Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
  • A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/04—Preserving or maintaining viable microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
  • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
  • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
  • A23L29/212—Starch; Modified starch; Starch derivatives, e.g. esters or ethers
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
  • A23P20/00—Coating of foodstuffs; Coatings therefor; Making laminated, multi-layered, stuffed or hollow foodstuffs
  • A23P20/10—Coating with edible coatings, e.g. with oils or fats
  • A23P20/12—Apparatus or processes for applying powders or particles to foodstuffs, e.g. for breading; Such apparatus combined with means for pre-moistening or battering
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• the present invention relates to the use of poly-y-glutamic acid ( ⁇ -PGA) in improving the viability of probiotic microorganisms.
• ⁇ -PGA poly-y-glutamic acid
• the invention also relates to methods of manufacturing ⁇ -PGA, in particular ⁇ -PGA with specific properties.
• Probiotics were defined by the World Health Organization in 2002 as live microorganisms that when administered in adequate amounts confer a health benefit on the host. Accordingly probiotic microorganisms have been introduced into a variety of food and drink products for administration to a human or animal. However, many of the probiotic microorganisms used in food and drink products do not survive for long enough to confer a health benefit on the host. The processes that they must be subjected to to get them into the food or drink product and the human or animal body result in many becoming unviable before they have the chance to affect the host in any way.
• the probiotic microorganisms are subjected to a freeze drying process.
• the probiotic microorganisms are incorporated into a food or drink product and then stored for a period of time that depends on the product they have been incorporated into.
• the probiotic microorganisms are ingested by a human or animal with the food or drink product; the conditions of the mouth and stomach in particular being adverse to the viability of the probiotic microorganisms. Accordingly having passed through all of the above processes there are only relatively few microorganisms remaining viable and able to exert some effect on the host.
• the present invention provides an ingestible product comprising a probiotic microorganism and a poly glutamic acid.
• the poly glutamic acid is preferably poly-y-glutamic acid ( ⁇ -PGA).
• the ⁇ -PGA preferably has a molecular weight of from 10,000 to 1 ,000,000 Daltons (Da).
• the ⁇ -PGA may have a molecular weight of from 20,000 to 800,000 Da or 20,000 to 700,000 Da or 20,000 to 600,000 Da. More preferably the ⁇ -PGA may have a molecular weight of from 50,000 to 500,000 Da, for example 100,000 to 300,000 Da or 200,000 to 260,000 Da.
• the ⁇ -PGA may be produced in any suitable bacteria, for example Bacillus subtilis and Bacillus licheniformis.
• the ⁇ -PGA is produced in Bacillus subtilis, for example Bacillus subtilis natto.
• the probiotic microorganism may be a bacteria.
• examples of possible bacteria include Bifidobacterium longum, Bifidobacterium breve and Lactobacillus casei.
• All or part, of some or all, of the probiotic microorganisms may be coated with biopolymer, for example by suspending the probiotic microorganisms in biopolymer with subsequent freeze drying.
• probiotic microorganisms may be coated with the poly glutamic acid, for example by suspending the probiotic microorganisms in the polyglutamic acid with subsequent freeze drying
• the ingestible product may be a food or beverage product.
• the ingestible product may be milk based, for example yogurt or a yogurt drink, or non-milk based, for example fruit based such as a fruit juice.
• probiotic microorganism at least partially coated with a poly glutamic acid.
• the poly glutamic acid is preferably poly-y-glutamic acid ( ⁇ -PGA).
• the ⁇ -PGA preferably has a molecular weight of from 10,000 to 1 ,000,000 Daltons (Da).
• the ⁇ -PGA may have a molecular weight of from 20,000 to 800,000 Da or 20,000 to 700,000 Da or 20,000 to 600,000 Da. More preferably the ⁇ -PGA may have a molecular weight of from 50,000 to 500,000 Da, for example 100,000 to 300,000 Da or 200,000 to 260,000 Da.
• the ⁇ -PGA may be produced in any suitable bacteria, for example Bacillus subtilis and Bacillus licheniformis.
• the ⁇ -PGA is produced in Bacillus subtilis, for example Bacillus subtilis natto.
• the probiotic microorganism may be a bacteria.
• examples of possible bacteria include Bifidobacterium longum, Bifidobacterium breve and Lactobacillus casei.
• Also provided is method of at least partially coating a probiotic microorganism with a poly glutamic acid comprising mixing the microorganism with a solution of a poly glutamic acid.
• the poly glutamic acid may be provided in a concentration of from 2 to 15% (w/v), for example from 5 to 12% (w/v), such as 10%> (w/v).
• the microorganism may be provided in cell pellet form.
• the probiotic microorganism may be a bacteria.
• examples of possible bacteria include Bifidobacterium longum, Bifidobacterium breve and Lactobacillus casei.
• the poly glutamic acid is preferably poly-y-glutamic acid ( ⁇ -PGA).
• the ⁇ -PGA preferably has a molecular weight of from 10,000 to 1 ,000,000 Daltons (Da).
• the ⁇ -PGA may have a molecular weight of from 20,000 to 800,000 Da or 20,000 to 700,000 Da or 20,000 to 600,000 Da. More preferably the ⁇ -PGA may have a molecular weight of from 50,000 to 500,000 Da, for example 100,000 to 300,000 Da or 200,000 to 260,000 Da.
• the ⁇ -PGA may be produced in any suitable bacteria, for example Bacillus subtilis and Bacillus licheniformis. In one embodiment the ⁇ -PGA is produced in Bacillus subtilis, for example Bacillus subtilis natto. Also provided is a method of making poly-y-glutamic acid ( ⁇ -PGA) comprising the steps of:
• starter culture comprises one or more colonies of an appropriate strain of bacteria, preferably one or more highly mucoid colonies, inoculated in a growth medium, for example, TSB and incubated, for example at 37°C for 24h.
• the bacterial colony may be B. subtilis or B. licheniformis .
• the bacterial colony is preferably B. subtilis natto.
• the fermentation broth may be any suitable broth, for example tryptone soy broth (TSB).
• TLB tryptone soy broth
• the growth media may be any suitable growth media, for example growth media E or growth media GS.
• Growth medium GS generally produces a higher yield of ⁇ -PGA than growth medium E.
• the growth media can affect the crystallinity of the ⁇ -PGA with the ⁇ -PGA produced in medium E being amorphous and the ⁇ -PGA produced in medium GS being crystalline.
• the growth medium has an effect on the formation of the salt or free acid form of the ⁇ -PGA.
• In growth medium GS most of the ⁇ -PGA produced was the sodium salt whereas in growth medium E a considerable amount of the acid form was produced. Molecular weight is affected by both growth medium and strain of bacteria.
• the bacterial colony may be highly mucoid.
• the fermentation step may take place at a temperature of 35 to 39°C, for example 37°C.
• the fermentation time may be from 18 to 30 hours, for example from 20 to 28 hours, such as 24 hours.
• the growth step may take place at a temperature of 35 to 39°C, for example 37°C.
• the growth time may be from 90 to 100 hours, for example from 94 to 98 hours, such as 94 hours.
• the growth step may include the steps of agitating in any suitable fermenter vessel for all or part of the growth time. Agitation can range from 100 rpm to 1000 rpm.
• Any suitable fermenter vessel could be used including, for example, shake flasks, aerated stirred tank reactors and solid state fermenters.
• the poly-y-glutamic acid may be isolated from the growth media.
• the poly- ⁇ - glutamic acid in the growth media may first be subjected to centrifugation.
• Any suitable alcohol based solvent such as ethanol, may then be added to the cell free supernatant resulting from centrifugation, for example at a ratio of 2: 1 to 6: 1 , for example 4: 1 , alcohol to supernatant.
• the alcohol/supernatant mixture may be incubated at 2 to 6°C, for example 4°C for 70 to 75 hours, for example 72 hours.
• the poly-y-glutamic acid may be removed from the alcohol/supernatant mixture by centrifugation and/or filtration.
• the poly-y-glutamic acid may be subjected to lyophilization.
• the poly-y-glutamic acid may be frozen before being subjected to lyophilization.
• Figure 2 shows the growth of the Bacillus strains in E medium
• Figure 3 shows FT-IR spectra for the ⁇ -PGA produced by the Bacillus strains compared to that of a commercially available ⁇ -PGA sample
• Figure 4 shows crude yield of ⁇ -PGA from different Bacillus strains in growth media E and GS
• Figure 5 shows the XRD spectra for the strain - B. subtilis ATCC 23856 in GS and E medium;
• Figure 6 shows ICP-AES results showing % salt composition of ⁇ -PGA produced by the different Bacillus strains in GS medium;
• Figure 7 shows ICP-AES results showing % salt composition of ⁇ -PGA produced by the different Bacillus strains in E medium
• Figures 9a and b show the effect of ⁇ -PGA on the viability of B. longum and B. breve in orange juice; and Figures 10a and b show the effect of ⁇ -PGA on the viability of B. longum and
• This experiment describes the production of ⁇ -PGA with 8 different strains of bacteria - B.subtilis natto, B. subtilis ATCC 23856, B. subtilis ATCC 23857, B. subtilis ATCC 23858, B. subtilis ATCC 23859, B. licheniformis 9945a, B. licheniformis NCIMB 1525 and B. licheniformis NCIMB 6816 in shake flasks.
• subtilis EMG53 subtilis EMG53
• three strains of Bacillus licheniformis - B. licheniformis 9945a, B. licheniformis NCIMB 1525 (also known as B. licheniformis 1229) and B. licheniformis NCIMB 6816 (also known as B. licheniformis Glaxo417) were investigated for the production of ⁇ -PGA.
• Tryptone soya agar (TSA), tryptone soya broth (TSB) and one-quarter strength ringer solution were prepared according to the manufacturer's protocol (Lab M, UK).
• the composition of GS medium and Medium E has been given in Table 1 & 2 below.
• the pH of both media was adjusted to 7.2 using 3 M NaOH and 1 M HC1.
• Iron (III) chloride hexahydrate FeCl 3 -6H 2 0 0.2g/L Sigma- Aldrich ®
• the cell suspension was centrifuged at 17000g for 30 minutes (Hermele 2 300K).
• Four volumes of cold 90% (v/v) ethanol was added to the cell free supernatant and incubated at 4°C for 72 h.
• Wet ⁇ -PGA powder was obtained as sediment.
• the sediment was separated from the supernatant by centrifugation at 17000g for 30 mins.
• the obtained polymer was prepared for lyophilisation by dissolving it in 10 ml of deionised water in round bottom flasks. The flasks were then rotated gently on a mixture of 90%> (v/v) ethanol at -20°C and dry ice to freeze the biopolymer in the form of a thin film.
• the frozen biopolymer was then lyophilized to obtain dry ⁇ -PGA powder (Edward Modulo).
• the dried powder was weighed to calculate yield in g/1 and stored in a desiccator for further analysis.
• CFU/ml Colony Forming Units /ml
• Isolated biopolymer was analyzed using Fourier Transformed Infra Red spectroscopy (FTIR) with an Impact 404 Nicolet spectrometer (UK) with KBr pellet in conjunction with OMNIC software.
• FTIR Fourier Transformed Infra Red spectroscopy
• UK Impact 404
• KBr pellet in conjunction with OMNIC software.
• the FTIR spectra of the produced ⁇ -PGA were compared with the spectra of a commercially available ⁇ -PGA sample.
• Aqueous based gel permeation chromatography was used to determine molecular weight (Mw), molecular number (Mn) and polydispersity (Smithers Rapra) using a MZ Hema guard plus 2 x Hema Linear column.
• Mw molecular weight
• Mn molecular number
• Polydispersity Smithers Rapra
• MZ Hema guard plus 2 x Hema Linear column 0.2 M NaN03, 0.01 M NaH2P04 at pH 7 was used as the eluent with a flow rate of 1.0 ml/min at 30°C and an RI detector.
• the data was collected and analyzed using Polymer Laboratories "Cirrus" software.
• GPC system used for this work was calibrated with sodium polyacrylate calibrants obtained from Polymer Laboratories. Nutrient consumption analysis
• Nutrient consumption analysis was performed using High Performance Liquid Spectroscopy (HPLC) with an HP series 1 100 HPLC machine at the University of Reading, U.K.
• HPLC High Performance Liquid Spectroscopy
• a Prevail Organic Acid 5u column with a UV detector was used for analyzing L-glutamic acid and citric acid whereas a Phenomenex carbohydrate column (Rezex RCM - monosaccharide Ca++ - 8%) was used for sucrose and glycerol determination.
• Filtered deionised water was used as the eluent for sucrose analysis.
• 25 mM KH2P04 pH 2.5 adjusted with phosphoric acid was used as the eluent.
• the samples were filtered using 0.45 ⁇ filters and were diluted 10 fold.
• Crystallinity was assessed using powder X-Ray Diffraction (XRD) analysis. Data was collected at room temperature with a Phillips PW1700, 40kV/40mA, CuKa instrument. Statistical analyses
• Figures l and 2 show the growth of all bacterial strains in both GS and E medium. At 0 h, all the strains had a cell count of ⁇ 5-6 CFU/ml. In GS medium, B. subtilis 23858 reached a maximum cell count of log 9.58 at 72 h. In medium E, B. Ucheniformis 1525 reached a maximum cell count of log 8.57 at 72 h. The maximum cell counts for the B. subtilis strains ( ⁇ log 7.5-8.1 CFU/ml) did not reach as high as that of the B. Ucheniformis strains ( ⁇ log 8.5 CFU/ml) in medium E. B. subtilis 23858 & 23859 and B.
• Tables 3 and 4 below show utilization of nutrients by bacterial strains in both GS and E media after 96 h.
• GS medium except B. subtilis 23859 (75.71 %), all strains utilized more than 85% of provided sucrose at the end of 96 h.
• the C source in Medium E i.e. glycerol
• B. subtilis strains consumed more L-glutamic acid than B. /zcheniformis strains.
• B. subtilis natto consumed the most L-glutamic acid at the end of 96 h ( ⁇ 95%>) .
• B. subtilis natto also produced the highest yield of ⁇ -PGA in GS medium ( ⁇ 17.7g/l).
• B. licheniformis strains consumed more L-glutamic acid than B. subtilis strains. This could possibly be reflected in the fact that the B. licheniformis strains produced a slightly higher yield of ⁇ -PGA in medium E than most of the B. subtilis strains at the end of 96 h.
• subtilis subtilis subtilis subtilis lichenlichen23856 23857 23858 23859 natto iformis iformis iformis
• the yield of ⁇ -PGA could not only be dependent on the consumption of exogenous L-glutamic acid, but also on the ability of the bacteria to produce endogenous L-glutamic acid for production of ⁇ -PGA.
• Medium E is known to be most suitable for production of ⁇ -PGA with B. licheniformis 9945a, which is probably the most studied strain for the production of ⁇ -PGA. All B. licheniformis strains produced more ⁇ -PGA in medium E when compared to the B. subtilis strains, except B. subtilis 23859. B. subtilis 23859 consumed more glycerol in medium E than the other B. subtilis strains and this could be the reason why its yield of ⁇ -PGA was slightly better. Since nutrient consumption could be affected by pH of the medium, the yield could also be affected. 3.5 Crystallinity
• Elemental analysis was performed to identify whether the salt or the acid form of ⁇ - PGA was produced.
• ICP-AES analysis breaks down the crude polymer and measures the concentration of individual elements that make up the polymer. Hence impurities, if present, in the sample would also be detected.
• ICP-AES results showed that most of the ⁇ -PGA obtained with cells grown in GS medium was in fact the sodium salt of ⁇ - PGA (Na-y-PGA) with some ⁇ -PGA also in its P, Mg and K salt form, see Figure 6. None of the strains produced the acid form of ⁇ -PGA (H+-y-PGA) in GS medium.
• strains grown in medium E produced considerable amount of H+-y-PGA (37-57%) along with Na-y-PGA, see Figure 7.
• the pH of medium E was adjusted with the help of 3M NaOH.
• licheniformis NCIMB 6816 produced ⁇ -PGA weighing 856500 Da (polydispersity - 1.2) and B. licheniformis 9945a produced ⁇ -PGA weighing 760000 Da (polydispersity - 1 .2) while the other strains produced a lower molecular weight product ( ⁇ ⁇ 3000 Da).
• Table 5 Properties of ⁇ -PGA produced from 8 Bacillus strains in GS medium
• Table 6 Properties of ⁇ -PGA produced from 8 Bacillus strains in medium E.
• the cells are grown in GS medium, they produce a crystalline salt form of ⁇ -PGA. In contrast, when they are grown in Medium E, an amorphous acid form of ⁇ -PGA is produced. Crystallinity and form of ⁇ -PGA seem to be Bacillus strain independent and these properties could be manipulated with the medium of production.
• B. licheniformis 6816 produced a very high molecular weight ⁇ -PGA in both Medium E and GS, but B. subtilis natto produced a high molecular weight product only in GS medium.
• B. licheniformis 9945a produced a high molecular weight polymer in Medium E, but not in GS medium.
• Bacillus subtilis natto was chosen to produce ⁇ -PGA for tests with probiotic bacteria.
• the increased viscosity decreases the volumetric oxygen mass transfer, leading to oxygen limitation.
• the supply of oxygen was maintained above 40% by controlling the agitation speed and air flow rate.
• the cell suspension was centrifuged at 17000g for 30 minutes.
• Four volumes of cold 90% (v/v) ethanol was added to the cell free supernatant and incubated at 4°C for 72 h.
• Wet ⁇ -PGA powder was obtained as sediment.
• the sediment was separated from the supernatant by centrifugation at 17000g for 30 mins.
• the wet crude polymer was then dissolved in water and dialyzed to eliminate impurities lower than 10,000 Da.
• the obtained pure polymer was prepared for lyophilisation in round bottom flasks.
• Bifidobacterium longum Three probiotic bacteria, Bifidobacterium longum, Bifidobacterium breve and Lactobacillus casei were used for the tests.
• the effect of 10%> Na-y-PGA was tested on viability of the bacteria before and after freeze drying.
• Bifidobacteria were inoculated in TPY broth (22 h for B. breve and 16 h for B. longum) and Lactobacillus casei was inoculated in MRS broth (for 48 h) at 37 °C anaerobically.
• the effect of 2.5% Na-y-PGA was tested on viability of the two Bifidobacteria strains when stored in orange juice for 39 days and simulated gastric juice for 4 h.
• bacteria were inoculated in TPY broth (22 h for B. breve and 16 h for B. longum) at 37 °C. The culture was then centrifuged and washed with PBS to obtain cell pellets. Cells were then mixed thoroughly in a 10%> Na-y-PGA solution ( 1 gm Na-y-PGA in 9 ml of deionised water). This mixture was frozen at -80 °C and freeze dried to obtain a dry powder with cells coated with Na-y-PGA.
• Na-y-PGA can be used to noticeably increase the shelf life of the food probiotic product.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Biotechnology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Genetics & Genomics (AREA)
• Microbiology (AREA)
• Food Science & Technology (AREA)
• Polymers & Plastics (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Virology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Medicinal Chemistry (AREA)
• Biomedical Technology (AREA)
• Nutrition Science (AREA)
• Mycology (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Dispersion Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Molecular Biology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Coloring Foods And Improving Nutritive Qualities (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (1)

Family

ID=44882090

Family Applications (1)

Country Status (6)

Cited By (1)

Families Citing this family (5)

Citations (2)

Family Cites Families (7)

• 2011
  • 2011-09-02 GB GBGB1115143.8A patent/GB201115143D0/en not_active Ceased
• 2012
  • 2012-08-31 WO PCT/GB2012/052149 patent/WO2013030596A1/en not_active Ceased
  • 2012-08-31 US US14/342,188 patent/US20140248396A1/en not_active Abandoned
  • 2012-08-31 DK DK12772120.7T patent/DK2751254T3/da active
  • 2012-08-31 JP JP2014527740A patent/JP2014525259A/ja active Pending
  • 2012-08-31 EP EP12772120.7A patent/EP2751254B1/en active Active

Patent Citations (2)

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events