Alicyclobacillus acidoterrestris / gram / spore-forming
bacillus / Thermo-acidophilic Bacilli / guaiacol |
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Back Ground
Alicyclobacillus acidoterrestris is a gram-positive, spore-forming bacillus that occurs widely in nature. This species have been studied substantially after the incident of turbidity of transparent apple juice in 1984. The Alicyclobacillus genus bacteria, which peculiarly grow under relatively high temperature and acid conditions, are called Thermo-acidophilic Bacilli (TAB) [1, 2, 3].
TAB are not destroyed by pasteurization and may remain in final products of fabricated food and beverages. TAB are not known to be harmful to health but are known to degrade the quality of juices and other products by producing guaiacol, causing off-flavor. Even without the off-flavor problem, product values may be degraded by slight deterioration during distribution.
It is very difficult to avoid contamination of TAB, which are common in soil. Fortunately, however, A. acidocaldarius and other main species that cause contamination are known to be harmless, and damage caused by TAB can be minimized by monitoring the contamination of A. acidoterrestris, which is known to be harmful. This species, however, cannot be detected by usual isolation culture methods because it grows only under acid and relatively high temperature conditions.
Because of frequent occurrence of fruit juice deterioration incidents in Europe and other regions, Japan Fruit Juice Association developed and publicized a unified test method for thermo-acidophilic bacilli in March 2003 [4]. To allow easy performance of this test method, we developed a test kit to differentiate A. acidoterrestris in terms of productivity of guaiacol, which causes off-flavor.
Scheme of the unified test method for TAB
prease click
Scheme of the unified test method for TAB
TAB test:
TAB are detected selectively by culturing in an acid medium (pH 3.7±1) at temperatures best suited for their growth.
A. acidoterrestris differentiation:
After the TAB test, A. acidoterrestris is differentiated from the other Alicyclobacillus species by using either of the following methods: 1)The differential growth temperature method, using differences in breeding ability at best suited temperatures, and 2)The peroxidase method, using tetraguaiacol production.
*This product adopts the peroxidase method to differentiate A. acidoterrestris from TAB. The method allows quick measurement of productivity of guaiacol, which causes off-flavor.
*Direct testing after the preliminary culture is also under consideration.
Contents
Va-YSG Medium (2mL) 100 tubes(Catalog number: 08901)
| Vanillic acid added YSG medium for guaiacole
formation |
2 mL x 100 tubes |
Guaiachole Detection Kit 100 tests(Catalog number: 08921)
| Reagent 1: 50mM Potassium hydrogen phthalate Buffer |
60 mL x 2 bottles |
| Reagent 2: 1.3 % hydrogen peroxide solution |
2.5 mL x 1 tube |
| Reagent 3: Peroxidase-Phosphoric Acid Buffer |
2.5 mL x 1 tube |
| Positive Control: Guaiachole (1050 ppm) |
2.5 mL x 1 tube |
Method
1. Take with single inoculation loop, 10 µL, of young colony
that has been isolated on YSG agar plate and that has not formed
spores. Suspend the colony in a tube of “Va-YSG medium
(2mL), ” (≥1 x 10 8cfu/mL).
2. Incubate at 45±1°C for 3 hours.
3. Add 1.0 mL of Reagent 1 of “Guaiacole Detection Kit”.
4. Add 20 uL of Reagent 2 of “Guaiacole Detection Kit”.
5. Add 20 uL of Reagent 3 of “Guaiacole Detection Kit”.
6. Mix well.
7. Let it stand for 5-10 minutes.
8. Check the color change of the solution.
*Read the result within 10 minutes
*Blank (Negative Control)
Use "Va-YSG medium (2mL)" that has not been inoculated and follow steps (2)-(8).
*Positive Control
Mix well 2 mL of distilled water and 100 µL of Positive Control. Follow steps (3)-(8).
Interpretation of Results
Darker brown than blank = Positive *If the color change is not significant and difficult to determine, retest with 24 hours incubation instead of 3 hours, step(2)
Same color as blank = Negative
Verify the test results by checking the reading of positive control; positive control result should have significantly darker brown than blank.
Handling instructions
Please dispose used medium after high pressure sterilization(121C
for more than 30 min)with appropriate care.
Please strictly keep the storage condition ( 2-10 degrees Celsiusand
and dark place).
Expiration date : 12 month
Va-YSG Medium
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Guaiachole Detection Kit
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