COLORED AND FLUORESCENT MAGNETIC BACTERIA AND THEIR USE FOR ANIMAL FEED APPLICATION

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COLORED AND FLUORESCENT MAGNETIC BACTERIA AND THEIR USE FOR ANIMAL FEED APPLICATION

Сообщение автор Admin в Вт Фев 25 2014, 18:02

Nikolay Barashkov
Micro Tracers, Inc, San Francisco, CA;
Arash Komeili; Olga Draper
UC Berkeley, Berkeley, CA

COLORED AND FLUORESCENT MAGNETIC BACTERIA AND THEIR USE FOR ANIMAL FEED APPLICATION

The worldwide formula feed industry manufactures millions of tons of liquid feed annually. Currently there are no tracers that suspend stably in liquid feeds that can be easily analyzed quantitatively. As a result, feed manufacturers assay for active ingredients to establish their feeds are completely mixed, to validate cross contamination control procedures, or to verify microingredients are added at formulated levels. Such assays are often expensive, require waiting, and have significant inherent analytical error.
Periodic mixer testing is important because feed manufacturers waste labor, energy and capital when they mix feeds longer than necessary [1,2]. Excess mixing may also degrade expensive vitamins and medications. If feeds are not completely mixed, portions will contain either too much or too little of the formulated ingredients. This variability may negatively impact the efficacy of the feed and may increase the incidence of illegal drug residues. The EU has required mixer performance validation since 1995. The US-FDA will likely require such validation as it implements the Food Safety Modernization Act of 2010 (FSMA).
Feedmills often manufacture formulations for many species using the same mixing equipment. Contamination of medications to non-target feeds is inevitable but controllable. Medicated feed assays are often of limited value because: they are expensive, they cannot be performed immediately, and they are often inaccurate at contamination levels of concern (i.e. 1% or less of formulated levels) [3,4]. The EU has established maximum levels of cross-contamination for coccidiostats of 3% of formulated levels into any non-target feed and 1% into milk or egg feed or finisher feed for the intended species. Though the US-FDA does not currently require validation of cross-contamination control, it may require such validation as it implements FSMA.
In this study we developed and investigated new magnetically retrievable tracers based on ferromagnetic bacteria colored with a food grade dye FD&C Red #3 which can be used to evaluate mixing and cross-contamination of molasses based liquid feeds, and to code critical microingredients, such as drugs and enzymes added in liquid feeds. These tracers will suspend stably in liquid feeds, be easily retrievable from liquid and dry feeds, be safe for use in feeds, be formulated at low ppm (parts per million) levels, and be simple and inexpensive to analyze both qualitatively and quantitatively.
General information about magnetic bacteria
Magnetic bacteria (MB) were first observed by S.Bellini in 1963 [5,6] . He noticed that certain bacteria accumulated at the edge of a water droplet corresponding to the magnetic North. Using various experimental setups, including simple bar magnets and magnetic coils, he showed definitively that the bacteria of interest were ‘magnetosensitive’. Independently in 1975 the MB were re- discovered by R. Blakemore [7]. Using various techniques, he also showed that these bacteria contained chains of crystal-like inclusions that were rich in iron, thus providing a mechanistic explanation for their ability to respond to magnetic fields
The early experiments by Blakemore and others established that MB respond to external magnetic fields through the use of specialized organelles termed magnetosomes [8]. The magnetosome consists of a magnetic mineral, either the iron oxide magnetite or the iron sulfide greigite, surrounded by a lipid-bilayer membrane (Fig. 1). Individual magnetosomes, which usually measure 50–70 nm in diameter, are organized into one to several chains within the cell that provide the means for alignment with magnetic fields. In fact, the magnetic properties of the magnetosome chain are sufficient to provide the sensitivity required to stably align with the weak magnetic field of the earth [9].
The unique properties of MB, in particular their uniform size and shape, purity, and their production under ambient conditions, have made them an attractive choice for potential biotechnological and biomedical applications. For instance, magnetite particles can make great contrast agents for nuclear magnetic resonance imaging, and they have been proposed as potential therapeutic agents in the hyperthermic treatment of tumors. Magnetic bacteria have also been thought of as potential tools for bioremediation of toxic metals. Under one scenario, MB would sequester toxic compounds within magnetosomes and would then be separated from the water sample with magnets
Experimental
Food grade dye FD&C Red#3 (erythrosine) has been purchased from Emerald Performance Materials.
Molasses-based liquid feed for laboratory trials has been received from ZINPRO, Inc
The AMB-1 bacteria were stained by submersion in a 0.1% aqueous solution of dimethyl ammonium salt of oleic acid (DMAOA) containing FD&C Red #3. The bacteria were dispersed in molasses (no signs of precipitation was observed after 72 hrs of storage at room temperatute) and then retrieved using a rare earth magnet, washed with DI water treated with 0.1% aqueous solution of DMAOA at 70oC for 15 min. Experimental set up for retrieving colored MB is presented in Fig 2. It included the burette with dispersion of MB in molasses, and a beaker covered with a screen made of stainless steel and a Neodimium magnet protected with a layer of ABC plastic which was purchased from Magnetics J&K. The rate of molasses-based feed coming through the magnet surface was selected around 10-12 ml/min.
After the process of retrieving was finished, the cellular structure of colored MB was destroyed by heating with 0.5% aqueous DMAOA solution. a
Amount of released FD&C Red #3 in solution has been estimated using the Genesys 5 spectrophotometer (Thermo-Fisher, Inc) at wavelength 520 nm. . This process released the dye from the bacteria which was then read spectrophotometrically (Table 1).
RESULTS AND DISCUSSION
Table 1 – Results of homogeneity test for trial with stable suspension of Red#3-containing magnetic bacteria AMB-1 in molasses-based liquid feed
Parameter ## analyzed samples
1 2 3 4 5
Concentration of Red #3 in sample (absorbance units at 525 nm) x 103 179 160 183 192 148
Mean value of concentration 172.4
Standard Deviation, +/- 17.95
Coefficient of variation, %, +/- 10.41
Table 1 shows the absorbance values of 5 investigated solutions. It was found that a coefficient of variation (CV) for the series of sample analyses is about 10.4%. Taking into consideration the value of CV we should conclude that a mixing is complete.
It is known that in order to be practically feasible ferromagnetic tracers should have the recovery at least 50%. In this respect the feasibility of proposed colored tracers based on magnetotactic AMB-1 bacteria which show a recovery of 85% of dye is obvious (the initial loading of FD&C Red #3 in molasses-based feed was as low as 5 ppm).
CONCLUSIONS
Prepared magnetotactic bacteria AMB-1 containing food grade dye FD&C Red #3, possesses an ability to form a stable dispersion in aqueous molasses- based liquid feeds. Investigated bacteria can be used as the harmless markers (microtracers) for evaluation of mixing efficiency in liquid feeds and for qualitative and quantitative evaluation of presence of certain liquid ingredients, like enzymes, in liquid premixes and their distribution in the volume of feed.

References: 1. Lee H.J., Kim B.C., Kim K.W., Kim Y.K., Kim J., and Oh M.K., A sensitive method to detect Escherichia coli based on immunomagnetic separation and real-time PCR amplification of aptamers // Biosensors and Bioelectronics, 24, 3550-3555, 2009. 2.Terazono H., Anzai Y., Soloviev M., Yasuda K. Labelling of live cells using fluorescent aptamers: binding reversal with DNA nucleases // J.Nanobiotechnol. 2010. V. 8. P. 8. 3.Ito et al., 2005; or Alphandery et al., 2011. 4.Lang et al., 2007;or Yoshino et al., 2010. 5.Bellini S. On a unique behavior of freshwater bacteria // Chin J Oceanol Limnol. 2009 v. 27, 3. 6.Bellini S. Further studies on “magnetosensitive bacteria // Chin. J. Oceanol. Limnol., 2009 v. 27, 6. 7. Blakemore R. Magnetotactic bacteria // Science, 1975, v. 190, 377. 8. Balkwill D.L. et al., Ultrastructure of a magnetotactic spirillum // J Bacteriol., 1980, v.141: 1399. 9.Frankel R.B., Blakemore R.P. Navigational compass in magnetic bacteria // J. Magn Magn. Mater., 1980 ,v. 15-8, 1562.

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