CN112899204A - Probiotic freeze-dried shell composite protective agent and application thereof - Google Patents
Probiotic freeze-dried shell composite protective agent and application thereof Download PDFInfo
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Abstract
The invention relates to a probiotic freeze-dried shell composite protective agent and application thereof, wherein the shell composite protective agent comprises the following components in percentage by weight: 0.7-1.8% of isolated whey protein powder, 0.5-1.5% of tyrosine, 0.7-1.3% of arginine, 0.1-1.1% of gellan gum, 0.2-1.0% of inulin, 0.2-0.8% of vitamin C, 0.1-0.5% of sodium alginate, 0.1-0.5% of gamma-polyglutamic acid and the balance of distilled water. The shell composite protective agent does not contain any chemical synthetic substance, so that the safety is high; the shell composite protective agent has very obvious protective effect on probiotics before and after a freeze drying process; the shell composite protective agent has wide application range, has good protective effect on lactobacillus plantarum and lactobacillus strains, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biological product preparation, and particularly relates to a probiotic freeze-dried shell composite protective agent and application thereof.
Background
Probiotics are widely studied and applied because of their long shelf life and high viable count. The freeze-drying process generally causes the change of the permeability of cell membranes of the probiotics, the denaturation and inactivation of proteins, the destruction of pH value, DNA damage, the change of the composition of membrane fatty acid and the like, thereby leading to the mass death of the probiotics in the freeze-drying process product. Therefore, in order to protect the activity of the probiotics during the production process of the probiotic preparation, in addition to controlling the freeze-drying process, it is most critical to select a suitable freeze-drying protective agent for the probiotics as the shell protective agent.
Chinese patent CN 103333840A discloses a probiotic ultralow temperature freezing process, a full-salt cryoprotectant formula suitable for the process and probiotic ultralow temperature frozen particles prepared by the probiotic ultralow temperature freezing process. The probiotic ultralow temperature freezing process comprises the following steps: (1) preparing a fermentation culture medium; (2) culturing probiotics; (3) centrifuging; (4) preparing a protective agent; (5) adding a protective agent; (6) and (5) quick-freezing and forming. Which provides a probiotic preparation prepared by using the probiotic ultralow-temperature frozen particles and a preparation method thereof. The probiotic ultralow-temperature frozen particles are probiotic frozen strains, do not need drying and dewatering, and have the advantages of high freezing speed, high viable bacteria rate, good rehydration performance and the like.
Many studies have found that the addition of suitable substances before the freeze-drying of lactic acid bacteria can play a protective role, and the key to the success of freeze-drying of most probiotics lies in the use of a shell protective agent. The existing shell protective agent has a certain protective effect on the activity of probiotics, but still causes certain damage to cells during freeze drying or rehydration, and the loss of living cells of the probiotics in the freeze drying process is large, so that various original physiological and biochemical characteristics and biological activities cannot be well maintained.
In order to improve the survival rate of cells of probiotics in a freeze drying process, Chinese patent CN 108676721A discloses a composite protective agent for probiotic low-temperature freeze drying, which comprises the following components in percentage by weight: 3.5-5.5% of rhamnolipid, 1.5-2.2% of malonaldehyde, 1.0-1.6% of glutamic acid, 0.8-1.2% of tyrosine, 2.0-4.0% of 7-decene-4-lactone, 0.8-1.5% of disodium hydrogen phosphate, 0.8-1.5% of sodium dihydrogen phosphate, 0.3-0.5% of vitamin C and the balance of distilled water. After being mixed with probiotic solid, the protective agent can greatly improve the survival rate of probiotics, thereby having wide application prospect. But the malondialdehyde in the raw material composition selected by the method has cytotoxicity and potential carcinogenicity, and the use safety is still to be further improved.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a probiotic freeze-dried shell composite protective agent and application thereof. The shell composite protective agent used in the invention can change the physical and chemical environment of the biological sample during freeze drying, reduce or prevent the damage of freeze drying or rehydration to cells, and keep various original physiological and biochemical characteristics and biological activity as far as possible; and no chemical synthetic substance is added into the shell composite protective agent, so that the safety is higher.
In order to achieve the purpose, the invention adopts the technical scheme that:
the probiotic freeze-dried shell composite protective agent comprises the following components in percentage by weight: 0.7-1.8% of isolated whey protein powder, 0.5-1.5% of tyrosine, 0.7-1.3% of arginine, 0.1-1.1% of gellan gum, 0.2-1.0% of inulin, 0.2-0.8% of vitamin C, 0.1-0.5% of sodium alginate, 0.1-0.5% of gamma-polyglutamic acid and the balance of distilled water.
The invention well overcomes the technical defect of great loss of living cells of probiotics in the freeze drying process in the prior art, and each component in the shell composite protective agent has obvious protective effect on improving the survival rate of the probiotics in the freeze drying process, and has better component safety and wide application prospect.
Preferably, the weight ratio of the separated whey protein powder to the inulin is 3: 1. the shell composite protective agent obtained under the control of the weight ratio has the best protective effect on probiotic cells.
Preferably, the composite protective agent consists of the following components in percentage by weight: 1.5% of isolated whey protein powder, 1.0% of tyrosine, 1.0% of arginine, 0.6% of gellan gum, 0.5% of inulin, 0.5% of vitamin C, 0.2% of sodium alginate, 0.2% of gamma-polyglutamic acid and the balance of distilled water. With this formulation ratio, the cell loss rate of the probiotic cells in the freeze-drying process is minimal.
The invention also provides application of the shell composite protective agent in a probiotic freeze-drying process.
Preferably, the probiotic freeze-drying process comprises the following steps:
(1) controlling the temperature of probiotic fermentation liquor to be 9-18 ℃, the centrifugal rotating speed to be 15000-20000 rpm, and the centrifugal time to be 10-30 mins, removing supernatant, preliminarily dehydrating, and collecting dehydrated probiotic bacteria;
(2) and (3) uniformly mixing the shell composite protective agent with the obtained dehydrated probiotic bacteria, and putting the mixture into a vacuum freeze dryer for freeze drying.
Preferably, the freeze-drying in the step (2) is specifically as follows: and reducing the temperature to-30 to-40 ℃ at a cooling speed of 10 ℃ per half hour, maintaining the vacuum degree at 150-200 Pa for 8-12 hours, increasing the temperature to 30 ℃ at a heating speed of 8 ℃ per half hour, controlling the vacuum degree at 100-150 Pa, and continuously drying for 8 hours, namely completing the freeze drying.
Preferably, before the probiotic preparation is freeze-dried, the shell composite protective agent and the dehydrated probiotic thallus are mixed according to the mass ratio of 1-5: 1, and mixing.
Preferably, before freeze-drying the probiotic preparation, the shell is compounded with the protective agent and the dehydrated probiotic bacteria according to the ratio of 3: 1, and mixing.
Preferably, the probiotic fermentation broth is a fermentation broth of lactobacillus plantarum, a fermentation broth of lactobacillus acidophilus, or a cell fermentation broth of lactobacillus fermentum and lactobacillus paracasei.
The invention is matched with a probiotic freeze-dried shell composite protective agent with a certain proportion, and viable bacteria freeze-dried powder (probiotic freeze-dried preparation) is obtained by freeze drying of fermentation liquor of lactobacillus plantarum, fermentation liquor of lactobacillus acidophilus and cell fermentation liquor of lactobacillus fermentum and lactobacillus paracasei. The probiotic freeze-dried preparation has the advantages of stable quality, small dosage, easy carrying, administration and storage, no addition of any chemical synthetic substance, high safety, unique function and good stability.
The invention has the beneficial effects that:
(1) the shell composite protective agent does not contain any chemical synthetic substance, so that the safety is high;
(2) the composite protective agent for the shell can reduce or prevent the damage of freeze drying or rehydration on cells, keeps various original physiological and biochemical characteristics and biological activities as far as possible, and has very obvious protective effect on probiotics before and after a freeze drying process;
(3) the shell composite protective agent has wide application range, has good protective effect on lactobacillus plantarum and lactobacillus strains, and has wide application prospect.
Detailed Description
The invention is further illustrated by the following examples, without limiting the scope of the invention.
The raw materials selected by the invention are purchased from the market unless specified otherwise.
The probiotic fermentation broth is a fermentation broth of lactobacillus plantarum, a fermentation broth of lactobacillus acidophilus, or a cell fermentation broth of lactobacillus fermentum and lactobacillus paracasei.
Example 1
A composite protective agent for probiotic low-temperature freeze drying is composed of the following components in percentage by weight: 1.5% of isolated whey protein powder, 1.0% of tyrosine, 1.0% of arginine, 0.6% of gellan gum, 0.5% of inulin, 0.5% of vitamin C, 0.2% of sodium alginate, 0.2% of gamma-polyglutamic acid and the balance of distilled water.
The freeze drying process comprises the following steps:
(1) controlling the temperature of the probiotic fermentation liquor to be 12 ℃, the centrifugal speed to be 18000rpm, and the centrifugal time to be 30mins, removing supernatant, preliminarily dehydrating, and collecting probiotic thalli;
(2) and (3) mixing the composite protective agent for freeze drying with dehydrated probiotic bacteria 3: 1 proportion, placing the mixture into a vacuum freeze dryer for freeze drying, reducing the temperature to-33 ℃ at the temperature reduction speed of 10 ℃ per half hour, maintaining the vacuum degree at 190Pa for 10 hours, raising the temperature to 30 ℃ at the temperature rise speed of 8 ℃ per half hour, controlling the vacuum degree at 140Pa, and continuing to dry for 8 hours, namely completing the freeze drying.
Example 2
A composite protective agent for probiotic low-temperature freeze drying is composed of the following components in percentage by weight: 1.1% of isolated whey protein powder, 0.5% of tyrosine, 0.7% of arginine, 0.3% of gellan gum, 0.2% of inulin, 0.4% of vitamin C, 0.3% of sodium alginate, 0.3% of gamma-polyglutamic acid and the balance of distilled water.
The freeze drying process comprises the following steps:
(1) controlling the temperature of the probiotic fermentation liquor to be 15 ℃, the centrifugal speed to be 19000rpm, the centrifugal time to be 30mins, removing supernatant, preliminarily dehydrating, and collecting probiotic thalli;
(2) and (2) mixing the composite protective agent for freeze drying and dehydrated probiotic bacteria 2.2: 1 proportion, placing the mixture into a vacuum freeze dryer for freeze drying, reducing the temperature to-40 ℃ at the temperature reduction speed of 10 ℃ per half hour, maintaining the vacuum degree at 180Pa for 10 hours, increasing the temperature to 30 ℃ at the temperature increase speed of 8 ℃ per half hour, controlling the vacuum degree at 140Pa, and continuing to dry for 8 hours, namely completing the freeze drying.
Example 3
A composite protective agent for probiotic low-temperature freeze drying is composed of the following components in percentage by weight: 1.1% of isolated whey protein powder, 0.5% of tyrosine, 0.7% of arginine, 0.3% of gellan gum, 0.2% of inulin, 0.4% of vitamin C, 0.3% of sodium alginate, 0.2% of gamma-polyglutamic acid and the balance of distilled water.
The freeze drying process comprises the following steps:
(1) controlling the temperature of the probiotic fermentation liquor to be 10 ℃, the centrifugal speed to be 20000rpm, centrifuging for 20mins, removing supernatant, preliminarily dehydrating, and collecting probiotic bacteria;
(2) and (3) mixing the composite protective agent for freeze drying and dehydrated probiotic bacteria 3.7: 1 proportion, placing the mixture into a vacuum freeze dryer for freeze drying, reducing the temperature to-30 ℃ at the temperature reduction speed of 10 ℃ per half hour, maintaining the vacuum degree at 200Pa for 10 hours, increasing the temperature to 30 ℃ at the temperature increase speed of 8 ℃ per half hour, controlling the vacuum degree at 150Pa, and continuing to dry for 8 hours, namely completing the freeze drying.
Example 4
A composite protective agent for probiotic low-temperature freeze drying is composed of the following components in percentage by weight: 1.3% of isolated whey protein powder, 0.5% of tyrosine, 0.7% of arginine, 0.3% of gellan gum, 0.5% of inulin, 0.4% of vitamin C, 0.3% of sodium alginate, 0.2% of gamma-polyglutamic acid and the balance of distilled water.
The freeze drying process comprises the following steps:
(1) controlling the temperature of the probiotic fermentation liquor to be 12 ℃, the centrifugal speed to be 18000rpm, and the centrifugal time to be 30mins, removing supernatant, preliminarily dehydrating, and collecting probiotic thalli;
(2) and (2) mixing the composite protective agent for freeze drying and dehydrated probiotic bacteria 2.6: 1 proportion, placing the mixture into a vacuum freeze dryer for freeze drying, reducing the temperature to-33 ℃ at the temperature reduction speed of 10 ℃ per half hour, maintaining the vacuum degree at 190Pa for 10 hours, raising the temperature to 30 ℃ at the temperature rise speed of 8 ℃ per half hour, controlling the vacuum degree at 140Pa, and continuing to dry for 8 hours, namely completing the freeze drying.
Example 5
A composite protective agent for probiotic low-temperature freeze drying is composed of the following components in percentage by weight: 1.3% of isolated whey protein powder, 0.5% of tyrosine, 0.7% of arginine, 0.3% of gellan gum, 0.5% of inulin, 0.4% of vitamin C, 0.3% of sodium alginate, 0.3% of gamma-polyglutamic acid and the balance of distilled water.
The freeze drying process comprises the following steps:
(1) controlling the temperature of the probiotic fermentation liquor at 18 ℃, the centrifugal speed at 15000rpm, the centrifugal time for 10mins, removing supernatant, preliminarily dehydrating, and collecting probiotic thalli;
(2) and (2) mixing the composite protective agent for freeze drying and dehydrated probiotic bacteria 2.8: 1 proportion, placing the mixture into a vacuum freeze dryer for freeze drying, reducing the temperature to-35 ℃ at the temperature reduction speed of 10 ℃ per half hour, maintaining the vacuum degree at 150Pa for 10 hours, increasing the temperature to 30 ℃ at the temperature increase speed of 8 ℃ per half hour, controlling the vacuum degree at 100Pa, and continuing to dry for 8 hours, namely completing the freeze drying.
Example 6
A composite protective agent for probiotic low-temperature freeze drying is composed of the following components in percentage by weight: 1.2% of isolated whey protein powder, 0.5% of tyrosine, 0.7% of arginine, 0.3% of gellan gum, 0.2% of inulin, 0.4% of vitamin C, 0.4% of sodium alginate, 0.3% of gamma-polyglutamic acid and the balance of distilled water.
The freeze drying process comprises the following steps:
(1) controlling the temperature of the probiotic fermentation liquor to be 12 ℃, the centrifugal speed to be 18000rpm, and the centrifugal time to be 30mins, removing supernatant, preliminarily dehydrating, and collecting probiotic thalli;
(2) and (2) mixing the composite protective agent for freeze drying and dehydrated probiotic bacteria 2.4: 1 proportion, placing the mixture into a vacuum freeze dryer for freeze drying, reducing the temperature to-33 ℃ at the temperature reduction speed of 10 ℃ per half hour, maintaining the vacuum degree at 190Pa for 10 hours, raising the temperature to 30 ℃ at the temperature rise speed of 8 ℃ per half hour, controlling the vacuum degree at 140Pa, and continuing to dry for 8 hours, namely completing the freeze drying.
Example 7
A composite protective agent for probiotic low-temperature freeze drying is composed of the following components in percentage by weight: 1.1% of isolated whey protein powder, 0.5% of tyrosine, 0.7% of arginine, 0.3% of gellan gum, 0.2% of inulin, 0.4% of vitamin C, 0.4% of sodium alginate, 0.3% of gamma-polyglutamic acid and the balance of distilled water.
The freeze drying process comprises the following steps:
(1) controlling the temperature of the probiotic fermentation liquor to be 12 ℃, the centrifugal speed to be 18000rpm, and the centrifugal time to be 30mins, removing supernatant, preliminarily dehydrating, and collecting probiotic thalli;
(2) and (2) mixing the composite protective agent for freeze drying and dehydrated probiotic bacteria 2.3: 1 proportion, placing the mixture into a vacuum freeze dryer for freeze drying, reducing the temperature to-33 ℃ at the temperature reduction speed of 10 ℃ per half hour, maintaining the vacuum degree at 190Pa for 10 hours, raising the temperature to 30 ℃ at the temperature rise speed of 8 ℃ per half hour, controlling the vacuum degree at 140Pa, and continuing to dry for 8 hours, namely completing the freeze drying.
Comparative example 1
A composite protective agent for probiotic low-temperature freeze drying is composed of the following components in percentage by weight: 2.0% of isolated whey protein powder, 1.7% of tyrosine, 1.5% of arginine, 1.3% of gellan gum, 1.2% of inulin, 0.9% of vitamin C, 0.7% of sodium alginate, 0.6% of gamma-polyglutamic acid and the balance of distilled water. The freeze-drying process was the same as in example 1.
Comparative example 2
The freeze-drying was carried out by the same procedure as in example 1 without adding the composite protectant for freeze-drying.
According to the process, a sample 1, a sample 2 and a sample 3 are respectively processed, and the quantity change of probiotics before and after freezing is respectively measured, wherein the sample 1 is fermentation liquor of lactobacillus plantarum, the sample 2 is fermentation liquor of lactobacillus acidophilus, and the sample 3 is cell fermentation liquor of lactobacillus fermentum and lactobacillus paracasei. The colony count is carried out according to the method described in the food safety national standard food microbiology test lactobacillus test of GB 4789.35-2010, and the survival rate is calculated, wherein the survival rate is the ratio of the total number of the freeze-dried bacteria to the total number of the bacteria before freeze-drying. The measurement results (the protective effect of the composite protective agent for the shell on probiotics before and after the freeze-drying process) are shown in table 1.
TABLE 1
| Sample 1 survival (%) | Sample 2 survival (%) | Sample 3 survival (%) | |
| Example 1 | 93.2 | 94.7 | 96.9 |
| Example 2 | 83.1 | 85.8 | 76.6 |
| Example 3 | 80.8 | 85.2 | 73.9 |
| Example 4 | 89.9 | 90.8 | 93.6 |
| Example 5 | 89.5 | 80.1 | 87.7 |
| Example 6 | 84.2 | 85.7 | 86.8 |
| Example 7 | 82.7 | 84.8 | 85.1 |
| Comparative example 1 | 63.7 | 67.8 | 65.4 |
| Comparative example 2 | 23.1 | 20.9 | 24.2 |
As can be seen from the table above, the composite protective agent for the shell has very obvious protective effects on probiotics before and after the freeze drying process, and has very obvious difference compared with the process without adding any protective agent. As can be seen from the different examples, the dosage ratio of the components, the control of the process conditions and the like have obvious influence on the effect of the shell compound protective agent, wherein the protective effect of the shell compound protective agent described in example 1 is the best.
Claims (9)
1. The probiotic freeze-dried shell composite protective agent is characterized by comprising the following components in percentage by weight: 0.7-1.8% of isolated whey protein powder, 0.5-1.5% of tyrosine, 0.7-1.3% of arginine, 0.1-1.1% of gellan gum, 0.2-1.0% of inulin, 0.2-0.8% of vitamin C, 0.1-0.5% of sodium alginate, 0.1-0.5% of gamma-polyglutamic acid and the balance of distilled water.
2. The probiotic freeze-dried shell composite protective agent according to claim 1, characterized in that: the weight ratio of the separated whey protein powder to the inulin is 3: 1.
3. the probiotic freeze-dried shell composite protective agent according to claim 1, characterized in that: the shell composite protective agent comprises the following components in percentage by weight: 1.5% of isolated whey protein powder, 1.0% of tyrosine, 1.0% of arginine, 0.6% of gellan gum, 0.5% of inulin, 0.5% of vitamin C, 0.2% of sodium alginate, 0.2% of gamma-polyglutamic acid and the balance of distilled water.
4. Use of the shell composition protecting agent as claimed in any one of claims 1 to 3 in a probiotic freeze-drying process.
5. Use according to claim 4, characterized in that: the probiotic freeze-drying process comprises the following steps:
(1) controlling the temperature of probiotic fermentation liquor to be 9-18 ℃, the centrifugal rotating speed to be 15000-20000 rpm, and the centrifugal time to be 10-30 mins, removing supernatant, preliminarily dehydrating, and collecting dehydrated probiotic bacteria;
(2) and (3) uniformly mixing the shell composite protective agent and the dehydrated probiotic bacteria, and putting the mixture into a vacuum freeze dryer for freeze drying.
6. Use according to claim 5, characterized in that the lyophilization in step (2) is in particular: and reducing the temperature to-30 to-40 ℃ at a cooling speed of 10 ℃ per half hour, maintaining the vacuum degree at 150-200 Pa for 8-12 hours, increasing the temperature to 30 ℃ at a heating speed of 8 ℃ per half hour, controlling the vacuum degree at 100-150 Pa, and continuously drying for 8 hours, namely completing the freeze drying.
7. Use according to claim 5, characterized in that: the shell composite protective agent and the dehydrated probiotic bacteria according to the mass ratio of 1-5: mixing at a ratio of 1.
8. Use according to claim 7, characterized in that: before freeze drying of the probiotic preparation, the shell composite protective agent and dehydrated probiotic thallus are mixed according to the ratio of 3: 1, and mixing.
9. Use according to claim 5, characterized in that: the probiotic fermentation liquid is fermentation liquid of lactobacillus plantarum, fermentation liquid of lactobacillus acidophilus or cell fermentation liquid of lactobacillus fermentum and lactobacillus paracasei.
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