Sugarcane tail vegetable protein beverage rich in amino acid and preparation method thereof

Abstract

The invention belongs to the field of vegetable protein beverages, and particularly relates to a sugarcane tail tip vegetable protein beverage rich in amino acid and a preparation method thereof, and the preparation method comprises the following steps: 1, preparing sugarcane tail tip crude protein through alkali dissolution and acid precipitation; 2, constructing recombinant lactobacillus bulgaricus, wherein the recombinant lactobacillus bulgaricus expresses neutral protease with an amino acid sequence shown as SEQ ID NO: 2 or a variant of the neutral protease; 3, recombinant lactobacillus bulgaricus is inoculated to the sugarcane tail crude protein according to the inoculation amount of 0.8%, the initial pH is 7.0, the fermentation temperature is 40 DEG C, fermentation is conducted on a lactic acid bacteria fermentation medium for 11 h, strains are filtered, fermentation liquor rich in amino acid is prepared, a sugarcane fruity additive is added, pasteurization and refrigeration are conducted, and the sugarcane tail drink rich in amino acid is obtained. Recombinant lactobacillus bulgaricus is constructed, the content of free amino acid in the sugarcane tail tip beverage is increased, the sugarcane tail tip beverage is easier to absorb by a human body, and the fermented beverage does not contain alcohol and is healthier.

Description

Technical Field

[0001] The invention belongs to the field of plant protein beverages, and particularly relates to a sugarcane tail plant protein beverage rich in amino acids and a preparation method thereof. Background Art

[0002] As people's living standards improve, their expectations for beverages are also becoming increasingly sophisticated. The global beverage market is currently trending towards natural beverages, particularly those rich in vitamins and amino acids, such as bamboo shoots and pollen. Currently, the global annual sugarcane production is approximately 1.06 billion tons. Because sugarcane straw is useless in the sugar industry, most of it is discarded. Only a small portion is reused, after simple processing, for return to farmland or as livestock feed. However, this processing yields low added value. Sugarcane straw, in fact, is rich in nutritional value and an underappreciated treasure of nature. This is especially true of the sugarcane tip, located at the growing point of the sugarcane stem. It is rich in inorganic elements, protein, starch, vitamins, amino acids, and enzymes essential for growth and development, as well as various minerals and trace elements such as potassium, calcium, magnesium, zinc, and iron. Furthermore, the tip contains dietary fiber, which promotes intestinal motility, improves digestive health, and helps control blood sugar levels. Even more surprising, it also contains a small amount of natural sugars and various vitamins, which can promote skin health and enhance the body's immune system. It is reported that sugarcane tails contain more than 20 kinds of amino acids and anti-cancer ingredients, which have unexpected effects on treating hypertension and preventing and treating cancer. Therefore, making discarded sugarcane tails into drinks rich in natural plant protein can create good economic value.

[0003] Currently, beverages made from sugarcane tips are typically processed by juicing. Due to the high fiber content and relatively low sugar content of sugarcane tips, the protein and cellulose in the tips are combined, making it difficult to separate the rich nutrients using simple mechanical processing. This results in low nutritional value and a loss of commercial value. Currently, plant-based protein beverages are mostly produced through fermentation using lactic acid bacteria or yeast, but this also presents several drawbacks. Yeast secretes extracellular alkaline proteases, which break down crude protein in the tips, particularly casein, into amino acids, producing an amino acid-rich fermentation broth. However, beverages produced using yeast fermentation contain ethanol as a byproduct. Alcohol is a direct toxin to the liver, impairing liver function. It can also suppress nervous system function, leading to slowed thinking, poor reaction times, and memory loss. It can also damage the cardiovascular and digestive systems. Using separation technology to separate the alcohol increases costs and can also result in the loss of other nutrients during the ethanol separation process. Although the use of lactic acid bacteria for fermentation can reduce the production of ethanol, especially the use of positive lactic acid bacteria for fermentation, which theoretically does not produce ethanol, the range of substrates for the enzymatic hydrolysis of its extracellular protease is relatively narrow, and it is unable to comprehensively degrade the crude protein in the sugarcane tail.

[0004] Based on the above background, the present invention first selected the positive lactic acid bacterium Lactobacillus bulgaricus as the fermentation strain. To enhance its ability to degrade crude protein in sugarcane tails, a heterologous neutral protease encoding gene was introduced into Lactobacillus bulgaricus. Enzyme mutation was performed to target the decreased enzyme activity in acidic fermentation environments, and pH-stable enzyme variants were screened. As a result, a sugarcane tail plant protein beverage rich in amino acids was successfully prepared. Summary of the Invention

[0005] In view of the above-mentioned defects of the prior art, the present invention provides a method for preparing a sugarcane tail plant protein beverage rich in amino acids, which comprises the following steps: 1. Preparation of crude protein from sugarcane tail by alkali dissolution and acid precipitation; 2. Constructing a recombinant Lactobacillus bulgaricus, wherein the recombinant Lactobacillus bulgaricus expresses a neutral protease having an amino acid sequence as shown in SEQ ID NO: 2 or a variant thereof; 3. The recombinant Lactobacillus bulgaricus was inoculated onto the sugarcane tail crude protein at an inoculum rate of 0.8%, with an initial pH of 7.0 and a fermentation temperature of 40°C. The fermentation was carried out on a lactic acid bacteria fermentation medium for 11 hours. The strain was filtered to prepare an amino acid-rich fermentation broth. A sugarcane fruit flavor additive was added, the broth was pasteurized, and the broth was refrigerated to obtain an amino acid-rich sugarcane tail beverage.

[0006] Preferably, the nucleic acid coding sequence of the neutral protease is shown in SEQ ID NO: 1.

[0007] Preferably, the Lactobacillus bulgaricus is CICC 6047.

[0008] Preferably, the neutral protease variant is T81R, Q149A, V159D, E185R.

[0009] Preferably, the amino acid sequences of the neutral protease variants T81R, Q149A, V159D, and E185R are shown in SEQ ID NOs: 3-6, respectively.

[0010] Preferably, the lactic acid bacteria fermentation medium contains (in percentage by weight): 0.5-1% glucose, 0.2-1% yeast extract, 1-8% molasses, 0.2-1% sodium acetate, 0.1-1% diammonium hydrogen citrate, 0.1-1% dipotassium hydrogen phosphate, 0.01-0.1% magnesium sulfate, 0.005-0.02% manganese sulfate, 0.01-0.5% Tween, and the balance is water.

[0011] Preferably, the method for preparing crude protein from sugarcane tails comprises the following steps: Enzymatic hydrolysis of sugarcane tails with cellulase; The sugarcane tail solution after cellulase treatment was dissolved by adding pH 8.5 NaOH solution; Centrifuge to separate impurities and collect the supernatant; Acidic solution was added to the supernatant to adjust the pH to 4.6, and the precipitate was collected.

[0012] Preferably, the sugarcane tail is further subjected to the following pretreatment steps: 1. Select fresh sugarcane tips and cut them into small pieces; 2. Crush the sugarcane tail to 40-60 mesh; 3. Alkali treatment of crushed sugarcane tails; In another preferred embodiment of the present invention, the present invention provides an engineered strain, wherein the engineered strain expresses a neutral protease having an amino acid sequence as shown in SEQ ID NO: 2 or a variant thereof.

[0013] In another preferred embodiment of the present invention, the present invention provides a sugarcane tail plant protein beverage rich in amino acids, which is prepared by the preparation method of the sugarcane tail plant protein beverage rich in amino acids described in the present invention.

[0014] The present invention constructs recombinant Lactobacillus bulgaricus, which increases the free amino acid content in the sugarcane tail beverage, making it easier for human body to absorb. In addition, the fermented beverage does not contain alcohol and is healthier.

[0015] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the test result of tyrosine content in the fermentation broth of crude protein from sugarcane tails fermented with recombinant Lactobacillus bulgaricus.

[0017] Figure 2 These are the test results for tyrosine content in crude protein hydrolysates of sugarcane tail tips enzymatically hydrolyzed by T81R, Q149A, V159D, and E185R variants. DETAILED DESCRIPTION

[0018] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0019] Experimental Materials The fermentation strain used was the commercially available Lactobacillus delbrueckii subsp. bulgaricus CICC 6047, strain number: CICC 6047, Latin name: Lactobacillus delbrueckii subsp. bulgaricus CICC 6047.

[0020] The lactic acid bacteria fermentation medium contains (in percentage by weight): 0.5-1% glucose, 0.2-1% yeast extract powder, 1-8% molasses, 0.2-1% sodium acetate, 0.1-1% diammonium hydrogen citrate, 0.1-1% dipotassium hydrogen phosphate, 0.01-0.1% magnesium sulfate, 0.005-0.02% manganese sulfate, 0.01-0.5% Tween, and the balance is water.

[0021] Example 1: Construction of recombinant Lactobacillus bulgaricus The specific operations are as follows: 1. Construction and assembly of plasmid vectors expressing neutral protease Using the gene of GenBank: X76986.1 as a template, amplification primers were designed, and the amplified product was inserted into the pET28 plasmid backbone to obtain the expression vector pET28-NPR.

[0022] 2. Construction of engineered bacteria expressing neutral protease Add a small amount of the plasmid vector solution to 100 μL of competent Lactobacillus bulgaricus. Mix gently and place on ice for 30 minutes. Then, heat shock at 42°C for 1.5 minutes. Finally, cool the mixture to 0°C on ice for 2 minutes. Add 900 μL of antibiotic-free LB medium to the mixture, and resuscitate at 37°C, 220 rpm, for 30 minutes. Then, spread the culture onto an LB plate containing kanamycin and incubate at 37°C for 12-14 hours to obtain a monoclonal strain. Amplified products were verified by Sanger sequencing to ensure sequence accuracy.

[0023] Experimental Example 2: Extraction of crude protein from sugarcane tips and preliminary fermentation with recombinant Lactobacillus bulgaricus Extraction process of crude protein from sugarcane tail: 1. Select fresh sugarcane tips as raw materials and cut them into small pieces of 2-3 cm; 2. Use a grinder to further grind the sugarcane tail obtained in step 1 until it can pass through a 40-60 mesh sieve to ensure uniform particle size; 3. Soak the crushed sugarcane tips in a dilute alkali solution (such as 0.1 mol / L sodium hydroxide) for 6-8 hours to break the connection between lignin, cellulose, and hemicellulose. After treatment, rinse the sugarcane tips thoroughly with clean water until they are neutral. 4. Dry the crushed sugarcane tips in a drying oven at 60-80°C until the moisture content is less than 10%; 5. Add cellulase (9012-54-8) solution to the dried sugarcane tips, adjust the cellulase concentration to 420 U / g (sugarcane tips substrate), and react at 38°C for 48 hours; 6. Add the cellulase-treated sugarcane tail solution to a pH 8.5 NaOH solution at a ratio of 1:10 and dissolve at 45°C at a rotation speed of 150 rpm for 4 hours; 7. Centrifuge at 8000 rpm for 8 min to further filter out insoluble matter and collect the supernatant; 8. Add acidic solution to the supernatant, adjust the pH to around 4.6, and stir at 150 rpm for 2 hours to fully precipitate the protein; 9. Collect the protein precipitate by centrifugation.

[0024] The process of preparing amino acid-rich fermentation broth by fermenting crude protein from sugarcane tail with recombinant Lactobacillus bulgaricus: According to the inoculation amount of 0.8% (strain mass / sugarcane tail crude protein), the initial pH was 7.0, the fermentation temperature was 40°C, and the fermentation was carried out on the lactic acid bacteria fermentation medium for 11 hours. The strain was filtered to prepare the fermentation liquid rich in amino acids.

[0025] Starting from 3 h of fermentation, 1 ml of the fermentation broth was collected every 1 h and the tyrosine content was determined by high-performance liquid chromatography. The reaction conditions were: Waters C18 column, 8 mmol / L potassium dihydrogen phosphate solution-methanol (9:1) as the mobile phase, detection wavelength at 280 nm, flow rate at 1.0 mL / min, and column temperature at 35°C.

[0026] The results are as follows Figure 1 As shown in the figure, with Lactobacillus bulgaricus CICC 6047 as the control, after 8 hours of reaction, the tyrosine accumulation leveled off and reached 8.2 mg / kg (crude protein from sugarcane tails), while the tyrosine accumulation in the control group was stable at 2.1 mg / kg (crude protein from sugarcane tails). It can be seen that the protein decomposition ability of Lactobacillus bulgaricus after recombinant neutral protease was significantly enhanced.

[0027] An analysis was conducted on the situation in which the tyrosine decomposition rate was significantly reduced after 8 hours of fermentation with the recombinant Lactobacillus bulgaricus. It was found that the accumulation of lactic acid led to a significant decrease in pH, which was caused by a decrease in the activity of the neutral protease expressed by the recombinant Lactobacillus bulgaricus.

[0028] Experimental Example 3: Construction of neutral protease variants In order to improve the stability of neutral protease under low pH conditions, the neutral protease encoded by the nucleic acid sequence shown in GenBank: X76986.1 was used as the starting amino acid sequence for mutant construction. Primers for T81R, Q149A, V159D, and E185R variants were designed by site-directed mutagenesis. The amplified encoding gene was ligated into the vector pET28a, and the pET28a was introduced into Escherichia coli BL21 (DE3). High-expressing strains were first screened by small-scale culture, and then expanded and induced for expression. The cells were collected, washed, and ultrasonically disrupted in an ice bath to release the enzyme protein. The liquid was centrifuged at low temperature to remove the bacterial precipitate and obtain the supernatant of the T81R, Q149A, V159D, and E185R variants. The supernatant was purified by affinity chromatography and dialyzed and lyophilized to obtain the pure enzyme protein.

[0029] After 8 h of fermentation, the pH of the recombinant Lactobacillus bulgaricus was approximately 5. Under the condition of pH = 5, T81R, Q149A, V159D, and E185R variants were added at 1.25% of the substrate weight. After 5 h of enzymatic hydrolysis, the tyrosine content was detected.

[0030] like Figure 2 As shown, the T81R variant has a 5-fold improvement over the wild-type neutral protease, and the E185R variant has a 7-fold improvement over the wild-type neutral protease.

[0031] Experimental Example 4: Construction of recombinant T81R and E185R variants of Lactobacillus bulgaricus and preparation of amino acid-rich sugarcane tip beverages Recombinant Lactobacillus bulgaricus expressing the T81R and E185R variants was constructed according to the method of Example 1. Fermentation was carried out on a lactic acid bacteria fermentation medium at an inoculum size of 0.8% (strain mass / sugarcane tail crude protein), an initial pH of 7.0, and a fermentation temperature of 40°C for 11 hours. The strain was filtered to prepare an amino acid-rich fermentation broth. A sugarcane fruit flavor additive was added, the broth was pasteurized, and the broth was refrigerated to obtain an amino acid-rich sugarcane tail beverage.

[0032] The amino acid composition of the sugarcane tip beverage was tested using an HX-1800 fully automatic amino acid analyzer. The test results are shown in Table 1, and the unit of amino acid content is mg / kg.

[0033] Table 1 Amino acid composition of sugarcane tip beverage The pH stability of the recombinant T81R and E185R variants of the neutral protease of Lactobacillus bulgaricus variants T81R and E185R is significantly enhanced, and they have a stronger ability to decompose crude protein in sugarcane tails, which can increase the content of free amino acids in sugarcane tail beverages and are easier for the human body to absorb.

[0034] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for preparing a sugarcane tail plant protein drink rich in amino acids, comprising the following steps: (1) Preparation of crude protein from sugarcane tail by alkali dissolution and acid precipitation; (2) constructing a recombinant Lactobacillus bulgaricus, wherein the recombinant Lactobacillus bulgaricus expresses a neutral protease having an amino acid sequence as shown in SEQ ID NO: 2 or a variant thereof; (3) The recombinant Lactobacillus bulgaricus was inoculated onto the sugarcane tail crude protein at an inoculum rate of 0.8%, with an initial pH of 7.0 and a fermentation temperature of 40°C. The fermentation was carried out on a lactic acid bacteria fermentation medium for 11 h. The strain was filtered to prepare a fermentation broth rich in amino acids. A sugarcane fruit flavor additive was added, the broth was pasteurized, and the broth was refrigerated to obtain a sugarcane tail beverage rich in amino acids.

2. The preparation method according to claim 1, characterized in that The nucleic acid coding sequence of the neutral protease is shown in SEQ ID NO:

1.

3. The preparation method according to claim 1, characterized in that The Lactobacillus bulgaricus is CICC 6047.

4. The preparation method according to claim 1, characterized in that The neutral protease variants are T81R, Q149A, V159D, and E185R.

5. The preparation method according to claim 4, characterized in that The amino acid sequences of the neutral protease variants T81R, Q149A, V159D, and E185R are shown in SEQ ID NOs: 3-6, respectively.

6. The preparation method according to claim 1, characterized in that The lactic acid bacteria fermentation medium comprises, by weight percentage, 0.5-1% glucose, 0.2-1% yeast extract powder, 1-8% molasses, 0.2-1% sodium acetate, 0.1-1% diammonium hydrogen citrate, 0.1-1% dipotassium hydrogen phosphate, 0.01-0.1% magnesium sulfate, 0.005-0.02% manganese sulfate, 0.01-0.5% Tween, and the balance is water.

7. The preparation method according to claim 1, characterized in that The method for preparing crude protein from sugarcane tails comprises the following steps: (1) Enzymatic hydrolysis of sugarcane tails using cellulase; (2) The sugarcane tail solution treated with cellulase was dissolved in a pH 8.5 NaOH solution; (3) Centrifuge to separate impurities and collect the supernatant; (4) Add acidic solution to the supernatant, adjust the pH to 4.6, and collect the precipitate.

8. The preparation method according to claim 7, characterized in that The sugarcane tail is also subjected to the following pretreatment steps: (1) Select fresh sugarcane tips and cut them into small pieces; (2) Crush the sugarcane tail to 40-60 mesh; (3) Alkali-treated crushed sugarcane tips.

9. An engineered bacterium, which expresses a neutral protease having an amino acid sequence as shown in SEQ ID NO: 2 or a variant thereof.

10. A sugarcane tail plant protein drink rich in amino acids, prepared by the preparation method according to any one of claims 1 to 8.

Images