In this investigation, 1% of essential oils from three different oregano species and the oil of  Foeniculum vulgare (fennel) were tested for their antimicrobial activity against 4-chloro-3-methylphenol and commercial bactericides commonly used in the leather industry.  
The results showed that three essential oils of oregano had much stronger bactericidal activity than the commercial bactericides or the fennel. The findings suggest that these oregano essential oils could be used as bactericidal agents in the leather industry.
Microbial activity is responsible for the degradation of organic substances leading to some undesirable consequences. To avoid such activity in processing, which can have severe economic consequences for the tanner, specific care must be exercised2.
Soaking is the first process which rehydrates and cleans hides and for a short soaking period bacteria aren’t a huge problem. But, when the hides are too dry and longer soaking periods are needed, this can lead to exponential microbial growth which may also cause permanent damage.
Various researchers have reported that a wide variety of bacteria can be isolated from the soaking water, including species of Bacillus, Chromobacter, Pseudomonas, Clostridium, Lactobacillus and Serratia marcescens3. Since the raw material is not sterile4 during the soaking process, bacterial attack can lead to putrefactive odours and hair slip and, depending on the extent of the damage, tiny abrasions known as ‘pin prick’ may develop. The resultant leather may have grain loss3 and with further damage to the epidermal layer, a matt and lustreless grain, loss of ubstance etc, can also occur.
The bactericidal agents currently used in the industry are generally harmful to human health and nature, and their use has been either restricted or banned in certain countries, for example, pentachlorophenol (PCP)5, while the use of polyhalogenated phenolic compounds (TCP/TBP) has also been restricted6.
As can be seen from these particular examples, it is essential to discover new chemicals that can be used safely in the leather industry to counter undesirable effects of microorganisms. It has been known  for many years that essential oils are 100% natural chemicals displaying varying degrees of antimicrobial activity. These oils are volatile compounds of a plant’s secondary metabolism, and may be used as phytoproctective agents7,8.
The objective of this study was to determine whether the essential oils could be used as alternative bactericides in the leather industry. Essential oils of three different oregano species plus an organic fennel oil were tested for their bactericidal activity against a commonly used antimicrobial agent. This preference was based on the earlier reports pointing to their use in the food industry and health related issues as antimicrobial agents 1,9,10,11,12,13,14.
Materials and Methods
Raw skin
Five native sheepskins (salted-dry) were selected with six pieces (weighing about 100 grams each) used in soaking tests, which contained a known bactericidal agent (phenol, 4-chloro-3 methylphenol, sodium salt), three different essential oils and the fennel oil.
Essential oils and bactericidal agents
The essential oils: Origanum onites (Turkish oregano, potmarjoram), Origanum sp (1) and Origanum sp (2) and fennel oil were purchased from a commercial company. The oils were prepared in ethanol in equal volumes and the final concentration was adjusted to 1% in the soak water. 1% of 4-chloro-3 methylphenol was chosen as the control.
The percentage constituents of the essential oils within the three oregano species and F. vulgare (fennel oil) are presented in Table 115,16,17,18.
Soaking process
All washing procedures were performed in sterile distilled water (DW). Pre-washing steps (for 1 hour) of the samples and the consecutive soaking steps (8-24 hours) took place in 1 litre aliquots of sterile DW. The aim of the pre-washing process was to let the water diffuse through the skin, removing the dirt, blood, conservation salt, dust etc. The bactericides were added prior to secondary soaking steps and the samples were mechanically agitated for ten minutes per hour.
Growth media
PCA (Plate Count Agar-Oxoid) medium was used for total
bacterial counts (aerobic mesophyllics). 1 ml aliquots were taken from each of the soak waters after eight hours and 24 hours respectively. The aliquots were further diluted (101 to 106) in sterile saline and 0.1ml samples in duplicates were poured into PCA medium (55°C in a water bath). The Petri plates were incubated for 48 hours at 37ºC and the bacterial counts were averaged and expressed as cfu/ml.
Results and dıscussıon
Bacterial counts obtained from six different soaking regimes of the five sets with eight and 24 hour intervals were averaged.  As can be seen, Origanum onites (O0), Origanum sp (O1) and Origanum sp (O2) displayed varying degrees of antibacterial activity at concentrations around 1% compared with the control (C).
The data, when examined statistically on the logarithmic scale (Figure 5), indicated that the dimension or direction of bacterial growth was not the same for all groups between eight and 24 hour soaking periods. As time proceeded, the number of bacteria increased for the bactericide, control and the fennel sample, and it decreased for Origanum onites, Origanum sp (1) and Origanum sp (2) samples respectively. This can be attributed to a meaningful group-time interaction (p<0.05).
The changes observed from eight to 24 hours in each group were subjected to the Paired Samples t-test. The increase in bacterial numbers for the control, the bactericide and fennel oil and, a decrease for Origanum  sp (1) and Origanum sp (2) groups were statistically significant, whereas the Origanum onites group were not (p=0.196 and p>0.05).
The differences between the groups, by the end of the eighth hour, were evaluated with Oneway Anova and the differences between the groups by the end of 24th hour were organized according to the values of the eighth hour, and evaluated with Ancova (Analysis of Covariance) analysis respectively. In conclusion, the difference among the groups from the 8th to 24th hour has been found significant.
Examining the variations between the groups at the eighth hour, a significant difference was observed reciprocally between the following: the control and Origanum onites, the bactericide and fennel oil, Origanum onites and the control, Origanum sp (2)-fennel oil, Origanum  sp (1)-fennel oil, Origanum sp (2)-Origanum onites and fennel oil, and finally between fennel oil and all other groups (p<0.05).
The differences among the groups at the 24th hour were as follows: a significant difference was found among all the groups except that of the control and fennel oil (p<0.05).
The differences scored as significant among those groups were between the bactericide and all other groups, Origanum onites and the control, the bactericide and fennel oil, Origanum  sp (1) and the control, the bactericide and fennel oil, Origanum  sp (2) and the control, the bactericide and fennel oil.
The use of fennel oil as an antimicrobial agent has been reported19. Our results were also supported by this report. Although fennel oil seemed to increase the bacterial growth during an eight hour soaking period, at the end of the 24 hour period the bacterial counts were less than the control groups.
The study indicated that all oregano oils used during the experiments had a greater effect than that of the bactericide and the fennel oil. The most effective oil was Origanum onites, of which carvacrol content was the highest (84.48%).
As a result of the study, it can be concluded that Oregano essential oils displayed both antifungal and antibacterial activities. Further investigation as a bactericide in leather production is recommended.
The authoress would like to express her gratitude to Prof  Dr N Gürdal Alaeddinoglu for the information he provided and for the interest he has shown in the study. The author’s heartfelt thanks are also due to Türer Tarım for providing essential oils and to Ege University Research Fund Department for the financial support.
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