Restoration of kefir grains subjected to different treatments

ORIGINAL

RESEARCH Restoration of ke?r grains subjected to different treatments

PIOTR KO?AKOWSKI*and MAGDALENA OZIMKIEWICZ

Danisco Biolacta sp.z o.o.,Innovation Department,Tuwima1A Str.,10-747Olsztyn,Poland

*Author

for correspondence.

E-mail:piotr.kolakowski@ https://www.360docs.net/doc/8c3877753.html,

ó2011Society of

Dairy Technology The aim of the study was to?nd a way to recover the quality of ke?r grains that had been subjected to the following treatments:homogenisation,rinsing the grains with water,freeze-drying and milling,freez-ing in liquid nitrogen and then frozen storage,and cool storage.The grains were studied in respect of their later replication in milk,their size and their microbiota composition.The daily transfer of treated ke?r grains,except freeze-dried ones,into fresh milk was effective in respect of the recovery of their growth dynamics,size and microbiota balance.The growth dynamics of grains in milk seems to be a very good indicator of their vital and technological functions.

Keywords Ke?r grains,Recovery,Storage,Lactic acid bacteria,Yeasts.

I N T R O D U C T I O N

Ke?r grains constitute the natural dairy culture for

ke?r manufacture.The grains are elastic,irregular

in shape,occur in clusters resembling cauli?ower

?orets,range in size from0.3to2.0cm or more in

diameter and are white or slightly yellow in colour.

They consist of complex communities of some30

species of lactic acid bacteria(LAB)and yeasts.

Depending on their origin,acetic acid bacteria may

be present.The microbiota of ke?r grains is

embedded in a gelatinous spongy matrix composed

of bacteria,yeasts,polysaccharides and other prod-

ucts of bacterial metabolism,together with a milk-

protein curd(Farnworth1999,2005;Wszolek

et al.2006).Grains are kept viable by daily trans-

fer into fresh milk(Halle et al.1994).

In dairy plants,ke?r grains are multiplied in

skimmed pasteurised milk or pasteurised milk

reconstituted from skimmed milk powder at room

temperature.The growth is a continuous process

of a daily transfer of ke?r grains into freshly pre-

pared https://www.360docs.net/doc/8c3877753.html,ually,dairy plants utilise the same

ke?r grains for many years.The grains are

replaced in extraordinary cases because of prob-

lems concerning their growth or milk fermentation

ability,contamination with foreign microbiota,

unbalanced microbiota and for taste issues with

ke?r(Koroleva1982).This happens seldom

because it is usually possible to overcome these

problems in a longer time perspective by subject-

ing ke?r grains to special treatment(Hattowska

1984).Inoculation of milk with ke?r grains results

in biomass increases of5–7%per day and in milk

acidi?cation(Libudzisz and Pi a?tkiewicz1990).

The composition of microbiota of fermented milk

and ke?r grains is similar.However,ke?r grains

can be recreated and grown only from the pre-

existing grains(Steinkraus1996).Attempts to

reproduce ke?r grains from their constituent mic-

robiota or from microbiota released into milk dur-

ing cultivation failed.Old and dried grains also

showed very little or no ability to replicate them-

selves(La Riviere et al.1967).The mechanism of

grain formation and the conditions of treatment

when they lose the ability to replicate themselves

are little known.In unfavourable conditions,ke?r

grain growth is disturbed,their appearance deteri-

orates and they lose their resilience.They shrink,

and their microbiological balance is disrupted,

whereas in favourable conditions,after multiple

passages into milk,they retrieve their typical

appearance,physiological functions and techno-

logical properties.In some cases,however,irre-

versible changes take place,which cause constant

degeneration of grains and?nally result in their

disintegration.In industrial terminology,this phe-

nomenon is described as a sickness.To obtain

and maintain high-quality ke?r,it is of utmost

importance to ensure long-term activity of the

ke?r grains,simultaneously retaining their micro-

biological balance and technological properties.

Studies of cultivation conditions of different ke?r

grains and their in?uence on grain growth dynam-

ics and?or microbiota composition have been

reported(Rea et al.1996;Garrote et al.2001;

doi:

10.1111/j.1471-0307.2011.00746.x

Schoevers and Britz2003;Ninane et al.2005;Witthuhn et al. 2005).The microbiota composition and dynamics of mass increase in ke?r grains may depend on their origin as well as media composition and cultivation conditions.For example, ke?r grains replicated in soy milk have been reported to be smaller in size than those replicated in cows’milk(Liu et al. 2002).

There are limited data describing growth ability and microbi-ota balance of ke?r grains subjected to different treatments (Garrote et al.1997).It is important to know the pretreatment conditions of ke?r grains which limit or prevent their ability to grow in milk.The aim of the study was to evaluate the recovery ability of ke?r grains subjected to different treatments followed by their daily transfer into milk.Ke?r grains were subjected to the following treatments:homogenisation,rinsing with water, deep-freezing in liquid nitrogen and then1,5and10years fro-zen storage at)50°C,storage at4°C as well as freeze-drying and milling.The growth dynamics of ke?r grains in milk,size and their microbiota composition were studied.Additionally, the microbiota composition of the fermented milk,after ke?r grains were removed,was evaluated.

M A T E R I A L S A N D M E T H O D S

Ke?r grains

Fresh active ke?r grains manufactured by Danisco Biolacta, Poland,were added to reconstituted pasteurised(95°C for 30min)skim milk(100g?L)and incubated at18°C.After 24h,the grains were sieved out and placed again in1000mL of freshly prepared pasteurised milk.This treatment was repeated for?ve subsequent days until ke?r grains doubled their weight(200g?L).The studies were carried out on sieved-out ke?r grains that are dried on tissue paper and obtained after ?ve subsequent days of their incubation in milk.

Ke?r grains treatment

Rinsing

The grains were rinsed with ultra-pure water at18°C in1:5 ratio(w?v)and separated by?ltration through a plastic sieve. The procedure was repeated four times and,last of all,the grains were dried on tissue paper.

Storage at cool temperature

The grains were gently mixed with sterilised0.9%(w?v) sodium chloride solution in1:2ratio and stored at4°C for 14days.Before use,the grains were separated from solution with a plastic sieve.

Freezing

The grains were gently mixed with50%sterilised glucose solu-tion and10%sterilised reconstituted skim milk in2:1:2ratio (w?w?w),frozen in liquid nitrogen in200g portions and stored at)50°C for1,5and10years.The grains were defrosted in a water bath at ambient temperature and separated before use from solution with a plastic sieve.

Freeze-drying and milling

The grains were gently mixed with50%sterilised glucose solu-tion and15%sterilised reconstituted skim milk in2:1:2ratio (w?w?w),poured onto trays and freeze-dried.The parameters of freeze-drying were as follows:prefreezing to)45°C,pri-mary drying(sublimation)at vacuum0.4mbar,secondary dry-ing(desorption)at?nal vacuum0.001mbar and maximum temperature of product25°C(Telstar freeze-dryer,Spain). Before use,freeze-dried grains were milled and stored at4°C. Homogenisation

The grains were mixed with10%sterilised reconstituted skim milk in1:1ratio(w?w)and were homogenised in a Stomacher (BagMixer,Interscience,France)for20min at maximum speed.Before use,the grains were separated from solution with a plastic sieve.

Characteristics of ke?r grains subjected to treatment Determination of replication ability in milk

One hundred grams of wet grains or equivalent amount of freeze-dried grains subjected to treatment as well as nontreated fresh ke?r grains was used for inoculation of1000mL of pas-teurised milk.After each24h of incubation,the ke?r grains were separated by?ltration with a plastic sieve with double layer of gauze and were dried on tissue paper.Then the grains were weighed and added to freshly pasteurised milk.This pro-cess was repeated until ke?r grains doubled their mass.Then the grains were divided in two samples.The100g sample of the ke?r grains was incubated again in freshly prepared milk to duplicate the yield of grains.The second sample was used for further analysis.The multiplication of ke?r grains was contin-ued to obtain a double mass after?ve subsequent transfers into milk.

The number of culturing days,number of mass duplication cycles essential for the restoration of growth dynamics as well as the increase in grains mass expressed in g?L were recorded. Microbiota composition

The microbiota of ke?r grains and the postharvest broth was determined.For enumeration of viable micro-organisms,10g of ke?r grains were homogenised in90mL of sterile saline solution(9g?L)for15min at maximum speed in a Stomacher (BigMixer,Interscience,France).The postharvest broth was used without any pretreatment.The concentrations of the viable LAB and yeasts in the suspensions were determined in the mass by serial plating dilutions on the proper media.Nickels-Leesment medium(N-L)in own modi?cation for the total count of LAB(aerobic incubation,at temperature25°C for 72h)(Nickels and Leesment1964)and Nickels-Leesment medium with200l g?mL?lter-sterilised vancomycin(Sigma)

for Leuconostoc and mesophilic Lactobacillus(aerobic incuba-tion,at25°C for5days)were used(Kolakowski et al.2004). Yeast counts were determined on yeast extract chloramphenicol agar(aerobic incubation,at25°C for120h)as described by Rea et al.(1996).The results were expressed as colony-form-ing units(cfu)per1g of ke?r grains or per1mL of postharvest broth.

R E S U L T S A N D D I S C U S S I O N

Multiplication of pretreatment ke?r grains in milk

The active untreated ke?r grains double their weight on a regu-lar basis after?ve subsequent transfers into milk.The weight of ke?r grains increased with?ve successive transfers(linear func-tion of the transfers–Figure1).The mass of ke?r grains was higher in?ve subsequent transfers into milk by21.9,16.5, 15.2,13.8and12.9%,respectively(Figure1).The data clearly indicated that growth dynamics was correlated with the ratio of ke?r grains amount to milk sample volume.Changes in size and appearance of ke?r grains did not vary signi?cantly during subsequent transfers into milk.

Only the process of rinsing with water,among the applied treatments,accelerated the growth dynamics of the ke?r grains. Four transfers were enough to obtain a double increase in ke?r grains mass in comparison with the5days needed for the grains not subjected to rinsing(Figure1).This can be explained by the fact that rinsing water removes the surface microbiota and its metabolites,which can facilitate the access of grains microbiota for limited milk nutrients.

The frozen grains,after defrosting in a water bath at ambient temperature,retained their size and appearance,and they were only a little bit less elastic.However,the growth of defrosted grains was retarded.The longer the storage time,the more incu-bation days to double the mass and longer period for growth recovery dynamics were needed.The grains frozen in liquid nitrogen and then stored at)50°C for1,5and10years dupli-cated their weight after7,9and12successive transfers into milk,respectively(Figure1).The number of days needed to duplicate ke?r grains’mass in milk systematically diminished with the time of incubation.The growth dynamics,resulting in the duplication of mass after5days,was reached after18,33 and45days of incubation for the grains stored at)50°C for1, 5and10years,respectively(Figure2).These observations con?rmed the recovery mechanism of ke?r grains’structure subjected to freezing in liquid nitrogen even after10years of storage at)50°C.

Unexpectedly,storage of ke?r grains at4°C for14days in 0.9%sodium salt solution showed very strong negative impact on the recovery time of growth dynamics.Those treatment con-ditions were unfavourable for the grains in spite of invisible changes in their appearance.A?rst double increase in the mass was observed after12successive transfers of ke?r grains into milk(Figure1).However,full growth recovery resulting in mass duplication in?ve transfers was observed only

after Figure1Number of incubation days in milk essential to duplicate the mass of ke?r grains subjected to different treatments.

49days of incubation.In this case,recovery time was 4days longer than for 10-year stored frozen grains (Figure 2).Our results are in agreement with those previously published by Garrote et al.(1997)and Pintado et al.(1996)teams.Garrote et al.(1997)found that ke?r grains kept frozen at )20and )80°C for 120days increased their weight with subsequent transfers into milk,however with lower dynamics compared with fresh grains in ?rst transfers.Grains stored at 4°C showed negligible growth in eight subsequent passages.Pintado et al.(1996)showed that ke?r grains,stored at room temperature or at 4°C for 3months,had microbiologically different pro?les in comparison with fresh ones.

Homogenised grains duplicated their weight after eight sub-sequent transfers into milk.The growth recovery resulting in mass duplication in ?ve transfers was noted after 19subsequent days of incubation (Figure 2).The presented data showed that ke?r grains’structure was mechanically stable,and even the treatment in a Stomacher for 20min at maximum speed did not give a fully homogenised suspension.The homogenisation pro-cess reduced the size of grains from 0.5–2.5cm to 0.1–0.3cm in diameter,respectively.The phenomenon of rebuilding the size of grains was noted,and it was relatively fast.Just after three subsequent transfers,ke?r grains went back to the size of 0.6–1.2cm (Figures 3and 4).They totally recovered their size just after two subsequent duplications of mass (14days of incu-bation),and after further transfers into milk,their behaviour did not differ from that of untreated ke?r grains in respect of the size,appearance and growth dynamics.

Cultivation of grains in the fermentation tank during ke?r production process requires stirring.Shear forces created by the

stirrer can mechanically damage ke?r grain structure and can in?uence their growth dynamics.This factor should be taken into consideration when constructing the stirring system in tanks intended for cultivation of ke?r grains.

The results obtained during freezing,cool storage and homogenisation showed that some ke?r grains broke up and redissolved in milk.After the ?rst day of incubation,the weight of 10-year grains stored at )50°C,the grains stored at 4°C for 14days and the homogenised grains decreased from 100

to

Figure 2Number of incubation days in milk essential to recover the growth dynamics of ke?r grains subjected to different treatments.1,No treatment;2,Rinsing with water;3,Homogenisation;4,Storage at 4°C for 14days’;5,Freezing in liquid nitrogen and storage at )50°C for 1year;6,Freezing in liquid nitrogen and storage at )50°C for 5years;7,Freezing in liquid nitrogen and storage at )50°C for 10

years.

Figure 3Ke?r grains before

homogenisation.

Figure 4Ke?r grains after three successive transfers into milk.

83.2,75.6and69.7g,respectively(Figure1).The weight of grains stored for5year and1year increased merely from100 to106.4and111.2g,respectively,while untreated ke?r grains increased their weight from100to121.9g.Homogenisation con?rmed the dissolving phenomenon of some grains,not only old ones but also those that are mechanically damaged.

Ke?r grains subjected to freeze-drying and then milling did not form grains in milk anymore.Prolongation of incubation time in milk from24to48h as well as?ve subsequent trans-fers into freshly prepared pasteurised milk was not successful as regards the restoration of these grains.Incubation of the freeze-dried milled grains in milk was found to diminish the pH value,and it resulted in milk clotting.This con?rms that the microbiota present in freeze-dried and milled ke?r grains is active but presumably is not symbiotic anymore,or it is not mutually stimulating enough to produce some components for the construction of the grains matrix.The freeze-dried and milled grains lost their fundamental vital functions that are characteristic for untreated grains.

Microbiota of ke?r grains

Among dairy cultures,the ke?r grains’microbiota composition is the most complex one.If the culture is complex,higher prob-ability of microbiota composition changes during culture manu-facture and the fermentation process in dairy plant can occur and have in?uence on the quality of the?nal product.In gen-eral,lactic acid bacteria are more numerous(108–109),while yeasts(105–106)are less dominating in ke?r grains(Koroleva 1991).Vancomycin-tolerant LAB are characteristic for ke?r grains,and their ratio to total count of LAB is quite constant (Kolakowski et al.2004).The count of total lactic acid bacte-ria,vancomycin tolerant(Leuconostoc and mesophilic Lactoba-cillus)and yeasts in fresh active grains were 1.9·108, 2.2·107and2.3·107cfu?g,respectively(Table1).In1mL of postharvest broth,after removing the ke?r grains,the num-bers for total lactic acid bacteria and vancomycin tolerant ones were twice as high and approximately?ve-fold lower for yeasts (Table2).More than90%of colonies on N-L medium with vancomycin formed clear zone characteristics for Leuconostoc and mesophilic heterofermentative Lactobacillus.For the grains and the postharvest broth,the proportion of vancomycin toler-ant to total count of LAB was at the same level.In both cases, approximately10%of total lactic acid bacteria count consti-tuted the vancomycin tolerant ones(Tables1and2).Ke?r grains subjected to rinsing with water,freezing in liquid nitro-gen and storage at)50°C for1,5and10years,storage for 14days at4°C as well as homogenisation that have reverted to typical size and growth dynamics were characterised by approximate numbers and proportions between the determined groups of micro-organisms as opposed to untreated ke?r grains. The counts of total LAB varied in a very narrow range between

2.2·108cfu?g for grains subjected to homogenisation to 1.4·108cfu?g for grains subjected to freezing in liquid nitro-gen and stored at)50°C for a year vs1.9·108cfu?g for fresh active grains.The ratio of total LAB count,vancomycin tolerant and yeasts was10:1:1for all treated and untreated ke?r grains samples,respectively(Table1).These data have Table1Microbiota of ke?r grains,subjected to different treatments, after recovery of growth dynamics in milk

Type of ke?r

grains treatment

Microbiota(cfu?g)

Total count

of lactic

acid

bacteria

Vancomycin

tolerant

(Leuconostoc,

mesophilic

Lactobacillus)Yeasts

No treatment 1.9·108 2.2·107 2.3·107 Rinsing with water 1.6·108 2.0·107 1.8·107 Homogenisation 2.2·108 3.1·107 2.1·107 Storage at4°C for

14days

1.7·108

2.0·107 1.2·107 Freezing in liquid

nitrogen and storage

at–50°C for1year

1.4·108 1.9·107 1.4·107

Freezing in liquid

nitrogen and storage

at–50°C for5years

1.8·108 1.2·107 3.0·107

Freezing in liquid

nitrogen and storage

at–50°C for10years

1.7·108 1.4·107 1.6·107 Data represent mean value of two independent trials.

Table2Microbiota of postharvest broth obtained after recovery of growth dynamics of ke?r grains subjected to different treatments

Type of ke?r grains

treatment

Microbiota(cfu?g)

Total count

of lactic acid

bacteria

Vancomycin

tolerant

(Leuconostoc,

mesophilic

Lactobacillus)Yeasts

No treatment 4.1·108 4.2·107 5.8·106 Rinsing with water 2.9·108 3.4·107 6.1·106 Homogenisation 3.6·108 3.2·107 5.7·106 Storage at4°C for

14days

3.1·108 2.8·107 6.4·106

Freezing in liquid

nitrogen and storage

at)50°C for1year

4.4·108 4.0·107 6.0·106

Freezing in liquid

nitrogen and storage

at)50°C for5years

4.9·108 3.2·107 4.5·106

Freezing in liquid

nitrogen and storage

at)50°C for10years

3.8·108

4.0·107 6.0·106 Data represent mean value of two independent trials.

provided clear evidence that microbiota balance of ke?r grains as well as postharvest broth is probably correlated with grains growth recovery dynamics.Growth dynamics of ke?r grains seems to be a very good indicator of their vital functions.

C O N C L U S I O N S

Daily transferring of treated ke?r grains,except freeze-dried ones,into fresh milk was a very effective way to recover their growth dynamics,size and microbiota balance.The recovery time depended on the type of treatment and varied widely. Growth dynamics of grains in milk seems to be a very good indicator of their vital and technological functions.Low storage temperature allowed the preservation of ke?r grains’quality for long periods.The phenomenon of grains size rebuilding ability was distinctly noted for ke?r grains mechanically destroyed in a Stomacher.But the freeze-drying process clearly revealed the existence of critical treatment conditions that can cause the irre-versible loss of the ability to recreate ke?r grains matrix.Rins-ing the grains with water signi?cantly accelerated their growth dynamics without any negative impact on the microbiota balance.

The obtained results can be useful in respect of retaining ke?r grains’quality in long production perspective in dairy plants.

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