CN110563835A - Lactoferrin freeze-drying protective agent and application thereof - Google Patents
Lactoferrin freeze-drying protective agent and application thereof Download PDFInfo
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- CN110563835A CN110563835A CN201810567103.4A CN201810567103A CN110563835A CN 110563835 A CN110563835 A CN 110563835A CN 201810567103 A CN201810567103 A CN 201810567103A CN 110563835 A CN110563835 A CN 110563835A
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- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 title claims abstract description 184
- 102000010445 Lactoferrin Human genes 0.000 title claims abstract description 183
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- 235000021242 lactoferrin Nutrition 0.000 title claims abstract description 183
- 229940078795 lactoferrin Drugs 0.000 title claims abstract description 183
- 238000004108 freeze drying Methods 0.000 title claims abstract description 138
- 239000003223 protective agent Substances 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 claims abstract description 72
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- 239000000243 solution Substances 0.000 claims description 15
- 238000010438 heat treatment Methods 0.000 claims description 13
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 9
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 9
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 7
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 7
- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 claims description 7
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- 239000004472 Lysine Substances 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 229930091371 Fructose Natural products 0.000 claims description 3
- 239000005715 Fructose Substances 0.000 claims description 3
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 2
- 229920002774 Maltodextrin Polymers 0.000 claims description 2
- 239000005913 Maltodextrin Substances 0.000 claims description 2
- 229920000881 Modified starch Polymers 0.000 claims description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 2
- 235000014633 carbohydrates Nutrition 0.000 claims description 2
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 claims description 2
- JCQLYHFGKNRPGE-FCVZTGTOSA-N lactulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-FCVZTGTOSA-N 0.000 claims description 2
- 229960000511 lactulose Drugs 0.000 claims description 2
- PFCRQPBOOFTZGQ-UHFFFAOYSA-N lactulose keto form Natural products OCC(=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O PFCRQPBOOFTZGQ-UHFFFAOYSA-N 0.000 claims description 2
- 229940035034 maltodextrin Drugs 0.000 claims description 2
- 230000001012 protector Effects 0.000 claims 3
- 229920002307 Dextran Polymers 0.000 claims 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 claims 1
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- 241000588724 Escherichia coli Species 0.000 abstract description 17
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- 238000011156 evaluation Methods 0.000 abstract description 2
- 238000001035 drying Methods 0.000 description 21
- 102000004169 proteins and genes Human genes 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 13
- 239000007788 liquid Substances 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 10
- 230000002401 inhibitory effect Effects 0.000 description 10
- 239000004471 Glycine Substances 0.000 description 8
- 238000007710 freezing Methods 0.000 description 8
- 230000008014 freezing Effects 0.000 description 8
- 238000003756 stirring Methods 0.000 description 7
- 230000004071 biological effect Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000004806 packaging method and process Methods 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
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- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 229920001503 Glucan Polymers 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
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- 102000007544 Whey Proteins Human genes 0.000 description 2
- 108010046377 Whey Proteins Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- FFBHFFJDDLITSX-UHFFFAOYSA-N benzyl N-[2-hydroxy-4-(3-oxomorpholin-4-yl)phenyl]carbamate Chemical compound OC1=C(NC(=O)OCC2=CC=CC=C2)C=CC(=C1)N1CCOCC1=O FFBHFFJDDLITSX-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
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- 239000008273 gelatin Substances 0.000 description 2
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- 229910052739 hydrogen Inorganic materials 0.000 description 2
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- 239000000843 powder Substances 0.000 description 2
- 238000004904 shortening Methods 0.000 description 2
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- 229920001817 Agar Polymers 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
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- 230000009102 absorption Effects 0.000 description 1
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- 238000001914 filtration Methods 0.000 description 1
- 235000013350 formula milk Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000012792 lyophilization process Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000003988 neural development Effects 0.000 description 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000031337 regulation of inflammatory response Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a lactoferrin freeze-drying protective agent and application thereof, wherein the lactoferrin freeze-drying protective agent comprises saccharides and amino acids, the total weight of the lactoferrin is 100%, the amount of the saccharides is 0.01% -5%, and the amount of the amino acids is 0.01% -2%. The antibacterial activity of the lactoferrin on escherichia coli is taken as an evaluation index, the bacteriostatic rate of the freeze-dried lactoferrin added with the freeze-drying protective agent is obviously higher than that of the lactoferrin freeze-dried without the freeze-drying protective agent, and the freeze-drying protective agent provided by the invention can effectively reduce the activity loss of the lactoferrin in the freeze-drying process.
Description
Technical Field
The invention relates to a lactoferrin freeze-drying protective agent and application thereof, belonging to the technical field of biology.
Background
Lactoferrin (LF) is a glycoprotein naturally present in the external secretions of milk, tears, saliva, etc. with bioactive functions including broad-spectrum antibacterial activity, enhancement of iron transmission and absorption, immunomodulation, regulation of inflammatory responses, and anti-oxidant action. The biological activity of lactoferrin is closely related to the structure of lactoferrin, and the mechanism of action of some lactoferrin structures on the biological activity is researched and confirmed that various excellent biological activities of lactoferrin must depend on the normal maintenance of protein structures at all levels. However, lactoferrin is sensitive to external environments such as heat, pH, ionic strength, and the like, and the structure thereof is easily changed to lose activity.
At present, commercial lactoferrin is mainly extracted from cow milk or whey, the final step of the lactoferrin production process is to dry a liquid lactoferrin solution into a solid state, and due to the high sensitivity of lactoferrin, the main drying methods at present are freeze drying and low-temperature spray drying.
Freeze drying (freeze drying for short) is a drying method in which a water-containing substance is frozen at a low temperature and then ice is sublimated by heating the freeze-dried substance under a vacuum condition, thereby removing water. Because the material is always at a lower temperature in the freeze-drying process, the change of the physical and chemical properties of the material is smaller, the quality of the freeze-dried material is higher, and the freeze-dried material has a loose and porous structure, thereby being very beneficial to the re-dissolution of the material. It is clear that freeze-drying is the most suitable method for drying structurally unstable proteins such as lactoferrin. Compared with drying modes such as roller drying, spray drying and the like, the structural change and activity loss of the lactoferrin caused by freeze drying are minimum. However, the lyophilization process is a complex phase transition process, and there are many factors inducing protein denaturation during freezing, primary drying, secondary drying, and the like. Thus, in fact, lactoferrin still suffers a relatively slight loss of its biological activity after freeze-drying. Besides adjusting the process parameters such as pre-freezing temperature, cooling rate and the like, the freeze-drying protective agent is also a particularly effective method for maintaining the biological activity of the lactoferrin.
The inventor of the present invention has found through practical research that the bioactivity of the lactoferrin reconstituted after conventional freeze-drying is reduced, which indicates that the lactoferrin is denatured to some extent during the freeze-drying process.
Therefore, the technical problem to be solved in the art is to provide a freeze-drying protective agent which is suitable for lactoferrin and can effectively reduce the activity loss of lactoferrin in the freeze-drying process.
Disclosure of Invention
In order to solve the above disadvantages and shortcomings, the present invention aims to provide a freeze-drying protective agent for lactoferrin. The lactoferrin freeze-drying protective agent provided by the invention can greatly reduce the activity loss of lactoferrin in the freeze-drying process.
the invention also aims to provide the application of the lactoferrin freeze-drying protective agent in lactoferrin freeze drying.
The invention also aims to provide a lactoferrin freeze-drying method, which adopts the lactoferrin freeze-drying protective agent.
In order to achieve the above object, in one aspect, the present invention provides a lactoferrin freeze-drying protective agent, wherein the lactoferrin freeze-drying protective agent includes saccharides and amino acids, the saccharides are used in an amount of 0.01% to 5% and the amino acids are used in an amount of 0.01% to 2% based on the total weight of the lactoferrin as 100%.
The lactoferrin freeze-drying protective agent is preferably used in an amount of 0.1% -2% of the saccharides and 0.05% -1% of the amino acids.
The sugar has two main protection mechanisms, one is that the sugar with high viscosity surrounds the protein molecules, limits the diffusion of substances, and reduces the extension degree of the protein molecules, for example, sucrose has a relatively high glass transition temperature, so that the sucrose can be concentrated with the protein in a frozen state to dilute the protein, thereby preventing polymerization and maintaining the structure of the protein molecules; secondly, after the protein loses water, hydrogen bonds originally combined with water molecules are combined with sugar molecules, so that the hydrogen bonds and polar groups on the surface of the protein cannot be denatured due to exposure.
according to the lactoferrin freeze-drying protective agent, preferably, the saccharide comprises one or more of sucrose, trehalose, lactose, fructose, lactulose, fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, glucan, maltodextrin and pregelatinized starch.
When the amino acid is used as a protein freeze-drying protective agent, the glass transition temperature of the amino acid is relatively higher than that of a non-amino acid buffer agent, so that the amino acid plays a certain role in the stability of a freeze-drying preparation; in the freezing and freeze-drying processes, specific interaction occurs between amphoteric amino acid molecules and protein molecules, so that the protein invariance can be protected; in addition, amino acids can also protect proteins by preventing crystallization of buffer salts. Therefore, the freeze-drying protective agent claimed by the invention can significantly reduce the loss of lactoferrin activity in the freeze-drying process by adopting the combination of the saccharides and the amino acids.
According to the lactoferrin freeze-drying protective agent, preferably, the amino acid comprises one or a combination of arginine, lysine, tryptophan and glycine.
According to the lactoferrin freeze-drying protective agent, preferably, when the amino acid comprises arginine and lysine, the molar ratio of the arginine to the lysine is 20:1-1: 20.
According to the application field and relevant regulations of lactoferrin, different combinations of saccharides and amino acids can be selected as a lactoferrin freeze-drying protective agent in the freeze-drying process, for example, for lactoferrin in infant formula milk powder, the freeze-drying protective agent can be selected from fructo-oligosaccharide and/or galacto-oligosaccharide and tryptophan, wherein the fructo-oligosaccharide and the galacto-oligosaccharide are prebiotics which are beneficial to intestinal health of infants, and the tryptophan is important for neural development of infants; and for lactoferrin for energy supplement use, a freeze-drying protective agent used for the lactoferrin can be fructose and various amino acids in combination; the freeze-drying protective agent used for the lactoferrin used for the yoghourt and the liquid milk can be selected from lactose and/or a combination of glucan and glycine with relatively low cost.
On the other hand, the invention also provides the application of the lactoferrin freeze-drying protective agent in lactoferrin freeze drying.
In another aspect, the present invention also provides a freeze-drying method of lactoferrin, which uses the freeze-drying protective agent of lactoferrin, the method comprising the following steps:
(1) Adding the lactoferrin freeze-drying protective agent into the lactoferrin aqueous solution, and uniformly mixing at room temperature;
(2) freeze-drying the mixed solution obtained in the step (1); wherein the freeze drying comprises the following four stages:
The first stage is as follows: the freeze drying temperature is in the range of-50 to-30 deg.C, and the pressure is 0.3bar for 0.5-3 h;
And a second stage: rapidly heating to-20 deg.C to-15 deg.C, maintaining for 3-8h, and keeping pressure at 0.3 bar;
And a third stage: heating to 0 deg.C, maintaining for 1-3h under 0.3 bar;
a fourth stage: heating to 25-35 deg.C, and maintaining at 0.3bar for 5-10 h.
According to the lactoferrin freeze-drying method, the four-stage freeze-drying method can greatly reduce freeze-drying time and freeze-drying cost, wherein the second stage is mainly adopted, and the temperature is increased to be within the range of-20 to-15 ℃ by adopting rapid heating. The main purposes of this operation are: since the water of the freeze-dried product is mainly discharged at a low temperature, i.e. below the eutectic point of the solution, the eutectic point of the lactoferrin is between-14 and-12 ℃, and therefore, the supply of more heat at the eutectic point temperature during freeze-drying is an important factor for shortening the freeze-drying time. The moisture is removed as much as possible in the early stage, and the later drying time can be shortened.
according to the lactoferrin freeze-drying method of the present invention, preferably, the mixing time is 1-60 min.
According to the lactoferrin freeze-drying method, the thickness of the feed liquid is preferably controlled to be 1-5cm in the freeze-drying process. Wherein, in the freeze drying process, if the thickness of the feed liquid is too large, the feed liquid can not be completely freeze-dried, so the thickness of the feed liquid needs to be controlled to be 1-5cm in the lactoferrin freeze drying method provided by the invention.
According to the lactoferrin freeze-drying method of the present invention, preferably, the second stage is to rapidly raise the temperature to-15 ℃.
according to the lactoferrin freeze-drying method, preferably, the temperature rise rate of the rapid temperature rise is 1-1.5 ℃/min.
According to the lactoferrin freeze-drying method of the present invention, preferably, the concentration of lactoferrin is 1 to 10%, more preferably 5%, based on the total weight of the lactoferrin aqueous solution as 100%.
According to the lactoferrin freeze-drying method of the present invention, preferably, the pH of the lactoferrin aqueous solution is 6.5 to 7.0.
According to the lactoferrin freeze-drying method of the present invention, preferably, the conductivity of the lactoferrin aqueous solution is 1.5mS/cm or less. Wherein, in the freeze drying process of the lactoferrin, the amount of salts contained in the lactoferrin solution can be reduced by controlling the conductivity of the lactoferrin solution to be less than 1.5mS/cm, and the finally obtained lactoferrin has higher purity.
The lactoferrin freeze-drying method provided by the invention is characterized in that the lactoferrin aqueous solution is prepared by chromatography and membrane filtration technologies, and specifically, the preparation method comprises the following steps: carrying out ion exchange chromatography on a milk raw material containing lactoferrin, such as skim milk or whey protein, to obtain a lactoferrin crude extract, wherein the concentration of the lactoferrin is about 1% by taking the total weight of the lactoferrin crude extract as 100%;
And carrying out ultrafiltration, concentration and desalination on the lactoferrin crude extract to obtain a lactoferrin water solution with the pH value of 6.5-7.0, the conductivity of less than 1.5mS/cm and the concentration of 1-10%.
If the concentration of the lactoferrin is more than 10%, the lactoferrin aqueous solution can precipitate to block the membrane filter, so that the concentration speed is influenced, and further the concentration cannot be continued.
Adding the lactoferrin freeze-drying protective agent provided by the invention into the lactoferrin aqueous solution, uniformly stirring, and freeze-drying the lactoferrin according to the freeze-drying program. The antibacterial activity of the lactoferrin on escherichia coli is taken as an evaluation index, and the antibacterial rate of the freeze-dried lactoferrin added with the freeze-drying protective agent is obviously higher than that of the lactoferrin freeze-dried without the freeze-drying protective agent, which shows that the freeze-drying protective agent provided by the invention can effectively reduce the activity loss of the lactoferrin in the freeze-drying process, namely the freeze-drying protective agent can effectively reduce the damage of the lactoferrin in the freeze-drying process, and the biological activity preservation rate of the lactoferrin in the production and processing is improved to a certain extent.
Detailed Description
In order to clearly understand the technical features, objects and advantages of the present invention, the following detailed description of the technical solutions of the present invention will be made with reference to the following specific examples, which should not be construed as limiting the implementable scope of the present invention.
the procedures and equipment used in the present invention, which are not specifically mentioned in the present invention, can be performed according to the conventional procedures in the art or by referring to the specifications of the equipment used.
Example 1
the embodiment provides a lactoferrin freeze-drying method, which comprises the following steps:
Adding a freeze-drying protective agent into 5L of lactoferrin aqueous solution with the concentration of 5 wt% (the pH value is 6.5-7.0, and the conductivity is less than 1.5 mS/cm), wherein the protective agent comprises 12.5g of lactose and 5g of glycine, namely 5 wt% of lactose and 2 wt% of glycine (based on the mass of lactoferrin), gently stirring until the protective agent is dissolved, pouring the obtained mixed solution into a freeze-drying tray, and enabling the thickness of the feed liquid to be 2 cm.
The lactoferrin aqueous solution was subjected to the lyophilization operation according to the following procedure: pre-freezing for 1h at minus 40 ℃; the temperature is raised to-20 ℃ within 20min and maintained for 8h by primary drying, and the heating rate is 1 ℃/min; then raising the temperature to 0 ℃ and keeping the temperature for 1 h; the secondary drying was completed by continuing to raise the temperature to 30 ℃ for 10h, maintaining the vacuum at about 0.3 mbar. And (3) crushing and packaging the freeze-dried lactoferrin under aseptic conditions.
Example 1 the method for detecting inhibitory activity of lactoferrin against escherichia coli prepared in the preparation method comprises the following steps:
Strain: escherichia coli K99 strain: c83312 (national veterinary collection of microorganisms).
Culture medium: buffered peptone water, crystal violet neutral red bile salt agar.
Preparing bacterial liquid: selecting a small amount of escherichia coli preserved on the inclined plane, culturing and activating the escherichia coli in 10mL of peptone water at 37 ℃ at 60 rpm for 16h, transferring the first-generation activated bacterium solution into new peptone water according to the inoculation amount of 1%, and culturing the escherichia coli in the peptone water at 37 ℃ at 60 rpm for 1h to ensure that the concentration of the bacterium solution reaches 106-107And then diluted by 10 times for standby.
Preparation of lactoferrin solution: dissolving the lactoferrin freeze-dried powder by pure water, preparing a lactoferrin solution with the concentration of 2mg/mL, and filtering the lactoferrin solution by a 0.2-micrometer needle type filter membrane.
Bacteriostatic experiments: 0.4mL of the lactoferrin solution with a concentration of 2mg/mL was added to 8.6mL of peptone water, and 1mL of 105-106The E.coli cells were cultured at 37 ℃ for 24 hours to count plates.
And (3) detection results:
The results of the inhibitory activity of lactoferrin using the lyoprotectant provided by the present invention and lactoferrin without the lyoprotectant (the same process as in example 1 except that no lyoprotectant was added) against escherichia coli in example 1 are shown in table 1 below.
TABLE 1
| Sample set | Number of bacteria 24h | Rate of inhibition of bacteria |
| Control | 4.72×108 | — |
| Conventional lyophilized (without lyoprotectant) lactoferrin | 3.13×107 | 93.37％ |
| Example 1 Lactoferrin Using lyoprotectants | 8.51×106 | 98.2％ |
From the results in table 1, it can be seen that the bacteriostatic activity of lactoferrin is improved by 4.83% after the freeze-drying protective agent provided by the present invention is used in the freeze-drying process of lactoferrin.
Example 2
The embodiment provides a lactoferrin freeze-drying method, which comprises the following steps:
Adding a freeze-drying protective agent into 5L of lactoferrin aqueous solution with the concentration of 5 wt% (the pH value is 6.5-7.0, and the conductivity is less than 1.5 mS/cm), wherein the protective agent comprises 5g of lactose and 2.5g of glycine, namely 2 wt% of lactose and 1 wt% of glycine (based on the mass of the lactoferrin), gently stirring until the protective agent is dissolved, pouring the obtained mixed solution into a freeze-drying tray, and enabling the thickness of the feed liquid to be 2 cm.
the lactoferrin aqueous solution was subjected to the lyophilization operation according to the following procedure: pre-freezing for 1h at minus 40 ℃; the temperature is raised to-20 ℃ within 20min and maintained for 8h by primary drying, and the heating rate is 1 ℃/min; then raising the temperature to 0 ℃ and keeping the temperature for 1 h; the secondary drying was completed by continuing to raise the temperature to 30 ℃ for 10h, maintaining the vacuum at about 0.3 mbar. And (3) crushing and packaging the freeze-dried lactoferrin under aseptic conditions.
The method for detecting inhibitory activity of lactoferrin obtained in example 2 against E.coli was the same as in example 1.
And (3) detection results:
The results of the inhibitory activity of lactoferrin using the lyoprotectant provided by the present invention and lactoferrin without the lyoprotectant (the same process as in example 2 except that no lyoprotectant was added) against escherichia coli in example 2 are shown in table 2 below.
TABLE 2
from the results in table 2, it can be seen that the bacteriostatic activity of lactoferrin is improved by 5.41% after the freeze-drying protective agent provided by the present invention is used in the freeze-drying process of lactoferrin.
Example 3
The embodiment provides a lactoferrin freeze-drying method, which comprises the following steps:
Adding a freeze-drying protective agent into 5L of lactoferrin aqueous solution with the concentration of 5 wt% (pH value is 6.5-7.0, and the conductivity is less than 1.5 mS/cm), wherein the protective agent comprises 0.0125g of fructo-oligosaccharide, 0.0125g of galacto-oligosaccharide, 0.0125g of arginine and 0.0125g of tryptophan, namely 0.01 wt% of carbohydrate and 0.01 wt% of amino acid (based on the mass of lactoferrin), gently stirring until the protective agent is dissolved, pouring the obtained mixed solution into a freeze-drying tray, and the thickness of the material liquid is 2 cm.
The lactoferrin aqueous solution was subjected to the lyophilization operation according to the following procedure: pre-freezing for 1h at minus 40 ℃; the temperature is raised to-20 ℃ within 20min and maintained for 8h by primary drying, and the heating rate is 1 ℃/min; then raising the temperature to 0 ℃ and keeping the temperature for 1 h; the secondary drying was completed by continuing to raise the temperature to 30 ℃ for 10h, maintaining the vacuum at about 0.3 mbar. And (3) crushing and packaging the freeze-dried lactoferrin under aseptic conditions.
Example 3 the method for detecting inhibitory activity of lactoferrin against E.coli prepared in example 1 was the same.
And (3) detection results:
The results of the inhibitory activity of lactoferrin using the lyoprotectant provided by the present invention and lactoferrin without the lyoprotectant (the same process as in example 3 except that no lyoprotectant was added) against escherichia coli in example 3 are shown in table 3 below.
TABLE 3
| Sample set | Number of bacteria 24h | rate of inhibition of bacteria |
| Control | 4.72×108 | — |
| Conventional lyophilized (without lyoprotectant) lactoferrin | 3.13×107 | 93.37％ |
| Example 3 Lactoferrin Using lyoprotectants | 9.53×106 | 97.98％ |
from the results in table 3, it can be seen that the bacteriostatic activity of lactoferrin was improved by 4.61% after the freeze-drying protective agent provided by the present invention was used in the freeze-drying process of lactoferrin.
Example 4
The embodiment provides a lactoferrin freeze-drying method, which comprises the following steps:
adding a freeze-drying protective agent into 5L of lactoferrin aqueous solution with the concentration of 5 wt% (pH value is 6.5-7.0, and the conductivity is less than 1.5 mS/cm), wherein the protective agent comprises 0.125g of fructo-oligosaccharide, 0.125g of galacto-oligosaccharide, 0.0625g of arginine and 0.0625g of tryptophan, namely 0.1 wt% of saccharide and 0.05 wt% of amino acid (based on the mass of the lactoferrin), gently stirring until the protective agent is dissolved, pouring the obtained mixed solution into a freeze-drying tray, and the thickness of the feed solution is 2 cm.
The lactoferrin solution was subjected to the lyophilization procedure as follows: pre-freezing for 1h at minus 40 ℃; the temperature is raised to-20 ℃ within 20min and maintained for 8h by primary drying, and the heating rate is 1 ℃/min; then raising the temperature to 0 ℃ and keeping the temperature for 1 h; the secondary drying was completed by continuing to raise the temperature to 30 ℃ for 10h, maintaining the vacuum at about 0.3 mbar. And (3) crushing and packaging the freeze-dried lactoferrin under aseptic conditions.
Example 4 the inhibitory activity of lactoferrin against E.coli prepared in example 1 was examined.
and (3) detection results:
The results of the inhibitory activity of lactoferrin using the lyoprotectant provided by the present invention and lactoferrin without the lyoprotectant (the same process as in example 4 except that no lyoprotectant was added) against escherichia coli in example 4 are shown in table 4 below.
TABLE 4
| Sample set | Number of bacteria 24h | Rate of inhibition of bacteria |
| Control | 4.72×108 | — |
| Conventional lyophilized (without lyoprotectant) lactoferrin | 3.13×107 | 93.37％ |
| Example 4 Lactoferrin Using lyoprotectants | 3.91×106 | 99.17％ |
from the results in table 4, it can be seen that the bacteriostatic activity of lactoferrin was improved by 5.8% after the freeze-drying protective agent provided by the present invention was used in the freeze-drying process of lactoferrin.
Example 5
The embodiment provides a lactoferrin freeze-drying method, which comprises the following steps:
Adding a freeze-drying protective agent into 5L of lactoferrin aqueous solution with the concentration of 5 wt% (pH value is 6.5-7.0, and the conductivity is less than 1.5 mS/cm), wherein the protective agent comprises 5g of lactose, 2.5g of glycine, 0.5g of mannitol and 0.5g of gelatin, namely 2 wt% of lactose, 1 wt% of glycine, 0.2 wt% of mannitol and 0.2 wt% of gelatin (based on the mass of lactoferrin), gently stirring until the protective agent is dissolved, pouring the obtained mixed solution into a freeze-drying tray, and enabling the thickness of the feed liquid to be 2 cm.
The lactoferrin aqueous solution was subjected to the lyophilization operation according to the following procedure: pre-freezing for 1h at minus 40 ℃; the temperature is raised to-20 ℃ within 20min and maintained for 8h by primary drying, and the heating rate is 1 ℃/min; then raising the temperature to 0 ℃ and keeping the temperature for 1 h; the secondary drying was completed by continuing to raise the temperature to 30 ℃ for 10h, maintaining the vacuum at about 0.3 mbar. And (3) crushing and packaging the freeze-dried lactoferrin under aseptic conditions.
the inhibitory activity of lactoferrin obtained in example 5 against E.coli was determined in the same manner as in example 1.
And (3) detection results:
The results of the inhibitory activity of lactoferrin using the lyoprotectant provided by the present invention and lactoferrin without the lyoprotectant (the same process as in example 5 except that no lyoprotectant was added) against escherichia coli in example 5 are shown in table 5 below.
TABLE 5
| sample set | Number of bacteria 24h | rate of inhibition of bacteria |
| Control | 4.72×108 | — |
| Conventional lyophilized (without lyoprotectant) lactoferrin | 3.13×107 | 93.37％ |
| Example 5 Lactoferrin Using lyoprotectants | 7.92×106 | 98.32％ |
From the results in table 5, it can be seen that the bacteriostatic activity of lactoferrin was improved by 4.95% after the freeze-drying protective agent provided by the present invention was used in the freeze-drying process of lactoferrin.
Example 6
The embodiment provides a lactoferrin freeze-drying method, which comprises the following steps:
Adding a freeze-drying protective agent into 5L of lactoferrin aqueous solution with the concentration of 5 wt% (pH value is 6.5-7.0, and the conductivity is less than 1.5 mS/cm), wherein the protective agent comprises 0.125g of fructo-oligosaccharide, 0.125g of galacto-oligosaccharide, 0.0625g of arginine and 0.0625g of tryptophan, namely 0.1 wt% of saccharide and 0.05 wt% of amino acid (based on the mass of the lactoferrin), gently stirring until the protective agent is dissolved, pouring the obtained mixed solution into a freeze-drying tray, and the thickness of the feed solution is 2 cm.
The aqueous lactoferrin solution was lyophilized according to the freeze-drying procedure shown in table 6 below, and the lyophilized lactoferrin was aseptically crushed and packaged. The effect duration and the water content of the obtained lactoferrin are also shown in table 6 below for different freeze-drying procedures.
TABLE 6
Note: the temperature rise rate of the second stage of the rapid temperature rise in the processes 1 and 2 is 1 ℃/min.
As can be seen from table 6, the freeze-drying of lactoferrin according to "comparative process 1" requires 26 hours for freeze-drying, and the independent shortening of the freeze-drying time in the fourth stage in "comparative process 2" results in an increase in the water content, which can reach 6.25%, indicating that the freeze-drying of lactoferrin by using "comparative process 2" does not achieve the drying purpose. And the temperature of the second stage is increased, for example, the drying time of the second stage can be reduced by adopting the 'process 1' and 'process 2' provided by the invention, and meanwhile, the drying time of the fourth stage can also be greatly reduced. Therefore, the freeze drying procedure provided by the invention can be used for freeze drying lactoferrin, so that the freeze drying time can be obviously shortened, and the freeze drying cost is further reduced.
Claims (10)
1. The freeze-drying protective agent for the lactoferrin is characterized by comprising saccharides and amino acids, wherein the total weight of the lactoferrin is 100%, the saccharides are 0.01-5%, and the amino acids are 0.01-2%.
2. A lactoferrin freeze-drying protector according to claim 1, characterized in that the carbohydrate is used in an amount of 0.1-2% and the amino acid in an amount of 0.05-1%.
3. A lactoferrin freeze-drying protector according to claim 1 or 2, characterized in that the sugar comprises one or a combination of several of sucrose, trehalose, lactose, fructose, lactulose, fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, dextran, maltodextrin and pregelatinized starch.
4. A lactoferrin freeze-drying protector according to claim 1 or 2, characterized in that the amino acids comprise one or a combination of arginine, lysine, tryptophan and glycine;
preferably, when the amino acids include arginine and lysine, the molar ratio of arginine to lysine is 20:1 to 1: 20.
5. use of a lactoferrin freeze-drying protectant according to any one of claims 1 to 4 in lactoferrin freeze-drying.
6. a method for freeze-drying lactoferrin, wherein the method uses the lactoferrin freeze-drying protectant according to any one of claims 1 to 4, the method comprising the steps of:
(1) Adding the lactoferrin freeze-drying protective agent into the lactoferrin aqueous solution, and uniformly mixing at room temperature;
(2) Freeze-drying the mixed solution obtained in the step (1); wherein the freeze drying comprises the following four stages:
the first stage is as follows: the freeze drying temperature is in the range of-50 to-30 deg.C, and the pressure is 0.3bar for 0.5-3 h;
and a second stage: rapidly heating to-20 deg.C to-15 deg.C, maintaining for 3-8h, and keeping pressure at 0.3 bar;
and a third stage: heating to 0 deg.C, maintaining for 1-3h under 0.3 bar;
A fourth stage: heating to 25-35 deg.C, maintaining for 5-10 hr at 0.3 bar;
Preferably, the temperature rise rate of the rapid temperature rise is 1-1.5 ℃/min.
7. Lactoferrin freeze-drying process according to claim 6, characterized in that the mixing time is 1-60 min.
8. the freeze-drying method of lactoferrin according to claim 6, wherein the thickness of the control solution during the freeze-drying process is 1-5 cm.
9. A lactoferrin freeze-drying process according to claim 6, characterised in that the second stage is a rapid warming to a temperature of-15 ℃.
10. A lactoferrin freeze-drying process according to any of claims 6-9, characterized in that the concentration of lactoferrin is 1-10%, preferably 5%, based on 100% total weight of the lactoferrin aqueous solution;
Also preferably, the pH of the lactoferrin aqueous solution is 6.5-7.0;
Also preferably, the conductivity of the lactoferrin aqueous solution is 1.5mS/cm or less.
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