Preparation method of casein hydrolysate and application of casein hydrolysate in improvement of spray drying survival rate of lactic acid bacteria

Abstract

The invention discloses a preparation method of casein hydrolysate and application of the casein hydrolysate in improving the spray drying survival rate of lactic acid bacteria, and belongs to the technical field of lactic acid bacteria. According to the preparation method of the casein hydrolysate and the application of the casein hydrolysate in improving the survival rate of the lactic acid bacteria after spray drying disclosed by the invention, the survival rate of the lactic acid bacteria after spray drying is improved by more than 5 times by accurately controlling the enzymolysis time, the addition amount and molecular weight screening compared with a conventional process.

Description

Technical Field

[0001] The present invention relates to the technical field of lactic acid bacteria, and more specifically, to a preparation method of a casein hydrolysate and its application in improving the spray drying survival rate of lactic acid bacteria. Background Art

[0002] Lactic acid bacteria, as probiotics, are widely used in the fields of food, medicine and functional foods. Their preparation technology directly affects the viability of the bacteria and industrial applications. The current mainstream process is freeze-drying technology, which avoids thermal damage through low-temperature sublimation, and the survival rate can reach 60-80%. However, it has significant defects such as poor continuity, high cost and long cycle. Spray drying technology is regarded as an ideal alternative due to its low cost (only 15-20% of freeze-drying), continuous production and short-time high efficiency. However, its core bottleneck lies in the damage of the cell membrane structure and enzyme inactivation caused by high-temperature air flow, and the survival rate is generally lower than 5%.

[0003] At present, how to reduce the death and activity reduction of lactic acid bacteria caused by drying is the main technical difficulty in the preparation of highly active lactic acid bacteria starters, lactic acid bacteria powders and probiotic powders. Although casein can be used as a protective agent to improve the survival rate after drying, its protective effect is significantly affected by the hydrolysis time, addition amount and molecular weight distribution.

[0004] Therefore, providing a preparation method of a casein hydrolysate and its application in improving the spray drying survival rate of lactic acid bacteria is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a preparation method of a casein hydrolysate and its application in improving the spray drying survival rate of lactic acid bacteria. By optimizing the enzymatic hydrolysis conditions (hydrolysis time, addition amount and molecular weight parameters) of the casein hydrolysate, the spray drying survival rate and functional activity of lactic acid bacteria are significantly improved, and it is mainly used in the production of probiotic powders, lactic acid bacteria preparations and lactic acid bacteria powders.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A preparation method of a casein hydrolysate, the specific steps are as follows:

[0008] (1) For every 4 g of casein, add 0.02 g of sodium carbonate, dissolve in 80 ml of distilled water, add a rotor and dissolve at room temperature for 2 h to obtain a casein solution; preheat the casein solution at the optimal temperature of papain, 37°C, for 30 min and then adjust the pH to 6.0;

[0009] (2) Under the conditions of a temperature of 37 °C and a pH of 6.0, papain is added to the casein solution. The addition amount of papain is 3000 U / g of casein, and the enzymolysis time is 3 hours. The enzymolyzed solution is centrifuged to separate the supernatant, and then ultrafiltered and fractionated to retain the fraction with a molecular weight > 10,000 Da, resulting in a casein hydrolysate.

[0010] Furthermore, the casein hydrolysate prepared by the above method.

[0011] Furthermore, the application of the casein hydrolysate in improving the spray-drying survival rate of lactic acid bacteria.

[0012] Furthermore, the application of the casein hydrolysate in improving the spray-drying survival rate of lactic acid bacteria is as follows:

[0013] (1) Cultivate lactic acid bacteria in a medium until the late logarithmic phase and the early stationary phase;

[0014] (2) Centrifuge to collect the bacterial cells, and wash the bacterial cells twice with physiological saline or 0.01 M phosphate buffer; centrifuge again to collect the bacterial cells to obtain a bacterial cell paste.

[0015] (3) Using 30% w / v maltodextrin as the drying matrix, add 10% w / v of the casein hydrolysate, mix well, and then mix with the bacterial cell paste at a mass ratio of 5:1, and spray-dry to make a bacterial powder.

[0016] When the casein hydrolysate is used as a protective agent, the synergistic regulation of its enzymolysis time, molecular weight distribution, and addition amount plays a decisive role in the protective performance. By precisely controlling the enzymolysis time to 3 hours, the degree of hydrolysis and the formation efficiency of the protective layer can be balanced, avoiding the attenuation of the protective performance caused by over-hydrolysis in the conventional process; directionally screening casein fragments with a molecular weight > 10,000 Da, whose macromolecular structure forms a dense protective layer on the surface of the bacterial cells through hydrogen bonding, significantly reducing thermal damage; the addition amount is 10%, which not only ensures the complete coverage of the protective layer on the bacterial cells but also avoids the cost waste caused by excessive addition. In the prior art, although the casein hydrolysate has been used as a protective agent, problems such as too long enzymolysis time, broad molecular weight selection, and large randomness of the addition amount have led to significant fluctuations in the survival rate. Through the precise matching of the above parameters, the present invention has for the first time realized the systematic optimization of the protective performance of the casein hydrolysate, providing an efficient and stable solution for the spray-drying of lactic acid bacteria.

[0017] From the above technical solutions, compared with the prior art, the present invention discloses a preparation method of a casein hydrolysate and its application in improving the spray-drying survival rate of lactic acid bacteria. By precisely controlling the enzymolysis time, addition amount, and molecular weight screening, compared with adding an equal amount of unhydrolyzed casein, the survival rate of lactic acid bacteria after spray-drying is increased by more than 5 times. Detailed implementation methods

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0019] Example 1

[0020] By adjusting the enzymatic hydrolysis time, the protective effect of casein hydrolysate on Lactobacillus delbrueckii subsp. sp1.1 (L. bulgaricus sp1.1) was explored.

[0021] For every 4 g of casein, 0.02 g of sodium carbonate was added, dissolved in 80 ml of distilled water, and dissolved at room temperature for 2 h with a rotor to obtain a casein solution; the casein solution was preheated at the optimal temperature of papain, 37 °C, for 30 min and then the pH was adjusted to 6.0. Under the conditions of a temperature of 37 °C and a pH of 6.0, papain was added to the casein solution. The addition amount of papain (enzyme activity 50000 U / g) was 3000 U / g of casein, and hydrolysis was carried out at 37 °C for 0, 1.5 h, 3 h, 4 h, and 6 h respectively; after terminating the enzymatic hydrolysis, centrifugation was carried out (6000 r / min, 10 min), and the supernatant was taken as the protective agent.

[0022] L. bulgaricus sp1.1 after two generations of activation was inoculated into MRS at an inoculation amount of 2% (v / v). After culturing for 24 h, the cells were collected by centrifugation at 4 °C and 6000 r / min for 10 min. After washing the cells twice with physiological saline, the cells were collected again by centrifugation at 4 °C and 6000 r / min for 10 min to obtain cell sludge for standby.

[0023] A laboratory-scale spray drying device was used to prepare L. bulgaricus sp1.1 bacterial powder: 30% (w / v) maltodextrin of the feed liquid was used as the drying matrix, 10% (w / v) of the supernatant was added respectively, and after thorough mixing, it was mixed with the cell sludge according to a mass ratio of 5:1.

[0024] The spray drying conditions were as follows: 30% (w / v) maltodextrin of the feed liquid, inlet air volume: 380 m 3 / h, inlet air / outlet air temperature: 120 / 70 °C, atomization pressure 0.9 m 3 / h, and the feed rate was adjusted to make the inlet and outlet air temperatures meet the preset values. The results of the survival rate and moisture content after spray drying are shown in Table 1.

[0025] Table 1 Effects of different enzymatic hydrolysis times on the survival rate, moisture content and acid production performance of lactic acid bacteria

[0026]

[0027]

[0028] The results in Table 1 show that the survival rate of the group enzymolyzed for 3 hours was significantly higher than that of other groups, and the moisture content met the standard.

[0029] Example 2 Molecular Weight Screening and Protection Performance Verification

[0030] By screening the molecular weight, the protective effect of casein hydrolysate on Lactobacillus delbrueckii subsp. sp1.1 (L. bulgaricus sp1.1) was explored. The casein hydrolysate enzymolyzed for 3 hours in Example 1 was taken, centrifuged at a low temperature and high speed for 10 min (6000 r / min, 4 °C), and the supernatant was taken. It was separated into 4 groups by ultrafiltration membrane: <1000 Da, 1000 - 3000 Da, 3000 - 10000 Da, >10000 Da.

[0031] Each group of hydrolysates was added to a 30% maltodextrin matrix at 10% (w / v) and mixed with the bacterial sludge (mass ratio 5:1); bacterial powder was prepared under the same spray drying conditions, and the survival rate and moisture content were measured. The results are shown in Table 2.

[0032] Table 2 Effects of Casein Hydrolysates with Different Molecular Weights on the Survival Rate and Moisture Content of Lactic Acid Bacteria

[0033] Molecular weight range (Da) Number of viable bacteria before drying Number of viable bacteria after drying Survival rate (%) Moisture content (%) ＜1000 <![CDATA[2.61×10 10 CFU / g dry matter]]> <![CDATA[2.17×10 8 CFU / g dry matter]]> 0.83 3.72 1000-3000 <![CDATA[2.61×10 10 CFU / g dry matter]]> <![CDATA[2.77×10 8 CFU / g dry matter]]> 1.06 3.89 3000-10000 <![CDATA[2.61×10 10 CFU / g dry matter]]> <![CDATA[5.53×10 8 CFU / g dry matter]]> 2.12 4.49 ＞10000 <![CDATA[2.61×10 10 CFU / g dry matter]]> <![CDATA[3.49×10 9 CFU / g dry matter]]> 13.37 4.91

[0034] The results in Table 2 show that the survival rate of the enzymolyzed solution group with a molecular weight >10000 Da was the highest, and the moisture content was positively correlated with the protection performance.

[0035] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a casein hydrolysate, characterized in that, The specific steps are as follows: (1) Add 0.02 g of sodium carbonate to every 4 g of casein, dissolve it in 80 ml of distilled water, add a rotor and dissolve at room temperature for 2 h to obtain a casein solution; preheat the casein solution at 37 °C for 30 min and then adjust the pH to 6.0; (2) Under the conditions of a temperature of 37 °C and a pH of 6.0, add papain to the casein solution. The addition amount of papain is 3000 U / g of casein, and the enzymolysis time is 3 hours. The enzymolyzed solution is centrifuged to separate the supernatant, and then ultrafiltered and fractionated to retain the fraction with a molecular weight > 10,000 Da to prepare a casein hydrolysate.

2. The casein hydrolysate prepared by the method according to claim 1.

3. Use of the casein hydrolysate according to claim 2 in improving the spray-drying survival rate of lactic acid bacteria.

4. The application according to claim 3, wherein The specific steps are as follows: (1) Culture lactic acid bacteria in a medium until the late logarithmic phase and the early stationary phase; (2) Centrifuge to collect the bacterial cells, and wash the bacterial cells twice with physiological saline or 0.01 M phosphate buffer solution; centrifuge again to collect the bacterial cells to obtain a bacterial cell paste; (3) Using 30% w / v maltodextrin as the drying matrix, add 10% w / v of the casein hydrolysate, mix well, and then mix with the bacterial cell paste according to a mass ratio of 5:1, and spray-dry to prepare a bacterial powder.

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