Contents lists available at ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Novel frozen yogurt production fortified with sea buckthorn berries andprobiotics

Antonia Terpoua,b,∗, Aikaterini Papadakib, Loulouda Bosneac, Maria Kanellakia,Nikolaos Kopsahelisb

a Food Biotechnology Group, Department of Chemistry, University of Patras, GR-26500, Patras, GreecebDepartment of Food Science and Technology, Ionian University, Argostoli, 28100, Kefalonia, GreececHellenic Agricultural Organization DEMETER, Dairy Research Institute, Katsikas, 45221, Ioannina, Greece

A R T I C L E I N F O

Keywords:Frozen yogurtSea buckthorn berriesProbioticsGastrointestinal digestion

A B S T R A C T

The objective of the present study was to produce a novel frozen yogurt fortified with sea buckthorn berriessupported probiotic cells. Berries originated from Hippophae rhamnoides L. were used as immobilization carrier ofthe probiotic strain Lactobacillus casei ATCC393. A commercial yogurt culture was used as starter culture whilefree and immobilized probiotic cells were added as adjuncts for frozen yogurt production. The viability of theimmobilized probiotic cells was maintained in high levels during 90 storage days (−18 °C), while the viability offree probiotic cells decreased (∼10%). L. bulgaricus counts reduced by approximately 3 log cycles and no countsof S. thermophilus were detected by the end of freezing storage. Gastrointestinal simulation showed that cellimmobilization offer protection to probiotic cells against the harsh environmental conditions of the gastro-intestinal tract and help in maintaining the minimum viable cell counts required to offer health benefits to theconsumers (> 107 CFU g−1). Finally, frozen yogurt with the immobilized biocatalyst showed superior proper-ties, exceptional appearance and an enhanced citrus aroma as described by the assessors.

1. Introduction

Over the past decade, interest has risen on functional food productsdue to consumers concern about the influence of food and food com-ponents to their health. As a result, consumers have nowadays turnedtheir preference on foods with alleged health benefits (Brown, Caligiuri,Brown, & Pierce, 2018). This trend is also reported by Research andMarkets which has estimated that the global market of health-clammingfunctional foods, which was approximately $168 billion in 2013, willexceed $300 billion by year 2020 (Source: Research and Markets).

Dairy products represent one of the biggest market segments amongfunctional foods as they have been proposed as the most promisingvehicles for the delivery functional ingredients such as probiotic, pre-biotics, proteins, vitamins and minerals (Akin & Ozcan, 2017). Pro-biotics are viable microorganisms that can confer a beneficial effect tothe consumer when administered in appropriate quantities (higher than106 CFU g−1 or mL−1). The development of a suitable technologies andprocesses for the maintenance of viable probiotic cells are considered askey steps in the production of functional foods. Therefore, in order toenhance the viability of probiotic bacteria various methods have beenproposed such as specific strain selection, addition of prebiotics, cell

immobilization, microencapsulation, stress adaptation etc. (Patrignaniet al., 2017; Schoina, Terpou, Bosnea, Kanellaki, & Nigam, 2018; Shori,2015, 2017; Terpou et al., 2018a).

Among lactic acid bacteria (LAB), Lactobacillus casei is commonlyused in probiotic dairy products production due to its possible bene-ficial effects to the host. In vitro and in vivo studies have demonstratedtheir ability to survive the GI tract, to provide adhesion to the intestineand modulation of the intestinal microflora in rats and resistance duringlow temperature storage (Bosnea et al., 2009; Saxami et al., 2012;Terpou et al., 2018a). Specifically, the strain L. casei ATCC 393 has beenused in many studies for probiotic food production such as yogurt(Bosnea, Kopsahelis, Kokkali, Terpou, & Kanellaki, 2017), ice cream(Farias et al., 2019), fermented milk (Abdel-Hamid et al., 2019; Terpouet al., 2017b), cheese (Terpou et al., 2017c), bread (Plessas et al., 2007)and sausages (Sidira, Karapetsas, Galanis, Kanellaki, & Kourkoutas,2014).

Among dairy products with live cultures, probiotic frozen dessertsare also gaining popularity nowadays and can be categorized as icecreams and frozen yogurt. (Akalın et al., 2018; Cruz, Antunes, Sousa,Faria, & Saad, 2009). Ice cream is a partially-frozen milk mixture withcream, sugar, stabilizers and emulsifiers (Cruz et al., 2009). On the

https://doi.org/10.1016/j.lwt.2019.02.024Received 14 September 2018; Received in revised form 6 February 2019; Accepted 7 February 2019

∗ Corresponding author. Food Biotechnology Group, Department of Chemistry, University of Patras, GR-26500, Patras, Greece.E-mail address: a.terpou@upatras.gr (A. Terpou).

LWT - Food Science and Technology 105 (2019) 242–249

Available online 08 February 20190023-6438/ © 2019 Elsevier Ltd. All rights reserved.

T

other hand, frozen yogurt is a fermented frozen dairy product whichcombines the physical characteristics of ice cream with the sensory andnutritional properties of fermented milk products (Goff, 2008). Frozenyogurt fermentation bioprocess is achieved by the addition of com-mercial yogurt culture parted of Streptococcus thermophilus and Lacto-bacillus delbrueckii ssp. Bulgaricus (Abdelazez et al., 2017). These mi-croorganisms are claimed to confer health benefits; however, they arenot natural inhabitants of the intestine (Wasilewska, Zlotkowska, &Wroblewska, 2019). In order to confer beneficial effects to the con-sumer, frozen desserts can be supplemented with probiotics or pre-biotics or a combination of both (Di Criscio et al., 2010; Farias et al.,2019; Rezaei, Khomeiri, Kashaninejad, Aalami, & Mazaheri-Tehrani,2018). However, the freezing processes of frozen desserts manufactureis reported to cause a loss of over than 50% of viable bacterial counts(Davidson, Duncan, Hackney, Eigel, & Boling, 2000). Thus, a need forprobiotic cell protection techniques like immobilization and micro-encapsulation is emerged to reinsure probiotic cell viability duringfreezing processes of frozen desserts production and storage.

Subsequently, the aims of the present study focus in: (1) productionof a novel functional frozen yogurt with enhanced probiotic char-acteristics by the use of free and immobilized on sea buckthorn berriesL. casei ATCC393 cells; (2) evaluation of the survival of the probioticcells during storage and through simulated gastrointestinal conditions,and (3) evaluation of the microbiological stability and the sensorycharacteristics of produced frozen yogurts at freezing storage condi-tions.

2. Materials and methods

2.1. Microbial cultures

As a starter culture for yogurt production was used the commercialfreeze-dried ready-to-use mixed bacterial culture consisting of S. ther-mophilus and L. bulgaricus at a proportion 2:1 (FD-DVS CH-1 – Yo-Flex,Chr. Hansen, Horsholm, Denmark). The bacterial starter culture wasactivated by incubation in 10mL of sterile skim milk at 40 °C for ap-proximately 1 h.

The probiotic bacterial strain Lactobacillus casei ATCC 393 (DSMZ,Braunschweig, Germany) was used as an adjunct culture for probioticfrozen yogurt manufacture. L. casei was grown at 37 °C in de Man-Rogosa-Sharpe (MRS) liquid medium (LabM, UK) for 48–72 h underanaerobic conditions (AnaeroGen TM, Oxoid, Ltd., Hamphire, UK). Thebiomass of L. casei was harvested by centrifugation (Sigma 3K12,Bioblock Scientific, France) at 5000 rpm for 10min at 20 °C (Bosneaet al., 2017). The harvested biomass was either added as a free probioticculture or used for the immobilization bioprocess. All media were au-toclaved at 120 °C at 1–1.5 atm for 15min prior to use.

2.2. Immobilized biocatalyst preparation

Berries from Hippophae rhamnoides L. (Fig. 1 A) so named seabuckthorn berries, were obtained by organic farmers of Western Greeceand used as an immobilization carrier of the probiotic strain L. caseiATCC393. According to Basu, Banerjee, Chowdhury, and Bhattacharya(2018) the approximate weight of 109 L. casei cells is 1 mg. The im-mobilization bioprocess was performed by mixing 10 g of berries and4 g of L. casei biomass in 500mL of pasteurized deproteinized cheesewhey (Terpou et al., 2017b, 2017c). The initial cheese whey (5% lac-tose, 0.8% protein) was obtained by a local cheese manufacturer as aby-product of Feta cheese production. The liquid mixture was in-oculated at 37 °C for 24–48 h for cell immobilization to occur with aparallel maintenance of pH at 5.0 ± 0.3 by the addition of Na2CO3

solution at various time internal. Subsequently, the liquid medium wasdecanted and the immobilized biocatalyst was washed (x3) with100mL of sterile Ringer's solution (1/4 strength) for the removal of freecells (Terpou et al., 2017c).

2.3. Scanning electron microscopy

Samples of the immobilized biocatalyst as well as samples ofHippophae rhamnoides L. berries were examined by Scanning ElectronMicroscopy (SEM) to verify the immobilization of probiotic cells. Allsamples were freeze-dried on a Freeze Dry System, FreeZone 4.5(Labconco, USA) and coated with gold in a Balzers SCD 004 Sputtercoater (Bal-Tec, Germany) for 2–3min. Then the samples were ex-amined on a JSM-6300 scanning electron microscope (Jeol, Japan)which operated at accelerating voltage of 20 kV.

2.4. Functional frozen yogurt manufacture

Pasteurized homogenized semi-skim cow's milk (1.5% fat, 4.7%carbohydrates, 3.3% protein, 0.12% salt) supplied from a local dairycompany (Kalavryta association, Achaia, Western Greece) and pas-teurized heavy cream 35% fat (Nestlé, Greece) were used for frozenyogurt production. The dry matter content in the milk was adjusted byadding skim milk powder (5%, LabM, U.K.) and guar gum (E 412) asstabiliser. All above ingredients were mixed with glucose syrup byconstant stirring (1000 rpm) at 60 °C for 20min. The obtained mixturewas rapidly cooled and retained at 10 °C for 24 h for further ripening.

Yogurt was produced by pasteurized homogenized cow's milk (pH6.8 ± 0.1) used as a fermentation media and the commercial yogurtculture added at an inoculum of 1% w/v. The mixture of milk andstarter yogurt culture was incubated at 45 °C for 5 h until pH drop at4.6 ± 0.1. To produce probiotic yogurt, the above procedure was usedwith one specific modification: milk was initially heated at 37 °C andfree or immobilized L. casei cells (2% w/w) were added, mixed well andleft for 60min undistributed (Bosnea et al., 2017; Terpou, Bekatorou,Kanellaki, Koutinas, & Nigam, 2017a). Subsequently, milk with free orimmobilized probiotic cells was inoculated at 45 °C by the commercialyogurt culture as described above.

The aged mixture (milk, heavy cream, skim milk powder, glucoseand stabiliser) was blended with each produced yoghurt (proportion1:1). Subsequently, three different frozen yogurt samples were pre-pared: Commercial frozen yogurt containing only the classic yogurtculture (Frozen yogurt 1; FYC), frozen yogurt prepared by the com-mercial yogurt culture and free L. casei cell culture 1% w/v (Frozenyogurt 2; FYF), and frozen yogurt prepared by the commercial yogurtculture and sea buckthorn berries supported probiotic cells – im-mobilized biocatalyst 2% w/v (Frozen yogurt 3; FYB). Each yogurtmixture was submitted to freezing individually by a domestic ice creamfreezer (SECM 12 A1 domestic ice cream freezer, 1 L capacity,Hamburg, Germany) with parallel mixing for 90min at a final drawtemperature of −5.5 °C. Frozen yogurt samples were packaged in cupsof 100mL and eventually hardened and stored under quiescent freezingconditions at −18 °C for 90 days of storage.

2.5. Physicochemical analysis

The pH values of the frozen yogurt were determined using a digitalpH meter (Hanna HI99161). The total solids of frozen yogurts weredetermined by drying the samples at 110 °C overnight until constantweight. Ash was determined according to AOAC (1995). Fat and proteincontents were determined by the Soxlet and Kjeldahl methods, re-spectively (Terpou et al., 2018b). The overrun of the final product wasdetermined using the following formula (Akın, Akın, & Kırmacı, 2007).

=−

×

OverrunWeight of unit mix weight of equal volume of frozen yogurt

Weight of equal volume of frozen yogurt

%

100

A. Terpou, et al. LWT - Food Science and Technology 105 (2019) 242–249

243

2.6. Microbiological assessment and probiotic cell counts

For the enumeration of viable cell counts, 10 g samples were col-lected from each frozen yogurt at various time intervals (1, 3, 7, 14, 21,35, 60 and 90 days after manufacture) during storage at−18 °C (T0: theday of manufacture). The samples were serially diluted in 90mL sterileRinger's solution of 1/4 strength, homogenized in a Bagmixer (400Model VW, Interscience), diluted and plated on selective media.

L. casei viable cells were enumerated on MRS-V agar (LaBM, U.K.)containing 1% vancomycin antibiotic that promotes the growth of L.casei against L. bulgaricus (Tharmaraj & Shah, 2003). The MRS-V agarwas incubated for selective enumeration at 37 °C for 72 h. The survivalrate (SR%) of free and immobilized probiotic cells during frozen yogurtstorage under freezing conditions for 90 days was determined accordingto the equation given below:

= ×SRCFU NCFU No

%loglog

100

The calculation was based on cell counts (log CFU/g) of the pro-biotic cells enumerated in each sample where N0 and N are the popu-lation numbers from the T0 day of manufacture and after each testedday during storage, respectively.

L. bulgaricus was enumerated on MRS agar with pH adjusted to 5.2and incubated at 45 °C for 72 h and S. thermophilus was enumerated onM17 medium (LaBM, U.K.) containing 1% lactose after incubation at45 °C for 72 h (Terpou et al., 2017a). Coliform counts were enumeratedon Violet red bile agar (LaBM, U.K.) after incubation at 30 °C for 24 h.Total Enterobacteriaceae were enumerated on Violet red bile glucoseagar (LaBM, U.K.) after incubation at 37 °C for 24 h. Staphylococcuscounts were determined on Baird Parker agar (LaBM, U.K.) after in-cubation at 37 °C for 48 h. Yeasts and fungi were determined by platingon Potato Dextrose Agar (LaBM, U.K.) after incubation at 30 °C for 72 h(Terpou et al., 2018c).

All above media were prepared according to the instructions of themanufacturer and were sterilized at 135 °C for 15min before use. Cellcounts were expressed as log CFU g −1 of frozen yogurt.

2.7. Resistance of free and immobilized probiotic cells to simulatedgastrointestinal conditions

The test was performed, in vitro, following the method describedpreviously by Terpou et al. (2017a) with small modifications. For eachtest, samples of frozen yogurt were collected by the end of the estimatedshelf-life (90th day) during products freezing storage (−18 °C). To in-vestigate the influence of stomach pH on the survival of living probioticbacteria (free or immobilized on SBB), simulated gastric juice wasprepared by suspending 3mg/mL of pepsin (Sigma-Aldrich) in ster-ilized saline water and pH adjusted to 3 with concentrated HCl. Salinewater (Sigma-Aldrich), commonly known as salt water, was used due toits high concentration of dissolved salts (0.9% NaCl). Simulated small-intestinal juices were prepared by suspending 1mg/mL of pancreatin(Sigma-Aldrich) in sterilized saline water and 0.3% bile salts (Sigma-Aldrich) while the pH was adjusted to 7.0 with concentrated 0.1 NNaOH (González-Córdova et al., 2016).

Samples contained the following: 25 g of frozen yogurt diluted in225mL saline solution (10−1) and the mixtures were then blended in astomacher (Bagmixer 400, Model VW, Interscience) for 10min. Fromeach sample, 10 mL of 10−1 dilution was transferred to each sterile vialand the addition of the simulated gastric and enteric juice was per-formed according to each treatment. The flasks were incubated in awater bath at 37 °C with periodic shaking. The enumeration of L. caseiwas performed at zero time (before the in vitro assay) and respectivelyafter 30, 60, 90, and 120min. Viable probiotic cell counts were en-umerated on MRS-V agar after incubation at 37 °C for 72 h (Terpouet al., 2017a).

2.8. Sensory evaluation: consumers acceptance test

A consumer acceptance test was conducted by 10 adults (18–55years old) concerning attributes of citrus taste, dairy-sour taste,sweetness, bitterness, dairy flavour, crumbly texture, smoothness,colour and overall acceptability. Assessors were non-smokers and theywere familiar with the consumption of both frozen dairy desserts andfermented milk products. Prior, evaluation a 2 h session was held to testtheir descriptive profiling resilience. The sensory evaluation sessionwas conducted in individual booths and consumers were asked to

Fig. 1. (A) Sea buckthorn berries (Hippophae rhamnoides L.) obtained from organic farms of western Greece, (B) Surface of sea buckthorn berries illustrated byscanning electron microscopy (40 μm) and (C) Immobilized L. casei ATCC393 cells within sea buckthorn berries illustrated by scanning electron microscopy (10 μm).

A. Terpou, et al. LWT - Food Science and Technology 105 (2019) 242–249

244

evaluate each product based on 0–10 preference scales as presented inTable 1. Frozen yoghurt samples (30 g) were evaluated from the 1st dayof production and they were served at −15 °C in 50mL transparentplastic cups numbered randomly with 3-digit codes. Assessors had ac-cess to unsalted crackers and low mineral content water to neutraliseand clean their palates between samples and were unaware of thesamples they tasted (Schoina et al., 2018). All analyses were conductedat ambient room temperature under standard fluorescent light. Asses-sors were encouraged to provide, when necessary, additional commentsrelating to the tested frozen yogurt samples.

2.9. Statistical analysis

All experiments were carried out in triplicate. Significance was es-tablished at P < 0.05. The results were analysed for statistical sig-nificance with one-way-ANOVA. Principal component analysis (PCA)used for identifying flavour and aroma attributes of frozen yogurtsamples was computed using PanelCheck.

3. Results and discussion

3.1. Rational

Sea buckthorn berries (Hippophae rhamnoides L.), an ancient cropwith modern virtues as presented in Fig. 1A, has recently gainedworldwide attention mainly due to their high concentration in valuablenutrients and antioxidant compounds that in addition show anti-microbial (Guo, Guo, Li, Fu, & Liu, 2017) and prebiotic characteristics(Bal, Meda, Naik, & Satya, 2011; Gunenc et al., 2016). Attri, Sharma,Raigond, and Goel (2018) suggested that sea buckthorn berries can be areliable source of prebiotics in terms of proliferation of the beneficialgut microbiota. In addition, sea buckthorn berries have been proposedas reinforcement substrates of probiotic bacterial cells when admini-strated in yogurt (Gunenc et al., 2016) and cheese products (Terpouet al., 2017c). Regarding future market insights, it has been reportedthat the global market for plant-based frozen desserts which reached avalue of a billion dollars by the end of 2017, will grow to the estimated$2.45 billion by the end of 2018 (Future Market Insights, 2018). Con-cerning the above data, the present research has been organized tostudy the effect of sea buckthorn berries supported probiotic cells onfrozen yogurt biochemical, microbiological and sensory characteristicsthrough freezing storage.

3.2. Physicochemical properties of functional frozen yogurt

The potential probiotic frozen yoghurt samples with free (FYF) orimmobilized (FYB) cells of L. casei ATCC393 had lower pH valuescompared to control frozen yogurt (FYC) as presented in Table 2. Thisresult was expected and is compatible with previous studies of yogurt(Terpou et al., 2017a) and frozen yogurt (Bezerra, Araujo, Santos, &

Correia, 2015) samples with incorporated probiotic cultures. A ten-dency of lower pH, in dairy products containing adjunct probioticcultures compared to commercial dairy products, is generally observedas a result of an enhanced acid production (Bezerra et al., 2015; Schoinaet al., 2015; Terpou et al., 2017a). Acid coagulation of milk caseinmicelles occurs mainly due to lactic acid production during incubationthat lowers the pH (Aryana & Olson, 2017). Likewise, enhanced acidproduction during milk fermentation for frozen yogurt production de-pends upon the growth, viability and ability of L. casei to ferment themilk carbohydrates (Terpou et al., 2017b). Overall, the pH of frozenyogurt samples with incorporated free (FYF) or immobilized (FYB)probiotic cells ranged between 4.46 and 4.43 (Table 2) which is withinaccepted levels of frozen yogurt products (Bezerra et al., 2015; Pintoet al., 2012).

The incorporation of air in frozen desserts is commonly referred asoverrun. Overrun is one of the most important quality parameters offrozen desserts and affects the texture and consequently the price ofthese products (Goff, 2008). Overrun has a crucial impact on the phy-sical and sensory properties of frozen desserts as the texture is depen-dent on the composition (mainly protein and sugar content) and theeffectiveness of the freezing process (e.g. overrun % and freezing time)(Ferraz et al., 2012; Goff, 2008). Frozen yogurt samples retrievedoverruns between 22 and 28% (w/w) (Table 2) while significant dif-ferences (p < 0.05) were observed between control samples and frozenyogurt with the incorporated immobilized biocatalyst. Therefore, it isconcluded that the addition of the sea buckthorn berries supportedprobiotic cells affected the overrun of frozen yogurt products providinghigher overrun results. In general, low overrun levels of the presentstudy (Table 2) is comparable to overrun of 14.2–22.6% of frozen yo-gurt samples produced with jambolana fruit pulp and Bifidobacteriumanimalis subsp. lactis as reported previously by Bezerra et al. (2015).

Table 1List of sensory attributes that were studied regarding consumers acceptance test of produced frozen yogurts.

Sensory attribute Definition Reference/intensity (low = 0, high = 10)

Citrus taste Taste associated with sour feeling from citrus fruit 0 = no citrus, 10 = citrus fellingDairy-sour taste Refreshing sourness perception associated with cultured dairy products 0 = Ice cream mix, 10 = yoghurtSweetness taste associated with sweet feeling 0 = not sweet, 10 = very sweetBitterness Basic taste associated with caffeine or alkaloids 0 = 0.05% caffeine solution, 10 = 0.5% caffeine solutionDairy (milk fat) flavour Flavour like high fat dairy products e.g. buttermilk 0 = Non-fat milk, 10 = full fat creamCrumbly texture A crumble considerably dry ice cream which falls apart during scooping 0 = Premium commercial ice cream, 10 = crumbly textured ice cream (1, ice

cream mix: 4, water)Smoothness Ice cream is characterized by soft, balanced and foamy texture free of

recrystallisation defects.The product melts uniformly to a thick and homogenous fluid

0 = Nonfat ice cream subjected to heat shock, 10 = mouthcoating ice cream

Appearance/colour Assessment of white colour intensity 0 = Egg custard, 10 = yoghurt-whiteOverall acceptance Assessment of general opinion/total acceptance 0 = not accepted, 10 = highly accepted

Table 2Frozen yogurt characteristics concerning protein, total solids, ash, fat, pH andoverrun from the 1st day of storage at −18 °C.

Composition FYC a FYF b FYB c

Protein (%) 3.25 ± 0.06 3.27 ± 0.05 3.39 ± 0.06Total solids (%) 16.01 ± 0.02 16.13 ± 0.07 17.58 ± 0.07Ash (%) 0.75 ± 0.09 0.71 ± 0.03 0.95 ± 0.01Fat (%) 3.38 ± 0.03 3.36 ± 0.04 3.31 ± 0.03Overrun (%) 22 ± 0.50 23 ± 0.51 28 ± 0.51pH 4.51 ± 0.05 4.46 ± 0.03 4.43 ± 0.05

*The results are expressed as mean (n = 3) ± standard deviations.a Frozen yogurt 1; FYC: commercial frozen yogurt containing only the classic

yogurt culture.b Frozen yogurt 2; FYF: frozen yogurt prepared with free L. casei ATCC393

culture.c Frozen yogurt 3; FYB: frozen yogurt prepared with the immobilized bio-

catalyst.

A. Terpou, et al. LWT - Food Science and Technology 105 (2019) 242–249

245

3.3. Microbial stability and probiotics’ survival rate in frozen yogurt duringstorage

Microorganisms in dairy products have very important roles and areessential for physicochemical and organoleptic characteristics of thefinal product (Cocolin, Dolci, Alessandria, & Rantsiou, 2018). Likewise,microbial stability is of crucial role for maintenance of products’characteristics. Viable cell counts of S. thermophilus, L. bulgaricus and L.

casei during frozen yogurts freezing storage for 90 days, are presentedin Table 3. It is noteworthy that no spoilage or possible pathogenicmicroorganisms as Staphylococci, coliforms, enterobacteria, yeasts orfungi were detected during product freezing storage (data not shown).

There was observed a decrease in S. thermophilus counts which werenot detected in any frozen yogurt sample after the 20th day of storage.This result may have occurred initially due to the antagonistic effect ofother bacteria as well as the effect of temperature as S. thermophilus is athermophilic bacterium with optimum culture conditions of 40–45 °Cwhile its viability has been reported to decrease also in yogurt culturesenhanced by adjunct Lactobacilli (Ng, Yeung, & Tong, 2011; Terpouet al., 2017a). Likewise, the addition of the probiotic strain in frozenyogurt (FYF, FYB) correlates with the decreased S. thermophilus counts.In addition, according to Beal, Skokanova, Latrille, Martin, and Corrieu(1999) S. thermophilus could also have been inhibited by the productsenvironment.

L. bulgaricus cell counts also showed a considerable continuous de-crease during 90 days freezing storage in all samples. More specifically,the counts of L. bulgaricus were reduced by approximately 3 log cyclesthroughout freezing storage in all cases. This result may have occurreddue to freeze injuries in cells, which eventually lead to the reduction ofbacterial cell counts as also reported by other studies (Akın et al., 2007;Pinto et al., 2012).

Table 1 presents the population of L. casei in frozen yogurts duringstorage for 90 days at −18 °C while in Fig. 2 presents the survival rateof L. casei. The viable probiotic cell counts of the frozen yogurt with theincorporated immobilized biocatalyst (FYB) remained above 9 log cfu/gthroughout the whole storage period, which is in accordance to re-commended levels of viable probiotic cell counts in food at the time ofconsumption (Boylston, Vinderola, Ghoddusi, & Reinheimer, 2004;Shori, 2015). In addition, the frozen yogurt with free probiotic cells(FYF) remained above 6 log cfu/g throughout the whole storage periodwhich is also within the recommended level of viable probiotic cells atthe time of food consumption (Shori, 2015). As also illustrated in Fig. 2,there was observed a significant reduction in probiotic cell counts infrozen yogurt with free probiotic cells (FYF) compared to frozen yogurtwith the incorporated immobilized biocatalyst. More specifically, thesurvival rate (SR%) of free probiotic cells (FYF) was reported at 90% bythe end of storage period (90 days, −18 °C) while in the case of frozenyogurt with the incorporated immobilized biocatalyst no significant(P > 0.05) reduction in probiotic cell counts was observed maintaininga survival rate of over 97%. In the case of free cells (FYF) lower survivalrates were observed. This result might be attributed to the fact thatfrozen yogurt is a whipped product while L. casei is a preferentially

Table 3Viable cell counts of L. bulgaricus, S. thermophilus and L. casei (log CFU g −1) infrozen yogurt during 90 storage days (−18 °C).

FrozenYoghurt

Storagetime(days)

Streptococcusthermophilus (logCFU g−1)

Lactobacillusbulgaricus (logCFU g−1)

Lactobacillus caseiATCC 393 (log CFUg−1)

FYC a 1 2.66 ± 0.15 5.90 ± 0.10 -3 1.56 ± 0.15 4.86 ± 0.15 -7 1.34 ± 0.10 4.35 ± 0.17 -14 1.03 ± 0.15 3.04 ± 0.16 -21 nd 3.97 ± 0.15 -35 nd 3.95 ± 0.05 -60 nd 3.71 ± 0.16 -90 nd 3.23 ± 0.14 -

FYF b 1 2.12 ± 0.18 5.87 ± 0.16 9.59 ± 0.103 1.34 ± 0.15 4.66 ± 0.15 9.16 ± 0.107 1.11 ± 0.11 4.14 ± 0.08 8.85 ± 0.1214 nd 4.02 ± 1.10 8.32 ± 0.1121 Nd 3.67 ± 0.10 7.29 ± 0.1635 nd 3.16 ± 0.15 7.17 ± 0.1060 nd 3.14 ± 0.16 7.16 ± 0.1090 nd 3.07 ± 0.04 6.97 ± 0.10

FYB c 1 1.56 ± 0.11 6.21 ± 0.16 9.98 ± 0.133 1.25 ± 0.08 5.96 ± 0.14 9.93 ± 0.107 nd 4.67 ± 0.15 9.86 ± 0.1014 nd 4.25 ± 1.10 9.79 ± 0.1621 nd 4.02 ± 0.15 9.65 ± 0.1035 nd 3.56 ± 0.06 9.48 ± 1.1060 nd 3.06 ± 0.05 9.44 ± 0.1290 nd 2.98 ± 0.16 9.40 ± 0.15

* The results are expressed as mean (n = 3) ± standard deviations.a Frozen yogurt 1; FYC: commercial frozen yogurt containing only the classic

yogurt culture.b Frozen yogurt 2; FYF: frozen yogurt prepared with free L. casei ATCC393

culture.c Frozen yogurt 3; FYB: frozen yogurt prepared with the immobilized bio-

catalyst.

Fig. 2. SR% (survival rate) of Lactobacillus casei ATCC393 in frozen yogurt with free (————X———, FYF) or immobilized (… …….○……., FYB) cells during 90 daysof freezing storage (−18 °C).

A. Terpou, et al. LWT - Food Science and Technology 105 (2019) 242–249

246

anaerobic microorganism (Schoina et al., 2015). Hence, high oxygenavailability at the beginning of the production process, together withfreezing conditions’ unavoidable stress, may have strongly contributedin the obtained decrease of probiotic cells viability. In addition, as al-ready reported (Bosnea et al., 2017; Terpou et al., 2017a) the acidicenvironment of yogurt itself, affects mostly free cells than the im-mobilized ones, leading to a high decrease of free L. casei cells duringproduction and storage. In several studies there has been observed thatsupports of immobilization, especially ones that contain prebiotic in-gredients, may conduct to the protection of probiotic cells againstacidic and harsh environmental conditions during production and sto-rage of dairy products (Bosnea et al., 2017; Schoina et al., 2018; Shori,2015; Terpou et al., 2018c). Likewise, in the present study sea buck-thorn berries that were used as immobilization carrier of probiotic cellstargeting functional frozen yogurt production are illustrated in Fig. 1.More specifically, Fig. 1 B presents the surface of sea buckthorn berrieswhile Fig. 1C illustrates the immobilized probiotic cells within theberries, verifying the successful immobilization process.

The above findings are in accordance with previous studies whichreport that sea buckthorn berries used as a prebiotic source in yogurtand feta type cheeses can enhance the viability of the probiotic cultureduring products refrigerated (4 °C) storage (Gunenc et al., 2016; Terpouet al., 2017c). In addition, according to Hong and Marshall (2001)natural exopolysaccharides can enhance the survival of lactic acidbacteria in frozen dairy desserts. In the frame of that, buckthorn berriesprebiotic character might contribute in maintaining the observed highprobiotic survival rates.

3.4. Survival of probiotic cells in simulated gastrointestinal conditions

Synbiotic is a combination of probiotic and prebiotics that affectsthe host beneficially by maintaining or enhancing the survival andimplantation of probiotic bacterial strains in gastrointestinal tract(Fazilah, Ariff, Khayat, Rios-Solis, & Halim, 2018). Thus, in the presentstudy, a combination of the probiotic bacterial strain L. casei ATCC393was enforced by the prebiotic effect of sea buckthorn berries targetingfunctional frozen yogurt production. As it has been established, thesuccessful incorporation of probiotics in functional dairy products isconfirmed by their ability to survive through the harsh digestive con-ditions applied during passage through the gastrointestinal tract ofhumans (Shori, 2015). In line with these findings, simulated gastro-intestinal conditions were conducted at the 1st day of freezing storagein order to assess the protective effect of sea buckthorn berries as im-mobilization carrier on the survival of L. casei after consumption. Inparallel, samples of frozen yogurt prepared by free L. casei cells was alsoassessed for comparison reasons. The immobilized and free L. caseipopulations were monitored under simulated gastrointestinal condi-tions and the results of viable cell counts are presented in Table 4. Theuse of immobilized biocatalysts in frozen yogurt (FYB) allowed main-tenance of higher viable L. casei cell numbers compared to frozen yo-gurt with free cells (FYF). This finding indicates a protective effect ofsea buckthorn berries on L. casei cells, and possible migration of pre-biotic constituents to the final product that stimulated survival throughgastrointestinal simulation. This result is also in accordance with pre-vious studies that described the suitability of prebiotic substrates asimmobilization carriers to deliver probiotic bacteria to the human gut(Romano, Tymczyszyn, Mobili, & Gomez-Zavaglia, 2016; Terpou et al.,2017a).

3.5. Sensory evaluation

Given the expanding influence of world cultures, today's consumersare more open to trying new combinations of flavours, especially withingredients that may confer beneficial health effects (Aguiar, Geraldi,Betim Cazarin, & Maróstica Junior, 2019). In the same manner, frozendesserts and frozen yogurt, have the capacity to offer textures beyond

just frozen and soft and provide an expanded area for innovative tex-tures.

The sensory scores of the frozen yogurt samples analysed by PCA arepresented in Fig. 3. The PCA was used to investigate the correlationsamong the attributes of samples. The PCA was carried out on 2 prin-cipal components that were chosen by the common rules. The firstprincipal component (PC1; X-axis) explained 93.6% of the total varia-tion in the data set while the second principal component (PC2; Y-axis)explained 6.4% of the total variation. According to the values of thePCA some of the attributes could be correlated as they cluster together.For example, dairy sour taste and crumbly texture were found to be inthe same region indicating that these two characteristics could becorelated in the mouth feel. In general, all products were characterizedby dairy flavour, smoothness and sweetness on similar basis as also il-lustrated on Fig. 4. Regarding citrus taste, this attribute was found to beapart from others on PCA hit-map, while it was only detected in frozenyogurt samples with the incorporated immobilized biocatalyst. Hence,it can be assumed that sea buckthorn berries provided this hint flavourto the product.

In general, all samples received high scores of preferences whilefrozen yogurts with sea buckthorn berries supported probiotic cells(FYB) were characterized by a citrus aroma and taste, compared tocontrol (FYC) and frozen yogurt samples with free probiotic cells (FYF)that had no such character. In addition, frozen yogurt samples withincorporated sea buckthorn berries supported probiotic cells werecharacterized by exceptional taste and good aroma while consumersshowed a significant preference compared to other frozen yogurt sam-ples. Regarding some general extra comments, a probiotic flavour wasnot found to be particularly noticeable while on the other hand allproducts were characterized by a yogurt-like favour. In addition, allevaluators referred to a high instrumental hardness especially in controlfrozen yogurt samples which can be attributed to their lower overruns(%) and more specifically to the lower air incorporated in the product,compared to commercial ice-cream products in general. The lowoverruns may have also occurred due to low storage temperatures andlack of vacuum sealing of the final products (Marshall, Goff, & Hartel,2003). Finally, all frozen yogurt samples gave high total acceptancescores and were characterized by a good total impression while nomarked off-flavours were detected.

4. Conclusions

Sea buckthorn berries were successfully incorporated as a probioticcell immobilization carrier for the production of functional frozen yo-gurt. Both frozen yogurts with incorporated free or immobilized pro-biotic cells maintained high survival rates during 90-days of frozenstorage. As a matter of fact, the achieved viabilities remained in higher

Table 4Viable cells counts of L. casei (log CFU g −1) throughout passage under simu-lated gastrointestinal conditions of frozen yogurt samples prepared by free orimmobilized on sea buckthorn berries probiotic cells from the 1st day of storageat −18 °C.

Time of incubation (min) FYB a FYF b

0 9.91 ± 0.09 9.43 ± 0.0115 9.13 ± 0.04 7.65 ± 0.0530 8.65 ± 0.04 7.06 ± 0.0460 7.96 ± 0.01 6.97 ± 0.04120 7.73 ± 0.11 6.68 ± 0.04180 7.47 ± 0.07 6.01 ± 0.13

*The results are expressed as mean (n = 3) ± standard deviations.a Frozen yogurt 3; FYB: frozen yogurt prepared with the immobilized bio-

catalyst.b Frozen yogurt 2; FYF: frozen yogurt prepared with free Lactobacillus casei

ATCC393.

A. Terpou, et al. LWT - Food Science and Technology 105 (2019) 242–249

247

levels than the threshold (106–107 CFU/mL) required, in order to conferhealth effects of a particular probiotic food during the time of con-sumption. Moreover, the novel frozen yogurt with the incorporatedimmobilized biocatalyst was characterized by exceptional sensory at-tributes as well as microbiological safety. Sea buckthorn berries infrozen yogurt not only satisfied the demand as functional ingredient,but also improved the mouthfeel of produced frozen yogurts. As a re-sult, the produced functional frozen yogurts show high commerciali-zation potential in the dairy industry as they combine the beneficialeffects of both prebiotic and probiotic characteristics.

Acknowledgments

Dr. Terpou A. would like to thank the Greek State ScholarshipsFoundation (IKY– SIEMENS) for the financial support in the frame ofher post-doc research.

References

Abdel-Hamid, M., Romeih, E., Gamba, R. R., Nagai, E., Suzuki, T., Koyanagi, T., et al.(2019). The biological activity of fermented milk produced by Lactobacillus caseiATCC 393 during cold storage. International Dairy Journal, 91, 1–8.

Abdelazez, A., Muhammad, Z., Zhang, Q. X., Zhu, Z. T., Abdelmotaal, H., Sami, R., et al.(2017). Production of a functional frozen yogurt fortified with Bifidobacterium spp.

Fig. 3. Principal component analysis (PCoA) of sensory attributes of frozen yogurt (FYC: commercial frozen yogurt as control sample, FYF: frozen yogurt preparedwith free L. casei culture, and FYB: frozen yogurt prepared with sea buckthorn berries supported probiotic cells).

Fig. 4. Sensory attributes presented as a spider chart of the sensory data analysis of frozen yogurt (FYC: commercial frozen yogurt as control sample, FYF: frozenyogurt prepared with free L. casei culture, and FYB: frozen yogurt prepared with sea buckthorn berries supported probiotic cells).

A. Terpou, et al. LWT - Food Science and Technology 105 (2019) 242–249

248

BioMed Research International, 2017, 10. https://doi.org/10.1155/2017/64385286438528.

Aguiar, L. M., Geraldi, M. V., Betim Cazarin, C. B., & Maróstica Junior, M. R. (2019).Chapter 11 - functional food consumption and its physiological effects. In M. R. S.Campos (Ed.). Bioactive compounds (pp. 205–225). Woodhead Publishing.

Akalın, A. S., Kesenkas, H., Dinkci, N., Unal, G., Ozer, E., & Kınık, O. (2018). Enrichmentof probiotic ice cream with different dietary fibers: Structural characteristics andculture viability. Journal of Dairy Science, 101(1), 37–46.

Akin, Z., & Ozcan, T. (2017). Functional properties of fermented milk produced with plantproteins. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 86,25–30.

Akın, M. B., Akın, M. S., & Kırmacı, Z. (2007). Effects of inulin and sugar levels on theviability of yogurt and probiotic bacteria and the physical and sensory characteristicsin probiotic ice-cream. Food Chemistry, 104(1), 93–99.

Aryana, K. J., & Olson, D. W. (2017). A 100-Year Review: Yogurt and other cultured dairyproducts. Journal of Dairy Science, 100(12), 9987–10013.

Attri, S., Sharma, K., Raigond, P., & Goel, G. (2018). Colonic fermentation of poly-phenolics from Sea buckthorn (Hippophae rhamnoides) berries: Assessment of effectson microbial diversity by Principal Component Analysis. Food Research International,105, 324–332.

Bal, L. M., Meda, V., Naik, S. N., & Satya, S. (2011). Sea buckthorn berries: A potentialsource of valuable nutrients for nutraceuticals and cosmoceuticals. Food ResearchInternational, 44(7), 1718–1727.

Basu, S., Banerjee, D., Chowdhury, R., & Bhattacharya, P. (2018). Controlled release ofmicroencapsulated probiotics in food matrix. Journal of Food Engineering, 238, 61–69.

Beal, C., Skokanova, J., Latrille, E., Martin, N., & Corrieu, G. (1999). Combined effects ofculture conditions and storage time on acidification and viscosity of stirred yogurt.Journal of Dairy Science, 82(4), 673–681.

Bezerra, M., Araujo, A., Santos, K., & Correia, R. (2015). Caprine frozen yoghurt producedwith fresh and spray dried jambolan fruit pulp (Eugenia jambolana Lam) andBifidobacterium animalis subsp. lactis BI-07. Lebensmittel-Wissenschaft und-Technologie- Food Science and Technology, 62(2), 1099–1104.

Bosnea, L. A., Kopsahelis, N., Kokkali, V., Terpou, A., & Kanellaki, M. (2017). Productionof a novel probiotic yogurt by incorporation of L. casei enriched fresh apple pieces,dried raisins and wheat grains. Food and Bioproducts Processing, 102, 62–71.

Bosnea, L. A., Kourkoutas, Y., Albantaki, N., Tzia, C., Koutinas, A. A., & Kanellaki, M.(2009). Functionality of freeze-dried L. casei cells immobilized on wheat grains.Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 42(10),1696–1702.

Boylston, T. D., Vinderola, C. G., Ghoddusi, H. B., & Reinheimer, J. A. (2004).Incorporation of bifidobacteria into cheeses: Challenges and rewards. InternationalDairy Journal, 14(5), 375–387.

Brown, L., Caligiuri, S. P. B., Brown, D., & Pierce, G. N. (2018). Clinical trials usingfunctional foods provide unique challenges. Journal of Functional Foods, 45, 233–238.

Cocolin, L., Dolci, P., Alessandria, V., & Rantsiou, K. (2018). Microbiology of fermenteddairy products. Reference module in life sciences. Elsevier.

Cruz, A. G., Antunes, A. E. C., Sousa, A. L. O. P., Faria, J. A. F., & Saad, S. M. I. (2009). Ice-cream as a probiotic food carrier. Food Research International, 42(9), 1233–1239.

Davidson, R. H., Duncan, S. E., Hackney, C. R., Eigel, W. N., & Boling, J. W. (2000).Probiotic culture survival and implications in fermented frozen yogurt character-istics. Journal of Dairy Science, 83(4), 666–673.

Di Criscio, T., Fratianni, A., Mignogna, R., Cinquanta, L., Coppola, R., Sorrentino, E., et al.(2010). Production of functional probiotic, prebiotic, and synbiotic ice creams.Journal of Dairy Science, 93(10), 4555–4564.

Farias, T. G. S.d., Ladislau, H. F. L., Stamford, T. C. M., Medeiros, J. A. C., Soares, B. L. M.,Stamford Arnaud, T. M., et al. (2019). Viabilities of Lactobacillus rhamnosus ASCC 290and Lactobacillus casei ATCC 334 (in free form or encapsulated with calcium alginate-chitosan) in yellow mombin ice cream. Lebensmittel-Wissenschaft und -Technologie-Food Science and Technology, 100, 391–396.

Fazilah, N. F., Ariff, A. B., Khayat, M. E., Rios-Solis, L., & Halim, M. (2018). Influence ofprobiotics, prebiotics, synbiotics and bioactive phytochemicals on the formulation offunctional yogurt. Journal of Functional Foods, 48, 387–399.

Ferraz, J. L., Cruz, A. G., Cadena, R. S., Freitas, M. Q., Pinto, U. M., Carvalho, C. C., et al.(2012). Sensory acceptance and survival of probiotic bacteria in ice cream producedwith different overrun levels. Journal of Food Science, 77(1), S24–S28.

Future Market Insights (2018). Ice-cream and frozen dessert market: Global industry analysisand opportunity assessment 2015–2025.

Goff, H. D. (2008). 65 Years of ice cream science. International Dairy Journal, 18(7),754–758.

González-Córdova, A. F., Beltrán-Barrientos, L. M., Santiago-López, L., Garcia, H. S.,Vallejo-Cordoba, B., & Hernandez-Mendoza, A. (2016). Phytate-degrading activity ofprobiotic bacteria exposed to simulated gastrointestinal fluids. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 73, 67–73.

Gunenc, A., Khoury, C., Legault, C., Mirrashed, H., Rijke, J., & Hosseinian, F. (2016).Seabuckthorn as a novel prebiotic source improves probiotic viability in yogurt.Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 66, 490–495.

Guo, R., Guo, X., Li, T., Fu, X., & Liu, R. H. (2017). Comparative assessment of phyto-chemical profiles, antioxidant and antiproliferative activities of Sea buckthorn(Hippophaë rhamnoides L.) berries. Food Chemistry, 221, 997–1003.

Hong, S. H., & Marshall, R. T. (2001). Natural exopolysaccharides enhance survival oflactic acid bacteria in frozen dairy desserts. Journal of Dairy Science, 84(6),1367–1374.

Marshall, R. T., Goff, H. D., & Hartel, R. W. (2003). Packaging, labeling, hardening andshipping. Ice cream (pp. 225–252). Boston, MA: Springer US.

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(1),169–175.

Patrignani, F., Siroli, L., Serrazanetti, D. I., Braschi, G., Betoret, E., Reinheimer, J. A., et al.(2017). Microencapsulation of functional strains by high pressure homogenization fora potential use in fermented milk. Food Research International, 97, 250–257.

Pinto, S. S., Fritzen-Freire, C. B., Muñoz, I. B., Barreto, P. L. M., Prudêncio, E. S., &Amboni, R. D. M. C. (2012). Effects of the addition of microencapsulatedBifidobacterium BB-12 on the properties of frozen yogurt. Journal of Food Engineering,111(4), 563–569.

Plessas, S., Trantallidi, M., Bekatorou, A., Kanellaki, M., Nigam, P., & Koutinas, A. A.(2007). Immobilization of kefir and Lactobacillus casei on brewery spent grains for usein sourdough wheat bread making. Food Chemistry, 105(1), 187–194.

Rezaei, R., Khomeiri, M., Kashaninejad, M., Aalami, M., & Mazaheri-Tehrani, M. (2018).Steady and dynamic rheological behaviour of frozen soy yogurt mix affected by re-sistant starch and β-glucan. International Journal of Food Properties, 20, S2688–S2695.

Romano, N., Tymczyszyn, E., Mobili, P., & Gomez-Zavaglia, A. (2016). Chapter 10 -prebiotics as protectants of lactic acid bacteria. In R. R. Watson, & V. R. Preedy (Eds.).Probiotics, prebiotics, and synbiotics (pp. 155–163). Academic Press.

Saxami, G., Ypsilantis, P., Sidira, M., Simopoulos, C., Kourkoutas, Y., & Galanis, A.(2012). Distinct adhesion of probiotic strain Lactobacillus casei ATCC 393 to rat in-testinal mucosa. Anaerobe, 18(4), 417–420.

Schoina, V., Terpou, A., Angelika-Ioanna, G., Koutinas, A., Kanellaki, M., & Bosnea, L.(2015). Use of Pistacia terebinthus resin as immobilization support for Lactobacilluscasei cells and application in selected dairy products. Journal of Food Science &Technology, 52(9), 5700–5708.

Schoina, V., Terpou, A., Bosnea, L., Kanellaki, M., & Nigam, P. S. (2018). Entrapment ofLactobacillus casei ATCC393 in the viscus matrix of Pistacia terebinthus resin forfunctional myzithra cheese manufacture. Lebensmittel-Wissenschaft und -Technologie-Food Science and Technology, 89, 441–448.

Shori, A. B. (2015). The potential applications of probiotics on dairy and non-dairy foodsfocusing on viability during storage. Biocatalysis and Agricultural Biotechnology, 4(4),423–431.

Shori, A. B. (2017). Microencapsulation improved probiotics survival during gastrictransit. HAYATI Journal of Biosciences, 24(1), 1–5.

Sidira, M., Karapetsas, A., Galanis, A., Kanellaki, M., & Kourkoutas, Y. (2014). Effectivesurvival of immobilized Lactobacillus casei during ripening and heat treatment ofprobiotic dry-fermented sausages and investigation of the microbial dynamics. MeatScience, 96(2), 948–955 Part A.

Terpou, A., Bekatorou, A., Bosnea, L., Kanellaki, M., Ganatsios, V., & Koutinas, A. A.(2018a). Wheat bran as prebiotic cell immobilisation carrier for industrial functionalFeta-type cheese making: Chemical, microbial and sensory evaluation. Biocatalysisand Agricultural Biotechnology, 13, 75–83.

Terpou, A., Bekatorou, A., Kanellaki, M., Koutinas, A. A., & Nigam, P. (2017a). Enhancedprobiotic viability and aromatic profile of yogurts produced using wheat bran(Triticum aestivum) as cell immobilization carrier. Process Biochemistry, 55, 1–10.

Terpou, A., Bosnea, L., Kanellaki, M., Plessas, S., Bekatorou, A., Bezirtzoglou, E., et al.(2018b). Growth capacity of a novel potential probiotic Lactobacillus paracasei K5strain incorporated in industrial white brined cheese as an adjunct culture. Journal ofFood Science, 83(3), 723–731.

Terpou, A., Gialleli, A.-I., Bekatorou, A., Dimitrellou, D., Ganatsios, V., Barouni, E., et al.(2017b). Sour milk production by wheat bran supported probiotic biocatalyst asstarter culture. Food and Bioproducts Processing, 101, 184–192.

Terpou, A., Gialleli, A.-I., Bosnea, L., Kanellaki, M., Koutinas, A. A., & Castro, G. R.(2017c). Novel cheese production by incorporation of sea buckthorn berries(Hippophae rhamnoides L.) supported probiotic cells. Lebensmittel-Wissenschaft und-Technologie- Food Science and Technology, 79, 616–624.

Terpou, A., Mantzourani, I., Galanis, A., Kanellaki, M., Bezirtzoglou, E., Bekatorou, A.,et al. (2018c). Employment of L. paracasei K5 as a novel potentially probiotic freeze-dried starter for feta-type cheese production. Microorganisms, 7(1), 3.

Tharmaraj, N., & Shah, N. P. (2003). Selective enumeration of Lactobacillus delbrueckii ssp.bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, bifidobacteria,Lactobacillus casei, Lactobacillus rhamnosus, and propionibacteria. Journal of DairyScience, 86(7), 2288–2296.

Wasilewska, E., Zlotkowska, D., & Wroblewska, B. (2019). Yogurt starter cultures ofStreptococcus thermophilus and Lactobacillus bulgaricus ameliorate symptoms andmodulate the immune response in a mouse model of dextran sulfate sodium-inducedcolitis. Journal of Dairy Science, 102(1), 37–53.

A. Terpou, et al. LWT - Food Science and Technology 105 (2019) 242–249

249