WO2024077846A1 - 一种基于界面调控的具有良好风味与功能活性亚麻籽植物乳及其制备方法与应用 - Google Patents

一种基于界面调控的具有良好风味与功能活性亚麻籽植物乳及其制备方法与应用 Download PDF

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WO2024077846A1
WO2024077846A1 PCT/CN2023/079484 CN2023079484W WO2024077846A1 WO 2024077846 A1 WO2024077846 A1 WO 2024077846A1 CN 2023079484 W CN2023079484 W CN 2023079484W WO 2024077846 A1 WO2024077846 A1 WO 2024077846A1
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flaxseed
milk
plant
plant milk
functional activity
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PCT/CN2023/079484
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English (en)
French (fr)
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邓乾春
陈亚淑
杨陈
禹晓
周琦
张珊
向霞
万楚筠
陈洪建
彭登峰
郝倩
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中国农业科学院油料作物研究所
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Publication of WO2024077846A1 publication Critical patent/WO2024077846A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
    • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
    • A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk

Definitions

  • the invention belongs to the field of food technology, and in particular relates to a flaxseed plant milk with good flavor and functional activity based on interface regulation, and a preparation method and application thereof.
  • Plant milk has many favorable factors in terms of safety, nutrition, humanity and carbon emissions, and has huge market size and industry development potential.
  • the first generation of traditional plant milk (milk) beverages such as soy milk, mainly supplement protein.
  • plant milk (milk) will inevitably develop in a segmented way with high nutritional value and meet the market needs of nutrition and application scenarios for different groups of people.
  • flaxseed plant milk (milk) has gradually attracted the attention of consumers because it is suitable for different groups of people to consume and has comprehensive nutrients and extremely high nutritional value.
  • Flaxseed is not only rich in ⁇ -linolenic acid (ALA, ⁇ 59%), the only essential polyunsaturated fatty acid in the n-3 series, but also contains high-quality plant protein, dietary fiber such as flaxseed gum, lignans, phenolic acid, vitamin E and other biologically active substances.
  • the methionine content of flax protein (1.86g/100g) is about twice that of soy protein (0.93g/100g), and its biological value (BV) (77.4) is higher than that of soy protein (74) and close to casein (80).
  • the current domestic flaxseed milk processing technology uses shelled flaxseed kernels (sauce) as raw materials, resulting in 1) low flaxseed kernel yield after shelling, high cost, and large energy and raw material losses; 2) low utilization rate of flaxseed nutrients, loss of flaxseed gum, flax lignans, total phenols, dietary fiber and other nutrients in flaxseed skin; 3) high temperature baking is used for flaxseed cooking, which has low thermal efficiency, and combined with the grinding process, the natural emulsified structure of the oil body is destroyed, the oil and water are separated, and ALA is easily oxidized to produce a fishy smell with poor flavor, and toxic and harmful oxidation products may be produced; 4) a large amount of emulsifiers are required to emulsify the oil sauce during the milking process, which further increases the cost and is not conducive to clean label properties; 5) the milk system lacks natural antioxidants, needs to be added exogenously, and has a short shelf life.
  • the present invention breaks through the key technical bottlenecks of full-value and high-value processing of flaxseed, such as precise degumming, detoxification, aroma production, wall breaking, and synergy enhancement.
  • the dry precise degumming is coupled with low-consumption microwave and bio-enzyme hydrolysis technology, and the physical and chemical properties of plant emulsion droplets are regulated based on interface regulation technology. It improves stability, changes the particle size distribution and the charge distribution on the droplet surface, and significantly reduces grassy flavor substances, providing flaxseed plant milk with roasted, coffee, and cocoa aroma.
  • This method is green, energy-saving and water-saving, and significantly improves the nutrition (nutrient content), stability (particle size, potential, storage stability), and health attributes (ALA bioavailability, functional activity evaluation) of flaxseed plant milk, and ensures the clean label attributes of the plant milk (no exogenous additives are used).
  • the present invention provides a long-lasting flaxseed plant milk with good flavor and functional activity based on interface regulation, and a preparation method and application thereof.
  • the present invention is based on the principle of interface regulation.
  • Softening microwave conditioning not only efficiently removes cyanogenic glycosides, but also directly improves the interface stability of plant milk oil droplets and the content of each nutrient in the system based on interface regulation, and greatly promotes the flavor and nutritional health activity of the plant milk.
  • the process is green and the long-term storage stability of the product can be maintained without adding exogenous additives.
  • animal experimental studies have found that the plant milk developed by the technology of the present invention has outstanding functional advantages in improving intestinal microecological function and immune test function compared with other commercially available plant milks, and the prepared flaxseed long-lasting plant milk is safe, nutritious and delicious, and is suitable for promotion and application.
  • the present invention adopts the following technical solution:
  • a method for preparing flaxseed plant milk with good flavor and functional activity based on interface regulation specifically comprising the following steps:
  • flax seeds are subjected to pretreatments of degumming, microwave cooking and soaking and softening in sequence, and then set aside;
  • the flaxseed plant milk A is subjected to a first high-pressure homogenization, enzyme inactivation and sterilization, and a second high-pressure homogenization in sequence, thereby obtaining a long-lasting flaxseed plant milk with good flavor and functional activity based on interface regulation.
  • the present invention uses microwave, bio-enzymatic hydrolysis, post-aseptic homogenization and other processes, without adding exogenous additives, and can achieve the enrichment of ⁇ -linolenic acid, protein, lignans and total phenols in flaxseed plant milk while saving energy and protecting the environment.
  • Commercial aseptic conditions can meet the shelf life of plant milk products.
  • the technology of the present invention can be expanded to a variety of flaxseed-based plant milks, such as flaxseed-sesame, flaxseed-hemp, flaxseed-peanut, flaxseed-soybean and other plant milk processing, which can meet the health promotion and nutritional supplement needs of different consumer groups with good flavor and functional activity based on interface regulation.
  • the degumming in step (1) is carried out by dry degumming
  • the present invention When preparing the flaxseed permanent plant milk, the present invention first performs dry degumming treatment on the flaxseed raw seeds, which can avoid the plant milk (milk) system being too viscous and having low fluidity due to excessive flaxseed gum.
  • the by-product flaxseed gum powder can be used to extract and prepare flaxseed gum, flaxseed oligosaccharides, flax lignans, etc.
  • microwave temperature is 115-145° C.
  • microwave time is 3-12 min
  • flaxseed water content is 8-20%
  • the solid-liquid mass ratio of the soaking softening is 1:(5-10), and the soaking time is 2-24h.
  • the present invention can remove toxic substances and anti-nutritional factors such as cyanogenic glycosides contained in raw flax seeds by water-adjusting coupled microwave flax seed treatment, generate aroma molecules, and ensure the safety and nutritional quality of the product; on the other hand, it can improve the dissolution of nutrients such as lignans in the flax seed milk production process.
  • the colloid grinding time in step (2) is 10-210 min, and the solid-liquid mass ratio of flaxseed to water is 1:(5-10);
  • the deslagging method used is horizontal screw deslagging with a rotation speed of 2500-3000rpm.
  • the enzyme used for the enzymatic hydrolysis in step (2) is any one of cellulase, saccharifying enzyme, protease, pectinase and phytase.
  • the enzymatic hydrolysis temperature in step (2) is 45-55°C, the time is 30-120 min, and the addition amount is 0.01-2%.
  • the flax seed seed coat contains 8 layers of cells, of which the second layer from the outside to the inside is colloid cells.
  • the protein bodies storing flax seed protein and the oil bodies storing oil bodies are restricted in the plant cell walls.
  • the present invention can effectively promote the dissolution of nutrients such as endogenous protein, fat, total phenols, lignans, etc. that would otherwise be restricted in flax seeds through soaking softening, colloid mill circulation refining coupled with biological enzymatic hydrolysis.
  • the pressure of the first high-pressure homogenization in step (3) is 5-20 MPa, and the pressure of the second high-pressure homogenization is 50-200 bar.
  • the temperature for inactivating the enzyme in step (3) is 90-115°C and the time is 15-300S;
  • the sterilization is carried out by UHT sterilization at a temperature of 135-140° C. and a time of 8-30 seconds.
  • Flaxseed itself is rich in protein, and flaxseed gum polysaccharide can be used as an emulsifier stabilizer. Therefore, the ultra-high temperature sterilization coupled with aseptic homogenization in the present invention can achieve the commercial aseptic effect of flaxseed plant milk without adding additional exogenous emulsifiers and stabilizers, and can be stored for a long time at room temperature, meeting the shelf life requirements of commercially available plant milk.
  • the flaxseed plant milk with good flavor and functional activity is aseptically filled, the canning temperature is 25-40°C, and the canning includes any one of paper bags, PET bottles, glass jars and aluminum cans.
  • the present invention adopts dry degumming, microwave, circulating pulping, biological enzymolysis, aseptic homogenization and other steps to collaboratively prepare commercial aseptic flaxseed plant milk, which can achieve that the quality of the plant milk is basically not deteriorated when stored at room temperature for 6 months.
  • the protein content of the obtained flaxseed plant milk is as high as 1.6g/100g
  • the flaxseed oil content is as high as 3.5g/100g
  • the ALA content is 1.9g/100g
  • the total phenol content is 674mg/100g
  • the lignan content is 176mg/100g.
  • it has significant advantages in nutrient content and plot labeling.
  • the present invention does not require any additives, has low energy and water consumption, and significantly reduces production costs, and is a new green processing technology.
  • the flavor of the plant milk is greatly improved, and the content of pyrazine compounds and furan compounds increases, indicating that the flavor gradually changes from grass flavor to roasted flavor and milk flavor; not only that, the plant milk has high self-stability, the product particle size is 3.81 ⁇ m, the potential is -21.4mv, and the accelerated oxidation results at 37 degrees Celsius show that it still maintains high stability after 6 months.
  • the results of animal experiments show that after taking the flaxseed plant milk prepared by the technology provided by the present invention, the proportion of DHA in the jejunal tissue of rats continues to increase and is significantly greater than that of the flaxseed milk in the control group; at the same time, compared with the oat milk and soy milk in the control group, the flaxseed milk manufactured by the technology of the present invention has a significant effect on the repair of animal colon tissue damage, and can promote the abundance of Parabacteroides in the antibiotic mouse model; the results of animal immunity experiments show that the low-dose group of flaxseed plant milk manufactured by the technology of the present invention can enhance the delayed-type hypersensitivity (DTH) induced by DNFB in mice, with an increase of 27.4%, which is 31.3% higher than that of oat milk; compared with the negative control group, the high-dose group of flaxseed plant milk can enhance the ConA-induced spleen lymphocyte proliferation ability of mice by 21.6%; compared with the negative control group, soy milk and oat
  • FIG1 shows the particle size distribution of flaxseed vegetable milk with different degumming rates according to the present invention
  • FIG2 is a graph showing the contents of various lignans in flaxseed plant milks with different water contents according to the present invention
  • FIG3 is a diagram showing the regulation of the interface appearance of fat bodies in flaxseed vegetable milk by microwaves of the present invention
  • FIG4 is a diagram showing the regulation of the particle size potential of fat bodies in flaxseed vegetable milk by microwaves of the present invention.
  • FIG5 is a diagram showing the regulation of total phenols and flavonoids in oil bodies of flaxseed plant milk by microwaves of the present invention
  • FIG6 is a diagram showing the regulation of ALA bioavailability in fat bodies in flaxseed vegetable milk by microwaves of the present invention.
  • FIG7 is a graph showing the total solid content of different enzymatically hydrolyzed flaxseed plant milks of the present invention.
  • FIG8 is a graph showing the viscosity of different enzymatically hydrolyzed flaxseed plant milks of the present invention.
  • FIG9 is an appearance diagram of different enzymatically hydrolyzed flaxseed plant milks of the present invention.
  • FIG10 is a graph showing the particle size and volume components of different enzymatically hydrolyzed flaxseed plant milks of the present invention.
  • FIG11 is a diagram showing the particle sizes of different enzymatically hydrolyzed flaxseed plant milks of the present invention.
  • FIG12 is a graph showing the potential of different enzymatically hydrolyzed flaxseed plant milks of the present invention.
  • FIG13 is a graph showing the particle size and volume components of flaxseed vegetable milk at different process steps of the present invention.
  • FIG14 is a graph showing particle sizes of flaxseed vegetable milk at different process steps of the present invention.
  • FIG15 is a graph showing the potential of flaxseed plant milk at different process steps of the present invention.
  • FIG16 is a diagram showing the influence of different process steps of the flaxseed vegetable milk TSI of the present invention.
  • FIG17 is a graph showing the particle size and volume components of flaxseed vegetable milk at different storage temperatures of the present invention.
  • FIG18 is a graph showing particle sizes of flaxseed vegetable milk at different storage temperatures of the present invention.
  • FIG19 is a graph showing the potential of flaxseed plant milk at different storage temperatures according to the present invention.
  • FIG20 is a diagram showing the effect of different storage temperatures on TSI of flaxseed vegetable milk of the present invention.
  • FIG21 is a graph showing the influence of different storage temperatures on the centrifugal sedimentation rate of flaxseed vegetable milk of the present invention.
  • FIG22 is a graph showing the effect of different storage temperatures on the fatty acid composition of flaxseed vegetable milk of the present invention.
  • FIG23 is a HE staining image of the blind-end small intestine segment of the experimental group of the present invention.
  • FIG24 is a graph showing the ⁇ -diversity index of the intestinal flora of the experimental group of the present invention.
  • FIG25 is a PCoA diagram of the weighted UniFrac distance based on the ⁇ -diversity of the intestinal flora in the experimental group of the present invention.
  • FIG. 26 is a graph showing the relative abundance of representative bacteria of the experimental group of the present invention at the genus level.
  • a method for preparing a long-lasting flaxseed plant milk with good flavor and functional activity based on interface regulation specifically comprising the following steps:
  • flax seeds are sequentially subjected to pretreatments of degumming, microwave cooking and soaking for use; wherein, the flax seeds are degummed by dry method, and the degumming rate is 7.6%; the water adjustment range of the microwave-cooked flax seeds is 20%, the microwave temperature is 145°C, and the microwave time is 8 minutes; the solid-liquid mass ratio during soaking is 1:7, and the soaking time is 2 hours;
  • flax seeds obtained by soaking and softening are subjected to colloidal grinding, enzymatic hydrolysis and slag removal in sequence to obtain flax seed plant milk A; wherein the colloidal grinding time is 90 minutes, the solid-liquid mass ratio of flax seeds to water is 1:9; the enzyme used for enzymatic hydrolysis is cellulase, the enzymatic hydrolysis temperature is 50° C., the time is 60 minutes, and the addition amount is 0.5wt%; the slag removal is carried out by horizontal screw slag removal at a rotation speed of 2770rpm;
  • the flaxseed plant milk A is subjected to the first high-pressure homogenization, enzyme inactivation and sterilization, and the second high-pressure homogenization in sequence to obtain a long-lasting flaxseed plant milk with good flavor and functional activity based on interface regulation; wherein the pressure of the first high-pressure homogenization is 5 MPa; the temperature of enzyme inactivation is 90°C, and the time is 15S; UHT sterilization is adopted for sterilization, the temperature is 135°C, and the time is 8S; the pressure of the second high-pressure homogenization is 50 bar; the obtained flaxseed plant milk has a particle size of 3 ⁇ m, a potential of -20 mV, a protein content of 1.6 g/100 g, and an ALA content of 1.9 g/100 g.
  • the outer skin of flax seeds is rich in flaxseed gum polysaccharides.
  • the viscosity of the system be too high, but also the dissolution of endogenous proteins in flax seeds will be affected.
  • too much flaxseed gum will also cause flocculation of flaxseed vegetable milk, resulting in increased particle size and instability. Therefore, through the dry grinding process, while solving the above problems, the by-product flaxseed gum powder is obtained, which can be used to prepare food additive flaxseed gum and flaxseed lignans with high biological activity, etc., which meets the requirements of full-value processing and utilization.
  • Flaxseed; other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.;
  • TJFL-18S flaxseed degumming machine Malvern 3000 laser particle size analyzer - Malvern Instruments, UK; Shanghai Hengping Instruments SNB-1 digital viscometer;
  • Sample preparation Fresh, dry, and non-rotten flax seeds were selected and degummed using dry degumming equipment to obtain degumming rates of 0.00%, 2.70%, 4.00%, 6.30%, and 7.60%; microwave cooking was performed using a closed microwave oven.
  • the digestion instrument enclosed microwave rapid extraction system
  • the digestion instrument was used to microwave for 6 minutes at a power of 720 W to obtain microwave cooked flax seeds; each group of cooked dry degummed flax seeds were soaked in pure water at room temperature for 2 hours according to a solid-liquid mass ratio of 1:7, and ground for 3 minutes to obtain flax seed plant milk;
  • dry degumming can effectively reduce the viscosity of plant milk; dry degumming can effectively increase the protein content in flaxseed plant milk, indicating that dry degumming promotes the dissolution of protein during the pulping process.
  • Flax seeds are added to a reactor, steam is introduced, and the flax seeds are taken out after being fully stirred for different times.
  • the steam intake and stirring time are adjusted to obtain different gradients of flax seed moisture content, ranging from 11% to 23%.
  • Microwaves as an ultra-high frequency electromagnetic wave, promote high-frequency reciprocating motion of dipole molecules to generate "internal friction heat", which will be absorbed by food and water to generate heat. It can achieve simultaneous heating and temperature increase without the need for heat conduction.
  • the speed is fast and uniform, and the energy consumption is a few or several tenths of traditional heating.
  • flax seed detoxification 2) Maillard reaction flavor enhancement 3) passivation of endogenous oxidase, change cell wall structure, promote the depolymerization of macromolecular lignans and dissolution of polyphenols, and improve quality.
  • flax seed detoxification 2) Maillard reaction flavor enhancement 3
  • passivation of endogenous oxidase, change cell wall structure promote the depolymerization of macromolecular lignans and dissolution of polyphenols, and improve quality.
  • the interface structure and interface composition of flax seed oil bodies including phospholipids and proteins, change, and microwaves promote phenol
  • the substances migrate into the interface, which effectively improves the chemical stability of flaxseed oil and promotes the bioavailability of ALA during digestion.
  • Flaxseed; other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.;
  • Flaxseed degumming machine Malvern 3000 laser particle size analyzer - Malvern Instruments, UK; Shanghai Hengping Instruments SNB-1 digital viscometer;
  • Sample preparation Degummed flax seeds were selected, added to a reactor, steam was introduced, and the flax seeds were taken out after being fully stirred for different times; the steam intake and stirring time were adjusted to obtain different gradients (11%-20%) of flax seed moisture content, the gradient was from 11%-23%, microwaved at a power of 720 W for 9 minutes to obtain microwave-cooked flax seeds, and each group of cooked dry degummed flax seeds were soaked in pure water at room temperature for 2 hours at a solid-liquid mass ratio of 1:7, and slurried in a colloid mill for 12 minutes to obtain flax seed plant milk;
  • the effect of microwave on the particle size distribution of flaxseed oil was determined by laser diffraction technology using a laser particle size analyzer.
  • the measurement parameters were as follows: wet dispersion method was used for analysis, the sample refractive index was 1.480, the water refractive index was 1.330, the stirring rate was 2000 rpm/min, and the test temperature was 25°C.
  • the results are shown in FIG4A .
  • the emulsion was diluted with deionized water at a ratio of 1:250, and the ⁇ potential of the emulsion under different enzymatic hydrolysis conditions was measured using a Malvern nanoparticle size analyzer.
  • the results are shown in FIG4B .
  • FIG5A is a regulation diagram of flavonoids
  • FIG5B is a regulation diagram of total phenols in oil bodies.
  • the contents of total phenols and flavonoids in oil bodies increased with the extension of microwave time, indicating that microwaves promote the migration of antioxidant molecules in the plant milk system to the interface, showing the promotion of the chemical stability of the plant milk.
  • Microwave interface regulation promotes the improvement of ALA bioavailability in flaxseed milk:
  • Flax seeds adjusted to 20% moisture content were microwaved at 700W for 1-5 minutes to obtain microwave cooked flax seeds, the microwaved flax seeds were soaked in pure water at room temperature for 2 hours at a solid-liquid mass ratio of 1:10, and the flax seed plant milk was obtained after shear mill circulation grinding for 3 minutes, and then filtered with a 200-mesh filter bag and kept for animal experiments;
  • mice Male SD rats (220–250 g) were obtained from Fuhe Biotechnology Co., Ltd. (Shanghai, China). After one week of adaptive feeding in a temperature- and humidity-controlled environment with a 12-h light-dark cycle, the rats were randomly divided into four groups of 15 rats each. After fasting for one night, five rats in each group were gavaged with 2.5 mL of flaxseed milk. Intestinal tissues were collected and snap-frozen in liquid nitrogen before the rats were killed 1, 2, and 4 h after gavage. The collected specimens were immediately stored at ⁇ 80°C until analysis. Jejunal tissues were dispersed into precooled saline at a ratio of 1:9 (w/v) by high-speed shearing.
  • Total lipids were extracted with chloroform-methanol (2:1, v/v), centrifuged at 10,000 rpm for 10 min, and the supernatant chloroform layer was removed and dried with nitrogen.
  • Fatty acid methyl esters were prepared and analyzed using an Agilent 6890GC with a flame ionization detector (FID) and a silica capillary column (30 m ⁇ 0.25 mm, 0.25 ⁇ m). The temperature started at 175°C, was maintained for 10 minutes, and then increased to 250°C at a rate of 1°C/min. The temperature of both the injector and detector was set to 250°C. Helium was used as the carrier gas with a flow rate of 1.5mL/min. The injection volume was 2 ⁇ L with a split ratio of 10:1. The temperature of the injector and detector was set to 250°C. Fatty acid methyl esters were identified by comparison with authentic standards (GLC-463), and the relative content was expressed by area normalization.
  • A, B, C, and D in Figure 6 are the proportions of ALA and its conversion products EPA, DPA, and DHA in the jejunum tissue 1 hour after taking microwave flaxseed milk. It can be seen that the proportions of ALA and its conversion products EPA, DPA, and DHA in the jejunum tissue are significantly higher than those in the non-microwaved flaxseed milk 1 hour after taking microwave flaxseed milk.
  • the ratios of EPA, DPA and DHA in rats were 3.82%, 0.26%, 0.76% and 1.29% respectively.
  • Headspace solid phase microextraction was used to extract volatile compounds from the headspace above the enzymatic hydrolyzed vegetable milks of unhydrolyzed, Flavourzyme 500MG, novo, bromelain solarbio, hemicellulase XS, ⁇ -glucanase XS, cellulase CTS novo, and CELLUCLAST 1.5L novo.
  • the samples were numbered 0-6 in sequence.
  • Gas chromatography-mass spectrometry (Agilent 7890A-5975C) and HP-5MS column (60m ⁇ 0.25mm ⁇ 0.25 ⁇ m, Agilent Technologies, catalog number 122-5532) were used to determine the types and concentrations of volatiles. The results are shown in Table 2.
  • the inlet temperature was set at 250°C
  • the ion source temperature was set at 230°C
  • the interface temperature was set at 280°C
  • the carrier gas flow rate was 1.5mL/min.
  • the temperature ramp used in the process was: hold at 40°C for 2 min; heat to 200°C, 4°C/min; hold at 200°C for 2 min; then heat to 280°C, 8°C/min; the injection volume was set to 1 ⁇ L; the mass spectrometer was operated in impact mode at 150°C and 70 eV voltage; the mass spectrometer scan range was 40-400 amu, and the solvent delay was 7 min; individual compounds were identified and quantified by MS-library search (Wiley 138K, John Wiley and Sons, Hewlett Packard, USA); the effect of enzymatic hydrolysis on volatile flavor compounds in vegetable milk was analyzed by headspace solid phase microextraction-gas chromatography-mass spectrometry;
  • the flaxseed milk prepared from flaxseed that has not been treated with microwaves has a bland flavor
  • the main manifestation is that with the increase in the moisture content of the flaxseed, the content of pyrazine compounds and furan compounds gradually increases, especially after 6 minutes, showing a significant upward trend.
  • microwaves can greatly increase the aroma molecules of plant milk and improve the flavor.
  • the principle of targeted enzymatic hydrolysis is based on the targeted and efficient hydrolysis of glycosidic bonds, peptide bonds, and ester bonds, which can achieve 1) promoting the cleavage of cell wall constituent fibers such as cellulose, hemicellulose, and pectin, and improving the dissolution of endogenous proteins, polyphenols, and oils; 2) reducing the molecular weight of plant macromolecular polysaccharides, which can improve the dissolution of endogenous dietary fiber on the one hand and reduce the viscosity of the system on the other hand; 3) improving the flavor and taste of the system, and improving the emulsification stability;
  • Flaxseed different enzymes and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.;
  • Flaxseed degumming machine Malvern 3000 laser particle size analyzer - Malvern Instruments, UK; Shanghai Hengping Instruments SNB-1 digital viscometer;
  • Volatile compounds were extracted from the headspace of unhydrolyzed plant milks, Flavourzyme 500MG, novo, bromelain solarbio, hemicellulase XS, ⁇ -glucanase XS, cellulase CTS novo, and CELLUCLAST 1.5L novo by headspace solid phase microextraction.
  • the samples were numbered 0-6.
  • the volatile ... by headspace solid phase microextraction (Agilent 7890A-5975C), HP-5MS column (60m ⁇ 0.25mm ⁇ 0.25 ⁇ m, Agilent Technologies, catalog number 122-5532) was used to determine the types and concentrations of volatiles. The results are shown in Table 2.
  • the inlet temperature was set to 250°C
  • the ion source temperature was set to 230°C
  • the interface temperature was set to 280°C
  • the carrier gas flow rate was 1.5mL/min;
  • the temperature ramp used in the process was: hold at 40°C for 2 min; heat to 200°C, 4°C/min; hold at 200°C for 2 min; then heat to 280°C, 8°C/min; the injection volume was set to 1 ⁇ L; the mass spectrometer was operated in impact mode at 150°C and 70 eV voltage; the mass spectrometer scan range was 40-400 amu, and the solvent delay was 7 min; individual compounds were identified and quantified by MS-library search (Wiley 138K, John Wiley and Sons, Hewlett Packard, USA); the effect of enzymatic hydrolysis on volatile flavor compounds in vegetable milk was analyzed by headspace solid phase microextraction-gas chromatography-mass spectrometry;
  • a high-resolution camera was used to photograph and record the appearance of unhydrolyzed, Flavourzyme 500MG novo, bromelain solarbio, hemicellulase XS, ⁇ -glucanase XS, cellulase CTS novo, and CELLUCLAST 1.5L novo hydrolyzed vegetable milk, samples numbered 0-6, and the results are shown in Figure 9;
  • enzymatic hydrolysis has different effects on the appearance of flaxseed plant milk, among which enzymatic hydrolyzed flaxseed plant milk No. 1, 2, and 4 show obvious browning, while the color change of No. 3, 5, and 6 is not obvious;
  • the emulsion was diluted with deionized water at a ratio of 1:250, and the ⁇ potential of the emulsion under different enzymatic hydrolysis conditions was measured using a Malvern nanoparticle size analyzer.
  • the results are shown in Figure 12, where the samples of unenzymatically hydrolyzed plant milk, Flavourzyme 500MG, novo, bromelain solarbio, hemicellulase XS, ⁇ -glucanase XS, cellulase CTS novo, and CELLUCLAST 1.5L novo were numbered 0-6 in sequence;
  • enzymatic hydrolysis increased the particle size of flaxseed plant milk to varying degrees, and reduced the absolute value of the surface charge of the oil droplets, indicating that enzymatic hydrolysis may have changed the interface of the oil droplets in the flaxseed plant milk.
  • composite homogenization is that after enzymatic hydrolysis, enzyme inactivation and sterilization, the molecular structure, adsorption and rearrangement of proteins, phospholipids, polysaccharides and other molecules on the interface of oil droplets in flaxseed plant milk change.
  • Composite high-pressure homogenization is used to promote the rearrangement of active substances on the interface of oil droplets in flaxseed plant milk, promote the redispersion of flocculated oil droplets, and significantly improve the stability of plant milk.
  • Flaxseed; cellulase, saccharifying enzyme, and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.;
  • Flaxseed degumming machine Malvern 3000 laser particle size analyzer - Malvern Instruments, UK; Shanghai Hengping Instruments SNB-1 digital viscometer Co., Ltd. viscometer;
  • the emulsion was diluted with deionized water at a ratio of 1:250, and the zeta potential of the emulsion under different process conditions was measured using a Malvern nanoparticle size analyzer. The results are shown in Figure 15;
  • the particle size of flaxseed plant milk increased significantly after the first high-pressure homogenization coupled with enzyme inactivation, and the absolute value of potential decreased, indicating that the first high-pressure homogenization coupled with enzyme inactivation caused the plant milk oil droplet interface to be partially unstable due to the influence of heat.
  • the particle size decreased again and the absolute value of potential was also improved, indicating that post-aseptic homogenization significantly improved the adsorption and distribution of active substances on the oil droplet interface in the plant milk, such as proteins, phospholipids, and polysaccharides, and promoted the further stabilization of the oil droplets.
  • the phase separation stability of plant milk was determined by laser diffraction scanning.
  • the equipment consists of a detector head equipped with a near-infrared light source (880nm), which scans the height of the sample and collects transmission and backscattering data every 40 ⁇ m; the light source scans the sample from top to bottom every 30 seconds, and measures the percentage of light backscattering or transmission within 15 minutes at 25°C; the stability of the plant milk is evaluated by TSI (Turbiscan stability Index) parameters calculated by Turbisoft 2.1 software. The results are shown in Figure 16. The TSI results show that homogenization promotes the stability of plant milk.
  • the flaxseed plant milk After ultra-high temperature instantaneous sterilization, post-aseptic homogenization, and aseptic filling, the flaxseed plant milk has reached commercial aseptic conditions and can be stored in a closed container for a long time without worrying about spoilage caused by microorganisms.
  • the flaxseed plant milk is a multiphase system, and oil droplets, protein/carbohydrate macromolecules, insoluble solid particles, etc. are prone to flocculation, aggregation, precipitation, floating and other instability due to gravity.
  • the oxidation of ALA in the system is also likely to cause the stability of the flaxseed plant milk to decrease. Therefore, in order to verify that the plant milk can be stored for a long time, a storage stability experiment is specially carried out;
  • the particle size distribution of flaxseed plant milk was determined by laser diffraction technology using a laser particle size analyzer, and the results are shown in 17-18;
  • the measurement parameters are as follows: wet dispersion method was used for analysis, the sample refractive index was 1.480, the refractive index of water was 1.330, the stirring rate was 2000 rpm/min, and the test temperature was 25°C;
  • the emulsion was diluted with deionized water at a ratio of 1:250, and the zeta potential of the emulsion under different compounding ratios was measured using a Malvern nanoparticle size analyzer. The results are shown in FIG19 ;
  • the phase separation stability of the plant milk was determined by laser diffraction scanning.
  • the device consists of a probe equipped with a near-infrared light source (880 nm). The probe scans the height of the sample and collects transmission and backscattering data every 40 ⁇ m. The light source scans the sample from top to bottom every 30 seconds and measures the percentage of light backscattering or transmission within 15 minutes at 25°C.
  • the TSI (Turbiscan stability Index) parameter calculated by Turbisoft 2.1 software is used to evaluate the stability of the plant milk. The results are shown in Figure 20. When the storage time is 6 months and the temperature is 4°C and 37°C, the TSI change value of the flaxseed plant milk is small, indicating that it is more stable.
  • Centrifugal sedimentation rate (%) sediment weight (g) / centrifugal sample weight (g) ⁇ 100%.
  • the test results are the average of 3 parallel measurements. The results are shown in Figure 21. When the storage time is 6 months and the temperature is 4°C and 37°C, the centrifugal sedimentation rate of the flaxseed plant milk is relatively small, indicating that it has strong stability.
  • GB 5009.168-2016 weigh about 1.5000g of vegetable milk, put it in a 10mL plastic centrifuge tube, add 2mL of n-hexane, sonicate in an ultrasonic instrument for 20min, add 3mL of 0.5M methanol-sodium solution to the original test tube together with the residue, mix it on a vortex mixer for 5min, place it in a high-speed centrifuge, centrifuge it at 5000rpm for 10min, and take the supernatant for testing;
  • Chromatographic conditions chromatographic column HP-INNOWAX 30m ⁇ 0.32mm ⁇ 0.25 ⁇ m; carrier gas is nitrogen, flow rate is 1.5mL/min; injection volume is 1 ⁇ L; injection port temperature is 260°C, split ratio is 80:1, split flow rate is 120mL/min; heating program: 210°C for 9min, 20°C/min to 250°C, hold for 10min, no post-run;
  • mice Sixty SPF C57BL/6 male mice were fed with basic feed for 7 days at 20 ⁇ 4°C, 12h light/dark cycle, and 40-55% relative humidity. Then they were randomly divided into 6 groups (10 mice in each group): normal control group (NC), natural recovery group (CS), high and low dose flaxseed milk groups (FML (approximately equal to the recommended daily ALA intake for humans (1.6g/60kgBW/day)), FMM (2 times the recommended dose, using 2 times concentrated milk), oat milk group (OM), soy milk group (SM), and the number and weight of each mouse were recorded;
  • NC group drank distilled water freely;
  • the CS group and the plant milk group were gavaged with 400 mg/mL ceftriaxone sodium, 0.2 mL per mouse per day, for 8 days; 8 days later, the plant milk group was gavaged with plant milks of equal energy (FML-450uL, FMM-450uL, OM-400uL and SM-330uL),
  • mice were free to eat and drink, and the food intake and body weight were recorded every day.
  • the mice were killed by cervical dislocation on the 30th day of feeding, and the blood samples, intestinal samples, intestinal contents, etc. of the mice were collected for subsequent experiments;
  • mice treated with CS Compared with the mice treated with CS, the OM group and the SM group also showed the characteristics of inflammatory cell infiltration and disappearance of goblet cells.
  • the FMM group of mice treated with twice concentrated flaxseed milk was more able to restore the mucosal structure to health.
  • Fecal samples of mice were collected and immediately stored at -80°C for bioinformatics analysis.
  • Bacterial DNA in fecal samples was isolated using a DNA kit.
  • Primers 338 (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGTWTCTAAT-3') were used to PCR amplify the V3-V4 variable region.
  • the amplified products were sent to the Illumina MiSeq platform for sequencing.
  • the obtained sequences were classified into the same operational taxonomic unit (OUT), where the sequence similarity was ⁇ 97%;
  • Alpha diversity was evaluated using Shannon, Simpson, and Observed Species indicators to infer the diversity within the sample group. The larger the Shannon index and Simpson index, the richer the diversity composition of the sample. Beta diversity was used to compare the diversity between sample groups. Unweighted UniFrac was used to measure ⁇ diversity (i.e., PCoA analysis), using the two most important factors to distinguish between samples. The large difference characteristics were used as coordinate axes for mapping analysis, and the results are shown in Figures 24-26. As shown in Figure 24, they are Simpson, Pielou_e, Shannon and Chao1 result diagrams, respectively. There was no statistically significant difference in the Simpson and Pielou_e indexes between the two groups.
  • Flaxseed plant milk is provided by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences.
  • the recommended daily intake for humans is 300 mL/person/d, i.e. 5 mL/kg BW (calculated based on the average adult body weight of 60 kg).
  • the animals were divided into five experimental groups, each with 50 animals.
  • the first batch of 150 animals were subjected to the following three experiments: the first group of immune experiments was used to test delayed-type hypersensitivity in mice, the second group was used to test carbon clearance in mice, and the third group was used to test the phagocytosis of chicken red blood cells by mouse peritoneal macrophages; the second batch of 100 animals were subjected to the following two experiments: the fourth group of immune experiments was used to determine serum hemolysin and antibody-producing cells; the fifth group was used to test ConA-induced mouse lymphocyte transformation and NK cell activity;
  • the recommended daily intake of the test substance for humans is 300 mL/person/d, i.e. 5 mL/kg BW (calculated based on the average body weight of 60 kg for adults).
  • Two flaxseed plant milk low and high dose groups were set up, and a negative control group (distilled water), a Doubendou soy milk control group, and an oat milk control group were also set up.
  • the protein content of the Doubendou soy milk control group and the oat milk control group was consistent with the protein content of the flaxseed plant milk dose group samples in the experimental design.
  • the oral gavage capacity of mice was 40 ml/kg BW, and the test was conducted after continuous oral gavage of the test substance for 28 days.
  • the flaxseed plant milk, the soy bean milk control group and the oat milk control group were all prepared with distilled water and used immediately after preparation.
  • the specific preparation methods are shown in Table 4.
  • DNFB Dinitrofluorobenzene
  • DTH delayed-type hypersensitivity
  • mice were sensitized with 1% DNFB (prepared with 1:1 acetone sesame oil solution), and then the right ear was challenged with DNFB on the 5th day. The animals were killed 24 hours later, and the left and right ear shells were cut off and an ear piece with a diameter of 8 mm was taken out with a puncher. The weight difference between the left and right ears was used to indicate the degree of DTH.
  • DNFB prepared with 1:1 acetone sesame oil solution
  • Jeme's modified slide method Take defibrinated sheep blood, wash it with physiological saline for 3 times, centrifuge it (2000r/min) for 10min, and inject 0.2mL of 2% (v/v) SRBC into each mouse through the peritoneal cavity; the mice immunized with SRBC for 4 days were killed by cervical dislocation, the spleen was taken out, Hank's solution was added, the spleen was ground to make a cell suspension, filtered through a 200-mesh sieve, centrifuged (1000/min) for 10min, washed twice with Hank's solution, and finally the cells were suspended in 5mL RPMI1640 culture medium, counted and the cell concentration was adjusted to 5 ⁇ 10 6 cells/mL;
  • Plaque determination After the surface culture medium (1g agarose plus double distilled water to 100mL) is heated and dissolved, it is placed in a 45-50°C water bath for heat preservation, mixed with an equal amount of pH7.2-7.4, 2 ⁇ Hank's solution, and dispensed into small test tubes, 0.5mL per tube, and then 50 ⁇ L 10% SRBC (v/v, prepared with SA buffer) and 20 ⁇ L spleen cell suspension (5 ⁇ 106 /mL) are added to the tube, and the mixture is quickly mixed and poured onto a glass slide brushed with a thin layer of agarose to make parallel slices.
  • SRBC v/v, prepared with SA buffer
  • 20 ⁇ L spleen cell suspension 5 ⁇ 106 /mL
  • the glass slide is placed horizontally on a slide rack and placed in a carbon dioxide incubator for incubation for 1.5h, and then complement diluted with SA buffer (1:8) is added to the groove of the slide rack. After incubation for another 1.5h, the number of hemolytic plaques is counted.
  • Hemagglutination method sheep blood was collected and washed with saline for 3 times, each time centrifuged (2000r/min) for 3min, and the packed SRBC was prepared into a 2% (v/v) cell suspension with saline. 0.2mL was injected intraperitoneally into each mouse for immunization. After 4 days, the eyeballs were removed and blood was collected in a centrifuge tube. The tube was left for about 1 hour, and the coagulated blood was separated from the tube wall to allow the serum to be fully separated. The serum was collected by centrifugation at 2000r/min for 10min.
  • Agglutination reaction dilute the serum in multiples with physiological saline, place the serum of different dilutions in the micro-hemagglutination test plate, 100 ⁇ L per well, add 100 ⁇ L 0.5% (v/v) SRBC suspension, mix well, place in a moistened plate and cover, incubate in a 37°C incubator for 3 hours, observe the degree of hemagglutination, and calculate the anti-volume number according to the level of serum agglutination;
  • Lactate dehydrogenase (LDH) assay Lactate dehydrogenase
  • Target cell subculturing 24 h before the experiment, target cells were subcultured, washed three times with Hank's solution, and the cell concentration was adjusted to 4 ⁇ 10 5 cells/mL with RPMI1640 complete culture medium;
  • spleen cell suspension Effector cells: Aseptically remove spleen, place in a small dish containing an appropriate amount of sterile Hank's solution, gently grind spleen to make single cell suspension, filter through a 200-mesh sieve, wash twice with Hank's solution, centrifuge for 10 min each time (1000 r/min), discard supernatant, flick up cell slurry, add 0.5 mL sterile water for 20 seconds, add 0.5 mL 2 ⁇ Hank's solution after lysing red blood cells, centrifuge (1000 r/min) for 10 min, discard supernatant, resuspend with 1 mL RPMI1640 complete culture medium containing 10% calf serum, count viable cells with trypan blue staining (should be above 95%), and finally adjust cell concentration to 2 ⁇ 107 /mL with RPMI1640 complete culture medium;
  • NK cell activity detection Take 100 ⁇ L of target cells and effector cells (effector-target ratio 50:1), add them to a U-shaped 96-well culture plate: add 100 ⁇ L of target cells and culture medium to the target cell natural release well, add 100 ⁇ L of target cells and 1% NP40 to the target cell maximum release well, set three parallel wells for each of the above items, culture in a 37°C, 5% CO2 incubator for 4 h, then centrifuge the 96-well culture plate at 1500 r/min for 5 min, aspirate 100 ⁇ L of supernatant from each well and place it in a flat-bottomed 96-well culture plate, add 100 ⁇ L of LDH matrix solution at the same time, react for 3-10 min depending on the room temperature, add 30 ⁇ L of 1 mol/L HCL to each well, and measure the optical density (OD) at 490 nm on an enzyme reader;
  • MTT method aseptically remove spleen, place in a plate containing an appropriate amount of sterile Hank's solution, grind the spleen gently with tweezers to make a single cell suspension, filter through a 200-mesh sieve, wash twice with Hank's solution, centrifuge 1.8 gin (1000r/min) each time, then suspend the cells in 1 mL of complete culture medium, count the number of live cells (should be above 95%) with trypan blue staining, adjust the cell concentration to 3 ⁇ 10 6 cells/mL, add each spleen cell suspension into two wells of a 24-well culture plate, 1 mL per well, add 75 ⁇ L Con A solution (equivalent to 7.5 ⁇ g/mL) to one well, and use the other well as a control, place at 5% CO 2 , 37°C CO 2 incubator for 72 h; 4 h before the end of the culture, gently aspirate 0.7 mL of supernatant from each well, add 0.7 mL
  • SPSS software one-way analysis of variance and pairwise comparison of the means of multiple experimental groups and a control group were used to compare whether there were differences between the dose groups and the control group. If any dose group showed significant and enhanced differences compared with the control group (P ⁇ 0.05), the experiment was positive.
  • variable transformation For non-normal or uneven variance data, appropriate variable transformation is performed. After the normality or variance homogeneity requirements are met, the transformed data are used for statistics. If the normality or variance homogeneity is still not achieved after the variable transformation, the rank sum test is used for statistics instead.
  • test samples had no significant effect on the body weight of mice in each dose group, and the differences were not significant (P>0.05).
  • the results are shown in Tables 5-9.
  • the low-dose flaxseed plant milk group could enhance the DTH response of mice induced by DNFB (P ⁇ 0.05).
  • the low-dose and high-dose flaxseed plant milk groups could enhance the DTH response of mice induced by DNFB (P ⁇ 0.05), as shown in Table 11.
  • the high-dose flaxseed plant milk group could significantly enhance the proliferation of spleen lymphocytes induced by ConA in mice (P ⁇ 0.05), as shown in Table 12;
  • the high-dose flaxseed plant milk group could significantly enhance the NK cell activity of mice (P ⁇ 0.05), while there were no significant changes in the other dose groups, as shown in Table 12.
  • SPF-grade KM female mice were selected as the experimental system to conduct a study on the function of enhancing immunity.
  • BW calculated according to the average body weight of 60kg for adults
  • two dose groups of flaxseed plant milk, low and high were designed, and a negative control group (distilled water), a soy bean milk control group, and an oat milk control group were set up.
  • the mice were tested after continuous gavage for 28 days, and the experimental results were judged as significant differences with P ⁇ 0.05.

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Abstract

一种基于界面调控的具有良好风味与功能活性亚麻籽植物乳的制备方法及应用，制备方法包括以下步骤：(1)将亚麻籽依次经过脱胶、微波熟制和浸泡软化的预处理后，备用；(2)将浸泡软化得到的所述亚麻籽依次经过胶体研磨、酶解和除渣后，得到亚麻籽植物乳A；(3)将所述亚麻籽植物乳A依次经过第一次高压均质、灭酶灭菌和第二次高压均质后，即得到一种基于界面调控的具有良好风味与功能活性的常存亚麻籽植物乳。本发明工艺绿色，无需添加外源性添加剂即可维持产品稳定性，制备的亚麻籽常存植物乳安全、营养、美味，适于推广应用。

Description

一种基于界面调控的具有良好风味与功能活性亚麻籽植物乳及其制备方法与应用 技术领域
本发明属于食品技术领域，尤其涉及一种基于界面调控的具有良好风味与功能活性的亚麻籽植物乳及其制备方法与应用。
背景技术
“健康和可持续”是国内外食品行业发展的主要驱动因素，植物乳(奶)在安全性、营养性、人道性和碳排放等方面存在着诸多有利因素，且市场规模与产业发展潜力巨大。传统第一代植物乳(奶)饮料如豆浆等，以补充蛋白为第一需求，第二代植物乳(奶)如燕麦乳等，以植物乳(奶)的独特风味、宣传某一项营养功效如补充膳食纤维等为主，未来植物乳(奶)将势必向着高营养价值和满足不同人群营养、应用场景的市场需求来细分发展。近年来，由于亚麻籽植物乳(奶)适合不同人群摄入且营养素全面、营养价值极高，逐渐受到消费者关注。亚麻籽不仅富含n-3系列唯一必需多不饱和脂肪酸α-亚麻酸(α-linolenic acid，ALA，～59％)，更含有优质植物蛋白、亚麻籽胶等膳食纤维、木酚素、酚酸、维生素E等生物活性物质，其中亚麻蛋白的甲硫氨酸含量(1.86g/100g)约为大豆蛋白的2倍(0.93g/100g)，其生物价BV值(77.4)高于大豆蛋白(74)，接近酪蛋白(80)。
近年来，膳食摄入富含多重营养素的亚麻籽对于改善肥胖、糖尿病、心血管疾病、肠道炎性疾病、肿瘤疾病、神经退行性疾病等的作用正逐步被证实。使用亚麻籽全籽制备植物乳(奶)饮料不仅可满足人们对健康生活的需要，还可在一定程度上促进相关产业与市场发展。然而目前国内亚麻籽乳加工现行工艺均以脱壳亚麻籽仁(酱)为原料，导致1)亚麻籽脱壳后亚麻籽仁得率低，成本高，能源和原料损耗大；2)亚麻籽营养成分利用率不高，损失了亚麻籽皮中亚麻籽胶、亚麻木酚素、总酚、膳食纤维等营养素；3)亚麻仁熟制使用高温烘烤，热效率低，且与磨酱过程联合导致天然乳化结构油脂体被破坏，油水分离，ALA极易氧化产生鱼腥味风味不佳，并可能产生有毒有害氧化产物；4)制乳过程需用大量乳化剂对油酱进行乳化，进一步导致成本提高，并不利于清洁标签属性；5)乳体系缺乏天然抗氧化剂，需要外源添加，且保质期较短。因此，如何基于界面调控技术创新一种具有良好风味与功能活性亚麻籽植物乳的制备方法是本领域亟待解决的问题。基于此，本发明突破了亚麻籽精准脱胶、脱毒、生香、破壁、增效等全值化、高值化加工关键技术瓶颈，干法精准脱胶耦合低耗微波与生物酶解技术，基于界面调控技术调控植物乳油滴物理、化 学自稳定性，改变粒径分布与液滴表面电荷分布，同时显著降低青草味风味物质，提供亚麻籽植物乳焙烤、咖啡、可可香气，该方法绿色节能节水，显著提高了亚麻籽植物乳的营养(营养素含量)、稳定(粒径、电位、储藏稳定性)、健康属性(ALA生物利用度、功能活性评价)，并保证了植物乳的清洁标签属性(无外源添加剂使用)。
发明内容
有鉴于此，本发明提供了一种基于界面调控的具有良好风味与功能活性的常存亚麻籽植物乳及其制备方法与应用，本发明基于界面调控原理，首先通过自动化干法精准脱胶，实现植物乳体系稳定性和流动性的定量把控，不仅节约能耗，更避免了水洗脱胶所带来的水资源与人力资源的浪费，软化微波调质不仅高效脱除生氰糖苷，更基于界面调控，直接提高了植物乳油滴界面稳定性以及体系中各营养素含量，同时对植物乳的风味与营养健康活性起到极大促进作用，且工艺绿色，无需添加外源性添加剂即可维持产品长期储藏稳定性，同时，通过动物实验研究发现，本发明技术所研发的植物乳在改善肠道微生态功能、免疫试验功能方面，与市售其他植物乳相比，具有突出明显的功能优势，且制备的亚麻籽常存植物乳安全、营养、美味，适于推广应用。
为了实现上述目的，本发明采用如下技术方案：
一种基于界面调控的具有良好风味与功能活性的亚麻籽植物乳的制备方法，具体包括以下步骤：
(1)将亚麻籽依次经过脱胶、微波熟制和浸泡软化的预处理后，备用；
(2)将浸泡软化得到的所述亚麻籽依次经过胶体研磨、酶解和除渣后，得到亚麻籽植物乳A；
(3)将所述亚麻籽植物乳A依次经过第一次高压均质、灭酶灭菌和第二次高压均质后，即得到一种基于界面调控的具有良好风味与功能活性的常存亚麻籽植物乳。
本发明使用微波、生物酶解、无菌后均质等工序，无需添加外源添加剂，在节能、环保的同时，可实现亚麻籽植物乳中α-亚麻酸、蛋白、木酚素、总酚的富集。商业无菌条件可满足植物乳产品货架期，同时本发明技术可拓展至多种亚麻籽基植物乳，如亚麻籽-芝麻、亚麻籽-火麻、亚麻籽-花生、亚麻籽-大豆等植物乳加工，能满足不同消费人群提供具有基于界面调控的具有良好风味与功能活性的促健康、营养补充需求。
优选的，步骤(1)所述脱胶采用干法脱胶；
本发明在制备亚麻籽常存植物乳时，先对亚麻籽原籽进行干法脱胶处理，可避免由于亚麻籽胶过多导致植物乳(奶)体系过于黏稠，流动性低，同时副产物亚麻籽胶粉可用于提取制备亚麻籽胶、亚麻籽低聚糖、亚麻木酚素等。
所述微波熟制的条件为：微波温度为115-145℃，微波时间为3-12min，亚麻籽调水范围为8-20％；
所述浸泡软化的固液质量比为1：(5-10)，浸泡时间2-24h。
本发明通过调水耦合微波亚麻籽处理一方面可除去生亚麻籽含有的生氰糖苷等毒性物质和抗营养因子，生成香气分子，保证产品的安全性和营养品质；另一方面可提高亚麻籽制乳过程中营养素如木酚素等的溶出。
优选的，步骤(2)所述胶体研磨时间为10-210min，亚麻籽与水的固液质量比为1：(5-10)；
所述除渣采用的为卧螺除渣，转速为2500-3000rpm。
优选的，步骤(2)所述酶解采用的酶为纤维素酶、糖化酶、蛋白酶、果胶酶和植酸酶中的任意一种。
优选的，步骤(2)所述酶解的温度为45-55℃，时间为30-120min，添加量为0.01-2％。
亚麻籽自身种皮含有8层细胞，其中由外及内的第二层为胶质细胞，同时储藏亚麻籽蛋白的蛋白体与储藏油脂体的油脂体被限制在植物细胞壁内，本发明通过浸泡软化、胶体磨循环磨浆耦合生物酶解，可有效促进会限制亚麻籽内源蛋白质、脂肪、总酚、木酚素等营养素的溶出。
优选的，步骤(3)所述第一次高压均质的压力为5-20MPa，所述第二次高压均质的压力为50-200bar。
优选的，步骤(3)所述灭酶的温度为90-115℃，时间为15-300S；
所述灭菌采用UHT灭菌，温度为135-140℃，时间为8-30S。
亚麻籽自身富含蛋白质、亚麻籽胶多糖可作为乳化稳定剂，因此，本发明超高温灭菌耦合无菌后均质，可实现在不额外添加外源乳化剂、稳定剂的前提下，实现亚麻籽植物乳商业无菌效果，并可在室温下长期储藏，达到市售植物乳的货架期要求。
优选的，还包括良好风味与功能活性的常存亚麻籽植物乳进行无菌灌装，罐装温度为25-40℃，罐装包括纸包、PET瓶、玻璃罐和铝罐中的任意一种
如上述所述制备方法得到的基于界面调控的具有良好风味与功能活性的亚麻籽植物 乳。
如上述所述的亚麻籽植物乳在食品加工中的应用
经由上述的技术方案可知，与现有技术相比，本发明的有益效果如下：
1、本发明采用干法脱胶、微波、循环磨浆、生物酶解、无菌均质等步骤协同制备商业无菌亚麻籽植物乳，可实现常温储藏6个月时植物乳品质基本无劣变，所得亚麻籽植物乳中蛋白质含量高达1.6g/100g，亚麻籽油含量高达3.5g/100g，ALA含量达1.9g/100g，总酚含量达674mg/100g，木酚素含量达176mg/100g，与市售亚麻籽植物乳相比在营养素含量与情节标签方面相比具有显著优势。
2、与常规技术相比，本发明无需使用任何添加剂，能耗与水耗低，生产成本明显下降，属于绿色新型加工技术。同时，植物乳的风味得到大幅提高，吡嗪类化合物和呋喃类化合物含量增加，提示风味逐渐从青草味转变为烤香味、奶香味；不仅如此，植物乳自稳定性较高，产品粒径为3.81μm，电位为-21.4mv，且37摄氏度加速氧化结果显示其在6个月仍保持较高稳定性。
3、动物实验结果表明，摄入本发明提供的技术所制备的亚麻籽植物乳后，大鼠空肠组织中DHA的比例不断上升并显著大于对照组亚麻籽乳；同时与对照组燕麦奶、豆奶相比，本发明技术制造的亚麻籽乳对动物结肠组织损伤修复效果显著，并可促进抗生素小鼠模型Parabacteroides菌属的丰度；对动物免疫力实验结果表明，本发明技术制造亚麻籽植物乳低剂量组能增强小鼠对DNFB诱发的迟发性超敏反应(DTH)，增幅达27.4％，与燕麦乳相比，提高31.3％；与阴性对照组比较，亚麻籽植物乳高剂量组增强小鼠ConA诱导的脾淋巴细胞增殖能力可达21.6％；与阴性对照组、豆奶、燕麦奶相比，增强小鼠NK细胞活力分别达19.6％、10％和12.2％，功能活性显著。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，本描述中的附图仅仅是本发明的实施例。
图1为本发明不同脱胶率亚麻籽植物乳的粒径分布情况；
图2为本发明不同含水量亚麻籽植物乳的各种木酚素含量图；
图3为本发明微波对亚麻籽植物乳中油脂体界面外观的调控图；
图4为本发明微波对亚麻籽植物乳中油脂体粒径电位的调控图；
图5为本发明微波对亚麻籽植物乳中油脂体总酚和类黄酮的调控图；
图6为本发明微波对亚麻籽植物乳中油脂体中ALA生物利用度的调控图；
图7为本发明不同酶解亚麻籽植物乳总固形物含量图；
图8为本发明不同酶解亚麻籽植物乳黏度图；
图9为本发明不同酶解亚麻籽植物乳外观图；
图10为本发明不同酶解亚麻籽植物乳粒径体积分量图；
图11为本发明不同酶解亚麻籽植物乳粒径图；
图12为本发明不同酶解亚麻籽植物乳电位图；
图13为本发明不同工艺步骤亚麻籽植物乳粒径体积分量图；
图14为本发明不同工艺步骤亚麻籽植物乳粒径图；
图15为本发明不同工艺步骤亚麻籽植物乳电位图；
图16为本发明不同工艺步骤亚麻籽植物乳TSI的影响图；
图17为本发明不同储藏温度亚麻籽植物乳粒径体积分量图；
图18为本发明不同储藏温度亚麻籽植物乳粒径图；
图19为本发明不同储藏温度亚麻籽植物乳电位图；
图20为本发明不同储藏温度亚麻籽植物乳TSI的影响图；
图21为本发明不同储藏温度亚麻籽植物乳离心沉淀率的影响图；
图22为本发明不同储藏温度亚麻籽植物乳亚麻乳脂肪酸组成的影响图；
图23为本发明实验组盲端小肠肠段HE染色图；
图24为本发明实验组肠道菌群的α-多样性指数图；
图25为本发明实验组基于肠道菌群的β-多样性的weightedUniFrac距离的PCoA图；
图26为本发明实验组代表细菌在属水平上的相对丰度图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
一种基于界面调控的具有良好风味与功能活性的常存亚麻籽植物乳的制备方法，具体包括以下步骤：
(1)将亚麻籽净选后依次经过脱胶、微波熟制和浸泡软化的预处理，备用；其中，亚麻籽脱胶采用干法脱胶，脱胶率为7.6％；微波熟制的亚麻籽调水范围为20％，微波温度为145℃，微波时间为8min；浸泡时固液质量比为1：7，浸泡时间2h；
(2)将浸泡软化得到的亚麻籽依次经过胶体研磨、酶解和除渣后，得到亚麻籽植物乳A；其中，胶体研磨时间为90min，亚麻籽与水的固液质量比为1：9；酶解采用的酶为纤维素酶，酶解的温度为50℃，时间为60min，添加量为0.5wt％；除渣采用的为卧螺除渣，转速为2770rpm；
(3)将所述亚麻籽植物乳A依次经过第一次高压均质、灭酶灭菌和第二次高压均质后，即得到一种基于界面调控的具有良好风味与功能活性的常存亚麻籽植物乳；其中，第一次高压均质的压力为5MPa；灭酶的温度为90℃，时间为15S；灭菌采用UHT灭菌，温度为135℃，时间为8S；第二次高压均质的压力为50bar；所得亚麻籽植物乳粒径3μm，电位-20mV，蛋白质1.6g/100g，ALA 1.9g/100g。
条件摸索实验
一、亚麻籽脱胶预处理
亚麻籽外表皮中富含亚麻籽胶多糖，制备植物乳过程中不仅会使体系黏度过大，还会使亚麻籽中内源蛋白质溶出受到影响，此外过多的亚麻籽胶也会使亚麻籽植物乳发生絮凝，导致粒径增大失稳。因此通过干法磨胶工艺，在解决上述问题的同时，得到副产物亚麻籽胶粉，可用于制备食品添加剂亚麻籽胶，以及具有高生物活性的亚麻籽木酚素等，符合全值化加工利用要求。
1.材料与试剂
亚麻黄籽；其他试剂购自国药集团化学试剂有限公司；
2.主要仪器与设备
TJFL-18S亚麻籽脱胶机；马尔文3000激光粒度分析仪--英国马尔文仪器公司；上海衡平仪器仪表SNB-1数字粘度计；
3.实验方法及结果
(1)样品制备：选择新鲜、干燥、无腐烂变质的亚麻籽，使用干法脱胶装备进行脱胶处理，得到脱胶率为0.00％、2.70％、4.00％、6.30％、7.60％；微波熟制使用密闭式微波 消解仪(密闭式微波快速萃取系统)，在720W功率下微波6min，获得微波熟制亚麻籽；将各组熟制干法脱胶亚麻籽按照1:7的固液质量比使用纯水室温浸泡2h，磨浆3min，得到亚麻籽植物乳；
(2)利用激光粒度分析仪通过激光衍射技术确定各组亚麻籽植物乳的粒径分布情况，结果见图1，其中，D[3,2]为表面积动量平均径，D[4,3]为体积或质量动量平均径；测定参数：使用湿分散法分析，样品折射率为1.480，水的折光率为1.330，搅拌速率为2000rpm/min，测试温度为25℃，结果见图1；
如图1所示，随着亚麻籽干法磨胶程度的提高，亚麻籽植物乳的粒径呈现下降趋势，表明经过干法脱胶后，亚麻籽植物乳中油滴尺寸下降，利用体系稳定；
(3)亚麻籽植物乳的粘度的测定：取30mL亚麻籽植物乳于50mL平底离心管中，使用数显粘度计，用3号转子，设置转速为60rpm，记录其粘度，结果见表1；
(4)按照GB 5009.5-2016第一法进行亚麻籽植物乳蛋白质含量的测定，结果见表1；
表1不同脱胶率亚麻籽植物乳检测结果
由表1数据可以看出，干法脱胶可以有效降低植物乳黏度；干法脱胶可以有效提高亚麻籽植物乳中蛋白质含量，说明干法脱胶促进了提浆过程中蛋白质的溶出。
二、基于界面调控的亚麻籽微波处理
在反应釜中加入亚麻籽，通入蒸汽，充分搅拌不同时间后取出亚麻籽，调整蒸汽进气量和搅拌时间，获得不同梯度的亚麻籽含水量，梯度从11％-23％；微波作为一种超高频电磁波，促使偶极分子高频往复运动产生"内摩擦热"，会被食物和水等吸收从而使自身发热，不须热传导过程即可实现同时加热、同时升温，速度快且均匀，能耗为传统加热的几分之一或几十分之一，并可实现1)亚麻籽脱毒2)美拉德反应增香3)钝化内源氧化酶，改变细胞壁结构，促使大分子木酚素解聚与多酚溶出，实现提质；从微观角度分析，微波处理后，亚麻籽油脂体的界面结构与界面组成包括磷脂、蛋白质等发生变化，同时微波促使酚 类物质向界面内迁移，有效提高了亚麻籽油脂体的化学稳定性，并对消化过程中ALA的生物利用度起到促进作用。
1.材料与试剂
亚麻黄籽；其他试剂购自国药集团化学试剂有限公司；
2.主要仪器与设备
亚麻籽脱胶机；马尔文3000激光粒度分析仪--英国马尔文仪器公司；上海衡平仪器仪表SNB-1数字粘度计；
3.实验方法及结果
(1)样品制备：选择脱胶亚麻籽，在反应釜中加入亚麻籽，通入蒸汽，充分搅拌不同时间后取出亚麻籽；调整蒸汽进气量和搅拌时间，获得不同梯度(11％-20％)的亚麻籽含水量，梯度从11％-23％，在720W功率下微波9min，获得微波熟制亚麻籽，将各组熟制干法脱胶亚麻籽按照1:7的固液质量比使用纯水室温浸泡2h，胶体磨循环磨浆12min，得到亚麻籽植物乳；
(2)亚麻籽植物乳木酚素分量的含量：称取1.5g亚麻籽植物乳样品，加入8mL 80％(v/v)甲醇水溶液，超声提取30min，再振荡提取30min，在5000rpm条件下离心10min，收集上清液；从中吸取5mL，加入NaOH，使其终浓度为20mmol/L，50℃水浴震荡碱解12h，再加入HCl中和至pH值6.8，用0.22μm滤头过滤后装入上样小瓶，使用配备PDA检测器的Agilent 1290超高效液相色谱仪(UPLC)分析亚麻籽中SDG、CouAG和FeAG含量，结果见图2；色谱条件：RP18色谱柱(100mm×2.1mm，1.7μm)；流动相A，100％甲醇；流动相B，0.5％醋酸水溶液；流速0.10mL/min；检测器波长为280nm，进样量2μL；流速0.1mL/min；梯度洗脱条件：15％A，0～8min；15～28％A，8～12min；28～55％A，16～24min；55～85％A，24～28min；85～15％A，32～33min；15％A，33～35min；
由图2可知，随着亚麻籽调水程度的增加，亚麻籽植物乳中SDG、Cou-AG分量呈现升高趋势，表明调水对亚麻籽植物乳中木酚素的溶出解聚起到促进作用；
(3)微波对界面调控的作用：使用调至20％含水量的亚麻籽，在700W功率下微波1-5min，获得微波熟制亚麻籽，将微波后亚麻籽按照1:10的固液质量比使用纯水室温浸泡2h，剪切磨循环磨浆3min后得到亚麻籽植物乳，再用120目滤袋过滤后，于4℃下10000g转速离心30min，取上层亚麻籽油脂体层作为界面分析材料；采用低温制备系统结合高分辨率场发射扫描电子显微镜对亚麻油脂体的微观形貌进行表征，结果见图3，由图3可知， 微波前亚麻籽油脂体界面光滑，经微波后亚麻籽油脂体界面结构发生明显变化，表面粗糙化提示其界面组成发生改变；
利用激光粒度分析仪通过激光衍射技术确定微波对亚麻籽油脂体粒径分布情况，测定参数：使用湿分散法分析，样品折射率为1.480，水的折光率为1.330，搅拌速率为2000rpm/min，测试温度为25℃，结果见图4A；将乳液以1:250的比例使用去离子水进行稀释，使用马尔文纳米粒径分析仪测定在不同酶解条件下乳液的ζ电位，结果见图4B；
由图4可知，随着微波时间延长，油脂体的粒径先升高再降低，电位逐渐升高，这进一步证实了微波对油脂体界面进行了调控；
总酚和类黄酮测定分别使用福林酚和硝酸铝测定法，结果如图5所示，图5A为类黄酮的调控图，图5B为油脂体总酚的调控图，油脂体中总酚和类黄酮的含量均随着微波时间延长而增加，提示微波促使植物乳体系中抗氧化分子向界面迁移，表现出对植物乳化学稳定性的促进；
(4)微波界面调控促进亚麻籽乳中ALA生物利用率提高：
使用调至20％含水量的亚麻籽，在700W功率下微波1-5min，获得微波熟制亚麻籽，将微波后亚麻籽按照1:10的固液质量比使用纯水室温浸泡2h，剪切磨循环磨浆3min后得到亚麻籽植物乳，再用200目滤袋过滤后留待做动物实验；
雄性SD大鼠(220-250g)来自富和生物技术有限公司(中国上海)，大鼠在控制温度和湿度、12h明暗循环的环境中适应性喂养一周后，随机分为4组，每组15只。禁食一晚后，每组5只大鼠灌胃2.5mL亚麻籽乳。灌胃1、2、4h后处死大鼠前，收集肠组织液氮速冻。将采集的标本立即保存于-80℃，直至分析。高速剪切将空肠组织按1:9(w/v)的比例分散到预冷盐水中。用氯仿-甲醇(2:1,v/v)提取总脂质，10000rpm离心10min，取下清液氯仿层，用氮气干燥。制备脂肪酸甲酯并使用Agilent 6890GC与火焰电离检测器(FID)和二氧化硅毛细管柱(30m×0.25mm,0.25μm)进行分析。温度从175℃开始，保持10分钟，然后以1℃/min的速度增加到250℃。注入器和检测器的温度均设置为250℃。氦气作为载气，流速为1.5mL/min。注入量为2μL，分流比为10:1。注入器和检测器温度设置为250℃。脂肪酸甲酯通过与正品标准(GLC-463)比较进行鉴别，相对含量采用面积归一化法表示。
大鼠在摄入亚麻籽植物乳后，空肠组织的主要N-3系多不饱和脂肪酸分布如下图6所示，图6中A,B,C,D依次为摄入微波亚麻籽乳1h后，空肠组织中ALA及其转化产物EPA、DPA、DHA的比例图，可以看出在摄入未微波亚麻籽乳1h后，空肠组织中ALA及其转 化产物EPA、DPA、DHA的比例分别达到3.82％、0.26％、0.76％和1.29％；当亚麻籽经微波照射1～3min时，空肠组织组织中ALA和EPA的比例分别增加了11.24％和25.90％(p<0.05)；随后，亚麻籽微波照射至5min后，空肠组织中ALA和EPA的比例保持不变，但在亚麻籽微波照射1～5min后，大鼠空肠组织中DPA和DHA的比例呈现同步比例增加。摄入亚麻籽植物乳4h后，随着微波照射时间的延长(1-5min)，大鼠空肠组织中ALA和EPA的积累呈线性减少(-17.42％，-24.38％；p<0.05)。食用未经处理的亚麻籽和微波照射1min的亚麻籽制成的植物乳后，大鼠空肠组织中DPA的比例趋于一致，微波照射3～5min时DPA的比例明显提高(+21.27％，+15.19％；p<0.05)。值得注意的是，亚麻籽微波处理1～5min后，空肠组织中DHA的比例不断上升，然后下降，仍大于未处理的亚麻籽(p<0.05)。总的来说，上述实验结果证明微波处理调控亚麻籽植物乳中油脂体界面，促进了ALA的生物利用度提高。
(5)不同微波时间亚麻籽植物乳的风味
采用顶空固相微萃取法从未酶解、Flavourzyme 500MG，novo、菠萝蛋白酶solarbio、半纤维素酶XS、β-葡聚糖酶XS、纤维素酶CTS novo、CELLUCLAST 1.5L novo酶解植物乳上方顶空提取挥发性化合物，样品依次标号0-6；采用气相色谱-质谱联用技术(Agilent 7890A-5975C)，HP-5MS柱(60m×0.25mm×0.25μm,Agilent Technologies，目录号122-5532)测定挥发物的种类和浓度，结果见表2，入口温度设置为250℃，离子源温度设置为230℃，界面温度设置为280℃，载气流量为1.5mL/min；
过程中使用的温度斜坡为：保持在40℃2min；加热至200℃，4℃/min；保持在200℃2min；然后加热到280℃，8℃/min；注射体积设为1μL；质谱仪在150℃和70eV电压的冲击模式下工作；质谱仪扫描范围40-400amu，溶剂延迟7min；单个化合物通过MS-library搜索(Wiley138K,John Wiley and Sons,HewlettPackard,USA)进行鉴定和量化；通过顶空固相微萃取-气相色谱-质谱联用技术分析酶解对植物乳中挥发性风味化合物的影响；
从表2可知，未经过微波处理的亚麻籽制备的亚麻籽乳风味寡淡，主要呈现随着亚麻籽水分含量的增加，吡嗪类化合物和呋喃类化合物含量逐渐增加，尤其是6min后呈现大幅上升趋势。上述结果表明，微波可大幅提高植物乳香味分子，改善风味。
表2不同水分含量对亚麻籽微波后制备乳液的风味(μg/kg)

三、亚麻籽植物乳靶向酶解处理
靶向酶解原理是基于糖苷键、肽键、酯键的靶向高效水解，实现1)促进细胞壁组成纤维如纤维素、半纤维素、果胶等裂解，提高内源蛋白质、多酚、油脂的高校溶出；2)降低植物大分子多糖的分子量，一方面提高内源膳食纤维的溶出，另一方面降低体系黏度；3)改善体系风味、口感，并提高乳化稳定性；
1.材料与试剂
亚麻黄籽；不同酶，其他试剂购自国药集团化学试剂有限公司；
2.主要仪器与设备
亚麻籽脱胶机；马尔文3000激光粒度分析仪--英国马尔文仪器公司；上海衡平仪器仪表SNB-1数字粘度计；
3.实验方法及结果
(1)样品制备：使用微波脱胶亚麻籽1：7浸泡2h后，按照1：9比例加入纯水，胶体磨循环12min后收集植物乳；添加0.5wt％酶，于50℃酶解1h后收集酶解后植物乳，将全部样品于2770rpm转速下离心3min取上清获得亚麻籽植物乳；
(2)按照GB/T 30885-20146.2对亚麻籽植物乳总固形物含量测定，结果见图7；
由图7可知，随着亚麻籽酶解，亚麻籽植物乳中固形物含量呈现不同的变化趋势，总得来说，蛋白酶、纤维素酶、细胞壁裂解酶对植物乳固形物的提高效果最明显，最高可使固形物超过40％；
(3)亚麻籽植物乳的粘度的测定：取30mL亚麻籽植物乳于50mL平底离心管中，使用SNB-1数字粘度计，用2#转子，设置转速为60rpm，记录其粘度，结果见图8；
由图8可知，随着亚麻籽酶解，亚麻籽植物乳中黏度呈现不同的变化趋势，总得来说，纤维素酶、细胞壁裂解酶、碱性蛋白酶对植物乳黏度的降低效果最明显，最高可使黏度下降超过70％。
(4)酶解亚麻籽植物乳香气成分分析
采用顶空固相微萃取法从未酶解、Flavourzyme 500MG，novo、菠萝蛋白酶solarbio、半纤维素酶XS、β-葡聚糖酶XS、纤维素酶CTS novo、CELLUCLAST 1.5L novo酶解植物乳上方顶空提取挥发性化合物，样品依次标号0-6；采用气相色谱-质谱联用技术(Agilent  7890A-5975C)，HP-5MS柱(60m×0.25mm×0.25μm,Agilent Technologies，目录号122-5532)测定挥发物的种类和浓度，结果见表2，入口温度设置为250℃，离子源温度设置为230℃，界面温度设置为280℃，载气流量为1.5mL/min；
过程中使用的温度斜坡为：保持在40℃2min；加热至200℃，4℃/min；保持在200℃2min；然后加热到280℃，8℃/min；注射体积设为1μL；质谱仪在150℃和70eV电压的冲击模式下工作；质谱仪扫描范围40-400amu，溶剂延迟7min；单个化合物通过MS-library搜索(Wiley138K,John Wiley and Sons,Hewlett Packard,USA)进行鉴定和量化；通过顶空固相微萃取-气相色谱-质谱联用技术分析酶解对植物乳中挥发性风味化合物的影响；
表3酶解对植物乳风味影响结果



结果如表3所示，与0号相比，1、3、5号酶解亚麻籽植物乳新增脂味、甜味、果味，5号新增奶油味、果味、糖浆味、柑橘、黄瓜、青草、柑橘、清新绿色，上述数据显示不同酶解处理对亚麻籽植物乳风味产生了不同影响，总得来看，5号纤维素酶处理后风味较佳；
(5)酶解亚麻籽植物乳的外观检测
使用高分辨率相机对未酶解、Flavourzyme 500MG，novo、菠萝蛋白酶solarbio、半纤维素酶XS、β-葡聚糖酶XS、纤维素酶CTS novo、CELLUCLAST 1.5L novo酶解植物乳，样品依次标号0-6的外观进行拍摄记录，结果见图9；
由图9可知，酶解对亚麻籽植物乳的外观表现出了不同程度的影响，其中1、2、4号酶解亚麻籽植物乳表现出明显褐变，3、5、6号颜色变化不明显；
(5)酶解对亚麻籽植物乳的粒径与电位的影响
利用激光粒度分析仪通过激光衍射技术确定各组亚麻籽植物乳的粒径分布情况，结果见图10-11；
测定参数：使用湿分散法分析，样品折射率为1.480，水的折光率为1.330，搅拌速率为2000rpm/min，测试温度为25℃；
将乳液以1:250的比例使用去离子水进行稀释，使用马尔文纳米粒径分析仪测定在不同酶解条件下乳液的ζ电位，结果见图12，其中未酶解、Flavourzyme 500MG，novo、菠萝蛋白酶solarbio、半纤维素酶XS、β-葡聚糖酶XS、纤维素酶CTS novo、CELLUCLAST 1.5L novo酶解植物乳，样品依次标号0-6；
由图10-12可知，酶解对亚麻籽植物乳的粒径表现出了不同程度的增加效果，同时对油滴表面电荷绝对值产生了降低效果，表明酶解可能使亚麻籽植物乳中油滴界面发生了变化；
四、亚麻籽植物乳复合均质处理
复合均质原理是基于酶解、灭酶、灭菌后，亚麻籽植物乳中油滴界面蛋白质、磷脂、多糖等分子结构、吸附、重排等发生变化，通过复合高压均质促使亚麻籽植物乳中油滴界面活性物质重排，促使絮凝油滴重新分散，显著提高植物乳稳定性；
1.材料与试剂
亚麻黄籽；纤维素酶、糖化酶，其他试剂购自国药集团化学试剂有限公司；
2.主要仪器与设备
亚麻籽脱胶机；马尔文3000激光粒度分析仪--英国马尔文仪器公司；上海衡平仪器仪表SNB-1数字粘度计有限公司粘度计；
3.实验方法及结果
(1)样品制备：使用微波脱胶亚麻籽1：7浸泡2h后，按照1：9比例加入纯水，胶体磨循环12min后收集植物乳；添加1wt％的纤维素酶与2wt％糖化酶，于50℃酶解2h后收集酶解后植物乳，将全部样品于2770rpm转速下离心5min，对亚麻籽植物乳C进行第一次高压均质的压力为20MPa，在无菌条件下对亚麻籽植物乳C进行第二次高压均质的压力为200bar；
(2)亚麻籽植物乳粒径与电位检测
利用激光粒度分析仪通过激光衍射技术确定亚麻籽植物乳的粒径分布情况，结果见图13-14；
测定参数：使用湿分散法分析，样品折射率为1.480，水的折光率为1.330，搅拌速率为2000rpm/min，测试温度为25℃；
将乳液以1:250的比例使用去离子水进行稀释，使用马尔文纳米粒径分析仪测定在不同工艺条件下乳液的ζ电位，结果见图15；
如图13-15所示，亚麻籽植物乳粒径在讲过第一次高压均质耦合灭酶后，出现了大幅增大，电位绝对值出现降低，表明第一次高压均质耦合灭酶处理使植物乳油滴界面受到热的影响产生了部分失稳，但经过UHT耦合第二次高压均质后，粒径重新下降，电位绝对值也得到提高，说明无菌后均质显著改善了植物乳中油滴界面活性物质如蛋白质、磷脂、多糖的吸附与分布情况，促使油滴进一步稳定；
(3)亚麻籽植物乳相分离稳定性检测
用激光衍射扫描测定植物乳的相分离稳定性，该设备由一个装有近红外光源(880nm)的探测头组成，该探测头扫描样品的高度，每40μm采集一次传输和后向散射数据；光源从上到下每隔30秒扫描样品，并测量在25℃下15分钟内光背散射或透射的百分比；使用Turbisoft 2.1软件计算的TSI(Turbiscan stability Index)参数评估植物乳的稳定性，结果如图16所示，TSI结果显示均质对植物乳稳定性均起到促进作用。
五、亚麻籽植物乳的储藏稳定性
亚麻籽植物乳在经过超高温瞬时灭菌、无菌后均质、无菌灌装后，达到了商业无菌条件，可在密闭容器内实现长期储藏且无需担心微生物引起的腐败变质问题，但亚麻籽植物乳属于多相体系，油滴、蛋白质/碳水化合物大分子、不溶性固体颗粒等由于引力的作用，易发生絮凝、聚结、沉淀、上浮等失稳现象，同时ALA在体系中的氧化，也容易导致亚麻籽植物乳稳定性下降，因此为了验证本植物乳可以长时储藏，特开展储藏稳定性实验；
1.材料与试剂
试剂购自国药集团化学试剂有限公司；
2.主要仪器与设备
马尔文3000激光粒度分析仪--英国马尔文仪器公司；上海衡平仪器仪表SNB-1数字粘度计；
3.实验方法及结果
(1)亚麻籽植物乳粒径与电位检测
利用激光粒度分析仪通过激光衍射技术确定亚麻籽植物乳的粒径分布情况，结果见17-18；
其中测定参数：使用湿分散法分析，样品折射率为1.480，水的折光率为1.330，搅拌速率为2000rpm/min，测试温度为25℃；
将乳液以1:250的比例使用去离子水进行稀释，使用马尔文纳米粒径分析仪测定在不同复配比条件下乳液的ζ电位，结果见图19；
由图17-19所示，显示储藏时间为6个月，温度为4℃和37℃时，亚麻籽植物乳的粒径与电位的改变值均较小，说明其稳定性较强；
(2)亚麻籽植物乳相分离稳定性检测
用激光衍射扫描测定植物乳的相分离稳定性，该设备由一个装有近红外光源(880nm)的探测头组成，该探测头扫描样品的高度，每40μm采集一次传输和后向散射数据，光源从上到下每隔30秒扫描样品，并测量在25℃下15分钟内光背散射或透射的百分比，使用Turbisoft 2.1软件计算的TSI(Turbiscan stability Index)参数评估植物乳的稳定性，结果如图20所示，储藏时间为6个月，温度为4℃和37℃时，亚麻籽植物乳的TSI改变值均较小，说明其稳定性较强；
(3)亚麻籽植物乳离心沉淀率的测定
植物乳饮料放置相应时间后，将饮料摇匀后准确称取10g样品，以3000r/min离心15min，记离心管底部的沉淀物重量计算离心沉淀率：离心沉淀率(％)＝沉淀物重量(g)/离心样品重量(g)×100％，试验结果取3次平行测定的平均值，结果如图21所示，储藏时间为6个月，温度为4℃和37℃时，亚麻籽植物乳的离心沉淀率均较小，说明其稳定性较强。
(4)亚麻籽植物乳脂肪酸组成的测定
参考GB 5009.168-2016，称取植物乳1.5000g左右，于10mL塑料离心管中，加2mL正己烷，于超声仪中超声20min，连同残渣在原试管中加入加3mL 0.5M的甲醇-钠溶液，于漩涡混合仪上混合5min后，置于高速离心机中，在5000rpm下离心10min，取上清液待测；
GC测定条件：Agilent 6890型气相色谱仪，Agilent 7683B自动进样器，氢火焰离子化检测器(FID)；
色谱条件：色谱柱HP-INNOWAX 30m×0.32mm×0.25μm；载气为氮气，流速为1.5mL/min；进样量为1μL；进样口温度为260℃，分流比为80:1，分流流量为120mL/min；升温程序：210℃保持9min，20℃/min升至250℃，保持10min，无后运行；
结果如图22所示，亚麻籽植物乳储藏时间为6个月，温度为4℃和37℃时，其脂肪酸组成与原始植物乳相比，为表现明显差异，特别是ALA的组成仍然占比在55％以上，表明了亚麻籽植物乳的化学稳定性较强。
六、亚麻籽植物乳的改善肠道微生态功能评价
1实验方法
SPF级C57BL/6雄性小鼠60只，在20±4℃下，12h的光照/黑暗循环交替、相对湿度为40-55％的环境，给予基础饲料自由适应性饲养7天，然后随机分组(6组，每组10只)设置：正常对照组(NC)，自然恢复组(CS)，亚麻籽奶高低剂量组(FML(约等于人体每日推荐ALA摄入剂量(1.6g/60kgBW/day))、FMM(2倍推荐剂量，使用2倍浓缩奶)，燕麦奶组(OM)、大豆奶组(SM)，各小鼠标号、记录称重；其中，NC组自由饮用蒸馏水；CS组和植物奶组进行400mg/mL头孢曲松钠灌服，每天每只小鼠0.2mL，灌服8天；8日后，植物奶组分别灌胃等能量的植物奶(FML-450uL、FMM-450uL，OM-400uL和SM-330uL)，NC、CS组用400uL生理盐水灌胃，每组灌服14天。实验过程中，小鼠自由进食饮水，每天记录进食量和体重。于饲养第30天颈椎脱臼处死小鼠，收集小鼠血液样本、肠道样本、肠内容物等用于后续实验；
2实验结果
2.1植物奶对结肠组织损伤修复效果
取近盲端小肠肠段0.5cm，用预冷无菌生理盐水冲洗干净后，放入4％中性甲醛固定24h，常规脱水，石蜡包埋，切片(厚4μm)，HE染色，最后使用光学显微镜进行镜检并且对图像进行采集分析，结果见图23，结果表明：CS组杯状细胞减少，隐窝破坏和缺失，固有层和粘膜下层有炎性细胞浸润，NC组结肠组织形态正常，结肠黏膜完整，隐窝结构健康，CS处理的小鼠相比，OM组与SM组也表现出炎性细胞浸润、杯状细胞消失的特点，而给予浓缩两倍亚麻籽奶处理的小鼠FMM组更能使黏膜结构恢复健康；
2.2植物奶对肠道菌群的影响
收集小鼠的粪便样本并立即储存在-80℃进行生物信息学分析，使用DNA试剂盒分离粪便样本中的的细菌DNA，用引物338(5'-ACTCCTACGGGAGGCAGCA-3')和806R(5'-GGACTACHVGGTWTCTAAT-3')PCR扩增V3-V4可变区域，扩增的产物送到IlluminaMiSeq平台上进行测序，得到的序列被分到同一个操作分类单元(OUT)，其中序列的相似度≥97％；
使用Shannon、Simpson和Observed Species等指标评测Alpha多样性(Alphadiversity)，从而用于推测样本组内的多样性；其中，Shannon指数和Simpson指数的数值越大，则代表样本的多样性组成越丰富，而Beta多样性(Beta diversity)用于样本组之间多样性的比较，使用未加权的UniFrac衡量β多样性(即PCoA分析)，采用区分样本之间的两个最 大差异特征作为坐标轴进行作图分析，结果见图24-26，如图24所示，依次为Simpson、Pielou_e、Shannon和Chao1结果图，Simpson和Pielou_e指数两组比较无统计学意义，与NC组相比，经抗生素处理后小鼠的Chao1指数和Shannon指数明显下降(P＜0.05)，说明抗生素处理可以降低群落的丰度，且在四种植物奶的影响下，群落丰富度无明显变化，可能是由于抗生素干预时间较短；如图25所示，经抗生素处理的小鼠CS组离NC组相距较远，说明差异较大，在四种植物奶影响下SM组合OM组相距较近，FML和FMM组相距较近，说明其物种组成相近；从图26中，发现与NC组相比，经抗生素处理后的CS组中[Eubacterium]，Enterococcus，Akkermansia、[Eubacterium]菌属相对丰度升高，但在四种植物奶的影响下降低了Akkermansia、Bacteroides菌属的相对丰度，OM组和SM组Enterococcus菌属的相对丰度升高了；Bacteroides菌属经抗生素处理后相对丰富升高，但在OM组和SM组影响下，可以降低其相对丰度。Bifidobacterium、Lactobacillus经抗生素处理CS组丰度显著降低，在四种植物奶影响下也未见变化；在抗生素小鼠模型上，服用亚麻籽奶可以促进Parabacteroides菌属的丰度，服用豆奶和燕麦奶可以促进Lachnospiraceae_Clostridium的菌属丰度。
七、亚麻籽植物乳的免疫力增强试验
1材料和方法
1.1样品来源及处理
亚麻籽植物乳由中国农业科学院油料作物研究所委托提供；人体推荐日摄入量为300mL/人/d，即5mL/kg BW(按照成人60kg体重均值计算)；
1.2实验动物
SPF级KM雌性小鼠，18-22g，第一批动物150只，湖北省实验动物研究中心提供，生产许可证号为SCXK(鄂)2020-0018；实验动物质量合格证号：NO.42000600047363；第二批动物100只，湖北省实验动物研究中心提供，生产许可证号为SCXK(鄂)2020-0018，实验动物质量合格证号：NO.42000600048366；
动物饲料：武汉万千佳兴生物科技有限公司提供，许可证号为SCXK(鄂)2021-0011；
饲养环境：本中心SPF级动物实验室，温度20-26℃，湿度40-70％，使用许可证号为SYXK(鄂)2017-0065；第一批实验动物设施合格证号：NO.00295306；第二批实验动物设施合格证号：NO.00295999；
1.3主要仪器与试剂
酶标仪，Multiskan GO1510型CO2培养箱，MCO-18AIC(UV)生物显微镜，OLYMPUS CX41
1.4剂量设计与分组
动物分为五个实验组，每组50只，第一批动物150只分别进行以下三组实验：免疫实验一组进行小鼠迟发型超敏反应试验，实验二组进行小鼠碳廓清实验，实验三组进行小鼠腹腔巨噬细胞吞噬鸡红细胞实验；第二批动物100只分别进行以下两组实验：免疫实验四组进行血清溶血素的测定和抗体生成细胞检测；实验五组进行ConA诱导的小鼠淋巴细胞转化实验和NK细胞活性测定；
受试物人体推荐日摄入量为300mL/人/d，即5mL/kg BW(按照成人60kg体重均值计算)，设置亚麻籽植物乳低、高两个剂量组，另设阴性对照组(蒸馏水)、豆本豆大豆乳对照组和燕麦乳对照组，实验设计豆本豆大豆乳对照组和燕麦乳对照组的蛋白质含量与亚麻籽植物乳剂量组样品的蛋白质含量一致，小鼠灌胃容量为40ml/kg BW，连续灌胃给予受试物28天后进行测试；
1.5实验方法
1.5.1样品的配制及给予
亚麻籽植物乳、豆本豆大豆乳对照组和燕麦乳对照组均用蒸馏水配制，并现配现用，具体配制方法，见表4，
表4亚麻籽植物乳浓缩液的配制方法
1.5.2二硝基氟苯(DNFB)诱导迟发型变态反应(DTH)
采用耳肿胀法：用1％DNFB(以1:1的丙酮麻油溶液配制)致敏小鼠后，第5天再用DNFB攻击右耳，24h后处死动物剪下左右耳壳用打孔器取下直径8mm的耳片，称重，以左右耳的重量之差来表示DTH的程度；
1.5.3抗体生成细胞检测
Jeme改良玻片法：取脱纤维的羊血，用生理盐水洗涤3次，离心(2000r/min)10min，每只小鼠经腹腔注射2％(v/v)SRBC 0.2mL；将SRBC免疫4后的小鼠颈椎脱臼处死，取出脾脏，加入Hank’s液，磨碎脾脏，制成细胞悬液，经200目筛网过滤，离心(1000/min)10min，用Hank’s液洗2遍，最后将细胞悬浮在5mLRPMI1640培养液中，计数并将细胞浓度调整为5×106个/mL；
空斑的测定：将表层培养基(1g琼脂糖加双蒸水至100mL)加热溶解后，放入45-50℃水浴保温，与等量pH7.2～7.4、2×Hank’s液混合，分装小试管，每管0.5mL，再向管内加50μL 10％SRBC(v/v，用SA缓冲液配制)，20μL脾细胞悬液(5×106个/mL)，迅速混匀，倾倒于己刷琼脂糖薄层的玻片上，做平行片，待琼脂凝固后，将玻片水平扣放在片架上，放入二氧化碳培养箱中孵育1.5h，然后用SA缓冲液稀释的补体(1:8)加入玻片架凹槽内，继续温育1.5h后，计数溶血空斑数；
1.5.4血清溶血素的测定
血凝法：取羊血，用生理盐水洗涤3次，每次离心(2000r/min)3min，将压积SRBC用生理盐水配成2％(v/v)的细胞悬液，每只鼠腹腔注射0.2mL进行免疫，4天后，摘除眼球取血于离心管内，放置约lh，将凝固血与管壁剥离，使血清充分析出，2000r/min离心10min，收集血清；
凝集反应：用生理盐水将血清倍比稀释，将不同稀释度的血清分别置于微量血凝实验板内，每孔100μL，再加入100μL 0.5％(v/v)SRBC悬液，混匀，放入湿润的平盘内并加盖，于37℃温箱孵育3h，观察血球凝集程度，根据血清凝聚程度的级别计算出抗体积数；
1.5.5 NK细胞活性测定
乳酸脱氢酶(LDH)测定法；
靶细胞的传代(YAC-1细胞)：实验前24h将靶细胞进行传代培养，用前Hank’s液洗3次，用RPMIl640完全培养液调整细胞浓度为4×105个/mL；
脾细胞悬液的制备(效应细胞)：无菌取脾，置于盛有适量无菌Hank’s液的小平皿中，轻轻将脾磨碎，制成单细胞悬液，经200目筛网过滤，用Hank’s液洗2次，每次离心10min(1000r/min)，弃上清将细胞浆弹起，加入0.5mL灭菌水20秒，裂解红细胞后再加入0.5mL 2×Hank’s液，离心(1000r/min)10min，弃上清，用lmL含10％小牛血清的RPMIl640完全培养液重悬，用台酚兰染色计数活细胞数(应在95％以上)，最后用RPMll640完全培养液调整细胞浓度为2×107个/mL；
NK细胞活性检测：取靶细胞和效应细胞各100μL(效靶比50:1)，加入U型96孔培养板中：靶细胞自然释放孔加靶细胞和培养液各100μL，靶细胞最大释放孔加靶细胞和1％NP40各100μL，上述各项均设三个平行孔，于37℃、5％CO2培养箱中培养4h，然后将96孔培养板以1500r/min离心5min，每孔吸取上清100μL置平底96孔培养板中，同时加入LDH基质液100μL，根据室温不同反应3-10min，每孔加入lmol/L的HCL30μL，在酶标仪490nm处测定光密度值(OD)；
1.5.6 ConA诱导的小鼠脾淋巴细胞转化试验
采用MTT法：无菌取脾，置于盛有适量无菌Hank’s液平皿中，用镊子轻轻将脾磨碎，制成单个细胞悬液，经200目筛网过滤，用Hank’s液洗2次，每次离心1.8gin(1000r/min)，然后将细胞悬浮于1mL的完全培养液中，用台酚兰染色计数活细胞数(应在95％以上)，调整细胞浓度为3×106个/mL，将每一份脾细胞悬液分两孔加入24孔培养板中，每孔1mL，一孔加75μL Con A液(相当于7.5μg/mL)，另一孔作为对照，置5％CO2，37℃CO2孵箱中培养72h；培养结束前4h，每孔轻轻吸去上清液0.7mL，加入0.7mL不含小牛血清的RPMIl640培养液，同时加入MTT(5mg/mL)50μL/孔，继续培养4h，培养结束后，每孔加入1mL酸性异丙醇，吹打混匀，使紫色结晶完全溶解，然后分装到96孔培养板中，每个孔作2个平行孔(100μL/孔)，用酶标仪以570nm波长测定光密度值；
1.6数据处理和结果判定
用SPSS软件，单因素方差分析方法及多个实验组和一个对照组均数的两两比较的方法，比较剂量组和对照组是否有差异，如果任何一个剂量组与对照组比较差异具有显著性且增强(P<0.05)，则该实验为阳性；
一般采用方差分析，但需按方差分析的程序先进行方差齐性检验，方差齐，计算F值，F值＜F0.05，结论：各组均数间差异无显著性：F值≥F0.05，P≤0.05，用多个实验组和一个对照组间均数的两两比较方法进行统计；
对非正态或方差不齐的数据进行适当的变量转换，待满足正态或方差齐要求后，用转换后的数据进行统计；若变量转换后仍未达到正态或方差齐的目的，改用秩和检验进行统计。
2结果
2.1对小鼠体重的影响
五个实验组，与阴性对照组比较，受试样品对各剂量组小鼠体重均无明显影响，差异均无显著性(P＞0.05)，结果见表5-9，
表5亚麻籽植物乳对第一组小鼠体重的影响(均数±标准差)

注：与阴性对照组比较，P＞0.05。
表6亚麻籽植物乳对第二组小鼠体重的影响(均数±标准差)

注：与阴性对照组比较，P＞0.05。
表7亚麻籽植物乳对第三组小鼠体重的影响(均数±标准差)

注：与阴性对照组比较，P＞0.05。
表8亚麻籽植物乳对第四组小鼠体重的影响(均数±标准差)


注：与阴性对照组比较，P＞0.05。
表9亚麻籽植物乳对第五组小鼠体重的影响(均数±标准差)

注：与阴性对照组比较，P＞0.05。
2.2对小鼠抗体生成细胞检测的影响
与阴性对照组比较，亚麻籽植物乳各剂量组不能明显提高小鼠溶血空斑数(P＞0.05)，见表10；
表10对抗体生成细胞功能和溶血素滴度水平的影响(均数±标准差)

注：与阴性对照组比较，P＞0.05。
2.3对小鼠血清溶血素滴度水平的影响
与阴性对照组比较，亚麻籽植物乳各剂量组不能明显提高小鼠血清抗体积数(P＞0.05)，见表10；
2.4对小鼠迟发型变态反应的影响
与阴性对照组比较，亚麻籽植物乳低剂量组能增强小鼠对DNFB诱发的DTH反应(P<0.05)，与燕麦乳对照组比较，亚麻籽植物乳低、高剂量组能增强小鼠对DNFB诱发的DTH反应(P<0.05)，见表11；
表11亚麻籽植物乳对DNFB诱发的DTH耳重的影响(均数±标准差)

注：*：与阴性对照组比较P<0.05；#：与燕麦乳对照组比较P<0.05，##：与燕麦乳对照组比较P<0.01
2.5对小鼠脾淋巴细胞转化的影响
与阴性对照组比较，亚麻籽植物乳高剂量组能明显增强小鼠ConA诱导的脾淋巴细胞增殖能力(P<0.05)，见表12；
表12对抗Con A诱导脾淋巴细胞增殖和NK细胞活性的影响(均数±标准差)

注：*：与阴性对照组比较P<0.05。#：与豆本豆大豆乳对照组比较P<0.05。
2.6对小鼠NK细胞活性的影响
与阴性对照组、豆本豆大豆乳对照组比较，亚麻籽植物乳高剂量组能明显增强小鼠NK细胞活性(P<0.05)，其余各剂量组无明显变化，见表12。
3结论
选用SPF级KM雌性小鼠作为实验体系，进行了增强免疫力功能试验研究。根据亚麻籽植物乳的人群日推荐摄入量为300mL/人/d，即5mL/kg BW(按照成人60kg体重均值计算)，设计亚麻籽植物乳低、高(分别相当于人群推荐量的10、20倍)两个剂量组，同时设阴性对照组(蒸馏水)、豆本豆大豆乳对照组和燕麦乳对照组。小鼠连续灌胃给予28天后开始测试，实验结果以P<0.05判断为显著性差异。结果显示：亚麻籽乳，豆本豆乳、燕麦乳均对小鼠体重无明显影响；与阴性对照组比较，亚麻籽植物乳低剂量组能增强小鼠对DNFB诱发的迟发性超敏反应(DTH)，增幅达27.4％，与燕麦乳相比，提高31.3％； 与阴性对照组比较，亚麻籽植物乳高剂量组增强小鼠ConA诱导的脾淋巴细胞增殖能力可达21.6％；与阴性对照组、豆本豆乳、燕麦乳相比，增强小鼠NK细胞活力分别达19.6％，10％，12.2％。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种基于界面调控的具有良好风味与功能活性亚麻籽植物乳的制备方法，其特征在于，具体包括以下步骤：
    (1)将亚麻籽依次经过脱胶、微波熟制和浸泡软化的预处理后，备用；
    (2)将浸泡软化得到的所述亚麻籽依次经过胶体研磨、酶解和除渣后，得到亚麻籽植物乳A；
    (3)将所述亚麻籽植物乳A依次经过第一次高压均质、灭酶灭菌和第二次高压均质后，即得到一种基于界面调控的具有良好风味与功能活性的常存亚麻籽植物乳。
  2. 根据权利要求1所述的一种基于界面调控的具有良好风味与功能活性的亚麻籽植物乳的制备方法，其特征在于，步骤(1)所述脱胶采用干法脱胶；
    所述微波熟制的条件为：微波温度为115-145℃，微波时间为3-12min，亚麻籽调水范围为8-20％；
    所述浸泡软化的固液质量比为1：(5-10)，浸泡时间2-24h。
  3. 根据权利要求1所述的一种基于界面调控的具有良好风味与功能活性的亚麻籽植物乳的制备方法，其特征在于，步骤(2)所述胶体研磨时间为10-210min，亚麻籽与水的固液质量比为1：(5-10)；
    所述除渣采用的为卧螺除渣，转速为2500-3000rpm。
  4. 根据权利要求1所述的一种基于界面调控的具有良好风味与功能活性的亚麻籽植物乳的制备方法，其特征在于，步骤(2)所述酶解采用的酶为纤维素酶、糖化酶、蛋白酶、果胶酶和植酸酶中的任意一种。
  5. 根据权利要求1所述的一种基于界面调控的具有良好风味与功能活性的亚麻籽植物乳的制备方法，其特征在于，步骤(2)所述酶解的温度为45-55℃，时间为30-120min，添加量为0.01-2％。
  6. 根据权利要求1所述的一种基于界面调控的具有良好风味与功能活性的亚麻籽植物乳的制备方法，其特征在于，步骤(3)所述第一次高压均质的压力为5-20MPa，所述第二次高压均质的压力为50-200bar。
  7. 根据权利要求1所述的一种基于界面调控的具有良好风味与功能活性的亚麻籽植物乳的制备方法，其特征在于，步骤(3)所述灭酶的温度为90-115℃，时间为15-300S；
    所述灭菌采用UHT灭菌，温度为135-140℃，时间为8-30S。
  8. 根据权利要求1所述的一种基于界面调控的具有良好风味与功能活性的亚麻籽植 物乳的制备方法，其特征在于，还包括具有良好风味与功能活性的常存亚麻籽植物乳进行无菌灌装，罐装温度为25-40℃，罐装包括纸包、PET瓶、玻璃罐和铝罐中的任意一种
  9. 如权利要求1-8任一项所述制备方法得到的基于界面调控的具有良好风味与功能活性的亚麻籽植物乳。
  10. 如权利要求9所述的亚麻籽植物乳在食品加工中的应用。
PCT/CN2023/079484 2022-10-14 2023-03-03 一种基于界面调控的具有良好风味与功能活性亚麻籽植物乳及其制备方法与应用 WO2024077846A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105918610A (zh) * 2016-04-20 2016-09-07 武汉天天好生物制品有限公司 一种具有降胆固醇作用的亚麻籽肽及其制备方法
CN108308585A (zh) * 2018-02-13 2018-07-24 广州利众生物科技有限公司 一种即食膳食亚麻籽粉及其制备方法
CN111000189A (zh) * 2019-10-14 2020-04-14 杭州耐雀生物科技有限公司 一种亚麻籽仁浆料及其制备方法和应用
CN113785881A (zh) * 2021-09-09 2021-12-14 中国农业科学院油料作物研究所 一种无添加自稳定的亚麻籽鲜食植物乳的制备方法及应用
CN115553341A (zh) * 2022-10-14 2023-01-03 中国农业科学院油料作物研究所 一种基于界面调控的具有良好风味与功能活性亚麻籽植物乳及其制备方法与应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2333656C1 (ru) * 2007-02-26 2008-09-20 Государственное научное учреждение Всероссийский научно-исследовательский, проектно-технологический институт механизации льноводства Россельхозакадемии (ГНУ ВНИПТИМЛ Россельхозакадемии) Способ приготовления растительного молока из семян льна
CN108485798A (zh) * 2018-03-30 2018-09-04 广州利众生物科技有限公司 一种不含氰化物的冷榨亚麻籽油生产方法
CN110973557A (zh) * 2019-11-01 2020-04-10 杭州耐雀生物科技有限公司 一种干法酶解制备多肽亚麻籽酱的方法
CN114376134A (zh) * 2022-01-24 2022-04-22 河南农业大学 富含开环异落叶松脂酚和异黄酮的亚麻胚芽乳及其生产方法
CN114568533A (zh) * 2022-02-24 2022-06-03 青岛德慧海洋生物科技有限公司 一种亚麻籽仁饮品及其制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105918610A (zh) * 2016-04-20 2016-09-07 武汉天天好生物制品有限公司 一种具有降胆固醇作用的亚麻籽肽及其制备方法
CN108308585A (zh) * 2018-02-13 2018-07-24 广州利众生物科技有限公司 一种即食膳食亚麻籽粉及其制备方法
CN111000189A (zh) * 2019-10-14 2020-04-14 杭州耐雀生物科技有限公司 一种亚麻籽仁浆料及其制备方法和应用
CN113785881A (zh) * 2021-09-09 2021-12-14 中国农业科学院油料作物研究所 一种无添加自稳定的亚麻籽鲜食植物乳的制备方法及应用
CN115553341A (zh) * 2022-10-14 2023-01-03 中国农业科学院油料作物研究所 一种基于界面调控的具有良好风味与功能活性亚麻籽植物乳及其制备方法与应用

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