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CN-121975708-A - Sweet protein expressed by recombinant bacillus subtilis and application of sweet protein in sealwort milk tea

CN121975708ACN 121975708 ACN121975708 ACN 121975708ACN-121975708-A

Abstract

The invention discloses sweet protein expressed by recombinant bacillus subtilis and application thereof in sealwort milk tea, belonging to the technical fields of bioengineering and food. In order to improve the efficiency of further expressing the protein by the bacillus subtilis, the invention further screens different signal peptides and realizes the efficient secretory expression of the sweet protein in the bacillus subtilis through amino acid mutation optimization. Meanwhile, the recombinant secreted expressed sweet protein is added into the milk tea substrate containing the rhizoma polygonati, so that the rhizoma polygonati sweet protein milk tea with low calorie, high nutritive value and rich taste is prepared, and the application and development of the sweet protein and the rhizoma polygonati in the field of food and beverage industry are promoted.

Inventors

  • ZHAO JINSONG
  • JIN PENG
  • SI JINPING
  • HUANG LEI
  • CHEN LIJIE
  • LUO CHENGRUI

Assignees

  • 浙江农林大学
  • 苍南县中医院

Dates

Publication Date
20260505
Application Date
20260123

Claims (8)

  1. 1. A recombinant strain capable of secreting and expressing a sweet protein Brazzein is characterized in that a gene encoding the sweet protein Brazzein is connected to a bacillus subtilis expression vector pMA05 and is transformed into bacillus subtilis to obtain recombinant bacillus subtilis capable of secreting and expressing the sweet protein Brazzein, and a nucleotide sequence encoding LEHHHHHH eight amino acids is fused at the carboxyl end of the gene encoding the sweet protein Brazzein.
  2. 2. The recombinant strain capable of secreting and expressing the sweet protein Brazzein according to claim 1, wherein the pMA05 carries a signal peptide for secreting and expressing the sweet protein Brazzein, the amino acid sequence of the signal peptide is shown as any one of SEQ ID NO. 4-10 and SEQ ID NO.12-30, and the pMA05 expression vector further comprises a constitutive promoter PHpaII.
  3. 3. The recombinant strain capable of secreting and expressing the sweet protein Brazzein according to claim 2, wherein the nucleotide sequence of the signal peptide is shown as SEQ ID NO.6 or SEQ ID NO. 16.
  4. 4. A recombinant strain capable of secreting and expressing a sweet protein Brazzein according to any of claims 1 to 3 wherein said bacillus subtilis is Bacillus subtilis, bacillus subtilis WB, 600 or Bacillus subtilis WB.
  5. 5. The method for constructing the recombinant strain according to any one of claims 1 to 4, wherein the gene encoding the sweet protein Brazzein is linked to a Bacillus subtilis expression vector pMA05 and transformed into Bacillus subtilis.
  6. 6. A method for secreting and expressing the sweet protein Brazzein by using the recombinant strain according to any one of claims 1 to 4, wherein the recombinant strain is cultured in a culture medium for a period of time, fermentation supernatant is collected by centrifugation, then the fermentation supernatant is separated and purified by nickel ion affinity chromatography, dialyzed in ultrapure water, and finally the sweet protein Brazzein is obtained by freeze drying.
  7. 7. The application of the recombinant strain in preparing milk tea according to any one of claims 1-4, wherein the milk tea is obtained by compounding sweet protein secreted and expressed by the recombinant strain and a milk tea substrate according to a certain proportion.
  8. 8. The use according to claim 7, wherein the milk tea is a milk tea of Polygonatum sibiricum, further comprising an extract of Polygonatum sibiricum.

Description

Sweet protein expressed by recombinant bacillus subtilis and application of sweet protein in sealwort milk tea Technical Field The invention relates to sweet protein expressed by recombinant bacillus subtilis and application of sweet protein in sealwort milk tea, belonging to the technical field of bioengineering and food. Background Sweet protein Brazzein is a natural high sweetness low calorie protein originally isolated from the fruit of western african shrubs PENTADIPLANDRA BRAZZEANA. The sweetness of the sweet potato is 500-2000 times of that of the sucrose, and the sweet perception is close to that of the sucrose, so that the sweet potato has the characteristics of refreshing sweet taste curve, quick onset and no afterbitterness. Brazzein activates the sweet taste signal by binding to a specific domain of the human taste receptor T1R2/T1R3, producing a strong sweet feel at very low concentrations. In addition, studies have shown that Brazzein retains stable sweetness activity at high temperatures, over a wide pH range and over long storage periods, which provides a basis for its wide use in food processing. Notably, brazzein is not metabolized by the human body, hardly generates heat, and does not cause blood glucose fluctuations, and is therefore considered as an ideal sucrose substitute for the diabetic, obesity, etc. population. Currently, brazzein has been gradually applied to low calorie foods, functional health products and oral care products, and with the development of protein engineering and flavor modification technology, the sweetness characteristics and processing adaptability of Brazzein are expected to be further optimized, and the Brazzein has a wide market prospect in the future health food industry. With the improvement of the living standard of modern people, the flavored milk tea pursuing low calories, nutrition and health is more and more favored. The rhizoma polygonati extract is added into milk tea production, and sweet protein is used for replacing sucrose, so that not only can the heat of the milk tea be reduced and the potential health risk be reduced, but also the nutritional value of the milk tea can be enriched to a certain extent. Although Brazzein is excellent in sweetness characteristics and stability, its practical use is subject to multiple limitations. Firstly, the natural source is seriously deficient, namely the Brazzein can only be extracted from fruits of the original plants in a trace way, the plant has long growth cycle and limited regional distribution, the yield of natural products is extremely low, the cost is high, and the industrial requirement cannot be met. Secondly, the efficiency of recombinant expression systems is insufficient, and heterologous expression in systems such as E.coli and yeast has been attempted, but there are general problems such as low expression level, abnormal protein folding, and loss of activity. Thus, achieving low cost, high yield and high sweetness biosynthesis of sweet proteins in food-grade hosts is critical to driving the commercial application of Brazzein. Disclosure of Invention Technical problem The invention aims to solve the technical problems that the yield of sweet protein recombinantly expressed by using escherichia coli is low, the structure is abnormal, the sweet taste is insufficient, potential safety hazards possibly exist and the like. Technical proposal The invention constructs a recombinant expression system of the sweet protein in the food-grade microorganism bacillus subtilis Bacillus subtilis for the first time, aims at producing the sweet protein with excellent sweetness in a safe host, and simultaneously, further screens different signal peptides for improving the efficiency of further expressing the protein by the bacillus subtilis, and realizes the efficient secretion expression of the sweet protein in the bacillus subtilis through amino acid mutation optimization. Meanwhile, the recombinant secreted expressed sweet protein is added into the milk tea substrate containing the rhizoma polygonati, so that the rhizoma polygonati sweet protein milk tea with low calorie, high nutritive value and rich taste is prepared, and the application and development of the sweet protein and the rhizoma polygonati in the field of food and beverage industry are promoted. The invention firstly provides a construction method of recombinant bacillus subtilis capable of secreting and expressing a sweet protein Brazzein, which is to connect a gene for encoding the sweet protein Brazzein to a bacillus subtilis expression vector pMA05 and transform the gene into the bacillus subtilis to obtain the recombinant bacillus subtilis capable of secreting and expressing the sweet protein Brazzein. The gene for encoding the sweet protein Brazzein is subjected to codon optimization, and the nucleotide sequence is shown as SEQ ID NO. 1. In one embodiment, the carboxy terminus of the gene encoding the sweet protein Brazzein is further fused