Study On HMB Microbial Inhibitory Activity
Two studies were conducted to determine the anti-microbial potential of DL-2-hydroxy-4-(methylthio)-butanoic acid (HMB). Results showed that HMB was effective in inhibiting pathogenic bacteria, including E coli O8, O149 and O157, Clostridium perfringens, Salmonella pullorum, and fungi Aspergillus flavus and Fusarium graminum. The degree of microbial inhibition was closely related to HMB concentration and the medium pH. Equi-molar comparison suggests that HMB is less effective as an anti-microbial agent than propionic acid and potassium sorbate.
DL-2-hydroxy-4-(methylthio)-butanoic acid (HMB) or liquid methionine analogue not only serves as a source of methionine but has other potential roles including microbial inhibition. HMB is a hydroxy acid, with four carbons and a methyl-thio radical, with a pKa of 3.86, similar to formic (pKa 3.75), acetic (pKa 4.76) and propionic (pKa 4.88) acid. This suggests that HMB has potential anti-microbial properties. Studies on the HMB anti-microbial role are reviewed by Dibner and Buttin (2002). With the present need to find alternatives to antibiotics, the potential role of HMB in this regard, needs to be evaluated.
II. STUDY ONE: HMB MINIMUM INHIBITION CONCENTRATION (MIC)
This study was conducted at the Microbiological Centre of Sichuan Agricultural University, China. In total five strains of pathogenic bacteria, i.e. E. coli O157, O8, O149, Salmonella pullorum, Clostridium perfringens, and two species of fungi, i.e. Aspergillus flavus and Fusarium graminum were examined. These organisms were obtained from the China National Veterinary Institute in Beijing, further cultivated and diluted with saline solution to 106 organisms/ml, and kept in 4oC prior to testing.
For the inhibition test, culture broth was prepared to nurture the organisms and liquid HMB (Rhodimetâ„¢ AT 88) was introduced into the broth in gradient ppm (Table 1). 0.1 ml test organisms were inoculated into the broth and the tubes were incubated in 37oC for 24 h, then the broth was checked for minimum inhibitory concentration (MIC). A clear broth indicated no microbial growth or total inhibition whilst a perturbed broth suggested a growth or presence. After MIC check the incubation continued for an additional 24 h, no growth represented minimum lethal concentration (MLC). For fungi, the incubation was at 28oC for 72 h for MIC and 96 h for MLC. Results of bacteria inhibition are presented in Table 1.
Table 1. Effect of HMB concentration (ppm) on microbial growth
(N=3, + means growth or presence and -means no growth)
Clearly, at HMB concentration of 2400 ppm, all bacteria stopped multiplication. For fungi, results in Table 2 showed 2,000 ppm HMB was able to inhibit growth of Fusarium whilst 4000 ppm was required to stop growth of Aspergillus.
Table 2. Effect of HMB concentration (ppm) on growth of fungi
(N=3, + means growth or presence, -means no growth)
The effect of propionic acid (PPA) was tested under the same conditions, in which PPA at 1000 ppm stopped growth of both bacteria and fungi, its MIC and MLC should fall between 500-1000 ppm. Table 3 lists a summary of the MIC results of HMB and PPA. It should be noted that there were no PPA test concentrations between 500 and 1000 ppm. As such, the actual MIC of PPA appears to lie somewhere between 500 and 1000 ppm.
Table 3. MIC comparison between HMB and Propionic acid (PPA)
III. STUDY TWO: HMB INHIBITION ZONE BY PETRI DISH TEST
The Oxford Cup method was employed to determine inhibition zone. HMB and other materials were from the same source as Study One. Petri dishes with potato dextrose agar (PDA) culture medium were prepared and sterilized. 0.2 ml inoculants were evenly sprayed onto the culture gel surface, the dishes were then dried at 37oC for 30 min. HMB was diluted with buffer solution to obtain concentrations of 0, 1.0, 2.0 and 4.0 mg/g at a pH of either 4.5 or 6.5. DMSO and Flavomycin (both 500 ppm) were used as negative and positive controls.
A sterilized Oxford Cup was placed vertically on the gel surface, with no gap between the cup and the gel surface. Test solutions (HMB, DMSO and Flavomycin) were poured into the cup. The cup was full but no spillage was permitted. Since the cup had no bottom the test liquids (HMB, DMSO and Flavomycin) had a direct access to gel surface. The dishes were carefully transferred into the oven and incubated at 37oC for 12 h. The inhibition zone was measured around the edge of the cup. The strength of inhibition was proportional to the diameter of inhibited organism growth around the cup.
Table 4 shows the inhibition results from five bacterial inoculants at pH 4.5. The degree of inhibition exerted by HMB was concentration dependent, with 4.0 mg/g achieving the highest efficacy especially against E coli O149 and S. pullorum. Whilst Flavomycin displayed moderate inhibition with the E coli strains and S. pullorum, this antibiotic seemed to have lost its efficacy against C. perfringens.
Table 4. Inhibition zone (mm) of HMB, DMSO and Flavomycin at pH 4.5 (N=3)
Inhibition results at pH 6.5 were less effective and less pronounced than at pH 4.5. Positive responses were observed against E. coli O149, but no inhibition was observed against other strains. Flavomycin inhibited 4 strains but had little effect against E. coli O8.
Fungal inhibition was tested using petri dish. The gel was prepared to contain graded levels of HMB, propionic acid and potassium sorbate and pH was adjusted to either 4.5 or 6.5. Two holes of 7 mm diameter were drilled in each dish, and fixed amounts of fungi inoculants were introduced into the holes. The dishes were incubated at 28oC for 48 h, and inhibition zones were measured by diameter, the smaller the diameter, the stronger the inhibition was. Each treatment was repeated 3 times.
Table 5. Comparison of fungi inhibition in petri dish test (mm)
Data in Table 5 demonstrated that increase in HMB dosage led to a reduction in fungi growth zone, and higher inhibition was achieved at pH 4.5. HMB 4.0 mg/g did not completely eliminate fungal growth. In contrast, propionic acid at 2.0 mg/g achieved a total elimination of fungi regardless of the medium pH. Potassium sorbate displayed an more effective inhibition at pH 4.5 than pH 6.5, but was less effective than propionic acid.
IV. DISCUSSION AND CONCLUSION
Some short chain organic acids (SCOA) possess the ability to penetrate cell membranes by simple diffusion. Upon entry, the SCOA can release H+ due to low pKa. Once in the bacterial cell, the higher pH of its cytoplasm causes dissociation of the acid, reducing the internal pH, hence disrupting the enzymatic reactions and nutrient transport systems, and impairing the normal functioning of the cytoplasm. The cells hence divert energy to pump out the extra H+ , which finally exhausts the cells, resulting in death (Cherrington et al., 1991). Formic, lactic, propionic and sorbic acid are widely used alone or in combination, for feed or food hygiene purposes.
The present results agree with Enthoven (2002) who studied the effect of HMB on Salmonella enteritidis at pH 4.5 or 6.75 in comparison with formic acid. His results showed inhibitory effects at pH 6.75, and a bactericidal effect at pH 4.5. The response was dose dependent at each pH level for both HMB and formic acid.
These studies demonstrate that HMB possesses anti-microbial properties but that in this role HMB is less effective than propionic acid and potassium sorbate. In general, commercial inclusion of propionic acid as a mould inhibitor is between 0.2-0.5 kg/t feed, whilst HMB is widely applied as a methionine source at 1.5-3.0 kg. The higher dosage of HMB may act as a partial substitute for commercial mould inhibitor. Further study is warranted to quantify HMB's efficacy in microbial inhibition.
Cherrington C.A., M. Hinton, G.C. Mead and I. Chopra. (1991) Advanced Microbiological Physiology. 32:87-108.
Dibner J.J. and P. Buttin (2002). Journal of Applied Poultry Research 11:453-463.
Enthoven P., van den Hoven S., Wiltenburg R. (2002). 11th European Poultry Conference. Germany, Bremen, Aug. 6-10-2002.