DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-10 are pending (claim set as filed on 03/03/2026). Claims 1-10 are currently under examination and were examined on their merits.
Information Disclosure Statement
The Information Disclosure Statement (IDS) filed on 03/05/2026 has been received and considered.
Withdrawn Objections/Rejections
The objection to the Specification as set forth in the previous Office action is withdrawn in light of Applicant’s amendment filed on 03/03/2026.
The rejection of claim 2 under 35 U.S.C. 112(a) as set forth in the previous Office action is withdrawn in light of the provided Statement of Availability filed on 03/03/2026.
Claim Rejections - 35 USC § 112 (b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-10 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1, line 7, recites “the incubated media” which is indefinite for lacking antecedent basis because ‘media’ is not recited within the claim. One of ordinary skill in the art would not be able to determine the metes and bounds of the claim, and thus, could not clearly determine how to avoid infringement of claim 1.
In the interest of compact prosecution, claim 1 is interpreted to the broadest embodiment claimed.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4 and 8-10 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Kirtel et al. (“Fructanogenic traits in halotolerant Bacillus licheniformis OK12 and their predicted functional significance”, published on 09/01/2021, Journal of Applied Microbiology, Vol. 131, pages 1391-1404), hereinafter ‘Kirtel’, in view of Kekez et al. (“High Levan Production by Bacillus licheniformis NS032 Using Ammonium Chloride as the Sole Nitrogen Source”, published on 01/16/2015, Appl Biochem Biotechnol, Vol. 175, pages 3068-3083), hereinafter ‘Kekez’, in view of Kumar et al. (“Characterization of an extracellular biopolymer flocculant from a haloalkalophilic Bacillus isolate”, published in 2004, World Journal of Microbiology & Biotechnology, Vol. 20, pages 837-843), hereinafter ‘Kumar’, and in view of Dos Santos et al. (“An improvement of surfactin production by B. subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor”, published on 07/14/2016, Bioresource Technology, Vol. 218 (2016), pages 944–952), hereinafter ‘Dos Santos’.
Kirtel’s general disclosure relates to isolating a novel bacterial source of fructan from a saltern and analysis of its genome (see entire document, including abstract).
Regarding claims 1-4, and 10, pertaining to a process for production of a levan, Kirtel teaches a process for production of a levan (“Exopolysaccharide accumulated in the presence of sucrose by isolate OK12 was purified and chemically characterized via HPLC, FT-IR and NMR, which revealed that it was a levan-type fructan”; see abstract), comprising the steps of:
preparing a medium comprising sucrose (“Production and purification of EPS … For EPS production with B. licheniformis OK12, medium with the following composition was used (…): sucrose, 100 g l-1; NaCl, 100 g l-1; peptone from meat, 1 g l-1; yeast extract, 0.6 g l-1; K2HPO4.3H2O, 3 g l-1; KH2PO4, 3 g l-1; MgSO4.7H2O, 0.2 g l-1; FeSO4.7H2O, 0.001 g l-1”; page 1393, right column, paragraph 2);
inoculating a marine Bacillus sp. in the medium and incubating at 37 °C (“a halotolerant B. licheniformis strain was isolated from hypersaline Camalti Saltern in Western Turkey. Bacillus licheniformis OK12 was shown to be able to accumulate levan in saline medium”, “Camalti Saltern is a coastal solar saltern in Turkey where seawater is pumped into evaporation ponds to obtain salt via evaporation”, “A loopful of cells from nutrient agar plates grown overnight at 37°C were used to inoculate 50 ml of media in each 250 ml Erlenmeyer flasks, which were then left to incubation at 37°C”; page 1393, right column, paragraph 2; page 1401, left column, paragraph 3; page 1401, right column, paragraph 2; see Fig. 5).
centrifuging the incubated media to obtain a cell-free supernatant (“To purify EPS, samples withdrawn were centrifuged at 8000 g for 15 min to pellet the cells.”; page 1393, right column, paragraph 2).
precipitating the cell free supernatant using an absolute ethanol solvent to obtain precipitated exopolysaccharide (EPS) (“Supernatants were added two volumes of ethanol to precipitate polymers”; page 1393, right column, paragraph 2; it is noted by the Examiner that Kirtel’s ethanol reads on absolute ethanol since no percentage is indicated);
separating the precipitated EPS in absolute ethanol by centrifugation (“Supernatants were added two volumes of ethanol to precipitate polymers overnight at -20°C, followed by centrifugation 7000 g for 10 min”; page 1393, right column, paragraph 2).
dissolving EPS of step (e) in a solvent to make a solution containing levan (“Polymer pellets were then re-dissolved in warm dH2O”; page 1393, right column, paragraph 2). It is noted that Kirtel teaches that “[e]xopolysaccharide accumulated in the presence of sucrose by isolate OK12 was purified and chemically characterized via HPLC, FT-IR and NMR, which revealed that it was a levan-type fructan (β-2,6 linked homopolymer of fructose)” (see abstract), thereby indicating that Kirtel’s redissolved EPS pellets obtained from strain OK12 contain levan.
Regarding claim 2, pertaining to marine Bacillus sp. SGD-03 with accession no. MCC 0243, Kirtel teaches a marine Bacillus sp. strain that produces levan in the presence of sucrose (“a halotolerant B. licheniformis strain was isolated from hypersaline Camalti Saltern in Western Turkey. Bacillus licheniformis OK12 was shown to be able to accumulate levan in saline medium”, “Camalti Saltern is a coastal solar saltern in Turkey where seawater is pumped into evaporation ponds to obtain salt via evaporation”, “Exopolysaccharide accumulated in the presence of sucrose by isolate OK12”; page 1401, left column, paragraph 3; page 1401, right column, paragraph 2; see abstract), wherein the Bacillus sp. strain is a B. licheniformis strain (“The isolate was taxonomically classified as Bacillus licheniformis OK12”; see abstract). Kirtel teaches wherein “B. licheniformis OK12 was found to be a halotolerant since it grew faster in the absence of NaCl” (page 1394, right column, paragraph 3).
The Examiner notes that the instantly recited assigned strain number SGD-03 and accession number MCC 0243 of the claimed strain do not further characterize the instant strain. The claimed strain was isolated from marine samples (specification paragraphs [0079]-[0080]), has the ability to produce levan in the presence of sucrose (specification, paragraphs [0002], [0056]), and was identified as a Bacillus licheniformis strain (specification, paragraph [0080]). The specification further discloses: “It was observed that the Bacillus sp. SGD-03 is growing well in salt conditions. An inverse relationship of growth to salt concentration was observed. As the concentration of salt increases, the growth of an organism decreases.” (paragraph [0080]).
Regarding claim 9, pertaining to dissolving EPS, Kirtel teaches wherein the EPS is dissolved using solvent H2O (“Polymer pellets were then re-dissolved in warm dH2O”; page 1393, right column, paragraph 2).
In addition, Kirtel teaches the use of ultrapure water as the mobile phase for chemical characterization of EPS using HPLC (page 1394, left column, paragraph 1).
Regrading claims 1, 3-4, and 10, claim 1 recites a levan essentially free of a contaminating polysaccharide obtained from the process recited in claim 1, claim 3 recites the yield and purity of levan obtained in the process of claim 1, claim 4 recites the molecular weight of levan obtained in the process of claim 1, and claim 10 recites the fiber diameter of levan obtained in the process of claim 1. It is noted that the recited purities (claims 1 and 3), yield (claim 3), molecular weight (claim 4), and fiber diameter (claim 10) of levan obtained in claim 1 naturally result from performing the instantly claimed method steps, and therefore, the recited yield, purity, molecular weight, and fiber diameter of levan, are inherent to the method.
Kirtel does not teach
preparing and optimizing a medium comprising 4% of sucrose using a microtiter plate in a micro-bioreactor (instant claim 1),
incubating at 28 °C for 18-24 hours (instant claim 1),
centrifuging the incubated media at 10000 rpm (instant claim 1),
wherein precipitated EPS is washed with a solvent (instant claim 1),
lyophilizing the solution containing levan (instant claim 1),
wherein the solvent for precipitation in step (d) is three-volume of pre-chilled absolute ethanol (4°C) and the solvent for washing in step e) is 70% ethanol (instant claim 8).
wherein the EPS is dissolved using ultrapure H2O (instant claim 9)
Kekez’s general disclosure relates to “levan production by Bacillus licheniformis NS032 isolated from a petroleum sludge sample” (see entire document, including abstract).
Regarding claim 1, pertaining to optimizing a medium in step (a), Kekez discloses: “Optimization of Levan Production by the OFAT Method … For determination of the effect of substrate concentration, the concentration of sucrose in BM was varied in the range of 60–500 g/L and for effect of N source, the BE/YE mixture from BM was replaced with the following components: YE, BE, peptone, ammonium sulfate, ammonium chloride and sodium nitrate” (page 3070, paragraph 7; note OFAT, one-factor-at-a-time; YE, yeast extract; BE, beef extract; BM, basal medium; page 3069, paragraph 4, and page 3070, paragraph 3).
Pertaining to the incubation temperature in step (b), Kekez teaches incubating of a Bacillus licheniformis strain at 28 °C for 24 hours (see Fig. 1(a)), and that “[g]enerally, optimal temperature for levan production for most microbial levan producers is below than 30 °C, while the maximum growth rate of these strains occurs at 35–37 °C […]. This is in accordance with the reports that bacteria favor lower temperatures for EPS production compared with their optimum temperature for cell growth” (page 3072, paragraph 6).
Pertaining to step (c), Kekez teaches centrifuging incubated medium at 10000 rpm (“bacterial biomass was separated by centrifugation at 10,000 rpm”; page 3070, paragraph 5).
Pertaining to step (g), Kekez teaches lyophilizing the solution containing levan (“the sediment was suspended in distilled water and dialyzed against running tap water for at least 3 days. The dialysate was lyophilized to afford crude EPS”; page 3070, paragraph 5).
It is noted that Kekez teaches wherein the EPS contains levan (“The 13C NMR spectrum (…) represents the final confirmation of a levan type structure of EPS”; page 3072, paragraph 2), indicating that the solution in steps (g) contains levan.
Regarding claims 1 and 8, pertaining to step (d), Kekez teaches “the supernatant was mixed with two volumes of ice cold ethanol, stirred vigorously and kept at 4 °C overnight” (page 3070, paragraph 5). It is noted by the Examiner that Kekez’s ethanol reads on ‘absolute ethanol’ since no ethanol percentage is indicated.
Additionally, Kekez discloses: “In the food industry, microbial levan is used as a source for production of fructooligosacharides (FOS) and hence is also used as a prebiotic, while other uses are as a stabilizer, emulsifier, flavor or fragrance carrier, and encapsulating agent. Of special interest to the pharmaceutical and cosmetics industries are its antioxidant, antitumor, immunomodulating, cholesterol-lowering as well as skin-moisturizing and irritation-alleviating effects” (page 3068, paragraph 1 – page 3069, paragraph 1).
Kekez further discloses: “In vitro or industrial production of EPS is highly influenced by the concentration of nutrients in fermentation medium used as well as cultivation conditions. For the development of any industrial-scale process, it is necessary to find optimal conditions specific for the particular producer microorganism” (page 3069, paragraph 4). Kekez adds wherein “[s]ucrose concentration, temperature, aeration, nitrogen source type, pH of medium and mineral requirements have been identified by various authors as factors influencing the polysaccharide yield […]. However, each microbial strain shows different responses toward these environmental factors.” (page 3072, paragraph 3).
Kekez teaches wherein “changes in fermentation conditions also affect the molecular weight of levan. Bacteria often produce two types of levan of low and high molecular weight simultaneously; sucrose concentration is the main factor determining the final ratio of high/low molecular weight levan” (page 3074, paragraph 4).
Kumar’s general disclosure relates to “[a] biopolymer flocculant, produced by a haloalkalophilic Bacillus sp. I-471” (see entire document, including abstract).
Regarding claims 1 and 8, pertaining to step (e), Kumar teaches washing precipitated polysaccharide with a solvent (“The precipitated polysaccharide was recovered by centrifugation (…), washed with 70% ethanol and vacuum dried”; page 838, right column, paragraph 2) (instant claim 1), wherein the solvent for washing in step (e) is 70 % ethanol (“washed with 70% ethanol”; page 838, right column, paragraph 2) (instant claim 8).
Dos Santos’s general disclosure relates to enhancing “surfactin yield of a
constitutive strain of B. subtilis by optimizing medium composition in microbioreactors” (see entire document, including page 945, right column, paragraph 3).
Regarding claim 1, pertaining to using a microtiter plate in a microbioreactor in step (a), Dos Santos teaches “[b]atch fermentations in microbioreactors” wherein “[c]ultures for the optimization of medium composition were performed in 48-well microtiter Flowerplates (…) in a BioLector® device (…) which allows an online monitoring of biomass, pH and dissolved oxygen tension, non-invasively and separately in each well.” (page 946, left column, paragraph 2), and further discloses wherein “a central composite design was performed in microplate bioreactors using a BioLector®, in which variations of these impressive parameters, glucose, glutamic acid and tryptophan concentrations were selected for optimization of product-biomass yield (YP/X).” (see abstract).
While Kirtel does not teach incubating at 28 °C for 18-24 hours (instant claim 1), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined modified Kirtel’s method with Kekez’s teachings on levan production optimization and on incubating at 28 °C for 24 hours, to have created a process for production of levan wherein the inoculated medium is incubated at 28 °C for 24 hours. One would have been motivated to do so for further increasing the yield of produced levan and/or of a levan with a specific molecular weight. A skilled artisan would have reasonably expected success since individual microbial strains respond differently to process parameters (see Kekez, page 3072, paragraph 3), and since optimal levan production is generally achieved below 30 °C (see Kekez, page 3072, paragraph 6).
While modified Kirtel does not teach wherein a medium comprises 4% sucrose, the recited concentration would have been within the realm of routine experimentation, since Kirtel teaches a sucrose concentration of 10 % (page 1393, right column, paragraph 2), and Kekez teaches sucrose concentrations of 6-50% (see Fig. 1(c)). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to determine the optimal sucrose concentration for increased yield and/or for producing levan with a specific molecular weight. Further one would have expected success, since Kekez discloses wherein “[s]ucrose concentration, temperature, aeration, nitrogen source type, pH of medium and mineral requirements have been identified by various authors as factors influencing the polysaccharide yield” (page 3072, paragraph 3), and wherein “sucrose concentration is the main factor determining the final ratio of high/low molecular weight levan” (page 3074, paragraph 4). Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA1955). See MPEP § 2144.05 part II A.
While modified Kirtel does not teach centrifuging the incubated media at 10000 rpm (instant claim 1), wherein precipitated EPS is washed with a solvent (instant claim 1), wherein the solvent for precipitation in step (d) is pre-chilled absolute ethanol (4°C) and the solvent for washing in step e) is 70% ethanol (instant claim 8), dissolving the EPS using ultrapure H2O (instant claim 9), and lyophilizing the solution containing levan (instant claim 1), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have combined modified Kirtel’s process with Kirtel’s teachings on ultrapure water, Kekez’s and Kumar’s teachings on polysaccharide isolation from a culture, to have created a process for levan production comprising centrifuging the incubated media at 10000 rpm, wherein the solvent for precipitation in step(d) is pre-chilled absolute ethanol, wherein precipitated EPS is washed with a solvent, wherein the solvent for washing in step e) is 70% ethanol, wherein the EPS is dissolved using ultrapure H2O, and wherein the solution containing levan is lyophilized. One would have been motivated to do so to increase the yield and purity of the obtained levan. A skilled artisan would have reasonably expected success in combining Kirtel’s teachings with Kekez’s, and Kumar’s teachings since all teachings are directed to isolation of polysaccharides produced by Bacillus cultures.
While modified Kirtel does not teach wherein three volumes of ethanol are used for EPS precipitation and wherein the pre-chilled ethanol has a temperature of 4°C (instant claim 8), the instantly recited ethanol amount and temperature would be within the realm of routine experimentation since Kirtel teaches two volumes of ethanol for EPS precipitation (page 1393, right column, paragraph 2) and Kekez teaches precipitation of EPS at 4°C (page 3070, paragraph 5). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have determined the optimal ethanol amount and temperature for maximal yield and purity of the obtained EPS. Further, Kekez teaches EPS analysis for further characterization of the obtained EPS (page 3070, paragraph 6), and therefore, manipulation of the EPS preparation conditions would be within the purview of an artisan. Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA1955). See MPEP § 2144.05 part II A.
While modified Kirtel does not teach wherein preparing and optimizing a medium comprising 4% sucrose using a microtiter plate in a micro-bioreactor (instant claim 1), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined modified Kirtel’s process with Dos Santos’s teachings on optimizing medium composition in microbioreactors, in order to have developed a process comprising preparing and optimizing a medium comprising 4% sucrose using a microtiter plate in a micro-bioreactor. One would have been motivated to do so in order to test multiple medium compositions with different medium components and concentrations simultaneously, for identifying an optimized medium allowing for maximal production of a levan and/or of a levan with a desired molecular weight. A skilled artisan would have reasonably expected success in combining modified Kirtel’s teachings with Dos Santo’s teachings since both references are directed to optimizing medium compositions.
Modified Kirtel does not expressly teach wherein the marine Bacillus sp. strain is
SGD-03 having accession no. MCC 0243 (instant claim 2).
However, based on Kirtel’s teachings, it is highly likely that Kirtel's strain and
Applicant's strain are the same strain since both strains are marine Bacillus licheniformis strains and share the ability to produce levan in the presence of sucrose. Still, if there should be a slight variation between Kirtel's strain and the instantly recited strain, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have used the instant strain in lieu of Kirtel’s strain since both strains, Kirtel's strain and the instant strain, are Bacillus licheniformis strains of marine origin, and share the ability to produce levan in the presence of sucrose.
Regarding claims 1, 3-4, and 10, the combination of Kirtel’s, Kekez’s, Dos Santos’s, and Kumar’s teachings would implicitly result in the same or similar yield, purity, molecular weight, and fiber diameter. Since Kirtel teaches the use of a marine Bacillus sp. for producing a levan, Kekez teaches medium optimization for improved levan production and impact of fermentation conditions including sucrose concentrations on the yield and molecular weight of levan, Dos Santos teaches improved medium optimization using a microbioreactor, and Kirtel, Kekez, and Kumar teach isolation of Bacillus derived polysaccharides using ethanol precipitation, it is highly expected that the method taught by Kirtel, in view of Kekez, Dos Santos, and Kumar, would result in the same or similar, i.e. obvious, yield, purity, molecular weight, and fiber diameter as described in claims 1, 3-4, and 10.
Claims 1 and 5-6 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Kirtel et al. (“Fructanogenic traits in halotolerant Bacillus licheniformis OK12 and their predicted functional significance”, published on 09/01/2021, Journal of Applied Microbiology, Vol. 131, pages 1391-1404), hereinafter ‘Kirtel’, in view of Kekez et al. (“High Levan Production by Bacillus licheniformis NS032 Using Ammonium Chloride as the Sole Nitrogen Source”, published on 01/16/2015, Appl Biochem Biotechnol, Vol. 175, pages 3068-3083), hereinafter ‘Kekez’, in view of Kumar et al. (“Characterization of an extracellular biopolymer flocculant from a haloalkalophilic Bacillus isolate”, published in 2004, World Journal of Microbiology & Biotechnology, Vol. 20, pages 837–843), hereinafter ‘Kumar’, in view of Dos Santos et al. (“An improvement of surfactin production by B. subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor”, published on 07/14/2016, Bioresource Technology, Vol. 218 (2016), pages 944-952), hereinafter ‘Dos Santos’, and in view of Tanaka et al. (“Synthesis of Levan by Levansucrase”, published in 1979, J. Biochem., Vol. 85, pages 287-293), hereinafter ‘Tanaka’.
Kirtel’s, Kekez’s, Kumar’s, and Dos Santos’ teachings have been set forth above.
As discussed above, modified Kirtel teaches medium comprising 4% of sucrose.
Regarding claims 5 and 6, pertaining to the medium, Kirtel discloses growing B. licheniformis OK12 and producing EPS in a medium comprising 0.1 % peptone, and 10 % NaCl, (“For EPS production with B. licheniformis OK12, medium with the following composition was used (…): sucrose, 100 g l-1; NaCl, 100 g l-1; peptone from meat, 1 g l-1; yeast extract, 0.6 g l-1; K2HPO4.3H2O, 3 g l-1; KH2PO4, 3 g l-1; MgSO4.7H2O, 0.2 g l-1; FeSO4.7H2O, 0.001 g l-1”; page 1393, right column, paragraph 2). Additionally, Kirtel teaches wherein “B. licheniformis OK12 was found to be a halotolerant since it grew faster in the absence of NaCl” (page 1394, right column, paragraph 3).
Regarding claims 5 and 6, pertaining to the medium, Kekez discloses a medium for levan production comprising 0.1 % beef extract, 0.1 % NaCl, and water (“For levan production, the basal medium (BM) (100 g sucrose, 1 g beef extract (BE), 0.6 g yeast extract (YE), 3 g K2HPO4·3H2O, 3 g KH2PO4, 1 g NaCl, 0.2 g MgSO4·7H2O, 0.001 g FeSO4·7H2O in 1000 mL of water”; page 3070, paragraph 3), and further teaches testing the effect of nitrogen sources beef extract and peptone on levan production (“Optimization of Levan Production by the OFAT Method …for effect of N source, the BE/YE mixture from BM was replaced with the following components: YE, BE, peptone,”; page 3070, paragraph 7; note, YE, yeast extract, BE, beef extract; BM, basal medium; page 3070, paragraph 3).
Additionally, Kekez discloses: “In vitro or industrial production of EPS is highly influenced by the concentration of nutrients in fermentation medium used as well as cultivation conditions. For the development of any industrial-scale process, it is necessary to find optimal conditions specific for the particular producer microorganism” (page 3069, paragraph 4), and teaches that “[s]ucrose concentration, temperature, aeration, nitrogen source type, pH of medium and mineral requirements have been identified by various authors as factors influencing the polysaccharide yield […]. However, each microbial strain shows different responses toward these environmental factors.” (page 3072, paragraph 3).
Kekez further discloses wherein “changes in fermentation conditions also affect the molecular weight of levan. Bacteria often produce two types of levan of low and high molecular weight simultaneously; sucrose concentration is the main factor determining the final ratio of high/low molecular weight levan” (page 3074, paragraph 4).
Modified Kirtel does not teach wherein the medium is a composition of polysaccharides, peptone, beef extract and salt (instant claim 5), wherein the medium comprises 0.4 % of peptone, 0.4 % of beef extract, 0.5 % of NaCl, and water (instant claim 6).
Tanaka’s general disclosure relates to “the addition of levan as a so-called "acceptor"” that “accelerated the rate of polymerization of levan catalyzed by levansucrase” (see entire document, including abstract).
Regarding claim 5, pertaining to the medium, Tanaka teaches that “ [t]he
synthesis of the high molecular weight levan was significantly increased by the addition of either high or low molecular weight levan (page 291, left column, paragraph 1). Tanaka further discloses wherein NaCl concentration affects the stimulative effect of levan on levan synthesis (“the stimulative effect of levan decreased with increasing concentration of phosphate buffer, and became negligibly small over a certain concentration of the buffer. A similar result was obtained for the enzyme reaction performed with various concentrations of NaCl instead of phosphate”; page 290, left column, paragraph 2 – right column, paragraph 1).
While modified Kirtel does not teach wherein the medium is a composition of polysaccharides, peptone, beef extract and salt (instant claim 5), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined modified Kirtel’s process with Kirtel’s and Kekez’s teachings on medium composition, with Kekez’s teachings on the effects of nitrogen sources on levan production, and further with Tanaka’s teachings on the effect of added levan on levan biosynthesis, to have created a medium wherein the medium is a composition of levan polysaccharides, peptone, beef extract, NaCl, and water. One would have been motivated to do so in order to increase production of levan and/or of a levan with a specific molecular weight. A skilled artisan would have reasonably expected success since Kekez teaches that “[f]or the development of any industrial-scale process, it is necessary to find optimal conditions specific for the particular producer microorganism” (page 3069, paragraph 4).
While modified Kirtel does not teach wherein the medium comprises 0.4 % of peptone, 0.4 % of beef extract, and 0.5 % of NaCl (instant claim 6), the recited concentrations would be within the realm of routine experimentation since Kirtel teaches 0.1 % peptone (page 1393, right column, paragraph 2), and Kekez teaches 0.1% beef extract and 0.1 % NaCl (page 3070, paragraph 3; see Fig 1(c)). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have determined the optimal medium composition for maximizing yield of the produced levan and/or of produced levan with a specific molecular weight. Further, one would expect success since modified Kirtel’s teachings are directed to analyzing levan production of a Bacillus culture, and therefore, manipulation of the culturing conditions including medium composition based on the teachings of the references would be within the purview of an artisan. Generally, differences in concentration will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA1955). See MPEP § 2144.05 part II A.
Claims 1 and 7 are newly rejected as necessitated by amendment under 35 U.S.C. 103 as being unpatentable over Kirtel et al. (“Fructanogenic traits in halotolerant Bacillus licheniformis OK12 and their predicted functional significance”, published on 09/01/2021, Journal of Applied Microbiology, Vol. 131, pages 1391--1404), hereinafter ‘Kirtel’, in view of Kekez et al. (“High Levan Production by Bacillus licheniformis NS032 Using Ammonium Chloride as the Sole Nitrogen Source”, published on 01/16/2015, Appl Biochem Biotechnol, Vol. 175, pages 3068–3083), hereinafter ‘Kekez’, in view of Kumar et al. (“Characterization of an extracellular biopolymer flocculant from a haloalkalophilic Bacillus isolate”, published in 2004, World Journal of Microbiology & Biotechnology, Vol. 20, pages 837–843), hereinafter ‘Kumar’, in view of Dos Santos et al. (“An improvement of surfactin production by B. subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor”, published on 07/14/2016, Bioresource Technology, Vol. 218 (2016), pages 944–952), hereinafter ‘Dos Santos’, and in view of Marajan et al. (“The Effect of Incubation Time, Temperature and pH Variations on the Surface Tension of Biosurfactant Produced by Bacillus spp.”, published on 10/05/2018, AIP Conf. Proc. 2020, 020047 (2018), pages 1-7), hereinafter ‘Marajan’.
Kirtel’s, Kekez’s, Kumar’s, and Dos Santos’ teachings have been set forth above.
Regarding claim 7, pertaining to the inoculum, Kirtel teaches that “[a] loopful of cells from nutrient agar plates grown overnight at 37°C were used to inoculate 50 ml of media” (page 1393, right column, paragraph 2).
Modified Kirtel does not teach wherein the marine Bacillus sp. is grown in a rotatory shaker at 150 rpm for 12-18h to prepare the inoculum (instant claim 7).
Marajan’s general disclosure relates to “the effect of incubation time, temperature and pH variations on the surface tension of biosurfactant produced by Bacillus subtilis and Bacillus tequilensis” (see entire document, including abstract).
Regarding claim 7, pertaining to an inoculum, Marajan teaches wherein a Bacillus strain is grown for 12 hrs in a rotatory shaker at 150 rpm to prepare an inoculum (“Biosurfactant-producing bacteria culture was prepared by inoculating a loopful of stock solution into 25 mL of nutrient broth contained within a universal bottle. This was incubated at 37 °C and 150 rpm for 12 hours. An aliquot (1.0 mL) of inoculums was transferred to a 50 mL centrifuge tube containing sterile nutrient broth”; page 3, paragraph 2).
While modified Kirtel does not teach wherein the marine Bacillus sp. is grown in a rotatory shaker at 150 rpm for 12-18h to prepare the inoculum (instant claim 7), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention, to have combined modified Kirtel’s process for production of a levan with Marajan’s teachings on preparing an inoculum, to have created a process for production of a levan wherein the marine Bacillus sp. is grown in a rotatory shaker at 150 rpm for 12h to prepare the inoculum. One would have been motivated to do so in order to provide a superior inoculum of the marine Bacillus for improved production of levan in the inoculated medium. A skilled artisan would have reasonably expected success since modified Kirtel’s and Marajan’s teachings are both directed to Bacillus production cultures.
Response to Arguments
Applicant has traversed the previous rejections of claims 1-10 under 35 U.S.C. 103 in the reply filed on 03/03/2026 (remarks, pages 7-10). Applicant's arguments filed have been fully considered but they are not persuasive.
In response to Applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, regarding claims 1-4 and 8-10, Kirtel provides a method for producing levan using a marine Bacillus. Kekez teaches culturing conditions for levan production and provides motivation for optimizing the conditions including sucrose concentration. Kekez further teaches centrifugation rpm, pre-chilled ethanol for EPS precipitation, and levan lyophilization. Kumar provides a wash step with 70% ethanol. Dos Santos teaches a micro-bioreactor using microtiter plates for optimizing culturing conditions. Regarding claims 5-6, Kumar and Kekez provide medium components, and Tanaka teaches on the effect of added levan on levan synthesis. Regarding claim 7, Marajan provides culturing conditions for preparing a Bacillus inoculum.
Applicant states that Dos Santos is non-analogous art (remarks, page 8-9), The Examiner responds that it has been held that a prior art reference must either be in the field of the inventor’s endeavor or, if not, then be reasonably pertinent to the particular problem with which the inventor was concerned, in order to be relied upon as a basis for rejection of the claimed invention. See In re Oetiker, 977 F.2d 1443, 24 USPQ2d 1443 (Fed. Cir. 1992). In this case, the inventor was concerned with optimizing a medium for culturing a Bacillus strain (see claim 1, lines 3-6). As discussed above, Dos Santos teaches a micro-bioreactor using microtiter plates for optimizing bacterial culturing conditions. One would have been motivated to use Dos Santo’s microbioreactor set-up in order to test multiple medium compositions with different medium components and concentrations simultaneously, for identifying an optimized medium allowing for maximal production of a levan and/or of a levan with a desired molecular weight.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Correspondence Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SANDRA ZINGARELLI whose telephone number is (703)756-1799. The examiner can normally be reached M-F 9-5.
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/SANDRA ZINGARELLI/ Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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