DESTRUCTION OF GRAM NEGATIVE BACTERIA

Abstract: Techniques for the destruction of both Gram-negative and Gram-positive bacteria are disclosed comprising treatment involving hypo-osmotic shocks combined with exposure to lysozyme and/or cold shock. Such procedures appear suitable for the treatment of animal carcasses, since no addition of toxic chemicals (e.g. EDTA) is required. Application of these procedures to meat treatment may, therefore, lead to reduced levels of pathogens and improve keeping qualities. Both immersion and spray techniques may be used to apply the treatment media.

Description: OBSTRUCTION OF GRAM NEGATIVE BACTERIA. Recent reports on food-borne illness clearly indicate the economic and public health significance of salmonellosis and campylobacteriosis. In 1989 t the number of confirmed salmonella cases in England and Wales rose to 29,998 (Cooke, M E.(1990). The Lancet 336:790-793-) while those of campylobacter rose to 32,359 (Skirrow M B.(a) (1990). Proceedings of the l4th International Symposium of the ICFMH. Telemark, Norway; (b) (1990). The Lancet 336: 921-23.) with estimated average tangible costs per case of £2,240 (Yule, B F et al (1988) Epidemiology and Infection 100: 35-42. ) and £273 (Skirrow 1990 (a) (b)) respectively. Similar data exist for the USA where approximately 40,000 salmonella cases are reported annually, with average hospitalization or treatment costs rising up to $4,350 per case (Roberts T. (1988). Poultry Science 67: 936-43.). The incidence of campylobacter is also high and, for example, in the state of Washington, has been estimated at 100,000/150,000 (Todd, E. (1990). The Lancet 336: 788-90. ). Confirmed cases of disease probably represent only 1 to 10# of the total number of clinically significant cases (Aserkoff B et al (1970). American Journal of Epidemiology 92: 13-24. ; Oostero (1990) Procedings of the 14th International Symposium of the ICFMH. Telemark, Norway. ; Skirrow 1990a, b). Following the compulsory heat treatment of milk in 1983 poultry meat has become the most incriminated food vehicle of salmonellosis and campylobacteriosis in the UK. The Public Health Laboratory Service ((1989) PHLS Microbiology Digest 6: 1-9) found that 60 to Bθ% of retail chickens in the UK were contaminated with salmonella while reports from other countries indicate levels ranging between 5 and 73#. The incidence of campylobacter may be even higher, and in some studies all chicken carcasses examined were contaminated (Hood, A M et al (1988) Epidemiology and Infection 100: 17-25- ϊ Lammerding, A M et al (1988). Journal of Food Protection 51: 7- 2. ). In addition, contamination of red meats leads to sporadic cases of both salmonella and campylobacter disease. Thus there is considerable pressure on the meat and poultry industries to improve the bacteriological quality of their products by developing and applying decontamination processes. Considerable effort has been devoted to the development of chemical decontamination techniques. However, although a large number of chemical treatments have been tested (Table 1), these have in general proven either unsuccessful on application, or have had adverse effects on the appearance, odour or taste of meat, occasionally leaving undesirable residues. Chlorine is the only chemical currently in use in poultry processing operations and maximum levels of 20 ppm in the spray wash are recommended by the EC, although higher concentrations (40 ppm) may be required to reduce bacterial populations in both carcasses and equipment. Chlorine, however, can damage processing equipment and leads to the formation of potential carcinogens such as chlorinated hydrocarbons when contacted with organic matter. There has been for many years a considerable interest in using the enzyme lysozyme as a food preservative. Lysozyme is a naturally occuring antimicrobial agent, has no adverse effects on man and is present, for example, in tears and milk. It can also easily be recovered through industrial processes from egg-white and is approved for food use in Europe, Japan and the USA (Hughey, V L et al (I987) • Applied and Environmental Microbiology 3: 2165-2170). Table 2 lists the variety of food products that may be preserved by treatments involving lysozyme derived from milk or egg-white. Lysozyme may cause rapid lysis of Gram-positive bacteria but unless subjected to modifying treatments, cells of Gram-negative bacteria are resistant. Lysozyme hydrolyses peptidoglycan, a polymer present in the cell walls of Gram-positive and Gram-negative bacteria which maintains rigidity of the wall. In Gram-positive organisms, peptidoglycan is present throughout the cell wall, which consists of a more or less homogeneous matrix of peptidoglycan and other polymers. However, in Gram-negative bacteria, peptidoglycan exists as a discrete layer which is protected from the environment by a lipid outer membrane which acts as a permeability barrier against large molecules, such as lysozyme (MW 14,900 D) . Thus, in the absence of procedures for modifying the outer membrane, only foods dominated by a Gram-positive bacterial flora may be preserved by lysozyme. The outer membrane of Gram-negative bacteria may be disrupted by heat (Becker M E et al (1954) Archives of Biochemistry and Biophysics 53: 402-410; freezing and thawing (Kohn, N R. (I960) Journal of Bacteriology 79: 697-706. ); extraction of the lipopolysaccharide component of the outer membrane with lipid solvents or alkali (Becker et al (1955) Archives of Biochemistry and Biophysics 55: 257-269. ), starvation at extreme pH environments (Nakamura, 0. (1923) Immunitatsforschrift 38: 425-449 ; Grula, E A et al (1957) Canadian Journal of Microbiology 3: 13-21), treatments with EDTA (Repaske, R. (1956) Biochemica et Biophysica Acta 22: 189-191 and (1958) Biochemica et Biophysica Acta 30: 225-232), detergents (Colobert L. (1957) Comptes Rendues 245: 1674-1676. ), or polybasic antibiotics ( Warren G H (1957) Journal of Bacteriology 74: 788-793- )• Hypo-osmotic shock in the presence of lysozyme (Birdsell, D C et al 1967- Journal of Bacteriology 93: 427-437 ; Witholt, B H et al (1976) Biochimica et Biophysica Acta 443: 534-44. ) has also been demonstrated to kill Escherichia coli ( E. coli ) cells suspended in Tris-EDTA buffer and plasmolysed by the addition of sucrose. Procedures involving EDTA and lysozyme have been tested on shrimp (Chandler R et al (1980) Applied Microbiology and Biotechnology 10: 253-258. ) and poultry (see Table 2), but although some reduction in contamination levels was observed the use of EDTA makes the technique generally inapplicable to food-treatment. Osmotic shock procedures (Withholt B H et al 1976) might also be acceptable in food processing if the requirement for EDTA could be eliminated. The transfer of bacteria from typical growth media (a w 0.999) to media made hypertonic by the addition of solutes which do not penetrate cells, such as sucrose or NaCl, is accompanied by an abrupt loss of cell water. Gram-negative bacteria subjected to such hyper-osmotic shock undergo "plas olysis" which is characterised by loss of turgor pressure, shrinkage of the protoplast (Witter L (1987) Vol. 1: 1-35. In T J Montville (ed) , Food Microbiology. CRC Press, Florida. ), retraction of the cytoplasmic membrane from the outer membrane ( Scheie, P 0. (1969) Journal of Bacteriology 98: 335-40. ), or contraction of the whole cell (Alemohammad M M et al (1974) Journal of General Microbiology 82: 125-142. ). Subsequent survival, growth rate and maximum population density then depends upon the a w of the medium and the rate and extent to which the osmoregulatory mechanisms (Booth, I R, et al (1988) Journal of Applied Bacteriology Symposium Supplement PP. 35~49; Csonka L N. (1989) Microbiological Reviews 53: 121-147) of the organism may be restored to regain cell water ( Dhavises, G et al (1979) Microbios Letters 7 105-115- and (1979) Microbios Letters 7: 149-59- )- Water uptake is achieved by 'deplasmolysis' , which in contrast to plasmolysis requires the presence of an energy source in the medium and is characterised by uptake and accumulation of K+ ions and uptake and/or synthesis of certain organic osmolytes, referred to as compatible solutes or osmoprotectants. In contrast, transfer of cells from media of low to high a w (water activity) , thus effecting hypo-osmotic shock, results in an instantaneous influx of water and a concomitant increase in the cytoplasmic volume. However, cell volume increase in bacteria is generally limited by the presence of the cell wall which is relatively rigid and may withstand pressures of up to 100 atmospheres . Although hypo-osmotic shock does not generally result in cell lysis, it may cause membrane disruption which can be demonstrated by the loss of intracellular solutes, such as ions, neutral and anionic sugars and phosphate esters (Leder, I G (1972) Journal of Bacteriology 111: 211-19; Tsapis A et al (1976) Biochimica et Biophysica Acta 469: 1-12. ). Such loss has been described at optimum growth temperatures (30-37 °C) and at 5 °C, as well as in combination with cold shock. Cold shocks are achieved by rapidly lowering the temperature of cell suspensions, for example from 37°C to 0°C ( Sherman, J M et al (1923) Journal of Bacteriology 8: 127-139. ). The shock may result in cell death and cells from the exponential phase of growth are most susceptible (Jay, J. (1986) Modern Food Microbiology. 3rd ed Van Nostrand Reinhold Co Inc, NY. ). Lysozyme has been reported to enhance lysis of exponential phase E. coli cells suspended in Tris-HCl buffer and subjected to cold shock (Scheie, P 0. (1982) Biochimica et Biophysica Acta 716: 420-23. ), though Tris-HCl may itself aid lysis of Gram-negative cells (Schindler, H et al (1979) American Chemical Society 18: 4425-30. ). The present inventors have developed novel techniques which rapidly kill both Gram-negative and Gram-positive bacteria, being particularly useful in the destruction of those bacteria of significance in the food industry such as Salmonella tvphimurium. E. coli and the common meat spoilage organism, Pseudomonas fluorescens. The techniques are based on combined treatments involving hypo-osmotic shocks combined with exposure to lysozyme and/or cold shock. Such procedures appear suitable for the treatment of animal carcasses, since no addition of toxic chemicals (eg EDTA) is required. Application of these procedures to meat treatment may, therefore, lead to reduced levels of pathogens and improve keeping qualities. Both immersion and spray techniques may be used to apply the treatment media. The advantages of the treatment are several-fold: (i) It is non-toxic, making use of lysozyme, a naturally occuring anti-microbial enzyme, already approved for food use in both Europe and USA. Low concentrations of egg-white may also be used as an effective substitute for purified enzyme preparations. (ii) It is economically viable, using only low cost materials (NaCl, sucrose, egg-white) . (iii) It does not necessitate major alterations in current processing-plant technology, as the washing procedures required may be carried out using existing washing tanks and sprayers. (iv) It does not cause corrosion as a consequence of pH change, or scaling of metal equipment. (v) No additional effluents of environmental consequence are produced and cross-contamination of carcasses is potentially reduced due to lower numbers of organisms being found in wash solutions. (vi) It is potentially effective against all Gram-negative organisms; of particular interest in this group are salmonellae and campylobacters. (vii) It is suitable for processing plants, catering establishments and possibly households. (viii)It is relatively simple and may be carried out using unskilled labour. Thus the present invention provides a method for the destruction of bacterial cells of both Gram-negative and Gram-positive classes comprising subjecting said cells to hypo-osmotic shock in combination with a further treatment selected from the group comprising (a) exposure of the cells to lysozyme and (b) subjecting the cells to cold-shock. Preferably the hypo-osmotic shock is applied by exposure of the cells to a first solution having a water activity (a w ) of 0.997 or less and then exposing them to a solution of a w higher than that of said first solution. More preferably the first solution has a water activity (a w ) of 0.992 to O.96 and most preferably of 0.974 to O.96. It is particularly convenient that the solution of higher water activity contains the lysozyme for the further treatment (a) . The solution having water activity of 0.997 or less is preferably applied for between seconds and 3 minutes, more preferably for between 30 seconds and 20 minutes and most preferably is applied for between 1 and minutes. In this first aspect of the present invention providing a method for the destruction of bacterial cells of both Gram-negative and Gram-positive classes comprising subjecting the cells to hypo-osmotic shock combined with exposure to lysozyme, it is preferred that the cells are first exposed to a nutrient containing medium. Particularly preferred is a method wherein the solution of water activity of 0.997 or less is a nutrient containing medium. The present inventors have found that when stationary phase Gram -negative cells are suspended in a media (eg. a foodstuff compatible media such as sucrose or NaCl) of low a w they undergo a rapid dehydration (plasmolysis) followed by a relatively slow rehydration (deplasmolysis) which is dependent upon the a w and nutrient composition of the medium in which the cells were suspended, temperature, and the presence of osmoprotectants (eg proline, betaine) . When the partly or fully deplasmolysed cells are transferred to deionised water containing lysozyme, high kill rates are observed. Kill rates of the order of $0% can be achieved using media lacking in nutrients and it is thought that this is enabled by deplasmolysis using the organisms internal energy reserves. Best results however are obtained where optimal growth conditions are provided in the application of the first treatment. Thus any nutrient capable of supporting deplasmolysis of the target organism may be employed to achieve enhanced bactericidal effect of the present method; examples of these being given in the Tables and Examples provided herein. Simple media such as glucose or lactalbumin hydrolysate (casein hydrolysate - hydrolysed milk protein) at eg. about 5 E l "1 can be successfully used. Ideally the first treatment is applied at a temperature optimised for deplasmolysis to take place in the target organism. Thus for Salmonella or Shigella media at about 37°C optimally are used while for Pseudomonas about 30°C is preferred. Using these optimised media and temperatures kill rates in excess of 99-99# "n y be achieved (see Table 3)- The lysozyme may conveniently be provided as commercially available lysozyme (eg. 10 μg ml "1 or more) or lysozyme in the form of pasteurised (eg. at 63 °C, 4 min) freeze-dried egg-white (eg 0.5 ωg ml "1 or more) , prepared in the laboratory Preferably the lysozyme is provided at a concentration of 5 μg ml "1 or more and more preferably is in the solution of higher a . More preferably the lysozyme is provided at a concentration of 10 μg ml "1 or more, most preferably at a concentration of 50 μg ml "1 or more. Conveniently the lysozyme is in the form of a solution of pasteurised freeze-dried egg-white wherein the concentration of the egg-white in the solution is 0.1 mg ml "1 or more, more preferably 0.5 mg ml "1 or more. In a second aspect of the present invention there is provided a method for the destruction of bacterial cells of both Gram-negative and Gram-positive classes by subjecting the cells to a combination of cold shock and hypo-osmotic shock. The treatment may be applied in the presence or absence of lysozyme, but preferably with lysozyme. The present inventors have found that when stationary phase Gram-negative cells are suspended at low temperature (eg. 0 to 10°C) in deionised water containing solute (eg.NaCl at 0.2 to 0.8 mol/1) but no nutrients deplasmolysis does not take place. If, after temperature equilibration, cells are transferred to deionised water (at the same temperature with or without lysozyme cell destruction occurs. This further treatment comprises exposure to cold shock wherein said shock comprises exposure to a temperature of 10°C or below, more preferably comprising exposure to an aqueous liquid at 10°C or below. Particularly preferred are treatments where this aqueous liquid is at 8°C or below, most preferably 0°C or below. Preferably the exposure is for a period sufficient to equilibrate the temperature of the cells to that of the exposure temperature, preferably being for about 10 minutes. Preferably the temperature of the bacteria prior to shock is at from 15°C to 37°C Maximum cell destruction (100#) has been observed by following a 30 in incubation in NaCl medium at 0°C with deionised water/lysozyme treatment. Such deionised water/lysozyme treatment is as described for the lysozyme treatment in the first aspect of this invention but is carried out using a cold shock inducing solution. The extent of cell destruction for E. coli and S. typhimurium at 8 and 0 °C was investigated and results are shown in Table 4 below. By way of comparison, the ability of lysozyme to kill cells during cold shock (eg. on sudden transfer from between deplasmolysis enabling temperatures (eg. 15°C-37°C) to 10°C-0°C) in deionised water with or without NaCl was confirmed, however, maximum cell destruction was less than 80% without the hypo-osmotic treatment. The method of the present invention will now be illustrated further with regard to the following non-limiting examples which are provided for the purpose of assisting a man skilled in the art to determine suitable conditions for given situations. Other embodiments falling within the scope of the present invention will occur to the man skilled in the art in the light of these examples. The in vitro methods described above were adapted for the decontamination of artificially contaminated red meat and poultry skin but can equally be used on any meat and may be particularly applied to treatment of fish meats such as eg. prawns and shrimps. EXAMPLE 1. Meat/poultry skins were dipped in aqueous sucrose or NaCl medium (a w 0.979) for 10 min at 20-37°C and then washed in an aqueous solution of pasteurised egg white or pure lysozyme. EXAMPLE 2. Meat/poultry skins were dipped in an aqueous solution of NaCl (0.8 M; a w 0.974) for up to 30 min at 0°C and then sprayed with or dipped in an aqueous solution of pasteurised egg white or pure lysozyme for up to 30 min at 0°C. Results The (%) recovery of S. typhimurium cells from artificially contaminated meat/chicken skin treated according to the above procedures of Examples 1 and 2 is given in Tables and 6 respectively. The number of organisms recovered ie. still attached to the sample of meat or skin, was less than 10# of the population initially applied. Also, the number of organisms recovered in the washing solutions was reduced by low temperature NaCl/lysozyme treatment (Table 7 a-b) • This may of significance in poultry processing where it is believed that cross-contamination of carcasses may occur via washing solutions. Bacterial contamination for laboratory tests was simulated by immersion of fresh chicken pieces in Brain Heart Infusion Broth containing 5 10 9 cell/ml of the organism to be destroyed. The pieces were removed from the broth and air dried prior to use. When the treatment is applied to the surfaces of artificially contaminated meat a reduction in numbers of Gram Negative Bacteria of over 90 is achieved. The efficacy of the procedure at both higher and low temperatures allows for the application of the treatment at a number of sites within a typical meat processing factory. It will be appreciated that certain applications will require particular techniques for improving the contact of the shock and lysozyme media with the surfaces upon which the bacteria are located. Such surfaces may for example be animal skin upon which there are located many pores in which the bacteria might be located. Any technique which will allow improved access of the media might be used as long as it is acceptable for food product use. Thus electrostatic spray techniques, where a charge is applied to the media prior to application, or the inclusion of acceptable surfactant in said media might be used. Acceptable surfactants would include those e ulsifiers or detergents that are suitable for food processing, eg. the emulsifier lysolecithin. Table 1: Chemicals evaluated for the decontamination of fresh meat and poultry. Compound Reference Ammonia Smol'skii et al I985 Acetic acid Goepfert & Hicks 1969; Eustace 1981 Chlorine Sanders & Blackshear 1971; Marshal et al 1 Table 2: Food preservation using lysozyme Specifically Food Targeted Organisms Reference Fresh vegetables, Kanebo Ltd 1973 fruit and fish meat Seafood Eisai Co 1971; 1972; Decadt & Debevere 1990 Sushi, noodles Yashitake &. Shnichirio 1977 pickles, cream custard Kamaboko Akashi & Oono 1968 Vienna-type sausage Akashi 1970 Salami sausage Akashi 1971 Sake Lactobacilli Yajima et al 1968 Infant food Nishihava & Isoda 1967; Morigana Milk Industry Co 1970 Cheese Clostridia Wasserfall & Teuber 1979; Ferrari Dell'Agua 1979 Table 3: Optimum conditions for maximum destruction of stationary phase E. Coli B/r/1 cells incubated in chemically defined or nutrient rich media of reduced a w and subsequently diluted in deionised water containing lysozyme. Key: (a) Defined Medium A (as per Poole et al (1974) , Biochemical Journal 144 : 77-85: (b) Brain Heart Infusion broth (as commercially available) (*) NaCl = Sodium chloride In all cases the amount of NaCl or sucrose is varied to achieve the desired a w , the nutrient medium composition remaining constant in all other respects. All treatments were carried out using solutions at between 20 and 37°C. Table 4: The effect of lysozyme on E. coli and S. typhimurium cells subjected to hypo-osmotic shock at 0 or 8 °C. 99 S. typhimurium 99 88 99 Key: (a) - Cells were incubated in NaCl (0.8 M; a w 0-974) for 10 min and subsequently transferred to and incubated for 30 min in deionised water in the presence (10 μg ml), or absence of lysozyme. Table 5: Decontamination of meat at ambient temperture using hypo-osmotic wash and a further wash in the presence or absence of lysozyme. Key: (*) - Meat was contaminated with S. typhimurium (2.24 x 10 6 colony forming units (c.f.u.) g "1 , and subjected to a washing procedure at 23 °C. The first wash was of NaCl-BHI medium and the second was of water, with or without lysozyme. Table 6: Decontamination of poultry skin at low temperature in the presence or absence of lysozyme. Washing procedure Salmonella recovered 1st wash 2nd wash c. f.u. g- l ( « ) % of total applied bacteria water water 1.03 x ιo 7 10.20 Control (- lysozyme) NaCl water 1.11 x 10 6 1.10 (- lysozyme) NaCl water 9.69 x 10 5 0.96 (+ lysozyme) Key: (*) - Skin was contaminated with S. typhimurium (1.01 x 10 8 c.f.u g "1 ) and subjected to a washing procedure at 0 °C. The first wash was of water or NaCl (0.8 M) , and the second wash was of water, with or without lysozyme. Table 7a Organisms recovered in the first washing solution. Table 7b Organisms recovered in the second washing solution.

Claims: 1. A method for the destruction of bacterial cells of both Gram-negative and Gram-positive classes comprising subjecting said cells to hypo-osmotic shock in combination with a further treatment selected from the group comprising (a) exposure of the cells to lysozyme and (b) subjecting the cells to cold-shock. 2. A method as claimed in claim 1 wherein the hypo-osmotic shock is applied by exposure to a first solution having a water activity (a w ) of 0.997 or less and then exposing them to a solution of a w higher than that of said first solution. 3. A method as claimed in claim 1 wherein the hypo-osmotic shock is applied by exposure to a first solution having a water activity (a w ) of 0.992 to O.96 and then exposing them to a solution of a higher than that of said first solution. 4. A method as claimed in claim 1 wherein the hypo-osmotic shock is applied by exposure to a first solution having water activity (a w ) of 0.974 to O.96 and then exposing them to a solution of a w higher than that of said first solution. 5. A method as claimed in any one of claims 2 to 4 wherein solution of higher water activity is that containing the lysozyme for the further treatment (a). 6. A method as claimed in any one of claims 2 to 4 wherein the solution having water activity of 0.997 or less is applied for between seconds and 30 minutes. 7. A method as claimed in claim 6 wherein the solution having water activity of 0.997 or less is applied for between 30 seconds and 20 minutes. 8. A method as claimed in claim 6 wherein the solution having water activity of 0.997 or less is applied for between 1 and 5 minutes. 9. A method as claimed in claim 6 wherein the solution of water activity of 0.997 or less is a nutrient containing medium. 10. A method as claimed in any one of claims 1 to 9 wherein the further treatment comprises exposure to lysozyme wherein said lysozyme is provided as a solution of commercially available lysozyme. 11. A method as claimed in claim 10 wherein the lysozyme is provided at a concentration of μg ml -1 or more. 12. A method as claimed in claim 10 wherein the lysozyme is provided at a concentration of 10 μg ml "1 or more. 13. A method as claimed in claim 10 wherein the lysozyme is provided at a concentration of 50 μg ml "1 or more. 14. A method as claimed in any one of claims 1 to 9 wherein the further treatment comprises exposure to lysozyme wherein the lysozyme is in the form of a solution of pasteurised freeze-dried egg-white. 15- A method as claimed in claim 14 wherein the concentration of the egg-white in the solution is 0.1 mg ml "1 or more. 16. A method as claimed in claim 14 wherein the concentration of the egg-white in the solution is 0.5 mg ml "1 or more. 17- A method as claimed in any one of claims 1 to 16 wherein the further treatment comprises exposure to cold shock wherein said shock comprises exposure to a temperature of 10°C or below. 18. A method as claimed in claim 17 wherein said cold shock comprises exposure to an aqueous liquid at 10°C or below. 19. A method as claimed in claim 17 wherein the liquid is at 8°C or below. 20. A method as claimed in claim 17 wherein the liquid is at 0°C or below. 21. A method as claimed in any one of claims 17 to 20 wherein the exposure is for a period sufficient to equilibrate the temperature of the cells to that of the exposure temperature. 22. A method as claimed in claim 21 wherein the exposure is for about 10 minutes. 23. A method as claimed in any one of claims 16 to 22 wherein the temperature of the bacteria prior to shock is from 15°C to 37°C 24. A method as claimed in any one of claims 2 to 23 wherein one or more of the solutions are applied in the form of a spray. 25. A method as claimed in any one of claims 2 to 24 wherein one or more of the solutions are applied by immersion of the bacteria therein. 26. A method as claimed in claim 1 as hereinbefore described in any one of the Examples 1 and 2 with reference to the Tables 3 to 8.

Inventors: ANAGNOSTOPOULOS GERASIMOS, CHATZOPOULOU ATHINA, MILES ROGER JOSEPH, ANAGNOSTOPOULOS, GERASIMOS, CHATZOPOULOU, ATHINA, MILES, ROGER, JOSEPH
Applicants: MINI AGRICULTURE & FISHERIES, THE MINISTER OF AGRICULTURE, FISHERIES AND FOOD IN
Application year: 1992
Main classification: A23B4
IPC codes: A23B4, A23L3, A61K35