Background technology
The non-fermentation converted products of red bayberry mainly contains quick-frozen arbutus, canned arbutu, red bayberry preserved fruit, gooseberry jam and cranberry juice.Because it is higher to be used to process the raw material arbutus purchasing price of these products, makes that the market price of red bayberry processed goods is also high.The red bayberry processed goods no longer keeps original shape, only depends on visual inspection and sense of taste identification can not accurately judge its feedstock property as red bayberry preserved fruit, gooseberry jam and red bayberry juice, carries out easily thereby make both to mingle in the red bayberry processed goods, and is lucrative again.
Red bayberry is a special product of China, does not also differentiate the detection method of the non-fermentation converted products of the red bayberry true and false at present abroad.Domesticly do not see corresponding report yet.
Haematochrome in the red bayberry is an anthocyanin.The main at present difference pH method (Wrolstad that adopts classics of the mensuration of anthocyanin content, R.E. (1993) .Color and pigment analyses in fruit products.Oregon State University Agricultural Experimental Station.Bulletin, No.464 (reprinted)).This method adopts anthocyanin at the different content that calculate in raw material anthocyanin of pH1.0 with near 4.5 o'clock absorbances 510nm.But the kind of anthocyanin has the hundreds of kind in the plant, in all red, purple, blue plants anthocyanin is arranged all, and such plant also has hundreds and thousands of kinds, so only can only detect the total amount (can understand the use amount of raw material in the processed goods thus) of anthocyanin with this chemical analysis method, and can not determine raw material properties in the processed goods, can not determine promptly this product with which kind of raw material is processed.
High performance liquid chromatography-mass spectrometry method is a kind of quick, easy, analysis and detection technology accurately and efficiently, has been widely used in the analysis of multiple natural component in the plant, identifies comprising the structure of anthocyanin in the plant, as be used for european cranberry (Mullen, W., Lean, M.E.J.﹠amp; Crozier, A. (2002) .Rapidcharacterization of anthocyanins in red raspberry fruit by high-performance liquidchromatography coupled to single quadrupole mass spectrometry.Journal ofChromatography A, 996,63-70.) and grape in anthocyanin (
V., Monagas, M., Gomez-Cordov é s, M.C.﹠amp; Bartolom é, B. (2004) .Vitis vinifera L.cv.Gracianograpes characterized by its anthocyanin profile.Postharvest Biology and Technology, 31, structure 69-79.) is identified.But this method is not having under the situation of standard specimen, the content of anthocyanin in can not analytic sample (promptly quantitative), can not distinguish isomers (as cyanidin 3-glucoside and cyanidin 3-galactoside) again, and the acquisition of standard specimen also is not easy, even external at present existing certain commodity standard specimen, but (the cyanidin 3-glucoside that provides as the Extrasynthses company of France is 88 dollars/milligram) also costs an arm and a leg, also be difficult for preserving (in 5 ℃ of refrigerators, can only preserve one month behind the wiring solution-forming), make that the detection cost is very high, be unsuitable for applying of China.
Summary of the invention
The purpose of this invention is to provide the quality detection technology that a kind of chemical analysis method and high performance liquid chromatography-mass spectrum and gas chromatography coupling check whether the non-fermentation waxberry processed goods is mingled, relate to the fruit-vegetable food technical field of quality detection, can be used for differentiating the true and false of non-fermentation waxberry processed goods.With the anthocyanin process acid hydrolysis of basic purifying, and, can determine the structure of anthocyanin glycosyl, can distinguish isomers according to the difference of its glycosyl, thereby accurately judge the structure of anthocyanin in the red bayberry through the laggard promoting the circulation of qi chromatography of acetate anhydridization (GC) detection.Give sample quantitative in conjunction with difference pH method, whether HPLC-DAD-MS and GC are qualitative, just can identify to have in the non-fermentation waxberry processed goods under not having the situation of standard specimen and mingle.
The composition of anthocyanin is similar in the close plant of each class sibship, and composition difference very big (Jackman, the R.L.﹠amp of anthocyanin between the sibship different plants far away; Smith, J.L. (1996) .Anthocyanins and betalains.In:B.S.Henry, Natural food colorants (pp.244-310) .Glasgow:Chapman and Hall.).We with this integrated processes analyzed anthocyanin in three kinds of red bayberries content and structure of (wherein a kind of for present China output the highest, one of kind that quality is best water chestnut kind, two kinds are respectively eastern chief and Fragrance Hill in addition), anthocyanin content in them is between 83.25-156.7mg/100g as a result, and all has only a kind of structure, be cyanidin-3-glucoside, its content accounts for more than 97% of total pigment.And at present we also do not have to find by the anthocyanin in which kind of plant form to red bayberry in anthocyanin form similar report.
Technical solution
The quality testing process whether the non-fermentation waxberry processed goods is mingled is:
1. raw material sampling, acidifying methyl alcohol/acidifying alcohol extract, 35-40 ℃ following vacuum concentrate remove methyl alcohol, be dissolved in 0.01% the aqueous hydrochloric acid solution, anthocyanin content in the anthocyanin crude extract (A1), difference pH method test sample product, determine whether be added with other raw material that does not contain anthocyanin in the sample, promptly whether contain the colourless substance except that red bayberry.
2. get part A 1, successively respectively with the ethyl acetate of the sherwood oil of 2 times of volumes and 2 times of volumes separates the water that removes organic phase, collection be adsorbed on the carbon 18 solid phase pillars (200 milligrams of the amounts of filling out) of methyl alcohol activation go up, with 0.01% aqueous hydrochloric acid solution wash-out of 2 times of volumes of A1 solution, with ultraviolet feature and the molecular weight of anthocyanin in 0.01% hydrochloric acid methanol recovery anthocyanin (A2), the HPLC-DAD-MS mensuration red bayberry.
3. get in the tool plug test tube that part A 2 (containing 1 milligram of anthocyanin approximately) adds 20 milliliters, add 15 milliliters of 2M hydrochloric acid solution, charge into that hydrolysis 45 minutes in nitrogen, the 100 ℃ of water-baths, cooled hydrolyzate are adsorbed on the carbon 18 solid phase pillars, glycosyl structure that anthocyanin is determined in the collection of 0.01% aqueous hydrochloric acid solution wash-out, vacuum drying below 50 ℃, acetate anhydridization, vapor-phase chromatography.
4. in conjunction with the structure of anthocyanin in 2 and 3 judgement samples, thereby determine whether raw material in the sample whether only from red bayberry, contains the coloring matter except that red bayberry.
5. judging from qualitative and quantitative angle whether the red bayberry processed goods has in conjunction with 1 and 4 mingles.
Concrete detection method is: with the anthocyanin in the red bayberry as index, with of the national standard sampling of red bayberry processed goods by food sampling, sample through or without with acidifying methyl alcohol or acidifying alcohol extract, at concentrated methyl alcohol or the ethanol of removing of 35-40 ℃ of following vacuum, residue is dissolved in 0.01% the aqueous hydrochloric acid solution, and this solution A 1 usefulness difference pH method is surveyed anthocyanin content: get buffer solution that a part of A1 adopts pH1.0 and pH4.5 on ultraviolet-visible pectrophotometer in 510nm place its content of mensuration; Another part A1 separates with the ethyl acetate of the sherwood oil of 2 times of volumes and 2 times of volumes respectively and removes organic phase, the water of collecting is adsorbed on the carbon 18 solid phase pillars that activate with methyl alcohol, the amount of filling out is 200 milligrams, use 0.01% aqueous hydrochloric acid solution wash-out of 2 times of volumes of A1 solution earlier, reclaim anthocyanin A2 with 0.01% hydrochloric acid methanol again, last its uv-spectrogram of HPLC-DAD-MS instrument detecting of part A2 and molecular weight are with the basic structure of determining anthocyanin, the A2 that another part contains 1 milligram of anthocyanin adds in 20 milliliters the tool plug test tube, the hydrochloric acid solution that adds 15 milliliters of 2M more immediately, charged into behind the nitrogen in 100 ℃ of water-baths hydrolysis 45 minutes, cooled hydrolyzate is adsorbed on the carbon 18 solid phase pillars, the glycosyl part of anthocyanin is collected with 0.01% aqueous hydrochloric acid solution wash-out, the acetate anhydridization carries out GC mensuration after vacuum drying, to determine the glycosyl structure of anthocyanin, in conjunction with above-mentioned uv-spectrogram and molecular weight, thereby accurately judge the structure of anthocyanin in the red bayberry; Difference pH quantitative analysis is measured anthocyanin content in the processed goods, and to determine whether to contain the colourless substance except that red bayberry, HPLC-DAD-MS and GC are used for qualitative analysis to determine whether to contain the coloring matter except that red bayberry.
The non-fermentation waxberry processed goods comprises that all are the non-fermented product that no longer has new arbutus shape that primary raw material processes with the red bayberry.Solid sample needs through acidifying methyl alcohol or alcohol extract, and fluid sample does not need acidified methyl alcohol or acidifying alcohol extract.
The present invention is under the condition that does not need standard model (cyanidin-3-glucoside), adopt a kind of unite difference pH method survey in the red bayberry processed goods anthocyanin content and HPLC-DAD-MS and GC measure wherein the method for anthocyanin kind the anthocyanin in the red bayberry processed goods is carried out qualitative and quantitative, thereby whether mingle in definite red bayberry processed goods.
Get a certain amount of red bayberry processed goods 5-20 gram earlier, doubly the acidifying methyl alcohol of (volume/mass) or alcohol extract (formic acid or acetate or hydrochloric acid or phosphoric acid or the trifluoroacetic acid that contain 0.01%-4% in methyl alcohol or ethanol) extracted 30 minutes to 24 hours not being higher than under 30 ℃ the temperature with 5-10.Extract can extract once with a small amount of acidifying alcohol liquid after filtration or centrifugal removing slag in case of necessity again.Extract is merged, with Rotary Evaporators at reduction vaporization below 40 ℃ to doing, residue is with 0.01% acidifying water dissolving, again with 0.01% acidified aqueous solution constant volume to certain volume (A1), use anthocyanin content in the difference pH method working sample.
Measure the kind time-like of anthocyanin in the red bayberry, desirable part A 1, separate with the ethyl acetate of the sherwood oil of 2 times of volumes and 2 times of volumes respectively and remove organic phase, the water of collecting is adsorbed on the carbon 18 solid phase pillars (200 milligrams of the amounts of filling out) that activate with methyl alcohol, use 0.01% aqueous hydrochloric acid solution wash-out of 2 times of volumes of A1 solution again, reclaim anthocyanin (A2) with 0.01% hydrochloric acid methanol, after using the micro-pore-film filtration of 0.45 μ m at last, sample introduction 10 μ L are used for ultraviolet feature and the molecular weight that HPLC-DAD-MS analyzes the red bayberry anthocyanin.Other gets in the tool plug test tube of 20 milliliters of part A 2 (containing 1 milligram of anthocyanin approximately) addings, and add the hydrochloric acid solution of 15 milliliters of 2M, charged into behind the nitrogen in 100 ℃ of water-baths hydrolysis 45 minutes, be adsorbed on the carbon 18 solid phase pillars after the hydrolyzate cooling, collect with 0.01% aqueous hydrochloric acid solution wash-out, in vacuum drying below 50 ℃ and behind the acetate anhydridization, determine the glycosyl structure of anthocyanin with vapor-phase chromatography.
In the difference pH method working sample during anthocyanin content, adopt the damping fluid that is made into pH1.0 and pH4.5 by sodium acetate, potassium chloride and hydrochloric acid to be undertaken by method.
When HPLC-DAD-MS analyzed, HPLC-DAD adopted anti-phase C-18 post, mineral acid (formic acid or acetate or trifluoroacetic acid)-methanol-water or mineral acid-acetonitrile-water gradient elution, and DAD is full scan in the 200-600nm scope, and 520nm is for detecting wavelength.Mass spectrometer adopts positive ion mode (EIS
+), capillary voltage 3.87kVolts, taper hole voltage 25Volts, 120 ℃ of ion source temperatures, 300 ℃ of desolventizing temperature degree, mass charge ratio range 200-1000m/z, photomultiplier cell voltage 700Volts, instrument pressure 2.6e-5mBar, flow rate of carrier gas 4.2lit/hr.The red bayberry anthocyanin is having only a tangible absorption peak when 520nm detects on the HPLC chromatogram, its uv-spectrogram is for to have two places to absorb by force at 280nm and 520nm, under positive ion mode, its mass spectrogram has the fragmention m/z=287 of a less false molion m/z=449 of an intensity and an intensity maximum.Be the uv-spectrogram and the fragment ion pattern of cyanidin-3-glucose.
When GC analyzes, acetate anhydridization process is for taking by weighing dry monose sample 50-100mg, add the 0.5ml pyridine, the 10mg oxammonium hydrochloride, quantitatively add 3~5mg inositol as interior mark, kept 30 minutes in 90 ℃ of water-baths, taking-up cooling back adds the 0.5ml acetic anhydride and kept 30 minutes in 90 ℃ of water-baths, treats to carry out the GC analysis after the sample cooling.Analytical instrument and condition are day island proper Tianjin (Shimadzu) GC-14A, OV-1701 quartz capillary column 30m * 0.32mm i.d., flame ion (FID) detecting device, 260 ℃ of temperature of vaporization chamber and detector temperatures, temperature programme: 185 ℃, kept 3 minutes; 185-240 ℃, per minute rises 3 ℃; 240 ℃ kept 20 minutes down; Nebulizer gas pressure (N
2) 0.60kg/cm
2Gaseous-pressure (H
2) 0.65kg/cm
2Combustion-supporting gas (air) 0.5kg/cm
2Split ratio 30: 1; Sample size 1 μ l.Rhamnose, arabinose, wood sugar, mannose, glucose and galactose are external standard in the same old way, and the glycosyl part of anthocyanin only can detect tangible glucose in the red bayberry.
For judging the true and false of red bayberry processed goods, to process genuine piece to red bayberry earlier and detect, preserve the content in stage with anthocyanin in definite different processed goods in difference, and set up a database.If being inconsistent with above feature with the GC collection of illustrative plates, the HPLC-DAD-MS in the checking matter (do not conform to for when HPLC-DAD-MS analyzes, near 520nm, having only a tangible absorption peak, uv-spectrogram is for to have two places to absorb by force at 280nm and 520nm, false molion m/z=449, fragmention m/z=287; GC then only detects tangible glucose when analyzing), then be mixed with the coloring matter except that red bayberry in the checking matter.If the HPLC-DAD-MS in the checking matter conforms to above feature with the GC collection of illustrative plates, but being lower than red bayberry, anthocyanin content processes genuine piece at the content in corresponding storage stage, then be mixed with the colourless substance except that red bayberry in the checking matter.
Beneficial effect of the present invention:
The present invention has proposed a kind of no standard specimen analyzing detecting method whether the non-fermentation waxberry processed goods is mingled that is used for first.Adopt chemical analysis method and high performance liquid chromatography-mass spectrum and gas chromatography joint-detection, have few, the characteristics fast and accurately of sampling amount, can be widely used in the supervision and check whether departments such as customs, commodity inspection, technical supervision and health and epidemic prevention are mingled the red bayberry converted products.
Embodiment
Embodiment 1: be the analyzing detecting method of whether mingling in the cranberry juice of raw material production with the water chestnut kind.
Earlier the cranberry juice of not mingling is preserved the content of stage anthocyanin with difference pH method working sample in difference, and set up the anthocyanin content database of different phase.Owing to is fluid sample, can carry out acidifying methyl alcohol or alcohol extract and directly carry out difference pH method and measure the wherein content of anthocyanin by the sample cranberry juice.Cranberry juice successively separates with the ethyl acetate of the sherwood oil of 2 times of volumes and 2 times of volumes respectively and removes organic phase, the water of collecting is adsorbed on the carbon 18 solid phase pillars (200 milligrams of the amounts of filling out) that activate with methyl alcohol, use 0.01% aqueous hydrochloric acid solution wash-out of 2 times of volumes of cranberry juice again, reclaim anthocyanin (A2) with 0.01% hydrochloric acid methanol, after using the micro-pore-film filtration of 0.45 μ m at last, sample introduction 10 μ L are used for ultraviolet feature and the molecular weight that HPLC-DAD-MS analyzes the red bayberry anthocyanin.Other gets in the tool plug test tube of 20 milliliters of part A 2 (containing 1 milligram of anthocyanin approximately) addings, and add the hydrochloric acid solution of 15 milliliters of 2M, charged into behind the nitrogen in 100 ℃ of water-baths hydrolysis 45 minutes, be adsorbed on the carbon 18 solid phase pillars after the hydrolyzate cooling, collect with 0.01% aqueous hydrochloric acid solution wash-out, in vacuum drying below 50 ℃ and behind the acetate anhydridization, determine the glycosyl structure of anthocyanin with vapor-phase chromatography.
If being inconsistent with above feature with GC, the HPLC-DAD-MS in the checking matter (do not conform to for when HPLC-DAD-MS analyzes, near 520nm, having only a tangible absorption peak, uv-spectrogram is for to have two places to absorb by force at 280nm and 520nm, false molion m/z=449, fragmention m/z=287; GC then only detects tangible glucose when analyzing), then be mixed with the coloring matter except that red bayberry in the checking matter.If the HPLC-DAD-MS in the checking matter conforms to above feature with GC, but being lower than red bayberry, anthocyanin content processes genuine piece at the content in corresponding storage stage, then be mixed with the colourless substance except that red bayberry in the checking matter.
Embodiment 2: be the analyzing detecting method of whether mingling in the gooseberry jam of raw material (corresponding collection of illustrative plates slightly) with the Fragrance Hill red bayberry.
Earlier the gooseberry jam of not mingling is preserved the content of stage anthocyanin with difference pH method working sample in difference, and set up the anthocyanin content database of different phase.Method is when sample extraction, gets 5g jam, uses the hydrochloric acid methanol of 100mL 0.1% to stir extraction 1 hour at 25 ℃, and extract filter paper filtering suction filtration, residue stir extraction once with the hydrochloric acid methanol of 50mL 0.1% at 25 ℃ again.Extract is merged, with Rotary Evaporators at reduction vaporization below 45 ℃ to doing, residue dissolves with 0.1% acidifying water, again with 0.1% acidified aqueous solution constant volume to 5mL (A1), use the content of anthocyanin in the difference pH method working sample then.Then with A1 carry out HPLC-DAD-MS and GC when analyzing then with embodiment 1.
If being inconsistent with above feature with GC, the HPLC-DAD-MS in the checking matter (do not conform to for when HPLC-DAD-MS analyzes, near 520nm, having only a tangible absorption peak, uv-spectrogram is for to have two places to absorb by force at 280nm and 520nm, false molion m/z=449, fragmention m/z=287; GC then only detects tangible glucose when analyzing), then be mixed with the coloring matter except that red bayberry in the checking matter.If the HPLC-DAD-MS in the checking matter conforms to above feature with GC, but being lower than red bayberry, anthocyanin content processes genuine piece at the content in corresponding storage stage, then be mixed with the colourless substance except that red bayberry in the checking matter.