CN112980817A - Novel process for extracting bromelain - Google Patents

Abstract

The invention discloses a new process for extracting bromelain; the invention is realized by the following steps: extracting bromelain from fresh pineapple waste peels, purifying and preparing the bromelain by an ultrasonic freezing technology, and measuring the absorbance value of the bromelain obtained by purification under the wavelength of 280 nm; compared with the common stirring and purifying preparation method, the sulfate ion residual quantity and the enzyme specific activity in the solution are compared. The obtained bromelain has high purity and high specific activity when dialyzed by an ultrasonic ice bath mode. Can be used for treating edema and various inflammations, and has effects of invigorating stomach and resolving food stagnation; it can also be used as food additive for tenderizing meat, hydrolyzing protein, clarifying beer, and producing cheese.

Description

Novel process for extracting bromelain

The technical field is as follows: the invention belongs to the field of purification and preparation of biochemical enzymes, and particularly relates to a purification and preparation method of bromelain.

Background art: bromelain (EC 3, EC 4, bromelain 22, and bromelain 3) is a proteolytic enzyme containing cysteine residue in pineapple plant, and can be used for treating edema, various inflammations, and invigorating stomach and promoting digestion; it can also be used as food additive for tenderizing meat, hydrolyzing protein, clarifying beer, and producing cheese. Bromelain is a relatively unstable enzyme, and the process steps in the extraction process have a great influence on the activity of the product.

Bromelain is generally extracted from waste pineapple peels, and the fruits, stems, stalks and leaves of the bromelain contain bromelain; the bromelain has a relative molecular mass of 33000, is a pale yellow powder, has an isoelectric point of 9.5, and is slightly soluble in water.

The invention content is as follows: the invention relates to a purification preparation method of bromelain.

The invention is realized by the following steps: extracting bromelain from fresh pineapple waste peels, purifying and preparing the bromelain by an ultrasonic freezing technology, and measuring the absorbance value of the bromelain obtained by purification under the wavelength of 280 nm; compared with the common stirring and purifying preparation method, the sulfate ion residual quantity and the enzyme specific activity in the solution are compared. The obtained bromelain has high purity and high specific activity when dialyzed by an ultrasonic ice bath mode.

The purification preparation process comprises the following steps:

squeezing 300g of weighed pineapple peel raw material, adding 600mL of 0.1mol/L phosphate buffer solution with pH7.5, and stirring in a stirrer for 15min to fully and uniformly stir; filtering with 4 layers of gauze. Centrifuging the filtrate at 4000rpm for 5min, collecting supernatant, adding ammonium sulfate to precipitate; transferring the precipitate into a dialysis bag, dialyzing in an ultrasonic ice bath mode for 15min to remove inorganic ions and organic micromolecules, repeating for three times, and detecting residual sulfate ions by barium ions; and measuring the absorbance of the enzyme solution by using an ultraviolet-visible spectrophotometer at the wavelength of 280nm, and detecting the specific activity of the enzyme.

The invention is further illustrated by the following examples, which do not limit the scope of the invention.

Example 1:

preparing enzyme solutions with the concentration of 10 percent respectively, taking 1mL, adding 0.1mol/L phosphate buffer solution (pH7.5) and 4.5mL of 0.5 percent sodium thiosulfate and 4.5mL of 0.5 percent EDTA4.5mL to fix the volume to 100 mL; preheating at 37 deg.C for 10min, adding 1% casein solution 10mL, reacting for 10min, adding 10% trichloroacetic acid 3mL to terminate enzymatic reaction, and measuring absorbance of the supernatant at 280nm wavelength.

Example 2

And (3) drawing a protein standard curve of 0-1000 mu g/mL.

0.0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of bovine serum albumin standard solution (1000 mu g/mL) is respectively sucked, 1.0, 0.8, 0.6, 0.4, 0.2 and 0mL of water are sequentially added, after uniform mixing, the mixture is respectively mixed with 5.0mL of 5% Coomassie brilliant blue, and the absorbance value is measured at 595nm after shaking. And drawing a standard curve by taking the absorbance as an abscissa and the concentration of the bovine serum albumin as an ordinate.

And (5) absorbing 1mL of 10% bromelain, reading the absorbance value by the same method, and searching the corresponding concentration in the standard curve.

Example 3

2mL of barium chloride solution with the concentration of 0.01mol/L is put into a test tube, and 2-3 drops of dialysis external liquid are added into the test tube; the ultrasonic ice bath is carried out for 15min, no white precipitate is generated after three water changes, and turbidity is still generated after 4 water changes by a common stirring method.

Claims (2)

1. A new process for extracting bromelain is characterized in that an ultrasonic ice bath mode is adopted during dialysis, so that the purification efficiency can be improved; squeezing 300g of weighed pineapple peel raw material, adding 600mL of 0.1mol/L phosphate buffer solution with pH of 7.5, putting into a stirrer, stirring for 15min, and fully and uniformly stirring; filtering with 4 layers of gauze; centrifuging the filtrate at 4000rpm for 5min, collecting supernatant, adding ammonium sulfate to precipitate; transferring the precipitate into a dialysis bag, dialyzing in an ultrasonic ice bath mode to remove inorganic ions, and measuring absorbance at 280 nm.

2. The method of claim 1, wherein the bromelain obtained by dialysis in an ultrasonic ice bath mode has high purity and high specific activity.