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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 08/03/2026 has been entered.
Priority
This application is a 371 of PCT /J P2021/043705 filed 11/29/2021. Applicant's claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Acknowledgment is made of applicant's claim for foreign priority under 35 U.S.C. 119 (a)-(d) based on JP 2020-198420 filed 11/30/2020. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 06/18/2026 complies with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Status of the Claims
Claims 1 and 6 are amended. Claims 2-5 are cancelled.
Claims 1 and 6-9 are pending (claim set filed 08/03/2026) and are examined on the merits herein.
Withdrawal of Rejections
The response and amendment filed on 08/03/2026 are acknowledged. All of the amendment and arguments have been thoroughly reviewed and considered.
For the purposes of clarity of the record, the reasons for the Examiner's withdrawal and/or maintaining if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner's response to arguments section.
Claim Rejections - 35 USC § 103
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 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. 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, 6, 8 and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Nishino (Nishino et al. Heliyon, 2018, e00597, 1-8 on record in IDS) in view of Chung (US 20180333441 A1) as evidenced by Ba (WO 2018130630 A1), Miller (Miller et al. Biochemistry, 2001, 40, 14215-14223), Kucernak (Kucernak et al. ACS Energy Lett., 2024, 9, 3939-3946) and Millipore Sigma (Millipore Sigma, 1x Phosphate-Buffered Saline (PBS) [retrieved on 03/26/2026]. Retrieved from the Internet: <1X Phosphate-Buffered Saline (PBS) Recipe Calculator>).
Regarding claims 1 and 6, Nishino teaches separation of viable lactic acid bacteria from fermented milk (Abstract). Nishino describes diluting specimen of the fermented milk with buffer, phosphate-buffered saline (PBS, pH 6.8) in the ratio of 1/10 (1 ml of sample plus 9 ml of buffer) (p. 3, 2nd paragraph). The diluted specimen is subjected to density gradient centrifugation to pellet the separated cells which are then resuspended in PBS (p. 3, 2nd paragraph). The method does not include contacting the microorganisms with a polymer-degrading enzyme.
Thus, Nishino teaches the claimed steps of dilution of the specimen containing fermented milk with a buffer, centrifugation and collecting microorganism. Centrifugation in Nishino teaching includes laying the diluted specimen on top of Percoll gradient, however, since the claimed method has an open language with transitional phrase "comprising", it does not exclude additional steps. Nishino does not teach the protein concentration after dilution. However, the fermented milk product typically contains protein in a level of between 2.0% and 3.5% by weight as evidenced by Ba (p. 16, lines 26-27). Additionally, Ba mentions that fermented milk product may also be low protein product with a protein level of between 1.0% and 2.0%. Nishino teaches dilution of the specimen 10 times (p. 3, 2nd paragraph). Considering the typical protein level of Ba teaching, the 10-fold dilution of the specimen will provide protein level of 0.2% to 0.35% and protein concentration of 2 mg/ml to 3.5 mg/ml that reads on claim 1 limitation. However, if Nishino sample has protein concentration different from disclosed by Ba, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to optimize the protein dilution rate. One would have been motivated to do that with reasonable expectation of success to increase the solubilization of proteins of fermented milk specimen and prevent aggregation to achieve efficient separation of microorganisms.
Nishino does not teach buffer with concentration of 25 mM to 1000 mM and does not teach washing of the centrifugation pellet prior to collection of the separated microorganism.
Chung teaches pharmaceutical and food probiotic compositions for alcohol degradation (paragraphs 0013, 0015). Chung describes collection of bacteria from the bacterial culture by centrifugation followed by washing bacterial pellets three times with 100 mM potassium phosphate buffer at pH 7.4 to remove impurities and recover the bacterial cells (paragraph 0069). The obtained bacteria were shown by Chung to degrade alcohol and acetaldehyde (Example 1 and 2, paragraphs 0072, 0077) and hence are viable.
The ionic strength of 100 mM potassium phosphate buffer at pH 7.4 used by Chung is 0.19 as evidenced by Miller (p. 14220, left column, last paragraph). The ionic strength of standard 1x PBS buffer used by Nishino is 0.156 M as evidenced by Kucernak (p. 3939, left column, 2nd paragraph). The PBS buffer includes potassium phosphate as evidenced by Millipore Sigma.
First, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add step of washing the bacterial pellet as described by Chung to bacterial pellet obtained after centrifugation during the method of microorganism recovery from the fermented milk based on Nishino teaching. One would have been motivated to do so to remove impurities and components of the fermented milk other than bacteria. A skilled artisan would have reasonably expected success in that combination since both Nishino and Chung describe methods involving recovery of viable bacteria from the specimen.
Second, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that the same buffer, i.e. 100 mM potassium phosphate buffer at pH 7.4 taught by Chung can be used as dilution buffer and washing buffer for method of collecting microorganisms from the fermented milk taught by Nishino. One would have been motivated to suggest that with reasonable expectation of success since PBS used by Nishino and 100 mM potassium phosphate buffer used by Chung have potassium phosphate, have neutral pH and similar ionic strength, i.e. 0.156 for PBS as evidenced by Kucernak and 0.19 for 100 mM potassium phosphate buffer at pH 7.4 as evidenced by Miller. Additionally, substitution of PBS in Nishino teaching will allow to avoid buffer exchange for washing step after centrifugation.
Thus, Nishino and Chung teachings as evidenced by Ba, Miller, Kucernak and Millipore Sigma render claims 1 and 6 obvious.
Regarding claim 8, Nishino teaches collected lactic acid bacteria (LAB) to be viable: "The separated LAB actively proliferated, and their colony-forming ability was nearly equal to that of nontreated LAB." (p. 5, 1st paragraph). Thus, Nishino and Chung teachings as evidenced by Ba, Miller, Kucernak and Millipore render claim 8 obvious.
Regarding claim 9, Nishino teaches separating microorganisms from several specimen of commercially fermented milk, including apple yogurt containing juice and acidifier and therefore representing acidic beverage (p. 5, Table 2). Thus, Nishino and Chung teachings as evidenced by Ba, Miller, Kucernak and Millipore render claim 9 obvious.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Nishino (Nishino et al. Heliyon, 2018, e00597, 1-8 on record in IDS) in view of Chung (US 20180333441 A1) as evidenced by Ba (WO 2018130630 A1), Miller (Miller et al. Biochemistry, 2001, 40, 14215-14223), Kucernak (Kucernak et al. ACS Energy Lett., 2024, 9, 3939-3946) and Millipore Sigma (Millipore Sigma, 1x Phosphate-Buffered Saline (PBS) [retrieved on 03/26/2026]. Retrieved from the Internet: <1X Phosphate-Buffered Saline (PBS) Recipe Calculator>) as applied to claim 1 above, and further in view of Dewan (Dewan and Tamang Antonie van Leeuwenhoek, 2007, 92, 343-352).
Teaching of Nishino and Chung have been set forth above.
Nishino and Chung do not teach the recited selected treatments following dilution step.
Regarding claim 7, Dewan teaches isolation of lactic acid bacteria from the Himalayan ethnic fermented milk products (Abstract). Dewan describes that samples are diluted with saline and homogenized in a stomacher lab-blender prior to further culturing and isolation of bacterial strains (p. 345, left column, 2nd paragraph). The obtained bacteria were identified and characterized (Abstract) and most of them caused coagulation of milk with significant pH drop and hence were alive and active (p. 347, right column, 1st paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add step of homogenization of a fermented milk sample in a stomacher described by Dewan to the method of microorganism recovery from the fermented milk based on Nishino and Chung teachings and include this step after diluting the sample and prior to centrifugation. One would have been motivated to do so to increase solubilization of ingredients of fermented milk product such as casein protein for more efficient separation of microorganisms and because Dewan produced alive and active bacterial strains after application of homogenization step. A skilled artisan would have reasonably expected success in that combination since both Nishino and Dewan teach isolation of microorganisms from the fermented milk specimen. Thus, Nishino, Chung and Dewan teachings as evidenced by Ba, Miller, Kucernak and Millipore render claim 7 obvious.
Response to Arguments
Applicant's arguments filed 08/03/2026 have been fully considered but they are not persuasive.
Applicant argues (addressing p. 4-6 of the Remarks) that Nishino discloses a 10-fold dilution of the sample and does not focus on or teach controlling the protein concentration to a specific parameter and discloses dilution with distilled water that according to Applicant’s additional data (not provided) only yields a protein removal rate of 56.6%. Applicant further argues that the cited references do not provide motivation or teaching to intentionally target and adjust the protein concentration to 2.0 mg/mL or less to achieve such a drastic improvement in the protein removal rate and provide no reason to adjust the ionic strength to completely solubilize proteins without enzymes. Applicant argues that the unexpected chemical synergy (unexpected results) results uniquely from the combination of the specific buffer species, specific concentration (ionic strength), and dilution level. These arguments are not persuasive because:
First, Nishino does not teach dilution of the sample with distilled water but uses PBS buffer at pH 6.8 (p. 3, 2nd paragraph). The PBS buffer contains potassium phosphate as evidenced by Millipore Sigma and has ionic strength of 0.156 M as evidenced by Kucernak (p. 3939, left column, 2nd paragraph) and described in the rejection above. Nevertheless, the current rejection is based on combination of prior art of Nishino and Chung in which Chung teaches 100 mM potassium phosphate buffer at pH of 7.4 for washing probiotic bacteria during their collection (paragraph 0069) providing motivation to use the same buffer for dilution and washing of bacteria since these buffers have neutral pH, both contain potassium phosphate and have close ionic strength as described above.
Second, although prior art does not teach intentionally targeting protein concentration to 2 mg/ml for protein removal, the combination of prior art teaches the same method steps, the same buffer and dilution level. MPEP 2145 states: ""The fact that appellant has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious." Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985) (The prior art taught combustion fluid analyzers which used labyrinth heaters to maintain the samples at a uniform temperature. Although appellant showed that an unexpectedly shorter response time was obtained when a labyrinth heater was employed, the Board held this advantage would flow naturally from following the suggestion of the prior art.). See also Lantech Inc. v. Kaufman Co. of Ohio Inc., 878 F.2d 1446, 12 USPQ2d 1076, 1077 (Fed. Cir. 1989), cert. denied, 493 U.S. 1058 (1990) (unpublished — not citable as precedent) ("The recitation of an additional advantage associated with doing what the prior art suggests does not lend patentability to an otherwise unpatentable invention.")”. Nishino teaches the claimed steps of dilution of the specimen containing fermented milk with a buffer, centrifugation and collecting microorganism (p. 3, 2nd paragraph). Besides, Nishino teaches 10-fold dilution of fermented milk (p. 3, 2nd paragraph) providing the claimed protein concentration as evidenced by Ba and described in the Office Action. Chung teaches washing the bacterial pellet during bacterial recovery and provides buffer with the claimed type and concentration (paragraph 0069). Therefore, the combination of Nishino and Chung teaches all the claimed steps for collecting microorganisms from the specimen containing fermented milk and is expected to provide high removal of protein from the specimen.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to LIOUBOV G KOROTCHKINA whose telephone number is (571)270-0911. The examiner can normally be reached Monday-Friday: 8:00-5:30.
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/L.G.K./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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