CN107212421B

CN107212421B - A method for preparing nutritional composition rich in milk protein active peptide and phosphatidylserine

Info

Publication number: CN107212421B
Application number: CN201710360598.9A
Authority: CN
Inventors: 袁永红; 郭旭; 杨贺
Original assignee: Lixing Tianjin Biotechnology Co ltd
Current assignee: Lixing Tianjin Biotechnology Co ltd
Priority date: 2017-05-21
Filing date: 2017-05-21
Publication date: 2021-09-14
Anticipated expiration: 2037-05-21
Also published as: CN107212421A

Abstract

The invention relates to a method for preparing a nutritional composition rich in milk protein active peptide and phosphatidylserine by adopting a microchannel reactor, which comprises the following steps: pumping whey protein reaction liquid rich in MFGM and protease solution into a microchannel reactor to prepare a milk protein active peptide system; step two, inactivating protease activity at high temperature; pumping the serine solution containing the phospholipase D solution into a microchannel reactor, and fully mixing the serine solution with the reaction system prepared in the second step to obtain a mixed reaction system; step four, carrying out phospholipase D catalytic reaction; step five, inactivating the activity of phospholipase D at high temperature, and stopping the enzymolysis reaction; and step six, quickly cooling to the temperature required by the process to obtain the product. The invention has the advantages of high reaction speed, high yield and good product quality.

Description

A method for preparing nutritional composition rich in milk protein active peptide and phosphatidylserine

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for preparing a nutritional composition rich in milk protein active peptide and phosphatidylserine in a microchannel reactor.

Background

The bioactive peptide refers to peptide chain fragments which are derived from animals, plants and microorganisms and have a bioactive effect. Cow's milk is the main source of bioactive peptides. The bioactive peptides contained in the cow milk are divided into free bioactive peptides and bioactive peptides to be subjected to enzymolysis. The free active peptide in cow milk includes insulin, insulin-like growth factor, transforming growth factor, epidermal growth factor, free nerve growth factor, etc. The active peptide to be subjected to enzymolysis in the cow milk mainly comprises various biological active peptides such as casein phosphopeptide, morphoidin, antihypertensive peptide, immunoregulation peptide, antithrombotic peptide, antibacterial peptide and the like. The milk protein active peptide has various physiological activity functions and becomes an important research and development hotspot in the food industry.

Milk Fat Globule Membrane (MFGM) refers to a membrane or membrane-associated material that surrounds fat globules in mammalian Milk. The milk fat globule membrane contains specific glycoproteins such as lactoferrin, mucin, lactadherin and xanthine oxidase, and complex lipids such as glycerophospholipids and sphingolipids. In mammalian milk, fat is about 40g/L, consisting mainly of triglycerides (96%, W/W of total fat), diglycerides (3%, W/W of total fat) and complex lipids (1%, W/W of total fat).

Phosphatidylserines are a ubiquitous class of phospholipids, usually located in the inner layers of cell membranes, and are involved in a range of membrane functions. Phosphatidylserine can increase the number of brain spurs, the fluidity of brain cell membranes and promote the glucose metabolism of brain cells, so that the brain cells are more active, and nowadays, the phosphatidylserine is increasingly applied to the fields of medicine and health care.

Various ingredients rich in MFGM are commercially available. MFGM can be present in cream, buttermilk and whole milk. For example Lacprodan MFGM-10, which is a MGFM rich whey protein fraction produced by Arla Food Ingredient amba (Denmark). As the MFGM product rich in milk fat globule membrane usually contains rich protein and phospholipid (containing about 50 percent of protein and about 12 percent of phospholipid, including about 3 percent of phosphatidylcholine and about 2.7 percent of sphingomyelin, W/W), the MFGM product can be used as a basic raw material and developed into a nutritional composition rich in milk protein active peptide and phosphatidylserine by an enzymatic process, and the nutritional composition is applied to the fields of food, nutrition and health care, cosmetics and the like.

The current methods for preparing milk protein active peptides are mainly divided into the following two categories:

1, chemical hydrolysis method: mixing lactoprotein with acid or alkali solution of certain concentration, breaking peptide chain in a sealed container at certain temperature and pressure, and separating and purifying the hydrolysate. However, this method requires the use of a large amount of acid and base, and the active peptide products obtained by the preparation have an unpleasant odor, so that the chemical hydrolysis method is gradually replaced by a biological method.

2, biological method: the method for preparing the bioactive peptide by hydrolyzing the protein at a certain temperature by using the protease or directly using the microorganism (containing the compound protease) has the advantages of mild operation conditions, no use of toxic chemical reagents, good product quality and the like, and is a main method for preparing the bioactive peptide at present.

In the prior art, various methods for preparing phosphatidylserine are available, and extraction method and enzyme conversion method are mainly used. The extraction method is to extract phosphatidylserine from animal phospholipids, and mainly takes animal brains as raw materials. Patent CN1583766A is to extract phosphatidylserine from animal brain. However, the safety of the product is suspected due to mad cow disease and the like, and the product is eliminated. In recent years, enzyme catalysis techniques are widely applied to preparation of phosphatidylserine, for example, patent CN101230365A is to use soybean lecithin as a raw material to prepare phosphatidylserine by an enzyme method, patent CN100402656C is to use phospholipid extracted from fish liver as a raw material, patent CN101157946A is to use phospholipid extracted from squid as a raw material, and phospholipase D is used to catalyze phosphatidylcholine in fish liver or phosphatidylcholine in squid to react with serine to prepare phosphatidylserine rich in polyunsaturated double-bond fatty acyl group.

However, the prior art for preparing phosphatidylserine by enzyme catalysis faces the problem of low product purity, and the main reasons are that the prior enzyme catalysis reaction adopts a traditional kettle type reactor, and due to the characteristics of interface reaction, the catalytic reaction is not complete because of slow heat and mass transfer of the kettle type reactor and difficulty in full contact of reactants and enzyme, and the product phosphatidylserine is easily hydrolyzed by phospholipase D to generate phosphatidic acid and other byproducts after reaching a certain concentration, so that the content of phosphatidylserine in the final product is not high, the residue of the substrate is high, and the product phosphatidylserine contains high amount of phosphatidic acid and other byproducts. Therefore, how to effectively increase the mass transfer effect of the reaction system, improve the efficiency of the catalytic reaction and accelerate the reaction process, thereby shortening the reaction time and reducing the generation of the hydrolysis by-product of the phosphatidic acid is always a key problem to be solved by the technology for preparing the phosphatidylserine by enzyme catalysis.

The microchannel reactor is a continuous flow pipeline reactor, wherein the inner diameter of the pipeline is controlled to be 10-1000 microns, and is a device for controlling chemical or enzyme catalytic reaction in a tiny reaction space.

So far, no process study on preparation of nutritional compositions rich in milk protein active peptides and phosphatidylserine by taking MGFM-rich whey protein as a raw material and applying a microchannel reactor and using an enzyme catalysis technology is available.

The present invention has been made in view of the above circumstances.

The invention content is as follows:

the invention aims to provide a method for preparing a nutritional composition rich in milk protein active peptide and phosphatidylserine by using MGFM-rich whey protein as a raw material and applying a microchannel reactor and using an enzyme catalysis technology aiming at the defects of the prior art.

The purpose of the invention is realized by the following technical scheme: a process for the preparation of a nutritional composition enriched in milk protein active peptides and phosphatidylserine, the preparation being carried out by the steps of:

pumping MGFM-rich whey protein reaction liquid and protease solution into a first mixing module of a microchannel reactor, mixing the MGFM-rich whey protein reaction liquid and the protease solution, then feeding the mixture into the first reaction module, and rapidly heating the mixture to perform a proteolysis reaction to prepare a milk protein active peptide system;

secondly, allowing a milk protein active peptide system to enter a first high-temperature module, inactivating the activity of protease at high temperature, stopping the enzymolysis reaction, allowing the reaction system to flow through a first low-temperature module and then enter a second mixing module;

pumping the serine solution containing the phospholipase D solution into a second mixing module of the microchannel reactor, and fully mixing the serine solution with the reaction system prepared in the second step to obtain a mixed reaction system;

step four, the mixed reaction system in the step three enters a second reaction module, the temperature is rapidly increased to the temperature required by the process, and the phospholipase D catalytic reaction is carried out;

after the catalytic reaction of the phospholipase D is finished, the reaction system enters a second high-temperature module, the activity of the phospholipase D is passivated at high temperature, and the enzymolysis reaction is stopped;

and step six, after leaving the second high-temperature module, the reaction system enters a second low-temperature module, is rapidly cooled to the temperature required by the process, and finally flows out of the microchannel reactor to obtain a nutritional composition product rich in lactoprotein active peptide and phosphatidylserine.

Further, the microchannel reactor comprises a mixing module, a reaction module, a high-temperature module and a low-temperature module, wherein the length of a fluid channel of each module is 0.1-300 m, preferably 1-60 m; the width and depth of the fluid channel of each module is 0.5 mu m-10 mm, preferably 0.1 mm-1 mm; the width and depth of the fluid channel of each module part link are 0.5-100 μm.

Further, the proportion of each substance in the reaction solution in the first step is as follows: the MGFM-rich whey protein solution is obtained by uniformly mixing MGFM-rich whey protein and water according to the weight ratio of 1: 10-15; the protease solution is obtained by uniformly mixing a complex enzyme and water according to a weight ratio of 1: 100-500.

Further, in the first step, the temperature of the first mixing module is 0-20 ℃, preferably 10-15 ℃; the residence time of the reaction system in the mixing module is 30 to 1200 seconds, preferably 30 to 300 seconds.

Further, in the first step, the reaction temperature in the first reaction module is 35-55 ℃, preferably 45-50 ℃; the residence time of the reaction system in the first reaction module is 30 to 1200 seconds, preferably 60 to 300 seconds.

Further, in the second step, the temperature of the first high-temperature module is 75-105 ℃, preferably 85-90 ℃; the residence time of the reaction system in the first high temperature module is 10 to 1200 seconds, preferably 15 to 120 seconds.

Further, in the second step, the temperature of the first low-temperature module is 0-20 ℃, preferably 10-15 ℃; the residence time of the reaction system in the first low temperature module is 30 to 1200 seconds, preferably 30 to 300 seconds.

Further, in the third step, the serine solution is obtained by uniformly mixing serine and water according to the weight ratio of 1: 10-25, preferably 10-15.

The weight ratio of the mass of serine to the raw material (MGFM-rich whey protein) was 1: 2-10, preferably 4-5; adding calcium salt into the solution, wherein the weight part of the calcium salt in each liter of reaction solution is 0.05-0.3, preferably 0.1-0.2; adding phospholipase D to the solution, wherein the weight portion of phospholipase D in each liter of the reaction solution is 0.01-0.1, preferably 0.05-0.06.

Further, in the fourth step, the temperature of the second mixing module is 0-20 ℃, preferably 10-15 ℃; the residence time of the reaction system in the mixing module is 30 to 1200 seconds, preferably 30 to 300 seconds.

Further, in the fourth step, the reaction temperature of the second reaction module is 35-55 ℃, preferably 45-50 ℃; the residence time of the reaction system in the second reaction module is 30 to 1200 seconds, preferably 60 to 300 seconds.

Further, in the fifth step, the temperature of the second high-temperature module is 75-105 ℃, preferably 85-90 ℃; the residence time of the reaction system in the second high temperature module is 10-1200 seconds, preferably 15-120 seconds.

Further, in the sixth step, the temperature of the second low-temperature module is 0-20 ℃, preferably 10-15 ℃; the residence time of the reaction system in the second low temperature module (8) is 30-1200 seconds, preferably 30-300 seconds.

The invention also provides application of the prepared nutritional composition in the fields of food and nutritional health care.

Compared with the prior art, the invention has the following advantages:

1-the efficiency of catalytic reaction can be greatly improved, the reaction time is shortened, the catalytic rate can reach more than 95% under the same condition compared with the reaction in a kettle type reactor, and the time of the microchannel reactor can be saved by more than 50%;

2-the milk protein active peptide and the phosphatidylserine can be prepared step by step through a continuous flow preparation process, and the generation of phosphatidic acid which is a byproduct of the hydrolysis of the phosphatidylserine in a final product is greatly reduced;

3-the reaction temperature and the post-treatment temperature can be quickly and accurately controlled, the reaction temperature can be kept accurately and constantly, the generation of byproducts is reduced, and the stable quality of the product is ensured;

4-the invention has no amplification effect by using the microchannel reactor, is easy to upgrade and amplify and can realize seamless amplification from research and development to production.

The final product rich in milk protein active peptide and phosphatidylserine can be used in high-grade nutritional products and health products, and can also be added into food, medicines or cosmetics.

Description of the drawings:

fig. 1 is a process flow diagram of the present invention for preparing a nutritional composition enriched in milk protein active peptides and phosphatidylserine.

Detailed Description

For a better understanding of the present invention, reference is made to the following description taken in conjunction with the accompanying drawing 1.

In the present invention, MGFM refers to a substance named Milk Fat Globule Membrane (MFGM).

Fig. 1 is a process flow diagram for producing a nutritional composition enriched in milk protein active peptides and phosphatidylserine using a continuous microchannel reactor, where the modules within the continuous microchannel reactor may be a series of multiple sub-modules. A method for preparing a nutritional composition enriched in milk protein active peptides and phosphatidylserine using a microchannel reactor, comprising the steps of:

pumping the MGFM-rich whey protein reaction liquid and the protease solution into a first mixing module of a microchannel reactor, mixing the MGFM-rich whey protein reaction liquid and the protease solution, then feeding the mixture into the first reaction module, and rapidly heating the mixture to perform proteolysis reaction to prepare the active peptide system of the milk protein.

Step one, preparing MGFM-rich whey protein reaction solution and protease solution. The whey protein reaction liquid is obtained by uniformly mixing MGFM-rich whey protein and water according to the weight ratio of 1: 10-15, and the mixture is treated by a high-pressure homogenizer under the pressure of 200-500 bar to obtain a protein reaction liquid; the protease solution is obtained by uniformly mixing a complex enzyme and water according to the weight ratio of 1: 100-500, wherein the complex enzyme consists of neutral protease and papain or bromelain, and the weight ratio of the neutral protease to the papain or bromelain is 1: 1-1.5; the weight part of the protease solution in each liter of the mixed reaction solution is 1-20, preferably 5-10.

The MGFM rich whey protein material may be derived from commercial processed products of cream, buttermilk and whole Milk powders, such as Lacprodan MFGM rich whey protein powder Lacprodan MFGM-10 from Arla Food Ingredients amba (Denmark) and MGFM rich whey protein powder SMI Lipidex Concentrate 100 from Synlait Milk Ltd (New Zealand), or may be prepared by means of techniques commonly used in the art.

The complex enzyme consists of food-grade neutral protease and papain or bromelain. Neutral proteases, papain and bromelain are available from various commercial sources, such as, but not limited to, Novistin enzyme preparations.

The reaction system composed of the MGFM-rich whey protein reaction solution and the protease solution enters the first mixing module 1 of the microchannel reactor to be fully mixed and then enters the first reaction module 2 to perform enzymolysis reaction.

The MGFM-rich lactalbumin reaction solution and the enzyme solution are conveyed at high pressure through a high-pressure pump at 0.5bar, 1bar, 5bar, 20bar, 50bar, 100bar or 500bar, namely 0.5-500 bar pressure, preferably 2-50 bar pressure, pumped into a first mixing module 1 of a microchannel reactor for sufficient mixing, wherein the mixing temperature can be 0 ℃, 10 ℃, 15 ℃ or 20 ℃, namely the mixing temperature range is 0-20 ℃, the residence time in the mixing module 1 is 30 seconds, 40 seconds, 50 seconds, 60 seconds, 100 seconds, 150 seconds, 250 seconds or 300 seconds, namely the residence time range is 30-300 seconds, so as to increase the mixing effect. Here, a plurality of first mixing modules 1 can be used, or a plurality of applications of the first mixing modules 1 can be carried out.

After being mixed, the reaction system enters a first reaction module 2 of the microchannel reactor for reaction, the reaction temperature is 35 ℃, 40 ℃, 45 ℃, 50 ℃ or 55 ℃, namely the reaction temperature range is 35-55 ℃, the residence time of the reaction system in the reaction module 2 is 30 seconds, 50 seconds, 100 seconds, 200 seconds, 400 seconds, 800 seconds or 1200 seconds, namely the residence time range is 30-1200 seconds, and a plurality of reaction modules 2 can be used or the reaction modules 2 can be used for multiple times.

After the proteolysis reaction is finished, the reaction system flows through the first high-temperature module 3 to passivate the activity of the compound protease. The reaction system is rapidly heated to 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃ or 105 ℃ within 30 seconds, 50 seconds, 100 seconds, 200 seconds or 300 seconds, namely within 30 to 300 seconds, namely within 75 to 105 ℃, preferably within 85 to 90 ℃ to passivate the activity of the complex enzyme, and the reaction system stays in the first high-temperature module 3 for 30 seconds, 50 seconds, 100 seconds, 200 seconds or 300 seconds, namely within 30 to 300 seconds. Then the proteolysis reaction system enters a first low-temperature module 4 to be rapidly cooled. The cooling temperature of the first low-temperature module 4 is 0 ℃, 10 ℃, 15 ℃ or 20 ℃, that is, the cooling temperature range is 0-20 ℃, the residence time of the reaction system in the first low-temperature module 4 is 100 seconds, 200 seconds, 300 seconds, 400 seconds, 800 seconds, 1200 seconds, 1500 seconds, 1800 seconds or 2200 seconds, that is, the residence time range is 100-2200 seconds, in order to ensure sufficient and thorough cooling, a plurality of low-temperature modules 4 can be used here, or the low-temperature modules 4 can be used for multiple times.

Step three, preparing a serine solution containing a phospholipase D solution, wherein the quantity ratio of all substances in the serine solution is as follows:

if the weight part of the MGFM-rich whey protein raw material is 100, the weight part of the serine is 10, 20, 30, 40 and 50, namely the weight part range is 10-50, and preferably 20-25;

the weight portion of the added serine is 100; the water can be added in the range of 1000, 1500, 2000 and 2500 parts by weight, namely 1000 to 2500 parts by weight, and preferably 1000 to 1500 parts by weight. The weight parts of the calcium salt are 5, 10, 20 and 30, namely the weight parts range is 5-30, preferably 10-20. The weight parts of the added phospholipase D are 1, 4, 6 and 10, namely the weight parts range is 1-10, and 4-6 is preferred.

In this step, the source of serine is: a commercial biological product; calcium salt: is a soluble calcium salt, such as calcium chloride, and may be present in the water at a concentration of 0.1 grams per liter to 20 grams per liter; phospholipase D is an enzyme, commercially available from Streptomyces and/or plants, such as phospholipase D (phosphoipase D) sold by Sigma-Aldrich, St.Louis, MO, U.S. A., which may also be purified from Streptomyces fermentation broth.

In the third step, the serine solution and the reaction solution obtained in the second step are pumped into the second mixing module 5 of the microchannel reactor by a pressure of 0.5-500 bar, preferably 2-50 bar, and are fully mixed, wherein the mixing temperature can be 0 ℃, 10 ℃, 15 ℃ or 20 ℃, namely the mixing temperature range is 0-20 ℃, and the residence time in the mixing module 1 is 30 seconds, 40 seconds, 50 seconds, 60 seconds, 100 seconds, 150 seconds, 250 seconds or 300 seconds, namely the residence time range is 30-300 seconds, so as to increase the mixing effect. Here, a plurality of second mixing modules 5 may be used, or a plurality of applications of the second mixing modules 5 may be performed.

And step four, the reaction system is mixed and then enters a second reaction module 6 of the microchannel reactor for reaction, the reaction temperature is 35 ℃, 40 ℃, 45 ℃, 50 ℃ or 55 ℃, namely the reaction temperature range is 35-55 ℃, the residence time of the reaction system in the reaction module 2 is 30 seconds, 50 seconds, 100 seconds, 200 seconds, 400 seconds, 800 seconds or 1200 seconds, namely the residence time range is 30-1200 seconds, and in order to ensure the full and thorough reaction, a plurality of reaction modules 6 can be used, or the reaction modules 6 can be used repeatedly.

And step five, after the phospholipase reaction is finished, enabling the reaction system to flow through the second high-temperature module 7 to passivate the activity of the phospholipase D. The reaction system is rapidly heated to 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃ or 105 ℃ within 30 seconds, 50 seconds, 100 seconds, 200 seconds or 300 seconds, namely within 30 to 300 seconds, namely within 75 to 105 ℃, preferably within 85 to 90 ℃ to passivate the activity of the complex enzyme, and the reaction system stays in the second high-temperature module 7 for 30 seconds, 50 seconds, 100 seconds, 200 seconds or 300 seconds, namely within 30 to 300 seconds.

And step six, the phospholipase reaction system rapidly enters a second low-temperature module 8 after passing through a second high-temperature module 7, and is rapidly cooled. The cooling temperature of the second low-temperature module 8 is 0 ℃, 10 ℃, 15 ℃ or 20 ℃, that is, the cooling temperature range is 0-20 ℃, the residence time of the reaction system in the second low-temperature module 8 is 100 seconds, 200 seconds, 300 seconds, 400 seconds, 800 seconds, 1200 seconds, 1500 seconds, 1800 seconds or 2200 seconds, that is, the residence time range is 100-2200 seconds, in order to ensure sufficient and thorough cooling, a plurality of low-temperature modules 8 can be used here, or the low-temperature modules 8 can be used for multiple times. And finally, allowing the reaction system to flow out of the microchannel reactor and enter a product collecting device 9, and refining and drying the obtained reaction liquid to obtain a nutritional composition product rich in lactoprotein active peptide and phosphatidylserine.

The nutritional composition prepared by the method can be used as a functional ingredient and applied to the fields of food, health care nutriment, cosmetics and the like.

The following detailed description of the preferred embodiments of the invention in its specific application is provided to illustrate and not to limit the scope of the invention as defined by the claims.

The micro-channel reactor used in the invention has no uniform regulation on the fluid channel of each module, the length is 0.1-300 m, preferably 1-60 m, and the width and the depth of the fluid channel are 0.5-10 mm, preferably 0.1-0.5 mm. The material from which the modules are made may be stainless steel, super hard steel, copper, ruby, diamond, ceramic, silicon carbide, cermet composite, or other polymeric material. The microchannel reactor of the present invention may be a commercial microchannel reactor, such as the glass high throughput microchannel reactor of heart type structure G1 manufactured by corning incorporated, usa, or other commercial or non-commercial microchannel reactors prepared according to the same principles.

Example 1: preparation of nutritional composition rich in milk protein active peptide and phosphatidylserine using microchannel reactor

(1) The device comprises the following steps: the microchannel device adopts a self-made high-pressure microchannel reactor, the length of the microchannel is determined according to the flow velocity and the reaction residence time, the length of each module channel used in the microchannel device is 10m, the pipe diameter is 0.1mm, and the heat exchange medium is heat conduction oil.

(2) Preparation of the nutritional composition:

a-preparation of microchannel reactor:

the residence time of the reaction system in each module is controlled by adjusting the flow rate of the delivery pump and the channel length of the microchannel, wherein the residence time in the first mixing module 1 is 30 seconds, the residence time in the first reaction module 2 is 120 seconds, the residence time in the first high temperature module 3 is 15 seconds, and the residence time in the first low temperature module 4 is 100 seconds. The residence time in the second mixing module 5 was 30 seconds, the residence time in the second reaction module 6 was 120 seconds, the residence time in the second high temperature module 7 was 15 seconds, and the residence time in the second low temperature module 8 was 100 seconds.

The temperature of each module of the reactor is controlled by an external circulation refrigeration and heating oil bath system, wherein the temperature of the first mixing module 1 is 0 ℃, the temperature of the first reaction module 2 is 45 ℃, the temperature of the first high-temperature module 3 is 80 ℃, and the temperature of the first low-temperature module 4 is 0 ℃. The temperature of the second mixing module 5 is 0 degree centigrade, the temperature of the second reaction module 6 is 50 degrees centigrade, the temperature of the second high temperature module 7 is 80 degrees centigrade, and the temperature of the second low temperature module 8 is 0 degree centigrade.

B-preparation of reaction System:

preparation of a proteolysis reaction system: 100g of MGFM-rich whey protein (wherein the protein content is 50%, the phosphatidylcholine content is 2.7%, the phosphatidic acid content is 0.2%, and the phosphatidylserine content is 1.1%) is put into 1000ml of water, fully mixed, homogenized by a high-pressure homogenizer under the pressure of 500bar, and the homogenized liquid is collected and poured into a material mixing tank (1).

Preparation of composite protease: in the batching tank (2), 1ml of neutral protease and 1ml of papain are added into 3500ml of water.

Preparation of serine reaction system: in the compounding tank (3), 20g of serine, 0.8g of calcium chloride and 10ml of phospholipase D were put into 200ml of water and mixed well.

C-enzyme-catalyzed reaction: opening a high-pressure metering pump which connects a batching tank (1) and a batching tank (2), carrying out high-pressure conveying under the pressure of 35bar, so that a protease reaction system and a compound protease solution are forced to enter a first mixing module 1 of a microchannel reactor for full mixing, the temperature of the first mixing module 1 is 0 ℃, the mixed solution then enters 50 groups of first reaction modules 2 which are connected in series, the temperature of the first reaction modules 2 is 45 ℃, the reaction solution then enters a first high-temperature module 3, the temperature of the first high-temperature module 3 is 5 modules, the temperature of the first high-temperature module 3 is 85 ℃, the reaction solution then enters a first low-temperature module 4, the temperature of the first low-temperature module 4 is 2 modules which are connected in series, the temperature of the first low-temperature module 4 is 0 ℃, then, the reaction solution enters a second mixing module 5, and meanwhile, opening the high-pressure metering pump which connects the batching tank (3), under 35bar pressure, high-pressure conveying, make serine solution get into the second mixing module 5 of microchannel reactor, carry out intensive mixing with the enzyme reaction system, the temperature of second mixing module 5 is 0 degrees centigrade, the mixed liquor then gets into 50 series connection second reaction modules 6, the temperature of second reaction module 6 is 45 degrees centigrade, the reaction liquid then gets into second high temperature module 7, second high temperature module 7 is 5 modules, the temperature of second high temperature module 7 is 85 degrees centigrade, the reaction liquid then gets into second low temperature module 8, second low temperature module 8 is that 2 modules are established ties, the temperature of second low temperature module 8 is 0 degrees centigrade, finally, the reaction product gets into product collection device 9.

D-refining: the resulting reaction product was freeze-dried to obtain 120g of a pale yellow solid. The product is tasted without bitter taste, and the HPLC liquid phase detection shows that the product contains 37% of active peptide, 3.3% of phosphatidylserine, 0.3% of phosphatidylcholine and 0.9% of phosphatidic acid.

Example 2: preparation of nutritional composition rich in milk protein active peptide and phosphatidylserine using microchannel reactor

(1) The device comprises the following steps: the microchannel device adopts a self-made high-pressure microchannel reactor, the length of the microchannel is determined according to the flow velocity and the reaction residence time, the length of each module channel used in the microchannel device is 10m, the pipe diameter is 0.3mm, and the heat exchange medium is heat conduction oil.

(2) Preparation of the nutritional composition:

a-preparation of microchannel reactor:

The temperature of each module of the reactor is controlled by an external circulation refrigeration and heating oil bath system, wherein the temperature of the first mixing module 1 is 20 ℃, the temperature of the first reaction module 2 is 50 ℃, the temperature of the first high-temperature module 3 is 90 ℃, and the temperature of the first low-temperature module 4 is 20 ℃. The temperature of the second mixing module 5 is 20 degrees centigrade, the temperature of the second reaction module 6 is 45 degrees centigrade, the temperature of the second high temperature module 7 is 85 degrees centigrade, and the temperature of the second low temperature module 8 is 20 degrees centigrade.

B-preparation of reaction System:

Preparation of composite protease: in the compounding tank (2), 1ml of neutral protease, 1ml of bromelain was added to 3500ml of water.

C-enzyme-catalyzed reaction: opening a high-pressure metering pump which connects a batching tank (1) and a batching tank (2), carrying out high-pressure conveying under the pressure of 25bar, so that a protease reaction system and a compound protease solution are forced to enter a first mixing module 1 of a microchannel reactor for full mixing, the temperature of the first mixing module 1 is 20 ℃, the mixed solution then enters 50 groups of first reaction modules 2 which are connected in series, the temperature of the first reaction modules 2 is 50 ℃, the reaction solution then enters a first high-temperature module 3, the temperature of the first high-temperature module 3 is 5 modules, the temperature of the first high-temperature module 3 is 90 ℃, the reaction solution then enters a first low-temperature module 4, the temperature of the first low-temperature module 4 is 2 modules which are connected in series, the temperature of the first low-temperature module 4 is 20 ℃, then, the reaction solution enters a second mixing module 5, and meanwhile, opening the high-pressure metering pump which connects the batching tank (3), under 25bar pressure, high-pressure conveying, make serine solution get into the second mixing module 5 of microchannel reactor, carry out intensive mixing with the enzyme reaction system, the temperature of second mixing module 5 is 20 degrees centigrade, the mixed liquor then gets into 50 series connection second reaction modules 6, the temperature of second reaction module 6 is 45 degrees centigrade, the reaction liquid then gets into second high temperature module 7, second high temperature module 7 is 5 modules, the temperature of second high temperature module 7 is 85 degrees centigrade, the reaction liquid then gets into second low temperature module 8, second low temperature module 8 is that 2 modules are established ties, the temperature of second low temperature module 8 is 20 degrees centigrade, finally, the reaction product gets into product collection device 9.

D-refining: the resulting reaction product was freeze-dried to obtain 120g of a pale yellow solid. The product is tasted without bitter taste, and the HPLC liquid phase detection shows that the product contains 38% of active peptide, 3.4% of phosphatidylserine, 0.2% of phosphatidylcholine and 0.8% of phosphatidic acid.

Example 3: preparation of nutritional composition rich in milk protein active peptide and phosphatidylserine using microchannel reactor

(1) The device comprises the following steps: the microchannel device adopts a self-made high-pressure microchannel reactor, the length of the microchannel is determined according to the flow velocity and the reaction residence time, the length of each module channel used in the microchannel device is 10m, the pipe diameter is 0.35mm, and the heat exchange medium is heat conduction oil.

(2) Preparation of the nutritional composition:

a-preparation of microchannel reactor:

the residence time of the reaction system in each module is controlled by adjusting the flow rate of the delivery pump and the channel length of the microchannel, wherein the residence time in the first mixing module 1 is 40 seconds, the residence time in the first reaction module 2 is 140 seconds, the residence time in the first high temperature module 3 is 25 seconds, and the residence time in the first low temperature module 4 is 150 seconds. The residence time in the second mixing module 5 was 40 seconds, the residence time in the second reaction module 6 was 140 seconds, the residence time in the second high temperature module 7 was 25 seconds, and the residence time in the second low temperature module 8 was 150 seconds.

The temperature of each module of the reactor is controlled by an external circulation refrigeration and heating oil bath system, wherein the temperature of the first mixing module 1 is 15 ℃, the temperature of the first reaction module 2 is 40 ℃, the temperature of the first high-temperature module 3 is 90 ℃, and the temperature of the first low-temperature module 4 is 15 ℃. The temperature of the second mixing module 5 is 20 degrees centigrade, the temperature of the second reaction module 6 is 50 degrees centigrade, the temperature of the second high temperature module 7 is 90 degrees centigrade, and the temperature of the second low temperature module 8 is 15 degrees centigrade.

B-preparation of reaction System:

C-enzyme-catalyzed reaction: opening a high-pressure metering pump which connects a batching tank (1) and a batching tank (2), carrying out high-pressure conveying under the pressure of 15bar, so that a protease reaction system and a compound protease solution are forced to enter a first mixing module 1 of a microchannel reactor for full mixing, the temperature of the first mixing module 1 is 15 ℃, the mixed solution then enters 50 groups of first reaction modules 2 which are connected in series, the temperature of the first reaction modules 2 is 40 ℃, the reaction solution then enters a first high-temperature module 3, the temperature of the first high-temperature module 3 is 5 modules, the temperature of the first high-temperature module 3 is 90 ℃, the reaction solution then enters a first low-temperature module 4, the temperature of the first low-temperature module 4 is 2 modules which are connected in series, the temperature of the first low-temperature module 4 is 15 ℃, then, the reaction solution enters a second mixing module 5, and meanwhile, opening the high-pressure metering pump which connects the batching tank (3), under 15bar pressure, high-pressure conveying, make serine solution get into the second mixing module 5 of microchannel reactor, carry out intensive mixing with the enzyme reaction system, the temperature of second mixing module 5 is 20 degrees centigrade, the mixed liquor then gets into 50 series connection second reaction modules 6 of group, the temperature of second reaction module 6 is 50 degrees centigrade, the reaction liquid then gets into second high temperature module 7, second high temperature module 7 is 5 modules, the temperature of second high temperature module 7 is 90 degrees centigrade, the reaction liquid then gets into second low temperature module 8, second low temperature module 8 is that 2 modules are established ties, the temperature of second low temperature module 8 is 15 degrees centigrade, finally, the reaction product gets into product collection device 9.

D-refining: the resulting reaction product was lyophilized to obtain 120g of a pale yellow solid. The product is tasted without bitter taste, and the HPLC liquid phase detection shows that the product contains 35% of active peptide, 3.2% of phosphatidylserine, 0.2% of phosphatidylcholine and 0.6% of phosphatidic acid.

Example 4: comparative example corresponding to example 1.

(1) The device comprises the following steps: a traditional kettle type reactor and an anchor type stirring paddle are adopted, a jacket is arranged, and a heat exchange medium is heat conduction oil.

(2) Preparation of the nutritional composition:

preparation of a proteolysis reaction system: 100g of MGFM-rich whey protein (wherein the protein content is 50%, the phosphatidylcholine content is 2.7%, the phosphatidic acid content is 0.2%, and the phosphatidylserine content is 1.1%) is put into 1000ml of water, fully mixed, homogenized by a high-pressure homogenizer under the pressure of 500bar, and the homogenized liquid is collected and poured into a first mixing tank (1).

Preparation of composite protease: in the second compounding tank (2), 1ml of neutral protease and 1ml of papain are added to 3500ml of water.

Preparation of serine reaction system: in the third blending tank (3), 20g of serine, 0.8g of calcium chloride and 10ml of phospholipase D were put into 200ml of water and mixed well.

Reaction: the metering pump of joining batching jar (1) and batching jar (2) is opened for reaction system and catalyst protease solution system get into kettle-type reactor, the agitator rotational speed is 70rpm, and reaction temperature is 45 degrees centigrade, and the reaction stirring maintains 12h, then, the metering pump of joining batching jar (3) is opened and is got into the kettle-type reactor with serine reaction system, and the agitator rotational speed is 70rpm, and reaction temperature is 45 degrees centigrade, and the reaction stirring maintains 12h, gets into product collection device 9 with the reaction product at last.

Refining: the resulting reaction product was lyophilized to obtain 120g of a pale yellow solid. The product tastes bitter, and the HPLC liquid phase detection shows that the product contains 39% of active peptide, 2.3% of phosphatidylserine, 0.5% of phosphatidylcholine and 1.7% of phosphatidic acid.

The product of example 4, which tasted a bitter taste relative to example 1, had a low phosphatidylserine content of 2.3%, a phosphatidylcholine content of 0.5%, and a high phosphatidic acid content of 1.7%. The product of example 1, on the other hand, tasted no bitter taste, with a phosphatidylserine content of 3.3%, a phosphatidylcholine content of 0.3%, and a phosphatidic acid content of 0.9%. The method shows that the microchannel enzyme catalysis technology can accurately control the proteolysis degree, avoid the bitter taste generated by excessive hydrolysis, simultaneously obtain more target products of phosphatidylserine from the same raw material and lower the content of the by-product phosphatidic acid.

In this specification, the invention has been described with reference to specific embodiments thereof. It is apparent, however, that various modifications and changes may be made by one skilled in the art without departing from the spirit and scope of the invention, and that other embodiments may be practiced within the scope of the invention by those skilled in the art without departing from the teachings of this patent, and it is intended that all such modifications, equivalents and changes to the above embodiments as come within the true spirit of the invention be embraced therein.

Claims

1. A method for preparing a nutritional composition rich in milk protein active peptide and phosphatidylserine is prepared in a microchannel reactor, the microchannel reactor comprises a first mixing module (1), a first reaction module (2), a first high temperature module (3), a first low temperature module (4), a second mixing module (5), a second reaction module (6), a second high temperature module (7) and a second low temperature module (8), the length of a fluid channel of each module of the microchannel reactor is 1-60 m, and the width and depth of the fluid channel are 0.1-0.5 mm; the preparation method is characterized by comprising the following steps:

pumping a whey protein reaction solution rich in MFGM and a protease solution into a first mixing module (1) of a microchannel reactor, mixing the whey protein reaction solution and the protease solution, then feeding the mixture into a first reaction module (2), and rapidly heating to perform a proteolysis reaction to prepare a milk protein active peptide system;

secondly, the lactoprotein active peptide system enters a first high-temperature module (3), protease activity is passivated at high temperature, the enzymolysis reaction is stopped, and the reaction system flows through a first low-temperature module (4) and then enters a second mixing module (5);

pumping the serine solution containing the phospholipase D solution into a second mixing module (5) of the microchannel reactor, and fully mixing the serine solution with the reaction system prepared in the second step to obtain a mixed reaction system;

step four, the mixed reaction system in the step three enters a second reaction module (6), the temperature is rapidly increased to the temperature required by the process, and the phospholipase D catalytic reaction is carried out;

after the catalytic reaction of the phospholipase D is finished, the reaction system enters a second high-temperature module (7), the activity of the phospholipase D is passivated at high temperature, and the enzymolysis reaction is stopped;

sixthly, after leaving the second high-temperature module (7), the reaction system enters a second low-temperature module (8), is rapidly cooled to the temperature required by the process, and finally flows out of the microchannel reactor to obtain a nutritional composition product rich in milk protein active peptide and phosphatidylserine; the proportion relationship of all substances in the reaction liquid in the step one is as follows: the MGFM-rich whey protein solution is obtained by uniformly mixing MGFM-rich whey protein and water according to the weight ratio of 1: 10-15; the protease solution is obtained by uniformly mixing a complex enzyme and water according to a weight ratio of 1: 100-500; the temperature of the first mixing module (1) is 0-20 ℃; the residence time of the reaction system in the mixing module is 30-1200 seconds; the reaction temperature in the first reaction module (2) is 35-55 ℃; the residence time of the reaction system in the first reaction module (2) is 30-1200 seconds; in the second step, the temperature of the first high-temperature module (3) is 75-105 ℃; the reaction system stays in the first high temperature module (3) for 10-1200 seconds; the temperature of the first low-temperature module (4) is 0-20 ℃; the residence time of the reaction system in the first low-temperature module (4) is 30-1200 seconds; in the third step, the serine solution is obtained by uniformly mixing serine and water according to the weight ratio of 1: 10-25; in the fourth step, the temperature of the second mixing module (5) is 0-20 ℃; the residence time of the reaction system in the mixing module is 30-1200 seconds; the reaction temperature of the second reaction module (6) is 35-55 ℃; the residence time of the reaction system in the second reaction module (6) is 30-1200 seconds; in the fifth step, the temperature of the second high-temperature module (7) is 75-105 ℃; the reaction system stays in the second high temperature module (7) for 10-1200 seconds; in the sixth step, the temperature of the second low-temperature module (8) is 0-20 ℃; the residence time of the reaction system in the second low temperature module (8) is 30-1200 seconds.