A low-fat, low-calorie, algae-free protein drink and a preparation method thereof

Abstract

The invention relates to the technical field of food processing, and in particular to a method for preparing a low-fat, low-calorie, algae-free protein drink, comprising: S1, the algae raw material is a chlorophyll-deficient high-protein microalgae, the algae cell protein content is ≥35%, the oil content is ≤15%, and the starch content is ≤30%; S2, water is added to adjust the dry weight of the microalgae cells to 50-150 g / L, and a high-pressure homogenizer is used to crush the cells to obtain a cell slurry; if the microalgae raw material is a slender euglensis without a cell wall or a Chlamydomonas reinhardtii with a cell wall as a polysaccharide protein, the cells are homogenized 1-2 times; if the microalgae raw material is a microalgae with a cell wall and the main component of the cell wall is cellulose, high-pressure homogenization is required until the particles below 1 μm in the cell slurry account for more than 90% of the total number of particles, and the particles below 300 nm account for more than 50% of the total number of particles; S3, one or more of cellulase, protease, amylase and saccharifying enzyme are added according to the type of microalgae for enzymolysis, and a pH regulator is added after the enzymolysis is completed; S4, homogenization is performed again; S5 sterilization and filling. The microalgae protein beverage of the present invention can better meet the needs of people who are allergic to milk or lactose intolerant or who pursue low calorie intake.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a low-fat, low-calorie, algae-odor-free microalgae protein beverage and a preparation method thereof. Background Art

[0002] At present, the plant milk beverage products on the market mainly include soy milk, peanut milk, nut milk such as almond or almond kernel, cereal milk such as oat or rice, and coconut milk. Soy milk has a high protein content (about 4.0g / 100mL), but it often contains anti-nutritional factors such as chymosin inhibitors, and many people do not accept the beany smell of soy milk; peanut milk has a low protein content (Yinlu peanut milk 1.2g / 100mL, Daliyuan peanut milk 1.1g / 100mL), and the current peanut milk products are basically compounded with milk, which still contain milk ingredients (some people who are allergic to milk or lactose intolerant cannot drink it). In addition, some people are allergic to peanut protein. Nuts such as almonds and almond kernels have a high fat content, and their dairy products generally have a high fat content, but a low protein content (almond milk 0.6g / 100mL, almond milk 1.2g / 100mL), and the nutrition is relatively simple. The protein content of cereal milk such as oatmeal and rice is relatively low (the protein content of products without milk is below 1.0g / 100mL), and the main component is starch, so the carbohydrate content is high, the protein to beverage calorie ratio is low, and the nutrition is relatively simple. The protein content of coconut milk is even lower (less than 1%), and the saturated fat content of oil is high.

[0003] As for higher plants such as peanuts, nuts, oats, and rice, the ratios of protein, oil, and starch content are relatively fixed. For example, peanuts contain 24-35g / 100g of protein and 40-50g / 100g of fat or oil; almond kernels contain 28g / 100g of protein and 55-61g / 100g of fat (oil); oats contain 19.43g / 100g of protein and 50-65g / 100g of starch; rice contains 6.7-7.4g / 100g of protein and 77.6g / 100g of starch. As a result, the above-mentioned plant milks all have a common problem, that is, either the protein content is low, or the carbon water or oil content is high relative to the protein content. For some people who are controlling their calorie or sugar intake, the protein / oil or protein / carbon water ratios in these dairy products are not reasonable enough. For example, peanuts and tree nuts have a high fat content. Increasing the protein content in dairy products will inevitably increase the fat content of the products, which is not conducive to the stability of the beverage and easily causes the calorie content of the beverage to be too high. For example, oats and rice have a high carbohydrate content. Increasing the protein content in oat milk and rice milk products will also increase the starch content in the products, increase the carbohydrate content, and increase the calories.

[0004] As more and more microalgae species are included in the food list, downstream foods or nutritional supplements made from microalgae are gradually becoming known to people, such as Spirulina, Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella protothecoides, Chlamydomonas, Euglena, Haematococcus pluvialis, etc. As a single-cell plant, microalgae have the advantages of a wide variety, short growth cycle, rapid cultivation, and the ability to easily change the composition ratio of protein, oil and starch in cells through culture conditions. At the same time, microalgae are single-cell organisms, and it is relatively easy to induce mutant cell strains with certain metabolic preferences. These advantages allow protein drinks produced from microalgae to reduce the fat or sugar content while maintaining a high protein content to meet the needs of the relevant population.

[0005] Nevertheless, the current technology only uses microalgae as a source of certain ingredients, such as algae-derived DHA, algae-derived EPA, algae-derived protein, algae-derived lutein, etc. Microalgae can not only provide edible oils or proteins, but also provide nutrients such as polysaccharides, dietary fiber, carotenoids, etc. However, in these technologies, when using microalgae as raw materials, the target components in the microalgae biomass must be extracted and purified, which will not only increase the processing cost of microalgae-related products, but also lead to other nutrients in the microalgae being wasted and some extractants remaining. In addition, most algae cells are dark green and have an algae smell, so they need to be decolorized using organic solvents or some decolorizing agents such as activated carbon / diatomaceous earth, but this will also lead to the loss of some nutrients. If the food is directly processed without decolorization, it is necessary to use colorants or flavoring agents to cover the color or smell brought by the microalgae, which means that a large amount of additives are needed. On the one hand, a large amount of additives may not meet food standards, and on the other hand, it is also contrary to the advocacy of healthy eating. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a low-fat, low-calorie, algae-free microalgae protein beverage and a preparation method thereof, which mainly uses chlorophyll-deficient high-protein microalgae as raw materials to make a beverage from whole algae. On the one hand, the beverage can have a better protein / oil ratio or protein / carbon water ratio, and on the other hand, the whole algae can be utilized, thereby solving the technical problems of high cost of existing microalgae utilization, biomass waste, extractant residue, unpleasant color, algae smell, etc.

[0008] (II) Technical solution

[0009] In a first aspect, the present invention provides a method for preparing a low-fat, low-calorie, algae-free microalgae protein beverage, comprising:

[0010] S1. Selecting microalgae raw materials: The microalgae raw materials are chlorophyll-deficient high-protein microalgae, with a protein content of ≥35%, a fat content of ≤15%, and a starch content of ≤30% in the algae cells;

[0011] S2. Cell disruption: add water to adjust the dry weight of microalgae cells to 50-150 g / L, and use a high-pressure homogenizer to disrupt them to obtain cell slurry;

[0012] If the microalgae raw material is cell wall-free Euglena gracilis or Chlamydomonas reinhardtii whose cell wall is polysaccharide protein, only homogenize 1-2 times;

[0013] If the microalgae raw material is a microalgae with a cell wall and the main component of the cell wall is cellulose, high-pressure homogenization treatment is required until the particles below 1 μm in the cell slurry account for more than 90% of the total number of particles, and the particles below 300 nm account for more than 50% of the total number of particles;

[0014] S3, enzymatic hydrolysis: adding water to the cell paste to dilute the cell paste;

[0015] If the microalgae raw material is microalgae with cell walls, 600-5000 units / kg of cellulase, 5000-8000 units / kg of protease, 500-1000 units / kg of amylase and 1000-3000 units / kg of saccharifying enzyme are added to carry out enzymatic hydrolysis reaction based on the weight of the cell slurry after adding water;

[0016] If the microalgae raw material is Euglena gracilis without cell walls, only 5000-8000 units / kg of protease is added for enzymatic hydrolysis based on the weight of the cell paste after adding water;

[0017] After the reaction is completed, a pH adjuster is added, and the mixture is stirred and mixed to obtain a mixture;

[0018] S4, homogenization: heat the mixture to 50-80°C, transfer it into a high-pressure homogenizer for homogenization, repeat 1-4 times to obtain a homogenized mixture;

[0019] S5. Sterilization and filling: The homogenized mixture is sterilized and then filled or filled and then sterilized to obtain the finished product.

[0020] According to a preferred embodiment of the present invention, in S1, the microalgae is yellow protein nucleus Chlorella, the deposit number of which is CGMCC NO.40458, the deposit date is April 27, 2023, and the protein content of the yellow protein nucleus Chlorella is 50-60%; or, the microalgae is white protein nucleus Chlorella, the deposit number of which is CGMCC NO.40787, the deposit date is September 9, 2023, and the protein content of the white protein nucleus Chlorella is 40-50%; or, the microalgae is white slender Euglena, the deposit number of which is CGMCC NO.40699, the deposit date is June 5, 2023. The protein content of the white slender Euglena is 35-45%.

[0021] According to a preferred embodiment of the present invention, in S2, the pressure of the high pressure homogenizer is 60-130 MPa;

[0022] When the microalgae is Chlorella pyrenoidosa, a pressure of 100-130 MPa is used for homogenization; when the dry weight of the microalgae cells is below 80 g / L, a pressure of 100-105 MPa is used for homogenization, when it is 80-100 g / L, a pressure of 106-115 MPa is used for homogenization, and when it is 100-130 g / L, a pressure of 116-130 MPa is used for homogenization;

[0023] When the microalgae is Euglena gracilis, homogenization is performed 1-2 times at a pressure of 60-80 MPa.

[0024] According to a preferred embodiment of the present invention, in S3, during the enzymatic hydrolysis process, water is added to control the dry matter content of the microalgae to 1-10 g / 100 mL (i.e., 10-100 g / L) and the protein content to ≥1.0 g / 100 mL; the enzymatic hydrolysis conditions are: reacting at 50±5° C. for 45-90 min.

[0025] According to a preferred embodiment of the present invention, in S3, the cellulase is food grade cellulase; the protease is one or a combination of food grade alkaline protease, neutral protease, papain, bromelain, trypsin and pepsin; the amylase is one or a combination of α-amylase and pullulanase; the saccharifying enzyme is one or a combination of glucoamylase or β-amylase.

[0026] According to a preferred embodiment of the present invention, in S3, when the microalgae raw material is a microalgae with a cell wall, 3000 units / kg of cellulase, 6000 units / kg of protease, 1000 units / kg of amylase and 1500 units / kg of saccharifying enzyme are added to carry out enzymatic hydrolysis for 60 minutes based on the weight of the cell slurry after adding water. When the algae species is Chlorella vulgaris and contains 20-30% starch, after the addition of the saccharifying enzyme and the reaction is completed, the resulting beverage has a certain sweetness, the most moderate taste, and good smoothness and consistency.

[0027] According to a preferred embodiment of the present invention, in S3, the pH regulator is one or more of phosphate, carbonate or citrate, which is used to adjust the pH of the reaction solution after enzymatic hydrolysis to 7.0-8.0. Most of the microalgae-derived proteins are alkaline-soluble gluten proteins, which have poor solubility in common neutral or weakly acidic beverage systems and are prone to protein instability; therefore, it is preferred to add an appropriate amount of sodium carbonate or sodium citrate to help adjust the pH value of the microalgae protein beverage to a neutral or alkaline environment, improve the taste and extend the shelf life.

[0028] According to a preferred embodiment of the present invention, in S4, the pressure of the homogenization treatment is 60-100 MPa, and the homogenization is repeated 1-3 times. Through the homogenization treatment, the microalgae beverage can be kept stable for a long time, with uniform phase and no stratification without adding an emulsifier.

[0029] According to a preferred embodiment of the present invention, in S5, the sterilization conditions are selected from one or a combination of the following three sterilization methods: 135-150°C treatment for 2-10s, 75-100°C pasteurization and 100-121°C treatment for 10-25min; sterilization is performed before or after filling.

[0030] Filling can be done in 1000mL or 250mL Tetra Pak boxes.

[0031] In a second aspect, the present invention provides a low-fat, low-calorie, algae-free microalgae protein beverage, which is prepared by the preparation method of any of the above embodiments.

[0032] (III) Beneficial effects

[0033] The main technical effects of the present invention are:

[0034] (1) The microalgae are rich in protein, have a short growth cycle, are cultured quickly, are easy to screen mutants with specific metabolic preferences, and can easily accumulate more protein in stress cells and increase the protein / carbohydrate or protein / oil ratio by adjusting the culture medium components (such as nitrogen source concentration and form, C / N ratio) or culture conditions (such as autotrophic light intensity, light-dark cycle, flow acceleration, pH, dissolved oxygen in heterotrophic culture medium), etc., which have advantages that higher plants cannot match. As a high-protein, low-fat, and low-calorie food raw material, there is currently no higher biomass food raw material comparable to microalgae.

[0035] The invention adopts high-protein microalgae as the production raw material for plant milk drinks, and utilizes the diversity of microalgae species, short microalgae culture cycle, rapid culture, and easy adjustment of microalgae nutritional composition structure to produce plant milk drink products with better protein / fat or protein / starch content ratios, thereby making up for the unreasonable protein / fat or protein / carbohydrate ratios in existing plant milk drinks such as peanut milk, nut milk, oat milk, and rice milk.

[0036] (2) The present invention uses chlorophyll-deficient high-protein microalgae as processing raw materials, and uses whole algae to make the beverage of the present invention. The technical effects are: on the one hand, there is no need to pre-extract the microalgae, which can save production costs, reduce extractant residues and pollution, and reduce the waste of microalgae biomass. It can also make full use of the rich carotenoids (including but not limited to: lutein for protecting eyesight, antioxidant astaxanthin, fucoxanthin for promoting lipid metabolism and protecting the liver, etc.), lipids, vitamins, polysaccharides, dietary fiber and trace elements contained in the microalgae; on the other hand, since it does not contain dark green chlorophyll, the prepared beverage does not need to add colorants or flavoring agents to cover the color or taste of the product, making the product more natural, green and healthy.

[0037] (3) Chlorella cells have tough cell walls (the main component is cellulose) and cell membranes. Although naked algae do not have cell walls, they have cell membranes that wrap the cell substances. The cells must be broken to dissolve the cell contents and complete the enzymatic hydrolysis reaction, so that the nutrients can enter the beverage. Although traditional wall breakers used in homes or factories can break large-grained crops such as soybeans and peanuts, they are not suitable for Chlorella. Chlorella is a single-cell organism with small cells and tough cell walls. Traditional wall breakers cannot effectively break the cell walls. In the present invention, a high-pressure homogenizer is used to break the cells of Chlorella at a pressure of 100-130MPa, and an efficient cell breaking effect can be achieved in a short time.

[0038] (4) The main component of most algae cell walls is cellulose. The cell wall debris is easy to flocculate and precipitate and stratify. During the precipitation process, protein will be adsorbed and precipitated and stratified, which is not conducive to the uniformity and stability of the beverage. The present invention adjusts the conditions and number of treatments of high-pressure homogenization to achieve the detailed requirements of "more than 90% of particles below 1 μm and more than 50% of particles below 300 nm" to maintain the uniformity and stability of the beverage phase. At the same time, after the enzymatic hydrolysis reaction is completed, the high-pressure homogenization treatment is repeated to further stabilize the cellulose substances in the beverage, without the need to use a large amount of thickeners to prevent the precipitation of cellulose substances like peanut milk.

[0039] (5) Most algae-derived proteins are alkali-soluble gluten. In particular, most of the protein of Chlorella vulgaris is gluten. The present invention adds a pH adjuster such as dipotassium hydrogen phosphate, sodium carbonate or sodium citrate to maintain the pH of the milk beverage in a neutral to alkaline environment, thereby promoting the dissolution of algae-derived proteins and enhancing the stability of the beverage. DETAILED DESCRIPTION

[0040] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods.

[0041] The present invention provides a method for preparing a low-fat, low-calorie, algae-free protein beverage, comprising the following steps:

[0042] (1) Selection of microalgae raw materials: The microalgae raw materials are chlorophyll-deficient high-protein microalgae, with the protein content in the algae cells being ≥35%, the oil content being ≤15%, and the starch content being ≤30%.

[0043] Preferably, the microalgae is yellow protein-nuclear Chlorella, whose preservation number is CGMCC NO.40458, and the preservation date is April 27, 2023, and the protein content of the yellow protein-nuclear Chlorella is 50-60%; or, the microalgae is white protein-nuclear Chlorella, whose preservation number is CGMCC NO.40787, and the preservation date is September 9, 2023, and the protein content of the white protein-nuclear Chlorella is 40-50%; or, the microalgae is white slender Euglena, whose preservation number is CGMCC NO.40699, and the preservation date is June 5, 2023; the protein content of the white slender Euglena is 35-45%.

[0044] (2) Cell disruption: Add water to adjust the dry weight of microalgae cells to 50-150 g / L, and use a high-pressure homogenizer to disrupt them to obtain cell slurry;

[0045] If the microalgae raw material is a cell wall-free Euglena gracilis or a cell wall of Chlamydomonas reinhardtii, it is homogenized only 1-2 times to obtain a cell paste;

[0046] If the microalgae raw material is microalgae with cell walls and the main component of the cell walls is cellulose, high-pressure homogenization treatment is required until the particles below 1 μm in the cell slurry account for more than 90% of the total number of particles, and the particles below 300 nm account for more than 50% of the total number of particles.

[0047] Before using a high-pressure homogenizer to break the algae powder / algae mud, water is added to make the algae cell dry matter concentration 50-150g / L. When the raw material is dry algae powder, the amount of water added is relatively large. When the raw material is algae mud collected by a centrifuge, the amount of water added can be reduced because the algae mud itself contains water.

[0048] Preferably, when the microalgae is Chlorella vulgaris, a pressure of 100-130 MPa is used for homogenization; when the dry weight of the microalgae cells is below 80 g / L, a pressure of 100-105 MPa is used for homogenization, when it is 80-100 g / L, a pressure of 106-115 MPa is used for homogenization, and when it is 100-130 g / L, a pressure of 116-130 MPa is used for homogenization; when the microalgae is Euglena gracilis, a pressure of 60-80 MPa is used for homogenization 1-2 times to achieve the effect of breaking the cell membrane of Euglena gracilis.

[0049] (3) Enzymatic hydrolysis: Add water to the cell paste to dilute the cell paste;

[0050] If the microalgae raw material is a microalgae with a cell wall, based on the weight of the cell slurry after adding water, 600-5000 units / kg of cellulase, 5000-8000 units / kg of protease, 500-1000 units / kg of amylase and 1000-3000 units / kg of saccharifying enzyme are added for enzymatic hydrolysis; if the microalgae raw material is a gracilis algae without a cell wall, based on the weight of the cell slurry after adding water, only 5000-8000 units / kg of protease are added for enzymatic hydrolysis. After the reaction is completed, a pH adjuster is added, and the mixture is stirred and mixed to obtain a mixture.

[0051] Preferably, when the microalgae raw material is microalgae with cell walls (such as Chlorella), 3000 units / kg of cellulase, 6000 units / kg of protease, 1000 units / kg of α-amylase and pullulanase and 1500 units / kg of saccharifying enzyme are added to the weight of the cell slurry after adding water for enzymatic hydrolysis and saccharification reaction for 60 minutes. When Chlorella with a starch content of 20-30% is used as the raw material, after the addition of the saccharifying enzyme and the reaction is completed, the sweetness and taste of the resulting beverage are the most moderate, and the smoothness and thickness are both good.

[0052] The gracilis algae does not contain cell walls, so no cellulase is added. When the cells are broken, there is no need to break the cell walls, only the cell membrane of the gracilis algae needs to be destroyed, so a smaller homogenization pressure treatment can be used. The starch component of gracilis algae is mainly β-glucan, which is not easy to hydrolyze. There is no need to hydrolyze and saccharify the starch of gracilis algae, so no amylase and saccharifying enzyme are added. When using gracilis algae to prepare the protein drink of the present invention, only an appropriate amount of protease needs to be added for hydrolysis. The cell wall of Chlamydomonas reinhardtii is a polysaccharide protein, not a tough cellulose cell wall, so no cellulase or only a small amount of cellulase may be added during the enzymatic hydrolysis process.

[0053] The added cellulase is food grade cellulase, the added protease is one or a combination of food grade alkaline protease, neutral protease, papain, bromelain, trypsin and pepsin, the amylase is one or a combination of α-amylase and pullulanase, and the saccharifying enzyme is one or a combination of glucoamylase or β-amylase.

[0054] When adding water to the cell paste, the amount of water is preferably such that the dry matter content of the microalgae is 1-10 g / 100 mL (10-100 g / L) and the protein content is ≥1.0 g / 100 mL. The enzymatic hydrolysis conditions are: reacting at 50±5° C. for 45-90 min.

[0055] After the saccharification reaction, a small amount of starch in the algae slurry is hydrolyzed to generate smaller dextrins, oligosaccharides, maltose or glucose to improve the taste of food, increase sweetness or promote digestion and absorption, without adding sucrose or other sweeteners. In the low-fat, low-calorie, algae-free microalgae protein beverage of the present invention, no additional sweetener is added, and saccharifying enzymes are directly used to hydrolyze starch to produce a light sweetness, and sweet and non-obvious sweet drinks can be obtained by controlling the amount of saccharifying enzyme added and the hydrolysis time. In addition, since chlorophyll-deficient high-yield protein algae strains are used as production raw materials, the beverage of the present invention does not require any flavoring or colorant, and the finished product has the natural color and smell / flavor of the beverage.

[0056] After the enzymatic hydrolysis reaction is completed, a small amount of pH regulator is added to adjust the pH of the reaction solution after the enzymatic hydrolysis to 7.0-8.0. The pH regulator is one or more of dipotassium hydrogen phosphate, carbonate or sodium citrate. In addition, a water retaining agent may be appropriately added to improve the lubricity and softness of the product, such as phosphate; phosphate can also protect the beverage and reduce protein denaturation and aggregation precipitation during high-temperature sterilization. However, the present invention does not add sweeteners, nor does it add any flavoring agents and colorants for correcting odor or taste.

[0057] Most of the microalgae-derived proteins are alkaline-soluble gluten proteins, which have poor solubility in common neutral or weakly acidic beverage systems and are prone to protein instability. Therefore, adding an appropriate amount of sodium carbonate or sodium citrate can help adjust the pH value of microalgae protein drinks to a neutral or alkaline environment to improve the taste and extend the shelf life.

[0058] (4) Homogenization: The mixture obtained in step (3) is heated to 50-80° C. and transferred to a high-pressure homogenizer for homogenization at least once. The pressure of the homogenization is 60-100 MPa, 80-100 MPa when Chlorella is used as the raw material, and 60-80 MPa when Euglena is used as the raw material. The homogenization is repeated 1-3 times. After the high-pressure homogenization, the enzymatic reaction products and additives are evenly mixed, and the particles such as the cell wall are further refined, so that the taste of the beverage is more delicate, lubricated and soft, and the physical phase of the beverage is more stable.

[0059] (5) Sterilization and filling: The mixture after homogenization in step (4) is sterilized and then immediately filled to obtain the finished product.

[0060] Ultra-high temperature instantaneous sterilization conditions are 135-150℃ for 2-10s; filling can be aseptically filled in 1000mL or 250mL Tetra Pak-style packaging boxes. In addition, pasteurization can be performed after filling, or high temperature sterilization can be performed at 100-121℃ for 10-25min.

[0061] In addition, in addition to the above steps, microalgae culture and algae cell collection steps may also be included. Microalgae culture is mainly carried out by heterotrophic culture. For example, SE medium or BG-11 medium can be used as the basic formula for the culture of Chlorella, the C / N ratio in the fermentation medium and the feed medium can be controlled, and ammonium nitrogen that is more easily absorbed and utilized is used as the nitrogen source. Heterotrophic-light-induced serial culture, high dissolved oxygen induction, appropriate pH induction, hydrogen peroxide induction, plant growth regulator induction and other methods are used to increase the protein and carotenoid content in the algae cells as much as possible, and reduce the oil and starch content, thereby providing algae raw materials with better nutritional component structure for microalgae protein drinks.

[0062] The present invention is described below with reference to specific embodiments.

[0063] Example 1

[0064] This example uses Chlorella vulgaris (accession number CGMCCNO.40458) as the raw material for processing microalgae protein beverage. The steps are as follows:

[0065] (1) Through heterotrophic fermentation, 80 L of fermentation liquid with an algae dry matter concentration of 100 g / L and a cell protein content of 52% (w / w, dry matter) is obtained, and algae cells are separated by a disc centrifuge to obtain about 20 L of algae slurry with an algae cell dry matter concentration of about 400 g / L. The yellow protein core chlorella has an oil content of 10%, a starch content of 25%, a β-carotene content of 3.2 mg / 100 g, and a lutein content of 120 mg / 100 g.

[0066] (2) Place the algae slurry in a 100L storage tank, add drinking water to make the total volume of the algae slurry 80L, and the algae cell dry matter concentration 100g / L, and homogenize it three times with a high-pressure homogenizer at 120MPa to obtain cell wall slurry. In the cell wall slurry, particles below 1μm account for 93%, and particles below 300nm account for 60%.

[0067] (3) Place the broken cell slurry in a 200L mixing tank and add water to 160L. At this time, the dry matter content of the microalgae is 50g / L (5.0g / 100mL). Stir and mix while heating until all substances are evenly dispersed or dissolved. When the temperature reaches 55°C, add 1000 units / kg of cellulase, 6000 units / kg of protease, 500 units / kg of medium-temperature α-amylase, 500 units / kg of pullulanase and 1500 units / kg of saccharifying enzyme based on the total weight of the cell slurry. Stir for 60 minutes for enzymatic hydrolysis and saccharification. After completion, use sodium carbonate to adjust the pH to 7.0-8.0 to promote protein dissolution and beverage stability.

[0068] (4) The temperature was adjusted to 80°C and the mixture was again transferred to the homogenizer and homogenized three times at 100 MPa.

[0069] (5) After homogenization, the product is transported to an ultra-high temperature instantaneous sterilizer and sterilized at 143°C for 5 seconds. It is then immediately filled in an aseptic filling system and packaged in 250 mL Tetra Pak packages.

[0070] The drink contains 2.6g / 100mL of protein, 0.16mg / 100mL of beta-carotene, 6.0mg / 100mL of lutein, 1.25g / 100mL of carbohydrates, and 0.50g / 100mL of fat.

[0071] Example 2

[0072] This example uses Chlorella vulgaris (CGMCC No. 40787) as the raw material for the processing of microalgae protein beverage. The steps are as follows:

[0073] (1) Through heterotrophic fermentation, 75 L of fermentation liquid with an algae dry matter concentration of 120 g / L and a cell protein content of 42% (w / w, dry matter) was obtained, and algae cells were separated by a disc centrifuge to obtain about 20 L of algae slurry with an algae cell dry matter concentration of about 400 g / L. The white protein core chlorella has an oil content of 15%, a starch content of 30%, a β-carotene content of 4.5 mg / 100 g, and a lutein content of 90 mg / 100 g.

[0074] (2) Place the algae slurry in a 100L storage tank, add drinking water to make the total volume of the algae slurry 80L, and the algae cell dry matter concentration 100g / L, and homogenize it three times with a high-pressure homogenizer at 110MPa to obtain cell wall slurry. In the cell wall slurry, particles below 1μm account for 92%, and particles below 300nm account for 58%.

[0075] (3) Place the broken cell slurry in a 200L mixing tank, add water to 160L, at which point the dry matter content of the microalgae is 50g / L (5.0g / 100mL), stir and mix while heating until all substances are evenly dispersed or dissolved, add 800 units / kg of cellulase, 7000 units / kg of protease, 500 units / kg of medium-temperature α-amylase, 500 units / kg of pullulanase and 2000 units / kg of saccharifying enzyme based on the total weight of the cell slurry when the temperature reaches 50°C, and stir for 75 minutes for enzymatic hydrolysis and saccharification. Afterwards, add sodium carbonate to adjust the pH to 7.0-8.0 to promote protein dissolution and beverage stability.

[0076] (4) The temperature was adjusted to 75°C and the mixture was transferred to the homogenizer again and homogenized twice at 80 MPa.

[0077] (5) After homogenization, the product is transported to an ultra-high temperature instantaneous sterilizer and sterilized at 135°C for 8 seconds. It is then immediately filled in an aseptic filling system and packaged in 250 mL Tetra Pak packages.

[0078] The drink contains 2.1g / 100mL of protein, 0.225mg / 100mL of beta-carotene, 4.5mg / 100mL of lutein, 1.5g / 100mL of carbohydrates, and 0.75g / 100mL of fat.

[0079] Example 3

[0080] This example uses white slender euglena (preservation number CGMCC NO.40699) as the raw material for processing microalgae protein beverage. The steps are as follows:

[0081] (1) Through heterotrophic fermentation, 75 L of fermentation liquid with an algae dry matter concentration of 80 g / L and a cell protein content of 43% (w / w, dry matter) was obtained, and algae cells were separated by a disc centrifuge to obtain about 15 L of algae slurry with an algae cell dry matter concentration of about 400 g / L. The wild-type euglena has an oil content of 10%, a paramylon (β-glucan) content of 10%, and a carotenoid content of 160 mg / 100 g.

[0082] (2) Place the algae slurry in a 100 L storage tank, add drinking purified water to make the total volume of the algae slurry 60 L and the algae cell dry matter concentration 100 g / L, and homogenize twice with a high-pressure homogenizer at 80 MPa to obtain broken cell slurry.

[0083] (3) Place the broken cell slurry in a 200L mixing tank and add water to 120L. At this time, the dry matter content of the microalgae is 50g / L (5.0g / 100mL). Stir and mix while heating until all substances are evenly dispersed or dissolved. When the temperature reaches 56°C, add 5000 units / kg of protease based on the total weight of the cell slurry and stir for 45 minutes for enzymatic hydrolysis. Then, add sodium citrate to adjust the pH to 7.0-8.0 to promote protein dissolution and beverage stability.

[0084] (4) The temperature was adjusted to 75°C and the mixture was transferred to the homogenizer again and homogenized twice at 80 MPa.

[0085] (5) After homogenization, the product is transported to an ultra-high temperature instantaneous sterilizer and sterilized at 140°C for 5 seconds. It is then immediately filled in an aseptic filling system and packaged in 250 mL Tetra Pak packages.

[0086] The drink contains 2.15g / 100mL of protein, 8mg / 100mL of carotenoids, about 0g / 100mL of carbohydrates (paramylon is β-glucan, which cannot be detected according to the food carbohydrate detection standard), 0.5g / 100mL of fat, and 0.5g / 100mL of dietary fiber (β-glucan).

[0087] Example 4

[0088] This example uses the yellow pyrenoid Chlorella (deposit number CGMCCNO.40458) of Example 1 as a germplasm resource, changes the culture conditions of heterotrophic fermentation, adjusts the structure of the nutritional components in the pyrenoid Chlorella, and uses the pyrenoid Chlorella as a raw material to prepare a microalgae protein beverage.

[0089] The fermentation culture conditions in Example 1 are: BG-11 is used as the fermentation medium, and 50 g / L of glucose is supplemented. 50 L of the prepared medium is dissolved in a 100 L fermenter, sterilized at 121°C for 30 min, cooled to 25-30°C, and inoculated with 5 L of pyrenoid chlorella cultured in a fermenter. After 5 days of fermentation, fed-batch culture is started, and the feeding rate is adjusted according to the sugar consumption. The feed medium formula is 60 g / L of glucose and 1 g / L of sodium nitrate. After 8 days of fermentation, 80 L of fermentation liquid with an algae dry matter concentration of 100 g / L and a cell protein content of 52% (w / w, dry matter) is obtained.

[0090] In this embodiment, the conditions of heterotrophic fermentation culture are changed as follows: BG-11 is used as the fermentation medium, and 40 g / L of glucose is supplemented. 50 L of the prepared culture medium is dissolved in a 100 L fermenter, sterilized at 121 ° C for 30 min, cooled to 25-30 ° C, and inoculated with protein nucleus chlorella cultured in a 5 L fermenter. After 5 days of fermentation, feed culture is started, and the feed rate is adjusted according to the sugar consumption. The feed culture medium formula is 60 g / L of glucose and 1.5 g / L of ammonium chloride. During the culture process, ammonia water is used to adjust the pH of the fermentation system to be stable at 6.2-6.5, and the temperature is 36-37 ° C. After 8 days of fermentation, 75 L of fermentation liquid with an algae dry matter concentration of 96 g / L and a cell protein content of 61% (w / w, dry matter) is obtained. The oil content of the yellow protein nucleus chlorella is 10%, the starch content is 20%, the β-carotene content is 3.5 mg / 100 g, and the lutein content is 100 mg / 100 g. The algae cells were separated by a disc centrifuge to obtain about 20 L of algae slurry with an algae cell dry matter concentration of about 350 g / L.

[0091] (2) Place the algae slurry in a 100L storage tank, add drinking water to make the total volume of the algae slurry 50L and the algae cell dry matter concentration 140g / L, and homogenize it three times with a high-pressure homogenizer at 130MPa to obtain cell wall slurry. In the cell wall slurry, particles below 1μm account for 95%, and particles below 300nm account for 65%.

[0092] (3) Place the broken cell slurry in a 200L mixing tank and add water to 120L. At this time, the dry matter content of the microalgae is 58.3g / L (5.83g / 100mL). Stir and mix while heating until all substances are evenly dispersed or dissolved. When the temperature reaches 50°C, add 1200 units / kg of cellulase, 6000 units / kg of protease, 800 units / kg of α-amylase, 500 units / kg of pullulanase and 1500 units / kg of saccharifying enzyme based on the total weight of the cell slurry. Stir for 90 minutes for enzymatic hydrolysis and saccharification. After completion, use sodium citrate to adjust the pH to 7.0-8.0 to promote protein dissolution and beverage stability.

[0093] (4) The temperature was adjusted to 80°C and the mixture was transported to the homogenizer again and homogenized twice at 100 MPa.

[0094] (5) After homogenization, fill the mixture into 250 mL aluminum cans and sterilize the mixture at 121° C. for 20 min. Cool the mixture immediately after sterilization, dry the surface, and inspect the mixture to obtain the product.

[0095] The drink contains 3.56g / 100mL of protein, 0.204mg / 100mL of beta-carotene, 5.83mg / 100mL of lutein, 1.17g / 100mL of carbohydrates, and 0.58g / 100mL of fat.

[0096] Example 5

[0097] In this example, in Example 1, step (3) is changed to: based on the total weight of the cell slurry, 1000 units / kg of cellulase, 6000 units / kg of protease, 1000 units / kg of medium-temperature α-amylase, 1000 units / kg of pullulanase and 3000 units / kg of saccharifying enzyme are added, and the mixture is stirred for 90 minutes for enzymatic hydrolysis and saccharification.

[0098] Example 6

[0099] In this example, in Example 1, step (3) is changed to: based on the total weight of the cell slurry, 1000 units / kg of cellulase, 6000 units / kg of protease, 500 units / kg of medium-temperature α-amylase and 500 units / kg of pullulanase are added, and the mixture is stirred for 45 minutes for enzymatic hydrolysis and saccharification.

[0100] Comparative Example 1

[0101] This comparative example is based on Example 1 except that step (4) is omitted.

[0102] Comparative Example 2

[0103] This comparative example is based on Example 1 except that the pH regulator is omitted.

[0104] Comparative Example 3

[0105] In this comparative example, when homogenization was performed in step (2) of Example 1, the homogenizer pressure was 70 MPa and homogenization was performed 3 times to obtain cell wall-broken slurry. In the cell wall-broken slurry, particles below 1 μm accounted for 30%, and particles below 300 nm accounted for less than 10%.

[0106] Comparative Example 4

[0107] This comparative example adopts a household wall-breaking machine for 30 minutes, instead of homogenizing three times at 100 MPa with a high-pressure homogenizer in Example 1.

[0108] The protein content, fat content, starch content, and energy value of the microalgae protein beverages in the above embodiments were calculated or tested, and the results are shown in Table 1:

[0109] Table 1:

[0110]

[0111] Note: *This indicator is the total energy intake caused by consuming 1g of protein while consuming fat and carbohydrates.

[0112] Sensory tests were performed on the microalgae protein beverages of Examples 1-6 and Comparative Examples 3 and 4. Ten sensory assessors performed descriptive tests on the same batch of samples of the examples, and the results are shown in Table 2.

[0113] Table 2:

[0114] color Overall smell Sweetness Smoothness Thickness Example 1 golden No algae smell 4.0 5.5 4.8 Example 2 White No algae smell 4.5 6.0 5.2 Example 3 White No algae smell 0 6.4 5.4 Example 4 golden No algae smell 5.5 5.4 4.0 Example 5 golden No algae smell 7.0 5.0 6.0 Example 6 golden No algae smell 1.0 5.0 5.8 Comparative Example 3 golden No algae smell 2.8 4.0 3.6 Comparative Example 4 Light yellow No algae smell 1.8 2.9 2.7

[0115] Scoring rules: sweetness score 0-9 points, representing from not sweet to sweet, the higher the score, the higher the sweetness, 3.0 points or more represent sweetness; smoothness score 0-9 points, representing the sensory perception from not smooth to smooth, 6.5 ± 0.2 points represent the best smoothness; consistency score 0-9 points, representing the sensory perception from thin to thick, 5.0 ± 0.2 points represent the best consistency; the lower the starch content in the algae cells, the less saccharification substrate, the lower the sweetness of the drink. In Example 3, the slender Euglena, no saccharification reaction was performed, the carbohydrate was about 0g / 100mL, and the sweetness was 0.

[0116] Compared with Example 1, the pressure of the high-pressure homogenizer used in Comparative Example 3 was insufficient, the cell disruption effect was poor, and thus the enzymatic hydrolysis and saccharification reactions were insufficient, the sweetness was low, there was a grainy feeling, and the smoothness and consistency were average. The cell disruption method used in Comparative Example 4 was not suitable for microalgae, the cell disruption effect was poor, the cellulose cell wall was not destroyed, the enzymatic hydrolysis effect and saccharification effect were poor, the sweetness was basically not felt, the grainy feeling was obvious, the taste was poor, the smoothness was poor, and the consistency was thin.

[0117] Comparison between Example 1 and Examples 5-6 shows that when the microalgae dry matter content is 5.0 g / L, the addition amount and reaction time of amylase and saccharifying enzyme in Example 1 are optimal, and the resulting beverage has a light sweet taste and the most moderate taste, and the smoothness and thickness are both good. The sweetness of Example 5 is more obvious and suitable for people who like sweetness, while Examples 3 and 6 have almost no sweetness and are suitable for people who do not like sweetness.

[0118] Comparison between Example 1 and Example 4 shows that even for the same microalgae germplasm resources, only the fermentation culture conditions need to be adjusted, such as increasing the content of ammonium nitrogen in the feed medium, and using the method of adding ammonia water to keep the pH of the fermentation system neutral and slightly acidic, which can promote the algae cells to accumulate more protein, increase the protein content from 52% to 61%, and relatively reduce the accumulation of oil and starch in the algae cells, and reduce the oil and starch content in the algae cells. It can be seen that compared with the method of producing protein drinks using higher plants as raw materials, the present invention can obtain microalgae biomass with high protein, low starch, and low oil composition through customized culture conditions, and use the microalgae biomass to prepare low-fat, low-carbohydrate, and low-calorie protein drinks.

[0119] The microalgae milk products prepared in Examples 1-6 and Comparative Examples 1-4 were placed at room temperature for 21 days, poured into a glass and left to stand for 2 hours, and the stratification was observed. The results are shown in the following table:

[0120] Table 3:

[0121] Stability (whether it is layered) Example 1 Stable, no stratification Example 2 Stable, no stratification Example 3 Stable, no stratification Example 4 Stable, no stratification Example 5 Stable, no stratification Example 6 Stable, no stratification Comparative Example 1 With slight layering Comparative Example 2 Slightly delaminated Comparative Example 3 Layered Comparative Example 4 Unstable, with obvious delamination

[0122] As shown in Table 3, the cell disruption methods of Comparative Examples 3-4 are not conducive to obtaining a microalgae protein beverage with uniform and stable physical phase; while the microalgae protein beverages prepared in Examples 1-6 can maintain uniform and stable physical phase without adding thickeners and stabilizers.

[0123] In summary, the present invention provides a low-fat, low-sugar, and low-energy plant protein dairy product using microalgae as raw materials. The plant protein dairy product contains high protein and can be used as a substitute for milk, providing an ideal protein drink for people who are allergic to milk or lactose intolerant, who pursue low calorie intake, and who control their body fat percentage.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a low-fat, low-calorie, and algae-free microalgae protein beverage. It is characterized in that include: S1. Select microalgae raw materials: the microalgae raw materials are chlorophyll-deficient high-protein microalgae, with a protein content of ≥35%, a fat content of ≤15%, and a starch content of ≤30% in the algae cells; the microalgae raw materials are microalgae with cell walls whose main component is cellulose; S2. Cell disruption: adding water to adjust the dry weight of microalgae cells to 50-150 g / L, and using a high-pressure homogenizer to disrupt the cells to obtain a cell paste; the pressure of the high-pressure homogenizer is 60-130 MPa; high-pressure homogenization is performed until the particles below 1 μm in the cell paste account for more than 90% of the total number of particles, and the particles below 300 nm account for more than 50% of the total number of particles; S3, enzymatic hydrolysis: adding water to the cell paste to dilute the cell paste; Based on the weight of the cell paste after adding water, 600-5000 units / kg of cellulase, 5000-8000 units / kg of protease, 500-1000 units / kg of amylase and 1000-3000 units / kg of saccharifying enzyme are added to carry out enzymatic hydrolysis reaction; After the reaction is completed, a pH adjuster is added, and the mixture is stirred and mixed to obtain a mixture; the pH adjuster is one or more of phosphate, carbonate or citrate, and is used to adjust the pH of the reaction solution after the enzymatic hydrolysis to 7.0-8.0; S4, homogenization: heat the mixture to 50-80°C, transfer it into a high-pressure homogenizer for homogenization, the homogenization pressure is 60-100MPa, repeat 1-4 times to obtain a homogenized mixture; S5. Sterilization and filling: The homogenized mixture is sterilized and then filled or filled and then sterilized to obtain the finished product.

2. The preparation method according to claim 1, It is characterized in that In S1, the microalgae is yellow protein-core Chlorella, its preservation number is CGMCC NO.40458, and the preservation date is April 27, 2023; or, the microalgae is white protein-core Chlorella, its preservation number is CGMCC NO.40787, and the preservation date is September 9, 2023.

3. The preparation method according to claim 1, It is characterized in that In S2, when the microalgae is Chlorella vulgaris, a pressure of 100-130 MPa is used for homogenization; when the dry weight of the microalgae cells is below 80 g / L, a pressure of 100-105 MPa is used for homogenization, when it is 80-100 g / L, a pressure of 106-115 MPa is used for homogenization, and when it is 100-130 g / L, a pressure of 116-130 MPa is used for homogenization.

4. The preparation method according to claim 1, It is characterized in that In S3, during the enzymatic hydrolysis process, water was added to control the dry matter content of the microalgae to be 1-10 g / 100 mL and the protein content to be ≥1.0 g / 100 mL; the enzymatic hydrolysis conditions were: reaction at 50±5°C for 45-90 min.

5. The preparation method according to claim 1, It is characterized in that In S3, the cellulase is food grade cellulase; the protease is one or a combination of food grade alkaline protease, neutral protease, papain, bromelain, trypsin and pepsin; the amylase is one or a combination of α-amylase and pullulanase; and the saccharifying enzyme is one or a combination of glucoamylase or β-amylase.

6. The preparation method according to claim 5, It is characterized in that In S3, based on the weight of the cell paste after adding water, 3000 units / kg of cellulase, 6000 units / kg of protease, 1000 units / kg of α-amylase and 1500 units / kg of saccharifying enzyme were added to carry out enzymatic hydrolysis reaction for 60 minutes.

7. The preparation method according to claim 1, It is characterized in that In S5, the sterilization conditions are selected from one or a combination of the following three sterilization methods: 135-150° C. treatment for 2-10 s, 75-100° C. pasteurization, and 100-121° C. treatment for 10-25 min; and the sterilization is performed before or after filling.

8. A low-fat, low-calorie, algae-free microalgae protein drink. It is characterized in that The method is prepared by the preparation method according to any one of claims 1 to 7.

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