U.S. patent application number 13/519112 was filed with the patent office on 2012-12-13 for biodegradation process and composition.
This patent application is currently assigned to Agrinos AS. Invention is credited to Jaime Lopez-Cervantes, Karl Reiner Fick Rochin, Dalia Isabel Sanchez-Machado.
Application Number | 20120315668 13/519112 |
Document ID | / |
Family ID | 43639923 |
Filed Date | 2012-12-13 |
United States Patent
Application |
20120315668 |
Kind Code |
A1 |
Lopez-Cervantes; Jaime ; et
al. |
December 13, 2012 |
Biodegradation Process and Composition
Abstract
Disclosed are novel microbial compositions and biodegradation
processes to treat marine animal or marine animal by-products to
produce solid, liquid and lipid fractions that contain useful
compounds.
Inventors: |
Lopez-Cervantes; Jaime;
(Obregon, MX) ; Sanchez-Machado; Dalia Isabel;
(Obregon, MX) ; Rochin; Karl Reiner Fick; (Los
Naranjos, MX) |
Assignee: |
Agrinos AS
Lysaker
NO
|
Family ID: |
43639923 |
Appl. No.: |
13/519112 |
Filed: |
June 25, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/EP2010/070825 |
Dec 20, 2010 |
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13519112 |
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61289706 |
Dec 23, 2009 |
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61299869 |
Jan 29, 2010 |
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61355365 |
Jun 16, 2010 |
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Current U.S.
Class: |
435/42 ;
435/252.4 |
Current CPC
Class: |
C11B 1/025 20130101;
C12R 1/00 20130101; C12N 1/14 20130101; C12N 1/20 20130101 |
Class at
Publication: |
435/42 ;
435/252.4 |
International
Class: |
C12P 39/00 20060101
C12P039/00; C12N 1/20 20060101 C12N001/20 |
Claims
1. A microbial composition comprising ATCC Patent Deposit
Designation PTA-10861
2. A microbial composition comprising (a) one or more lactic acid
bacteria (LAB) and (b) one or more microorganisms selected from the
group of genera consisting of Bacillus, Azotobacter, Trichoderma,
Rhizobium, Clostridium, Pseudomonas, Streptomyces., Micrococcus,
Nitrobacter and Proteus.
3. The microbial composition of claim 2 wherein said LAB is
selected from the genera consisting of Lactobacillus, Pediococcus,
Lactococcus, and Streptococcus.
4. The microbial composition of claim 2 wherein said LAB is
selected from the group consisting of Lactobacillus acidophilus and
Lactobacillus casei.
5. The microbial composition of claim 2 wherein said LAB is
selected from the group consisting of Lactobacillus acidophilus
(Bioderpac, 2008) and Lactobacillus casei (Bioderpac, 2008).
6. The microbial composition of claim 2 wherein said Bacillus is
selected from the group consisting of Bacillus subtilis, Bacillus
cereus, Bacillus megateriu, Bacillus licheniformis and Bacillus
thuringiensis, said Azotobacter is Azotobacter vinelandii, said
Trichoderma is Trichoderma harzianum, said Rhizobium is Rhizobium
japonicum, said Clostridium is Clostridium pasteurianu and said
Pseudomonas is Pseudomonas fluorescens.
7. The microbial composition of claim 2 wherein said Bacillus is
selected from the group consisting of Bacillus subtilis
(SILoSil.RTM. BS), Bacillus cereus (Bioderpac, 2008), Bacillus
megaterium (Bioderpac, 2008), Bacillus licheniformis (Bioderpac,
2008) and Bacillus thuringiensis (strains HD-1 and HD-73
(SILoSil.degree. BT)), said Azotobacter is Azotobacter vinelandii
(Bioderpac, 2008), said Trichoderma is Trichoderma harzianum
(TRICHOSIL), said Rhizobium is Rhizobium japonicum (Bioderpac,
2008), said Clostridium is Clostridium pasteurianu (Bioderpac,
2008) and said Pseudomonas is Pseudomonas fluorescens (Bioderpac,
2008).
8. The microbial composition of claim 2 wherein at least one of
said Bacillus, Azotobacter, Trichoderma, Rhizobium, Clostridium,
Pseudomonas, Streptomyces., Micrococcus, Nitrobacter and Proteus is
a chitinolytic strain.
9. A biodegradation process comprising: mixing a marine animal or
marine animal by-product with the microbial composition of any of
claims 1 through 11 to form a mixture; fermenting said mixture; and
separating said mixture into solid, aqueous and lipid
fractions.
10. The process of claim 9 wherein said marine animal is a marine
arthropod.
11. The process of claim 10 wherein said marine arthropod is
selected from the group consisting of shrimp, crab and krill.
12. The process of claim 9 wherein said marine animal is fish.
13. The process of claim 9 wherein said microbial composition
comprises ATCC Patent Deposit Designation PTA-10861.
14. The process of claim 9 wherein said fermenting is by
facultative aerobic fermentation.
15. The process of claim 9 wherein said separating is by
centrifugation.
16. The process of claim 9 wherein said fermenting is at a
temperature of about 30.degree. C. to 40.degree. C.
17. The process of claim 16 wherein said fermenting is carried out
at a pH between about 4.3 and 5.0
18. The process of claim 16 wherein said fermentation is carried
out for about 24-96 hours.
19. The process of claim 14 wherein said aqueous fraction comprises
amino acids, chitosan and glucosamine
20. The process of claim 19 wherein said aqueous fraction further
comprises trace elements.
21. The aqueous fraction made according to claim 19 or 20.
22. The process of claim 12 wherein said solid fraction comprises
chitin.
23. The solid fraction made according to claim 22.
24. A composition comprising HYTb.
25. A composition comprising HYTc.
Description
[0001] This application is a U.S. National Stage application under
37 C.F.R. .sctn.3.71, claiming priority to PCT application serial
no. PCT/EP2010/070285 filed Dec. 20, 2010, claiming the benefit of
U.S. Provisional Application Ser. Nos. 61/289,706, filed Dec. 23,
2009, 61/299,869, filed Jan. 29, 2010, and 61/355,365, filed Jun.
16, 2010 under 35 U.S.C. .sctn.119(e) and are expressly
incorporated herein by reference.
TECHNICAL FIELD
[0002] Disclosed are novel microbial compositions and microbial
processes to treat marine animal by-products and in some cases the
entire marine animal to produce solid, liquid and lipid fractions
that contain useful compounds.
BACKGROUND OF THE INVENTION
[0003] The processing of fish and marine arthropods, such as
shrimp, crab and crayfish, produces large quantities of marine
by-products. Most are used in low end products such as fertilizers,
fish silage or pet food but the unused by-products pose an economic
burden for the marine product processing industries because of the
need to dispose of such residues in an environmentally sound way.
By some estimates such by-products represent 25% of the total
production captured by fisheries.
[0004] For example, during the processing of shrimp for its
subsequent freezing and marketing, a large amount of remains are
generated since 35% of the animal is inedible and must be
discarded. These remnants or by-products are composed of the
shrimp's cephalothorax and exoskeleton. However, these shrimp
by-products are rich in high-value substances, such as chitin,
protein, lipids, carotenoid pigments (astaxanthine) and minerals.
The majority of the inedible by-products are disposed at landfills
or dumped back into the ocean, thus causing serious environmental
problems and considerable losses to the shrimp processing industry.
At present, only a small amount of these by-products are used as a
supplement for animal feed.
[0005] The most common technique for shrimp by-product utilization
is sun drying. This technique has low hygienic control and the
products are used primarily for animal consumption. Other methods
employ chemical acids and alkalis at different concentrations,
temperatures and times for the extraction of chitin and recovery of
protein hydrolysates. However, these methods cause a
depolymerization and partial deacetylation of the chitin. Moreover,
these methods complicate the recovery of other products, such as
protein and pigment.
[0006] Enzymatic methods have been developed for the extraction of
chitin, liquid hydrolysates and pigments. Such methods use
enzymatic extracts or enzyme isolates. Other studies have reported
the use of microbial enzymes, such as commercial alcalase, for the
extraction of proteins from shrimp and marine animal by-products.
The combination of alcalase and pancreatin has been reported for
the extraction of chitin, hydrolyzed protein and pigmented
lipids.
[0007] Lactic fermentation processes have been used as a substitute
for the above chemical and enzymatic processes. Fermentation
represents a cost effective technique which stabilizes and retains
the nutritional quality of the by-products. The optimal conditions
for fermentation depend on several factors including the choice and
concentration of carbohydrates, pH, temperature, time, and the
choice of aerobic or anaerobic conditions. Another important factor
is the choice of microorganism and initial inoculum concentration.
To facilitate the fermentation process of shrimp by-products pure
cultures of lactic acid bacteria (LAB) have been used. Such LAB
include Lactobacillus plantarum (Rao, M. S., Stevens, W. F., 2006,
"Fermentation of shrimp biowaste under different salt
concentrations with amylolytic and non-amylolitic Lactobacillus
strains for chitin production," Food Technology and Biotechnology
44, 83-87; Rao, M. S., Munoz, J., Stevens, W. F., 2000, "Critical
factors in chitin production by fermentation of shrimp biowaste,"
Applied Microbiology and Biotechnology 54, 808-813; Bhaskar, N.,
Suresh, P. V., Sakhare, P. Z., Sachindra, N. M., 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. 82
(Circ, L. A., Huerta, S., Hal, G. M., 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., Gonzalez, R.
O., Hall, G. M., 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,
W. J., Jo, G. H., Kuk, J. H., Kim, Y. J., Oh, K. T., Park, R. D.,
2007, "Production of chitin from red crab shell waste by successive
fermentation with Lactobacillus paracasei KCTC-3074 and Serratia
marcescens FS-3," Carbohydrate Polymers 68, 746-750), Lactobacillus
pentosus (Bautista, J., Jover, M., Gutierrez, J. F., 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 84495 and Lactobacillus lactis (Bhaskar 2007),
Lactobacillus salvarus (Beaney 2005), Enteroccus facium (Beaney
2005), Pedioccoccus acidilactici (Bhaskar 2007) and Pedioccoccus
sp. L1/2 (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 LAB has
been used (Bhaskar 2007) and there are reports using 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 the poor performance of commercial inoculants.
[0008] Lactic fermentation of shrimp by-products produces protein
hydrolysates, chitin, minerals, and lipids. Chitin and its
deacetylated derivatives have many applications in agriculture,
biomedicine, food and the paper industry, while liquid hydrolysate
is an excellent source of essential amino acids that can be used
for human or animal consumption. The lipidic paste contains
sterols, vitamin A and E, and carotenoid pigments such as
astaxanthin which can be used in feed for salmonoids or as a
natural coloring in the food industry.
[0009] Chitin is a natural polysaccharide found particularly in the
exoskeleton of crustaceans, the cuticles of insects, and the cell
walls of fungi. Because chitin is one of the most abundant
biopolymers, much interest has been paid to its biomedical,
biotechnological and industrial applications. Chitosans are
poly-(.beta.-1-4)-N-acetyl-D-glucosamine compounds produced by the
deacetylation of chitin (.beta.-1-4)-N-acetyl-D-glucosamine.
Glucosamine is an amino monosaccharide obtained by
de-polymerization of chitosan. It participates in the constitution
of glycosaminoglycans, a major class of extracellular complex
polysaccharides. Glucosamine sulphate, glucosamine hydrochloride
and N-acetyl-glucosamine are commonly used alone or as part of a
mixture.
[0010] Generally, the liquid hydrolysate 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 nitrogen source in growth media for
microorganisms. Additionally, these hydrolysates are a source of
free amino acids and can be used for nutrition in plants as a
biostimulant.
[0011] Astaxanthine (3,3''-dihydroxy-8,8-carotene-4,4'-dione), a
ketocarotenoid oxidized from .beta.-carotene, naturally occurs in a
wide variety of marine and aquatic organisms. Due to its attractive
pink color, its biological functions as a vitamin A precursor, and
antioxidative activity, astaxanthine can be used as a colorant in
food and in medicine. In the structure of astaxanthine, two
identical asymmetric carbon atoms at C3 and C3' are found. However
trans-asthaxanthine is the quantitatively most prevalent carotenoid
in crustacean species.
[0012] References disclosing these and other products from lactic
fermentation include: Sanchez-Machado et al. "Quantification of
organic acids in fermented shrimp waste by HPLC" Food Technology
and Biotechnology, volume 46, 456 (2008); Sanchez-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); Lopez-Cervantes et al., "Quantitation of glucosamine from
shrimp waste using HPLC" Journal of Chromatographic Science, volume
45, 1 (2007); Lopez-Cervantes et al., "Quantification of
astaxanthin in shrimp waste hydrolysate by HPLC" Biomedical
Chromatography, volume 20, 981 (2006); Lopez-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); Lopez-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).
SUMMARY OF THE INVENTION
[0013] Disclosed are microbial compositions and biodegradation
processes.
[0014] One microbial composition comprises (a) one or more lactic
acid bacteria (LAB) and (b) one or more or two or more
microorganisms selected from the group of genera consisting 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 (e.g. endochitinase and/or exochitinase). In
some microbial compositions the LAB is selected from the genera
consisting of Lactobacillus, Pediococcus, Lactococcus, and
Streptococcus. When the LAB is Lactobacillus, it is preferred that
the LAB is Lactobacillus acidophilus and/or Lactobacillus casei,
more preferably Lactobacillus acidophilus (Bioderpac, 2008) and
Lactobacillus casei (Bioderpac, 2008).
[0015] The Bacillus in this composition is preferably selected from
the group consisting of Bacillus subtilis, Bacillus cereus,
Bacillus megaterium, Bacillus licheniformis and Bacillus
thuringiensis, more preferably Bacillus subtilis (SILoSil.RTM. BS),
Bacillus cereus (Bioderpac, 2008), Bacillus licheniformis
(Bioderpac, 2008) and Bacillus thuringiensis strains HD-1 and HD-73
(SILoSil.RTM.BT).
[0016] The Azotobacter in this composition is preferably
Azotobacter vinelandii, more preferably Azotobacter vinelandii
(Bioderpac, 2008).
[0017] The Trichoderma in this composition is preferably
Trichoderma harzianum, more preferably Trichoderma harzianum
(TRICHOSIL)
[0018] The Rhizobium in this composition is preferably Rhizobium
japonicum, more preferably Rhizobium japonicum (Bioderpac,
2008).
[0019] The Clostridium in this composition is preferably
Clostridium pasteurianu, more preferably Clostridium pasteurianu
(Bioderpac, 2008).
[0020] The Pseudomonas in this composition is preferably
Pseudomonas fluorescens, more preferably Pseudomonas fluorescens
(Bioderpac, 2008).
[0021] Another microbial composition comprises one or more or two
or more microorganisms selected from the group consisting of
Bacillus subtilis ((SILoSil.RTM. 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), Bacillus
thuringiensis strains HD-1 and HD-73 (SILoSil.RTM.BT), Streptomyces
(Bioderpac, 2008), Micrococcus (Bioderpac, 2008), Nitrobacter
(Bioderpac, 2008) and Proteus (Bioderpac, 2008).
[0022] Another embodiment of a microbial composition of comprises
Lactobacillus acidophilus (Bioderpac, 2008) and/or Lactobacillus
casei (Bioderpac, 2008).
[0023] A particularly preferred microbial composition comprises
Bacillus subtilis (SILoSil.RTM. 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), Bacillus
thuringiensis strains HD-1 and HD-73, Streptomyces (Bioderpac,
2008), Micrococcus (Bioderpac, 2008), Nitrobacter (Bioderpac, 2008)
and Proteus (Bioderpac, 2008).
[0024] A preferred microbial composition is HQE. HQE was deposited
with the American Type Culture Collection (ATCC) Manassas, Va., USA
on Apr. 27, 2010 and given Patent Deposit Designation
PTA-10861.
[0025] Also disclosed are isolated microorganisms selected from the
group consisting of Bacillus subtilis (SILoSil.RTM.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), Bacillus thuringiensis strains HD-1 and HD-73
(SILoSil.RTM.BT), Streptomyces (Bioderpac, 2008), Micrococcus
(Bioderpac, 2008), Nitrobacter (Bioderpac, 2008) and Proteus
(Bioderpac, 2008).
[0026] The biodegradation process comprises mixing a marine animal
or marine animal by-product with any of the aforementioned
microbial compositions to form a mixture; fermenting the mixture;
and separating the mixture into solid, aqueous and lipid fractions.
Unlike prior art biodegradation processes, the disclosed
biodegradation process produces chitosan and glucosamine which can
be found in the aqueous fraction. The marine animal is preferably a
marine arthropod, such as shrimp, crayfish, crab or krill. In some
embodiments the marine animal is fish or a fish by product such as
fish skin, muscle or organ.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 schematically shows a preferred degradation process
for shrimp by-products.
[0028] FIG. 2 depicts the pH and total titratable acidity during
lactic fermentation of the shrimp by-products.
DETAILED DESCRIPTION OF THE INVENTION
[0029] As used herein, the term "marine animal" refers to any
animal that lives in oceans, seas or fresh water. Marine animals
include fish and marine arthropods.
[0030] As used herein the term "marine arthropod" refers to an
invertebrate marine animal having an exoskeleton that lives in
oceans, seas or fresh water. Generally marine arthropods have a
segmented body, and jointed appendages. Marine arthropods are
members of the subphylum Crustacea. Preferred classes of Crustacea
include Branchiopoda (e.g. brine shrimp, Cladocera and Triops),
Cephalocardia (e.g. horseshoe shrimp), Maxillopoda (e.g. barnacles
and copepods (zooplankton)), Ostracoda (e.g. ostracods) and
Malacostraca (e.g. crab, lobsters, shrimp, krill etc.).
[0031] As used herein, the term "by-product" refers to any part of
a marine animal. In some embodiments, the by-product is produced by
the commercial processing of the marine animal. For example, in the
shrimp industry, the cephalothorax and exoskeleton are shrimp
by-products. In the crab and lobster processing industry the
exoskeleton (shell) is a by-product.
[0032] In some embodiments, the entire arthropod can be used in the
biodegradation process. For example, many krill are about 1-2
centimeters (0.4-0.8 in) long as adults but a few species grow to
sizes on the order of 6-15 centimeters (2.4-5.9 in). Krill oil
contains at least three components: (1) omega-3 fatty acids similar
to those in fish oil, (2) omega-3 fatty acids conjugated to
phospholipids and (3) the antioxidant astaxanthin. In addition, the
exoskeleton contains fluoride. Accordingly, these components can be
separated in the biodegradation process with the oil components
being isolated in the lipid fraction and the fluoride in the liquid
fraction.
[0033] As used herein the term "microbial composition" refers to a
liquid, solid or gelatinous medium containing or physically
supporting 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
inoculums which are generally used to start a fermentation broth
and which contain a higher concentration of the microorganisms than
which are 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.
[0034] As used herein the term "isolated microorganism" refers to a
liquid, solid or gelatinous medium containing or physically
supporting one microorganism.
[0035] The disclosed microbial compositions or isolated
microorganisms can be combined with other microorganisms to form a
new microbial composition which can be used for processes other
than those specifically disclosed herein. The selection of
disclosed microorganism(s) will depend on its biological properties
(e.g. production of protein or carbohydrate or chitin degrading
enzymes), the other microorganisms selected and the process in
which the combination is to be used. The selection of such
components and processes will be apparent to the skilled artisan
following the disclosure herein.
[0036] One microbial composition comprises (a) one or more lactic
acid bacteria (LAB) and (b) one or more or two or more
microorganisms selected from the group of genera 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 Proteus is a chitinolytic strain that produces a chitinase
(e.g. endochitinase and/or exochitinase). In some microbial
compositions the LAB is selected from the genera consisting of
Lactobacillus, Pediococcus, Lactococcus, and Streptococcus. When
the LAB is Lactobacillus, it is preferred that the LAB is
Lactobacillus acidophilus and/or Lactobacillus casei, more
preferably Lactobacillus acidophilus (Bioderpac, 2008) and
Lactobacillus casei (Bioderpac, 2008).
[0037] The Bacillus in this composition is preferably selected from
the group consisting of Bacillus subtilis, Bacillus cereus,
Bacillus megaterium, Bacillus licheniformis and Bacillus
thuringiensis, more preferably Bacillus subtilis (SILoSil.RTM. BS),
Bacillus cereus (Bioderpac, 2008), Bacillus licheniformis
(Bioderpac, 2008) and Bacillus thuringiensis strains HD-1 and HD-73
(SILoSil.RTM.BT).
[0038] The Azotobacter in this composition is preferably
Azotobacter vinelandii, more preferably Azotobacter vinelandii
(Bioderpac, 2008).
[0039] The Trichoderma in this composition is preferably
Trichoderma harzianum, more preferably Trichoderma harzianum
(TRICHOSIL).
[0040] The Rhizobium in this composition is preferably Rhizobium
japonicum, more preferably Rhizobium japonicum (Bioderpac,
2008).
[0041] The Clostridium in this composition is preferably
Clostridium pasteurianu, more preferably Clostridium pasteurianu
(Bioderpac, 2008).
[0042] The Pseudomonas in this composition is preferably
Pseudomonas fluorescens, more preferably Pseudomonas fluorescens
(Bioderpac, 2008).
[0043] Another microbial composition comprises one or more or two
or more microorganisms selected from the group consisting of
Bacillus subtilis ((SILoSiI.RTM. 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), Bacillus
thuringiensis strains HD-1 and HD-73 (SILoSil.RTM.BT), Streptomyces
(Bioderpac, 2008), Micrococcus (Bioderpac, 2008), Nitrobacter
(Bioderpac, 2008) and Proteus (Bioderpac, 2008).
[0044] Another embodiment of a microbial composition of comprises
Lactobacillus acidophilus (Bioderpac, 2008) and/or Lactobacillus
casei (Bioderpac, 2008).
[0045] A particularly preferred microbial composition comprises
Bacillus subtilis ((SILoSil.RTM. 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), Bacillus
thuringiensis strains HD-1 and HD-73 (SILoSil.RTM.BT), Streptomyces
(Bioderpac, 2008), Micrococcus (Bioderpac, 2008), Nitrobacter
(Bioderpac, 2008) and Proteus (Bioderpac, 2008).
[0046] The preferred microbial composition for use in the
biodegradation process is HQE. HQE was deposited with the American
Type Culture Collection (ATCC) Manassas, Va., USA on Apr. 27, 2010
and given Patent Deposit Designation PTA-10861. HQE is a microbial
consortium made up of microorganisms derived from fertile soils and
microorganisms from commercial sources. The microorganisms form
commercial sources include Bacillus subtilis (SILoSil.RTM. BS),
Bacillus thuringiensis strains ND-1 and HD-73 (SILoSil.RTM.BT) and
Trichoderma harzianum each obtained from Biotecnologia
Agroindustrial S.A. DE C.V., Morelia, Michoacan, Mexico
Microorganisms designated "Bioderpac 2008" by strain or species and
strain can be derived from HQE. However, subsets of the
microorganisms in HQE can also be used. The Bacillus subtilis
(SILoSil.RTM. BS), Bacillus thuringiensis (SILoSil.RTM. BT) and
Trichoderma harzianum (TRICHOSIL) microorganisms produce
chitinolytic enzymes that are especially important at the beginning
of the biodegradation. The chitinolytic enzymes help to degrade
chitin containing solids which can constitute a barrier to further
digestion. These organisms also produce proteases, lipases and
other enzymes that facilitate the breakdown of proteins, lipids and
carbohydrates.
[0047] As used herein, the term "HYTb refers to the aqueous
fraction obtained from the biodegradation process. HYTb contains
typically contains amino acids (about 3 wt % to 12 wt %, usually
about 12 wt %), chitosan (about 1.2 wt %), glucosamine (about 1 wt
%) and trace elements (about 6 wt %) including calcium, magnesium,
zinc, copper, iron and manganese. The amount of chitosan can range
between about 0.5 wt % and 1.5 wt %, more preferably between about
1.0 wt % and 1.5 wt %. The amount of glucosamine can range between
about 0.5 wt % and 1.5 wt %, more preferably between about 1.0 wt %
and 1.5 wt %. The total of chitosan and glucosamine is about 2.0 to
2.5 wt %. HYTb also contains enzymes such as lactic enzymes,
proteases, lipases, chitinases, lactic acid, polypeptides and other
carbohydrates. The specific gravity of HYTb is typically about
1.050-1.054. The average amino acid content in HYTb for certain
amino acids is set forth in Table 1.
TABLE-US-00001 TABLE 1 Amino acid profile dry powder hydrolysates
(mg per g dry weight) Dry powder Amino acid hydrolysates Aspartic
acid 38 Glutamic acid 39 Serine 16 Histidine* 9 Glycine 28
Threonine* 14 Alanine 36.1 Proline 25.8 Tyrosine* 70 Arginine 22.2
Valine* 20 Methionine* 16.4 Isoleucine* 18.3 Tryptophan* 3.1
Leucine* 23 Phenylalanine* 39 Lysine* 13 Total 431 *Essential amino
acids 226
[0048] HYTb is typically produced by the centrifugation of the
fermentation product formed by the biodegradation product. As the
biodegradation process proceeds, nutrients for the microorganisms
used for the biodegradation process, e.g. HQE, are depleted and the
pH drops due to the acid produced during the fermentation. This
causes the microorganisms in the fermentation product to die or
become dormant. Depending on the g force and time of the
centrifugation of the fermentation product, such microorganisms can
be found in HYTb. Accordingly, HYTb can include any one or more of
the above identified components, e.g. chitosan and glucosamine, in
combination with all or part of the microbial component of the
fermentation process that is present when it is stopped.
Alternatively, the centrifugation may proceed to a point where
substantially all of the microbial component is depleted from HYTb.
In such cases the microbial component can be centrifuged into the
HYTc fraction. Alternatively, HYTc can be separated from HYTb by
low g centrifugation. The HYTb can then be centrifuged to form a
pellet of microorganisms and a microorganism free-HYTb aqueous
solution.
[0049] As used herein, the term "HYTc" refers to the solid fraction
obtained from the biodegradation process. The primary component of
HYTc is chitin. It typically has an average molecular weight of
about 2300 daltons and constitutes about 64 wt % of the
composition. About 6% of HYTc contains minerals including calcium,
magnesium, zinc, copper, iron and manganese, about 24 wt % protein
and 6% water. It has a specific gravity of about 272 Kg/m.sup.3.
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 chitinolytic microorganisms for
the chitin substrate. Accordingly, HYTc can also contain
chitinolytic microorganisms unless steps are taken to remove them.
In the case of HQE, such chitinolytic microorganisms include one or
more of the chitinase and/or exochitinase producing microorganisms
discussed herein. Such microorganism organisms include but are not
limited to Bacillus subtilis (SILoSil.RTM. BS) Bacillus
thuringiensis strains HD-1 and HD-73 (SILoSil.RTM.BT), and
Trichoderma harzianum (TRICHOSIL). The chitinolytic microorganisms
can be removed from the solid chitin by sterilization,
pasteurization or washing the chitin with anti-microbial compounds
sucg as soaps or chlorine. HYTc may also contain additional
microorganisms present at the end of the biodegradation process
because of the presence of residual fermentation product or the
centrifugation of HYTb.
HQE Consortium
[0050] The following are the microorganisms in HQE which are
believed to be involved in the biodegradation process and their
known properties. In some cases the strain is identified as
"Bioderpac, 2008". Where the species is not known, the species and
strain are identified as "Bioderpac, 2008"
[0051] HQE was deposited with the ATCC on Apr. 27, 2010 and given
Patent Deposit Designation PTA-10861.
[0052] Bacillus subtilis ((SILoSil.RTM. BS) is a Gram positive
bacterium which is mesophilic and grows at an optimum temperature
between 25 and 35.degree. C. It is aerobic and can grow in
anaerobic conditions and utilizes a wide variety of carbon sources.
It contains two nitrate reductases, one of which is utilized for
nitrogen assimilation. It is capable of secreting amylase,
proteases, pullulanases, chitinases, xilanases and lipases.
[0053] Bacillus thuringiensis (Strains HD-1 and HD-73 (SILoSil.RTM.
BT)) are Gram Positive anaerobic facultative bacteria, in the form
of a peritrichous flagella. Strains HD-1 and HD-73 synthetizes
crystals with diverse geometric forms of proteic and insecticide
activity during the spore period. Strains HD-1 and HD-73 secret
exochitanases when in a chitin containing medium and can be
utilized for the degradation of the crustacean residues during the
production of chitooligosaccharides.
[0054] Bacillus cereus (Bioderpac, 2008) is an aerobic facultative
bacterium, gram positive, and spore forming. It is mesophilic and
grows at an optimum temperature between 20 and 40.degree. C. It
produces the antibiotics zwittermicin A and kanosamin.
[0055] Bacillus licheniformis (Bioderpac, 2008) is a Gram-positive,
motile, spore forming and facultative anaerobic bacterium. It
produces bacitracin, alpha amylases, lactamases, proteases and
alkaline phosphatases. This is a non-pathogen microorganism that is
associated with plants or plant materials.
[0056] Bacillus megaterium (Bioderpac, 2008) is a Gram-positive
aerobic bacterium. It is considered a saprophyte. It produces
glucose dehydrogenase, penicillin amydase, beta-amidase and neutral
proteases.
[0057] 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 utilizes lactose as a principal source of carbon
to produce energy. It grows with or without the presence of oxygen
in an acidic medium (pH 4-5). It produces the bactereocins named
lactacin B, organic acids, diacetyls and hydrogen peroxide.
[0058] Lactobacillus casei (Bioderpac, 2008) is a mesophilic,
facultative anaerobic which is Gram positive and non-spore forming.
It has the ability to adapt to cold temperatures. The optimum pH
for its growth is 5.5. It ferments galactose, glucose, fructose,
manose, manitol, and acetylglucosamine. This species can be grown
over a wide range of pH and temperature. It produces amylase
enzymes. It inhibits the growth of pathogenic bacteria such as H.
pylori by reducing pH through the production of (1) organic acids
such as acetic, proprionic or lactic acid or (2) hydrogen peroxide.
This microorganism secrets bacterocines.
[0059] Pseudomonas fluorescens (Bioderpac, 2008) is a bacteria with
multiple flagellum, forced aerobic and its optimal temperature for
growth is between 25 and 35.degree. C. It produces thermostable
lipases and proteases. It is antagonist towards a large number of
soil fungus strains. It produces secondary metabolites such as
antibiotics, iron chelates, and cyanides. It produces endochitanase
and cellulase in mediums with different glucose concentrations.
[0060] Trichoderma harzianum (TRICHOSIL) is a saprophyte fungus. It
exhibits antibiotic action and biological competition and for this
reason has biological control properties. It produces enzymes that
degrade cell walls or a combination of such activities. It produces
glucanases, chitinases, lipases, and extracellular proteases when
it interacts with some pathogenic fungi, such as Fusarium.
[0061] Rhizobium japonicum (Bioderpac, 2008) is a nitrogen fixating
bacteria. It synthesizes a hydrogenase system that participates in
the recycling of hydrogen to avoid its loss during nitrogen
fixation.
[0062] Azotobacter vinelandii (Bioderpac, 2008) is an aerobic
bacterium. It produces nitrogenases and is capable of nitrogen
fixation.
[0063] Clostridium pasteurianum (Bioderpac, 2008) is a Gram
positive bacteria, anaerobic obligated. It produces ferroxine (an
electron transporting protein) that acts as a direct electron donor
in the reduction of proteic iron.
[0064] Proteus vulgaris (Bioderpac, 2008) Is a gram positive
bacteria, anaerobic, facultative that grows at temperatures close
to 23.degree. C. It proteolytically degrades proteins to free amino
acids by the enzymes it produces.
[0065] Streptomyces sp. (Bioderpac, 2008) is a Gram-positive soil
bacterium. It produces multiple enzymes that metabolize diverse
nutrients. It can survive significant changes in temperature,
humidity and nutrient sources. The extracellular enzymes produced
by these bacteria utilize chitin and chitosan as substrates at a pH
of 4.5 to 6.5 and at 60.degree. C. These are conditions generated
at the beginning and at the end stages of lactic fermentation in
the biodegradation process.
[0066] Nitrobacter sp. (Bioderpac, 2008) is Gram negative bacteria,
aerobic, which converts nitrites into nitrates. It grows at a pH
between 6 and 9 and at temperatures between 10 to 34.degree. C. The
bacteria degrade organic polymers such as chitin into compounds
that are utilized by other organisms, such as Pseudomonas
fluorescens ( ) and Rhizobium japonicum (Bioderpac2008).
[0067] Micrococcus sp. (Bioderpac, 2008) is a spheric Gram positive
bacterium. This microorganism in association with Streptomyces sp (
) is capable of degrading colloidal chitin derivatives.
Groups and Enzymatic Activity of Microorganisms in HQE
[0068] The biodegradation of the components of marine animals or
by-products requires hydrolytic enzymes such as proteases, lipases,
and chitinases. The disclosed microbial compositions contain one or
more of such enzymes.
[0069] The primary group of microorganisms in HQE are Lactobacillus
acidophilus (Biodepac 2008), Bacillus subtilis (SILoSil.RTM. BS),
Pseudomonas fluorescens (Biodepac 2008), Bacillus licheniformis
(Biodepac 2008) and Trichoderma harzianum (TRICHOSIL). These
microorganisms are capable of biodegrading arthropod or arthropod
by-products. One or more of the members of this primary group also
have a synergistic action when combined with other microorganisms
from HQE.
[0070] The first group of microorganisms includes microorganisms
which cause the reduction of pH and which stabilize fermentation
due to the production of organic acids and hydrogen peroxide. This
group includes Lactobacillus acidophilus (Biodepac 2008) and
Lactobacillus casei (Biodepac 2008). Their activity is important at
the start of fermentation and during the final stages of
fermentation to produce the optimum pH for the hydrolytic enzymes.
Their activity also creates a culture environment which prevents
the growth of unwanted microorganisms and favors the
demineralization of the chitin residues. Lactobacillus acidophilus
(Biodepac 2008) is a member of the primary group.
[0071] The second group of microorganisms includes microorganisms
which produce extracellular enzymes. This second group includes
Bacillus subtilis (SILoSil.RTM. BS), Bacillus cereus (Biodepac
2008), Trichoderma harzianum (Biodepac 2008), Rhizobium japonicum
(Biodepac 2008) and Azotobacter vinelandii (Biodepac 2008). The
chitin chains in arthropod or arthropod by-products are associated
with protein molecules. The separation of such polymers requires
the hydrolytic action obtained from the chitinolytic and
proteolytic enzymes produced by these microorganisms. Both types of
enzymes break the chains on the internal portion of the polymer to
produce oligomers of diverse sizes. The action from these enzymes
occurs in a successive manner within the intermediate and final
phases of the fermentation process when the appropriate pH
conditions are achieved. The microorganisms on this group and the
environmental conditions they produce facilitate the liberation of
pigments and the lipid fraction adhered to these residues. Bacillus
subtilis (SILoSil.RTM. BS) and Trichoderma harzianum (Biodepac
2008) are members of the primary group.
[0072] 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
oligomers (chito-oligosaccharides 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.
[0073] In preferred embodiments, one or two of the first, second
and third groups of microorganisms can be combined. Alternatively,
all of the first, second and third groups can be combined.
[0074] A fourth group of microorganisms includes Bacillus
thuringiensis (strains HD-1 and/or HD-73), 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 primary 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 of the first second and third groups. The addition of this
fourth group results in a synergistic effect which enhances the
biodegradation process.
[0075] Each of these groups, including the primary group, are
separately useful and can be combined with prior art microbial
compositions to enhance their performance. In this regard, the
fourth group is particularly preferred.
[0076] Table 2 sets forth some of the aforementioned combinations.
Column 1 is a list of the known microorganisms in HQE that are
believed to be active in the biodegradation process. Column 2 lists
the microorganisms from column 1 without the microorganisms in the
fourth group of microorganisms. Column 3 shows the combination of
the primary 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 groups 2 and 3. Other useful
combinations are set forth in columns 7-10.
TABLE-US-00002 TABLE 2 Culture Composition Microorganism 1 2 3 4 5
6 7 8 9 10 Bacillus subtilis X X X X X X X X Bacillus cereus X X X
X X X Bacillus X X megaterium Azotobacter X X X X X X vinelandii
Lactobacillus X X X X X X X X acidophilus Lactobacillus X X X X X X
casei Trichoderma X X X X X X X X harzianum Rhizobium X X X X X X
japonicum Clostridium X X X X X X pasteurianum Bacillus X X X X X X
X X licheniformis Pseudomonas X X X X X fluorescens Bacillus X X X
X X X thuringiensis Streptomyces X X X X X X X Nitrobacter X X X X
X Micrococcus X X X X X Proteus vulgaris X X X X X
[0077] The activity of the enzymatic extracts produced by the
microorganisms within HQE is complex, but has permitted the
degradation of the chitinous residues of arthropods such as
crustaceans. The microorganisms in HQE are activated in a
successive manner according to the environment generated by the
organisms used.
Methods for Identification and Isolation of Microbes in HQE
[0078] It is important to obtain pure isolates before attempting to
characterize or identify a species. A few bacteria are
morphologically unique and can be identified without isolation, but
nearly all require isolation. The following describes the isolation
of pure cultures from a mixture of species contained within HQE for
Bacillus subtilis (SILoSil.RTM. 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 (strains HD-1
and HD-73), Rhizobium japonicum (Bioderpac, 2008), Azotobacter
vinelandii (Bioderpac, 2008), Clostridium pasteurianum (Bioderpac,
2008), Proteus vulgaris (Bioderpac, 2008), Bacillus thuringiensis
(SILoSil.RTM. BT), Streptomyces sp. (Bioderpac, 2008), Nitrobacter
sp. (Bioderpac, 2008) and Micrococcus sp. (Bioderpac, 2008).
[0079] The first steps include: [0080] (1) Dilution streaking and
differential Incubations [0081] (2) Identification and separation
of colony types [0082] (3) Narrowing down the collection, and
[0083] (4) Determining the initial characteristics of specific
colonies and the preservation of Isolates.
Dilution Streaking and Differential Incubations
[0084] Once a specimen is removed from the HQE sample it should be
cultured immediately. Any liquid sample must be thoroughly vortexed
prior to preparation of the plates because non-motile bacteria may
settle to the bottom of a sample if they are associated with
particulate matter. Unfortunately, bacteria do not segregate
homogeneously and replicate samples from the same mixture may
contain different quantities of bacteria. The object of thorough
vortexing and subsequent dilution streaking is to spread out
individual CFUs (Colony Forming Units) so as to obtain discrete
colonies that may be sub-cultured.
[0085] All of the mentioned microorganism can be separately
subjected to the following procedures for identification and
isolation with the exception of Nitrobacter sp. which will be
explained separately.
[0086] A loop full of thoroughly vortexed sample from HQE is
obtained aseptically and applied to one edge of the agar surface.
With back and forth movements about one-fourth of the surface
should be streaked while drawing the loop toward the middle of the
plate. Streaking should not break the surface of the agar, and
there should be (20 or more) streak lines produced. To dilute or
spread out the sample the loop must be flamed to destroy all viable
material, touched to a clean part of the agar to cool it, then
streaks made perpendicular to the original inoculum, overlapping
that part of the plate once or twice. The second section should
cover one-half of the remaining sterile surface. This spreads out a
small part of the original inoculum possibly diluting it
sufficiently to result in the appearance of individual colonies
after incubation. A third section is then streaked perpendicular to
the second section, flaming and cooling the loop and overlapping
the previous section as before, to further dilute the inoculum.
[0087] In the preparation of isolates from each batch of product,
the next phase is to prepare replicate streak plates and incubate
them under different conditions in an inverted position, to
maximize opportunities to differentiate colony types for Bacillus
subtilis (SILoSiI.RTM. 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), Bacillus thuringiensis (strains HD-1 and HD-73,
SILoSil.RTM.BT), Streptomyces sp. (Bioderpac, 2008), and
Micrococcus sp. (Bioderpac, 2008). The temperature of incubation is
varied (typically 25, 30, and 37.degree. C.) and incubated under
both aerobic and anaerobic conditions. This approach increases the
chances of separating individual species, since different
species/bacterium have different optimum temperature ranges for
growth and different requirements for oxygen. The aerobic organisms
should be checked after one day of incubation, since some of our
featured bacteria being tested grow very fast and crowd out the
others.
Approaches to Identifying and Separating Colony Types
[0088] A dissecting microscope with a trans-illuminator can be used
to distinguish individual colonies. Plates should remain inverted
during examination. Colonies are distinguishable by size, shape,
opacity, and texture regarding afore the mentioned microorganisms.
Upon examination, it is best to indicate the colonies to be sampled
by putting a small mark next to them on the bottom of the plate. It
will be necessary to turn over the plate lid to collect colony
material. Caution should be taken to carefully insert the sterile
loop only for the time it takes to obtain an inoculum.
[0089] For color, surface characteristics and profile (raised,
flat, etc.), it is necessary to examine the colonies with incident
light, through the transparent lid. Lids should be left on
otherwise the plates will become contaminated. Before turning the
plate remove and invert the lid to remove the moisture. The old lid
should be replaced with a new one for viewing.
[0090] In the event two colonies overlap and still can be
distinguished, then at least two colonies are present. Colonies
usually have a fairly simple, uniform texture. If an area resembles
a mosaic, there are probably at least two species. Each unique type
of colony should be sampled by taking a needle inoculum and
performing a three way dilution streak on a fresh plate. Care
should be taken to sample only the colony of interest. Incubate
each new streak plate under aerobic conditions at the temperature
which the original plate was incubated.
[0091] Species will exhibit temperature optima, indicated by faster
growth and/or larger colonies at temperatures closest to ideal. Any
colony that is sampled from an anaerobically incubated plate will
likely be a facultative anaerobe.
Narrow Down the Collection
[0092] Duplicates of the same species and strain are likely to be
isolated from different streak plates. Many different species and
different strains of the same species produce very similar colony
types. To narrow the number of isolates to unique species/strains,
culture suspected duplicate isolates on the same plate. On two
thirds of the surface conduct two "mini-dilutions" to obtain
individual colonies for each culture, and on the remaining
one-third mix them. After incubation, if the two isolates grow on
the same plate and/or mixed inoculum produce two distinguishable
colony types, two unique isolates have been identified.
Initial Characterization of Colonies and Preservation of
Isolates
[0093] Most of the characteristic of the organisms under
consideration should be determined using incident, not transmitted,
light.
[0094] Once an isolate is obtained and purity established by both
colony examination and microscopic examination, an agar slant tube
should be inoculated and incubated at an appropriate temperature
with the cap loose to allow gas exchange. After growth appears, the
culture should be described, the cap tightened, and the tube kept
at room temperature as a source of pure culture for assays.
[0095] In addition to gross descriptive characterization, a young
(<18 h) culture should be gram stained and the results recorded
including cell shape and size, sheaths or capsules if evident, and
any evidence of spores or similar structures. Relationship to
oxygen is the next step with which to narrow possible categories.
After that, it is this particular combination of Gram stain
results, cell type, and relationship to oxygen that determines the
next series of steps toward characterizing the isolate.
The Deletion and Counting of Nitrobacter sp. Population in the
Product
[0096] There have been several studies on the metabolism, survival,
and growth of nitrite oxidizers in pure cultures and on their
nitrifying activity in various environments, but fewer studies have
dealt with 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 methods of detection and counting.
[0097] This failure is due in part to the unfavorable physiological
characteristics of these bacteria, namely slow growth, small
biomass, and susceptibility of cultures to contamination. There is
a way to count Nitrobacter population in the soil but it is time
consuming and selective.
Detailed Description of the Growth Process
[0098] Bacillus cereus (Bioderpac, 2008), Bacillus megaterium
(Bioderpac, 2008),
[0099] Bacillus subtilis (SILoSil.RTM. BS) and Bacillus
licheniformis (Bioderpac, 2008)
[0100] Media ingredient: purified water and plate medium nutrient
agar.
[0101] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of purified water, (2) commence a serial dilution range up to 1-10,
(3) incubate a replicate plate series with 1 ml of the dilution
range, the plate medium is nutrient agar, (4) incubate plates at
37.degree. C. for 24-48 hours, (5) colony forming units should be
1,000,000 per 1 ml of product.
[0102] Pseudomonas fluorescens and Proteus vulgaris
[0103] Media ingredient: purified water and plate medium nutrient
agar.
[0104] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of purified water, (2) commence a serial dilution range up to 1-10,
(3) incubate a replicate plate series with 1 ml of the dilution
range, the plate medium is nutrient agar, (4) incubate plates at
37.degree. C. for 24-48 hours, (5) colony forming units should be
1,000,000 per 1 ml of product.
[0105] Lactobacillus acidophilus and Lactobacillus casei
[0106] The Agar M.R.S. was developed by Man, Rogosa and Sharpe to
provide means that could demonstrate a good growth of lactobacillus
and other lactic acid bacteria. The culture medium allows an
abundant development of all the species of lactobacillus. Peptona
and glucose constitute the nitrogen source, carbon and of other
necessary elements for the bacterial growth. The sorbitan
monoleate, magnesium, manganese and acetate, contribute cofactors
and can inhibit the development of some microorganisms. The
ammonium citrate acts like an inhibiting agent of the growth of
negative Gram bacteria. See Table 3.
TABLE-US-00003 TABLE 3 Formula (in grams per liter) Instructions
Proteose peptone N.sup.o 3 10.0 Suspend 64 g of the medium in a
liter Meat extract 8.0 of distilled water. Let it rest 5 minutes
Yeast extract 4.0 and mix warming up to boiling point Glucose 20.0
during 1 or 2 minutes. Sterilize in Sorbitan Monoleate 1 ml
sterilizer during 15 minutes to 121.degree. C. Dipotassium
Phosphate 2.0 Sodium Acetate 5.0 Ammonium Citrate 2.0 Magnessium
Sulfate 0.2 Manganese Sulfate 0.05 Agar 13.0 Final pH: 6.4 .+-.
0.2
[0107] Media ingredient: purified water and plate medium nutrient
agar M.R.S substrate.
[0108] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of purified water, (2) commence a serial dilution range up to 1-10,
(3) incubate into aerobic chamber with 5-10% CO.sub.2 a replicate
plate series with 1 ml of the dilution range, the plate medium is
agar M.R.S., (4) incubate plates at 33-37.degree. C. for 72 hours
or 30.degree. C. for 5 days, (5) colony forming units should be
1,000,000 per 1 ml of product.
[0109] Results. Colony forming units should be 1,000,000 per 1 ml
of product.
[0110] Characteristics of the colonies: generally small,
white-grayish, smooth or rough.
[0111] Characteristics of the medium: Prepared medium is
yellow.
[0112] Lactobacillus Identification:
TABLE-US-00004 TABLE 4 Growth at Acid NH.sub.3 Growth in 4%
15.degree. C. 45.degree. C. la su sal mn so xi Arginine NaCl broth
L. acidophilus - + + + + - - - - - L. casei + V +/- +/- + + + - -
+
[0113] Azotobacter vinelandii (Bioderpac, 2008)
[0114] Media ingredient: purified water and plate medium nutrient
agar substrate (Burk's, Asbhy, Jensen's).
[0115] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of purified water, (2) incubate this solution at 25.degree. C. for
48 hours, (3) commence a serial dilution range up to 1-10, (4)
incubate a replicate plate, with nutrient agar substrate, series
with 1 ml of the dilution range, the plate medium is nutrient agar
(5) incubate plates at 25.degree. C. for 48-72 hours, (6) colony
forming units should be 1,0000,000 per 1 ml of product.
[0116] Clostridium pasteurianum (Bioderpac, 2008)
[0117] Media ingredient: phosphate buffered water and plate medium
standard methods agar substrate (TYG).
[0118] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of phosphate buffered water, (2) make appropriate serial dilutions
(3) plate appropriate aliquot for desired dilution into Petri
dishes, (4) add tempered standard methods agar and mix of dish, (5)
place inverted dried plates into anaerobic chamber, (6) incubate
plates at 35-37.degree. C. for 48-72 hours, (7) after incubation
period, removed plates from anaerobic chamber and count plates,
record dilutions used and the total number of colonies counted for
each dilution, (8) colony forming units should be 1,000,000 per 1
ml of product.
[0119] The identification of this species of Clostridium uses a
typical microscopic morphology of a colony which allows a fast and
presumptive identification of some species of Clostridium
frequently isolated. In addition, along with the use of simple
biochemical tests such as the study of the production of lecitinase
and lipase in agar egg yolk, the hydrolysis of the gelatin and urea
and the production of indol through the fast method
(p-dimetil-amino-cinnamaldehide), constitute an easy and
inexpensive method for the identification, even definitive, for
some of them.
[0120] Micrococcus sp. (Bioderpac, 2008)
[0121] Media ingredient: purified water and plate medium nutrient
agar.
[0122] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of purified water, (2) commence a serial dilution range up to 1-10,
(3) incubate a replicate plate series with 1 ml of the dilution
range, the plate medium is nutrient agar, (4) incubate plates at
37.degree. C. for 24 hours, (5) colony forming units should be
1,000,000 per 1 ml of product.
[0123] If positive Gram coccos are found, perform antibiotic
sensitivity tests. Micrococcus is sensitive to Bacitracin and
resistant to furazolidone.
[0124] Rhizobium japonicum (Bioderpac, 2008)
[0125] Media ingredient: purified water and plate medium ALM (agar
yeast extract mannitol).
[0126] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of purified water, (2) commence a serial dilution range up to 1-10,
(3) incubate a replicate plate series with 1 ml of the dilution
range, the plate medium is ALM (agar yeast extract mannitol), (4)
incubate plates at 28.degree. C. for 96 hours, (5) colony forming
units should be 1,000,000 per 1 ml of product.
[0127] To confirm Rhizobium japonicum (Bioderpac, 2008), the
isolated colony is used to infect a leguminosae aseptically to
cause the formation of nodules.
[0128] Trichoderma harzianum (TRICHOSIL)
[0129] Media ingredient: purified water and malt extract agar
medium (2% wt/vol) substrate supplemented with chloramphenicol,
streptomycin sulfate, and nystatin.
[0130] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of purified water, (2) commence a serial dilution range up to 1-10,
(3) incubate a replicate plate series with 1 ml of the dilution
range, the plate medium is malt extract agar (4) incubate plates at
25.degree. C. for 4 days, (5) colony forming units should be
1,000,000 per 1 ml of product.
[0131] Bacillus thuringiensis (Strains HD-1 and HD-73 (SILoSil.RTM.
BT))
[0132] Media ingredient: purified water and plate Superbroth medium
agar substrate supplemented with 2 g/litro de D-glucosa and 50
.mu.g/ml erythromycin.
[0133] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of purified water, (2) commence a serial dilution range up to 1-10,
(3) incubate a replicate plate series with 1 ml of the dilution
range, the plate medium is Superbroth agar (4) incubate plates at
28.degree. C. for 10-14 days, (5) colony forming units should be
1,000,000 per 1 ml of product.
[0134] Streptomyces sp. (Bioderpac, 2008)
[0135] Media ingredient: purified water and plate actinomycete
isolation agar medium.
[0136] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of purified water, (2) commence a serial dilution range up to 1-10,
(3) incubate a replicate plate series with 1 ml of the dilution
range, the plate medium is actinomycete isolation agar (4) incubate
plates at 28.degree. C. for 2-3 days, (5) colony forming units
should be 1,000,000 per 1 ml of product.
[0137] Nitrobacter sp. (Bioderpac, 2008)
[0138] Media ingredient: purified water and plate medium nutrient
agar substrate.
[0139] Procedure: (1) place 0.1 ml liquid sample of HQE in 9.9 ml
of purified water, (2) commence a serial dilution range up to 1-10,
(3) incubate a replicate plate series with 1 ml of the dilution
range, the plate medium is plate nutrient agar substrate, (4)
incubate plates at 30.degree. C. for 10-14 days.
Biodegradation Process
[0140] In a preferred embodiment, the marine arthropod is a
crustacean and the preferred crustacean is shrimp. Shrimp
by-product comprises shrimp cephalothorax and/or exoskeleton.
[0141] In the biodegradation process, it is preferred that the
fermentation be facultative aerobic fermentation. It is also
preferred that the fermentation is carried out at a temperature of
about 30.degree. C. to 40.degree. C. The pH is preferably less than
about 6, more preferably less than about 5.5. However, the pH
should be maintained above about 4.3. The fermentation is carried
out for about 24-96 hours. In some embodiments, the fermentation is
carried out for about 24-48 hours and more preferably 24-36 hours.
These fermentation times are far shorter than the typical prior art
fermentation times of 10 to 15 days to achieve substantially the
same amount of digestion, albeit without detectable formation of
chitosan and glucosamine.
[0142] The separation of the mixture is preferably by
centrifugation. (e.g. about 920 g). Gravity separation can also be
used but is not preferred because of the time required to achieve
separation.
[0143] The mixture separates in to three fractions: solid, aqueous
and lipid. The solid fraction comprises chitin and is designated
HYTc. The aqueous fraction comprises protein hydroysate, amino
acids, chitosan and glucosamine and is designated HYTb. The lipid
fraction comprises sterols, vitamin A and E and carotenoid pigments
such as astaxanthine.
[0144] Any of the microbial compositions identified herein 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 the
fermentation broth. As described above, HYTb contains amino acids,
chitosan, glucosamine and trace elements including 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 can also
contain dormant microorganisms from a prior biodegradation process.
Such microorganisms can become reactivated and, in combination with
HQE, contribute to a more robust biodegradation process as compared
to when HQE is used by itself as otherwise described herein
[0145] More particularly, the process includes the following steps:
[0146] a. Activation of the microbial cells in a sugar base
solution to enhance its growth and the biomass formation. [0147] b.
Milling of the shrimp by-products (cephalthorax and exosqueleton)
to make a homogeneous paste. [0148] c. Homogeneous mixing of the
shrimp by-product paste with at least 10% of the activated
inoculum. [0149] d. Adjustment of the pH values to less than 6.0 in
the mixture using a citric acid solution to inhibit the growth of
micro organisms and to promote the development of microbial cells
that constitute the inoculum. [0150] e. Fermentation of the mixture
in a non continuous agitated system at temperatures within a range
of 30 to 40.degree. C. at least for at least 96 hours maintaining
pH at less than 5.0. The pH is monitored periodically. If the pH
rises above 5.0, a citric acid buffer is added in an amount to
maintain the pH below 5.0. [0151] f. Centrifugation of the ferment
to separate the three principal fractions: chitin, liquid
hydrolysate and pigmented paste. [0152] g. Rinsing of the crude
chitin and recollection of the rinse water to recuperate fine
solids or minerals. [0153] h. Drying of the chitin and storage.
[0154] i. Drying and storage of the liquid hydrolysate. [0155] j.
The pigmented paste (lipid fraction) is stored in closed recipients
for conservation.
[0156] The process and operational fundamentals are better
understood with reference to FIG. 1 and the following detailed
description.
[0157] Activation of Microbial Cells
[0158] A microbial composition as disclosed herein is used as
inoculum. The inoculum of HQE has a concentration of microbes of
about 2.5 to 3.0% (w/v). HQE is activated by dilution to 5% in
sugar cane solution (3.75% final concentration of sugar cane), and
incubated at 37.degree. C. for 5 days. HYTb (10 ml per liter of
culture) is preferably added to provide a source of minerals and
naturally derived amino acids. The cellular growth of the
microorganisms was estimated by optical density measured at 540 nm.
The activation is complete at an optical density of about 1.7. The
concentration of microbes after activation is about 1.9 to 3.0%
(w/v).
[0159] Preparation of Samples
[0160] The shrimp by-products samples are obtained from shrimp
processing plants. Slightly thawed and minced residue (1500 g by
batch) is mixed with 99 grams of sugar cane (final concentration
6.6% wt %) and 85.5 ml of activated HQE 5% (v/w) (optical density
of cell=1.7). Then the pH is adjusted to 5.5 using 2 M citric
acid.
[0161] Fermentation Control
[0162] The mixture is incubated at 36.degree. C. with a non
continuous agitation for 96 h. During the fermentation process, the
pH is monitored by using 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. The TTA is expressed as a percentage of
lactic acid.
[0163] Conditions of Separation
[0164] The fermentation product is a viscous silage which has an
intense orange color, due to the astaxanthine presence. The
ensilage is centrifuged (5.degree. C.) at 1250 rpm (930 g) for 15
min to obtain the chitin, the liquid hydrolysates, and the pigment
paste. The upper phase (pigment paste) is separated manually. The
liquid hydrolysates are separated by decantation, and the sediment
that constitutes the raw chitin is washed with distilled water to
separate fine solids. The resulting liquid is collected and dried.
The raw chitin, liquid hydrolysates and fine solids are dried at
60.degree. C. All the fractions are stored to protect them from
light.
[0165] The above protocol was carried out using HQE in three
fermentation batches in duplicate as set forth in the following
examples.
Example 1
Fermentation Control by Measurement of pH and Total Titratable
Acidity (TTA, %)
[0166] The average initial values of pH and TTA were 7.31.+-.0.10
and 0.53.+-.0.09, respectively. As shown in FIG. 2, the pH was
initially reduced to 6.5 by the addition of 2 M citric acid. Then,
due to proteolysis and the release of ammonium, the pH increased
again to 7.11.+-.0.08 during the first 2 h, and later, the pH
diminished by 28% (up to 5.28.+-.0.01) during the 12 hours of
fermentation. At 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 of the average values of TTA were 1%, and then these
values increased gradually to an average value of 3.33.+-.0.23 at
24 hours, as shown in FIG. 2.
Example 2
Products of the Fermentation and Chemical Composition
[0167] After the fermentation process, the silage was centrifuged
to separate the three principal products (chitin, liquid
hydrolysate, and pigment paste). The other product, the fine solids
were retained with the raw chitin wash. Table 5 shows the
proportion recovered in each fraction. In dry weight, the bigger
fraction corresponded to the liquid hydrolysate (55%), then the
fine solids (29%), raw chitin (10%) and pigment paste (5%). In the
fermented batch, the average value of dry weight was 32%
(481.1.+-.5.6 g).
[0168] 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 high protein content (42.34%)
quantified in the liquid hydrolysates. This facilitated the
recovery of a pigment paste, consisting mainly of total lipids
(42.67%), and produced a fraction abundant in ash (16.72%) in the
fine solids.
TABLE-US-00005 TABLE 5 Products separated from fermentation Lipidic
Dry matter Raw chitin Liquid hydrolysate paste Fine solids (g) (g)
(g) (g) (g) 480.0 48.0 264.4 22.9 141.2 487.5 50.2 268.8 24.0 140.8
475.7 51.0 265.7 23.7 131.7 481.1 .+-. 5.6 49.5 .+-. 2.4 266.3 .+-.
4.6 23.3 .+-. 0.5 141.9 .+-. 5.5
TABLE-US-00006 TABLE 6 Chemical composition (dry weight) of the
fermentation products Products % Protein % Ash % Total lipid Raw
chitin 18.12 .+-. 0.15 4.36 .+-. 0.26 2.02 .+-. 0.46 Liquid
hydrolysate 42.34 .+-. 0.03 7.96 .+-. 0.16 4.28 .+-. 0.28 Lipidic
paste 30.80 .+-. 0.25 5.11 .+-. 0.16 42.67 .+-. 0.63 Fine solids
31.62 .+-. 0.10 16.72 .+-. 0.37 Nd
Example 3
Amino Acid Profile in the Dry Powder Hydrolysates
[0169] The amino acid content of dried hydrolysate was determined
as described by Lopez-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 dry
powder hydrolysates. The proportion of essential amino acids was of
52.5% to dry powder hydrolysates.
TABLE-US-00007 TABLE 7 Amino acid profile dry powder hydrolysates
(mg per g dry weight) Amino acid Dry powder hydrolysates Aspartic
acid 38 Glutamic acid 39 Serine 16 Histidina* 9 Glycine 28
Threonine* 14 Alanine 30 Proline 8 Tyrosine* 70 Arginine 18 Valine*
20 Methionine* 4 Isoleucine* 15 Leucine* 23 Phenylalanine* 39
Lysine* 13 Total 394 * Essential amino 207 acids
Example 4
Quantification of Glucosamine in Raw Chitin
[0170] In the chitin, the content of glucosamine was quantified as
an index of purity. The contents of glucosamine 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 quantification of
glucosamine was reported by Lopez-Cervantes et al., "Quantitation
of glucosamine from shrimp waste using HPLC" Journal of
Chromatographic Science, volume 45, 1 (2007).
Example 5
Contents of Astaxanthin and Profile of Fatty Acids in Pigment
Paste
[0171] Astaxanthin is the main pigment in the lipidic paste
obtained from fermented shrimp waste. The content of astaxanthin
ranged from 1.98 to 2.25 mg g.sup.-1 of dry lipidic paste, and the
average is 2.11 mg g.sup.-1 of dry lipidic paste. Astaxanthin was
determined by a version of the method of Lopez-Cervantes et al.,
"Quantification of astaxanthin in shrimp waste hydrolysate by HPLC"
Biomedical Chromatography, volume 20, 981 (2006).
[0172] In the pigment paste, fourteen fatty acids were identified.
The palmitic acid (C16:0), and the oleic acid (C18:1n9) were found
in higher quantity.
* * * * *