tube was measured at 595 nm using a spectrophotometer. A scatter plot was plotted with the standard concentration on the x-axis and the absorbance on the y-axis, and linear regression was performed to obtain the regression equation Y. 吸光度 =3.94X 蛋白浓度 +0.51, its R 2 =0.99, Figure (6). Furthermore, a linear relationship between the dry and wet weight of *Kluyveromyces lactis* cells was established, as follows: different amounts of centrifuged cells were taken and their wet weight was measured. Then, the cells were dried in an oven and their dry weight was measured. The linear relationship between wet and dry weight was plotted as follows: Figure 7 As shown: Y 干重 =0.15387X 湿重 -0.01787 ( R 2 =0.99).

[0049] The yeast (1g wet weight yeast) was disrupted using the optimal high-pressure homogenization conditions of Kluyveromyces lactis in Example 5. The specific steps were as follows: a buffer solution with a material-to-liquid ratio of 0.04 g / mL was added to the wet yeast cells to resuspend the cells. The pH of the resuspension buffer was adjusted to pH 7.37, and the cells were disrupted under high-pressure homogenization for 46 seconds.

[0050] Centrifuge and collect the supernatant. Adjust the pH of the supernatant to its isoelectric point of 2.5 to precipitate bacterial proteins. Let it stand for 1 hour, centrifuge again to obtain the protein precipitate, and freeze-dry for 12 hours. Redissolve the dried protein powder in water, determine its concentration using the Bradford method, and calculate the obtained protein amount as 11.6 mg using a standard curve. Therefore, the extraction rate of Kluyveromyces lactis protein obtained by this method is: 11.6 mg / [(1g × 0.15387 - 0.01787) × 21%] = 40.6%.

[0051] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for extracting Kluyveromyces lactis cell protein, comprising: Culture Kluyveromyces lactis, collect the cells, and prepare a bacterial suspension with a material-to-liquid ratio of 0.025-0.05 g / mL. Homogenize the bacterial suspension under high pressure for 30-50 seconds.

2. The method according to claim 1, characterized in that, The pH of the bacterial suspension is 7-8.

3. The method according to claim 1 or 2, characterized in that, The feed-to-liquid ratio was controlled at 0.041 g / mL, the high-pressure homogenization time was 46 seconds, and the resuspension buffer pH was 7.

37.

4. The method according to any one of claims 1 to 3, characterized in that, The culture involves culturing the Kluyveromyces lactis in YPD medium at 28-30°C for at least 12 hours.

5. The method according to claim 4, characterized in that, The cultivation process includes: culturing *Kluyveromyces lactis* in YPD medium at 28–30 °C for 16–24 h to obtain a primary seed culture; then inoculating the primary seed culture into YPD medium and culturing at 28–30 °C for 8–12 h to obtain a secondary seed culture; inoculating the secondary seed culture into a fermentation medium and fermenting at 28–30 °C, pH 5.0–5.5, DO 20 ± 0.1%, stirring speed 300–600 rpm, and aeration rate of 2 vvm; when the glucose concentration is below 1 g / L, fermentation is carried out at a rate of 5 g / L·h. - ¹Fed at a rate of 10-28 h, and collect the cells after fermentation.

6. The method according to claim 5, characterized in that, After high-pressure homogenization, the supernatant was adjusted to the isoelectric point for acid precipitation. The supernatant was discarded, the precipitate was dissolved and the pH was adjusted to neutral. The precipitate was then freeze-dried to obtain Kluyveromyces lactis protein.

7. The method according to claim 6, characterized in that, The method involves adjusting the pH of the supernatant after high-pressure homogenization to 2.

5.

8. The method according to any one of claims 1 to 7, characterized in that, The Kluyveromyces lactis is Kluyveromyces lactis CICC 1772.

9. Yeast protein prepared by applying the method described in any one of claims 1 to 8.

10. Use in products containing yeast protein prepared by any one of claims 1 to 8.

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