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 .
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 statements (IDS) submitted on 10/24/2025 and 12/19/2025 comply 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, 7 and 8 are amended.
Claims 1-9 are pending (claim set filed 12/24/2025) and are examined on the merits herein.
Withdrawal of Rejections
The response and amendment filed on 12/24/2025 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.
The previous claim 7 objection has been withdrawn necessitated by amendment of claim 7.
The previous claims 1-9 rejections under 35 U.S.C. 103 have been withdrawn necessitated by amendment of claim 1 and Applicant’s arguments.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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 Taouji (FR 2873386 A1) as evidenced by Ba (WO 2018130630 A1) and MilliporeSigma (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 2, 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 claims 1 and 2 limitations. However, if Nishino sample has protein concentration different from discloses 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 to increase the solubilization of proteins of fermented milk specimen and prevent aggregation to achieve efficient separation of microorganisms. A skilled artisan would have reasonably expected success in this optimization because selection of dilution rate to reach low protein concentration is routine and conventional.
Nishino does not teach the washing of the centrifugation pellet prior to collection of the separated microorganism.
Taouji teaches preparation of pharmaceutical composition based on Rhodococcus equi extract (Abstract). Taouji describes centrifugation of bacteria to remove impurities and recover only the bacterial cells (p. 5, 9th paragraph). Taouji mentions that the bacterial pellet is washed at least once with washing buffer which can be Tris acetate buffer at a concentration of 10 mM or 25 mM (p. 5, 10th and 11th paragraphs). In a preferred embodiment Taouji describes two successive washes of bacterial pellet followed by the third washing in a 25 mM Tris acetate pH 7.5 buffer (p. 5, 12th 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 washing the bacterial pellet as described by Taouji 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 Taouji describe methods involving recovery of bacteria from the specimen. Thus, Nishino and Taouji teachings as evidenced by Ba render claims 1 and 2 obvious.
Regarding claim 3, Nishino teaches buffer solution to have pH of 6.8 (p. 3, 2nd paragraph). Thus, Nishino and Taouji teachings as evidenced by Ba render claim 3 obvious.
Regarding claim 4, Nishino teaches PBS buffer that includes potassium phosphate as evidenced by MilliporeSigma. Thus, Nishino and Taouji teachings as evidenced by Ba and MilliporeSigma render claim 4 obvious.
Regarding claims 5 and 6, Taouji teaches the concentration of Tris acetate buffer for washing bacterial pellet of 10 mM and 25 mM (p. 5, 10th and 11th paragraphs) and describes several washing steps with final washing in a 25 mM Tris acetate pH 7.5 buffer (p. 5, 12th paragraph).
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 such as Tris-based buffer with concentration of 10 mM or 25 mM as taught by Taouji 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 to avoid buffer exchange for washing step after centrifugation and since Taouji describes buffer at neutral pH for washing bacterial pellet and Nishino teaches buffer at neutral pH for dilution of fermented milk and because selection of buffer for dilution and washing of bacterial cells is within the skills of the artisan in the field. Thus, Nishino and Taouji teachings as evidenced by Ba and MilliporeSigma render claims 5 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 non-treated LAB.” (p. 5, 1st paragraph). Thus, Nishino and Taouji teachings as evidenced by Ba 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 Taouji teachings as evidenced by Ba 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 Taouji (FR 2873386 A1) as evidenced by Ba (WO 2018130630 A1) 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, Taouji and Ba have been set forth above.
Nishino, Taouji and Ba do not teach the recited selected treatments following dilution step.
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 Taouji 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, Taouji and Dewan teachings as evidenced by Ba render claim 7 obvious.
Response to Arguments
Applicant’s arguments, see p. 5-7 of the Remarks, filed 12/24/2025, with respect to the written description support for negative limitation of claim 1 based on MPEP 2173.05 and Court decisions have been fully considered and are persuasive. Applicant argues that the Specification describes that contacting the microorganisms with a polymer-degrading enzyme affects the state of the cells in the fermented milk, particularly the surface layer structure (paragraph 0005) and therefore the Specification shows the reason to exclude that limitation.
Applicant’s arguments, see p. 8-9 of the Remarks, filed 12/24/2025, with respect to rejection under 35 U.S.C. 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made necessitated by amendment of claims as described above.
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.
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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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Sharmila G Landau can be reached at (571)272-0614. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/L.G.K./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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