1 Scope
This standard specifies methods for determination of inositol in foods.
Method I in this standard is applicable to the determination of inositol in foods and Method II in this standard is applicable to the determination of inositol in modified milk products and beverages.
Method I Microbiological Method
2 Principle
The content of to-be-determined substance in specimen is quantitatively determined by utilizing the specificity and sensitivity of saccharomyces uvarum to inositol. In the medium containing all the nutrient contents except for the to-be-determined substance, the growth of microorganisms is linearly related to the content of to-be-determined substance, so the content of the to-be-determined substance in specimen can be calculated by comparing the transmittance and standard work curve.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade II water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 Sodium chloride (NaCl).
3.1.2 Sodium hydroxide (NaOH).
3.1.3 Hydrochloric acid (HCl).
3.1.4 Phosphorus pentoxide (P2O5).
3.1.5 Saccharomyces uvarum (ATCC 9080), or other equivalent standard bacterial strain.
3.2 Preparation of reagents
3.2.1 Sodium chloride solution (9g/L): weigh 9.0g of sodium chloride and dissolve it into 1 000mL of water, divide the solution into test tubes and 10mL for each tube, and then sterilize for 15min at 121℃.
3.2.2 Hydrochloric acid solution (1mol/L): measure 82mL of hydrochloric acid and bring the volume to 1 000mL after cooling.
3.2.3 Hydrochloric acid solution (0.44mol/L): measure 36.6mL of hydrochloric acid and bring the volume to 1 000mL after cooling.
3.2.4 Sodium hydroxide solution (600g/L): weigh 300g of sodium hydroxide, dissolve it into water, and bring the volume to 500mL after cooling.
3.2.5 Sodium hydroxide solution (1mol/L): weigh 40g of sodium hydroxide, dissolve it into water, and bring the volume to 1 000mL after cooling.
3.3 Standards
Inositol standard (C6H12O6): purity ≥99%, or reference material approved and awarded with reference material certificate by the nation.
3.4 Preparation of standard solutions
3.4.1 Inositol standard stock solution (0.2mg/mL): put the inositol standard into the dryer of phosphorus pentoxide to dry for more than 24h, weigh 50mg of inositol standard (accurate to 0.1mg), sufficiently dissolve it with water, bring the volume into a 250mL brown volumetric flask and store the solution in a 4℃ refrigerator.
3.4.2 Inositol standard intermediate solution (10μg/mL): pipet 5.00mL of inositol standard stock solution, bring the volume with water into a 100mL brown volumetric flask and store the solution in a 4℃ refrigerator.
3.4.3 Inositol standard working solutions (1μg/mL and 2μg/mL): pipet 10mL of inositol standard intermediate solution for twice, respectively bring the volume with water into a 100mL volumetric flask and a 50mL volumetric flask. This working solution shall be prepared immediately before use.
3.5 Materials
3.5.1 Medium
3.5.1.1 Malt extract agar: it may be prepared according to Appendix A.
3.5.1.2 Medium for determining inositol: it may be prepared according to Appendix A.
Note: Some merchandized synthetic media are effective and they are prepared according to descriptions on the label.
3.5.2 Glass bead
With diameter of about 5mm.
4 Apparatuses
Note: In addition to the apparatuses for conventional sterilization and cultivation of microorganism, other apparatuses and materials are as follows:
4.1 Balance: with sensitivity of 0.1mg.
4.2 pH meter: with precision of ±0.01.
4.3 Spectrophotometer.
4.4 Vortex oscillator.
4.5 Centrifuge: with rotation speed ≥ 2 000r/min.
4.6 Thermostatic incubator: 30℃±1℃.
4.7 Incubator shaker: 30℃±1℃, with oscillating speed of 140 ~ 160 times/min.
4.8 Pressure steam sterilizer: 121℃(0.10MPa~0.12MPa).
Note: Prior to use of glass apparatus, clean the hard glass determination tubes and other necessary glassware with active agent (made by adding 1-dodecane sulfonic acid sodium salt or household detergent into washing water), and subject them to dry heat (200℃) for 2h.
5 Analysis Procedures
5.1 Preparation of inoculant suspension
5.1.1 Strain recovery
Inoculate the bacterial strain after activation on malt extract slant agar, after culturing for 16h~24h at 30℃±1℃, transfer the second or third generation to enhance activity and make stock strain; and then store the strain in a 4℃ refrigerator and the storage life shall not exceed two weeks. The strain is inoculated to new malt extract slant agar before use.
5.1.2 Preparation of inoculant suspension
Transfer the stock strain to newly prepared malt extract slant agar on the day before use and culture for 20h~24h at 30℃±1℃. Scrape the lawn with inoculating loop and place it into a test tube containing sodium chloride solution (9g/L). Centrifuge for 15min at 2 000r/min and rinse in this way for three or four times. Pipet a certain amount of this bacterium solution and transfer it into a test tube containing 10mL of sodium chloride solution (9g/L) to make inoculant suspension.
Determine the transmittance of this inoculant suspension with spectrophotometer at wavelength of 550nm by taking sodium chloride solution as blank; adjust the volume of added bacterium solution or add into a certain amount of sodium chloride solution to make the transmittance of this bacterial suspension within 60%~80%.
5.2 Specimen preparation
Specimens of grain and milk powder need to be smashed, grinded and sieved (mesh size of the sieve plate is 0.3mm ~ 0.5mm); those of meat and meat products need to be made into chyme with beating crusher; those of fruits and vegetables need to be made into homogenate and mixed well; and liquid specimens need to be shaken for mixing before use. These specimens shall be preserved in a 4℃ refrigerator and determined within 1 week.
5.3 Extraction of specimen
Accurately weigh specimens containing 0.5mg ~2.0mg of inositol; 1g (accurate to 0.01g) of common milk powder, fresh fruit and vegetables, viscera and raw meat specimens; 5g (accurate to 0.01g) of grain, bean and natural food specimens with low inositol content; 0.1g~0.5g of common nutrient supplements and composite and nutrient supplements; 5g~10g of liquid beverage or liquid and semifluid specimens; and then put them into a 250mL conical flasks. Add into 80mL of hydrochloric acid solution (0.44mol/L) for dry powder specimen, 100mL of hydrochloric acid solution (0.44mol/L) for liquid specimen, and then mix them well.
Cover the conical flask with aluminium foil and hydrolyze the solution in a 125℃ autoclave to hydrolyze for 1h. Take out the conical flask, cool it to room temperature, add into about 2mL of sodium hydroxide solution (600g/L) and then cool it. Adjust the pH value to 5.2 with sodium hydroxide solution (1mol/L) or hydrochloric acid solution (1mol/L), transfer the solution to a 250mL volumetric flask and bring the volume to the scale. Mix the solution well, filter and collect the filtrate, which is the to-be-determined solution. Adjust the dilutability and make the concentration of inositol in to-be-determined solution within the range of 1μg/mL~10μg/mL.
5.4 Plotting of standard curve
Add water, inositol standard working solution and medium for determining inositol into culture tube in triplicate according to the sequence in Table 1.
Table 1 Plotting of Standard Curve
Test tube No. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10
Water/mL 5 5 4 3 2 1 0 2 1 0
Inositol standard working solution (1μg/mL)/mL 0 0 1 2 3 4 5 0 0 0
Inositol standard working solution (2μg/mL)/mL 0 0 0 0 0 0 0 3 4 5
Medium mL 5 5 5 5 5 5 5 5 5 5
5.5 Preparation of to-be-determined solution
Add water, to-be-determined solution and medium for determining inositol into culture tube in triplicate according to the sequence in Table 2.
Table 2 Preparation of To-be-determined Solution
Test tube No. 1 2 3 4
Water/mL 4 3 2 1
Specimen extracting solution/mL 1 2 3 4
Medium /mL 5 5 5 5
5.6 Sterilization
Add one glass bead into each test tube, put on the test tube cap and sterilize for 5min at 121℃ (the sterilization for commercial culture medium is carried out in accordance with the description on label).
5.7 Inoculation
Fix the test tube in incubator shaker and carry out shaking culture for 22h~24h at 30℃±℃ with the shaking speed of 140~160 times/min.
5.8 Determination
Note: Carry out visual inspection for each test tube; the culture solution in inoculated blank tube S1 shall be clear; if turbidity appears, the result is invalid.
5.8.1 Take out the test tube from incubator shaker and put it inside the autoclave for sterilization and keep for 5min at 100℃ to make microorganism stop growing.
5.8.2 Use inoculated blank test tube S1 as blank, adjust the transmittance of spectrophotometer to 100% (or absorbance to 0), and then read out the reading of inoculated blank tube S2. Then use inoculated blank tube S2 as blank, adjust the transmittance to 100% (or absorbance to 0), read out the transmittance (or absorbance) of other test tubes successively.
5.8.3 After each test tube is thoroughly mixed by vortex oscillator (or one drop of defoamer is added), immediately transfer the culture solution into cuvette for determination, the wavelength is 550nm~660nm, and then read out the transmittance after the reading is stable for 30s; and stabilization duration for each test tube shall be same. Plot the standard curve by taking the concentration of inositol standard series as horizontal coordinate and the transmittance as longitudinal coordinate.
5.8.4 The concentration of inositol in this to-be-determined solution is obtained from the standard curve according to the transmittance of to-be-determined solution, and then the content of inositol in specimen is calculated according to the dilution factor and sampling weight. Specimen tubes with transmittance beyond the range of standard curve tubes S3~S10 shall be rejected.
5.8.5 For test tube with to-be-determined solution of each number, calculate the concentration (per milliliter) of inositol in to-be-determined solution of that number by the transmittance of each test tube and calculate the average value of this concentration; the concentration measured for each test tube shall not exceed ±15% of the average value and those exceeding that shall be rejected. If the determination result of one test tube fails to meet the above-mentioned requirements, the result of that test tube is rejected and the average value is recalculated; if the determination results of two test tubes fail to meet the above-mentioned requirements, reinspection is required.
Note: Either transmittance or absorbance may be read when plotting the standard curve.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Apparatuses
5 Analysis Procedures
6 Expression of Analysis Results
7 Accuracy
8 Others
9 Principle
10 Reagents and Materials
11 Apparatuses
12 Analysis Procedures
13 Expression of Analysis Results
14 Accuracy
15 Others
Appendix A Media and Reagents
Appendix B Gas Chromatogram of Inositol Standard Derivative
