CN115868570A - Lactoferrin processing method and device - Google Patents

Abstract

The invention mainly provides a method and a device for processing lactoferrin, the processing technology is based on a double-layer membrane filtration technology, can replace a thermal sterilization technology adopted in the traditional industrial preparation process, effectively reduces the denaturation rate of the lactoferrin, improves the extraction rate to more than 80 percent, and protects the quality of the lactoferrin in the raw materials to the maximum extent. Furthermore, the invention also provides an online continuous double-pressure-maintaining test verification scheme aiming at the double-layer membrane filtration technology to ensure the integrity of the sterilization membrane in the whole production process. In addition, the invention also provides equipment for carrying out the lactoferrin treatment process.

Description

Lactoferrin processing method and device

This application is a divisional application of the Chinese invention patent application with the application number "CN202211269779.8", the application date is October 18, 2022, and the title is "lactoferrin processing method, device and effectiveness verification method".

technical field

The present invention relates to the field of food processing, in particular, to the processing or treatment process of protein substances, more specifically, to a treatment method in the preparation of lactoferrin and the device used therein.

Background technique

The pollution and reproduction of microorganisms in food are important factors that cause food deterioration, so sterilization has become the most critical link in food processing. Cold sterilization technology is a new technology, which is quite different from traditional heat sterilization technology. It not only It can kill microorganisms in food, protect the physiological activity of food function, and maintain the freshness, original flavor and nutritional content of food.

Membrane filtration technology is recognized as an excellent "cold sterilization" technology in academia, and has been widely used in pharmaceutical and food fields in recent years, such as purified water, beer, vaccines and various intravenous injections.

Lactoferrin is one of the main glycoproteins in the udder glandular secretions of dairy cows during colostrum formation, lactation, dry-up and mastitis. It has a variety of biologically active functions, such as broad-spectrum antibacterial properties, enhancing the body's anti-virus, anti-oxidation and immune capabilities, preventing and treating infectious diseases, stimulating the growth of bifidobacteria, maintaining the balance of intestinal flora, and enhancing iron delivery And absorption, etc., have become the focus of the food industry, especially the dairy industry.

However, for a long time, researchers believed that lactoferrin is easily inactivated by heat, which is not conducive to dairy processing. Because lactoferrin is a glycoprotein and is unstable to heat, some studies have found that bovine lactoferrin begins to inactivate when the pH value is 6.6 and the temperature is 65-69°C. Since then, it has been reported that heating at 70°C for 15 to 30 minutes has completely degraded lactoferrin. Another study found that the denaturation rate of lactoferrin was 85.81% at 63°C for 30 minutes; at 72°C for 15s, the denaturation rate was 17.34%; at 120°C for 4s, the denaturation rate was 90.1%. It can be seen that the common heat sterilization process of dairy products has a great influence on the denaturation rate of lactoferrin, but it is stipulated in GB1903.17-2016 "Food Nutritional Enhancer Lactoferrin" that the preparation process must be separated, sterilized, extracted, Refining, drying, etc., among which the sterilization process is indispensable. Therefore, efficient and stable lactoferrin cold sterilization technology has always been a technical difficulty in the current industry and has become a development bottleneck that needs to be solved urgently.

At present, the preparation method of lactoferrin in the industry generally adopts thermal sterilization process. For example, New Zealand TATUA brand adopts separation degreasing, pasteurization (72°C, 15s), ion exchange, elution, and concentration process; New Zealand SYNLAIT brand adopts degreasing, chromatography, pasteurization Degreasing (72°C, 15s), ultrafiltration, and spray drying processes; the Australian SAPUTO brand uses degreasing, pasteurization (72°C, 15s), chromatography, ultrafiltration, and drying processes.

Citation 1 discloses a method for preparing lactoferrin, which includes the following steps: using a cation exchange resin to perform ion exchange treatment on liquid milk with a temperature not exceeding 60 degrees Celsius, and obtaining a salt solution containing lactoferrin by elution; and The salt solution containing lactoferrin is subjected to desalination treatment so as to obtain the lactoferrin. In addition, a 0.2-micron membrane can be further used for sterilization treatment.

Citation 2 discloses a method for preparing lactoferrin, which separates lactoferrin from peroxidase through repeated cationic resin chromatography separation methods to obtain more pure lactoferrin.

Citation 3 discloses a method for producing lactoferrin and lactoperoxidase, which retains lactoferrin and peroxidase on a macroporous cationic resin column, and then controls the elution conditions (temperature and eluent concentration ) to collect peroxidase and lactoferrin eluted under different conditions.

Citation 4 discloses a method for extracting and preparing high-purity low-iron saturation lactoferrin from cow's milk, by successively using cation exchange resin and insoluble Fe ³⁺ chelating resin for treatment and concentration to reduce the production of lactoferrin The content of iron ions (Fe ³⁺ ).

Citation 5 discloses a method for directly extracting lactoferrin from skimmed milk, which includes (a) passing fresh skimmed milk directly through a chromatography column packed with a cation exchange packing, which has a certain Particle size and pore size; (b) washing residual milk and impurities with water and an aqueous solution containing a first concentration of salt; (c) eluting with an aqueous solution or a buffer solution containing a second concentration of salt at a flow rate of 200-1000cm/h to obtain Lactoferrin.

Although in the prior art, research has been carried out on the technology of isolating or concentrating lactoferrin from cow's milk or from whey protein liquid, the processing method for the deep processing of lactoferrin, such as sterilization treatment process, etc. Due to the consideration of production efficiency, most of them are still limited to heat sterilization. Therefore, there is still room for further improvement in the discussion of the processing methods in the production of lactoferrin, especially such as filtration or sterilization technology.

Citation:

Citation 1: CN104926936A

Citation 2: CN113105542A

Citation 3: CN106008704A

Citation 4: CN106008703B

Citation 5: CN113372437A.

Contents of the invention

The problem to be solved by the invention

As mentioned above, the current traditional further processing of lactoferrin concentrate or purification is still mainly in accordance with the processing method of ordinary protein products. Typically, for example, heat treatment sterilization (such as pasteurization) can be used, which is generally considered effective. It is beneficial to the standardized production of protein products. Although it is known that lactoferrin is more sensitive to temperature, it is generally hoped in the art to control the heat treatment temperature and time to deal with it.

For Citation 2 to Citation 5, it is expected to efficiently separate high-purity lactoferrin products from milk sources or meet specific requirements through different separation processes and separation conditions. However, there is no specific discussion on the sterilization process of isolated or purified lactoferrin.

Furthermore, citing the preparation method of lactoferrin disclosed in Document 1, it is disclosed that after the concentrated lactoferrin product is obtained, a 0.2 micron membrane can be further used for sterilization treatment. However, in actual operation, the following problems were also found:

i. The efficiency of using the membrane for sterilization treatment is not high, and the membrane is prone to blockage during the sterilization process, resulting in an excessive pressure difference between the inlet and outlet of the equipment and the sterilization process is substantially stopped. Therefore, it is often necessary to clean the filter sterilization equipment to prevent the disinfection failure caused by the blockage of the membrane pores;

ii. There is almost no feasible method for evaluating the effect of membrane filtration sterilization, for example, even for the sterilization failure caused by accidental membrane rupture during use, there is no good monitoring method. Although final product testing can be used to assess the level of bacteria in the product, even if an abnormal flora is present, it may be difficult to determine whether it is due to a lethality failure (membrane rupture) or another problem or process.

Therefore, based on the above-mentioned problems in the prior art, the present invention firstly provides a method for deep processing of lactoferrin. In some specific embodiments, it can be a method of filtering or sterilizing, for example, it can be A cold sterilization process. The method of the present invention is based on double-layer membrane filtration technology, which can replace the heat sterilization technology used in the traditional industrial preparation process, effectively reduce the denaturation rate of lactoferrin, increase the extraction rate to more than 80%, and protect the raw materials to the greatest extent. quality of lactoferrin.

Further, the present invention also provides an online continuous double-holding pressure test verification scheme for the above-mentioned double-layer membrane filtration technology to ensure the integrity of the sterilization membrane in the entire production process.

In addition, the present invention also provides a device for performing the above lactoferrin treatment process.

solutions to problems

Through the long-term research of the inventors of the present invention, it is found that the above-mentioned technical problems can be solved through the implementation of the following technical solutions:

[1]. A method for processing lactoferrin, wherein the method comprises:

filtering raw materials containing lactoferrin through a filter element comprising a double-layer membrane to obtain a processed lactoferrin product,

The filter element comprising a double membrane comprises:

A porous membrane A having a pore diameter of 0.35 to 0.55 μm facing the side containing the lactoferrin raw material, and,

A porous membrane B with a pore diameter of 0.22 μm or less on the side facing the lactoferrin product.

[2]. The method according to [1], wherein the lactoferrin-containing raw material is derived from skim milk or whey protein solution.

[3]. The method according to [1] or [2], wherein the temperature T of the filtration treatment is 20°C to 55°C.

[4]. The method according to any one of [1] to [3], wherein, in the filtration process, the pressure on the raw material side containing lactoferrin of the filter element is set to P1, and the The pressure on one side of the lactoferrin product is P2, then the pressure difference ΔP=P1-P2 of the filtration treatment, and then, the relationship between ΔP and the temperature T of the filtration treatment is:

△P＝0.0002T ² -0.0646T+6.1826

where ΔP is in bar and temperature T is in °C.

[5]. The method according to any one of [1] to [5], wherein the pressure of the lactoferrin-containing raw material side of the filter element is P1, and P1 does not exceed 2.5 bar.

[6]. An apparatus for performing the method according to any one of [1] to [5], wherein the apparatus includes:

A box body, including the filter element, the filter element divides the box body into a raw material storage part and a processed product storage part, and the raw material storage part and the processed product storage part only perform mass transfer through the filter element ;

entrance and exit,

Wherein the raw material containing lactoferrin enters the raw material storage part from the inlet, and the lactoferrin product is discharged from the outlet connected to the processing product storage part.

[7]. The device according to [6], wherein the ratio of the volume V1 of the raw material storage part in the box to the volume V2 of the processed object storage part is V1:V2=1 to 100:1

[8]. The device according to [6] or [7], wherein an inlet and an outlet of the device have pressure detection elements.

[9]. The device according to any one of [6] to [8], wherein the double-layer membrane in the filter element is an organic membrane or an inorganic membrane.

[10]. A sterilization method according to any one of [1] to [5], wherein the verification method includes:

i. the porous membrane in the filter element is fully wetted with water;

ii. keep the gas pressure on one side of the porous membrane A of the filter element constant as P, and measure the gas escape volume _VD1 of the filter element in the test time T;

Said gas slip quantity VD ₁ is compared with the theoretical gas slip value VD ₀ under the same conditions.

[11] A verification method for the validity of the device according to any one of [6] to [9], wherein the verification method includes:

i. the porous membrane in the filter element is fully wetted with water;

ii. keep the gas pressure in the raw material holding part of the device constant as P, and measure the gas escape volume _VD of the filter element in the test time T;

Said gas slip quantity VD ₁ is compared with the theoretical gas slip value VD ₀ under the same conditions.

[12]. The method according to [10] or [11], wherein the P is 2.5 bar, the t is 5 min, and the VD ₀ =1.8H, wherein the unit of VD ₀ is mL/min, The H is the vertical height of the double-layer membrane in the filter element, and its unit is cm.

The effect of the invention

Through the implementation of the above technical solutions, the present invention can obtain the following technical effects:

1) The present invention provides a new treatment process for lactoferrin, which can be a filtration or sterilization process, especially a cold sterilization process, which can replace the heat sterilization technology used in the traditional industrial preparation process, and effectively reduce the lactoferrin Denaturation rate, increase the extraction rate to over 80%, and maximize the protection of lactoferrin in raw milk.

2) The present invention is different from the previous treatments. It adopts double-layer composite sterilization membranes with different pore diameters, and adding a layer of large-pore sterilization membranes can provide protection for small-pore sterilization membranes. The microbial filtration accuracy challenge test conducted And the bacterial retention test, the results show that the microbial removal efficiency is >10 ¹² , and the LRV (log reduction value, the logarithmic reduction value of the microbial load before and after filtration) is 12, which can achieve a better sterilization effect, and the final product microbial indicators all meet National standard requirements. At the same time, compared with the single-layer pore size sterilization film, the double-layer composite pore size sterilization film can intercept more impurities and larger microorganisms in advance, and the service life of the sterilization film is extended by more than 30%.

3) This patent also innovates a lactoferrin treatment process, especially a method for verifying the effectiveness of filtration or sterilization, so as to ensure its continuous, stable and effective operation during use. As a key quality control point of the entire preparation process, if its effectiveness cannot be guaranteed in real time, there will be a huge food safety risk.

Description of drawings

Figure 1: Schematic diagram of the structure of the sterilization device described in a specific embodiment of the present invention

1: Inlet; 2: Outlet; 3: Box; 4: Porous membrane A (pore size 0.45 μm); 5: Porous membrane B (pore size 0.20 μm)

Detailed ways

Hereinafter, the content of the present invention will be described in detail. The description of the technical features described below is based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted that, unless otherwise defined, then:

In this specification, the numerical range represented by "numerical value A - numerical value B" means the range which includes numerical value A and B of an end point.

In this specification, the use of "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, more preferably within 1%.

In this specification, the meaning expressed by "may" includes the meaning of performing certain processing and not performing certain processing.

In this specification, the use of "normal temperature"/"room temperature" refers to a room temperature of 23±2°C.

In this specification, "pure water" is used to mean water having a surface tension of 70 dynes/cm or less.

In this specification, the "treatment temperature T" when using a filter element for filtration treatment should be understood as the corresponding temperature reached by the material to be treated.

In this specification, "bar" is used to represent the unit of pressure, wherein 1 bar (bar) = 100 kilopascals (kPa) = 0.1 MPa.

In this specification, "D" is used to represent the unit "Dalton" of molecular weight, that is, "Dalton".

In this specification, "optional" or "optionally" means that the next described event or situation may or may not occur, and that the description includes situations where the event occurs and situations where the event does not occur.

In this specification, references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments" and the like refer to specific elements described in relation to the embodiments (for example, A feature, structure, property and/or characteristic) is included in at least one embodiment described herein and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

<first aspect>

In the first aspect of the present invention, a method for processing lactoferrin is mainly provided, especially a method for filtering or sterilizing lactoferrin. The present invention filters raw materials containing lactoferrin through a double-layer composite porous membrane. deal with.

Specifically, the processing method of the present invention, especially the filtering or sterilization method includes: filtering the lactoferrin-containing raw material through a filter element including a double-layer membrane to obtain a processed lactoferrin product.

The filter element comprising a double-layer membrane includes: a porous membrane A with a pore diameter of 0.35-0.55 μm facing the side containing the lactoferrin raw material, and a 0.22 μm pore diameter facing the lactoferrin product. Porous membrane B below μm.

(source of raw material containing lactoferrin)

In the present invention, the original source of the lactoferrin-containing raw material is not particularly limited in principle, and it may be defatted animal milk or whey protein liquid.

For skimmed animal milk, it can usually be partly skimmed or completely skimmed. In some specific embodiments of the present invention, for skimmed animal milk, its fat content is less than 1% (mass) of animal milk, preferably 0.8%. (mass) or less, more preferably 0.5% (mass) or less.

There is no particular limitation on the type of the above-mentioned animal milk, which may be one or more of cow's milk, goat's milk, horse's milk, camel's milk and the like. In some preferred embodiments of the present invention, the preferred animal milk is cow's milk or goat's milk, more preferably, cow's milk.

For whey protein liquid, in some specific embodiments of the present invention, it can be the whey protein liquid formed by whey protein and water to be further or re-sterilized, or it can be the above-mentioned animal milk protein after removing casein liquid. For the method of removing casein from animal milk, it is typical to adjust the animal milk to the vicinity of the isoelectric point of casein at a suitable temperature, so that the casein is precipitated, and the precipitated part is removed to obtain a whey protein liquid .

In some preferred embodiments of the present invention, the above-mentioned whey protein liquid is preferably used as the lactoferrin-containing raw material of the present invention.

(purification/concentration treatment)

For the original source of the above-mentioned raw material containing lactoferrin, wherein as the defatted animal milk, it contains a large amount of casein, whey protein and other components, and there is also a large amount of whey protein in the whey protein liquid. Therefore, In order to further obtain components enriched in lactoferrin, it is necessary to perform purification or concentration treatment on the above-mentioned raw materials containing lactoferrin.

There is no particular limitation on the above-mentioned purification or concentration treatment method, and typically, purification or concentration for whey protein can be performed by chromatography.

In some specific embodiments, cationic resins can be used as stationary phases for chromatography. As the cation resin that can be used, there may be cited cation exchange resins of acrylic (ester) type, polystyrene type cation exchange resins, and agarose type cation exchange resins. For these resins, cationic groups can be endowed on the surface of the resins by means of chemical modification, preferably, these cationic groups include sulfonic acid groups.

Regarding the use form of the above-mentioned cationic resin, in some specific embodiments of the present invention, resin particle aggregates may be used for filling. Furthermore, the particle diameter of the resin particles may be 100-500 μm, preferably 150-300 μm, and more preferably 180-250 μm, from the viewpoint of facilitating the retention of lactoferrin.

When using the above-mentioned cationic resin for chromatography, the cationic resin can be impregnated with a buffer solution in advance, and then the raw material containing lactoferrin is contacted with the cationic resin and kept for a period of time. The retention time is not particularly limited, but usually from the viewpoint of treatment efficiency, it may be less than 30 min, preferably less than 20 min, such as 1-20 min, 2-15 min, 3-10 min, etc.

After the retention time has elapsed, the unadsorbed components can be eluted with an ion-free mobile phase. For such a mobile phase, typically deionized water may be used.

Furthermore, the components including lactoferrin adsorbed on the surface of the cationic resin can be eluted by means of an eluent with a certain ionic strength to obtain the eluted components. As the eluent, typically, sodium salt or potassium salt, such as sodium chloride or potassium chloride, etc., can be used. The ion concentration of the eluent may be 0.05-0.8 mol/L, preferably 0.1-0.5 mol/L.

The above-mentioned elution component is used to elute the lactoferrin component adsorbed on the cationic resin into the elution component, so as to complete the primary purification or concentration of the lactoferrin.

Further, in order to improve the purity of lactoferrin in the eluted fraction, the repeated process can be carried out several times, that is, the eluted fraction is contacted with the above-mentioned cationic resin again, and the eluent with a suitable ion concentration is used for elution to obtain Reconcentrated lactoferrin fraction.

In some preferred embodiments of the present invention, the process of cationic resin adsorption-eluent elution can be performed more than twice, preferably, this repeated process can be performed 2-3 times.

In addition, the present invention is not particularly limited to the method for one-step separation of lactoferrin and peroxidase, and the purification or concentration method provided in Chinese patent application (publication number CN113105542A) can typically be used.

Further, the above-mentioned purification or concentration is usually carried out at 5 to 55°C, preferably at 15 to 30°C, more preferably at room temperature. Under some extreme conditions, too high a temperature may be unfavorable for cationic resin Adsorption of lactoferrin leads to undesired protein denaturation, and too low temperature may lead to longer purification or concentration operation time.

The lactoferrin fraction obtained after the cationic resin adsorption-elution treatment can be further desalted to obtain a desalted lactoferrin fraction. The present invention has no particular limitation on the desalination treatment method, and typically, an ultrafiltration membrane can be used for ultrafiltration treatment. Usually, the molecular weight cut-off of the ultrafiltration membrane can be 25-50KD, so as to ensure that the salt in the lactoferrin component is removed without losing the lactoferrin.

The lactoferrin-containing raw material to be sterilized in the present invention is obtained through the above cationic resin adsorption-elution-desalination treatment. The raw material containing lactoferrin In some specific embodiments of the present invention, the purity of lactoferrin may be above 90% (mass), preferably above 92% (mass), more preferably above 95% (mass).

(filtered or sterilized)

The lactoferrin-containing raw material of the present invention obtained from the above-mentioned cationic resin adsorption-elution-desalination treatment of lactoferrin can be further treated by the filtration or sterilization method of the present invention. Optionally, in order to effectively perform the filtration or sterilization treatment of the present invention, the above-mentioned lactoferrin-containing raw materials can also be diluted or concentrated according to actual needs, so that the lactoferrin raw materials have suitable solid content and viscosity and other rheological properties. In some specific embodiments of the present invention, the solid content of the lactoferrin-containing raw material to be sterilized is 3% (mass) to 15% (mass), preferably 5% (mass) to 13% ( mass), more preferably 7% (mass) to 11% (mass).

The filtration or sterilization treatment of the present invention is mainly carried out using membrane filtration. The membrane filtration in the present invention refers to the filtration treatment of the lactoferrin-containing raw material through a filter element comprising a double-layer membrane, so as to obtain a filtered or sterilized lactoferrin product.

For the above-mentioned filter element, it mainly includes a double-layer membrane and optional supporting or sealing auxiliary parts. For the bilayer membrane, the porous membrane A and the porous membrane B are included in the present invention.

As for the porous membrane A, it may have a pore size of 0.35-0.55 μm, preferably 0.38-0.52 μm, more preferably 0.40-0.50 μm. The porous membrane B may have a pore diameter of 0.22 μm or less, preferably 0.17 to 0.21 μm, more preferably 0.19 to 0.21 μm.

Among them, the porous membrane A has a larger pore size, which can pre-intercept components with higher molecular weights to reduce the blocking of these components on the porous membrane B, that is, adding a layer of large-pore bactericidal membrane can provide a small pore-diameter sterilizing membrane. Protection, which is conducive to improving the filtration or sterilization effect and efficiency of the entire filtration or sterilization process.

For the above two porous membranes, in some specific embodiments of the present invention, they are bonded in contact with each other, and the thickness of each porous membrane is not particularly limited, typically 1.2-1.4 μm. The mutually stacked double-layer membranes are set in such a way that the porous membrane A faces the lactoferrin-containing raw material to be filtered, and the porous membrane B faces the filtered or sterilized lactoferrin product.

In addition, the materials of the above two porous membranes are not particularly limited, and may be organic resin films or thin films formed of inorganic substances, such as porous ceramic membranes.

In addition, for the optional auxiliary components, it can be enumerated that the supporting components that play a supporting role can provide sufficient support for the double-layer membrane during filtration or sterilization treatment; the components that play a fixing role can be used to The double-layer membrane is laminated and fixed or the double-layer membrane is fixed in the filter element.

When performing the filtration or sterilization process of the present invention, the raw material containing lactoferrin passes through the filter element under certain temperature and pressure conditions. in particular:

The pressure on the raw material side containing lactoferrin of the filter element can be set as P1, and the pressure on the side of the lactoferrin product is P2, then the pressure difference of the filtration process ΔP=P1-P2, and then , the relationship between ΔP and the filtration temperature T is:

△P＝0.0002T ² -0.0646T+6.1826

where ΔP is in bar and temperature T is in °C.

In some specific embodiments of the present invention, from the perspective of ensuring safe filtration, the pressure of P1 is no more than 2.5 bar. Preferably, from the perspective of ensuring safe filtration and ensuring filtration efficiency, the pressure of P1 can be 1.8 to 2.5 bar bar, more preferably 2.0 to 2.4 bar, more preferably 2.1 to 2.3 bar.

For the treatment temperature T, in some specific embodiments of the present invention, it can be 20°C to 55°C, preferably 30°C to 55°C, or 40°C to 55°C, or 45 to 55°C, more preferably 50 to 55°C °C, more preferably 50-53 °C. Therefore, the above-mentioned treatment of the present invention is especially suitable for low-temperature/cold sterilization treatment.

For the lactoferrin-containing products that have undergone the above-mentioned filtration or sterilization treatment, protein products can be directly prepared according to needs, or undergo further processing, such as through drying processes (such as spray drying, freeze-drying) to obtain final lactoferrin solids (powder) products.

<Second aspect>

Although there is no particular limitation on the device itself that executes the processing described in the above <first aspect>, as long as the device has a configuration of the functions described above. However, from the viewpoint of good operability, the present invention further provides a membrane filtration or sterilizing device that can be applied to the above-mentioned sterilizing treatment.

Specifically, the device includes:

A box body, including the filter element, the filter element divides the box body into a raw material storage part and a processed product storage part, and the raw material storage part and the processed product storage part only perform mass transfer through the filter element ;and

An inlet and an outlet, wherein the lactoferrin-containing raw material enters the raw material storage part from the inlet, and the lactoferrin product is discharged from the outlet connected to the treatment product storage part.

As for the box body, it may have an optional pressure-resistant material, which may be metal, inorganic, organic or composite. The shape of the box may be a cylinder from the perspective of pressure resistance. Preferably, the top of the cylinder is a circular dome to further increase the pressure resistance. There is no particular limitation on the volume of the box. In some specific embodiments of the present invention, the cylindrical box shall have an internal diameter of 10 cm to 20 cm, preferably 14 cm to 18 cm, and more preferably 15 cm to 17 cm. , its internal height (calculated at the highest point on the top) may be 55cm-74cm, preferably 57cm-72cm, more preferably 60cm-70cm.

As for the box body, its exterior has an inlet and an outlet provided with materials, and, in some specific embodiments of the present invention, the inlet and outlet are connected with a pressure detection element to provide pressure detection for the corresponding area .

Further, the filter element is arranged inside the box body, and the inside of the box body is divided into two physically disconnected storage parts by the filter element: a raw material storage part and a processed product storage part. Moreover, the raw material storage part is connected to the inlet of the material, and the processed product storage part is connected to the outlet of the material.

There is no special setting for the shape of the filter element, but it can be a cylinder from the perspective of pressure resistance. Preferably, the top of the cylinder is a circular dome to further increase the pressure resistance.

In addition, in some specific embodiments of the present invention, the volume of the raw material storage part formed by the filter element is greater than or equal to the volume of the processed product storage part. In some preferred embodiments, the ratio of the volume V1 of the raw material storage part in the box to the volume V2 of the processed product storage part is V1:V2=1-100:1, more preferably 2-50:1 , more preferably 5 to 20:1.

In addition, in some other preferred embodiments of the present invention, the above-mentioned device of the present invention may also have other optional auxiliary elements such as heating, temperature control or temperature detection elements.

<Third aspect>

In a third aspect of the present invention, there is provided a method for testing the processing method of the present invention, and a verification method for testing the effectiveness of the operation of the processing device of the present invention.

Through the verification method, it is possible to provide an effective judgment or detection for the filtration or sterilization device, especially for the filter element thereof, in a continuous or semi-continuous filtration or sterilization process.

Specifically, the verification methods include:

i. the porous membrane in the filter element is fully wetted with water;

ii. keep the porous membrane A side of the filter element or keep the gas pressure in the raw material holding part of the device constant as P, and measure the gas escape volume _VD of the filter element in the test time T;

iii. Comparing said gas escape amount VD ₁ with the theoretical value of gas escape VD ₀ under the same conditions.

Wherein, in step i, the pure water is sent into the inside of the filter or sterilizer from the inlet of the filter or sterilizer under the action of compressed air, and the pressure of the compressed air can be less than or equal to that of the filter or sterilizer When working, the pressure P1 of the raw material side of the filter element containing lactoferrin, preferably, the pressure of the compressed air can be 0.9 times to 1 times the pressure P1, more preferably, the pressure of the compressed air can be P1.

Further, with the help of compressed air under the pressure, pure water is used to flush the double-layer membrane of the filter element, so that the double-layer membrane is completely wetted (the side facing the porous membrane B is completely permeated with water). The flushing time is not particularly limited, and is generally more than 5 minutes, preferably 5-10 minutes. The amount of flushing water is not particularly limited, and is related to the volume of the device.

In step ii, the supply of pure water is stopped, and only compressed air is injected into the raw material storage part through the inlet, so that the pressure of the raw material storage part (that is, the porous membrane A side) is kept constant. Such a constant pressure is preferably equal to the maximum pressure P1 of the lactoferrin-containing raw material side of the filter element during operation of the filter or sterilizer, ie 2.5 bar.

Further, in the case of maintaining a pressure of 2.5 bar, the holding time t may be 5 min. Furthermore, the gas escape volume VD ₁ (mL/min) of the filter element is calculated within t time by detecting the pressure of the gas entering the processing object storage part.

After VD ₁ is obtained, it can be compared with the theoretical value VD ₀ of the gas escape volume of the filter element. If VD ₁ is greater than VD ₀ , it indicates that the filter element may be damaged. Otherwise, it indicates that the filter element is in normal condition.

For the measurement method of VD ₀ of the filter element, in principle, the present invention is not particularly limited. In some specific embodiments of the present invention, for example, in the case shown in Figure 1, the VD ₀ =1.8H, the H is the vertical height of the double-layer membrane in the filter element (with the highest point of the double-layer membrane, the vertical distance from the bottom plane of the filter element), which is in cm.

After the verification method is completed, the sterilization device can be heated and steam sterilized at a temperature of 110-140°C, preferably at a temperature of 120-135°C.

Through the verification method provided by the third aspect of the present invention, the verification program can be added to the continuous or semi-continuous filtration or sterilization process for lactoferrin to ensure the integrity of the membrane during the entire production process and ensure the effectiveness of the filtration or sterilization process In particular, the verification method of the present invention can be used to detect the bactericidal effectiveness of lactoferrin on-line.

Example

Hereinafter, the technical solution of the present invention will be described through specific examples.

The following examples are processed according to the device shown in Figure 1 , wherein the box of the device is in the shape of a cylindrical structure with a dome having a round shape at the top. And, the shape of the filter element is a cylindrical structure, and has a rounded dome at the top.

Example 1:

1. At the beginning of the test, use 2.5bar compressed air to push pure water (pure water ≤ 70dynes/cm) into the cavity from the inlet end, _flushing time t = 5min, flushing volume V = 150L, so that the sterilization membrane is completely wetted ;

2. After the sterilization film is completely wetted, open the outlet valve and inject compressed air into the chamber from the inlet. When the pressure P at the inlet _reaches 2.5bar, close the inlet valve and perform a pressure-holding test for 5 minutes. In the process, if the pressure P at the inlet end is _lower than 2.5bar, open the valve at the inlet end to replenish air until it reaches 2.5bar. Calculate the gas escape volume VD ₁ value per unit time. When VD ₁ ≤ VD ₀ , the first step of validity verification is passed. When VD ₁ > VD ₀ , the validity verification fails, the sterilization membrane fails, and the verification activity is terminated;

The corresponding relationship between the gas escape volume VD ₀ per unit time and the height of the sterilization film is as follows:

H=10cm

VD ₀ ＝18mL/min;

3. After the system is depressurized, use 121°C steam to sterilize for 30 minutes, P _at the inlet port = 2.3 bar, and P _out at the outlet port = 2 bar;

4. After the sterilization is completed, enter the normal cold sterilization process, the material temperature T = 20 ° C, the inlet port P _in = 2.5 bar, the outlet port P _out = 0.5 bar;

5. After the cold sterilization process is over, execute the cleaning procedure to clean the sterilization film;

6. Use 2.5bar compressed air to push pure water (pure water ≤ 70dynes/cm) into the cavity from the inlet end, _flushing time t = 5min, flushing volume V = 150L, so that the sterilization membrane is completely wetted;

7. After the sterilization film is completely wetted, open the outlet valve and inject compressed air into the chamber from the inlet. When the inlet pressure P _reaches 2.5bar, close the inlet valve and perform a pressure-holding test for 5 minutes. In the process, if the pressure P at the inlet end is _lower than 2.5bar, open the valve at the inlet end to replenish air until it reaches 2.5bar. Calculate the gas escape volume VD ₁ value per unit time. When VD ₁ ≤ VD ₀ , the first step of validity verification is passed. When VD ₁ > VD ₀ , the validity verification fails, the sterilization membrane fails, and the verification activity is terminated;

The corresponding relationship between the gas escape volume VD ₀ per unit time and the height of the sterilization film is as follows:

H=10cm

VD ₀ =18 mL/min.

Example 2:

1. At the beginning of the test, use 2.5bar compressed air to push pure water (pure water ≤ 70dynes/cm) into the cavity from the inlet end, _flushing time t = 5min, flushing volume V = 200L, so that the sterilization membrane is completely wetted ;

2. After the sterilization film is completely wetted, open the outlet valve and inject compressed air into the chamber from the inlet. When the pressure P at the inlet _reaches 2.5bar, close the inlet valve and perform a pressure-holding test for 5 minutes. In the process, if the pressure P at the inlet end is _lower than 2.5bar, open the valve at the inlet end to replenish air until it reaches 2.5bar. Calculate the gas escape volume VD ₁ value per unit time. When VD ₁ ≤ VD ₀ , the first step of validity verification is passed. When VD>VD ₀ , the validity verification fails, the sterilization membrane fails, and the verification activity is terminated;

The corresponding relationship between the gas escape volume VD ₀ per unit time and the height of the sterilization film is as follows:

H=20cm

VD ₀ =36mL/min;

3. After the system is depressurized, use 121°C steam to sterilize for 30 minutes, P _at the inlet port = 2.1 bar, and P _{out at} the outlet port = 1.9 bar;

4. After the sterilization is completed, enter the normal cold sterilization process, the material temperature T = 55 ° C, the inlet port P _in = 2.5 bar, the outlet port P _out = 0.5 bar;

5. After the cold sterilization process is over, execute the cleaning procedure to clean the sterilization film;

6. Use 2.5bar compressed air to push pure water (pure water ≤ 70dynes/cm) into the cavity from the inlet end, flushing time _t = 5min, flushing volume V = 200L, so that the sterilization membrane is completely wetted;

7. After the sterilization film is completely wetted, open the outlet valve and inject compressed air into the chamber from the inlet. When the inlet pressure P _reaches 2.5bar, close the inlet valve and perform a pressure-holding test for 5 minutes. In the process, if the pressure P at the inlet end is _lower than 2.5bar, open the valve at the inlet end to replenish air until it reaches 2.5bar. Calculate the gas escape volume VD ₁ value per unit time. When VD ₁ ≤ VD ₀ , the first step of validity verification is passed. When VD ₁ > VD ₀ , the validity verification fails, the sterilization membrane fails, and the verification activity is terminated;

The corresponding relationship between the gas escape volume VD ₀ per unit time and the height of the sterilization film is as follows:

H=20cm

VD ₀ =36 mL/min.

Example 3:

1. At the beginning of the test, use 2.5bar compressed air to push pure water (pure water ≤ 70dynes/cm) into the cavity from the inlet end, _flushing time t = 5min, flushing volume V = 150L, so that the sterilization membrane is completely wetted ;

2. After the sterilization film is completely wetted, open the outlet valve and inject compressed air into the chamber from the inlet. When the pressure P at the inlet _reaches 2.5bar, close the inlet valve and perform a pressure-holding test for 5 minutes. In the process, if the pressure P at the inlet end is _lower than 2.5bar, open the valve at the inlet end to replenish air until it reaches 2.5bar. Calculate the gas escape volume VD ₁ value per unit time. When VD ₁ ≤ VD ₀ , the first step of validity verification is passed. When VD>VD ₀ , the validity verification fails, the sterilization membrane fails, and the verification activity is terminated;

The corresponding relationship between the gas escape volume VD ₀ per unit time and the height of the sterilization film is as follows:

H=30cm

VD ₀ =54mL/min;

3. After the system is depressurized, use 134°C steam to sterilize for 30 minutes, P _at the inlet port = 2.3 bar, and P _out at the outlet port = 2 bar;

4. After the sterilization is completed, enter the normal cold sterilization process, the material temperature T = 53 ° C, the inlet port P _in = 2.5 bar, the outlet port P _out = 0.5 bar;

5. After the cold sterilization process is over, execute the cleaning procedure to clean the sterilization film;

6. Use 2.5bar compressed air to push pure water (pure water ≤ 70dynes/cm) into the cavity from the inlet end, _flushing time t = 5min, flushing volume V = 150L, so that the sterilization membrane is completely wetted;

7. After the sterilization film is completely wetted, open the outlet valve and inject compressed air into the chamber from the inlet. When the inlet pressure P _reaches 2.5bar, close the inlet valve and perform a pressure-holding test for 5 minutes. In the process, if the pressure P at the inlet end is _lower than 2.5bar, open the valve at the inlet end to replenish air until it reaches 2.5bar. Calculate the gas escape volume VD ₁ value per unit time. When VD ₁ ≤ VD ₀ , the first step of validity verification is passed. When VD ₁ > VD ₀ , the validity verification fails, the sterilization membrane fails, and the verification activity is terminated;

The corresponding relationship between the gas escape volume VD ₀ per unit time and the height of the sterilization film is as follows:

H=30cm

VD ₀ =54 mL/min.

Example 4:

1. At the beginning of the test, use 2.5bar compressed air to push pure water (pure water ≤ 70dynes/cm) into the cavity from the inlet end, _flushing time t = 5min, flushing volume V = 150L, so that the sterilization membrane is completely wetted ;

2. After the sterilization film is completely wetted, open the outlet valve and inject compressed air into the chamber from the inlet. When the pressure P at the inlet _reaches 2.5bar, close the inlet valve and perform a pressure-holding test for 5 minutes. In the process, if the pressure P at the inlet end is _lower than 2.5bar, open the valve at the inlet end to replenish air until it reaches 2.5bar. Calculate the gas escape volume VD ₁ value per unit time. When VD ₁ ≤ VD ₀ , the first step of validity verification is passed. When VD>VD ₀ , the validity verification fails, the sterilization membrane fails, and the verification activity is terminated;

The corresponding relationship between the gas escape volume VD ₀ per unit time and the height of the sterilization film is as follows:

H=40cm

VD ₀ =72mL/min;

3. After the system is depressurized, use 134°C steam to sterilize for 30 minutes, P _at the inlet port = 2.3 bar, and P _out at the outlet port = 2 bar;

4. After the sterilization is completed, enter the normal cold sterilization process, the material temperature T = 38 ° C, the inlet port P _in = 2.5 bar, the outlet port P _out = 0.5 bar;

5. After the cold sterilization process is over, execute the cleaning procedure to clean the sterilization film;

6. Use 2.5bar compressed air to push pure water (pure water ≤ 70dynes/cm) into the cavity from the inlet end, _flushing time t = 5min, flushing volume V = 150L, so that the sterilization membrane is completely wetted;

7. After the sterilization film is completely wetted, open the outlet valve and inject compressed air into the chamber from the inlet. When the inlet pressure P _reaches 2.5bar, close the inlet valve and perform a pressure-holding test for 5 minutes. In the process, if the pressure P at the inlet end is _lower than 2.5bar, open the valve at the inlet end to replenish air until it reaches 2.5bar. Calculate the gas escape volume VD ₁ value per unit time. When VD ₁ ≤ VD ₀ , the first step of validity verification is passed. When VD ₁ > VD ₀ , the validity verification fails, the sterilization membrane fails, and the verification activity is terminated;

The corresponding relationship between the gas escape volume VD ₀ per unit time and the height of the sterilization film is as follows:

H=40cm

VD ₀ =72 mL/min.

The specific embodiment 1-4 group of normal products, 1 group of abnormal products (damaged antibacterial film, film perforation) and no antibacterial film are carried out cold sterilization process effectiveness verification (gas escape amount per unit time), according to GB1903. 17-2016 "Food Nutritional Fortifier Lactoferrin" requires that microbiological results be tested and reviewed, and the specific data are as follows:

Table 1

It can be seen from the above results that when the antibacterial film is damaged, the VD value can immediately feedback abnormality, and the product microbial result is abnormal; when the antibacterial film is normal, the VD value meets the theoretical requirements, and the product microbial index meets the national standard requirements, which proves that the lactoferrin The cold sterilization process is effective.

It should be noted that although the technical solution of the present invention is described with specific examples, those skilled in the art can understand that the present disclosure should not be limited thereto.

Having described various embodiments of the present disclosure above, the foregoing description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and alterations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principle of each embodiment, practical application or improvement of technology in the market, or to enable other ordinary skilled in the art to understand each embodiment disclosed herein.

Industrial availability

The sterilizing method and device for lactoferrin and the method for validating the effectiveness provided by the invention can be applied in industry.

Claims (9)

1. A method of lactoferrin treatment, the method comprising:

filtering a lactoferrin containing raw material through a filter element comprising a double-layer membrane to obtain a treated lactoferrin preparation,

the filter element comprising a bilayer membrane comprises:

a porous membrane A having a pore diameter of 0.35 to 0.55 μm on the side facing the lactoferrin-containing raw material,

a porous membrane (B) having a pore diameter of 0.22 [ mu ] m or less on the side facing the lactoferrin product.

2. The method according to claim 1, wherein the lactoferrin containing material is derived from skim milk or whey protein liquid.

3. The method according to claim 1 or 2, characterized in that the temperature T of the filtration treatment is between 20 ℃ and 55 ℃.

4. Method according to claim 1 or 2, characterized in that in the filtration process, assuming that the pressure on the lactoferrin containing feed side of the filter element is P1 and the pressure on the lactoferrin product side is P2, the pressure difference Δ P = P1-P2 of the filtration process, and further that Δ P is related to the temperature T of the filtration process by:

△P＝0.0002T ² -0.0646T+6.1826

where Δ P is in bar and the temperature T is in C.

5. Method according to claim 1 or 2, characterized in that the pressure on the lactoferrin containing feed side of the filter element is P1 and P1 does not exceed 2.5bar.

6. An apparatus for performing the method according to any one of claims 1 to 5, characterized in that the apparatus comprises:

a tank including the filter element therein, the filter element dividing the tank into a raw material containing part and a processed matter containing part, and the raw material containing part and the processed matter containing part performing mass transfer only through the filter element;

an inlet and an outlet, wherein the inlet is provided with a plurality of holes,

wherein the lactoferrin containing material enters the material containing portion from an inlet and the lactoferrin product is discharged from the outlet connected to the treatment containing portion.

7. The apparatus according to claim 6, wherein a ratio of a volume V1 of the raw material accommodating portion to a volume V2 of the processed object accommodating portion in the tank is V1: v2=1 to 100.

8. A device according to claim 6 or 7, wherein the inlet and outlet of the device are provided with pressure sensing means.

9. The device according to claim 6 or 7, wherein the double membrane in the filter element is an organic or inorganic membrane.

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