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.

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet

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.

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet

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)