CN102329759B - Method for improving freeze-drying survival rate of Lactobacillus acidophilus - Google Patents
Method for improving freeze-drying survival rate of Lactobacillus acidophilus Download PDFInfo
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- CN102329759B CN102329759B CN201110310289.3A CN201110310289A CN102329759B CN 102329759 B CN102329759 B CN 102329759B CN 201110310289 A CN201110310289 A CN 201110310289A CN 102329759 B CN102329759 B CN 102329759B
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Abstract
The invention aims to provide a method for improving the freeze-drying survival rate of Lactobacillus acidophilus. The method comprises the following steps of: culturing the Lactobacillus acidophilus for 18h at the temperature of 37 DEG C, performing sublethal treatment on the Lactobacillus acidophilus, centrifuging for 10 to 15min at the temperature of 4 DEG C at the rotating speed of 7,000rpm, adding skimmed milk and a phosphate buffer solution into bacterial sludge of the Lactobacillus acidophilus, pre-freezing for 8 to 12h at the temperature of -40 DEG C, and putting into a freeze dryer and freeze-drying for 18 to 24h at the temperature of between -50 and -60 DEG C under the vacuum degree of between 4 and 6Pa to obtain bacterial powder of the Lactobacillus acidophilus. In the method for improving the freeze-drying survival rate of the Lactobacillus acidophilus, a Lactobacillus acidophilus culture solution is subjected to cold shock, heat shock or acid shock treatment first so as to ensure that the Lactobacillus acidophilus induces to synthesize cold shock protein and heat shock protein under adverse circumstances or changes the composition of fatty acid of cell membranes, so that the resistance of the Lactobacillus acidophilus in the subsequent freeze-drying process is improved, and the freeze-drying survival rate and the viable count of the bacterial powder are improved.
Description
Technical field
The invention belongs to Lactobacterium acidophilum production of articles technical field, relate to a kind of method that improves freeze-drying survival rate of Lactobacillus acidophilus.
Background technology
Lactobacterium acidophilum (Lactobacillus acidophilus) belongs to the lactobacillus in lactobacillaceae, and form is elongated rod-shaped, and cell size is generally 0.6～0.9*1.5～6.0 μ m, Cheng Dan, become two or 2-3 become short chain arrangement.Atrichia, does not move, Gram-positive, and amphimicrobian, is present in the gi tract of humans and animals natively, in people's oral cavity, vagina and traditional fermented-milk.Optimum growth temperature is 35 ℃～38 ℃, do not grow below for 20 ℃, and 43 ℃～48 ℃ of maximum growth temperatures, poor heat resistance, can utilize glucose, fructose, lactose, sucrose to carry out homofermentation and produce DL type lactic acid.
Lactobacterium acidophilum is one of probiotic bacterium conventional in milk-product and preparation thereof, it has the lactose intolerance of alleviation and irritable bowel syndrome, pre-anti-diarrhea or constipation, reconcile human 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, therefore prepared Bifidobacteria powder necessary as the raw material of starter or protective foods.
The preparation of Lactobacterium acidophilum bacterium powder at present mainly adopts vacuum freeze-drying method, but in freezing dry process, Lactobacterium acidophilum will be stood the effect of freezing and dry two kinds of fierce factors, causes thalline death.The domestic screening aspect that the research of freeze-dried Lactobacillus acidophilus powder is mainly concentrated on to lyophilized vaccine in preparation process at present, and obtained various frozen-dried protective agent prescriptions, but do not improve the method for freeze-drying survival rate of Lactobacillus acidophilus by sub-lethal processing.
Summary of the invention
The object of the present invention is to provide a kind of method that improves freeze-drying survival rate of Lactobacillus acidophilus, it is low that the method can solve the freeze-drying survival rate that Lactobacterium acidophilum exists in freeze-drying process, the problem that viable count is few.
For achieving the above object, the present invention has adopted following technical scheme:
Lactobacterium acidophilum is cultivated to 18h in 37 ℃ in MRS meat soup, after stopping cultivating, Lactobacterium acidophilum is carried out to sub-lethal processing, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 10-15min of 7000rpm, abandoning supernatant obtains Lactobacterium acidophilum bacterium mud, in Lactobacterium acidophilum bacterium mud, add after the 10-20% skimming milk of bacterium shale amount and the phosphoric acid buffer of bacterium shale amount 80-90%, pH6.8 pre-freeze 8-12h at-40 ℃, then be placed in freeze drier in temperature-50-60 ℃, vacuum tightness is freeze-drying 18-24h under 4-6Pa, obtains Lactobacterium acidophilum bacterium powder.
The total mass of described skimming milk and phosphoric acid buffer equates with the quality of Lactobacterium acidophilum bacterium mud.
The lethal processing in described Asia refers to cold shock.
Described cold shock refers to Lactobacterium acidophilum at 8-10 ℃ of standing 0.5-1.5h.
The lethal processing in described Asia refers to heat-shocked.
Described heat-shocked refers to Lactobacterium acidophilum at 38-42 ℃ of standing 1h.
The lethal processing in described Asia refers to acid shock.
Described acid shock refers to and regulates the pH of nutrient solution to make Lactobacterium acidophilum in 5-8 ℃ of standing 30min after 4.0-5.5 with sulfuric acid.
The method of raising freeze-drying survival rate of Lactobacillus acidophilus of the present invention is by the first lethal processing through Asia of Lactobacterium acidophilum, make Lactobacterium acidophilum in adverse circumstance, induce synthetic cold shock protein, heat shock protein(HSP) or change cell membrane fat acid composition, thereby improve the resistibility of Lactobacterium acidophilum in follow-up freeze-drying process, improve freeze-drying survival rate and bacterium powder viable count.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
Design of the present invention is such: Lactobacterium acidophilum nutrient solution is first processed through cold shock, heat-shocked or acid shock, make Lactobacterium acidophilum in adverse circumstance, induce synthetic cold shock protein, heat shock protein(HSP) or change cell membrane fat acid composition, thereby improve the resistibility of Lactobacterium acidophilum in follow-up freeze-drying process, improve freeze-drying survival rate and bacterium powder viable count.Concrete grammar is as follows: (1) is first by Lactobacterium acidophilum 37 ℃ of cultivation 18h in MRS meat soup, then stop cultivating, and by nutrient solution at 8-10 ℃ of standing 0.5-1.5h, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 10min-15min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, after bacterium mud adds skimming milk and phosphoric acid buffer at-40 ℃ pre-freeze 8-12h, be then placed in freeze drier and carry out freeze-drying, at temperature-50--60 ℃, vacuum tightness is freeze-drying 18-24h under 4-6Pa, can obtain Lactobacterium acidophilum bacterium powder.(2) first by Lactobacterium acidophilum 37 ℃ of cultivation 18h in MRS meat soup, then stop cultivating, and by nutrient solution at 38-42 ℃ of standing 1h, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 10min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, after bacterium mud adds skimming milk and phosphoric acid buffer at-40 ℃ pre-freeze 8-12h, be then placed in freeze drier and carry out freeze-drying, at temperature-50-60 ℃, vacuum tightness is freeze-drying 18-24h under 4-6Pa, can obtain Lactobacterium acidophilum bacterium powder.(3) first by Lactobacterium acidophilum 37 ℃ of cultivation 18h in MRS meat soup, then with sulfuric acid, the pH of nutrient solution is adjusted to 4.0-5.5,5-8 ℃ of standing 30min, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 10min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, add skimming milk and phosphoric acid buffer pre-freeze 8-12h at-40 ℃ at bacterium mud, be then placed in freeze drier and carry out freeze-drying, at temperature-50-60 ℃, vacuum tightness is freeze-drying 18-24h under 4-6Pa, can obtain Lactobacterium acidophilum bacterium powder.
Embodiment 1-1
By MRS meat soup sterilizing 15min at 118 ℃, be cooled to room temperature, then by 4% (V/V) inoculum size access Lactobacterium acidophilum, 37 ℃ of constant temperature culture 18h, then by nutrient solution at 9 ℃ of standing 1.0h, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 10min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, the bacterium mud of weighing, add the skimmed milk powder of bacterium mud weight 20% (W/W), add again the phosphoric acid buffer of the pH6.8 of bacterium mud weight 80% (W/W), then pre-freeze 8h at-40 ℃, then be placed in freeze drier and carry out freeze-drying, 4-6Pa, freeze-drying 24h at-50 ℃, can obtain Lactobacterium acidophilum bacterium powder, its freeze-drying survival rate is 70.45% (control group is 46.11%), viable count is 2.1 × 10
11((control group is 1.10 × 10 to cfu/g
11cfu/g)).
Embodiment 1-2
By MRS meat soup sterilizing 15min at 118 ℃, be cooled to room temperature, then by 4% (V/V) inoculum size access Lactobacterium acidophilum, 37 ℃ of constant temperature culture 18h, then by nutrient solution at 8 ℃ of standing 0.5h, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 15min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, the bacterium mud of weighing, add the skimmed milk powder of bacterium mud weight 10% (W/W), add again the phosphoric acid buffer of the pH6.8 of bacterium mud weight 90% (W/W), then pre-freeze 12h at-40 ℃, then be placed in freeze drier and carry out freeze-drying, 6Pa, freeze-drying 21h at-60 ℃, can obtain Lactobacterium acidophilum bacterium powder.
Embodiment 1-3
By MRS meat soup sterilizing 15min at 118 ℃, be cooled to room temperature, then by 4% (V/V) inoculum size access Lactobacterium acidophilum, 37 ℃ of constant temperature culture 18h, then by nutrient solution at 10 ℃ of standing 1.5h, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 11min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, the bacterium mud of weighing, add the skimmed milk powder of bacterium mud weight 16% (W/W), add again the phosphoric acid buffer of the pH6.8 of bacterium mud weight 84% (W/W), then pre-freeze 11h at-40 ℃, then be placed in freeze drier and carry out freeze-drying, 4Pa, freeze-drying 18h at-52 ℃, can obtain Lactobacterium acidophilum bacterium powder.
Embodiment 2-1
By MRS meat soup sterilizing 15min at 118 ℃, be cooled to room temperature, then by 4% (V/V) inoculum size access Lactobacterium acidophilum, 37 ℃ of constant temperature culture 18h, then by nutrient solution at 39 ℃ of standing 1.0h, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 10min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, the bacterium mud of weighing, add the skimmed milk powder of bacterium mud weight 15% (W/W), add again the phosphoric acid buffer of the pH6.8 of bacterium mud weight 85% (W/W), then pre-freeze 12h at-40 ℃, then be placed in freeze drier and carry out freeze-drying, 4-6Pa, freeze-drying 20h at-50 ℃, can obtain Lactobacterium acidophilum bacterium powder, its freeze-drying survival rate is 84.25% (control group is 49.64%), viable count is 2.61 × 10
11(control group is 1.13 × 10 to cfu/g
11cfu/g).
Embodiment 2-2
By MRS meat soup sterilizing 15min at 118 ℃, be cooled to room temperature, then by 4% (V/V) inoculum size access Lactobacterium acidophilum, 37 ℃ of constant temperature culture 18h, then by nutrient solution at 38 ℃ of standing 1.0h, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 13min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, the bacterium mud of weighing, add the skimmed milk powder of bacterium mud weight 20% (W/W), add again the phosphoric acid buffer of the pH6.8 of bacterium mud weight 80% (W/W), then pre-freeze 9h at-40 ℃, then be placed in freeze drier and carry out freeze-drying, 6Pa, freeze-drying 18h at-52 ℃, can obtain Lactobacterium acidophilum bacterium powder.
Embodiment 2-3
By MRS meat soup sterilizing 15min at 118 ℃, be cooled to room temperature, then by 4% (V/V) inoculum size access Lactobacterium acidophilum, 37 ℃ of constant temperature culture 18h, then by nutrient solution at 42 ℃ of standing 1.0h, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 15min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, the bacterium mud of weighing, add the skimmed milk powder of bacterium mud weight 10% (W/W), add again the phosphoric acid buffer of the pH6.8 of bacterium mud weight 90% (W/W), then pre-freeze 8h at-40 ℃, then be placed in freeze drier and carry out freeze-drying, 4Pa, freeze-drying 24h at-60 ℃, can obtain Lactobacterium acidophilum bacterium powder.
Embodiment 3-1
By MRS meat soup sterilizing 15min at 118 ℃, be cooled to room temperature, then by 4% (V/V) inoculum size access Lactobacterium acidophilum, 37 ℃ of constant temperature culture 18h, then with the sulfuric acid of 2mol/L, the pH of nutrient solution is adjusted to 4.5, 5 ℃ of standing 30min, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 10min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, the bacterium mud of weighing, add the skimmed milk powder of bacterium mud weight 10% (W/W), add again the phosphoric acid buffer of the pH6.8 of bacterium mud weight 90% (W/W), then pre-freeze 10h at-40 ℃, then be placed in freeze drier and carry out freeze-drying, 4-6Pa, freeze-drying 22h at-60 ℃, can obtain Lactobacterium acidophilum bacterium powder, its freeze-drying survival rate is 87.22% (contrast is 49.64%), viable count is 4.09 × 10
11(control group is 1.07 × 10 to cfu/g
11cfu/g).
Embodiment 3-2
By MRS meat soup sterilizing 15min at 118 ℃, be cooled to room temperature, then by 4% (V/V) inoculum size access Lactobacterium acidophilum, 37 ℃ of constant temperature culture 18h, then with the sulfuric acid of 2mol/L, the pH of nutrient solution is adjusted to 4.0, 7 ℃ of standing 30min, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 12min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, the bacterium mud of weighing, add the skimmed milk powder of bacterium mud weight 14% (W/W), add again the phosphoric acid buffer of the pH6.8 of bacterium mud weight 86% (W/W), then pre-freeze 8h at-40 ℃, then be placed in freeze drier and carry out freeze-drying, 4Pa, freeze-drying 24h at-50 ℃, can obtain Lactobacterium acidophilum bacterium powder.
Embodiment 3-3
By MRS meat soup sterilizing 15min at 118 ℃, be cooled to room temperature, then by 4% (V/V) inoculum size access Lactobacterium acidophilum, 37 ℃ of constant temperature culture 18h, then with the sulfuric acid of 2mol/L, the pH of nutrient solution is adjusted to 5.5, 8 ℃ of standing 30min, viable count is surveyed in sampling, then at 4 ℃, the centrifugal 15min of 7000rpm, abandoning supernatant, obtain Lactobacterium acidophilum bacterium mud, the bacterium mud of weighing, add the skimmed milk powder of bacterium mud weight 20% (W/W), add again the phosphoric acid buffer of the pH6.8 of bacterium mud weight 80% (W/W), then pre-freeze 12h at-40 ℃, then be placed in freeze drier and carry out freeze-drying, 6Pa, freeze-drying 18h at-53 ℃, can obtain Lactobacterium acidophilum bacterium powder.
In above-described embodiment, control group refers to that other condition is identical, does not just pass through sub-lethal processing.
Claims (1)
1. a method that improves freeze-drying survival rate of Lactobacillus acidophilus, is characterized in that: comprise the following steps:
Lactobacterium acidophilum is cultivated to 18h in 37 ℃ in MRS meat soup, after stopping cultivating, Lactobacterium acidophilum is carried out to sub-lethal processing, then at 4 ℃, the centrifugal 10-15min of 7000rpm, abandoning supernatant obtains Lactobacterium acidophilum bacterium mud, in Lactobacterium acidophilum bacterium mud, add 10-20% skimming milk and the bacterium shale amount 80-90% of bacterium shale amount, after the phosphoric acid buffer of pH6.8 at-40 ℃ pre-freeze 8-12h, then be placed in freeze drier in temperature-50--60 ℃, vacuum tightness is freeze-drying 18-24h under 4-6Pa, the lethal processing in described Asia refers to cold shock, described cold shock refers to Lactobacterium acidophilum at 8-10 ℃ of standing 0.5-1.5h, the total mass of described skimming milk and phosphoric acid buffer equates with the quality of Lactobacterium acidophilum bacterium mud.
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| CN102757923B (en) * | 2012-07-30 | 2014-04-02 | 陕西省科学院酶工程研究所 | Method for preparing bifidobacterium bifidum powder with high viable count |
| CN108935704A (en) * | 2018-07-16 | 2018-12-07 | 陕西科技大学 | A kind of probiotics goat milk powder and preparation method thereof |
| CN108935703A (en) * | 2018-07-16 | 2018-12-07 | 陕西科技大学 | A kind of compound probiotic goat milk powder and preparation method thereof |
| CN108935709A (en) * | 2018-07-16 | 2018-12-07 | 陕西科技大学 | A kind of probiotics goat milk piece and preparation method thereof |
| CN111575223B (en) * | 2020-05-20 | 2022-04-15 | 江南大学 | Method for reducing secretion of surface substances of lactobacillus rhamnosus |
| CN114686407B (en) * | 2022-05-13 | 2022-09-06 | 微康益生菌(苏州)股份有限公司 | Preparation method of lactobacillus acidophilus powder for improving culturable cell content |
| CN116396904A (en) * | 2023-04-04 | 2023-07-07 | 陕西科技大学 | Preparation of probiotics powder and application of probiotics powder in goat milk powder |
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