Biodegradation process and composition.

(54) Title: PROCESS AND COMPOSITION OF BIODEGRADATION. (54) Title: BIODEGRADATION PROCESS AND COMPOSITION.

(57) Summary

Novel microbial compositions and biodegradation processes are described for treating marine animals or marine animal by-products to produce solid, liquid and lipid fractions containing useful compounds.

(57) Abstract

Disclosed are novel microbial compositions and biodegradation processes to treat marine animal or marine animal byproducts to produce solid, liquid and lipid fractions that contain useful compounds.

BIODEGRADATION PROCESS AND COMPOSITION

Field of the Invention

Novel microbial compositions and microbial processes are described for treating marine animal by-products and, in some cases, the entire marine animal to produce solid, liquid and lipid fractions containing useful compounds.

Background of the Invention

Processing of marine fish and arthropods, such as shrimp, crab, and crayfish, produces large amounts of marine by-products. Most are used in low-cost products, such as fertilizers, fish silage, or pet food, but unused by-products represent an economic burden on marine product processing industries, due to the need to dispose of such residues. in an environmentally sound way. According to some estimates, by-products of this type represent 25% of the total production captured by the fisheries.

For example, during the processing of the shrimp for its subsequent freezing and commercialization, a large quantity of remains is generated since 35% of the animal is not edible and must be discarded. These remains or by-products are made up of the shrimp's cephalothorax and exoskeleton. No Ref.: 231006 However, these shrimp by-products are rich in high-value substances such as chitin, protein, lipids, carotenoid pigments (astaxanthin) and minerals. Most of the inedible by-products are dumped in landfills or dumped back into the ocean, thereby causing serious environmental problems and considerable losses to the shrimp processing industry. Currently, only a small amount of these by-products is used as a supplement as an animal feed.

The most common technique for using the shrimp by-product is solar drying. This technique has little hygienic control and the products are mainly used for animal feed. Other methods employ chemical acids and alkalis at different concentrations, temperatures, and times for chitin extraction and recovery of protein hydrolyzates. However, these methods produce a partial depolymerization and deacetylation of chitin. Furthermore, these methods complicate the recovery of other products, such as protein and pigment.

Enzymatic methods for the extraction of chitin, hydrolyzates from liquids and pigments were developed. Methods of this type use enzyme extracts or enzyme isolates. Other studies reported the use of microbial enzymes, such as commercial alcalase, for the extraction of proteins from by-products from shrimp and marine animals. The combination of alcalase and pancreatin was reported for the extraction of chitin, hydrolyzed protein and pigmented lipids.

Lactic fermentation processes were used as a substitute for previous chemical and enzymatic processes. Fermentation represents a profitable technique that stabilizes and preserves the nutritional quality of by-products. Optimal fermentation conditions depend on several factors, including the selection and concentration of carbohydrates, pH, temperature, time, and the selection of aerobic or anaerobic conditions. Another important factor is the selection of the microorganism and initial concentration of the inoculum. To facilitate the fermentation process of the shrimp by-products, pure cultures of the lactic acid bacteria (LAB) were used. Such LABs include Lactobacillus plantarum (Rao, MS, Stevens, WF, 2006, Fermentation of shrimp biowaste under different salt concentrations with amylolytic and non-amylolitic Lactobacillus strains for chitin production, Food Technology and Biotechnology 44, 8'3-87; Rao, MS, Muñoz, J., Stevens, WF, 2000, Critical factors in chitin production by fermentation of shrimp biowaste, Applied Microbiology and Biotechnology 54, 808-813; Bhaskar, N. , Suresh, PV, Sakhare, PZ, Sachindra, NM, 2007, Shrimp biowaste fermentation with Pediococcus acidolactici CFR2182:

optimization of fermentation conditions by response surface methodology and effect of optimized conditions on deproteination / demineralization and carotenoid recovery, Enzyme and Microbial Technology 40, 1427-1434), Lactobacillus sp. B2 (Cira, LA, Huerta, S., Hal, GM, Shirai, K., 2002, Pilot scale lactic acid fermentation of shrimp waste for chitin recovery, Process Biochemistry 37, 1359-1366; Shirai, K., Guerrero, I ., Huerta, S., Saucedo, G., Castillo, A.,

González, RO, Hall, GM, 2001, Effect of initial glucose concentration and inoculation level of lactic acid bacteria in shrimp waste ensilation, Enzyme and Microbial Technology 28, 446-452), Lactobacillus casei (Shirai 2001), Lactobacillus paracasei (Jung, WJ, Jo, GH, Kuk, JH, Kim, YJ, Oh, KT, Park, RD, 2007, Production of chitin from red crab Shell waste by successive fermentation with Lactobacillus paracasei KCTC-3074 and Serratia marcescens FS3, Carbohydrate Polymers 68, 746-750), Lactobacillus pentosus (Bautista, J., Jover, M., Gutiérrez, JF, Corpas, R., Cremades, O., Fontiveros, E., Iglesias, F., Vega, J., 2001, Preparation of crayfish chitin by in situ lactic acid production, Process Biochemistry 37, 229-234; Shirai 2001), Lactobacillus acidophilus B4495 and Lactobacillus lactis (Bhaskar 2007), Lactobacillus salvation (Beaney 2005), Enteroccus facium (Beaney 2005), Pedioccoccus acidilactic Bhaskar 2007) and Pedioccoccus sp. Ll / 2 (Choorit, W.,

Patthanamanee, W., Manurakchinakorn, S., 2008, Use of response surface method for the determination of demineralization efficiency in fermented shrimp shells,

Biores. Technol. 99, 6168-6173). In addition, a mixture of four LABs has been used (Bhaskar 2007) and there are reports of the use of Lactobacillus in combination with Serratia marcescens

FS-3 (Jung 2007) or Staphylococcus carnosus (Shirai 2001). However, the industrialization of such fermentation processes has not been successful due to the poor performance of commercial inoculants.

Lactic fermentation of shrimp by-products produces protein, chitin, mineral and lipid hydrolyzates. Chitin and its deacetylated by-products have many applications in agriculture, biomedicine, food, and the paper industry, while liquid hydrolyzate is an excellent source of essential amino acids that can be used for human or animal consumption. The lipid paste contains sterols, vitamins A and E, and carotenoid pigments such as astaxanthin that can be used in salmon feed or as a natural dye in the food industry.

Chitin is a natural polysaccharide found particularly in the exoskeleton of crustaceans, insect cuticles, and fungal cell walls. Because chitin is one of the most abundant biopolymers, much interest has been paid in its biomedical, biotechnological, and industrial applications. Chitosans are compounds of poly- (β-1-4) -N-acetyl-D-glucosamine that are produced by deacetylation of chitin (β-1-4) -Nacetyl-D-glucosamine. Glucosamine is an amino monosaccharide that is obtained by the depolymerization of chitosan. It participates in the constitution of glycosaminoglycans, an important class of complex extracellular polysaccharides. Glucosamine sulfate, glucosamine hydrochloride, and Nacetyl glucosamine are normally used alone or as part of a mixture.

In general, the liquid hydrolyzate has a high content of essential amino acids, indicating a high nutritional value that justifies its use as a supplement for animal and aquaculture nutrition or as a source of nitrogen in growth media for microorganisms. Furthermore, these hydrolyzates are a source of free amino acids and can be used as a biostimulant for plant nutrition.

Astaxanthin (3,3'-dihydroxy-β, β-carotene-4,4¹-dione), an oxidized ketocarotenoid of β-carotene, occurs naturally in a wide variety of marine and aquatic organisms. Due to its attractive pink color, its biological functions as a precursor to vitamin A, and antioxidant activity, astaxanthin can be used as a food and medicine dye. In the astaxanthin structure, two identical asymmetric carbon atoms are found at C3 and C3 '. However, trans-astaxanthin is the most frequent quantitative carotenoid in crustacean species.

References describing these and other products of lactic fermentation include: Sánchez-Machado et al., Quantification of organic acids in fermented shrimp waste by HPLC Food Technology and Biotechnology, volume 46, 456 (2008); Sánchez-Machado et al. High-performance liquid chromatography with fluorescence detection for quantitation of tryptophan and tyrosine in a shrimp waste protein concentrate, Journal of Chromatography B, volume 863, 88 (2008); López-Cervantes et al., Quantitation of glucosamine from shrimp waste using HPLC Journal of Chromatographic Science, volume 45, 1 (2007); López-Cervantes and others,

Quantification of astaxanthin in shrimp waste hydrolysate by HPLC Biomedical Chromatography, volume 20, 981 (2006);

López-Cervantes et al., High-performance liquid chromatography method for. the simultaneous quantification of retinol, alpha-tocopherol, and cholesterol in shrimp waste hydrolysate Journal of Chromatography A, volume 1105, 1-2 (2006); López-Cervantes et al., Analysis of free amino acids in fermented shrimp waste by 'high-performance liquid chromatography, Journal of Chromatography A, volume 1105, 1 (2006)

Brief Description of the Invention

<td>Compositions are described</td><td>microbial</td><td>and</td><td>processes</td><td>of</td>

<td>biodegradation.</td><td></td><td></td><td></td><td></td>

<td>A microbial composition</td><td>understands</td><td>(to)</td><td>one or</td><td>plus</td>

<td>lactic acid bacteria (LAB)</td><td>and (b) one or</td><td>plus</td><td>or two or</td><td>plus</td>

<td colspan="2">selected microorganisms from group i</td><td>of the</td><td>gender</td><td>than</td>

It consists of Bacillus, Azotobacter, Trichoderma, Rhizobium,

Clostridium, Pseudomonas, Streptomyces, Micrococcus,

Nitrobacter and Proteus. In preferred embodiments, at least one of the Bacillus, Azotobacter, Trichoderma, Rhizobium,

Clostridium, Pseudomonas, Streptomyces, Micrococcus,

Nitrobacter and Proteus is a chitinolytic strain that produces a chitinase (for example, endochitinase and / or exochitinase). In some microbial compositions LAB is selected from the genus consisting of Lactobacillus,

Lactococcus, and Streptococcus. When .

Lactobacillus, it is preferred that the LAB is Lactobacillus acidophilus and / or Lactobacillus casei, most preferably Lactobacillus acidophilus (Bioderpac, 2008) and Lactobacillus casei (Bioderpac, 2008).

The Bacillus in this composition is selected

Pediococcus, LAB is preferably from the group consisting of Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus licheniformis, and Bacillus thuringiensis, most preferably Bacillus subtilis (SILoSil BSJ, Bacillus cereus (Bioderpac, 2008), Bacillus lichenc. ) and HD-1 and HD-73 strains of Bacillus thuríngiensis (SILoSil® BT).

The Azotobacter in this composition is preferably Azotobacter vinelandii, most preferably Azotobacter vinelandii (Bioderpac, 2008).

The Trichoderma in this composition is preferably Trichoderma harzianum, most preferably Trichoderma harzianum (TRICHOSIL)

The Rhizobium in this composition is preferably Rhizobium japonicum, most preferably Rhizobium japonicum (Bioderpac, 2008).

The Clostridium in this composition is preferably Clostridium pasteurianu, more preferably Clostridium pasteurianu (Bioderpac, 2008).

The Pseudomonas in this composition is preferably Pseudomonas fluorescens, most preferably Pseudomonas fluorescens (Bioderpac, 2008).

Another microbial composition comprises one or more or two or more microorganisms selected from the group consisting of Bacillus subtilis (3ΙΕο3ί1^Φ BS), Bacillus cereus (Bioderpac, 2008), Bacillus megaterium (Bioderpac, 2008), Azotobacter vinelandii (Bioderpac, 2008), Lactobacillus acidophilus (Bioderpac, 2008), Lactobacillus casei (Bioderpac, 2008), Trichoderma harzianum (TRICH japonicum acidophilus (Bioderpac,

Rhizobium (Bioderpac, 2008), Clostridium pasteurianum (Bioderpac, 2008), Bacillus licheniformis (Bioderpac, 2008), Pseudomonas fluorescens (Bioderpac, 2008), HD-1 and HD-73 strains of Bacillus thuríngiensis (SILoSil® BT), Streptces Bioderpac, 2008), Micrococcus (Bioderpac, 2008), Nitrobacter (Bioderpac, 2008) and Proteus (Bioderpac, 2008).

Another embodiment of a microbial composition comprises Lactobacillus acidophilus (Bioderpac, 2008) and / or Lactobacillus casei (Bioderpac, 2008).

A particularly preferred microbial composition comprises Bacillus subtilis (SILoSil¹⁸ BS), Bacillus cereus (Bioderpac, 2008), Bacillus megaterium (Bioderpac, 2008), Azotobacter vinelandii (Bioderpac, 2008), Lactobacillus (Bioderpac, 2008), Lactobacillus casei 2008), Trichoderma harzianum (TRICHOSIL), japonicum (Bioderc ), Clostridium pasteurianum (Bioderpac, 2008), Bacillus licheniformis (Bioderpac, 2008), Pseudomonas fluorescens (Bioderpac, 2008), Bacillus thuríngiensis strains HD-1 and HD-73, Streptomyces (Bioderpac, 2008), Micrococcus (Bioderpac, 2008) , Nitrobacter (Bioderpac, 2008) and Proteus (Bioderpac, 2008).

A preferred microbial composition is HQE. HQE was deposited with the American Type Culture Collection (ATCC) Manassas, VA, United States on April 27, 2010 and was given the patent filing designation PTA-10861.

In addition, isolated microorganisms selected from the group consisting of Bacillus subtilis (SILoSil® BSJ, Bacillus cereus (Bioderpac, 2008), Bacillus megaterium (Bioderpac, 2008), Azotobacter vinelandii (Bioderpac, 2008), Lactobacillus acidophilus (Bioderpac, 2008) are described. ), Lactobacillus casei (Bioderpac, 2008), Trichoderma harzianum (TRICHOSIL), Rhizobium japonicum (Bioderpac, 2008), Clostridium pasteurianum (Bioderpac, 2008), Bacillus licheniformis (Bioderpac, 2008), Pseudomonas fluorescens (Bioderpac, 2008), Bacillus thuringiensis strains HD-1 and HD-73 (SILoSil® BT), Streptomyces (Bioderpac, 2008), Micrococcus (Bioderpac, 2008), Nitrobacter (Bioderpac, 2008) and Proteus (Bioderpac, 2008).

The biodegradation process comprises mixing a marine animal or marine animal by-product with any of the aforementioned microbial compositions to form a mixture; ferment the mixture, and separate the mixture into solid, liquid and lipid fractions. Unlike the biodegradation processes of the prior art, the described biodegradation process produces chitosan and glucosamine that can be found in the aqueous fraction. The marine animal is preferably a marine arthropod, such as shrimp, crayfish, crab, or Antarctic shrimp. In some embodiments, the marine animal is fish or a by-product of fish, such as the skin, muscle, or organ of the fish.

Brief Description of the Figures

Figure 1 schematically shows a preferred degradation process for shrimp by-products.

Figure 2 depicts the pH and total titratable acidity during lactic fermentation of shrimp by-products.

Detailed description of the invention

As used herein, the term marine animal refers to any animal that lives in the oceans, seas, or freshwater. Marine animals include fish and marine arthropods.

As used herein, the term marine arthropod refers to an invertebrate marine animal that has an exoskeleton, that lives in oceans, seas, or freshwater. Marine arthropods usually have a segmented body and jointed appendages. Marine arthropods are members of the subphylum Crustacea. Preferred classes of Crustacea include Eranchiopoda (for example, sea shrimp, Cladócero and Triops), Cephalocardia (for example, horseshoe shrimp), Maxillopoda (for example, barnacles and copepods (zooplankton)), Ostracoda (for example, ostracods) and Malacostraca (for example, crabs, lobsters, shrimp, Antarctic shrimp etc.).

As used herein, the term by-product refers to any part of a marine animal. In some modalities', the by-product is produced through commercial processing of the marine animal. For example, in the shrimp industry, cephalothorax and exoskeleton are by-products of shrimp. In the crab and lobster processing industry, the exoskeleton (shell) is a by-product.

In some embodiments, the entire arthropod can be used in the biodegradation process. For example, many Antarctic shrimp are about 1-2 centimeters (0.4-0.8 inches) long as adults, but some species grow to sizes on the order of 6-15 centimeters (2.4-5.9 inches). Antarctic shrimp oil contains at least three components: (1) omega-3 fatty acids similar to those of fish oil, (2) phospholipid-conjugated omega-3 fatty acids, and (3) the antioxidant astaxanthin. Also, the exoskeleton contains fluoride. As a consequence, these components can be separated in the biodegradation process with the oil components that are isolated in the lipid fraction and the fluoride in the liquid fraction.

As used herein, the term "microbial composition" refers to a liquid, solid, or gelatinous medium that physically contains or supports one or more microorganisms, preferably two or more. Microbial compositions include, but are not limited to, fermentation broths containing one or more microorganism (s) and inocula generally used to initiate a fermentation broth and containing a concentration of microorganisms greater than is present in the fermentation broth. Microbial compositions containing species / strains are sometimes referred to as Bioderpac microbial compositions which refers to a composition containing one or more of the microorganisms or a combination of one or more microorganisms with other microorganisms.

As used herein the term "isolated microorganism" refers to a liquid, solid, or gelatinous medium that physically contains or supports a microorganism.

The described microbial compositions or isolated microorganisms can combine with other microorganisms to form a new microbial composition that can be used for processes other than those specifically described in this document. The selection of the described microorganism (s) will depend on their biological properties (for example, production of protein or carbohydrate or enzymes that degrade chitin), the other selected microorganisms and the process in which the combination is going to use. Selection of such components and processes will be apparent to one skilled in the art by following the description herein.

A microbial composition comprises (a) one or more lactic acid bacteria (LAB) and (b) one or more or two or more microorganisms selected from the genus group consisting of Bacillus, Azotobacter, Trichoderma, Rhizobium , Clostridium, Pseudomonas, Streptomyces., Micrococcus, Nitrobacter and Proteus. In preferred embodiments, at least one or more, two or more or three or more of the Bacillus, Azotobacter, Trichoderma, Rhizobium,

Clostridium, Pseudomonas, Streptomyces., Micrococcus, Nitrobacter and Proteuses a chitinolytic strain that produces a chitinase (eg endochitinase and / or exochitinase). In some microbial compositions LAB is selected from the genus consisting of Lactobacillus, Pediococcus, Lactococcus, Lactococcus, When LAB is Lactobacillus, it is preferred that LAB be Lactobacillus acidophilus and / orLactobacillus casei, most preferably Lactobacillus acidophilus (Bioderpac, 2008) and Lactobacillus casei (Bioderpac, 2008).

The Bacillus in this composition is preferably selected from the group consisting of Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus licheniformis and Bacillus thuringiensis, most preferably Bacillus subtilis (SILoSil ”⁵ BS), Bacillus cereus (Bioderpac, 2008), Bacillus licheniformis (Bioderpac, 2008) and Bacillus thuringiensis strains HD-1 and HD-73 (SILoSil BT).

The Azotobacter in this composition is preferably

Azotobacter vinelandii, most preferably Azotobacter vinelandii (Bioderpac, 2008).

The Trichoderma in this composition is preferably

Trichoderma harzianum, most preferably Trichoderma harzianum (TRICHOSIL).

The Rhizobium in this composition is preferably Rhizobium japonicum, most preferably Rhizobium japonicum (Bioderpac, 2008).

The Clostridium in this composition is preferably

Clostridium pasteurianu, more preferably Clostridium pasteurianu (Bioderpac, 2008).

The Pseudomonas in this composition is preferably

Pseudomonas fluorescens, most preferably Pseudomonas fluorescens (Bioderpac, 2008).

Another microbial composition comprises one or more or two or more microorganisms selected from the group consisting of Bacillus subtilis ((SILoSil® BS), Bacillus cereus (Bioderpac, '2008), Bacillus megaterium (Bioderpac, 2008), Azotobacter vinelandii (Bioderpac, 2008 ), Lactobacillus acidophilus (Bioderpac, 2008), Lactobacillus casei (Bioderpac, 2008), Trichoderma harzianum (TRICHOSIL), Rhizobium japonicum (Bioderpac, 2008), Clostridium pasteurianum (Bioderpac,

2008), Bacillus licheniformis (Bioderpac, 2008), Pseudomonas fluorescens (Bioderpac, 2008), HD-1 and HD-73 strains of Bacillus thuringiensis (SILoSil¹"BT), Streptomyces (Bioderpac, 2008),

Micrococcus (Bioderpac, 2008), Nitrobacter (Bioderpac, 2008) and Proteus (Bioderpac, 2008).

Another embodiment of a microbial composition comprises Lactobacillus acidophilus (Bioderpac, 2008) and / or Lactobacillus casei (Bioderpac, 2008),

A particularly preferred microbial composition comprises Bacillus subtilis ((SILoSil® BS), Bacillus cereus (Bioderpac, 2008), Bacillus megaterium (Bioderpac, 2008), Azotobacter vinelandii (Bioderpac, 2008), Lactobacillus (Bioderpac, 2008), Lactobacillus casei 2008) , Trichoderma harzianum (TRICHOSIL), japonicum (Bioderpac, 2008), Clostridium pasteurianum (Bioderpac, 2008), Bacillus licheniformis (Bioderpac, 2008), Pseudomonas fluorescens (Bioderpac, 2008), HD-1 and HD-73 strains of Bacillus thuringiensis (SILoSil '”BT) Streptomyces (Bioderpac, 2008), Micrococcus (Bioderpac, 2008), Nitrobacter (Bioderpac, 2008) and Proteus (Bioderpac,

2008).

acidophilus (Bioderpac,

Rhizobium

The preferred microbial composition for use in the biodegradation process is HQE. HQE was filed with the American Type Culture Collection (ATCC) Manassas, VA, United States on April 27, 2010 and was given the patent filing designation PTA-10861. HQE is a microbial consortium made up of microorganisms derived from fertile soils and microorganisms from commercial sources. Microorganisms from commercial sources include Bacillus subtilis (SILoSil⁸ .BS), Bacillus thuringiensis strains ND-1 and HD-73 (SILoSil⁸ BT) and Trichoderma harzianum each obtained from Biotecnología Agroindustrial SA DE CV, Morelia, Michoacan, México. The microorganisms that

<td colspan="2">they can</td><td>derive</td><td>of</td><td>HQE</td><td>I know</td><td>they named</td><td colspan="2">Bioderpac 2008 by</td>

<td>strain</td><td>or</td><td>species</td><td>and</td><td>strain.</td><td>Without</td><td>However, it</td><td>can use</td><td>too</td>

<td>the</td><td colspan="3">subsets</td><td>of</td><td>the</td><td colspan="2">microorganisms in HQE.</td><td>The</td>

Φ microorganisms Bacillus subtilis (SILoSil BS), Bacillus thuringiensis (SILoSil⁸ BT) and Trichoderma harzianum (TRICHOSIL) produce chitinolytic enzymes that are especially important in the beginning of biodegradation. Chitinolytic enzymes help to break down chitin containing solids that can be a barrier to further digestion. These organisms also produce proteases, lipases, and other enzymes that facilitate the breakdown of proteins, lipids, and carbohydrates.

As used herein, the term HYTb refers to the aqueous fraction that is obtained from the biodegradation process. HYTb typically contains amino acids (from about 3% by weight to 12% by weight, usually about 12% by weight), chitosan (about 1.2% by weight), glucosamine (about 1% by weight), and trace elements (about 6% by weight) including calcium, magnesium, zinc, copper, iron and manganese. The amount of chitosan can be in the range of between about 0.5% by weight and 1.5% by weight, more preferably between about 1.0% by weight and 1.5% by weight. The amount of glucosamine can be in the range of between about 0.5% by weight and 1.5% by weight, more preferably between about 1.0% by weight and 1.5% by weight. The total of chitosan and glucosamine is approximately 2.0 to 2.5% by weight. HYTb also contains enzymes such as lactic enzymes, proteases, lipases, chitinases, lactic acid, polypeptides, and other carbohydrates. The specific gravity of HYTb is typically approximately 1,050-1,054. The average amino acid content in HYTb for certain amino acids is indicated in

Table 1.

TABLE 1

Amino acid profile of hydrolyzates in dry powder (mg per g dry weight)

<td>Amino acid</td><td>Hydrolyzed dry powder</td>

<td>Aspartic acid</td><td> 38</td>

<td>Glutamic acid</td><td> 39</td>

<td>Serine</td><td> 16</td>

<td>Amino acid</td><td>Hi drolized dry powder</td>

<td>Histidine *</td><td> 9</td>

<td>Glycine</td><td> 28</td>

<td>Threonine *</td><td> 14</td>

<td>To the girl</td><td> 36.1</td>

<td>Proline</td><td> 25.8</td>

<td>Tyrosine *</td><td> 70</td>

<td>Arginine</td><td> 22.2</td>

<td>Valine *</td><td> 20</td>

<td>Methionine *</td><td> 16.4</td>

<td>Isoleucine *</td><td> 18.3</td>

<td>Tryptophan *</td><td> 3.1</td>

<td>Leucine *</td><td> 23</td>

<td>Phenylalanine *</td><td> 39</td>

<td>Lysine *</td><td> 13</td>

<td>Total</td><td> 431</td>

<td>*Amino acids</td><td> 226</td>

<td>essential</td><td></td>

<td></td><td></td>

HYTb is typically produced by centrifugation of the fermentation product formed by. the biodegradation product. As the biodegradation process progresses, the nutrients used for microorganisms in the biodegradation process, for example

HQE, I know. They deplete and the pH drops due to the acid produced during fermentation. This causes the microorganisms in the fermentation product to die or become inactive. Depending on the force g and the time of centrifugation of the fermentation product, microorganisms of this type can be found in HYTb. As a consequence, HYTb can include any one or more of the components identified above, eg, chitosan and glucosamine, in combination with all or part of the microbial component of the fermentation process that is present when stopped. Alternatively, centrifugation could proceed to a point where substantially all of the microbial component is depleted of HYTb. In such cases, the microbial component can be centrifuged in the HYTc fraction. Alternatively, HYTc can be separated from HYTb by low gravity centrifugation. The HYTb can then be centrifuged to form a microorganism precipitate and a microorganism free of aqueous HYTb solution.

As used herein, the term HYTc refers to the solid fraction that is obtained from the biodegradation process. The main component of HYTc is chitin. It typically has an average molecular weight of approximately 2,300 daltons and constitutes approximately

64% by weight of the composition. Approximately 6% HYTc contains minerals including calcium, magnesium, zinc, copper, iron, and manganese, approximately 24% by weight of protein, and 6% of water. It has a specific gravity of approximately 272 Kg / m³. The chitin in HYTc typically has microorganisms from the fermentation product associated with it. Chitinolytic microorganisms have a propensity to associate with solid chitin. This is based on the affinity of the chitinolytic microorganisms for the chitin substrate. Consequently, HYTc may also contain chitinolytic microorganisms unless steps are taken to eliminate them. In the case of HQE, chitinolytic microorganisms of this type include one or more of the chitinase and / or exochitinase producing microorganisms discussed herein. Such microorganisms include but are not limited to Bacillus subtilis (SILoSil⁰ BS) HD-1 and HD73 strains of Bacillus thuringiensis (SILoSil® BT), and Trichoderma harzianum (TRICHOSIL). Chitinolytic microorganisms can be removed from the solid chitin by sterilization, pasteurization, or by washing the chitin with antimicrobial compounds such as soaps or chlorine. HYTc may also contain additional microorganisms present at the end of the biodegradation process due to the presence of residual fermentation product or centrifugation of HYTb.

HQE Consortium

The following are the HQE microorganisms and their known properties believed to be involved in the biodegradation process. In some cases, the strain is identified as Bioderpac, 2008. When the species is unknown, the species and the strain are identified as Bioderpac, 2008.

HQE was filed with the ATCC on April 27, 2010 and issued the patent filing designation PTA-10861.

Bacillus subtilís ((SILoSil® BS) is a Gram-positive bacterium that is mesophilic and grows at an optimal temperature between 25 and 35 ° C. It is aerobic and can grow in anaerobic conditions and uses a wide variety of carbon sources. It contains two nitrates reductases , one of which is used for nitrogen assimilation, is capable of secreting amylase, proteases, pullulanases, chitinases, xylanases and lipases.

Bacillus thuringiensis (strains HD-1 and HD-73 (SILoSil® BT)) are facultative anaerobic Gram positive bacteria, in the form of a peritric flagellum. Strains HD-1 and HD-73 during the spore period synthesize crystals with various geometric forms of protein and insecticidal activity. Strains HD-1 and HD-73 secrete exochitinases in a medium when it contains chitin and can be used for the degradation of crustacean residues during the production of chitooligosaccharides.

Bacillus cereus (Bioderpac, 2008) is a gram-positive, aerobic, facultative aerobic bacterium. It is mesophilic and grows at an optimal temperature between 20 and 40 ° C. Produces the antibiotics zwittermicin A and kanosamine.

Bacillus licheniformis (Bioderpac, 2008) is a bacterium

Gram positive, mobile, spore forming and facultative anaerobia. Produces bacitracin, alpha amylases, lactamases, proteases, and alkaline phosphatases. This is a non-pathogenic microorganism that is associated with plants or plant materials.

Bacillus megaterium (Bioderpac, 2008) is a Gram-positive aerobic bacterium. It is considered a saprophyte. Produces glucose dehydrogenase, penicillin amidase, beta-amidase, and neutral proteases.

Lactobacillus acidophilus (Bioderpac, 2008) is a member of one of the eight species of lactic acid bacteria. It is Gram positive, non-sporulating and produces lactic acid during fermentation that uses lactose as a primary source of carbon to produce energy. It grows with or without the presence of oxygen in an acidic medium (pH 4-5).

Produces bacteriocins, called lactazin B, organic acids, diacetyls, and hydrogen peroxide. .

Lactobacillus casei (Bioderpac, 2008) is a mesophyll, facultative anaerobe, that 'is Gram positive and does not form spores. It has the ability to adapt to cold temperatures. The optimal pH for growth is 5.5. Ferments galactose, glucose, fructose, mannose, mannitol, and acetylglucosamine. This species can be grown in a wide range of pH and temperature. Produces amylase enzymes. Inhibits the growth of pathogenic bacteria such as H. pylori reducing the pH through the production of (1) organic acids such as acetic, propionic or lactic acid or (2) of hydrogen peroxide. This microorganism secretes bacteriocins.

Pseudomonas fluorescens (Bioderpac, 2008) is a bacterium with multiple flagellum, strict aerobic and its optimal growth temperature is between 25 and 35 ° C. Produces thermoset lipases and proteases. It is antagonistic to a large number of strains of soil fungi. It produces secondary metabolites such as antibiotics, iron chelates, and cyanides. Produces endochitinase and cellulase in media with different glucose concentrations.

Trichoderma harzianum (TRICHOSIL) is a saprophytic fungus. It has antibiotic action and biological competition and for this reason it has biological control properties. Produces enzymes that degrade cell walls, or a combination of activities. It produces glucanases, chitinases, lipases and extracellular proteases when it interacts with some pathogenic fungi, such as Fusarium.

Rhizobium japonicum (Bioderpac, 2008)) is a nitrogen-fixing bacterium. It synthesizes a hydrogenase system that participates in the recycling of hydrogen to avoid its loss during nitrogen fixation.

Azotobacter vinelandii (Bioderpac, 2008) is an aerobic bacterium. It produces nitrogenase and is capable of fixing nitrogen.

Clostridium pasteurianum (Bioderpac, 2008) is a strict anaerobic, gram positive bacterium. Produces ferroxin (an electron transporting protein) that acts as a direct electron donor in reducing iron protein.

Proteus vulgaris (Bioderpac, 2008) is a facultative, anaerobic, Gram-positive bacterium that grows at temperatures close to 23 ° C. Proteolytically breaks down proteins to free amino acids with the enzymes it produces.

Streptomyces sp. (Bioderpac, 2008) is a Gram positive soil bacterium. Produces multiple enzymes that metabolize various nutrients. It can survive significant changes in sources of temperature, humidity and nutrients. The extracellular enzymes that are produced by these bacteria use chitin and chitosan as substrates at a pH of 4.5 to 6.5 and at 60 ° C. These are the conditions generated in the initial and final stages of lactic fermentation in the biodegradation process.

Nitrobacter sp. (Bioderpac, 2008) is an aerobic, Gram negative bacterium that converts nitrites to nitrates.

It grows at a pH between 6 and 9 and at temperatures between .10 to 34 ° C. The bacterium breaks down organic polymers such as chitin into compounds that are used by other organisms, such as Pseudomonas fluorescens () and Rhizobium japonicum (Bioderpac2008).

Micrococcus sp. (Bioderpac, 2008) is a spherical Gram positive bacterium. This microorganism in association with Streptomyces sp () is capable of degrading colloidal chitin derivatives.

Groups and enzymatic activity of microorganisms in the

HQE

Biodegradation of animal components or marine by-products requires hydrolytic enzymes such as proteases, lipases, and chitinases. The described microbial compositions contain one or more of such enzymes.

The primary group of microorganisms in HQE are Lactobacillus acidophilus (Biodepac 2008), Bacillus subtilis (SILoSil¹”BS), Pseudomonas fluorescens (Biodepac 2008), Bacillus licheniformis (Biodepac 2008) and Trichoderma harzianum (TRICHOSIL). These microorganisms are capable of biodegrading arthropods or arthropod by-products. One or more of the members of this main group also have a synergistic action when combined with other HQE microorganisms.

The first group of microorganisms includes the microorganisms that cause pH reduction and stabilize fermentation due to the production of organic acids and hydrogen peroxide. This group includes Lactobacillus acidophilus (Biodepac 2008) and Lactobacillus casei (Biodepac 2008). Its activity is important at the start of fermentation and during the final stages of

1Q fermentation to produce the optimal pH for hydrolytic enzymes. Its activity also creates a culture environment that prevents the growth of unwanted microorganisms and favors the demineralization of chitin residues. Lactobacillus acidophilus (Biodepac 2008) is a member of the primary group.

The second group of microorganisms includes microorganisms that produce extracellular enzymes. This second group includes Bacillus subtilis (SILoSil¹⁵ BS), Bacillus cereus (Biodepac 2008), Trichoderma harzianum (Biodepac

2008), Rhizobium japonicum (Biodepac 2008) and Azotobacter vinelandii (Biodepac 2008). Chitin chains in arthropods or arthropod by-products associate with protein molecules. The separation of polymers of this type requires the hydrolytic action that is obtained from the chitinolytic and proteolytic enzymes produced by these microorganisms. Both types of enzymes break the chains in the internal region of the polymer to produce oligomers of various sizes. The action of these enzymes occurs successively within the intermediate and final phases of the fermentation process when the appropriate pH conditions are reached. The microorganisms in this group and the environmental conditions they produce facilitate the release of pigments and the lipid fraction adhered to these residues. Bacillus subtilis (SILoSil® BS) and Trichoderma harzianum (Biodepac 2008) are members of the primary group.

The third group of microorganisms includes the microorganisms Bacillus licheniformis (Biodepac 2008), Pseudomonas flourescens (Biodepac 2008), Sptreptomyces, (Biodepac 2008) and Clostridium (Biodepac 2008). These microorganisms hydrolyze the oligomers (chitooligosaccharides and peptides) to produce chitobioses, glucosamine, and free amino acids. Bacillus licheniformis (Biodepac 2008) and Pseudomonas flourescens (Biodepac 2008) are members of the primary group.

In preferred embodiments, one or two of the first, second, and third groups of microorganisms can be combined. As an alternative, all first, second and third groups can be combined.

A fourth group of microorganisms includes Bacillus thuringiensis (HD-1 and / or HD-73 strains), Streptomyces (Bioderpac, 2008), Micrococcus (Bioderpac, 2008), Nitrobacter (Bioderpac, 2008) and Proteus vulgaris (Bioderpac, 2008). The fourth group of microorganisms can be combined with (1) the main group of microorganisms (2) any of the first, second and third groups of microorganisms (3) the combination of one or two of the first, second and third groups of microorganisms or (4) the combination of all the first, second and third groups. The addition of this fourth group gives rise to a synergistic effect that improves the biodegradation process.

Each of these groups, including the main group, is useful separately and can be combined with the prior art microbial compositions to improve their performance. In this regard, the fourth group is particularly preferred.

Table 2 sets out some of the combinations mentioned above. Column 1 is a list of the microorganisms known in the HQE that are believed to be active in the biodegradation process. Column 2 lists the microorganisms in column 1, without the microorganisms in the fourth group of microorganisms. Column 3 shows the combination of the main microorganisms while columns 4, 5 and 6 identify the combination of microorganisms from the first, second and third groups. Column 4 is the combination of groups 1 and 2; column 5 of groups 1 and 3 and column 6 of groups 2 and 3. Other useful combinations are set forth in columns 7-10.

Table 2 Composition of the culture

<td>Microorganism</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>Bacillus subtilis</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td>

<td>Bacillus cereus</td><td>X</td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td>

<td>Bacillus megaterium</td><td>X</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Azotobacter vinelandii</td><td>X</td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td>

<td>Lactobacillus acidophilus</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td><td></td>

<td>Lactobacillus casei</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td></td><td></td><td>X</td><td>X</td><td></td>

<td>Trichoderma harzianum</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td>

<td>Rhizobium japonicum</td><td>X</td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td>X</td>

<td>Clostridium pasteurianum</td><td>X</td><td>X</td><td></td><td></td><td>X</td><td>X</td><td></td><td></td><td>X</td><td>X</td>

<td>Bacillus licheniformis</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td>

<td>Microorganism</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>Pseudomonas fluorescens</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td><td>X</td><td></td><td></td><td></td><td></td>

<td>Bacillus thuringi ensi s</td><td>X</td><td></td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Streptomyces</td><td>X</td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Nitrobacter</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Micrococcus</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Proteus vulgaris</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td>X</td><td>X</td><td>X</td><td>X</td>

The activity of the enzyme extracts produced by the microorganisms within HQE is complex, but has allowed the degradation of chitinous residues of arthropods such as crustaceans. The microorganisms in HQE are activated successively according to the environment generated by the organisms used.

Methods for the identification and isolation of microbes in the HQE

It is important to obtain pure isolates before attempting to characterize or identify a species. Some bacteria are morphologically unique and can be identified without isolation, but almost all require isolation. The following describes the isolation of pure cultures from a mixture of species contained in the HQE for Bacillus subtilis (SILoSil® BS), Bacillus cereus (Bioderpac, 2008), Bacillus licheniformis (Bioderpac, 2008), Bacillus megaterium (Bioderpac, 2008), Lactobacillus acidophilus (Bioderpac, 2008), Lactobacillus casei (Bioderpac, 2008), Pseudomonas fluorescens (Bioderpac, 2008), Trichoderma harzianum (HD-1 and HD-73 strains), Rhizobium japonicum (Bioderpac, 2008), Azotobacter vinelandii (Bioderpac, 2008), Clostridium pasteurianum (Bioderpac, 2008), Proteus vulgaris (Bioderpac, 2008), Bacillus thuringiensis (SILoSil ”BT), Streptomyces sp. (Bioderpac, 2,008), Nitrobacter sp. (Bioderpac, 2008) and Micrococcus sp. (Bioderpac, 2008).

The early stages include:

(1) Dilution of seeding by striae and differential incubations (2) Identification and separation of colony types (3) Reduce collection, and (4) Determine initial characteristics of specific colonies and preservation of isolates.

Dilution of seeding by striae and differential incubations

Once a specimen is removed from the HQE sample it should be cultured immediately. Any liquid sample should be shaken carefully before preparing the plates because the immobile bacteria could settle to the bottom of a sample if they are associated with particulate matter. Unfortunately, the bacteria do not secrete homogeneously, and replicates of samples from the same mixture may contain different amounts of bacteria. The purpose of careful agitation and subsequent dilution of streak seeding is to expand the individual CFUs (Colony Forming Units) to obtain discrete colonies that can be subcultured.

All the mentioned microorganisms can be submitted separately to the following procedures for identification and isolation, with the exception of Nitrobacter sp. to be explained separately.

A full loop of carefully shaken HQE sample is aseptically obtained and applied to one edge of the agar surface. With back and forth movements about a quarter of the surface should be scored while drawing the loop to the center of the plate. Streak seeding should not break the agar surface, and there should be (20 or more) streak lines produced. To dilute or expand the sample, the loop must be flamed to destroy all viable material, contact a clean part of the agar to cool it, then the streaks are made perpendicular to the initial inoculum, overlapping that part of the plate once or twice . The second section should cover one half of the remaining sterile surface. This expands a small part of the initial inoculum possibly diluting it enough to give rise to the appearance of individual colonies after incubation. A third section is then, fluted perpendicular to the second section, flaming and cooling the loop and overlapping the previous section, as before, to further dilute the inoculum.

In the preparation of the isolates from each batch of product, the next phase is to prepare the replication of the striated plates and incubate under different conditions in an inverted position, to maximize the opportunities to differentiate the types of Bacillus subtilís colonies (SILoSil ® BS), Bacillus cereus (Bioderpac, 2008), Bacillus licheniformis (Bioderpac, 2008), Bacillus megaterium (Bioderpac, 2008), Lactobacillus acidophilus (Bioderpac, 2008), Lactobacillus casei (Bioderpac, 2008), Pseudomonas fluorescens (Bioderpac, 2008), Trichoderma harzianum (TRICHOSIL), Rhizobium japonicum (Bioderpac, 2008), Azotobacter vinelandii (Bioderpac, 2008), Clostridium pasteurianum (Bioderpac, 2008), Proteus vulgaris (Bioderpac, 2008) thuringiensis (strains HD-1 and HD-73, SILoSil® BT), Streptomyces sp. (Bioderpac, 2008), and Micrococcus sp. (Bioderpac, 2008). The incubation temperature is diverse (typically 25, 30, and 37 ° C) and is incubated under both aerobic and anaerobic conditions. This approach increases the chances of separating individual species, since different species / bacteria have different optimal temperature ranges for growth and different oxygen needs. Aerobic organisms should be checked after a day of incubation, as some of the bacteria that are tested grow very fast and displace the others.

Approaches to identifying and separating colony types

A dissecting microscope with a transilluminator can be used to distinguish individual colonies. The plates must remain inverted during the exam. Colonies are distinguished by size, shape, opacity, and texture with respect to the aforementioned microorganisms. During the examination, it is best to indicate the colonies that are sampled by placing a small mark next to them at the bottom of the plate. It will be necessary to turn the cover of the plate to collect the cologne material. Care must be taken to carefully insert the sterile loop only for the time it takes to obtain an inoculum.

Due to the characteristics of color, surface and profile (raised, flat, etc.), it is necessary to examine the colonies in direct light, through the transparent cover. The lids should be left, otherwise the plates will become contaminated. Before turning the plate over, remove the cover and invert the cover to remove moisture. The old cap should be replaced with a new one for viewing.

In the event that two colonies overlap and can still be distinguished, then at least two colonies are present. The colonies generally have a fairly simple, uniform texture. If an area resembles a mosaic, there are likely to be at least two species. Each unique type of colony should be sampled by taking an inoculum with the needle and performing a three-way dilution of the streak on a fresh plate. Care should be taken when only sampling the colony of interest. Incubate each new striated plate under aerobic conditions at the temperature that the original plate was incubated.

Species that are indicated with faster growth and / or larger colonies at temperatures closer to ideal, will present the optimum temperature. Any colony that is sampled from an anaerobically incubated plate will likely be a facultative anaerobe.

Collection reduction

Duplicates of the same species and strain are likely to be isolated from different striated plates. Many different species and different strains of the same species produce very similar types of colonies. To reduce the number of single species / strain isolates, culture with duplicate isolates from the same plate was suspected. Two mini-dilutions are carried out on two thirds of the surface to obtain individual colonies for each culture, and in the remaining third a third mixture of these. After incubation, if the two isolates grow on the same plate and / or the mixed inoculum. produces two types of distinguishable colonies, two unique isolates have been identified.

Initial characterization of the colonies and conservation of the isolates

Most of the characteristics of the organisms under study should be determined using direct light, not transmission.

Once an isolation is obtained and establishes purity by both colony examination and microscopic examination, a slant agar tube should be inoculated and incubated at an appropriate temperature with the cap removed to allow gas exchange. After growth appears, the culture should be described, tightening the cap, and keeping the tube at room temperature as a pure culture source for testing.

In addition to the large descriptive characterization, a young culture (&lt;18 h) should be stained with gram, and record the results including cell shape and size, septum or capsule if evident, and any evidence of spores or similar structures. The relationship with oxygen is the next stage with which to reduce the possible categories. After that, it is this particular result of Gram-type cell staining, and oxygen relationship that determines the next series of steps toward characterization of the isolation.

The suppression and counting of the Nitrobacter sp. in the product

There have been several studies on the metabolism, survival and growth of nitrite oxidants in pure cultures and on their nitrifying activity in various environments, but few studies have dealt with the natural Nitrobacter population. Although the biological conversion of nitrite to nitrate is a well-known process, studies and procedures of the Nitrobacter population are currently hampered by inadequate detection and counting methods.

This failure is due in part to the unfavorable physiological characteristics of this bacterium, called slow growth, small biomass, and susceptibility of crops to contamination. There is a way to count the Nitrobacter population in the soil, but it is time consuming and selective.

Detailed description of the growth process

Bacillus cereus (Bioderpac, 2008), Bacillus megaterium (Bioderpac, 2008), Bacillus subtilis (SILoSil® BS) and Bacillus licheniformis (Bioderpac, 2008)

Medium ingredient: purified water and medium on nutrient agar plate.

Procedure: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of purified water, (2) start a serial dilution in a range up to 1-10, (3) incubate a series of the duplicate plate with 1 ml of the range dilution, the plate medium is nutrient agar, (4) incubate the plates at 37 ° C for 24-48 hours, (5) the colony forming units should be 1,000,000 per 1 ml of product.

Pseudomonas fluorescens and Proteus vulgaris

Medium ingredient: purified water and medium on nutrient agar plate.

Procedure: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of purified water, (2) start a serial dilution in a range up to 1-10, (3) incubate a series of the duplicate plate with 1 ml of the range dilution, the plate medium is nutrient agar, (4) incubate the plates at 37 ° C for 24-48 hours, (5) the colony forming units should be 1,000,000 per 1 ml of product.

Lactobacillus acidophilus and Lactobacillus casei

MRS agar was developed by Man, Rogosa, and Sharpe to provide media that could demonstrate good growth of lactobacilli and other lactic acid bacteria. The culture medium allows an abundant development of all the lactobacillus species. Peptone and glucose constitute the source of nitrogen, carbon, and other elements necessary for bacterial growth. Sorbitan, magnesium, manganese and acetate monoleate contribute cofactors and may inhibit the growth of some microorganisms. Ammonium citrate acts as a growth inhibitory agent for Gram negative bacteria.

See Table 3.

TABLE 3

<td colspan="2">Formula (in grams per liter)</td><td>Instructions</td>

<td>Proteose peptone No. 3</td><td> 10.0</td><td rowspan="11">Suspend 64 g of medium in a liter of water distilled. Leave it rest 5 minutes and mix by heating up the boiling point for 1 or 2 minutes. Sterilize in a sterilizer for 15 minutes at 121 ° C.</td>

<td>Excerpt from meat</td><td> 8.0</td>

<td>Excerpt from yeast</td><td> 4.0</td>

<td>Glucose</td><td> 20.0</td>

<td>Monoleate sorbitan</td><td>1 mi</td>

<td>Phosphate dipotassium</td><td> 2.0</td>

<td>Sodium acetate</td><td> 5.0</td>

<td>Citrate ammonium</td><td> 2.0</td>

<td>Sulfate magnesium</td><td> 0.2</td>

<td>Sulfate manganese</td><td> 0.05</td>

<td>Agar</td><td> 13.0</td>

<td colspan="3">Final pH: 6.4. + 0.2</td>

Media ingredient: purified water and substrate on MRS nutrient agar medium plate

Procedure: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of purified water, (2) start a serial dilution in a range up to 1-10, (3) incubate in an aerobic chamber with

5-10% CO₂ a duplicate plate from the series with 1 ml of the dilution interval, the plate medium is MRS agar, (4) incubate the plates at 33-37 ° C for 72 hours or 30 ° C for 5 days, (5) colony forming units must be

1,000,000 for 1 ml of product.

Results. Colony forming units must be

1,000,000 for 1 ml of product.

Characteristics of the colonies: generally small, grayish-white, smooth or rough.

Characteristics of the medium: The prepared medium is yellow.

Lactobacillus identification:

TABLE 4

<td rowspan="2"></td><td colspan="2">Growth to a</td><td colspan="6">acid</td><td rowspan="2">nh₃Arginin to</td><td rowspan="2">I grew up- I lie in broth of culture 4% NaCl</td>

<td>fifteen 'C</td><td>Four. Five ° C</td><td>the</td><td>its</td><td>Salt</td><td>mn</td><td>SW</td><td>xi</td>

<td>L. acidophilus</td><td></td><td> +</td><td> +</td><td> +</td><td> +</td><td></td><td></td><td></td><td></td><td></td>

<td>L. casei</td><td> +</td><td>V</td><td> +/</td><td> +/</td><td></td><td></td><td> +</td><td></td><td></td><td></td>

Azotobacter vinelandii (Bioderpac, 2008)

Ingredient of medium: purified water and substrate in nutrient agar medium plate with (from Burk, Asbhy, Jensen).

Procedure: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of purified water, (2) incubate this solution at 25 ° C for 48 hours, (3) start a serial dilution in a range up to 1-10, ( 4) incubate a duplicate plate, with nutrient agar substrate, of the series with 1 ml of dilution interval, the medium of the plate is nutrient agar (5) incubate the plates at 25 ° C for 48-72 hours, (6 ) Colony forming units must be 1,000,000 per 1 ml of product.

Clostridium pasteurianum (Bioderpac, 2008)

Media ingredient: aqueous phosphate buffer and standard methods for agar medium plate substrate (TYG).

Process: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of aqueous phosphate buffer, (2) prepare appropriate serial dilutions (3) seed the appropriate aliquot for the desired dilution in Petri dishes, (4) add the agar tempered by standard methods and mixing on the plate, (5) place the inverted dry plates in the anaerobic chamber, (6) incubate the plates at 35-37 ° C for 48-72 hours, (7) after the incubation period, remove the plates from the anaerobic chamber and count the plates, record the dilutions used and the total number of colonies counted for each dilution, (8) the colony forming units should be 1,000,000 per 1 ml of product.

Identification of this Clostridium species uses a typical colony microscopic morphology that allows rapid and presumptive identification of some frequently isolated Clostridium species. Furthermore, together with the use of simple biochemical tests such as the study of the production of lecithinase and lipase in egg yolk agar, the hydrolysis of gelatin and urea and the production of indole through the rapid method (p-dimethyl-amino - cinnamaldehyde), constitute an easy and cheap method for the identification, even definitive, for some of them.

Micrococcus sp. (Bioderpac, 2008)

Medium ingredient: purified water and medium on nutrient agar plate.

Procedure: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of purified water, (2) start a serial dilution in a range up to 1-10, (3) incubate a series of the duplicate plate with 1 ml of the range dilution, the medium in the plate is nutrient agar, (4) incubate the plates at 37 ° C for 24 hours, (5) the colony forming units should be 1,000,000 per 1 ml of product.

If Gram-positive cocci are found, perform antibiotic sensitivity tests. Micrococcus is sensitive to bacitracin and resistant to furazolidone.

Rhizobium japonicum (Bioderpac, 2008)

Media ingredient: purified water and ALM plate medium (mannitol yeast extract agar).

Procedure: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of purified water, (2) start a serial dilution in a range up to 1-10, (3) incubate a series of the duplicate plate with 1 ml of the range dilution, plate medium is ALM (Mannitol Yeast Extract Agar) (4) incubate plates at 28 ° C for 96 hours, (5) colony forming units should be 1,000,000 per 1 ml product.

To confirm Rhizobium japonicum (Bioderpac, 2008), the isolated colony is used to infect a legume aseptically to cause nodule formation.

Trichoderma harzianum (TRICHOSIL)

Media ingredient: purified water and media substrate malt extract agar (2% w / v) supplemented with chloramphenicol, streptomycin sulfate, and nystatin.

Procedure: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of purified water, (2) start a serial dilution in a range up to 1-10, (3) incubate a series of the duplicate plate with 1 ml of the range dilution, plate medium is malt extract agar (4) incubate plates at 25 ° C for 4 days, (5) colony forming units should be 1,000,000 per 1 ml product.

Bacillus thuringiensis (HD-1 and HD-73 strains (SILoSil® BT))

Medium ingredient: purified water and substrate in Superbroth agar medium plate supplemented with 2 g / liter of D-glucose and 50 pg / ml of erythromycin.

Procedure: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of purified water, (2) start a serial dilution in a range up to 1-10, (3) incubate a series of the duplicate plate with 1 ml of the range dilution, the plate medium is Superbroth agar (4) incubate the plates at 28 ° C for 10-14 days, (5) the colony forming units should be 1,000,000 per 1 ml of product.

Streptomyces sp. (Bioderpac, 2008)

Media ingredient: purified water and agar medium plate for isolation of actinomycetes.

Procedure: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of purified water, (2) start a serial dilution in a range up to 1-10, (3) incubate a series of the duplicate plate with 1 ml of the range dilution, the plate medium is actinomycete isolation agar (4) incubate the plates at 28 ° C for 2-3 days, (5) the colony forming units should be 1,000,000 per 1 ml of product.

Nitrobacter sp. (Bioderpac, 2008)

Ingredient of the medium: purified water and substrate in nutrient agar medium plate.

Procedure: (1) place 0.1 ml of HQE liquid sample in 9.9 ml of purified water, (2) start a serial dilution in a range up to 1-10, (3) incubate a series of the duplicate plate with 1 ml of the range dilution, the plate medium is substrate on nutrient agar plate, (4) incubate the plates at 30 ° C for 10-14 days.

Biodegradation process

In a preferred embodiment, the marine arthropod is a crustacean and the preferred crustacean is shrimp. The shrimp by-product comprises cephalothorax and / or shrimp exoskeleton.

In the biodegradation process, it is preferred that the fermentation is facultative aerobic fermentation. It is also preferred that the fermentation is carried out at a temperature of about 30 ° C to 40 ° C. The preferred pH is less than about 6, more preferably less than about 5.5. However, the pH should remain above approximately 4.3. Fermentation takes place for approximately 24-96 hours. In some embodiments, fermentation takes place for approximately 24-48 hours, and most preferably 24-36 hours. These fermentation times are much shorter than the typical prior art fermentation times of 10 to 15 days to achieve substantially the same amount of digestion, albeit with no detectable formation of chitosan and glucosamine.

The separation of the mixture is preferably by centrifugation (for example, about 920 g). Gravity separation can also be used, but is not preferred due to the time required to achieve separation.

The mixture is separated into three fractions: solid, aqueous and lipid. The solid fraction comprises chitin and is called HYTc. The aqueous fraction comprises hydrolyzed protein, amino acids, chitosan and glucosamine and is called HYTb. The lipid fraction comprises sterols, vitamins A and E, and carotenoid pigments such as astaxanthin.

Any of the microbial compositions identified in this document can be used in the biodegradation process. In some embodiments, it is preferred that HQE be used in the biodegradation process. In other embodiments, it is preferred that HYTb be added to HQE or to the fermentation broth. As previously described, HYTb contains amino acids, chitosan, glucosamine, and trace elements that include calcium, magnesium, zinc, copper, iron, and manganese. HYTb also contains enzymes such as lactic enzymes, proteases, lipases, chitinases, lactic acid, polypeptides, and other carbohydrates. HYTb may also contain latent microorganisms from a previous biodegradation process. Microorganisms of this type can be converted into reagents and, in combination with HQE, contribute to a more robust biodegradation process compared to when used contrary as described in this document, HQE alone

More particularly, the process includes the following steps:

to. Activation of microbial cells in a base solution with sugar to improve their growth and the formation of biomass.

b. Shrimp by-product grinding (cephalothorax and exoskeleton) to prepare a homogeneous paste.

c. Homogeneous mixture of the shrimp by-product paste with at least 10% of the activated inoculum.

d. Adjust the pH value to less than 6.0 in the mixture using a citric acid solution to inhibit the growth of miero-organisms and promote the development of microbial cells that constitute the inoculum.

and. Fermentation of the mixture in a discontinuous agitation system at temperatures within a range of at least 30 to 40 ° C for at least 96 hours, keeping the pH below 5.0. The pH is monitored periodically. If the pH rises above 5.0, a citric acid buffer is added in an amount to keep the pH below 5.0.

F. Centrifugation of the ferment to separate the three main fractions: chitin, liquid hydrolyzate and pigmented paste.

g. Chitin crude oil washing and washing water collection to recover fine solids or minerals.

h. Chitin drying and storage.

i. The drying and storage of the liquid hydrolyzate.

j. The pigmented paste (lipid fraction) is stored in closed containers for preservation.

The process and operational fundamentals are best understood with reference to Figure 1 and the following detailed description.

Microbial cell activation

A microbial composition as described herein is. use as inoculum. The HQE inoculum has a microbe concentration of approximately 2.5 to 3.0% (w / v). HQE is activated by dilution to 5% in cane sugar solution (3.75% of final concentration of cane sugar), and incubates at 37 ° C for 5 days. HYTb (10 ml per liter of culture) is preferably added to provide a source of naturally occurring minerals and amino acids. The cell growth of the microorganisms was estimated by the optical density measured at 540 nm. Activation is completed at an optical density of approximately 1.7. The concentration of microbes after activation is approximately 1.9 to 3.0% (w / v).

Sample preparation

Shrimp and by-product samples were obtained from shrimp processing plants. Samples derived from shrimp are obtained from mind thawed and minced (1500 g per batch) mixed with 99 grams of cane sugar (final concentration 6.6% by weight) and 85.5 ml of 5% activated HQE (v / p ) (optical cell density = 1.7). The pH is then adjusted to 5.5 using 2M citric acid.

Fermentation control

The mixture is incubated at 36 ° C with discontinuous shaking for 96 hours. During the fermentation process, the pH will be monitored through the use of a potentiometer, and the total titratable acidity (TTA,%) was determined by titration with 0.1 N NaOH until a pH of 8.5 is obtained. TTA is expressed as a percentage of lactic acid.

Separation conditions

The fermentation product is a viscous silage that has a deep orange color, due to the presence of astaxanthin. The silage is centrifuged (5 ° C) at 1250 rpm (930g) for 15 minutes to obtain chitin, liquid hydrolyzates, and pigment paste. The upper phase (pigment paste) is separated manually. The liquid hydrolyzates are separated by decantation, and the sediment that constitutes the crude chitin is washed with distilled water to separate the fine solids. The resulting liquid. collected and dried. Crude chitin, liquid and fine solid hydrolyzates are dried at 60 ° C. All fractions are stored to protect them from light.

The above protocol was carried out with HQE in three duplicate fermentation batches as shown in the following examples.

Example 1

Fermentation control by measuring pH and total titratable acidity (TTA,%)

The initial mean values ​​of pH and TTA were 7.31 + 0.10 and 0.53 ± 0.09, respectively. As shown in

Figure 2, the pH was initially lowered to 6.5 by the addition of 2M citric acid. Then, due to proteolysis and ammonium release, the pH increased again to 7.11 ± 0.08 during the first 2 h, and later, the pH decreased by 28% (to 5.28 + 0.01) during the 12 hours of fermentation. In approximately 24 hours of fermentation, the final pH was 4.57 + 0.15. In parallel to the decrease in pH, a similar behavior was observed in the mean values ​​of the TTA. During the first 2 hours the average TTA values ​​were 1%, and then these values ​​gradually increased to an average value of 3.33 +

0.23 at 24 hours, as shown in Figure 2.

Example 2

Fermentation products and chemical composition

After the fermentation process, the silage is centrifuged to separate the three main products (chitin, liquid hydrolyzate and pigment paste). The other product, the fine solids were maintained by washing the crude chitin. Table 5 shows the proportion recovered in each fraction. In dry weight, the largest fraction corresponded to the liquid hydrolyzate (55%), then the fine solids (29%), crude chitin (10%) and pigment paste (5%). In the fermented batch, the average dry weight value was 32% (481.1 ± 5.6 g).

Table 6 shows the chemical characterization of each of the four main products that were obtained through fermentation. The chitinous product (chitin) shows a partial demineralization reflected as ash. It also reveals the high protein content (42.34%) that was quantified in the liquid hydrolyzates. This facilitates the recovery of a pigment paste, mainly consisting of total lipids (42.67%), and produced an abundant ash fraction (16.72%) in fine solids.

Table 5 Products separated from fermentation

<td>Matter</td><td>Chitin</td><td>Hydrolyzed</td><td>Paste of</td><td>Solids</td>

<td>dry</td><td>raw (g)</td><td>liquid</td><td>1ipids</td><td>fine</td>

<td> (9)</td><td></td><td> (9)</td><td> (9)</td><td> (9)</td>

<td> 480.0</td><td> 48.0</td><td> 264.4</td><td> 22.9</td><td> 141.2</td>

<td> 487.5</td><td> 50.2</td><td> 268.8</td><td> 24.0</td><td> 140.8</td>

<td> 475.7</td><td> 51.0</td><td> 265.7</td><td> 23.7</td><td> 131.7</td>

<td> 481.1 ±</td><td> 49.5 ±</td><td> 266.3 ± 4.6</td><td> 23.3 ± 0.5</td><td> 141.9 ±</td>

<td> 5.6</td><td> 2.4</td><td></td><td></td><td> 5.5</td>

Table 6 Chemical composition (dry weight) of fermentation products

<td>products</td><td>% Protein</td><td>% Ash</td><td>% Total of lipids</td>

<td>Raw chitin</td><td> 18.12 ± 0.15</td><td> 4.36 ± 0.26</td><td> 2.02 ± 0.46</td>

<td>Hydrolyzed</td><td> 42.34 + 0.03</td><td> 7.96 ± 0.16</td><td> 4.28 ± 0.28</td>

<td>liquid</td><td></td><td></td><td></td>

<td>Paste of</td><td> 30.80 ± 0.25</td><td> 5.11 + 0.16</td><td> 42.67 ± 0.63</td>

<td>lipid</td><td></td><td></td><td></td>

<td>Fine solids</td><td> 31.62 ± 0.10</td><td> 16.72 ± 0.37</td><td>ND</td>

Example 3

Amino acid profile in powdered hydrolyzates

The amino acid content of the dry hydrolyzate was determined as described by López-Cervantes et al., Analysis of free amino acids in fermented shrimp waste by high-performance liquid chromatography, Journal of Chromatography A, volume 1105, 1 (2006). Table 6 shows the total amino acid profile of the powdered hydrolyzates. The proportion of essential amino acids was 52.5% for the hydrolyzed powders.

Table 7 Amino acid profile of hydrolyzates in dry powder (mg per g dry weight)

<td>Amino acid</td><td>Hydrolyzed powder dry</td>

<td>Aspartic acid</td><td> 38</td>

<td>Glutamic acid</td><td> 39</td>

<td>Serine</td><td> 16</td>

<td>Histidine *</td><td> 9</td>

<td>Glycine</td><td> 28</td>

<td>Threonine *</td><td> 14 .</td>

<td>To the girl</td><td> 30</td>

<td>Proline</td><td> 8</td>

<td>Tyrosine *</td><td> 70</td>

<td>Arginine</td><td> 18</td>

<td>Valine *</td><td> 20</td>

<td>Methionine *</td><td> 4</td>

<td>Isoleucine *</td><td> 15</td>

<td>Leucine *</td><td> 23</td>

<td>Phenylalanine *</td><td> 39</td>

<td>Lysine *</td><td> 13</td>

<td>TOTAL</td><td> 394</td>

<td>♦ Amino acids essential</td><td> 207</td>

Example 4

Glucosamine quantification in crude chitin

In chitin, glucosamine content was quantified as a purity index. The glucosamine contents in chitin were 516, 619 and 640 mg per g (dry weight), these values ​​correspond to the results of three fermentation batches carried out in duplicate. Therefore, the average amount of glucosamine in this study was 591 mg per g dry weight of chitin. The method for the quantification of glucosamine was reported by López-Cervantes et al., Quantitation of glucosamine from shrimp waste using HPLC Journal of Chromatographic Science, volume 45, 1 (2007). Example 5

Astaxanthin content and fatty acid profile in pigment paste

Astaxanthin is the main pigment in the lipid paste obtained from the fermented shrimp residue. The astaxanthin content was found in the range of 1.98 to 2.25 mg g '¹ of dry lipid paste, and the average is 2.11 mg g '¹ of dry lipid paste. Astaxanthin was determined by a variant of the López-Cervantes et al. Method, Quantification of astaxanthin in shrimp waste hydrolysate by HPLC Biomedical Chromatography, volume 20, 981 (2006).

Fourteen fatty acids were identified in the pigment paste. Palmitic acid (C16: 0), and oleic acid (C18: ln9) were found in higher quantity.

It is noted that in relation to this date, the best method known by the applicant to put the aforementioned invention into practice is the one that is clear from the present description of the invention.