Inhibitory substances

Agar, which is low in metabolizable or inhibitory substances, debris, and thermoduric spores, is ideal for the propagation and pure culture of yeasts, molds, and bacteria. Agar also meets the other requirements of ready solubiUty, good gel firmness and clarity, and a gelation temperature of 35—40°C and a gel melting temperature of 75—85°C. A clarified and purified form of the bacterial polysaccharide, geUan gum, is the only known satisfactory substitute. 

The unique properties and actions of an inhibitory substance can often help to identify aspects of an enzyme mechanism. Many details of electron transport and oxidative phosphorylation mechanisms have been gained from studying the effects of particular inhibitors. Figure 21.29 presents the structures of some electron transport and oxidative phosphorylation inhibitors. The sites of inhibition by these agents are indicated in Figure 21.30. 

Various assay methods have been used to detect the presence of inhibitory substances. These include some of the classical tests used by investigators of growth-promoting substances—i.e., the various Avena coleoptile assays which utilize intact, decapitated, or isolated cylinders and the split pea stem test. Effects on seed germination and seedling shoot or root growth and development have also been measured in addition to other visible expressions of inhibition. Details of many of these tests have been compiled by Mitchell et al. (99). Tests have been carried out in Petri dishes, with various solution culture techniques, and by sand and soil culture. Effects so measured may or may not be similar to those obtained under field situations— i.e., the establishment of inhibition under controlled conditions pro-... 

Preservatives may inelude disinfeetant and antiseptic chemicals together with eertain compounds used almost exclusively as preservatives. They are added to mar industrial, including pharmaceutical, products which may, by their nature, support the growth of bacteria and moulds causing spoilage of the product and possibly infection of the user. In the field of pharmaceutical preservation, addition of an inhibitory substance to a multidose injection (Chapter 21) or the prevention of growth in aqueous suspensions ofdmgs intended for oral administration (Chapter 18) are prime examples. 

Relating the effects caused by specific allelochemicals to those caused by an allelopathic plant is complicated because the inhibitory substances released from a plant are often unknown, and generally several different compounds are involved. However, the actions of the weeds studied in our investigations have certain parallels to those found from pCA and FA. The allelopathic nature of Kochi a, Jerusalem artichoke, and cocklebur was established, since both aque-ous extracts and weed residues reduced sorghum growth. The data show a concentration dependency characteristic of allelopathy, and some difference in toxicity among the three weeds was observed with cockle-bur the most toxic. 

Other kinds of bloassays have been used to detect the presence of specific allelochemical effects (8), effects on N2 fIxatlon (9), the presence of volatile compounds (10) and of Inhibitory substances produced by marine microalgae (11). Putnam and Duke (12) have summarized the extraction techniques and bioassay methods used In allelopathy research. Recent developments In high performance liquid chromatography (HPLC) separation of allelochemlcals from plant extracts dictates the need for bloassays with sensitivity to low concentrations of compounds contained In small volumes of eluent. Einhellig at al. (13) described a bloassay using Lemna minor L. growing In tissue culture cluster dish wells that maximizes sensitivity and minimizes sample requirements. 

The cellular actions of cannabinoids clearly support the proposal that the cannabinoid receptor is inhibitory and, consequently, reduces the firing rate of target neurons. However, this is not wholly confirmed by electrophysiological measurements, which suggest that cannabinoid compounds can stimulate neurons in the hippocampus. This apparent discrepancy may be due to the ability of cannabinoids to inhibit the release of an inhibitory substance in the hippocampus and, thus, produce a net excitation. 

In this connexion we will stress again that, although there is often a correspondence between toxic action of organo-phosphorus insecticides and anti-cholinesterase activity (p. 67), the relationship is not always simple. Thus parathion (p. 178), not itself an esterase inhibitor, is converted in vivo into an enzyme inhibitor.1 On the other hand, Aldridge2 has shown that the inhibitor paroxan can be hydrolysed enzymically to produce non-inhibitory substances. 

Significant parameters to be considered in designing a treatment and disposal facility for pharmaceutical wastewater are given in Table 12. Biochemical oxygen demand measurements of the waste have been reported to increase greatly with dilution, indicating the presence of toxic or inhibitory substances in some pharmaceutical effluents. The toxicity impact upon various biological treatments by various antibiotics, bactericidal-type compounds, and other pharmaceuticals has been described in the literature [21-24]. 

The system discussed, commercially known as the Delvotest (9-11), utilizes a strain of stearothermophilus which grows at a very rapid rate and produces acid from the nutrients in the medium in the absence of inhibitory substances. When inhibitory substances such as the 3-lactam antibiotics are present, acid production is inhibited. By incorporating bromcresol purple in the medium, it is easy to observe acid production, a change from purple to yellow. Incubation is performed at 65 C for approximately 3 h. 

Inhibitors of lactic dehydrogenase have been reported in commercial preparations of NAD+ and NADH (B4, M6, S28). The concentration of inhibitory substances varied from lot to lot. In a serum lactic dehydrogenase study with NAD+ from 8 sources, activities were found to vary from 145 to 75 units (B4). Inhibitors of lactic dehydrogenase activity have also been observed in dialyzates in uremic patients (W8) and in human urine (G8). The purity of available substrate can also effect enzyme activity. Schwartz and Bodansky observed that, in 6 batches of fructose 6-phosphate, all weighed to a 0.5 mM concentration, the actual concentration varied from 0.13 mAf to 0.55 mM (S14). 

In addition to the cholinergic and adrenergic receptors on autonomic ganglion cells, there also appear to be receptors for a variety of excitatory and inhibitory substances, including angiotensin, bradykinin, histamine, 5-hydroxytryptaimine (serotonin), and substance P. The... 

Many unit processing operations have been shown to influence protein quality. Heat, commonly applied to protein to increase digestibility, can not only make the protein intrinsically more digestible but can inactivate inhibitors to protein digestion. Denaturation of protein is thought to be the mechanism for the increased digestibility and the inactivation of inhibitory substances. 

Owing to the variability in results of the early developed microbiological procedures, standardized materials were introduced and are currently used for routine quality control. Depending on the format of the test, the presence of an inhibitory substance is indicated by zones of growth inhibition or a change in the color of the medium with pH and redox indicators. Examples of commercial... 

A disadvantage of both competitive assays is that the enzyme conjugate solution has to be mixed with the sample solution. A sample solution, however, may contain inhibitory substances such as proteases that can alter the activity of the antibody and/or the enzyme-label used. Where enzyme-labeled antibodies are employed, such problems may be circumvented by using an unlabeled antibody in the competition phase, followed by incubation with an enzyme-labeled second antibody that is directed against the primary antibodies (109). 

In assays in which bromocresol purple indicator is incorporated into the medium (disc assay for penicillin, disc assay with indicator), a clear bluish zone around the disk appears if samples contain inhibitory substances. When addition of penicillinase to positive milk samples eliminates the zone, the inhibitors can be roughly identified as -lactams. Among these tests, the 3 h Bacillus stearothermophilus disc assay was selected in 1981 as the regulatory test for detecting antibiotic residues in milk. Although particularly sensitive to -lactams, this test is relatively insensitive to a number of commonly used antibiotics (20). 

In the Accusphere test a freeze-dried sphere containing the test organism Streptococcus thermophilus and bromocresol purple as indicator disperses into the test milk sample. The acidification test is very similar the milk sample is heated to be further inoculated with a Streptococcus thermophilus culture containing yeast extract, bromocresol purple indicator, and trimethoprim. It is then incubated for 2.5 h at 45 C (33). In the presence of inhibitory substances, the organism growth is suppressed, acid production is reduced or eliminated, and the color of the indicator remains unchanged. Addition of penicillinase to a positive milk sample results in change of the color of the indicator from purple to yellow when only -lactams are present. 

In contrast to tire previous multiresidue tests, the Valio TlOl test uses a freeze-dried Streptococcus thermophilus TIO l-strain dispersed into the milk. During incubation, the test organisms grow and produce acid, which causes the pH indicator to change color from blue to yellow. In the presence of any inhibitory substances, the indicator remains blue or turns to a greenish color depending on the concentration of inhibitors. 

The BR-test has been modified several times (37, 38). One of its versions, known as the BR-test AS, contains antifolates that make it possible to detect sulfones and sulfonamides in addition to the usual inhibitory substances. Another version, which is called BR-test Blue Star, has been officially accepted and used in Canada. By decreasing the spore concentration in the detection medium, a sensitivity of less than 40 ng sulfamethazine/ml milk is possible with this test (38). 

Since yogurt cultures are about 10 times more sensitive to penicillin than other starters, a yogurt inhibitor test has been also developed to detect this drug in milk (45, 46). In this test, the milk is heated and inoculated with a yogurt culture. Following incubation, inhibitory substances are considered to be present when the increase of the Soxhlet-Henkel acidity of the test sample is lower than half the increase of acidity of an inhibitory-free confiol milk sample. 

Detectable concentrations of various antibacterials in milk attained by different microbiological tests are presented in Table 27.2. Milk constitutes a matrix that, apart from heating to destroy natural inhibitory substances, does not generally necessitate further sample treatment. Some antibiotics, however, exhibit some instability to heat treatment (54-56) and, therefore, if further confirmation is required reference frozen samples should always be available. When raw milk is directly analyzed, critical evaluation is generally required because natural inhibitors such as somatic cells, immunoglobulins, and metabolites may cause zones of inhibition (56, 57). Furthermore, several factors including marked pH-devia-tions, use of paper disks that contain inhibitory substances, and work with forceps that are too hot or have not been cleaned properly can readily lead to falsepositive readings (56, 58). 

Hirsch, A., McClintock, M. and Mocquot, G. 1952. Observations on the influence of inhibitory substances produced by the lactobacilli of Gruyere cheese on the development of anaerobic spore-formers. J. Dairy Res. 19, 179-186. 

Mattick, A. T. R. and Hirsch, A. 1944. A powerful inhibitory substance produced by group N streptococci. Nature 154, 551. 

---