Evaluation of Antibacterial properties of the suspension of Ginger, Black Pepper, Vinegar, Honey and its application in Shelf life extension of Agaricus bisporus

Nitin Jain, Himanshu Karaiya*, Kumari Amrita, Swapnil Tiwari, Vishakha Dubey and Dr. C. Ramalingam
School of Bio Science and Technology, Vellore Institute of Technology University, Vellore-14
Corresponding Author: Himanshu Karaiya , Email: [email protected]
Date of Submission: 03-02-2013
Date of Acceptance: 06-03-2013
Conflict of Interest: NIL
Source of Support: NONE
Citation: Nitin Jain, Himanshu Karaiya*, Kumari Amrita, Swapnil Tiwari, Vishakha Dubey and Dr. C. Ramalingam “Evaluation of Antibacterial properties of the suspension of Ginger, Black Pepper, Vinegar, Honey and its application in Shelf life extension of Agaricus bisporus” Int. J. Drug Dev. & Res., April-June 2013, 5(2): 179-186. doi: doi number
Copyright: © 2013 IJDDR, Himanshu Karaiya 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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Suspensions of vinegar, honey, black pepper and ginger were examined for antibacterial activity against Salmonella sp., Pseudomonas sp. and E.coli by agar well diffusion technique. The prepared suspensions were applied on Agaricus bisporus to evaluate suspensions effect for increasing the shelf life of it. Vinegar suspension showed better antibacterial effect and good result on Agaricus bisporus to increase its shelf life as compared to honey suspension. Enzymatic spoilage of Agaricus bisporus were studied by applying vinegar suspension on both blanched and unblanched Agaricus bisporus at 4oC and 27oC. Treated blanched Agaricus bisporus which were stored at 4oC showed least microbial load as compared to other blanched and unblanched Agaricus bisporus kept at 4oC and 27oC. Based on survey analysis minimal change was observed in sensory quality, color and texture on Agaricus bisporus due to applied suspension.


Agaricus bisporus, vinegar, black pepper, ginger, honey, shelf life.


Herbs and spices have several roles in the human diet and their part is not replaceable. They have been used for thousands of years to enhance the flavor, color and aroma of food. In Ayurveda there are many spices that are described for improving health and taste of foods. In addition to boosting flavor, herbs and spices are also known for their preservative and medicinal value[1] which forms one of the oldest sciences. Now days many scientist are trying to prove their biological properties like antibacterial[2], anticancerous, anti-inflammatory, and anti-oxidative properties[3]. Vinegar which is a fermented product of starches or sugars can be produced from rice, wheat, malt, apple, onion[4] and various agriculture materials. Sugar or starch fermented first to alcohol and then the alcohol is oxidized by certain bacteria to acetic acid. It is now a relatively common practice to bottle vegetables and herbs and spices in either oil, vinegar or a mixture of both. All plant materials contain various pigments some of which change color as the pH of the plant tissue is changed by the addition of vinegar or other acids. The most common of these pigments are the anthocyanin which may be blue, colorless or red depending on the pH. Agaricus bisporus commonly known as button mushroom forms a large group of edible mushrooms. Mushrooms can be preserved by canning, freezing, drying, and pickling-using vinegar-either alone or in combination with herbs/spice extracts. Honey is the natural sweet substance produced by honey bees from nectar or blossoms from the secretion of living parts of plants or excretions of plants which honey bees collect transform and combine with specific substances of their own to ripen and mature. One of the most important features of honey is that it can be kept for a long period of time without becoming spoiled. The anti-microbial activity varies depending on the type of spice or herb. The need to which a spice material is used as a spice is dictated primarily by its essential oils or oleoresins. Black pepper is native to India and a major spice crop of Kerala. It has been proved that it have antimicrobial activity[5]. Piperine ([1-[5-[1,3-benzodioxol-5-yl]-1-oxo-2,4, pentadienylpiperidine), a pungent alkaloid present in black pepper, responsible for the therapeutically and biological activity. Ginger a common substance found increasingly in the diets of the global population, have known antibacterial effects and are commonly used together in teas. Many studies have implicated Staphylococcus aureus, and Streptococcus pyogeneas leading causative agents of both community and hospital acquire infections[6]. Ginger has been valued for its antibacterial properties for thousands of years in Asian cultures[7]. Ginger (Zingiber officinale) is one of those plants that were seriously investigated over the years. It has been used in centuries to fight infection. Ginger compounds are active against a form of diarrhoea which is leading cause of infant death in developing countries. The most well-known member of Zingiber (ginger) is Zingiber officinale[8]. In many parts of the world, Z. officinale has medicinal and culinary values. The volatile oil gingerol and other pungent principles not only give ginger its pungent aroma, but are the most medically powerful because they inhibit prostaglandin and leukotriene formation, which are products that influence blood flow and inflammation[8]. Honey produced by Appis mellifera is a sweet food made from the synthesis of nectar from flowers, plant saps and man waste products. Honey is a mixture of sugars mainly fructose and glucose having the highest percentage among other carbohydrates present. Antimicrobial agents with selective toxicity are especially useful as a chemotherapeutic agent in treating infectious diseases and may be a function of specific receptor requirement for drug attachment or it may depend on the inhibition of biochemical events essential to the pathogen but not to the host[9]. A member of Zingiberaceae family has shown significant antibacterial activity on pathogenic bacteria. The volatile oils are responsible for the aroma and taste of most spices. These volatile oils contain terpenes, sesquiterpenes, alcohols, esters, aldehydes, ketones, phenols etc.[10]. The application of extracts of spice could possibly control the microbial activities associated with food samples while retaining the nutritive and economic quality. The acid from the vinegar tends to neutralize the base earthiness and hence, have special applications in mushroom preservation. In our work, we have tried to investigate the antimicrobial and preservative properties of honey, ginger and vinegar with special reference to the shelf life extension of commonly edible button mushroom Agaricus bisporus.


Preparation of Extracts:

Ginger, Black Pepper and Honey

Sample of ginger bulb 500gm, black pepper 100gm, honey and vinegar were purchased from retail food store Vellore, Tamil Nadu, India. Ethanol and distilled water were used as solvent for extraction from ginger bulb and black pepper. The extract of ginger root was prepared by washing thoroughly under running tap water, aseptically cut into small pieces with a sterile knife and was blended making use of an electric blender[1]. The extracts were then filtered using whatman grade no.1 filter paper.
Black pepper extract was prepared by blending seeds using an electric blender. 10gm powder was boiled in 100ml distilled water in a water bath above 80o C for 15 minutes. The flasks were then plugged and removed from heat and allowed to cool[7]. After cooling the contents of flasks were filtered using the same method as that for ginger blend.
The filtrate of ginger and black pepper were used as aqueous stocks for antimicrobial activity and stored in refrigerator for further use. The filtrates were used for preparing ethanolic extract by adding equal amount of 70% ethanol, ginger and black pepper respectively in two different conical flask which were properly plugged with foil paper, the flask were then kept on regular shaker at 120rpm for 72 hrs and ambient temperature for proper mixing of extracts. After 3 days the extracts were filtered and holes were made in foil paper for removal of ethanol by Similarly, aqueous[9] and ethanolic extract of honey was prepared by adding equal ratio of distilled water and 70% of ethanol to honey respectively in two different flasks. For ethanolic extract, same procedure was followed as above.

Preparation of Suspensions:

Two different suspensions S1 and S2 were prepared from aqueous and ethanolic extracts by mixing equal ratio of ginger, black pepper and vinegar extracts (S1). Similarly, S2 was prepared with equal volumes of ginger, black pepper and honey extracts.

Antimicrobial Activity of Extracts and Suspensions Using Agar Well diffusion Assay:

Seeded culture of E.coli, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus and Bacillus subtilis were obtained from MTCC and used as test organisms. The bacterial cultures were maintained in Peptone Saline Water, after 1 h 100 μl of each test culture were spread on Nutrient Agar Media plates. In Nutrient Agar plates, 3 wells were made in each plate with a 6mm sterile cork borer. 100 μl extract of ginger, black pepper, honey and vinegar respectively were added in each well out 3 wells. Similarly, 100 μl of aqueous and ethanolic suspensions of ginger, black pepper, vinegar and ginger, black pepper, honey were added in each well. Plates were then incubated at 37o C for 24 h.

Treatment to increase mushroom shelf life

Fresh mushrooms were purchased from Vellore Vegetable store, Tamil Nadu, India. Before use mushrooms were thoroughly washed with running distilled water. Out of 20 mushrooms, 10 were steam blanched for 1-2 min and dipped into chilled water for 5 min[10]. After this step, both blanched and unblanched mushroom were cut into two equal halves under dry condition at room temperature (fig 1a). Each half, both blanched and unblanched was dipped in beakers containing 5 ml of aqueous and ethanolic suspensions of S1 and S2 respectively for 1-2 min, followed by air drying (fig.1b). After complete drying of mushrooms, each suspension dipped mushroom half and control were packed in separate plastic covers (fig.1c) and kept at 37oC and 4oC respectively for 7 days.

Organisms identification

Microbial spoilage of test mushrooms was monitored on 2nd, 4th and 6th day by mixing mushroom tissue samples with distilled water without surface sterilization. The diluted samples were further serially diluted and spread plated on newly prepared MacConkey Agar plates. These plates were incubated at 37oC for 24h. The grown bacterial colonies were counted to determine the number of colony forming units per gram (cfu/g) of mushroom. Extent of spoilage was determined by comparing number of colonies formed on test sample and control plates. Organisms responsible for mushroom spoilage were identified by biochemical test and microscopic observation.

Survey of Mushrooms Samples for Sensory Quality

Three mushroom samples were prepared, Sample 2 and sample 3 containing test mushrooms whereas Sample 1 was a control. Sample 2 was dipped into S1 suspensions and sample 3 dipped into S2 suspension for 1-2 minute followed by air drying for 2h. After this, sample 2 and 3 mushrooms were washed in running distilled water. All Sample1, 2 and 3 were boiled in hot water and completely air dried. Later all the samples were fried separately with 1 teaspoon each of butter and table salt which were purchased from food store Vellore, Tamil Nadu, India. Finally, Samples were surveyed on 30 people with proper questionnaires to check overall sensory quality of mushrooms.


Positive control and suspension S1 showed comparatively similar and maximum zone of inhibition against Salmonella sp., Pseudomonas sp. and E.coli as compared to suspension S2 and individual vinegar, honey, ginger and black pepper extracts (Table 1). In mushrooms mostly Pseudomonas species are responsible for spoilage. In this project the shelf life of sliced mushroom was extended by applying vinegar suspension (S1) over blanched mushroom that were stored at 4oC, it also control the microbial growth of other commonly food born pathogen such as Salmonella and E.coli.
This study emphasized that mixture of vinegar, honey, ginger and black pepper as suspension has more inhibitory action against pathogen as compare to individual effect of vinegar, honey, ginger and black pepper. This experiment also showed that inhibitory potency of vinegar S1 suspension was greater than honey S2 suspension.
Furthur, the cfu/g result at different temperature reveals that blanched mushroom samples with suspensions S1 and S2 at 4oC showed minimum spoilage by microbes as compare to unblanched mushroom samples which were stored at 27oC. Here also less number of colonies were seen in S1 suspension compare to S2 supension (fig 3 and 4). In mushroom enzymatic browning occurs when theenzyme, tyrosinase, makes contact with its substrate and initiates a series of reactions which produces brown melanin pigments. Microbial spoilage of mushrooms is usually due to the growth of pseudomonad bacteria. As these bacteria grow, they break down the mushroom fibres which softens the mushroom and leads to enzymatic browning[10]. Chilled storage (4oC) of blanched mushroom samples treated with suspension showed less growth of microbial colonies for one week and the browning was also less when compare to unblanched mushroom samples.
Survey result helped to understand the effect of suspension on the sensory quality of mushroom. Except color and odor there was less variation in votes for sensory parameters: taste, softness, sensation in mouth and overall sensory quality between control and test samples of mushroom. For both vinegar (S1) and honey (S2) suspensions the effects on sensory quality of mushroom samples were similar.


It was concluded that the suspension made from ginger, black pepper, vinegar (S1) and ginger, black pepper, honey (S2) have antimicrobial properties and able to suppress the food borne pathogens like E.coli, Salmonella and Pseudomonas which are major causative agent for mushrooms spoilage, hence both suspensions are effective in increasing the shelf life of mushrooms but suspension 1 is more affecting than suspension 2. Therefore, it can be used in industries for preservation and storing. The suspension didn’t affect the overall sensory quality of mushrooms. Thus these extracts and suspension can be used as an economic and eco-friendly tool to increase shelf life of mushrooms and prevent growth of microbes. Instead of the chemicals this would be a cheaper and easily available treatment that would reduce the processing cost in preservation technique.
Further studies of blanching and suspensions at varied optimize concentration in different temperature and conditions can be done to prevent browning and enzymatic degradation of mushrooms, so that to increase the shelf life more than 1-2 weeks and to enhance the sensory parameters; color and odor.


The authors wishes to express their gratitude to Dr. C. Ramalingam, Deputy Director, Research VIT University, who gave us the opportunity to do project under him and grateful for his offered invaluable assistance, support and guidance by which we have done it successfully. We are also thankful to Jemimah Naine for her valuable suggestion. We also would like to thank VIT University for providing us with a good environment and facilities to complete this project.

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