A method for reducing bitterness of rice protein peptide by lactobacillus casei

Abstract

This invention provides a method for reducing the bitterness of rice protein peptides using Lactobacillus casei, comprising: inoculating Lactobacillus casei JAAS-N-101 into a sterilized rice protein peptide solution, fermenting at 37°C for 20-48 h, and after fermentation, separating and sterilizing the mixture, and collecting the fermentation supernatant of the rice protein peptides. This invention can efficiently remove bitterness while maximizing the preservation of the biological activity of rice protein peptides, and is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of plant protein deep processing technology, specifically relating to a debittering preparation technology for rice protein peptides, and more particularly to a method for reducing the bitterness of rice protein peptides using Lactobacillus casei. Background Technology

[0002] Rice protein peptides are prized for their small molecular weight, good solubility, and easy absorption, and they possess various physiological activities such as antioxidation, blood pressure reduction, and immune regulation. However, during the production process, a large number of hydrophobic amino acid residues are exposed and form hydrophobic bitter peptides, resulting in a distinctly bitter taste in the rice protein peptide liquid. This significantly impacts the product's palatability and application scope. This bitterness issue has become a key technical challenge restricting the industrial production and market promotion of rice protein peptides in functional foods and beverages.

[0003] Currently, debittering technologies for protein peptides are mainly divided into non-enzymatic and enzymatic methods. Non-enzymatic methods include adsorption, encapsulation / masking, extraction, chromatographic separation, Maillard reaction, and protein-like reactions. While these methods have some debittering effect, they generally have drawbacks. For example, adsorption can simultaneously lose active peptides and essential amino acids; encapsulation / masking cannot completely remove bitterness and may introduce other off-flavors; extraction and chromatographic separation are costly, involve safety hazards with organic solvents, and are unsuitable for industrial production. Enzymatic debittering mainly utilizes exopeptidases such as aminopeptidase and carboxypeptidase or microbial fermentation. This method is mild and highly specific, but still suffers from drawbacks such as high enzyme costs, complex processes, difficulty in controlling multi-step enzymatic hydrolysis, and the potential for over-hydrolysis leading to loss of biological activity. Compared to enzymatic methods, microbial fermentation is less costly and results in less loss of biological activity. However, this method still carries the risk of contamination and unstable effectiveness. Therefore, in microbial fermentation debittering technology, screening for a microorganism capable of stable and significant debittering is crucial.

[0004] Therefore, developing a microbial fermentation process for debittering protein peptides that is efficient, safe, does not affect the bioactivity of the product, and is suitable for industrial production needs remains a key technological bottleneck that needs to be overcome. Meanwhile, the screening of functional microorganisms that meet the needs of protein peptide debittering remains a core research and development direction in this field for a long time. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a method for reducing the bitterness of rice protein peptides using Lactobacillus casei, which can efficiently remove bitterness while maximizing the preservation of the bioactivity of rice protein peptides, and is suitable for industrial production.

[0006] The technical solution adopted by this invention to achieve its technical objectives is as follows:

[0007] This invention provides a method for reducing the bitterness of rice protein peptides using Lactobacillus casei, comprising: inoculating Lactobacillus casei JAAS-N-101 into a sterilized rice protein peptide solution, fermenting and culturing at 37°C for 20-48 h, and after fermentation, separating and sterilizing the supernatant of rice protein peptide fermentation and collecting it.

[0008] Further, a bacterial suspension of Lactobacillus casei JAAS-N-101 was inoculated into a sterilized rice protein peptide solution at an inoculation rate of 1% to 10% (v / v), wherein the viable count of the bacterial suspension was 1 to 2 × 10⁻⁶. 8 CFU / mL.

[0009] Furthermore, the concentration of rice protein peptides in the rice protein peptide solution is 19~50 mg / mL.

[0010] Furthermore, the sterilization process involves sterilizing at 105°C for 20 minutes.

[0011] Further, it was placed in a constant temperature shaker incubator with a rotation speed of 80 r / min for fermentation.

[0012] Furthermore, after fermentation, the fermentation broth was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The supernatant was then filtered and sterilized using a 0.22 μm aqueous microporous membrane.

[0013] The present invention also provides a low-bitter rice protein peptide, which is prepared by any of the methods described above.

[0014] The present invention has the following beneficial effects:

[0015] The Lactobacillus casei JAAS-N-101 of this invention can significantly reduce the bitterness of rice protein peptides without affecting their antioxidant properties.

[0016] Experiments showed that after fermentation with *Lactobacillus casei* JAAS-N-101 for 24 hours, the bitterness value of rice protein peptide stock solution prepared on a small scale in the laboratory decreased from 4.76 to 2.64, a reduction of 44.54%. After fermentation with *Lactobacillus casei* JAAS-N-101 for 40 hours, the bitterness value of industrially produced rice protein peptide stock solution decreased from 1.62 to 0.88, a reduction of 45.68%. After fermentation with *Lactobacillus casei* JAAS-N-101 for 48 hours, the bitterness value decreased from 1.31 to 0.37, a reduction of 71.75%, with no significant effect on its antioxidant activity. After reconstitution of industrially produced rice protein peptide powder and fermentation with *Lactobacillus casei* JAAS-N-101 for 36 hours, the bitterness value decreased from 2.64 to 1.46, a reduction of 44.70%. Detailed Implementation

[0017] To more clearly illustrate the present invention, the following detailed description is provided in conjunction with embodiments. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0018] Example 1

[0019] Preparation of *Lactobacillus casei* JAAS-N-101 bacterial suspension: Activated and rejuvenated *Lactobacillus casei* JAAS-N-101 plate strain was inoculated into 100 mL of sterile liquid culture medium and incubated in a constant temperature shaker at 37 ℃ and 100 r / min for 24 h to prepare the seed culture. The seed culture was then inoculated at a rate of 1% into 300 mL of liquid culture medium and cultured for another 36 h at 37 ℃ and 100 r / min. The liquid culture medium consisted of: 4 g yeast extract, 5 g peptone, 10 g brown sugar, 10 g glucose, and deionized water to a final volume of 1 L. After thorough mixing, the mixture was sterilized at 115 ℃ for 20 min. The *JAAS-N-101* bacterial suspension was collected and centrifuged at 4000 r / min for 5 min. The supernatant was discarded. Sterile pure water was added to the precipitate at the bottom, and the cells were resuspended by refluxing. After centrifugation again, the supernatant was discarded, completing the cell washing process. Repeat the washing and precipitation process described above once. After adding 30 mL of sterile water, the OD600 value of the bacterial suspension was measured at 600 nm using a UV spectrophotometer; the value was 29.2. Verification using plate plating showed that the viable count of *Lactobacillus casei* JAAS-N-101 suspension was 1–2 × 10⁻⁶. 8 CFU / mL. The *Lactobacillus casei* JAAS-N-101 used in the following examples was prepared according to this method. The *Lactobacillus casei* (… Lactobacillus casei JAAS-N-101 was deposited on November 19, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 65505. This strain and its deposit information have been disclosed in patent application No. 202511130366.5, entitled "A Composite Microecological Preparation and its Preparation Method and Application".

[0020] Preparation of Saccharomyces cerevisiae JAAS-N-110 suspension: The procedure was the same as described above, using Saccharomyces cerevisiae JAAS-N-110. Viable cell counts in the Saccharomyces cerevisiae JAAS-N-110 suspension were verified by plate plating and found to be 1~2 × 10⁻⁶. 8 CFU / mL. The brewer's yeast ( Saccharomyces cerevisiaeJAAS-N-110 was deposited on November 19, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 65507. This strain and its deposit information have been disclosed in patent application No. 202511130366.5, entitled "A Composite Microecological Preparation and its Preparation Method and Application".

[0021] Preparation of *Lactobacillus plantarum* JAAS-N-102 bacterial suspension: The procedure was the same as described above, using *Lactobacillus plantarum* JAAS-N-102 as the bacterial strain. Viable cell counts in the *Lactobacillus plantarum* JAAS-N-102 bacterial suspension were verified by plate plating and found to be 1~2 × 10⁻⁶. 8 CFU / mL. Lactobacillus plantarum ( Lactiplantibacillus plantarum JAAS-N-102 was deposited on November 19, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 65506. This strain and its deposit information have been disclosed in patent application No. 202511130366.5, entitled "A Composite Microecological Preparation and its Preparation Method and Application".

[0022] Rice protein peptides improve bitterness during fermentation experiment:

[0023] The experiment included a control group and an experimental group. Rice protein peptide stock solution, provided by Anhui Shunxin Shengyuan Biological Food Co., Ltd., was sterilized at 105℃ for 20 minutes before being packaged. Rice protein peptide stock solution refers to the crude liquid of rice protein peptides obtained by filtration after protease hydrolysis of rice protein; it is an orange-yellow, transparent, clear liquid with a distinctive odor.

[0024] Control group: Take 100mL of rice protein peptide stock solution, add 1mL of sterile water, place it in a constant temperature shaker incubator, and incubate at 37℃ and 80r / min for 20 and 40 hours;

[0025] Lactobacillus casei fermentation group: Take 100 mL of rice protein peptide stock solution, add 1 mL of Lactobacillus casei JAAS-N-101 bacterial suspension, place in a constant temperature shaker incubator, and incubate at 37℃ and 80 r / min for 20 and 40 h.

[0026] Fermentation group of Lactobacillus plantarum: Take 100mL of rice protein peptide stock solution, add 1mL of Lactobacillus plantarum JAAS-N-102 bacterial suspension, place it in a constant temperature shaker incubator, and incubate at 37℃ and 80r / min for 20 and 40h.

[0027] Saccharomyces cerevisiae fermentation group: Take 100mL of rice protein peptide stock solution, add 1mL of Saccharomyces cerevisiae JAAS-N-110 bacterial suspension, place in a constant temperature shaker incubator, and incubate at 37℃ and 80r / min for 20 and 40h.

[0028] After fermentation, the fermentation broth was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm aqueous microporous membrane for sterilization, finally obtaining sterile rice protein peptide filtrate. Three parallel experimental groups were set up.

[0029] The bitterness of rice protein peptide filtrate was detected using the Japanese INSENT TS-SA402B intelligent sensory evaluation system to determine the changes in bitterness. The experimental results showed that fermentation of rice protein peptides with different strains resulted in a 45.68% reduction in bitterness after 40 hours of fermentation with Lactobacillus casei, while fermentation with Saccharomyces cerevisiae increased the bitterness of rice protein peptides. Detailed data are shown in Table 1.

[0030] Table 1. Bitterness Values ​​of Rice Protein Peptide Extract

[0031]

[0032] Example 2: Bitterness optimization experiment of rice protein peptide stock solution prepared on a small scale in the laboratory.

[0033] Weigh an appropriate amount of rice protein and prepare a suspension with a substrate concentration of 10% (w / v, g / mL). Pre-treat the suspension by heating it at 50℃ for 30 min, then adjust the pH to the optimal value for trypsin reaction (pH 7.5) using NaOH solution. Add trypsin at an enzyme-to-substrate mass ratio of 2.0%, and enzymatically hydrolyze at 55℃ and pH 7.5 for 4.0 h. After enzymatic hydrolysis, inactivate the enzyme in a 95℃ water bath for 10 min, centrifuge the fermentation broth at 4000 r / min for 20 min, and retain the supernatant to obtain the rice protein peptide stock solution. Then sterilize at 105℃ for 20 min.

[0034] Fermentation experiment to optimize the bitterness of rice protein peptides: The experiment was set up with a control group and an experimental group.

[0035] Control group: Take 100mL of rice protein peptide stock solution, add 1mL of sterile water, place it in a constant temperature shaker incubator, and incubate for 24h at 37℃ and 80r / min.

[0036] Experimental group: Take 100 mL of rice protein peptide stock solution, add 1 mL of Lactobacillus casei JAAS-N-101 bacterial suspension, place in a constant temperature shaker incubator, and incubate for 24 h at 37℃ and 80 r / min.

[0037] After fermentation, the fermentation broth was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm aqueous microporous membrane for sterilization, finally obtaining sterile rice protein peptide filtrate. Three parallel experimental groups were set up.

[0038] The bitter taste of rice protein peptide filtrate was detected using the Japanese INSENT TS-SA402B intelligent sensory evaluation system to determine the change in bitterness. It was found that the bitterness value of rice protein peptide decreased by 44.54% after fermentation with Lactobacillus casei for 24 hours. Detailed data are shown in Table 2.

[0039] Table 2. Bitterness Values ​​of Rice Protein Peptide Stock Solution

[0040]

[0041] Example 3

[0042] Take 100 g of rice protein peptide powder provided by Anhui Shunxin Shengyuan Biological Food Co., Ltd., add it to 2 L of deionized water, and stir thoroughly until completely mixed to obtain a rice protein peptide powder reconstituted solution.

[0043] Experiment on improving bitterness in fermentation with rice protein peptide powder: The experiment set up a control group and an experimental group.

[0044] Control group: Take 100mL of rice protein peptide powder reconstituted solution, add 1mL of sterile water, place it in a constant temperature shaker incubator, and incubate at 37℃ and 80r / min for 48h;

[0045] Experimental group: Take 100 mL of rice protein peptide powder reconstituted solution, add 1 mL of Lactobacillus casei JAAS-N-101 bacterial suspension, place in a constant temperature shaker incubator, and incubate for 36 h at 37℃ and 80 r / min.

[0046] After fermentation, the fermentation broth was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm aqueous microporous membrane for sterilization, finally obtaining sterile rice protein peptide filtrate. Three parallel experimental groups were set up.

[0047] The bitter taste of rice protein peptide filtrate was detected using the Japanese INSENT TS-SA402B intelligent sensory evaluation system to determine the change in bitterness. It was found that the bitterness value of rice protein peptide decreased by 44.70% after fermentation with Lactobacillus casei for 36 hours. Detailed data are shown in Table 3.

[0048] Table 3. Bitterness Values ​​of Rice Protein Peptide Powder Reconstituted Solution

[0049]

[0050] Example 4

[0051] I. Fermentation Experiment to Optimize Bitterness of Rice Protein Peptide Stock Solution: The experiment included a control group and an experimental group. The rice protein peptide stock solution provided by Anhui Shunxin Shengyuan Biological Food Co., Ltd. was sterilized at 105℃ for 20 min before being packaged.

[0052] Control group: Take 100mL of rice protein peptide stock solution, add 1mL of sterile water, place it in a constant temperature shaker incubator, and incubate at 37℃ and 80r / min for 48h;

[0053] Experimental group: Take 100 mL of rice protein peptide stock solution, add 1 mL of Lactobacillus casei JAAS-N-101 bacterial suspension, place in a constant temperature shaker incubator, and incubate for 48 h at 37℃ and 80 r / min.

[0054] After fermentation, the fermentation broth was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm aqueous microporous membrane for sterilization, finally obtaining sterile rice protein peptide filtrate. Three parallel experimental groups were set up.

[0055] The bitter taste of rice protein peptide filtrate was detected using the Japanese INSENT TS-SA402B intelligent sensory evaluation system to determine the change in bitterness. It was found that the bitterness value of rice protein peptide decreased by 71.75% after fermentation with Lactobacillus casei for 48 hours. Detailed data are shown in Table 4.

[0056] Table 4. Bitterness Value of Rice Protein Peptide Stock Solution

[0057]

[0058] II. Determination of the DPPH free radical scavenging ability of rice protein peptides. Rice protein peptide sample preparation: The rice protein peptide filtrate obtained in step one was diluted with deionized water to different concentrations as sample solutions. The rice protein peptide concentrations were 0.5, 1.0, 1.5, 2.0, and 2.5 mg / mL. Experimental procedure: An appropriate amount of DPPH was dissolved in anhydrous ethanol to prepare a 0.1 mM DPPH alcohol solution (stored in the dark). Three reaction systems were set up: the sample group consisted of 500 µL sample solution + 500 µL DPPH alcohol solution; the blank group consisted of 500 µL sample solution + 500 µL anhydrous ethanol; and the control group consisted of 500 µL DPPH alcohol solution + 500 µL deionized water. After mixing each system, the reaction was carried out at room temperature in the dark for 30 min. The absorbance was measured at 517 nm (zeroed with anhydrous ethanol), and these were recorded as Sample A, Blank A, and Control A, respectively. The scavenging rate of the sample against DPPH free radicals was calculated using the formula: scavenging rate (%) = [1 - (A sample - A blank) / A control] × 100%. The color interference of the sample itself was subtracted by the blank group. Each group was set up with 3 parallels. The final result is expressed as scavenging rate, as shown in Table 5.

[0059] Table 5. Rice protein peptide DPPH free radical scavenging rate

[0060]

[0061] Therefore, it can be seen that the method of using Lactobacillus casei JAAS-N-101 to reduce the bitterness of rice protein peptides can effectively remove bitterness without significantly affecting its antioxidant activity.

[0062] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for reducing bitterness of rice protein peptides using Lactobacillus casei, characterized by, include: Lactobacillus casei JAAS-N-101 was inoculated into a sterilized rice protein peptide solution and fermented at 37°C for 20-48 h. After fermentation, the rice protein peptide fermentation supernatant was collected after separation and sterilization.

2. The method of claim 1, wherein, A bacterial suspension of Lactobacillus casei JAAS-N-101 was inoculated into a sterile rice protein peptide solution at an inoculation rate of 1% to 10%, wherein the viable count of the bacterial suspension was 1 to 2 × 10⁻⁶. 8 CFU / mL.

3. The method of claim 1, wherein, The concentration of rice protein peptides in the rice protein peptide solution is 19~50 mg / mL.

4. The method of claim 1, wherein, The sterilization process involves sterilizing at 105°C for 20 minutes.

5. The method according to claim 1, characterized in that, Fermentation culture was carried out in a constant temperature shaker incubator with a rotation speed of 80 r / min.

6. The method of claim 1, wherein, After fermentation, the fermentation broth was centrifuged at 4000 r / min for 10 min, and the supernatant was collected.

7. The method of claim 6, wherein, The supernatant was filtered and sterilized using a 0.22 μm aqueous microporous membrane.

8. A low-bitter rice protein peptide, prepared by the method described in any one of claims 1 to 7.

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