CN102358884B - Bifidobacterium bifidum lyophilization composite protection agent and application method thereof - Google Patents
Bifidobacterium bifidum lyophilization composite protection agent and application method thereof Download PDFInfo
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- CN102358884B CN102358884B CN 201110310363 CN201110310363A CN102358884B CN 102358884 B CN102358884 B CN 102358884B CN 201110310363 CN201110310363 CN 201110310363 CN 201110310363 A CN201110310363 A CN 201110310363A CN 102358884 B CN102358884 B CN 102358884B
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Abstract
The invention provides a bifidobacterium bifidum lyophilization composite protection agent and application method thereof. A formula of the bifidobacterium bifidum lyophilization composite protection agent is composed of xylooligosaccharide of 3.0 to 5.0 g, synanthrin of 6.0 to 8.0 g, stachyose of 5.0 to 7.0 g, glycine 2.0 to 4.0 g, arginine of 2.0 to 4.0 g, sodium bicarbonate of 0.4 to 0.6 g and sodium ascorbate of 0.7 to1.2 g. the composite protection agent is utilized to lyophilize bifidobacterium bifidum, lyophilization survival reaches more than 85%, and viable count of bacterium powder is larger than 2.0 * 1011 cfu/g. Prebiotic of the xylooligosaccharide, synanthrin and stachyose is added in the composite protection agent so as to further improving a health protection function of the bacterium powder of the bifidobacterium bifidum. The composite protection agent resolves the important key technology problems in processes of develop high activity bacterium powder of the bifidobacterium bifidum. The prepared high activity bacterium powder can serve as addition agents and leavening agents to be used for developing functionality food and pharmaceutical preparation of the bifidobacterium bifidum.
Description
Technical field
The invention belongs to bifidus bacillus production of articles technical field, relate to a kind of freeze-drying composite protectant and application method thereof that improves bifidus bacillus freeze-drying survival rate.
Background technology
Genus bifidobacterium Gram-positive, anaerobism, do not move, nonspore-bearing bacillus, its dyeing is irregular, catalase is negative, form is changeable, often presents the variforms such as crooked rod, F or G shape because cell age and growing environment are different, and bacterium colony is smooth, dome, the edge is complete.That has found at present has 30 kinds approximately, comprises bifidumbacterium bifidum (B.bifidum), bifidobacteria infantis (B.infantis), bifidobacterium adolescentis (B.adolescentis), bifidus longum bb (B.longum) and bifidobacterium breve (B.breve) etc.
Bifidus bacillus is one of probiotic bacterium commonly used in milk-product and the preparation thereof, it has the lactose intolerance of alleviation and irritable bowel syndrome, prevention diarrhoea or constipation, the middleman puies forward the intestinal microflora balance, reduce blood cholesterol, enhancing body immunizing power suppresses the functions such as tumour generation, be widely used at present the production of functional dairy product, protective foods and probiotics, it is necessary as the raw material of starter or protective foods therefore to prepare Bifidobacteria powder.
The preparation method of Bifidobacteria powder mainly contains spray drying technology, vacuum drying technique and Vacuum Freezing ﹠ Drying Technology.Wherein spray drying technology unit product thermal losses is large, and the thermo-efficiency of equipment is low; Inlet temperature and air outlet temperature are high, cause the thalline survival rate low, if reduce temperature, then go out easily damp powder; Tripping device for the bacterium powder is had relatively high expectations, and easily makes product loss and pollution; But spray-dired treatment capacity is large, and cost is low.And vacuum drying technique need to dispose vacuum system, the vacuum dynamic seal structure of drive disk assembly also need to be set at import and export on the Large Scale and Continuous production unit, and this structural element easily causes leakage, and maximum problem is that treatment capacity is little in addition, cost is high, is only applicable to high value-added product.The survival rate of the viable bacteria that these two kinds of methods obtain is very low, does not reach cryodesiccated level, and the bacterium powder rehydration that obtains simultaneously is lower.By contrast, Vacuum Freezing ﹠ Drying Technology has the unrivaled advantage of other drying meanss: bacterial classification is dry at low temperatures, and the loss of activity of various compositions, enzyme is few; Form, the color and luster that can keep bacterial classification; Rehydration is good, can absorb water soon to be reduced into dry front fresh and alive state; Long preservative period stores transportation, sells conveniently.
But in the vacuum lyophilization process; bifidus bacillus will be stood the effect of freezing and dry two kinds of fierce factors; cause thalline dead; affect in the factor of its freeze-drying survival rate and bacterium powder viable count; lyophilized vaccine is the most complicated, the key factor of difficult selection; it not only affects the cell survival rate of bifidus bacillus in freeze-drying process, also affects the cell stability between preservation term.
A kind of composite protectant of high activity bifidobacteria dry powder formulation is disclosed among the ZL200610012689.5, its prescription consists of beta-cyclodextrin 10g, lactose 2-5g, trehalose 0.5-2.0g, yeast extract paste 0.5-2.0g, vitamin-E 0.05-0.1g and distilled water 100ml; A kind of composite protectant of freeze dried fermenting preparation of directly thrown bifidobacterium is also disclosed among the ZL200610012690.8, its prescription consists of skimmed milk powder 10g, lactose 3-6g, trehalose 1.0-2.0g, tween 80 0.05-1.0g, yeast extract paste 0.5-1.0g, serum 0.5-1g, vitamin-E 0.05-0.2g and distilled water 100ml; A kind of bifidobacteria viable bacteria preparation and special-purpose protecting agent thereof are also disclosed among the ZL200710006529.4; its prescription consists of glycerine 0.01-5.0 part, polyvinylpyrrolidone 0.1-6.0 part, polyoxyethylene glycol 0.1-5.0 part, skim-milk 1.0-10.0 part, trehalose 0.5-15.0 part, Sodium Glutamate 0.1-5.0 part, L-Aspartic acid 0.1-5.0 part, sodium ascorbate 0.1-5.0 part, ornithine hydrochloride 0.1-5.0 part.All added trehalose in above-mentioned three protective materials; the water-absorbent of trehalose is very strong; the easy moisture absorption of bacterium powder; cause in the storage process dead speed fast; next two protective materials have also added skimmed milk powder; the bacterium powder of producing is restricted such as the application of the aspects such as medicament in non-dairy product, in addition, does not all add the prebiotics composition in three protective materials.
Summary of the invention
The object of the present invention is to provide a kind of freeze-drying composite protectant and application method thereof that can significantly improve bifidumbacterium bifidum freeze-drying survival rate and bacterium powder viable count; it is low to have solved the freeze-drying survival rate that bifidumbacterium bifidum bacterium powder exists in freeze-drying process; viable count is few; easily suction becomes the problems such as tide, has improved the nourishing function of bacterium powder.
In order to achieve the above object, the present invention has adopted following technical scheme:
A kind of bifidumbacterium bifidum freeze-drying composite protectant, the prescription of this composite protectant forms and comprises: 3.0-5.0g xylo-oligosaccharide, 6.0-8.0g synanthrin, 5.0-7.0g stachyose, 2.0-4.0g glycine, 2.0-4.0g arginine, 0.4-0.6g sodium bicarbonate and 0.7-1.2g sodium ascorbate.
The prescription of described composite protectant forms and comprises: 3.5g xylo-oligosaccharide, 6.5g synanthrin, 5.5g stachyose, 2.8g glycine, 2.2g arginine, 0.4g sodium bicarbonate and 1.2g sodium ascorbate.
The prescription of described composite protectant forms and comprises: 5.0g xylo-oligosaccharide, 8.0g synanthrin, 6.0g stachyose, 3.0g glycine, 4.0g arginine, 0.5g sodium bicarbonate and 0.8g sodium ascorbate.
Described composite protectant adopts the following steps preparation:
(1) glycine, arginine and the sodium ascorbate that takes by weighing is dissolved in 20mL 0.85% stroke-physiological saline solution, then gets solution A with 0.22 μ m membrane filtration degerming;
(2) xylo-oligosaccharide, synanthrin, stachyose and sodium bicarbonate are dissolved in the 80mL distilled water, then in 100-115 ℃ of sterilization 10-20min, are cooled to room temperature after the sterilization and get solution B;
(3) solution A and solution B mixing is for subsequent use.
The application method of above-mentioned bifidumbacterium bifidum freeze-drying composite protectant may further comprise the steps: bifidumbacterium bifidum bacterium mud and the described composite protectant equal-volume of centrifugal collection are mixed; then in cryogenic refrigerator in-40 ℃ of pre-freeze 3-12h; then put into Freeze Drying Equipment in temperature-50--60 ℃; vacuum tightness is lyophilize 18-24h under the 4-6Pa, namely gets Bifidobacteria powder.
The preparation method of described bifidumbacterium bifidum bacterium mud is: bifidumbacterium bifidum activation three times is accessed in the MRS substratum that contains the 0.5g/L cysteine hydrochloride by the inoculum size of 3% (V/V) afterwards cultivate 18h, then the centrifugal 10min of 7000r/min gets bifidumbacterium bifidum bacterium mud, with 0.85% stroke-physiological saline solution bifidumbacterium bifidum bacterium mud viable count is adjusted to (0.8-2.0) * 10
11Cfu/mL.
Bifidumbacterium bifidum freeze-drying composite protectant of the present invention be with xylo-oligosaccharide, synanthrin, stachyose, glycine, arginine, sodium bicarbonate and sodium ascorbate proportionally organic assembling form; utilize this composite protectant that bifidumbacterium bifidum is carried out lyophilize; the freeze-drying survival rate reaches more than 85%, and bacterium powder viable count is greater than 2.0 * 10
11Cfu/g; solved the key technology problem in exploitation high reactivity bifidumbacterium bifidum freeze-dried vaccine powder process; the high reactivity bacterium powder of preparation can be used as additive, starter is used for exploitation bifidumbacterium bifidum functional foodstuff and pharmaceutical preparation; bifidumbacterium bifidum freeze-drying composite protectant of the present invention does not contain skimmed milk powder and trehalose; and the prebioticses such as xylo-oligosaccharide, synanthrin and stachyose have been added; solved the problems such as easy suction change tide that bifidumbacterium bifidum bacterium powder exists in freeze-drying process, the simultaneously interpolation of prebiotics has further improved the nourishing function of bacterium powder.
Embodiment
The present invention will be further described below in conjunction with embodiment.
Embodiment 1
Accurately take by weighing 3.5g xylo-oligosaccharide, 6.5g synanthrin, 5.5g stachyose and 0.4g sodium bicarbonate, add in the 80mL distilled water, heating for dissolving and mix after in 110 ℃ of sterilization 15min, be cooled to room temperature.Accurately taking by weighing 2.8g glycine, 2.2g arginine and 1.2g sodium ascorbate is dissolved in 20ml 0.85% stroke-physiological saline solution again; carry out filtration sterilization with the aseptic filter membrane of 0.22 μ m again; aseptic technique mixes both in Bechtop at last, namely gets freeze-drying composite protectant.
Behind activated three times of bifidumbacterium bifidum, it is contained by the access of inoculum size of 3% (V/V) in the MRS substratum of 0.5g/L cysteine hydrochloride cultivate 18h, then the centrifugal 10min of 7000r/min, obtain bifidumbacterium bifidum bacterium mud, with 0.85% stroke-physiological saline solution bifidumbacterium bifidum bacterium mud viable count is adjusted to 2.0 * 10
11Cfu/mL; equal-volume adds above-mentioned freeze-drying composite protectant; in cryogenic refrigerator in-40 ℃ of pre-freeze 9h; then put into Freeze Drying Equipment in-52 ℃; vacuum tightness is to carry out lyophilize 20h under the 4-6Pa; namely get bifidumbacterium bifidum bacterium powder, its freeze-drying survival rate can reach 85.15%, and bacterium powder viable count is 2.87 * 10
11Cfu/g.
Embodiment 2
Accurately take by weighing 5.0g xylo-oligosaccharide, 8.0g synanthrin, 6.0g stachyose and 0.5g sodium bicarbonate, add in the 80mL distilled water, heating for dissolving and mix after in 110 ℃ of sterilization 15min, be cooled to room temperature.Accurately taking by weighing 3.0g glycine, 4.0g arginine and 0.8g sodium ascorbate is dissolved in 20ml 0.85% stroke-physiological saline solution again; carry out filtration sterilization with the aseptic filter membrane of 0.22 μ m again; aseptic technique mixes both in Bechtop at last, namely gets freeze-drying composite protectant.
Behind activated three times of bifidumbacterium bifidum, it is contained by the access of inoculum size of 3% (V/V) in the MRS substratum of 0.5g/L cysteine hydrochloride cultivate 18h, then the centrifugal 10min of 7000r/min, obtain bifidumbacterium bifidum bacterium mud, with 0.85% stroke-physiological saline solution bifidumbacterium bifidum bacterium mud viable count is adjusted to 1.2 * 10
11Cfu/mL, equal-volume add above-mentioned freeze-drying composite protectant, in cryogenic refrigerator in-40 ℃ of pre-freeze 6h; then put into Freeze Drying Equipment in-60 ℃, vacuum tightness is to carry out lyophilize 18h under the 6Pa, namely gets bifidumbacterium bifidum bacterium powder; its freeze-drying survival rate is 89.70%, and bacterium powder viable count is 2.45 * 10
11Cfu/g.
Embodiment 3
Accurately take by weighing 3.0g xylo-oligosaccharide, 6.0g synanthrin, 7.0g stachyose and 0.6g sodium bicarbonate, add in the 80mL distilled water, heating for dissolving and mix after in 100 ℃ of sterilization 20min, be cooled to room temperature.Accurately taking by weighing 2.0g glycine, 2.0g arginine and 0.7g sodium ascorbate is dissolved in 20ml 0.85% stroke-physiological saline solution again; carry out filtration sterilization with the aseptic filter membrane of 0.22 μ m again; aseptic technique mixes both in Bechtop at last, namely gets freeze-drying composite protectant.
Behind activated three times of bifidumbacterium bifidum, it is contained by the access of inoculum size of 3% (V/V) in the MRS substratum of 0.5g/L cysteine hydrochloride cultivate 18h, then the centrifugal 10min of 7000r/min, obtain bifidumbacterium bifidum bacterium mud, with 0.85% stroke-physiological saline solution bifidumbacterium bifidum bacterium mud viable count is adjusted to 1.0 * 10
11Cfu/mL, equal-volume add above-mentioned freeze-drying composite protectant, in cryogenic refrigerator in-40 ℃ of pre-freeze 3h; then put into Freeze Drying Equipment in-50 ℃, vacuum tightness is to carry out lyophilize 24h under the 4Pa, namely gets bifidumbacterium bifidum bacterium powder; its freeze-drying survival rate reaches more than 85%, and bacterium powder viable count is greater than 2.0 * 10
11Cfu/g.
Embodiment 4
Accurately take by weighing 3.0g xylo-oligosaccharide, 6.0g synanthrin, 5.0g stachyose and 0.6g sodium bicarbonate, add in the 80mL distilled water, heating for dissolving and mix after in 115 ℃ of sterilization 10min, be cooled to room temperature.Accurately taking by weighing 4.0g glycine, 2.0g arginine and 0.7g sodium ascorbate is dissolved in 20ml 0.85% stroke-physiological saline solution again; carry out filtration sterilization with the aseptic filter membrane of 0.22 μ m again; aseptic technique mixes both in Bechtop at last, namely gets freeze-drying composite protectant.
Behind activated three times of bifidumbacterium bifidum, it is contained by the access of inoculum size of 3% (V/V) in the MRS substratum of 0.5g/L cysteine hydrochloride cultivate 18h, then the centrifugal 10min of 7000r/min, obtain bifidumbacterium bifidum bacterium mud, with 0.85% stroke-physiological saline solution bifidumbacterium bifidum bacterium mud viable count is adjusted to 0.8 * 10
11Cfu/mL, equal-volume add above-mentioned freeze-drying composite protectant, in cryogenic refrigerator in-40 ℃ of pre-freeze 12h; then put into Freeze Drying Equipment in-53 ℃, vacuum tightness is to carry out lyophilize 24h under the 5Pa, namely gets bifidumbacterium bifidum bacterium powder; its freeze-drying survival rate reaches more than 85%, and bacterium powder viable count is greater than 2.0 * 10
11Cfu/g.
Claims (4)
1. bifidumbacterium bifidum freeze-drying composite protectant, it is characterized in that: the prescription of this composite protectant consists of: 3.0-5.0g xylo-oligosaccharide, 6.0-8.0g synanthrin, 5.0-7.0g stachyose, 2.0-4.0g glycine, 2.0-4.0g arginine, 0.4-0.6g sodium bicarbonate and 0.7-1.2g sodium ascorbate;
Described composite protectant adopts the following steps preparation:
(1) glycine, arginine and sodium ascorbate are dissolved in the 20mL stroke-physiological saline solution, then get solution A with 0.22 μ m membrane filtration degerming;
(2) xylo-oligosaccharide, synanthrin, stachyose and sodium bicarbonate are dissolved in the 80mL distilled water, then in 100-115 ℃ of sterilization 10-20min, are cooled to room temperature after the sterilization and get solution B;
(3) solution A and solution B mixing is for subsequent use.
2. described a kind of bifidumbacterium bifidum freeze-drying composite protectant according to claim 1, it is characterized in that: the prescription of described composite protectant consists of: 3.5g xylo-oligosaccharide, 6.5g synanthrin, 5.5g stachyose, 2.8g glycine, 2.2g arginine, 0.4g sodium bicarbonate and 1.2g sodium ascorbate.
3. described a kind of bifidumbacterium bifidum freeze-drying composite protectant according to claim 1, it is characterized in that: the prescription of described composite protectant consists of: 5.0g xylo-oligosaccharide, 8.0g synanthrin, 6.0g stachyose, 3.0g glycine, 4.0g arginine, 0.5g sodium bicarbonate and 0.8g sodium ascorbate.
4. application method of bifidumbacterium bifidum freeze-drying composite protectant as claimed in claim 1, it is characterized in that: may further comprise the steps: bifidumbacterium bifidum bacterium mud and described composite protectant equal-volume are mixed, then in cryogenic refrigerator in-40 ℃ of pre-freeze 3-12h, then put into Freeze Drying Equipment in-50 ~-60 ℃ of lyophilize 18-24h;
The preparation method of described bifidumbacterium bifidum bacterium mud is: the by volume inoculum size access of per-cent 3% after the bifidumbacterium bifidum activation three times is contained in the MRS substratum of 0.5g/L cysteine hydrochloride and cultivate 18h, then the centrifugal 10min of 7000r/min gets bifidumbacterium bifidum bacterium mud, with stroke-physiological saline solution bifidumbacterium bifidum bacterium mud viable count is adjusted to (0.8-2.0) * 10
11Cfu/mL.
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| CN105012155A (en) * | 2015-08-13 | 2015-11-04 | 山东龙力生物科技股份有限公司 | Composition of arginine and xylo-oligosaccharide and application thereof |
| CN107475117A (en) * | 2017-07-03 | 2017-12-15 | 桐乡恒基生物股份有限公司 | A kind of freeze drying protectant for lactobacillus reuteri and lyophilized store method |
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| CN108546647A (en) * | 2018-04-26 | 2018-09-18 | 九江牧威利元科技中心（普通合伙） | A kind of Bifidobacterium lyophilized preparation and its special-purpose protecting agent |
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| CN111621443B (en) * | 2020-06-17 | 2022-07-22 | 江南大学 | Freeze-drying protective agent and application thereof in freeze-drying lactobacillus reuteri |
| CN111647510A (en) * | 2020-06-18 | 2020-09-11 | 科兴生物制药股份有限公司 | Bifidobacterium infantis freeze-dried powder, preparation method and composite protective agent used by same |
| CN112586609B (en) * | 2020-12-15 | 2022-09-27 | 浙江艾杰斯生物科技有限公司 | Lysozyme freeze-dried feed additive |
| CN113005040B (en) * | 2021-01-22 | 2023-07-25 | 武汉微康益生菌研究院有限公司 | Bifidobacterium lactis freeze-drying protective agent and application method thereof |
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| CN100400649C (en) * | 2006-05-12 | 2008-07-09 | 河北农业大学 | Compound protective agent of direct-vat-set bifidobacterium pure-breed freeze-dried leaven |
| CN101606552B (en) * | 2009-05-06 | 2013-09-11 | 任发政 | Bifidobacterium deep freezing direct vat starter culture and composite cryoprotectant thereof |
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