Method for determining lipids

Commonly used measurement methods are:

(1) Soxhlet extraction method (2) Babcock method (3) Yiler method (4) Ross-Gothic method (5) Acid decomposition method

In the past, the so-called extraction method was generally used for the determination of fat. This method is still considered as a representative method for determining the lipid content of various foods, but the results of some samples are often low, and the Babcock method. The Yeller method and the Ross-Gothic method are mainly used for the determination of lipids in milk and dairy products, while the fats measured by the acid hydrolysis method are free lipids and all lipids of the binding lipids.

First, the cable extraction method (classical method)

1, the principle

After the sample is pretreated, it is placed in a cylindrical filter paper, and the filter paper tube is placed in a cable extraction tube, and heated and refluxed in a water bath with diethyl ether or petroleum ether to allow the fat in the sample to enter the solvent, and the solvent is recovered. The residue is fat (crude fat). The free fat is measured by this method. In addition, it contains phospholipids, pigments, waxes, volatile oils, glycolipids, etc., so the fat measured by Soxhlet extraction is thick. fat.

2. Scope and characteristics

The Soxhlet extraction method is suitable for the determination of samples with high lipid content, low binding lipid content, dry and fine grinding, and no moisture absorption and agglomeration. This method can only measure free fat, but the combined fat can not be measured. To determine the bound fat, it needs to be hydrolyzed under certain conditions to become free fat. In addition, this method is a classical method, and the results of most samples are relatively reliable, but it requires a long cycle and a large amount of solvent.

3, method

(1) Preparation of filter paper tube

Cut the filter paper into a rectangular shape of 8×15cm, roll it into a cylinder, and have a diameter of 6cm. Seal the bottom of the cylinder, and put some cotton wool on it to avoid leakage.

(2) Weigh the sample, dry the sample and grind it, weigh a certain amount and seal it with the paper tube, and use the sample to measure the water.

(3) Preparation of cable extractor

The Soxhlet extractor consists of three parts, a reflux condenser, an extraction tube, and a grease bottle. The grease bottle should be dried and weighed to constant weight before use. Others should be dry.

(4) Extraction

Put the loaded paper tube into the extraction tube, pour in ether, add the amount of ether from the extraction tube, add 2/3 of the volume of the extraction bottle, connect the condensing device, extract in a constant temperature water bath, and bath temperature It is about 55 °C and can be tested with filter paper. The theoretical value is extracted for 6-8 hours, and the actual value is 3-4 hours, but it is also determined according to the nature of the sample.

(5) Recovering ether

When the ether is about to be siphoned in the extraction tube, the extraction tube is immediately removed, and the lower mouth is placed in an ether recovery bottle to be tilted, and then the extraction bottle is placed in an oven at 100-150 ° C to a constant weight.

(6) Calculation

Fat%= (W2-W1)/W x 100

W2 - bottle and sample weight (g) W1 - bottle weight (g) W - sample weight (g)

or:

% fat = (filter paper and sample weight after extraction - pumping filter paper weight) / sample weight × 100

The filter paper cylinder should be placed in a beaker and baked in a 100-105 ° C oven to constant weight.

3, note:

(1) The sample should be dried and then ground. The filter paper tube containing the sample must be tight, and the sample cannot be leaked outside, otherwise it will be redone.

(2) The height of the filter paper can not exceed the return bend, otherwise the ether will not penetrate the sample, so that the fat can not be all raised, causing errors.

(3) Samples containing polysaccharides and dextrin should be treated with cold water first, and then dried and placed together with filter paper in the extractor.

(4) The temperature of the water bath during extraction should not be too high. Generally, the ether should start to boil (about 45 °C), and the reflux rate should be 8-12 times/hour.

(5) The ether used must be anhydrous ether. If it contains water, the sugar and inorganic substances in the sample may be extracted, causing errors.

(6) If the dry sample is used to measure fat, the fat content of the original sample can be calculated by the following formula:

Fat (%) = (W1-W2) (100-A) / W

A—100 g sample moisture content (g)

(7) The upper end of the condenser tube is connected to a calcium chloride drying tube, which not only prevents moisture from entering the air, but also prevents the ether from volatilizing in the air. This can prevent the pollution of the laboratory's tiny ambient air. If there is no such device, a dry cotton wool ball can also be plugged.

(8) If there is no anhydrous ether, it can be prepared by itself. The preparation method is as follows: 50 g of anhydrite is added to 100 ml of diethyl ether, shaken several times, static for more than 10 hours, distilled, and the distillate below 35 ° C is collected. application.

(9) When the extraction bottle is dried in an oven, the mouth of the bottle is inclined by 45 degrees to one side to prevent the volatile ether from forming a convection with the air, so that the drying is rapid.

(10) If there is no ether or absolute ethanol, it can be extracted with petroleum ether, and the petroleum ether has a boiling point of 30-60 ° C.

(11) When using volatile ether or petroleum ether, avoid heating directly with fire source, apply electric heating sleeve, electric water bath, electric bulb, etc.

(12) Here, the concept of constant weight differs. It indicates the low weight of zui initially reached by Zui, that is, the constant weight when the solvent and water are completely volatilized. If the heating is continued after that, the weight will increase due to oxidation of the grease or the like.

(13) The cooling time in the dryer is generally the same.

Second, the Babcock Act (Babcock Act)

The Babcock method is a method for determining milk and dairy products. Before we measure milk, we first understand the nutrients of milk.

Average composition of milk:

100% milk: 87.5% moisture, 12.2% total solids, vitamins, immune bodies and enzymes;

12.2% of total solids: 8.8% non-fat solids, 3.4% milk fat;

Non-fat solids 8.8%: protein 3.5%, lactose 4.6%, mineral Ca.p.Fe.K, etc.;

Protein 3.5%: casein 3.0%, albumin 0.4%, globulin 0.1%.

In the ingredient list, 3.4% milk fat is required for our testing. Protein is higher in milk than human milk protein. The lactose content is less than that of human milk. The content of Ca in minerals is more than that of human milk, and the content of Fe is less than that of human milk.

The fat content of milk is as follows

Fresh milk:

(1) Special grade ≥ 3.2%

(2) Level ≥ 3.0%

(3) Level ≥ 2.8%

Disinfecting milk ≥ 3.0 %

It is well known that milk is an emulsion. Its lipids are not present in the milk, but in the form of a fat globule, there is a film around it that allows the fat globule to maintain the stability of the emulsion in the milk. This film contains protein. Many substances such as phospholipids are usually dissolved in non-fat components when concentrated H2SO4, and the fat globule membrane is softened and destroyed, so the emulsion is destroyed and the fat can be separated. This is a recognized standard analytical method.

The two methods of Babcock and Gabriel were developed by two scientists. One was Babcock in the United States, which studied the fat of cow's milk in 1890. Later, after 2 years, in 1892 by the British Gabriel Liquid research to measure the fat of milk.

Both methods are used to extract the fat in dairy products. This method is also called wet extraction. Because the sample does not need to be dried beforehand, the fat exists in the form of latex in the milk. To determine the fat, it is necessary to destroy the latex fat. Separated from other non-fat components, the separated non-fat components are generally decomposed with concentrated H2SO4, quantified by volumetric method, and easy to operate, and are used for routine analysis of dairy products in many countries.

Babcock principle

The non-fat component such as lactose bath protein in the milk is dissolved in sulfuric acid to destroy the fat globule membrane, the fat is released, and the fat layer is directly read in the cream bottle, thereby quickly obtaining the fat percentage in the milk to be tested.

2, method

Accurately absorb 17.6ml of milk → in the cream bottle → add 17.5ml of sulfuric acid (measured by the measuring tube) → mix → centrifuge for 5 minutes (1000 rpm) → 60 ° C water to the bottleneck → centrifuge for 2 minutes (1000 rpm) → Add 60 ° C water to 4% mark line → Centrifuge for 1 minute → 60 ° C water bath → Stabilize the fat column → Read

Add the role of H2SO4:

(1) Dissolving protein

(2) lactose lactose

(3) reduce the adsorption of fat

Because the non-fat component is dissolved in H2SO4, this increases the specific gravity of the digestive juice (H2SO4 specific gravity 1.820-1.825, fat specific gravity is less than 1), that is, the specific gravity is greater than 1.820-1.825, and the specific gravity of fat is less than 1, so that the fat is rapid. Completely with non-lipid precipitation, the effect of centrifugation is the very clear separation of fat, the purpose of heating, the fat adsorption is reduced, the speed of floating is accelerated, which is the Babcock method for measuring milk fat.

Recently, in some scientific books, the Babcock method has been improved to measure meat products and cereal samples.

Because Babcock uses concentrated sulfuric acid as a protein solubilizer, carbonization occurs in meat products. These carbides are suspended between the interface between the fat layer and the water layer, which results in inaccurate readings. Therefore, they studied various alternatives to sulfuric acid. The reagents were later determined to use a perchloric acid-acetic acid mixture instead of sulfuric acid to determine the fat of meat and meat products. The measured values ​​were consistent with the AOAC method, and the precision was expressed as 0.2% by standard deviation.

Reagents used:

Perchloric acid-acetic acid mixture

6% perchloric acid is mixed with an equal volume of acetic acid.

specific method:

Weigh 9 grams of minced meat in a pasteurized bottle, add 30m of mixed acid, heat in a boiling water bath for 15min (to make the sample fully soluble), add acid to make it reach the scale of the bottle

Centrifuge for 2 min (1000 rpm) 60 ° C water bath for 10 min to read

Calculation: Fat% = H x 0.95

H—Read the value of the fat column

0.95—acetic acid correction value dissolved in the fat layer

Improved Babcock method: For the solute quality heated sample, it can be completely dissolved by heating in a water bath for 15 minutes. For the raw meat sample, since the heat is not degenerated, after mixing the acid, the meat protein is solidified in a certain period of time. The dissolution time takes approximately 25 minutes.

Third, the Gerber method (Gerb method)

This method is not suitable for samples with high sugar content, which are easily coked by this method, resulting in large error in results.

1, the principle:

The addition of sulfuric acid to the milk can destroy the colloidality of the milk, make the casein calcium salt in the milk into a soluble heavy casein compound, and can reduce the adsorption of the fat globule, and at the same time increase the specific gravity of the digestive juice. To make the fat easier to float out of the liquid, in the operation also need to add isoamyl alcohol, reduce the surface tension of the fat globule, promote the segregation of the fat globule, but the solubility of isoamyl alcohol is very small, so in the operation, can not be added too More, if too much is added, isoamyl alcohol will enter the fat, so that the fat volume will increase, and there will be a part of isoamyl alcohol and sulfuric acid to form sulfate, the reaction is as follows:

2C5H11OH + H2SO4 → (C5H11O)2SO2 + 2H2O

Heating 65-70 ° C and centrifugation during the operation, the purpose is to quickly and completely separate fatty acids.

2, method

Take 10ml H2SO4 in the cream bottle - accurately measure 11.0ml milk - add 1ml isoamyl alcohol - mix - 65 ° C water bath 5min

Centrifuge for 5 min (1000 rpm) - 65 ° C water bath for 5 min - read immediately

Description:

(1) In the Pap method, a 17.6 ml pipette is used. In fact, only 17.5 ml is injected into the Papillon bottle, and the specific gravity of the milk is 1.030 g/ml, so the sample quality is

17.5 x 1.030 = 18g

The Papillon bottle has a total of 10 large grids (0-10%), each volume is 0.2ml, the fat has an average specific gravity of 0.9, and its fat mass is 0.2 x 10 (10 large cells) x 0.9 (fat specific gravity). = 1.8 (g), 18 g of sample contains 1.8 g of fat, that is, the full scale of the bottleneck represents 10% of the fat content, and each large cell represents 1% of fat, so the Papillon bottleneck scale reading is directly the percentage of fat.

(2) The concentration and dosage of sulfuric acid should strictly abide by the requirements specified in the method. If the concentration of sulfuric acid is too high, the milk will be turned into a black solution and affect the reading. If the concentration is too small, the casein will not dissolve completely, which will make the measured value low or fat. The layer is cloudy.