Journal of Herbal Medicine and Toxicology 4 (2) 167-175 (2010) ISSN : 0973-4643

Original Article

ENHANCEMENT OF ANTIMICROBIAL POTENTIAL OF PHYLLANTHUS NIRURI BY FERMENTATION

Vaishnavi Venugopalan, Dinesh M.S.* and Geetha K.S.
Department of Biotechnology, PES Institute of Technology, 100 feet Ring Road, BSK III Stage, Bangalore *Corresponding author - E-mail: dineshms@pes.edu Received - 5th April, 2010; Revised - 4th May, 2010; Accepted - 28th May, 2010

ABSTRACT: The use of plants products for medicinal value is common in Indian system of medicine. We can enhance the medicinal property of plants through bioprocesses. In the present study, bioprocess such as fermentation was used to enhance the antimicrobial potential of the crude herbal extract of Phyllanthus niruri. Fermentation was carried out using the standard/commercial isolates of Lactobacillus acidophilus and by the isolates of the same bacterium from the herb surface separately. The fermented product obtained from both these procedures were compared for their antimicrobial property against common human pathogens like Escherichia coli, Staphylococcus aureus, Salmonella typhi, Psuedomonas aeruginosa, Bacillus cereus, Bacillus subtilus and Klebsiella species. The variation of the antimicrobial property of the fermented extracts along the fermentation time period was also studied. The results indicated that the antimicrobial potential of the fermented herb is more than that of the crude herbal extract. The antimicrobial property of the fermented herb increases by about 80% -170% when compared to the crude herbal extract. Also, the fermented product obtained using Lactobacillus isolates from the herbal surface is more potent against tested human pathogens when compared to the product obtained using commercial L.acidophilus isolates. The potency improves by 49% if Lactobacillus species from the herbal surface are used for fermentation. The antimicrobial capabilities of the fermented product increases along the fermentation period by about 65%-95% irrespective of the source of lactic acid bacteria used. Another salient feature of the study is that E.coli is the most sensitive while Klebsiella species is the least sensitive to both the crude as well as the fermented extracts. Key Words: Phyllanthus niruri, Lactobacillus isolates, Fermentation, Antimicrobial activity

INTRODUCTION
Plants have always been a common source of medicaments, either in the form of traditional preparations or as pure active principles. Medicinal plants are considerably useful and economically essential. They contain active constituents that are used in the treatment of many human diseases [1]. Many plant extracts have been developed and proposed for use as antimicrobial substances. Plants used in traditional medicine contain a vast array of substances that can be used to treat chronic and infectious diseases [2]. Plants have an almost limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen-substituted derivatives. Most are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total [3]. In many cases, these substances serve as plant defense mechanisms against predation by microorganisms, insects, and herbivores. Some, such as terpenoids, give plants their odors; others (quinones and tannins) are responsible for plant pigment. Many compounds are also responsible for plant flavor while some of the herbs and spices used by humans to season food yield useful medicinal compounds [4]. Phyllanthus niruri is a

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herb belonging to Euphorbiaceae family. It is known for a wide variety of phytochemicals and pharmacological properties. It is an ingredient of almost 175 ayurvedic formulations. Fruits are commonly used in the treatment of hemorrhages, diarrheas, dysentery, jaundice, cough and anaemia. It is also used for the preparation of various health care and personal products like chavanprash, hair oil, dye, face cream, tooth powder etc [5]. The active phytochemicals, flavonoids, alkaloids, terpenoids, lignans, polyphenols, tannins, coumarins and saponins, have been identified from various parts of P. niruri. Extracts of this herb have been proven to have therapeutic effects in many clinical studies [6]. Fermentation of herbs produces beverages which are considered to be healthy by people who consume it. They contain high nutritional content and bioactive compounds formed from the raw material and the fermentation reactions [7]. Lactic acid fermentations is regarded the most popular used for fermenting of herbs. Lactic acid bacteria (LAB) produces organic acids, diacetyl, hydrogen peroxide and bacteriocins that improve the shelf life of the fermented product by controlling spoilage organisms and potential pathogens. LAB is also known to possess antimicrobial properties thereby improving human health [8, 9]. In the present study, a crude extract of P.niruri was tested against common human pathogens using a standard antibiotic as positive control and sterilized distilled water as a negative control. Further to this, the herb was separately fermented using Lactobacillus species isolated from two different sources standard or commercially available Lactobacillus acidophilus and the Lactobacillus sp. isolated from the herbal surface. The antimicrobial activities of the fermented products obtained from both these bacterial strains were then compared and evaluated. The activities along the fermentation period (25 days) were also studied comparatively. Preparation of the crude extract: 20g of clean and fresh herbs were crushed using a pestle and mortar and added to 100 ml sterile water to obtain an aqueous extract of the herb (200mg/ml). The extraction was done at room temperature (24 C). Muslin cloth was then used to filter the plant residues and the filtrate thus obtained was further purified by filtration through Whatman No 1 filter paper [10, 11].This crude extract was tested against common human pathogens for antimicrobial properties. Culturing of Lactobacillus isolates for fermentation of the herb: Standard or commercial Lactobacillus acidophilus isolates were obtained as pure cultures from the Department of Microbiology, G.K.V.K, Bangalore, and stored in semi solid medium at 4 C for further utilization in the study. Before use, there were thawed and maintained in Mann Rogosa Sharpes media [12]. In order to isolate Lactobacillus species from the herbal surfaces, clean and washed leaves of the herb were placed on Mann Rogosa Sharpes agar (15 ml) in petri plates under sterile procedures [13]. MRS agar is used for isolation of lactic acid bacteria species since the medium restricts growth of other species [12]. The plates were incubated at 37C for 2 days. On observing the petri plates, uniform colonies of bacteria were seen to be grown. These colonies were similar in morphology and other physical characteristics as that of standard Lactobacillus acidophilus colonies maintained. The colonies isolated from the herbal surfaces were identified and characterized using standard methods such as gram staining, test for catalase, gelatin hydrolyses activities and ability to assimilate a range of carbon sources [13, 14]. Hence, the standard and the herbal isolates of Lactobacillus species were separately used to ferment the herb extract of Phyllanthus niruri to know their efficacies. The characteristics of the two lactic acid bacterial isolates are outlined in table 1. The isolates used in the study are shown in figure 1. Lactic acid fermentation of the herb [15-17]: The following sequential steps were followed. (i) Preparation of P.niruri based fermentation media: The pest and disease free leaves of the herb were harvested after eliminating its surface microbial load using running water. 20% of the medicinal herb was considered for fermentation. 20g of leaves were

MATERIAL AND METHODS

Plant material: The fresh herbs were collected from the aromatic crop section, Division of Horticulture, UAS (University of Agriculture Sciences), GKVK, Bangalore, India, for the present study. Fresh herbs that are not infected by pests and disease damage were harvested. The parts of the herbs like leaves and fruits were detached and washed thoroughly with clean water.

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Table 1: Characteristics of the Lactic acid bacteria (LAB) isolates collected from two different sources
Characters tics studies Source of LAB Isolates Standard / Commercially available LAB Cell Shape and Arrangement Gram Reaction Catalase Reaction Gelatin Hydrolysis Growth on MRS broth after 24 hrs of incubation (OD at 600nm) Growth on MRS broth after 48 hrs of incubation (OD at 600nm) Assimilation of Glucose Assimilation of Sucrose Assimilation of Galactose Assimilation of Lactose Organism Rods Positive Negative Positive 1.25 1.71 Good growth Medium growth Medium growth Medium growth Lactobacillus acidophilus LAB isolated from herbal surface of Phyllanthus niruri Rods occurring in short chain Positive Negative Positive 1.23 1.72 Good growth Medium growth Medium growth Medium growth Lactobacillus Sp.

The above table shows the different characteristics studied for the two sources of Lactic acid bacteria (LAB) isolated in the present research work one commercially available and the other from the surfaces of the herb Phyllanthus niruri.

Table 2: Antimicrobial Activities of crude herbal extract of P.niruri and fermented products against common human pathogens
Bacterial Pathogens Zone of Inhibition (mm) for NC Zone of Inhibition (mm) for CEx Zone of Inhibition (nm) at different fermentation times Zone of Inhibition (mm) for PC

Day 5 A E. Coli S. aureus S. thphi P.aeruginosa B.cereus B.subtilus Klebsiella sp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.0 5.5 4.0 2.0 1.5 1.5 0.5 6.5 6.0 4.0 2.5 2.0 1.5 1.0 B 8.0 7.5 4.5 3.0 2.5 2.0 1.5

Day 10 A 8.5 7.5 5.0 3.5 2.0 2.0 1.5 B 9.5 8.5 5.5 4.0 3.5 2.5 2.0

Day 15 A 9.0 8.0 6.0 3.5 3.0 2.5 2.0 B 11.5 9.5 7.0 6.5 5.0 3.5 2.5

Day 20 A 10.5 9.0 6.5 4.0 3.5 3.0 2.5 B 14.0 12.0 9.0 8.5 6.0 4.5 3.5

Day 25 A 10.5 9.1 6.5 4.1 3.5 3.0 2.6 B 14.0 12.0 9.2 8.5 6.1 4.5 3.5 2.5 20.5 2.5 1.5 14.0 15.1 6.1

NC: Negative control (distilled water); CEx: Crude Extract of the herb Phyllanthus niruri; PC: Positive control (Ampicillin taken 10 g/disc); A: Fermented products obtained using Standard/commercially available LAB isolates ; B: Fermented products obtained using LAB isolated from the herbal surfaces of Phyllanthus niruri. The above table shows the antimicrobial activities of the controls and the samples against common human pathogens. Fermented samples are tested every 5 days for a period of 25 days of the fermentation period. The tests were performed in triplicates and the average value has been entered in the above table.

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crushed with pestle and mortar and added to 100ml of sterile distilled water. The extract was filtered using muslin cloth and Whatman No 1 filter paper to remove the plant debris present. 400 ml of this herbal extract was made and stored in conical flasks (100 ml each), to be used for preparing starter cultures and for lactic acid fermentations. The Lactobacillus species (standard isolates and herbal surface isolates) collected and maintained previously were used separately to ferment the herbal extract prepared. Before subjecting the herbal extract to fermentation, each of the Lactobacillus isolates were multiplied and made to adapt to herbal environment by preparing respective starter cultures. (ii) Preparation of starter culture of Lactobacilli strains: To prepare a starter culture, a test tube with 5ml of sterilized MRS broth was added with one loop inoculums of the lactic acid bacterial culture intended to be multiplied and kept overnight at 25 C for growth. The culture grown in the test tube was then added to 100ml of herbal extract in a conical flask. The flask was kept at 37 C in an incubator with shaking facility of 100rpm for 24 hrs. Dilution was done using MRS broth to obtain a size of 106 cfu/ml. Adjusted cultures was tested for right size by plate count method on MRS agar. 5ml (v/v) of the adjusted starter culture was used for further study. Respective starter cultures were used for respective fermentations keeping the number of lactic acid bacterial cells added to herbal extract constant. (iii) Setting up the fermentation process: 100 ml of the herbal extract in a conical flask was fermented using standard Lactobacillus acidophilus starter culture and 100 ml of herbal extract in another conical flask was fermented using starter culture of Lactobacillus isolates from the herbal surface. About 2g of dextrose was added to enhance the fermentation process. Fermentation process was carried out at room temperature using a shaker incubator (100rpm). The fermented products from each of these were compared for antimicrobial activity at every 5 days interval for a period of 25 days. A flow chart of the fermentation procedure is shown in fig 1. Collection and culturing of human pathogens: The microorganisms to be tested against the crude herbal extract and the fermented products of P.niruri were obtained from the Department of Microbiology, G.K.V.K., Bangalore, as pure cultures maintained in nutrient agar medium at 4C for timely use. The human pathogens collected were Escherichia coli, Staphylococcus aureus, Salmonella typhi, Psuedomonas aeruginosa, Bacillus cereus, Bacillus subtilis and Klebsiella species. Suspension cultures of the pathogens were made prior to testing the antimicrobial potential of the fermented products. Nutrient broth was used for the preparation of bacterial culture suspension [18]. Using a sterile loop, 3-5 colonies of the bacterial cells were transferred from the stock culture plate into a 10ml of nutrient broth taken in test. The test-tube was incubated at 37C for 24 hours. Using sterile nutrient broth, a population of 10 6 cfu/ml was obtained for each pathogen. Appropriate dilution was determined after spread plating the adjusted culture on Plate Count Agar (PCA). 100 l of 24 hr incubated test organism was used for further studies [19]. All the growth media used in the experiment was purchased from Himedia Laboratories Pvt. Ltd., Mumbai. Screening the crude herbal extract and fermented products for antimicrobial activity: To test the antimicrobial potential of fermented products, modified agar diffusion method (filter paper disc method) was employed [20, 21]. On solid nutrient agar medium (15 ml taken in petri plate), the pathogenic culture suspension was swabbed uniformly. A sterile filter paper impregnated with 0.1ml of the sample to be tested for antimicrobial activity was placed on the agar surface and kept for incubation at 37C for 24 hrs. After the stipulated time, presence of a clear zone of inhibition around the filter paper disc indicated the sensitivity of the pathogen to the sample tested [22, 23]. Sterile distilled water was taken as negative control while ampicillin (10g/ml) was taken as the positive control. The zone of inhibition was calculated by subtracting the diameter of the zone formed due to application of the sample on the filter paper disc from the diameter of filter paper disc used for testing sensitivity of the pathogens to the sample [21]. The experiment was done in triplicates and the zones of inhibition seen were reported in mm.

RESULTS AND DISCUSSION

(i) Isolation and collection of Lactobacillus species: The characteristics of Lactobacillus species collected from standard sources and the ones isolated from herbal surfaces were studied by growth on MRS agar. These include features like colony morphology, gram

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Venugopalan et al. staining, catalase activity, gelatin hydrolysis activity and capability to assimilate various sugars. The results of the same are outlined in table 1. From the table it can be seen that species isolated from the herbal surfaces bears same features as that of the commercially available L.acidophilus species. Hence it was reported that the species isolated from the herb on MRS agar is Lactobacillus species. Literature shows that studies of isolating lactic acid bacteria from different herbs gave similar results as the present study. J. Orvin Mundt and James L. Hammer isolated Lactobacillus casei, L.leichmanii, L.platarum from chickpeas and cowpeas in 1968 [24]. Cereals and millets contain lactic acid bacteria species which were successfully isolated on MRS media [25, 26]. (ii) Screening and evaluation of antimicrobial activity of crude extract P.niruri: The antimicrobial assay done by filter paper disc procedure showed that the crude herbal extract of Phyllanthus niruri exhibits antibacterial activity against all the tested pathogens. During the assay, sterile distilled water was used as negative control while ampicillin was used as positive control. The zone of inhibition in mm for the crude extract is shown in table 2. From the table it can be seen that the zones of inhibition formed by the crude extract and standard antibiotic (ampicillin) are comparable. Results showed that out of the 7 microorganisms tested, E.coli, S.typhi and P.aeruginosa were most sensitive to the crude extract when compared to the standard antibiotic. The herb Phyllanthus niruri is a rich source of phytochemicals, including many which have been found only in the Phyllanthus genus [27]. Many of the constituents are attributed to biologically active lignans, glycosides, flavonoids, alkaloids, ellagitannins, and phenylpropanoids found in the leaf, stem, and root of the plant. Common lipids, sterols, and flavonols also occur in the plant. The main plant chemicals in it include alkaloids, astragalin, brevifolin, carboxylic acids, corilagin, cymene, ellagic acid, ellagitannins, gallocatechins, geraniin, hypophyllanthin, lignans, lintetralins, lupeols, methyl salicylate, niranthin, nirtetralin, niruretin, nirurin, nirurine, niruriside, norsecurinines, phyllanthin, phyllanthine, phyllanthenol, phyllochrysine, phyltetralin, repandusinic acids, quercetin, quercetol, quercitrin, rutin, saponins, triacontanal, and tricontanol [28]. (iii) Enhancement of antimicrobial activity: During fermentation of P.niruri, the antimicrobial potency was seen to be enhanced when the herbal extract was fermented using lactic acid bacteria. This was evident through comparing the zones of inhibition (mm) formed against the tested pathogens by crude extract and the fermented products (Table 2). Fermentation was carried out separately by using standard L.acidophilus isolates and Lactobacillus species isolated from the herbal surfaces. Fermented products of both the fermentation procedures gave antimicrobial potential better than that of the crude unfermented extract. In comparison to the crude extract, fermentation of the herb increased the antimicrobial activity by 80% when commercial L.acidophilus were used and by 170% when herbal isolates of Lactobacillus species were used. Many phytochemicals important for antimicrobial activity in a herb are less freely available and mainly exist bound to the cell surface [30, 31]. Fermentation process causes release of microbial enzymes which in turn produce more freely available forms of plant chemicals like flavonoids, alkaloids and phenylpropanoids [32]. The increase in the levels of free (non-bound) plant chemicals may be responsible for the improvement in antimicrobial activities. Also, the fermented products formed by the Lactobacillus species from the herb surfaces gave better zones of inhibition than the products formed by standard L.acidophilus isolates. The antimicrobial property was enhanced by about 49% when herbal isolates of lactic acid bacteria was used for fermentation. This could be because the Lactobacillus species isolated from Phyllanthus niruri are native to the herb and hence more adapted to the herb than the commercially available L.acidophilus. Due to this, the potential to ferment the herbal extract is better by former than the latter thereby releasing more active fermentation products that are better potent against the human pathogens. The fermented products (irrespective of the source of lactic acid bacteria) have a potential to be commercialized since they showed better activities against E.coli, S.typhi and P.aeruginosa than the standard antibiotic that was used as positive control. A very important result through the present study is the increase in antimicrobial activity along the fermentation period. Assaying was done every 5 days to a period of 25 days, during which increase in zones of inhibition can be clearly seen from table 2. This is regardless of the source of lactic acid bacteria used.

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As the fermentation time progressed, antimicrobial activity was increased by about 65% when commercial L.acidophilus were used and by 95% when herbal isolates of Lactobacillus species were used. The activity, estimated in terms of the zone of inhibition, reached a peak on the 20 th day of fermentation and remained steady thereafter. Since all the available raw materials get converted to fermented products by the 20th day of fermentation process, the zones of inhibition formed by the samples from then on remains constant [31]. A graphical representation has been shown in figures 3, 4 and 5. Among the organisms tested, the sensitivity of the pathogens varies as follows: E.coli>S.aureus>S.typhi>P .aeruginosa>B.cereus>B.subtilis>Klebsiella species. The outer membrane of bacteria and its structure are responsible for differences of sensitivity that may occur between different bacteria [33, 34].
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[3].

[4]. [5].

[6]. [7]. [8].

[9].

CONCLUSION
The results of the present study show that fermentation of the herb Phyllanthus niruri evidently enhanced the antimicrobial properties of the herb. Also, using the Lactobacillus species growing on the herbal surface if used for the fermentation process gives better results for antimicrobial property. The fermented P.niruri obtained using isolates of Lactobacilli from the leaf surface have high potential of being developed as a neutraceutical to curb infectious diseases caused by the pathogens used in the study.

[10]. [11]. [12]. [13]. [14].

[15]. [16]. [17]. [18].

ACKNOWLEDGMENT
The authors thank the Principal, Dr. K.N. Balasubramanium, and the Management of PES Institute of Technology, Bangalore India, supporting the study through PESIT Internal project. We also thank Dr. V. Krishnamurthy, Head of the Department, Biotechnology, for providing useful suggestions to improve our work. Lastly we thank the Department of Microbiology and Horticulture, G.K.V.K., Bangalore for providing us the materials required for the study.

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