Process for bacterial decontamination of vegetable foods

Process for the decontamination of vegetable foods from Listeria-sp. which consists in adding solutions of lysozyme, non-toxic and physiologically compatible salts thereof, to said foods, with or without adjuvant substances, at concentrations suitable for affording decontamination.

Object of the present invention is to provide a process of use in the 
industry of vegetable food-stuffs, usually included in the diet, in order 
to decontaminate them from highly pathogenic bacteria for human beings. 
This technological process consists in the treatment of vegetable foods, 
particularly those which are commonly called "vegetables", with a solution 
at varying concentrations of lysozyme enzyme (E.C. 3.2.1.17) or its 
non-toxic and physiologically compatible salts, with optional addition of 
adjuvant or synergistic substances, in order to have said foods 
decontaminated from Listeria-genus bacteria. The Listeria genus has called 
attention in recent years in consequence of some cases of death by 
infection caused by the species Listeria monocytogenes, which is highly 
pathogenic indeed for same animals and the man, as recent epidemiological 
data published in U.S.A. also confimed (Ciesielski C.A. et al. Arch. 
Internal. Med. 148, 1416, 1988). 
Such micro-organism, broadly occurring in the natural world, both in the 
animal and in the vegetable ones, is gram-positive and belongs to "cocci", 
although it could also assume the form of a little stick. When observed 
under a microscope, it is similar to the diphteric bacillus and often it 
appears in pairs, having a size of 0.5.times.2 u. It is a very mobile 
micro-organism, which hydrolyzes esculin and is catalase-positive. It 
causes listeriosis which presents tragic aspects in the breast-fed child, 
when attacking the meninges. 
In addition to the monocytogenes genus, it must be remembered Listeria 
ivanovii too, present in the green vegetable foods and very pathogenic for 
animals, which causes problems in zootechny, particularly the mortal 
infection of pregnant sheep. 
Listeria monocytogenes grow very well in a borad range of temperatures, 
f.e. between 25.degree. C. and 42.degree. C., but it has been reported 
that Listeria can grow even at lower temperatures (f.e. 4.degree. C.) and 
over 42.degree. C. 
Furthermore, these bacteria can tolerate even high concentrations of NaCl, 
f.e. 25%. 
The Listeria genus can also grow in a broad pH range, f.e. between 5.0 and 
9.5. 
As far as the epidemiological problem is concerned Listeria monocytogenes 
attacks, particularly some classes at risk, such as babies, pregnant 
women, immuno-compromised people and wasted away elders. The transmission 
through food seems to be in natural relation with environmental 
contamination, particularly through feces or liquids. Although Listeria 
was once regarded as a classical zoonoses, its ubiquituous diffusion 
always through food is now established. 
The microbiological control of foods and transformation plants has pointed 
out the possible mutual contamination of the same; thus Listeria 
monocytogenes has been found in the milk at the stable, cheese, eggs, 
vegetables, ensilated products, sausages, meat, sea-food and poultry. 
Furthermore, it has been proved that Listeria monocytogenes easily 
contaminates again already industrially treated foods, along the packaging 
line as in the case of ice-creams, vegetables in bags and salted meats. 
As a matter of fact, a 1986 FDA Control regarding 357 plants of food 
industries pointed out that 2,5% of plants were Listeria contaminated. 
The certainty of the decontamination can depend on three factors: 
removal of the pathogenic bacterium by means of specific technologies 
(pasteurization, etc . . . ); 
protection against re-contamination of the finished food-stuff; 
addition of a specific anti-microbial protection based on one or more 
synergistic agents, which protection can also be repeated for fear of 
re-contamination to occur. 
We have now found that lysozyme of white of egg is active on pathogenic 
strains of Listeria monocytogenes, both in broth and in buffer, for at 
least four strains, (Scott A. California, V7, 20A.sub.2 OHIO). 
It is known that lysozyme is a basic polypeptide, naturally occurring both 
in the vegetable and animal kingdom, where it performs complex; f.e. 
immunological and metabolic, activities. In the industry lysozyme is 
obtained from the white of hen egg, wherein it is contained in amounts 
ranging between 0.3% and 0.5%. 
Its use in the food field is already known, as f.e. in the late swelling of 
some types of cheese, which phenomenon is due to milk contamination by the 
spores of particular bacteria, such as Clostidrium tyrobutirricum, 
followed by their growth and germination during the seasoning process of 
cheese. 
This use of lysozyme has been protected (G.B. Patent 2.014.032). 
Always in the food field, particularly the dairy farming, the 
decontamination of milk or other animal products from Listeria was the 
object of our U.S. patent application No. 07-113,068 of Oct. 27, 1987. As 
far as the activity of lysozyme on the Listeria genus is concerned, 
although in vitro activities were previously reported, the possibility of 
a practical industrial application for decontaminating vegetable foods, 
which is the object of the present invention, was never envisaged, 
particularly in the case of Listeria monocytogenes and the resulting 
danger of human, even mortal, infection caused by the same bacterium. 
In addition, the present invention also comprises the utilization of other, 
lysozyme-synergistic substances, such as, for example, the alkali salts of 
ethylenediaminotetracetic acid, or other chelating, non-toxic substances, 
such as conalbumin, lactoferrin, transferrins, ceruloplasmin, etc. 
As a matter of fact, in the literature Listeria are described as sensitive 
also to other anti-bacterial substances, such as ampicillin and 
gentamicin, but the utilization in the food field of these latters, being 
antibiotics of therapeutic use, cannot be envisaged. 
Furthermore, the legislation of many countries forbids the utilization of 
these and other antibiotics in the field on grounds of toxicity, 
sensitization or formation of microbial resistances, whereas the 
utilization of lysozyme in the industry is already allowed in many 
countries, on account also of its innocuity. 
In addition it does not give the vegetable food-stuffs any particular taste 
or other undesirable organoleptic properties. 
Obviously, the possibility of employing lysozyme for the treatment of 
vetable foods also refers to its salts with non-toxic and pharmaceutically 
acceptable acids, such as hydrochloride, lactate, phosphate, 
glycerophosphate, citrate, ascorbate etc. which all suit the purpose of 
the present invention. 
As far as the utilization of lysozyme and its salts jointly with 
synergistic or adjuvant substances is concerned, some of these substances 
are already incidentally known in consequence of in vitro microbiologial 
screenings, but in no case at all their synergistic activity wa observed 
in tests carried out on vegetable substances, such as lettuce, cabbage, 
corn, peas, carrots, asparagus, artichokes, etc.

Referring to the methods employed for setting up the process of the present 
invention and for the appropriate extension and experimental trials, tests 
were carried out for determining the activity of lysozyme on various 
strains of Listeria sp., in particular monocytogenes is suitable culture 
media and on vegetable food-stuffs. 
By analytical assays, the stability of lysozyme and its salts in form 
microbiologically suitable for performing its activity Listeria sp. has 
been proved for the time elapsing from the crop to the consumption of the 
vegetable food; as target-bacterium, we chose Listeria monocytogenes Scott 
A., from the Collection of Wisconsin Madison University, on account of its 
utilization also by International Dairy Federation in the methods for 
identification and counting of Listeria monocytogenes in foods. 
Listeria monocytogenes was cultured in DHI broth static culture of DIFCO 
Laboratories (Brain-Heart Infusion) at 37.degree. C. The growth in culture 
medium was measured from the optical density (OD at 500 nm) using a Bausch 
and Lomb Spectronic 20 spectrophotometer. With the aim of investigating 
the growth of Listeria monocytogenes (Scott A.) in vegetable food-stuffs, 
this bacterium was firstly grown in BHI broth at 37.degree. C. and then 
inoculated in the test-vegetables at a predetermined cell concentration; a 
concentration of 10.sup.4 cells per gram of vegetables was employed for an 
adequate monitoring of its survival capability. All tests on foods were 
carried out in duplicate. 25 g food samples were analyzed for the presence 
of Listeria. 
The Listeria monocytogenes colonies were counted by seeding them directly 
on agar lithium chloride-phenylethanol-moxalactane medium, which contained 
glycin. 
The counting of colonies (CFU) per gram of vegetables was carried out by 
placing 25 g samples of vegetables in sterile bags of stomacher plastic 
with the following addition of 225 ml LEB (Listeria Enrichment Broth) 
having the following Composition, per liter; 
______________________________________ 
Protease Peptone Difco: 5 g 
Triptone Difco: 5 g 
Yeast extract Difco: 5 g 
NaCl: 20 g 
Lab. Lemco Powder (OXOID) 
5 g 
Na.sub.2 H PO.sub.4 12 g 
KH.sub.2 PO.sub.4 1.35 g 
Esculin (Sigma) 1 g 
Seriflavine HCl 12 mg 
Nalidixic acid, Sodium salt: 
20 mg 
______________________________________ 
(Sigma) 
The samples were then homogenized for 2 minutes in a Stomacher apparatus 
(Lab. Blender, mod. 400). 0.1 ml portions were than seeded on 10 lithium 
chloride - phenylethanol - moxalactame plates. Samples with a too high 
cell number (10.sup.4 /9) were repeatedly diluted with 0.01 M phosphate 
buffer (pH 7.2) and plated as 0.01 ml duplicated samples. The plates were 
incubated at 20.degree. C. for 5-7 days. 
With a too low number of Listeria monocytogenus cells an enrichment method 
was employed. 
The Mc Clain and Lee eurichment method was modified by incubating the 
cultures for 5-7 days at 30.degree. C. in Stomach bags. 
A first eurichment was carried out by incubating food homogenates in 
Stomacher bags at 30.degree. C. for 5-7 days. 
Afterwards, 0,1 ml portions of the euriched extract were inoculated in 10 
ml of broth for Listeria containing acriflavine hydrochloride (25 mg/1) 
and incubated for 5-7 days for a further eurichment. These samples were 
directly smeared on lithium chloride-phenylethanol-moxalactane plates and 
treated with a solution of potassium hydroxide to select Listeria 
monocytogenes. 
In order to prove the identification, five supposed colonies were analyzed 
for various phenotypic characters including the absence of pigmentation, 
the morphological appearance on agar tryptose and the 
catalase-positiveness. Three colonies were then assayed for the 
characteristic end-over-end motility in tryptose broth at room temperature 
and for the "umbrella" motility just beneath the surface of Difco medium 
at room temperature and 37.degree. C. A colony for each sample was then 
assayed for positiveness in methyl red reaction on MRVP medium (Difco), 
for esculin hydrolysis in agar bile-esculin (Difco) and for reduction of 
"litmus-milk" (Difco) after 2 days at 30.degree. C. 
Moreover, a serological analysis was carried out, by using Listeria type 4 
antiserum. As positive control, Listeria o antigen (Difco type 4) was 
used. 
Strains isolated from vegetables were also assayed on Purple Broth Difco 
containing 0,5% (w/v) gelactase, sorbitol mannitol, dulcitol, maltose, 
rhamnose, xylose, glucose, lactose and (+) mellitose and the acid 
formation mechanism was found to be the same as with Listeria Scott A. 
Treatments of vegetables 
As substrate for Listeria monocytogenes various vegetables were used, some 
of them being indicated in the following Examples. For all types of food 
were carried out four tests in duplicate: 
controls (without treatment) 
lysozyme (100 mg/kg) 
EDTA (5 mM) 
Lysozyme+EDTA 
In each test the foods (vegetable) were specially prepared and individually 
placed in containers to be incubated. Two containers were tested 
(duplicate samples) at each time of the sampling. All vegetables were 
incubated with or without experimental inoculation of Listeria 
monocytogenes for determining also the spontaneous contamination of 
vegetables. 
At the completion of the experimental treatments, foods were analyzed for 
determining the activity of lysozyme. Dried cells of M. Lutens Sigma (0.25 
mg/ml) were suspended in 2.9 ml portions of 0.067 M phosphate buffer (pH 
6.6). 
Samples of vegetables with lysozyme were diluted ten times with phosphate 
buffer and added to the M. Lutens suspension. 
Two hours later the absorbance variations (A 540) was measured. The 
presence of still active lysozyme was indicated by a marked decrease of 
absorbance, as compared with not lysozyme-treated samples. 
A solution, just prepared, of lysozyme (1-3 ppm) in buffer was used as 
test. The same solution was then used to established the possible presence 
of lysozyme activity in not lysozyme-treated vegetables. 
The pH of each samples of vegetables was measured after homogenization of 
10 g of substance in 90 ml of distilled H.sub.2 O. 
In the following are reported some examples of our studies, which are meant 
as merely illustrative and by no means limitative of the practical scope 
of our invention. 
The results of each example are reported in form of graphic for sake of 
brevity and evidence. 
EXAMPLE 1 
Small bunches of fresh lettuce were carefully cut in stripes and divided in 
1800 g portions. Lysozyme was dissolved in 25 ml of 67 mM phosphate 
buffer, pH 6.6, and EDTA (tetrasodium salt) dissolved in 25 ml of 
distilled water. The solutions were then sprayed on the 1800 g portions of 
lettuce. 
The final concentrations of lysozyme and EDTA were 100 mg/kg and 5 mM, 
respectively. Listeria monocytogenes was diluted and place in 25 ml of 67 
mM phosphate buffer for a final concentration of 1 .times.10.sup.4 cfu/g 
lettuce. Lettuce was then accurately blended. 100 g portions of the 50 
treated lettuce were placed in 250 ml polystirene containers and incubated 
at 5.degree. C. 
Lettuce samples were analyzed for Listeria monocytogenes after 0,3, 7, 12 
days (two containers per test were examined at each time of control. The 
counting of Listeria monocytogenes was carried out on two different 
amounts drawn from each bag. The variability of the two countings was less 
than twice as much). 
The Listeria monocytogenes values of the incubated material are reported in 
FIG. 1. 
The lysozyme-EDTA association proved markedly listericide as compared with 
lysozyme alone, which proved listeriostatic. 
Control and samples with EDTA alone showed an increase of Listeria 
monocytogenes up to 3.4.times.10.sup.5 and 1.6.times.10.sup.5, 
respectively; on the contrary, samples with EDTA+lysozyme did not present 
Listeria monocytogenes after 12 days. 
Samples with lysozyme showed about 2.5.times.10.sup.3 Listeria 
monocytogenes, the decrease being 50% of the initial counting. 
The experiments on lettuce were carried out for only 12 days, owing to 
obvious deterioration of vegetables. In samples of lettuce, small 
variations of pH were observed, with the exception of controls, which 
showed a lower final pH (4.8) than the treated samples (pH between 5.6 and 
5.8). 
EXAMPLE 2 
Fresh cabbages were prepared as the lettuce of Example 1. 
Samples were analyzed on 0, 3, 7, 12, 20, 27, 34, 41, 48 days. The results 
are reported in FIG. 2. 
Listeria monocytogenes grows well in chopped cabbages; in the absence of 
inhibiting substances, the pathogenic micro-organism reaches quickly 
3.5.times.10.sup.6 cfu/g and then increases up to 10.sup.8 cfu/g. In 
incubated samples containing cabbage+100 mg/kg lysozyme or lysozyme+5 mM 
EDTA, Listeria monocytogenes decreases approximately by 10 and 100 times, 
respectively, after 7 days of incubation at 5.degree. C. As to the samples 
treated with lysozyme alone, an increase after 7 days was followed by a 
decrease of the counting of the colonies; on the contrary, samples 
incubated with lysozyme+EDTA showed a constant decrease throughout the 
experiment. 
Samples treated with EDTA alone also showed for 30 days a bacterial 
increase similar to the not treated controls, but afterwards the colonies 
decreased rapidly. 
The unusual increase of Listeria monocytogenes in cabbages treated with 
EDTA alone was often observed also in other foods. 
The viability loss of Listeria monocytogenes in cabbages, in the presence 
of lysozyme, with or without EDTA, does not seem to be bound to pH 
variations, because in all four tests the same trend of pH variations was 
observed. 
The initial pH of cabbages was 4.8, which increased to 6.6-6.8 after 7 
days, these values remaining constant up to 20 days from incubation. 
Afterwards, pH decreased to 4.3-4.9 in samples containing lysozyme as 
compared to controls which showed, after 40 days, final pH values between 
5.8 and 5.85. 
Samples treated with EDTA alone showed, after 40 days, pH values ranging 
between 5.0 and 5.5. 
The usual pH variations can be ascribed to alterations caused by Listeria 
monocytogenes or bacterial flora at the refrigeration temperature, the 
lactic fermentation having been inhibited. 
After 1-2 weeks, cabbage appearance changes were observable, such as 
browning and texture loss. These results suggest that lysozyme+EDTA 
possesses listericide activity, whereas lysozyme alone is listeriostatic. 
EXAMPLE 3 
Fresh green beans were chopped in about 2.5-3.8 cm piece. 
The following treatment was the same as with lettuce of Example 1. Samples 
were examined on 0, 2, 5, 7 and 9 days. 
Results are graphically reported in FIG. 3. 
Lysozyme with EDTA proved bactericide on these vegetables, in fact a 100 
times decrease of Listeria monocytogenes was observed after one week. In 
the other tests increases were observed; however, lysozyme slows down this 
tendency to increase of Listeria monocytogenes, as (compared to control 
and EDTA alone. 
Lysozyme with or without EDTA appears on these vegetables less active than 
on cabbages or lettuce. 
Bean initial pH ranged between 5.94 and 6.07. Final pH for all samples was 
less than 0.4 units different from the initial pH. Therefore, it was the 
absence of marked pH variations which possible allowed some degree of 
survival of Listeria monocytogenes. Alternatively, one could think that 
the pieces of the beans were of such size as to binder lysozyme 
penetration in depth and, therefore, colonies of Listeria monocytogenes 
could have been formed in the center and grown without contacting 
lysozyme. 
EXAMPLE 4 
Maize grains, freshly husked, were used. Sampling and lysozyme addition 
were carried out as in Example 1. 
The examinations were carried out on 0, 2, 4, 9, 11, and 15 days. 
Results are reported in FIG. 4. 
The treatment of fresh maize with lysozyme, with or without EDTA, addition, 
gave results similar to green beans. 
As compared with lettuce and cabbages, the decrease rate of Listeria 
monocytogenes grew up to 1.0.times.10.sup.7 and 1.8.times.10.sup.5, 
respectively, after 9 days. 
Initial pH of samples ranged between 6.6 and 6.7 and did not change during 
the first 9 days, but a decrease to 4.5-5.0 was observed in samples not 
containing EDTA. 
In the presence of EDTA, however, pH did not decrease below 6.0, even after 
15 days. EDTA seems to have some influence on the deterioration caused by 
bacteria, but no influence at all on Listeria monocytogenes survival. It 
results from above that lysozyme has a negative influence on the growth of 
Listeria monocytogenes on fresh maize. 
The lower rate of decreased of Listeria monocytogenes could be due to the 
different composition of this vegetable food-stuff, such as, for example, 
the higher content of carbohydrates which possibly aids the growth of 
Listeria monocytogenes. 
EXAMPLE 5 
Fresh carrots were treated as the lettuce, with the exception that inoculum 
of Listeria monocytogenes was 1.times.10.sup.3 cfu/g. Carrots were 
examined on 0, 2, 5, 9 and 16 days. 
The obtained results are reported in FIG. 5. 
In chopped carrots, the incubation with lysozyme, with or without EDTA, and 
Listeria monocytogenes gave anomalous responses, as far as the microbial 
growth and the bactericide activity are concerned. 
In these experiments, Listeria monocytogenes was employed at lowed intial 
levels (about 100 colonies per gram). 
Lysozyme alone or lysozyme+EDTA quickly removed Listeria monocytogenes from 
the carrots. The not treated controls showed that initially the bacterial 
colonies were quickly doubled, with disappearance however after 9 days. 
On the contrary, EDTA alone caused an initial increase of the bacterial 
counting, which then decreased after 9 days. 
One could assume the presence in carrots of either chelating agents for 
metallic ions or listericide substances, synergistic with lysozyme. 
The sample initial pH ranged between 6.2 and 6.4. After 9 days a gradual 
decrease to 4.1-4.4 was diserved. The pH change does not seem to have any 
influence on Listeria monocytogenes survival.