A method for low temperature preparation of betacyanins

Abstract

The application discloses a low-temperature preparation method of betalain, and comprises the following steps: after color protection of red beet pulp beating, low-temperature enzymolysis is carried out on the red beet pulp by using a composite enzyme to break the cell wall, and supernatant is obtained by centrifugation; the supernatant is pre-deoxidized by using a polyethylene glycol / ammonium sulfate aqueous two-phase system, and then refined by using a macroporous resin, and eluate is collected; inoculation of lactobacillus plantarum is carried out for low-temperature fermentation to remove the earthy smell, and then the betalain powder is obtained by vacuum concentration and spray drying. The whole process is carried out at low temperature, the extraction rate is more than 85%, the color value E535nm is more than 88, and the smell score is more than 4, which is significantly better than the prior art.

Description

Technical Field

[0001] This invention relates to the field of natural pigment preparation technology, specifically to a low-temperature preparation method for betalain. Background Technology

[0002] Beetroot red pigment is a water-soluble nitrogenous pigment extracted from the roots of red beets. It boasts vibrant color, high safety, and excellent antioxidant and free radical scavenging activities, making it widely used in food, beverages, cosmetics, and health products. However, beetroot red pigment is sensitive to temperature, light, and oxygen, and is prone to degradation and fading during processing and storage. Furthermore, red beets have a strong earthy taste, limiting its application.

[0003] In the related technology, CN202010942596A discloses a process for preparing deflavored beet red pigment, including the following steps: S1, crushing red beets and extracting them with warm water to obtain crude beet juice; S2, filtering the crude juice through a nanofiltration membrane to remove some macromolecular impurities to achieve preliminary deflavoring; S3, the nanofiltration permeate enters a macroporous adsorption resin column for pigment adsorption; S4, eluting the resin with ethanol to obtain a high-color-value beet red pigment concentrate.

[0004] However, this process relies solely on nanofiltration membranes for pre-deodorization, which has a weak ability to retain small-molecule earthy odor impurities, resulting in limited deodorization effects. Furthermore, the process only uses resin purification without a two-phase pre-sugar removal step. The large amount of sugar impurities in the feed solution will significantly increase the resin adsorption load and reduce the number of resin cycles. The existing process cannot integrate two-phase pre-sugar removal with resin purification in series, resulting in deficiencies in both product purity and production economy.

[0005] CN202411661187A discloses a processing technology for removing the earthy taste of red beets through microbial fermentation, comprising the following steps: S1, crushing and extracting red beets to obtain crude beet juice, and adjusting the pH value and soluble solids content of the liquid; S2, simultaneously inoculating a mixed strain of Bacillus subtilis and yeast, and performing constant temperature and closed fermentation; S3, after fermentation, filtering to remove the bacteria, and obtaining deodorized beet juice.

[0006] However, this process relies on yeast to participate in fermentation and metabolism, and alcohol byproducts are easily produced during fermentation, which can introduce new off-flavors into beet juice, affecting the flavor and quality of the finished pigment product, and the deodorization effect has obvious defects. Summary of the Invention

[0007] To address the problems mentioned in the prior art as described in the background section, this invention provides a low-temperature preparation method for betaine, comprising the following steps:

[0008] (1) Raw material pretreatment: Take fresh red beet root, wash, peel, cut into pieces to 5~10 mm, add 0.5~1.0 times the weight of raw material in pre-cooled deionized water (4℃), pulp, and add 0.1% ascorbic acid for color protection.

[0009] (2) Low-temperature enzymatic hydrolysis of compound enzyme: Add pectinase and cellulase compound enzyme to the slurry (the amount of enzyme added is 0.02%~0.05% of the weight of raw materials), stir and enzymatically hydrolyze at 15~20℃ and pH 4.5~5.0 for 60~120 min, and centrifuge to take the supernatant.

[0010] (3) Aqueous two-phase-resin series purification: Polyethylene glycol 4000 and ammonium sulfate are added to the supernatant to make the mass concentration of polyethylene glycol 18%~25% and ammonium sulfate 12%~18%. After stirring and extraction, the mixture is allowed to stand and separate into layers. The upper phase of polyethylene glycol is collected. After dilution, the upper phase is loaded onto a macroporous adsorption resin column, washed with water, and eluted with 30%~50% ethanol. The eluent is collected and polyethylene glycol is recovered by ultrafiltration.

[0011] (4) Fermentation, deodorization and concentration drying: Inoculate Lactobacillus plantarum (inoculation amount 2%~4%) into the eluent, ferment at 25~30℃ for 20~30 h, inactivate and then concentrate under vacuum at 45~50℃ to a solid content of 20%~35%, add maltodextrin and spray dry, with an inlet air temperature of 130~145℃ and an outlet air temperature of 65~75℃ to obtain betalain powder.

[0012] Preferably, in step (2), the mass ratio of pectinase to cellulase in the compound enzyme is 1:1.5 to 1:2.5, the pectinase activity is ≥30000 U / g, and the cellulase activity is ≥20000 U / g.

[0013] Preferably, in step (3), the macroporous adsorption resin is AB-8 or X-5 type non-polar macroporous resin, the column loading flow rate is 1~2 BV / h, the elution flow rate is 0.5~1 BV / h, and the ultrafiltration membrane has a molecular weight cutoff of 3000~5000 Da.

[0014] Preferably, in step (4), the number of viable Lactobacillus plantarum bacteria is ≥1×10⁻⁶. 9 CFU / mL, nitrogen gas was introduced during fermentation to maintain a micro-aerobic environment, and after fermentation, the temperature was raised to 60℃ and held for 10 min to inactivate the enzyme.

[0015] Preferably, in step (4), the spray drying feed rate is 15~25 mL / min, the atomizer speed is 20000~30000 r / min, and the moisture content of the resulting beet red powder is ≤5%.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The aqueous two-phase resin tandem purification process is adopted. The aqueous two-phase process can remove more than 80% of sugar impurities in advance, reduce the adsorption load of the resin, extend the service life of the resin, and the color value of the product at 535nm can reach more than 88 after tandem purification, effectively improving the purity of the pigment.

[0018] 2. Low-temperature fermentation with Lactobacillus plantarum is used for deodorization. Under conditions of 25~30℃, it can decompose earthy odor substances. The process hardly destroys betalains and does not generate alcohol byproducts. The odor score after deodorization can reach 4.6 points. The deodorization is mild and efficient.

[0019] 3. The overall process flow is smooth, with the temperature controlled below 20℃ during the cell breaking and refining stages and no higher than 50℃ during the concentration stage, which can reduce the thermal degradation of betalains; all raw materials used are food-grade and are equipped with conventional food production equipment, which is convenient for large-scale processing. Detailed Implementation

[0020] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0021] Example 1

[0022] (1) Raw material pretreatment: Fresh red beetroot (from Jiangsu Province), washed and peeled. Cut red beetroot into 8 mm pieces, take 1000g and add 500 mL of pre-cooled deionized water (4℃), pulp, and add 1.0 g of ascorbic acid for color protection.

[0023] (2) Low-temperature enzymatic hydrolysis of the compound enzyme: Add 0.8 g of pectinase (enzyme activity 35000 U / g) and 1.6 g of cellulase (enzyme activity 25000 U / g) to the slurry, and stir at 200 rpm for 90 min at 18℃ and pH 4.8. Centrifuge at 10000 r / min for 15 min and collect about 1400 mL of supernatant.

[0024] (3) Aqueous two-phase-resin tandem purification: Add PEG 4000 to the supernatant to a concentration of 22% and ammonium sulfate to a concentration of 15%, stir and extract for 25 min, then let stand at room temperature for 3 h to separate the layers, and collect the PEG supernatant. Dilute the supernatant 4 times and load it onto an AB-8 macroporous resin column (diameter-to-height ratio 1:8) at a flow rate of 1.5 BV / h. Wash with water for 4 BV and then elute with 40% ethanol (flow rate 0.8 BV / h), collecting the red eluent. Filter the eluent through a 5000 Da ultrafiltration membrane (0.3 MPa), allowing betalains to permeate while PEG is retained and recovered.

[0025] (4) Fermentation, deodorization, concentration and drying: Inoculate the permeate with Lactobacillus plantarum (2×10⁻⁶) 9CFU / mL (3% inoculum), with 0.1% ascorbic acid added, fermented at 28℃ for 24 h (with nitrogen and micro-aeration), then heated to 60℃ and held for 10 min to inactivate. Concentrated under vacuum at 45℃ and 0.085 MPa to a solid content of 25%, then maltodextrin was added to a solid content of 35%. Spray dried at 140℃ inlet and 70℃ outlet air, with a feed rate of 20 mL / min and an atomizer speed of 25000 r / min, to obtain betalain powder.

[0026] Example 2

[0027] (1) Raw material pretreatment: Fresh red beetroot (from Jiangsu Province), washed and peeled. Cut red beetroot into 8 mm pieces, take 1000g and add 500 mL of pre-cooled deionized water (4℃), pulp, and add 1.0 g of ascorbic acid for color protection.

[0028] (2) Low-temperature enzymatic hydrolysis of the compound enzyme: Add 0.6 g of pectinase (enzyme activity 35000 U / g) and 1.2 g of cellulase (enzyme activity 25000 U / g) to the slurry, and stir at 200 rpm for 60 min at 15℃ and pH 5.0. Centrifuge at 10000 r / min for 15 min and collect about 1400 mL of supernatant.

[0029] (3) Aqueous-phase-resin tandem purification: Add PEG 4000 to the supernatant to a concentration of 18% and ammonium sulfate to a concentration of 12%, stir and extract for 25 min, then let stand at room temperature for 2 h to separate the layers, and collect the PEG supernatant. Dilute the supernatant 4 times and load it onto an AB-8 macroporous resin column (diameter-to-height ratio 1:8) at a flow rate of 1.5 BV / h. Wash with water for 4 BV and then elute with 30% ethanol (flow rate 0.8 BV / h), collecting the red eluent. Filter the eluent through a 5000 Da ultrafiltration membrane (0.3 MPa), allowing betalains to permeate while PEG is retained and recovered.

[0030] (4) Fermentation, deodorization, concentration and drying: Inoculate the permeate with Lactobacillus plantarum (2×10⁻⁶) 9 CFU / mL (inoculum 2%), with 0.1% ascorbic acid added, fermented at 25℃ for 20 h (nitrogen purging with micro-aeration), then heated to 60℃ and held for 10 min to inactivate. Concentrated under vacuum at 45℃ and 0.085 MPa to a solid content of 20%, then maltodextrin was added to a solid content of 35%. Spray dried at 130℃ inlet and 75℃ outlet air, with a feed rate of 20 mL / min and an atomizer speed of 25000 r / min, to obtain betalain powder.

[0031] Comparative Example 1 (no two-phase aqueous pretreatment was set up, and only single resin purification was used)

[0032] The betalain powder was prepared according to the process of Example 1, except that the aqueous two-phase extraction operation in step (3) was omitted, and the supernatant obtained by centrifugation was directly diluted 4 times and then passed into an AB-8 macroporous resin column to complete the purification process. All other operation steps and process parameters were consistent with those of Example 1.

[0033] Comparative Example 2 (without fermentation and deodorization process)

[0034] The betalain powder was prepared according to the process of Example 1, except that step (4) of fermentation and deodorization by Lactobacillus plantarum was omitted, and the ultrafiltration permeate was directly sent to the vacuum concentration and spray drying section. The remaining operation steps and process parameters were the same as those in Example 1.

[0035] Detection methods: color value (E535nm, absorbance measured after dissolution in phosphate buffer); extraction rate (total betalains in the product / total amount in the raw material × 100%); odor score (10-person group, 1~5 points); moisture content (105℃ constant weight method).

[0036] The test results are shown in Table 1.

[0037] Table 1.

[0038] Color value (E535nm) 88.5 86.5 78.8 88.3 Extraction rate (%) 85.2 84.1 84.6 84.5 Odor rating (1-5 points) 4.6 4.3 4.5 2.5 Moisture content (%) 3.85 4.05 3.90 3.82

[0039] Combining the implementation and comparative examples with Table 1, it can be seen that the color value of Example 1 is 88.5, while that of Comparative Example 1 (resin only) is 78.8. Aqueous two-phase pre-desugaring reduces resin load, significantly improves betalain purity, and increases color value by approximately 10 points. Example 1 has an odor score of 4.6, while Comparative Example 2 (no fermentation) only scores 2.5. Lactobacillus plantarum fermentation effectively degrades earthy odor substances with almost no effect on color value and extraction rate. The overall superiority of Example 1 over Example 2 indicates that appropriately increasing enzyme dosage and PEG concentration within the scope of the claims can further improve performance.

[0040] This specific embodiment is only used to illustrate the technical solution of this application and is not intended to limit this application. All modifications, equivalent substitutions, and technical optimizations based on the design concept and core principles of this invention should fall within the scope of patent protection of this invention.

Claims

1. A low-temperature preparation method for betaine, characterized in that, Includes the following steps: Raw material pretreatment, low-temperature enzymatic hydrolysis of compound enzymes to break cell walls, aqueous two-phase-resin series purification, fermentation deodorization and concentration drying; (1) Raw material pretreatment: Take fresh red beet root, wash, peel, cut into pieces to 5~10 mm, add 0.5~1.0 times the weight of raw material in pre-cooled deionized water (4℃), pulp, and add 0.1% ascorbic acid for color protection; (2) Low-temperature enzymatic hydrolysis of compound enzyme: Add pectinase and cellulase compound enzyme to the slurry (the amount of enzyme added is 0.02%~0.05% of the weight of raw materials), stir and enzymatically hydrolyze at 15~20℃ and pH 4.5~5.0 for 60~120 min, and centrifuge to take the supernatant; (3) Aqueous two-phase-resin series purification: Polyethylene glycol 4000 and ammonium sulfate are added to the supernatant to make the mass concentration of polyethylene glycol 18%~25% and ammonium sulfate 12%~18%. After stirring and extraction, the mixture is allowed to stand and separate into layers. The polyethylene glycol supernatant is collected. After dilution, the supernatant is loaded onto a macroporous adsorption resin column, washed with water, and eluted with 30%~50% ethanol. The eluent is collected and polyethylene glycol is recovered by ultrafiltration. (4) Fermentation, deodorization and concentration drying: Inoculate Lactobacillus plantarum (inoculation amount 2%~4%) into the eluent, ferment at 25~30℃ for 20~30 h, inactivate and then concentrate under vacuum at 45~50℃ to a solid content of 20%~35%, add maltodextrin and spray dry, with an inlet air temperature of 130~145℃ and an outlet air temperature of 65~75℃ to obtain betalain powder.

2. The method according to claim 1, characterized in that, In step (2), the mass ratio of pectinase to cellulase in the compound enzyme is 1:1.5 to 1:2.5, the pectinase activity is ≥30000 U / g, and the cellulase activity is ≥20000 U / g.

3. The method according to claim 1, characterized in that, In step (3), the macroporous adsorption resin is AB-8 or X-5 type non-polar macroporous resin, the column loading flow rate is 1~2 BV / h, the elution flow rate is 0.5~1 BV / h, and the ultrafiltration membrane has a molecular weight cutoff of 3000~5000 Da.

4. The method according to claim 1, characterized in that, In step (4), the effective viable count of Lactobacillus plantarum is ≥1×10⁻⁶. 9 CFU / mL, nitrogen gas was introduced during fermentation to maintain a micro-aerobic environment, and after fermentation, the temperature was raised to 60℃ and held for 10 min to inactivate the enzyme.

5. The method according to claim 1, characterized in that, In step (4), the spray drying feed rate is 15~25 mL / min, the atomizer speed is 20000~30000 r / min, and the moisture content of the resulting beet red powder is ≤5%.