A Statistical study of effects of bacterial Decaffeination on Beverages

Siddharth Sharan*, Naina Thangaraj, Bishwambhar Mishra, Suneetha V.
School of Biosciences and Technology, VIT University, Vellore – 632014, Tamil Nadu, India
Corresponding Author: Siddharth Sharan , Email: [email protected]
Received:06 August 2011 Accepted: 02 October 2011
Citation: Siddharth Sharan*, Naina Thangaraj, Bishwambhar Mishra, Suneetha V. “A Statistical study of effects of bacterial Decaffeination on Beverages” Int. J. Drug Dev. & Res., April-June 2013, 5(2): 138-144. doi: doi number
Copyright: © 2013 IJDDR, Siddharth Sharan et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Caffeine degradation has been popularized due to its promising scope in food applications and environmental safety. Hence we conducted a study of bacterial caffeine degradation and its effect on sensory qualities through a survey and reported the data using Correspondence Analysis- Symmetrical Model of SPSS software to identify key sensory attributes and study what factors could lead to them. The decaffeinated beverages showed undesirable sensory attributes, especially those containing milk and other extraneous substances and hence it was concluded that decaffeination is most desirable during raw material processing. Also a background study of the bacterial growth curve and its relation with caffeine degradation and N-Methyl Urea formation was done using Origin software and ways to ensure the release of non-toxic products were hypothesized. An alternate glyoxylate pathway was considered to avoid the release of toxic metabolites, which is possible by media starvation. Thus avoiding degradation of sensory qualities and alternating the production of N-methylurea would reflect positively on human consumption and acceptance and this can be a scope of future study.


Caffeine, Caffeine degradation, Correspondence Analysis, Glyoxylate pathway.


Coffee, cocoa, tea and soft drinks contain the plant producing alkaloid derivative (1,3,7 trimethyl xanthine), caffeine, a mild stimulant and therapeutic by nature.[1] It has commercialized importance in pharmaceutical preparations and in beverages. It stimulates the central nervous system, it has also proved to be toxic when taken in excess.[2] On prolonged consumption, caffeine not only leads to addiction and subsequent withdrawal effects like headache, nausea and drowsiness[3], but it also has deleterious effects including adrenal stimulation, irregular muscular activity[4, 5], cardiac arrhythmias[6] and increased heart output. Thus, due to these adverse effects, it is necessary to develop methods to remove caffeine from caffeinated beverages.
Decaffeination has several advantages including reduction of long term side effects of caffeine, provision of coffee husk and pulp as animal feed and as a source of carbohydrates and proteins, [7, 8] and also reduction of toxicity in water bodies.[9] Decaffeination is frequently carried out by chemical or physical techniques that are not specific, costly and also pose a risk to the surroundings in terms of release of toxic substances.[10] Alternatively, microbial and enzymatic techniques for caffeine removal have been found advantageous than other methods[10].
Sensory perception is one of the means to a flavorful and natural image that beverages continue to enjoy with the consumer. Due to the fundamental nature of sensory perception with beverages, a sensory measurement is often the final step in many experiments or applications.[11, 12] Also statistical techniques have extensively been used in sensory analysis and thus serve as ideal means of studying and analyzing food attributes. Out of the five perceived senses viz. taste, smell, touch, sight and hearing; only smell and sight were given consideration here and taste was not studied in account for the use of crude enzyme for decaffeination process and also because instability and labile nature of purified caffeinase[13] (caffeine demythylase or oxidase).[14]
Our earlier study had reported the action of Paenibacillus marcens (Sharan et al, 2012) in degradation of caffeine and the production of caffeinase. In this report, beverage decaffeination using Paenibacillus marcens bacteria was tested and a sensory survey of decaffeinated beverages by 50 candidates was analyzed statistically using Correspondence Analysis (CA)[15] – Symmetrical Model and a basic study of the bacterial growth curve and its relation with the substrate (caffeine) and its possible metabolic product was done. Possible hypothesis of the factors leading to the results were made.


Beverage Decaffeination

The bacterial culture was grown at 37°C for 24 hours using Caffeine enriched media(CEM)[13] and then centrifuged at 12,000 rpm for 12 minutes to yield the supernatant containing caffeinase enzyme. Six different beverage samples known to contain caffeine, viz. coffee, tea, chocolate drink, ice-tea, a soft drink and black coffee were taken from VIT University Cafeteria and 1 part was added with the 4 parts of supernatant having crude caffeinase and then were incubated at 37°C for 24 hours. The samples were checked for absorbance at 275 nm[16] using UV-Visible spectrophotometer, keeping the hypothesized caffeinated samples as blank.

Sensory Survey

The decaffeinated beverages were refrigerated at 10°C in the dark for storage. A qualitative study of these beverages was done using survey of 50 participants aged between 20- 25. The survey was conducted in a period of 24 hours after the incubation of the sample beverages. The candidates received the samples in the order of Latin Square Design to prevent any carryover effects [17, 18]. The samples were studied for relevant sensory descriptors in visual, olfactory and aesthetic-impact parameters (Table 1), under constant light source and temperature conditions and candidates were given coffee beans before each sample to cleanse their palates [19].

Sensory Data Analysis

Univariate statistical analysis using SPSS software Version 16.0 for Windows statistical package (SPSS Inc.) was employed to identify exact significant sensory descriptors of each sample [18]. To investigate on the association between sensory parameters and the type of sample, correspondence analysis[18] was conducted using SPSS version 16.0 – Symmetrical Analysis for individual samples and was studied using a scatter plot. Bar graphs of visual ranking and sample identification were also plotted using Microsoft Excel.

Growth Curve Analysis

At first a 24 hour culture of the bacterium in caffeine enriched medium (CEM)[13] was observed to give maximum absorbance at 600 nm. The bacterial culture was then grown in CEM and its growth was monitored at 600 nm using colorimeter, for 24 hours. To study simultaneous caffeine degradation and formation of metabolites, 4ml of the culture was continuously withdrawn and centrifuged at 12,000 rpm for 12 minutes to obtain the supernatant containing the metabolites and caffeine. The caffeine reduction was checked at 275 nm[16] with CEM (without caffeine component) as blank, using UVVisible Spectrophotometer whereas N-Methyl Urea metabolite formation was studied at 265 nm[20] with the same blank. The experiment was repeated three times to obtain mean and standard deviation of each set of values.


Beverage Decaffeination

All the samples showed increase in the absorbance when the decaffeinated samples were taken as blank, thus showing decreased caffeine levels in the samples (Fig. 1).

Sensory Evaluation

Results of the sensory study showed that 46% and 49% of the candidates could identify chocolate and black coffee respectively (Fig. 2). Only 1% of the candidates identified coffee and none could identify soft drink or iced tea (Fig. 2). This may have caused since the sensory attributes were completely changed in decaffeinated samples. The Correspondence Analysis results showed key sensory attributes in each of the samples, given by Fig. 3. The darkened circle separates the plot into the attributes that showed at least 50% significance and those that showed more than 80% significance. In the symmetrical model, the most significant attributes were plotted away from the centre, outside the darkened circle (Table 2).
The samples containing chocolate drink (Fig. 3c) and black coffee (Fig. 3f) showed significant parameters that were present in the original samples (before decaffeination) and hence could be identified by the survey candidates. Whereas those of coffee, tea, iced tea and soft drinks did not. Presence of milk and other extraneous substances in the other samples with the crude enzyme used for decaffeination could have yielded in an undesirable reaction, thereby giving such sensory attributes and making it difficult for the candidates to identify samples. Thus decaffeination will be ideal at the stage of production of raw coffee and tea leaves, as it would avoid reaction with other food components.

Growth Curve Analysis

The data was evaluated using Microcal Origin (Version 5.0) using the mean and standard deviation values of each set of readings to obtain the following graph (Fig. 4) representing the range of values that could be formed. The figure shows gradual degradation of caffeine at the beginning of the log phase, with simultaneous formation of N-methyl urea, which is seen to behave as a primary metabolite. After 16 hours, it was observed that the formation of the metabolite and degradation of caffeine had decreased. This could be due to two reasons: One where the media would have scarce amounts of caffeine left, thus allowing no further degradation. Another explanation to this is that the microbes could have followed a different pathway of Krebs cycle[21] change in environmental conditions. Since the bacteria is known to exhibit an alternate glyoxylate pathway in caffeine metabolism[13] it could have followed this pathway due to starvation of the media and competition, analogous to that of Saccharomyces cerevisiae[22] and rats[23]. The latter could provide a breakthrough in diverting the metabolic pathway towards glyoxylate pathway and not towards alkaloid metabolites that may potentially be toxic and harmful to human consumption and the environment.[10] Methods of induction of the glyoxylate pathway by metabolic engineering or by continuous media starvation over generations can be a scope for future study.


Caffeine degradation on food beverages was found to change their sensory parameters due to undesirable reactions with intrinsic substances, thus making it difficult for the survey candidates to identify the samples. Hence decaffeination would result in lesser effects on sensory attributes, if done during raw material processing of the food. Also the products and by-products can possibly be changed by altering the metabolic pathway which would yield a less toxic and hence a much safer product, thus making it acceptable for consumption. Further analysis and research on the factors leading to the sensory effects and metabolic manipulation, on a molecular and metabolic level is required to ensure safety and acceptability of food decaffeination.


Conflict of interest declared none


We would like to express our sincere gratitude to Vellore Institute of Technology University for providing us laboratory and infrastructural facilities.
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