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Easy test kit for the detection of Alicyclobacillus acidoterrestris in fruit juice

Guaiacol Detection Kit


Alicyclobacillus acidoterrestris is a gram-positive, spore-forming bacillus that occurs widely in nature. This specie has been studied substantially after the incident of turbidity of transparent apple juice in 1984. The Alicyclobacillus genus bacteria, which peculiarly grows under relatively high temperatures and acid conditions, is 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, 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. These species, however, cannot be detected by usual isolation culture methods as they grow only under acidic 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 TAB in March 2003 [4]. To allow easy performance of this test, we developed a kit that differentiates A. acidoterrestris in terms of productivity of guaiacol, which causes off-flavor.

Guaiacol Detection Kit ReferencePDF

Va-YSG Medium

Guaiacol Detection Kit

Guaiacol Detection Kit Flyer
PDF Download [419 kb]

Kit component

YSG Agar Plate 20 plates (Cat. No. KYO-06810)
YSG agar medium for TAB isolation 10 x 2 plates/1 box
Va-YSG Medium (2mL) 100 tubes (Cat. No. KYO-08901)
Vanillic acid added YSG medium for guaiacol formation 2 mL x 100 tubes
Guaiacol Detection Kit 100 tests (Cat. No. KYO-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: Guaiacol (1050 ppm) 2.5 mL x 1 tube

Scheme of TAB Test -Japan Fruit Juice Association-

Experimental Procedure

1. Take with single inoculation loop, 10 L, 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 mL of Reagent 1 of “Guaiacol Detection Kit”.
4. Add 20 μL of Reagent 2 of “Guaiacol Detection Kit”.
5. Add 20 μL of Reagent 3 of “Guaiacol Detection Kit”.
6. Mix well.
7. Leave on rack for 5-10 minutes.
8. Check the color change of the medium. Read the result within 10 minutes.

Brown color indicates contamination of AAT

*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 L of Positive Control, and follow steps (3)-(8).

Handling instructions

 Please dispose used medium after high pressure sterilization (121 Celsius for more than 30 min) with appropriate care.
 Storage condition: 2-10 degrees Celsius and, protect from light
 Shelf life: 12 months from date of manufacture.

Please note that we will ship out products with shelf life of 3 months to 10 months depending on batch available.

Product List

Product Name Cat# Quantity Price

YSG Agar Plate

KYO-06810 20PLATE

¥ 5,000
$ 67
€ 50

Va-YSG Medium

KYO-08901 100PC

¥ 20,000
$ 267
€ 200

Guaiacol Detection Kit

KYO-08921 100TEST

¥ 25,000
$ 334
€ 250


> G.Deinhard, et al., Bacillus acidoterrestris sp. nov., a new thermotolerant acidophile isolated from different soils. Syst. Appl. Microbiol. 10, 47-53 (1987)

> GOTO, M., Spore-forming thermo-acidophilic bacilli of the genus Alicyclobacillus, Bokin Bobai, 28 (8), 499-508 (2000), in Japanese

> GOTO, K., Lecture on "Classification and identification of microbes" 1. Classification and properties of new thermo-acidophilic bacilli, January 2001 issue of Soft Drink Technical Data, 9-26 (2001), in Japanese

> Niwa, M., et al., Development of a rapid detection method of A.acidoterrestris, hazardous bacteria to acidic beverage. Fruit processing, 13, 102-107 (2003)

> Japan Fruit Juice Association, On the unified test method for thermo-acidophilic bacilli, Report of Japan Fruit Juice Association, 535, 4-12 (2003), in Japanese

> Niwa, M., et al., A.acidoterretris rapid detection kit, Fruit processing, 13, 328-331 (2003)

> Niwa, M., Control of deleterious bacteria in acidic beverages by using a guaiacol detection kit (peroxidase method), Japan Food Science, 2004-2, 23-28 (2004), in Japanese

> Japan Laid-Open P2003-259A “Detection of guaiacol forming bacteria and/or its identification method”

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.