(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2015/0223481 A1
`Larsen et al.
`(43) Pub. Date:
`Aug. 13, 2015
`
`US 20150223481A1
`
`(54) POLYPEPTIDES HAVING
`TRANSGALACTOSYLATING ACTIVITY
`(71) Applicant: DuPont Nutrition Biosciences APS,
`Copenhagen (DK)
`
`(72) Inventors: Morten Larsen, Krog (DK); Jacob
`Cramer, Flyvholm (DK)
`
`(73) Assignee: DUPONT NUTRITION
`BIOSCIENCES APS, Copenhagen
`(DK)
`
`(21) Appl. No.:
`
`14/.405,072
`
`(22) PCT Filed:
`
`Jun. 7, 2013
`
`(86). PCT No.:
`S371 (c)(1),
`(2) Date:
`
`PCT/EP2013/061819
`
`Dec. 2, 2014
`
`Related U.S. Application Data
`(60) Provisional application No. 61/657,180, filed on Jun.
`8, 2012.
`Foreign Application Priority Data
`
`(30)
`
`Jun. 8, 2012 (EP) .................................. 1217 1335.8
`
`Publication Classification
`
`(51) Int. Cl.
`A23C 9/12
`CI2N 9/38
`(52) U.S. Cl.
`CPC ............ A23C 9/1206 (2013.01); C12N 9/2471
`(2013.01)
`
`(2006.01)
`(2006.01)
`
`ABSTRACT
`(57)
`The present invention relates to polypeptides, specifically
`polypeptides having transgalactosylating activity and nucleic
`acids encoding these, and their uses in e.g. dairy product.
`
`

`

`Patent Application Publication
`
`Aug. 13, 2015 Sheet 1 of 5
`
`US 2015/0223481 A1
`
`HindIII(1)
`BPN' term
`| R4
`// Term region
`Bam-Dead Hindli Linker
`f'ss. ha
`Banh (254)
`M Cld (276)
`2BN-BR-Cla
`Apali (630)
`V bla
`- pBR322
`\
`is
`pMB1 origin
`ApaLI (1876)
`
`
`
`
`
`pBNSpeBIF1326
`
`Figure 1
`
`BIF1326
`
`
`
`
`
`9492bp
`
`S
`HindIII (5876)
`so Y
`EcdRI (4986)
`2BS-BN-RI/
`BN-BS-RI
`
`X
`
`Apal. (2374)
`Neo
`
`\
`
`...
`
`Y.
`
`Ned (a69)
`
`pUB110 (ori)
`
`

`

`Patent Application Publication
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`Aug. 13, 2015 Sheet 2 of 5
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`US 2015/0223481 A1
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`Figure 2
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`
`
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`Patent Application Publication
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`Aug. 13, 2015 Sheet 3 of 5
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`US 2015/0223481 A1
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`Figure 3
`
`3OO
`
`250 -
`
`2OO .
`
`SO
`
`OO .
`
`50 -
`
`0
`
`-o--on.....
`ox.re
`II
`Bif 917 Bif 995 Bif 1068 Bif 1124 Bif 1241 Bif 1326 Bif 1400 Bif 1478
`
`I
`
`

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`Patent Application Publication
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`Aug. 13, 2015 Sheet 4 of 5
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`US 2015/0223481 A1
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`Figure 4
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`
`
`

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`Patent Application Publication
`
`Aug. 13, 2015 Sheet 5 of 5
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`US 2015/0223481 A1
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`Figure 5
`
`A B A2 B2 A3 B3 A4 B4 A5 B5 A6 B6 A7 B7 A8 B8 A9 B9 AO BO
`
`
`
`
`
`BF 1068
`u-Y
`
`e-D BIF 995
`
`BIF 917
`
`

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`US 2015/0223481 A1
`
`Aug. 13, 2015
`
`POLYPEPTDES HAVING
`TRANSGALACTOSYLATING ACTIVITY
`
`FIELD OF THE INVENTION
`0001. The present invention relates to polypeptides, spe
`cifically polypeptides having transgalactosylating activity
`and nucleic acids encoding these, and their uses in e.g. dairy
`product.
`
`BACKGROUND OF THE INVENTION
`0002 Galactooligosaccharides (GOS) are carbohydrates
`which are nondigestable in humans and animals comprising
`two or more galactose molecules, typically up to nine, linked
`by glycosidic bonds. GOS's may also include one or more
`glucose molecules. One of the beneficial effects of GOSs is
`their ability of acting as prebiotic compounds by selectively
`stimulating the proliferation of beneficial colonic microor
`ganisms such as bacteria to give physiological benefits to the
`consumer. The established health effects have resulted in a
`growing interest in GOSS as food ingredients for various
`types of food.
`0003. The enzyme B-galactosidase (EC 3.2.1.23) usually
`hydrolyses lactose to the monosaccharides D-glucose and
`D-galactose. In the normal enzyme reaction of B-galactosi
`dases, the enzyme hydrolyses lactose and transiently binds
`the galactose monosaccharide in a galactose-enzyme com
`plex that transfers galactose to the hydroxyl group of water,
`resulting in the liberation of D-galactose and D-glucose.
`However, at high lactose concentrations some B-galactosi
`dases are able to transfer galactose to the hydroxyl groups of
`D-galactose or D-glucose in a process called transgalactosy
`lation whereby galacto-oligosaccharides are produced. Also
`at high lactose concentrations some B-galactosidases are able
`to transfer galactose to the hydroxyl groups of lactose or
`higher order oligosaccharides.
`0004. The genus Bifidobacterium is one of the most com
`monly used types of bacteria cultures in the dairy industry for
`fermenting a variety of diary products. Ingestion of Bifido
`bacterium-containing products furthermore has a health-pro
`moting effect. This effect is not only achieved by a lowered
`pH of the intestinal contents but also by the ability of Bifido
`bacterium to repopulate the intestinal flora in individuals who
`have had their intestinal flora disturbed by for example intake
`of antibiotics. Bifidobacterium furthermore has the potential
`of outcompeting potential harmful intestinal micro-organ
`1SS.
`0005 Galacto-oligosaccharides are known to enhance the
`growth of Bifidobacterium. This effect is likely achieved
`through the unique ability of Bifidobacterium to exploit
`galacto-oligosaccharides as a carbon source. Dietary Supple
`ment of galacto-oligosaccharides is furthermore thought to
`have a number of long-term disease protecting effects. For
`example, galacto-oligosaccharide intake has been shown to
`be highly protective against development of colorectal cancer
`in rats. There is therefore a great interest in developing cheap
`and efficient methods for producing galacto-oligosaccharides
`for use in the industry for improving dietary Supplements and
`dairy products.
`0006 An extracellular lactase from Bifidobacterium bifi
`dum DSM20215 truncated with approximately 580 amino
`acids (BIF3-d3) has been described as a transgalactosylating
`enzyme in a solution containing lactose solubilised in water
`(Jorgensen et al. (2001), Appl. Microbiol. Biotechnol. 57:
`
`647-652). WO 01/90317 also describes a truncation variant
`(OLGA347) as being a transgalactosylating enzyme and in
`WO 2012/010597 OLGA347 was shown to transfer a galac
`tose moiety to D-fucose, N-acetyl-galactosamine and Xylose.
`0007. In WO 2009/071539 a differently truncated frag
`ment compared to BIF3-d3 is described as resulting in effi
`cient hydrolysis and very low production of GOS when tested
`in milk.
`0008. The Bifidobacterium bifidum lactase enzymes
`described above have the drawback of either requiring high
`lactose concentrations such as above 10% (w/w) in order to
`produce GOS, or a high Surplus of another acceptor molecule
`to generate heterooligosaccharides. Furthermore, a molecule
`has been described that predominately having beta-galacto
`Sylase (hydrolase) activity.
`0009. There is still a need to develop enzymes that are
`efficient at producing GOS in applications with low lactose
`Substrate levels such as in milk.
`
`SUMMARY OF THE INVENTION
`0010. It is an object of embodiments of the invention to
`provide a polypeptide which has a useful ratio of transgalac
`tosylation to hydrolysis activity and thus are efficient produc
`ers of GOS when incubated with lactose even at low lactose
`levels such as in a milk-based product. It is a further object of
`embodiments of the invention to provide a method for pro
`duction of galacto-oligosaccharides (GOS) in situ in dairy
`products. It is a further object of embodiments of the inven
`tion to provide a method for developing a cheaper and more
`efficient method for production of galacto-oligosaccharides
`(GOS) for use in the industry. It is further object of embodi
`ments of the invention to provide polypeptides which are
`stable against further truncation Such as by proteolytic deg
`radation when produced in a Suitable organism Such as Bacil
`lus subtilis e.g. Bacillus subtilis strain BG3594. It is yet a
`further object of embodiments of the invention to provide
`polypeptides which are stable against further truncation dur
`ing storage after final formulation.
`0011. The present inventors have surprisingly found that
`the polypeptides disclosed herein are efficient producers of
`galacto-oligosaccharides for example in situ when incubated
`in a lactose containing composition Such as milk, wherein
`they have an efficient conversion of lactose into GOS result
`ing in a lower amount of free lactose. The presence of galacto
`oligosaccharides in diary products or other comestible prod
`ucts has the advantage of enhancing the growth of beneficial
`microbial strains (probiotics) such as the health-promoting
`Bifidobacterium sp. in the product itself and/or in the human
`or animal that consumes the product.
`0012. In one aspect, disclosed herein is a polypeptide hav
`ing transgalactosylating activity, which comprises an amino
`acid sequence having at least 90% sequence identity with
`SEQID NO: 1, and wherein said polypeptide, when being an
`expression product in a Bacillus subtilis strain BG3594 of a
`nucleic acid sequence, which encodes said polypeptide, is the
`only polypeptide expression product of said nucleic acid
`sequence that exhibits transgalactosylating activity.
`0013. In one aspect, disclosed herein is a polypeptide hav
`ing transgalactosylating activity selected from the group con
`sisting of:
`0014 a. a polypeptide comprising an amino acid
`sequence having at least 90% sequence identity with
`SEQID NO: 1, wherein said polypeptide consists of at
`most 980 amino acid residues,
`
`

`

`US 2015/0223481 A1
`
`Aug. 13, 2015
`
`0015 b. a polypeptide comprising an amino acid
`sequence having at least 97% sequence identity with
`SEQID NO: 2, wherein said polypeptide consists of at
`most 975 amino acid residues,
`0016 c. a polypeptide comprising an amino acid
`sequence having at least 96.5% sequence identity with
`SEQID NO:3, wherein said polypeptide consists of at
`most 1300 amino acid residues,
`0017 d. a polypeptide encoded by a polynucleotide that
`hybridizes under at least low stringency conditions with
`i) the nucleic acid sequence comprised in SEQID NO: 9.
`10, 11, 12 or 13 encoding the polypeptide of SEQ ID
`NO: 1, 2, 3, 4, or 5; or ii) the complementary strandofi),
`0018 e. a polypeptide encoded by a polynucleotide
`comprising a nucleotide sequence having at least 70%
`identity to the nucleotide sequence encoding for the
`polypeptide of SEQID NO: 1, 2, 3, 4 or 5 or the nucle
`otide sequence comprised in SEQID NO: 9, 10, 11, 12
`or 13 encoding a mature polypeptide, and
`0019 f. a polypeptide comprising a deletion, insertion
`and/or conservative Substitution of one or more amino
`acid residues of SEQID NO: 1, 2, 3, 4 or 5.
`0020. In another aspect disclosed herein is a polypeptide
`having transgalactosylating activity selected from the group
`consisting of:
`0021 a. a polypeptide comprising an amino acid
`sequence having at least 96.5% sequence identity with
`SEQID NO:3, wherein said polypeptide consists of at
`most 1300 amino acid residues,
`0022 b. a polypeptide comprising an amino acid
`sequence having at least 90% sequence identity with
`SEQID NO: 1, wherein said polypeptide consists of at
`most 980 amino acid residues,
`0023 c. a polypeptide encoded by a polynucleotide that
`hybridizes under at least low stringency conditions with
`i) the nucleic acid sequence comprised in SEQID NO: 9.
`10, 11, 12 or 13 encoding the polypeptide of SEQ ID
`NO: 1, 2, 3, 4, or 5; or ii) the complementary strandofi),
`0024 d. a polypeptide encoded by a polynucleotide
`comprising a nucleotide sequence having at least 70%
`identity to the nucleotide sequence encoding for the
`polypeptide of SEQID NO: 1, 2, 3, 4 or 5 or the nucle
`otide sequence comprised in SEQID NO: 9, 10, 11, 12
`or 13 encoding a mature polypeptide, and
`0025 e. a polypeptide comprising a deletion, insertion
`and/or conservative Substitution of one or more amino
`acid residues of SEQID NO: 1, 2, 3, 4 or 5.
`0026. In one aspect, disclosed herein is polypeptide which
`is a C-terminal truncated fragment of SEQID NO:22 having
`transgalactosylating activity and which are stable against fur
`ther truncation Such as by proteolytic degradation when pro
`duced in a suitable organism Such as Bacillus subtilis e.g.
`Bacillus subtilis strain BG3594 and/or which are stable
`against further truncation during storage after final formula
`tion.
`0027. In one aspect, disclosed herein is a polypeptide com
`prising an amino acid sequence having at least 90% sequence
`identity with SEQID NO: 1, wherein said polypeptide con
`sists of at most 980 amino acid residues.
`0028. In one aspect, disclosed herein is a polypeptide com
`prising an amino acid sequence having at least 97% sequence
`identity with SEQID NO: 2, wherein said polypeptide con
`sists of at most 975 amino acid residues, is provided.
`
`0029. In one aspect, disclosed herein is a polypeptide com
`prising an amino acid sequence having at least 96.5%
`sequence identity with SEQID NO:3, wherein said polypep
`tide consists of at most 1300 amino acid residues.
`0030. In one aspect, disclosed herein is a nucleic acid
`capable of encoding a polypeptide as described herein.
`0031. In one aspect, disclosed herein is an expression vec
`tor and/or a plasmid comprising a nucleic as described herein,
`or capable of expressing a polypeptide as described herein.
`0032. In one aspect, disclosed herein is a cell capable of
`expressing a polypeptide as described herein.
`0033. In one aspect, disclosed herein is a method of
`expressing a polypeptide, the method comprising obtaining a
`cell as described herein and expressing the polypeptide from
`the cell, and optionally purifying the polypeptide.
`0034. In one aspect, disclosed herein is a composition
`comprising a polypeptide as described herein, preferably a
`food composition, more preferably a dairy product.
`0035. In one aspect, disclosed herein is a method for pro
`ducing a food product such as a dairy product by treating a
`milk-based substrate comprising lactose with a polypeptide
`as described herein.
`0036. In one aspect, disclosed herein is a galacto-oligosac
`charide or composition thereof obtained by treating a Sub
`strate comprising lactose with a polypeptide as described
`herein.
`
`LEGENDS TO THE FIGURE
`0037 FIG. 1 shows a plasmid map for the BIF 1326
`variant for recombinant expression in Bacillus subtilis.
`0038 FIG. 2 shows SDS-PAGE showing truncation vari
`ants purified using HyperQ column eluted with a NaCl gra
`dient.
`0039 FIG.3 shows the ratio of transgalactosylation activ
`ity. Ratio is calculated as ratio between Abs420 with acceptor
`present divided by Abs420 without acceptor present times
`100. Variants at or below index 100 are purely hydrolytic
`variants, whereas the level above reflects relative transgalac
`tosylating activity.
`0040 FIG. 4 shows Galacto-oligosaccharides (GOS) gen
`erating efficacy of selected variants in a yoghurt matrix at 30°
`C. for 3 hours. In this example GOS is the accumulative
`amount oligosaccharides at and above DP3.
`004.1
`FIG. 5 shows SDS-PAGE gel showing the different
`variants from table 2 expressed and the degradation frag
`ments detected. Lower panel shows magnification and iden
`tification of degradation bands.
`
`SEQUENCE LISTING
`0042 SEQID NO: 1 (also named (BIF 917) herein) is a
`887 amino acid truncated fragment of SEQID NO: 22.
`0043 SEQID NO: 2 (also named (BIF 995) herein) is a
`965 amino acid truncated fragment of SEQID NO: 22.
`0044 SEQID NO:3 (also named (BIF 1068) herein) is a
`1038 amino acid truncated fragment of SEQID NO: 22.
`0045 SEQID NO: 4 (also named (BIF 1172) herein) is a
`1142 amino acid truncated fragment of SEQID NO: 22.
`0046 SEQID NO:5 (also named (BIF 1241) herein) is a
`1211 amino acid truncated fragment of SEQID NO: 22.
`0047 SEQID NO: 6 (also named (BIF 1326) herein) is a
`1296 amino acid truncated fragment of SEQID NO: 22.
`0048 SEQID NO: 7 is Bifidobacterium bifidum glycoside
`hydrolase catalytic core
`
`

`

`US 2015/0223481 A1
`
`Aug. 13, 2015
`
`0049 SEQID NO: 8 is a nucleotide sequence encoding an
`extracellular
`lactase from Bifidobacterium
`bifidum
`DSM2O215
`0050 SEQ ID NO: 9 is nucleotide sequence encoding
`BIF 917
`0051 SEQ ID NO: 10 is nucleotide sequence encoding
`BIF 995
`0052 SEQ ID NO: 11 is nucleotide sequence encoding
`BIF 1068
`0053 SEQ ID NO: 12 is nucleotide sequence encoding
`BIF 1172
`0054 SEQ ID NO: 13 is nucleotide sequence encoding
`BIF 1241
`0055 SEQ ID NO: 14 is nucleotide sequence encoding
`BIF 1326
`0056 SEQID NO: 15 is forward primer for generation of
`above BIF variants
`0057 SEQID NO: 16 is reverse primer for BIF917
`0.058 SEQID NO: 17 is reverse primer for BIF995
`0059 SEQID NO: 18 is reverse primer for BIF1068
`0060 SEQID NO: 19 is reverse primer for BIF1241
`0061 SEQID NO: 20 is reverse primer for BIF1326
`0062 SEQID NO: 21 is reverse primer for BIF1478
`0063 SEQID NO: 22 is extracellular lactase from Bifido
`bacterium bifidum DSM20215.
`0064 SEQID NO: 23 is signal sequence of extracellular
`lactase from Bifidobacterium bifidum DSM20215
`
`DETAILED DISCLOSURE OF THE INVENTION
`
`Definitions
`0065. In accordance with this detailed description, the fol
`lowing abbreviations and definitions apply. It should be noted
`that as used herein, the singular forms “a,” “an.” and “the
`include plural referents unless the context clearly dictates
`otherwise. Thus, for example, reference to “a polypeptide'
`includes a plurality of Such polypeptides, and reference to
`“the formulation' includes reference to one or more formu
`lations and equivalents thereof known to those skilled in the
`art, and so forth.
`0.066 Unless defined otherwise, all technical and scien
`tific terms used herein have the same meaning as commonly
`understood by one of ordinary skill in the art. The following
`terms are provided below.
`0067. “Transgalactosylase' means an enzyme that, among
`other things, is able to transfer galactose to the hydroxyl
`groups of D-galactose or D-glucose whereby galacto-oli
`gosaccharides are produced. In one aspect, a transgalactosy
`lase is identified by reaction of the enzyme on lactose in
`which the amount of galactose generated is less than the
`amount of glucose generated at any given time.
`0068. In the present context, the term “transgalactosylat
`ing activity” means the transfer of a galactose moiety to a
`molecule other than water. The activity can be measured as
`glucose-galactose generated at any given time during
`reaction or by direct quantification of the GOS generated at
`any given time during the reaction. This measurement may be
`performed in several ways such as by a HPLC method as
`shown in the examples. When comparing measurements of
`transgalactosylating activity, they have been performed at a
`given initial lactose concentration, such as e.g. 3, 4, 5, 6, 7, 8,
`9 or 10% (w/w).
`
`0069. In the present context, the term “B-galactosidase
`activity” means the ability of an enzyme to hydrolyse B-ga
`lactosides Such as for example lactose into monosaccharides,
`glucose and galactose.
`0070. In the context of calculating transgalactosylating
`activity: B-galactosidase activity, the B-galactosidase activity
`is measured as galactose generated at any given time during
`reaction. This measurement may be performed in several
`ways such as by a HPLC method as shown in the examples.
`0071. In the present context, the term “ratio of transgalac
`tosylation activity” using ortho-nitrophenol-3-D-galactopy
`ranoside (ONPG) was calculated as follows: Ratio is calcu
`lated as ratio between Abs420 with acceptor present divided
`by Abs420 without acceptor present times 100. Variant at or
`below index 100 are purely hydrolytic variants, whereas the
`level above depicts relative transgalactosylating activity.
`(0072) Ratio of transgalactosylation activity=(Abs420" '
`base/Abs420°)*100%, where Abs420"-"
`is
`the absorbance read at 420 nm using the described method 3
`below including cellobiose in the reaction and Abs420".
`biose is the absorbance read at 420 nm using the described
`method 3 below but without cellobiose in the reaction. The
`equation above is only valid for dilutions where the absor
`bance is between 0.5 and 1.0.
`0073. In one aspect, the activity is measured after 15 min.
`reaction, 30 min. reaction, 60 min. reaction, 90 min. reaction,
`120 min. reaction or 180 min. reaction. Thus in one aspect, as
`an example the relative transgalactosylation activity is mea
`sured 15 minutes after addition of enzyme, such as 30 minutes
`after addition of enzyme, such as 60 minutes after addition of
`enzyme. Such as 90 minutes after addition of enzyme. Such as
`120 minutes after addition of enzyme or such as 180 minutes
`after addition of enzyme.
`0074. In the present context, the term “ratio of transgalac
`tosylating activity:B-galactosidase activity” means (Glu
`cose-Galactose/Galactose).
`0075. In the present context, the term Glucose means the
`glucose concentration in 96 by weight as measured by HPLC.
`0076. In the present context, the term Galactose means
`the galactose concentration in % by weight as measured by
`HPLC.
`0077. In the present context, the term “lactose has been
`transgalactosylated” means that a galactose molecule has
`been covalently linked to the lactose molecule such as for
`example covalently linked to any of the free hydroxyl groups
`in the lactose molecule or as generated by internal transgala
`tosylation for example forming allolactose.
`0078. In the present context, the evaluation of perfor
`mance of polypeptides disclosed herein in galactooligosac
`charide (GOS) production were tested in a “milk-based
`assay' (yogurt application mimic). Batch experiments with a
`volume of 100 ul were performed in 96 well MTP plates using
`a yogurt mix, consisting of 98.60%(w/v) fresh pasteurized
`low-fat milk (Arla Mini-maelk) and 1.4% (w/v) Nutrilac
`YQ-5075 whey ingredient (Arla). To completely hydrate
`Nutrilac YQ-5075 the mixture was left with agitation for 20h.
`and afterwards added 20 mMNaPhosphate pH 6.5 to ensure
`a pH of 6.5. This yogurt-base was either used plain or with
`various Supplements such as additional lactose, fucose, mal
`tose, xylose or salts. 90 ul of the yogurt was mixed with 10 ul
`purified enzyme or crude ferment, sealed with tape and incu
`bated at 43°C. for 3 hours. The reaction was stopped by 100
`ul 10 Na2CO3. Samples were stored at -20° C. Quantifica
`tion of galactooligosaccharides (GOS), lactose, glucose and
`
`

`

`US 2015/0223481 A1
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`Aug. 13, 2015
`
`galactose were performed by HPLC. Analysis of samples was
`carried out on a Dionex ICS 3000. IC parameters were as
`follows: Mobile phase: 150 mM NaOH, Flow: Isochratic,
`0.25 ml/min, Column: Carbopac PA1, Column temperature:
`RT. Injection volume: 10 uI, Detector: PAD, Integration:
`Manual, Sample preparation: 100 times dilution in Milli-Q
`water (0.1 ml sample+9.9 ml water) and filtration through
`0.45 im Syringe filters, Quantification: Peak areas in percent
`of peak area of the standard. A GOS syrup (Vivanal GOS,
`Friesland Campina) was used as standard for GOS quantifi
`cation. Results of such an evaluation is shown in FIG. 4, and
`further described in example 2.
`0079. In the present context, the term “which polypeptide
`is freeze-dried” means that the polypeptide has been obtained
`by freeze-drying a liquid of the polypeptide at an appropriate
`pressure and for an appropriate period removing the water.
`0080. In the present context, the term “which polypeptide
`is in solution relates to a polypeptide which is soluble in a
`solvent without precipitating out of solution. A solvent for
`this purpose includes any millieu in which the polypeptide
`may occur. Such as an aqueous buffer or salt solution, a
`fermentation broth, or the cytoplasm of an expression host.
`0081. In the present context, the term “stabilizer” means
`any stabilizer for stabilizing the polypeptide e.g., a polyol
`Such as, e.g., glycerol or propylene glycol, a Sugar or a Sugar
`alcohol, lactic acid, boric acid, or aboric acid derivative (e.g.,
`an aromatic borate ester). In one aspect, the stabilizer is
`glycerol.
`I0082 In the present context, the term "carbohydrate sub
`strate” means an organic compound with the general formula
`C(HO), that is, consisting only of carbon, hydrogen and
`oxygen, the last two in the 2:1 atom ratio Such as a disaccha
`ride.
`0083. In the present context, the term “disaccharide' is
`two monosaccharide units bound together by a covalent bond
`known as a glycosidic linkage formed via a dehydration reac
`tion, resulting in the loss of a hydrogen atom from one
`monosaccharide and a hydroxyl group from the other. The
`formula of unmodified disaccharides is CHO. In one
`aspect, the disaccharide is lactulose, trehalose, rhamnose,
`maltose, Sucrose, lactose, fucose or cellobiose. In a further
`aspect, the disaccharide is lactose.
`0084. The term "isolated” means that the polypeptide is at
`least Substantially free from at least one other component
`with which the sequence is naturally associated in nature and
`as found in nature. In one aspect, "isolated polypeptide' as
`used herein refers to a polypeptide which is at least 30% pure,
`at least 40% pure, at least 60% pure, at least 80% pure, at least
`90% pure, and at least 95% pure, as determined by SDS
`PAGE.
`0085. The term “substantially pure polypeptide' means
`hereina polypeptide preparation which contains at most 10%,
`preferably at most 8%, more preferably at most 6%, more
`preferably at most 5%, more preferably at most 4%, at most
`3%, even more preferably at most 2%, most preferably at
`most 1%, and even most preferably at most 0.5% by weight of
`other polypeptide material with which it is natively associ
`ated. It is, therefore, preferred that the substantially pure
`polypeptide is at least 92% pure, preferably at least 94% pure,
`more preferably at least 95% pure, more preferably at least
`96% pure, more preferably at least 96% pure, more preferably
`at least 97% pure, more preferably at least 98% pure, even
`more preferably at least 99%, most preferably at least 99.5%
`pure, and even most preferably 100% pure by weight of the
`
`total polypeptide material present in the preparation. The
`polypeptides disclosed herein are preferably in a substantially
`pure form. In particular, it is preferred that the polypeptides
`are in “essentially pure form', i.e., that the polypeptide prepa
`ration is essentially free of other polypeptide material with
`which it is natively associated. This can be accomplished, for
`example, by preparing the polypeptide by means of well
`known recombinant methods or by classical purification
`methods. Herein, the term “substantially pure polypeptide' is
`synonymous with the terms "isolated polypeptide' and
`"polypeptide in isolated form.”
`I0086. The term “purified’ or “pure” means that a given
`component is present at a high level state—e.g. at least about
`51% pure, such as at least 51% pure, or at least about 75%
`pure such as at least 75% pure, or at least about 80% pure such
`as at least 80% pure, or at least about 90% pure such as at least
`90% pure, or at least about 95% pure such as at least 95%
`pure, or at least about 98% pure such as at least 98% pure. The
`component is desirably the predominant active component
`present in a composition.
`I0087. The term “microorganism’ in relation to the present
`invention includes any “microorganism' that could comprise
`a nucleotide sequence according to the present invention or a
`nucleotide sequence encoding for a polypeptide having the
`specific properties as defined herein and/or products obtained
`therefrom. In the present context, “microorganism” may
`include any bacterium or fungus being able to ferment a milk
`substrate.
`I0088. The term “host cell” in relation to the present
`invention includes any cell that comprises either a nucleotide
`sequence encoding a polypeptide having the specific proper
`ties as defined herein or an expression vector as described
`above and which is used in the production of a polypeptide
`having the specific properties as defined herein. In one aspect,
`the production is recombinant production.
`I0089. The term “milk”, in the context of the present inven
`tion, is to be understood as the lacteal secretion obtained from
`any mammal. Such as cows, sheep,goats, buffaloes or camels.
`In the present context, the term “milk-based substrate” means
`any raw and/or processed milk material or a material derived
`from milk constituents. Useful milk-based substrates include,
`but are not limited to Solutions/suspensions of any milk or
`milk like products comprising lactose, such as whole or low
`fat milk, skim milk, buttermilk, reconstituted milk powder,
`condensed milk, solutions of dried milk, UHT milk, whey,
`whey permeate, acid whey, or cream. Preferably, the milk
`based Substrate is milk or an aqueous solution of skim milk
`powder. The milk-based substrate may be more concentrated
`than raw milk. In one embodiment, the milk-based substrate
`has a ratio of protein to lactose of at least 0.2, preferably at
`least 0.3, at least 0.4, at least 0.5, at least 0.6 or, most prefer
`ably, at least 0.7.
`0090 The milk-based substrate may be homogenized and/
`or pasteurized according to methods known in the art.
`0091) “Homogenizing” as used herein means intensive
`mixing to obtain a soluble Suspension or emulsion. It may be
`performed so as to break up the milkfat into Smaller sizes so
`that it no longer separates from the milk. This may be accom
`plished by forcing the milk at high pressure through Small
`orifices.
`0092 “Pasteurizing” as used herein means reducing or
`eliminating the presence of live organisms, such as microor
`ganisms, in the milk-based substrate. Preferably, pasteuriza
`tion is attained by maintaining a specified temperature for a
`
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`US 2015/0223481 A1
`
`Aug. 13, 2015
`
`specified period of time. The specified temperature is usually
`attained by heating. The temperature and duration may be
`selected in order to kill or inactivate certain bacteria, Such as
`harmful bacteria, and/or to inactivate enzymes in the milk. A
`rapid cooling step may follow. A “food product' or “food
`composition' in the context of the present invention may be
`any comestible food or feed product suitable for consumption
`by an animal or human.
`0093. A "dairy product” in the context of the present
`invention may be any food product wherein one of the major
`constituents is milk-based. Preferable, the major constituent
`is milk-based. More preferably, the major constituent is a
`milk-based substrate which has been treated with an enzyme
`having transgalactosylating activity.
`0094. In the present context, "one of the major constitu
`ents' means a constituent having a dry matter which consti
`tutes more than 20%, preferably more than 30% or more than
`40% of the total dry matter of the dairy product, whereas “the
`major constituent’ means a constituent having a dry matter
`which constitutes more than 50%, preferably more than 60%
`or more than 70% of the total dry matter of the dairy product.
`0095. A “fermented dairy product” in present context is to
`be understood as any dairy product wherein any type of
`fermentation forms part of the production process. Examples
`of fermented dairy products are products like yoghurt, but
`termilk, creme fraiche, quark and fromage frais. Another
`example of a fermented dairy product is cheese. A fermented
`dairy product may be produced by any method known in the
`art
`0096. The term “fermentation” means the conversion of
`carbohydrates into alcohols or acids through the action of a
`microorganism Such as a starter culture. In one aspect, fer
`mentation comprises conversion of lactose to lactic acid.
`0097. In the present context, “microorganism” may
`include any bacterium or fungus being able to ferment a milk
`substrate.
`0098. In the present context the term “Pfam domains”
`means regions within a protein sequence that are identified as
`either Pfam-A or Pfam-B based on multiple sequence align
`ments and the presence of Hidden Markov Motifs (“The Pfam
`protein families database': R. D. Finn, J. Mistry, J. Tate, P.
`Coggill, A. Heger, J. E. Pollington, O. L. Gavin, P. Guneseka
`ran, G. Ceric, K. Forslund, L. Holm, E. L. Sonnhammer, S. R.
`Eddy, A. Bateman Nucleic Acids Research (2010) Database
`Issue 38:D211-222.). As examples of Pfam domains mention
`may be made of Glyco hydro2N (PF02837), Glyco hydro
`(PF00703), Glyco hydro 2C(PF02836) and Bacterial Ig-like
`domain (group 4) (PF07532).
`0099. As used herein “a position corresponding to posi
`tion” means that an alignment as described herein is made
`between a particular query polypeptide and the reference
`polypeptide. The position corresponding to a specific position
`in the reference polypeptide is then identified as the corre
`sponding amino acid in the alignment with the highest
`sequence identity.
`0100. A “variant' or “variants’ refers to either polypep
`tides or nucleic acids. The term “variant may be used inter
`changeably with the term “mutant. Variants include inser
`tions, Substitutions, trans