extfieldsofstudy: Biology Medicine

provenance: AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2011.jsonl.gz.gz

text:
Intestinal infection following aerosol challenge of calves with Mycobacterium avium subspecies paratuberculosis

A challenge experiment was performed to investigate whether administration of Mycobacterium avium subsp. paratuberculosis (MAP) via the respiratory route leads to MAP infection in calves. Eighteen calves from test negative dams were randomly allocated to four groups. Six calves were challenged with MAP nasally and six calves were challenged by transtracheal injection; three orally challenged calves served as positive controls, and three non challenged calves as negative controls. The challenge was performed as a nine-fold trickle dose, 107 CFU in total. Blood and faecal samples were collected frequently. Calves were euthanized three months post-challenge and extensively sampled. Blood samples were tested for the presence of antibodies and interferon gamma producing cells by ELISA. Faecal and tissue samples were cultured in a liquid culture system and the presence of MAP was confirmed by IS900 realtime PCR. Fourteen out of fifteen calves had no MAP antibody response. The negative controls remained negative; all positive controls became infected. Two nasally challenged calves showed a Purified Protein Derivative Avian (PPDA) specific interferon gamma response. In all nasally challenged calves, MAP positive intestinal samples were detected. In three calves of the nasal group MAP positive retropharyngeal lymph nodes or tonsils were detected. In all calves of the transtracheal group MAP positive intestinal tissues were detected as well and three had a MAP positive tracheobronchial lymph node. These findings indicate that inhalation of MAP aerosols can result in infection. These experimental results may be relevant for transmission under field conditions since viable MAP has been detected in dust on commercial dairy farms.

Introduction
Paratuberculosis or Johne's disease (JD) is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). The well-accepted transmission route of MAP is the oral uptake of bacteria by susceptible calves via colostrum, milk, water or food contaminated with faeces from MAP-shedding cattle [1]. In addition, intrauterine transmission has been described [2]. Due to limited effectiveness of control programs and the fact that eradication could not be achieved, other nonidentified transmission routes have been suggested [3][4][5].
Recently, MAP has been identified in settled dust samples collected on dairy farms under experimental and field conditions [6,7]. Currently, JD prevention programs do not include management practices to reduce dust production, spread of dust or exposure of susceptible calves to dust. All existing recommendations to decrease the risk of new infections of MAP in dairy operations are directly aimed at reducing the infection rate in calves by decreasing the contact with faeces of adult cows [8,9]. MAP containing dust may cause infection in susceptible calves via ingestion due to normal calf behaviour (exploring the environment by licking and suckling). In addition, inhalation of MAP containing dust may also represent a route of transmission. In sheep, experimental intratracheal infection has been successful in the past [10]. However, inhalation of MAP by cattle has only been hypothesized as a possible route of transmission [4].
The current study was designed as a proof of principle experiment to investigate whether inhalation of MAP can establish infection in dairy calves. Particle size of dust determines how deep particles can penetrate into the lungs. In humans it has been determined that inhaled particles of approximately 5 μm will be cleared by the mucociliary system of the respiratory tract and subsequently ingested (inhalable dust). Most particles < 5 μm can reach the alveoli and are called respirable dust [11]. A similar deposition and clearing pattern of aerosols was found in the lungs of calves [12]. Therefore, the influence of particle size on clearing in the respiratory tract of calves was assumed to be similar to humans. Two inoculation routes were tested in this study, intratracheal inoculation mimicking the uptake of respirable dust and aerosolized MAP mimicking the uptake of inhalable dust particles. In early MAP infection (< 13 weeks) humoral immune responses as well as macroscopic and histological lesions are unlikely to occur [13,14]. After experimental MAP inoculation in calves, specific cellular immune responses could be detected early by interferon gamma assays and tissue culture could confirm infection status [15][16][17]. Therefore, colonization of tissue determined by culture was used to confirm successful intestinal infection in this study.

Animals
Fourteen newborn Holstein Friesian male calves, one male twin and one male/female twin (total of 18) were obtained from nine dairy farms located around Calgary, Alberta, over a period of five weeks. Donor-herds were identified as low MAP prevalent (< 5%) in a MAP prevalence study in 2009 by testing individual faecal, serum and milk samples of cows over 36 months of age. Faecal samples were cultured by para-JEM automated MAP culturing (para-JEM ® , TREK Diagnostic Systems, Cleveland, OH, USA) and serum and milk samples were analyzed for MAP antibodies by Pourquier ELISA (IDEXX Laboratories, Inc, Westbrook, Maine, USA). Only calves of first and second lactation cows were included in the study. Faecal and serum samples of dams were collected at the day of parturition to confirm individual animal negative MAP status by both liquid culture and ELISA. All samples of dams tested negative. Calves were separated from their dam directly after birth and a pre-colostral serum sample was collected and used to check for BVD carriers. The calves were transported to the research facility and fed 6 L of gamma-irradiated colostrum (Hamilton McMaster Nuclear Reactor, Ontario, Canada) within 6 h, followed by milk replacer and calf starter grain without antimicrobial additives.
Calves were housed individually on sawdust bedding in a biosecurity level 2 barn. The barn was heated and the temperature was maintained at a minimum of 14°C for the duration of the trial. The health status was monitored on a daily base by clinical inspection. The protocol was approved by the Health Sciences Animal Care Committee (M09083) at the University of Calgary and performed according to Canadian Council of Animal Care regulations.

Inoculum
A virulent cattle type MAP strain isolated from a clinical JD case in Alberta (Cow 69), with an IS900-RFLP profile identical to the K10 reference strain recommended in the literature, was used [18]. To mimic field conditions the dosage chosen was close to one of the lowest described to be infective when administered orally [16] which was also plausible for uptake through respiration. Data about dust concentrations in cattle housing [19,20] and the tidal volume of calves (8 mL/kg bodyweight) were used to estimate that a calf approximately inhales 700 mg of dust in the first 3 months of life via respiration alone excluding exploratory licking and suckling behaviour. It was shown that 10 5 CFU of viable MAP/mg dust can be detected in dust samples on commercial dairy farms [7]. The challenge dose was 8 × 10 5 CFU/dose administered on 3 consecutive days for 3 weeks in a row (trickle dose) leading to exposure of calves to approximately 1 × 10 7 CFU which is one of the lowest dosages described to cause infection after oral inoculation [16].
MAP colonies were grown in 7H9/mycobactin/glycerol/OADC broth at 37°C shaking at 100 RPM. The inoculum was tested for contamination with Gram staining and subculture to blood agar. The inoculum was vortexed with 3-4 1 mm glass beads to eliminate clumps and checked for viability using a fluorescent Live/Dead ® BacLight™ Bacterial Viability kit (Invitrogen, Burlington, ON, Canada). Subsequently, MAP was quantified using the "pelleted wet weight method" as previously described, where the pelleted wet weight averaged approximately 1 × 10 7 CFU/mg [18]. Aliquots of 1.7 mg of MAP/tube were frozen at -80°C to be used as inoculum for each inoculation series. A separate aliquot of MAP was used for each week of inoculation.
Prior to each inoculation, a tube was thawed and inoculated in 100 mL of 7H9/mycobactin/glycerol/ OADC medium at 37°C shaking at 100 RPM for 5 days. MAP dose per mL was assessed using an in-house quantitative realtime PCR with a standard curve based on the 16 s rRNA gene of Mycobacterium smegmatis confirming the presence and the quantity of the 16 s rRNA gene using primers p882 (5'-aggattagataccctggtag-3') and p1100 (5'-gctgacgacatccatgc-3'). The inoculum was diluted to achieve the desired concentration of 8 × 10 5 CFU/mL and was stored at 4°C until just prior to the inoculation for a maximum of three days.

Study design
Calves were assigned randomly to one of the four groups. Six calves were inoculated by transtracheal inoculation, six by nasal aspiration of aerosolized MAP, three were inoculated orally, and three did not receive a MAP challenge and served as negative control. Transtracheal inoculation with 5 mL of inoculum was performed using a technique described in the literature for the collection of bronchoalveolar fluid in calves without sedation [21]. Aerosolized inoculums (5 mL) with a variable size were administered using a nasal spray pump during inspiration of the calf directly into the nostril [22]. Oral inoculation was performed to prove pathogenicity of the administered strain at the dosage used for the other challenge routes by allowing calves to suckle a syringe containing 5 mL of MAP suspension. Negative controls were used to support the negative status of the collected calves and to detect unexpected MAP transmission during the trial. In addition, dust samples were collected with an electrostatic dust collector (EDC) to detect environmental contamination with MAP.
An overview of all samples collected during the trial is given in Table 1. At week 12 after the first challenge, euthanasia was performed using intravenous injection of barbiturate (pentobarbital sodium 540 mg/mL, Euthanyl Forte ® , DIN 00241326, Bimeda-MTC Animal Health, Cambridge, Ontario, Canada) and necropsy was performed immediately afterwards.

Necropsies
During necropsies no other ruminants were allowed in the necropsy room. One calf was opened at a time. At post-mortem, a total of 18 tissue samples were collected per calf with separate sterile instruments for each sample. Three parts of the ileum were sampled with adjacent lymph nodes (ileocaecal valve, middle and proximal parts of the ileum) as well as four parts of the jejunum with adjacent lymph nodes (0.5, 1, 1.5 and 2 m proximal from the proximal ileum). Additionally, the tonsils, the retropharyngeal lymph nodes and right cranioventral lobe of the lung with tracheobroncheal lymph nodes were collected. In case of macroscopic lung pathology the abnormal lung tissue was sampled additionally. To minimize cross contamination, samples were collected in the following order: tonsils, retropharyngeal lymph nodes and lung tissue were collected first, then parts of interest of the intestinal tract were identified, marked and adjacent lymph nodes were collected before opening and sampling the intestinal tissue. Equipment and the necropsy room were cleaned and disinfected between necropsies.

Sample analysis
Serum samples of the calves were analysed for MAP specific antibodies using Pourquier ELISA following the manufacturer's instructions. The results were expressed as sample to positive ratio (S/P-ratio) using the kit positive control to correct for inter-plate variation as per the manufacturers instruction. Whole blood was analyzed by Bovigam ® (Prionics, Lavista, USA) to detect T-cell sensitization. Samples were stimulated within 8 hours after blood collection in a 24-well tissue culture plate. Commercially available antigens were used to incubate cells. Purified Protein Derivative Avian (PPDA; Prionics) and Purified Protein Derivative Bovis (PPDB; Prionics) included with the Bovigam ® assay were used as specific antigens. Pokeweed mitogen (PMT; Sigma Aldrich, Oakville, Ontario) was used as a positive control and PBS (pH 7.4) as a negative control. The supernatant was stored at -20°C until analysis. The results were expressed as the S/N ratio using the PBS stimulated sample to correct as per the manufacturer's instruction.
Faecal samples of calves were analysed as described in Table 1 and dust samples were tested individually. Dust samples were decontaminated and prepared as described previously [7]. All faecal samples were decontaminated and prepared for culturing in liquid medium (TREK para-JEM ® ) according to the instructions provided by the manufacturer.
Intestinal samples (4 × 4 cm) were rinsed in PBS to remove intestinal contents and the mucosa was scraped off the intestinal wall using slides. The tissue was put into 50 mL tubes prefilled with 20 mL of 1/2 strength Brain Heart Infusion (BHI) containing 0.9% hexadecylpyridinium chloride (HPC). The lymph nodes (2 g) and pieces of lung were cut open and put into stomacher bags with 5 mL of NaCl. Samples were homogenized with a Stomacher ® (Stomacher ® 80 Biomaster, Seward Laboratory Systems Inc., Bohemia, New York, United States) for 1 min at high speed. Fluid containing debris was poured into a 50 mL tube containing 20 mL 1/2 strength BHI with 0.9% HPC. Samples were incubated overnight at 35°C. The next day, tubes were centrifuged at 1700 g for 20 min, the supernatant was discarded, the pellet was resuspended in 1 mL 1/ 2 strength BHI containing antibiotics (vancomycin and naladixic acid 100 μg/mL, amphotericin 50 μg/mL) and incubated overnight.
After these initial decontamination and preparation steps faecal, dust and tissue samples were incubated at 35°C for 42 days in para-JEM ® liquid culture medium. MAP presence was confirmed by IS900 realtime PCR on culture medium as previously described [7]. Culture results were considered as a binominal outcome (MAP detected/not detected).

Data analysis
Interferon gamma results were analyzed by ANOVA analysis comparing the S/N-ratios of the 4 experimental groups. The number of positive tissue cultures per calf was recorded and presented per location. An inoculation route was considered to be successful if at least one calf per group had at least one culture positive intestinal tissue.

Clinical findings
During the study no severe clinical abnormalities were detected. Several calves had a dry cough throughout the experimental period. It occurred in all groups before and also several weeks after inoculation. One calf in the nasal group was diagnosed with and treated for pneumonia within the first week of life before inoculation. Slight diarrhoea was detected in 3 calves; in one it occurred before inoculation, and in the other 2 in week 6 post inoculation (pi).
Calves showed no reaction after nasal inoculation. Some calves inoculated trans-tracheally reacted with a slight cough to the injection of fluid into the trachea. Twice traces of blood were detected in the nostrils after transtracheal injection.

Immune parameters
Serum samples of calves at day 3 of life and D82 after inoculation were negative for MAP specific antibodies in the ELISA with the exception of one calf from the oral inoculation group.
In all calves, interferon gamma production of cells stimulated with PBS and PPDB were negligible. Stimulation with PMT showed a clear interferon gamma response in all animals. Two calves of the nasal inoculation group had a more prominent interferon gamma response to PMT. After stimulation with PPDA the interferon gamma production was comparably low in the negative control, the positive control and the transtracheal inoculated group. In the nasal inoculation group, the two calves with the prominent interferon gamma response to PMT also showed a high response to PPDA. A significant (p = 0.002) difference was found between the nasal inoculated and the positive control group.

Necropsy
No macroscopic intestinal lesions were observed in any of the calves at necropsy. The calf in the nasal group treated for pneumonia showed a few pleural adhesions. No MAP could be detected in an additional piece of lung tissue collected underneath the adhesions.

Culture results
All faecal samples of the calves collected throughout the trial were culture negative. All 18 dust samples collected 8 weeks after the last inoculation were also culture negative. Culture results of tissue samples are summarized in Table 2

Discussion
This study supports the hypothesis that MAP might be transmitted via bioaerosols since a low trickle dose administered via nasal and transtracheal routes could establish MAP infection in all inoculated calves. Both respiratory routes caused infection of intestinal tissue and intestinal lymph nodes.
In sheep intratracheal MAP exposure has been previously described as a potential route of infection [10]. The number of calves used in this study was too small to statistically compare results for tissue locations between groups, but the goal was a proof of principle. The presence of MAP in cultured intestinal tissue and lymph node samples of all challenged calves (Table 2) supports that MAP uptake via the respiratory tract can induce MAP infection in susceptible calves. Recent detection of MAP in bioaerosols on commercial dairy farms indicates that this route has to be considered as a possible route of transmission [6].
Intermittent faecal shedding due to infection has been described in experimental trials as early as 146 days post challenge [13]. Since calves in this study were inoculated with a lower dose per inoculation day compared to literature and were euthanized at 12 weeks pi, no presence of MAP in any faecal sample was expected. In early MAP infection, no macroscopic lesions occur and histological lesions containing acid-fast bacteria can be detected sporadically if at all [13,14]. Therefore in this study, confirmation of MAP infection in tissue samples was performed by MAP culture. The detection of seroconversion against MAP using commercially available ELISA tests has occurred earlier in experimental infection studies than in naturally infected cattle, but not before 406 days after inoculation [13,23,24]. The absence of MAP specific antibodies shortly after birth showed the absence of maternal antibodies in colostrum administered to the calves. Not detecting antibodies in 14 out of 15 challenged calves in week 12 after inoculation was in agreement with published studies indicating seroconversion so early after challenge to be a rare event. The interferon gamma assay detects cellular immune responses and was deemed useful for early paratuberculosis detection in cattle [15,25]. In experimental studies in calves, interferon gamma production has been detected as early as day 7 after intra-peritoneal inoculation [15]. However, after oral inoculation (4 times of 1-2 × 10 10 CFU/dose) interferon gamma production did not increase before day 90 pi and was considered a robust measure only after 6 months of age [15]. In the present study, calves were inoculated with a considerably lower dose and were euthanised before 90 days post inoculation (dpi) explaining the low specific interferon gamma response in the oral inoculation group. Surprisingly, two calves inoculated through the nasal route showed a clear interferon gamma response after specific antigen stimulation as early as 12 weeks pi. These findings and negligible responses to stimulation with PBS and PPDB indicate a specific sensitization of peripheral blood mononuclear cells to MAP after nasal inoculation.
Faecal-oral transmission of MAP is believed to be the most common route of transmission under field conditions [1,26]. In addition, this route of inoculation has been frequently confirmed to be effective in experiments [18]. Therefore, the positive control group was inoculated orally with the same inoculation scheme and dose as the experimental groups. All 3 positive control calves became infected (Table 2), although the total dose (1 × 10 7 CFU/dose) and the dose given per inoculation day (8 × 10 5 CFU/dose) was low [18]. A dose of 1.5 × 10 6 CFU/dose for 2 consecutive days reliably induced detectable infection, whereas a dose of 2 × 10 5 CFU/ dose did not [16]. In this study the daily oral dosage was lower than 10 6 CFU/dose and probably only led to an infection because it was administered several times as a trickle dose adding up to a total dose around 10 7 CFU.
In humans, aerosol routing after aspiration has been described to be dependent on droplet size [11,27]. Similar estimates for particle penetration into the respiratory tract have been performed in calves [12]. To mimic aerosol infection by droplets in the upper respiratory tract a device was used to administer the inoculum to the nasal group which was designed for intranasal application in humans. It produced aerosols with a variable size which were mainly trapped in the mucociliary system of the nose [22]. Thus, most MAP would have reached only the nasal cavity passing the nasal associated lymphoid tissue (NALT) before being cleared by the mucociliary system and ingested [28]. NALT contains M-cells which have been shown to facilitate MAP uptake in Peyers patches of gut associated lymphoid tissue (GALT) after oral uptake [29,30]. After nasal inoculation, MAP would come into direct contact with NALT and thus with M cells; it would also pass by the palatine tonsils before being swallowed and diluted by saliva. Therefore, when presented to M cells in NALT, uptake of MAP might occur via a similar mechanism to that which occurs during oral infection; respiratory uptake may be even more efficient since in this case MAP is not diluted in intestinal contents before reaching the target tissue. In addition, when inhaled, surplus MAP will be swallowed and M-cells in the Peyers patches subsequently will be exposed. Confirmation of MAP in retropharyngeal lymph nodes and in tonsils of calves of the nasal inoculation group supports that the nose can act as a portal of entry for MAP.
Transtracheal application of MAP was performed, mimicking the scenario of MAP attached to respirable dust. Particles and bacteria entering alveoli will be taken up by pulmonary-alveolar macrophages and removed by the mucociliary system, passing the tonsils before being swallowed; alternatively they could pass the mucosal epithelial barrier [12,31,32]. M-cells in bronchus-associated lymphoid tissue (BALT) have been described as a portal of entry for Mycobacterium bovis in mice [33]. BALT is not present in the neonatal bovine lung, but can be detected after 4 months of age [34] indicating that BALT could play a role in MAP uptake in older young stock but not in the present study. In this study, MAP was possibly cleared by pulmonary-alveolar macrophages and partially passed the mucosal epithelial barrier indicated by confirmed positive tracheobroncheal lymph nodes in 3 calves of the transtracheal group. These findings support that the lung can act as a portal of entry for MAP.
After oral inoculation and uptake by M cells of the Peyers patches, macrophages are the target cells since MAP is capable of interfering with bactericidal mechanisms of these cells [35]. Within macrophages, MAP migrates to the regional lymph nodes [14] and in later stages of disease the whole body is colonized [36][37][38]. Older oral infection studies detected MAP in lymphoid tissue of the pharyngeal area shortly after inoculation, followed later on by the intestinal tract and other organs indicating dissemination via the reticuloendothelial system to reach the intestinal tissue [39,40]. This theory was supported by the finding that instillation of MAP in the tonsils of calves' leads to infected intestinal tissue [13]. In addition, intraperitoneal inoculation proved to be effective for inducing infection [41]. In this study MAP lymph nodes close to the inoculation site were positive, as well as intestinal tissue and lymph nodes, indicating that migration occurred as well. Intestinal colonization when inoculated orally, intraperitoneally and directly into the tonsils and now also nasally and transtracheally supports that migration of MAP has to occur once it has passed the epithelial barrier [16,41,42].
In conclusion, intestinal MAP infection can be induced via the respiratory route when administered into the nose or the trachea of young calves. If MAP uptake after inoculation occurred through the nose and lung tissue directly as hypothesized or due to ingestion after mucociliary clearance maintains unclear. Nevertheless intestinal infection occurred indicating that bioaerosols can be a route of transmission. Since MAP has been detected in bioaerosols on dairy farms, the transmission of MAP by bioaerosols will need to be considered in MAP control programs in addition to the faecaloral route.


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Protective Effects of Milk Thistle ( Silybum marianum ) against Aflatoxin B1 in Broiler Chicks

: Aflatoxin-contaminated feed cause mortality, suppression of the immune system, reduced growth rates and losses in feed efficiency. This research study was planned to investigate the immunomodulatory and growth promoting effect of milk thistle as feed additive against aflatoxin B 1 in broiler chicks at NWFP Agricultural University Peshawar, Pakistan. Two hundred and forty (240) day old broilers chicks were randomly assigned into four major groups AfF, aflatoxin free feed; Aflatoxin B 1 was present in the feed at the levels of 80-520 μ g/kg of the feed in the remaining three groups. Aflatoxin contaminated feed was provided for 5 weeks. Group AfB was supplemented with toxin binder “Mycoad” at 3 g/kg of feed and group AfT was supplemented with milk thistle at10 g/kg of feed. Each group was further sub divided into two sub-groups, vaccinated against ND (Newcastle disease), IB (Infectious bronchitis) and IBD (Infectious bursal diseases) according to recommended schedule of vaccination or non vaccinated. Each sub group carried three replicates with 10 chicks per replicate. Chicks were reared in pens in an open sided house. Supplementary heat was provided to all the chicks during brooding period. Mean body weight gain and dressing percentage were significantly (p<0.05) higher in group AfF, followed by AfT, AfB and Af. Weight gain and dressing percentage was the same in group AfB and AfT, while it was significantly lower in group Af. Feed intake, breast, thigh and leg weight were found significantly (p<0.05) higher in group AfF, followed by AfB, AfT and Af. Significantly lower (better) FCR value was recorded in group AfT. Water intake was significantly (p<0.05) higher in group AfT and AfF as compared to other groups. Mortality was significantly (p<0.05) higher in group Af. Mean bursa and thymus weights were found significantly (p<0.05) higher in group AfF, AfB and AfT followed by Af, while higher spleen weight was recorded in group AfT. Mean antibody titer against ND, IB and IBD was significantly (p<0.05) higher in group AfT, as compared to other groups. It is concluded that milk thistle at 10 g/kg of feed could effectively be utilized as immunostimulant and growth promotant in the presence of immunosuppressant aflatoxin B 1 in the feed. (

INTRODUCTION
Chicken meat is an important source of nutrients for human consumption. Broiler feed is exposed to various contaminants during the process of production, transportation and storage. Moisture is the most important factor favoring the growth of fungus during these stages while the hygienic condition and quality control measure are normally poor in the area under study. One of the important injurious agents is mycotoxins as it has hepatotoxic, hepatocarcinogenic, mutagenic and teratogenic effects in many animal species (IARC, 1987). Mycotoxins are metabolites from moulds. Aflatoxins, a group of closely related biologically active mycotoxins, are produced by strains of Aspergillus flavus and Aspergillus parasiticus. Even though, 18 different Aflatoxins have been identified, only Aflatoxins B 1 , B 2 , G 1 and G 2 have been detected as natural contaminants of feedstuffs. In a previous study Tedesco et al. (2004) the peanuts and cereals have been reported as having afore-mentioned natural contaminants.
Keeping in view the medicinal value of Milk thistle, this research study was conducted to investigate the effect of Milk thistle on the growth and immune performance of broiler chicks against Newcastle disease (ND), infectious bursal disease (IBD) and infectious bronchitis (IB).

MATERIALS AND METHODS
The research study was conducted at NWFP Agricultural University, Peshawar poultry farm to investigate the immunomodulatory and growth promoting effect of milk thistle against aflatoxin B 1 in broiler chicks. Moreover, detection of aflatoxin B l and their level in feed was carried out in the Nutrition Laboratory of Directorate of Livestock Research and Development, Peshawar.

Experimental design
The experiment was conducted in completely randomized block design (CRBD) with two factors i.e. i) level of milk thistle and ii) level of vaccine.
Two hundred and forty (240) starbrow, commercial dayold broiler chicks of approximately the same weight were purchased from the local market. Chicks were divided into four groups AfF, Af, AfB and AfT and each group was further divided into two sub-groups for different vaccination. Each sub-group carried three replicates with 10 chicks per replicate. The birds were raised in 4×4 square foot wooden pens on conventional deep litter, open sided housing system. All the pens were located in the same house in order to have identical environment, where each pen was provided with separate feeder and drinker. Chicks were brooded in the same pens and artificial supplementary heat was provided during the brooding period to meet the requirements of the chicks. Feed and water was offered on daily basis and the refusal was measured next morning. Birds were examined for health status on daily basis. Temperature was recorded and maintained on daily basis. Group AfF was kept as control while group Af was fed contaminated feed, group AfB was fed contaminated feed along with Toxin binder "Mycoad" at the dose rate of 3 g/kg feed and group AfT was provided contaminated feed along with milk thistle at the dose rate of 10 g/kg feed.

Production of aflatoxin B 1 contaminated poultry feed
The aflatoxigenic strain of Aspergillus flavus isolated previously on Sabouraud agar from field cases of aflatoxicosis as well as from contaminated feed samples was used for the production of aflatoxins during the course of the experiment. The field cases comprised aflatotoxicosis infected broilers received at Veterinary Research Institute, Peshawar, for diagnosis purposes, while the feed samples were provided by the owners of the infected birds and/or collected from the feed suppliers in the relevant market. Broth culture of the aflatoxigenic stain of Aspergillus flavus was evenly inoculated aseptically on freshly prepared Sabouraud agar plates with the help of sterile cotton swabs. The plates were incubated in incubator at 28°C for 7 days. A briefly thick growth of spores was produced during this period. The 50 kg poultry feed bag was inoculated with the above culture and was thoroughly mixed. The feed was moistened with water and again mixed thoroughly. The feed bags were kept in store at minimum of 24°C and moisture level above 17.5% to enhance the fungal growth. Contaminated feed was prepared for each week separately. Keeping the amount of both the inoculum and feed constant and a representative feed sample for every week was subjected to analysis for aflatoxins (Rahim et al., 1999). Analysis of experimentally contaminated feed by TLC method showed that aflatoxin B 1 was present in the feed at the levels of 80-520 μg/kg of the feed. The TLC plate was coated with slurry of silica gel to a thickness of 750 μm, dried in air and activated at 100°C for two hours in oven. Chloroform extract of feed, 200 μl, was spotted on the plate in the form of 10 μl spots spreading over an area of 6 cm in width at a height of 2 cm from the baseline. The spot was air dried. The plate was developed using diethylether as developer to a liquid front of 12 cm from the baseline in an equilibrated tank. The TLC plate was removed from the tank, air dried and examined under UV light. The plate was developed in an equilibrated tank using chloroform method developer (50+1 ratio) to a liquid front of 12 cm from the baseline. It was removed from the tank, dried and redeveloped to the same liquid front in the same developer. The plate was viewed under UV light and the position of blue and green fluorescing spot of B1, B2 and G1, G2 were marked. The spots were scrapped and silica gel of each component was collected in a separate glass dish. The gel was extracted with cold methanol method for 3 minutes and the extract filtered into a small beaker. The gel was thrice washed with methanol, making the combined methanol filtrates up to 5 ml. The methanolic extract was examined in a Double Beam spectrophotometer using methanol as blank. The densities of aflatoxins were measured at 363 and 420 Angstrum. The corrected optical density was calculated by subtracting the value at 420 Angstrum from 363 Angstrum. The quantity of aflatoxin was then calculated.

Preparation of milk thistle powder
The seeds of milk thistle were collected from Warsak and adjoining tribal areas of Michini, Mohmand Agency of Northern Pakistan in summer season. After drying, it was ground to powder form with the help of electric grinder. Powdered milk thistle was mixed in feed at the rate of 10 g/kg feed. Toxin binder (Mycoad) was mixed in feed at the rate of 03 g/kg feed.
Data was recorded for body weight gain (weekly basis), feed intake (daily basis), dressing percentage (at the end of experiment), water consumption (daily basis) and mortality. Feed intake was calculated by subtracting feed refusal from feed offered while water intake was calculated by subtracting water refused from water offered on daily basis. For determination of dressing percentage, one bird from each replicate was live weighed and slaughtered, head, feet, and all internal visceral organs including abdominal fat were removed and then the dressed body was weighed. The dressed body weight was then expressed in terms of dressing percentage by using the following formula: Dressing percentage = (Live weight/Dressed weight)× 100 Feed conversion ratios (FCR) were calculated at the end of experiment. ND, HI antibodies were estimated using the haemagglutination inhibition tests described by Alexander and Chettle (1997), whereas antibody response against IB and IBD was estimated using ELISA (Marquardt et al., 1980). Tissue samples (bursa of fabricius, thymus and spleen) from chicks in each sub-group were collected on last day of experiment.

Statistical analysis
The data was statistically analyzed with the standard procedures of analysis of variance (ANOVA), using completely randomized block design as suggested by Steel and Torrie (1981). The statistical packages SPSS-10 (1999) were used to perform the above analysis on computer.

RESULTS AND DISCUSSION
The results obtained are presented under various sections as follows:

Body weight gain
Mean body weight gain per chick for the four experimental groups and for vaccinated and non vaccinated sub-groups are presented in Table 1. The data when subjected to statistical analysis, showed significant (p<0.05) difference among the groups and non significant between vaccinated and non vaccinated chicks. Interaction effects were also non-significant. Maximum body weight gain was observed in group AfF, receiving normal feed, followed by group AfT (80-520 μg/kg), receiving aflatoxin B 1 contaminated feed with milk thistle. The body weight gain Table 1. Mean water intake (ml), feed intake (g), body weight gain (g) and FCR in broiler chicks fed normal feed, aflatoxin contaminated feed, toxin binder "Mycoad" and milk thistle* was severely depressed in birds receiving aflatoxin contaminated feed without milk thistle and toxin binder (Group-B). Non significant differences were observed between group AfB and AfT. It is evident from the above observations that body weight gain was the lowest in aflatoxin B 1 treated birds. In the Aflatoxin B 1 and milk thistle treated birds, body weight gain was higher with respect to birds receiving AFB 1 alone and not different from the toxin binder treated birds. The results of the present study are in agreement with the findings of Tedesco et al. (2004), who reported that addition of silymarin phytosome in the feed at 600 mg/kg of body weight resulted in an increase of 14.83% in body weight. This increase was lower than the present value of 31.12%. Similarly, Gowda and Sastry (1998) also confirmed significant (p<0.05) improvement of milk thistle on body weight gain and attributed the effect to antioxidant activity that stimulated protein synthesis by bird's enzymatic system. Higher weight gain was also reported by Chakarverty and Parsad (1991), in milk thistle supplemented group. The exact mechanism of improving body weight is not well established; however this effect might be due to the improved immune function of the birds (Table 3) receiving milk thistle. Relative weight of the lymphoid organs and antibody titers against ND, IB and IBD were improved in milk thistle fed group, which are indicative of improved function of the immune system. Avian thymus and bursa of fabricius play an important role in the development of immunological competence as neonatal surgical or embryonic hormonal bursectomy in chicken greatly reduced subsequent antibody forming capacity (Mueller et al., 1960).

Feed and water intake
Mean feed and water intake per chick are presented in Table 1. Significant (p<0.05) differences were recorded among the treated groups and non significant between vaccinated and non-vaccinated groups. Interaction effects were also non-significant. Water intake was significantly higher in group AfF and AfT than other groups. Feed intake was significantly higher in control group AfF and was similar to group AfB. Feed intake was lowest in group Af. Presence of aflatoxin B 1 in feed depressed feed intake (group Af). On the other hand, presence of milk thistle in aflatoxin contaminated feed resulted in improved feed intake, which is at par with control. The higher feed intake in control group did not caused proportionate increase in body weight gain therefore, resulted in lower efficiency of feed utilization. The results of this study support the findings of Kalorey et al. (2005), who reported that milk thistle improved feed intake in presence of aflatoxin B 1 in feed. Our results are correlated with Tedesco et al. (2004), who observed improved feed intake in milk thistle treated group as compared to birds raised on aflatoxin contaminated feed only.
In the present study feed intake was improved by 12.45% in milk thistle fed group (group AfT) as compared 12.06% in the toxin binder group and 18.06% in the toxin free group. This shows sufficient improvement in performance of broilers with the supplementation of milk thistle. Similarly, Tedesco et al. (2004) reported 22.29% improvement in feed intake, having silymarin phytosome at 600 mg/kg of body weight. The greater improvement in feed intake may be due to the pure form of silymarine phytosome while in the present study milk thistle seeds as whole were used which may contain anti-nutritional factors leading to depressed appetite.

Feed conversion ratio
Average feed conversion ratio (FCR) was 2.39, 2.84, 2.44 and 2.37 for the four experimental treated groups AfF, Af, AfB and AfT, while it was 2.52 and 2.50 for vaccinated and non vaccinated groups, respectively (Table 1). FCR was significantly (p<0.05) affected by the treatments but non significant differences were observed between vaccinated and non vaccinated chicks. Interaction effects were also non-significant. FCR values were significantly higher (poor) in group Af and were the same in all other groups. Numerically feed efficiency was the best in milk thistle fed group. The results of our study are in agreement with the findings of Tedesco et al. (2004), who observed better feed efficiency response (2.527) in aflatoxin B 1 contaminated feed with milk thistle with respect to birds receiving aflatoxin B 1 contaminated feed alone (2.929), during the last two weeks. Similar results were also reported by Chakaraverty et al. (1991) and Zahid and Durrani (2007), who fed milk thistle to broilers and observed efficient feed conversion ratio in broilers. Zahid and Durrani (2007) fed milk thistle to broilers at the rate of 15 g/kg feed and found better FCR (2.2) as compared to the control group (2.4).

Dressing percentage
Mean dressing percentage was significantly affected by treatments, while vaccination and interaction effects were non significant (Table 2). Mean dressing percentage was lower (p<0.05) in birds raised on aflatoxin contaminated feed alone (group Af), while no significant (p<0.05) difference was observed in group AfF, AfB and AfT. Zahid and Durrani (2007) reported similar findings, who fed different levels of milk thistle to broilers and found significantly higher dressing percentage at the level of 15 g/kg feed. In the present study an increase of 5.96% in dressing percentage was recorded while Zahid and Durrani (2007) reported 3.92% improvement in dressing percentage.

Weight of different body organs
Differences in breast, thigh and leg muscles were significant among the groups and non-significant between vaccinated and non vaccinated chicks. Interactive effects were also non-significant (Table 2). Zahid and Durrani (2007) reported similar findings, who fed different levels of milk thistle to broilers and found significantly higher breast and thigh weights at the level of 15 g/kg feed. They reported higher (33.65 and 64.89%) improvement in breast and thigh muscles, respectively which are higher than our values. The greater improvement in breast and thigh muscles may be due to the high level of milk thistle while in the present study milk thistle seeds were used at the level of 10 g/kg feed.

Weight of lymphoid organs
Bursa weight : Bursa weight data showed significant (p<0.05) differences among treatments and non significant Table 3. Mean weight of lymphoid organs (g) and antibody titer against ND, IB and IBD in broiler chicks fed normal feed, aflatoxin contaminated feed, toxin binder "Mycoad" and milk thistle*  between vaccinated and non vaccinated groups (Table 3). Interaction effects were also non-significant. Bursa weight was significantly lower in group Af, while non significant differences were observed in groups AfF, AfB and AfT. Aflatoxin contaminated feed significantly reduced bursa weight (group Af), causing interference with antibody production. However, milk thistle supplemented feed restored the normal weight of bursa of fabricius (group AfT). The findings of the present study are supported by Tedesco et al. (2004), who reported reduced bursa weight in broilers by feeding aflatoxin contaminated feed, while increased weight and morphology of bursa in milk thistle feeded groups. Kalorey et al. (2005) also reported the protective role of milk thistle against aflatoxicosis on weight of Bursa of fabricius. Milk thistle has been reported to support the immune system through its powerful antioxidant, free-radical scavenging action, its ability to preserve the supply of another important antioxidant, glutathione, as well as direct effects on immune cells (Basaga et al., 1997). Silymarin, as antioxidant, has protective action against the oxidative damages on the immune organs (Bursa, spleen and thymus) and immune cells which cause immunosuppression.
Spleen weight : Spleen weight was significantly higher in group AfT, followed by AfB, AfF and Af, respectively ( Table 3). As evident from the observations, aflatoxins significantly (p<0.05) reduced spleen weight and milk thistle was more efficient to protect spleen against adverse effect of aflatoxin as compared to synthetic toxin binder. Similar findings have been reported by Kalorey et al. (2005) and Tedesco et al. (2004) by feeding aflatoxin contaminated feed and milk thistle to broilers.
Thymus weight : Mean thymus weight was significantly affected by treatments and non significantly by vaccination. Interactive effects were also non-significant (p>0.05) ( Table  3). Lowest thymus weight was observed in birds raised on aflatoxin contaminated feed alone (group Af), while non significant differences were recorded in groups AfF, AfB and AfT (Table 3). Findings of the present study are supported by Tedsco et al. (2004), who reported reduced weight of thymus by feeding Aflatoxin B 1 contaminated feed to broilers and protective role of milk thistle on thymus weight in aflatoxicosis. Similar findings were also reported by Kalorey et al. (2005), who reported that feed added milk thistle preserved morphology and weight of lymphoid organs (thymus, bursa and spleen) in aflatoxicosis.

Antibody titer
Mean antibody titer of broilers against ND, IB and IBD is given in Table 3. Serum antibody titer for ND, IB and IBD was significantly higher in group AfT (milk thistle treated group), while lowest titers were recorded in group Af. It is clear from the above observations that aflatoxin caused severe immuno-suppression due to reduction in phagocytic activity of blood monocytes, depressed complement activity, hence depressed opsonization and phagocytic activity. However, treatment with milk thistle protected the reduction of humoral immune response in broiler due to aflatoxin B 1 in feed. Milk thistle treated birds also revealed better antibody titer than toxin binder treated group. The results of the present study are supported by Grizzle et al. (1999), Tedsco et al. (2004 and Kalorey et al.     * Group AfF was kept as control while group; Af was fed contaminated feed; group AfB was fed contaminated feed along with Toxin binder "Mycoad" at the dose rate of 3 g/kg feed; group AfT was provided contaminated feed along with milk thistle at the dose rate of 10 g/kg feed. (2005), who reported the immunomodulatory role of milk thistle in the presence of immunosuppressant aflatoxin in feed. Similarly Wilasrusmee et al. (2002) and Thyagarajan et al. (2002) also reported the immunostimulatory effect of milk thistle. Basaga et al. (1997) reported that milk thistle supports the immune system through its powerful antioxidant, free-radical scavenging action, its ability to preserve the supply of another important antioxidant, glutathione, as well as direct effects on immune cells. Silymarin protect the immune cells and organs (Bursa, spleen and thymus) against the oxidative damages which cause immunosuppression.

Mortality
Mean mortality of broilers is presented in Table 1. The data when subjected to statistical analysis, showed significant (p<0.05) difference among the groups and non significant between vaccinated and non vaccinated chicks. Interaction effects were also non-significant. Significantly higher mortality was observed in group Af, followed by AfB, AfT and AfF, respectively. The results of the present study are supported by by the findings of Siddique and Javed (1989), who reported 10.6 percent mortality in broiles, raised on feed with 30-50 ppb of aflatoxins. Findings of the present research trial are also in line with those of Sabri et al. (1989), who reported 12.47 percent mortality in broiles, raised on feed with 22-96 ppb of aflatoxins.

Post-mortem lesions
Post-mortem lesions included pale, enlarged (swollen), yellow friable livers with pinpointed haemorrhages, swollen kidneys and atrophy of bursa and thymus in broiler suffering form aflatoxicosis. Heart showed hydro pericardium, intestines revealed haemorrhagic enteritis. These findings are in conformity with Rahim et al. (1999). Pathological changes in liver and other organs were of milder degree in milk thistle fed birds as compared to birds raised on contaminated feed only and those raised on toxin binder. Similar findings were recorded by Arshad et al. (1992); they conducted clinico-pathological studies of experimentally induced aflatoxicosis in broiler chicks. Similar results were also observed by Sabri et al. (1989); who studied prevalence and pathology of mycontoxicosis in broiler chicks in and around Faisalabad.
It is concluded that milk thistle at 10 g/kg of feed may effectively stimulate the immune function and growth performance in the presence of immunosuppressant aflatoxin B 1 in the feed.


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Utilization of Soybean as Food Stuffs in Korea

1.1 Origin of soybean The origin of soybean is considered as Manchuria and Korean Peninsula and these areas are also the living base of ancient Dongyi tribes. In general, in the estimation of the origin of crops, the presence of its wild species is the most important index in which lots of wild soybeans are found at the present time in these areas. In archaeology, the cultivation year of soybeans is estimated to be about 4,000 years ago. In Korean Peninsula, carbonized soybeans were found at some historic sites in the Bronze Age and that provides evident for such cultivation (Kwon et al., 2005 a). Table 1 shows the historic sites of soybean excavated in Korean Peninsula according to their era.

Origin of soybean
The origin of soybean is considered as Manchuria and Korean Peninsula and these areas are also the living base of ancient Dongyi tribes. In general, in the estimation of the origin of crops, the presence of its wild species is the most important index in which lots of wild soybeans are found at the present time in these areas. In archaeology, the cultivation year of soybeans is estimated to be about 4,000 years ago. In Korean Peninsula, carbonized soybeans were found at some historic sites in the Bronze Age and that provides evident for such cultivation   It has been known that the cultivation of soybean was originated from the era of the middle period in the agricultural age of the New Stone Age to the Bronze Age (around BC 1,500). Some pieces of earthenware studded with soybean in the Bronze Age were found at the Paldang submerged area in the suburb of Seoul. Lee Sung-Woo (1988) proposed that the first people that cultivated soybean as a food in the history of mankind is around BC 4000∼2000. Also, it is assumed that the first cultivation of soybeans was started in arable fields at the hub of Mountain Baekdu.

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It is considered that soybean have been contributed to the table of Dongyi tribes as an important protein source in such primitive ages that show insufficient nutrients. Thus, it can be seen that soybean play an important role in forming the early countries in Northeast Asia due to the increase in military powers on the basis of improving their nutrients  ).

Strain of soybeans and its production
The strains of soybean cultivated in Korea are classified according to purpose that includes soybean for soybean sauces and pastes, soybean sprouts, boiled rice, early-ripening beans, unripe beans, and premium strain. The strains of soybean for soybean sauces and pastes are a total of 43 varieties and distributed from Jangeup in 1978 to Joongmo 3003 in 2009 (National Institute of Crop Science, Rural Development Administration, Korea). As new strains of soybean have been distributed every year as a similar way to that of red peppers it is necessary to continuously conduct studies on the proper strains of soybean for soybean sauces and pastes. The production of soybean is about 130~180 thousand tons per year even though it is varied according to its harvest (Table 2).
Year Area ( The essential ingredients of soybean are protein (30~50%) and fat (14~24%) where the protein covers the largest portion of such ingredients. About 63~90% of the soybean protein are glycinin included in globulin, and the other components are phaseolin (17%) and legumelin. These are a type of water insoluble protein that is solved in a salt solution and represents no specific taste (Snyder et al., 1987a;Liu, 1997a). Although a sulfur containing amino acid is a limiting factor in an amino acid that is an ingredient of protein, it has abundant lysine, which is insufficient in cereal protein, and helps to intake balanced nutrients as it is taken with some cereals due to their interaction. Although the protein of soybean has no specific component in presenting its own taste, it represents several savory tastes as it is dissolved into the types of amino acid and peptide and that may be used as various seasoning sources. A fermentation process that multiplies molds or bacteria after cooking soybean dissolves soybean protein using protease in order to obtain amino acids and peptides for resenting such savory tastes.
Regarding the uses of soybean in Korea, these uses are divided into two large different ways such as a direct use and a dissolving process of the protein of soybean through a fermentation process. In the dietary life in Southeast Asian countries, the soybean fermentation technology that obtains seasonings are already developed very well. As mentioned above, the fermentation technology using soybean has been used since a great while ago and that represents a rich variety of products in Southeast Asian countries. The fermented soybean products in Korea are largely classified as soybean sauces and pastes such as Ganjang(soy sauce), Doenjang(fermented soybean paste), Gochujang(fermented red pepper soybean paste), Dambukjang(admixture of soybean paste with other seasonings), and Cheonggukjang(fermented soybean paste by Bacillus). Thus, such fermented soybean www.intechopen.com products are fairly-well known as a common name in the foods of soybean sauces and pastes. Fig. 1 shows the classification of the uses of soybean in Korea.  (Hwang, 1995). Regarding the characteristics of soybean sprout, it can be cultivated without sunlight and shows a short period of growth within seven days at 20~22℃. Also, soybean sprout can be cultivated regardless of locations and season if possible growth conditions are configured. In particular, soybean sprout have been used as one of the most familiar vegetables for Koreans due to its low price and nutritional aspects, which include abundant protein, fat, vitamins, active minerals, and so on (Park, 1991;Kim et al., 1993). Table 3 shows the comparison of the nutrition facts in soybean sprout.  Table 3. Nutritional value of soybean and soybean sprout (Kim, et al, 1993) The Vitamin C contents in the special nutrition facts of soybean and soybean sprout are presented in Fig. 2 (Kim et al., 1993). As shown in Fig. 2, the vitamin C contents largely increase during the growth period of soybean sprout in which it is not detected in the early stage of the growth but shows the maximum level at the seventh day as 18㎎/100g. In the results, it shows the vitamin C can be newly produced through its entire growth period.

Soybean
In addition, changes in the generation of asparagines, which is one of the particular ingredients in soybean sprout, are presented in Fig. 3 (Yang et al., 1977). Growth period (day) Vitamin content ((mg/100g) www.intechopen.com As illustrated in Fig. 3, the asparagines contents in the growth period of soybean sprout significantly increase after six days of starting the growth and show up to 22% of the amount of dried one after 10 days of starting the growth. In addition, the isoflavone contents largely increase during the sprouting period of soybean sprout . Based on these results, it shows that the special functional ingredients in soybean are newly created during its growth period. Soybean sprout in Korea have been used as sub-materials in various cuisines, such as boiled rice with assorted mixtures, boiled rice ball with soybean sprouts, stews, and soups, as exquisite foods (Lee, 2005).

Soybean curd (Tofu)
Soybean curds show a long history in its dietary uses in Korea, China, and Japan and have currently been sold in western markets. In particular, soybean curds have been playing roles in supplying excellent protein sources to the consumers who are lack of animal proteins in these countries. Literatures on soybean curds were recorded about 2,100 years ago at the era of Hoenam King, Han dynasty, China, and showed the propagation of soybean curds to Korean Peninsula before the end of the era of Goryeo Dynasty (Kim, 2005). Then, the production technology of soybean curds was propagated to other neighbor countries since the era. The reason that the soybean curds have been used in recent times even though it has such a long history is considered as its unique taste and particular texture (Jang, 1993 b ) The chemical composition of soybean curds are about 80~85% of water, 8.5% of protein, 5.5% of fat, and 1.5% of ash in which protein covers a high portion of about 42~52% in its solid part. The nutrients included in soybean curds are essential amino and fatty acids required by the human body. Also, soybean curds include some active minerals such calcium, iron, and so on. Fig. 4 shows the process of producing soybean curds (Jang et al., 2008 The major soybean curd products produced in Korea are regular soybean curds, coagulated bean curds, and soft soybean curds. The most largely produced regular soybean curds represent soft texture and rectangular shapes, and have the characteristics of simple taste and flexible resilience. The soft soybean curds represent satiny surfaces and very soft textures. The coagulated bean curds partially include some solid parts in a liquid phase. Although such soybean curds are directly taken, soybean curds are usually processed as stews, soups, soybean curds pancakes, and soybean curds dumplings. The daily intake of soybean curds in Korea has been recorded as 24.5g per person (National Health and Nutrition Examination Survey, 2005). Thus, a lot of people in Korean take soybean curds every day as a large sum.

Creamy soybeans (Soy milk)
Soybean curds are produced using coagulants after grinding and filtering soybeans, and the phase before applying the gelling agent is called creamy soybeans or soybean juice (Kim, 2005). As the creamy soybeans were mentioned in the Hyangyakgugeupbang, which was published in King Gojong (1236), Goryeo Dynasty, it is estimated that the creamy soybeans had already produced by average people in the era of the Unified Shilla(an old country in Korean peninsula) .
Although creamy soybeans are produced by grinding steamed soybeans or by grinding just after applying water immersion, it can be regarded that the grinding and filtering of soybeans are used in the early stage of producing such creamy soybeans  ). The creamy soybeans include several functional substances and then considered as good nutritious foods for preventing chronic diseases (Lee et al., 1997). Creamy soybeans are directly taken as a type of beverage and have been largely known as Korean exquisite foods that are usually prepared with other foods including noodles, such as creamy soybean noodles. Fig. 5 illustrates a simple process for producing such creamy soybeans (Jang et al., 2008

Other soybean products
There are many soybean used as foods in Korea. Some foods use soybeans directly such as soybean Gangjeong(a kind fried glutinous rice or soybeans), Jorim(boiled soybean with seasonings) and roasted soybean powder for making rice cake. Also, soybeans have been used as several purposes for increasing tastes by grinding and applying it to other rice www.intechopen.com products such as soybean rice cakes. In addition, there are lots of homemade soybean used foods (Lee, 2005). Furthermore, soybeans have been used to compensate the nutrition of rice by mixing it with other different foods including boiled rice and boiled rice with different grains. Soybean Gangjeong mixed soybean that has been taken as one of the most popular soybean foods is produced by mixing some puffed grains or soybeans with a concentrate malt syrup. Then, the mixtures of soybeans, sesames, walnuts, pine nuts, peanuts, and so on are cut by proper sizes. The concentrate malt syrup mixed with soybean is taken as a snack because they show nutty, sweet, and other particular tastes. Jorim(boiled soybean with seasonings) are made using black soybeans and boiled in soybean sauces with some starch syrup after immersing soybeans into water. The properly Jorim are taken as a side dish of boiled rice. Soybeans applied to rice cakes contribute to softening its texture and to improving taste. Roasted soybean flours are used to produce rice cakes, such as glutinous rice cakes, using the dough of glutinous rice by mashing it using a wooden hammer. Then, the rice cakes are coated with roasted soybean flours and cut by proper sizes. Rice cakes have been contributed to all types of ceremonies including some happy events and sacred rites and then the cakes are distributed to families and relatives. In addition, rice cakes have been recently taken as good snacks. Roasted soybean flours are produced using a fine mesh in order to obtain fine flours after roasting it with high temperature and grinding it. Also, oil extraction using soybeans is performed applying a modern method. The soybean oil products have been produced and distributed using imported soybeans, which are about one million ton annually. The remains of soybeans after extraction it are used as feeds or soybean sauces and pastes.

Fermented soybean products
Korean traditional foods use agricultural products from a longstanding heritage of agrarian society, and such agricultural products provided by nature have been largely used in practical life due to distinctive four seasons. In particular, the dietary life was determined based on the principle food of rice and then cultures with boiled rice were already settled in B.C. Also, some side dishes were also introduced as the major elements in the dietary life. In addition, some subsidiary food materials that provide specific flavors to boiled rice, which has no particular taste, were required and then subsidiary foods were also developed. However, vegetables were not properly used as such side dishes for boiled rice because these vegetables showed no flavors. Thus, certain brining methods for these vegetables were introduced and developed. By introducing brining methods in Korea, it was an occasion that the dietary life in Korea was changed to fermentation based diets.
Fermented foods in Korea have been formed as the principle foods in Korean dietary life where there are no foods that do not use fermentation methods in diets directly or indirectly. Also, fermented products play important roles in determining the tastes of foods.
In particular, such brining and fermentation methods have been used as a way for spending a winter season, which has no vegetables, and are the chances to form the characteristics of dietary cultures in Korean people. In addition, for presenting tastes, savory, sour, and sweet tastes are created based on the fermentation by salts and that contributed to represent the originality of Korean foods (Shin, 2008).
Therefore, the food cultures in Korea cannot be considered by excluding the fermented foods, and Korean traditional foods should be discussed on the basis of the characteristics of fermented foods. That is, the dietary culture of fermentation becomes the basic fabric of Korean foods and takes a large part of life. Table 4 shows the products that can be produced using fermentation methods.  Table 4, lots of products can be produced using such fermentation methods. This section describes fermented products produced by using soybean in Korea.

Traditional Ganjang (soybean sauces) and Doenjang (fermented soybean pastes)
Soybean sauces are classified into traditional soybean sauces and improved soybean sauces. These two soybean sauces show very different production processes. For producing traditional soybean sauces, it is necessary to produce Meju (fermented soybean lump). Traditional Mejus are made by housewives in average households and handed down to the present time.

Production of Ganjang (soybean sauce)
1. After preparing Mejus followed by the mentioned process, Mejus are to be washed using water and dried again. Then, dried Mejus are soaked in brine . The ratio of Mejus:salt: water is determined as 1:1:3. For maintaining a uniform quality, the concentration of brine and the ratio of Mejus to brine are to be specifically determined. Also, Mejus should be immersed under the brine because it floats on the surface of brine. 2. After soaking Mejus in brine, some red peppers and charcoals are applied to the brine in order to prevent bad smells and germs. 3. Mejus soaked in brine are to be matured for about 60 days at a sunny place and occasionally open the cover of the jar for scorching sun light. 4. After soaking Mejus in brine for 70~80 days, Mejus are to be separated from brine using a filtering patch or mesh. The separated solution is used as raw Ganjang, and the separated solid matter is used to produce Doenjang.

Production of Doenjang (fermented soybean paste)
1. Separated Meju masses are to be mashed and mixed with some soybean sauces, Meju flours, and steamed rice or barley according to needs and preferences. 2. Well mixed paste type raw soybean pastes are stored in a jar or pottery and pushed.
Then, the post maturation of the stored Doenjang are processed for 3~6 months before using it on the table. Fig. 6 shows the production process of Doenjang.

Improved Ganjang and Doenjang
Korean Ganjang and Doenjang are classified into traditional and improved products. In producing improved products, bacteria and fermentation conditions are fully controlled. Fermentation methods are simply presented in Fig. 7

Gochujang (Fermented hot pepper soybean paste)
For producing fermented hot pepper pastes (Gochujang), the mixture of soybeans and grains (rice and others) are cooked and mashed. Then, the mashed mixture is shaped and fermented by molds and bacteria introduced to make Gochujang Mejus. Then, the mixture of Gochujang Mejus, hot pepper flours, and a digested rice syrup by malt, which is produced using some barley malts, is to be fermented for a long time . Gochujang products are divided into two categories such as traditional Gochujangs produced by using traditional fermentation methods and improved Gochujangs produced by improved methods for mass production. Traditional Gochujangs are usually produced by natural fermentation, and improved Gochujangs represent different ways in managing microbesa and fermentation conditions.

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Recently, Gochujang products are made by housewives in average households and produced by middle and large factories based on traditional methods. Improved Gochujangs are produced in a mass production system and cover almost 80~90% of Gochujang markets in Korea. Traditional Gochujangs are usually sold to middle aged consumers, and improved Gochujangs are largely consumed because its major markets are young people and food service businesses.

Production of traditional Gochujangs 1. Production of Gochujang Meju
The mixture with the mixing ratio of soybeans to rice as 6:4 is to be cooked and mashed. Then, the mashed mixture is formed as a donut shape and to be matured and fermented at room temperature for 2~3 months. The fermented Gochujang Mejus are to be dried and powdered for storing it. It has been known that Gochujang Mejus are processed and fermented by Aspergillus species and Bacillus subtilis  3.2.2 Production process of Gochujang 1. Traditional Gochujang Fig. 9 shows a general process for producing traditional Gochujangs.  9. Process of traditional Gochujang making 2. Improved Gochujang A basis for Gochujang is to be prepared and mixed with some flours, rice, and starch sources. Then, the mixture is to be fermented. Pure microorganisms are used to produce the basis of Gochujang, and Aspergillus oryzae and Bacillus subtilis are used to improve its taste. Fig. 10 simply shows the production process of improved Gochujangs. 3. Quality characteristics of Gochujang The taste of Gochujang represents a spicy taste caused by hot peppers, a sweet taste presented by sugars from starch hydrolysis, and a savory taste due to amino acids, peptides and nucleic acid related substances generated by the dissolution of soybeans. Then, these tastes become a fermented spice mixed with a salty taste generated by salts. Several molds, bacteria, and yeasts are contributed to the fermentation of traditional Gochujangs, and taste facts are generated by protease and amylase Shin et al., 1996).  Therefore, the quality characteristics of Gochujang represent a complex taste with various tastes and are very different according to microorganisms related, fermentation conditions, and materials used. In addition, various odors of Gochujang including 3-methyl butanol are generated during its fermentation process and that plays an important factor for presenting flavors according to products .

Cheonggukjang (Fermented soybean paste by Bacillus)
Cheonggukjang is a traditional soybean fermented food that is fully fermented within 2~3 days. Cheonggukjang represent a particular quality characteristic caused by some microorganisms that shows a palatable taste and a unique smell due to the protein generated by protease, which is produced from the proliferation of Bacillus sp. In addition, it produces a sticky viscous material. Although there is a similar product in Japan, Natto, to the Cheonggukjang, it shows different fermentation bacteria and dietary usages. Korean Cheonggukjang are usually used in stews and mixed with other foods instead of taking it directly. However, Natto are directly taken.
Various physiological activities in Cheonggukjang have been largely known, and Cheonggukjang have been produced using traditional methods in average households. Also, it has been taken as a side dish in boiled rice. Also, it can easily be produced in households due to its easy production.

Production process of Cheonggukjang
Although fermentation conditions in Cheonggukjang are almost same for traditional and improved products, the differences in these products are whether they use natural fermentation or manage bacteria using a specific way. The production process of Cheonggukjang is simply presented as follows.
1. Soaking soybean in water Soybeans are soaked in water for 24~30 hours in winter (0~5℃), 16~24 hours in spring and fall (10~16℃), and 10~16 hours in summer (18~25℃). 2. Cooking soybeans Soybeans are to be cooked using a pot in households for about 5~6 hours. Also, the steaming can be performed using high pressure steam (1.5~2kg) for about 30 minutes. If soybeans are not fully cooked, the quality of Cheonggukjang is not guaranteed. Also, over cooking shows bad colors and physical properties.

Fermentation
Cooked soybeans are to be fermented in a jar. Traditionally some rice straws are inserted to soybeans or used at the bottom and upper sections of the jar for inoculating bacteria. It has been known that rice straws have bacteria, which have high protease activities (Lee et al., 1971). The use of rice straws represents a certain way of natural inoculation in which Bacillus sp., which has been known as B. subtilis, contributes to the process Ju et al., 2009). Also, the fermentation takes 30~35 hours at 40~43℃. The fermentation time are largely varied according to temperature and applied bacteria. Also, a humidity condition is important to the fermentation. Tastes and odors are different according to its fermentation period. Recently a two-stage fermentation method that uses two fermentation stages at 42~43℃ and 50~53℃ is used to remove a disgusting smells. After completing the fermentation of cooked soybeans, some sticky and viscous materials are presented at the surface of soybeans and a unique smell is also generated. 4. Maturation Fermented Cheonggukjang masses are to be roughly mashed and mixed with some salts and garlic according to needs and preferences. Then, finished products are matured and stored. The distribution of finished products is distributed under refrigeration. The taste of Cheonggukjang is not changed as it is frozen. 5. Quality characteristics of Cheonggukjang Thermophilic bacteria are usually proliferated in Cheonggukjang according to the fermentation of soybeans at high temperature (higher than 40℃) in which Bacillus sp. is participated. These bacteria represent very particular and unique smells by dissolving soybean proteins. The smells are due to the trimethyl pyrazin and tetra methyl pyrazin newly generated from the fermentation with the 3-methyl-1-butanol in soybeans itself (Choi et al., 1989). It is considered that a large amount of free amino acids are generated in its fermentation process and that affects the taste of Cheonggukjang (Joo, 1971). In addition, soluble proteins are largely increased as much as 40~80kDa and differed from that of Natto (Santos et al., 2007).
The viscous substance that is a particular material in Cheonggukjang represents 61% of crude proteins. Also, the glutamic acid in amino acids shows the highest content, 32%, and a fibrinolytic activity is presented . In the fermentation process of Cheonggukjang, the viscous substance are different according to applied bacteria , and the molecular weight of the substance is about 15,000~65,000. The viscous substance usually consists of glutamate and fructose, and the composition of polymers is varied according to their proliferation conditions (Lee et al., 1992). In addition, there are some reports that the odors, compositions, and threshold values in Cheonggukjang are changed according to bacteria applied to the fermentation of Cheonggukjang Seo et al., 1983).

Other fermented products
In addition to the fermented soybean products mentioned above, other various soybeans fermented products have been used in Korean dietary life. For instance, Doenjang dipping that are produced by mixing some Gochujang, garlic, ginger, hot pepper, and etc. to traditional Doenjang are taken with some wrapping vegetables including lettuces and cabbages. The Doenjang dipping are different types of improved and mixed Doenjang and enjoyed in Korea.
In addition, there are some soybean pastes, such as Eoyukjang made of dried beefs and fishes and Jeupjang made of Meju and dried vegetables. The Jeupjang show a fast fermentation process and can be taken as a side dish of boiled rice during summer season. Such soybean fermented pastes produced by using various sub-materials based on Meju represent different tastes and preservations.

Functionalities of fermented soybean products
Various functional ingredients are already included in soybeans. In particular, a large amount of isoflavones (genistin, genestein, daidzin, and daidzein), which are a type of phytochemical, represent some functionalities in itself (Liu, 1997b). In addition, phenolic compounds (syringic, vanillic, chlorogenic, ferulic, and cinnamic acid), lignan, and carotenoids (lutein, and -and -carotene) including linoleic acid, linolenic acid, licithin, choline, and saponin are included in soybean . As these ingredients in soybeans represent their own particular physiological activities, the value of soybean is reevaluated by the people that have no concerns to take soybean as foods.
As mentioned above, there are many types of physiological active ingredients in soybean. In addition, according to some related studies on such issues, it has been known that fermented soybean products create new functional substances through various microbes. This section proposes the summary of the functionalities of fermented soybean products in Korea, such as Doenjang, Gochujang, and Cheonggukjang.

Functionalities of Doenjang (Fermented soybean paste)
Doenjang are one of the traditional fermented soybean foods in Korea and produced with some other sub-materials including salts based on Meju produced by using soybeans as a principal material through its fermentation process. Meju represent its own physiological activities by generating new functional ingredients during its fermentation process in addition to the functional substances presented in soybeans.
The functionalities of Doenjang are deeply related to the isoflavones of soybeans, and antioxidant, anti-mutagenicity, and anti-cancer effects and ACE functions caused by fermented products have also been known.

Changes in the isoflavone content in Doenjang
It has been known that isoflavones are the glycoside of phenolic compounds and represent effects of preventing breast cancer and prostate diseases (Pagliacci, 1994;Severson, 1989). Also, various functionalities of soybeans presented in Korean Doenjang have been recognized . Based on this study, it can be seen that the ingredients of isoflavones are newly generated during the fermentation process of soybean (  As shown in Table 5, the fermented Meju and Doenjang represent increases in the ingredients of isoflavones. In particular, the Doenjang show a high increase rate in these ingredients. In addition, the change of these ingredients to aglycone, which has a high absorption rate in the body, during its fermentation process by -glucosidase is also presented . The overall isoflavone contents in traditional and improved Doenjang are 370~723µg/g and 170~537µg/g, respectively, where the traditional Doenjang show higher level in the contents (Lee et al., 2010). The isoflavones of Doenjangs are presented as a type of aglycone.

Anti-oxidant effects
In general, anti-oxidant activities are related to anti-cancer and anti-mutagenicity characteristics. It is verified that lots of anti-oxidant substances exist in Doenjang. Doenjang extracts show an increase in oxidation delaying effects according to increases in the addition of the extracts as noted in Table 6 based on the results of the measurement of peroxide values by adding the extracts to linoleic mixtures (Cheigh et al., 1990).  Table 6. Peroxide value of linoleic acid mixture(LA) with the addition of Doenjang powder(DP) during oxidation reaction at 50℃ for 24 and 48hrs.
Anti-oxidant effects in Meju and Doengjang show that it has relation to the amount of browning substances generated from phenolic compounds and its fermentation process .
In the results of the comparison of the anti-oxidant capabilities in browning chromatic substances, which are divided into fat and water soluble substances, significant anti-oxidant effects are presented and the structure of the primary amine is also verified . Also, a strong anti-oxidant activity is presented in water soluble browning substances in which the activity is varied according to its fermentation period . Peroxide values are largely decreased according to the fermentation period of Meju and Doengjang (Table 7) (Kim, 1994 b  In other studies, fermentation processes of Doenjang show increases in phenolic compounds and improve anti-oxidant capabilities in the comparison of peroxide values. Thus, it is concluded that the results are obtained by phenolic compounds and browning substances .

Lowering angiotensin converting enzyme (ACE)
The angiotensinⅡ generated by ACE increases blood pressure and that makes possible to constrain hypertension by interrupting the effect. In the fraction obtained by dissolving Doenjang, the fractions that show ACE lowering effects of 90% and 70% and their structures are determined as arginin and proline as a type of dipeptide . In other studies, physiological activity peptides are separated from Doenjang and their activities are compared in which the histidine content in amino acids show the highest level (Shin et al., 1995). In addition, in the results of the comparison of the ACE lowering activity using methanol extracted from traditional Doenjang, the lowering effects are about 23.6~74.5% where the activity of -glucosidase is also compared (Hwang et al., 2009). Also, in the results of the identification of the strains separated the microorganisms, which contribute to the fermentation of Doenjang, that show excellent ACE activities, the strains are verified as Bacillus pumilus. In other studies, by extracting B. subtilis SCB-3, which represents an effect of lowering ACE, the ACE lo wering rate of the bacterium is 61% by applying it to the fermentation of Doenjang, and IC 50 shows a high value of 0.02mg/mL (Hwang et al., 1997).

Anti-cancer and anti-mutagenicity of Doenjang
In several studies on Doenjang, it has been known that Doenjang have anti-cancer and antitumer effects. Table 8 shows the results of the comparison of anti-cancer effects using SRB assays with respect to cancer cells through extracting the raw materials of Doenjang and methanol extracts (Lim et al., 1999). Survival rate(%) = OD510 of treated cells × 100 OD510 of control cells Table 8. Inhibitory effect of methanol extracts(2mg/assay) of doenjang, other soybean products and soybean on the growth of AGS human gastric adenocarcinoma cells in sulforhodamine B(SRB) assay that determined after 2 days of incubation at 37℃

Samples
As presented in Table 8, Doenjang products represent higher anti-cancer effects than that of the raw materials of Doenjang. In the similar experiments of anti-cancer effects for liver and www.intechopen.com large intestine cancer cells, Doenjang products show higher anti-cancer effects than other raw materials. (Lim et al., 2004). In the results of the comparison of anti-cancer effects for liver and large intestine cancer cells using the Doenjang produced from Sunchang, which is a representative region of producing Korean traditional Doenjang, it shows higher lowering effects in the fractions of acetate and hexane more than 75% (Choi et al., 1999). In addition, in the results of the studies (Hwang, 2007;, it is considered that the peptides and other substances generated during the fermentation of Doenjang contribute to increase such anti-cancer effects. Moreover, as the water used in producing Doenjang is replaced by germanium contained water or maple sap, it show high anti-mutagenicity effects. Also, nine times burned bamboo salts represent an increase in the effects . In the case of the Doenjangs produced by applying mushroom mycelium hyphaes, it shows higher anti-tumor and anticancer effects than that of traditional Doenjang .

Other functions
In traditional Doenjang, other various functions in addition to the above mentioned functions are verified by experiments. The excellent bacteria isolated from Doenjang are used to produce the Doenjang that can prevent thromboses and have high -glucosidase contents (Ra et al., 2004), In addition, Doenjang show the lowering effects of hyaluronidase activities, which have anti-inflammation and anti-allergic effects, about 56~70% (Ahn et al., 2005). Also, Doenjang represent anti-bacterial effects for B. cereus, E. coil, L. monocytognese, and S .aureus (Yi et al., 1999).

Functionalities of Gochujang
Gochujang are a type of fermented seasoning by mixing some rice products dissolved by using hot pepper powders and barley malts based on Gochujang Meju. The functionality of this seasoning is deeply related to the ingredients of capsaicin and fermented products in red peppers. In the classification of the studies on the functionalities of Gochujang, studies are focused on anti-obesity, anti-cancer, anti-tumor, and cardiovascular improvement effects. Also, some studies are focused on the anti-cancer effects on the basis of anti-oxidant effects.

Anti-obesity effects
The capsaicinoid included in Gochujang shows anti-obesity effects by promoting fat metabolism in the body. Based on the effects, a study on the anti-obesity effects using Gochujang is performed through animal and clinical tests ). In the results (a 9.5% of the diet weight) of the measurement of anti-obesity effects in rats, which intake high fat diet, their weights and fat accumulation decrease as 13% and 30%, respectively (Fig. 11). The results show increases in energy consumption (Choo, 2000). In the results of the comparison of anti-obesity effects with that of hot pepper powders, the effects are caused by not only hot pepper powders but the fermentation of Gochujang (Fig. 12). In the results of the comparison of the experiments performed by using various Gochujang, which show different production methods, for rats with high fat diet and Gochujang diet, the fermented rice Gochujang diet shows the highest effects of reducing fat tissues (Lee, 2002). Also, in the results of the comparison of the changes in weight for SD rats with high fat diet and Gochujang diet in order to verify the differences before and after the fermentation of Gochujang, the rats with fermented Gochujang represent high weight-loss effects. In particular, the rats with traditional Gochujangs show large decreases in their weights. It shows that the fermentation process of Gochujangs increases weight-loss effects and decreases fats in epididymis and kidney . In similar studies, in the results of the animal tests by applying traditional and improved Gochujang and hot pepper powders, the fermented Gochujang represent significantly lower increases in their weights and improve the effects of its fermentation based on low intestine weights, low triglyceride contents, and low cholesterol contents (Kim, 2004). The results of the actual clinical tests using Gochujang are presented (Kim, 2009). Gochujang are made as pills and applied to 54 subjects by 32g per day over 12 weeks. In the results, visceral areas and the ratio of visceral/subcutaneous fats are improved as a significant level. Also, the Gochujang intake group shows decreases in triglycerides and improves in serum lipids, fat proteins, altherogenic indexes, and coronary calcium indexes. Thus, it is estimated that the intake of Gochujang for a long period in obese people can prevent obesities and coronary diseases by improving serum lipids and fat proteins.

Anti-cancer and anti-tumor effects
The largest numbers of studies have been conducted are on the functionalities of Gochujang for anti-cancer and anti-tumor effects. In particular, there are many studies on the anticancer functions of Gochujang in which the results of positive effects are verified. In addition, traditional Gochujang products represent higher effects than other products (Park, 2002). The substances for the anti-cancer effects are not determined as a single substance but a complex substance. In the results of the anti-cancer tests for the cells of stomach cancer using the samples extracted from methanol extracts, which are obtained by a freeze drying method from commercially distributed Gochujang with wheat koji, and other materials, the Gochujang fully fermented by using wheat koji represent the most excellent effects (Kim et al., 2005). For measuring anti-cancer and immune activities, onion added Gochujang are produced and processed using a freeze drying method in order to extract methanol extracts . The results show the effects of anti-mutagenicity as a concentration dependent way of Salmonella typhimurium, and onion added Gochujang represent higher effects than others. Also, in the case of MCF-7 cancer cell lines, the onion added Gochujang represent higher anti-cancer effects than others. The generation of NO in macrophages shows increases as a concentration dependent way and that also represents higher level than regular Gochujang. In the results of the comparison of the weight of tumors since 32 days after transplanting Sacroma-180 tumor cells using the samples obtained from freeze dried traditional Sunchang Gochujang and factory made Gochujang by extracting them using methanol, the control group shows 6.0 g and the fermented traditional Gochujang shows 3.3 g. Thus, the traditional Gochujang represents an anti-tumor effect of 45%. However, unfermented Gochujang shows an anti-tumor effect of 17% only (Table 9). Although factory made Gochujang represents an anti-tumor effect of 23%, it represents lower effects than traditional Gochujang . Using the ethanol extracts of traditional Gochujang, the anti-mutagenicity characteristic is verified. Also, in Meju, it is verified that the extracts constrain the mutagenicity caused by aflatoxin B 1 . By obtaining the ethanol extracts of sea tangle powder added Gochujang, the tests of mutagenicity and cell toxicity are performed (Cui et al., 2002). The results show that fermented Gochujang represent significant anti-cancer effects and increase such effects as the fermented Gochujang are mixed with other anti-cancer materials.

Other functions
In the case of the extracts of green teas or natural plants that are added to Gochujang, the extracts added Gochujang show increases in anti-oxidant capabilities and anti-bacterial effects . Also, garlic porridge added Gochujang show anti-oxidant and anticancer effects (Song et al., 2008).
In other experiments, as applying orotic acids, which have been known as a cause substance in accumulating triglycerides to the liver, the adding of capsaicin prevents the generation of such triglycerides and largely decreases the activity of phosphatidate phosphohydrolase, which contributes to the composition of triglycerides, and that leads to constrain triglycerides to the liver (Cha et al., 2004). Studies on the bacteria, which are separated from Gochujang, represent various functionalities in which the strains that produce a polymer of poly--glutamate are Bacillus similar species. This substance has been largely used to control viscosity, to constrain bitter tastes, and to produce various medicines and show a possibility of economic mass productions (Kang et al., 2005). In addition, by isolating the bacteria that have the effects of thromboses dissolution, immunity activation, and cell toxicity from Gochujang, the possibility of the bacteria contributed to the fermentation of Gochujang is considered as a different way through identifying it into Bacillus stearothermophilus and B. amyloliquefacience (Seo et al., 2007).

Functionalities of Cheonggukjang
Cheonggukjang are a pure soybean fermented product that can be used in the table within a short period of time after fermenting cooked soybeans at high temperature and require a small amount of salts and seasonings as in the table. Cheonggukjang have a particular viscous substance and represent a unique small. Also, it shows specific functionalities due to the substances created during its fermentation including the functionalities of isoflavones presented in soybeans itself.
Effects of preventing hypertension, improving fat metabolism, thromboses dissolution, antioxidant, and preventing osteoporosis including physiological effects have been known Lee et al., 2005).

Anti-cancer effects of Cheonggukjang
In the results of the tests of the lowering effects for cancer cells using the Cheonggukjang methanol extracts, the lowering rate is 65% and that is a higher level than that of Miso. Also, it shows the lowering effects for liver cancer cells in which the lowering rate is 60% (Lim et al., 2004). In addition, it shows an effect of constraining other cancer cells (Lim et al., 1999). In particular, by adding the porphyran extracted from lavers to Cheonggukjang, the prophyran added Cheonggukjang represent high constraint effects for cancer cells such as bowel cancer (Min et al., 2008). The results represent that although regular Cheonggukjang show the constraint effects of proliferation as 19~27%, the prophyran added Cheonggukjang represent increases in the constraint effects of proliferation about 27~32%. Thus, it is verified that Cheonggukjang show significant constraint effects on cancer cells.

Lowering Angiotensin converting enzyme (ACE)
Studies on the constraint of blood pressure increases by lowering ACE activities are performed. Cheonggukjang show specific ACE lowering effects (Kil et al., 1998) and peptides are known as its cause factor (Cho et al., 2000). The peptide contents are the order of alanine (30.84%), phenylalanine (30.3%), and histidine (20.24%). In the results of the calculation of the lowering rate of ACE, a 1mg of peptides represents 74.74%. Although traditional Cheonggukjang use straws as a starter (Kim et al., 1982), some superior bacteria are separated from Cheonggukjang and used to the fermentation of Cheonggukjang in order to manage the fermentation (Kil et al., 1998;Hwang, 2010). In these results, the fibrinolytic activity generated by such superior bacteria represents a high level (Table 10). Also, it is verified that the threshold value of enzyme shows increases as Meju are only used as a matrix. In addition, although the microorganisms that contribute to the fermentation of Cheonggukjang are usually Bacillus sp., some strains that strongly secret fibirinolytic enzymes employed in the Bacillus sp. (Kim et al., 1995).

Other functions
In the results of the animal tests, white rats, by inoculating the extracted superior bacteria with respect to high cholesterol and Cheonggukjang intake groups, the effects that constrain www.intechopen.com the generation of peroxide fats in liver tissues and protects liver damages are verified . Regarding other effects, it removes sodium nitrides including anti-oxidant effects and verifies the effects on the constraint of inflammations (Ahn et al., 2005). In addition, the application of Cheonggukjang effectively constrains increases in blood pressures from animal tests (Yang et al., 2003). Also, the sticky viscous substance shows decreases in blood sugars and serum lipids .

Conclusion
The origin of soybeans has been known as Manchuria, Far East Asia. Then, soybeans were propagated to various countries of the world. In some Southeast Asian countries, in particular Korea, China, and Japan, soybeans have been variously used in their dietary life and contributed to improve nutrients as protein sources. The history of using soybeans goes back to B.C. On the other hand, in Western countries, soybeans have been largely used as feeds and oil source. However, recently these countries also use soybeans as food materials and produce various soybean processing products. Soybeans are an important position in Korean dietary life as one of the five major grains and have been taken by soybeans itself with boiled rice. Also, soybeans have been variously used by processing it or by growing it as vegetables. Then, soybeans have been taken as vegetables, powders, and other various types of foods differed from other countries. In particular, fermentation methods using molds, bacteria, and yeasts are developed using cooked soybeans and that leads to produce Ganjang(soybean sauces,) Doenjang (fermented soybean pastes), Cheonggukjang, and Gochujang(fermented hot pepper soybean paste). These products have been used in Korean dietary life as the primary seasonings and side dishes. Also, these soybean products play important roles in differentiating Korean foods from other foods.
Recently phytochemicals including isoflavones in soybeans have been known as functional ingredients. Also, it is verified that soybean proteins have physiological activities from the results of various studies. Therefore, people around the world represent interests on the use of soybeans as foods. In addition, lots of soybean processing products have been produced and distributed due to their effects on preventing various chronic diseases through simple pharmacological treatments only. In Korea, regarding the characteristics of using soybeans, various soybean products have been developed using specific fermentation processes. It has been scientifically proven that various functionalities are newly created during several fermentation processes. Also, studies on this issue have been continuously conducted. In the specific functionalities of soybean products, it contributes to present effects on anti-cancer, anti-tumor, improving blood circulation, and preventing chronic diseases. In the case of Gochujang, it has been known that Gochujangs represent significant effects on constraining obesities.