Evaluation, Enumeration and Control of Bacteria in Rawstock.

Transcription

1 Evaluation, Enumeration and Control of Bacteria in Rawstock. K Hartung, AJ Long, (BLC Leather Technology Centre, UK) C Dodd, S, Movahedi, A Mauger, (University of Nottingham, UK) N Pritsos, (ELKEDE Technology and Design Centre, Greece) Abstract Research has recently been carried out under a European CRAFT funded project named MICROSTAT. This project is related to the evaluation and enumeration of bacteria in hides and skins with the aim of developing alternative control and detection methods. The research has focused on key problem areas within the industry related to the prevention of damage to raw hides and skins due to putrefaction. Using techniques such as DNA extraction of the bacteria obtained from hides at different stages of putrefaction it has been possible to identify some of the key bacterial species inhabiting hides. One interesting conclusion is that 90% of the species isolated from salted skins so far have been found to have collagenolytic activity. This indicates that salting alone may be insufficient to control microorganisms that may be detrimental to the condition of the skin. The control of the bacterial populations on the hide is critical to hide quality. Microbial control techniques which are being investigated include UV light, chilling, trisodium phosphate (TSP, a detergent) and bacteriocins. BLC has had a bespoke UV sterilisation system built which has been used to subject hides to different levels of exposure to UV light (at 254nm wavelength), which is known to have bacteriocidal properties. Initial trials have shown that this is a promising technique for the rapid control of bacterial populations from hides and skins. Combining the control techniques with detection techniques is another challenging aspect of this project; an ATP bioluminescence system is currently being evaluated to monitor hide quality. The system can detect ATP of microbial origin; the greater the quantity of microbial ATP found, the larger the bacterial community present. This technique is an excellent indicator of microbial levels and is used widely within the food sector, and it appears that technology cross-over is achievable. Electronic nose technology is also being investigated as a means of identifying the volatile gases given off by bacteria that result in putrefaction of hides. By relating the change in profile of the volatile compounds detected by the electronic nose to bacteria number and skin condition, it will be possible to determine the sensitivity of the electronic nose for the early detection of bacterial action. A sufficiently sensitive system would be capable of detecting the activity of bacteria before the manifestation of the physical signs of putrefaction and, consequently, before damage to the skin had occurred.

2 Introduction The raw material used in the leather industry is hide and skin, which is subject to attack by microorganisms from the point of slaughter. The degradation of this raw material results in excessive waste in the tannery leading to re-working, with the associated added costs of chemicals, energy and processing times, and often leading to an inferior product. The estimated cost of this lost potential to the leather industry is approximately 40M Euro annually within Europe alone. Currently, the most widely used form of preservation for raw skins is salt curing. Curing with salt has a bacteriostatic effect (i.e. it inhibits the growth of bacteria but does not destroy them) by reducing the moisture content of the skin. In addition there are various factors related to the state of curing and storage of she skins that may lead to proliferation of bacteria. Salt curing, although economically viable, is not an effective method for the control of all bacteria. Salt tolerant bacteria, termed halophiles, continue to proliferate. Reduced grain quality has been reported on leather manufactured from brine-cured hides and this has been linked to the growth of halophiles on the skin during storage. As a result of these factors, tanners often receive raw material with a significant microbial loading, which may give rise to putrefaction damage during subsequent storage, which is often several weeks or even months. Several key indicators do exist which are characteristic to hides with bacteria damage. These include hair slip, pitting and red heat (characteristic of halophilic bacteria damage). The main objectives of the MICROSTAT project are to measure and improve the microbiological quality of raw material through the use of novel techniques of quantification and preservation. The aim is thus to limit bacterial damage and improve the quality of the end leather product. This will be achieved through better understanding of the methodology of bacterial control and by using alternatives in place of traditional curing. The methods evaluated include nisin, ultra violet (UV) light, boric acid, trisodium phosphate (TSP) and chilling. In conjunction with this, there is a need for a simple but accurate method for quantifying the microbiological quality of raw material, thus enabling the tanner to identify inferior raw materials. Two techniques were evaluated; namely an electronic nose system and an ATP bioluminescence kit. Both methods have been used successfully in the food industry as accurate procedures for predicting bacterial numbers. Characterisation of Bacteria Populations On British and Greek Hides Work carried out within the project aimed to both identify, using Denaturing Gradient Gel Electrophoresis (DGGE) and count (using standard plate count techniques) bacteria on hides supplied by a Greek tannery over a 9-month period. Results showed that salting led to the elimination of E.coli, coliforms, lactic acid bacteria and Pseudomanas species, but there was a dramatic increase in bacteria classed as collagenase producers after storage in excess of 1 month. In parallel to this there was an increase in halophilic bacteria populations, which increased to 10 4 colony forming units

3 (cfu) cm 2 (see figure 1). DGGE analysis showed that after this period, Bacillus species dominated (Data not shown). 6 Log bacterial count (cfu/cm2) aerobic 37 aerobic 15 anaerobic 37 collagenase 15% salt 25% salt 35% salt Staphylococci Pseudomonads Lactococci Lactobacilli Yeasts & moulds E.coli Coliforms Sep Nov Feb May Figure 1. Analysis of microflora of Greek hides on different culture media. Salt concentrations of 15, 25 and 35% were used to determine the presence of halotolerent and halophilic bacteria. In addition to analysis of Greek hides a similar analysis was carried out on British hides, (over 6-month sampling and analysis period). The results showed similar trends and are illustrated in Figure 2.

4 8 7 6 Log CFU/cm aerobic 37 aerobic 15 anaerobic 37 Collagen 15% salt 25% salt 35% salt Y&M Staphylococci Pseudomonads Month 2 Month 3 Month 4 Figure 2. Analysis of microflora of British hides in months 2, 3 and 4 of a 6- month trial, on different culture media. 15, 25 and 35% concentrations were used to determine halotolerent and halophilic bacteria species. The results obtained from this analysis are of considerable significance for the leather industry. Colleganase producing bacteria are known to cause the degradation of hides as they result in enzymatic hydrolysis of the substrate 1. Populations of these species are shown to increase over time on Greek hides whilst remaining reasonably constant on British hides. From this work it has been shown that salting alone fails to preserve the hide from potentially damaging bacteria although physical tests would have to be completed on finished leather to show this. In response to these results, the salt sources from the Greek and British tanneries were investigated. Salt samples, as used by the British and Greek industrial sectors, were analysed for the presence of bacteria. Extreme halophiles were isolated from both British and Greek salt sources, which in itself could be a source of hide damage. Similar investigation of Finnish salt sources along with British Pure Dried Vacuum (PDV) sources showed that no halophiles were detected. This indicates that in the experiments that were carried out the salt sources themselves are the main bacteria source Trials were therefore carried out with the aim of identifying the role of these isolates in bacterial damage of hides. Isolates from various salts were applied to hides and were incubated at 37 C for a week. During this time levels of hair-slip, pitting and red heat was monitored and graded according to their extent. The results showed that the extreme halophiles isolated from the salt, caused red heat but little pitting or hair slip. The use of laboratory grade salt with or without laboratory grade boric acid (another preservation method for the reduction of halophilic bacteria) on unsalted hides caused little or no pitting but when boric acid was used, more significant pitting was evident. The results

5 have highlighted that the salt source itself may be a contributing factor to hide quality. They also suggest that the application of boric acid causes an increase in pitting (due either to bacterial or chemical action) although this requires further investigation. The use of ATP bioluminescence kit to predict bacterial numbers on hides One method which has been developed for rapidly predicting total microbial loads, particularly in the food industry, is adenosine triphosphate (ATP) bioluminescence. The basis of the method is the isolation of microbial ATP from samples under investigation (in this case hides) by using a bacterial releasing agent solution. Following this a subsequent quantification can be made using a luciferase/luciferin complex (obtained from fireflies). The resulting reaction generates inference light, the amount of which is proportional to the concentration of bacteria present on the (hide) sample. A summary of the chemical processes involved is as follows: Step 1 Luciferin + luciferase + ATP = luciferyl adenylate-luciferase + pyrophosphate Step 2 Luciferyl adenylate-luciferase + Oxygen = Oxyluciferin + luciferase + ATP + light Initial trials were carried out to determine the accuracy of the method for evaluating bacterial numbers on rawstock for the leather industry. When tested on 35 hides, the ATP assay results correlated well with the traditional viable count results obtained using both standard plate count methods (SPC) and collagen agar. The correlation coefficients for fresh hides were and on SPC and collagen agar respectively. For salted hides, the coefficients were on SPC agar and on collagen agar. Possible factors for the lower correlation observed for salted hides included Salt may interfere with the components of the assay, Some spores present in the samples may not be giving an ATP reading but may still contribute to plate counts Haleophilic bacteria may produce less ATP. Trials were completed at two European tanneries to analyse samples of hides of varying age and thus varying bacterial condition using this method. In addition 10 hides were sampled from two stockpiles, which had been stored for differing times. The aim here was not to monitor predicted bacteria numbers as hides from different sources will naturally contain higher or lower populations, but to see if the variation in the samples could be distinguished using the method. The results of the trial from two tanneries are shown in Tables 1 and 2 below.

6 Table 1. ATP Results from Stored Hides - Trial 1. Hide storage ATP count (cfu/cm 2 ) Medium storage (3-4 weeks) Long term storage (3-4 months) day storage-medium salted day storage-heavy salted day storage-medium salted day storage-heavy salted 75.4 Sample with red heat day storage-salted (average) day storage-salted (average) Table2. ATP Results from Stored Hides - Trial 2 Hide storage ATP Count (cfu/cm 2 ) 1 day storage week storage week storage week storage (average) month storage (average) 52 Estimates of CFU/cm 2 were made by comparison of the ATP data with a standard curve of the ATP data with a standard curve of RLU (the measurement of bioluminescence test) verses CFU (determined by plate counts) of known bacterial populations. Although it is not possible to draw any direct conclusions from these trials related to bacterial populations following storage (as the hides came from different sources) it is clear that different hides can be distinguished in terms of their ATP count. Results in table 2 though, indicate initial bacteria numbers increase over a 1-week period, probably due to high sources of available nutrients. As these source become scarce bacteria numbers then decline due to the lack of nutrients and subsequent mutalsitic competition. Electronic Nose Electronic noses are a relatively new technology and are currently finding uses in a wide range of industries such as the wine, dairy and meat sectors for measuring odours given off by a sample. Typically the technology utilises stationary gas chromatography phase in association with a sensor. Several sensors are available including metal oxide, conductive polymers and quartz crystal sensors. Like ATP bioluminescence, the electronic nose is being investigated for its use in predicting the bacterial status of raw hide. Some work has been carried out historically to evaluate the technology for application in the leather industry. The main problems associated with the previous work include obtaining sensors, which have the ability to distinguish relevant volatile compounds and separate these from irrelevant interference.

7 In partnership with a German engineering company BLC set out to see if an electronic nose system could be used to distinguish between hides, in various bacterial condition, and from various different sources. Successful work of this nature would provide an excellent foundation to developing a system, which could be applied in the tannery. The initial aim of trials carried out was to see if different samples could be distinguished. Samples evaluated during the ATP analyses were also subjected to trials using an electronic nose system. At this stage the trials look promising. The results show clustering occurs between points obtained from the same samples indicating that the electronic nose can distinguish between samples of varying status (Figure 4). Although currently the system give little indication of the condition of the hide these results along with detailed discussions with electronic engineers indicate there is a firm foundation for the use of electronic nose to predict hide condition. Salted medium term (4-6 weeks) Long term storage (3-4 months) Salted-2 days Salted-1 week Salted-2 week Figure 3. An example of electronic nose readings obtained from trial 2. It is clear that replicate analysis of samples show clustering. This indicates that the electronic nose can distinguish between samples of different bacterial status. Alternative Preservation Techniques Curing of hides currently involves using huge amounts of salt, often in the region of 40% based on green weight and, therefore, the effluent discharge from tanning operations contains large amounts of total dissolved solids (TDS) and chlorides. Soaking contributes to 40% of the TDS that is generated from the whole leather processing procedure 2 and this is mainly due to the salt content.

8 The complete replacement or partial substitution of the total offer of salt could significantly reduce the costs of tannery effluent treatment. Various alternative preservation techniques were therefore evaluated and summarised below. Nisin The use of nisin as a preservation technique, particularly in the food and dairy industry has long been known 3. Nisin has been shown to work as a preservation technique as it targets the cytoplasmic membrane thus disrupting bacterial replication 4. Work carried out at the University of Nottingham, using nisin, as a preservation has proven to be positive, although full data will not be available for publication until a later date. Ultra Violet (UV) Light UV light has been shown to eliminate certain bacteria, especially with respect to drinking water. The aim of this treatment would not be the total replacement of salt but would hopefully lead to a reduction of total offer required. An investigation to assess the suitability of UV-sterilisation technology within the leather industry to was completed. It was conducted using a UV unit containing 10 low-pressure lamps emitting a bactericidal wavelength of 254nm. Exposure limits were investigated to identify the maximum level of exposure required for sterilisation of the hide whilst avoiding damage to the raw material. In order to assess the suitability of UV light, small scale trials were completed, with bacteria numbers being estimated using the ATP bioluminescence kit. The results indicated that 30 seconds was the optimum exposure time to reduce bacteria numbers. Following this initial evaluation, further trials were carried out on two hides to assess the potential of using UV light in conjunction with salting. Each hide was divided into two down the backbone and then into six sub sections. After exposing one side of each hide to UV light for 30 seconds, the hides were salted with either 20% or 40% salt. The hides were then stored and sampled for total bacteria (using nutrient agar with 0, 10 or 20% salt) at regular intervals over three months. The results (only 40% salted hide data available) are shown below:

9 Table 3. Total bacteria counts seen on nutrient agar after one and two months storage from hides stored with 40% salt with or without UV treatment. 0, 10 and 20% salt concentrations were also used with the nutrient agar to isolate halophilic and halotolerant species. Month1 Nutrient agar (NA) NA + 10% NaCl NA + 20% NaCl Month2 Nutrient agar (NA) NA + 10% NaCl NA + 20% NaCl UV 1.45E E E E E+04 Control 1.88E E E E E E+04 The results indicate that at 30 seconds exposure at 254nm UV is an ineffective method at reducing bacteria numbers from raw hide. Although the results do indicate that halophilic and halotolerent species are lower after 1 month when exposed to UV in comparison with a control. This suggests that UV may be an effective measure for eliminating initial halophilic bacteria populations if it does not contain any halophilic bacteria in the subsequently applied salt. Trisodium Phosphate (TSP) Trisodium phosphate (TSP) is a widely used detergent/disinfectant chemical, which has been applied to the control of bacteria on a variety of foodstuffs including vegetables, meat and poultry. A commercial rinse, AvGard, for the decontamination of carcasses and based on TSP is available and has been shown to be effective for the control of human pathogens on poultry carcasses. TSP acts by inhibiting the adhesion of bacteria to surfaces. Therefore, the use of TSP for reducing the contamination of raw skins was investigated. Trials are currently underway to evaluate raw skins that have been sprayed at concentrations of 10,20 and 30% TSP and sampled for total bacteria numbers and skin condition over time. Chilling Temperature control through chilling with ice has been shown to be a cheap and effective means of controlling bacterial growth and proliferation on skins. Previous studies have shown that skins treated with flo-ice or carbon dioxide snow were effectively preserved in insulated boxes for up to 10 days and would be adequate for short lead time producers. Indeed, ice is used in many tanneries where storage time is short. The trials carried out and reported below were completed to provide data related to the changes in bacteria numbers over a 24-hour period. Results from BLC for putrefaction trials at 4, 20 and 37 C are presented below (analysis carried out on NA agar).

10 1.00E+09 bacterial count cm2 hide 1.00E E E E+05 t0 t3 t6 t9 t12 t24 time (hours) Figure 4. Bacterial populations detected on raw bovine hide after storage at 4 C. Plate counts were carried out on nutrient agar on both the left and right side of the hide. 7.10E E E+07 log cfu cm E E E+07 Mean Left hand flank Right hand flank 1.10E E Time (h) Figure 5. Bacterial Populations Detected at 20 C. Plate counts were carried out on nutrient agar on both the left and right side of the hide.

11 1.00E E+08 log CFU cm E+07 Mean Left hand flank Right hand flank 1.00E E Time (h) Figure 6. Bacterial Populations Detected at 37 C. Plate counts were carried out on nutrient agar on both the left and right side of the hide. Comparison of the results shown in Figures 4, 5 and 6 illustrate that reducing the temperature increases the length of time at which the bacterial numbers remain relatively constant. At 37 o C, the bacterial numbers proliferate immediately after slaughter. Reducing the temperature to 20 o C provides a window of up to 8 hours before any significant increase in the bacteria. Cooling to 4 o C extends this time to in excess of 24- hours. Conclusions The preliminary results from both the ATP bioluminescence trials and electronic nose, indicate that, using both methods, differences can be illustrated between hides from various storage conditions. Although further work is required before the methods can be used to illustrate the microbiological status of the hide. From the various trials evaluating alternative preservation techniques, it is apparent that UV light currently does not seem to work as a viable method to reduce total bacteria numbers although there is some evidence that UV initially reduces halotolerant and halophilic bacteria numbers. Results from the chilling trials show that total bacteria numbers are reduced over a 24-hour period whilst the nisin and TSP trials are still on going.

12 Analysis of industrial salt as well as the results from the UV trials suggests that the industrial salt sources used to preserve the hides, may indeed be a key source of bacterial hide contamination within the leather industry. References 1. Mozersky. S et al. Viscometric Determination of Collegenase Activity in the Presence of High Salt Concentration. Journal of the American Leather Chemists Association. Vol 94 (1999) 2. Kanagaraj. J et al. A New Approach to Less-Salt preservation of Raw Skin/Hide. Journal of the American Leather Chemists Association. Vol 95 (2000) 3. Donaldson et al. Beta-Lysin of Platlet Origin. Bacteria Review Vol 41 (1977) 4. Ruhr E et al. Mode of Action of the Peptide Nisin and Influence of the Membrane. Antimicrobial Agents and Chemotherapy. Vol 27 (1985) Acknowledgements BLC would like to acknowledge the European Union for the funding of the MICROSTAT project (Contract Number: QLK5-CT ) as well as all the partners who have contributed throughout the completion of the work