Chapter 12 Food additives analysis. Determination of Antibiotics. 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. 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 essentially have two consequences:
1. Selection of resistant strains.
2. Disequilibrium of the normal flora of the digestive tract.
In processing operations, the presence of residues with antibiotic activity in milk or meat makes them unsuitable for some 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 methods of determination used at present can be classified into three groups:
The most frequently used detection methods are those that exploit the sensitivity of certain bacterial strains vis-à-vis one or several antibiotics. The manifestation of this inhibition is affected either in liquid (e.g., acidification method) or solid media (e.g., agar diffusion method).
This technique is widely used for the detection of antibiotics in milk. After pasteurization, the sample is cultured with a strain 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 also be measured by nephtelometry.
Several 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 Lactobacillus 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 incubation.
4. Measurement of Medium Turbidity
Measure the growth of the test germ by recording the variations in medium turbidity over time.
By 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 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.
These techniques have been employed in antibiotic analyses in all food products.
When one 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 appearance 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: Healtey’s agar; Chabbert medium; Bacto Whey Agar medium. The pH must be adjusted to 6.6 or 7.8, depending on the antibiotic studied.
1. 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 extraction (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 recommended.
The following strains are used most often:
·Bacillus stearothermophilus var calidolactis strain
·Bacillus subtilis ATCC 6633,
·Sarcina lutea ATCC 9341:
·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.
1. The agar medium, 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 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 agar surface.
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 performed 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 properties 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 other than penicillin, then an inhibition zone appears around the disk saturated with penicillinase.
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” technique 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, however, 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 will 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 inhibition 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.
1. 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 elimination 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 before, 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 microbiological method. )
1. 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 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, 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 antibiotics
This technique is rarely used in rountine work. It is an excellent reference method for situations requiring expertise.
2. Physicochemical Methods
These techniques of antibiotic detection have developed considerably in the past few years. They are special applications of the analytical principles 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 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 theoretical sensitivity, this technique cannot reach detection limits lower than 1 pg/ml of milk (Flavigny, 1980)
5. Determinations by Fluorimetry
Hamann and Heeschen (1975) have desrcibed a determination technique using immunofluorescence, the principle of which consists of labeling the antibiotic with fluoresceine. The determination occurs by competition in the presence of an unlabeled antibiotic and a specificserum. The measurement is affected by fluorimetry in polarized or nonpolarized light. This technique is used for the detection of tetracylines, which form fluorescent compounds after being heated in neutral or alkaline medium (detection limit: 2 µg/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 accompanies the reaction, is measured by a photometer. This technique therefore involves a bacteriological step.
Used in the ultraviolet, visible, and infrared spectra, spectrometry permits the identification and the determination of penicillin (detection limit: 10 µg/g), streptomycin, tetracyclines, and the like.
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 detection limits are:
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 Bacillus
cereus, Bacillus subtilis, Micrococcus flavrs, and Sareina lutea.
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:
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 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 determination 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 increased 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.
The presence of antiseptic residues in foods is due essentially 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 disinfectant 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.
The presence of antiseptic substances in food poses problems 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).
These technique are based on the fact that the growth of certain microorganisms in inhibited by the presence of antiseptic residues.
1. General Method: Fermentation Test
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 Saccharomnyces 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.
Liquid nutrients do not require any special preparation, with the possible exception of dilutions for products 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-solid 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 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%.
In 100 ml of a sterile 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 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℃.
Eliminate the carbon dioxide gas from the second culture by filtering it on sterile hydrophilic cotton. In a sterile Erlenmeyer flask, aspetically introduce 10 ml of the yeastsuspension thus prepared, and 50 ml of the sample or sample preparation. Mix carefully and decant the sample thus cultured in a sterile Einhorn’s endiometer in such a way that the bowl is one-third filled and the tube is filled completely.
If possible, prepare a control with a sample or an extract free from antiseptics under the same conditions.
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 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 volumes of gas is greater than 50%. One must always be prudent in the interprepation of results, and keep in mind thatcertain natural substances can have inhibiting effects vis-à-vis the test strain. Chemical characterization of the antiseptic provides the confirmation of positive results.
1. Milk and Milk Products: Kluyver Method
The sample to be analyzed is enriched in glucose, and then cultured with a dilution of Saccharomyces cerevisiae in peptonated water. A reference free from antiseptic is prepared under the same conditions. After incubation at 25℃ for 48 hours, if the sample examined has gaseous release of lower than 50% of that of the reference, then one may conclude that it contains antiseptics like hydrogen peroxide, quaternary ammoniums, and by-products of monobromacetic acid at concentrations that are likely to cause manufacturing flaws. This method can detect the antiseptics mentioned previously to minimal concentrations of 0.02.-0.05 and 0.001%, repectively (Serres et al.).
2.1 Official French method for detection of antiseptics in wines.
Sulfur anhydride is first removed from the wine by adding a requisite quantity of acetaldehyde (… the wine must contain less than 20 mg/L of free SO2). The wine is brought back to 10% alcohol and the quantity of
glucose is enhanced (20-50 g/L). A nutrient solution is added and the wine is cultured with a suspension of Saccharomyces oviformis accustomed to alcohol (10²*10³ yeast/ml of wine). The intensity of the fermentation, measured in a Stella-Garoglio (Lecoq, 1965) zygometer, enable one to drow conclusions about the presence or absence of antiseptics.
It is necessary to be able to compare the sample’s rate of fermentation with that of a wine with a composition as close as possible to that of the sample that is free of antiseptics. In normal wines, the release of dioxiode begins after 48 hours of incubation at 25℃. The presence of antiseptics can be confirmed if th commencement of fermentation is delayed by 48 hours as compared with that of the reference test; however, the interpretation of results with always be difficult with wines made from rotted grape. A delay in fermentation could be possible, which suggests the content of antiseptic traces.
This method consists of culturing the sample enriched in nutrients with a Saccharomyces cerevisiae suspension, the growth of which can be studied by measuring the volume of carbon dioxide gas released. With a drink free of antiseptics, it is found that the curve representing the volume of CO2 as a function of time is S-shaped, which represents a latency phase, a logarithmic growth phase, and a slowed growth antiseptics, the latency phase is more significant than that of the reference. By plotting the different curves of growth on a graph, it can be seen that the presence of an antiseptic residue in the samples creates a shift of the growth curve toward the right.
For the methods to produce good results, the authors recommend:
1. the use of very active yeasts with concentration of between 50 and 100 cells/µl of sample liquid;
2. the elimination of sulfurous anhydride and alcohol by a stream of water vapor;
3. conducting a reference test with a sample free of antiseptic.
2.4 Baetsle, Mossel, and Verheyden method. This is the official method of the Benelux countries (Baetsle et al., 1955) for the detectiong of antiseptics in beer. Its principle is also based on the use of Saccharomyces cerevisiae, and the magnitude of the carbon dioxide gas released is measured in the Einhorn tube.
This method consists of incorporating the suspect sample in a medium of agar culture. The presence of the antiseptic is indicated by the total or partical inhibition of inoculated germs on the surface of th culture medium, as compared with a reference cultured under the same conditions.
The technique consists of introducing 10 ml of the sample and 0.1 ml if 1% triphenyltetrazolium chloride in 10 ml of double concentration nutrient agar. Homogenize and pour this mixture in a Petri dish. Under the same conditions, prepare a reference Petri dish using a sample free of antiseptics. After setting the agar, divide each dish into four parts and inoculate by streaking with four species of bacteria:
Incubate at 30-37℃ for 24-28 hours. In the same manner, preparea second series of dishes that use four species that belong to the group of yeasts and molds.
·Penicillium glaucum;·Aspergillus niger;
Incubate at 20-25℃ for 4-5 days. Compare the growth of the germs after incubation of the dishes. The absence of culture or a partical inhibition in the dish that contains the sample to be examined reflects bactericidal or fungicical effect vis-à-vis the species considered.
This method (Serres et al.) can be used to detect antiseptics in butter. This is performed on the aqueous phase of butter that is enriched in glucose and yeast extract. The mixture prepared in this manner is mixed with nutrient agar and poured into a Petri dish. As in the previous technique, affect a reference test and divide the dishes into two parts. Each part is cultured, one with a strain of Candida The ever-increasing number of chemical substances with antiseptic action considered, it is not possible to provide an exhaustive list accompanied by their methods of detection. This chapter is therefore limited to detection and determination techniques that concern:
a) principle antiseptic substances, used as such and authorized by regulation for the treatment of certain foods;
b) products with antiseptic action that are often used in the operations of cleaning and disinfeceting;
c) a few antiseptics that are sometimes used illicitly.
The colorimetric method:
which was published by Schmidt in 1960, is applicable to all foods. First, ascorbic acid is distilled by vapor. It is collected in the distillate and oxidized by an acid solution of potassium bichromate. It particularly forms malonic dialdehyde, which with thiobarbituric acid produces a red complex with a maximum absorption at 532 mm. The sorbic acid concentration of the sample is read on a standard curve that is formed from a solution of potassium sorbate. The other antiseptics do not produce interference. The precision of this method is on the order of ±5%.
High performance liquidchromatography:
can be used for the determination of sorbic acid.
A number of authors have published papers on the determination of benzoic acid and its derivatives (e.g., orthochlorobenzoic, parachlorobenzoic, salicylic, and parahydroxybenzoic acids, and methyl, propyl, and butyl esters of parahydroxybenzoic acid). Parkinson advocates a chromatographic technique after extraction with chloroform or a chloroform-ethanol mixture.
Benzoic acid is extracted by ethyl eher. The solvent is evaporated, and the odor and the points of melting and sublimation are determined with one part of the residue. The other part is redissolved in hot water. The liquid is then neutralized and iron perchloride is added, which produces a characteristic precipitate in the presence of benzoic acid.
This method (Ribereau-Gayon and Peynaud, 1958) modifies the preceding technique by effecting destruction between benzoic acid and succinic acid using the characteristic aspect of their crystalline deposits.
Guillaume and Mehl
The Guillaume and Mehl (1951) Method uses ethyl extraction and the determination of the antiseptic by borometric titration.
Sulfurous anhydride is introduced into food liquids in the from of sulfurous gas, which combines with sugars, ethanol, and coloring matter. A fraction of free SO2 still remains and is responsible for the characteristic odor and the antiseptic action.
After the sample is heated, and water and phosphoric acid are added, sulfurous anhydride can be detected in two ways (Diemair and Postel, 1970):
·chemically by using a narrow starched band of paper saturated with potassium. In the presence of SO2, the paper takes on a blue tint that disappears if the duration of the test is prolonged.
Liquid Products: Wines, Beers, Ciders, Fruit Juices, and Meads
The method, which is employed most often (Brun, 1963; Ribereau-Gayon and Peynaud, 1958), is based on the oxidation of sulfrous anhydride by iodine. The free SO2 is determined diretely by iodine in an acid medium. On the other hand, sulfurous anhydride is not oxidized by cold iodine in an acid medium, and therefore, it is necessary to displace it by distillation and titrate it with iodine.
Semi-Liquid and Solid Products: Mustard, Dry Fruits, and Fish
Monier-Williams and Shipton (1954) have described methods in which sulfurous anhydride reacts with hydrogen peroxide to form sulfuric acid, which is then determined with a titrated alkaline solution.
Many other methods have been described (Coeur et al., 1961; Jaulmes and Hamelle, 1961) among which are colorimetric methods (Brun, 1963), gravimentric techniques (Code de la Charcuterie, 1980), or that of Stone and Laschiver (1957), which are based on the purple color produced by rosaniline with SO2
Halogenated Acetic Acids – Monobromacetic Acid
Formaldehyde and Substances Producing Formaldehyde
Fluorinated Compounds: Sodium Fluoride and
Hypochlorites and Chloramines
A general diagnosis that affirms that a food product contains antiseptic residues is difficult to make using chemical methods. Microbiological techniques, although nonspecific, offer the advantage of being able to make a rapid distinction between products that can cause microbial inhibition and those that do not affect the growth of sensitive strains. Unfortunately, the results can be distorted by the presence of certain natural substances that are harmful to the development of the test strain, always necessitating a reference sample free of antiseptics. Chemical methods can be used to verify the microbiological results. They are affected, before the biological test, every time it is necessary to verify whether the adition of a parmissible antiseptic conforms to regulations, or when there is a strong presumption of the presence of a particular antiseptic.