Patent Application: US-29673202-A

Abstract:
the present invention relates to a method for the production of yoghurt or other fermented milk products , comprising the following steps : inoculation of a starter medium with a starter culture , said starter culture comprising an exopolysaccharide - producing micro - organism . an exopolysaccharide production step wherein the ph of said starter medium is kept stable at a predetermined ph value at a suitable temperature , and an acidification step to allow clotting of the starter medium .

Description:
this invention deals with a new process technology for yoghurt production with an exopolysaccharide - producing starter culture , namely a two - step process including an exopolysaccharide production step and an acidification step . to avoid a low and unstable exopolysaccharide production , and to maximally exploit the exopolysaccharide production capacity of the yoghurt starter strain , the following new , two - step , process technology for yoghurt production was invented : 1 ) a first step or exopolysaccharide production step : inoculation of the milk with the yoghurt starter culture including an exopolysaccharide - producing s . thermophilus strain and on line control of the ph of the fermentation medium at a fixed value situated between 5 . 5 - 6 . 5 and of the temperature at 37 - 44 ° c . usual agents as sodium hydroxide , ammonium hydroxide , calcium hydroxide , potassium hydroxide , sodium carbonate , calcium carbonate , etc . can be used for maintaining the set ph value . 2 ) an acidification step to allow clotting of the milk and improve aroma and taste formation . acidification can take place either spontaneously through prolonged fermentation or by the addition of an acidifying agent . the acidification contributes to the stability of the exopolysaccharides produced during the first process step . during a laboratory experiment , a 10 l fermentor with enriched milk medium ( 10 . 0 % ( m / v ) skimmed milk powder , 1 . 0 % ( m / v ) peptone , 0 . 5 % ( m / v ) yeast extract ) was inoculated with 1 . 0 % ( v / v ) of a freshly grown culture of s . thermophilus lmg p - 19262 . the milk was sterilised in situ by heating according to the following profile : heating of the milk at 95 ° c . during 20 min , cooling down to 20 ° c . and keeping it at 20 ° c . for 1 h , and then another heating step of 95 ° c . during 20 min , after which the milk was cooled down to the fermentation temperature of 42 ° c . the peptone and yeast extract was sterilised separately ( 20 min at 121 ° c .) and aseptically added to the fermentor . optimised fermentation conditions for maximum exopolysaccharide production were applied , i . e . a temperature of 42 ° c . and an agitation rate of 100 rpm ( to keep the fermentation broth homogenous ). the ph was kept constant at ph 6 . 2 through on line control by automatic addition of 10 n naoh . bacterial growth was followed by on line registration of the amount of naoh required to neutralize the lactic acid accumulated in the fermentation broth and was hence also a measure of the amount of lactose consumed . also cell numbers were determined to follow the course of living cells . after 7 . 5 h of growth , 135 ml of naoh was added , indicating that approximately 50 . 0 % of the lactose present in the fermentation medium was consumed . at this stage , ph control was switched off and the second step of the process ( the acidification step ) started . the ph drop was followed on line . the fermentation was stopped after 24 h when a final ph of 4 . 6 was reached . the same fermentation strategy was also applied to perform a two - step fermentation process in milk without peptone and yeast extract . during all fermentations , samples of 550 ml were taken at regular time intervals to determine the cell number , the amount of exopolysaccharides , and the viscosity of the fermentation broth . for all fermentations , the maximum specific growth rate and maximum acidification rate were estimated by linear regression from the plots of in cfu / ml versus time and in ph versus time , respectively . as a control fermentation , the same experiment was done without the first , ph - controlled process step . a 10 l fermentor with sterilised enriched milk ( see above ) was inoculated with 1 . 0 v ( v / v ) of a freshly grown culture of s . thermophilus p - 19262 , and run at a controlled temperature of 42 ° c . and an agitation rate of 100 rpm ( to keep the fermentation broth homogenous ), but without ph - control . the ph was measured on line . the fermentation was stopped after 24 h when a final ph of 4 . 2 was reached . the same fermentation strategy was also applied to perform a control fermentation in milk without peptone and yeast extract . during all fermentations , samples of 550 ml were taken at regular time intervals to determine the cell number , the amount of exopolysaccharides , and the viscosity of the fermentation broth . for all fermentations , the maximum specific growth rate and maximum acidification rate were estimated by linear regression ( see above ). cell numbers ( cfu . ml − 1 ) were determined by plate counting on solid lapt ( yeast extract , 10 g . l − 1 ; peptone , 15 g . l − 1 ; tryptone , 10 g . l − 1 ; tween 80 , 1 ml . l − 1 ; glucose , 10 g . l − 1 ; agar , 20 g . l − 1 ). isolation of exopolysaccharides was carried out using 500 ml of milk medium in four consecutive steps . first , ( milk ) proteins were removed from the fermentation liquor by precipitation with one volume of 20 % trichloroacetic acid ( tca ), followed by centrifugation of cells and proteins ( 25000 × g , 20 min , 4 ° c .). second , exopolysaccharides were precipitated overnight with an isovolume of chilled aceton , followed by centrifugation of the precipitate ( 25000 × g , 30 min , 4 ° c . ), after which the pellet was redissolved in ultrapure water . third , residual proteinaceous material was precipitated with tca and removed by centrifugation as described above . fourth , the exopolysaccharides were finally isolated by chilled aceton precipitation ( one volume ) and harvested . the exopolysaccharides were redissolved in ultrapure water , washed with aceton and then dried to determine the polymer dry mass ( pdm ). apparent viscosity measurements were carried out on 0 . 5 ml samples , using a cone - plate brookfield digital rheometer model dv iii ( brookfield engineering laboratories inc ., stoughton , mass ., usa ) equipped with a flat spindle , type cp 40 , rotating in a sample - containing chamber connected to a temperature controlled cryostat water bath . the rheometer was steered by the brookfield rheocalc software ( brookfield engineerling laboratories , inc .). to perform the apparent viscosity measurements of the fermentation samples , spindle speeds of 50 rpm were used during 45 s . the spindle speed of 50 rpm was chosen because the torque to rotate the spindle in the fluid was then between 10 . 0 % and 90 . 0 %. it corresponed with a shear rate of 375 / s . values out of this range were not valid and lead to misinterpretation of the results . the measurements were done at a constant temperature of 42 ° c . values were expressed in mpa · s . the results of the fermentation profile of both the two - step fermentation process and of the control fermentation , carried out in enriched milk medium , are represented in fig1 ( course of ph and living cell numbers ) and fig2 ( course of eps and apparent viscosity ). fig1 represents a fermentation profile of the exopolysaccharide - producing streptococcus thermophilus lmg p - 19262 strain in enriched milk medium using a two - step and a classical ( one - step ) fermentation strategy . the fermentations were carried out at a constant temperature of 42 ° c . and a constant agitation rate of 100 rpm . the ph of the first step of the two - step fermentation process was kept constant at 6 . 2 through automatic addition of 10 n naoh . values of ph , cell number ( cfu . ml − 1 ) and added naoh ( ml . l − 1 ) are displayed . all values linked to the two - step fermentation process are marked with ‘*’. the vertical line indicates the switch between the first and the second step . fig2 shows a fermentation profile of streptococcus thermophilus lmg p - 19262 in enriched milk medium using a two - step and a classical ( one - step ) fermentation strategy . the fermentation was carried out at a constant temperature of 42 ° c . and a constant agitation rate of 100 rpm . values of apparent viscosity ( mpa · s ) and amount of eps ( mg pdm . l − 1 ) are displayed . all values linked to the two - step fermentation process are marked with ‘*’. the vertical line indicates the switch between the first ( ph - controlled ) and the second ( acidification ) step . the fermentations carried out in unsupplemented milk medium are represented in fig3 ( course of ph and living cell numbers ) and fig4 ( course of eps and apparent viscosity ). fig3 is a fermentation profile of streptococcus thermophilus lmg p - 19262 in unsupplemented milk medium using a two - step and a classical ( one - step ) fermentation strategy . the fermentations were carried out at a constant temperature of 42 ° c . and a constant agitation rate of 100 rpm . the ph of the first step of the two - step fermentation process was kept constant at 6 . 2 through automatic addition of 10 n naoh . values of ph , cell number ( cfu . ml − 1 ) and added naoh ( ml . l − 1 ) are displayed . all values linked to the two - step fermentation process are marked with ‘*’. the vertical line indicates the switch between the first and the second step . fig4 depicts a fermentation profile of streptococcus thermophilus lmg p - 19262 in unsupplemented milk medium using a two - step and a classical ( one - step ) fermentation strategy . the fermentations were carried out at a constant temperature of 42 ° c . and a constant agitation rate of 100 rpm . values of apparent viscosity ( mpa · s ) and amount of eps ( mg pdm . l − 1 ) are displayed . all values linked to the two - step fermentation process are marked with ‘*’. the vertical line indicates the switch between the first ( ph - controlled ) and the second ( acidification ) step . for both fermentations carried out in enriched milk medium ( fig1 ), the maximum specific growth rates μ max were calculated as 1 . 12 h − 1 ( r 2 = 0 . 930 ) and 0 . 84 h − 1 ( r 2 = 0 . 931 ) for the ph - controlled part of the two - step fermentation and for the one - step fermentation , respectively . although a comparable maximum specific growth rate for both fermentation processes , a higher living cell number was obtained for the two - step ( ph - controlled ) fermentation experiment . for the second part ( acidification step ) of the two - step fermentation and for the one - step ( ph - free ) fermentation process , the r max values were calculated as 0 . 18 h − 1 ( r 2 = 0 . 999 ) and 0 . 21 h − 1 ( r 2 = 0 . 981 ), respectively , indicating a parallel course of ph for both fermentation experiments . however , the acidification stopped at a higher ph value for the two - step fermentation experiment as compared to the one - step control fermentation , possibly due to lactose depletion . a delay of almost 5 h was observed for the two - step fermentation process to reach a final ph and a maximum cell number . fermentations were also carried out in unsupplemented milk medium , a medium simulating a non - fortified one that may be used for commercial yoghurt production ( fig3 ). for the first ( ph - controlled ) part of the two - step fermentation process , bacterial growth was almost linear ( μ max = 0 . 14 h − 1 ; r 2 = 0 . 987 ). for the one - step fermentation , μmax was calculated as 0 . 17 h − 1 ( r 2 = 0 . 984 ); however , a higher living cell number was obtained for the two - step ( ph - controlled ) fermentation experiment as was already seen in the fermentation with enriched milk medium . it was demonstrated again that the maximum acidification rates were comparable for the second part of the two - step fermentation process and for the one - step fermentation experiment . the rmax values were calculated as 0 . 01 h − 1 ( r 2 = 0 . 999 ) and 0 . 01 h − 1 ( r 2 = 0 . 995 ), respectively . eps yields and apparent viscosity ( fig2 ) show a signifcant difference between both fermentation experiments carried out in enriched milk medium . indeed , while a maximum of 114 mg pdm . l − 1 and a corresponding apparent viscosity of 3 . 2 mpa · s was observed for the fermentation experiment with free ph - course , an increased maximum exopolysaccharide yield of 432 mg pdm . l − 1 and a concomitant apparent viscosity of 5 . 2 mpa · s was observed for the two - step fermentation process . the maximum exopolysaccharide amounts were clearly stable upon prolonged fermentation . for both experiments , the apparent viscosity of the fermentation medium parallelled the eps yields . the two - step fermentation process took a few hours more to achieve the increased eps production level and almost doubled apparent viscosity . similar observations were made for both fermentations carried out in unsupplemented milk medium ( fig4 ). a maximum of 108 mg pdm . l − 1 and a corresponding apparent viscosity of 1 . 9 mpa · s was observed for the fermentation experiment with free ph - course , while an increased maximum eps yield of 221 mg pdm . l − 1 and a concomitant apparent viscosity of 4 . 2 mpa · s was observed for the two - step fermentation process . these increased eps yields and apparent viscosities were once more at the expense of the time needed to reach the maximum values . as seen for the bacterial growth , an almost doubled fermentation time was needed . no syneresis was observed upon storage of the fermented ( un ) supplemented milk using the two - step production process as compared to the one - step fermentation process where clear water separation took place . the two - step fermentation process thus results in a product with a rather high , stable exopolysaccharides content and an almost doubled viscosity . a deposit has been made on feb . 18 , 2000 according to the budapest treaty for the microorganism streptococcus thermophilus imdo 61001 under deposit number lmg p - 19262 at the bccm / lmg culture collection , laboratorium voor microbiologie , k . ledeganckstraat 35 , b - 9000 gent ( belgium ). beal , c ., skokanova , j ., latrille , e ., martin , n . and g . corrieu . 1998 . combined effects of culture conditions and storage time on acidification and viscosity of stirred yoghurt . j . dairy sci . 82 , 673 - 681 . cerning , j . and v . m . marshall . 1999 . exopolysaccharides produced by the dairy lactic acid bacteria . rec . res . dev . microbiol . 3 , 195 - 209 . davis , j . g . 1971 . standars for yoghurt . dairy ind . 111 , 465 - 472 . degeest , b . and l . de vuyst . 1999 . indication that the nitrogen source influences both amount and size of exopolysaccharides produced by streptococcus thermophilus ly03 and modelling of bacterial growth and exopolysaccharide production . appl . environ . microbiol . 65 , 2863 - 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