阅读说明：本技术 一种利用大蒜种植及加工废弃物发酵制备蒜氨酸的方法 (Method for preparing alliin by fermenting garlic planting and processing wastes ) 是由 蔡俊 戴慧敏 于 2021-09-28 设计创作，主要内容包括：本发明公开了一种利用大蒜种植及加工过程中的废弃物来发酵制备蒜氨酸的方法,以大蒜叶、杆、茎、蒂、须等为原料,将大蒜种植及加工废弃物加热使蒜酶失活,粉碎后用纤维素酶酶解处理,在水解液中接种酿酒酵母(Saccharomyces cerevisiae)CCCG进行发酵,菌体破壁后即可提取蒜氨酸。该方法利用大蒜种植及加工过程中的废弃物通过微生物合成法制备蒜氨酸,实现了变废为宝的目的,反应条件温和,并且避免了蒜酶对蒜氨酸的分解作用。(The invention discloses a method for preparing alliin by fermentation by using wastes in garlic planting and processing processes, which takes garlic leaves, stems, stalks, whiskers and the like as raw materials, heats the wastes in garlic planting and processing to inactivate allinase, pulverizes the wastes, uses cellulase for enzymolysis, inoculates saccharomyces cerevisiae (yeast) Saccharomyces cerevisiae ) CCCG is fermented, and alliin can be extracted after cell wall breaking. The method utilizes the waste in the garlic planting and processing process to prepare the alliin by a microbial synthesis method, realizes the purpose of changing waste into valuable, has mild reaction conditions, and avoids the decomposition effect of alliinase on the alliin.)

1. A method for preparing alliin by fermenting garlic planting and processing wastes is characterized by comprising the following steps:

(1) heating the waste of garlic planting and processing to inactivate allinase, pulverizing, performing enzymolysis with cellulase, and collecting hydrolysate;

(2) inoculating Saccharomyces cerevisiae (II) to the hydrolysateSaccharomyces cerevisiae) The fermentation is carried out on the CCCG,

the alliin can be extracted after the cell wall is broken.

2. The method for preparing alliin by fermenting garlic planting and processing waste as claimed in claim 1, wherein the garlic planting and processing waste comprises garlic leaves, stems, stalks and whiskers.

3. The method for preparing alliin by fermenting garlic planting and processing waste according to claim 1, wherein the garlic planting and processing waste in the step (1) is subjected to dry heat treatment at 60-70 ℃.

4. The method for preparing alliin by fermenting garlic planting and processing waste according to claim 1, wherein the cellulase in the step (1) has a treatment temperature of 30-40 ℃ and a pH of 4.5-7.5.

5. The method for preparing alliin by fermenting garlic planting and processing waste as claimed in claim 1, wherein the fermentation medium in step (2) further comprises glucose, yeast powder, (NH)₄)₂SO₄，KH₂PO₄，MgSO₄。

Technical Field

The invention belongs to the field of microbial fermentation, and particularly relates to a method for synthesizing alliin by fermenting wastes in garlic planting and processing processes.

Background

Alliin is a relatively stable non-protein sulfur-containing amino acid existing in bulbels of Allium plants in Liliaceae, is odorless and tasteless, is easily soluble in water but insoluble in ethanol, has the functions of reducing cholesterol, preventing obesity, resisting tumors and the like, can inhibit the growth of Staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Helicobacter pylori (Helicobacter pylori), Shigella dysenteriae (Shigella dysenteriae) and Bacillus subtilis (Bacillus subtilis), inhibits the toxin production effect of Aspergillus flavus, is non-toxic to human bodies, can be used as a good food preservative to prolong the preservation period of fruits and vegetables, can also be developed into a pharmaceutical preparation, and has a good application prospect.

The prior production method of alliin mainly comprises a chemical synthesis method, a tissue culture method and a plant extraction method. The chemical synthesis and plant extraction methods are widely accepted by the public at present. The chemical synthesis method takes allyl halide, cysteine, alkali and oxidant as raw materials, and the alliin is obtained by reaction under certain conditions, for example, CN103483231A takes cysteine, allyl chloride, sodium hydroxide and hydrogen peroxide as raw materials, and the alliin is synthesized by continuously stirring under the environment of low temperature and high pressure. The plant extraction method comprises crushing Bulbus Allii, extracting, and separating with column, and comprises organic solvent extraction and water extraction. Since alliinase is present to cause decomposition of alliin, it is usually necessary to exclude the influence of alliinase during the extraction process. For example, CN112028798A uses microwave high fire enzyme-extinguishing device, and uses microwave to inactivate alliinase in the extraction process; CN108164611A mixing garlic raw material with water for heat extraction, inactivating alliinase by using high temperature, and sequentially performing water extraction, salt extraction, alkali extraction, alcohol extraction, adsorbent adsorption and vacuum freezing concentration treatment to obtain crystalline alliin; CN110615748A rapidly inactivates alliinase by ethanol, and obtains crude alliin by vacuum distillation concentration and alternate treatment of ion exchange column; CN110963949A is prepared by mixing any one of 3 to 10 mass percent of sodium hydroxide, 3 to 10 mass percent of calcium hydroxide, 3 to 10 mass percent of potassium carbonate, 25 to 45 mass percent of acetic acid, 25 to 45 mass percent of hydrochloric acid, 25 to 45 mass percent of hydrobromic acid and 25 to 45 mass percent of phosphoric acid with a garlic raw material, crushing and extracting under the protection of inert gas, and extracting by an organic solvent. Therefore, in the existing production method of alliin, the chemical synthesis method has the disadvantages of severe reaction conditions, low reaction yield, environmental pollution, troublesome post-treatment, complex extraction process in the plant extraction method, and the need of reducing the influence of alliinase on the decomposition of alliin, thereby further increasing the cost.

The waste of garlic planting and processing, including leaves, stems, stalks, whiskers and the like, contains precursor substances synthesized by alliin such as alliin and allyl mercaptan, and a large amount of garlic waste is generated in garlic planting and processing bases every year, so that the environment is polluted, and the waste of resources is caused.

Disclosure of Invention

The invention aims to make up the defects of the prior art and provides a method for preparing alliin by fermenting wastes including garlic leaves, stems, stalks, whiskers and the like in the garlic planting or processing process. The invention takes the biosynthetic pathway of alliin as reference, garlic waste as raw material and saccharomyces cerevisiae CCCG (CCTCC NO: M2012215) from laboratory as production bacteria, and the alliin is obtained by extraction and separation from fermentation liquor of the strain. The microbial synthesis method has mild reaction conditions, is green and efficient, and avoids the decomposition effect of alliinase on alliin.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing alliin by fermenting garlic planting and processing wastes comprises the following steps:

(1) heating the waste of garlic planting and processing to inactivate allinase, pulverizing, performing enzymolysis with cellulase, and collecting hydrolysate;

(2) inoculating Saccharomyces cerevisiae CCCG to the hydrolysate, fermenting, and breaking cell wall to extract alliin.

Further, the garlic planting and processing waste comprises garlic leaves, stems, stalks and whiskers.

Further, the garlic planting and processing waste in the step (1) is treated by dry heat at the temperature of 60-70 ℃ to inactivate allinase.

Further, the treatment temperature of the cellulase in the step (1) is 30-40 ℃, and the pH value is 4.5-7.5.

Further, the fermentation medium in the step (2) also contains glucose, yeast powder, (NH)₄)₂SO₄，KH₂PO₄，MgSO₄。

Compared with the prior art, the invention has the advantages that:

1. the method utilizes the garlic straw waste to prepare the alliin, realizes the purpose of changing waste into valuable, has low cost and has obvious social and economic benefits.

2. Compared with a chemical synthesis method, the whole reaction process is carried out at normal temperature and normal pressure, toxic substances such as allyl bromide and the like are not needed as raw materials, the energy consumption is low, the environmental pollution is low, and the post-treatment is simple.

3. The saccharomyces cerevisiae is used as a production strain, so that the safety of the product is ensured, and a theoretical basis is laid for the industrial scale production of the saccharomyces cerevisiae.

4. The alliin is synthesized by adopting a microbiological method, so that the decomposition effect of alliinase on the alliin is avoided, the yield of the alliin is obviously improved compared with an extraction method, the production cost is reduced, and the obtained alliin has stable property and is easy to separate and purify.

Drawings

FIG. 1 is an electrospray ionization source-mass spectrogram of alliin, (a) and (b) are excimer ion flow diagrams of alliin standard and a saccharomyces cerevisiae sample after wall breaking, respectively, and (c) is a mass spectrogram of the saccharomyces cerevisiae sample after wall breaking.

FIG. 2 is a high performance liquid chromatogram of alliin, wherein (a) is alliin standard product, and (b) is sample obtained after wall breaking of Saccharomyces cerevisiae.

FIG. 3 is a graph showing the standard curve of alliin.

Detailed Description

The foregoing aspects of the present invention are described in further detail below by way of examples, but it should not be construed that the scope of the subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above aspects of the present invention are within the scope of the present invention.

Example 1: preparation of the starting materials

Collecting 1-2kg of waste (including garlic leaves, stems, stalks, and whiskers) from garlic planting and processing, deactivating allinase by dry heat treatment at 60-70 deg.C, sun drying, and pulverizing. Since the histiocytes of garlic leaves, stems, stalks, whiskers and the like contain alliin and precursors synthesized by alliin, the crushed particles are treated by cellulase, so as to destroy the cell walls of the histiocytes of garlic leaves, stems, stalks, whiskers and the like, and to make more contents enter the extracting solution. And (3) enzymolysis treatment conditions: the ratio of the materials to the liquid is 1:6-1:10, the temperature is 30-40 ℃, the pH is 4.5-7.5, the enzyme dosage is 0.1-0.5% (W/W), the enzymolysis time is 1-2 h, then the hydrolysate is centrifuged and washed, the supernatant is collected, then the filter residue is soaked in water at the temperature of 30-50 ℃ for 1-2 h and then filtered, the filtrate is collected and mixed with the supernatant, and the volume is fixed to 1L for standby.

Example 2: preparation of fermentation broth

(1) Bacterial strains

Saccharomyces cerevisiae (Saccharomyces cerevisiae) CCCG, with a collection number CCTCC NO: m2012215

(2) Preparation of the Medium

Preparation of solid PDA medium (1L): mixing 20g glucose, 20g peptone, 10g yeast extract powder and 20g agar uniformly, adjusting pH to 6.0, and sterilizing at 115 deg.C for 30 min.

Preparation of seed medium (1L): mixing 20g glucose, 20g peptone and 10g yeast extract powder, adjusting pH to 6.0, and sterilizing at 115 deg.C for 30 min.

Preparation of fermentation medium (1L): mixing 20g glucose, 10g yeast powder, 8g (NH)₄)₂SO₄，3g KH₂PO₄，0.15g MgSO₄After being mixed uniformly with 1L of the raw material prepared in example 1, the mixture was adjusted to pH 5.0 and sterilized at 115 ℃ for 30 min.

(3) Preparation of fermentation broth

Dipping a ring of strains from a glycerol tube, streaking the strains on a fresh solid PDA culture medium for activation, and culturing the strains in a constant-temperature incubator at the temperature of 20-35 ℃ for 18-24 hours; taking a ring of activated strains from a inoculating ring, and culturing for 12-30 h under the conditions that the temperature is 20-35 ℃, the rotating speed of a shaking table is 140-200 r/min, and the bottle-shaking loading is 50mL/250 mL; and (3) sucking 1-5 mL of the primary seed culture solution by using a sterile 10mL pipette, inoculating into a new seed culture medium, and performing shaking culture for 12-30 h under the conditions of a culture temperature of 20-35 ℃ and a shaking table rotating speed of 140-200 r/min. And (3) sucking 1-5 mL of the seed solution by using a sterile 10mL pipette according to the inoculation amount of 2-10%, inoculating the seed solution into a fermentation culture medium, fermenting for 12-24 hours under the conditions that the temperature is 20-35 ℃, the rotating speed of a shaking table is 140-200 r/min and the bottle shaking loading is 50mL/250mL, and then finishing the culture and sampling.

Example 3: alliin extraction and analysis detection

(1) Determination of moisture content of cells

Centrifuging the bacterial suspension at 4500-8000 r/min to remove supernatant, washing the bacteria with sterile water for three times, weighing wet bacteria weight M1, placing in a 65 ℃ oven to dry to constant weight, and weighing dry bacteria weight M2. Moisture content (%) - (M1-M2)/M1 × 100%.

(2) Ultrasonic method for processing cells and calculating wall breaking rate

Centrifuging the bacterial suspension at 4500-8000 r/min to remove supernatant, washing with sterile water for 3 times, weighing wet weight of the bacteria, and re-suspending the wet bacteria to a constant volume. Diluting the bacterial suspension with a proper amount after constant volume, counting on a blood counting plate, and taking the parallel counting result of 3 times as the cell number N1 before wall breaking. And (3) carrying out ultrasonic disruption for 30-60 min (setting working time for 1s and interval for 1s) under an ice bath condition by adopting an ultrasonic wall breaking method with the set power of 100-300W, taking a proper amount of the bacterial suspension subjected to wall breaking for dilution, counting on a blood counting chamber, and taking 3 times of parallel counting results to record as the cell number N2 after wall breaking. The wall breaking rate (%) - (N1-N2)/N1 × 100%.

(3) ESI-MS analysis of alliin

Centrifuging the bacterial suspension after wall breaking at 8000r/min to obtain supernatant, and performing mass spectrometry after the supernatant is subjected to membrane filtration by using a microfiltration membrane (0.22 μm). Mass spectrum conditions: ion mode: electrospray positive ionization (ESI +), capillary voltage: 3.5 KV; taper hole voltage: 25V; ion source temperature: 100 ℃; desolventizing gas temperature: 300 ℃; the mass range is as follows: 100-200 m/z; selective ion mode detection (SIM) m/z 178.

As shown in FIG. 1a and FIG. 1b, the excimer current graphs of alliin sample and Saccharomyces cerevisiae after wall breaking both appeared at about 2.88min, and FIG. 1c shows that the sample appeared at 2.887min as M/z 178.00, which is the excimer ion of alliin [ M + H ] +.

(4) HPLC analysis of Alliin

Centrifuging the bacterial suspension after wall breaking at 4500-8000 r/min to obtain supernatant, and analyzing the supernatant by using a high performance liquid chromatograph after the supernatant is subjected to membrane filtration by using a microfiltration membrane (0.22 mu m). Chromatographic conditions are as follows: chromatography column, Inertsil ODS SP C18 column (4.6mm х 150mm, 5 μm); the mobile phase is V (methanol): v (formic acid aqueous solution) ═ 5:95, ultraviolet detector, detection wavelength: 214 nm; the column temperature is 30 ℃; flow rate: 0.8mL/min, sample size: 20 μ L.

As shown in FIG. 2a and FIG. 2b, the peak-off time of the alliin standard was 5.336min, and the peak-off time of the cell disruption solution was similar to that of the standard at 5.36min, indicating that the product alliin was produced.

(5) Drawing standard curve of alliin

Preparing standard alliin solutions with the concentrations of 200, 400, 600, 800 and 1000 mug/mL respectively. Taking the concentrations of different alliin standard solutions as abscissa and peak area value measured by HPLC as ordinate, and plotting to obtain standard alliin curve (see FIG. 3); the regression equation for alliin was obtained as: 7232.5x +39674 (R)²＝0.9998)。

(6) Determination of intracellular alliin content

Centrifuging the bacterial suspension after wall breaking to obtain supernatant at 4500-8000 r/min, filtering the supernatant with microfiltration membrane (0.22 μm), calculating by high performance liquid chromatography to obtain the concentration of alliin in the extractive solution, and calculating to obtain the content of alliin in each g of dry cells

Through calculation, the content of the alliin in the raw materials is 50mg in each 1kg of garlic planting and processing waste, and the content of the alliin in each g of yeast dry cells is 102.66mg after the hydrolysate of the garlic planting and processing waste is fermented by saccharomyces cerevisiae. Because the tissue cells of garlic leaves, stems, stalks, whiskers and the like contain alliin and precursor substances synthesized by the alliin, a method of hot water extraction and enzymolysis mixed extraction is adopted, the precursor substances synthesized by the alliin are released from the tissue cells of the garlic leaves, stems, stalks, whiskers and the like, and the alliin is synthesized by fermentation of saccharomyces cerevisiae. The method has the advantages of short production period, low cost, easy separation and purification of alliin, and significantly improved yield of alliin after fermentation.