A method for preparing a low-allergy fermented milk peptide beverage

Abstract

The application discloses a preparation method of low-sensitivity fermented milk beverage and belongs to the field of fermented milk, and specifically comprises the following steps: adding konjac glucomannan and distilled water into skim milk powder and dissolving; performing desensitization treatment on the milk by means of dynamic high pressure microfluidization, glycosylation and lactic acid bacteria fermentation; the application mainly utilizes the synergistic effect of the co-fermentation of two strains to degrade milk protein, destroy allergen epitopes, and combines dynamic high pressure microfluidization treatment and glycosylation reaction, so that the allergenicity of main milk allergens is effectively reduced. The application provides a new idea for the development of low-sensitivity dairy products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food processing, and in particular to a preparation method of low-sensitivity fermented milk peptide beverage. BACKGROUND

[0002] Food allergy, also known as food allergy or digestive system allergy, allergic gastroenteritis, etc., is an IgE-mediated and non-IgE-mediated immune reaction caused by the ingestion of certain foods or food additives, etc., thereby causing an allergic reaction in the digestive system or the whole body. At present, the incidence of adult food allergy and the incidence of children's food allergy are both increasing year by year. The recognized allergic foods mainly include milk, eggs, fish, shellfish, peanuts, soybeans, nuts and wheat. Among them, epidemiological studies have shown that milk allergy is the most common allergic reaction in the population, especially in children, which seriously affects the life of allergic people and the growth and development of children.

[0003] The protein in milk is the main cause of allergy, and there are more than 20 proteins in milk that can cause allergic reactions, among which the main allergens are casein, alpha-lactalbumin and beta-lactoglobulin. Exploring the technical method to reduce the allergenicity of milk protein has become the focus and hotspot of current domestic and foreign research. At present, there are many methods for reducing the allergenicity of milk, most of which will have adverse effects on food, reduce the sensory quality of food, and may also face problems such as high cost and poor safety.

[0004] At present, the commonly used milk desensitization methods include physical method, chemical method and biological method. However, the physical method has problems such as damage to the quality of milk, pH reduction of milk, protein denaturation, vitamin loss, etc.; the chemical method has problems such as single method, loss of valuable nutrients, adverse reactions such as melanin browning, etc.; the biological method has problems such as incomplete elimination of milk allergy, and the process also inevitably produces bitter peptides and seriously affects the flavor of dairy products, etc.

[0005] Therefore, a new method for preparing low-sensitivity fermented milk peptide beverage needs to be developed. SUMMARY

[0006] The present application provides a preparation method of low-sensitivity fermented milk peptide beverage, which uses whole milk as the main raw material, adopts dynamic high-pressure microfluidization treatment, biological enzyme engineering, pulse electric field combined with glycosylation modification technology, reduces the allergenicity of milk and main allergen proteins by shielding and destroying the allergic epitopes, to overcome the key technology of reducing milk allergy; combines with modern food processing technologies such as compounding and homogenization to develop a desensitization milk peptide beverage; the desensitization milk peptide beverage of the present application achieves the effectiveness of desensitization through enzymolysis combined with glycosylation treatment, and maintains good stability in the beverage compounding and seasoning process.

[0007] The application provides a preparation method of a low-sensitivity fermented milk peptide beverage.

[0008] S1, konjac glucomannan is added to skimmed milk powder and dissolved in water to obtain solution one;

[0009] S2, the solution one is subjected to desensitization treatment to prepare the low-sensitivity fermented milk peptide beverage; the desensitization treatment comprises dynamic high-pressure microfluidization, glycosylation and lactic acid bacteria fermentation; the lactic acid bacteria comprises Lactobacillus helveticus with a preservation number of CICC NO.20275 and Lactobacillus plantarum with a preservation number of CICC NO.20261.

[0010] Optionally, the mass ratio of the skimmed milk powder, the konjac glucomannan and the water is (10-14):(3-5):(87-81).

[0011] Optionally, in the step S2, the solution one is treated by using a microfluidization nanometer homogenizer under the condition of a pressure of 0-120 MPa, and the treatment is circular for 3 times to obtain solution two.

[0012] Optionally, in the step S2, the glycosylation treatment comprises the following steps: raspberry tannin is added to the solution two, and the mass percentage of the raspberry tannin in the solution two is 0.05-0.1%; after vacuum stirring, the glycosylation reaction is performed at a temperature of 90-100 ℃ for 10-30 s to obtain solution three.

[0013] Optionally, in the step S2, the lactic acid bacteria fermentation comprises the following steps:

[0014] The solution three is subjected to homogenization, sterilization and cooling after the fermentation;

[0015] The Lactobacillus helveticus and the Lactobacillus plantarum are inoculated into the solution after cooling, and the fermentation is performed under anaerobic conditions at 30-38 ℃ for 12-36 h; the inoculation amount of the Lactobacillus helveticus is 8×10 6 -8×10 7 CFU / mL; and the inoculation amount of the Lactobacillus plantarum is 5×10 6 -5×10 7 CFU / mL.

[0016] After the fermentation is completed, the solution is subjected to vacuum stirring and cooling to prepare the low-sensitivity fermented milk peptide beverage.

[0017] In the second aspect, the application provides a low-sensitivity fermented milk peptide beverage.

[0018] Optionally, the beverage comprises mogroside, sea buckthorn concentrated juice and vitamin B 12 .

[0019] The present application provides the biological preservation information:

[0020] Lactobacillus helveticus Lactobacillus helveticus , Latin name is Lactobacillus helveticus, Preserved in China Industrial Microbial Culture Collection Center, the preservation number is CICC NO.20275.

[0021] Lactobacillus plantarum Lactiplantibacillus plantarum , Latin name is Lactiplantibacillus plantarum Preserved in China Industrial Microbial Culture Collection Center, the preservation number is CICC NO.20261.

[0022] The present application has the beneficial effects including:

[0023] (1) the preparation method provided by the present application uses dynamic high pressure microfluidization treatment before glycosylation modification, uses high pressure, cavitation shearing and other effects to change the tertiary structure of the allergenic protein, promotes glycosylation reaction and lactic acid bacteria fermentation, and reduces the allergenicity of the product;

[0024] (2) the preparation method provided by the present application adds raspberry tannin before glycosylation modification, effectively inhibits the generation of product (AGEs) at the end of glycosylation reaction;

[0025] (3) in the preparation method provided by the present application, konjac glucomannan is added in glycosylation modification, which is a natural food gum and also a water-soluble dietary fiber, can inhibit Maillard browning in the glycosylation process, and is conducive to the growth of lactic acid bacteria in the subsequent step and the inhibition of the proliferation of harmful bacteria;

[0026] (4) in the preparation method provided by the present application, lactic acid bacteria fermentation is carried out by using Lactobacillus plantarum, which can hydrolyze allergen proteins and has immune regulation effect on the body, so that the allergenicity of the final product can be effectively reduced;

[0027] (5) in the low-sensitivity milk peptide beverage provided by the present application, mogroside, sea buckthorn concentrated juice, compound stabilizer, vitamin B 12 ; mogroside is used instead of sucrose, which can improve the nutritional value of the product while providing rich sweetness, has the functions of regulating blood sugar, reducing lipids, and resisting oxidative stress; sea buckthorn concentrated juice is conducive to subsequent lactic acid bacteria fermentation, has high vitamin C and vitamin E content, can improve human immunity, prevent respiratory and digestive system diseases, and sea buckthorn concentrated juice can be used as an auxiliary drug for diabetes, has the functions of inhibiting tumor cells, reducing cholesterol, resisting oxidation, and resisting aging; the compound stabilizer includes locust bean gum, pectin, and carrageenan, has good gel synergistic effect, reduces the amount of stabilizer, and improves the viscosity and stability of the product; vitamin B12 It not only increases the nutritional value of the product, but also promotes the growth of lactobacilli, reduces the mortality rate of the strain, and increases the activity of enzymes required for lactic acid biosynthesis, thereby increasing lactic acid production. Attached Figure Description

[0028] Figure 1 IC50 of the hypoallergenic milk peptide beverages prepared in the embodiments and comparative examples of this invention. 50 A comparison chart of values. Detailed Implementation

[0029] The present invention will be further described in conjunction with the accompanying drawings and through the following embodiments.

[0030] In a first aspect, embodiments of the present invention provide a method for preparing a hypoallergenic fermented milk peptide beverage, comprising the following steps:

[0031] S1. Add konjac glucomannan to skim milk powder and dissolve it in water to obtain solution one;

[0032] S2. Desensitize Solution 1 to obtain a low-sensitivity fermented milk peptide beverage; the desensitization process includes dynamic high-pressure microfluidic treatment, glycosylation, and lactic acid bacteria fermentation; the lactic acid bacteria include Lactobacillus helveticus with accession number CICC NO.20275 and Lactobacillus plantarum with accession number CICC NO.20261.

[0033] In some embodiments, the mass ratio of the skim milk powder, konjac glucomannan, and water is (10-14):(3-5):(87-81).

[0034] In some embodiments, the processing conditions of the dynamic high-pressure microjets in step S2 are as follows: under a pressure of 0-120 MPa, the solution one is treated using a microjets nano-homogenizer, and the cycle is repeated 3 times to obtain solution two.

[0035] In some embodiments, the glycosylation treatment in step S2 includes the following steps: adding raspberry tannin to solution two, wherein the raspberry tannin accounts for 0.05-0.1% of the mass of solution two; after vacuum stirring, performing a glycosylation reaction at a temperature of 90-100 °C for 10-30 s to obtain solution three.

[0036] In some embodiments, the lactic acid bacteria fermentation in step S2 includes the following steps:

[0037] The solution is fermented after homogenization, sterilization, and cooling.

[0038] The *Lactobacillus helveticus* and *Lactobacillus plantarum* were inoculated into the cooled solution and fermented under anaerobic conditions at 30-38 °C for 12-36 h; the inoculum size of *Lactobacillus helveticus* was 8 × 10⁻⁶. 6 -8×10 7 CFU / mL; the inoculum size of *Lactobacillus plantarum* is 5 × 10⁻⁶. 6 -5×10 7 CFU / mL;

[0039] After fermentation, the mixture is vacuum stirred and cooled to obtain a hypoallergenic fermented milk peptide beverage.

[0040] Secondly, the present invention provides a low-allergen fermented milk peptide beverage.

[0041] In some embodiments, the beverage includes mogrosides, sea buckthorn concentrate, and vitamin B. 12 .

[0042] Biological preservation information provided by this invention:

[0043] Lactobacillus helveticus Lactobacillus helveticus Latin name Lactobacillus helveticus, It is deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC NO.20275.

[0044] Lactobacillus plantarum Lactiplantibacillus plantarum Latin name Lactiplantibacillus plantarum It is deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC NO.20261.

[0045] The culture media used in the embodiments of this invention are all from Merck Co., USA, and other reagents are also commercially available. Example

[0046] This invention provides a method for preparing a hypoallergenic milk peptide beverage, comprising the following steps:

[0047] S1. Mix 12% (w / w) of skim milk powder, 4% (w / w) of konjac glucomannan and 84% (w / w) of distilled water until dissolved to obtain solution one.

[0048] S2. Desensitize solution 1. The desensitization process includes dynamic high-pressure microfluidization, glycosylation, and lactic acid bacteria fermentation, including the following sub-steps.

[0049] S21. Solution 1 was circulated three times at 80 MPa using a microfluidic nano-homogenizer to obtain solution 2.

[0050] S22. Add raspberry tannin at a mass ratio of 0.075% to solution 2, stir under vacuum at 45 ℃ for 7 min, and then perform glycosylation treatment in an electric thermostatic water bath at 95 ℃ for 20 s to obtain a glycosylated solution.

[0051] S23. Cool the glycosylation solution to 45 °C, add 5% mogroside, 0.75% sea buckthorn concentrate, and 0.13% compound stabilizer (compound stabilizer includes 0.05% pectin, 0.05% carrageenan, and 0.03% locust bean gum) at a mass percentage of the glycosylation solution. Stir under vacuum at 45 °C for 15 min to completely dissolve all additives, and homogenize in a homogenizer at 65 °C and 20 MPa.

[0052] S24. After homogenizing the solution, sterilize it at 95 °C for 5 min, then cool it to 35 °C. Aseptically inoculate it with 0.5% Lactobacillus helveticus suspension and 0.5% Lactobacillus plantarum suspension, and add vitamin B at a mass ratio of 0.07% to the solution. 12 Anaerobic culture at 35 ℃ for 24 h; after culture, vacuum stirring at 35 ℃ for 7 min, and cooling to 25 ℃ to obtain a low-sensitivity fermented milk peptide beverage;

[0053] The preparation of Lactobacillus helveticus culture in step S24 includes the following steps:

[0054] Dissolve the lyophilized Lactobacillus helveticus powder in 0.3 mL of sterile water, and use an inoculation loop to streak a loopful onto MRS solid medium. Incubate anaerobically at 37 °C for 16 h.

[0055] After the culture is complete, pick a single colony and put it into 10 mL of MRS liquid medium. Use a vortex shaker to evenly disperse the colony in the MRS liquid medium and anaerobic culture at 37 ℃ for 16 h.

[0056] After cultivation, the cells were inoculated into MRS liquid medium at a volume ratio of 2%, and anaerobically cultured at 37 °C for 16 h. After centrifugation at 7000 rpm for 10 min, the supernatant was discarded. The cells were washed twice with sterile distilled water and then resuspended in the same volume of sterile distilled water as the inoculated MRS liquid medium to obtain the seed culture with a concentration of 5 × 10⁻⁶. 8 CFU / mL;

[0057] The preparation of Lactobacillus plantarum bacterial solution in step S24 includes the following steps:

[0058] Add 0.2 mL of sterile water to dissolve the lyophilized Lactobacillus plantarum powder, and use an inoculation loop to streak a loopful onto MRS solid medium. Incubate anaerobically at 37 °C for 36 h.

[0059] After the culture is completed, pick a single colony and put it into 10 mL of MRS liquid medium. Use a vortex shaker to evenly disperse the colony in the MRS liquid medium and anaerobic culture at 37 ℃ for 36 h.

[0060] After cultivation, the cells were inoculated into MRS liquid medium at a volume ratio of 2%, and anaerobically cultured at 37 °C for 36 h. After centrifugation at 7000 rpm for 10 min, the supernatant was discarded. The cells were washed twice with sterile distilled water and then resuspended in the same volume of sterile distilled water as the MRS liquid medium used for inoculation to obtain the seed culture with a concentration of 4 × 10⁻⁶. 8 CFU / mL. Example

[0061] Example 2 of this invention provides a method for preparing a hypoallergenic milk peptide beverage, which differs from Example 1 in that: in step S1, 3% by mass of konjac glucomannan is added; in step S21, the pressure of the dynamic high-pressure microjet is 120 MPa; in step S22, the amount of raspberry tannin added is 0.05%, and the glycosylation condition is 90 ℃ for 30 s; in step S23, the amount of mogroside added is 2%, and the amount of compound stabilizer added is 0.1%, which includes 0.04% pectin, 0.04% carrageenan, and 0.02% locust bean gum; in step S24, the inoculum amount of Lactobacillus helveticus is 0.3%, the amount of Lactobacillus plantarum added is 0.7%, and vitamin B... 12 The addition amount is 0.05%; other conditions and steps remain the same, and a hypoallergenic milk peptide beverage is obtained in step S24. Example

[0062] Example 3 of this invention provides a method for preparing a hypoallergenic milk peptide beverage, which differs from Example 1 in that: in step S1, 10% by mass of skim milk powder is added; in step S21, the pressure of the dynamic high-pressure microjet is 40 MPa; in step S22, the amount of raspberry tannin added is 0.1%, and the glycosylation condition is 100 °C for 10 s; in step S23, the amount of mogroside added is 8%, and the amount of compound stabilizer added is 0.16%, which includes 0.06% pectin, 0.06% carrageenan, and 0.04% locust bean gum; in step S24, the inoculum amount of Lactobacillus helveticus is 0.7%, the amount of Lactobacillus plantarum added is 0.3%, and vitamin B... 12 The addition amount is 0.1%; other conditions and steps remain the same, and a hypoallergenic milk peptide beverage is obtained in step S24.

[0063] Comparative Example 1

[0064] Comparative Example 1 of the present invention provides a method for preparing a hypoallergenic milk peptide beverage. The difference from Example 1 is that only Lactobacillus helveticus is added for fermentation in step S24, while other conditions and steps remain the same; a hypoallergenic milk peptide beverage is obtained.

[0065] Comparative Example 2

[0066] Comparative Example 2 of this invention provides a method for preparing a hypoallergenic milk peptide beverage. The difference from Example 1 is that only Lactobacillus plantarum is added for fermentation in step S24, while other conditions and steps remain the same; a hypoallergenic milk peptide beverage is obtained.

[0067] Comparative Example 3

[0068] Comparative Example 3 of the present invention provides a method for preparing a hypoallergenic milk peptide beverage. The difference from Example 1 is that fermentation is not carried out in step S24, while other conditions and steps remain the same; a hypoallergenic milk peptide beverage is obtained.

[0069] Comparative Example 4

[0070] Comparative Example 4 of this invention provides a method for preparing a hypoallergenic milk peptide beverage. The difference from Example 1 is that dynamic high-pressure microfluidic treatment is not performed in step S21, while other conditions and steps remain the same; a hypoallergenic milk peptide beverage is obtained.

[0071] Comparative Example 5

[0072] Comparative Example 5 of this invention provides a method for preparing a hypoallergenic milk peptide beverage. The difference from Example 1 is that in step S24, the inoculation amount of Lactobacillus helveticus is 0.25%, the addition amount of Lactobacillus plantarum is 0.25%, and other conditions and steps remain the same; a hypoallergenic milk peptide beverage is obtained.

[0073] 1. Determination of beverage viscosity and viable bacteria count:

[0074] The viscosity of the milk peptide beverages in each embodiment and comparative example was measured by rotational viscometer method, and the results are shown in Table 1.

[0075] The viable bacterial count of the milk peptide beverages in each example and comparative example was detected by the dilution plating method, and the results are shown in Table 1.

[0076] Table 1. Results of viscosity and viable cell count in the examples and comparative examples.

[0077]

[0078] 2. Sensory testing

[0079] Twenty taste evaluators were selected to score the samples. Evaluators were required to have no particular taste preferences, be in the same evaluation environment, and rinse their mouths before each evaluation.

[0080] The scores are based on three aspects: taste, color and flavor, and texture. The scoring criteria are shown in Table 2. The evaluation results are shown in Table 3.

[0081] Table 2 Sensory Test Evaluation Criteria

[0082]

[0083] Table 3 Sensory evaluation results

[0084]

[0085] Referring to Table 3, reducing the inoculum size, adding excessive skim milk powder, or excessive mogrosides will all affect the taste score. When using only a single inoculum, the sensory score of the fermented milk will decrease. When the inoculum size of one strain increases, the corresponding fermentation time and temperature will change drastically, making the fermentation conditions unsuitable for the other strain and adversely affecting the sensory evaluation of the final product.

[0086] 3. Allergenicity test of milk peptide beverages

[0087] The IC50 of the milk peptide beverages prepared in the examples and comparative examples was determined. 50 The determination of the allergenicity of milk peptide beverages using the value includes the following steps:

[0088] F1. Take enzyme-labeled plate A, coat it with antigen at 4 ℃ for 12 h, wash with PBST and block with skim milk for 1 h;

[0089] F2. Take ELISA plate B, block it with skim milk at 37 ℃ for 1 h, wash with PBST, add competitive antigen and 60 μL of primary antibody (human serum diluted with PBST, 1:20), and incubate at 37 ℃ for 1 h.

[0090] F3. Take the ELISA plate C, block it with skim milk at 37 ℃ for 1 h, wash with PBST, add the same volume of PBST as the competing antigen and 60 μL of primary antibody (human serum diluted with PBST, 1:20), and incubate at 37 ℃ for 1 h.

[0091] F4. Competitive reaction: Take 100 μL of the reaction solution from ELISA plate A and ELISA plate B and ELISA plate C respectively and transfer them into ELISA plate A (to form a control). Incubate at 37 ℃ for 1 h and wash with PBST.

[0092] After washing, add 100 μL of biotin-labeled secondary antibody (goat anti-human IgE, 1:5000) and incubate at 37 ℃ for 1 h;

[0093] F5. After the competitive reaction is complete, wash the microplate with PBST and dry it. Add 100 HRP-labeled streptavidin (1:60) to each well and incubate at 37 °C for 1 h. Wash with PBST.

[0094] F6. After washing, add 100 μL of TMB colorimetric solution to each well, react at 37 ℃ for 15 min, then add 50 μL of 2 mol / L H2SO4 solution to each well to terminate the colorimetric reaction, and measure the OD. 450nm .

[0095] IgE inhibition rate (IC) 50 The calculation formula is as follows:

[0096]

[0097] IC measured in each embodiment and comparative example 50 Values ​​such as Figure 1 As shown;

[0098] Among them, OD 竞争蛋白 The enzyme-labeled plate B, which represents the normal experimental group with the addition of the competing protein in step F2, shows the OD value measured at a wavelength of 450 nm.

[0099] OD 阳性 The enzyme-labeled plate C, which represents the positive control group in step F3 with PBST added to replace the competing protein, shows the OD value measured at a wavelength of 450 nm.

[0100] See Figure 1 Comparative Example 3 was an unfermented product, whose IC 50 The IgE value was significantly lower than that of the examples and other comparative examples, indicating that the milk peptide beverage prepared in Comparative Example 3 had the strongest binding ability to IgE, i.e., the strongest potential allergenicity, proving that lactic acid bacteria fermentation can effectively reduce the allergenicity of major milk allergens; among them, the fermented milk in Comparative Example 5 had the lowest inoculum amount, and its IC50 value was significantly lower. 50 The value was the lowest among other fermented products, and its potential allergenicity also showed a high level, proving that the inoculation amount of lactic acid bacteria has a significant impact on the allergenicity of the final product; Comparative Example 4 did not undergo dynamic high-pressure microfluidic treatment, and its potential allergenicity was relatively higher than that of Example 1, proving that dynamic high-pressure microfluidic treatment can promote glycosylation reaction and lactic acid bacteria fermentation; Comparative Examples 1 and 2 were fermented milk inoculated with only a single lactic acid bacteria, and their potential allergenicity also showed a high level, proving that the synergistic effect of co-fermentation of two strains can more effectively reduce the antigenicity of the main allergenic proteins and their binding ability to IgE compared with fermentation of a single strain.

[0101] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for preparing a low-allergenic fermented milk peptide beverage, characterized in that, Includes the following steps: S1. Add konjac glucomannan to skim milk powder and dissolve it in water to obtain solution one; S2. Desensitize solution 1 to obtain a low-sensitivity fermented milk peptide beverage; the desensitization steps are dynamic high-pressure microfluidic treatment, saccharification, and lactic acid bacteria fermentation; the lactic acid bacteria include Lactobacillus helveticus with accession number CICC NO.20275 and Lactobacillus plantarum with accession number CICC NO.20261. The processing conditions for the dynamic high-pressure microjets in step S2 are as follows: under a pressure of 40-120 MPa, the solution one is processed using a microjets nano-homogenizer, and the cycle is repeated 3 times to obtain solution two. The glycosylation process described in step S2 includes the following steps: Raspberry tannin is added to solution two, wherein the raspberry tannin accounts for 0.05-0.1% of the mass of solution two; After vacuum stirring, a glycosylation reaction was carried out at 90-100℃ for 10-30 seconds to obtain solution three; The lactic acid bacteria fermentation described in step S2 includes the following steps: The solution is fermented after homogenization, sterilization, and cooling. The *Lactobacillus helveticus* and *Lactobacillus plantarum* were inoculated into the cooled solution, and fermented under anaerobic conditions at 30-38℃ for 12-36 hours; the inoculation amount of *Lactobacillus helveticus* was 8 × 10⁻⁶. 6 -8×10 7 CFU / mL; the inoculum size of *Lactobacillus plantarum* was 5 × 10⁻⁶. 6 -5×10 7 CFU / mL; After fermentation, the mixture is vacuum stirred and cooled to obtain a hypoallergenic fermented milk peptide beverage.

2. The method according to claim 1, characterized in that, The mass ratio of the skim milk powder, konjac glucomannan, and water is (10-14):(3-5):(81-87).

3. The hypoallergenic fermented milk peptide beverage prepared by the method according to any one of claims 1-2.

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