GB 1903.80-2025National Food Safety Standard - Food Nutritional Fortification Substance - Yeast Beta-Glucan (English PDF)

1 Scope

This standard applies to the food nutritional fortification substance yeast beta-glucan, whose main component is beta-1,3 and beta-1,6 glucan, obtained from brewing yeast (Saccharomyces cerevisiae) as raw material through cell wall disruption and extraction, acid and alkali treatment, separation and purification, drying and other operations.

Note: the species name Saccharomyces cerevisiae is rendered in Chinese as brewing yeast; the baker's yeast referred to in Ministry of Health Announcement No. 6 of 2012, in the wording made from baker's yeast (Saccharomyces cerevisiae) as raw material, is a common English name for that same yeast.

2 Molecular formula, structural formula and relative molecular mass

2.1 Molecular formula. (C6H10O5)n, where n is the degree of polymerisation, from 125 to 25000 inclusive.

2.2 Structural formula. Printed in the standard as a drawing, which the digitised text does not carry.

2.3 Relative molecular mass. From 20000 to 4000000, based on the 2022 international relative atomic masses.

3 Technical requirements

3.1 Sensory requirements. The sensory requirements shall comply with Table 1. Table 1 fixes the colour as pale yellow to yellowish brown, the state as powder, and the odour as the odour characteristic of the product. The test method given against all three is the same: take a suitable amount of the sample, place it in a clean, dry white porcelain dish, observe its colour and state under natural light, and smell it.

3.2 Physical and chemical indexes. The physical and chemical indexes shall comply with Table 2. Table 2 fixes the following limits, each with the test method shown against it. Yeast beta-glucan content, mass fraction not less than 75 percent, by A.3 of Annex A. Protein, mass fraction not more than 3.5 percent, by the Kjeldahl method of GB 5009.5. Fat, mass fraction not more than 10.0 percent, by the acid hydrolysis method of GB 5009.6. Moisture, mass fraction not more than 8.0 percent, by the direct drying method of GB 5009.3. Ash, mass fraction not more than 3.0 percent, by the determination of total ash in foods of GB 5009.4. Lead, not more than 0.5 mg/kg, by GB 5009.12 or GB 5009.75. Total arsenic, expressed as As, not more than 0.5 mg/kg, by GB 5009.11 or GB 5009.76. Total mercury, expressed as Hg, not more than 0.05 mg/kg, by GB 5009.17. Cadmium, not more than 0.5 mg/kg, by GB 5009.15.

3.3 Microbiological limits. The microbiological limits shall comply with Table 3, which is written as a sampling plan with the number of sample units n, the maximum allowable number of sample units between m and M given as c, and the two limits m and M, expressed in CFU per gram unless otherwise stated. For aerobic plate count, n is 5, c is 2, m is 10000 and M is 50000, by GB 4789.2. For coliforms, n is 5, c is 2, m is 10 and M is 100, by the plate count method of GB 4789.3. For Staphylococcus aureus, n is 5, c is 0 and m is zero in 25 g, with no M given, by GB 4789.10. For Salmonella, n is 5, c is 0 and m is zero in 25 g, with no M given, by GB 4789.4. A footnote to the table states that the collection and handling of the samples follow GB 4789.1.

A.1 General provisions

Unless other requirements are stated, the reagents and the water used in this standard are analytically pure reagents and grade three water as laid down in GB/T 6682.

Unless other requirements are stated, the standard solutions, the standard solutions for the determination of impurities, and the preparations and products used in the tests are prepared as laid down in GB/T 601, GB/T 602 and GB/T 603.

Where the solvent used to prepare a solution is not stated, an aqueous solution is meant.

A.2 Identification test

Identification of yeast beta-glucan by its characteristic infrared spectrum is carried out by the potassium bromide pellet method following GB/T 6040. The infrared spectrum of the yeast beta-glucan shall match the features of the infrared spectrum of the yeast beta-glucan reference substance given in Annex B, with a broad, fairly strong absorption band near 3419 reciprocal centimetres, which is the O-H stretching absorption of sugars, and weaker absorption bands near 2923 reciprocal centimetres, which is the C-H stretching absorption of sugars, and near 889 reciprocal centimetres, which is the absorption characteristic of the beta configuration.

A.3 Determination of yeast beta-glucan content

A.3.1 Acid hydrolysis method (first method). Dissolve the sample in water, filter off the insoluble matter, wash the filter residue with water so that it is completely separated from the bulk of the sample, dry it, and weigh the mass of the water insoluble impurities on the balance.

A.3.1.1 Principle of the acid hydrolysis method. After the yeast beta-glucan sample has been hydrolysed with acid, the glucose and the other components in the sample are separated on a high performance liquid chromatographic column, detected with a differential refractive index detector, and determined quantitatively by the external standard method. Note: during the hydrolysis of yeast beta-glucan the hydrolysis may be incomplete, and the glucose produced may partly undergo other side reactions at high temperature, so that the yeast beta-glucan content found is lower than the true value.

A.3.1.2 Apparatus and equipment. Electronic balance, readability 0.001 g. Constant temperature drying oven, 100 degrees Celsius plus or minus 2 degrees Celsius. Constant temperature water bath, 30 degrees Celsius plus or minus 1 degree Celsius. Autoclave. Vortex mixer. High performance liquid chromatograph with differential refractive index detector.

A.3.1.3 Reagents and materials. Water: grade one water of GB/T 6682. Hydrochloric acid: 37 percent. Anhydrous glucose (CAS number 50-99-7), purity analytically pure, not less than 99.5 percent. Glucose standard solution, 2.0 g/L: weigh 0.2 g, to the nearest 0.001 g, of glucose that has been dried at 98 degrees Celsius to 100 degrees Celsius for 2 h, dissolve it in water and make up to 100 mL, then shake well. Yeast beta-glucan reference substance: of known purity, the purity being not less than 75 percent. Sodium hydroxide solution, 300 g/L: weigh 300 g of sodium hydroxide to the nearest 0.01 g, make up to 1000 mL with water and shake well. Cellulose acetate membrane: pore size 0.22 micrometres.

A.3.1.4 Treatment of the sample. Weigh 0.4 g, to the nearest 0.001 g, of the sample or of the yeast beta-glucan reference substance into a 20 mL screw capped tube, add 6.0 mL of hydrochloric acid, cap tightly and shake to obtain a uniform suspension. Keep the tube in a water bath at 30 degrees Celsius for 45 min, mixing on the vortex mixer once every 15 min. Transfer the suspension from the water bath into a 200 mL heat resistant screw capped bottle, washing the tube several times with 100 mL to 120 mL of water and adding the washings to the bottle. Place the screw capped heat resistant bottle in the autoclave and sterilise at 121 degrees Celsius for 60 min. Take it out, cool it to room temperature, adjust the pH to between 6 and 7 with the sodium hydroxide solution, transfer to a 200 mL volumetric flask, make up to the mark with water and mix. Filter through the cellulose acetate membrane of 0.22 micrometre pore size before use.

A.3.1.5 Reference chromatographic conditions. Chromatographic column: a sugar column, 6.5 mm by 300 mm, or a separation column of equivalent analytical performance. Mobile phase: pure water. Column temperature: 80 degrees Celsius. Flow rate: 0.5 mL/min. Injection volume: 20 microlitres.

A.3.1.6 Drawing of the calibration curve. Measure 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL and 10.0 mL of the glucose standard solution into 10 mL volumetric flasks, make up to the mark with water and shake well, giving a series of standard solutions with glucose mass concentrations of 400 mg/L, 800 mg/L, 1200 mg/L, 1600 mg/L and 2000 mg/L. Inject 20 microlitres under the chromatographic conditions of A.3.1.5 and draw the calibration curve from the chromatographic peak areas and the glucose concentrations.

A.3.1.7 Determination of the sample and of the reference substance. Under the same chromatographic conditions, inject the sample solution and the yeast beta-glucan reference substance solution prepared by the method of A.3.1.4 into the chromatograph and record the retention time and the peak area of each chromatographic peak. Identify by the retention time of the glucose standard solution peak and quantify by the peak area of the glucose standard solution peak.

A.3.1.8 Calculation of the result. The yeast beta-glucan content is calculated by formula (A.1). In the formula: X1 is the yeast beta-glucan content of the sample, in grams per 100 grams; c1 is the glucose content of the sample solution calculated from the peak area of the sample solution by means of the calibration curve, in milligrams per litre; 0.2 is the volume to which the sample or the reference substance was made up after treatment, in litres; 100 is the conversion factor for percentage content; m1 is the mass of sample taken, in grams; 1000 is the conversion factor between milligrams and grams; 0.9 is the factor for converting glucose to yeast beta-glucan; and F1 is the empirical compensation factor for the low result caused by destruction of glucose during acid hydrolysis of the sample. The value of F1 is calculated by formula (A.2), in which P1 is the purity of the yeast beta-glucan reference substance, taken from the test report supplied by its manufacturer, in grams per 100 grams; 100 is the conversion factor for percentage content; W is the moisture of the yeast beta-glucan reference substance, taken from the test report supplied by its manufacturer, in grams per 100 grams; m2 is the mass of yeast beta-glucan reference substance taken, in grams; 1000 is the conversion factor between milligrams and grams; c2 is the glucose content of the reference substance solution calculated from the peak area of that solution by means of the calibration curve, in milligrams per litre; 0.2 is the volume to which the sample or the reference substance was made up after treatment, in litres; and 0.9 is the conversion factor between glucose and yeast beta-glucan. The result is given as the arithmetic mean of two independent determinations obtained under repeatability conditions, kept to the nearest whole number.

A.3.1.9 Precision. The absolute difference between two independent results obtained under repeatability conditions shall not exceed 5 percent of their arithmetic mean.

A.3.2.1 Principle of the enzymatic hydrolysis method. The yeast beta-glucan is gelatinised with potassium hydroxide solution and then hydrolysed in several stages with lyticase, beta-(1,6)-glucanase, beta-(1,3)-glucanase and beta-glucosidase, so that it is finally hydrolysed to glucose. Glucose oxidase catalyses the oxidation of glucose in the presence of oxygen, giving D-glucono-delta-lactone and hydrogen peroxide. The hydrogen peroxide, catalysed by peroxidase, reacts with 4-aminoantipyrine and p-hydroxybenzoic acid to form a red quinoneimine; this is the glucose oxidase and peroxidase method. The absorbance of the quinoneimine is measured on a spectrophotometer at a wavelength of 510 nm, the glucose content of the sample is calculated, and from it the yeast beta-glucan content is obtained.

A.3.2.2 Apparatus and equipment. Vortex mixer. Constant temperature water bath. Spectrophotometer. Electronic balance, readability 0.001 g. pH meter, accuracy 0.1 pH.

A.3.2.3 Reagents and solutions. Yeast beta-glucan reference substance: of known purity, not less than 75 percent. Lyticase: enzyme activity not less than 200 units per milligram. Beta-(1,6)-glucanase: enzyme activity not less than 2 units per milligram. Mixed beta-(1,3)-glucanase and beta-glucosidase: enzyme activities not less than 100 units per millilitre and 20 units per millilitre respectively. Series of glucose standard solutions: measure 0.6 mL, 1.0 mL, 2.5 mL, 4.0 mL and 5.0 mL of the glucose standard solution into 10 mL volumetric flasks, make up with water and mix, giving glucose mass concentrations of 0.12 g/L, 0.20 g/L, 0.50 g/L, 0.80 g/L and 1.00 g/L. Potassium hydroxide solution, 2 mol/L: weigh 11.2 g of potassium hydroxide to the nearest 0.01 g, dissolve it thoroughly in water, make up to 100 mL, mix and store refrigerated at 4 degrees Celsius. Sodium hydroxide solution, 1 mol/L: weigh 4 g of sodium hydroxide to the nearest 0.01 g, dissolve it thoroughly in water, make up to 100 mL and mix. Hydrochloric acid solution, 1 mol/L: measure 8.33 mL of hydrochloric acid to the nearest 0.01 mL, add water, make up to 100 mL and mix. Sodium acetate buffer solution A, pH 5: weigh 5.25 g of anhydrous sodium acetate and 2.16 g of glacial acetic acid, each to the nearest 0.01 g, into 400 mL of water, adjust the pH to 5 with the sodium hydroxide solution or with glacial acetic acid, make up to 500 mL with water and mix. Sodium acetate buffer solution B, pH 3.8: weigh 4.96 g of anhydrous sodium acetate and 32.32 g of glacial acetic acid, each to the nearest 0.01 g, into 400 mL of water, adjust the pH to 3.8 with the sodium hydroxide solution or with glacial acetic acid, make up to 500 mL with water and mix. Buffer solution C, pH 7.5: dissolve 1.212 g of tris(hydroxymethyl)aminomethane, 1.169 g of sodium chloride and 0.416 g of EDTA tetrasodium dihydrate, each to the nearest 0.001 g, in 90 mL of water; adjust the pH to 7.5 with the hydrochloric acid solution or the sodium hydroxide solution, make up to 100 mL with water and mix. Lyticase solution, 10 units per microlitre: transfer a suitable amount of lyticase into 10 percent buffer solution C so that the final concentration of lyticase is 10 units per microlitre; it keeps for one year at minus 15 degrees Celsius and shall not be repeatedly frozen and thawed. Beta-(1,6)-glucanase solution: dissolve a suitable amount of beta-(1,6)-glucanase in sodium acetate buffer solution A to a final concentration of 1 unit per 300 microlitres; it is a suspension, keeps for 60 days at minus 15 degrees Celsius, and shall not be repeatedly frozen and thawed. Mixed enzyme solution: transfer a suitable amount of the mixed beta-(1,3)-glucanase and beta-glucosidase, add a suitable amount of sodium acetate buffer solution A, dilute and mix to final concentrations of 20 units per millilitre and 4 units per millilitre respectively; keep it in an ice bath while in use and use it the same day, and store any unused portion frozen at minus 15 degrees Celsius for up to 2 years without repeated freezing and thawing. Glucose oxidase and peroxidase buffer: add about 160 mL of water to a 200 mL volumetric flask, then add 27.2 g of potassium dihydrogen phosphate, 8.4 g of sodium hydroxide and 6.0 g of p-hydroxybenzoic acid, each to the nearest 0.01 g, stir until completely dissolved, adjust the pH to 7.4, finally add 0.8 g of sodium azide and, when it has dissolved, make up to the mark with water. Store at 4 degrees Celsius; valid for 4 years. To dilute it, measure 48 mL of the buffer into a 1000 mL volumetric flask, make up with water and mix; prepare it fresh for use. Glucose oxidase and peroxidase mixed enzyme powder: glucose oxidase not less than 400 units, peroxidase not less than 1000 units, and 4-aminoantipyrine. Glucose oxidase and peroxidase working solution: measure 20 mL of the diluted buffer into the mixed enzyme powder and swirl gently until it is fully dissolved, then add the remaining 980 mL of diluted buffer; store away from light at 4 degrees Celsius, valid for 3 months, or at minus 20 degrees Celsius, valid for 12 months, without repeated freezing and thawing. Note: the solutions from buffer solution C to the glucose oxidase and peroxidase working solution may also be commercial reagents or reagent kits. Cellulose acetate membrane: pore size 0.22 micrometres.

A.3.2.4 Treatment of the sample. Weigh 15 mg to 20 mg, to the nearest 0.001 g, of the sample or of the reference substance into a centrifuge tube, add 0.4 mL of cold potassium hydroxide solution, and vortex in an ice water bath for 20 min, vortexing briefly several times during that period until all the precipitate is dispersed and no lumps are visible. Add 1.6 mL of sodium acetate buffer solution B and 600 microlitres of lyticase solution, record the total volume of the solution at this point as V1, incubate the reaction mixture at 50 degrees Celsius for 12 h to 18 h and cool it to room temperature. Pipette 130 microlitres, recorded as V2, of the cooled hydrolysate into a 2 mL centrifuge tube, add 25 microlitres of potassium hydroxide solution and 300 microlitres of beta-(1,6)-glucanase solution, incubate at 80 degrees Celsius for 15 min and cool to room temperature. Then add 390 microlitres of the mixed enzyme solution, record the total volume of the solution at this point as V3, incubate at 40 degrees Celsius for 1 h and cool to room temperature. Take a suitable amount of the solution, filter it through the membrane and carry out the determination.

A.3.2.5 Enzymatic hydrolysis blank solution. Transfer 20 microlitres of cold potassium hydroxide solution into a 5 mL centrifuge tube, add 80 microlitres of sodium acetate buffer solution B, mix, then add 30 microlitres of lyticase solution and mix. Incubate at 50 degrees Celsius for 12 h to 18 h and cool to room temperature. Add 25 microlitres of potassium hydroxide solution and 300 microlitres of beta-(1,6)-glucanase solution, incubate at 80 degrees Celsius for 15 min and cool to room temperature. Finally add 390 microlitres of the mixed enzyme solution, incubate at 40 degrees Celsius for 1 h and cool to room temperature. Take a suitable amount of the solution, filter it through the membrane and carry out the determination.

A.3.2.6 Drawing of the calibration curve. Transfer 100 microlitres of each of the series of glucose standard solutions in turn into 5 mL centrifuge tubes, add 3 mL of the glucose oxidase and peroxidase working solution and hold in a water bath at 40 degrees Celsius for 20 min. Remove from the water bath, cool to room temperature, filter through the membrane, transfer the whole into a 1 cm cell and, with the working solution as the blank, measure the absorbance at 510 nm on the spectrophotometer. Draw the calibration curve with the absorbance on the vertical axis and the concentration of the series of standard solutions on the horizontal axis.

A.3.2.7 Determination of the sample solution. Measure 100 microlitres each of the sample solution and the reference substance solution treated as in A.3.2.4 and of the enzymatic hydrolysis blank solution treated as in A.3.2.5 into 5 mL centrifuge tubes, add 3 mL of the glucose oxidase and peroxidase working solution to each and hold in a water bath at 40 degrees Celsius for 20 min. Remove from the water bath, cool to room temperature, filter through the membrane, transfer the whole into a 1 cm cell and, with the working solution as the blank, measure the absorbance at 510 nm on the spectrophotometer, then calculate the glucose concentration of the sample solution from the calibration curve. The solution may be diluted according to the particular sample.

A.3.2.8 Calculation of the result. The content of the yeast beta-glucan sample is calculated by formula (A.3). In that formula: X2 is the purity of the glucan in the sample, in grams per 100 grams; 100 is the conversion factor for percentage content; c3 is the measured final glucose content of the sample hydrolysate, in grams per litre; c0 is the measured glucose content of the enzymatic hydrolysis blank solution, in grams per litre; 0.9 is the conversion factor between glucose and yeast beta-glucan; F2 is the compensation factor for the reference substance; V1 is the volume of the hydrolysate after hydrolysis with lyticase, in millilitres; V3 is the final volume after all the enzymatic hydrolysis steps, in millilitres; f is the dilution factor used in determining the glucose content; m3 is the mass of the sample, in milligrams; and V2 is the volume of hydrolysate taken from the lyticase hydrolysate, in millilitres. The content of the yeast beta-glucan reference substance is calculated by formula (A.4), in which P prime is the purity of the reference substance calculated from the final glucose content cs of its hydrolysate, in grams per 100 grams, and cs is the measured final glucose content of the reference substance hydrolysate, in grams per litre, the other symbols having the meanings already given, with ms the mass of the reference substance in milligrams. The factor F2 in formula (A.3) is calculated by formula (A.5) as the ratio of P2 to P prime, where P2 is the purity of the reference substance taken from the purity report supplied by its manufacturer, in grams per 100 grams. When the values of V1, V2, V3 and f are exactly the same for the sample and for the reference substance in the procedure of A.3.2.4, formula (A.3) may be simplified to formula (A.6). The result is given as the arithmetic mean of two independent determinations obtained under repeatability conditions, kept to the nearest whole number.

A.3.2.9 Precision. The absolute difference between two independent results obtained under repeatability conditions shall not exceed 5 percent of their arithmetic mean.

Annex B Infrared spectrum of the yeast beta-glucan reference substance

The infrared spectrum of yeast beta-glucan shall match the features of the infrared spectrum of the yeast beta-glucan reference substance, with a broad, fairly strong absorption band near 3419 reciprocal centimetres and weaker absorption bands near 2923 reciprocal centimetres and near 889 reciprocal centimetres, as shown in Figure B.1. The figure itself is a plate and is not carried by the digitised text.

Annex C Infrared spectrum of the glucose reference substance

The infrared spectrum of the glucose used in the test shall match the features of the infrared spectrum of the glucose reference substance: a broad, strong hydroxyl stretching band near 3300 reciprocal centimetres, several medium to weak C-H stretching bands near 2980 reciprocal centimetres, an aldehyde stretching band near 1650 reciprocal centimetres, several C-O stretching bands near 1100 reciprocal centimetres, a weak bending band of the anomeric carbon C-H near 930 reciprocal centimetres, and ring breathing bands between 700 and 500 reciprocal centimetres, as shown in Figure C.1. The figure is taken from the Collection of Infrared Spectra of Drugs compiled by the Pharmacopoeia Commission of the Ministry of Health of the People's Republic of China; sample preparation by the potassium bromide pellet method, spectrum number 464.