Preparation method and application of grifola frondosa protein capable of delaying fat digestion of emulsion

Abstract

The invention discloses a preparation method and application of grifola frondosa protein capable of delaying fat digestion of emulsion, and relates to deep processing and application of edible mushrooms. Grifola frondosa is used as an edible fungus source, protein in the Grifola frondosa is used as a natural emulsifier, and the prepared oil-in-water emulsion has low release amount of free fatty acid in the digestion process and can inhibit digestion of grease in the emulsion. The edible mushroom protein is used as a substitute emulsifier for egg yolk, and a novel mayonnaise which can not only inhibit grease digestion but also reduce cholesterol intake is developed. The adopted emulsifier grifola frondosa protein is subjected to modification treatment, the modification method is microgelation of the protein, the interface property of the protein is improved through microgelation treatment, cholate, lipase and the like are prevented from making contact with oil drops, and digestion of lipid in gastrointestinal tracts is inhibited. Through modification treatment and application of the edible mushroom grifola frondosa protein, the source of food protein is expanded, and the edible mushroom grifola frondosa protein conforms to the current concept of big food.

Description

Technical Field

[0001] The invention relates to preparing cholesterol-free vegetable mayonnaise with low fat digestibility by using edible fungus protein as a natural emulsifier, and belongs to the technical field of food processing. Background Art

[0002] Fat is one of the three major nutrients for the human body. It not only provides essential fatty acids to the human body, but also serves as a carrier for the transport of fat-soluble vitamins in the human body. However, excessive intake of fat, especially animal fat, may cause obesity, which in turn leads to a series of diseases such as diabetes, hypertension, and hyperlipidemia. In order to meet people's nutritional and health needs, limiting fat intake, developing low-fat foods, developing animal fat substitutes and other means of reducing fat intake are currently hot topics in the research of fat-containing foods.

[0003] The fats and oils in food mostly exist in the form of emulsions. After the emulsified fat particles enter the duodenum, their interface composition will be replaced by bile salts to form smaller new emulsified fat particles. Then, lipase and its coenzyme will adsorb on the oil-water interface of the bile salt emulsified fat particles, hydrolyzing the fatty acids of triglycerides in the fat into free fatty acids and monoglycerides. The products of fat digestion, free fatty acids and monoglycerides, will self-assemble and aggregate with bile salts to form micelles and be transported into the epithelial cells of the small intestine. Therefore, improving the stability of the emulsion and delaying the digestion of fat in the food emulsion through the structural design of food products is considered to be one of the effective strategies to reduce fat intake.

[0004] At present, the inhibition of lipid digestion is mainly achieved by inhibiting lipase activity and regulating the interfacial composition of the emulsion to reduce the adsorption of bile salts and lipase to lipids. Pickering emulsion is an emulsion stabilized by particles. Compared with traditional emulsions, it has high resistance to aggregation due to the irreversible adsorption of its particles. It can delay the digestion of fat by controlling the stability of the emulsion, resisting the competitive substitution of bile salts and inhibiting the diffusion adsorption of lipase.

[0005] As a type of protein-based Pickering particles, protein microgels are usually soft particles with viscoelasticity. They not only have micron or nanometer particle sizes, but also have the structural characteristics of gels. Compared with protein nanoparticles, protein colloidal particles are submicron particles formed by non-covalent or covalent bonds. They are soft and easy to deform. The particles can form a dense interfacial film through bridging, which can effectively avoid the contact between oil and lipase and have a good ability to control oil digestion. At present, some scholars have studied the differences in whey protein microgels with different hardness in improving emulsion stability and delaying lipid digestion. Studies have shown that softer microgel particles are more likely to denature and form an interfacial film when adsorbed to the oil-water interface, and have the best emulsion stability and lipid digestion delay rate. Therefore, protein microgels can be used in the development and utilization of low-fat foods. At present, most of the research on the properties of protein microgels is on animal-derived proteins such as whey protein. However, the use of animal protein cannot meet the needs of vegetarian consumers and is not conducive to reducing carbon emissions. At the same time, under the concept of big food, it is extremely important to build a diversified protein supply system and develop new protein sources through multiple channels.

[0006] Edible fungi are rich in resources, with a protein content of about 19%-40% (dry basis content) and a ratio of essential amino acids to non-essential amino acids of mostly higher than 0.4, making them a source of high-quality protein. At the same time, edible fungi protein has a variety of biological activities, such as anti-tumor, immunomodulatory, and antiviral. However, insufficient research on edible fungi protein processing technology and its application has affected the application of edible fungi protein in food products.

[0007] Grifola frondosa, commonly known as Maitake mushroom and chestnut mushroom, has a protein content of about 20%, containing 18 amino acids needed by the human body. It is a high-quality source of edible fungus protein. China's production of Grifola frondosa will be 49,000 tons in 2022, and the annual output continues to rise year by year. At present, Grifola frondosa is mainly eaten fresh, and there are few products developed with it as raw material. Products developed with Grifola frondosa as raw material are mostly based on its polysaccharide components and are mainly used in anti-cancer and anti-tumor drugs, such as Grifola frondosa polysaccharide capsules. There is still a lack of foods developed with Grifola frondosa protein as the main ingredient on the market. Therefore, the use of Grifola frondosa protein to prepare microgels to develop Pickering emulsions that can delay fat digestion has broad application prospects in food development.

[0008] Mayonnaise has a viscous texture, delicate and soft taste, and unique flavor. It combines sour, sweet, salty and egg yolk flavors. It is very popular in the United States, Western Europe, Japan and other countries. With the changes in people's lifestyles and consumption habits in recent years, mayonnaise as a seasoning sauce has gradually become popular in my country. The consumption in my country is increasing. In 2023, the market size of my country's mayonnaise industry will reach 12 billion yuan. Mayonnaise is a typical food emulsion system. Traditional mayonnaise mostly uses egg yolk as an emulsifier. The addition of egg yolk gives mayonnaise a special texture and rheological properties. However, as consumers have paid more and more attention to the nutritional and health attributes of food in recent years, they have begun to worry that excessive consumption of traditional mayonnaise products will lead to increased cholesterol intake, posing hidden dangers to their own health, and have generated a demand for traditional mayonnaise alternatives. In addition, people have also begun to pay attention to the adverse effects that the acquisition of animal-derived raw materials may have on environmental sustainability. Therefore, the use of maitake mushroom protein to replace egg yolk to develop a new type of mayonnaise can meet the people's diversified food consumption and nutritional and health needs with higher quality, which is in line with the current concept of big food and has good practical significance. Summary of the invention

[0009] The invention provides a method for preparing a Grifola frondosa protein emulsifier capable of delaying digestion of emulsion lipids, and uses the Grifola frondosa protein emulsifier as an alternative emulsifier for egg yolk in the development of novel mayonnaise.

[0010] The technical solution of the present invention uses cultivated edible fungi Grifola frondosa as raw material to prepare protein, and performs microgel treatment on the prepared protein to form Pickering particles. The formed microgel particles are used as an emulsifier to prepare a stable protein emulsion and achieve the effect of delaying the fat digestion of the emulsion. Then, Grifola frondosa protein microgel is used as a natural emulsifier to replace egg yolk to prepare a new cholesterol-free mayonnaise.

[0011] A preparation method of a Grifola frondosa protein emulsifier and an emulsion having the function of delaying the digestion of emulsion lipids is carried out according to the following steps:

[0012] (1) Using cultivated edible fungus Grifola frondosa as raw material, preparing Grifola frondosa protein by alkali dissolution and acid precipitation method;

[0013] (2) preparing an 8.6% solution of Grifola frondosa protein and subjecting it to heat denaturation treatment in a water bath at 70°C for 10 min;

[0014] (3) Add a certain amount of TG enzyme to the denatured protein solution and keep it in a 55°C water bath for 6 h;

[0015] (4) After the insulation is completed, the protein solution is heated at 90°C for 15 min, cooled to room temperature, and placed at 4°C overnight;

[0016] (5) adding water to the obtained protein gel to crush it, and using ultrasound to crush it to obtain a microgel solution;

[0017] (6) Preparation of Grifola frondosa protein emulsion: The obtained microgel solution and corn oil were dispersed at a mass ratio of 9:1 at 10,000 rpm for 1 minute to prepare a crude emulsion; the crude emulsion was further homogenized by ultrasonic equipment to obtain a final emulsion; the ultrasonic working parameters were: power 400 W, ultrasonic time 5 min, ultrasonic working 5 s and rest 5 s, the ultrasonic probe was placed 1 cm from the bottom of the beaker, and the ultrasonic temperature was controlled at 20 °C.

[0018] The amount of TG enzyme added in step (3) is 10 U / g.

[0019] The ultrasonic working parameters in step (5) are as follows: ultrasonic power 240W, total ultrasonic time 20min, working time 2min, rest time 2min. The temperature is controlled below 30°C during the ultrasonic process.

[0020] The preparation method of the novel mayonnaise without egg yolk is carried out according to the following steps:

[0021] (1) A 2.2% griseoside protein microgel solution was prepared according to the above method, mixed with vegetable oil at a mass ratio of 2:3, and dispersed at 15,000 rpm for 3 min using a high-speed disperser. The dispersed emulsion was subjected to a high-pressure homogenization treatment for one cycle under the conditions of a primary pressure of 200 bar and a secondary pressure of 50 bar to obtain a final emulsion.

[0022] (2) preparing an aqueous solution containing sucrose, sodium chloride and potassium sorbate and adjusting the pH to 4.0 with white vinegar; the mass fractions of sucrose, sodium chloride and potassium sorbate are 20%, 2% and 0.005% respectively.

[0023] (3) The homogenized emulsion and the aqueous solution are mixed and stirred in a mass ratio of 9:1 to obtain a mayonnaise product.

[0024] The vegetable oil in step (1) is soybean oil, peanut oil, corn oil, sunflower oil, rapeseed oil, linseed oil or olive oil.

[0025] Compared with untreated maitake protein, the physical and oxidative stability of the emulsion prepared by maitake protein microgel induced by TG enzyme synergistic heat treatment is significantly improved, and the release rate and final release amount of free fatty acids during gastrointestinal digestion are significantly reduced, which has the potential effect of reducing fat intake and can be used for the development of low-fat foods. By using edible fungus protein as a natural emulsifier to replace egg yolk to prepare a new mayonnaise, cholesterol intake is effectively reduced, reducing the potential health risks of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the average particle size of Grifola frondosa protein microgels formed in different ways;

[0027] Figure 2 Particle size distribution of Grifola frondosa protein microgels formed by different methods;

[0028] Figure 3 The emulsifying activity and emulsifying stability of Grifola frondosa protein microgels formed in different ways;

[0029] Figure 4 The oxidative stability of emulsions prepared for Grifola frondosa protein microgels formed by different methods;

[0030] Figure 5 The release of free fatty acids during the digestion of emulsions prepared from Grifola frondosa protein microgels formed by different methods;

[0031] Figure 6 The viscosity of emulsion mayonnaise was prepared for different oil phases;

[0032] Figure 7 viscoelastic properties of mayonnaise emulsions prepared for different oil phases;

[0033] Figure 8 Centrifugal stability of mayonnaise prepared with different oil phases (a, b, c, d, e, f, g correspond to soybean oil, peanut oil, corn oil, sunflower oil, rapeseed oil, linseed oil, and olive oil, respectively). DETAILED DESCRIPTION

[0034] 1. Extraction of Grifola frondosa protein

[0035] The fruiting bodies of the maitake mushroom dried in a constant temperature air drying oven at 40°C were crushed, the maitake mushroom powder was mixed with 95% ethanol at a solid-liquid ratio of 1:4 (g:mL), stirred at a constant temperature of 40°C for 2h, and degreased, and finally the solution was centrifuged to collect the precipitate. The degreasing process was repeated three times, and finally the obtained precipitate was dried in a blast drying oven at 40°C to collect the powder.

[0036] The defatted grisea frondosa powder was mixed with distilled water at a solid-liquid ratio of 1:40 (g:mL), heated to 50°C in a water bath, adjusted to pH 10 with 2mol / LNaOH solution, and stirred and extracted at 50°C for 2h. After leaching for 2h, centrifuged at 8000r / min for 15min, and the supernatant was taken. The supernatant was stirred at room temperature and adjusted to pH 3.0 with 2mol / LHCL, and allowed to stand at 4°C overnight. The solution was centrifuged at 8000r / min for 15min, and the precipitate was collected. The precipitate was re-dissolved at a solid-liquid ratio of 1:10 (g:mL). The above operation was repeated once, and the final precipitate was re-dissolved in distilled water, and the pH was adjusted to 7.0 with 1mol / L HCl, and the water was removed by vacuum freeze drying.

[0037] 2. Preparation of Grifola frondosa protein microgel

[0038] Example 1

[0039] Prepare 12% maitake mushroom protein powder, stir magnetically at 25°C for 1 hour, and place in a 4°C refrigerator overnight. Denature the fully hydrated protein solution in a 70°C water bath for 10 minutes. Cool the denatured protein solution to room temperature and heat it at 90°C for 15 minutes. Cool the heated protein solution to room temperature with tap water and place it in a refrigerator overnight. Add water to the gel formed overnight and crush it using an ultrasonic device. Ultrasonication was performed for 2 minutes and 2 minutes intermittently for a total of 20 minutes. The temperature was kept below 30°C during the ultrasonic process. Finally, the formed microgel solution was freeze-dried.

[0040] Example 2

[0041] Prepare 12% maitake mushroom protein powder, stir magnetically at 25°C for 1 hour, and place in a 4°C refrigerator overnight. Denature the fully hydrated protein solution in a 70°C water bath for 10 minutes. After cooling the denatured protein solution to room temperature, add TG enzyme (10U / g), stir evenly, and keep warm in a 55°C water bath for 6 hours. After the insulation is completed, place it in a water bath and heat it at 90°C for 15 minutes. After heating, cool the protein solution to room temperature with tap water and place it in a refrigerator overnight. Add water to the gel formed overnight and crush it using an ultrasonic device. Ultrasonication works for 2 minutes, with an interval of 2 minutes, for a total of 20 minutes. During the ultrasonic process, the temperature is controlled below 30°C. Finally, the formed microgel solution is freeze-dried.

[0042] The average particle size of the gristma-fotoxin microgels formed by different methods is shown in Figure 1 Compared with the untreated Grifola frondosa protein (117.3nm), the cross-linking treatment caused the average particle size of the protein to increase significantly. After heat treatment and enzyme-heat combined treatment, the Grifola frondosa protein increased to 231.8nm and 280.4nm, respectively, which were 0.98 and 1.39 times higher than the untreated Grifola frondosa protein. The particle size distribution of Grifola frondosa protein microgels formed by different methods was analyzed, and the results are as follows: Figure 2 The particle size distribution of Grifola frondosa protein microgels shifted to the right after heat treatment and enzyme-heat combined treatment, showing a trend of increasing particle size. The results showed that heat treatment and enzyme-heat combined treatment led to protein aggregation. Among them, protein aggregation was more significant after enzyme-heat combined treatment.

[0043] 3. Preparation of Grifola frondosa protein microgel emulsion

[0044] Example 3

[0045] Untreated Grifola frondosa protein powder was re-dissolved in water to prepare a 1% solution, and the pH of the solution was adjusted to 7.0. Corn oil and protein solution were mixed in a ratio of 1:9 (w / v), and the mixed solution was dispersed in a high-speed disperser at 10000rpm for 1min to prepare a crude emulsion. 50mL of the crude emulsion was placed in a beaker, and the beaker was placed in an ultrasonic cell disruptor for ultrasonication. Ultrasonic working conditions: power 400W, ultrasonic time 5min, ultrasonic working 5s intermittent 5s, and the ultrasonic probe was placed 1cm from the bottom of the beaker.

[0046] Example 4

[0047] The Grifola frondosa protein microgel powder formed by heat treatment was re-dissolved in water to prepare a 1% solution, and the pH of the solution was adjusted to 7.0. Corn oil and protein solution were mixed in a ratio of 1:9 (w / v), and the mixed solution was dispersed in a high-speed disperser at 10000r / min for 1min to prepare a crude emulsion. 50mL of the crude emulsion was taken into a beaker, and the beaker was placed in an ultrasonic cell disruptor for ultrasonication. Ultrasonic working conditions: power 400W, ultrasonic time 5min, ultrasonic working 5s intermittent 5s, and the ultrasonic probe was placed 1cm from the bottom of the beaker.

[0048] Example 5

[0049] The Grifola frondosa protein microgel powder formed by enzyme-heat composite treatment was re-dissolved in water to prepare a 1% solution, and the pH of the solution was adjusted to 7.0. Corn oil and protein solution were mixed in a ratio of 1:9 (w / v), and the mixed solution was dispersed in a high-speed disperser at 10000r / min for 1min to prepare a crude emulsion. 50mL of the crude emulsion was taken in a beaker, and the beaker was placed in an ultrasonic cell disruptor for ultrasonication. Ultrasonic working conditions: power 400W, ultrasonic time 5min, ultrasonic work 5s intermittent 5s, and the ultrasonic probe was placed 1cm from the bottom of the beaker.

[0050] Result analysis: Figure 3 and Figure 4 It can be seen that after the Grifola frondosa protein was treated with microgel, the physical stability and oxidative stability of the Grifola frondosa protein emulsion were significantly improved. Compared with the control group, the microgel formed by the enzyme-heat composite treatment had the best effect, the emulsification stability ESI increased by 41.32%, and the thiobarbituric acid value decreased by 30.56%. After the Grifola frondosa protein was treated with composite modification, the release rate and final release amount of free fatty acids in its stable emulsion during digestion were reduced. The results are as follows Figure 5 As shown in Table 1. The final release of free fatty acids was significantly reduced by 15.1%, indicating that the application of Grifola frondosa protein microgel significantly inhibited the digestion of fat in the intestine.

[0051] 4. Preparation of Novel Mayonnaise

[0052] Prepare 3% of the maitake protein microgel and mix it with soybean oil in a mass ratio of 40:60, and then disperse it at 15000r / min for 3min by a high-speed disperser. In order to obtain a more uniform and delicate emulsion, the dispersed sample was subjected to high-pressure homogenization, with a primary pressure of 200bar and a secondary pressure of 50bar, and homogenized for one cycle. The obtained emulsion was mixed with the prepared aqueous solution at a ratio of 9:1 (w / w), and dispersed at 2000r / min for 30s by a high-speed disperser to make it evenly mixed. The aqueous solution contained 20% sucrose, 2% sodium chloride and 0.005% potassium sorbate by mass, and the pH was adjusted to 4.0 with edible white vinegar.

[0053] Table 1. Fitting characteristic parameters of free fatty acid release (first order kinetic equation y = a(1-e -kt ))

[0054]

[0055] Example 6

[0056] The prepared 3% maitake protein microgel was mixed with soybean oil in a mass ratio of 40:60 and dispersed at 15000r / min for 3min by a high-speed disperser to obtain a crude emulsion. In order to obtain a more uniform and delicate emulsion, the dispersed sample was subjected to high-pressure homogenization, with a primary pressure of 50ba and a secondary pressure of 200ba, and homogenized for one cycle. The obtained emulsion was mixed with the prepared aqueous solution in a ratio of 9:1 (w / w), and dispersed at 2000r / min for 30s by a high-speed disperser to mix evenly. The aqueous solution contained 20% sucrose, 2% sodium chloride and 0.005% potassium sorbate by mass, and the pH was adjusted to 4.0 with edible white vinegar, and the pH was adjusted to 4.0 with edible white vinegar.

[0057] Example 7

[0058] The method of Example 7 is the same as that of Example 6, except that the oil phase is peanut oil.

[0059] Example 8

[0060] Example 8 is the same as the method of Example 6, except that the oil phase is corn oil.

[0061] Example 9

[0062] The method of Example 9 is the same as that of Example 6, except that the oil phase is sunflower oil.

[0063] Example 10

[0064] The method of Example 10 is the same as that of Example 6, except that the oil phase is rapeseed oil.

[0065] Example 6

[0066] The method of Example 11 is the same as that of Example 6, except that the oil phase is linseed oil.

[0067] Example 7

[0068] The method of Example 12 is the same as that of Example 6, except that the oil phase is olive oil.

[0069] Result analysis: Figure 6 and Figure 7 It can be seen that the rheological properties of emulsions prepared with different oil phases are different. Peanut oil has lower viscosity and viscoelasticity, while linseed oil and olive oil have higher viscosity and viscoelasticity. Figure 8 The results of centrifugal stability tests showed that mayonnaise prepared with peanut oil was the least stable, mayonnaise prepared with linseed oil was the most stable, and there was little difference between the other oils.

Claims

1. A preparation method of a Grifola frondosa protein emulsifier and an emulsion having the function of delaying the digestion of emulsion lipids, characterized in that Follow the steps below: (1) Using cultivated edible fungus Grifola frondosa as raw material, preparing Grifola frondosa protein by alkali dissolution and acid precipitation method; (2) preparing an 8.6% solution of Grifola frondosa protein and subjecting it to heat denaturation treatment in a water bath at 70°C for 10 min; (3) Add a certain amount of TG enzyme to the denatured protein solution and keep it in a 55°C water bath for 6 h; (4) After the insulation is completed, the protein solution is heated at 90°C for 15 min, cooled to room temperature, and placed at 4°C overnight; (5) adding water to the obtained protein gel to crush it, and using ultrasound to crush it to obtain a microgel solution; (6) Preparation of Grifola frondosa protein emulsion: The obtained microgel solution and corn oil were dispersed at a mass ratio of 9:1 at 10,000 rpm for 1 minute to prepare a crude emulsion; the crude emulsion was further homogenized by ultrasonic equipment to obtain the final emulsion.

2. The method for preparing a Grifola frondosa protein emulsifier and an emulsion having the function of delaying the digestion of emulsion lipids according to claim 1, characterized in that The amount of TG enzyme added in step (3) is 10 U / g.

3. The method for preparing a Grifola frondosa protein emulsifier and an emulsion having the function of delaying the digestion of emulsion lipids according to claim 1, characterized in that According to the ultrasonic working parameters described in step (5): ultrasonic power 240W, total ultrasonic time 20min, working time 2min, rest time 2min; the temperature is controlled below 30°C during the ultrasonic process.

4. The method for preparing a Grifola frondosa protein emulsifier and an emulsion having the function of delaying the digestion of emulsion lipids according to claim 1, characterized in that The ultrasonic working parameters in step (6) are: power 400 W, ultrasonic time 5 min, ultrasonic working 5 s and rest 5 s, the ultrasonic probe is placed 1 cm away from the bottom of the beaker, and the ultrasonic temperature is controlled at 20°C.

5. A method for preparing a novel yolk-free mayonnaise, characterized in that Follow the steps below: (1) A 2.2% gristma-fotoxin protein microgel solution was prepared according to the above method, mixed with vegetable oil at a mass ratio of 2:3, and dispersed at 15,000 rpm for 3 min using a high-speed disperser; the dispersed emulsion was subjected to a high-pressure homogenization treatment under the conditions of a primary pressure of 200 bar and a secondary pressure of 50 bar for one cycle to obtain a final emulsion; (2) preparing an aqueous solution containing sucrose, sodium chloride and potassium sorbate and adjusting the pH to 4.0 with white vinegar; the mass fractions of sucrose, sodium chloride and potassium sorbate are 20%, 2% and 0.005% respectively; (3) The homogenized emulsion and the aqueous solution are mixed and stirred in a mass ratio of 9:1 to obtain a mayonnaise product.

6. The method for preparing the novel yolk-free mayonnaise according to claim 5, characterized in that The vegetable oil in step (1) is soybean oil, peanut oil, corn oil, sunflower oil, rapeseed oil, linseed oil or olive oil.

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