WO2013030280A1 - Process for the production of a fermented milk product - Google Patents
Process for the production of a fermented milk product Download PDFInfo
- Publication number
- WO2013030280A1 WO2013030280A1 PCT/EP2012/066866 EP2012066866W WO2013030280A1 WO 2013030280 A1 WO2013030280 A1 WO 2013030280A1 EP 2012066866 W EP2012066866 W EP 2012066866W WO 2013030280 A1 WO2013030280 A1 WO 2013030280A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- diacetyl
- lactic acid
- cbs
- acetoin
- dahi
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 44
- 238000004519 manufacturing process Methods 0.000 title claims description 22
- 235000014048 cultured milk product Nutrition 0.000 title claims description 18
- ROWKJAVDOGWPAT-UHFFFAOYSA-N Acetoin Chemical compound CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 claims abstract description 80
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 58
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical group CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 claims abstract description 45
- GFAZHVHNLUBROE-UHFFFAOYSA-N hydroxymethyl propionaldehyde Natural products CCC(=O)CO GFAZHVHNLUBROE-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000004310 lactic acid Substances 0.000 claims abstract description 29
- 235000014655 lactic acid Nutrition 0.000 claims abstract description 29
- 238000012360 testing method Methods 0.000 claims abstract description 20
- 230000001580 bacterial effect Effects 0.000 claims abstract description 19
- 235000013336 milk Nutrition 0.000 claims description 23
- 239000008267 milk Substances 0.000 claims description 23
- 210000004080 milk Anatomy 0.000 claims description 23
- 238000000855 fermentation Methods 0.000 claims description 16
- 230000004151 fermentation Effects 0.000 claims description 16
- 235000001252 Lactococcus lactis subsp lactis bv diacetylactis Nutrition 0.000 claims description 14
- 241000168725 Lactococcus lactis subsp. lactis bv. diacetylactis Species 0.000 claims description 14
- 241000894006 Bacteria Species 0.000 claims description 10
- 241000194041 Lactococcus lactis subsp. lactis Species 0.000 claims description 9
- 241000194036 Lactococcus Species 0.000 claims description 6
- 235000014969 Streptococcus diacetilactis Nutrition 0.000 claims description 6
- 230000020477 pH reduction Effects 0.000 claims description 6
- 241000194020 Streptococcus thermophilus Species 0.000 claims description 4
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims description 2
- 101100099821 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cbs-1 gene Proteins 0.000 claims 1
- 239000000796 flavoring agent Substances 0.000 description 19
- 235000019634 flavors Nutrition 0.000 description 19
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 12
- 244000005700 microbiome Species 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 235000013618 yogurt Nutrition 0.000 description 10
- 238000011534 incubation Methods 0.000 description 7
- 239000007858 starting material Substances 0.000 description 7
- 235000015140 cultured milk Nutrition 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000003860 storage Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000000769 gas chromatography-flame ionisation detection Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- UIHCLUNTQKBZGK-UHFFFAOYSA-N 3-methyl-2-pentanone Chemical compound CCC(C)C(C)=O UIHCLUNTQKBZGK-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000192132 Leuconostoc Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000194034 Lactococcus lactis subsp. cremoris Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 240000000513 Santalum album Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 235000014962 Streptococcus cremoris Nutrition 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000020246 buffalo milk Nutrition 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000020247 cow milk Nutrition 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000013213 extrapolation Methods 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000021191 food habits Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000005350 fused silica glass Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 235000015141 kefir Nutrition 0.000 description 1
- 235000015138 kumis Nutrition 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 235000020129 lassi Nutrition 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 235000020200 pasteurised milk Nutrition 0.000 description 1
- 238000009928 pasteurization Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 235000020138 yakult Nutrition 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/02—Making cheese curd
- A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
- A23C19/0323—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1236—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using Leuconostoc, Pediococcus or Streptococcus sp. other than Streptococcus Thermophilus; Artificial sour buttermilk in general
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/219—Diacetilactis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
Definitions
- the present invention relates to a process for manufacturing fermented milk products as well as to lactic acid bacteria capable of producing diacetyl and/or acetoin at thermophilic temperature conditions.
- Lactic acid bacteria are extensively used for production of fermented foods and they greatly contribute to flavor, texture and overall characteristics of these products.
- An old and well known example is yoghurt which probably originated from the Middle East and which still makes up more than half of the fermented milk production - or approximately 19 million tons in 2008 (source: Euromonitor). Fermented milks as e.g. yoghurts are popular due to the healthy image and pleasant sensory properties.
- Dahi is a popular fermented milk in India, Nepal and other Asian countries It is still made in every household in villages as well as in urban areas using traditional methods. Dahi is also known as "Curd", in particular in the southern part of India. The product is typically made from pasteurized or boiled milk, inoculated with dahi as starter left over from the previous day. Incubation is carried out at ambient temperature usually overnight. From Manufacturing yogurt and fermented milks (Ramesh C, Chandan editor), 2006, Blackwell Publishing, Iowa, USA; E.R. Vedamuthu - chapter 6 and 19 and Nagenadra P. Shah - chapter 21 .
- Subsequent pasteurisation may be carried out at any suitable combination of time and temperature, for example at 85-90°C for 15-30 minutes or at 90-95°C for 5-10 min.
- the acidity of dahi reaches 0.6 to 0.7 percent and a firm curd is formed.
- the curd is cooled, for instance by circulating chilled water or air (a blast chiller) around the containers and then transferred to the cold room maintained at about 4-5°C, or the containers are transferred the cold room directly.
- Dahi is often described as the Indian equivalent of yogurt. In reality, however, there are distinct differences between the two products.
- yogurt is characterized by a sharp acid tartness and the characteristic "green" acetaldehyde flavour. Dahi on the other hand has a mild acidity and diacetyl is the prominent flavour component. The latter contributes to a buttery, nut-meat like flavour.
- Other flavour compounds such as acetoin, acetic acid, alcohol and carbon dioxide impart a balanced round flavour to dahi. The difference in the flavour between yogurt and dahi is caused by the different starter cultures used.
- mesophilic lactococci and in some instances leuconostocs are used as starter flora.
- the basic role of starter culture is to bring about acid coagulation of milk and impart the characteristic flavour. Presence of more at least one type of lactose fermenting microorganism in the starter culture is required for the production of diacetyl flavour in dahi.
- a lactic culture comprising of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis biovar. d i acetyl a ctis may be used for dahi production.
- Recent developments in the industrial production of dahi have led to production processes at higher temperatures (i.e. from ambient temperature to temperatures between 37 and 45°C) in combination with shorter incubation times (from overnight fermentation to 4-8 hours).
- thermophilic starters e.g. Streptococcus thermophilus and Lactococcus delbrueckii subsp. bulgaricus
- acetaldehyde as in yogurt.
- lactis biovar. diacetylactis strains do not grow sufficiently at the higher process temperatures and therefore the industrial dahi produced at high temperature and short incubation time contain hardly if any of the typical "dahi" flavour in terms of diacetyl and/or acetoin.
- the invention provides a process for making a fermented milk product comprising the following steps: (a) adding one or more thermophilic lactic acid bacterial strains to milk for the acidification of the milk; and (b) adding one or more lactic acid bacterial strains for the production of diacetyl and/or acetoin that it is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A; and (c) carrying out the fermentation process in step (a) and in step (b) at a temperature range of 37 to 45°C during a period of 8 hours or less.
- the fermented milk product is dahi or curd . Dahi and curd and other fermented milk products have been described in the background of the invention.
- the temperature range of the fermentation process in step (a) and in step (b) is between 37 to 45°C. These higher temperatures, in particular for step (b), are preferred because this allows the process of the invention to be carried out for shorter times which is an advantage for the dairy industry.
- the temperature range is of 38 to 45°C, more preferably the temperature range is of 39 to 45°C, more preferably the temperature range is of 40 to 45°C.
- the fermentation process of the invention comprising step (a) and step (b) is carried out during a period of 8 hours or less. The time should be sufficient to obtain the required acidification as expressed by the pH value of the milk after fermentation in step (a) and for the production of diacetyl and/or acetoin in step (b).
- the fermentation process comprising step (a) and step (b) may be carried out during a period from 2-8 hours, preferably 3-8 hours, more preferably 3-7 hours, more preferably 4-8 hours, more preferably 4-7 hours.
- the skilled person is very well capable of determining the optimal temperature and time, in combination with the dosage of the one or more thermophilic lactic acid bacteria strains in step (a) and the one or more lactic acid bacterial strains for the production of diacetyl and/or acetoin in step (b) in order to obtain sufficient acidification as well as sufficient formation of the flavours such as acetoin and diacetyl.
- thermophilic lactic acid bacteria For the acidification of the milk under the conditions of the process of the invention, regular thermophilic lactic acid bacteria may be used. These bacteria are well known in the art and commercially available. Preferably, thermophilic lactic acid bacterial strains are selected from the group consisting of Streptococcus thermophilus and Lactococcus delbrueckii subsp. bulgaricus. The dosages of said thermophilic lactic acid bacteria are known in the art.
- the process of the invention comprises adding one or more of the lactic acid bacterial strains in step (b) characterized in that the lactic acid bacterial strain is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A, i.e. at a temperature of 40°C which is the temperature of Test A.
- the acetoin is produced at least at 150 mg/l, more preferably at least at 200 mg/l, more preferably at least at 250 mg/l, more preferably at least at 300 mg/l more preferably at least at 350 mg/l.
- the diacetyl is produced at least at 9 mg/l, more preferably at least at 12 mg/l, more preferably at least at 15 mg/l and more preferably at least at 18 mg/l.
- These diacetyl values are in addition to the diacetyl that may be present in the milk used in Test A. Test A has been described in the MATERIALS and METHODS section.
- the lactic acid bacterial strain capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A and which is added in step (b) of the process of the invention may belong to the genera Lactococcus and is preferably a Lactococcus lactis subsp. lactis biovar diacetylactis.
- the advantage of these lactic acid bacterial strains is their capability of producing flavours at the temperature of the process of the invention, i.e. between 37 and 45°C; as measured in Test A. Most preferred are the following strains: Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130345, Lactococcus lactis subsp.
- the dosages of said one or more of the lactic acid bacterial strain added in step (b) of the process of the invention may be determined by the skilled person and will depend also on the fermentation temperature and time of the process of the invention.
- the fermented milk product that may be produced by the process of the invention, preferable dahi or curd, comprises preferably at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl.
- the acetoin is at least 150 mg/l, more preferably at least 200 mg/l, more preferably at least 250 mg/l, more preferably at least 300 mg/l more preferably at least 350 mg/l.
- the diacetyl is at least 9 mg/l, more preferably at least 12 mg/l, more preferably at least 15 mg/l and more preferably at least 18 mg/l.
- the invention provides Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130345 and Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130347.
- the invention provides a fermented milk product obtainable by the process of the invention.
- the fermented milk product is Dahi or Curd. Dahi and Curd and other fermented milk products have been described in the background of the invention.
- the fermented milk product that may be produced by the process of the invention, preferable dahi or curd, comprises preferably at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl.
- the acetoin is at least 150 mg/l, more preferably at least 200 mg/l, more preferably at least 250 mg/l, more preferably at least 300 mg/l more preferably at least 350 mg/l.
- the diacetyl is produced at least 9 mg/l, more preferably at least 12 mg/l, more preferably at least 15 mg/l and more preferably at least 18 mg/l.
- the invention provides the use of a lactic acid bacterial strain that it is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A for the production of diacetyl and/or acetoin in the process of the invention for the production of a fermented milk product, preferable dahi or curd, comprising preferably at least 1 00 mg/l acetoin and/or at least 6 mg/l diacetyl , preferably the acetoin produced is at least 150 mg/l, more preferably at least 200 mg/l, more preferably at least 250 mg/l, more preferably at least 300 mg/l more preferably at least 350 mg/l.
- the diacetyl produced is at least 9 mg/l, more preferably at least 12 mg/l, more preferably at least 15 mg/l and more preferably at least 18 mg/l.
- the invention provides a method for the selection of a lactic acid bacterial strain that it is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A subjecting a population of lactic acid bacteria to Test A.
- Standard solutions solutions of a known concentration of analyte were added to the sample (unknown solution).
- the initial concentration in the sample was determined by extrapolation .
- the analysis was done on an Agilent 7890 A GC equipped with a combi Pal liquid sampler, a split/splitless injector and an FID detector.
- the injector was operated in the in the split mode (split 1 :20) with an injection volume of 1 ⁇ and an injector temperature of 250 °C.
- the column used was a 30m x 0.25 mm i.d. fused-silica capillary column DB-624 (Jennings & Wilkins) with a stationary phase thickness of 1 .4 ⁇ .
- Hydrogen was used as the carrier gas with a pressure of 100 kPa.
- the temperature of the column was initially set at 40°C (3 min hold), followed by a linear temperature gradient of 26.67 °C/min to 240 °C, at which value it was held for 3.5 min.
- the detector temperature was set at 250°C.
- Dionex Chromeleon software was employed for data processing.
- the milk used for the Dahi application test was composed of 1 1 % buffalo milk powder, 4% skimmed cow milk powder and 85% water, resulting in 4.5% protein and 3% fat milk.
- a commercially available yoghurt culture DELVO ® -YOG SVV-1 1 (DSM Food Specialties - Delft, the Netherlands), comprising the thermohiles Streptococcus thermophilus and Lactococcus delbrueckii subsp. bulgaricus was used for the acidification and dosed to the milk at 3 U/1000 L final concentration.
- Lactococcus lactis subsp. lactis biovar. diacetylactis cultures were pregrown overnight (-16 hours) at 30°C in 10% (w/v) RSM. The cultures were dosed to the milk at 1 % (w/v) final concentration. Dahi fermentation was performed at 40°C until pH 4.8 is reached. In all cases the time to reach pH 4.8 was ⁇ 5 hours. Dahi products were cooled overnight at 13°C and stored at 9°C for 2 weeks. Diacetyl and acetoin concentrations were determined in Dahi products at day 1 and at 1 week and 2 weeks storage period using GC-FID test method described above.
- Table 2 Diacetyl, acetoin and acetaldehyde concentrations (mg/kg) determined in produced Dahi products after 1 day, 1 week and 2 weeks storage at 9°C. Cultures and inoculation percentages used to prepare Dahi are indicated.
- Dahi made with cultures CBS 130345 and CBS 130347 contain a high level of acetoin in the range of 151 up to 350 mg/kg at day 1 . Furthermore, in case of Dahi prepared with CBS 130345 the concentration of acetoin increased during storage up to 507 mg/kg.
- Diacetyl concentrations in Dahi products at day 1 were in the range of 14-19 mg/kg and again in case of Dahi prepared with CBS 130345 the diacetyl concentration increased up to 27 mg/kg at 2 weeks storage.
- the concentrations of acetaldehyde are constant in time and equal in both experiments.
- the acetaldehyde is produced by the yoghurt culture used in the experiments which is the same in both experiments.
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Abstract
The present invention relates to a lactic acid bacterial strain characterized in that it is capable of producing at least 100 mg/l acetoin and/or at least 6mg/l diacetyl in Test A. The invention relates further to a process for producing Dahi or Curd at 40°C using said lactic acid bacterial strain.
Description
PROCESS FOR THE PRODUCTION OF A FERMENTED MILK PRODUCT
Field of the invention
The present invention relates to a process for manufacturing fermented milk products as well as to lactic acid bacteria capable of producing diacetyl and/or acetoin at thermophilic temperature conditions.
Background of the invention
Lactic acid bacteria are extensively used for production of fermented foods and they greatly contribute to flavor, texture and overall characteristics of these products. An old and well known example is yoghurt which probably originated from the Middle East and which still makes up more than half of the fermented milk production - or approximately 19 million tons in 2008 (source: Euromonitor). Fermented milks as e.g. yoghurts are popular due to the healthy image and pleasant sensory properties.
Initially the souring of milk was done by natural fermentation. With the advent of the knowledge of bacteriology and nutrition, fermentation process emerged as a well developed controlled sequence of changes in milk with the use of selective microorganisms. Selective organism produces lactic acid and may impart other ben eficia l effect to the prod uct. Th e use of d ifferent microorgan isms led the development of a wide range of milk products viz. dahi, yoghurt, shrikhand, lassi, kefir, koumiss, yakult, laben, etc. In India, Dahi is being produced with varieties of taste varying with region-to-region and individual food habits ("METHODS OF MANUFACTURE OF FERMENTED DAIRTY PRODUCTS" taken from http://vedvadhara.iqnou.ac.in/wiki/imaqes/d/d8/MCS-023- Unit2(Block 2).pdf last modified July 201 1 ).
Dahi is a popular fermented milk in India, Nepal and other Asian countries It is still made in every household in villages as well as in urban areas using traditional methods. Dahi is also known as "Curd", in particular in the southern part of India. The product is typically made from pasteurized or boiled milk, inoculated with dahi as starter left over from the previous day. Incubation is carried out at ambient temperature usually overnight. From Manufacturing yogurt and fermented milks (Ramesh C, Chandan
editor), 2006, Blackwell Publishing, Iowa, USA; E.R. Vedamuthu - chapter 6 and 19 and Nagenadra P. Shah - chapter 21 .
In contrast to the household production, more controlled processes for the dahi making in the organized (industrial) sector have been developed. Fresh, sweet, good quality milk is received, pre-heated and subjected to filtration and clarification. The milk is standardized to 2.5 to 3.0 % fat and 8.5-10.5% solids not fat, pre-heated to 60-65°C and homogenized. Homogenization is typically a two-stage process, with a first stage at 1500 psi (100 bar) and a second stage at 500 psi (35 bar) both at 60-65°C
Subsequent pasteurisation may be carried out at any suitable combination of time and temperature, for example at 85-90°C for 15-30 minutes or at 90-95°C for 5-10 min. After cooling down to 22-25°C and the milk is inoculated with 1 -2 percent of specific dahi starter culture. It is then put in suitable packaging containers of the appropriate size and incubated at 22-25°C for 16-18 hours. After proper setting of the dahi, the acidity of dahi reaches 0.6 to 0.7 percent and a firm curd is formed. The curd is cooled, for instance by circulating chilled water or air (a blast chiller) around the containers and then transferred to the cold room maintained at about 4-5°C, or the containers are transferred the cold room directly.
Dahi is often described as the Indian equivalent of yogurt. In reality, however, there are distinct differences between the two products. In terms of flavour, yogurt is characterized by a sharp acid tartness and the characteristic "green" acetaldehyde flavour. Dahi on the other hand has a mild acidity and diacetyl is the prominent flavour component. The latter contributes to a buttery, nut-meat like flavour. Other flavour compounds such as acetoin, acetic acid, alcohol and carbon dioxide impart a balanced round flavour to dahi. The difference in the flavour between yogurt and dahi is caused by the different starter cultures used.
For the production of dahi at the lower temperatures (e.g. 22-25°C), mesophilic lactococci and in some instances leuconostocs are used as starter flora. The basic role of starter culture is to bring about acid coagulation of milk and impart the characteristic flavour. Presence of more at least one type of lactose fermenting microorganism in the starter culture is required for the production of diacetyl flavour in dahi. A lactic culture comprising of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis biovar. d i acetyl a ctis may be used for dahi production.
Recent developments in the industrial production of dahi have led to production processes at higher temperatures (i.e. from ambient temperature to temperatures between 37 and 45°C) in combination with shorter incubation times (from overnight fermentation to 4-8 hours).
Under these conditions thermophilic starters are used (e.g. Streptococcus thermophilus and Lactococcus delbrueckii subsp. bulgaricus) leads to the formation of acetaldehyde as in yogurt. For dahi this is not desirable. The known diacetyl producing mesophilic leuconostoc and Lactococcus lactis subsp. lactis biovar. diacetylactis strains do not grow sufficiently at the higher process temperatures and therefore the industrial dahi produced at high temperature and short incubation time contain hardly if any of the typical "dahi" flavour in terms of diacetyl and/or acetoin.
It is an object of the present invention to provide diacetyl and/or acetoin producing lactic acid bacteria which are sufficiently active at the conditions of the industrial dahi process and also to provide a process for the production of dahi with the typical dahi flavour in an industrial process at higher temperatures and shorter times.
Detailed description of the Invention
In a first aspect the invention provides a process for making a fermented milk product comprising the following steps: (a) adding one or more thermophilic lactic acid bacterial strains to milk for the acidification of the milk; and (b) adding one or more lactic acid bacterial strains for the production of diacetyl and/or acetoin that it is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A; and (c) carrying out the fermentation process in step (a) and in step (b) at a temperature range of 37 to 45°C during a period of 8 hours or less. In a preferred embodiment, the fermented milk product is dahi or curd . Dahi and curd and other fermented milk products have been described in the background of the invention.
The temperature range of the fermentation process in step (a) and in step (b) is between 37 to 45°C. These higher temperatures, in particular for step (b), are preferred because this allows the process of the invention to be carried out for shorter times which is an advantage for the dairy industry. Preferably the temperature range is of 38 to 45°C, more preferably the temperature range is of 39 to 45°C, more preferably the temperature range is of 40 to 45°C. The fermentation process of the invention comprising step (a) and step (b) is carried out during a period of 8 hours or less. The
time should be sufficient to obtain the required acidification as expressed by the pH value of the milk after fermentation in step (a) and for the production of diacetyl and/or acetoin in step (b). For example, the fermentation process comprising step (a) and step (b) may be carried out during a period from 2-8 hours, preferably 3-8 hours, more preferably 3-7 hours, more preferably 4-8 hours, more preferably 4-7 hours. The skilled person is very well capable of determining the optimal temperature and time, in combination with the dosage of the one or more thermophilic lactic acid bacteria strains in step (a) and the one or more lactic acid bacterial strains for the production of diacetyl and/or acetoin in step (b) in order to obtain sufficient acidification as well as sufficient formation of the flavours such as acetoin and diacetyl.
For the acidification of the milk under the conditions of the process of the invention, regular thermophilic lactic acid bacteria may be used. These bacteria are well known in the art and commercially available. Preferably, thermophilic lactic acid bacterial strains are selected from the group consisting of Streptococcus thermophilus and Lactococcus delbrueckii subsp. bulgaricus. The dosages of said thermophilic lactic acid bacteria are known in the art.
For the production of the flavours acetoin and diacetyl in the fermented milk product, the process of the invention comprises adding one or more of the lactic acid bacterial strains in step (b) characterized in that the lactic acid bacterial strain is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A, i.e. at a temperature of 40°C which is the temperature of Test A. Preferably the acetoin is produced at least at 150 mg/l, more preferably at least at 200 mg/l, more preferably at least at 250 mg/l, more preferably at least at 300 mg/l more preferably at least at 350 mg/l. Preferably, the diacetyl is produced at least at 9 mg/l, more preferably at least at 12 mg/l, more preferably at least at 15 mg/l and more preferably at least at 18 mg/l. These diacetyl values are in addition to the diacetyl that may be present in the milk used in Test A. Test A has been described in the MATERIALS and METHODS section.
The lactic acid bacterial strain capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A and which is added in step (b) of the process of the invention, may belong to the genera Lactococcus and is preferably a Lactococcus lactis subsp. lactis biovar diacetylactis. The advantage of these lactic acid bacterial strains is their capability of producing flavours at the temperature of the process of the invention, i.e. between 37 and 45°C; as measured in Test A. Most preferred are the
following strains: Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130345, Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130346 or Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130347. It was surprisingly found that these strains were able to produce flavours such as acetoin and/or diacetyl at the temperature of the process of the invention, i.e. between 37 and 45°C. The dosages of said one or more of the lactic acid bacterial strain added in step (b) of the process of the invention may be determined by the skilled person and will depend also on the fermentation temperature and time of the process of the invention.
The fermented milk product that may be produced by the process of the invention, preferable dahi or curd, comprises preferably at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl. Preferably the acetoin is at least 150 mg/l, more preferably at least 200 mg/l, more preferably at least 250 mg/l, more preferably at least 300 mg/l more preferably at least 350 mg/l. Preferably, the diacetyl is at least 9 mg/l, more preferably at least 12 mg/l, more preferably at least 15 mg/l and more preferably at least 18 mg/l.
In a second aspect the invention provides Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130345 and Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130347.
In a third aspect, the invention provides a fermented milk product obtainable by the process of the invention. Preferably the fermented milk product is Dahi or Curd. Dahi and Curd and other fermented milk products have been described in the background of the invention. The fermented milk product that may be produced by the process of the invention, preferable dahi or curd, comprises preferably at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl. Preferably the acetoin is at least 150 mg/l, more preferably at least 200 mg/l, more preferably at least 250 mg/l, more preferably at least 300 mg/l more preferably at least 350 mg/l. Preferably, the diacetyl is produced at least 9 mg/l, more preferably at least 12 mg/l, more preferably at least 15 mg/l and more preferably at least 18 mg/l.
In a fourth aspect, the invention provides the use of a lactic acid bacterial strain that it is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A for the production of diacetyl and/or acetoin in the process of the invention for the production of a fermented milk product, preferable dahi or curd, comprising preferably at least 1 00 mg/l acetoin and/or at least 6 mg/l diacetyl , preferably the
acetoin produced is at least 150 mg/l, more preferably at least 200 mg/l, more preferably at least 250 mg/l, more preferably at least 300 mg/l more preferably at least 350 mg/l. Preferably, the diacetyl produced is at least 9 mg/l, more preferably at least 12 mg/l, more preferably at least 15 mg/l and more preferably at least 18 mg/l.
In a fifth aspect, the invention provides a method for the selection of a lactic acid bacterial strain that it is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A subjecting a population of lactic acid bacteria to Test A.
MATERIALS AND METHODS
Cultures
All strains were deposited on 24 August 201 1 at the Centraalbureau voor Schimmelcultures (Fungal Biodiversity Centre), Uppsalalaan 8, 3508 AD, Utrecht, The Netherlands.
Test A
1 % (v/v) of a freshly prepared preculture that was grown overnight at 30°C in 10% (w/v) RSM (reconstituted skimmed milk powder) + 0.05% (w/v) yeast extract, was inoculated into fresh retail pasteurized milk and subsequently incubated for 24 hours at 40°C. As a control the test was carried out at 30°C. Diacetyl, acetoin and acetaldehyde concentrations in the fermentation samples were determined using the GC-FID method (below).
GC-FID method (Gas Chromatography I Flame Ionization Detector)
Of each fermentation sample, 250 mg was accurately weighed into a suitable vial and 1 ml of internal standard solution (50 ppm 3-methyl-2-pentanone in acetonitrile) was accurately added. The sample was homogenized by vortexing and stored in a refrigerator for 30 min. The samples were subsequently centrifuged at 2000 rpm and the supernatant was further analysed. The quantification of the components
acetaldehyde, diacetyl and acetoin in the fermentation samples was done by standard addition.
Standard solutions (solutions of a known concentration of analyte) were added to the sample (unknown solution). The initial concentration in the sample was determined by extrapolation . The analysis was done on an Agilent 7890 A GC equipped with a combi Pal liquid sampler, a split/splitless injector and an FID detector. The injector was operated in the in the split mode (split 1 :20) with an injection volume of 1 μΙ and an injector temperature of 250 °C. The column used was a 30m x 0.25 mm i.d. fused-silica capillary column DB-624 (Jennings & Wilkins) with a stationary phase thickness of 1 .4 μηη. Hydrogen was used as the carrier gas with a pressure of 100 kPa. The temperature of the column was initially set at 40°C (3 min hold), followed by a linear temperature gradient of 26.67 °C/min to 240 °C, at which value it was held for 3.5 min. The detector temperature was set at 250°C. Dionex Chromeleon software was employed for data processing.
EXAMPLES
Example 1
A large variety of different strains of Lactococcus lactis subsp. lactis biovar. diacetylactis were tested for their suitability for Dahi production at 40°C. For this purpose the strains were tested for their capacity to produce the flavour compounds diacetyl and acetoin in milk after incubation for 24 hours at 40°C according to Test A described above.
The results in Table 1 show that L. lactis subsp. lactis biovar. diacetylactis strains CBS 130345, CBS 130346 and CBS 130347 produce a high amounts of diacetyl and acetoin after 24 hour incubation in milk both not only at 30°C but also at 40°C. For these strains at 40°C incubation temperature, the acetoin concentration ranges from 148 to 244 mg/L. The diacetyl concentration ranges from 14 to 19 mg/L which is well above the values for the blank in which no strains were added.
In contrast, many strains were not able to produce the required amounts of diacetyl and acetoin after 24 hour incubation in milk both at 40°C and therefore were not selected as suitable candidates for Dahi application. Table 1 shows 2 examples of these "negative" strains: L. lactis subsp. lactis biovar. diacetylactis F-008 and M-77.
The relatively high diacetyl values in the blank experiment (no cultures added), indicates that the milk used may already contain diacetyl. Table 1 suggests that the "negative strains" F-008 and M-77 do not produce or at most very low amounts of additional diacetyl while the CBS 130345, CBS 130346 and CBS 130347 double the concentration to values between 14 and 19 mg/L.
Table 1. Diacetyl and acetoin concentration (mg/L) determined at end of fermentation (24 hours) in milk-grown cultures of the indicated L. lactis subsp. lactis biovar. diacetylactis strains. Fermented milks were incubated both at 30°C and 40°C. Data are the average of three independent fermentation experiments.
Example 2
Dahi application test at 40°C
The milk used for the Dahi application test was composed of 1 1 % buffalo milk powder, 4% skimmed cow milk powder and 85% water, resulting in 4.5% protein and 3% fat milk.
A commercially available yoghurt culture DELVO®-YOG SVV-1 1 (DSM Food Specialties - Delft, the Netherlands), comprising the thermohiles Streptococcus thermophilus and Lactococcus delbrueckii subsp. bulgaricus was used for the acidification and dosed to the milk at 3 U/1000 L final concentration.
The Lactococcus lactis subsp. lactis biovar. diacetylactis cultures were pregrown overnight (-16 hours) at 30°C in 10% (w/v) RSM. The cultures were dosed to the milk at 1 % (w/v) final concentration.
Dahi fermentation was performed at 40°C until pH 4.8 is reached. In all cases the time to reach pH 4.8 was ~ 5 hours. Dahi products were cooled overnight at 13°C and stored at 9°C for 2 weeks. Diacetyl and acetoin concentrations were determined in Dahi products at day 1 and at 1 week and 2 weeks storage period using GC-FID test method described above.
Table 2: Diacetyl, acetoin and acetaldehyde concentrations (mg/kg) determined in produced Dahi products after 1 day, 1 week and 2 weeks storage at 9°C. Cultures and inoculation percentages used to prepare Dahi are indicated.
The results show that Dahi made with cultures CBS 130345 and CBS 130347 contain a high level of acetoin in the range of 151 up to 350 mg/kg at day 1 . Furthermore, in case of Dahi prepared with CBS 130345 the concentration of acetoin increased during storage up to 507 mg/kg.
Diacetyl concentrations in Dahi products at day 1 were in the range of 14-19 mg/kg and again in case of Dahi prepared with CBS 130345 the diacetyl concentration increased up to 27 mg/kg at 2 weeks storage.
The concentrations of acetaldehyde are constant in time and equal in both experiments. The acetaldehyde is produced by the yoghurt culture used in the experiments which is the same in both experiments.
In addition to the measurements of the concentrations, also the flavour notes were analyzed and described. The typical Dahi flavour buttery/creamy/sweet was most pronounced in the Dahi's made with CBS 130345 followed by CBS 130347.
Applicant's or agent's file reference number 28272-WO-PCT | International application No,
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 13bis)
The indications made below relate to the microorganism referred to in the description
first mentioned on page 5 line 1.
Name of depositary institution
CENTRAAL BUREAU VOOR SCHIMMELCULTURES
Address of depositary institution (including postal code and country)
Uppsalalaan 8
P.O. Box 85167
NL-3508 AD Utrecht
The Netherlands
Date of deposit 24 August 2011 Accession Number CBS130345
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet
We inform you that the availability of the microorganism identified above, referred to Rule 13bis PCTshall be effected only by issue of a sample to an expert nominated by the requester until the publication of the mention of grant of the national patent oryvhere applicable, for twenty years from the date of filing if the application has been refused, wikdrawn or deemed to be withdrawn.
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADYfifthe indications are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS(¾ave blank if not applicable)
The indications listed below will be submitted to the International Bureau later(specify the general nature of the indicatit "Accession Number of Deposit")
For receiving Office use only For International Bureau use only
This sheet was received with the international This sheet was received by the International Bureau
application
Authorized officer Authorized officer
Form PCT/RO/134 (July 1992)
Applicant's or agent's file reference number 28272-WO-PCT | International application No,
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 13bis)
For receiving Office use only For International Bureau use only
This sheet was received with the international This sheet was received by the International Bureau
application
Authorized officer Authorized officer
Form PCT/RO/134 (July 1992)
Applicant's or agent's file reference number 28272-WO-PCT | International application No,
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 13bis)
The indications made below relate to the microorganism referred to in the description
first mentioned on page 5 line 3.
Name of depositary institution
CENTRAAL BUREAU VOOR SCHIMMELCULTURES
Address of depositary institution (including postal code and country)
Uppsalalaan 8
P.O. Box 85167
NL-3508 AD Utrecht
The Netherlands
Date of deposit 24 August 2011 Accession Number CBS130347
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet
We inform you that the availability of the microorganism identified above, referred to Rule 13bis PCTshall be effected only by issue of a sample to an expert nominated by the requester until the publication of the mention of grant of the national patent oryvhere applicable, for twenty years from the date of filing if the application has been refused, wikdrawn or deemed to be withdrawn.
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADYfifthe indications are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS(¾ave blank if not applicable)
The indications listed below will be submitted to the International Bureau later(specify the general nature of the indicatit "Accession Number of Deposit")
For receiving Office use only For International Bureau use only
This sheet was received with the international This sheet was received by the International Bureau
application
Authorized officer Authorized officer
Form PCT/RO/134 (July 1992)
Claims
1 . A process for making a fermented milk product comprising the following steps:
(a) Adding one or more thermophilic lactic acid bacteria strains to milk for the acidification of the milk; and
(b) Adding one or more lactic acid bacterial strains for the production of diacetyl and/or acetoin, that it is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A; and
(c) Carrying out the fermentation process in step (a) and in step (b) out at a temperature range of 37 to 45°C during a period of 8 hours or less.
2. A process according to claim 1 wherein the fermented milk product is dahi or curd.
3. A process accord in g to anyon e of clai ms 1 or 2 wherei n the one or more thermophilic lactic acid bacterial strains added in step (a) of the process are selected from the group consisting of Streptococcus thermophilus and Lactococcus delbrueckii subsp. bulgaricus
4 A process according to anyone of claims 1 to 3 wherein the one or more lactic acid bacterial strains added in step (b) of the process belongs to the genus Lactococcus.
5. A process according to claims 4 wherein the one or more lactic acid bacterial strains added in step (b) of the process is Lactococcus lactis subsp. lactis biovar d i acetyl a ctis.
6. A process according to claim 5 wherein the strain is selected from the group consisting of Lactococcus lactis subsp. lactis biovar diacetylactis CBS 1 30345, Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130346 and Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130347.
7. A process according to claim 6 wherein the strain is Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130345.
8. A process according to claim 6 wherein the strain is Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130346.
9. A process according to claim 6 wherein the strain is Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130347.
10. Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130345.
1 1 . Lactococcus lactis subsp. lactis biovar diacetylactis CBS 130347.
12. A fermented milk product obtainable by the process according to any of claims 1 -9 comprising at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl.
13. A fermented milk product according to claim 12 wherein the fermented milk product is dahi or curd.
14. Use of a lactic acid bacterial strain in the process of any of claims 1 -9 and that it is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A.
15. A method for the selection lactic acid bacterial strain that it is capable of producing at least 100 mg/l acetoin and/or at least 6 mg/l diacetyl in Test A subjecting a population of lactic acid bacteria to Test A.
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EP3228194A1 (en) * | 2016-04-07 | 2017-10-11 | DSM IP Assets B.V. | Fermented milk product with diacetyl produced with aid of lactase |
EP3804526A1 (en) * | 2016-04-07 | 2021-04-14 | DSM IP Assets B.V. | Fermented milk product with diacetyl produced with aid of lactase |
WO2018019656A1 (en) * | 2016-07-19 | 2018-02-01 | Fundacion Tecnalia Research & Innovation | Bacterial strain producing 2,3-butanediol and other metabolites |
US11111474B2 (en) | 2016-07-19 | 2021-09-07 | Fundacion Tecnalia Research & Innovation | Bacterial strain producing 2,3-butanediol and other metabolites |
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