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LWT - Food Science and Technology 57 (2014) 276e282

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LWT - Food Science and Technology

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Effect of lyoprotectants on b-glucosidase activity and viability ofBifidobacterium infantis after freeze-drying and storage in milk andlow pH juices

Mimoza Basholli-Salihu a,b, Monika Mueller a,*, Sharareh Salar-Behzadi c, Frank M. Unger a,Helmut Viernstein a

aDepartment of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090 Vienna, AustriabDepartment of Pharmacy, Faculty of Medicine, University of Prishtina, 10000 Prishtina, KosovacResearch Center Pharmaceutical Engineering GmbH, Inffeldgasse 13a, A-8010 Graz, Austria

a r t i c l e i n f o

Article history:Received 2 May 2013Received in revised form10 October 2013Accepted 11 January 2014

Keywords:Bifidobacteriumb-GlucosidaseFreeze-dryingViabilityFood matrix

* Corresponding author. Tel.: þ43 1427755414.E-mail address: monika.mueller@univie.ac.at (M. M

0023-6438/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.lwt.2014.01.011

a b s t r a c t

The benefit of disaccharide protectants for maintaining viability and b-glucosidase activity of Bifido-bacterium infantis UV16PR during freeze-drying and storage in different food matrices was investigated.Protectants used were cellobiose, lactose, sucrose and trehalose.

At 5% concentration, cellobiose showed the best protective effect during freeze-drying. All protectantsat 10% concentration significantly improved the viability and retention of b-glucosidase activity afterfreeze-drying and storage in food matrices. Regardless of the protectant used, no significant differenceswere observed after freeze-drying and storage for 2 weeks in milk, while in red-beet and grape juice,cellobiose and trehalose significantly enhanced b-glucosidase activity, viability and acid tolerance.Viability of cellobiose and trehalose protected cells in juices was comparable, whereas differences inretention of enzyme activity and acid tolerance after the storage in different juices were observed. Inconclusion, various protectants increased the retention of b-glucosidase activity, viability and acidtolerance of freeze-dried B. infantis during storage in different food matrices. In order to choose pro-tectants for probiotics for freeze-drying and food as probiotic carrier, not only their effect on surviv-ability, but also the enzyme activity retention should be considered.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Probiotics are defined as “live microorganisms, which whenadministered in sufficient numbers, confer a health benefit to thehost” (FAO/WHO, 2001). Thus, probiotics are more and morefrequently added to food. The most common food carriers for de-livery of probiotics to the human body are dairy products, such asfermented milk, yoghurt, cheese and ice cream (Fritzen-Freire,Muller, Laurindo, & Prudencio, 2010; Ramchandran & Shah, 2010;Ranadheera, Baines, & Adams, 2010). Much less often, juices areused as carriers and much less has been reported regarding thestability of probiotics in juices at low pH values (Champagne &Gardner, 2008; Saarela, Virkajãrvi, Alakomi, Sigvart-Mattila, &Mättö, 2006; Vinderola et al., 2012). Milk (non-fermented) and fruit

ueller).

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juices are advantageous as food carriers for probiotics because theydo not contain starter cultures which may compromise the survivalof the probiotics (Dave & Shah,1997; Ng, Yeung, & Tong, 2011). Fruitjuices have the further advantage of being suitable for people withlactose intolerance.

Probiotics are added to food mainly as frozen or freeze-driedcultures. Freeze-dried cells are preferred because they are easierto handle than frozen cells. However, during drying the cells areexposed to stress which may lead to several injuries such as mem-brane injury, protein denaturation (including that of enzymes) andDNA damage (Lievense, Verbreek, Noomen, & van’t Riet, 1994;Meng, Stanton, Fitzgerald, Daly, & Ross, 2008; Stummer et al.,2012). Incorporation of freeze-dried cells into food matrices ischallenging because of the exposure to additional stress such as pHand oxygen (Saarela et al., 2006; Vinderola et al., 2012; Champagneet al., 2005). For preservation of probiotics during freeze-drying,sucrose, trehalose and lactose are widely used (Li et al., 2011;Stummer et al., 2012; Vinderola et al., 2012). Their protective ef-fect during drying was shown to be strain-specific (Siaterlis,

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Deepika, & Charalampopoulos, 2009; Strasser et al., 2009). Sucroseand lactose also showed good protection of probiotics during stor-age in food matrices (Saarela et al., 2005; Vinderola et al., 2012).

Together with viability, the functionality of probiotics likeenzyme activity and acid tolerance has to be considered. Someprobiotic microorganisms including Bifidobacterium spp. exert b-glucosidase activity (Marotti et al., 2007; Otieno et al., 2006).Several groups reported that freeze-drying reduces enzyme activ-ities in probiotic bacteria (Heljo, Jouppila, Hatanpää, & Juppo, 2011;Li et al., 2011; Vasiljevic & Jelen, 2003). Addition of various pro-tectants may preserve enzyme activities during freeze-drying ofprobiotics to different extents (Li et al., 2011).

The b-glucosidase activity of different probiotics declines duringstorage in fermented soymilk at different temperatures; the extentof decline varies with the strain used (Otieno et al., 2005). To thebest of our knowledge, no studies have been reported on the sta-bility of b-glucosidase and the effect of different protectants onactivity retention after storage of freeze-dried Bifidobacterium indifferent food matrices.

Storage of probiotic bacteria in food matrices, especially in lowpH juices affects their survival in the gastric environment(Champagne & Gardner, 2008; Saarela et al., 2006; Vinderola et al.,2012). Lankaputhra, Shah, and Britz (1996) reported that Bifido-bacterium infantis lose more than 83% of their viability duringstorage in fermented milk at 4 �C. Therefore, reducing the loss ofviability of B. infantis is an important challenge when this species isto be incorporated into functional food.

The aim of the present study was to investigate the role ofdisaccharide protectants (cellobiose, trehalose, sucrose and lactose)on viability and b-glucosidase activity of the probiotic B. infantisduring freeze-drying and during storage in different food matrices.In addition, the influence of protectants on the acid tolerance ofB. infantis after storage in different food matrices was investigated.Furthermore, the role of cellobiose as new potential probiotic lyo-protectant, in comparison to other common protectants on thestability and functionality of B. infantiswas compared. To the best ofour knowledge, this is the first study to test cellobiose for pro-tecting viability and functionality of a probiotic strain.

2. Materials and methods

2.1. Growth of microorganisms

B. infantis (UV16PR) was kindly provided by Medipharm(Kågeröd, Sweden). The microorganisms were grown in ReinforcedClostridial Medium (RCM, Oxoid Ltd., Hampshire, UK) by incubationat 37 �C in an anaerobic jar using Anaerogen kits (Oxoid Ltd.,Hampshire, UK).

2.2. Freeze-drying

Fresh cells were harvested by centrifugation (10,000 � g, 4 �C,15 min) and washed twice with 50 mM phosphate buffer, pH 6.5.The pellets were resuspended in phosphate buffer solution con-taining 5 or 10% (w/v) sucrose (SigmaeAldrich, St. Louis, MO, USA),trehalose (Merck, Darmstadt, Germany), lactose (SigmaeAldrich)or cellobiose (Glycon Biochemicals, Luckenwalde, Germany). Thesuspensions were divided into 500 ml aliquots and frozen at �80 �Cprior to lyophilization. Freeze-drying was undertaken under vac-uum (0.3 mbar) at a condenser temperature of �54 �C using aHetoPowerDry LL3000 lyophilizer (Thermo Scientific, Waltham,MA, USA) according to previous studies (Li et al., 2011; Stummeret al., 2012). Freeze-dried cells without protectants were preparedas control.

2.3. Storage of freeze-dried bifidobacteria in juices and milk

B. infantis cells freeze-dried in the presence of different pro-tectants were incorporated into 100 ml of pasteurized low fat milkcontaining �1.5% fat prepared from skim milk powder (pH 6.7;Merck, Darmstadt, Germany) and commercial juices includinggrape juices (pH 3.4) and red-beet juice (pH 4.2) purchased fromlocal stores in Austria. The juices were stored at 4 �C for 4 weeksand the milk for 2 weeks.

2.4. Cell enumeration

Viable cells were determined using the plate count methodprior to freezing and after freeze-drying and storage in foodmatrices.

Dried cells were resuspended at initial volume with phosphatebuffer and left for rehydration at room temperature. 1 ml of rehy-drated cells or 1 ml of milk or juices were serially diluted withphosphate buffered saline (PBS) and plated onto Reinforced Clos-tridial Agar plates in triplicate. The plates were incubated underanaerobic conditions at 37 �C for 48e72 h. The viability wasexpressed as colony forming units (CFU) per millilitre. The survivalof cells after freeze-drying and storage was calculated in percent.

The pH of food matrix was measured before and after storageusing pH meter (Hanna Instruments, Woonsocket, RI, USA).

2.5. Cell free extract preparation

b-Glucosidase activity was determined in cell free extractsbefore and after freeze-drying, and before and after storage indifferent food matrices.

After freeze-drying cells were rehydrated and harvested bycentrifugation, washed twice with 50 mM phosphate buffer (pH6.5) and then resuspended in the same buffer. From food matricescells were harvested by centrifugation (10,000 � g, 4 �C, 10 min)and washed with 50 mM phosphate buffer pH 6.5. For preparingthe cell free extract, resuspended cells weremechanically disruptedby mixing (1:1.5 w/v) with sterile glass beads (200e300 mm) usinga vortex for 5 cycles of 2 minwith 2 min break on ice. Following celldisruption, the suspensions were centrifuged (17,000 � g, 30 min,4 �C) using a Sorvall� RC5C centrifuge (Thermo Scientific, Waltham,MA, USA) to remove the cell debris and glass beads. The superna-tant was then filtered using a 0.45 mm filter (Merck Millipore,Darmstadt, Germany).

2.6. Determination of b-glucosidase activity retention after freeze-drying and after storage in different food matrices

The cell free b-glucosidase activity was determined before andafter freeze-dying, and before and after storage for 4weeks in juicesand 2 weeks in milk. p-Nitrophenyl-b-D-glucopyranoside (p-NPG,SigmaeAldrich) was used as substrate. p-NPG solution (5 mM,150 ml) in 50 mM phosphate buffer pH 6.5 was mixed with 600 ml ofcell free enzyme and themixturewas incubated for 15 min at 37 �C,according to Dianawati and Shah (2011) with minor modification.The reaction was stopped on ice by adding 375 ml of cold NaOH(0.1 M). The amount of p-nitrophenol released was measured at410 nm using a Hitachi U-1100 spectrophotometer (Hitachi Ltd.Tokyo, Japan). The enzyme activity at baseline (before storage) wasdefined as 100% activity. The enzyme activity after storage wascompared to the initial enzyme activity at the baseline. Theremaining enzyme activity after storage in different food matriceswas expressed as relative retention in percent.

The b-glucosidase activity was expressed in units (1 unit is theamount of enzyme that releases 1 mM p-nitrophenol per ml per

Fig. 2. b-Glucosidase activity retention [%] of Bifidobacterium infantis after freeze-drying in the presence of 5% or 10% of cellobiose, trehalose, sucrose and lactose.Control (w/o) are cells freeze-dried without any protectant. Each bar representmeans � SD, n ¼ 3.

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min). The protein concentration was determined by the Bradfordmethod (1976) using Bradford reagent (SigmaeAldrich) and bovineserum albumin (SigmaeAldrich) as standard.

2.7. Acid tolerance

Acid tolerance was determined after 4 weeks of storage in juicesand after 2 weeks of storage in milk. In brief, 10 ml of food matrixwas added to 90 ml of phosphate buffered saline (PBS) pH 2 con-taining 0.3% (w/v) porcine pepsin (SigmaeAldrich). The pH wasadjusted to pH 2 with 0.1 M hydrochloric acid (Saarela et al., 2006;Vinderola et al., 2012).

2.8. Statistical analysis

All experiments were performed in triplicate. Statistical ana-lyses were performed using GraphPad Prism (GraphPad Software,San-Diego, CA, USA) using one-way ANOVA, and Tukey test cross-comparing all study groups. Values of p < 0.05 were consideredas significant.

3. Results

3.1. Viability of B. infantis after freeze-drying

The survival rate of cells during freeze-drying without anyprotectant was 15.8%. 5% Cellobiose showed the best protection onviability during freeze-drying, namely 86%, compared to other di-saccharides at the same concentration which ranged from 32% to54.7% (p < 0.001). At 10% concentration no significant differenceswere observed between all protectants used. The viability rangedfrom 81.2% to 88.9%. The best protection was offered by 10% cello-biose and 10% trehalose (Fig. 1).

3.2. b-Glucosidase activity retention after freeze-drying

The b-glucosidase activity retention expressed in percent isshown in Fig. 2. The remaining activity of cells without any pro-tectant was 78.9%. Cellobiose at 5% significantly (p < 0.001)enhanced the b-glucosidase activity retention to 93.4%. Theremaining enzyme activity was much higher compared to thatprovided by other protectants at the same concentration, which

Fig. 1. Viability of Bifidobacterium infantis after freeze-drying in the presence of 5% and10% of protectants. Control (w/o) are cells freeze-dried without any protectant. Eachbar represent means � standard deviation (SD), n ¼ 3.

ranged from 69.7% to 78.9%. At 10% concentration of protectants,the best protection was achieved using cellobiose and trehalose,namely 98.1% and 97.6% of activity retention, respectively. No sig-nificant differences were observed between 10% sucrose and 10%lactose (p > 0.05), which showed enzyme activity retentions of86.7% and 91.8%, respectively.

3.3. Viability of B. infantis in milk, red-beet juice and grape juice

B. infantis cells that were freeze-dried in the presence of 5% or10% cellobiose, 10% lactose, 10% trehalose and 10% sucrose wereadded to milk (pH 6.7), red-beet juice (pH 4.2) and grape juice (pH3.4). The pH value before and after storage did not change morethan 0.1 units. Due to poor protective effect on viability and b-glucosidase activity during freeze-drying, 5% trehalose, 5% sucroseand 5% lactosewere not used for the further stability studies in foodmatrices.

In milk, in the sample without any protectant a slight, but sig-nificant decrease in stability during storage was observed(p < 0.05,0.4 log, Table 1). Adding the protectants resulted in asignificant increase of the stability. Regardless of the protectantused, no significant differences were observed in the stability ofcells for 2 weeks of storage in milk (p > 0.05, Table 1). The log unitsreduction in cell viability ranged between 0.2 and 0.3.

In red-beet juice, the viability of freeze-dried cells without anyprotectant was reduced by 2.8 log units after 4 weeks of storage.

Table 1Survival of Bifidobacterium infantis, freeze-dried with different protectants, afterstorage for 2 weeks in milk (pH 6.7) at 4 �C.

Protectant Cell count (log CFU/ml)

Baseline (time 0) 2 weeks of storage

Control 7.9 � 0.1a 7.5 � 0.0b

5% Cellobiose 8.3 � 0.1c 8.1 � 0.2c

10% Cellobiose 8.3 � 0.1c 8.1 � 0.2c

10% Trehalose 8.4 � 0.1c 8.2 � 0.1c

10% Sucrose 8.3 � 0.1c 8.1 � 0.1c

10% Lactose 8.3 � 0.1c 8.1 � 0.1c

aecValues (means � SD) with different superscript letters are significantly different(p < 0.05), n ¼ 3.

Table 3Survival of Bifidobacterium infantis, freeze-dried with different protectants, afterstorage for 4 weeks in grape juice (pH 3.4) at 4 �C.

Protectant Cell count (log CFU/ml)

Baseline (time 0) 4 Weeks of storage

Control 7.9 � 0.1a 4.8 � 0.2b

5% Cellobiose 8.4 � 0.1c 7.3 � 0.1d

10% Cellobiose 8.4 � 0.1c 7.1 � 0.1d

10% Trehalose 8.4 � 0.0c 7.2 � 0.1d

10% Sucrose 8.4 � 0.0c 5.8 � 0.1e

10% Lactose 8.4 � 0.1c 5.7 � 0.1e

aeeValues (means � SD) with different superscript letters are significantly different(p < 0.05), n ¼ 3.

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Addition of protectants improved the storage stability so that theviability of the cells ranged between 1.1 and 1.7 log units (p< 0.001,Table 2). Cellobiose and trehalose provided significantly higherprotection of viability than sucrose (p < 0.001) and lactose. Therewere no significant differences between cells protected with 5% or10% cellobiose and 10% trehalose (p > 0.05). No significant differ-ences were observed between 10% lactose and 10% sucrose afterstorage in red-beet juice (p > 0.05).

In grape juice, the viability of the cells decreased more than inred-beet juice and milk. Without any protectant the viabilitydecreased by 3.1 log units. Cellobiose (5% or 10%) and 10% trehalosesignificantly (p < 0.001) increased the stability and offered the bestprotection whereas sucrose and lactose showed the least effect(Table 3). No significant differences were observed for cellobioseand trehalose protected cells when the viability was comparedbetween storage in grape juice and red-beet juice (p > 0.05).

3.4. b-Glucosidase activity retention during storage in milk, red-beet and grape juice

The retention of b-glucosidase activity of freeze-dried B. infantisafter storage in grape and red-beet juices (4 weeks at 4 �C) andmilk(2 weeks at 4 �C) is shown in Fig. 3AeC. For all foodmatrices tested,the preservation of b-glucosidase activity was enhanced by the useof protectants compared to controls without protectants.

In milk, the enzyme activity of non protected cells significantlydecreased, namely to 76.3%. Effects on increasing the retention ofenzyme activity during storage (p > 0.05) were similar for allprotectants, ranging from 93.7% to 95.1% (Fig. 3).

Without protectants, in red-beet juice the glucosidase activitydecreased to 69.2%. The retention of enzyme activity was signifi-cantly (p < 0.001) increased in the presence of protectants. Cello-biose (5% or 10%) and 10% trehalose showed the best protectiveeffect, 91.2%, 93.4 and 94.6% respectively. The remaining activitiesin cells protected by trehalose, sucrose or lactose were not signifi-cantly different (p > 0.05) (Fig. 3B).

After 4weeks of storage in grape juice, the b-glucosidase activitywas decreased significantly more than in red-beet juice and milk.The lowest remaining activity was observed for the cells withoutany cryoprotectant, namely 55.6%. The highest remaining activitywas found for cellobiose and trehalose protected cells, namely76.5%, 78.7% and 79.3%, respectively. The remaining activities forsucrose and lactose protected cells were 65.1% and 71.7%, respec-tively (Fig. 3C).

3.5. Acid tolerance

The acid tolerance of freeze-dried B. infantis after storage inmilk, red-beet juice and grape juice was determined by imitating agastric environment by using acidic conditions at pH 2 in thepresence of 0.3% porcine pepsine (Saarela et al., 2005). In milk,

Table 2Survival of Bifidobacterium infantis, freeze-dried with different protectants, afterstorage for 4 weeks in red-beet juice (pH 4.2) at 4 �C.

Protectant Cell count (log CFU/ml � SD)

Baseline (time 0) 4 Weeks of storage

Control 7.7 � 0.1a 4.9 � 0.1b

5% Cellobiose 8.4 � 0.0c 7.3 � 0.0d

10% Cellobiose 8.4 � 0.1c 7.2 � 0.1d

10% Trehalose 8.4 � 0.0c 7.3 � 0.1d

10% Sucrose 8.4 � 0.1c 6.9 � 0.1d

10% Lactose 8.4 � 0.1c 6.6 � 0.0d

aeeValues (means � SD) with different superscript letters are significantly different(p < 0.05), n ¼ 3.

protected cells showed comparable survival after acid exposure(0.2e0.9 log units reduction) which was significantly higher thanthat of unprotected cells (p < 0.01, Table 4).

In red-beet juice the sensitivity of cells to simulated gastricconditions was significantly increased (Table 4). Freeze-dried cellswithout any protectant showed no viability after exposure to pH 2for 90 min. Cellobiose increased the survival of cells under acidicconditions (0.4 and 0.8 log units reduction). 10% Cellobiose and 10%trehalose protected cells were significantly more resistant toexposure to acidic conditions (0.4 and 0.5 log units reduction,respectively) compared to cells protected with 5% cellobiose, 10%sucrose or lactose (0.7 and 0.8 log units reduction).

In grape juice the sensitivity of cells to simulated gastric con-ditions was higher compared to those stored in red-beet juice andmilk (Table 4). None of unprotected cells survived after exposure toacidic conditions (pH 2). Protected cells showed reductions in rangefrom 2.3 to 2.7 log units after exposure to simulated gastric con-ditions.10% Cellobiose showed the best protective effect in an acidicenvironment.

4. Discussion

Cellobiose and other disaccharide protectants reduce the lossesof viability and b-glucosidase activity that occur during freeze-drying of B. infantis UV16PR. Subsequent losses are also reducedas would occur during storage in different food matrices or in theacidic, gastric environment following storage in food matrices.

At 10% concentration, the effects of the protectants, cellobiose,lactose, sucrose and trehalose on viability and b-glucosidase ac-tivity retention during freeze-drying of B. infantis are comparable(Fig.1). This is in accordance to Vinderola et al. (2012) who reportedthat at 10% concentration, sucrose and lactose were similarlyeffective for viability protection during freeze-drying. Cellobiose isthe only disaccharide used in our study that significantly improvedstability of cells during freeze-drying in both concentrations used(5% and 10%). The protective effect of disaccharides has beenwidelydiscussed in the literature as due to stabilization of cell membranesand prevention of intracellular ice-formation (Heljo et al., 2011;Hubalek, 2003; Morgan et al, 2006). Among the protectants used,cellobiose offered the highest effect on retaining the b-glucosidaseactivity during freeze-drying. The higher effect of cellobiose can beexplained by possible binding of cellobiose to the active site of b-glucosidase (Pokusaeva et al., 2011; Roncaglia, Amaretti, Raimondi,Leonardi, & Rossi, 2011). These results are in accordance to previ-ously published studies which assume a protecting mechanism forb-galactosidase by lactose as substrate (Vasiljevic & Jelen, 2003).

Incorporation of the freeze-dried B. infantis in food matrices,especially in juices with lower pH, normally causes further detri-mental effects on viability and enzyme activity due to low pH andthe presence of oxygen (Saarela et al., 2006; Talwalkar, Miller,

Fig. 3. b-Glucosidase retention [%] of freeze-dried Bifidobacterium infantis (A) after 2weeks of storage inmilk (pH 6.7), (B) after 4weeks of storage in red-beet juice (pH 4.2) orafter 4weeks of storage in grape juice (pH3.4) at 4 �C.B. infantis cellswere freeze-dried inthe presence of 5% or 10% cellobiose, 10% trehalose, sucrose or lactose. Control (w/o) arecells freeze-dried without any protectant. Each bar represent means � SD, n ¼ 3.

Table 4Viability loss (log reduction) of Bifidobacterium infantis UV16PR after exposure tosimulated gastric conditions (PBS, pH 2 in the presence of 0.3% (w/v) pepsine at37 �C) after the storage in the food matrices.

Protectant Viability loss after acidic treatment

Grape juice Red-beet juice Milk

Control No cell detected No cell detected 0.9 � 0.15% Cellobiose 2.4 � 0.1 0.8 � 0.1 0.2 � 0.110% Cellobiose 2.3 � 0.1 0.4 � 0.1 0.2 � 0.110% Trehalose 2.5 � 0.1 0.5 � 0.1 0.2 � 0.110% Sucrose 2.7 � 0.1 0.8 � 0.2 0.3 � 0.110% Lactose 2.7 � 0.1 0.7 � 0.1 0.2 � 0.1

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Kailasapathy, & Nguyen, 2004; Vinderola et al., 2012). Most pro-biotics like Bifidobacterium spp. are sensitive to pH lower than 4(Boylston, Vinderola, Ghoddusi, & Reinheimer, 2004; Champagne &Gardner, 2008; Dave & Shah, 1997; Saarela, Alakomi, Puhakka, &

Mättö, 2009). It has been reported that the inhibitory effect offruit juices on the growth of lactic acid bacteria is strain dependent(Vinderola, Costa, Regenhardt, & Reinheimer, 2002; Yoon,Woodams, & Hang, 2005). Strains would express different func-tionality or stability when incorporated into different foodmatrices(Ranadheera et al., 2010; Vinderola et al., 2012). Since this is thefirst study with B. infantis UV16PR, it may be of importance whenthe strain is to be incorporated into food matrices. From our resultswe can observe that the factors that influence the viability of cellsduring storage are the type of protectant used during freeze-drying,pH (Boylston et al., 2004; Saarela et al., 2006), and the ingredientsof the food matrix (Ranadheera et al., 2010; Vinderola et al., 2012).We observed that storage in milk offered good protection of freeze-dried cells with different protectants. This can be attributed to thealmost neutral pH of milk, in accordance to the results reported bySaarela et al. (2006) and Vinderola et al. (2012). Small differences inviability of cells between red-beet juice and grape juice could bedue to influence of other ingredients of the food matrix on thestability of cells. This is in agreement with the study by Vinderolaet al. (2012) who reported that Bifidobacterium animalis subsp.lactis showed better stability in two juices with comparable pH butdifferent components.

According to the best of our knowledge this is the first study toshow the effect of trehalose and cellobiose for protecting Bifido-bacteria during storage in food matrices. Cellobiose as metaboliz-able sugar may activate the F1F0 ATP-ase through the glycolyticpathway which is in accordance to Corcoran, Stanton, Fitzgerald,and Ross (2005). The relation between the glycolysis, ATP genera-tion, ATP-ase activity and survival in low pH fluid has been reported(Corcoran et al., 2005; Duary, Batish, & Grover, 2010; Shabala et al.,2002). The effect of trehalose can be attributed the stabilizing ofmembrane integrity during freeze-drying (Li et al., 2011; Tang,Waring, & Hong, 2007) which can further make the cells moreresistant to other stress. Sucrose and lactose showed the least effecton protecting the cells during storage in low pH juices, especially ingrape juice. These results are in contrast to those reported bySaarela et al. (2006) who found that sucrose-protected cells sur-vived better in fruit juice compared to skim milk-protected cells,while in milk their effects were comparable. Vinderola et al. (2012)reported minor differences on viability of lactose protected cells indifferent food matrices with different pHs, but major differencesafter exposure to simulated gastric conditions of the stored cells.

To the best of our knowledge, this is the first studywhich reportsthe stability of Bifidobacterium b-glucosidase activity in juices andthe effect of different disaccharides on the b-glucosidase activity ofprobiotics after storage of freeze-dried probiotics in food matrices.

In our study, cellobiose protected cells retained b-glucosidaseactivity significantly better than unprotected controls during stor-age in milk, red-beet and grape juice. No significant differenceswere observed between storage in red-beet juice and milk for thecells protected with cellobiose. Storage in red-beet juice resulted ina significantly higher loss of b-glucosidase activity for the cells

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protectedwith sucrose and lactose than storage inmilk, while therewas no significant difference between storage in milk and red-beetjuice for the cells protected with 5% or 10% cellobiose and 10%trehalose.

The small differences in viability between red-beet and grapejuices for cells protected with cellobiose and trehalose accompa-nied by large differences in enzyme activity retention and acidtolerance confirm once more that viability alone is insufficient as apredictor of the functionality of cells (Saarela et al., 2005, 2006;Vinderola et al., 2012). The differences of enzyme activity forcellobiose and trehalose protected cells between storage in red-beet juice and grape juice, with no significant differences inviability, are in accordance to earlier published conclusions thatsome stress can cause metabolic injury even if they do not affectcell membranes (Wu, 2008). Furthermore, cellobiose as substrate ofglucosidase may improve the stability of the enzyme, by possiblebinding to the enzyme or through inducing the enzyme activityduring storage (Roncaglia et al., 2011). Our studies show that even ifthere are no significant differences in viability and enzyme activityafter freeze-drying of cells in the presence of 10% protectants, moresubstantial differences are observed after incorporation in foodmatrices.

The type of protectant and food matrix used influenced the acidtolerance of B. infantis to simulated gastric conditions. 10% Cello-biose or trehalose showed the highest increase in the tolerance toacidic conditions after the storage in different food matrices.

Small differences in viability were observed for cellobiose pro-tected cells stored in milk and red-beet juice. However, major dif-ferences have been observed in their tolerance to simulated gastricconditions. The differences in acid tolerance observed between thecells stored in red-beet juice and grape juice, regardless of pro-tectant used, are in accordance to those reported previously(Champagne & Gardner, 2008; Saarela et al., 2006; Stanton et al.,1998; Vinderola et al., 2012) that storage of probiotic bacteria infood matrices, especially in low pH juices, affects their survival in asimulated gastric environment.

In milk, regardless of protectant used, the acid tolerance of thecells after storage was comparable, but was significantly higherthan that of unprotected cells. Storage in red-beet juice wasaccompanied by a significant reduction in viability of unprotectedcells. Cells protected with 10% cellobiose showed significantlybetter acid resistance after storage for 4 weeks in red-beet juices.Following storage in grape juice, the resistance to acidic conditionswas decreased more than after storage in red-beet juice. Theseresults are in agreement to those reported by Saarela et al. (2006)who found significant differences in acid tolerance.

Cellobiose is a promising candidate protectant of Bifidobacte-rium cells and their b-glucosidase activity during freeze-drying andsubsequent storage in juices and milk. To the best of our knowl-edge, the beneficial effect of cellobiose on preservation of probioticsand b-glucosidase activity of probiotics has not been reported sofar. Cellobiose showed a protective effect during freeze-drying ofproteins like recombinant human growth hormone (Constantinoet al., 1998), b-galactosidase (Heljo et al., 2011), and the crude b-galactosidase from Lactobacillus delbrueckii.

Compared to the other disaccharides used in the present study,use of cellobiose as a lyoprotective provides several advantages. Incontrast to sucrose, cellobiose is not hydrolyzed by upper intestinalenzymes and thus does not influence glucose blood levels(Nakamura, Oku, & Ichinose, 2004; Nishimura et al., 2010). Cello-biose is advantageous compared to trehalose because of its potentialprebiotic effect (Sanz, Gibson, & Rastall, 2005). According to ourunpublished studies, cellobiose also functions as a carbon source forgrowth of B. infantis. In contrast to lactose, cellobiose will not causeproblems for persons with lactose intolerance. Furthermore, the b-

1,4-glycosidic bond present in cellobiose can induce b-glucosidaseactivity (Roncaglia et al., 2011).

The stabilizing effect of cellobiose on b-glucosidase activity is ofspecial interest when B. infantis is incorporated into a food matrixcontaining polyphenol glycosides like juices. The majority of poly-phenols in plants occurs in glycosidic form, so that a deglycosylationstep is necessary to make different polyphenols bioavailable andbioactive (Ávila et al., 2009; Roncaglia et al., 2011; Selma, Espín, &Tomás-Barberán, 2009). This may be of special relevance for peo-plewith variations in the levels of intestinal bacteria due to illnesses,diet or age, associated with a reduction in glycoside deconjugation(Hutt, Shchepetova, Loivukene, Kullisaar, & Mikelsaar, 2006).Therefore, when freeze-dried probiotics are incorporated indifferent foodmatrices, the level of b-glucosidase activity should beconsidered in addition to viability and acid tolerance.

5. Conclusion

In this study cellobiose is used as a new lyoprotectant for aprobiotic strain. Cellobiose at 5% concentration and other di-saccharides at 10% concentration significantly improve the viabilityof B. infantis UV16PR during freeze-drying and during storage inmilk and juices. All tested protectants, especially cellobiose,enhanced the acid tolerance of freeze-dried cells after storage infood matrices. Thus cellobiose can be used as an effective protec-tant for incorporation of B. infantis UV16PR into different foodmatrices. When choosing protectants for probiotics during freeze-drying and food as probiotic carrier, not only viability, but also b-glucosidase activity retention can be improved. b-Glucosidasecatalyzes the conversion of polyphenol glycosides to their agly-cones. In this manner, optimal conditions can be provided forpractical combinations of probiotics and polyphenol glycosides indifferent food matrices.

Acknowledgements

The authors are grateful to the Austrian Ministry of Science andTechnology (Bertha von Suttner program) for providing financialsupport to Mimoza Basholli-Salihu.

References

Ávila, M., Hidalgo, M., Sánchez-Moreno, C., Pelaez, C., Requena, T., & de Pascual-Teresa, S. (2009). Bioconversion of anthocyanin glycosides by Bifidobacteria andLactobacillus. Food Research International, 42, 1453e1461.

Boylston, T. D., Vinderola, C. G., Ghoddusi, H. B., & Reinheimer, J. A. (2004). Incor-poration of bifidobacteria into cheeses: challenges and rewards. InternationalDairy Journal, 14, 375e387.

Bradford, M. M. (1976). Rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry, 72, 248e254.

Champagne, C. P., & Gardner, N. J. (2008). Effect of storage in a fruit drink on sub-sequent survival of probiotic lactobacilli to gastro-intestinal stresses. FoodResearch International, 41, 539e543.

Champagne, C. P., Roy, D., & Gardner, N. (2005). Challenges in the addition of pro-biotic cultures to foods. Critical Reviews in Food Science and Nutrition, 45, 61e84.

Constantino, H. R., Carrasquillo, K. G., Cordero, R. A., Mumenthaler, M., Hsu, C. C., &Griebenow, K. (1998). Effect of excipients on the stability and structure oflyophilized recombinant human growth hormone. Journal of PharmaceuticalSciences, 87, 1412e1420.

Corcoran, B. M., Stanton, C., Fitzgerald, G. F., & Ross, R. P. (2005). Survival of probioticlactobacilli in acidic environments is enhanced in the presence of metabolizablesugars. Applied and Environmental Microbiology, 71, 3060e3067.

Dave, R. I., & Shah, N. P. (1997). Viability of probiotic bacteria in yoghurt made fromcommercial starter cultures. International Dairy Journal, 7, 707e715.

Dianawati, D., & Shah, N. P. (2011). Enzyme stability of microencapsulated Bifido-bacterium animalis ssp. lactis Bb12 after freeze-drying and during storage in lowwater activity at room temperature. Journal of Food Science, 76, 463e471.

Duary, R. K., Batish, V. K., & Grover, S. (2010). Expression of the atpD gene in pro-biotic Lactobacillus plantarum strains under in vitro acidic conditions using RT-qPCR. Research in Microbiology, 161, 399e405.

M. Basholli-Salihu et al. / LWT - Food Science and Technology 57 (2014) 276e282282

FAO/WHO. (2001). Health and nutritional properties of probiotics in food includingpowder milk with live lactic acid bacteria. Report of a Joint FAO/WHO ExpertConsultation on evaluation of health and nutritional properties of probiotics infood including powdermilk with live lactic acid bacteria. Córdoba, Argentina,1e4 October 2001 http://www.who.int/foodsafety/publications/fs_management/en/probiotics.pdf.

Fritzen-Freire, C. B., Muller, C. M. O., Laurindo, J. B., & Prudencio, E. S. (2010). Theinfluence of Bifidobacterium Bb-12 and lactic acid incorporation on the prop-erties of Minas Frescal cheese. Journal of Food Engineering, 96, 621e627.

Heljo, V. P., Jouppila, K., Hatanpää, T., & Juppo, A. M. (2011). The use of disaccharidesin inhibiting enzymatic loss and secondary structure changes in freeze-dried b-galactosidase during storage. Pharmaceutical Research, 28, 540e552.

Hubalek, Z. (2003). Protectants used in the cryopreservation of microorganisms.Cryobiology, 46, 205e229.

Hutt, P., Shchepetova, J., Loivukene, K., Kullisaar, T., & Mikelsaar, M. (2006).Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero-and uropathogens. Journal of Applied Microbiology, 100, 1324e1332.

Lankaputhra, W. E. V., Shah, N., & Britz, M. (1996). Survival of bifidobacteria duringrefrigerated storage in presence of acid and hydrogen peroxide. Milchwissen-schaft, 17, 65e70.

Li, B., Tian, F., Liu, X., Zhao, J., Zhang, H., & Chen, W. (2011). Effects of cryoprotectantson viability of Lactobacillus reuteri CICC6226. Applied Microbiology andBiotechnology, 92, 609e616.

Lievense, L. C., Verbreek, M. A. M., Noomen, A., & van’t Riet, K. (1994). Mechanism ofdehydration inactivation of Lactobacillus plantarum. Applied Microbiology andBiotechnology, 41, 90e94.

Marotti, I., Bonetti, A., Biavati, B., Catizone, P., & Dinelli, G. (2007). Biotransformationof common bean (Phaseolus vulgaris L.) flavonoid glycosides by bifidobacte-rium species from human intestinal origin. Journal of Agriculture and FoodChemistry, 16, 3913e3919.

Meng, X. C., Stanton, C., Fitzgerald, G. F., Daly, C., & Ross, R. P. (2008). Anhydro-biotics: the challenges of drying probiotic cultures. Food Chemistry, 106, 1406e1416.

Morgan, C. A., Herman, N., White, P. A., & Vesey, G. (2006). Preservation ofmicroorganisms by drying. A review. Journal of Microbiological Methods, 66,183e193.

Nakamura, S., Oku, T., & Ichinose, M. (2004). Bioavailability of cellobiose by toler-ance test and breath hydrogen excretion in humans. Nutrition, 20, 979e983.

Ng, E. W., Yeung, M., & Tong, P. S. (2011). Effects of yogurt starter cultures on thesurvival of Lactobacillus acidophilus. International Journal of Food Microbiology,145, 169e175.

Nishimura, T., Andoh, A., Hashimoto, T., Kobori, A., Tsujikawa, T., & Fujiyama, Y.(2010). Cellobiose prevents the development of dextran sulfate sodium (DSS)-induced experimental colitis. Journal of Clinical Biochemistry and Nutrition, 46,105e110.

Otieno, D. O., Ashton, J. F., & Shah, N. P. (2006). Evaluation of enzymic potential forbiotransformation of isoflavone phytoestrogen in soymilk by Bifidobacteriumanimalis, Lactobacillus acidophilus and Lactobacillus casei. Food Research Inter-national, 39, 394e407.

Otieno, D. O., Ashton, J. F., & Shah, N. E. (2005). Stability of b-glucosidase activityproduced by Bifidobacterium and Lactobacillus spp. In fermented soymilk duringprocessing and storage. Journal of Food Science, 70, 236e241.

Pokusaeva, K., O’Connell-Motherway, M., Zomer, A., MacSharry, J., Fitzgerld, G. F., &van Sinderen, D. (2011). Cellodextrin utilization by Bifidobacterium breveUCC2003. Applied and Environmental Microbiology, 77, 1681e1690.

Ramchandran, L., & Shah, N. P. (2010). Characterization of functional, biochemicaland textural properties of symbiotic low-fat yoghurts during refrigeratedstorage. LWT e Food Science and Technology, 43, 819e827.

Ranadheera, R. D. C. S., Baines, S. K., & Adams, M. C. (2010). Importance of food inprobiotic efficacy. Food Research International, 43, 1e7.

Roncaglia, L., Amaretti, A., Raimondi, S., Leonardi, A., & Rossi, M. (2011). Role ofbifidobacteria in the activation of the lignan secoisolariciresinol diglucoside.Applied Microbiology and Biotechnology, 92, 159e168.

Saarela, M. H., Alakomi, H.-L., Puhakka, A., & Mättö, J. (2009). Effect of thefermentation pH on the storage stability of Lactobacillus rhamnosus prepara-tions and suitability of in vitro analyses of cell physiological functions to predictit. Journal of Applied Microbiology, 106, 1204e1212.

Saarela, M., Virkajarvi, I., Alakomi, H.-L., Mattila-Sandholm, T., Vaari, A.,Suomalainen, T., et al. (2005). Influence of fermentation time, cryoprotectantand neutralization of cell concentrate on freeze-drying survival, storage sta-bility, and acid and bile exposure of Bifidobacterium animalis cells producedwithout milk-based ingredients. Journal of Applied Microbiology, 99, 1330e1339.

Saarela, M. H., Virkajãrvi, I., Alakomi, H.-L., Sigvart-Mattila, P., & Mättö, J. (2006).Stability and functionality of freeze-dried probiotic Bifidobacterium cells duringstorage in juice and milk. International Dairy Journal, 16, 1477e1482.

Sanz, M. L., Gibson, G. R., & Rastall, R. A. (2005). Influence of disaccharide structureon prebiotic selectivity in vitro. Journal of Agricultural and Food Chemistry, 53,5192e5199.

Selma, M. V., Espín, J. C., & Tomás-Barberán, F. A. (2009). Interaction betweenphenolics and gut microbiota: role in human health. Journal of Agricultural andFood Chemistry, 57, 6485e6501.

Shabala, L., Budde, B., Ross, T., Siegumfeldt, H., & McMeekin, T. (2002). Responses ofListeria monocytogenes to acid stress and glucose availability monitored bymeasurements of intracellular pH and viable counts. International Journal ofFood Microbiology, 75, 89e97.

Siaterlis, A., Deepika, G., & Charalampopoulos, D. (2009). Effect of culture mediumand cryoprotectants on the growth and survival of probiotic lactobacilli duringfreeze-drying. Letters in Applied Microbiology, 48, 295e301.

Stanton, C., Gardiner, G., Lynch, P. B., Collins, J. K., Fitzgerald, G., & Ross, R. P. (1998).Probiotic cheese. International Dairy Journal, 8, 491e496.

Strasser, S., Neureiter, M., Geppl, M., Braun, R., & Danner, H. (2009). Influence oflyophilization, fluidized bed drying, addition of protectants, and storage on theviability of lactic acid bacteria. Journal of Applied Microbiology, 107, 167e177.

Stummer, S., Toegel, S., Rabenreither, M., Unger, F. M., Wirth, M., Viernstein, H., et al.(2012). Fluidized-bed drying as a feasible method for dehydration of Entero-coccus faecium M74. Journal of Food Engineering, 111, 156e165.

Talwalkar, A., Miller, C. W., Kailasapathy, K., & Nguyen, M. H. (2004). Effect ofpackaging materials and dissolved oxygen on the survival of probiotic bacteriain yoghurt. International Journal of Food Science and Technology, 39, 605e611.

Tang, M., Waring, A. J., & Hong, M. (2007). Trehalose-protected lipid membranes fordetermining membrane protein structure and insertion. Journal of MagneticResonance, 184, 222e227.

Vasiljevic, T., & Jelen, P. (2003). Drying and storage of crude b-galactosidase extractsfrom Lactobacillus delbrueckii ssp. bulgaricus 11842. Innovative Food Science andEmerging Technologies, 4, 319e329.

Vinderola, C. G., Costa, G. A., Regenhardt, S., & Reinheimer, J. A. (2002). Influence ofcompounds associated with fermented dairy products on the growth of lacticacid starter and probiotic bacteria. International Dairy Journal, 12, 579e589.

Vinderola, G., Zacarías, M. F., Bockelmann, W., Neve, H., Reinheimer, J., & Heller, K. J.(2012). Preservation of functionality of Bifidobacterium animalis subsp. lactisINL1 after incorporation of freeze-dried cells into different food matrices. FoodMicrobiology, 30, 274e280.

Wu, V. C. H. (2008). A review of microbial injury and recovery methods in food. FoodMicrobiology, 25, 735e744.

Yoon, K. Y., Woodams, E. E., & Hang, Y. D. (2005). Fermentation of beet juice bybeneficial lactic acid bacteria. LWT e Food Science and Technology, 38, 73e75.