T 0715/11 (Lactic acid bacterial helper organisms/CHR. HANSEN) 11-05-2016

Reasons for the Decision

Rule 139 EPC - claim 6

1. Claim 6 according to the present main request is identical to the corresponding claim of both the patent as granted and the main request underlying the decision under appeal, except that the wording "the lactic add bacterial strain" has been corrected to read "the lactic acid bacterial strain" (emphasis added by the board). It is immediately apparent to a person skilled in the art, in the context of claim 6 as granted, that the word "add" does not fit and must, therefore, be a typographical error. Since claim 6 is dependent on claim 1, in which "a lactic acid bacterial strain" is specified (see section IV above), it is also immediately evident that the word "acid" offered instead in present claim 6 is what was actually intended.

2. For these reasons and in the absence of objections by the appellant, the board decides to allow the offered correction and admits into the proceedings the amended main request submitted together with the reply to the statement of grounds of appeal.

Article 123(2) EPC - claims 2, 17 to 25 and 30

3. Except for amended claim 6 (see point 1 above), the set of claims according to the present main request is identical to the claims of the main request underlying the decision under appeal.

4. In the decision under appeal, the opposition division found that the objections under Article 123(2) EPC raised by the opponent against claims 2, 17 to 25 and 30 were not justified (see section 9 of the decision). This finding is, in the board's view, correct.

5. A basis for present claim 2 (see point IV above) is found in claim 2 of the application as filed, read in the light of the passage on page 4, lines 20 to 24 of the description. Claim 2 of the application as filed is directed to a method of enhancing the growth rate and/or controlling the metabolic activity of a lactic acid bacterial strain, comprising the step of cultivating the strain in association with a lactic acid bacterial helper organism defective in its pyruvate metabolism, which results in enhanced the acid production by the lactic acid bacterial strain. On page 4, lines 20 to 24 of the application as filed, the expression "controlling the metabolic activity" is defined as referring to "... the increased or decreased production of any metabolite produced by the starter culture, including the production of acids,...". From this passage, a person skilled in the art can directly and unambiguously derive a link between the metabolic activity of the lactic acid bacterial strain and the increased production of acids, as specified in present claim 2.

6. Like the opposition division in the decision under appeal, the board regards claim 25 of the application as filed as a basis for the subject-matter of present claim 17 (see point IV above). Claim 25 of the application as filed is directed to a starter culture composition comprising a helper organism capable of enhancing the growth rate of a lactic acid bacterium also included in the composition and, alternatively or additionally, capable of controlling the metabolic activity of the lactic acid bacterium ("... being capable of enhancing the growth rate and/or controlling the metabolic activity of the lactic acid bacterium", emphasis added by the board). Undisputedly, a person skilled in the art reading claim 25 can derive from its wording three distinct embodiments of the starter culture composition. However, contrary to the appellant's view, the board holds that singling out one of these embodiments, in particular a starter culture composition comprising a helper organism capable of enhancing the growth rate of the lactic acid bacterial strain as claimed in present claim 17, does not present the skilled person with any information which is not directly and unambiguously derivable from claim 25 of the application as filed. Thus, the amendments in claim 17 do not contravene Article 123(2) EPC.

7. A lactic acid bacterium as claimed in present claim 30 (see point IV above) is directly and unambiguously derivable from the application as filed, inter alia from the disclosure on page 14, lines 12 to 30 taken together with the sequences according to SEQ ID NO:1 and 2. The passage on page 14, lines 12 to 18 reads:

"In accordance with the invention there is also provided a lactic acid bacterium that is defective in at least one enzyme involved in the pyruvate metabolism as it is described above and in which a gene capable of regenerating NAD**(+) is overexpressed, including a gene coding for an enzyme catalysing the reduction of O2 to H20 or H2O2 such as an NADH:H2O oxidase including the enzyme having the sequence SEQ ID NO:2."

From the sequence listing in the application it is immediately apparent that the amino acid sequence SEQ ID NO:2 is encoded by the nucleotide sequence in SEQ ID NO:1.

8. As regards variants or derivatives of SEQ ID NO:1, it is stated on page 14, lines 19 to 25 of the application as filed that:

"... the invention also provides an isolated DNA fragment derived from a lactic acid bacterium comprising a gene coding for a polypeptide having NADH:H2O oxidase activity such as a DNA fragment which is selected from the group consisting of the sequence shown in SEQ NO ID:1 and a variant or derivative hereof which is at least 50% e.g. at least 60% including at least 70% identical with said sequence ..."

9. In the board's view, the appellant's objection that the feature "... and has the same function" extends the subject-matter of present claim 30 beyond the content of the application as filed is not justified. There is no evidence whatsoever on file of any common general knowledge which a person skilled in the art at the relevant date might have had in mind while reading the application, and which would have made him/her doubt that variants or derivatives that were only 50% identical to SEQ ID NO:1 could still code for a polypeptide having NADH:H2O oxidase activity. In the absence of such evidence, the appellant's argument as to what the skilled person would derive from the passage on page 14, lines 19 to 25 quoted above with respect to variants of derivatives fails to convince the board.

10. Summarising the above, none of the objections raised by the appellant under Article 123(2) EPC is considered to be justified.

Article 84 EPC

11. The appellant contested the opposition division's adverse findings concerning the objection to claim 17 under Article 84 EPC (see last paragraph in section 10 of the decision under appeal).

12. Article 84 EPC requires the claims to be clear and concise and to be supported by the description. In the board's view, in particular as regards the feature "... and is capable of enhancing the growth rate of the lactic acid bacterial strain", claim 17 is clear within the meaning of Article 84 EPC. Whether or not a helper organism as specified in claim 17 enhances the growth rate of a lactic acid bacterial strain in a starter culture composition comprising both can be easily determined using methods that are well known in the art.

13. The experiments in the examples of the patent show that co-cultivation of a lactic acid bacterial strain with a lactic acid bacterial helper organism defective in its pyruvate metabolism results in a pH decrease due to increased acid production by the lactic acid bacterial strain (see Figures 4, 5, 6A, 7A and 8A). Since lactic acid bacteria normally produce lactic acid in proportion to the cell mass (see document (24), page 204, line 2), it can be inferred from the increased acid production that the growth rate of the lactic acid bacterium is enhanced. Thus, contrary to the appellant's argument, claim 17 is in fact supported by the examples of the patent.

14. For these reasons, the board holds that claim 17 fulfils the requirements of Article 84 EPC.

Article 83 EPC

15. In the decision under appeal, the opposition division found that the claimed invention was disclosed in the patent application in a manner sufficiently clear and complete for it to be carried out by a person skilled in the art (see last paragraph in point 11 of the decision).

16. In its statement of grounds of appeal, the appellant raised an objection to claims 1 to 9, 12 to 19 and 22 to 30 based on the allegedly extremely broad definition of the helper organism in the first sentence of paragraph [0023] of the patent (see point XII above).

17. The appellant's interpretation of the wording "In general, ..." in paragraph [0023] as meaning that the helper organism does not necessarily need to be derived from a lactic acid bacterium is not shared by the board. Nor can the board accept the appellant's argument that the technical information provided in the patent does not enable the skilled person to determine which helper organisms could be used to carry out the invention.

18. The meaning given in the patent to the feature "lactic acid bacterial" characterising the helper organism is explained in paragraph [0023], and specific examples of suitable helper organisms are given in paragraphs [0024] to [0026]. It is stated in paragraph [0023] that, in general, the lactic acid bacterial helper organism is a derivative of a lactic acid bacterium, derivatives including spontaneous mutants and mutants obtained by genetic modification in vitro of a lactic acid bacterium. Paragraphs [0024] to [0026] disclose lactic acid bacteria (e.g. Lactococcus lactis) from which a helper organism can be derived. In particular, DN223 and DN224 strains derived from Lactococcus lactis subspecies lactis are mentioned as suitable helper organisms for use in the invention.

19. The appellant raised a plausibility issue concerning the definition of a lactic acid bacterial helper organism defective in its pyruvate metabolism as an organism which, compared to the wild-type, has an increased or decreased production of one or more metabolites derived from pyruvate (see paragraph [0014] of the patent). In view of the fact that, in lactic acid bacteria, pyruvate acts as substrate in various metabolic pathways which are regulated differently by various environmental conditions (e.g. carbon source limitation or aeration; see document (8)), it is, in the board's view, not implausible that a lactic acid bacterial organism useful as a helper organism may be associated with increased production of a particular metabolite in one pathway, and another helper organism with decreased production of a different metabolite in a different pathway.

20. The appellant's objection of lack of disclosure in the application as to how to obtain a helper organism defective in its pyruvate metabolism, other than the DN223 and DN224 strains described in the examples, is not justified. Reference Example 2 illustrates how to obtain mutants of two different strains of Lactobacillus lactis which are defective in the Pfl gene encoding the enzyme pyruvate formate lyase, and in Example 3 double mutants defective in both the Pfl gene and the Ldh gene coding for lactate dehydrogenase are obtained, these two enzymes being involved in the pyruvate metabolism. It is not apparent to the board why a person skilled in the art following the technical instructions given in these examples could not obtain further mutants defective in the pyruvate metabolism, and the appellant has not provided any arguments or evidence to this effect. While the methods for obtaining mutants defective in their pyruvate metabolism disclosed in the application as filed use random mutagenesis, there is no evidence on file showing that a person skilled in the art could not identify and select the desired mutants without an undue burden of experimentation.

21. It should be noted that, at the relevant date, further genes coding for enzymes involved in the pyruvate metabolism had been described, and their nucleotide sequences were available from public databases (see e.g. documents (6) and (11)). As disclosed in the passage on page 8, lines 13 to 23 of the application as filed (paragraph [0024] of the patent), a person skilled in the art could modify these genes to construct the desired derivatives of a lactic acid bacterium, applying techniques known in the art at the relevant date, including mutation and DNA recombination techniques.

22. Moreover, the fact that the application as filed does not exemplify a lactic acid bacterial helper organism defective in the pyruvate metabolism which over-expresses a gene for an enzyme capable of catalysing the reduction of O2 and regenerating NAD**(+) does not mean that a person skilled in the art would not be able to obtain such a helper organism relying on the information provided in the application and methods well known in the art at the relevant date. The nox gene is mentioned in the application as a gene for a NADH oxidase capable of catalysing the reduction of O2 and regenerating NAD**(+). It has not been disputed that the sequence of the nox gene was available at the relevant date. There is also no evidence on file showing that finding other suitable genes coding for further NADH oxidases, as mentioned in the passage on page 10, lines 10 to 18 of the application as filed, would have involved an undue burden of experimentation or a need for inventive skills.

23. Contrary to the appellant's view, the examples of the application as filed (and of the patent as granted) show that cultivation of a lactic acid bacterial strain in association with a helper strain defective in its pyruvate metabolism, in particular strains DN223 and DN224, results in an enhancement of the acidification rate, i.e. an increased production of acids, presumably lactic acid, which is a metabolite derived from pyruvate. As stated in point 12 above, it can be inferred from the increase in acid production that the growth rate of the lactic acid bacterium is enhanced, as lactic acid bacteria produce lactic acid in proportion to the cell mass (see document (24), page 204, line 2).

24. Lastly, there is no evidence on file showing that the starter culture composition of claims 17 to 25 cannot be used to inoculate any culture media other than milk.

25. For these reasons, the appellant's objection under Article 83 EPC must fail.

Article 87 EPC - priority

26. The respondent accepts that the priority of the earlier DK and US applications filed on 30 May 1997 cannot be validly claimed for the subject-matter of claims 14 to 16, 22 to 24 and 26 to 30 (see respondent's reply to the statement of grounds of appeal, pages 15 and 16). Thus, for assessing whether or not the subject-matter of these claims is novel and involves an inventive step the relevant date is the filing date, i.e. 25 May 1998.

27. Thus, the content of document (4), an international application filed on 20 August 1997 claiming the priority of an earlier US application filed on 22 August 1996, forms part of the state of the art to be considered for the purposes of assessing novelty for claims 1 and 17 (Article 54(3) EPC) and novelty and inventive step for claims 14 to 16, 22 to 24 and 26 to 30 (Article 54(2) EPC).

Article 54(2) and (3) EPC - claims 1, 17 and 26

28. The appellant based its objection of lack of novelty on document (4). This document relates to methods for obtaining mutants or variants of lactic acid bacteria which, when they are used in the manufacture of fermented food products, produce increased amounts of desirable metabolites or reduced amounts of less desirable metabolites (see page 1, lines 3 to 9). Document (4) describes, in a first aspect, a method of isolating a pyruvate formate lyase (Pf1) defective lactic acid bacterium (see passage from page 6, line 3 to page 7, line 3) and, in a further aspect, also a method of isolating a Pfl and lactate dehydrogenase (Ldh) defective lactic acid bacterium. Lactose dehydrogenase (Ldh) and pyruvate formate lyase (Pfl) are two enzymes involved in the pyruvate metabolism of lactic acid bacteria.

29. In the examples, several Ldh or Ldh and Pfl defective mutants of Lactobacillus lactis obtained by these methods, inter alia the DN223 (Ldh**(-) Pf1**(-)) and DN224 (Ldh**(-)) mutants used in the examples of the patent in suit, are tested - individually - for growth (measured as 0D600 and/or pH of the medium) and production of various metabolites of the pyruvate metabolism (e.g. acetaldehyde, diacetyl, lactic acid or ethanol), under aerobic and anaerobic conditions (see e.g. Examples 3.2 and 4.3).

30. The appellant referred to the following passages of document (4) in support of its objection of lack of novelty:

"There is also provided a lactic acid bacterial starter culture composition comprising any of the above mentioned lactic acid bacteria." (page 8, lines 9 to 11).

"It will also be understood that the presently provided strains will be highly useful as production strains in the manufacturing of lactic acid bacterial metabolite compounds including the above aroma compounds. Accordingly, the invention encompasses in a still further aspect a method of producing a lactic acid bacterial metabolite. Such a method comprises cultivating one or more of the lactic acid bacteria as disclosed herein in a suitable medium under industrially feasible conditions where the metabolite is produced, and isolating, if required, the metabolite from the culture" (page 19, lines 1 to 10; emphasis added by the board).

31. In the board's view, neither passage anticipates the subject-matter of the claims on file. A person skilled in the art cannot derive, directly and unambiguously, from the first passage quoted above a starter culture composition which includes not only any of the Ldh**(-) or Ldh**(-) Pf1**(-) mutants described in document (4), in particular DN223 or DN224, but also - as required in claims 1 and 17 - a lactic acid bacterial strain. The second passage on which the appellant relied relates to the use of one or more of the Ldh**(-) or Ldh**(-) Pf1**(-)mutants described in document (4) as a production strain, rather than as a helper strain as specified in the present claims. Moreover, it is more than doubtful whether - as the appellant suggested - a skilled person can derive directly and unambiguously from this passage a starter culture composition comprising, specifically, the DN223 and DN224 mutants. But even if the board were to acknowledge that this specific combination of mutants could be derived from the passage in question, there is no evidence, either in the same document or in any other document on file, showing that DN223 is capable of enhancing the growth rate of DN224, or vice versa. Nor is there any evidence on file that either mutant is capable of controlling the metabolic activity of the other mutant. For these reasons, the board holds that the subject-matter of claims 1 and 17 must be regarded as novel over document (4).

32. As regards claim 26, the appellant cited Table 3.1 on page 28 of document (4) in support of its argument that, since the specific activity of the NADH oxidase - an enzyme capable of regenerating NAD**(+) - is higher in the DN223 mutant than in the parent strain CHCC373, the gene encoding this enzyme must be over-expressed in the mutant. This is, however, not necessarily the case. In Lactobacillus lactis, pyruvate metabolism is a metabolic network in which control of the pyruvate distribution within various pathways is subject to co-ordinated regulation by both gene expression mechanisms and allosteric modulation of enzyme activity (see document (8), last sentence of the abstract on page 157). Moreover, a modified level of lactose dehydrogenase (Ldh) and pyruvate formate lyase (Pfl) gene expression, as observed in the DN223 mutant, may have an effect on the regulation of oxygen metabolism and NAD**(+) regeneration, in which different NADH oxidising enzymes, inter alia the NADH oxidase, are involved (see document (8), chapter headed "Effect of aeration" starting on page 165). Thus, contrary to the appellant's view, it cannot be ascertained from the data in Table 3.1. of document (8) that a gene coding for an enzyme that is capable of catalysing the reduction of O2 to H2O or H2O2, and of regenerating NAD**(+), is over-expressed in the DN223 mutant.

33. In view of the above, the appellant's objection of lack of novelty based on document (4) must fail.

Article 56 EPC

Document (4) as the closest state of the art - claims 14 to 16, 22 to 24 and 26 to 30

34. Claims 14 and 22, which depend directly and/or indirectly of claims 1 and 17, respectively, as well as independent claim 26 (see point IV above) specify that the helper organism over-expresses a gene coding for an enzyme that is capable of catalysing the reduction of O2 to H2O or H2O2, and of regenerating NAD**(+). It is important to note that the subject-matter of claims 14 and 22 is further characterised by the features specified in the independent claims on which they depend.

35. The general content of document (4), which the appellant regards as the closest state of the art, has been outlined in points 28 and 29 above.

36. As stated above in the framework of assessing novelty, a person skilled in the art could not derive from document (4) a method in which a lactic acid bacterial strain is cultivated in association with a lactic acid bacterial helper organism defective in the pyruvate metabolism for the purpose of enhancing the growth rate and/or controlling the metabolic activity of the lactic acid bacterial strain. Nor could the skilled person derive from document (4) a composition comprising a lactic acid bacterium strain and a lactic acid helper organism capable of enhancing the growth rate of the lactic acid bacterial strain.Document (4) describes the use of a mutant defective in the pyruvate metabolism as a production strain rather than as a helper strain.

37. Thus, starting from document (4), the problem to be solved can be formulated as the provision of an alternative use of the lactic acid bacterial mutants described in this document. The board is convinced that this problem has been solved by the method and compositions proposed in the present claims.

38. The board is unable to find in document (4) any hint that would motivate the skilled person to use mutants in the pyruvate metabolism as helper strains to enhance the growth rate and/or control the metabolic activity of a lactic acid bacterial strain. There is also no suggestion of a further modification of the mutants in order to over-express a gene coding for an enzyme capable of catalysing the reduction of O2 to H2O or H2O2, and of regenerating NAD**(+).

39. The passage on page 13, lines 10 to 22 of document (4), to which the appellant referred in support of its objection, explains the strategy followed by the author for isolating and selecting a mutant of a lactic acid bacterium which is not only Pfl defective but also Ldh defective. This passage reads:

"Accordingly, it was hypothesized that a double mutant having the Pfl**(-) Ldh**(-) phenotype would be unable to grow under anaerobic conditions, i.e. such a strain would have the additional phenotype Ang**(-) (inability to grow anaerobically). This hypothesis was based on the assumption that such a double mutant would be unable to regenerate NAD**(+) from NADH under anaerobic conditions, since Pfl would be blocked by a mutation (whereas under aerobic conditions, NADH can be converted to NAD**(+) by NADH oxidase), PDC would be blocked due to inhibition by NADH and Ldh would be blocked by mutation. It was thus contemplated that a Pfl**(-) Ldh**(-) double mutant could grow under aerobic conditions but not under anaerobic conditions."

40. In the board's view, without knowledge of the present invention a person skilled in the art cannot derive from this passage that the over-expression of a gene coding for an enzyme capable of catalysing the reduction of O2 to H2O or H2O2 and of regenerating NAD**(+) would be in some way advantageous for the use of a mutant defective in the pyruvate metabolism as helper organism. Only with hindsight can such a measure be considered obvious.

41. Since the further claims objected to by the appellant are dependent on claims 14, 22 or 26, the same applies, mutatis mutandis, to those claims.

42. Hence, the board concludes that the objection of lack of inventive step based on document (4) is not justified.

Documents (6) and (2) as the closest state of the art - claims 1 to 30

43. Document (6) describes the genetic manipulation of the pathway for diacetyl metabolism in Lactococcus lactis, in particular the generation of mutants defective in the gene encoding alpha-acetolactate decarboylase (aldB), an enzyme involved in the pyruvate conversion to acetoin and 2,3-butanediol. Over-expression of genes encoding an alpha-acetolactate synthase in the defective strains lead to the production of higher levels of alpha-acetolactate, acetoin and diacetyl. Document (2) too reports a higher yield of diacetyl produced by mutants defective in the alpha-acetolactate decarboylase.

44. Starting from these documents, the problem to be solved is similar to that starting from document (4), i.e. the provision of an alternative use of the mutants described in the prior art.

45. The board holds that the use of such a mutant as helper organism in association with a lactic acid bacterial strain to enhance the growth rate and/or control the metabolic activity of the lactic acid bacterial strain was not obvious to a person skilled in the art. It is true that, as documents (15) and (16) show, the use of mixed starter cultures was common general knowledge at the priority date. Thus, a person skilled in the art could, in principle, have combined a mutant described in document (6) or (2) with a lactic acid bacterial strain. But without knowledge of the technical effect underlying the present invention, i.e. that in a mixed culture of a mutant defective in the pyruvate metabolism and a lactic acid bacterial strain the growth rate of the latter is enhanced and/or its metabolic activity controlled, he/she would have no motivation to do so.

46. It should also be stressed that the appellant's choice of documents relating to mutants defective in the pyruvate metabolism as the closest state of the art has been done with hindsight, because it introduces elements of the solution provided by the claimed invention. Already for this reason, the appellant's lines of argument in support of its objection of lack of inventive step are biased.

47. Summarising the above, the board is not persuaded that, in view of the documents brought forward by the appellant, the claimed subject-matter lacks inventive step.

Conclusion

48. The appellant's request that the patent be revoked fails.