As far as culture conditions are concerned, there is usually a dilemma between achieving maximal fungal growth rates and maximal antibiotic yields because conditions that allow fast cell growth could be unfavourable to metabolite production (Miao et al., 2006). Furthermore in submerged fermentation there is always production of metabolites which is not dependent to the fungal growth. The yield of metabolites can sometimes be substantially increased by the improvement of physical parameters such as incubation temperature, initial pH medium value, size of inoculum, agitation speed and light (Calvo et al. 2002; Llorens et al. 2004).

The maturity of the pre-culture seed was studied to determine its effect on fungal growth and production of their secondary metabolites. The current findings revealed that younger seed culture produced the highest anti-MRSA activity as well as fungal growth. Meanwhile, older pre-culture seed resulted in decrement of fungal growth as well as their secondary metabolite production. This condition could be due to the old culture that was in inactive condition which needs an extent cultivation period to reproduce and re-propagate new mycelia, whereas the young active culture just continues their growth phase in a new fresh medium. Brookman et al., (2000), did a comparative evaluation on fungal growth and the results revealed that the active growth of older cultures as inoculum was delayed compared to young cultures. Similarly, Villiers (1973), reported that older seed culture might delay the germination due to repairs and replacements mechanisms in the cell that might affected organelles and enzymes. Besides that, Herker et al., (2004) reported that apoptotic regulation occurred in older microbial cells that releasing a substance into medium to stimulate the survival of new younger and fitter cells. This resulted in delayed biomass production.

Endophytes are microorganisms that symbiotically colonized within plant tissues for all or a part of their life cycle without causing any harmful effect to their host (Hirsch and Braun, 1992; Wilson, 1995). Fungal endophytes benefit and offer several advantages to their host by preventing them from desiccation, promoting plant growth hormone, increasing nutrients acquisition, protecting from insect, pathogenic fungi and environmental harsh conditions (Dutta et al., 2014; Nair and Padmavathy, 2014). Meanwhile, the host plant would provide the endophytes with shelters, space for their colonization and also nutrients acquired by them (Saikkonen et al., 2004; Schardl et al., 2004). It was observed that culture medium supplemented with host plant extract enhanced the production of fungal metabolites and their growth. The current findings were in agreement with Yenn et al., (2012) who reported the anti-candidal activity was produced by Phomopsis sp. ED32 when the culture medium was supplemented with host plant extract. Host plant extract is believed to supply the endophytes with crucial needed nutrients or growth compounds that could be used in biosynthesis of cellular components resulting in increment of fungal secondary metabolite production (Tong et al., 2011; Padhi and Tayung, 2013).

The effects of light on the secretion of fungal bioactive metabolites and their growth were investigated and the result showed that the presence of light slightly decreased the anti-MRSA activity of L. pseudotheobromae IBRL OS-64. Schmidt-Heydt et al., (2011) reported the same observation in which the presence of light reduced the production of ochratoxin. Similarly, Soliman and Raizada, (2018) claimed that the presence of light exhibited inhibitory effect on the production of fungal secondary metabolites. In the Taxol-producing experiment, they observed that the complete loss of taxol production occurred when the endophytic fungus SSM001 exposed to light. Kim et al., (2013a), reported the inhibitory effect of the light might be due to unavailability of LaeA nuclear protein which was a regulator protein that involved in biosynthesis of fungal metabolites and thus leads to the inactivation of secondary metabolites production. Furthermore, Kim et al., (2013b) found that white light induced the carotenoid biosynthesis by Fusarium graminearum strains. They suggested this phenomenon might be due to the role of fgWC-1 and fgWC-2 that act as negative and positive regulators, respectively that monitored the light requirement of F. graminearum in its biological process. Furthermore, in filamentous fungus, the light-dependent gene was expressed with the light exposure and thus, enhancing secondary metabolite production by activating the light-mediated control of the DNA expression (Wang et al., 2014). However, Velmurugan et al., (2010) stated that the fungi may react differently according to light wavelength. For instant, certain light wavelength might enhance extracellular pigment production and some are not.

The initial medium pH of growth medium is a crucial factor for fungal growth and production of their secondary metabolite since it affect the function of several enzymes in catabolic reaction and also influences on complex physiological phenomena such as cell morphology and their cell membrane permeability (Guimaraes et al., 2004). In the present study, the slightly acidic growth medium (pH 6) was the best for the production of fungal secondary metabolite. The result was in agreement with previous study that reported pH 6 was the best for bioactive metabolite production of Streptomyces coeruleorubidus isolated from soil (Bundale et al., 2015). The current findings also revealed that production of fungal secondary metabolite of L. pseudotheobromae IBRL OS-64 was decreased with the increment of pH and the similar observation was reported by Keller et al., (1997). Drori et al., (2003) reported the increasing in pH of the growth medium from 4 to 6 was an ambient pH transduction pathway which increases the production of secondary metabolites by C. gloeosporiodes. Noaman et al., (2004) suggested the pH of growth medium is closely related to permeability of microbial cell wall and membrane and therefore, affected in ions uptake and loss in the growth medium. According to Rizk et al., (2007), the concentration of hydroxyl and hydrogen ion might have direct or indirect effects on the cell by varying dissociation level of substances in the medium. Thus, the change of pH in the medium was crucial for microbial enzyme activity especially for dissociation and solubility of intermediate products. Therefore, the extremely low and high pH of the growth medium is believed to disturb enzyme activity resulted in suppression of their secondary metabolite production.

Another cultural condition that has big effects on fungal secondary metabolite production is cultivation temperature (Rizk et al., 2007). In the present study, cultivation temperature of 30°C produced the highest anti-MRSA activity by L. pseudotheobromae IBRL OS-64. The results were in agreement with Bhattacharyya and Jha, (2011) that reported the fungal growth and their bioactive metabolite production increased with the increment of cultivation temperature from 25°C to 30°C. The current finding also revealed that lower cultivation temperature resulted in slightly decrement of bioactive compounds and the temperature higher than 40°C killed the growth of L. pseudotheobromae IBRL OS-64. Gogoi et al., (2008) stated that a fungal endophyte, Hypocrea spp. NSF-08 which was cultivated at temperature of 28°C produced the highest production of bioactive metabolite, and the fungus could not grow at temperature below 20°C and above 50°C. Agastian et al., (2013), stated the lower temperature might cease the fungal metabolite activity whilst at higher temperature, they might be killed.

Inoculum sizes are important in fermentation as well as secondary metabolites production by fungi. Low inoculum size may influence the time extension for cell proliferation to utilize substrate and produce the desired product of secondary metabolites (Darah et al., 2015). Moreover, low inoculum sizes might lead to prolongation of the lag phase of fungal growth and resulted in slow growing rate and greater fungal viability (Baert et al., 2008; Garcia et al., 2010). In the present study, this problem could be observed at inoculum size of 1 mycelial plug, where lower anti-MRSA activity was produced. On the other hand, higher inoculum size than 1 mycelial plug increased anti-MRSA activity. Thus, an inoculum size of 2 mycelial plugs was found to be optimal for the production of maximal anti-MRSA activity. The anti-MRSA activity declined slightly with further increment in inoculum sizes. Similar observation also reported by Sidhu et al., (2017) in which two mycelial discs was sufficient to stimulate higher laccase production by Scytalidium lignicola under submerged fermentation. Higher inoculum sizes than the optimal may stimulate too much production of microbial biomass which lead to nutrient depletion for their secondary metabolite production (Kumar et al., 2010). According to Ahsan et al., (2017), moderate inoculum size has a significant positive effect on the antibiotic production and growth yield. Limited space and less oxygen supply might occur if higher sizes of inoculum used in cultivation. However, if lower sizes of inoculum applied, the excess materials in the culture may turn toxic and thus lead to unfit fermentation products.

Microbial activity of aerobic cultures is markedly affected by the oxygen supply to the system. There are varying functional aspects of aeration in submerged fermentation including the maintenance of oxygen supply, removing carbon dioxide from the system and heat transfer. Aeration provides good transfer of nutrients and product gases in the submerged fermentation system. Agitation speed and dissolved oxygen are important factors in determining the successful of microbial cultivation whereby the sufficient dissolved oxygen in growth medium is crucial in mass transfer characteristics of microbial cells (Ibrahim et al., 2015). The present study revealed that the static condition produced the highest anti-MRSA activity and the activity was decreased with the increment of agitation speeds. The result obtained was in agreement with previous studies that reported the highest production of bioactive compound was obtained under static condition (Bringmann et al., 2007; Tamminen et al., 2014). The favorable effect of air flow on the secondary metabolite production could be attributed to the enhancement of product formation by microorganism under-forced aeration. The cell differentiation might be occurred under insufficient dissolved oxygen which leads to more formation of aerial hyphae and conidiophores and thus, increases the accumulation of bioactive compounds (Scott and Eaton, 2008; Tang et al., 2015). This statement was supported by Pearce, (1997) who postulated that the fungal are able to store bioactive compounds in their mycelia. According to Ahsan et al., (2017), agitation speed influenced the dissolved oxygen temperatures and oxygen availability and therefore affecting the antibiotic production. Moreover, oxygen availability is one of the crucial factors for cell growth and production of their secondary metabolites during fermentation process (Wang et al., 2010; Dou et al., 2013). Besides that, Song et al., (2012) postulated that proper ventilation should be considered during fermentation process since the culture media containing organic and inorganic compounds that lead to the decrement in dissolved oxygen level. In contrast, Noraziah and Izyani (2012) reported the agitation could increase biomass formation by supplementing more dissolved oxygen. However, the fungal growth and their fungal mycelium morphology is dependent on several factors including the growth medium, hydrodynamic in the bioreactor, fungal strains, and method of initiation of culture such as mycelium dispersion (Lopez et al., 2005). Interestingly, fungal growth of the L. pseudotheobromae IBRL OS-64 was not affected either under stationary or agitated condition since the value was almost the same even though the anti-MRSA activity was varies. According to Barborakova et al., (2012), the bioactive production is not dependent to the quantity of biomass. The agitated and static conditions of cultivation conditions might have their pros and cons, but the adaptation to the harsh environment is depends on the fungal strains and their response mechanisms to stress conditions. Even agitation is believed to provide adequate mixing especially for dissolved oxygen, heat and mass transfers, perhaps it also induce shear forces which resulted in morphological changes and damage of the cell structures (Darah et al., 2011; Zhu et al., 2012).

Cultivation time is also a crucial parameter in determining the success of fermentation process to gain fungal secondary metabolites. In the present study, the anti-MRSA activity was detected after about a week of cultivation time. The late detection of anti-MRSA activity might be due to the production of anti-MRSA compound as fungal secondary metabolites. According to Bu’Lock (1961), secondary metabolites are secreted during the late stage of microbial growth, at a stationary phase. It was noted in this study that the extended cultivation time (22^th day of cultivation time) produced maximal yield of anti-MRSA activity. The result was in agreement with Yamanaka et al., (2008) who reported the prolonged cultivation period could induce secondary metabolite production by Trametes versicolor. They also suggested that the cells are physiologically active at a long period of cultivation. Generally, the extended cultivation time would create unfavorable conditions which due to starvation of carbon or nitrogen, accumulation of self-toxic metabolites, translocation of nutrients, elevation of pH, increment of temperature and degeneration of fungal mycelium. These unfavorable conditions are believed to adversely affect the expression of microbial secondary metabolites (Griffin, 1995). According to Sejiny (1991), during the propagation of antibiotics producers, two phases were observed which were trophophase and idiophase. Trophophase is characterized by rapid growth of microbial cells producing biomass and during the idiophase, slow microbial growth was observed and maximal antibiotic production was obtained. In the present study, all the major physical parameters which were believed to affect the fermentation process for anti-MRSA production were investigated, and the results showed that the 16^th day of cultivation time produced maximal production of anti-MRSA activity.