A natural pigment of yam peel with antibacterial effect and its preparation method and application

Abstract

The present invention relates to a natural yam peel pigment with antibacterial effect and a preparation method and application thereof, belonging to the technical field of food waste separation and processing. The yam peel is taken as the main research object in this paper, and it is found that pigment secondary metabolites are produced under storage conditions. The pigment secondary metabolites are separated, extracted and applied, and it is found that the pigment has a typical antibacterial effect and can be used as a natural antibacterial agent for food preservation.

Description

Technical Field

[0001] The invention belongs to the technical field of food waste separation and processing, and relates to a natural pigment, in particular to a yam peel natural pigment with antibacterial effect, and a preparation method and application thereof. Background Art

[0002] Yam peel in Chinese yam ( Dioscorea opposita Thunb.) is often discarded as waste during the production and processing process, which not only pollutes the environment but also greatly wastes resources.

[0003] Existing research on yam peel mainly focuses on the extraction of polysaccharides and total phenols, and no research has been found on the pigments of yam peel secondary metabolites.

[0004] CN108559305A discloses a fresh-cut yam yellow pigment with antioxidant activity and a preparation method thereof, wherein natural fresh-cut yam yellow pigment is obtained by macroporous resin, gel resin chromatography and preparative liquid phase. However, it uses fresh-cut yam as a raw material, and points out that the provided fresh-cut yam yellow pigment has antioxidant properties and can be used to prepare high-performance antioxidant products. Summary of the invention

[0005] In order to solve the resource waste of the current yam peel waste and the lack of natural pigments with stable and antibacterial effects, the present invention discovered new pigments through the storage of yam peels, determined the optimal conditions for the maximum pigment production, and innovatively verified the inhibitory effect of the pigments on Gram-negative and Gram-positive bacteria.

[0006] The purpose of the present invention is to provide a method for preparing yam peel yellow pigment with antibacterial effect.

[0007] Another object of the present invention is to provide yam peel yellow pigment prepared by the above preparation method.

[0008] Another object of the present invention is to provide the application of the yellow pigment of the above-mentioned medicinal herb in food preservation. Furthermore, it is to provide a food preservation antibacterial agent.

[0009] In order to achieve the above object, the specific scheme adopted by the present invention is:

[0010] In a first aspect, a method for preparing yam peel yellow pigment having antibacterial effect comprises the following steps:

[0011] Step 1, raw material processing: take fresh yam peel for pigment enrichment, the conditions for pigment enrichment are: storage at 25°C, 90% N2, 10% O2 modified atmosphere packaging, storage time is 18 h; take out the yam peel, dry it, use a homogenizer to crush it, and obtain yam peel powder for later use;

[0012] Step 2: Separation of pigments:

[0013] Adding anhydrous ethanol to the yam peel powder obtained in step 1, ultrasonic extraction, and obtaining a crude extract after centrifugation;

[0014] The crude extract is filtered and then concentrated to obtain a clear yellow pigment concentrate;

[0015] The yellow pigment concentrate is separated and extracted by resin to obtain a pigment extract, and the pigment extract is concentrated by rotary evaporation and centrifugation to obtain an extract concentrate;

[0016] Step 3: Purification of the pigment: Use solid phase microextraction to purify the extract concentrate, first elute with distilled water until it is colorless, then elute the pigment with ethanol and collect it to obtain yam peel yellow pigment.

[0017] As a further optimization of the above preparation method, in step 2, the specific process of obtaining the crude extract is: using anhydrous ethanol to ultrasonically extract the yam peel powder, filtering the ultrasonic extract with gauze, taking out the filtrate, and centrifuging at 10000r / min for 15min to obtain the crude extract.

[0018] As a further optimization of the above preparation method, in step 2, the crude extract is filtered with filter paper to remove solid impurities in the solution to obtain a clear pigment solution, and then rotary evaporated at a vacuum pressure of 85 mbar, a temperature of 37°C, and a rotation speed of 45 r / min for 2.5 h, and then filtered to remove impurities to obtain a clear yellow pigment concentrate.

[0019] Furthermore, after the yellow pigment concentrate is centrifuged, it is drained to the resin surface with a glass rod, and 4 times the column volume of sterile water is passed through the column at a speed of 1-2 BV / hr, and then 2 / 3 of the column volume of anhydrous ethanol is passed through the column, and then the extract is started to be collected. When the color of the extract tends to be transparent, the collection is stopped, and the pigment extract is rotary evaporated at 85°C for 15 minutes using a rotary evaporator, and then treated in an ultracentrifuge for 15 minutes to finally obtain an extract concentrate.

[0020] As a further optimization of the above preparation method, in step 3, C 18 The solid phase microextraction column was activated by adding 5 column volumes of ethanol solution and distilled water in turn; 2 mL of the extract concentrate was added to the column, and then 6 column volumes of distilled water were added for elution; 6 column volumes of ethanol solution were added to elute the yellow pigment of yam peel and collected; finally, 6 column volumes of ethyl acetate were used to wash away the remaining pink impurities in the column for the next elution.

[0021] In the second aspect, the yam peel yellow pigment is prepared by the above preparation method.

[0022] The third aspect is the application of the above-mentioned yam peel yellow pigment in food preservation.

[0023] In a fourth aspect, a food preservative and antibacterial agent is prepared using the above-mentioned yam peel yellow pigment.

[0024] Beneficial effects: The present invention uses yam peel as raw material for the first time, extracts pigment secondary metabolites from yellowed yam peel, and verifies its typical antibacterial effect. On the one hand, it rationally utilizes yam resources, avoids the discarding of yam peel as food waste, and reduces environmental pollution; on the other hand, the new pigment produced by the separated and extracted yam peel under storage conditions has an antibacterial effect and can be used as a natural antibacterial agent for food preservation, increasing the added value of the yam peel. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a comparison of the yam skin before yellowing (left picture) and after yellowing (right picture);

[0026] Figure 2 The results of inhibition zones of YP against Escherichia coli (A) and Staphylococcus aureus (B); a, YP; b, 5-fold dilution of YP; c, 10-fold dilution of YP; d, 20-fold dilution of YP;

[0027] Figure 3 This is a comparison chart of the inhibition rates of YP and chloramphenicol against Staphylococcus aureus and Escherichia coli respectively. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0029] YP: Yam yellow pigment.

[0030] The instruments and materials used in the present invention are as follows.

[0031] Instruments: UV-1800 ultraviolet-visible spectrophotometer (Shimadzu Corporation, Japan), MB25 moisture analyzer (Ohaus Instrument (Shanghai) Co., Ltd.), DZKW-S-4 electronic constant temperature water bath (Beijing Zhongxing Weiye Instrument Co., Ltd.), AUW220D electronic balance (Shimadzu Corporation, Japan), ME204E / 02 electronic balance (Mettler Toledo Instrument (Shanghai) Co., Ltd.), RE52-98 vacuum rotary evaporator (Büchi Company, Switzerland), 101 electric heating blast drying oven (Tianjin Hongnuo Instrument Co., Ltd.), 3-18K high-speed refrigerated centrifuge (Sigma Company, Germany), homogenizer, high temperature and high pressure sterilizer, constant temperature incubator, dryer, ultrasonic cleaner, circulating water vacuum pump, constant temperature water bath.

[0032] Ingredients: Iron stick yam peel was purchased from Nanyang yam wholesale market.

[0033] Strains: Staphylococcus aureus, Escherichia coli.

[0034] Reagents: anhydrous ethanol, agar, peptone, beef extract, sodium chloride, hydrochloric acid, sodium hydroxide, sterile water.

[0035] Example 1 Extraction of natural pigment from yam peel

[0036] 1.1 Preparation of crude pigment extract

[0037] The invention effectively solves the resource waste of yam peel, prepares and extracts a new natural pigment, the optimal conditions for enriching the pigment are storage at 25°C, 90% N2, 10% O2 modified atmosphere packaging, storage time of 18 h, taking out the yam peel, placing it in a 50°C oven for drying for 8 h and storing it for later use; using a homogenizer to pulverize it for multiple times until the powder has no obvious particles, and then using it for later use.

[0038] Comparison of yam skin without yellowing and yellowing Figure 1 shown.

[0039] Separation of pigments

[0040] Take a 300 ml conical flask and add an appropriate amount of anhydrous ethanol to cover the yam peel powder. Put it in an ultrasonic cleaner, fix the conical flask with a rope, and perform ultrasonic extraction for 3 times for 8 hours. Filter the ultrasonic extract with gauze, take out the filtrate, and centrifuge it in an ultracentrifuge at 10000r / min for 15 minutes to obtain a crude extract, which is ready for use.

[0041] Use a suction filter to filter the solution with filter paper to remove solid impurities in the solution to obtain a clear pigment solution; the solution is heated to 85 mbar at a vacuum pressure of 37 o C, rotating speed 45 r / min, after rotary evaporation for 2.5 h, the impurities were removed by suction and a clear yellow pigment concentrate was obtained.

[0042] After centrifugation, the mixed solution was drained to the surface of the resin (XAD-7) with a glass rod, and 4 column volumes of sterile water were passed through the column at a rate of 1-2 BV / hr to wash away impurities, and then 2 / 3 column volumes of anhydrous ethanol were passed through the column, and then the extract was collected. When the color of the extract tends to be transparent, stop collecting. The pigment extract was evaporated by rotary evaporator at 85 o The concentrated solution was then subjected to ultracentrifugation for 15 min to obtain the final extract concentrate.

[0043] Pigment purification

[0044] C 18The solid phase microextraction column was activated by adding 5 column volumes (5×15 mL) of ethanol solution and distilled water, respectively. 2 mL of the concentrated fresh-cut yam yellow pigment solution was added to the column, and then 6 column volumes of distilled water were added for elution until only clear yellow remained on the column without other color impurities; 6 column volumes of ethanol solution were added to elute the fresh-cut yam yellow pigment and collect it; finally, 6 column volumes of ethyl acetate were used to wash away the remaining pink impurities on the column for the next elution.

[0045] Example 2 Pigment antibacterial test

[0046] 2.1 Sample dissolution

[0047] The color value of the pigment is 274.6 U / g; the absorbance at 630 nm is 0.379, which is a crude extract; take 300 μL of the sample, and make 5-fold dilution, 10-fold dilution and 20-fold dilution at the same time, mark them and set them aside.

[0048] Bacteria culture

[0049] Prepare 30 mL of LB liquid culture medium. After sterilization, divide 30 mL of liquid culture medium into three 10 mL centrifuge tubes, take a small amount of Staphylococcus aureus and Escherichia coli into the centrifuge tubes and shake them evenly, mark them, and place them in a 37 ℃ constant temperature shaker for 24 h.

[0050] Confrontation plate experiment of pathogenic microorganisms

[0051] Prepare three 400 mL LB agar medium and two 400 mL PDA medium. After sterilization, wait for the medium to cool to about 50 ℃, add 10 mL of Staphylococcus aureus and Escherichia coli to 400 mL LB agar medium, shake well, pour plates, and mark them. Add 1 mL of the shaken fungal spore suspension to 400 mL PDA medium, shake well, pour plates. After solidification, use tweezers to place four filter paper discs into the medium.

[0052] In the experimental group, 300 μL of YP stock solution, 5-fold dilution, 10-fold dilution and 20-fold dilution were added to four filter paper sheets, two in parallel in each group. In the control group, 300 μL of anhydrous ethanol stock solution, 5-fold dilution of anhydrous ethanol, 10-fold dilution of anhydrous ethanol and 20-fold dilution of anhydrous ethanol were added. Bacteria were cultured in a 37 ℃ constant temperature incubator for 24 h, and fungi were cultured in a 28 ℃ constant temperature incubator for 72 h.

[0053] Quantitative analysis of plate standoff experiment

[0054] According to the results of the confrontation plate experiment, the size of the dilution concentration gradient inhibition zone of each sample in the control group and the experimental group was measured, and the antibacterial efficiency was calculated according to the following formula:

[0055] Antibacterial efficiency (%) = R 1- R 2 / R 1×100%.

[0056] in: R 1 is the size of the inhibition zone of the dilution concentration gradient of each sample in the experimental group; R 2 is the size of the inhibition zone of each dilution concentration gradient in the control group.

[0057] The inhibition zone results of YP against Escherichia coli and Staphylococcus aureus are as follows Figure 2 shown.

[0058] The antibacterial zone of the original solution of the sample is more obvious, and its antibacterial effect is the strongest. Compared with the antibacterial zone of the original solution, the antibacterial zone of the 5-fold dilution is smaller, and its antibacterial effect is not obvious. The antibacterial zones of the 10- and 20-fold sample dilutions are similar to those of the control group, with almost no antibacterial effect, and the antibacterial effect decreases with the increase of the dilution multiple.

[0059] The antibacterial experiment showed that the inhibition rate of YP on Gram-negative model bacteria Staphylococcus aureus was 19.8%, and the antibacterial effect was significantly higher than 10 -7 The inhibition rate of chloramphenicol was 6.6%; the inhibition rate of YP on Gram-positive model bacteria Escherichia coli was 53.6%, which was higher than 10 -7 Inhibitory effect of chloramphenicol on Escherichia coli.

[0060] Depend on Figure 3 It can be seen that YP has a strong inhibitory effect on Escherichia coli and Staphylococcus aureus, with a wide range of inhibition, and the inhibitory effect decreases significantly after dilution; YP has been used in antibacterial treatment due to its high antibacterial activity, and has the ability to replace some antibiotics.

[0061] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing yam peel yellow pigment with antibacterial effect, characterized in that: The following steps are involved: Step 1, raw material processing: take fresh yam peel for pigment enrichment, the conditions for pigment enrichment are: storage at 25°C, 90% N2, 10% O2 modified atmosphere packaging, storage time is 18 h; take out the yam peel, dry it, use a homogenizer to crush it, and obtain yam peel powder for later use; Step 2: Separation of pigments: Adding anhydrous ethanol to the yam peel powder obtained in step 1, ultrasonic extraction, and obtaining a crude extract after centrifugation; The crude extract is filtered and then concentrated to obtain a clear pigment concentrate; After the pigment concentrate is centrifuged, it is drained to the resin surface with a glass rod, and 4 column volumes of sterile water are passed through the column at a speed of 1-2 BV / hr, and then 2 / 3 column volumes of anhydrous ethanol are passed through the column, and then the extract is collected. When the color of the extract tends to be transparent, the collection is stopped, and the pigment extract is evaporated by a rotary evaporator at 85° C. for 15 minutes, and then subjected to ultracentrifuge treatment for 15 minutes to finally obtain an extract concentrate; Step 3: Purification of the pigment: Use solid phase microextraction to purify the extract concentrate, first elute with distilled water until it is colorless, then elute the pigment with ethanol and collect it to obtain yam peel yellow pigment.

2. The preparation method according to claim 1, characterized in that: In step 2, the specific process of obtaining the crude extract is: ultrasonically extracting the yam peel powder with anhydrous ethanol, filtering the ultrasonic extract with gauze, taking out the filtrate, and centrifuging at 10000r / min for 15min to obtain the crude extract.

3. The preparation method according to claim 2, characterized in that: In step 2, the crude extract is filtered through filter paper to remove solid impurities in the solution to obtain a clear pigment solution, and then rotary evaporated at a vacuum pressure of 85 mbar, a temperature of 37° C., and a rotation speed of 45 r / min for 2.5 h, and then filtered to remove impurities to obtain a clear yellow pigment concentrate.

4. The preparation method according to claim 1, characterized in that: In step 3, C 18 The solid phase microextraction column was activated by adding 5 column volumes of ethanol solution and distilled water in turn; 2 mL of the extract concentrate was added to the column, and then 6 column volumes of distilled water were added for elution; 6 column volumes of ethanol solution were added to elute the yellow pigment of yam peel and collected; finally, 6 column volumes of ethyl acetate were used to wash away the remaining pink impurities in the column for the next elution.

5. Yam peel yellow pigment prepared by the preparation method described in any one of claims 1 to 4.

6. Use of the yam peel yellow pigment according to claim 5 in food preservation.

7. A food preservative and antibacterial agent prepared from the yam peel yellow pigment as claimed in claim 5.

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