A kind of sucrase and its microcapsule preparation and application in bread baking

Abstract

The present invention discloses an application of sucrase and its microcapsule preparation in baking. The sucrase sequence is shown in SEQ ID NO. 2. The enzyme and its microcapsule preparation can significantly increase the fructooligosaccharide content in baked products, providing a new method for improving the quality of baked foods.

Description

Technical Field

[0001] The invention relates to the field of enzyme engineering, in particular to an enzyme for food baking and a microcapsule preparation thereof. Background Art

[0002] Bread baking is a complex process involving a variety of physical, chemical and biological reactions. With the improvement of people's quality of life, how to reduce the health risks of high-sugar diets such as bread has become a focus of attention. Sucrase, as an important food enzyme, can decompose sucrose into glucose and fructose to form fructooligosaccharides (FOS). FOS is an excellent prebiotic that can be used by beneficial bacteria in the intestine (such as bifidobacteria and lactic acid bacteria) to promote the growth of these beneficial bacteria, thereby maintaining the balance of intestinal microorganisms. In addition, FOS also has multiple functions such as improving blood sugar control and enhancing the immune system. If the content of absorbable sugar can be reduced in high-sugar foods such as bread and the content of FOS can be increased, it will be of great significance to improve the quality of baked goods. However, sucrase is easily inactivated during high temperature and long baking, affecting its efficacy. Therefore, it is particularly important to develop an enzyme or enzyme preparation that can be stably used in the food baking process. Although there have been a few reports on the use of sucrase to increase the content of FOS in food, the enzymes available are still very limited. If new usable sucrase can be obtained from unknown microbial populations in fermented products, it will provide a new approach for developing healthy baked foods. Summary of the invention

[0003] The present invention aims to provide a novel sucrase and its microcapsule preparation, which can significantly increase the fructooligosaccharide content in baked foods and improve the taste of baked foods, and provide a new method for producing healthy baked foods. In order to achieve the above purpose, the present invention provides the following technical solutions.

[0004] Firstly, the present invention provides an enzyme applicable to food baking, characterized in that the amino acid sequence of the enzyme is as shown in SEQ ID NO.2.

[0005] Optionally, the preparation method of the enzyme includes but is not limited to genetic engineering expression, natural extraction, etc.

[0006] Secondly, the present invention provides a microcapsule that can be used in food baking, characterized in that the microcapsule contains the aforementioned enzyme.

[0007] Preferably, the microcapsules are prepared by spray drying, fluidized bed coating, solvent evaporation, or the like.

[0008] Optionally, the particle size of the microcapsule is 1-100 μm.

[0009] Preferably, the microcapsule further comprises other edible excipients that can be used in the microcapsule.

[0010] Preferably, the auxiliary material is one or more of edible polysaccharides, proteins, lipids, waxes, sugars, emulsifiers or composite materials.

[0011] Optionally, the polysaccharide auxiliary materials include but are not limited to gum arabic, sodium alginate, chitosan and the like.

[0012] Optionally, the protein excipients include but are not limited to gelatin, soy protein, whey protein, etc.

[0013] Optionally, the lipid excipients include but are not limited to stearic acid, lecithin, etc.

[0014] Optionally, the wax auxiliary materials include but are not limited to paraffin, beeswax, etc.

[0015] Optionally, the carbohydrate auxiliary material includes but is not limited to maltodextrin, sucrose and the like.

[0016] Optionally, the emulsifier includes but is not limited to monoglyceride, sucrose fatty acid ester and the like.

[0017] Preferably, the auxiliary material is used as a wall material to protect the activity of the enzyme during high temperature and long time baking.

[0018] Optionally, the wall material has a thickness of 1-10 μm.

[0019] Preferably, the mass ratio of the wall material to the enzyme is 2:1 to 3:1.

[0020] Application of the aforementioned enzyme or microcapsule in food baking.

[0021] Preferably, the food baking includes but is not limited to bread, cakes, biscuits and the like.

[0022] Optionally, the application method comprises adding the enzyme or microcapsule during the fermentation stage of the baked material.

[0023] Thirdly, the present invention provides a method for baking food, characterized in that the enzyme or microcapsule is catalytically acted during the fermentation stage of the baking material.

[0024] Preferably, the catalytic action time is 1-12 hours.

[0025] Optionally, the fermentation temperature is 25-40°C.

[0026] Preferably, the enzymes or microcapsules are added during the fermentation stage of the baked material or before the fermentation stage of the baked material.

[0027] Preferably, the adding method is mixing, spraying, soaking, etc.

[0028] Optionally, the added amount is 0.01%-5% of the total weight of the baking material.

[0029] Preferably, the baking material comprises sucrose.

[0030] Finally, the present invention provides genes encoding the aforementioned enzymes.

[0031] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO.1.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] 1) The present invention provides a new enzyme capable of producing fructooligosaccharides.

[0034] 2) The enzyme provided by the present invention can be used in food baking and can significantly increase the fructooligosaccharide content of baked foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In the following, in conjunction with the accompanying drawings and specific embodiments, a sucrase and its microcapsule preparation of the present invention, and the application thereof in bread baking and the beneficial effects thereof are described in detail.

[0036] Figure 1 Shown is a PAGE gel electrophoresis diagram (Marker size is 30-180 kDa), where the left side is the marker and the right lane A is the purified protein;

[0037] Figure 2 HPLC detection chart of the final reaction product. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0040] The nucleotide sequence of the gene encoding the enzyme in the embodiment is shown in SEQ ID NO.1:

[0041]

[0042] The amino acid sequence of the enzyme is shown in SEQ ID NO.2:

[0043] MNIKKFAKRATVLTFQTALLAGGATQAFAKENTQKPYKDTYGVPSHITRHDMLQIPKQTSSEKYQVPQFDQTIKNIESAKGLDVWDSWLQNADGTVAEYSGYHVVFAGSPKDADMTSIYMFY QKVGDNSIDSWKNAGRVFKDSTDKFDANDEILKEQTQEWSGSATFSSDGKIFSSKLEAQSQLTQAQVNVSKSDDTLKINGVEDHKTIFEGQNEFFRYTSGDNHTLRDPHYVEDKGRKYLAFE ANGTTDNGYQGESLFNKAYYGGSTNFFRKETREKYVMLVTTRKEGKGALARYTDYTLKKVMKPLITSNTVTDEIERANVFKMNGKWYLFTDSRGSKMTIDGINSNDIYMLSEDASGRHETFD GLYLYAAKTAGDGTNVTFTYSHFAVPGDGYSDRLVIATSYMTNRGFFEDKKATFAPSFLLNIKGKKTSVVKNSIFLEQGQLTVNNVVHATGEYTNASGITWSQFALSGQEDKLWIDKRALQA

[0044] Example 1 Identification and synthesis of sucrase gene

[0045] Collect fermentation-related metagenomic second-generation sequencing data from the NCBI database, assemble the metagenomics using SPAdes software, further annotate the genome, and preliminarily screen the gene sequences annotated as sucrase based on the annotation results. Align the second-generation sequencing data back to the preliminarily screened gene sequences, and select genes with relatively complete coverage by second-generation sequencing reads as candidate enzyme genes. Send the candidate gene sequence to the company for full gene synthesis to obtain the candidate gene sequence. The coding frame nucleotide sequence of the candidate gene is shown in SEQ ID NO.1:

[0046]

[0047] The amino acid sequence encoded by the gene is shown in SEQ ID NO.2:

[0048] MNIKKFAKRATVLTFQTALLAGGATQAFAKENTQKPYKDTYGVPSHITRHDMLQIPKQTSSEKYQVPQFDQTIKNIESAKGLDVWDSWLQNADGTVAEYSGYHVVFAGSPKDADMTSIYMFY QKVGDNSIDSWKNAGRVFKDSTDKFDANDEILKEQTQEWSGSATFSSDGKIFSSKLEAQSQLTQAQVNVSKSDDTLKINGVEDHKTIFEGQNEFFRYTSGDNHTLRDPHYVEDKGRKYLAFE ANGTTDNGYQGESLFNKAYYGGSTNFFRKETREKYVMLVTTRKEGKGALARYTDYTLKKVMKPLITSNTVTDEIERANVFKMNGKWYLFTDSRGSKMTIDGINSNDIYMLSEDASGRHETFD GLYLYAAKTAGDGTNVTFTYSHFAVPGDGYSDRLVIATSYMTNRGFFEDKKATFAPSFLLNIKGKKTSVVKNSIFLEQGQLTVNNVVHATGEYTNASGITWSQFALSGQEDKLWIDKRALQA

[0049] Example 2 Preparation and Activity Detection of Sucrase

[0050] Step 1: Preparation of linearized cloning vector for sucrase gene

[0051] Using the DNA synthesized in Example 1 as a template, the complete CDS was cloned into the pET28a plasmid and digested with EcoRI enzyme. The digestion product was recovered by gel and used as a linearized cloning vector for later use.

[0052] Step 2: Prepare E. coli BL21(DE3) competent cells

[0053] Add 5 μL of E. coli BL21 cells to a 5 mL LB test tube and culture at 37°C, 200 rpm for 12 h. Add 1 mL of culture solution to a shake flask containing 100 mL LB medium and culture at 37°C, 200 rpm. When the OD600 of the cells is 0.5-0.6, take out the shake flask, place it on ice for 10 min, centrifuge it at 4°C, 4100 rpm for 10 min, discard the supernatant, suspend the precipitate with 2 mL of 0.05 M CaCl2 solution containing 15% glycerol, and store it at -80°C after aliquoting for later use.

[0054] Step 3: Induction of expression and enzyme purification

[0055] Select positive clones and culture them in LB medium. When OD600 = 0.6-0.8 (with sterile LB medium as control), add isopropylthiogalactoside (IPTG) to a final concentration of 0.5mM and induce the expression of the target protein at 16°C overnight. After centrifugation of the fermentation broth, collect the bacterial precipitate, add citric acid-disodium hydrogen phosphate buffer to suspend it, and then ultrasonically disrupt it. The supernatant is collected as the crude enzyme solution. According to the His-Tag label on the fusion protein, the target protein is purified using a nickel affinity chromatography column, and the target protein is eluted using imidazole solutions of different concentrations. Finally, the molecular weight and purity of the target protein are detected by 10% SDS-PAGE electrophoresis. Figure 1 As shown, the purified protease solution has a single band in the electrophoresis diagram, and the size is consistent with the molecular weight of the theoretical protein plus the recombinant tag, indicating that the protein is successfully expressed in the supernatant and the purified protein can be used for subsequent experiments.

[0056] Example 3 Enzyme activity detection: 200μL reaction system contains 5% (w / v) sucrose and 20μg / mL pure enzyme, final concentration 50mM pH 6.0 phosphate buffer, react in a 35℃ water bath for 24h, after the reaction is completed, centrifuge at 15,000g for 10min, take the supernatant, add 3 times the volume of methanol to the supernatant, let stand overnight, centrifuge at 16,000g for 8min, and collect the precipitate. Determined by high performance liquid chromatography (GB / T23528.2-2021). The results are as follows Figure 2 It was found that the product was consistent with the standard fructooligosaccharide. The main fructooligosaccharides produced were GF2, GF3, and GF4, among which GF2 had the highest content.

[0057] Example 4: Preparation of microcapsules

[0058] Preparation of enzyme solution: Dissolve the fructooligosaccharide sucrase in the above example in water and adjust the concentration to 10 mg / mL.

[0059] Preparation of wall material solution: Dissolve gum arabic in water and stir well to ensure complete dissolution, with a concentration of 10% w / v.

[0060] Mixing: Mix the enzyme solution and the wall material solution in a volume ratio of 1:2 and stir thoroughly to ensure that the enzyme is evenly coated by the wall material.

[0061] Spray drying: The mixed solution was spray dried by a spray dryer. Inlet air temperature: 180°C, outlet air temperature: 80°C, spray flow rate: 10 mL / min, feed rate: 20 mL / min, to ensure the formation and drying of microcapsules.

[0062] Collection and treatment: Collect the spray-dried microcapsule powder, screen it to remove large particles and impurities, and use a standard sieve to collect the microcapsule powder that passes through a 40-mesh (mesh opening of about 425 μm) sieve but does not pass through a 100-mesh (mesh opening of about 150 μm) sieve.

[0063] Example 5: Application of enzymes and microcapsules in baking

[0064] Cake Preparation

[0065] 1. Prepare the ingredients: 4 eggs, 120g sugar, 120g low-gluten flour, 30g honey, 30ml milk, 30ml vegetable oil, 1 teaspoon vanilla extract (optional), a few drops of lemon juice (optional), 2 g salt, enzyme (microcapsule): no addition in the control group; 200ng enzyme or 500ng microcapsule in the experimental groups.

[0066] 2. Mix and beat: Put eggs, egg yolks, honey and sugar in a mixing bowl, add enzymes or microcapsules, and beat with a blender. Sift and add soft flour, then add milk heated to 36°C, salt, and vegetable oil, and mix thoroughly to form a batter.

[0067] 3. Baking: Pour the batter into a 6-inch round pan lined with baking paper, gently shake the pan a few times to remove large bubbles. Place the pan in a preheated oven and bake at 180°C for about 40-50 minutes.

[0068] 4. After cooling, analyze the fructo-oligosaccharide content in the finished product.

[0069] Preparation of sweet bread

[0070] 1. Prepare the ingredients: high-gluten flour: 300 grams, sugar: 60 grams, salt: 1 / 2 teaspoon, instant yeast: 3 grams (about 1 small packet), whole milk: 150 ml (warm, about 35°C), egg: 1 (room temperature), unsalted butter: 50 grams (softened at room temperature), enzyme (microcapsule): no addition in the control group; 200ng enzyme or 500ng microcapsule in the experimental groups respectively.

[0071] 2. Mix and ferment: Mix all the ingredients with the enzyme (or microcapsule). Place the dough in a lightly oiled fermentation bowl, cover with a damp cloth or plastic wrap, and leave in a warm place to ferment for about 1 hour, or until the dough expands to twice its original size. Take out the fermented dough, press it gently to release the air, divide it into several small doughs, roll them into balls, and place them on a baking tray.

[0072] 3. Secondary fermentation and baking: Place the baking tray in the oven, do not turn on the oven, place a bowl of hot water next to it to maintain humidity, and perform the second fermentation for about 30-40 minutes. After the second fermentation is completed, preheat the oven to 180°C and bake for 8 minutes.

[0073] Fructooligosaccharides and sucrose were extracted from the samples and quantitatively analyzed using high performance liquid chromatography (GB / T23528.2-2021). The results are shown in Table 1. The total content of fructooligosaccharides in cakes or breads containing enzymes or microcapsules was significantly higher than that in the control group (p≤0.01), and the content of fructooligosaccharides in cakes or breads containing microcapsules was also significantly higher than that in samples containing pure enzymes (p≤0.05), and the sucrose content was also significantly reduced accordingly. This may be caused by premature production and premature decomposition of fructooligosaccharides in the pure enzyme group, or by the rapid inactivation of the pure enzyme.

[0074] Table 1 Fructo-oligosaccharide and sucrose contents in different treatments

[0075]

[0076] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may 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 the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An enzyme used in food baking, characterized in that: The amino acid sequence of the enzyme is shown in SEQ ID NO.

2.

2. A microcapsule used in food baking, characterized in that: The microcapsule comprises the enzyme of claim 1.

3. The microcapsule according to claim 2, characterized in that The microcapsule further comprises edible auxiliary materials.

4. The microcapsule according to claim 3, characterized in that The auxiliary material is one or more of edible proteins, lipids or sugars.

5. The microcapsule according to claim 3, characterized in that The auxiliary material is an edible emulsifier.

6. The microcapsule according to claim 3, characterized in that The auxiliary material is used as wall material.

7. Use of the enzyme according to claim 1 or the microcapsule according to claim 2 in food baking.

8. A method for baking food, characterized in that: The enzyme according to claim 1 or the microcapsule according to claim 2 is catalyzed during the fermentation stage of the baked material.

9. The method according to claim 8, characterized in that The enzyme according to claim 1 or the microcapsule according to claim 2 is added during the fermentation stage of the baked material or before the fermentation stage of the baked material.

10. The method according to claim 8, characterized in that The baking material contains sucrose.

11. A gene encoding the enzyme according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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