1 Materials and methods
1.1 Experimental materials
1.1.1 Raw and auxiliary materials
Silk, kudzu, wolfberry, ebony All were purchased from Xuzhou market. Sucrose, neutral protease (130000 u/g), and trypsin (4000 u/g) were provided by the laboratory, and papain (650000 u/g) was provided by Guangxi Haifa Bioenzyme Products Factory.
1.1.2 Main reagents
Formaldehyde reagent, concentrated sulfuric acid, concentrated hydrochloric acid, sodium hydroxide, anhydrous sodium sulfate, anhydrous calcium chloride, chlorine water, hydrogen phthalate Potassium, methyl red, copper sulfate, boric acid, beef extract, peptone, agar, salt, 731 and 723 anion and cation exchange resins and mixed prompt agent: a mixture of 1 volume of methylene blue and 2 volumes of methyl red indicator.
1.1.3 Main equipment
Electric constant temperature drying oven, electric constant temperature incubator, multi-functional grinder, low-speed large-scale centrifuge, rack balance, magnetic stirrer, precision pH meter , electronic balance, digital constant temperature water bath, portable pressure steam sterilizer, induction cooker.
1.2 Experimental method
1.2.1 Process flow
(1) Process flow for preparing beverages using acidolysis solution
Degumming of silk →Acid hydrolysis→Cation exchange resin deacidification→Silk fibroin amino acids
Chinese herbal medicine→Cleaning→Extraction→Coarse filtration→Centrifugal blending→
Flavor, VC, sucrose, etc.
Filling→Sealing→Sterilization→Cooling→Sensory evaluation and hygienic testing→Finished product
(2) Process flow of beverage preparation using enzymatic solution
Silk degumming→CaCl2 Dissolve silk fibroin → enzymatic hydrolysis → enzyme inactivation → remove CaCl2 → silk fibroin amino acids
Chinese herbal medicine → cleaning → extraction → coarse filtration → centrifugation → preparation → filling
Flavor, VC, Sucrose, etc.
→Sealing→sterilization→cooling→sensory evaluation and hygienic testing→finished product
1.2.2 Method of silk degumming
Wash and dry The final silk was boiled (20min, 30min, 40min) in Na2CO3 solution (0.4%, 0.5%, 0.6%) to determine the optimal degumming conditions.
1.2.3 Method of acid hydrolysis of silk fibroin
Dry the degummed silk fibroin to do a single factor experiment. First, select an acidity of 3M, a solid-liquid ratio of 1:50, acidolysis at 111°C for 15 hours, and measure the amino acid content per hour to get a suitable range of acidolysis time; then select a solid-liquid ratio of 1:50, and the acidolysis time is between Within the above determined range, acidolysis at different concentrations such as 1.0M, 1.5M, 2.0M, 2.5M, 3.0M, 3.5M, 4.0M, 4.5M, 5.0M, etc., at 110°C can be used to select a suitable acid concentration range; Finally, the acid concentration and time were selected to be within the appropriate range, and the most suitable was determined by selecting different solid-liquid ratios of 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, and 1:80. Solid-liquid ratio range. The obtained acid concentration, solid-liquid ratio, and acidolysis time were selected from 3 levels within appropriate ranges to conduct an orthogonal experiment of 3 factors and 3 levels to find out the optimal acidolysis conditions.
1.2.4 Dissolution of silk fibroin in CaCl2
Prepare different concentrations (30%, 35%, 40%, 45%, 50%, 55%, 60%, 65 %,) CaCl2 solution, measure the dissolution effect of silk fibroin in it, and find out the optimal concentration of CaCl2 for dissolving silk fibroin.
1.2.5 Research methods of enzymatic hydrolysis process (1) Selection of the best hydrolase. The silk protein liquid was enzymatically hydrolyzed under the optimal enzyme dosage, enzymatic hydrolysis temperature, enzymatic hydrolysis pH value and the same enzymatic hydrolysis time of the three proteases. The enzymatic hydrolysis effects of the three enzymes were compared, and the hydrolysis effect was selected based on the amino acid yield. The best enzymes. (2) Determination of enzymatic hydrolysis conditions. Based on the optimal silk fibroin protease determined in the above experiments, orthogonal experiments were conducted on three levels of substrate concentration, pH value, and temperature to determine the optimal conditions for enzymatic hydrolysis.
The substrate concentrations were 4%, 5%, and 6% respectively, the pH values ??were 5.0, 5.5, and 6.0 respectively, and the enzymatic hydrolysis temperatures were 50°C, 55°C, and 60°C respectively.
1.2.6 Deacidification of the acidolysis solution
Use 732 type strong acid cation exchange resin to deacidify the acidolysis solution. When the effluent reacts to cause ninhydrin to turn purple, Stop loading the column, wash the ion exchange column with deionized water until the effluent is nearly neutral, and then use 0.5M ammonia water to elute the amino acids until the ninhydrin no longer appears purple.
1.2.7 Extraction of Chinese herbal medicine juice
Extract the washed Chinese herbal medicine and purified water in a 90°C water bath for 1 hour at a ratio of 1:10 to obtain the liquid. Add 8 times the amount of pure water to the residue and extract it in a 90°C water bath for 0.5 hours. Combine the filtrate and residue liquid and centrifuge them with a low-speed large-capacity centrifuge for later use.
1.2.8 Beverage formulation
Design an orthogonal experiment with 3 factors and 3 levels to select the beverage formula. In order to score beverages, a scoring standard needs to be determined based on the color, aroma, taste and organizational state of the product.
1.2.9 Beverage sterilization and hygiene testing
Beverage sterilization: Sterilize the finished product at 90°C for 15min, 20min, and 25min respectively, and then place it in a 37°C constant temperature water bath incubator for observation Its stability, choose the best sterilization time. The total number of bacteria was determined using the plate colony counting method.
1.3 Test items and methods
The crude protein in silk was determined using the micro Kjeldahl method. The amino nitrogen content was measured using the formaldehyde potentiometric method.
1.3.1 Test of the deacidification effect of silk fibroin enzymatic hydrolyzate
Use 732 type strong acid cation exchange resin, put it into a resin column with a height of 40cm and Φ15, wait for the effluent Make ninhydrin purple, indicating that the resin is saturated and the amino acid liquid flows out. At this time, stop loading the column, wash the ion exchange column with deionized water until the effluent is nearly neutral, and then use 0.5M ammonia water to elute the amino acid until no more The color reaction of ninhydrin indicates that all amino acids have been removed.
1.3.2 Detection of deionization effect of anion and cation exchange resin
Put the silk fibroin solution dissolved in CaCl2 solution through a cation exchange resin column with a column height of 40cm and Φ15. Ca2+, use NaOH solution to detect whether there is Ca2+ in the effluent. When no white precipitate appears, pass through an anion exchange resin with a column height of 40cm and Φ15 to remove Cl-. Use AgNO3 to detect whether there is Cl- in the effluent. When there is no white precipitate, Stop the column when it appears.
2 Results and Discussion
2.1 Determination of Silk Degumming Conditions
The minor components in silk are mainly distributed in sericin. In order to ensure the amino acid content of silk fibroin quality, degumming must be carried out. Silk fibroin is insoluble in water, while sericin is water-soluble. Although sericin is hydrophilic, it takes a long time to separate it from silk fibroin in an aqueous medium, and it requires high-temperature treatment. For this reason, Na2CO3 is used as the separation medium. Among them, the sericin removal rate (%) = protein content in the sericin liquid (g) × 100% total sericin protein (g). The test results are shown in Table 1.
Table 1 Orthogonal experimental design and results of Na2CO3 detachment of silk fibroin
Treatment time (min) Concentration (%) Sericin removal rate (%)
< p> 1 1 (20) 1 (0.4) 48.52 1 2 (0.5) 35.7
3 1 3 (0.6) 59.8
4 2 ( 30) 1 60.3
5 2 2 100
6 2 3 100
7 3 (40) 1 73.9
8 3 2 100
9 3 3 100
As can be seen from Table 1, boiling in 0.5% NaCO3 solution for 30 minutes has the best degumming effect. To prevent water from evaporating and affecting the NaCO3 concentration during boiling, a lid must be placed on the container. Silk becomes silk fibroin after degumming, and sericin exists in aqueous medium.
2.2 Acid hydrolysis single factor experiment
Use 1g of silk fibroin to be hydrolyzed in 3M H2SO4, solid-liquid ratio 1:60, and 110°C. From experiments, it was found that silk fibroin can be completely dissolved in H2SO4 for 3 hours. Measuring the hydrolysis situation from 4 to 15 hours showed that the amino acid content was higher from 8 to 10 hours, and changed little after 10 hours, or even began to decrease.
Use silk fibroin to acidolyze at a solid-liquid ratio of 1:60 and 110°C for 8 hours to compare the relationship between different H2SO4 concentrations and amino nitrogen content. The result is that acidolysis concentration that is too low or too high will affect the amino acid content. It is better to control it between 3M and 4M.
Use silk fibroin to acidolyze at 3M H2SO4 at 110°C for 8 hours. Measure the effect of solid-liquid ratio on acidolysis. Since silk fibroin can only be soaked with a solid-liquid ratio of at least 1:20, this is the minimum solid-liquid ratio. liquid ratio. The results showed that the hydrolysis effect was best when the solid-liquid ratio was between 1:50 and 1:70.
2.3 Acid hydrolysis orthogonal test
From the acid hydrolysis single factor experiment, it is known that the factors affecting acid hydrolysis are mainly acid concentration, solid-liquid ratio, and time, and each is selected accordingly. For the three levels of factors, the experimental results are shown in Table 2.
Table 2 Acid hydrolysis orthogonal test results and analysis
Column A B C
Factor time (h) Acidity (M) Solid-liquid amino acid yield (%)
Experiment 1 1 (8) 1 (3) 1 (1:50) 69.1
Experiment 2 2 (9) 2 (3.5) 2 (1:60) 73.2
Experiment 3 3 (10) 3 (4) 3 (1:70) 71.9
Experiment 4 2 1 2 87.2
Experiment 5 2 2 3 89.1
Experiment 6 2 3 1 86.5
Experiment 7 3 1 3 78.4
Experiment 8 3 2 1 80.3
Experiment 9 3 3 2 79.6
The average value 1 71.400 78.233 78.633
The average value of 2 87.600 80.867 80.000
Average 3 79.433 79.800
Extreme difference 16.200 2.634 1.367
From Table 2, the combination with the highest acidolysis yield is A2B2C3, but from Table 4, the ideal combination is A2B2C2, so A2B2C2 is compared again, and the result is that the silk fibroin amino acid yield is 89.8%. Slightly better than A2B2C3, so the optimal conditions for acidolysis are determined to be an acid concentration of 3.5M, a solid-liquid ratio of 1:60, and acidolysis at 110°C for 9 hours.
2.4 Results of CaCl2 dissolving silk fibroin
The dissolution characteristics of silk fibroin in CaCl2 solution are quite special. It is almost not dissolved when the concentration is lower than 35% or higher than 55%, as shown in Table 3. 40% is the best.
Table 3 The relationship between CaCl2 concentration and silk fibroin dissolution
1 2 3 4 5 6 7 8
CaCl2 concentration 30% 35% 40% 45% 50% 55% 60% 65%
Solubility of silk fibroin 0 14.5% 100% 32.5% 15.5% 0 0 0
2.5 Determination of the optimal hydrolase
Papain The pH is controlled at 6.0, the temperature is 50°C, E/S=10%, and the substrate concentration, that is, the silk fibroin concentration is 4%, for enzymatic hydrolysis. Experiments show that the enzymatic hydrolysis rate increases rapidly in the first hour, reaches 0.07 mg/ml in 3 hours, and then increases slowly. Trypsin is used for enzymatic hydrolysis at pH 8.0, temperature 40°C, E/S=2%, and silk fibroin concentration 4%. It reaches the maximum value of 0.053mg/ml in 7h.
Neutral protease enzymatically hydrolyzes silk fibroin at pH 7.0, temperature 50°C, E/S=2%, and silk fibroin concentration 4%, reaching 0.065 mg/ml in 3 hours. It can be seen that papain has the best hydrolysis effect.
2.6 Orthogonal experiment of papain hydrolyzing silk fibroin
Perform an orthogonal experiment to determine the best combination of conditions for papain to hydrolyze silk fibroin. The optimal enzymatic conditions for papain are pH 5.5, temperature 55°C, substrate concentration 5%, and enzyme amount 10%. In the end, 0.184g of amino acids can be obtained from 1.001g of silk fibroin. From the comparison of acid hydrolysis and enzymatic hydrolysis of silk fibroin, it can be concluded that acid hydrolysis is obviously better than enzymatic hydrolysis.
2.7 Beverage Preparation
When making beverages, attention should be paid to the adjustment of flavor. The main factors affecting the flavor of this drink are amino acid concentration, Chinese herbal medicine concentration, and sugar concentration. Therefore, a 3-factor 3 is designed. Horizontal orthogonal experiments to optimize beverage recipes. As shown in Table 4.
Table 4 Beverage preparation results and analysis
Column A B C
Factor amino acid Chinese herbal medicine sugar concentration (%) Experimental results
Concentration ( %) Concentration (%)
Experiment 1 1 (0.3) 1 (2) 1 (8) 7.8
Experiment 2 1 2 (3) 2 (9) 7.4
Experiment 3 1 3 (4) 3 (10) 7.2
Experiment 4 2 (0.4) 1 2 9
Experiment 5 2 2 3 8.5
< p> Experiment 6 2 3 1 8Experiment 7 3 (0.5) 1 3 7.6
Experiment 8 3 2 1 7
Experiment 9 3 3 2 7.5
Mean 1 7.467 8.133 7.600
Mean 2 8.500 7.633 7.967
Mean 3 7.367 7.567 7.767
Range 1.133 0.566 0.367
p>The optimal formula of the drink can be obtained from Table 4: the amino acid concentration is 0.4%, the concentration of Chinese herbal juice is 2%, the sugar concentration is 9%, and a small amount of peach flavor and VC are added to obtain the finished product.
2.8 Storage stability test of beverages
In this test, sterilization is performed before and after canning, and the Chinese herbal juice is centrifuged, so the beverage obtained is clear and transparent. After canning, observe its stability through different sterilization times. Sterilize at 90°C for 15min, 20min, and 25min respectively, then cool and place in a 37°C constant temperature incubator for observation. There was no change after 9 days, and it was still a clear and transparent yellow-brown solution.
In order to determine the optimal sterilization time, the finished product after 9 days of storage was subjected to a bacterial detection experiment. The result was that the total number of bacteria after sterilization at 90°C for 25 minutes was 56/ml, which met the national standard requirements.
3 Product quality standards
3.1 Sensory indicators
Color: yellowish brown, uniform. Flavor: sweet and sour, with slight bitterness of silk fibroin amino acids and Chinese herbal medicine, no peculiar smell. Tissue shape: The juice is transparent, without impurities, and no precipitation appears after being left for a long time.
3.2 Physical and chemical indicators
Acidity: pH 3.5 ~ 4.0, total bacterial count ≤ 100/ml, coliform count ≤ 3/ml, pathogenic bacteria must not be detected.
4 Conclusion
Acid hydrolysis of silk fibroin has a higher amino acid yield than enzymatic hydrolysis. The acid hydrolysis yield is 89.1%, while the enzymatic hydrolysis yield is only 18.1%. Since acid hydrolysis is easier to operate than enzymatic hydrolysis and the hydrolyzate has better color and smell, acid hydrolysis is used when making beverages. The optimal degumming condition determined is 0.5% Na2CO3 boiling treatment for 30 minutes. Under this condition, all sericin is removed without loss of silk fibroin.
After degumming, the silk fibroin was washed and dried and then acidolyzed. The optimal process conditions for acidolysis were 3.5MH2SO4, solid-liquid ratio 1:60, hydrolysis at 110°C for 9 hours, and the amino acid yield was as high as 89.8%. The amino acid solution deacidified by the cation exchange resin was mixed with Chinese herbal medicine. The optimal formula for the beverage was as follows: silk fibroin amino acid concentration 0.4%, Chinese herbal medicine concentration 2%, sugar concentration 9%, and VC concentration 0.2%. This produces a clear, sweet and sour tawny drink.