Direct spray drying and microencapsulation of probiotic Lactobacillus reuteri from slurry fermentation with whey.pdf

ORIGINAL ARTICLE

Direct spray drying and microencapsulation of probioticLactobacillus reuteri from slurry fermentation with wheyM. Jantzen, A. Gopel and C. Beermann

Department of Biotechnology, University of Applied Sciences Fulda, Fulda, Germany

Keywords

bacterial release, bacterial survival,

Lactobacillus reuteri, microencapsulation,

probiotic, spray drying, whey.

Correspondence

Christopher Beermann, Faculty of Food

Technology, Department of Biotechnology,

University of Applied Sciences Fulda, Mar-

quardstrasse 35, Fulda, 36039, Germany.

E-mail: [email protected]

2013/0751: received 19 April 2013, revised

10 June 2013 and accepted 20 June 2013

doi:10.1111/jam.12293

Abstract

Aims: Formulations of dietary probiotics have to be robust against process

conditions and have to maintain a sufficient survival rate during gastric transit.

To increase efficiency of the encapsulation process and the viability of applied

bacteria, this study aimed at developing spray drying and encapsulation of

Lactobacillus reuteri with whey directly from slurry fermentation.

Methods and Results: Lactobacillus reuteri was cultivated in watery 20% (w/v)

whey solution with or without 05% (w/v) yeast extract supplementation in asubmerged slurry fermentation. Growth enhancement with supplement was

observed. Whey slurry containing c. 109 CFU g1 bacteria was directly spray-dried. Cell counts in achieved products decreased by 2 log cycles after drying

and 1 log cycle during 4 weeks of storage. Encapsulated bacteria were

distinctively released in intestinal milieu. Survival rate of encapsulated bacteria

was 32% higher compared with nonencapsulated ones exposed to artificial

digestive juice.

Conclusions: Probiotic L. reuteri proliferate in slurry fermentation with yeast-

supplemented whey and enable a direct spray drying in whey. The resulting

microcapsules remain stable during storage and reveal adequate survival in

simulated gastric juices and a distinct release in intestinal juices.

Significance and Impact of the Study: Exploiting whey as a bacterial substrate

and encapsulation matrix within a coupled fermentation and spray-drying

process offers an efficient option for industrial production of vital probiotics.

Introduction

The Food and Agriculture Organization of the United

Nations and the World Health Organization underline in

their definition of probiotics that living micro-organisms

with health-promoting mechanisms have to be adminis-

tered in adequate amounts to be effective (FAO and

WHO 2001). Therefore, dietary formulations of probiotics

have to be robust against industrial production processes

in a cost-effective way and should maintain a sufficient

survival rate after oral uptake and gastrointestinal tract

(GIT) transit.

Commonly accepted bacterial counts for dietary prod-

ucts are at least 106 CFU ml1 (Kailaspathy and Chin2000). A wide range of health benefits have been

described, such as immune response-modulating proper-

ties, improving gut barrier function and protecting effects

of the colon against pathogens (Anal and Singh 2007).

Lactobacillus reuteri strains are probiotic lactic acid bacte-

ria (LAB) regularly applied to milk-related products but

also in meat, fruit and vegetable-based products. Further-

more, L. reuteri has been described to be a robust bacte-

rium feasible for large-scale cultivation techniques and

has high viability during storage and production (Casas

and Dobrogosz 2000).

Probiotic encapsulation is a multi-stage process which

includes fermentation, harvesting, resuspension in matrix

material and encapsulation. To increase process efficiency

and improve the survival of bacteria, this study aimed at

developing a microencapsulation process for L. reuteri by

spray drying directly from slurry fermentation using whey

as a culture substrate and encapsulation matrix. Several

encapsulation procedures for bacteria have been estab-

lished, predominantly emulsion, extrusion and spray

Journal of Applied Microbiology 115, 1029--1036 2013 The Society for Applied Microbiology 1029

Journal of Applied Microbiology ISSN 1364-5072

drying (Anal and Singh 2007; Rokka and Rantamaki

2010). Both extrusion and emulsion mainly generate wet

particles by cross-linking polymer systems which have to

be dried to enable handling and storage afterwards (Cook

et al. 2012).

Encapsulation by spray drying directly achieves optimal

product moisture content, which is between 4 and 7% for

storage stability (Ananta et al. 2005). Other main advanta-

ges of spray drying are its low cost and fast production of

large quantities of viable cells (De Castro-Cislaghi et al.

2012). Hereby, predominant factors for bacterial viability

loss in this process are heat, oxygen, as well as mechanical

and osmotic forces (Meng et al. 2008). In contrast, encap-

sulation with spray drying yields dried powder of small

particle sizes with a sufficient protection for the core (Anal

and Singh 2007; Cook et al. 2012).

Several spray-drying processes for dietary applications

have been established utilizing different polysaccharides

and protein compounds to protect probiotics. The pH

value profile of the gastrointestinal passage ranges from 19to 25 in the stomach, which is the most harmful for orallyapplied probiotics, up to pH values between 615 and 788in the small intestine and ending up with a slightly acidic

pH value in the colon (Cook et al. 2012). Also, digestive

enzymes, such as gastric pepsin and pancreatic trypsin and

chymotrypsin, pancreatin and bile salts are further antibac-

terial factors (Gbassi et al. 2011; Doherty et al. 2012).

Whey is a cheap cheese by-product containing 50% of

the milk nutrients, predominantly lactose (49% of totalwhey) and whey protein (07% of total whey) with themain protein fractions b-lactoglobulin and a-lactalbumin.With its dissimilar nutritional value, whey possesses

different interesting bio- and techno-functional proper-

ties. Aside from specific lipids, vitamins, minerals and

high-energetic lactose, whey has been discussed to be an

attractive source of functional proteins and peptides with

distinct amino acid profiles (Smithers 2008). To con-

tinue, whey is thermostable and possess excellent gelation,

water binding, emulsification as well as foam forming

properties and foam formation with thermal stability

(Foegeding et al. 2002; Smithers 2008). Recent studies

suggest that whey protein resists against acidic milieus

(Doherty et al. 2012). Gbassi et al. (2009) determined no

damaging effect at pH 18. The stability against pepsin ismore complex as a-lactalbumin breaks down in the pres-ence of pepsin, while the main whey protein b-lactoglob-ulin stays intact (Gbassi et al. 2009).

Considering the physiological and technological poten-

tial, whey might be useful as a bacterial substrate and

encapsulation matrix within a coupled fermentation and

spray-drying process to offer an efficient option for

industrial production of vital probiotics. For this, the

growth of L. reuteri in whey was characterized in batch

slurry fermentation process coupled with direct encapsu-

lation of bacteria by spray drying. At last, physical prod-

uct characteristics and bacterial protective properties

against GIT conditions were investigated in vitro.

Materials and methods

Bacterial strain

Probiotic Lactobacillus reuteri (DSM 20016) was obtained

from the German Collection of Microorganisms and Cell

Cultures Leibnitz Institute DSMZ and cultured aero-bically in 9 ml de ManRogosaSharpe (MRS) broth (pH62) at 37C for 24 h.

Growth of Lactobacillus reuteri in whey

To characterize the growth of L. reuteri in whey,

approximately 103 CFU ml1 bacteria were inoculated ina 200-ml-volume fermentation of watery 20% (w/v) whey

solution (pH 60) with or without 05% (w/v) yeastextract as supplement and cultivated in a agitating flask

culture at 37C. Before inoculation, whey solution wassterilized in 80C water bath for 20 min. Bacterial cellcount was measured after 0, 24, 48 and 72 h by direct

plate counting as described below.

Batch slurry fermentation of Lactobacillus reuteri in a

continuous stirred-tank reactor (CSTR)

The growth of L. reuteri was observed in a laboratory-scale

reactor (Biostat A, Sartorius Ltd., Melsungen, Germany).

Approximately 104 CFU ml1 bacteria were inoculated in1 l batch slurry fermentation of watery 20% (w/v) whey

solution with 05% (w/v) yeast extract as supplement,tested with adjustment at pH 50 and cultivated at 37Cwith agitation using one six-wing rotating disc at 200 rpm,

respectively. Samples of 5 ml were taken to determine cell

counts after 0, 24, 48 and 72 h by direct plate counting as

described below.

Microencapsulation of Lactobacillus reuteri in whey

For the spray-drying process, a 48-h-incubated 200-ml

slurry culture of L. reuteri with at least 108 CFU ml1 wasapplied. The fermented slurry was spray-dried by a labora-

tory-scale spray dryer (Buchi mini spray dryer B190; Flawil,

Switzerland) with two different outlet temperatures main-

tained at 55 2 and 65 2C and a flow rate of 500 Nlh1. Outlet temperatures were reached by adjusting differ-ent inlet temperatures (89 1 and 100 1C) and feedlevels from 2 to 4 ml min1. Spray-dried powder sampleswere collected from the cyclone and mixed gently.

1030 Journal of Applied Microbiology 115, 1029--1036 2013 The Society for Applied Microbiology

Encapsulation of Lactobacillus from fermentation with whey M. Jantzen et al.

Enumeration of surviving cells

To validate the survival rate of bacteria during drying

process, cell counts were observed before and after spray

drying. To detect the viable number of bacteria, direct

plate counting was used. Samples were serially diluted in

Ringers solution (pH 70) and plated on MRS agar (pH62). After 48-h incubation at 37C under anaerobic con-ditions, the cell counts were expressed in CFU g1.Spray-dried samples were previously rehydrated in

Ringers solution at a solid content of 20% (w/v), and

the solution was used to determine cell survival. Cell

counts were expressed as mean values of duplicate

measurements.

Moisture content and particle size of spray-dried

powders

The moisture content of spray-dried powders was deter-

mined using the moisture analyser (MA 40, Sartorius

AG, Gottingen, Germany) at 80C drying temperature.Data are expressed as mean value of double-tested 1-g

product.

Particle size was analysed using the Mastersizer 2000

(Malvern Instruments Ltd., Malvern, Worcestershire,

UK). Data are expressed as mean value of triple determi-

nations.

Survival of encapsulated Lactobacillus reuteri during

storage

The spray-dried product was stored in plastic tubes at

4C. Survival of encapsulated L. reuteri during storagewas determined directly following the drying process and

after 1 week and 4 weeks of storage, by performing direct

plate counting as described above.

Survival and release of encapsulated and

nonencapsulated Lactobacillus reuteri in simulated

gastrointestinal conditions

To estimate the tolerance of encapsulated cells to simu-

lated digestive conditions, an assay was adapted by using

a modified version of the method according to Picot

and Lacroix (2004). The cultures were exposed to simu-

lated gastric juice (pH 19) and afterwards to simulatedsmall intestinal juice (pH 75) at 37C. Simulated gastricjuice preparation contained pepsin (0304 g l1) (por-cine gastric mucosa, P7012, Sigma-Aldrich, Taufkirchen,

Germany) dissolved in sterile 01 mol l1 HCl/1 mol l1NaOH to adjust pH to 19. Simulated pancreatic juicepreparation contained pancreatin (195 g l1) (porcinepancreas, P1750, Sigma-Aldrich) dissolved in sterile

sodium phosphate buffer (002 mol l1, pH 75)adjusted with 01 mol l1 HCl/1 mol l1 NaOH to pH75. A concentrated bile salt solution contained bileextract powder (150 g l1) (bile bovine, B3883, Sigma-Aldrich) in sterile distilled water.

To examine the survival of encapsulated L. reuteri,

50 g of the dried product containing 107 CFU g1bacteria was gently mixed with 30 ml of pepsin prepara-

tion (026 g l1, 37C) in a 50-ml sterile plastic tube.The resulting dispersion was incubated at 37C in a250 rpm shaking thermo element (ThermoMixer and

BlockThermostate, HLC BioTech, Bovenden, Germany).

After 30 min, the reaction was stopped by raising the pH

to 75. A sample of 15 ml was taken and kept on icebefore viable cell counts were determined. A volume of

25 ml of concentrated sodium phosphate buffer(05 mol l1, pH 75) and 10 ml of the concentratedbile salt solution were added (333 g l1). After adjustingthe pH to 75 and filling the volume to 405 ml withsterile distilled water, 45 ml of the simulated pancreaticjuice (195 g l1) was added to make the final volume ofthe tube to 45 ml. At different time intervals (1, 2, 3,

5 h), 15-ml aliquots were taken and placed on ice beforebacterial enumeration. Except gentle shaking, no disper-

sion step was conducted to estimate the release proper-

ties of the microcapsules at different time intervals.

For comparison, nonencapsulated bacteria cultured in

MRS broth (37C, 24 h) were washed and resuspendedin sterile 085% saline. Five millilitres of cell suspensioncontaining approximately 106 CFU ml1 bacteria wasadded to 300 ml of pepsin preparation (026 g l1,37C) in a 50-ml sterile plastic tube and maintained at37C in a shaking thermo element, as described above.The rest of the procedure was same as that described for

the encapsulated cells.

Enumeration of bacteria from samples taken during

simulated digestion with encapsulated and nonencapsu-

lated cells was carried out by direct plate counting as

described above. The percentage surviving bacteria was

calculated as percentage survival = N/N0 9 100, whereN0 represents the number of bacteria in the inoculum

and N is the viable number of bacteria in the digestion

juice.

Statistical analysis

Data were evaluated by GraphPad 5 Prism software

(GraphPad Software, Inc., La Jolla, CA, USA). P-values

were determined by one-sided nonparametric WilcoxonMannWhitney U-test for tolerance to simulated gastricjuice and by two-sided nonparametric WilcoxonMannWhitney U-test for storage, moisture content and particle

size. P-values 005 were defined as significant.


M. Jantzen et al. Encapsulation of Lactobacillus from fermentation with whey

Results

Growth of Lactobacillus reuteri in whey

The initial experiment was accomplished to define the

growth capacity of L. reuteri (DSM 20016) in whey. For

this, a watery 20% (w/v) whey solution with or without

05% (w/v) yeast extract as supplement was tested. Underboth culture conditions, the stationary phase of bacterial

growth kinetics of batch culture was reached after 48 h of

fermentation in agitating flask at 37C. After 72-h cultur-ing, the end cell counts in pure whey solution increased 4

log cycles, whereas the bacterial counts increased 5 log

cycles by supplementing 05% yeast extract to whey.

Batch slurry fermentation of Lactobacillus reuteri in a

CSTR

Yeast extractsupplemented whey solution was testedusing batch slurry fermentation in a CSTR. As sufficient

agitation of whey slurry was reached at 200 rpm, station-

ary growth phase was reached after 24 h of cultivation

with 17 9 109 CFU g1 bacteria.

Survival of Lactobacillus reuteri in whey encapsulation

after spray drying

Lactobacillus reuteri was spray-dried at 55 and 65C tem-peratures using the whole slurry fermentation with whey

directly as feed. Loss of viable bacteria after spray drying

is illustrated in Fig. 1. For all experiments, whey solutions

with 16 ( 15) x 109 CFU g1 bacteria were applied tothe encapsulation process. Dried product contained 25( 17) x 107 CFU g1 bacteria directly after processingindependently from used outlet temperatures.

Characterization of encapsulated particles

To compare physical characteristics, moisture and particle

size of the spray-dried products were determined

(Table 1). The moisture content of dehydrated products

is of importance for product and bacterial stability while

storage. Optimal moisture content is between 4 and 7%

for storage (Ananta et al. 2005). Powder manufactured in

this study had a mean moisture content of about 65(07)%. There is no significant difference in moisturecontent of the powder produced at 55 or 65C outlettemperature of the process. The obtained particles had a

mean diameter of 55 (03) lm at 55C and 49 (03)lm at 65C again with insignificant differences withregard to the outlet temperatures. Achieved size is big

enough to encapsulate rod-shaped L. reuteri cells with

about 2 lm length (Muthukumarasamy et al. 2006).

To describe product stability during storage at 4C,survival of encapsulated bacteria was determined after

1 week and 4 weeks. The bacterial counts of the pro-

duced capsules decreased by 1 log cycle after a storage

period for 4 weeks (Fig. 1).

Survival and release of encapsulated and

nonencapsulated Lactobacillus reuteri in simulated

gastrointestinal conditions

To ensure the physio-functional benefits of L. reuteri, a

sufficient bacterial survival rate along the stomach and

intestine passage is essential to enable probiotic coloniza-

tion of the colon. Figure 2 examines the survival of free

and encapsulated cells after exposure to artificial digestive

juices, first 30 min in simulated gastric juice and after-

wards in simulated intestinal juice for a maximum of 5 h

in vitro.

Inoculation in artificial gastric juice resulted in a

decline in viable cell counts of L. reuteri. Observed cell

counts after 30 min were 12% of the applied bacteria for

free cells and about 26% for encapsulated ones. After

changing to simulated intestinal juice, the number of via-

ble cell counts for nonencapsulated bacteria remained

constant. In contrast, cell counts for encapsulated ones

Before After 7 days 28 days100

102

104

106

108

1010

Process Storage

CFU

g1

Figure. 1 Cell counts of Lactobacillus reuteri before and after spray-

drying process with ( ) 55C and ( ) 65C outlet temperatures

and during storage for 7 and 28 days at 4C storage temperature.

The data shown represent the mean of at least three measurements.

Table 1 Moisture content and particle size of spray-dried powder

directly from slurry fermentation in whey 20% (w/v) with 05% (w/v)yeast extract supplementation

Process outlet temperature (C) Moisture (%) Particle size (lm)

55 61 08 55 0365 68 03 49 03

Results for 55 and 65C are the mean of triplicate trials.



increased during exposure to intestinal juice due to a

distinct release of encapsulated bacteria reaching a sur-

vival rate of 54% after 3 h. Thus, after 5 h in digestive

juices, nonencapsulated L. reuteri revealed 86% loss of

living cells, whereas processed bacteria revealed a loss of

54% under simulated gastrointestinal conditions. There-

fore, survival rate of L. reuteri increased approximately

32% with encapsulation in whey matrix compared with

nonprocessed ones.

The statistical analysis testified the difference between

the reduction in cell counts of free and encapsulated cells

to be significant. Results show an improved survival rate

of L. reuteri in encapsulated forms.

Discussion

Aside lactose, vitamins and several minerals, whey con-

tains a complex protein composition predominated by

b-lactoglobulin and a-lactalbumin. These proteins possessa distinct amino acid profile and are a significant nitro-

gen source for bacteria, whereas whey lactose is a relevant

carbohydrate source. In this study, the nutritive value of

whey and the distinct characteristics of solubility, dena-

turation, dissociation and aggregation dependent on pH

value and temperature of these components were used to

cultivate probiotic L. reuteri (DSM 20016) for a coupled

encapsulation process within this matrix by spray drying

instead of harvesting the bacteria out of the fermentation

medium and resuspending in encapsulation material. In

this study, L. reuteri reached cell counts of 22 9108 CFU g1 in pure whey. On the one hand, bacterialb-galactosidase activity is necessary to metabolize lactose,which is generally shown by L. reuteri (Hidalgo-Morales

et al. 2011). Most LABs are able to digest whey due to

the high proteolytic activity (Pescuma et al. 2012).

However, L. reuteri possesses low proteolytic activity

(Hidalgo-Morales et al. 2011), and therefore, supplemen-

tation with extra nitrogen sources might improve growth.

Parente and Zottola (1991) suggested supporting LAB

cultivation in native whey with nutritive complex addi-

tives such as yeast extract. Furthermore, an increased

whey protein concentration to raise nitrogen source for

the bacteria has been described to be helpful (Bury et al.

1998). In this study, the bacterial growth could be

improved to maximum cell counts of up to 27 9109 CFU g1 with yeast extract supplementation.For encapsulation, bacteria in stationary phase with

reduced proliferation rate, less physiological activity and

increased resistance to stress seem to be most appropri-

ate for spray drying. Corcoran et al. (2004) revealed

approximately 50% higher survival rates of L. reuteri

within spray-drying processes if bacteria were taken

from stationary-phase cultures. Further, for spray-drying

processes, matrices with 20% solid content are recom-

mended (Desmond et al. 2001; Ananta et al. 2005). In

consequence, to couple cultivation and spray-drying pro-

cess, bacterial growth up to stationary phase should be

possible in 20% whey slurry fermentation. In this study,

L. reuteri reached the stationary phase at least after 24 h

in a slurry fermentation in CSTR.

Generally, spray drying is an effective way to produce

probiotic encapsulation with high survival rates and

improved resistance during gastric transit (Malmo et al.

2011; Paez et al. 2012). One critical factor for bacterial

survival within the drying process is the exposure to hot

air which leads to heat and osmotic stress for the cells.

Otherwise, high temperatures are required to facilitate

sufficient water evaporation along the process as dried

product contains at best moisture of 4 and 7% for good

stability of dried implementations throughout storage

(Ananta et al. 2005). Hereby, heat sensitivity of the bacte-

rial strain and hygroscopic properties of the encapsula-

tion material are important factors (Ananta et al. 2005;

Golowczyc et al. 2011). Survival of heat-sensitive L. para-

casei during spray drying ranged from 97% (outlet

temperature 7075C) to 0% (outlet temperature 120C)(Gardiner et al. 2000). Other Lactobacilli strains spray-

dried in skim milk at 85C outlet temperature reachedhigher survival rates (loss of 016095) (Paez et al.2012). Malmo et al. (2011) revealed a loss of 1631 logof L. reuteri (DSM 17938) after spray drying with alginate

matrix at 72C outlet temperature. Higher survival ratesof spray-dried L. reuteri (KUB-AC5) were measured at

70C outlet temperature with a variation of 8393% sur-vival depending on the growth media (Hamsupo et al.

2005). Taking the distinct heat sensitivity of bacteria into

consideration, the viability can be enhanced by lowering

the outlet temperature (Lian et al. 2002; Meng et al.

0 1 2 3 4 50

20

40

60

80

100

05 h

Surv

ival a

nd re

leas

e %

Gastric juice intestinal juice

Figure. 2 Percentage release and survival of () encapsulated and ()nonencapsulated Lactobacillus reuteri in simulated gastric juice for

30 min and afterwards in simulated intestinal juice for a maximum of

5 h. The data shown represent the mean of at least three measure-

ments.



2008). In this study, both outlet temperatures 55 and

65C lead to sufficient dry powder with a negligible viablecell decrease of 2 log cycles (213% mean survival rate)after spray drying due to dehydration and thermal

damages of bacterial cell structures (Fig. 1).

Although small-sized particles are less affecting textural

and sensorial food qualities, the particle size must at least

enable total bacterial encapsulation (Chen and Subirade

2006). In this study, the process produced small particles

sized 5 lm in mean (Table 1), which are large enough toencapsulate L. reuteri cells (Anal and Singh 2007). Gener-

ally, decreasing moisture content correlates with an

increasing temperature in the drying process (Lian et al.

2002; Anandharamakrishnan et al. 2008). In this study,

however, neither particle size nor moisture content of

capsule parameters were significantly affected by chosen

outlet temperatures of the drying process. Desmond et al.

(2001) described the possibility that milk proteins are

useful to protect probiotic cultures during heating. Fur-

thermore, lactose from whey protein might synergistically

improve the micro-organism-protecting properties of

these proteins. Young et al. (1993) discovered synergistic

effect of whey proteins and carbohydrates for microen-

capsulation by spray drying. They described higher

microencapsulation efficiency using the combination of

whey protein and carbohydrates. Additionally, an

increased thermotolerance of LAB resulting in high

survival rates during storage was observed if growth

media contained lactose (Carvalho et al. 2004). The pro-

tection of dehydrated biomaterials by sugars is mainly

due to hydrogen bonds with the proteins when water is

removed (Rokka and Rantamaki 2010). And lactose has

been discussed to stabilize the bacterial cell wall by induc-

ing a crusty glass phase (Rosenberg and Sheu 1996). Also,

Rokka and Rantamaki (2010) underlined that skim milk

and lactose, often in combination with milk proteins, are

widely used as protective agents within spray-drying

processes.

As dietary products contain at least 106 CFU ml1 bac-teria, the survival of probiotics during storage is impor-

tant to assess product applicability (Kailaspathy and Chin

2000). Different studies have illustrated that the bacterial

survival rate was most sufficient at 4C storage tempera-ture (Corcoran et al. 2004; Silva et al. 2011). In this

study, the examined loss in viable cells was at maximum

1 log cycle after a storage period of 4 weeks at 4C(Fig. 1). Depending on the outlet temperature, the viable

counts during storage decreased at 65C by about 1 logcycle and at 55C by

probably due to whey protein degradation by pancreatin

(Fig. 2). Accordingly, Chen and Subirade (2006)

described a release of whey protein capsule core material

into simulated intestinal juice with pancreatin.

In conclusion, whey exploited as growth substrate and

encapsulation matrix within a coupled fermentation and

spray-drying process might offer an efficient option for

industrial production of vital probiotics. The resulting

microcapsules remain stable during storage and reveal ade-

quate survival in simulated gastric juices and a distinct

release in intestinal juices. While lactose is supportive but

also problematic in this coupled process, like unwanted

caramelization or stickiness to the dryer wall, the influence

of the whey-protein-to-lactose ratio on the process and

product characteristics remains to be investigated.

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