Bioconversion of Major Ginsenoside Rb1 of Panax ginseng by Bacterial Isolates from Barley and their Characterization

Transcription

1 Bioconversion of Major Ginsenoside Rb1 of Panax ginseng by Bacterial Isolates from Barley and their Characterization by VEENA.V (06PB23) A Thesis Submitted To Avinashilingam University for Women, Coimbatore In Partial fulfillment of the Requirement for the Degree Of MASTER OF SCIENCE IN BIOCHEMISTRY MAY 2008

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3 ACKNOWLEDGEMENT First and foremost I bow before God Almighty to express my heartfelt respect and thanks for all His kind blessings and strength He has showered on me in all my endeavors. I owe my humble gratitude and sincere thanks to Dr.T.K.Shanmughanandam B.A, B.L., Chancellor, Avinashilingam University for Women, Coimbatore, for providing me with all the facilities to carry out the project in a smooth manner. I record my heartfelt thanks to Dr. Saroja Prabhakaran, Vice Chancellor, Avinashilingam University for Women, Coimbatore, for extending all possible help and support towards the completion of the study. I express my sincere thanks to Dr. Gowri Ramakrishnan, Registrar, Avinashilingam University for Women, Coimbatore, for providing the opportunity to carry out this piece of work with all facilities and support. I consider it a great privilege to have had the opportunity to work under the guidance of Dr.R.Parvatham, Dean, Faculty of Sciences, Head of the Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore, and am ever indebted for her constant motivation, encouragement, moral support and freedom to carry out the project successfully and on time. I humbly thank Dr.K.Kalaiselvi, Lecturer, Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore, for her able guidance, zealous cooperation, sustained encouragement, constant care, moral support, valuable time and suggestions during the course of the study.

4 I extend my heartfelt thanks to all the Staff members of the Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore, for their help and cooperation throughout the study. I deem it a pleasure to express my thanks to all my dear friends especially Neha.G.Wasnik, VijayaDevi, T.Ponmozhi, Sarasvathi.C, Poornima.P, Geetha.A and Mahalakshmi.M for all their timely help and support that helped me to complete my study successfully on time. I dedicate my work to my beloved parents and brother, for their prayers, mental and emotional support, motivation, loving care and time provided which has been my source of strength for the successful completion of the study. I am also thankful to everyone who has in any manner contributed directly or indirectly to the successful completion of this project work.

5 1.0 INTRODUCTION Plants have been an important source of medicine for thousands of years. Even today, the World Health Organization estimates that up to 80 per cent of people rely mainly on traditional remedies such as herbs for their medicines. The definition of Medicinal Plant has been formulated by WHO as "Any plant which, in one or more of its organ, contains substance that can be used for therapeutic purpose or which is a precursor for synthesis of useful drugs (Sofowora, 1982). Plants have been utilized for the discovery of new products of medicinal value for drug development. Several such distinct chemicals derived from plants are important drugs currently used in one or more countries in the world. Plants are now a source for many modern medicines. It is estimated that approximately one quarter of prescribed drugs contain plant extracts or active ingredients obtained from or modeled on plant substances. The most popular analgesic- aspirin was originally derived from species of Salix and Spiraea. Some of the most valuable anti-cancer agents such as paclitaxel and vinblastine are derived solely from plant sources (Tripathi and Tripathi., 2003). Plant secondary products have historically been defined as chemicals that do not appear to have a vital biochemical role in the process of building and maintaining plant cells, however, recent research has shown a pivotal role of these chemicals in the ecophysiology of plants (Wink and Schimmer, 1999). The evolving commercial importance of secondary metabolites has in recent years resulted in a great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites (Vanisree et al., 2004). The secondary products can have a variety of functions in plants and it is likely that their ecological function may have some bearing on potential medicinal effects for humans. For example, secondary products involved in plant defense through cytotoxicity toward microbial pathogens could prove useful as antimicrobial medicines in humans, if not too toxic (Briskin., 2000).They could be used in contemporary medicine or act as templates for the synthesis or semi-synthesis of potentially useful therapeutic compounds. Examples include Catharanthus roseus G.Don., the Madagascar periwinkle, Rauvolfia serpentina Benth., the Himalayan snakeroot, and Taxus spp., Among such plants is ginseng, esteemed by the Chinese for more than 5000 years. Reinvestigation of this forgotten medicinal plant

6 has proven its use as an alleviating agent or cure for the ills of modern stressful lifestyles (Court., 2000). Panax ginseng C. A. Meyer is one of the most commonly used plant in traditional medicines of China, Korea, Japan, and other Asian countries for the treatment of various diseases. The roots of the plant contain Ginseng saponins known as ginsenosides. These saponin secondary metabolites- ginsenosides have been regarded as the principal components responsible for the pharmacological activities of ginseng, and a large number of ginsenoside (G) derivatives have been identified in Panax ginseng and other Panax spp (Kim et al., 2005) With the development of new methods for ginsenosides isolation and better ginseng processing technology, a variety of minor ginsenosides has been discovered. The minor ginsenosides are found to have profound therapeutic properties and occur in minute amounts in natural habitat (Cheng et al., 2006). It is however, difficult to separate minor saponins from ginseng as they are rare or non-existent in most samples. The amounts of minor ginsenosides also vary according to growth conditions and from plant to plant. The contents of ginsenosides in Panax ginseng not only vary in different parts of the root, but also exhibit yearly variation (Wang et al., 2005). Conventional chemical methods, such as chemical synthesis, mild acid hydrolysis, or alkaline cleavage have been employed for the preparation of minor saponins and saponin metabolites. Side reactions such as epimerization, hydration and hydroxylation were inevitably produced and these methods could not be employed for the commercial preparation of the minor ginsenosides (Ko et al., 2003). A reliable and safe source for the production of minor ginsenosides is an enzymatic conversion using enzymes from micro organisms (Chen et al., 2007). In this respect, earlier attempts to convert major ginsenosides to minor ones using microbial enzyme sources isolated from animal or soil samples have been made (Bae et al., 2000; Kim et al., 2005). There are no reports on the use of bacteria isolated from food sources for their use in bioconversion of ginsenosides. Chi and Ji (2005) used commercially available food micro organisms like Lactobacillus, Bifidobacterium and Leuconostoc bacterial species and screened for the bioconversion of ginsenosides Rb1 and Re. The present study therefore

7 focuses on the isolation of bacteria from fermented food and the main objectives of the present study are: To isolate β-glucosidase producing bacterial cultures from fermented food sources To analyze the biochemical characteristics of the isolated bacteria To optimize the ph and growth media for the isolated bacterial strains To check for their conversion activity to minor ginsenosides when incubated with ginsenoside Rb1 by Thin Layer Chromatography (TLC) analysis To confirm the results obtained in TLC by HPLC analysis

8 2.0 REVIEW OF LITERATURE Herbal medicinal products are increasingly gaining popularity all over the world. With annual sales of more than $300 million accounting for a 15 to 20% market share in the United States ginseng is one of the most commonly used herbal medicinal remedies by American consumers (Tawab et al., 2003). Many plants have been investigated during the 20th century in order to assess their potential value as new medicinal agents or as sources of new organic molecules that could be used in contemporary medicine or could act as templates for the synthesis or semisynthesis of potentially useful therapeutic compounds. One among such plants is ginseng, the collective name for a group of plants esteemed by the Chinese for more than 5000 years (Court., 2000). The present study is focused on identifying a source for the production of minor ginsenosides which are proved to be potential sources of rejuvenation principles. The work done so far by various research groups have been reviewed here in this chapter under the following sub-heads: 2.1 Characteristics of ginseng plant 2.2 Importance of Ginseng Types of ginseng roots Ginsenoside variation based on extraction methods 2.3 Constituents of ginseng roots 2.4 Ginseng saponins- Ginsenosides Classification of ginsenosides Detection of ginsenosides 2.5 Fate of orally administered ginsenosides 2.6 Pharmaceutical activities Major Ginsenosides Minor ginsenosides 2.7 Synthesis of minor ginsenosides 2.8 Bioconversion of ginsenosides 2.9 Microbial sources of β- glucosidase

9 2.1 CHARACTERISTICS OF GINSENG PLANT True ginseng, Panax ginseng C.A.Meyer, is a small, inconspicuous, shade loving, perennial shrub attaining a height of about 60 cm and belonging to the ivy family Araliaceae. The generic name Panax was derived from Greek meaning all-heal or allcure and reflected the popular, traditional use of the plant as a panacea. The specific name ginseng or schinseng is a transliteration of the Chinese names Jin-chen, Jen-schen, Ren-shen, Schin-sen or Schan-shen (wild mountain ginseng). Cultivated or garden ginseng is known locally as Yuan-shen. According to the old Doctrine of Signatures, a theory apparently derived independently in many parts of the world, a plant would by its color, shape and characteristics indicate its potential medicinal uses (Court., 2000). Thus ginseng with its man-like appearance was quickly accepted as a tonic. The species of the Panax genus demonstrate a typical bicentric distribution. In North America ginseng plants can occur in a range from W longitude and 34 to 47 N latitude (Court., 2000), an area embracing the southern part of the Canadian provinces to the north and in the United States of America. In eastern Asia the range extends from 85 to 140 E longitude to 22 to 48 N latitude, an area including China with north east India, Nepal and Bhutan to the west, Burma, Laos and Vietnam to the south and Manchuria, Korea and Japan to the west (Court., 2000).

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11 Panax species can be grouped according to their rhizome and root characteristics although rhizome characteristics do vary according to the altitude at which the plants are growing. Thickened nodes and thinner internodes are observed in plants at higher elevations and at lower levels the nodes are less pronounced and the internodes thicker. Typical roots are persistent, thick, fleshy, fusiform and cream to pale yellowish buff in colour; the primary root is frequently irregularly branched. Older roots are wrinkled due to the annual contractile activity that maintains the position of the dormant bud at soil level. Root shape varies markedly according to the soil environment. Heavy stony soils cause short, thickened primary roots with many thickened secondary roots. Light, sandy soils produce longer, straight, carrot-like tap roots with less secondary branching. Fresh roots possess a strong taste that is bitter yet also sweet and a prominent, characteristic aroma that is gradually lost on storage (Court., 2000). The rhizome or underground stem, which is normally unbranched, usually bears adventitious roots which may become thick and fleshy. In wild P. ginseng the rhizome may be elongated but cultivated plants yield short, thick, compact, erect rhizomes (Baranov., 1966). Rhizomes bear leaf scars, the number of which gives an indication of the age of the plant. The small flowers of ginseng expand in June-July and are about 2 3 mm across (Court., 2000). 2.2 IMPORTANCE OF GINSENG: Ginseng, often refereed to as the King of Herbs was discovered over 5000 years ago in the mountains of Manchuria, China (Harding AR, 1936). Although there are many so called ginsengs on the market, the most commonly used ginseng species are Panax ginseng C.A.Meyer (Asian or Korean ginseng), Panax quinquefolius L (American ginseng), Panax notoginseng (Burki) F.H. Chen (Tianqi or Sanqi, Yunnan Province, China) and Panax japonicus C.A. Meyer (Chikusetsu, Japanese ginseng) (Court., 2000). Korean ginseng (Panax ginseng C.A.Meyer) is a typical medicinal plant, which has been widely used as a traditional medicine since ancient times, owing to its simulative and tonic properties. In Chinese, the word ginseng is directly translated as the essence of man. During the Han dynasty (from 206 B.C to 24 A.D.), a medical essay was written about a plant called shemg. Later, the plant became known as jen sheg, the root of heaven or

12 ginseng which literally means man-herb, because its root is shaped like a human body. Ginseng roots are commercially graded based primarily on their size, shape and overall appearance. Roots with an offwhite to beige color and smooth surface without blemishes are highly valued (Rahman and Punja., 2005). Ginseng products in the form of a variety of commercial health products including ginseng capsules, tablets, powder, syrup, drinks and cosmetics, etc. have gained popularity throughout the world, because of their strength-giving and rejuvenating power (Lou et al., 2005). Herbal medicines have extra ordinarily complex chemical compositions which may vary considerably depending on factors such as place of origin, growing conditions and the species used (Soldati and Tanaka., 1984; Shibata et al., 1985; Li et al., 1996). Ginseng, one of the most popular herbal medicines worldwide, is especially complex because a number of products from different genera and species ma be referred to as ginseng and therefore may be different chemical compositions. Traditionally, however ginsengs are dried roots of plants belonging to the genus Panax. Korean and Chinese ginsengs are prepared from the roots of Panax ginseng C.A.Meyer (Ji et al., 2001) TYPES OF GINSENG ROOTS: The roots of Panax ginseng are usually classified as white and red ginseng depending upon their usage method. The root is steamed and dried to prepare red ginseng, while the peeled roots dried without steaming are designated as white ginseng. It was reported that all of the saponins found in white ginseng were isolated in similar yields from red ginseng, while some partly deglycosylated saponins such as ginsenosides Rh1, Rh2 and Rg3 are obtained from red ginseng as artifacts produced during steaming (Kitagawa et al., 1983).

13 Figure 2.2. Root of Panax ginseng The binding of the sugar has been shown to have influence on biological activity. The biological activity of ginsenosides is found to be related to their structures (Wan et al., 2007) GINSENOSIDE VARIATION BASED ON EXTRACTION METHODS: The content of ginsenosides varies in ginseng roots and root extracts depending upon the method of extraction, subsequent treatment and the season of collection (Kim et al., 1987). The major components of fresh and dried ginsengs are malonyl ginsenosides Rb1, Rb2, Rc and Rd and ginsenosides Rb1, Rb2, Rc, Re, Rf, Rg and Rg2. However, red ginseng also contains ginsenosides Rg3, Rg5, Rh1 and Rh2 (Kitagawa et al., 1983).

14 Table 2.1 Variation in ginsenoside contents of red and white ginseng Designation Prosapogenin Recorded yield per cent White ginseng Red ginseng Ginsenoside Ra1 Protopanaxadiol Ra Ra Rb Malonyl Rb Trace ginsenoside Rb Malonyl Rb Trace ginsenoside Rb Rc Malonyl Rc 0.30 Trace ginsenoside Rd Malonyl Rd 0.12 Trace ginsenoside Re Protopanaxatriol Rf gluco Rf Trace Ginsenoside Rg Rg (R) -.. Rg Ginsenoside Rg3 Protopanaxadiol (S) -.. Rg Ginsenoside Rh1 Protopanaxatriol (R) -.. Rh Ginsenoside Rh2 Protopanaxadiol R0 Oleananae Rs1 Protopanaxadiol Rs Quinquenoside R Notoginsenoside R CONSTITUENTS OF GINSENG ROOTS Ginseng roots contain various components-ginsenosides, polyacetylenes, polyphenolic compounds and acidic polysaccharides (Kim et al., 2005). The most important non-saponin component of ginseng is the biophenols. In addition, ginseng contains many other valuable ingredients such as essential oil containing polyacetylenes and sesquiterpenes, polysaccharides, peptidoglycans, nitrogen containing compounds and

15 various ubiquitous compounds such as fatty aids, alcohols, vitamins, etc., which play an important role in improving the beauty of skin (Sivakumar et al., 2005). Table 2.2 Percentage saponin distribution in the main root of Panax ginseng MAIN ROOT 1 ST YEAR 2 ND YEAR 3 RD YEAR 4 TH YEAR 5 TH YEAR 6 TH YEAR Total saponins Diol saponins Triol saponins Ginsenoside Ro Free sugars composed of rhamnose, fructose, glucose, sucrose, maltose and an unidentified material. The principal sugar in fresh white ginseng root is sucrose, forming per cent in 2 year old roots but decreasing in older roots. In red ginseng root the main sugars are sucrose and rhamnose (Court., 2000). 2.4 GINSENG SAPONINS- GINSENOSIDES: The main bioactive constituents of ginseng drugs are considered to be triterpene saponins generally referred to as Ginsenosides also as panaxosides (Zhu et al., 2004). Among the components, ginsenosides are most pharmaceutically active (Kim et al., 2005). Saponin is a physiologically active substance found in many kinds of plants and has been used as the main active ingredient in oriental traditional drugs. The activity of saponin is closely related to the saponin sugar moiety. Sometimes, the structure of the natural saponin is not the best in physiological activity; when the saponin sugar moiety is changed the activity of the produced new saponin become higher.

16 Table 2.3 Percentage yields of ginsenosides in various parts of Panax ginseng CLASSIFICATION OF GINSENOSIDES: For many years, research on ginsenosides has been focused on their structures and biological activities. Upto now nearly 40 ginsenosides have been isolated and identified from ginseng (Sanada et al., 1974; Kim et al., 1996; Baek et al., 1996: Ryu et al., 1996, Baek et al 1997). Ginsenosides are classified into the following categories: Protopanxadiol (PPD), Protopanaxatriol (PPT) and Oleanolic acid, according to their chemical constituents (Luo et al., 2003: Cheng et al., 2006). Some literature state that ginsenosides are classified into four categories depending on their aglycone moiety namely PPD, PPT, ocotillol type and oleanolic acid (Fuzzati., 2004). Each type of ginsenosides has also at least three side chains at the C-3, C-6 or C-20 position. These side chains are free or coupled to sugar containing monomers, dimers or trimers. These sugar components might provide specificity for the cellular effects of each ginsenoside (Nah et al., 1995; Nah et al., 1997; Choi et al., 2001b; Rhim et al., 2002). However, ginsenosides are hydrophobic compounds are not water soluble (Nah et al., 2007). The biosynthetic pathway of the ginsenosides in plant cells is given below:

17 Figure 2.3 Biosynthetic pathways of ginsenosides in plant cells

18 Figure 2.4 Chemical structures of the major types of ginsenosides Numbers indicate the carbon in the glucose ring that links the two carbohydrates. Abbreviations for carbohydrates are as follows: Glc = glucopyranoside: Ara(fur) = arabinofuranose; Ara(pyr) = arabinopyranoside and Rha = rhamnopyranoside. PD and PT stand for protopanaxadiol and protopanaxatriol ginsenosides respectively. The 20(S)- Protopanaxadiol ginsenosides are ginsenoside Rb1, Rb2, Rb3, Rc, Rd and Rg3, the protopanaxatriol ginsenosides are ginsenoside Re. Rg1. Rg2 and Rh1 (Kim et al., 2007). The ginsenosides are named as Rx according to their mobility on TLC plates, with polarity decreasing from index a to h. The ginsenosides possess a dammarane triterpenoidal skeleton with a modified side chain at C-20 (Huang, 1999). They differ from one another by the type of sugar moieties, their number and their site of attachment (Park et al., 2005). The total amount of protopanaxadiol and protopanaxatriol ginsenosides as aglycones was found in human urine (Cui et al., 1997). The results showed that about 1.2% of the orally ingested dose of protopanaxatriol ginsenosides (3mg) and considerably

19 smaller amounts of the protopanaxadiol ginsenosides not exceeding 0.2% of the administered dose (7mg) were recovered. However, neither the individual ginsenosides nor their metabolites could be identified. The pharmacological properties of P.ginseng are generally attributed to its triterpene glycosides, ginsenosides with (20S)-protopanaxadiol and (20S)-protopanaxatriol aglycone moieties (Fuzzati et al., 1999). The malonyl derivatives Rh1. Rb2, Re and Rd and ginsenoside Ro of oleanolic acid-type are also called acidic ginsenosides while the other are usually named neutral ginsenosides (Fuzzati, 2004). Ginsenoside Rbl is the predominant ginsenoside in Panax ginseng, P. quinquefolium, P. japonicum and P. notoginseng (Cheng et al., 2006). Compound K, the main intestinal bacterial metabolite of protopanaxadiol ginsenosides was identified in human serum by a specific enzyme immunoassay, 8 hours after the oral administration of ginseng (Shibata., 2001). Unfortunately, since these active metabolites do not exist naturally, it is desirable to develop a method to prepare them (Tawab et al., 2003). These active metabolites cannot be prepared by chemical methods since both glycone moieties at C-20 are cleaved non-specifically under these conditions. Therefore, enzyme degradation, which selectively removes the glucose moieties sequentially is an effective method to prepare these active metabolites (Hu et al., 2007) DETECTION OF GINSENOSIDES: Many methods for quantifying ginsenosides in various types of ginseng samples have been developed, including thin layer chromatography. HPLC coupled with an ultraviolet (UV) detector, evaporative light scattering detector (ELSD), and mass spectrometry (MS) (Kim et al., 2007). HPLC has been used extensively to determine the ginsenosides in P. ginseng. The methods reported in the literatures used mainly C l8 columns. Water and acetonitrile mixtures were used as mobile phase in gradient elution mode and different detection techniques were applied (Chen et al., 2000; Court et al., 1996; Fuzzati., 2004; Lau et al., 2003; Kwon et al., 2001). Reversed phase separation of these constituents in Panax ginseng have been reported (Shi et al., 2006).

20 TLC is a very common technique for the finger print analysis of plant material and extract due to its easiness of use, low cost and versatility. Asian and American ginseng can be discriminated for their ginsenosides composition by two-dimensional TLC. Among all the classical techniques usually employed for phytochemical analyses high-performance liquid chromatography (HPLC) has been the method of choice for the analysis of ginsenosides in the last 20 years. HPLC, because of its speed, sensitivity and adaptability to non-volatile, polar compounds, is ideal for the analysis of saponins and sapogenins. Another advantage is versatility due to the possibility of using different detection techniques such us ultraviolet (UV), evaporative light scattering (ELSD), fluorescence and mass spectrometry (MS). Among the different techniques of detection of ginsenosides UV is the most employed since it is by far the most common detector found in phytochemical laboratories. Because of the weak UV absorption of ginsenosides, their detection is usually achieved at nm. The great majority of the literature methods use Cl8 columns with water or phosphate buffers and acetonitrile mixtures as solvent system either in isocratic or in gradient elution mode (Fuzzati., 2004). 2.5 FATE OF ORALLY ADMINISTERED GINSENOSIDES: If ginsengs are orally administered to humans, their constituents cannot be easily absorbed by the intestines due to their hydrophilicity (Akao et al., 1998: Hasegawa et al., 1997; Bae et al., 2002). In the intestinal tract they come in contact with and are metabolized by the intestinal micro flora. For example, Protopanaxadiol ginsenosides Rb1, Rb2 and Rc of fresh and white ginsengs are transformed to 20-O-β-D-glucopyranosyl- 20(S)- PD i.e., Compound K by human intestinal bacteria (Akao et al., 1998; Bae et al., 2000; Bae et al., 2002). The metabolites are then easily absorbed from the gastro intestinal tract, since most of the metabolites are non-polar compared to the parent components. For instance, when ginsenoside Rb1 is orally administered to rats, its metabolite, compound K and not ginsenoside Rb1 is absorbed into the circulation (Akao et al., 1998). When the metabolic pathway of conversion of ginsenosides by the intestinal micro flora was speculated it was found that the main metabolic pathway of ginsenoside Rb1

21 could be different according to the composition of human intestinal bacteria. The human intestinal bacteria should produce many kinds of glucosidases to produce the required ginsenoside (Bae et al., 2000). 2.6 PHARMACEUTICAL ACTIVITIES OF GINSENOSIDES: MAJOR GINSENOSIDES: The mail physical ailments for which ginseng is said to be effective include headaches, nausea, fatigue, dizziness, asthma, hemorrhage and impotence. It is said to generally strengthen the viscera, improve resistance to external disease causing agents and improve the general physical conditions and mental capacity (Park et al., 2005). Ginsenoside Rb1 is one of the major protopanaxadiol saponins from Panax species with various pharmacological activities. It is only slightly decomposed in rat stomach but is mainly degraded in rat large intestine. The metabolism begins with the cleavage of the terminal sugar moiety at the C-3 or C-20 hydroxyl group, and is followed by stepwise cleavage of the other sugars (Dong et al., 2003). Ginsenoside Rb1 and Rg1 possessed relaxation effects of on pulmonary vessels and discovered that it was eliminated by nitro-l-arginine, an inhibitor of NO synthase (Zhou et al., 2004). Ginsenoside Rb1 antagonizes lipid peroxidation and scavenges oxygen free radicals; these activities could reduce the potential for cerebral vascular accidents (Petkov et al., 1993) MINOR GINSENOSIDES: In the recent decades many studies have focused on the pharmaceutical activities of the minor ginsenosides such as ginsenoside Rd, Rg3, Rh2 and Compound-K as their activities are found to be superior to those of the major ginsenosides. These minor ginsenosides are present in only small percentages and known to be produced by hydrolysis of the sugar moieties of the major ginsenosides (Kim et al., 2005) The minor ginsenoside, Rd, can be produced by hydrolyzing and removing a sugar moiety from the major ginsenosides, Rb1, Rb2 and Rc. The minor ginsenoside, Rd, has been known to enhance the differentiation of neural stem cells, while other ginsenosides

22 induce no differentiation of neurons (Shi et al., 2005) and are known to protect neural systems against neurotoxicity by attenuating NO overproduction (Choi et al., 2003). Ginsenoside Rd has been known to decrease levels of urea nitrogen and creatinine in the kidney (Yokozawa et al., 1998), protect the kidney from apoptosis and DNA fragmentation caused by chemical drugs and cancer drugs (Yokozawa and Owada, 1999; Yokozawa and Liu, 2000; Yokozawa and Dong, 2001). Ginsenoside-Rd has been known to arrest the aging process of the suppressing antioxidative defence system and lipid peroxidation by elevating the GSH/GSSG ratio of glutathione and increasing the activities of glutathione peroxidase and glutathione reductase, which are both significantly lower in old organisms. Ginsenoside Rd is also known to help learning and memory functions in mice and prevent contraction of blood vessel; therefore, preventing blocking of blood circulation (Zeng et al., 2003). Rg3 and Rh2 which are rare ginsenosides belonging to 20(S)-protopanaxadiol type ginsenosides in ginseng have been found to have remarkable effects on cancer. Among the various plant glycosides tested, ginsenoside Rg3 was found to be a potent inhibitor of invasion by rat ascites hepatoma cells, B16FE7 melanoma cells, human small cell lung carcinoma (OC10), and human pancreatic adenocarcinoma (PSN-1) cells. Structurally analogous ginsenosides, Rb2, 20(R)-ginsenoside Rg2 and 20(S)-ginsenoside Rg3, showed little inhibitory activity. Neither Rh1, 20(R) - ginsenosides Rh1, Rb1, Rc nor Re had any effect (Shinkai et al., 1996). The underlying mechanism of ginsenoside induced protection against glutamate- or NMDA-induced excitotoxicity might be due to attenuation of the intracellular Ca2+ elevations induced by these excitatory amino acids in hippocampal neurons. Among various ginsenosides, Rg3 was the most potent inhibitor of NMDA-induced intracellular Ca2+ elevation in hippocampal neurons (Kim et al., 2002). Oral or intravenous administration of Rg3 exhibited significant neuroprotective effects against focal cerebral ischemic injury in rats. However, it is not yet known whether Rg3 exhibits a neuroprotective effect on HC-induced excitotoxicity in vitro and/or in vivo (Kim et al., 2007). Recently, ginsenoside compound K has received increasing attention, because in vivo or in vitro various biological actions, such as antigenotoxic activity, antiallergic effect

23 and the prevention of tumor invasion and metastasis, have shown to be mediated by this metabolite (Hasegawa and Uchiyama 1996; Lee et al., 2005b; Wakabayashi et al., 1998; Bae et al., 2002). Ginsenoside compound K shows potential hepatoprotective and antiinflammatory activities and the pharmacological effects of ginsenoside compound K appear to be stronger than those of known pharmaceutical drugs including diphenyl dimethyl bicarboxylate and indomethacin (Lee et al. 2005; Park et al. 2005). 2.7 SYNTHESIS OF MINOR GINSENOSIDES: With the development of new methods for ginsenoside isolation and better ginseng processing technology, a variety of minor active ginsenosides has also been discovered.however, it is quite difficult to separate such minor saponins from ginseng as they are rare or non-existent in most ginseng samples. Therefore, further research into the modification of ginsenosides has been conducted to convert major ginsenosides to minor saponins which may have more profound physiological properties. Many studies have aimed to convert major ginsenosides to the more active minor ginsenosides with methods such as heating, acid treatment and enzymatic conversion has been developed. Heating and acid treatment also degrade other minor ginsenosides and acidic polysaccharides by randomly hydrolyzing glycosidic bonds, which can remove the other pharmaceutical activities of ginseng (Kim et al., 2005). These chemical methods also produce side reactions, such as epimerization, hydration and hydroxylation (Chi and Ji., 2005). However, the enzymatic conversion for the appropriate sugar hydrolysis of a specific position is desirable for the production of the active minor ginsenosides. Many studies for the production of the active minor ginsenosides Rg3. Rh2 and ginsenoside K have been performed using microbial enzymes (Kim et al., 2005). 2.8 BIOCONVERSION OF GINSENOSIDES: Ginsenoside Rb1 is especially the most abundant (23%) of all the ginsenosides (Kim et al., 1987) and its structure can be easily converted to ginsenoside Rd by hydrolysis of one glucose moiety (Kim et al., 2005) and to other minor ginsenosides by subsequent hydrolysis of other glucose residues. Ginsenoside Rb1 is one of the major protopanaxadiol

24 saponins from Panax species with various pharmacological activities such as protection from free radical damage and maintaining normal cholesterol and blood pressure (Wang and Zhang., 2003) and rescuing hippocampal neurons from lethal ischemic damage (Lim et al., 1997). Figure 2.5 Bioconversion of some of the important ginsenosides Glc(1-6)glc O OH 2 0 G O OH 20 O H H 2 0 enzyme enzyme 3 Glc(1-2)glc O Rb1 3 Glc(1-2)gl c O Rd Glc(1-2)glc O 20(S)-Rg3 enzyme Glc Gl co OH 20 enzym Glc F 2 HO 3 C-K Ginsenoside Rb1 was found to decompose easily to four deglycosylated metabolites, Ginsenoside Rd, Rg3, F2, Rh2 or Compound-K and protopanxadiol when it was incubated with human and rat intestinal bacteria at 37 C. The metabolism and pharmacokinetics of ginsenoside Rb1 has been studied on rat and six deglycosylated metabolites ginsenoside Rd, gypenoside XVII, ginsenoside Rg3, F2, C-K and ginsenoside Rh2 have been reported to be the major metabolites of Rb1 (Chen et al., 2007). 2.9 MICROBIAL SOURCES OF β-glucosidase Microorganisms and their enzymes are increasingly being used as a useful tool in the structure modification and metabolism study of natural and synthetic organic compounds. Utilization of microbes as models for mammalian metabolism of xenobiotics was postulated in the early 1970s (Longhlin., 2000).

25 The use of microbial systems as models for drug metabolism has the advantages of saving the cost of large amount of animals, identifying trace metabolites, avoidance of ethical concerns and obtaining enough metabolites to investigate their activities. Therefore, it has been commonly used as in vitro model for obtaining enough amounts of in vivo metabolites of drug (Ma et al., 2006, 2007). The minor ginsenosides like ginsenoside compound K have been prepared via fermentation of protopanaxadiol-type ginsenosides in the presence of human intestinal bacteria under anaerobic conditions with saponins as their sole carbon source (Hasegawa et al. 1996, 1997; Bae et al. 2003). Additional studies have also attempted to produce ginsenoside compound K via transformation of ginsenosides using commercial enzyme preparations. Unfortunately, both of the above methods suffer from the facts that they are small scale, difficult, expensive and are low yielding. Many enzymes hydrolyzing the above glycosidic bonds are known, with β- glucosidase regarded as being the most useful (Bae et al., 2000; Park et al., 2001). There have been previous reports on microbial sources able to convert the major ginsenoside Rb1 to ginsenoside Rd. β-glucosidases from the human intestinal bacteria, Bifidobacterium sp., Eubacterium sp. and Fusobacterium sp., hydrolyzed ginsenoside Rb1 to ginsenoside Rd (Bae et al., 2000; Park et al., 2001). Fungal conversion, using fungi Rhizopus stolonifer and Curvularia lunata, has been also reported (Dong et al., 2003). Kim et al., 2005 reported the conversion of Ginsenoside Rb1 to Rd by bacteria isolated from soil of a ginseng field. Aerobic bacteria grow faster and produce enzymes in greater quantities than human intestinal bacteria (Coskun and Öndül, 2004; Yoon et al., 2004) and fungi, and therefore can be more effectively used for large scale enzyme preparation. Furthermore, the cultivation of human intestinal bacteria requires anaerobic space and a medium with a high concentration of nutrients, and their cultivation is not as simple as that of aerobic bacteria (Kim et al., 2005). The present study was a trial to obtain aerobic bacteria from fermented barley that were capable of converting ginsenoside Rb1. Earlier studies have focused only on bioconversion of ginsenosides by commercially available food grade micro organisms (Chi and Ji., 2005). In the present study, attempts were made to isolate and screen possibly new active β-glucosidase- producing bacteria that can efficiently transform Panax ginseng

26 saponins Rb1 into any minor ginsenoside under aerobic condition. Moreover, the growth conditions required for the biotransformation of saponins was optimized.

27 3. MATERIALS AND METHODS The study on Bioconversion of Major Ginsenoside Rb1 of Panax ginseng by bacterial cultures isolated from barley and their characterization was conducted at Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore. The bacterial cultures with β- glucosidase activity were isolated from fermented barley water, characterized and analyzed for their potential bioconversion activity. The major ginsenoside Rb1 with four glucose residues in β-orientation was used as the substrate and its bioconversion using bacterial β- glucosidase was followed by TLC and HPLC analysis. The present work was carried out at the following phases 3.1. Isolation of bacteria from fermented barley 3.2. Identification of bacteria with β-glucosidase activity 3.3. Biochemical characterization of the isolated bacterial cultures 3.4. Optimization of growth conditions for the selected bacterial isolates Optimization of growth media Optimization of ph 3.5 Localization and assay of the enzyme β-glucosidase in the isolated cultures Sample preparation Enzyme assay 3.6. Assessment of bioconversion capability of the isolated bacteria Preparation of reaction mixture for bioconversion Thin Layer Chromatography (TLC) Analysis HPLC Analysis

28 3.1. ISOLATION OF BACTERIA FROM FERMENTED BARLEY: The barley sample (10 grams) procured from local market was soaked in water (100ml) and allowed for fermentation for a period of 4 days. For the isolation of micro organisms from this source, varying volumes (10, 100, 200 and 1000µl) of the fermented supernatant was plated on R2A agar (Appendix 1). The plates were incubated at 37 C for a period of 7 days. Morphologically different colonies were identified IDENTIFICATION OF BACTERIA WITH β-glucosidase ACTIVITY The morphologically different bacterial colonies obtained were spotted on MRS agar (Appendix 2) with esculin (3 grams/liter) and ferric ammonium citrate (0.2grams/liter). The plates were incubated at 37 C for about 48 hours and colonies producing browning or blackening of the medium were noted as esculin hydrolyzing bacteria. The esculin positive bacterial colonies were identified and single colonies were obtained on MRS agar and their morphological features noted BIOCHEMICAL CHARACTERIZATION OF THE ISOLATED BACTERIAL CULTURES The five bacterial colonies with β-glucosidase activity were labeled as Bf1, Bf2, Bg, Bdt and Bdw. The cultures were streaked on MRS agar to obtain single colonies. The cultures were maintained at 4 C. The isolated single colonies were studied for their morphological, physiological and biochemical characteristics by performing various biochemical tests (Dubey and Maheshwari., 2002).

29 TABLE 3.1 Biochemical characteristics of the bacterial isolates S.NO BIOCHEMICAL TEST APPENDIX 1. Gram s staining 3 2. Indole production test 4 3. Methyl red- Voges Proskauer test 5 4. Citrate utilization test 6 5. Catalase test 7 6. Nitrate reduction test 8 7. Carbohydrate fermentation test 9 8. Starch hydrolysis test Urea hydrolysis test Triple sugar iron test OPTIMIZATION OF GROWTH CONDITIONS FOR THE SELECTED BACTERIAL ISOLATES Optimization of growth media The five bacterial cultures of interest were inoculated in four different bacterial growth media namely Luria Bertuni (LB) (Appendix 13), Nutrient Broth (NB) (Appendix 14), MRS and R2A. The cultures were incubated overnight at 37 C with rpm. The optical density of each culture was measured at 660 nm against its respective media blank Optimization of ph The isolated bacterial cultures were inoculated in MRS broth prepared at varying ph namely 4.5, 5.5, 6.5, 7.5 and 8.5. The tubes were maintained in shaking at rpm at 37 C and left for overnight growth. The optical density of each strain was measured at 660 nm against respective media blank.

30 3.5 LOCALIZATION AND ASSAY OF THE ENZYME β-glucosidase IN THE ISOLATED CULTURES The localization of the enzyme β-g1ucosidase was determined by assaying the amount of p-nitrophenol released from the substrate p-nitrophenol-β-d- glucopyranoside Sample preparation The isolated cultures were inoculated for overnight growth in MRS broth. 1ml of the grown culture was taken and centrifuged at 10,000 rpm for 3 minutes to pellet the cells. Three samples were prepared for the assay of the enzyme as follows: o The supernatant was used as sample 1. o The pelleted cells were resuspended in sodium acetate buffer (ph 4.6). The suspension was divided into two equal halves. One half of this pellet suspension was used as sample 2. o The other half was sonicated and used as sample Enzyme assay The β-g1ucosidase enzyme produced by the bacterial isolates were assayed by a method described by Gueguen et al., 1997 with some modification (Appendix 15). The enzyme was assayed in the overnight cultures. Also to test for the amount of the enzyme available during longer incubation periods, assay was performed for 1, 3, 5, 7, 9, 11, 13 and 15 day cultures ASSESSMENT OF POTENTIAL BIOCONVERSION CAPABILITY OF THE ISOLATED BACTERIA WITH β-glucosidase ACTIVITY To assess the ability of the isolated bacteria to convert the major Ginsenoside Rb1 to any of the more therapeutically valuable minor Ginsenosides, a single colony from the five esculin positive cultures was inoculated in 5ml of MRS broth and incubated at 37 C overnight at rpm PREPARATION OF REACTION MIXTURE FOR BIOCONVERSION

31 The reaction mixture was prepared with 200µl (1mM, 1000ppm) of Ginsenoside Rb1 and 200ul of bacterial suspension. The mixtures were incubated at 37 C for 1, 3, 5, 7, 9, 11, 13 and 15 days. At the end of its respective incubation period, 200ul of Butanol: Water in the ratio 7:3 (prepared one day before use) was added to this mixture, vortexed and centrifuged at 13000rpm for 5 minutes. The butanol layer was collected separately and the extraction repeated once again. The collected butanol extracts were pooled and stored in the refrigerator untl further analysis THIN LAYER CHROMATOGRAPHY (TLC) ANALYSIS: TLC was performed on Silica gel 60F 254 plates (Merck KGaA-Germany). A TLC plate of length 7cm and height 8 cm was taken and the origin was marked at about 0.8 cm from one end of the plate. The solvent mixture chloroform: methanol: water in the ratio 65:35:2.5 was prepared. The lower phase of the solvent was used. Butanol extract (10ul) of each culture collected through the varying incubation periods was spot on the TLC plate and allowed to dry. A 7ul standard containing the 6 Ginsenosides saponins Rb1, Rd, Rg3, F2, Rh2 and C-K was loaded for reference. The run was performed in a saturated chamber containing the solvent mixture. The plate was kept inside the chamber until the solvent reached the tip of the plate. The plate was then removed and dried. The plates were fixed by spraying 10% H 2 SO 4 (fixing solution) followed by drying. The spots were developed by heating and analyzed to assess the bioconversion capability of each bacterial culture. In order to confirm the results obtained, HPLC analysis was performed HPLC ANALYSIS: Distilled water (200ul) was added to the butanol extracts obtained from section The sample was evaporated using a 45 C water bath and a vacuum evaporator. The residue was redissolved in 200ul of HPLC grade methanol (Ranbaxy, India), filtered using PFTE membrane filter of diameter 13mm with pore size of 0.22um (MFC Advantec AFS,

32 Inc.). The sample (20ul) was injected using a Hamilton syringe (Hamilton Co., Reno, Nevada). HPLC analysis used a Shimadzu HPLC class VP series with two LC-6AD pumps, with CBM-20A variable wavelength photo diode array detector, CTO-10AS column oven and CBM-20A controller (Shimadzu). A Phenominex C18 reverse phase column (250 x 4.6mm) was used for all separations. Acetonitrile (Ranbaxy, India) and in house purified Milli Q water (Millipore Corporation, USA), filtered using 0.22 um filters were used as solvent A and B respectively. The solvent ratio A/B of, 25:75, 45:55,90:10, 25:75 and 25:75 was maintained at run times 0, 40, 50, 55 and 56 minute respectively. The flow rate of the solvent into the column was 1.6ml/min. Detection of the spectrum was at 203nm. Table 3.2 Solvent ratio for HPLC analysis at different time periods Total Time Time Acetonitrile Distilled Water (100%) (100%) The time periods of the peaks obtained were compared with the standard peaks and the results obtained were verified.

33 4. RESULTS AND DISCUSSION The present study Bioconversion of Major Ginsenoside Rb1 of Panax ginseng by bacterial cultures isolated from barley and their characterization was aimed at isolating a bacterial source for bioconversion of major ginsenosides and its characterization. The results obtained during the study are presented and discussed here under: 4.1. Isolation of bacteria from fermented barley 4.2. Identification of bacteria with β-glucosidase activity 4.3. Biochemical characterization of the isolated bacterial cultures 4.4. Optimization of growth conditions for the selected bacterial isolates Optimization of growth media Optimization of ph 4.5 Localization and assay of the enzyme β-glucosidase in the isolated cultures Sample preparation Enzyme assay 4.6. Assessment of bioconversion capability of the isolated bacteria Effect of culture age on bioconversion Butanol extraction and TLC analysis HPLC Analysis

34 4.1. ISOLATION OF BACTERIA FROM FERMENTED BARLEY In the present study, barley grains were allowed to ferment and screened for microorganisms. Barley grains were processed in the following methods for the isolation of microorganisms Sample A: Barley grains (10 grams) were soaked in water for a period of 3 days and the supernatant was used. Sample B: The above soaked barley grains were made into a fine paste and used. A loopful of the paste was suspended in 1ml of distilled water, vortexed and centrifuged. The supernatant was used. Sample C: Raw barley grains (2 grams) were finely powdered. A loopful of this powder was suspended in distilled water, vortexed and centrifuged. The supernatant was utilized. The sample plates were incubated at 37 C on R2A agar. At the end of the incubation period, the plates were observed for morphologically different bacteria. Eleven morphologically different microorganisms were identified from barley water (Sample A). It comprised of 4 different fungi and 7 bacterial colonies (Plate 4.1). Samples B and C did not show any growth of microorganisms on R2A agar even on longer incubation periods. Kim et al., (2005) used R2A agar for isolation of bacteria from soil samples as these strains will require only less amount of nutrients for their growth. In this study, R2A agar was used for isolation of bacteria from food sources as these bacteria will require minimum nutrients for their growth. The source of the enzyme β-glucosidase will be the bacterial cultures isolated from barley since, the sugars present in barley are in the β-glucan form (Robertson et al., 1996). When barley grains are allowed to ferment, the bacteria that grow on it during fermentation process can produce the enzyme β-glucosidase to utilize constituents of barley for their survival. Hence, in the present study, barley was chosen as a source for isolating bacterial strains with β-glucosidase activity. Samples B and C used the whole barley grains and no bacterial growth was observed in them. This shows the absence of any endophytic bacteria in barley.

35 After identification of morphologically different colonies from sample A (barley water) each colony was spotted to verify its growth on MRS (de Mann Rogosa Sharpe) agar. Previously, food grade bacterial cultures have been maintained on this medium and used for bioconversion experiments. MRS agar is specific for the growth of Lactobacillus and related species which are predominant in fermented food (Chi and Ji., 2005). The growth was observed after the plates were incubated at 37 C for a period of 2 days (Plate 4.2) and thereafter, pure cultures of the isolated bacteria were obtained on MRS agar and maintained at 4 C. The present study focused only on the isolation of bacteria for further analysis, therefore the fungal colonies were not considered. Earlier attempts to isolate bacteria for bioconversion have been made from soil samples in a ginseng field (Kim et al., 2005) and use of commercially available food grade micro organisms for the biotransformation of ginsenosides (Chi and Ji., 2005). There are no reports on the isolation of bacteria directly from food sources and employing them for bioconversion. Bacterial cultures identified from food sources when employed for bioconversion is considered to yield a safe product that is approved by the FDA. Hence, the present study focused on the isolation of bacteria from fermented barley.

36 4.2. IDENTIFICATION OF BACTERIA WITH β-glucosidase ACTIVITY In order to check for the β-glucosidase activity of the isolated bacteria, each individual bacterial colony was spotted on MRS agar supplemented with esculin and ferric ammonium citrate. The bacteria that are esculin positive possess β-glucosidase activity. The spotted plates were incubated at 37 C for 2 days and checked for the browning of the medium around the colony. From the isolated 7 bacterial strains, five strains were found to be esculin positive (Plate 4.3). These cultures were selected, streaked on MRS agar and pure cultures were maintained. The strains were labeled as Bf1, Bf2, Bg, Bdt and Bdw (Plate 4.4). Supplementing the growth media with esculin can identify the presence of the enzyme β-glucosidase. Esculin is cleaved by β-glucosidase to yield esculetin and glucose molecule. The hydrolytic product esculetin reduces the ferric ions (provided by ferric ammonium citrate in the medium) and produces iron, which results in the browning of the medium. Therefore, the colonies producing browning of the medium are esculin positive and possess β-glucosidase activity. The principle of esculin in MRS medium is represented in figure 4.1.

37 Figure 4.1 Principle of Esculin in MRS medium Esculin Ferric ions are reduced and brown or blackening of the medium due to production of Iron β-glucosidase Esculetin Glucose In the presence of Ferric ions Earlier, Kim et al (2005) isolated 77 bacterial colonies from soil of ginseng field with β-glucosidase activity by spotting the bacterial colonies on Esculin-R2A agar. Among the various chromogenic substances available like arbutin, alizarin, esculin etc., available to detect β-glucosidase, esculin was used in the present study. Though indoxylic substrates are highly effective, they are relatively difficult to prepare and have other limitations such as their requirement for oxygen in order for the colored complex to develop (Perry et al., 2006). The five esculin positive bacteria were used for further analysis. The major ginsenoside Rb1 of Panax ginseng possess glucose residues linked through β-linkages. The cleavage of the glucose residues at the C-3 or C-20 position results in the formation of minor ginsenosides. The esculin hydrolyzing

38 bacteria cleave these glucose residues resulting in the production of more therapeutically active minor ginsenosides (Kim et al., 2005).

39 4.3. BIOCHEMICAL CHARACTERIZATION OF THE ISOLATED BACTERIAL CULTURES To study the biochemical characteristics of the isolated cultures with esculin hydrolyzing activity, various biochemical tests were performed and the results are presented in Table 4.1. Table 4.1 Biochemical characterization of the isolated bacterial strains S.No Biochemical test Bf1 Bf2 Bg Bdw Bdt 1. Shape Rod Rod Rod Rod Rod 2. Grams staining Indole Production test Methyl Red Voges-Prousker Citrate Utilization test Starch hydrolysis test Urea hydrolysis Nitrate reduction test Catalase test Carbohydrate fermentation test Triple sugar Iron test Acid Acid Acid Acid Acid Body Pink Pink Yellow Yellow Y+P Slant Pink Yellow Yellow Yellow Pink Butt Pink Yellow Yellow Yellow Yellow

40 All the five bacterial cultures were rod shaped, citrate, starch, indole and urea hydrolysis negative. Therefore, the isolated cultures do not utilize citrate as the sole carbon source; do not contain α-amylases and cannot oxidize tryptophan. The bacteria were catalase positive and produced acids when allowed for fermentation with glucose as the carbon source. Except for the culture Bf2, the other four cultures were gram positive. From Methyl red and triple sugar iron test, it is noted that cultures Bf2, Bg and Bdw produce acids like acetic, lactic and succinic acids, thereby reducing the ph of the medium. The cultures Bf1 and Bdt produce neutral and alkaline end products. Pancheniak and Soccol., (2005) showed that the Lactobacillus species predominant in food are all gram positive. The anaerobic gram positive bacteria are all catalase negative. However, in the present study all the isolated cultures were aerobic in nature. It can be concluded that the 5 bacterial strains have similarities and differences among them. Therefore, the strains may belong to the same or related genus. From the morphological and biochemical analysis, it is obvious that the strains are distinct from each other OPTIMIZATION OF GROWTH CONDITIONS FOR THE SELECTED BACTERIAL ISOLATES Optimization of Growth Media For any bacterial strain to grow, proper nutrients need to be provided in the growth medium. These nutrients should be supplied in appropriate concentrations and in a form that can be utilized by the bacteria for its growth and survival. Four different growth media were used. The bacterial strains were inoculated in four individual media broth namely LB, NB, MRS and R2A and left for overnight growth at 37 C at rpm. The optical density of each strain was measured at 660nm against respective media blank. The results obtained are represented graphically in figure 4.2.

41 Figure 4.2 Growth of cultures in different media Optical Density at 660nm Media Optimization Bf1 Bf2 Bg Bdt Bdw Cultures MRS LB NB R2A Figure 4.2 shows that the cultures Bdt and Bdw showed a slow growth compared to others. The cultures Bf1, Bf2 and Bg showed maximum growth in MRS medium followed by R2A, NB and LB. Bdt showed a similar growth on R2A and LB media whereas Bdw had maximum growth on LB. MRS medium was chosen for the optimization of ph as the bioconversion analysis will be carried out only in MRS broth as used by Chi and Ji., (2005) for food grade microorganisms Optimization of ph The growth medium optimization is important to support the growth of bacteria. In this same regard, the acidity or basicity of the medium also plays an important role on bacterial growth. The results are presented in figure 4.3.

42 Figure 4.3 Growth of cultures at different ph of MRS broth ph Optimization Optical density at 660nm Bf1 Bf2 Bg Bdt Bdw ph From figure 4.3, it is observed that cultures Bf1, Bf2 and Bg showed a better growth profile in all ph range compared to Bdt and Bdw. A ph of 6.5 was found to be optimal for the culture Bdt and Bdw showed the best growth at the ph of 7.5. The bacteria Bdt and Bdw did not show any significant growth in the other ph ranges. On comparing the growth characteristics of the isolated bacteria on MRS media and different ph range, it can be noted that all the cultures except Bdw showed significant growth in ph 6.5, whereas Bdw had its maximum growth at a slightly alkaline ph of 7.5. For further analysis of the localization of the enzyme, the cultures were grown at ph LOCALIZATION OF THE ENZYME β-glucosidase IN THE ISOLATED CULTURES The bacterial cultures in the further analysis for bioconversion, it was necessary to determine the localization of the enzyme of β-glucosidase. This was determined by

43 assaying the amount of p-nitrophenol released from the substrate for the enzyme i.e., p- nitrophenol-β-d- glucopyranoside (pnpg) Sample preparation Three samples from the overnight grown cultures were prepared for the assay as mentioned in section o Sample 1- supernatant o Sample 2- Pelleted cells o Sample 3- sonicated pellet The pelleted and sonicated samples were resuspended in 500 µl of sodium acetate buffer (ph 4.6), centrifuged at 5000 rpm for 5 minutes and the supernatant was used as sample 2 and 3 respectively. Keshari et al., (2002) assayed the enzyme β-glucosidase in the culture supernatant. Whole cell preparations have been made and β-glucosidase was assayed to confirm the localization by Rodriguez et al., (2004) in yeast cultures. Gueguen et al., (1997) pelleted the cells and later sonicated and used it as a sample to determine the location of β- glucosidase. Measurement of the amount of p- nitrophenol released due to hydrolysis of pnpg in each of the above mentioned samples will give an indication about the location of the enzyme, i.e., if the enzyme is intra or extra cellular. If the enzyme β-glucosidase produced by the bacteria is extra cellular, maximum activity will be seen into supernatant as the enzyme will be secreted into the medium. However, if the enzyme is intra cellular, maximum activity would be observed in the sonicated sample Enzyme assay The assay was performed according to Gueguen et al., 1997 with some modification. There was no activity detected for all the five bacterial cultures in samples 2 and 3. The activity of the enzyme was observed only in sample1. Therefore, the enzyme β- glucosidase from these isolated cultures are only extra cellular in nature. The specific activities of the enzyme from the bacterial sources are indicated in table 4.2.

44 Table 4.2 Specific activity of β-glucosidase in supernatant of overnight grown cultures. S.No. Name of the bacterial culture (Overnight growth) Specific activity 1. Bf1 1.16x Bf2 1.35x Bg 1.96 x Bdt 8.96 x Bdw 1.82 x10-5 Earlier assays of β-glucosidase produced by bacteria used a neutral buffer like phosphate buffer of ph 7.0 (Otieno and Shah., 2007; Pyo et al., 2004; Gueguen et al., 1997). Therefore, the activity was measured with phosphate buffer of ph 7.0 (Pyo et al., 2005). However, no significant activity was detected in any of the samples. Since there were reports of using acetate buffer in the range of (Magalhaes et al., 2006; Rodriguez et al., 2004; Mc Cue et al., 2003; Tajima et al., 2001), sodium acetate buffer of ph 4.6 was used for the analysis. The temperature for the assay was maintained at 37 C as it was found to be optimum for the growth of the bacterial strains used in the present study. Also in most of the earlier research work on assay of β-glucosidase produced by bacteria; the samples were incubated at 37 C (Chi and Ji., 2005; Bae et al., 2000; Otieno and Shah., 2007). The enzyme was assayed in the three mentioned samples and the amount of the enzyme was expressed in terms of µg/ml/min. the specific activity of β-glucosidase was determined after measurement of total protein (Lowry s method). The specific activity of the enzyme β-glucosidase in units per mg of protein was calculated. The enzyme β-glucosidase present in the cultures Bf1, Bf2, Bg, Bdt and Bdw was found to be extra cellular as the supernatant was found to contain the maximum activity when compared with the other two samples.

45 To analyze the amount of the enzyme β-glucosidase available for the bioconversion of the major ginsenoside, the assay for the enzyme activity was carried out in the supernatant of the 1, 3, 5, 7, 9, 11, 13 and 15 day incubation period. The results obtained are shown in table 4.3. Table 4.3 β-glucosidase activity of bacterial cultures over 15 day incubation period Incubation period in days β-glucosidase activity of bacterial cultures (U/ml/min) Bf1 Bf2 Bg Bdt Bdw From the table 4.3, it is observed that, there is a significant increase in activity until 3 days of incubation after which the activity remained more or less the same through out. There was no decrease in activity observed on prolonged incubation periods. The activity of β-glucosidase was found to be on par in cultures Bf2, Bf1 and Bg, where as, the activity was less in cultures Bdt and Bdw. This may be due to slow growing nature of these two cultures ASSESSMENT OF BIOCONVERSION CAPABILITY OF THE ISOLATED BACTERIA The main objective of the study is the bioconversion of the major ginsenoside Rb1 to the minor ones by the bacterial cultures with β-glucosidase activity Effect of culture age on bioconversion Five ml of the 5 individual bacterial cultures were grown at 37 C for an overnight and 4 day period. After the specific growth period, 200µl of Rb1 was added to 200µl of

46 each individual culture and incubated for a two day period initially to check for bioconversion activity. It was found through TLC analysis that there was bioconversion activity in the overnight grown cultures. In continuation of the study, only overnight grown cultures were used. These were inoculated with 200µl of Rb1 and kept for 0 th, 1 st, 3 rd, 5 th, 7 th, 9 th, 11 th, 13 th and 15 th day period simultaneously at 37 C at rpm Butanol extraction and TLC analysis At the end of respective incubation period, the saponins were extracted from the reaction mixture for further analysis. Butanol extraction (Kim et al., 2005) was done to extract the saponins. The collected extracts of every individual culture was pooled and stored in the refrigerator and later subjected to TLC analysis. The lower phase of the solvent mixture Chloroform: Methanol: water in the ratio 65:35:10 was used for TLC analysis (Kim et al., 2005; Court., 2000). In the present study, the solvent mixture was used in the ratio 65:35:2.5 as there was no proper resolution of bands. The saponins did not get separated and only a single band was observed with high Rf value. The ratio of the solvents was standardized at 65:35:2.5. Earlier Dong et al., (2003) used a different ratio of the above solvent mixture in the ratio 5:5:1 for the separation of ginseng saponins. After the run was complete, the plate was developed as mentioned in section and the spots were observed. The spots were then compared with the standard saponins and the following observations were made. 1. The overnight grown cultures of all the five bacteria namely Bf1, Bf2, Bg, Bdt and Bdw converted the major ginsenoside Rb1 to minor ginsenoside Rd (Plate 4.5).

47 2. The 4 day grown cultures, followed by a 2 day period incubation with Rb1 also showed a conversion to ginsenoside Rd. Prolonged incubation periods with the cultures neither increased the conversion rate nor a conversion to higher more valuable minor ginsenoside was observed. Therefore, the study was performed at different incubation periods with only overnight grown cultures (Plate 4.6).