CN113956996A - Production method of freeze-dried lactobacillus acidophilus powder - Google Patents
Production method of freeze-dried lactobacillus acidophilus powder Download PDFInfo
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- CN113956996A CN113956996A CN202110887974.6A CN202110887974A CN113956996A CN 113956996 A CN113956996 A CN 113956996A CN 202110887974 A CN202110887974 A CN 202110887974A CN 113956996 A CN113956996 A CN 113956996A
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- 240000001046 Lactobacillus acidophilus Species 0.000 title claims abstract description 54
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 title claims abstract description 54
- 229940039695 lactobacillus acidophilus Drugs 0.000 title claims abstract description 54
- 239000000843 powder Substances 0.000 title claims abstract description 44
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 18
- 230000001580 bacterial effect Effects 0.000 claims abstract description 53
- 238000000855 fermentation Methods 0.000 claims abstract description 53
- 230000004151 fermentation Effects 0.000 claims abstract description 53
- 239000010802 sludge Substances 0.000 claims abstract description 49
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- 238000004945 emulsification Methods 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 22
- 230000008569 process Effects 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 238000000926 separation method Methods 0.000 claims abstract description 13
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 13
- 238000009630 liquid culture Methods 0.000 claims abstract description 9
- 230000001954 sterilising effect Effects 0.000 claims abstract description 8
- 239000000839 emulsion Substances 0.000 claims abstract description 7
- 238000007710 freezing Methods 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 230000008014 freezing Effects 0.000 claims abstract description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 39
- 239000003223 protective agent Substances 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- 241000894006 Bacteria Species 0.000 claims description 14
- 230000001804 emulsifying effect Effects 0.000 claims description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 244000068988 Glycine max Species 0.000 claims description 8
- 235000010469 Glycine max Nutrition 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 8
- 241001052560 Thallis Species 0.000 claims description 8
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 claims description 8
- 235000015278 beef Nutrition 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 8
- 229940099596 manganese sulfate Drugs 0.000 claims description 8
- 239000011702 manganese sulphate Substances 0.000 claims description 8
- 235000007079 manganese sulphate Nutrition 0.000 claims description 8
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000001632 sodium acetate Substances 0.000 claims description 8
- 235000017281 sodium acetate Nutrition 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 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 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- 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 5
- 239000008101 lactose Substances 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 5
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 5
- 229960005055 sodium ascorbate Drugs 0.000 claims description 5
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 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 4
- 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 4
- 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 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 4
- -1 diamine hydrogen citrate Chemical class 0.000 claims description 4
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 4
- 235000021255 galacto-oligosaccharides Nutrition 0.000 claims description 4
- 150000003271 galactooligosaccharides Chemical class 0.000 claims description 4
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 4
- 229940073490 sodium glutamate Drugs 0.000 claims description 4
- 239000008223 sterile water Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 239000001257 hydrogen Substances 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 11
- 230000003321 amplification Effects 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
- 239000006041 probiotic Substances 0.000 description 11
- 235000018291 probiotics Nutrition 0.000 description 11
- 238000004108 freeze drying Methods 0.000 description 9
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 8
- 230000036541 health Effects 0.000 description 7
- 241000186660 Lactobacillus Species 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 229940039696 lactobacillus Drugs 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 230000000529 probiotic effect Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 229960000448 lactic acid Drugs 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 239000004411 aluminium Substances 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-M 3-carboxy-2-(carboxymethyl)-2-hydroxypropanoate Chemical compound OC(=O)CC(O)(C(O)=O)CC([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-M 0.000 description 1
- 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 description 1
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- 201000010538 Lactose Intolerance Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002528 anti-freeze Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
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- 239000002775 capsule Substances 0.000 description 1
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- 125000000647 trehalose group Chemical group 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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Abstract
A production method of freeze-dried powder of lactobacillus acidophilus comprises the following process flows of lactobacillus acidophilus fermentation culture, fermentation liquor separation, bacterial sludge emulsification and emulsion vacuum freeze-drying, and is characterized in that the strain fermentation culture: inoculating lactobacillus acidophilus strain into a triangular flask liquid culture medium, and culturing for 16-24 hours at 35-40 ℃; then inoculating into a liquid fermentation tank for fermentation culture for 16-24 hours at the temperature of 35-40 ℃; sterilizing at 115 deg.C for 30 min; performing amplification culture in a strain fermentation tank for 8-24 hours, wherein the constant pH value of the fermentation liquor is 5.5 +/-0.1; and (3) vacuum freeze-drying the emulsion: pre-freezing for 4-8 hours at-55 to-45 ℃, and then carrying out vacuum freeze drying for 40-60 hours at-35 ℃ under the vacuum degree of 0.5-1.5 pa. The activity of the freeze-dried powder of lactobacillus acidophilus produced by the method is 2.0 multiplied by 1011cfu/g or more.
Description
Technical Field
The invention discloses a production method of lactobacillus acidophilus freeze-dried powder, which is applied to the technical field of food biology.
Background
Lactobacillus acidophilus (Lactobacillus acidophilus) is a Lactobacillus (Lactobacillus) which is a gram-positive, non-producing, non-motile, flagellate, capsular, spore-free, non-motile, single, bi-or short-chain form of slender bacteria, and can ferment sugars to produce lactic acid, ethanol, hydrogen peroxide, and other anaerobic or facultative anaerobic microorganisms. The optimum pH value is 5.5-6.0, the optimum growth temperature is 35-38 ℃, basically no growth is carried out at the temperature below 20 ℃, the heat resistance is poor, and the DL-lactic acid can be produced by homotypic fermentation by utilizing glucose, fructose, lactose and sucrose.
Lactobacillus acidophilus (Lactobacillus acidophilus) is an important microorganism in human intestinal tracts, is separated from feces of infants at first, can improve or regulate the balance of intestinal microflora, enhance the immunity of organisms, reduce the cholesterol level, relieve lactose intolerance, inhibit the formation of tumor cells and the like, and has a health promotion effect. Lactobacillus acidophilus has strong acid resistance and bile salt resistance, is few probiotics which can smoothly pass through the gastrointestinal tract environment and can play a role in health care, and products of the Lactobacillus acidophilus are widely developed and utilized in countries such as Europe, America, Japan and the like and are always popular.
In recent years, with the increasing health consciousness of people, probiotics and products thereof become important points for the future development of foods. Lactobacillus acidophilus is one of the strains in lactobacillus family which is regarded as the third generation lactic acid starter strain, and is an important microorganism in human intestinal tract and closely related to human health. When it reaches a certain amount, it can play a health promoting role. The study on the culture of lactobacillus acidophilus, one of the main strains commonly used in probiotic products, is of great significance.
In the forecast of early 1981, in the next twenty-three years, the development, research and application of lactobacillus acidophilus have strong vitality, and with the improvement of living standard of people and the gradual enhancement of health consciousness, the probiotics microecological preparation is researched and developed as a new field. At present, the market of products containing probiotics and containing bacteria is very active, and the products are more and more in variety, and comprise various putting forms such as food, capsules, tablets, powder, oral liquid and the like. Lactobacillus acidophilus is taken as a probiotic which has great importance on research and development values in the current lactobacillus family, is regarded as a third-generation lactic acid starter strain, and is one of the probiotics which is officially approved and approved by the U.S. food and drug administration and the Ministry of health of China.
The live lactobacillus preparation has active live activity of live bacteria to regulate the microecological balance of its host, and the live bacteria content is always one key index for evaluating the product quality. Scholars at home and abroad take the stability and survival of the maintenance bacteria as the standard for evaluating the quality of the bacteria. It has been found that the amount of probiotic bacteria must be sufficient to achieve the desired therapeutic effect. The number of probiotics to produce a therapeutic effect must be up to 106 cfu/ml, other studies have shown that 10 or more7cfu/ml and 108cfu/ml is a desirable level, referred to as the "minimum therapeutic amount", i.e., the consumer should have a minimum of 10 per day8cfu/ml intake. For therapeutic live bacteria preparation, the content of live bacteria should be not less than 109cfu/g. At present, commercial products appear abroad, and the viable bacteria content of the products is as high as 1010The formula composition is more than cfu/g, the commercial confidentiality is realized, the viable count of domestic probiotic products is always a difficult problem, the viable count of lactobacillus acidophilus probiotic products is improved, the lactobacillus acidophilus enriched culture is optimized, and the production method of the freeze-dried powder with high viable count is completed, so that the method is an important basic research work.
Disclosure of Invention
The invention aims to disclose a production method of freeze-dried powder of lactobacillus acidophilus, which is characterized in that the concentration of the thallus of fermentation liquor reaches 1.0 multiplied by 10 by culturing the lactobacillus acidophilus at the temperature of 35-40 ℃ for 8-24 hours at high density9More than cfu/ml, and the bacterial sludge concentration is up to 1.0 multiplied by 10 by the high-efficiency fermentation liquor separation technology10More than cfu/g, and obtaining 2.0 multiplied by 10 after optimized processes such as high-activity emulsification, vacuum freeze drying and the like11The production method of the lactobacillus acidophilus freeze-dried powder with more than cfu/ml can produce the lactobacillus acidophilus freeze-dried powder with high density, high stability and high activity.
On the basis of the characteristic research of lactobacillus acidophilus, the invention optimizes the high-density and high-activity fermentation culture, high-stability and high-activity separation, emulsification, freeze-drying and other technologies and processes of the lactobacillus acidophilus, optimizes the composition and concentration of the antifreeze protective agent, obtains the higher-density fermentation culture and activity stability protection functions to the maximum extent, reduces the death of thalli in the production process, improves the survival rate of cells, and provides the technology and process for the production of high-efficiency freeze-dried bacterial powder.
The technical scheme adopted by the invention is as follows:
a production method of freeze-dried powder of lactobacillus acidophilus comprises the following process flows of lactobacillus acidophilus fermentation culture, fermentation liquor separation, bacterial sludge emulsification and emulsion vacuum freeze-drying, and is characterized in that: the strain fermentation process comprises the steps of inoculating lactobacillus acidophilus strains into a triangular flask liquid culture medium for culture, wherein the weight percentage of the strains inoculated into the culture medium is 1.0-2.0%, and culturing for 16-24 hours at 35-40 ℃, and the triangular flask liquid culture medium comprises the following components in percentage by weight: soybean peptone 0.5-1.0%, glucose 2.0-3.0%, yeast extract powder 0.8-1.5%, beef extract powder 0.8-1.2%, magnesium sulfate 0.02%, manganese sulfate 0.005%, sodium acetate 0.5%, dihydrocitrates 0.2%, potassium dihydrogen phosphate 0.2%, Tween-800.1%, L-cysteine hydrochloride 0.05%, and water in balance, adjusting the pH value of the culture medium to 6.2-6.4 with 2.5mol/L food-grade sodium hydroxide solution, and sterilizing at 115 ℃ for 30 minutes. Then inoculating the mixture into a liquid fermentation tank for fermentation culture, wherein the inoculation weight percentage in a culture medium is 5-10%, and culturing the mixture for 8-30 hours at 35-40 ℃, wherein the fermentation culture medium of the liquid fermentation tank comprises the following components in percentage by weight: 0.5 to 1.0 percent of soybean peptone, 2.0 to 3.0 percent of glucose, 0.5 to 1.0 percent of sucrose, 0.5 percent of galacto-oligosaccharide, 0.8 to 1.5 percent of yeast extract powder, 0.8 to 1.2 percent of beef extract powder, 0.02 percent of magnesium sulfate, 0.005 percent of manganese sulfate, 0.5 percent of sodium acetate, 0.2 percent of citric acid hydrogen diamine, 0.2 percent of potassium dihydrogen phosphate, 800.1 percent of tween-L, 0.05 percent of L-cysteine hydrochloride and the balance of water, and sterilizing for 30 minutes at 115 ℃. Adding 2.5mol/L food-grade sodium hydroxide solution before inoculation to adjust the pH value of the culture medium to 6.5, inoculating the triangular flask strain, and after the pH value of the fermentation solution is reduced by 0.5, adding food-grade sodium hydroxide solution to maintain the constant pH value of the strain solution to be 5.5 +/-0.1 and OD600Stopping increasing or increasing negatively, performing microscopic examination on Lactobacillus acidophilus, stopping fermentation.
The fermentation liquor separation method comprises the steps of separating thalli by a tubular or disc centrifuge at 6000-13000 rpm, and washing the thalli for 1-2 times by using sterile water to obtain bacterial sludge.
The bacterial sludge emulsification process is characterized in that bacterial sludge and a bacterial sludge protective agent are mixed and emulsified according to the proportion of 10-1: 1-2 of the viable bacterial sludge and the bacterial sludge protective agent, the components of the bacterial sludge protective agent formula comprise, by weight, 0.5-2.0% of sodium glutamate, 0.1-0.5% of lactose, 8-12% of trehalose, 0.5-1% of sodium ascorbate, 10-20% of skim milk powder, 10-2.0% of tween-800.5 and the balance of water, and the bacterial sludge protective agent is sterilized at 115 ℃ for 30 minutes and then cooled to 5-20 ℃ for later use.
The bacterial sludge emulsification process is characterized in that bacterial sludge and a protective agent are emulsified and homogenized in an emulsification tank, the rotation speed of an emulsification motor of the emulsification tank is 1000-2000 rpm, and the rotation speed of a low-speed stirring motor is 80-100 rpm. Emulsifying for 20-30 min, and standing for 30-50 min at low temperature of 4 ℃ after the emulsification is finished.
The emulsion vacuum freeze-drying process is characterized in that the drying conditions are as follows: pre-freezing for 4-8 hours at-55 to-45 ℃, and then carrying out vacuum freeze drying for 40-60 hours at the temperature of-35 ℃ and the vacuum degree of 0.5-1.5 pa; drying to obtain viable count greater than 2.0 × 1011cfu/g freeze-dried powder of Lactobacillus acidophilus.
The operating environment conditions of the preparation of the liquid strain in the triangular flask, the separation of the fermentation liquor and the emulsification of the bacterial sludge are required to be carried out in a 10 ten thousand-level purification environment with the temperature lower than 25 ℃ and the humidity RH less than 35%.
Compared with the prior art, the invention has the following outstanding advantages:
1. aiming at the purposes of realizing the propagation culture of lactobacillus acidophilus and enriching thalli with high viable count for producing frozen and dry powder serving as food additives, different basic culture media and optimized culture media which are more suitable for different growth stages of the thalli are respectively adopted for the purification culture and the strain fermentation of the lactobacillus acidophilus, and the raw materials of the components adopted by the culture media are all food grade.
2. According to the invention, the bacterial sludge and the protective agent are emulsified and homogenized by the emulsifying tank, the protective agent is fully and uniformly wrapped with the bacterial sludge, the freezing resistance in the freeze dryer is enhanced, the low-temperature cooling circulating water is fed through the emulsifying tank jacket after emulsification is finished to enable live bacteria in the emulsion to be dormant, and the freeze drying capability of the freeze dryer is improved while the freezing resistance is enhanced.
3. The pH value is controlled to be constant in the fermentation process by feeding food-grade sodium hydroxide solution, a large amount of thalli are obtained in a short time, and the strains are stable and have less variation and degeneration.
4. The invention adopts a tubular centrifugal machine or a disc centrifugal machine to separate and collect the lactobacillus acidophilus bacterial sludge, the yield of the bacterial sludge of the tubular centrifugal machine can reach more than 2.0 percent, and the number of the viable bacteria of the bacterial sludge is as high as 1010cfu/g or more, and the rotary drum of the tubular centrifuge can be sterilized by steam or dry heat, so that the contamination of bacteria in the centrifugation process is effectively prevented. However, because the inner pipe diameter of the tubular centrifuge is limited, if a large-scale liquid fermentation tank is centrifuged, a plurality of tubular centrifuges are required to work together, and the investment cost of operators and equipment is high; and the separation capacity of the disc centrifuge is far beyond that of a tubular centrifuge, so that the slag can be discharged at regular time, and the disc centrifuge does not need to be disassembled to take the bacterial sludge after the rotating drum bacterial sludge is full like the tubular centrifuge, but the cost of the disc centrifuge is higher than that of the tubular centrifuge. The two kinds of separation equipment have the advantages and disadvantages respectively, and the centrifugal mode can be flexibly selected according to the actual situation.
5. The production of live bacterial preparations by freeze-drying techniques has a number of distinct advantages: the microorganism does not lose biological activity due to drying at low temperature; during the low-temperature drying process, the growth of microorganisms and the action of enzymes can hardly be carried out, and the original properties of the freeze-dried substance can be best maintained; the volume and shape are basically unchanged after drying; the freeze-dried product is in a spongy shape and is not dried and condensed, so that the contact area with water is large when the freeze-dried product is dissolved, and the original properties can be quickly recovered; can remove 95-99% of water in the substance and prolong the shelf life of the product.
6. In order to avoid the damage of the bacterial cell membrane caused in the freeze drying process, a freeze-drying protective agent is added, and the skim milk powder in the freeze-drying protective agent mainly plays a role of a protective layer on the cell surface; trehalose forms a glass state in the freeze drying process, and has extremely high viscosity in the glass state, so that the molecular diffusion coefficient is very low, the movement of macromolecular substances is blocked, and the permeability of cell walls is reduced, thereby playing a role in protection; the sodium ascorbate is an antioxidant with extremely strong water solubility, can prevent the lactobacillus acidophilus from being influenced by oxygen in the freeze-drying process, keeps the cell activity and improves the stability; the carbohydrate protective agent has a plurality of hydroxyl groups, so that the carbohydrate protective agent can form oxygen bonds with protein to replace water, and the stability of the protein is ensured. Each protective agent component in the compound protective agent system plays respective role in the freeze drying process, and simultaneously has synergistic effect with each other.
7. The lactobacillus acidophilus freeze-dried powder prepared by the technology has high viable count and high survival rate.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention, which is defined by the claims of the present application. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Proportions and percentages are based on weight units unless otherwise indicated.
Example 1:
inoculating the screened lactobacillus acidophilus strain into a triangular flask liquid culture medium, and then inoculating the lactobacillus acidophilus strain into a fermentation tank to be respectively cultured for 8 to 24 hours at 37 ℃ for step-by-step amplification fermentation culture, wherein the triangular flask liquid culture medium comprises the following components in percentage by weight: soybean peptone 0.5%, glucose 2.0%, yeast extract powder 1.5%, beef extract powder 0.8%, magnesium sulfate 0.02%, manganese sulfate 0.005%, sodium acetate 0.5%, dihydrogencitrate 0.2%, potassium dihydrogen phosphate 0.2%, tween-800.1%, L-cysteine hydrochloride 0.05%, and water in balance, adjusting the pH value of the culture medium to 6.4 with 2.5mol/L food-grade sodium hydroxide solution, and sterilizing at 115 ℃ for 30 minutes.
The fermentation medium of the liquid fermentation tank comprises the following components in percentage by weight: soybean peptone 0.6%, glucose 2.5%, sucrose 0.5%, galacto-oligosaccharide 0.5%, yeast extract powder 1.2%, beef extract powder 0.8%, magnesium sulfate 0.02%, manganese sulfate 0.005%, sodium acetate 0.5%, dihydrogenphosphate 0.2%, Tween-800.1%, L-cysteine hydrochloride 0.05%, and the balanceWater, sterilized at 115 ℃ for 30 minutes. Adding 2.5mol/L food-grade sodium hydroxide solution before inoculation to adjust the pH value of the culture medium to 6.5, inoculating the triangular flask strain, and after the pH value of the fermentation solution is reduced by 0.5, adding food-grade sodium hydroxide solution to maintain the constant pH value of the strain solution to be 5.5 +/-0.1 and OD600Stopping increasing or increasing negatively, performing microscopic examination on Lactobacillus acidophilus, stopping fermentation. The cells were separated by filtration using a tube centrifuge at 13000rpm to obtain a bacterial sludge. The bacterial sludge is washed by sterile water for 1 time for standby.
The bacterial sludge protective agent comprises 1.0 percent of sodium glutamate, 0.1 percent of lactose, 8 percent of trehalose, 0.5 percent of sodium ascorbate, 12 percent of skim milk powder, tween-800.5 and the balance of water by weight percent, and is sterilized at the temperature of 115 ℃ for 30 minutes and then cooled to 5 ℃ for later use.
Mixing and emulsifying the bacterial sludge and the bacterial sludge protective agent according to the ratio of the viable bacterial sludge to the bacterial sludge protective agent being 1: 1. During emulsification, the rotation speed of an emulsification motor of the emulsification tank is 1200rpm, and the rotation speed of a low-speed stirring motor is 100 rpm. Emulsifying for 30min, and standing at 4 deg.C for 30 min.
Pre-freezing at-55 deg.C for 4 hr, vacuum freeze-drying at-35 deg.C under vacuum degree of 0.8pa for 46 hr to obtain high-activity freeze-dried powder of Lactobacillus acidophilus.
The operating environment conditions of the preparation of the liquid strain in the triangular flask, the separation of the fermentation liquor and the emulsification of the bacterial sludge are required to be carried out in a 10 ten thousand-level purification environment with the temperature lower than 25 ℃ and the humidity RH lower than 35 percent.
The viable count of the produced product is 2.2 multiplied by 1011cfu/g, sealed and vacuum-packed with double aluminium films, 1000g per bag or packed according to the requirements of customers.
Example 2:
inoculating the screened lactobacillus acidophilus strain into a triangular flask liquid culture medium, and then inoculating the lactobacillus acidophilus strain into a fermentation tank to be respectively cultured for 8 to 24 hours at 37 ℃ for step-by-step amplification fermentation culture, wherein the triangular flask liquid culture medium comprises the following components in percentage by weight: soybean peptone 1.0%, glucose 2.0%, yeast extract powder 1.0%, beef extract powder 1.2%, magnesium sulfate 0.02%, manganese sulfate 0.005%, sodium acetate 0.5%, diamine hydrogen citrate 0.2%, potassium dihydrogen phosphate 0.2%, tween-800.1%, L-cysteine hydrochloride 0.05%, and water in balance, adjusting the pH value of the culture medium to 6.4 with 2.5mol/L food-grade sodium hydroxide solution, and sterilizing at 115 ℃ for 30 minutes.
The fermentation medium of the liquid fermentation tank comprises the following components in percentage by weight: soybean peptone 1.0%, glucose 2.0%, sucrose 1.0%, galacto-oligosaccharide 0.5%, yeast extract powder 1.5%, beef extract powder 0.8%, magnesium sulfate 0.02%, manganese sulfate 0.005%, sodium acetate 0.5%, dihydrogenphosphate 0.2%, Tween-800.1%, L-cysteine hydrochloride 0.05%, and water in balance, and sterilizing at 115 deg.C for 30 min. Adding 2.5mol/L food-grade sodium hydroxide solution before inoculation to adjust the pH value of the culture medium to 6.5, inoculating the triangular flask strain, and after the pH value of the fermentation solution is reduced by 0.5, adding food-grade sodium hydroxide solution to maintain the constant pH value of the strain solution to be 5.5 +/-0.1 and OD600Stopping increasing or increasing negatively, performing microscopic examination on Lactobacillus acidophilus, stopping fermentation. The bacterial cells were separated by filtration using a 6500 rpm disk centrifuge to obtain bacterial sludge. The bacterial sludge is washed by sterile water for 2 times for standby.
The bacterial sludge protective agent comprises 2.0 percent of sodium glutamate, 0.5 percent of lactose, 10 percent of trehalose, 0.5 percent of sodium ascorbate, 15 percent of skim milk powder, 801.5 percent of tween and the balance of water by weight percent, and is sterilized at the temperature of 115 ℃ for 30 minutes and then cooled to 5 ℃ for standby.
Mixing and emulsifying the bacterial sludge and the bacterial sludge protective agent according to the ratio of the viable bacterial sludge to the bacterial sludge protective agent being 10: 1. During emulsification, the rotation speed of an emulsification motor of the emulsification tank is 2000rpm, and the rotation speed of a low-speed stirring motor is 80 rpm. Emulsifying for 30min, and standing at 4 deg.C for 50 min.
Pre-freezing at-45 deg.C for 6 hr, vacuum freeze-drying at-35 deg.C under vacuum degree of 1.2pa, and vacuum freeze-drying for 60 hr to obtain high-activity freeze-dried powder of Lactobacillus acidophilus.
The operating environment conditions of the preparation of the liquid strain in the triangular flask, the separation of the fermentation liquor and the emulsification of the bacterial sludge are required to be carried out in a 10 ten thousand-level purification environment with the temperature lower than 25 ℃ and the humidity RH lower than 35 percent.
The viable count of the produced product is 3.0 multiplied by 1011cfu/g, sealed and vacuum-packed with double aluminium films, 500g per bag or packed according to the requirements of customers.
The scope of the invention is not limited to the specific embodiments described, the specific embodiments provided are only examples for further illustrating the invention, and various modifications and substitutions of components can be easily made by those skilled in the art with reference to the description of the specification, and such modifications and substitutions without inventive work are also within the scope of the appended claims.
Claims (7)
1. A production method of freeze-dried powder of lactobacillus acidophilus comprises the following process flows of lactobacillus acidophilus fermentation culture, fermentation liquor separation, bacterial sludge emulsification and emulsion vacuum freeze-drying, and is characterized in that: the strain fermentation process comprises the steps of inoculating lactobacillus acidophilus strains into a triangular flask liquid culture medium for culture, wherein the weight percentage of the strains inoculated into the culture medium is 1.0-2.0%, and culturing for 16-24 hours at 35-40 ℃, and the triangular flask liquid culture medium comprises the following components in percentage by weight: soybean peptone 0.5-1.0%, glucose 2.0-3.0%, yeast extract 0.8-1.5%, beef extract 0.8-1.2%, magnesium sulfate 0.02%, manganese sulfate 0.005%, sodium acetate 0.5%, diamine hydrogen citrate 0.2%, potassium dihydrogen phosphate 0.2%, tween-800.1%, L-cysteine hydrochloride 0.05%, and water in balance, adjusting the pH value of the culture medium to 6.2-6.4 with 2.5mol/L food-grade sodium hydroxide solution, and sterilizing at 115 ℃ for 30 minutes; then inoculating the mixture into a liquid fermentation tank for fermentation culture, wherein the inoculation weight percentage in a culture medium is 5-10%, and culturing the mixture for 8-24 hours at 35-40 ℃, wherein the fermentation culture medium of the liquid fermentation tank comprises the following components in percentage by weight: soybean peptone 0.5-1.0%, glucose 2.0-3.0%, sucrose 0.5-1.0%, galacto-oligosaccharide 0.5%, yeast extract powder 0.8-1.5%, beef extract powder 0.8-1.2%, magnesium sulfate 0.02%, manganese sulfate 0.005%, sodium acetate 0.5%, citric acid hydrogen diamine 0.2%, potassium dihydrogen phosphate 0.2%, tween-800.1%, L-cysteine hydrochloride 0.05%, and water in balance, sterilizing at 115 ℃ for 30 minutes; adding 2.5mol/L food-grade sodium hydroxide solution before inoculation to adjust the pH value of the culture medium to 6.5, inoculating the triangular flask strain, and after the pH value of the fermentation solution is reduced by 0.5, adding food-grade sodium hydroxide solution to adjust and maintain the constant pH value of the bacteria solution to be 5.5 +/-0.1.
2. The method for producing freeze-dried powder of lactobacillus acidophilus according to claim 1, wherein the method comprises the following steps: the liquid fermentation tank is used for fermentation culture, OD600Stopping increasing or increasing negatively, performing microscopic examination on Lactobacillus acidophilus, stopping fermentation.
3. The method for producing freeze-dried powder of lactobacillus acidophilus according to claim 1, wherein the method comprises the following steps: the fermentation liquor separation method is to separate thalli by a tubular or disc centrifuge under the condition of 6000-13000 rpm, and to wash the thalli for 1-2 times by using sterile water to obtain bacterial sludge.
4. The method for producing freeze-dried powder of lactobacillus acidophilus according to claim 1, wherein the method comprises the following steps: the bacterial sludge emulsifying process is characterized in that bacterial sludge and a bacterial sludge protective agent are mixed and emulsified according to the proportion of 10-1: 1-2 of the viable bacterial sludge and the bacterial sludge protective agent, the components of the bacterial sludge protective agent formula comprise, by weight, 0.5-2.0% of sodium glutamate, 0.1-0.5% of lactose, 8-12% of trehalose, 0.5-1% of sodium ascorbate, 10-20% of skim milk powder, 78-2.0% of tween-800.5 and the balance of water, and the bacterial sludge protective agent is sterilized at 115 ℃ for 30 minutes and then cooled to 5-20 ℃ for later use.
5. The method for producing freeze-dried powder of lactobacillus acidophilus according to claim 1, wherein the method comprises the following steps: the bacterial sludge emulsification process comprises the steps of emulsifying and homogenizing bacterial sludge and a protective agent in an emulsifying tank, wherein the rotating speed of an emulsifying motor in the emulsifying tank is 1000-2000 rpm, the rotating speed of a low-speed stirring motor is 80-100 rpm, the emulsifying time is 20-30 min, and standing is carried out for 30-50 min at a low temperature of 4 ℃ after emulsification is finished.
6. The method for producing freeze-dried powder of lactobacillus acidophilus according to claim 1, wherein the method comprises the following steps: the emulsion vacuum freeze-drying process is characterized in that the drying conditions are as follows: pre-freezing for 4-8 hours at-55 to-45 ℃, and then carrying out vacuum freeze drying for 40-60 hours at the temperature of-35 ℃ and the vacuum degree of 0.5-1.5 pa; drying to obtain viable count greater than 2.0 × 1011cfu/g freeze-dried powder of Lactobacillus acidophilus.
7. A method for producing freeze-dried powder of Lactobacillus acidophilus according to claims 1, 3, 4 and 5, characterized in that: the operating environment conditions of the preparation of the liquid strain in the triangular flask, the separation of the fermentation liquor and the emulsification of the bacterial sludge are required to be carried out in a 10 ten thousand-level purification environment with the temperature lower than 25 ℃ and the humidity RH lower than 35%.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101486986A (en) * | 2009-02-12 | 2009-07-22 | 上海谱莱生物技术有限公司 | Preparation of freeze-dried Lactobacillus acidophilus powder |
| CN105639124A (en) * | 2016-01-13 | 2016-06-08 | 河北省科学院生物研究所 | Microcapsule feed additive, and preparation method and application thereof |
| CN106701642A (en) * | 2017-03-01 | 2017-05-24 | 汉臣氏（沈阳）儿童制品有限公司 | Preparation method of lactobacillus acidophilus freeze-dried powder |
| CN209735494U (en) * | 2019-01-29 | 2019-12-06 | 润盈生物工程（上海）有限公司 | Emulsification tank capable of stirring materials by utilizing gas |
| CN112322531A (en) * | 2020-11-05 | 2021-02-05 | 苏州微克生活科技有限公司 | Production method and application of high-activity lactobacillus acidophilus freeze-dried powder |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101486986A (en) * | 2009-02-12 | 2009-07-22 | 上海谱莱生物技术有限公司 | Preparation of freeze-dried Lactobacillus acidophilus powder |
| CN105639124A (en) * | 2016-01-13 | 2016-06-08 | 河北省科学院生物研究所 | Microcapsule feed additive, and preparation method and application thereof |
| CN106701642A (en) * | 2017-03-01 | 2017-05-24 | 汉臣氏（沈阳）儿童制品有限公司 | Preparation method of lactobacillus acidophilus freeze-dried powder |
| CN209735494U (en) * | 2019-01-29 | 2019-12-06 | 润盈生物工程（上海）有限公司 | Emulsification tank capable of stirring materials by utilizing gas |
| CN112322531A (en) * | 2020-11-05 | 2021-02-05 | 苏州微克生活科技有限公司 | Production method and application of high-activity lactobacillus acidophilus freeze-dried powder |
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