CN108018317A - A kind of method for improving Lactococcus lactis resistance and the application in bacterium powder preparation - Google Patents

Abstract

The invention discloses a method for improving the stress resistance of Lactococcus lactis and its application in the preparation of bacterial powder, belonging to the technical field of food. The invention uses non-water-soluble dietary fiber as a solid phase carrier to ferment and produce Lactococcus lactis biofilm cells; after fermentation, the bacteria sludge is collected and mixed with a drying protective agent, and dried to prepare Lactococcus lactis bacteria powder. Lactococcus lactis using the method of the invention has a high drying survival rate, the freeze-drying survival rate can reach more than 85%, the vacuum-drying survival rate can reach more than 35%, the spray-drying survival rate can reach more than 40%, and the number of live bacteria in the bacterial powder can reach More than 5×10 ¹⁰ cfu/g. Compared with the dry survival rate of Lactococcus lactis in the non-biofilm state, it has increased by more than 10 times. The invention solves the key common technical problem of developing highly active Lactococcus lactis bacteria powder, and the prepared Lactococcus lactis bacteria powder can be applied to product forms such as starters and solid beverages.

Description

A method for improving the stress resistance of Lactococcus lactis and its application in the preparation of bacterial powder

technical field

The invention relates to a method for improving the stress resistance of Lactococcus lactis and its application in the preparation of bacterial powder, belonging to the technical field of food.

Background technique

Lactococcus lactis mainly includes Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. It is a typical dairy starter culture, mainly used in the production of cheese, butter and buttermilk, and has a long history of use. Lactococcus lactis can ferment lactose in milk to form lactic acid, and then produce flavor substances such as diacetyl and acetaldehyde. During the cheese maturation process, under the action of protease and peptidase, casein can be hydrolyzed into oligopeptides and amino acids. It plays an important role in the formation of mature cheese flavor substances.

The preparation of Lactococcus lactis bacteria powder usually adopts vacuum freeze-drying technology, and the survival rate of the freeze-dried strains can be improved by optimizing the formula of the protective agent. The problem greatly increases the preparation cost of Lactococcus lactis powder. Compared with freeze-drying, vacuum drying and spray drying have the advantages of simple operation, low equipment requirements, and low production cost. However, vacuum drying and spraying also have the disadvantage of being unable to prepare high-survival bacterial powder. Therefore, it is particularly important to improve the dry survival rate of Lactococcus lactis. Important, how to improve the dry survival rate of Lactococcus lactis to the greatest extent is the key limiting factor for the preparation of Lactococcus lactis starter.

Patent CN103396962A and other methods to improve the drying survival rate of Lactococcus lactis mostly focus on the research on the formula of the protective agent, and optimize the formula of the drying protective agent through the screening of sugar, protein, salt and other substances to improve the drying survival rate. The optimization of drying protectant formula, the improvement of drying process conditions, and the application of microencapsulation technology can improve the drying survival rate of Lactococcus lactis to a certain extent, but the improvement degree of the above-mentioned methods is limited, and the operation steps are cumbersome, which is not suitable for large-scale production. industrial applications. Compared with the study of protective agent formula, improving the stress resistance of Lactobacillus is the fundamental way to improve the dry survival rate of Lactococcus lactis.

Contents of the invention

The technical problem to be solved in the present invention is to overcome the low dry survival rate of Lactococcus lactis and provide a production process for Lactococcus lactis powder with high activity and strong stress resistance, which can significantly improve the dry survival rate and storage stability of Lactococcus lactis, A suitable preparation strategy for Lactococcus lactis powder is provided.

The first object of the present invention is to provide a preparation method of Lactococcus lactis bacteria powder, which is to prepare Lactococcus lactis biofilm first, and then freeze-dry, vacuum-dry or spray-dry the Lactococcus lactis biofilm.

In one embodiment of the present invention, the biofilm is an organized bacterial group formed by Lactococcus lactis cells.

In one embodiment of the present invention, the Lactococcus lactis bacterial film is obtained by culturing in a medium containing water-insoluble dietary fiber.

In one embodiment of the present invention, the water-insoluble dietary fiber is cellulose, lignin or hemifiber.

In one embodiment of the present invention, the water-insoluble dietary fiber includes one or more of cereal powder, bean powder, fruit and vegetable powder, mushroom powder and dietary homologous powder.

In one embodiment of the present invention, the water-insoluble dietary fiber includes one of oat bran powder, wheat bran powder, corn bran powder, walnut shell powder, kudzu root powder, Hericium erinaceus powder, fungus powder, and Jerusalem artichoke powder. one or more species.

In one embodiment of the present invention, the content of the water-insoluble dietary fiber is 10-30 g/L.

In one embodiment of the present invention, the medium contains: yeast powder 5-8g/L, beef extract 5-8g/L, anhydrous glucose 10-15g/L, tryptone 5-8g/L, soybean Peptone 5-8g/L, magnesium sulfate 0.2-0.3g/L, sodium glycerophosphate 15-25g/L, ascorbic acid 0.5-1.0g/L, water-insoluble dietary fiber 10-30g/L.

In one embodiment of the present invention, the method includes the following steps: (1) inoculating Lactococcus lactis into a medium containing water-insoluble dietary fiber; Cultivate at ℃ for 18-36 hours; (3) collect the bacteria by centrifugation; (4) add the drying protective agent to the bacteria slime according to the mass ratio of the bacteria slime: drying protective agent of 1:1-3, and mix well; (5) combine the step The mixture of (4) is freeze-dried, vacuum-dried or spray-dried.

In one embodiment of the present invention, the medium in the step (1) contains: yeast powder 5g/L, beef extract 5g/L, anhydrous glucose 10g/L, tryptone 5g/L, soybean peptone 5g /L, magnesium sulfate 0.2g/L, sodium glycerophosphate 20g/L, ascorbic acid 0.5g/L, soybean powder 10g/L.

In one embodiment of the present invention, the step (2) activates Lactococcus lactis for 3 generations, and transfers it to a 10L fermenter at an inoculation ratio of 3-5% by volume, with a constant pH of 30 degrees (pH = 6.5) Culture for 24 hours.

In one embodiment of the present invention, the step (3) collects the bacteria sludge by centrifuging the Lactococcus lactis biofilm fermentation broth at 4°C, the centrifugation conditions are: the rotation speed is 6000rpm/min, the centrifugation time is 10 minutes, Obtain Lactococcus lactis sludge.

In one embodiment of the present invention, the formulation of the dry protection agent in the step (4) is: the formulation of the protection agent is: at least 80-120 g/L skim milk powder, sodium caseinate, soybean protein One, at least one of 20-40g/L trehalose and sucrose, at least one of 0-0.5g/L cysteine hydrochloride, and vitamin C, with a pH of 6.5.

In one embodiment of the present invention, the step (5) is to spread the emulsion obtained in the step (4) on a flat plate, pre-freeze at -38～-40°C for 3～4h, and finally freeze the emulsion at 4～6Pa Freeze-dry for 25-35 hours.

In one embodiment of the present invention, the step (5) is to spread the emulsion obtained in the step (4) on a plate, and vacuum-dry at -0.1 MPa for 18-24 hours at room temperature.

In one embodiment of the present invention, the step (5) is to dry the bacterial suspension obtained in the step (4) through a spray dryer at a speed of 5ml/min, the inlet temperature is 130-160°C, and the outlet temperature It is 70-90°C.

The second object of the present invention is to provide the bacteria powder prepared by applying the method.

The third object of the present invention is to provide the application of the bacteria powder in the food field, which includes the use of Lactococcus lactis biofilm bacteria powder for fermentation to produce dairy products.

Beneficial effects: the present invention applies biofilm technology to the fermentation production of Lactococcus lactis, and uses non-water-soluble dietary fiber as a solid interface to form a biofilm on the solid interface to achieve the effect of improving its own stress resistance, thereby Significantly improve the drying survival rate of Lactococcus lactis, the survival rate of freeze-drying can reach more than 85%, the survival rate of vacuum drying can reach more than 35%, the survival rate of spray-drying can reach more than 40%, and the number of viable bacteria powder can reach 5×10 ¹⁰ More than cfu/g. The key technical problems in the development of highly active Lactococcus lactis powder are solved. The highly active and highly stress-resistant Lactococcus lactis powder prepared by the invention can be used in products such as solid beverages and starters.

Detailed ways

1. Determination of the number of viable bacteria: After gradient dilution of the sample to be tested, take 100 microliters and spread it on a solid plate, and after culturing for 36 hours, calculate the number of viable bacteria, in cfu/mL.

2. Calculation method of drying survival rate: measure the number of viable bacteria before drying as the initial bacterial concentration, after drying, redissolve the bacterial powder with sterile distilled water to the volume before drying, measure the number of viable bacteria as the final concentration of bacterial concentration, and calculate the drying survival rate.

Survival rate = 100% × final concentration / initial bacterial concentration

3. The bacterial powder is stored at 25°C for 12 weeks. The calculation method of the survival rate is: after vacuum drying, the bacterial powder is redissolved in sterile distilled water to the volume before vacuum drying, and the number of viable bacteria is determined as the initial bacterial concentration; Store at ℃ for 12 weeks, redissolve the bacteria powder with sterile distilled water to the volume before vacuum drying, and measure the number of viable bacteria as the final concentration.

Survival rate = 100% × final concentration / initial bacterial concentration

Embodiment 1: Freeze-drying of Lactococcus lactis

(1) Preparation of Lactococcus lactis membrane fermentation medium: yeast powder 5g/L, beef extract 5g/L, anhydrous glucose 10g/L, tryptone 5g/L, soybean peptone 5g/L, magnesium sulfate 0.2g/L , sodium glycerophosphate 20g/L, ascorbic acid 0.5g/L, and the amount of insoluble fiber added is shown in Table 1.

(2) Fermentation preparation of Lactococcus lactis bacterial film: Activate Lactococcus lactis for 3 generations, transfer it to a 10L fermenter according to the inoculation ratio of 3-5%, adjust the rotation speed, and cultivate at a constant pH (pH=6) at 30 degrees 24 hours.

(3) Collection of Lactococcus lactis membranes: Centrifuge the fermentation liquid of Lactococcus lactis bacteria membranes at 4°C to collect the bacteria sludge. The centrifugation conditions are: the rotation speed is 6000rpm/min, and the centrifugation time is 10 minutes to obtain Lactococcus lactis bacteria mud.

(4) Add drying protective agent: Mix the obtained bacteria slime and drying protective agent according to the weight-to-volume ratio (1:1). The formula of the above drying protectant is: 100g/L skimmed milk powder, 30g/L trehalose, 30g/L sucrose, 0.5g/L cysteine hydrochloride, and the pH is 6.5.

(5) Freeze-drying: pre-freeze the bacterial suspension obtained in step (4) at -38-40° C. for 3-4 hours, and finally freeze-dry at 4-6 Pa for 25-35 hours. The Lactococcus lactis powder can be obtained after freeze-drying.

Table 1 Effect of non-water-soluble fiber on the freeze-drying survival rate of Lactococcus lactis

According to Table 1, it can be seen that the amount of soybean powder added has a great influence on the freeze-dried survival rate of Lactococcus lactis. When the amount of soybean powder added is 10-30g/L, the freeze-dried survival rate of Lactococcus lactis is higher than 85%. , The survival rate of 12 weeks stored at 25°C was more than 60%, and the freeze-dried survival rate of Lactococcus lactis was low when no soybean powder was added, or when the soybean powder was added in a small amount.

Example 2

Follow the same steps as in Example 1, the difference is that soybean powder and oat bran powder are replaced by kudzu root powder and walnut shell powder to carry out bacterial film fermentation. The freeze-dried survival rate of the bacterial film was 81%, and the viable bacterial count of the bacterial powder was 6×10 ¹⁰ cfu/g. The bacterial powder was stored at 25 degrees for 12 weeks, and the survival rate was 60%.

Example 3

Follow the same steps as in Example 1, except that the freeze-drying protective agent is replaced by: 120g/L skimmed milk powder, 20g/L trehalose, 20g/L sucrose, 0.5g/L cysteine hydrochloride, The pH is 6.5. The freeze-drying survival rate of the pellicled Lactococcus lactis is 83%, which is significantly higher than the freeze-drying survivability rate of the bacterial cells in the non-bacteria state. The bacteria powder was stored at 25 degrees for 12 weeks, and the survival rate was 51%.

Embodiment 4: the vacuum drying of Lactococcus lactis

(1) Preparation of Lactococcus lactis film fermentation medium:

Yeast powder 5g/L, beef extract 8g/L, anhydrous glucose 10g/L, tryptone 4g/L, soybean peptone 6g/L, magnesium sulfate 0.25g/L, sodium glycerophosphate 25g/L, ascorbic acid 0.5g/L , soybean powder 10g/L, oat bran powder 10g/L.

(2) Fermentation preparation of Lactococcus lactis bacterial film: Activate Lactococcus lactis for 3 generations, transfer it to a 10L fermenter according to the inoculation ratio of 3-5%, adjust the rotation speed, and cultivate at a constant pH (pH=6.5) at 30 degrees 24 hours.

(3) Collection of Lactococcus lactis membranes: Centrifuge the fermentation liquid of Lactococcus lactis bacteria membranes at 4°C to collect the bacteria sludge. The centrifugation conditions are: the rotation speed is 6000rpm/min, and the centrifugation time is 10 minutes to obtain Lactococcus lactis bacteria mud.

(4) Add drying protective agent: Mix the obtained bacteria slime and drying protective agent according to the weight-to-volume ratio (1:1). The formula of the above drying protectant is: 100g/L soybean protein, 30g/L trehalose, 30g/L sucrose, 0.5g/L cysteine hydrochloride, and the pH is 6.5.

(5) Vacuum drying: vacuum-dry the emulsion obtained in step (4), the vacuum drying condition is: spread the obtained emulsion on a flat plate, and vacuum-dry at -0.1MPa at room temperature for 18-24h. Lactococcus lactis bacteria powder can be obtained after vacuum drying.

After testing, the vacuum-dried survival rate of the biofilmed Lactococcus lactis was 37%, and the number of viable bacteria in the bacterial powder was 4×10 ¹⁰ cfu/g. The bacteria powder was stored at 25 degrees for 12 weeks, and the survival rate was 49%.

Example 5

The bacterial powder was prepared in the same steps as in Example 4, the only difference being that soybean powder and oat bran powder were not added to the fermentation medium, and the rest of the conditions were the same as in Example 4. No bacteria film was formed during the fermentation process, and the vacuum-dried survival rate of the control group was less than 5% after testing. The bacteria powder was stored at 25 degrees for 12 weeks, and the survival rate was 29%.

Example 6

Follow the same steps as in Example 4, the difference is that fungus powder is used instead of soybean powder and oat bran powder to ferment the bacterial film, and the results of vacuum drying show that the vacuum-dried survival rate of the bacterial film formed by using fungus powder as a solid medium is 48%. %, which was significantly higher than the vacuum-dried survival rate of bacteria in the non-bacteria film state. The bacterial powder was stored at 25 degrees for 12 weeks, and the survival rate was 58%.

Example 7

Follow the same steps as in Example 4, except that the vacuum drying protective agent is replaced with: 120g/L skimmed milk powder, 20g/L trehalose, 20g/L sucrose, 0.5g/L vitamin C, and the pH is 6.5. The vacuum-drying survival rate of bacteria-filmed Lactococcus lactis is 49%, which is significantly higher than the vacuum-drying survival rate of bacteria in the non-biofilm state. The bacterial powder was stored at 25 degrees for 12 weeks, and the survival rate was 61%.

Embodiment 8: the spray drying of Lactococcus lactis

(1) Preparation of Lactococcus lactis membrane fermentation medium: yeast powder 6g/L, beef extract 7g/L, anhydrous glucose 10g/L, tryptone 5g/L, soybean peptone 5g/L, magnesium sulfate 0.3g/L , sodium glycerophosphate 20g/L, ascorbic acid 0.5g/L, soybean powder 10g/L.

(2) Fermentation preparation of Lactococcus lactis bacterial film: Activate Lactococcus lactis for 3 generations, transfer it to a 10L fermenter according to the inoculation ratio of 3-5%, adjust the rotation speed, and cultivate at a constant pH (pH=6) at 30 degrees 24 hours.

(3) Collection of Lactococcus lactis membranes: Centrifuge the fermentation liquid of Lactococcus lactis bacteria membranes at 4°C to collect the bacteria sludge. The centrifugation conditions are: the rotation speed is 6000rpm/min, and the centrifugation time is 10 minutes to obtain Lactococcus lactis bacteria mud.

(4) Add drying protective agent: Mix the obtained bacteria slime and drying protective agent according to the weight-to-volume ratio (1:1). The formulation of the above drying protectant is: 100g/L sodium caseinate, 30g/L trehalose, 30g/L sucrose, 0.5g/L cysteine hydrochloride, and the pH is 6.5.

(5) Spray drying: spray-dry the emulsion obtained in step (3), the spray-drying conditions are: the bacterial suspension is dried by a spray dryer at a speed of 5ml/min, the inlet temperature is 150°C, and the outlet temperature is 80°C . Lactococcus lactis bacteria powder can be obtained after spray drying.

After testing, the spray-dried survival rate of the bacteria-filmed Lactococcus lactis was 39%, and the number of live bacteria in the bacterial powder was 4×10 ¹⁰ cfu/g. The bacterial powder was stored at 25 degrees for 12 weeks, and the survival rate was 61%.

Example 8

The bacterial powder was prepared according to the same steps as in Example 7, the only difference being that soybean powder was not added to the fermentation medium, and the rest of the conditions were the same as in Example 7. The results showed that no biofilm was formed during the fermentation process, and the survival rate of Lactococcus lactis after spray drying was less than 4%. The bacteria powder was stored at 25 degrees for 12 weeks, and the survival rate was 27%.

Example 9

According to Example 7, corn bran powder was used to replace soybean powder for bacterial film fermentation, and the spray drying results showed that the spray-drying survival rate of the bacterial film formed with corn bran powder as a solid medium was 35%, which was significantly higher than that of the non-bacterial film state. Bacteria spray-dried survival rate. The bacteria powder was stored at 25 degrees for 12 weeks, and the survival rate was 55%.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. a kind of preparation method of Lactococcus lactis bacterium powder, it is characterised in that in the culture medium containing Water insoluble dietary fiber Middle culture Lactococcus lactis is to forming biomembrane, then Lactococcus lactis biomembrane is freeze-dried, is dried in vacuo and is sprayed It is dry.

2. according to the method described in claim 1, it is characterized in that, the Water insoluble dietary fiber includes cellulose, wooden Element and hemicellulose.

3. according to the method described in claim 2, it is characterized in that, the Water insoluble dietary fiber is included in food Grain dust, bean powder, fruit vegetable powder, the one or more of mushroom powder and meals homogenous foodstuff pulvis.

4. according to the method described in claim 1, it is characterized in that, the Water insoluble dietary fiber includes oat bran powder, Wheat bran, corn chaff powder, walnut shell powder, kudzu-vine root powder, hedgehog hydnum mushroom powder, auricularia auriculajudae powder, the one or more of jerusalem artichoke powder.

5. any methods of 1-4 are wanted according to right, it is characterised in that the content of the Water insoluble dietary fiber for 10~ 30g/L。

6. according to any methods of claim 1-4, it is characterised in that include the following steps：1) Lactococcus lactis is connect Kind is into the culture medium of the Water insoluble dietary fiber Han 10~30g/L；(2) in 6.0~6.5,28~30 DEG C of Anaerobic culturels 20 of pH ~30h；(3) bacterium mud is collected by centrifugation；(4) bacterium mud is pressed：Drying protectant is 1:1~3 mass ratio adds dry guarantor into bacterium mud Agent is protected, is mixed；(5) mixture of step (4) is freeze-dried, is dried in vacuo and is spray-dried.

7. the method according to right wants 1, it is characterised in that the protective agent agent prescription in the step (4) is：80~ At least one of 120g/L skimmed milk powders, casein sodium, soybean protein, at least one in 20~40g/L trehaloses, sucrose Kind, at least one of 0~0.5g/L cysteine hydrochlorides, vitamin C.

8. claim 1-4, bacterium powder prepared by 7 any the methods.

9. bacterium powder described in claim 8 prepares solid beverage, dairy products, probiotics, probiotics tablets in food, biological field Application in agent, probiotics capsule.

10. food or drink containing bacterium powder described in claim 8.