WO2006082267A1

WO2006082267A1 - Method of preserving crustaceans against melanosis

Info

Publication number: WO2006082267A1
Application number: PCT/ES2006/000039
Authority: WO
Inventors: Argimiro Llamas Marcos; Pedro Llamas Galilea; Jose Manuel Vargas Jimenez; Francisco Navarro Roldan; Francisco Cordoba Garcia; Manuel Jesus Borrero Romero
Original assignee: Universidad De Huelva
Priority date: 2005-01-28
Filing date: 2006-01-27
Publication date: 2006-08-10
Also published as: ES2257964A1; ES2257964B1

Abstract

The invention relates to a method of preserving a crustacean against melanosis, consisting in bringing the crustaceans into contact with a solution containing a suitable quantity of lactic acid bacteria and subsequently storing same under suitable conditions. The invention also relates to the use of lactic acid bacteria as melanosis-inhibiting agents in crustaceans and to crustaceans treated with lactic acid bacteria for food use.

Description

Method of preservation of crustaceans against melanosis

FIELD OF THE TECHNIQUE

The present invention relates to the use of lactic acid bacteria as inhibitors of melanosis. As well as to a method of preserving a crustacean against melanosis by the use of said bacteria and the crustaceans treated with them for food use.

STATE OF THE TECHNIQUE

Melanosis or blackening of crustaceans, fruits and vegetables and food in general, is an enzymatic process inherent in the physiology of the products of the animal and plant kingdom, and as such phenomenon manifests negatively, from the moment in that the animal dies or when the fruit or vegetable is deprived of its skin, its appearance is modified after a few minutes, appearing a brown hue for fruits and vegetables and black for seafood (enzymatic browning or melanosis). The fact is that a process of tissue oxidation has occurred.

Melanosis in crustaceans, is manifested by the appearance of dark spots, blackening in the cephalothorax and throughout the entire body. Said blackening occurs mainly as a consequence of the catalytic oxidation, caused by the presence of oxygen in the air, with the intervention of enzymes of the phenol oxidase type (PPO), which accelerates the oxidation process of organic phenolic compounds, such as tyrosine, amino acid essential part of the high protein content of shellfish, o-quinones which spontaneously polymerize to form a dark residue, high molecular weight.

Several methods have been developed to prevent blackening, including heat for the inhibition of PPO and various chemical treatments such as altering the pH of food. Heat inactivation is not an appropriate method for fresh foods. Likewise, lowering the pH by adding An acid, such as citric acid, phosphoric acid as described by Mc-Cord and Kilara in J. Food Science, 48: 1797-1483 (1983) can lead to the same effects.

The activity of the enzyme is high in different phases of the growth of the external tissue (skeleton) of the crustaceans and in the post mortem phase. The use of chemical products as an inhibitor of enzymatic and non-enzymatic oxidation processes is so far the most widespread method capable of alleviating the serious problem that the shellfish sector has when controlling the "blackening" of crustaceans.

Boric acid was widely used, although for two decades its use was banned for sanitary reasons. Since its prohibition, as a preservative agent of crustaceans against melanosis and the deterioration of shellfish, there have been many efforts aimed at finding alternatives of chemical compounds, non-toxic, capable of inhibiting this enzymatic activity.

The use of sulphites in the treatment of melanosis was described by Zawistowski et al., In Can. Inst. Food Sci.Tech. J., (1987) 20 (3): 162-165. However, its quantity is limited and regulated depending on the nature of the food, since its ingestion, inhalation and contact with the skin, and in its management, present potential health risks.

The usual form of application of the sulphites is by sprinkling, immediately after the capture of the crustaceans to delay the onset of melanosis. The dosage and homogeneity of the additive in the samples is problematic being difficult to achieve manually on board. There are many examples included in the scientific literature on the use of sulphites as inhibitors of melanosis in crustaceans (Knowles, ME Chem. Ind. (London), (1971), 910-911; McWeeny, DJ, et al. J. Sci. Food Agrie, (1974) 25, 735-746; Wedzicha, BL et al. Food Chem., (1988) 27, 259-71) and of patents that use them (ES 2,112,798, US 5,882,688, FR 2,696,077, EP 141,875, GB 2,222,509, etc.). However, none of these methods has proved to be fully satisfactory. Labuza and cois, in Cereal Foods World, (1989) 34 (4): 353 describes the use of proteases, especially ficin, in the enzymatic control in the browning of certain foods. This author attributes this effect to the attack on the PPO of this protease.

US Patent 4,981,708 describes the use of a free protease as an inhibitor of food browning. In this method, foods that are susceptible to browning, including for example, certain crustaceans, fruits, vegetables, and beverages, including fruit and wine juices, are treated with an extract of a sufficient amount of protease-free inhibiting the browning reaction. The protease-free extract that is extracted from latex (ficus) is as effective as sodium bisulfite.

Because the phenomenon of melanosis manifests itself within a few hours when crustaceans are not treated with traditional anti-melanosic agents, serious economic problems are caused to the fisheries sector and seafood marketers due to the depreciation of their market value.

It is therefore desirable to have a treatment of melanosis or browning of foods and especially crustaceans that minimizes the problems and disadvantages of the above products.

DESCRIPTION OF THE INVENTION

The inventors have surprisingly found that lactic acid bacteria (BAL) can be used as inhibitors of melanosis in crustaceans. Therefore, according to a first aspect of the present invention, this refers to a method of preservation of crustaceans against melanosis by its treatment with lactic acid bacteria.

In accordance with a second aspect of the present invention, this refers to the use of lactic acid bacteria as inhibitors of melanosis in crustaceans. According to a preferred embodiment, the lactic acid bacteria used are of the genera lactobacillus, lactococcus, leuconostoc and pediococcus. These bacteria have been used for many years, in fermentations, in which they are part of mixed natural crops along with other bacteria, yeasts and molds used for the production of various human or animal foods such as dairy products (cheeses, butter, yogurt, acid milks, etc.), fermented vegetables (pickles, olives, etc.), sausages, sour breads, pickles and silage.

According to a preferred embodiment, Lactobacillus helveticus, L. Lactis, C. piscícola, L. plantarun, and P. acidilacti are used as microbial starters.

Lactic acid bacteria

Lactic acid bacteria are immobile, bacillary, or spherical, coconut or bacillary bacteria. In preparations for the microscope they appear isolated or forming chains of coconuts or bacilli. The habitats are very varied; normal flora of the surface of plant material (fruits and vegetables), foods rich in sugars, milk and derivatives. They obtain energy exclusively by fermentation of sugars and require a large number of nutritional factors (amino acid, nitrogenous bases, some vitamins), having very limited anabolic possibilities, which contribute to reducing the yield of their growth.

They tolerate relatively high concentrations of acids and lower pH values well than other bacteria, which can displace them from the colonizing habitats.

Unable to breathe because they cannot synthesize porphyrin compounds, and therefore form an electron transport chain. They lack cycle of Functional Krebs and, are unable to synthesize ATP by breathing, which is a reflection of their inability to synthesize cytochromes.

Catalase (enzyme that destroys H2O2.) Needs a porphyrinic group and, therefore, this type of bacteria does not have this enzyme, which allows the identification of the group. Oxygen tolerance can be achieved by accumulating a large amount of manganese (Mn ²⁺ ) that acts as a superoxide dismutase.

All these organisms due to the limited biosynthetic capacity, have high nutritional requirements, needing growth factors, B vitamins, amino acids. As a consequence, LABs are grown in media containing peptone, yeast extract or other hydrolysates of plant or animal materials, with a fermentable carbohydrate that provides a source of energy.

The small size of the colonies is attributable, first of all, to the low growth yields due to their fermentative metabolism. Some species can produce large colonies when grown in media containing sucrose, as a result of the massive synthesis of extracellular polysaccharides such as dextrans and lévans, with a large part of the volume of the colony corresponding to these sugars. Another distinctive physiological characteristic of these bacteria is their high tolerance to acids, a necessary consequence of their metabolism mode. Although cocoa lactic acid bacteria can start their growth at neutral and even alkaline pHs, most bacillary forms cannot grow in media with an initial pH of less than 6. The growth of LABs can continue until the pH has dropped to 4.5 or even less. The ability of LABs to produce and tolerate a relatively high concentration of lactic acid has a great selective value, since it allows them to eliminate competition from most other bacteria, in nutrient-rich environments. As a result of their extreme physiological specialization, LABs are confined to a few characteristic natural environments. Some live in association with plants and grow to At the expense of nutrients released after death and decomposition of plant tissues.

S. Orla Jensen, in "Le clasication des lactiques bacteria. Lait 4. pag 468-474, pointed out that BALs could be divided into two biochemical subgroups, which are distinguished by products formed from glucose. Homofermentators convert glucose almost quantitatively in lactic acid, and the heteofermentators Ia convert into an equimolecular mixture of lactic acid, ethanol, acetic acid and CO2.

Homofermentators deamilate glucose via the Embden-Meyerhof route. However, heteofermenters cannot use this route since they lack the fructose diphosphate aldolase enzyme, therefore these organisms deamide glucose by way of pentose phosphate.

It is known that organic acids inhibit enzymatic reactions under certain pH ranges. Polyphenoloxldasa, like any other enzyme, has pH values under which it acts effectively, others in which it catalyzes less efficiently and, finally, in certain ranges the reaction is practically non-existent.

Tyrosinase (polyphenoloxidase) is known to be a key enzyme in the synthesis of melanin in plants, microorganisms and breast cells, and for having copper in its structure. Many compounds such as kojic acid and arbutin are inhibitors of tyrosinase.

The role of certain metabolites produced by LABs is known, for example:

(i) lactic acid: it can be synthesized as L (+), D (-), and in racemic form. Form D (-) is not metabolized by humans, so its intake is not recommended, especially in children and adolescents. Lactic acid, like other organic acids, produces at the level of the cytoplasmic membrane of the microorganisms mismatches in the membrane potential and inhibits transport processes. (ii) hydrogen peroxide: BALs do not possess catalase, so they cannot unfold hydrogen peroxide, excreting it into the medium. This molecule is a strong oxidant, producing oxidations in lipids and membrane proteins.

carbon dioxide: it is produced by heterofermentative BALs, creating an anaerobic environment in the environment, which could damage certain aerobic bacteria. It also produces a decrease in pH on both sides of the cytoplasmic membrane.

(iv) diacetyl: it is a product of the metabolism of citric acid, and is responsible for the characteristic flavor of some foods such as butters and other fermented dairy products being produced by certain bacteria of the genera Leuconostoc, Lactococcus, Pediococcus and Lactobacillus. Said production is inhibited when there are metabolizable hexoses in the medium. Gram- bacteria, fungi and yeasts are more sensitive than Gram + to diacetyl. Apparently its mechanism of action is based on interference in the use of the amino acid arginine by these microorganisms.

(v) reuterine: it is produced by Lactobacillus Reuteri in a medium with a mixture of glucose and glycerol or glyceraldehyde. It has a general antimicrobial spectrum affecting viruses, fungi, protozoa and bacteria. This activity occurs, by inhibiting ribonucleotide reductase.

In Enzyme and microbiological Technology, 1999, page 87-107, it is indicated that the medium used for the growth of LABs is decisive for the amount of lactic acid that will be produced.

BALs have the ability to withstand pH of 5 and lower, which gives them a selective advantage over other bacteria. The optimum growth temperature is between 20 and 45 ⁰ C. Many of them are considered beneficial for human consumption, but others can be toxic Like some streptococci. BALs have complex nutritional requirements, due to their limited ability to synthesize B vitamins, and amino acids.

BALs can use the carbohydrates by fermenting them to a single final product (homofermentadores) or several final products (heterofermentadores). In homofermentators the main and almost unique product is lactic acid, while heteofermenters synthesize lactic acid, ethanol, carbon dioxide (CO ₂ ), and acetates. The amount of ethanol and acetate formed depends on the redox potential of the medium. All LABs can use the pentose phosphate pathway, that is, carry out heterofermentation, except for lactobacilli type I, for example, Lactobacillus delbrueckii. A mixture of acids is produced by homofermentadores such as lactococci, when the concentration of glucose in the medium decreases, and during growth in other sugars such as Lactobacillus lactis in maltose, lactose and galactose, or when the pH increases and the temperature decreases.

For example, Lactobacilus amylophilus grows at 15 ⁰ C and 45 ⁰ C, but the higher production Ia leads to 25 and 37 ⁰ C. For Lactobacilus casei and Lactobacilus paracasei between 37 and 44 ⁰ C, which is contradictory, since these bacteria they grow at 15 ⁰ C and not at 45 ⁰ C. Lactobacilus lactis and Lactobacilus rhamnosus exhibited the highest productivity at 33-35 ⁰ C and 41-45 ⁰ C.

Given the competence that LABs can exert with bacteria of the genus pseudomonas, proteus, flavobacterium, enterobacter, shewanella etc. These bacteria would be displaced from the environment by the extreme conditions in which lactic bacteria can live.

Certain amines are formed in the shellfish after their capture, even when stored at low temperatures, because bacteria of the aforementioned genera produce the decarboxylation of free amino acids. The formation of amines such as histamine, putrescine and cadaverine, is parallel to the growth of these microorganisms, producing bad odors, gasification of the medium and producing allergic reactions in some consumers. According to a third aspect of the present invention, this refers to the use of lactic acid bacteria in the elimination of other bacteria that produce the formation of certain biogenic amines, not desirable in crustaceans.

In accordance with a fourth aspect of the invention, this refers to crustaceans treated with microbial starters consisting of BAL.

According to an embodiment of the present invention, the use of lactic acid bacteria such as Lactobacillus helveticus, L. lactis, C. piscícola, L. plantarun, and P. acidilacti, dramatically inhibits the appearance of melanosis in Crustaceans, post-mortem, once treated by immersion.

Other aspects and advantages of the immersion treatment of crustaceans with solutions of lactic acid bacteria are:,

(i) homogeneous distribution of the antimelanosic agent in crustaceans by immersion in the solution containing the BAL.

(ii) the method of immersion in the solution establishes the control of the effective concentration of the beneficial antimelanosic agent in the edible part of the crustaceans.

(iii) once the lactic acid bacteria are installed in the crustaceans, their action on the enzyme (PPO-Cu), protein substrate, etc., lasts once the process of immersion treatment of the crustaceans ceases.

Once the crustaceans are removed from the container containing the solution of lactic acid bacteria, the organoleptic properties (appearance, presence, color, taste) are practically not altered, which provides significant advantages over traditional sulphite sprinkling methods. , (aggressive products) which remain in contact with crustaceans during weeks, months, causing discoloration of the external skeleton, dryness, bad taste etc.,

According to a preferred embodiment, the concentration of lactic acid bacteria used is between approximately 80 CFU / ml and 140 CFU / ml. More preferably, between 100 CFU / ml and 110 CFU / ml, with 106 CFU / ml and 108 CFU / ml being even more preferred.

Suitable culture media for carrying out the method according to the present invention are those selective means of lactic bacteria, in which the bacteria grow satisfactorily. Among the most appropriate means are:

Minimum Essential Medium (MEM): Sodium chloride (NaCl) (0.7%) and D (+) glucose (2%), peptone (5%), magnesium sulfate. heptahydrate

(0.005%). Initial pH between 6-6.3.

Poor Medium (MP): Sodium Chloride (NaCl) (0.7%) and D (+) glucose (5%), initial pH between 6 and 6.5.

MRS medium: diammonic citrate broth trades; powdered meat extract, yeast extract, D (+) Glucose, magnesium sulfate, manganese (II) sulfate, bacteriological peptone, dipotassium phosphate, sodium acetate, and tween 80. This medium has an initial pH of approximately 6.2 .

In accordance with a preferred embodiment of the present invention, a medium selected from Minimal Essential Medium, Poor Medium, MRS and the culture medium synthesized by them, using glucose as a nutrient solution, is used as the culture broth. In a more preferred embodiment the culture medium is Poor Medium.

According to a preferred embodiment, the established time of immersion of the crustaceans in the solution of lactic acid bacteria is between 5 and 120 minutes. In a more preferred embodiment the immersion time is between 10 and 60 minutes According to an even more preferred embodiment, the immersion time is between 15 and 45 minutes, with an immersion time of approximately 30 minutes being especially preferred.

According to a preferred embodiment, the temperature at which the treatment of the crustaceans is carried out is between 0 ⁰ C and 35 ⁰ C. According to a more preferred embodiment, the treatment temperature is 3 ⁰ C.

Once the crustaceans are treated under the conditions set, the crustaceans are refrigerated and then distributed for fresh consumption, or they are frozen if their consumption expands over time. The microscopic suspension of lactic acid bacteria in the culture medium is suitable to repeat several cycles of treatment of crustaceans by immersion. This way of proceeding can be extrapolated indefinitely provided that the concentration of lactic acid bacteria is practically maintained so that the melanization process and the quality of crustaceans are not affected. The possibility of carrying out these immersion treatment cycles makes the process of using lactic acid bacteria as a melanosis inhibitor technologically profitable.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. The descriptions in the summary of this application are incorporated herein by reference. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Representation of the enzymatic activity of the enzyme Polyphenol Oxidase of Parapenaeus longirostis, by different absorbance curves (A) and pH as a function of time (t). It is observed in the graph that as the pH of the medium is more acidic, the absorbance is lower and therefore less color develops, which is the objective pursued with the production of lactic acid produced by lactic bacteria, in our case Lactobacillus helveticus.

FIG. 2. The percentage of P. longirostris PPO inhibition activity is compared comparatively, to which no treatment (in blue) is performed, compared to samples treated with the L. lactis (I), C. piscícola ( H), L. plantarum (III), L. helveticus (IV) and L. Pediococus acidilactici (V), in medium in poor medium (in red) and in minimal essential medium (in white).

FIG. 3. Graphical representation of the measure of absorbance (A) versus time (t) of the speed of appearance of Dopachrome.

The following embodiments are provided by way of illustration, and are not intended to be limiting of the present invention.

DETAILED EXHIBITION OF REALIZATION MODES

Example 1. Study of the native microflora of shellfish

One of the essential factors of action of LAB is competition with other bacteria that accelerate the melanosis process. For the microbiological study, the API strip system is used, which is a standardized identification system, which uses different miniaturized biochemical tests and a database. It consists of ten microtubes containing dehydrated substrates. These tests are inoculated with a bacterial suspension, which reconstitutes the media. During the incubation, spontaneous or revealed color changes occur by the addition of reagents.

After incubation for 18-24 hours at 35-37 ⁰ C, the reading is displayed contrasting reactions profile list, or with software identification. The extraction of the bacterial flora from the shellfish was carried out, using buffered peptone water, autoclaved 121 ⁰ C for 20 minutes, with 0.01% Triton X-100 on a magnetic stirrer, at 37 ⁰ C for 12 hours. At that time the bacteria take off from their host, and go to the solution of peptonated water. Next, we proceeded to isolate the strains that inhabit the liquid medium, using a solid, general, non-specific medium, where any bacteria can grow. This medium is the TSA (Tryptone Soy Agar). Were performed in petri dishes, sow "depletion" with triplicate, incubated at 37 ⁰ C for 24 hours. On the following day it was possible to observe how dozens of colonies have grown on the plates, in most cases between 3 and 4 different colonies, so it is expected that in the shellfish naturally decomposing, they are predominantly installed number of bacteria

Crustaceans, once captured, and after fifteen days of storage in the refrigerator at a temperature of 0-5 ⁰ C do not present melanosis and for an indefinite time (from 1 to 10 months) at the temperature of -18 ⁰ C, when were treated with microbial starters such as Lactobacillus helveticus, L. Lactis, C. piscícola, L. plantarun, and P. acidilacti.

Example 2. Inhibition of melanosis in crustaceans.

The studies are all based on the crustacean Parapenaeus longirostris, known as the "white prawn of Huelva". This arthropod has a cuticle and appearance with light tones, so that the effects of melanosis will be more pronounced. The specimens were always fresh and without chemical treatment, nor any other of any nature prior to our experiences.

Reconstitution of bacterial strains.

The Lactobacillus helveticus bacteria, CECT 4305 T. were entrusted to the Spanish Type Culture Center. These strains are lyophilized in small blisters, to which 0.3 milliliters of the MRS medium was added, and resuspended with Pasteur pipette, and inoculated with 200 mi of the same medium contained in volumetric flask. This broth was incubated for 24 hours at 37 ⁰ C, and to the end of this time, concentrations of Lactobacillus helveticus were obtained from 100 to 1000 million cells per milliliter in medium it reaches a pH between 4.20 and 4.50.

Enzymatic activity of the enzyme Polyphenol Oxidase of Parapenaeus lonαirostis. between a range of DH 3 v 9.

Cephalotorax samples were taken, where the enzyme is most abundant, the tissue extract taken was weighed and homogenization was carried out with liquid nitrogen and with 3 milliliters of the different buffers used. It was centrifuged at 13240 gs for 30 minutes and the precipitate was discarded, separating the soluble phase where the enzyme is found. A 5% trypsin solution in distilled water was then prepared, and a solution of 25 mM DL-Dopa in phosphate buffer pH 6.5.

The absorbance produced in the spectrophotometer was measured at the wavelength (λ of 390 manometers), every 2 minutes, for 20 minutes, of maximum activity of the PPO. The results are shown in Table 1.

Table 1

It was observed that after 8 minutes the enzymatic reaction ceased to be linear. The study of this area, in which the reaction of product formation with respect to time is linear allows us to calculate the slope of the line that represents the kinetics of the reaction, and which serves to quantify the enzymatic activity.

1. pH = 4. There is no reaction. The slope is equal to 0.

2. pH = 5. Y = 0.0042X + 0.058.

3. pH = 6. Y = 0.0084x + 0.011.

4. pH = 7. Y = 0.0108x + 0.0002. 5. pH = 8. Y = 0.0106X + 0.001.

6. pH = 9. Y = 0.0276X + 0.089.

Applying the equation of the Specific Enzyme Activity:

Abs. ^• VoI. Extraction buffer (mi)

Act. Enz. Esp. = E ^'1 • d ^"1

VoI sample (mi) - time (min) ^• fresh weight

e = molar extinction coefficient 3600 M ^"1 - cm ^{" 1}

d = optical path = 1 cm

We obtain the following results:

For pH 4, there is no enzymatic activity.

For pH 5, we have a specific enzymatic activity of 3.80 x 10 elevated to

The least 6 micromoles / min x gram fresh weight.

For pH 6, 7.60 x 10 raised to minus 6 micromoles / min x gram fresh weight For pH 7, 9.76 x 10 raised to minus 6 micromoles / min x gram fresh weight For pH 8, 9.58 x 10 raised to at least 6 micromoles / min x gram fresh weight.

For pH = 9, 24.95 x 10 raised to at least 6 micromoles / min x gram fresh weight.

For the development of the enzymatic activity "in situ" the following buffers were used:

For pH 4 and 5 we use the Acetic / Acetate buffered system.

For pH 6 and 7 we use the monobasic potassium phosphate / dibasic potassium phosphate buffered system.

For pH of 8 and 9 we use the tris buffered system.

All media are used at the concentration of 0.1 molar.

If we represent the data obtained we will see how there is a linear zone in which the variation of the absorbance is practically constant, it is in this area where we calculate the slope and with it the specific activity of the PPO, applying the equation of the Specific Enzymatic Activity.

The activity of the phenoloxidase (PPO) is expressed as moles of DL-DOPA transformed per minute and per mg at, 475 nm, at different pH and at room temperature under the conditions described. The oxidation of the DOPA to DOPACROMO, catalyzed by the PPO implies an appreciable color change, which is what we measured in the spectrophotometer at 475 nm.

The extract was obtained from a shrimp head sample, which was incubated with 1% trypsin for 15 minutes with stirring and the absorbance variation at 475 nm versus DL-DOP was measured over time.

The values presented in Table 2 are the average of 3 tests ± the standard deviation (SD), which we represent graphically and obtain the specific enzymatic activity of the linear zone of Figure 3 Table 2

If we represent the data obtained (see FIG. 3) we will see how there is a linear zone in which the variation of the absorbance is practically constant, it is in this area where we calculate the slope and with it the specific activity of the PPO, applying the following formula:

Slope of the straight line = 0.0339, R2 = 0.994

Specific activity = 30.65 x 10 ^"6 μmoles.m¡n-1.mg-1

Production of lactic bacteria and bacteriogenic means.

Different, selective cultures of lactic bacteria were tested, in which the bacteria grow satisfactorily such as Minimum Essential Medium (MEM), MRS Medium and Poor Medium (MP).

When these microorganisms were incubated at 37 ⁰ C, in the media, to the end of 24 hours concentrations were obtained between 100 and 1,000 million bacteria per milliliter of medium, being the new pH between 3.5 and 4.0, Io follows that the Bacterial metabolism has been the one that has generated that drastic drop in pH. Both MEM and MRS, provided adequate means for the development of the culture, however the MP medium is the one that granted greater enzymatic inhibition and absence of blackening of shellfish, and with excellent organoleptic properties of crustaceans.

Example of tests carried out with MEM media

A saline solution of 0.7% sodium chloride and D (+) glucose (2%), peptone (5%), magnesium sulfate heptahydrate (0.005%), initial pH 6.2 was taken. and it inoculated with lactic acid bacteria of Lactobacillus helveticus and incubated at 37 ⁰ C, observed slow growth of said bacteria. At 76 hours the solution presented desired concentrations for our experience, specifically 10 to 100 million bacteria per milliliter of medium. The initial pH of the medium was 5.82, while at 76 hours of incubation of the bacteria the pH is 3.3, in all experiences. The medium is odorless and colorless, not appreciating bad taste. Seafood was treated at refrigeration temperatures, specifically at 3 ⁰ C, at different residence times; 10, 20, 30, 60 and 120 minutes by immersion in the bacteriogenic medium described. Once the crustaceans have been removed from the solution, immersion cycles are repeated up to five consecutive times. Next, the evolution of melanosis was studied, observing that at 24 hours the treated shellfish remains unchanged, at room temperature 18-20 ⁰ C, while in the control shellfish (shellfish without any treatment) principles of melanosis in the head and even in the legs. After 12 days of keeping the crustaceans in the refrigerator at 3-5 ⁰ C no appearance of melanosis was observed. Figure 3 represents the percent inhibition of the bacteria tested in different culture media such as Poor Medium (MP) and Minimum Essential Medium (MEM).

Claims

1. A method of preserving a crustacean against melanosis, characterized in that it comprises the steps of contacting crustaceans with a solution containing an adequate amount of lactic acid bacteria and their subsequent storage under appropriate conditions.

2. The method according to the preceding claim, characterized in that the bacteria employed are of the genera lactobacillus, lactococcus, leuconostoc and pediococcus.

3. The method according to any of the preceding claims 1 to 2, characterized in that the bacteria are of the Lactobacillus helveticus, L. Lactis, C. piscícola, L. plantarun, and P. addilacti type.

4. The method according to any of the preceding claims 1 to 3, characterized in that the bacteria are suspended in a suitable culture medium.

5. The method according to any of the preceding claims 1 to 4, characterized in that said culture medium is selected from minimum essential medium (MEM), poor medium (MP), MRS medium and the culture medium synthesized by themselves, using glucose as a nutrient solution.

6. The method according to any of the preceding claims 1 to 5, characterized in that said culture medium is the poor medium (MP).

7. The method according to any of the preceding claims 1 to 6, characterized in that the concentration of lactic acid bacteria is between 80 CFU / ml and 140 CFUmI.

8. The method according to any of the preceding claims 1 to 7, characterized in that the concentration of lactic acid bacteria is between 100 CFU / ml and 110 CFU / ml.

9. The method according to any of the preceding claims 1 to 8, characterized in that the concentration of lactic acid bacteria is between 106 CFU / ml and 108 CFU / ml

10. The method according to any of the preceding claims 1 to

9, characterized in that the immersion time is between 5 and 120 minutes.

11. The method according to any of the preceding claims 1 to

10, characterized in that the immersion time is between 10 and 60 minutes.

12. The method according to any of the preceding claims 1 to 11, characterized in that the crustaceans are treated at a temperature between 0 ⁰ C and 35 ⁰ C.

13. The method according to any of the preceding claims 1 to 12, characterized in that the crustaceans are treated at a temperature of 3 ⁰ C.

14. Use of lactic acid bacteria (BAL) as inhibitors of crustacean melanosis.

15. The use according to the preceding claim characterized in that the bacteria employed are of the genera lactobacillus, lactococcus, leuconostoc and pediococcus.

16. The use according to any of the preceding claims 14 to 15, characterized in that the bacteria are of the Lactobacillus helveticus, L. Lactis, C. piscícola, L. plantarun, and P. acidilacti type.

17. Use of lactic acid bacteria in the elimination of other bacteria that produce the formation of certain biogenic amines, undesirable in crustaceans.

18. Crustaceans treated with lactic acid bacteria as melanosis inhibitors for food use.