1、 Chapter 12 Food additives analysis Introduction Antibiotics are substances of natural, semi-synthetic, or synthetic origin that exhibit antibacterial activity. Their presence in foods is essentially due either to therapeutic treatments or to the antibiotic-supplemented food gived to certain animals

2、. The consequences of the presence of antibiotic residues are as numerous to human health as they are to certain processing operations. Where human health is concerned, a number of dangers must be avoided, such as allergic effects and possibilities of microbial selections and of mutations that essen

3、tially have two consequences: 1. Selection of resistant strains. 2. Disequilibrium of the normal flora of the digestive tract. Determination of AntibioticsMethods of determination In processing operations, the presence of residues with antibiotic activity in milk or meat makes them unsuitable for so

4、me uses. For all these reasons, the problems related to the presence of the antibiotic residues in foods have been extensively studied. The detection and the determination of these residues are therefore essential elements in the study of antibiotic evolution and the protection of the consumer. The

5、methods of determination used at present can be classified into three groups: microbiological methods; eletrophoretic methods; physicochemical methods. The most frequently used detection methods are those that exploit the sensitivity of certain bacterial strains vis-vis one or several antibiotics. T

6、he manifestation of this inhibition is affected either in liquid (e.g., acidification method) or solid media (e.g., agar diffusion method). Microbiological MethodsPrinciple This technique is widely used for the detection of antibiotics in milk. After pasteurization, the sample is cultured with a str

7、ain sensitive to antibiotics (e.g., Bacillus, Strentococcus, etc.). After incubation, the production of latic acid, which results from the growth of test bacteria in the absence of antibiotic residues, is detected either by a pH indicator or by the coagulation of the milk. The bacterial growth can a

8、lso be measured by nephtelometry. Liquid Medium MethodsSeveral methods based on this principle are currently being used. 1. Reduction of Colored Indicators Methylene Blue test 2. Measurement of the Coagulation Time The test germ is yogurt fermenting agent (e.g., Streptococcus thermophilus and Lactob

9、acillus bulgaricus). The presence of inhibiting substances is detected through the absence of milk coagulation after a fixed culture period 3. Measurement of Acidity A technique that consists of adding a strain of Streptococcus thermophilus to milk and titrating the lactic acid produced after incuba

10、tion. 4. Measurement of Medium Turbidity Measure the growth of the test germ by recording the variations in medium turbidity over time.MethodologyBy using method studying antibiotics in liquid media, results can be obtained rapidly. They allow the analysis of large series of milk samples and can act

11、 as a primary selection method. All positive of questions samples must be subjected to a confirmation test by the agar diffusion method, which is to be described next. Sub-summaryAgar Diffusion MethodsThese techniques have been employed in antibiotic analyses in all food products.Principle When one

12、or more antibiotics in a solution are brought into contact with an agar medium, they diffuse into it. The diffusion is proportional to the logarithm of their concentrations. The growth of a test germ cultured in agar after incubation shows the presence of an inhibiting substance through the appearan

13、ce of a clear zone in the antibiotic diffusion zone, while everywhere else the growth of the microorganism is visible. Agar Culture Media The composition of the agar medium depends on the strain used and the antibiotic studied. For example, one may use: Healteys agar; Chabbert medium; Bacto Whey Aga

14、r medium. The pH must be adjusted to 6.6 or 7.8, depending on the antibiotic studied.Sample Preparation1. In its original state (milk);2. After mixing it aseptically in a small quantity of sterile physiological serum (e.g., curdled milk, cheese, or antibiotic supplemented food);3. After solvent extr

15、action (e.g., muscular tissues). For extraction, three solvents: pure methanol, methanol + pH 8 bicarbonate 1/1 buffer (V/V); or distilled water + pH 8 bicarbonate 3/7 buffer (V/V) are mended.MethodologyThe following strains are used most often:Bacillus stearothermophilus var calidolactis strainBaci

16、llus subtilis ATCC 6633,Sarcina lutea ATCC 9341: Staphylococcus aureus, Bacillus megaterium ATCC 9855: Micrococcus luteus and Bacillus cereus.* Other sensitive bacteria can also be used; including Micro flavus, Bacillus cereus, Sarcina lutea, and Escherichia coli. Test Microorganisms1. The agar medi

17、um, which is melted and then cooled to 45, is cultured with a diluted suspension of the test germ. It is homogenized and the mixture is poured into Petri dishes left to cool horizontally. After pasteurization, the sample is aseptically placed in contact with the agar according to two techniques:2. A

18、 filter paper disk is saturated with a fraction of the sample product or the extraction solution (in this case, drying is very important), and then deposited on the surface of the cultured agar;3. The sample is placed into hollow cavities in the agar or small stainless steel cylinders applied to the

19、 agar surface. Procedure Petri dishes prepared as discussed are incubated. A clear zone around the filter papar disk or the cavity indicates the presence of a substance in the sample with antibiotic activity. Otherwise, colonies propagate through the entire agar surface. A control must always be per

20、formed with a preparation that does not contain antibiotics. It is important that this test be conducted under conditions that are rigorously identical to the sample biological liquid. Most biological products (e.g., serum, liver, milk, muscle, urine, etc.) have enzyme binding or destructive propert

21、ies thar are likely to distort the results. It is possible to detect penicillin by performing an assay with a disk saturated with penicillinase. If the sample contains penicillin at the start, then no inhibition zone appears around the disk saturated with penicillinase. If it contains an antibiotic

22、other than penicillin, then an inhibition zone appears around the disk saturated with penicillinase. Interpretation of Results The agar diffusion method is relatively rapid and does not require much labortory equipment. However, just like the liquid medium methods, it is only an “all or none” techni

23、que that permits neither the determination nor the identification of the antibiotic (except for penicillin). It is especially well adapted when the sample antibiotic is known, as is the case with pharmacodynamic studies of a product in a particular animal. For a “regulatory type” control test, howev

24、er, there are other problems. One may work either with a whole “bank” of bacterial strains of varying sensitivity in such a way as to cover the largest range of antibiotics used, making it a cumbersome detection system, or else neglect the antibiotic and make do with a few sensitive bacteria that wi

25、ll not produce inhibition rings with the sample examined. Finally, it should always be kept in mind that there are natural antibiotics in the plant kingdom, and that in the animal world, substances like lactic acid, the lactoperoxidoses, the aglutinins, and the lactoransferinses can produce inhibiti

26、on rings. That is why it is so important to interpret the results carefully. In order to partially remedy these drawbacks, a modification was made in the agar diffiusion method. It consists of performing a preliminary electrophoretic separation of the sample or extract to be analyzed. Sub-summary1.

27、The principle of electrophoresis method: The sample to be tested is deposited in hollow cavities in the agar or in wells. Under the effect of an appropriate electric current, the antibiotics separate with different and specific speeds and directions of migration, which simultaneously permits the eli

28、mination of interfering substances and provides information on the nature of the antibiotics present in the sample. A second layer of agar cultured with one or more test microorganisms detects the diffusion of the antibiotics into the gel after incubation. A positive detection is indicated, as befor

29、e, by the formaiton of clear zones. In a certain sensitivity range the diameter of the inhibition zones is proportional to the concentration of the antibiotic present, which can be used in its determination. (*Billon and Taos (1979) used this electrophoretic technique by associating it with the micr

30、obiological method. )Other methods1. Elimination of “false positives” due to the seperation of interfering substances. Thus, the control sample, void of antibiotic, is no longer necessary, offering a great benefit to official regulatory bodies.2. Indentification of the antibiotics, which enables the

31、 recognition of those used the most in human therapeutics (e.g., penicillin, tetramycin, chlortetracycline, etc.) and which can potentially be the most dangerous (e.g., antibioticresistance) to be the consumer.3. Determination of the antibiotics. For fresh milk, when the content is particularly high

32、, the determination can aid in making seizure of the milk simpler so that the responsible suppliers can be penalized according to regulations.4. Noticeably lower detection limits. The detection limits are lower by four to ten times depending on the antibiotic or antibioticsThis technique is rarely u

33、sed in rountine work. It is an excellent reference method for situations requiring expertise. Advantages of Electrophoresis 2. Physicochemical Methods These techniques of antibiotic detection have developed considerably in the past few years. They are special applications of the analytical principle

34、s already employed for the determination of other types of molecules. 3. Radioenzymatic Determinations These are based on a specific biochemical reation between the antibiotic molecule and a cofactor labled with carbon 14. Acording to Charm (1979), the determination is effected in 10-15 minutes, and

35、 the lower sensitivity limit is on the order of 0.05 Ul/ml for penicillin (technique: “Charm test”). 4. Radioimmunological Determinations Several companies have commercialized a determination “kit” that includes the specific antibody and the antibody labeled with iodine 125. Despite a high theoretic

36、al sensitivity, this technique cannot reach detection limits lower than 1 pg/ml of milk (Flavigny, 1980) Other methods5. Determinations by Fluorimetry Hamann and Heeschen (1975) have desrcibed a determination technique using immunofluorescence, the principle of which consists of labeling the antibio

37、tic with fluoresceine. The determination occurs by competition in the presence of an unlabeled antibiotic and a specific serum. The measurement is affected by fluorimetry in polarized or nonpolarized light. This technique is used for the detection of tetracylines, which form fluorescent compounds af

38、ter being heated in neutral or alkaline medium (detection limit: 2 g/g). 6. Determinations by Bioluminescence Adenosine triphosphate (ATP) produced by bacterial cells is transformed into adenosine diphosphate (ADP) in the presence of luciferin luciferace. Photon emission, which panies the reaction,

39、is measured by a photometer. This technique therefore involves a bacteriological step.7. Spectrometry Used in the ultraviolet, visible, and infrared spectra, spectrometry permits the identification and the determination of penicillin (detection limit: 10 g/g), streptomycin, tetracyclines, and the li

40、ke.Other methods8. Chromatographic Techniques 8.1Thin Layer Chromatography (TLC) Hamann et al. (1979) used TLC to identify antibiotic residues in milk, with or without preliminary extraction. The antibiotics are identified on the plates by their RF. Determination can be affected by fluorescence. The

41、 detection limits are: tetracyline: 0.025g/ml chloramphenicol: 1g/ml neomycin: 15g/ml streptomycin: 0.5g/ml Bossuyt and Renterghem (1979) have also described a TLC determination method after extraction of the antibiotic residues with acetone. Identification was affected bye using test strains of Bac

42、illus cereus, Bacillus subtilis, Micrococcus flavrs, and Sareina lutea. 8.2 Gas Chromatography Hamann et al. (1979) have described different protocols for extractions, identifications, and determinations of antibiotics in milk by gas liquid chromatography. The detection limits are the following: tet

43、racycline: 0.5-10g/ml chloramphenicol: 0.01g/ml penicillin: 0.005Ul/ml 8.3 HPLC This technique is now widely used for the detection and determination of most antibiotics. In a number of methods, the extraction of antibiotics is followed by a purification (e.g., on Sephadex columns) before seperation

44、 by HPLC (Martinez and Shimoda, 1988; Pochard, 1987). Detection is often affected by UV spectrophotometry, but other methods can also be considered (Martinez and Shimoda, 1988). Research and testing laboratories have at their disposal a number of methods for the detection, identification, and determ

45、ination of the antibiotic residues in foods. However, all of these techniques are still not sufficiently classfied. The microbiological methods are most frequently used by all testing laboratories. The association of electrophoretic technique with microbiological detection has sighificantly increase

46、d reliability as well as the sensitivity of determination. The use of physicochemical methods is linked to the type of equipment used by the laboratories, and the complexity of the problems to be resolved. SummaryDetermination of AntisepticsThe presence of antiseptic residues in foods is due essenti

47、ally to: 1. the processing aimed at stablilizing certain foods (e.g., anhydrous sulphur in wines, biphenyls for citrus fruits, etc.) In this case, regulation indicate maximal quantities that must not be exceeded; 2. the contact of the products with inadequately rinsed surface after the use of disinf

48、ectant product (e.g., sodium hypochlorite, ammonium fluoride, formaldehyde, etc.); 3. the addition of antiseptics whose use is prohibited by law, in order to limit microbial growth harming the marketable quality of a product.Introduction-1The presence of antiseptic substances in food poses problems

49、similar to those concerning antibiotics: 1. The health of the consumer, due to the toxicity of the antiseptics; 2. At the technological level, certain industrial transformation operations (e.g., inhibition of lactic ferments in the dairy industry). Introduction-2These technique are based on the fact

50、 that the growth of certain microorganisms in inhibited by the presence of antiseptic residues.1. General Method: Fermentation Test Principle The food is introduced into a fermentation tube (eudiometer after Einhorn Diemair and Postel, 1970) and mixed with a standard quanity of a very active Sacchar

51、omnyces cerevisiae suspension (i.e., baking yeast). After incubation, the volume of carbon dioxide gas produced leads to a conclusion on the presence or absence of antiseptics.Sample preparation. Liquid nutrients do not require any special preparation, with the possible exception of dilutions for pr

52、oducts that are too concentrated. However, alcoholic drinks have to be dealcoholized by evaporation in a low vacuum at a low temperature (30-40). The initial volume is reestablished bye adding distilled water. The sugar content of the samples is raised to about 10% with glucose. For solid or semi-so

53、lid foods, two samples are prepared for analysis (about 10 g). One is mixed with 100 ml of 0.5% tartaric acid solution, wherease the other is mixed with 100 ml of 0.1% sodium hydroxide solution. Let them react for 10 min. The two extracts are filtered and adjusted to a pH between 3 and 4. The sugar

54、content is raised to about 10% with glucose. Pasteurize the two extracts by healting for 1 in at 80. In all cases, if the product is low in nitrogenated substances, then it is necessary to add the yeast extract to a concentration of 2.5%.Microbiological MethodsPreparation of Yeast. In 100 ml of a st

55、erile culture medium (glucose: 25 g; asparagine:1 g; KH2PO4: 0.5 g; MgSO4H2O: 0.5 g)(Diemair and Postel, 1970),use sterile technique to add 1 g of fresh baking yeast (test microorganism) taken from an unopened packet stored in the cold (+2). Agitate in such a way to disperse the yeast. Incubate for

56、24 hours at 25. Affect a second culture using the the same nutrient medium taking 10 ml from the first culture. Incubate for 48 hours at 25. Fermentation test. Eliminate the carbon dioxide gas from the second culture by filtering it on sterile hydrophilic cotton. In a sterile Erlenmeyer flask, aspet

57、ically introduce 10 ml of the yeast suspension thus prepared, and 50 ml of the sample or sample preparation. Mix carefully and decant the sample thus cultured in a sterile Einhorns endiometer in such a way that the bowl is one-third filled and the tube is filled completely. If possible, prepare a co

58、ntrol with a sample or an extract free from antiseptics under the same conditions. Results Incubate for 20-24 hours at 25. After this period, read the volume of carbon dioxide gas produced on the graduated scale.Assay without a reference. If the volume of carbon dioxide gas released after incubation

59、 is zero or less than 5 ml, then it may be presumed that the inhibition is due to an antiseptic (Diemair and Postel, 1970). Assay with a reference. If the sample examined release less gas than the reference test, then the test must be considered as positive when the difference between the two volume

60、s of gas is greater than 50%. One must always be prudent in the interprepation of results, and keep in mind that certain natural substances can have inhibiting effects vis-vis the test strain. Chemical characterization of the antiseptic provides the confirmation of positive results. 1. Milk and Milk