§114.90 测定酸化食品酸度的方法(Methodology)

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　     § 114.90   Methodology.

　　Methods that may be used to determine pH or acidity for acidified foods include, but are not limited to, the following:

　　（a） Potentiometric method for the determination of pH —（1） Principles. The term “pH” is used to designate the intensity or degree of acidity. The value of pH, the logarithm of the reciprocal of the hydrogen ion concentration in solution, is determined by measuring the difference in potential between two electrodes immersed in a sample solution. A suitable system consists of a potentiometer, a glass electrode, and a reference electrode. A precise pH determination can be made by making an electromotive force （emf） measurement of a standard buffer solution whose pH is known, and then comparing that measurement to an emf measurement of a sample of the solution to be tested.

　　（2） Instruments. The primary instrument for use in pH determination is the pH meter or potentiometer. For most work, an instrument with a direct-reading pH scale is necessary. Battery and line-operated instruments are available commercially. If the line voltage is unstable, line-operated instruments should be fitted with voltage regulators to eliminate drifting of meter-scale readings. Batteries should be checked frequently to ensure proper operation of battery operated instruments. An instrument using an expanded unit scale or a digital readout system is preferred since it allows more precise measurements.

　　（3） Electrodes. The typical pH meter is equipped with a glass membrane electrode and a reference electrode or a single probe combination electrode. Various types of electrodes designed for specific uses are available. The most commonly used reference electrode is the calomel electrode, which incorporates a salt bridge filled with saturated potassium chloride solution.

　　（i） Care and use of electrodes. Calomel electrodes should be kept filled with saturated potassium chloride solution or other solution specified by the manufacturer because they may become damaged if they are allowed to dry out. For best results, electrodes should be soaked in buffer solution, distilled or deionized water, or other liquid specified by the manufacturer for several hours before using and kept ready by storing with tips immersed in distilled water or in buffer solution used for standardization. Electrodes should be rinsed with water before immersing in the standard buffers and rinsed with water or the solution to be measured next between sample determinations. A lag in meter response may indicate aging effects or fouling of the electrodes, and cleaning and rejuvenation of the electrodes may be necessary and may be accomplished by placing the electrodes in 0.1 molar sodium hydroxide solution for 1 minute and then transferring them to 0.1 molar hydrochloric acid solution for 1 minute. The cycle should be repeated two times, ending with the electrodes in the acid solution. The electrodes should then be thoroughly rinsed with water and blotted with soft tissue before proceeding with the standardization.

　　（ii） Temperature. To obtain accurate results, a uniform temperature should be maintained for the electrodes, the standard buffer solutions, and the samples. Tests should be made at a temperature between 20° and 30 °C, the optimum being 25 °C. Any temperature determinations made without meter compensation may affect pH values. An automatic temperature compensator may be used.

　　（iii） Accuracy. The accuracy of most pH meters is stated to be approximately 0.1 pH unit, and reproducibility is usually ±0.05 pH unit or less. Some meters permit the expansion of any pH unit range to cover the entire scale and have an accuracy of approximately ±0.01 pH unit and a reproducibility of ±0.005 pH units.

　　（4） General procedure for determining pH. When operating an instrument, the operator should use the manufacturer's instructions and should observe the following techniques for pH determinations:

　　（i） Switch the instrument on and allow the electronic components to warm up and stabilize before proceeding.

　　（ii） Standardize the instrument and electrodes with commercially prepared standard 4.0 pH buffer or with freshly prepared 0.05 molar potassium acid phthalate buffer solution prepared as outlined in “Official Methods of Analysis of the Association of Official Analytical Chemists” （AOAC）, 13th Ed. （1980）, section 50.007（c）, under “Buffer Solutions for Calibration of pH Equipment—Official Final Action,” which is incorporated by reference. Copies may be obtained from the AOAC INTERNATIONAL, 481 North Frederick Ave., suite 500, Gaithersburg, MD 20877, or may be examined at the National Archives and Records Administration （NARA）. For information on the availability of this material at NARA, call 202–741–6030, or go to: http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html. Note the temperature of the buffer solution and set the temperature compensator control at the observed temperature （room temperature is near 25 °C）.

　　（iii） Rinse the electrodes with water and blot, but do not wipe, with soft tissue.

　　（iv） Immerse the tips in the buffer solution and take the pH reading, allowing about 1 minute for the meter to stabilize. Adjust the standardization control so that the meter reading corresponds to the pH of the known buffer （for example, 4.0） for the temperature observed. Rinse the electrodes with water and blot with soft tissue. Repeat procedure with fresh portions of buffer solution until the instrument remains in balance on two successive trials. To check the operation of the pH meter, check the pH reading using another standard buffer such as one having a pH of 7.0, or check it with freshly prepared 0.025 molar phosphate solution prepared as outlined in the AOAC, 13th Ed. （1980）, section 50.007（e）, which is incorporated by reference. The availability of this incorporation by reference is given in paragraph （a）（4）（ii） of this section. Expanded scale pH meters may be checked with pH 3.0 or pH 5.0 standard buffers. Buffers and instruments can be further checked by comparison with values obtained with a second properly standardized instrument.

　　（v） Indicating electrodes may be checked for proper operation by first using an acid buffer and then a base buffer. First standardize the electrodes using a pH 4.0 buffer at or near 25 °C. Standardization control should be adjusted so that the meter reads exactly 4.0. Electrodes should be rinsed with water, then blotted and immersed in a pH 9.18 borax buffer prepared as outlined in the AOAC, 13th Ed. （1980）, section 50.007（f）, which is incorporated by reference. The availability of this incorporation by reference is given in paragraph （a）（4）（ii） of this section. The pH reading should be within ±0.3 units of the 9.18 value.

　　（vi） The pH meter can be tested for proper operation by shorting the glass and reference electrode inputs, thereby reducing the voltage to zero. In some meters this shorting is done by switching the instrument to standby, and in other instruments by use of a shorting strap. With the instrument shorted out, standardization control should be turned from one extreme to another. This operation should produce a deflection greater than ±1.5 pH unit from center scale.

　　（5） Determining pH on samples. （i） Adjust the temperature of the sample to room temperature （25 °C）, and set the temperature compensator control to the observed temperature. With some expanded scale instruments, the sample temperature must be the same as the temperature of the buffer solution used for the standardization.

　　（ii） Rinse and blot the electrodes. Immerse the electrodes in the sample and take the pH reading, allowing 1 minute for the meter to stabilize. Rinse and blot the electrodes and repeat on a fresh portion of sample. Oil and grease from the samples may coat the electrodes; therefore, it is advisable to clean and standardize the instrument frequently. When oily samples cause fouling problems, it may become necessary to rinse the electrodes with ethyl ether.

　　（iii） Determine two pH values on the well-mixed sample. These readings should agree with one another to indicate that the sample is homogeneous. Report values to the nearest 0.05 pH unit.

　　（6） Preparation of samples. Some food products may consist of a mixture of liquid and solid components that differ in acidity. Other food products may be semisolid in character. The following are examples of preparation procedures for pH testing for each of these categories:

　　（i） Liquid and solid component mixtures. Drain the contents of the container for 2 minutes on a U.S. standard No. 8 sieve （preferably stainless steel） inclined at a 17- to 20-degree angle. Record weight of the liquid and solid portions and retain each portion separately.

　　（ a ） If the liquid contains sufficient oil to cause electrode fouling, separate the layers with a separatory funnel and retain the aqueous layer. The oil layer may be discarded. Adjust the temperature of the aqueous layer to 25 °C and determine its pH.

　　（ b ） Remove the drained solids from the sieve, blend to a uniform paste, adjust the temperature of the paste to 25 °C and determine its pH.

　　（ c ） Mix aliquots of solid and liquid fractions in the same ratio as found in the original container and blend to a uniform consistency. Adjust the temperature of the blend to 25 °C and determine the equilibriated pH. Alternatively, blend the entire contents of the container to a uniform paste, adjust the temperature of the paste to 25 °C, and determine the equilibriated pH.

　　（ii） Marinated oil products. Separate the oil from the solid product. Blend the solid in a blender to a paste consistency; it may become necessary to add a small amount of distilled water to some samples to facilitate the blending. A small amount of added water will not alter the pH of most food products, but caution must be exercised concerning poorly buffered foods. No more than 20 milliliters of distilled water should be added to each 100 grams of product. Determine the pH by immersing electrodes in the prepared paste after adjusting the temperature to 25 °C.

　　（iii） Semisolid products. Food products of a semisolid consistency, such as puddings, potato salad, etc., may be blended to a paste consistency, and the pH may be determined on the prepared paste. If more fluidity is required, 10 to 20 milliliters of distilled water may be added to 100 grams of product. Adjust the temperature of the prepared paste to 25 °C and determine its pH.

　　（iv） Special product mixtures. For special product mixtures such as antipasto, pour off the oil, blend the remaining product to a paste, and determine the pH of the blended paste. If more fluidity is required, add 10 to 20 milliliters of distilled water to each 100 grams of product and blend. Adjust the temperature of the prepared paste to 25 °C and determine its pH.

　　（7） Process pH determination. Obtain sample portions of material for pH determination.

　　（i） For process liquids, adjust the temperature of the liquid to 25 °C and determine the pH by immersing the electrodes in the liquid.

　　（ii） Drain solid materials on a sieve and blend to a workable paste. Adjust the temperature of the prepared paste to 25 °C and determine its pH.

　　（iii） If enough solid materials are available to make a paste, blend representative aliquots of liquid and solid materials to a workable paste. Adjust the temperature of the prepared paste to 25 °C and determine the equilibrated pH. Alternatively, blend the entire contents of the container to a uniform paste, adjust the temperature of the paste to 25 °C, and determine the equilibrated pH.

　　（b） Colorimetric methods for the determination of pH. This method may be used in lieu of the potentiometric method if the pH is 4.0 or lower.

　　（1） Principle. The colorimetric method for pH involves the use of indicator dyes in solutions that gradually change color over limited pH ranges. An indicator that has the greatest color change at approximately the pH of the sample being tested is selected. The pH is determined by the color of the indicator when exposed to the sample under test.

　　（2） Indicator solutions. Most indicator solutions are prepared as a 0.04 percent solution of the indicator dye in alcohol. In testing, a few drops of indicator solution are added to 10-milliliter portions of the sample solution. Colors should be compared using a bright background. Approximate determinations can be made on white porcelain spot plates, the test colors being compared thereon with a set of color standards. More accurate colorimetric tests can be made using a comparator block fitted with sets of tubes of standard indicator solutions of known pH.

　　（3） Indicator paper. A paper tape treated with indicator dye is dipped into the sample solution. Depending upon the pH of the solution, the tape will change color and an approximate pH can be determined by comparison with a standard color chart.

　　（c） Titratable acidity. Acceptable methods for determining titratable acidity are described in the AOAC, 13th Ed. （1980）, section 22.060, under “Titratable Acidity—Official Final Action,” for “Indicator Method,” and section 22.061 for “Glass Electrode Method—Official Final Action,” which is incorporated by reference. The availability of this incorporation by reference is given in paragraph （a）（4）（ii） of this section. The procedure for preparing and standardizing the sodium hydroxide solution is described in the AOAC, 13th Ed. （1980）, sections 50.032–50.035, under “Sodium Hydroxide—Official Final Action” by the “Standard Potassium Hydroxide Phthalate Method,” which is also incorporated by reference and available as set forth in paragraph （a）（4）（ii） of this section.

　　[44 FR 16235, Mar. 16, 1979, as amended at 47 FR 11822, Mar. 19, 1982; 49 FR 5609, Feb. 14, 1984; 54 FR 24892, June 12, 1989; 63 FR 14035, Mar. 24, 1998]

　　§114.90方法

　　用于测定酸化食品的pH或酸度方法如下（不仅限于此）：

　　（a）用电位法测定pH：

　　（1）原理　pH这一符号是用来表示酸的强度或酸度。pH是溶液氢离子浓度倒数的对数，它是用测量浸没在样品溶液中两个电极之间的电位差来测定的。一个合适的测量系统是由一个电位计、一个玻璃电极和一个参照电极组成。可以用下述方法精确测定pH：首先测量一种已知pH的标准缓冲溶液的电动势，然后将这一电动势与从需要测试的样品溶液中测得的电动势作比较。

　　（2）仪器　用以测定pH的主要仪器是pH表或电位计。在一般情况下，只需一只带有pH（读数）刻度尺的仪表。电池供电和线路供电的仪表都可以用。如果线路电压不稳定，线路供电仪表应配有稳压器，以防止刻度表上的读数偏差。电池要经常更换，以确保电池供电仪表的正常工作。最好采用带有放大的刻度尺或数字读数系统的仪表，因为这样可以测量得更精确。

　　（3）电极　典型的pH表装有一个玻璃护膜电极和一个参照电极或一个单一的探测组合电极。为特殊使用而设计的各种电极都有供应。最常用的参照电极是甘汞电极，甘汞电极中装有一个灌满饱和氯化钾溶液的盐桥。

　　（i）电极的使用和注意事项：甘汞电极应该保持充满饱和的氯化钾溶液或者制造厂规定的其它溶液。因为如果溶液干涸，电极可能损坏。为了达到最好的效果，电极在使用前应在缓冲溶液、蒸馏水或去离子水或制造厂规定的其它溶液中浸泡几小时，然后将顶端浸入蒸馏水或标准化的缓冲溶液中贮存备用。在浸入标准缓冲溶液前，电极要用水冲洗。在测量下一个样品之前，电极要用水或溶液冲洗，仪表反应滞后可能表示电极老化作用或者有污垢，有必要对电极进行清洗和复原处理。为此可将电极放入0.1mol/L NaOH溶液中1min，再移至0.1mol/L HCl中1min，这一过程需重复两次，结束时电极在酸溶液中。然后将电极用水彻底洗净，再用软质物品吸干，使其恢复标准化。

　　（ii）温度：为了获得正确的结果，电极、标准缓冲溶液和样品应该保持同一温度。测试温度应为20～30℃，最佳温度为25℃。如果温度表不作补偿调整，测得的温度会影响pH。可以采用自动温度补偿器。

　　（iii）精确度：pH表的精确度约为0.1pH单位，复制的通常为±0.05pH单位或更小的偏差。有的表将所有pH范围标注在整个刻度尺上，这种表的精确度约为±0.01pH单位，偏差为±0.005pH单位。

　　（4）测定pH的一般方法　操作人员应该按照制造商的说明使用仪表，并应按下述方法测定pH：

　　（i）打开仪表开关，在进入测定前使电子元件预热起来，并达到稳定。

　　（ii）用市售的标准pH 4.0缓冲溶液或现配的0.05mol/L（浓度）的邻苯二甲酸氢钾缓冲溶液将仪表和电极标准化。邻苯二甲酸氢钾缓冲溶液的配制见《公职分析化学家协会法定分析法》（AOAC），第13版（1980），50.007（c）节，题为"用于pH设备标定的缓冲溶液--法定最终操作"。该资料已列入参考文献，文本可按下列地址索取：Association of Official Analytical Chemists International, 481 North Frederick Ave., suite 500, Gaithersburg, MD 20877，或在下列地址查询：Office of the Federal Register， 800 North Capitol Street，NW.， Suite 700， Washington，DC.。注意缓冲溶剂的温度并将温度补偿器的调节控制设定在观察到的温度上（室温接近25℃）。

　　（iii）用水冲洗电极并用软质物品吸干，不要挥动。

　　（iv）将顶端浸入缓冲溶液约1min，使其达到稳定，记下pH读数。调节标准化操作，使表在所测得温度下的读数与已知缓冲剂的pH（如4.0）读数一致。用水冲洗电极并用软物品吸干。用新鲜缓冲溶液重复上述过程，直至在连续两次实验中仪表保持平衡。检查pH表的性能，用另一份标准缓冲剂，如pH为7.0的缓冲剂校对pH读数，或者用新配制的0.025mol/L磷酸盐溶液进行校对。磷酸盐溶液的配制见AOAC第13版（1980）50.007（e）节。该资料已列入参考文献，文本的索取或查询见本节（a）（4）（ii）。增长刻度的pH表可以用pH为3.0或5.0的标准缓冲剂校对。缓冲剂和仪表可以用与从第二次标准化的仪表获得的值作比较的方法进一步验证。

　　（v）校正指示电极是否正常可以先用酸性缓冲剂，然后用碱性缓冲剂。先用pH为4.0的缓冲剂在25℃左右校准电极。并将其调整到pH为4.0。电极应用水冲洗，然后吸干并浸在pH为9.18的硼砂缓冲剂中，硼砂缓冲剂的配制见AOAC第13版，（1980）50.007（f）节。该资料已列入参考文献。参考文献文本的索取或查阅见本节（a）（4）（ii）。pH的读数应该为9.18±0.3。

　　（vi）测试pH表是否正常的方法是将玻璃电极和参照电极的输入短路，使电压减到零。对于某些表，短路的方法是将仪表的开关旋到待用，有的仪表则用短路带。仪表短路之后，标准控制从一端转到另一端。这一操作应产生一个从中心刻度起±1.5pH单位的偏转。

　　（5）测定样品的pH

　　（i）将样品的温度调整到室温（25℃），将温度补偿控制定在所观察到的温度。采用扩大刻度的仪表，样品温度必须与用于标准化的缓冲溶液相同。

　　（ii）冲洗并吸干电极。将电极浸入样品中并记下pH读数，要让表有1min的稳定时间。冲洗并吸干电极，在另一份样品中重复测试。样品中的油或油脂可能覆盖在电极上，因此，建议经常清洗并校正仪表。如果沾染了油性样品，可能需要用乙醚冲洗电极。

　　（iii）在均匀混合的样品中测pH两次。两次的读数应该是一致的，这就表明样品是均质的。记下最接近pH 0.05的值。

　　（6）样品的准备　有些食品产品是由不同酸度的液体和固体成分混合组成的。另一些食品产品或呈半固体状。对于上述各种食品准备pH测试样品的方法举例如下：

　　（i）液体和固体成分的混合物：    将容器的内容物在美国标准8号筛（最好是不锈钢的）上沥干2min，筛子倾斜17°～20°角。记下液体和固体部分的重量，液体和固体分别保留。

　　（A）如果液体中含有太多的油，将沾染电极，用一只分液漏斗将油层和水层分开，保留水层。油层可以丢弃。将水层部分温度调节至25℃，测定它的pH。

　　（B）从筛中取出沥干的固体，混合成浆状，使浆的温度调整到25℃，测定其pH。

　　（C）按原来容器内的比例将整个等分液量的固体和液体部分混合成同一稠度。将混合物调节到25℃，测量其平衡的pH。也可用另一种方法：将容器内的全部内容物混合成均匀的浆，使浆的温度调节至25℃，测定浆的平衡pH。

　　（ii）浸泡在油中的产品：将油从固体产品中分离出。将固体放在混合器中混合成浆状稠度。样品中可能需要加少量的蒸馏水以利于混合。加入少量的水将不改变大多数食品的pH，但是对于缓冲性差的食品要注意，在100g产品中加入的蒸馏水不应该超过20mL。将准备好的浆状物温度调整至25℃后，插入电极测定pH。

　　（iii）半固体产品：半固状稠度的食品如布丁、土豆沙拉等，可以将它们混合成浆状稠度，用这种浆测定pH。如果需要将其流动性增大一点，可在100g产品中加10～20mL的蒸馏水。将配制好的浆料调整到25℃，测定其pH。

　　（iv）特殊产品的混合物：    对于特殊产品的混合物，如antipasto，将油倒去，留下的产品混合成浆状，测定浆状混合物的pH。如果需要增加流动性，在100g浆状混合物中加10～20mL的蒸馏水。将配制好的浆调节到25℃，然后测量其pH。

　　（7）加工过程中pH的测定　取出部分物料作为测定pH的样本（样品）。

　　（i）对于加工液体，将液体温度调至25℃，将电极插入液体中测得pH。

　　（ii）在筛上沥干的固体物料，将其混合成可测的浆状。将制备好的浆料调至25℃，测定其pH。

　　（iii）如果有足够的固体物料制成浆料，将整等分液量的液体和固体物料混合成可测浆料。将这浆料调节至25℃，测定其平衡pH。另一种方法是将容器内全部物料混合成均质的浆料，将其温度调至25℃，然后测得平衡pH。

　　（b）用比色法测定pH：如果pH等于或小于4.0，可用此法代替电位法。

　　（1）原理　用比色法测定pH是利用指示剂在溶液中着色的原理，这种指示剂在规定的pH范围内渐渐变色。要选择对测试样品的pH具有最大色泽变化的指示剂。在测试时，根据指示剂在样品中显示的颜色确定pH。

　　（2）指示剂溶液　大多数指示剂溶液是在乙醇中加0.04%指示剂染料溶液制成的。在测试时，在10mL样品溶液中滴入几滴指示剂溶液。应用明亮的背景来比较其颜色。近似的测定可以用白瓷点滴板进行，将测试的颜色与一组标准颜色作比较。更精确的比色测试可以用一个比较板，板上装有一组管子，管内盛有已知pH的标准指示剂溶液。

　　（3）指示剂试纸  经过指示剂染料处理过的试纸浸入样品溶液中，根据溶液中的pH，试纸改变颜色。将试纸颜色与标准颜色图表进行比较而获得近似的pH。

　　（c）可滴定酸度：测定可滴定酸度的方法见AOAC第13版（1980）22.00节，题为"可滴定酸度--法定最终操作"--"指示剂方法"和22.061节"玻璃电极法--法定最终操作"。这些资料都已列入参考文献。参考文献的索取或查询见本节（a）（4）（ii）。配制和调整氢氧化钠标准溶液的方法见AOAC（1980）50.032-50.035节，题为"氢氧化钠--法定最终操作" ，用标准氢氧化钾邻苯二甲酸盐法，该资料已列入参考文献。参考文献文本的索取或查询见本节（a）（4）（ii）。

　　［44 FR 16235, Mar. 16, 1979以47 FR 11822, Mar. 19, 1982； 49 FR 5609, Feb. 14, 1984； 54 FR 24892, Jun. 12, 1989修订］

　　更多关于美国FDA酸化食品HACCP法规，请详见美国FDA酸化食品HACCP法规第113和114部分汇总