CN115868570A - Lactoferrin treatment method and device - Google Patents
Lactoferrin treatment method and device Download PDFInfo
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- CN115868570A CN115868570A CN202310036075.4A CN202310036075A CN115868570A CN 115868570 A CN115868570 A CN 115868570A CN 202310036075 A CN202310036075 A CN 202310036075A CN 115868570 A CN115868570 A CN 115868570A
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
- A23J3/08—Dairy proteins
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/58—Multistep processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D65/00—Accessories or auxiliary operations, in general, for separation processes or apparatus using semi-permeable membranes
- B01D65/10—Testing of membranes or membrane apparatus; Detecting or repairing leaks
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Water Supply & Treatment (AREA)
- Dairy Products (AREA)
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
The application is a divisional application of Chinese patent application with the application number of 'CN 202211269779.8', the application date of 10-18/2022 and the name of 'lactoferrin processing method, device and validity verification method'.
Technical Field
The invention relates to the field of food processing, in particular to a processing or treating process of protein substances, and more particularly relates to a processing method in lactoferrin preparation and a device used by the same.
Background
The pollution and propagation of microorganisms in food are important factors causing food deterioration, so that sterilization becomes the most critical link in food processing, and the cold sterilization technology is a novel technology and has a great difference from the traditional hot sterilization technology, and can kill the microorganisms in the food, protect the physiological activity of food functions and keep the freshness, the original flavor and the original nutritional ingredients of the food.
The membrane filtration technology is known as an excellent cold sterilization technology in academia, and has been widely applied in the pharmaceutical and food fields in recent years, such as purified water, beer, vaccines and various intravenous injections.
Lactoferrin is one of the major glycoproteins in mammary gland secretions of cows during the colostrum formation phase, the lactation phase, the dry milk phase and during the onset of mastitis. It has many biological activity functions, such as broad-spectrum antibacterial property, enhancing the antiviral, antioxidant and immunological abilities of the body, preventing and treating infectious diseases, stimulating the growth of bifidobacteria, maintaining the balance of intestinal flora, enhancing the transmission and absorption of iron, etc., and has become a focus of attention in the food industry, especially in the dairy industry.
However, for a long time, researchers have considered lactoferrin to be easily inactivated by heat, which is detrimental to dairy processing. Since lactoferrin belongs to glycoprotein and is unstable to heat, it has been found that bovine lactoferrin starts to inactivate at a pH of 6.6 and a temperature of 65 to 69 ℃. It was reported that lactoferrin was completely degraded by heating at 70 ℃ for 15-30 min. In addition, the lactoferrin has a denaturation rate of 85.81% under the conditions of 63 ℃ and 30 min; the denaturation rate is 17.34 percent under the conditions of 72 ℃ and 15 s; the denaturation rate was 90.1% at 120 ℃ for 4 s. Therefore, common thermal sterilization processes of dairy products have great influence on the denaturation rate of lactoferrin, but the preparation processes of the lactoferrin have to be separated, sterilized, extracted, refined, dried and the like according to the stipulations in GB1903.17-2016 (food nutrition enhancer, lactoferrin), wherein the sterilization processes are indispensable. Therefore, the efficient and stable cold sterilization technology of lactoferrin is always a technical difficulty in the current industry, becomes a development bottleneck, and needs to be solved urgently.
Currently, the lactoferritin preparation method in the industry generally adopts a heat sterilization process, for example, the New Zealand TATUA brand adopts separation degreasing, pasteurization (72 ℃,15 s), ion exchange, elution and concentration processes; the new zealand SYNLAIT brand adopts degreasing, chromatography, killing (72 ℃,15 s), ultrafiltration and spray drying processes; australian SAPUTO brand adopts degreasing, pasteurizing (72 deg.C, 15 s), chromatography, ultrafiltration, and drying processes.
Citation 2 discloses a lactoferrin production method which separates lactoferrin from peroxidase by a repeated cationic resin chromatography separation method to obtain purer lactoferrin.
Citation 4 discloses a method for extracting and preparing high-purity low-iron-saturation lactoferrin from cow's milk by using a cation exchange resin and insoluble Fe in this order 3+ The chelating resin is treated and concentrated to reduce iron ion (Fe) in lactoferrin product 3+ ) The content of (a).
Although the prior art has been studied on the technology of separating or concentrating lactoferrin from milk or whey protein liquid, the methods for further processing lactoferrin, such as sterilization, are still limited to heat sterilization in terms of production efficiency in industrial production. Therefore, there is still room for further improvement in the treatment methods in the production of lactoferrin, particularly, for example, the filtration and sterilization techniques.
Cited documents:
cited document 1: CN104926936A
Cited document 2: CN113105542A
Cited document 3: CN106008704A
Cited document 4: CN106008703B
Cited document 5: CN113372437A.
Disclosure of Invention
Problems to be solved by the invention
As mentioned above, the conventional further processing of lactoferrin concentrates or purifications still mainly follows the processing of common protein products, typically heat treatment sterilization (e.g. pasteurization) may be used, for example, which is generally considered to be advantageous for the standardized production of protein products. Although lactoferrin is known to be more temperature sensitive, it is generally only a concern in the art to control the temperature and time of the heat treatment to do so.
With reference to cited documents 2 to 5, a lactoferrin preparation of high purity or meeting specific requirements is desired to be efficiently separated from a milk source by different separation processes and separation conditions. However, the sterilization process of isolated or purified lactoferrin is not discussed.
Further, the preparation method of lactoferrin disclosed in citation 1 discloses that after obtaining a concentrated lactoferrin product, sterilization treatment can be further performed using a 0.2 μm membrane. However, in actual practice, the following problems have been found:
i. the efficiency of using the membrane to carry out sterilization treatment is not high, the membrane is easy to block in the sterilization process, and the sterilization process is substantially stopped due to overlarge pressure difference of an inlet and an outlet of the equipment. It is therefore often necessary to clean the filter sterilization equipment to prevent sterilization failure due to clogging of the membrane pores;
there are few feasible methods for evaluating the effectiveness of membrane filtration sterilization, for example, even if there is no good monitoring method for sterilization failure due to accidental membrane rupture during use. Although the level of bacteria in the product can be assessed by inspection of the final product, even if a flora abnormality occurs, it may be difficult to identify whether it is due to sterilization failure (membrane rupture) or other problems or processes.
Therefore, based on the above problems in the prior art, the present invention provides a method for further processing lactoferrin, which may be a filtration or sterilization method, for example, a cold sterilization process, in some specific embodiments, the method of the present invention is based on a double-layer membrane filtration technology, can replace a thermal sterilization technology adopted in a traditional industrial preparation process, effectively reduce the denaturation rate of lactoferrin, increase the extraction rate to more than 80%, and maximally protect the quality of lactoferrin in the raw material.
Furthermore, the invention also provides an online continuous double pressure maintaining test verification scheme aiming at the double-layer membrane filtration technology, so that the integrity of the sterilization membrane in the whole production process is ensured.
In addition, the invention also provides equipment for carrying out the lactoferrin treatment process.
Means for solving the problems
Through long-term research by the inventor of the invention, the technical problems can be solved through implementation of the following technical scheme:
[1] a method of treatment of lactoferrin, wherein the method comprises:
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 μm or less on the side facing the lactoferrin product.
[2] The process according to [1], wherein the lactoferrin-containing raw material is derived from skim milk or whey protein liquid.
[3] The method according to [1] or [2], wherein the temperature T of the filtration treatment is 20 ℃ to 55 ℃.
[4] The method according to any one of [1] to [3], wherein, in the filtration process, assuming that the pressure on the lactoferrin-containing raw material side of the filter element is P1 and the pressure on the lactoferrin product side is P2, the pressure difference Δ P of the filtration process = P1-P2, and further, the relationship between Δ P and the temperature T of the filtration process is:
△P＝0.0002T 2 -0.0646T+6.1826
where Δ P is in bar and the temperature T is in C.
[5] The method according to any one of [1] to [5], wherein the pressure on the lactoferrin containing material side of the filter element is P1, and P1 is not more than 2.5bar.
[6] An apparatus for performing the method according to any one of [1] to [5], wherein 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 feedstock enters the feedstock 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 [6], wherein a ratio of a volume V1 of the raw material containing portion to a volume V2 of the processed object containing portion in the tank is V1: v2=1 to 100
[8] The device according to [6] or [7], wherein the inlet and the outlet of the device have pressure detecting elements.
[9] The apparatus 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] The sterilization method according to any one of [1] to [5], wherein the verification method includes:
i. wetting the porous membrane in the filter element with water throughout;
keeping the pressure of the gas at the side of the porous membrane A of the filter element constant at P, and measuring the gas escape VD of the filter element during a test time T 1 ；
The gas escape volume VD 1 Theoretical value VD of gas escape under the same conditions 0 A comparison is made.
[11] A verification method of validity of the apparatus according to any one of [6] to [9], wherein the verification method includes:
i. wetting the porous membrane in the filter element with water throughout;
maintaining the raw material holding part gas pressure of the apparatusConstant P, the gas escape VD of the filter element being determined over a test time T 1 ；
The gas escape volume VD 1 Theoretical value VD of gas escape under the same conditions 0 A comparison is made.
[12]According to [10]]Or [11]The method, wherein P is 2.5bar, t is 5min, and VD 0 =1.8H, wherein VD 0 In mL/min, H is the vertical height of the bilayer membrane in the filter element, which is in cm.
ADVANTAGEOUS EFFECTS OF INVENTION
Through the implementation of the technical scheme, the invention can obtain the following technical effects:
1) The invention provides a novel processing technology for lactoferrin, which can be a filtering or sterilizing technology, particularly a cold sterilizing technology, can replace a heat sterilizing technology adopted in the traditional industrial preparation process, effectively reduce the denaturation rate of the lactoferrin, improve the extraction rate to more than 80 percent, and protect the lactoferrin in the raw milk to the maximum extent.
2) Different from the treatment of the previous attempt, the invention adopts the double-layer composite sterilization membrane with different pore diameters, the addition of the large-pore diameter sterilization membrane can protect the small-pore diameter sterilization membrane, and the results show the microbial removal efficiency through the microbial filtration precision challenge test and the bacterial interception test>10 12 LRV (log reduction value, log reduction value of the microbial load before and after filtration) is 12, so that a better sterilization effect can be achieved, and all indexes of the final product microbes meet the national standard requirements. Meanwhile, compared with a single-layer aperture sterilization membrane, the double-layer composite aperture sterilization membrane can intercept more impurities and microorganisms with larger volume in advance, and the service cycle of the sterilization membrane is prolonged by more than 30%.
3) The patent also innovates a lactoferrin processing technology, in particular to a filtering or sterilizing validity verification method, so as to ensure that the lactoferrin processing technology runs continuously, stably and effectively in the using process. As a key quality control point of the whole preparation process, if the effectiveness can not be ensured in real time, huge food safety risks exist.
Drawings
FIG. 1: structural schematic of the sterilization apparatus in one embodiment of the present inventionDrawing (A)
1: an inlet; 2: an outlet; 3: a box body; 4: porous membrane A (pore size 0.45 μm); 5: porous Membrane B (pore size 0.20 μm)
Detailed Description
The present invention will be described in detail below. The technical features described below are explained based on typical embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It is noted that, unless otherwise defined:
in the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including an end point numerical value A, B.
In the present specification, the term "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.
In the present specification, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.
In the present specification, the term "normal temperature"/"room temperature" means an indoor temperature of 23 ± 2 ℃.
In the present specification, "pure water" is used to mean water having a surface tension of 70dynes/cm or less.
In this specification, "treatment temperature T" when the filtration treatment is performed using the filter element is to be understood as the corresponding temperature reached by the material being treated.
In the present specification, "bar" is used to represent a unit of pressure, wherein 1bar (bar) =100 kilopascals (kPa) =0.1MPa.
In the present specification, "D" is used to denote the unit of molecular weight "Dalton", i.e. "Dalton".
In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment 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 a first aspect of the present invention, a method for processing lactoferrin is mainly provided, and in particular, a method for filtering or sterilizing lactoferrin is provided.
In particular, the treatment method, in particular the filtration or sterilization method, according to the invention comprises: the lactoferrin containing feedstock is subjected to a filtration process by means of a filter element comprising a double membrane to obtain a treated lactoferrin preparation.
The filter element comprising a bilayer membrane comprises: a porous membrane A having a pore size of 0.35 to 0.55 μm on the side facing the lactoferrin-containing raw material, and a porous membrane B having a pore size of 0.22 μm or less on the side facing the lactoferrin product.
(sources of lactoferrin-containing raw materials)
In the present invention, the original source of the lactoferrin-containing raw material is not particularly limited in principle, and may be defatted animal milk or whey protein liquid.
The fat content of the defatted animal milk may be generally 1% by mass or less, preferably 0.8% by mass or less, more preferably 0.5% by mass or less of the animal milk, in some embodiments of the invention.
The type of the animal milk is not particularly limited, and may be one or more of cow milk, sheep milk, horse milk, camel milk, and the like. In some preferred embodiments of the invention, the preferred animal milk is bovine or goat milk, more preferably bovine milk.
For whey protein liquid, in some specific embodiments of the present invention, the whey protein liquid may be formed by whey protein and water to be further or again sterilized, or may be a protein liquid obtained by removing casein from the above-mentioned animal milk. As a method for removing casein from animal milk, typically, the animal milk is adjusted to the vicinity of the isoelectric point of casein at an appropriate temperature, and the casein is precipitated, and the precipitated portion is removed to obtain a whey protein liquid.
In some preferred embodiments of the present invention, it is preferred to use the whey protein liquid as the lactoferrin containing material of the present invention.
(purification/concentration treatment)
Since the raw material containing lactoferrin is originally a source of a defatted animal milk containing a large amount of components such as casein and whey protein, and a large amount of whey protein is present in a whey protein liquid, it is necessary to purify or concentrate the raw material containing lactoferrin in order to further obtain a component enriched in lactoferrin.
The method of the purification or concentration treatment is not particularly limited, and typically, purification or concentration of whey protein can be performed by chromatography.
In some embodiments, a cationic resin may be used as the stationary phase for chromatography. As the cation exchange resin which can be used, there may be mentioned acrylic acid (ester) type cation exchange resins, polystyrene type cation exchange resins, agarose type cation exchange resins. For these resins, the resin surface may be imparted with cationic groups by chemical modification, and preferably, these cationic groups include sulfonic acid groups.
As for 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. Further, the particle size of the resin particles may be 100 to 500. Mu.m, preferably 150 to 300. Mu.m, and more preferably 180 to 250. Mu.m, from the viewpoint of facilitating the retention of lactoferrin.
In the case of chromatography using the above-mentioned cationic resin, the cationic resin may be impregnated with a buffer solution in advance, and the lactoferrin-containing raw material may be contacted with the cationic resin and retained for a certain period of time. The retention time is not particularly limited, but may be usually 30min or less, preferably 20min or less, for example, 1 to 20min,2 to 15min,3 to 10min, and the like, from the viewpoint of the treatment efficiency.
After the retention time has ended, the non-adsorbed components can be eluted by means of a mobile phase which does not contain ions. For such mobile phase, typically deionized water may be used.
Further, the component including lactoferrin adsorbed on the surface of the cationic resin may be eluted with an eluent having a certain ionic strength to obtain an eluted component. For the eluent, typically, a sodium salt or a potassium salt, such as sodium chloride or potassium chloride, or the like, may be used. The ion concentration of the eluent may be 0.05 to 0.8mol/L, and preferably, may be 0.1 to 0.5mol/L.
And eluting the lactoferrin component adsorbed to the cationic resin into the elution component by using the elution component so as to complete one-time purification or concentration of the lactoferrin.
Further, in order to improve the purity of the lactoferrin in the eluted fraction, a repeated process may be performed for a plurality of times, that is, the eluted fraction is again contacted with the above cationic resin, and the elution is performed using an eluent having an appropriate ion concentration, to obtain a reconcentrated lactoferrin fraction.
In some preferred embodiments of the present invention, the cation resin adsorption-elution process may be performed twice or more, and preferably, the repeated process may be performed 2 to 3 times.
In addition, as for the method for separating lactoferrin from peroxidase in one step, the present invention is not particularly limited, and typically the method of purification or concentration provided in chinese patent application (publication No. CN113105542 a) can be used.
Further, the above purification or concentration is usually carried out at 5 to 55 ℃, preferably at 15 to 30 ℃, more preferably at room temperature, and under some extreme conditions, too high temperature may not be favorable for adsorption of lactoferrin by the cationic resin to cause undesired protein denaturation, and too low temperature may result in longer purification or concentration operation time.
The lactoferrin component obtained after the adsorption-elution treatment with the cationic resin may be further subjected to a desalting treatment to obtain a desalted lactoferrin component. The present invention is not particularly limited with respect to the method of desalting treatment, and typically, ultrafiltration treatment can be performed using an ultrafiltration membrane. Typically, ultrafiltration membranes may have a molecular weight cut-off of 25-50 KD to ensure removal of salts from the lactoferrin component without loss of lactoferrin.
The lactoferrin containing raw material to be sterilized is obtained through the cation resin adsorption-elution-desalination treatment. The lactoferrin-containing raw material in some specific embodiments of the present invention, the purity of lactoferrin may be 90% (by mass) or more, preferably 92% (by mass) or more, and further preferably 95% (by mass) or more.
(filtration or sterilization treatment)
The lactoferrin-containing raw material of the present invention obtained by adsorbing, eluting and desalting the lactoferrin with the cationic resin 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 lactoferrin-containing raw material may be diluted or concentrated according to actual needs so that the lactoferrin raw material has appropriate rheological properties such as solid content and viscosity. In some specific embodiments of the present invention, the solid content of the lactoferrin containing material to be sterilized is 3% by mass to 15% by mass, preferably 5% by mass to 13% by mass, and more preferably 7% by mass to 11% by mass.
The filtration or sterilization treatment of the present invention is mainly performed by a membrane filtration method. The invention relates to membrane filtration, which is to filter the raw material containing lactoferrin by a filter element comprising a double-layer membrane to obtain a lactoferrin product after filtration or sterilization treatment.
The filter element described above comprises essentially a double membrane and optionally a supporting, or sealing, auxiliary component. The two-layer film includes a porous film a and a porous film B in the present invention.
The porous membrane A may have a pore diameter of 0.35 to 0.55. Mu.m, preferably 0.38 to 0.52. Mu.m, and more preferably 0.40 to 0.50. Mu.m. The porous membrane B may have a pore diameter of 0.22 μm or less, preferably 0.17 to 0.21 μm, and more preferably 0.19 to 0.21. Mu.m.
The porous membrane A has larger pore size, and can intercept components with higher molecular weight in advance so as to reduce the blockage of the components on the porous membrane B, namely, a layer of large-pore degerming membrane is added to protect the small-pore degerming membrane, thereby being beneficial to improving the filtering or sterilizing effect and efficiency of the whole filtering or sterilizing process.
With respect to the above two porous films, in some specific embodiments of the present invention, they are combined in such a manner as to be laminated in contact with each other, and the thickness of each porous film is not particularly limited, and may typically be 1.2 to 1.4 μm. The two-layer membranes laminated to each other are disposed such that the porous membrane a faces a lactoferrin-containing raw material to be filtered, and the porous membrane B faces a lactoferrin product after filtration or sterilization treatment.
The material of the two porous films is not particularly limited, and may be an organic resin film, or a film formed of an inorganic substance, for example, a porous ceramic film.
Further, as optional auxiliary means, there may be enumerated, for example, a support means serving as a support to provide sufficient support to the bilayer membrane at the time of filtration or sterilization treatment; and a fixing component for fixing the double-layer membrane in a laminated manner or fixing the double-layer membrane in the filter element.
In carrying out the process of filtration or sterilisation treatment according to the invention, the lactoferrin containing feed is caused to pass through the filter element under conditions of temperature and pressure. Specifically, the method comprises the following steps:
if the pressure on the lactoferrin product-containing raw material side of the filter element is P1 and the pressure on the lactoferrin product side is P2, the pressure difference Δ P = P1-P2 in the filtration process, and further, the relationship between Δ P and the filtration process temperature T is:
△P＝0.0002T 2 -0.0646T+6.1826
where Δ P is in bar and the temperature T is in deg.C.
In some specific embodiments of the present invention, the pressure of P1 is not more than 2.5bar from the viewpoint of ensuring safe filtration, and preferably, the pressure of P1 may be 1.8 to 2.5bar, more preferably 2.0 to 2.4 bar, and still more preferably 2.1 to 2.3bar from the viewpoint of ensuring safe filtration and ensuring filtration efficiency.
As regards the treatment temperature T, it may in some particular embodiments of the invention be from 20 ℃ to 55 ℃, preferably from 30 ℃ to 55 ℃, or from 40 ℃ to 55 ℃, or from 45 ℃ to 55 ℃, more preferably from 50 ℃ to 55 ℃, and even more preferably from 50 ℃ to 53 ℃. Therefore, the above-described treatment of the present invention is particularly suitable for a low temperature/cold sterilization treatment.
For lactoferrin-containing products subjected to the above filtration or sterilization treatment, protein preparations may be prepared directly as desired, or subjected to further processing, for example, by means of a drying process (e.g., spray drying, freeze drying), etc., to obtain final lactoferrin solid (powder) preparations.
< second aspect >
Although there is no particular limitation on the apparatus itself that performs the processing described in < first aspect > above, as long as the apparatus has the configuration of the functions described above. However, the present invention further provides a membrane filtration or sterilization apparatus which can be applied to the above sterilization treatment from the viewpoint of good operability.
Specifically, 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; and
an inlet and an outlet, wherein the lactoferrin containing feedstock enters the feedstock containing portion from the inlet and the lactoferrin product is discharged from the outlet connected to the treatment containing portion.
The case may be made of any pressure-resistant material, and examples thereof include a metal material, an inorganic material, an organic material, and a composite material. As for the shape of the case, from the viewpoint of pressure resistance, it may be a cylinder, and preferably, the top of the cylinder is a circular dome to further increase the pressure resistance. The volume of the tank is not particularly limited, and in some specific embodiments of the present invention, the internal diameter of the tank may be 10cm to 20cm, preferably 14cm to 18cm, more preferably 15cm to 17cm, and the internal height (as measured by the top peak) may be 55cm to 74cm, preferably 57cm to 72cm, more preferably 60cm to 70cm, based on a cylindrical tank.
For the case, the outside thereof has an inlet part and an outlet part provided with the material, and, in some specific embodiments of the present invention, the inlet part and the outlet part are connected with pressure detecting elements to provide pressure detection of the corresponding areas.
Further, in the case body interior, the filter element is provided, and the case body interior is partitioned into two accommodation portions physically unconnected by the filter element: a raw material container and a processed object container. And the raw material containing part is connected with an inlet of the material, and the processed object containing part is connected with an outlet of the material.
There is no particular arrangement for the shape of the filter element, but from the viewpoint of pressure resistance, it may be a cylinder, preferably with the top of the cylinder being a circular dome to further increase pressure resistance.
Further, in some specific embodiments of the present invention, the volume of the raw material containing part formed by the filter element is greater than or equal to the volume of the treated material containing part. In some preferred embodiments, the ratio of the volume V1 of the raw material containing part in the tank to the volume V2 of the treatment substance containing part is V1: v2=1 to 100, more preferably 2 to 50, and still more preferably 5 to 20.
In addition, in some other preferred embodiments of the present invention, the above-mentioned device of the present invention may further comprise other optional auxiliary components such as heating, temperature control or temperature detection components.
< third aspect >
In a third aspect of the invention, a processing method for detecting the invention and a verification method for detecting the validity of the operation of the processing apparatus of the invention are provided.
By means of the validation method, an efficient determination or detection of the presence or absence of a failure of a filtration or sterilization device, in particular of its filter element, can be provided in a continuous or semi-continuous filtration or sterilization process.
Specifically, the verification method includes:
i. fully wetting the porous membrane in the filter element with water;
keeping the gas pressure of the porous membrane A side of the filter element or the raw material containing part of the device constant at P, and measuring the gas escape VD of the filter element within the test time T 1 ；
Escaping the gas by VD 1 Theoretical value VD of gas escape under the same conditions 0 A comparison is made.
Wherein, in step i, pure water is fed into the filtering or sterilizing device from the inlet of the filtering or sterilizing device under the action of compressed air, the pressure of the compressed air can be equal to or less than the pressure P1 of the side of the filtering element containing the lactoferrin raw material when the filtering or sterilizing device is in operation, preferably, the pressure of the compressed air can be 0.9 times to 1 times of the pressure P1, and more preferably, the pressure of the compressed air can be P1.
Further, with the aid of compressed air under this pressure, pure water was caused to flush the double-layer membrane of the filter element so that the double-layer membrane was completely wetted (completely permeated with water to the membrane B side). The time for flushing is not particularly limited, but is usually 5 minutes or more, preferably 5 to 10 minutes, and the amount of water for flushing is not particularly limited, depending on the volume of the apparatus.
In step ii, the supply of pure water is stopped, and compressed air is injected into the raw material holding part only through the inlet so that the pressure of the raw material holding part (i.e., the porous membrane a side) is kept constant. Such a constant pressure is preferably equal to the maximum value of the pressure P1, i.e. 2.5bar, on the side of the filter element containing the lactoferrin raw material during operation of the filtration or sterilisation apparatus.
Further, the holding time t may be 5min with a pressure of 2.5bar maintained. Further, the gas escape amount VD of the filter element in the time t is calculated by detecting the pressure of the gas entering the processing object accommodating part 1 (mL/min)。
To obtain VD 1 Then, it can be brought into contact with the theoretical value VD of the gas escape volume of the filter element 0 By contrast, if VD 1 Greater than VD 0 This indicates that the filter element may be broken, and vice versa indicates that the filter element is in a normal state.
VD for said filter element 0 In principle, the invention is not restricted to any particular method of measurement, and in some embodiments of the invention, for example in the case of FIG. 1, the VD is 0 H, which is the vertical height of the bilayer membranes in the filter element (in cm from the highest point of the bilayer membranes, which is the vertical distance from the bottom plane of the filter element), is 1.8H.
After the completion of the verification method, the sterilization apparatus may be heated and steam-sterilized at a temperature of 110 to 140 ℃, preferably 120 to 135 ℃.
By the verification method provided by the third aspect of the invention, the verification program can be added in the continuous or semi-continuous filtering or sterilizing process aiming at the lactoferrin so as to ensure the integrity of the membrane in the whole production process and the effectiveness of the filtering or sterilizing process, and particularly, the verification method can be used for detecting the effectiveness of the lactoferrin sterilization on line.
Examples
Hereinafter, the technical solution of the present invention will be explained by specific examples.
The following example was processed according to the apparatus shown in fig. 1, wherein the apparatus has a cylindrical configuration in the shape of a tank and a dome having a circular shape at the top end. Also, the filter element is shaped as a cylindrical structure and has a dome with a circular shape at the top end.
Example 1:
1. when the test is started, pure water (less than or equal to 70 dynes/cm) is pushed into the cavity from the inlet end by using compressed air with the pressure of 2.5bar, and the washing time t is Punching machine =5min, wash volume V =150L, make the sterile membrane completely wet;
2. after the sterilization film is completely wetted, the valve at the outlet end is opened, and compressed air is pumped into the cavity from the inlet end until the pressure at the inlet end is P Into When 2.5bar is reached, the inlet valve is closed, and pressure maintaining test is carried out for 5min, wherein the pressure maintaining process is such as inlet pressure P Into When the pressure is lower than 2.5bar, the inlet valve is opened to supplement air until 2.5bar is reached. Calculating the gas escape amount VD in unit time 1 Value when VD 1 ≤VD 0 When the first validation passes, when the VD 1 ＞VD 0 When the verification fails, the sterilization membrane fails, and the verification activity is terminated;
gas escape volume VD in unit time 0 The height of the degerming film corresponds to the following:
H＝10cm
VD 0 ＝18mL/min；
3. sterilizing with 121 deg.C steam for 30min after system pressure relief, and sterilizing at inlet end P Go into =2.3bar, outlet end P Go out ＝2bar；
4. After the sterilization is finished, the normal cold sterilization process is carried out, the material temperature T =20 ℃, and the inlet end P is Into =2.5bar, outlet end P Go out ＝0.5bar；
5. After the cold sterilization process is finished, a cleaning program is executed to clean the sterilization membrane;
6. pushing pure water (less than or equal to 70 dynes/cm) into the cavity from the inlet end by using compressed air of 2.5bar, and washing for a time t Punching machine =5min, washing volume V =150L, to make the sterilization membrane completely wet;
7. after the sterilization membrane is completely wetted, the outlet valve is opened, and compressed air is pumped into the cavity from the inlet end until the pressure P at the inlet end is reached Go into When 2.5bar is reached, the inlet valve is closed, and a pressure maintaining test is carried out for 5min, wherein the pressure maintaining process is the pressure P at the inlet end Go into When the pressure is lower than 2.5bar, the inlet valve is opened to make up air until 2.5bar is reached. Calculating the gas escape volume VD in unit time 1 Value when VD 1 ≤VD 0 When the first validation passes, when the VD 1 ＞VD 0 When the verification fails, the sterilization membrane fails, and the verification activity is terminated;
gas escape volume VD in unit time 0 The height of the degerming film corresponds to the following relationship:
H＝10cm
VD 0 ＝18mL/min。
example 2:
1. when the test is started, pure water (less than or equal to 70 dynes/cm) is pushed into the cavity from the inlet end by using compressed air with the pressure of 2.5bar, and the washing time t is Punching machine =5min, wash volume V =200L, make the sterilization membrane completely wet;
2. after the sterilization membrane is completely wetted, the outlet valve is opened, and compressed air is pumped into the cavity from the inlet end until the pressure P at the inlet end is reached Into When 2.5bar is reached, the inlet valve is closed, and a pressure maintaining test is carried out for 5min, wherein the pressure maintaining process is the pressure P at the inlet end Go into When the pressure is lower than 2.5bar, the inlet valve is opened to make up air until 2.5bar is reached. Calculating the gas escape volume VD in unit time 1 Value when VD 1 ≤VD 0 When the first step of validity verification passes, when VD is larger than VD 0 When the verification fails, the sterilization membrane fails, and the verification activity is terminated;
amount of gas escaping per unit time VD 0 The height of the degerming film corresponds to the following:
H＝20cm
VD 0 ＝36mL/min；
3. sterilizing with 121 deg.C steam for 30min after system pressure relief, and sterilizing at inlet end P Into =2.1bar, outlet end P Go out ＝1.9bar；
4. After the sterilization is finished, the normal cold sterilization process is carried out, the material temperature T =55 ℃, and the inlet end P is Into =2.5bar, outlet end P Go out ＝0.5bar；
5. After the cold sterilization process is finished, a cleaning program is executed to clean the sterilization membrane;
6. pushing pure water (less than or equal to 70 dynes/cm) into the cavity from the inlet end by using compressed air of 2.5bar, and washing for a time t Punching machine =5min, wash volume V =200L, make the sterilization membrane completely wet;
7. after the sterilization membrane is completely wetted, the outlet valve is opened, and compressed air is pumped into the cavity from the inlet end until the pressure P at the inlet end is reached Into When 2.5bar is reached, the inlet valve is closed, and pressure maintaining test is carried out for 5min, wherein the pressure maintaining process is such as inlet pressure P Go into When the pressure is lower than 2.5bar, the inlet valve is opened to make up air until 2.5bar is reached. Calculating the gas escape volume VD in unit time 1 Value when VD 1 ≤VD 0 When the first step validation is passed, when the VD is 1 ＞VD 0 When the effectiveness verification fails, the sterilization membrane fails, and the verification activity is terminated;
gas escape volume VD in unit time 0 The height of the degerming film corresponds to the following:
H＝20cm
VD 0 ＝36mL/min。
example 3:
1. when the test is started, pure water (less than or equal to 70 dynes/cm) is pushed into the cavity from the inlet end by using compressed air with the pressure of 2.5bar, and the washing time t is Punching machine =5min, washing volume V =150L, to make the sterilization membrane completely wet;
2. after the sterilization membrane is completely wetted, the outlet valve is opened, and compressed air is pumped into the cavity from the inlet end until the pressure P at the inlet end is reached Into When 2.5bar is reached, the inlet valve is closed, and pressure maintaining test is carried out for 5minProcess such as inlet end pressure P Go into When the pressure is lower than 2.5bar, the inlet valve is opened to make up air until 2.5bar is reached. Calculating the gas escape volume VD in unit time 1 Value when VD 1 ≤VD 0 When the first step of validity verification passes, when VD is larger than VD 0 When the verification fails, the sterilization membrane fails, and the verification activity is terminated;
gas escape volume VD in unit time 0 The height of the degerming film corresponds to the following relationship:
H＝30cm
VD 0 ＝54mL/min；
3. sterilizing with 134 deg.C steam for 30min after system pressure relief, and sterilizing at inlet end P Into =2.3bar, outlet end P Go out ＝2bar；
4. After the sterilization is finished, the normal cold sterilization process is carried out, the material temperature T =53 ℃, and the inlet end P is Into =2.5bar, outlet end P Go out ＝0.5bar；
5. After the cold sterilization process is finished, a cleaning program is executed to clean the sterilization membrane;
6. pushing pure water (less than or equal to 70 dynes/cm) into the cavity from the inlet end by using compressed air of 2.5bar, and washing for t Punching machine =5min, washing volume V =150L, to make the sterilization membrane completely wet;
7. after the sterilization membrane is completely wetted, the outlet valve is opened, and compressed air is pumped into the cavity from the inlet end until the pressure P at the inlet end is reached Into When 2.5bar is reached, the inlet valve is closed, and pressure maintaining test is carried out for 5min, wherein the pressure maintaining process is such as inlet pressure P Into When the pressure is lower than 2.5bar, the inlet valve is opened to make up air until 2.5bar is reached. Calculating the gas escape volume VD in unit time 1 Value when VD 1 ≤VD 0 When the first step validation is passed, when the VD is 1 ＞VD 0 When the verification fails, the sterilization membrane fails, and the verification activity is terminated;
gas escape volume VD in unit time 0 The height of the degerming film corresponds to the following:
H＝30cm
VD 0 ＝54mL/min。
example 4:
1. when the test is started, pure water (less than or equal to 70 dynes/cm) is pushed into the cavity from the inlet end by using compressed air with the pressure of 2.5bar, and the washing time t is Punching machine =5min, washing volume V =150L, to make the sterilization membrane completely wet;
2. after the sterilization membrane is completely wetted, the outlet valve is opened, and compressed air is pumped into the cavity from the inlet end until the pressure P at the inlet end is reached Into When 2.5bar is reached, the inlet valve is closed, and pressure maintaining test is carried out for 5min, wherein the pressure maintaining process is such as inlet pressure P Into When the pressure is lower than 2.5bar, the inlet valve is opened to supplement air until 2.5bar is reached. Calculating the gas escape amount VD in unit time 1 Value when VD 1 ≤VD 0 When the first step of validity verification passes, when VD is larger than VD 0 When the effectiveness verification fails, the sterilization membrane fails, and the verification activity is terminated;
gas escape volume VD in unit time 0 The height of the degerming film corresponds to the following:
H＝40cm
VD 0 ＝72mL/min；
3. sterilizing with 134 deg.C steam for 30min after system pressure relief, and sterilizing at inlet end P Into =2.3bar, outlet end P Go out ＝2bar；
4. After the sterilization is finished, the normal cold sterilization process is carried out, the material temperature T =38 ℃, and the inlet end P Into =2.5bar, outlet end P Go out ＝0.5bar；
5. After the cold sterilization process is finished, a cleaning program is executed to clean the sterilization membrane;
6. pushing pure water (less than or equal to 70 dynes/cm) into the cavity from the inlet end by using compressed air of 2.5bar, and washing for a time t Punching machine =5min, washing volume V =150L, to make the sterilization membrane completely wet;
7. after the sterilization membrane is completely wetted, the outlet valve is opened, and compressed air is pumped into the cavity from the inlet end until the pressure P at the inlet end is reached Into When 2.5bar is reached, the inlet valve is closed, and pressure maintaining test is carried out for 5min, wherein the pressure maintaining process is such as inlet pressure P Into When the pressure is lower than 2.5bar, beatingThe inlet valve was opened to allow gas to be added until 2.5bar was reached. Calculating the gas escape volume VD in unit time 1 Value when VD 1 ≤VD 0 When the first step validation is passed, when the VD is 1 ＞VD 0 When the verification fails, the sterilization membrane fails, and the verification activity is terminated;
gas escape volume VD in unit time 0 The height of the degerming film corresponds to the following:
H＝40cm
VD 0 ＝72mL/min。
the cold sterilization process validity verification (gas escape amount in unit time) is carried out on the normal products of the groups 1 to 4 in the specific examples, the abnormal products of the groups 1 (damaged sterilization film and film perforation) and the sterilization films which are not installed, and the detection and rechecking are carried out on the microorganism results according to the requirements of GB1903.17-2016 food nutrition fortifier lactoferrin, and the specific data are as follows:
TABLE 1
The results show that when the sterilization film is damaged, the VD value can be fed back to be abnormal immediately, and the microbial result of the product is abnormal; when the degerming film is normal, the VD value meets the theoretical requirement, and the microbial index of the product meets the national standard requirement, so that the lactoferrin cold sterilization process is proved to be effective.
It should be noted that, although the technical solutions of the present invention are described by specific examples, those skilled in the art can understand that the present disclosure should not be limited thereto.
Having described embodiments of the present disclosure, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations 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 in order to best explain the principles of the embodiments, the practical application, or improvements made to the technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.
Industrial applicability
The lactoferrin sterilization method and device and the validity verification method 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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