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 .
DETAILED ACTION
Claims 10-11, 20, 26-29, and 34-45 are pending and under examination on their merits. Claims 1-9, 12-19, 21-25, and 30-33 are cancelled.
Response to Arguments
Applicant's arguments filed 11/18/2025 have been fully considered but they are not persuasive.
Applicant argues against the rejection of claims under 35 U.S.C. 103 on the grounds that the claimed cysteine concentration range of 0.5% or more falls outside the range disclosed by Satya (i.e. 0.01-0.1%) and thus Satya’s range fails to establish a prima facie case of obviousness over the claimed range (Arguments, second to last paragraph on page 7). Applicant argues further that the claimed range of the cysteine or salt thereof at a concentration of 0.5 wt% to 5 wt% is critical in achieving thermal stability of the bacteria (Arguments, bottom paragraph on page 7). Applicant submits that the survival rate of the bacteria was significantly increased when the content of cysteine was 0.5 wt% of more (Example 1-1 and 1-2 in Table 6 of the specification).
In response, applicants should compare a sufficient number of tests both inside and outside the claimed range to show the criticality of the claimed range, to establish unexpected results over a claimed range (MPEP 716.02(d)(II)). Furthermore, in order to establish that results are unexpected, the burden is on the applicant to establish results are unexpected and significant: “the evidence relied upon should establish that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance” (MPEP 716.02(b) I). Applicant fails to provide sufficient evidence to overcome the rejections of claims as obvious over Choi in view of Satya and Hubalek. First, Applicant has not provided any evidence that the results are statistically significance. Second, Applicant has not compared a sufficient number of tests both inside and outside the claimed range. Example 1-1 is the only example outside the claimed range (0.1 wt% cysteine) and there are no examples above 5 wt% cysteine. Applicant has only provided three tests inside the claimed range and one test below the claimed range. The specification does not state whether any replicates were performed for the tested concentrations of cysteine. Independent claim 42 recites an even broader range (0.5 wt% to 10 wt% cysteine) than independent claim 1 and Applicant has not presented any results for cysteine concentrations above 5 wt%. Therefore, these examples are insufficient to establish that the results are unexpected or that the claimed range is critical.
Applicant argues further against the rejection of claims under 35 U.S.C. 103 on the grounds that Choi teaches away from the use of cysteine to increase the thermal stability of bacteria. Specifically, as shown in Table 1 of Choi, adding cysteine decreases the survival rate of freeze-dried bacteria from 21% to 13% (Arguments, bottom paragraph on page 8). Applicant concludes that the person of ordinary skill in the art would have had no reasonable expectation of success in increasing the thermal stability of a Lactobacillus plantarum based on the teachings of the cited references (Arguments, point (iv) on page 9).
In response, Choi is not relied on to teach cysteine. Although Choi teaches that the survival rate of Lactobacillus plantarum freeze-dried bacteria with only cysteine as a cryoprotectant is 13%, there is no teaching away in Choi that would have deterred the person of ordinary skill in the art from combining cysteine with other cryoprotectants, especially when Satya provides an explicit motivation to do so (Satya teaches in [0182] that cysteine is useful as an anti-oxidant and reducing agent, protecting probiotics from oxidative stress during lyophilization). Furthermore, nonpreferred embodiments still constitute prior art per MPEP 2123(II).
Claim Rejections - 35 USC § 112
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.
(New Rejection Necessitate by Amendment) Claims 29 and 40 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 29 recites “wherein the preparation shows a survival rate of Lactobacillus plantarum from 55% to 77.5% compared to an initial stage of the preservation when the prepared Lactobacillus plantarum preparation is preserved at 40°C for 4 weeks.” The step of preservation at 40°C for 4 weeks can reasonably be interpreted as either a required active step of the claimed method, or as an optional measurement of a characteristic property of the preparation, thus claim 29 is indefinite.
Claim 40 recites the limitation "the vitamin" in line 2. There is insufficient antecedent basis for this limitation in the claim. Applicant may consider amending to “the at least one vitamin.”
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 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.
The following rejections are necessitated by the amendment.
Claims 10-11, 29, 34-38, and 41-45 are rejected under 35 U.S.C. 103 as being unpatentable over Choi et al. (WO2016200048) in view of Satya et al. (US 20110217368 A1; cited in the Non-Final Action mailed on 8/10/2023) and Hubalek et al. (Cryobiology 2003, Vol. 46, No. 3: 205-229; cited in the Non-Final Action mailed on 8/10/2023) as evidenced by Oxoid (website, 2023; cited in the Non-Final Action mailed on 8/10/2023) and USGS (website, 2018).
Choi teaches mixing each of the 20 amino acids with a culture medium of Lactobacillus plantarum KCTC3108 to a final concentration of 5 g/L, freeze-drying the preparation, and then determining the survival rate by accelerated stability testing for 4 weeks at 40°C ([64], [77]).
Choi individually tests the effect of all amino acids on bacterial recovery, including cysteine ([71], Table 1).
Choi also tests the effect of the cryoprotectants trehalose and maltodextrin ([133]), which each offer excellent cryoprotection (see the accelerated stability testing survival rates in Table 10).
However, Choi does not combine cysteine with maltodextrin and trehalose.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to mix Choi’s Lactobacillus plantarum with both trehalose and maltodextrin in order to improve the stability of Lactobacillus plantarum to freeze-drying. The person of ordinary skill in the art would have been motivated by the high survival rates of L. plantarum with each of these cryoprotectants individually (82 and 83%, respectively, Table 10 in [135]). Combining trehalose and maltodextrin would have constituted combining art-recognized equivalents (cryoprotectants) for the same purpose (cryoprotection). See MPEP 2144.06(I).
Satya teaches that cysteine is useful as an anti-oxidant and reducing agent, protecting probiotics from oxidative stress during lyophilization ([0182]).
Pertaining to claims 10-11, Satya teaches a method of culturing a bacteria in media comprising a reducing agent and lyophilizing the bacteria with lyoprotectants, wherein the lyoprotectants comprise 0.2 to 10% maltodextrin and 0.01 to 0.1% cysteine (Satya claims 21-23). In one embodiment, Satya teaches culturing Lactobacillus bacteria under fermentation conditions comprising 0.01 to 0.1% cysteine ([0055]-[0056], [0062]) prior to lyophilization with 0.01 to 0.1% cysteine (Satya claim 23).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Choi by both including cysteine in the culture medium during the culturing of Lactobacillus plantarum as well as combining cysteine with Lactobacillus plantarum, trehalose, and maltodextrin prior to lyophilization. The person of ordinary skill in the art would have been motivated by Satya’s teaching that cysteine is useful as an anti-oxidant and reducing agent, capable of protecting probiotics from oxidative stress during lyophilization ([0182]). The person of ordinary skill in the art would have had a reasonable expectation of success in these modifications to the method of Choi.
Satya does not explicitly teach that cysteine and the bacteria are mixed prior to lyophilization. Satya teaches in [0160] that lyoprotectants are added to the free cell slurry of Lactobacillus reuteri at a final concentration of 10% maltodextrin and 0.33% yeast extract and that the slurries are then lyophilized. Therefore, Satya suggests that the lyoprotectants and bacteria are mixed. Otherwise, the concentration of maltodextrin and yeast extract would not be a final concentration but rather a local concentration.
It would have been further obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to mix Choi’s Lactobacillus plantarum with cysteine, trehalose, and maltodextrin in order to ensure a uniform concentration of components and maximize contact between all bacterial cells and the cryoprotectants prior to lyophilization.
Choi and Satya do not teach that the composition for cryoprotecting L. plantarum further comprises soy peptone.
Hubalek teaches that trehalose is a cryoprotectant used in microbiology (Table 1, page 207). Hubalek teaches that trehalose has been used as a cryoprotectant for Lactobacillus bulgaricus (page 211, right column, bottom paragraph). Hubalek teaches further that peptone soya (“soy peptone”) has cryoprotective effects when used as a diluent (page 215, left column, paragraph 3).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to mix soy peptone with Choi and Satya’s cysteine, maltodextrin, and trehalose prior to lyophilization in order to further protect Choi’s Lactobacillus plantarum. The person of ordinary skill in the art would have had a reasonable expectation of success that the combination of Choi and Satya’s composition containing trehalose, maltodextrin, and cysteine with soy peptone (a known cryoprotectant taught by Hubalek), would have further protected Choi’s Lactobacillus plantarum and resulted in greater stability of the preparation.
Regarding the concentration of cysteine in the composition, Satya teaches that cysteine is present in the lyoprotectant at a concentration of 0.01 to 0.1% (Satya claims 23), which is below the claimed range of 0.5 wt% to 5% wt%. Satya does not specify whether the percentages are weight percent or w/v%. However, Satya’s MRS culture media is aqueous ([0170]), as evidenced by Oxoid (see Direction page 2: “Add 52g to 1 litre of distilled water…”), and the density of water is approximately 1 g/mL as evidenced by USGS. Therefore, 0.01 to 0.1 w/v% cysteine would have been approximately equivalent to 0.01 to 0.1 w/w%, which is below the presently claimed ranges (0.5 to 5 wt% in claims 10 and 41 and 0.5 to 10 wt% in claim 42).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to optimize by routine experimentation the amount of cysteine in the method of Choi modified by Satya and Hubalek. The person of ordinary skill in the art would have been motivated to maximize the anti-oxidant effect of cysteine while minimizing the cost to perform the method (i.e. the amount of reagents required). The person of ordinary skill in the art would have had a reasonable expectation of success in the optimization.
Regarding claims 29 and 41, Choi teaches accelerated stability testing at 40°C for 4 weeks ([0077]).
The survival rate of Lactobacillus plantarum KCTC3108 after accelerated stability testing at 40 °C for 4 weeks is 82% and 84% for trehalose and maltodextrin, respectively (Table 10, [135]).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to incubate the preparation of Choi modified by Satya and Hubalek at 40°C for 4 weeks in order to perform an accelerated stability test. The person of ordinary skill in the art would have had a reasonable expectation of success in incubating the preparation at 40°C for 4 weeks. The person of ordinary skill in the art would also have had a reasonable expectation of success that the Lactobacillus plantarum preparation of Choi modified by Satya and Hubalek would have achieved a survival rate of at least 82%, which is approaching the claimed range of from 55% to 77.5%.
Regarding claims 34-35, Choi teaches that probiotic bacteria, which include lactic acid bacteria, are typically powders, granules, tablets, or capsules that are mixed with food ([7]-[8]).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to formulate the freeze-dried Lactobacillus plantarum in the method of Choi of modified by Satya and Hubalek as a powder in order to mix the bacteria with food. The person of ordinary skill in the art would have had a reasonable expectation of success in following Choi’s teaching.
Regarding claim 36, Choi does not teach the amount of maltodextrin in the composition added to L. plantarum. However, Satya teaches lyophilizing the bacteria with lyoprotectants, wherein the lyoprotectants comprise 0.2 to 10% maltodextrin and 0.01 to 0.1% cysteine (Satya claims 21-23).
0.2 to 10% maltodextrin overlaps with the claimed range of 0.1 wt% to 20 wt% maltodextrin.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply Satya’s teaching regarding the amount of maltodextrin to include in the composition and the person of ordinary skill in the art would have had a reasonable expectation of success in doing so.
Regarding claim 37, Choi does not teach the amount of soy peptone in the composition added to L. plantarum.
Hubalek teaches including 0.5%-5% soy peptone as a cryoprotectant (Hubalek page 215, left column, paragraph 3).
0.5%-5% soy peptone overlaps with the instantly claimed range of 0.1-20 wt%.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply Satya’s teaching regarding the amount of maltodextrin to include in the composition and the person of ordinary skill in the art would have had a reasonable expectation of success in doing so.
Regarding claim 38, Choi does not teach the ratio of L. plantarum to the composition is from 1:0.1 to 1:5 in terms of weight.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to optimize by routine experimentation the amount of cryoprotecting composition to the amount of L. plantarum in the method of Choi modified by Satya and Hubalek. The person of ordinary skill in the art would have had a reasonable expectation of success in a wide range of acceptable ratios so long as the cells were sufficiently covered by the cryoprotecting composition.
Regarding new claims 43-45, Choi does not teach the amount of trehalose in the composition mixed with L. plantarum.
Satya teaches adding 0.5 M trehalose to L. planaturm before freeze-drying ([0030]). 0.5 M trehalose (molar mass 342 g/mol) is approximately 17 wt%. 17 wt% is within the claimed range of 10 wt% to 40 wt% (claims 43-44) and 10wt% to 30 wt% (claim 45).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply Satya’s teaching regarding the amount of trehalose to include in the composition of Choi modified by Satya and Hubalek and the person of ordinary skill in the art would have had a reasonable expectation of success in doing so.
Regarding claims 44-45, Choi does not teach the amount of soy peptone in the composition added to L. plantarum.
Hubalek teaches including 0.5%-5% soy peptone as a cryoprotectant (Hubalek page 215, left column, paragraph 3).
0.5%-5% soy peptone overlaps with the instantly claimed ranges of 0.1-20 wt% (claim 44) and 1 wt% to 10 wt% (claim 45).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply Satya’s teaching regarding the amount of soy peptone to include in the composition of Choi modified by Satya and Hubalek and the person of ordinary skill in the art would have had a reasonable expectation of success in doing so.
Further regarding claims 44-45, Choi does not teach the amount of maltodextrin in the composition added to L. plantarum. However, Satya teaches lyophilizing the bacteria with lyoprotectants, wherein the lyoprotectants comprise 0.2 to 10% maltodextrin and 0.01 to 0.1% cysteine (Satya claims 21-23).
0.2 to 10% maltodextrin overlaps with the claimed range of 0.1 wt% to 20 wt% (claim 44) and 1 wt% to 10 wt% maltodextrin (claim 45).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply Satya’s teaching regarding the amount of maltodextrin to include in the composition and the person of ordinary skill in the art would have had a reasonable expectation of success in doing so.
Claims 20 and 28 are rejected under 35 U.S.C. 103 as unpatentable over Choi in view of Satya and Hubalek as evidenced by Oxoid and USGS, as applied to claims 10-11, 29, 34-38, and 41-45 above, further in view of Celik et al. (cited on IDS filed on 1/12/2023).
See discussion of Choi, Satya, and Hubalek above, which is incorporated into this rejection as well.
Satya is silent on the form of cysteine (i.e. whether or not it is a salt). Satya does not teach that lactic acid bacteria are mixed with a composition comprising a salt of cysteine, wherein the salt is cysteine hydrochloride.
Celik teaches a method for freeze-drying bifidobacteria in which strains of bifidobacteria are cultured with 0.05% L-cysteine HCl and subsequently freeze-dried (page 3507, right column, Preparation of Cultures for Freeze-Drying, paragraph 1). Celik teaches that Bifidobacterium and Lactobacillus are the most common probiotics used in food products (Introduction, right column, bottom of paragraph 1, page 3506).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the generic cysteine in the method of Choi modified by Satya and Hubalek with cysteine hydrochloride as taught by Celik. One of ordinary skill in the art would have recognized that the method of Celik and the method of Choi modified by Satya and Hubalek are both methods for the lyophilization of common probiotics that both rely on cysteine. Therefore, one of ordinary skill in the art would have had a reasonable expectation of success that cysteine hydrochloride would have been an acceptable form of cysteine in the method of Choi modified by Satya and Hubalek.
Claims 26 and 39-40 are rejected under 35 U.S.C. 103 as being unpatentable over Choi et al. ((WO2016200048A1) in view of Satya et al. (US 20110217368 A1; cited in the Non-Final Action mailed on 8/10/2023) and Hubalek et al. (Cryobiology 46.3 (2003): 205-229; cited in the Non-Final Action mailed on 8/10/2023) as evidenced by Oxoid and USGS, as applied to claims 10-11, 29, 34-38, and 41-45 above, further evidenced by Mount Sinai (cited in the Non-Final Action mailed on 8/10/2023).
See discussion of Choi, Satya, and Hubalek above, which is included in this rejection as well.
Regarding claim 26, Choi does not teach that the culture medium comprises vitamin B2. However, Satya teaches that the bacteria is cultured with a nitrogen source comprising yeast extract ([0023] and [0027]). Satya also teaches that the bacteria are lyophilized with lyoprotectants comprising maltodextrin, cysteine, and yeast extract ([0028]). Yeast extract contains riboflavin as evidenced by Hubalek (page 214, Complex compounds, right column, lines 1-4). Riboflavin is synonymous with vitamin B2, as evidenced by Mount Sinai (lines 1-3 on page 1).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply Satya’s teaching to include vitamin B2 in the form of yeast extract in the culture medium to the method of Choi modified by Satya and Hubalek. The person of ordinary skill in the art would have had a reasonable expectation of success given that both Choi and Satya were interested in cryoprotecting lactic acid bacteria.
Regarding claims 39-40, Choi does not teach that the ratio of L. plantarum to vitamin B2 in the culture medium is from 1:3 to 3:1 (claim 39) or that the amount of vitamin B2 is contained in the culture medium in an amount of 0.01 wt% to 30 wt% (claim 40).
Although Satya teaches culturing bacteria with yeast extract (Satya [0027]), Satya does not teach the ratio of L. plantarum to vitamin B2.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to optimize the amount of yeast extract (comprising riboflavin or vitamin B2) in the culture medium prior to lyophilization. The person of ordinary skill in the art would have been motivated to maximize growth prior to lyophilization. The person of ordinary skill in the art would have had a reasonable expectation of success in optimizing the amount of nutrients in culture medium through routine experimentation.
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Choi in view of Satya and Hubalek as evidenced by Oxoid and USGS, as applied to claims 10-11, 29, 34-38, and 41-45 above, further in view of Mohammadi et al. (Annals of microbiology 61 (2011): 411-424; cited in the Non-Final Action mailed on 8/10/2023).
See discussion of Choi, Satya and Hubalek above, which is incorporated into this rejection as well.
Choi, Satya, and Hubalek do not teach that the Lactobacillus plantarum in the preparation has an improved growth rate during culturing before freeze-drying.
Mohammadi teaches that protein derivatives such as L-cysteine (an amino acid) promote probiotic survival because of their nutritional value, their ability to reduce the redox potential of the medium, and their ability to increase the medium’s buffering capacity (page 418, right column, paragraph 1).
Therefore, it would have been obvious to the person of ordinary skill in the art before the effective filing date of the claimed invention that Lactobacillus plantarum in the method of Choi modified by Satya and Hubalek would have had an accelerated growth compared to Lactobacillus plantarum cultured without cysteine because of both the nutritional value of cysteine and its ability to improve the buffering capacity of the media. The person of ordinary skill in the art would have recognized that each of these factors would have improved (accelerated) the growth of Lactobacillus plantarum.
Conclusion
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 CANDICE LEE SWIFT whose telephone number is (571)272-0177. The examiner can normally be reached M-F 8:00 AM-4:30 PM (Eastern).
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/CANDICE LEE SWIFT/Examiner, Art Unit 1657